CN104994879A

CN104994879A - Methods of treating cancer and preventing drug resistance

Info

Publication number: CN104994879A
Application number: CN201480007964.7A
Authority: CN
Inventors: D·拉哈; J·赛特尔曼; T·R·威尔逊
Original assignee: F Hoffmann La Roche AG
Current assignee: F Hoffmann La Roche AG
Priority date: 2013-02-22
Filing date: 2014-02-21
Publication date: 2015-10-21
Also published as: MX2015010791A; EP2958592A1; CA2900097A1; JP2016509045A; BR112015018418A2; WO2014128235A1; KR20150118159A; HK1211235A1

Abstract

Provided herein are pharmaceutical products comprising therapeutically effective combinations of ALDH inhibitors (e.g., disulfiram and/or derivatives thereof) and targeted therapeutics, as well as methods of using said combinations for the treatment of cancer.

Description

The method of Therapeutic cancer and prophylactic agent resistance

Invention field

The therapy using ALDH inhibitor and target therapeutic agent treatment pathological condition (such as cancer) is provided herein.

Background of invention

Obtain the key obstacle resistance of cancer drug being remained successfully to cancer therapy relatively fast.The great efforts illustrating the molecular basis of this type of drug resistance discloses number of mechanisms, comprises medicine outflow, the acquisition of medicine binding deficient mutant of target thing, being involved in of alternative survival routes, rawly to change afterwards.Rare, the random resistance selected during it is generally acknowledged the inherent Drug therapy of this type of mechanism reflection tumor cell colonies give existence that sex-controlled inheritance changes people such as (, Cell 141 (1): 69-80 (2010)) Sharma.In cancer therapy, more and more a kind of phenomenon observed is so-called " treating response again ".Such as, some represent second response for the treatment of again EGFR TKI (people such as Kurata, Ann.Oncol.15:173-174 (2004) to nonsmall-cell lung cancer (NSCLC) patient that therapy failure is experienced better and after a while in EGFR (EGF-R ELISA) tyrosine kinase inhibitor (TKI) treatment response after " drug holiday "; The people such as Yano, Oncol.Res.15:107-111 (2005)).The similar response for the treatment of again (Cara and Tannock, Ann.Oncol.12:23-27 (2001)) is established preferably to several other anticarcinogen.This type of finds that the acquired resistance to cancer drug of prompting may involve reversible " drug resistance " state, and its manufacturing basis still needs to be established.

The existence of reversible in various human tumor cell line " Drug tolerance " cell colony has demonstrated being involved in and needing the change of the chromatin state of histone demethylase KDM5A to be maintained through the conduction of IGF-1 receptor signal.Although identify some specific resistances give property sudden change in many representing in the cancer patient of acquired drug resistance, still some is unclear to the effect of the Relative Contribution of drug resistance and each tumor cell subgroup for sudden change and not mutated mechanism.New Therapeutic Method is needed successfully to solve the intragroup heterogeneity of cancer cell and cancerous cell to the appearance of the resistance of Drug therapy.

Summary of the invention

Providing package contains the combination of ALDH inhibitor (such as disulfiram (disulfiram) and/or its derivant) and target therapeutic agent (such as TKI) herein.Be provided in the method for Therapeutic cancer in individuality herein, it comprises the target therapeutic agent of ALDH inhibitor to this individual concomitant administration effective dose and effective dose.In some embodiments, described ALDH inhibitor (such as disulfiram and/or its derivant) and the respective amount of described target therapeutic agent (such as TKI) effectively improve the effect of the treatment of cancer comprising target therapeutic agent (such as TKI).Such as, in some embodiments, with be included in there is no described ALDH inhibitor (such as disulfiram and/or its derivant) when (under described ALDH inhibitor (such as disulfiram and/or its derivant) lacks) use compared with the standard care of the described target therapeutic agent (such as TKI) of effective dose, described ALDH inhibitor (such as disulfiram and/or its derivant) and the respective amount of described target therapeutic agent (such as TKI) effectively improve effect.In some embodiments, with be included in there is no described ALDH inhibitor (such as disulfiram and/or its derivant) when (under described ALDH inhibitor (such as disulfiram and/or its derivant) lacks) use compared with the standard care of the described target therapeutic agent (such as TKI) of effective dose, described ALDH inhibitor (such as disulfiram and/or its derivant) and the respective amount of described target therapeutic agent (such as TKI) effectively improve response (such as totally linearization).In some embodiments of any described method, described ALDH inhibitor is disulfiram and/or its derivant.In some embodiments, described ALDH inhibitor is disulfiram.In some embodiments of any described method, described ALDH inhibitor is gossypol (gossypol) and/or its derivant.In some embodiments, described ALDH inhibitor is gossypol.

Also be provided in herein in individuality to improve and comprise the method for effect of the treatment of cancer of target therapeutic agent, it comprises the ALDH inhibitor of this target therapeutic agent to this individual concomitant administration effective dose and effective dose.

Be provided in the method for Therapeutic cancer in individuality herein, wherein treatment of cancer comprises the ALDH inhibitor of target therapeutic agent to this individual concomitant administration effective dose and effective dose, wherein this treatment of cancer have use this target therapeutic agent of effective dose with (under this target therapeutic agent lacks) when being included in not this target therapeutic agent standard care compared with effect of rising.In addition, be provided in herein in individuality and postpone and/or stop formation of cancer to the method for the resistance of target therapeutic agent, it comprises this target therapeutic agent of ALDH inhibitor to this individual concomitant administration effective dose and effective dose.

There is provided treatment to form the method with the individuality of cancer that raises the probability of the resistance of target therapeutic agent herein, it comprises this target therapeutic agent of ALDH inhibitor to this individual concomitant administration effective dose and effective dose.In addition, be provided in the method improving the sensitivity to target therapeutic agent in the individuality with cancer herein, it comprises this target therapeutic agent of ALDH inhibitor to this individual concomitant administration effective dose and effective dose.

In yet another aspect, be provided in the method for the period extending target therapeutic agent sensitivity in the individuality with cancer herein, it comprises the target therapeutic agent of ALDH inhibitor to this individual concomitant administration effective dose and effective dose.In addition, be provided in the method for the persistent period extending the response to target therapeutic agent in the individuality with cancer herein, it comprises this target therapeutic agent of ALDH inhibitor to this individual concomitant administration effective dose and effective dose.

In some embodiments of any described method, described ALDH inhibitor is micromolecule ALDH inhibitor.In some embodiments, described micromolecule ALDH inhibitor is disulfiram or its ALDH inhibition derivant or metabolite.In some embodiments, described ALDH inhibitor is N, N-diethyl [(diethyl thiocarbamoyl) disulphanes base] thioformamide or its pharmaceutical acceptable salts.In some embodiments, described ALDH inhibitor is N, N-diethyl [(diethyl thiocarbamoyl) disulphanes base] thioformamide.In some embodiments of any described method, described ALDH inhibitor is gossypol and/or its ALDH inhibition derivant or metabolite.In some embodiments, described ALDH inhibitor is gossypol.In some embodiments, described ALDH inhibitor is 2,2 '-two-(formoxyl-1,6,7-trihydroxy-5-isopropyl-3-methyl naphthalene) or its pharmaceutical acceptable salts.In some embodiments, described ALDH inhibitor is 2,2 '-two-(formoxyl-1,6,7-trihydroxy-5-isopropyl-3-methyl naphthalene).

In some embodiments of any described method, described target therapeutic agent is tyrosine kinase inhibitor (TKI).In some embodiments, described TKI is EGFR inhibitor, HER2 inhibitor, MET inhibitor, ALK inhibitor, BRAF inhibitor, ROS1 inhibitor and/or mek inhibitor.In some embodiments, described TKI is receptor tyrosine kinase inhibitors (RTKI).In some embodiments, described RTKI is EGFR inhibitor, HER2 inhibitor, MET inhibitor and/or ALK inhibitor.In some embodiments, described inhibitor is antibody inhibition, micromolecular inhibitor, Binding peptide inhibitor and/or polynucleotide antagonist.In some embodiments, described TKI is N-(3-ethynyl phenyl)-6,7-bis-(2-methoxy ethoxy) quinazoline-4-amine or its pharmaceutical acceptable salts (such as Erlotinib (erlotinib)).In some embodiments; described TKI is N-(4-(3-fluorine benzyloxy)-3-chlorphenyl)-6-(5-((2-(methyl sulphonyl) ethylamino) methyl) furan-2-base) quinazoline-4-amine, two 4-toluene sulfonic acide esters or its pharmaceutical acceptable salts (such as Lapatinib (lapatinib)).In some embodiments, described TKI is (S)-N-(2,3-dihydroxypropyl)-3-(the fluoro-4-idodophenylamino of 2-) Pyrazinamide or its pharmaceutical acceptable salts (such as AS703026).In some embodiments, described TKI is Wei Luofeini (vemurafenib).In some embodiments, described TKI is 3-((R)-1-(the chloro-3-fluorophenyl of 2,6-bis-) ethyoxyl)-5-(1-(piperidin-4-yl)-1H-pyrazoles-4-base) pyridine-2-amine or its pharmaceutical acceptable salts (such as gram azoles is for Buddhist nun (crizotinib)).

In some embodiments of any described method, described cancer is gastric cancer, pulmonary carcinoma (such as nonsmall-cell lung cancer (NSCL)), colorectal carcinoma (such as colon cancer and/or rectal cancer) or basal cell carcinoma.

In one embodiment, pharmaceutical products is provided, it comprises a) as the ALDH inhibitor of the effective dose of the first composition, and b) as the targeting agent (target therapeutic agent) of the effective dose of the second composition, for or sequentially to make for Therapeutic cancer.

In another embodiment, provide pharmaceutical products described above, wherein said ALDH inhibitor is micromolecule ALDH inhibitor.In another embodiment, provide pharmaceutical products described above, wherein said ALDH inhibitor is disulfiram or its ALDH inhibition derivant or metabolite.In another embodiment, provide pharmaceutical products described above, wherein said ALDH inhibitor is N, N-diethyl [(diethyl thiocarbamoyl) disulphanes base] thioformamide or its pharmaceutical acceptable salts.In another embodiment, provide pharmaceutical products described above, wherein said ALDH inhibitor is N, N-diethyl [(diethyl thiocarbamoyl) disulphanes base] thioformamide.In another embodiment, provide pharmaceutical products described above, wherein said ALDH inhibitor is 2,2 '-two-(formoxyl-1,6,7-trihydroxy-5-isopropyl-3-methyl naphthalene) or its pharmaceutical acceptable salts.In another embodiment, provide pharmaceutical products described above, wherein said ALDH inhibitor is 2,2 '-two-(formoxyl-1,6,7-trihydroxy-5-isopropyl-3-methyl naphthalene).

In another embodiment, provide pharmaceutical products described above, wherein said target therapeutic agent is tyrosine kinase inhibitor (TKI).In another embodiment, provide pharmaceutical products described above, wherein said TKI is EGFR inhibitor, HER2 inhibitor, MET inhibitor, ALK inhibitor, BRAF inhibitor, ROS1 inhibitor and/or mek inhibitor.In another embodiment, provide pharmaceutical products described above, wherein said TKI is receptor tyrosine kinase inhibitors (RTKI).In another embodiment, provide pharmaceutical products described above, wherein said RTKI is EGFR inhibitor, HER2 inhibitor, MET inhibitor and/or ALK inhibitor.In another embodiment, provide pharmaceutical products described above, wherein said inhibitor is antibody inhibition, micromolecular inhibitor, Binding peptide inhibitor and/or polynucleotide antagonist.In another embodiment, provide pharmaceutical products described above, wherein said TKI is N-(3-ethynyl phenyl)-6,7-bis-(2-methoxy ethoxy) quinazoline-4-amine or its pharmaceutical acceptable salts, particularly Erlotinib.In another embodiment; pharmaceutical products described above is provided; wherein said TKI is N-(4-(3-fluorine benzyloxy)-3-chlorphenyl)-6-(5-((2-(methyl sulphonyl) ethylamino) methyl) furan-2-base) quinazoline-4-amine; two 4-toluene sulfonic acide esters or its pharmaceutical acceptable salts, particularly Lapatinib.In another embodiment, pharmaceutical products described above is provided, wherein said TKI is (S)-N-(2,3-dihydroxypropyl)-3-(the fluoro-4-idodophenylamino of 2-) Pyrazinamide or its pharmaceutical acceptable salts, particularly AS703026.In another embodiment, provide pharmaceutical products described above, wherein said TKI is Wei Luofeini.In another embodiment, pharmaceutical products described above is provided, wherein said TKI is 3-((R)-1-(2, the chloro-3-fluorophenyl of 6-bis-) ethyoxyl)-5-(1-(piperidin-4-yl)-1H-pyrazoles-4-base) pyridine-2-amine or its pharmaceutical acceptable salts, particularly gram azoles be for Buddhist nun.

In another embodiment, provide pharmaceutical products described above, wherein said cancer is gastric cancer, pulmonary carcinoma, nonsmall-cell lung cancer (NSCL), colon cancer and/or rectal cancer or basal cell carcinoma.

Thering is provided beyond the improved treatment for cancer, compared with the quality of life experienced with the same patient accepting different treatment, using the quality of life that some combination described herein can improve patient.Such as, if only accept described target therapeutic agent as therapy with same patient, compared with the quality of life that can experience, the combination of using target therapeutic agent described herein (such as TKI) and ALDH inhibitor (such as disulfiram and/or its derivant) to individuality can provide the quality of life of improvement.Such as, the combination treatment of combination described herein can reduce the targeted therapy agent dose of needs, alleviates the side effect relevant with described therapeutic agent (such as Nausea and vomiting, alopecia, erythra, appetite reduce, weight saving, etc.) thus.Described combination also can cause tumor load reduce and adverse events of being correlated with (such as pain, organ dysfunction, weight saving, etc.) reduce/alleviate.Thus, one aspect of the present invention provides ALDH inhibitor (such as disulfiram and/or its derivant), and it is for the therapeutic use of improvement by the quality of life of the patient of target therapeutic agent (such as TKI) Therapeutic cancer.Thus, another aspect of the present invention provides ALDH inhibitor (such as disulfiram and/or its derivant), and it is for the therapeutic use of the quality of life of the individuality of improvement target therapeutic agent or its pharmaceutical acceptable salts Therapeutic cancer disease.

Accompanying drawing is sketched

Figure 1A-C.| Drug tolerance stomach cancer cell expresses high-caliber ALDH1A1.(A) Aldefluor algoscopy (Stem Cell Technology) is used to measure ALDH activity Kato II parent and Ke azoles in Buddhist nun (1uM) tolerant cells.Substrate emitting fluorescence when being oxidized to respective acids by ALDH of bodipy labelling.Photo display gram azoles processes rear ALDH for Buddhist nun ^highthe enrichment of cell.Arrow mark shows the ALDH in parental population ^highcell.(B) the rna expression level based on microarray assays 19 ALDH family members of oligonucleotide is used.Kato II parental cell and Aldefluor substrate one are arised from 37 DEG C of incubations 30, and by flow cytometry sorting ALDH ^highand ALDH ^lowcell.Use from ALDH ^highand ALDH ^lowthe RNA of cell separation implements gene expression analysis.Rod figure presents ALDH ^highthe differential expression of an only ALDH family member (ALDH1A1) in cell.(C) immunoblotting presents ALDH ^highcell neutralization gram azoles in Buddhist nun's toleration Kato II and GTL-16 cell respectively with ALDH ^lowcell compares the ALDH1A1 protein of more high expression level with parental cell.

Fig. 2 A-C.| ALDH inhibitor disulfiram eliminates drug resistance sexual cell.Parent Kato II (A) and GTL-16 (B) cell 25 days is processed for Buddhist nun with 1uM gram of azoles, and at the 1st day (d1) or gram azoles for the different time interval interpolation disulfiram during Buddhist nun's process, 200nM.(C) with Erlotinib and disulfiram, 200nM process parent PC9 cell, the different time points during Erlotinib process is added.The excellent figure of quantitative measurement presented from the often kind of process same form three hole shows the lethal effect of disulfiram to drug resistance sexual cell, as people such as (, 2011) Wilson that measured by Syto60 viability algoscopy.Data state the mark of non-processor contrast as, error bar reflection SEM value.

Fig. 3 A-B.| disulfiram kills the drug resistance sexual cell of kinds cancer type.(A) present disulfiram and targeted cancer drugs combination to originate to various tissue, depend on the effect of the cancerous cell of different carcinoma gene.Individually or with 200nM-300nM disulfiram in combination with the cancerous cell that suitable drugs process is responsive to Erlotinib (HCC827 and HCC4006), Lapatinib (HCC1419, SKBR3 and MDA-MB-175v2), mek inhibitor AS703026 (A549 and EBC-1) and BRAF inhibitor Wei Luofeini (Colo-205).The process persistent period, from 11 to 25 days, depends on that the time of nearly all drug resistance sexual cell needs is killed in the process of TKI+ disulfiram.(B) present targeted cancer drugs, the dependency of survival to ALDH that the excellent figure of quantitative measurement (the often kind of process same form three hole) (as by the measurement of Syto60 viability algoscopy) of effect of disulfiram and combination thereof presents generally speaking drug resistance sexual cell.Data state the mark of non-processor contrast as, error bar reflection SEM value.

Fig. 4 A-C.| the mitochondrial respiratory raised in drug resistance sexual cell and ROS level.(A) H based on fluorescein is used ₂dCFDA reagent (Molecular Probes) detects and measures ROS level by flow cytometry.Under TKI exists, process PC9 with disulfiram (200nM) and NAC (5mM) derive DTP and GTL-16 and derive DTP and reach 48 hours, and measure the effect of TKI, disulfiram and NAC.The excellent figure that the multiple presenting ROS level compared with untreated parental cell changes presents the effect of ALDH as ROS street cleaner.(B) use Seahorse XF 96 to measure GTL-16 and PC9 and derive the oxygen consumption rate (OCR) of DTP and thin outer acidification rate (ECAR) to measure respectively by mitochondrial respiratory and the generation of glucolytic energy.Rod figure presents the use rising that drug resistance sexual cell Mitochondria is breathed.(C) immunoblotting presents the double-strand DNA cleavage and the activation of DNA repair mechanism that raise in GTL-16 and PC9 drug resistance sexual cell, as the result of high ROS level.

Fig. 5 A-C.| ROS street cleaner's NAC reverses the effect of disulfiram.Process (A) PC9 and (B) GTL-16 parental cell 15 days with Erlotinib and gram azoles for Buddhist nun respectively in combination individually or with disulfiram, NAC and disulfiram+NAC.Describe these process and NAC redemption disulfiram is presented to the ability of the lethal effect of cell viability to the excellent figure of the effect of cell viability.(C1-2) 48 hours are reached with the DTP that disulfiram and NAC process GTL-16 derive.Immunoblotting data represents NAC to the reverse of effect of disulfiram to the PARP level of γ H2A.x, BimEL, BimS and cutting.Disulfiram improves ROS level and apoptosis-induced in DTP.

Fig. 6 A-B.| disulfiram postpones tumor recurrence.(A) individually or with disulfiram in combination with Erlotinib process PC9 parental cell.TKI process, after 6 days, allows that when being with or without disulfiram DTP grows in not containing the growth medium of Erlotinib.The PC9 that cell viability data exhibiting head accepts disulfiram for 6 days derives the growth delay of DTP, and those the growth delay continuing to accept disulfiram for follow-up 4 days is more of a specified duration.Rod figure shows DS derives DTP effect to PC9, states average +/-SD as from the same form three hole measurement.(B) in body data display Erlotinib with in the xenograft mouse model of disulfiram combined treatment compared with independent Erlotinib the PC9 derivative tumors of delay recur.

Fig. 7 A-C.| disulfiram pretreatment is not enough to kill all DTP.First with DS process PC9 (A) and GTL-16 (B) parental cell 3 or 6 days, then under DS disappearance with Erlotinib process PC9 cell, replace Buddhist nun to process GTL-16 cell with gram azoles.The of short duration DS of being exposed to of Syto60 cell viability dyeing display reduces DTP number but does not eliminate them.(C) in GLT16 cell, strike separately low ALDH1A1 have no significant effect for Buddhist nun's drug susceptibility gram azoles.Figure shows the relative expression of ALDH1A1 in the GTL16 cell using multiple shRNA.Table display gram azoles processes for Buddhist nun the relative percentage that rear ALDH1A1 strikes the DTP that GTL16 derives in low cell.

Fig. 8 A-C.| use multiple TKI inhibitor, drug treating induces multiple ALDH family member to express in various kinds of cell system.(A) GTL16 derives DTP (processing parental cell for Buddhist nun with gram azoles) derives the rna expression of ALDH family member in DTP (with Erlotinib process parental cell) relative change with PC9.(B) GTL16 parental cell and GTL16 derive the rna expression level that DTP (processing parental cell for Buddhist nun with gram azoles) and PC9 parental cell and PC9 derive ALDH family member in DTP (with Erlotinib process parental cell).(C) ALDH1A1 derives rise compared with GTL-16 parental cell in DTP (processing parental cell for Buddhist nun with gram azoles) at GTL-16.On the contrary, ALDH1A1 expresses to derive in DTP (with Erlotinib process parental cell) or PC9 parental cell at PC9 and does not significantly express, and the expression of ALDH1A1 derives in DTP (with Erlotinib process parental cell) at PC9 do not have significant change compared with PC9 parental cell.

Fig. 9.| ALDH inhibitor gossypol significantly reduces drug resistance sexual cell.(A) 2uM Erlotinib and 1.5uM gossypol process parent PC9 cell 8 days is used either individually or in combination.(B) either individually or in combination with 1uM gram of azoles for Buddhist nun and 1.5uM gossypol process parent GTL-16 cell 17 days.Present that to show gossypol by Syto60 algoscopy from the excellent figure of quantitative measurement of the cell viability in the often kind of process same form three hole similar to DS but more weak to the effect of drug resistance cell.

Detailed Description Of The Invention

I. define

As used in this article, term " ALDH " or " aldehyde dehydrogenase " refer to one or the class of enzymes that can be oxidized aldehyde.Aldehyde dehydrogenase (ALDH) (enzyme name 1.2.1.3) is responsible for aldehyde in oxidation born of the same parents and the enzyme played a role in alcohol metabolism, vitamin A, cyclophosphamide and other oxygen nitrogen phosphine (oxazaphosphorine).The example of the ALDH enzyme in people comprises ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, ALDH1L1, ALDH1L2, ALDH2, ALDH3A1, ALDH3A2, ALDH3B1, ALDH3B2, ALDH4A1, ALDH5A1, ALDH6A1, ALDH7A1, ALDH8A1, ALDH9A1, ALDH16A1, ALDH18A1.Term " wild type ALDH " refers generally to the polypeptide of the aminoacid sequence comprising natural generation ALDH protein.

Term " HER2 ", " ErbB2 " and " c-Erb-B2 " are used interchangeably.Except as otherwise noted, term " ErbB2 ", " c-Erb-B2 " and " HER2 " are referring to human protein for during this paper, and " erbB2 ", " c-erb-B2 " and " her2 " refer to people's gene.People erbB2 gene and ErbB2 protein are recorded in the people such as such as Semba, the people such as PNAS (USA) 82:6497-6501 (1985) and Yamamoto, Nature 319:230-234 (1986) (Genebank accession number X03363).ErbB2 comprises four territories (territory 1-4).Term " wild type HER2 " refers generally to the polypeptide of the aminoacid sequence comprising natural generation HER2 protein.

" EGFR " means receptor tyrosine kinase polypeptide EGF-R ELISA, and it is recorded in the people such as Ullrich, Nature (1984) 309:418425, or is called Her-1 and c-erbB gene outcome, and its variant, such as EGFRvIII.The variant of EGFR also comprises deletion, substitutes and inserts variant, the people (NEJM 2004,350:2129) such as such as Lynch, the people such as Paez (Science 2004,304:1497), record in the people such as Pao (PNAS 2004,101:13306) those.Term " Wild type EGFR " refers generally to the polypeptide of the aminoacid sequence comprising natural generation EGFR protein.

Except as otherwise noted, as used in this article, no matter natural or synthesis term " c-met " or " Met " refer to any natural or variation () c-met polypeptide.Term " wild type c-met " refers generally to the polypeptide of the aminoacid sequence comprising natural generation c-met protein.

Except as otherwise noted, as used in this article, no matter natural or synthesis term " BRAF " refer to any natural or variation () BRAF polypeptide.Term " wild type BRAF " refers generally to the polypeptide of the aminoacid sequence comprising natural generation BRAF protein.

Term " ALK " refers to anaplastic lymphoma kinase ALK Alk receptor tyrosine kinase.ALK (anaplastic lymphoma kinase ALK Alk receptor tyrosine kinase) (GenBank accession number: AB209477, UniProt accession number: Q9UM73) is a kind of receptor tyrosine kinase.This protein (it is long 1620 aminoacid in people) has the membrane-spanning domain in central part and has c-terminus tyrosine kinase district and amino terminal extracellular foreign lands (Oncogene, 1997Jan.30; 14 (4): 439-49).Relate to the comprehensive review of ALK see people such as Pulford, J.of Cellular Physiol., 199:330-358,2004.Total length ALK sequence is disclosed in U.S. Patent No. 5, and 770,421.Term " wild type ALK " refers generally to the polypeptide of the aminoacid sequence comprising natural generation alk protein matter.

" antagonist " of polypeptide of interest (interchangeable be called " inhibitor ") is activation or the function of interference polypeptide of interest, such as, partially or completely block, suppress or neutralize the medicament of the biologic activity mediated by polypeptide of interest.Such as, the antagonist of polypeptide X can refer to any molecule partially or completely blocking, suppress or neutralize the biologic activity mediated by polypeptide X.The example of inhibitor comprises antibody; Ligand antibody; Small molecular antagonists; Antisense and inhibitory RNA (such as shRNA) molecule.Preferably, inhibitor is antibody in conjunction with polypeptide of interest or micromolecule.In one particular embodiment, inhibitor has about 1,000nM or less binding affinity to polypeptide of interest (dissociation constant).In another embodiment, inhibitor has about 100nM or less binding affinity to polypeptide of interest.In another embodiment, inhibitor has about 50nM or less binding affinity to polypeptide of interest.In one particular embodiment, inhibitor is covalently bond to polypeptide of interest.In one particular embodiment, inhibitor is with 1,000nM or less IC ₅₀suppress the intracellular signaling of polypeptide of interest.In another embodiment, inhibitor is with 500nM or less IC ₅₀suppress the intracellular signaling of polypeptide of interest.In another embodiment, inhibitor is with 50nM or less IC ₅₀suppress the intracellular signaling of polypeptide of interest.In certain embodiments, antagonist is by the expression of polypeptide of interest or biologic activity reduction or suppression at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.

As used in this article, term " target therapeutic agent " refers in conjunction with polypeptide of interest and suppresses the activity of specific polypeptide of interest and/or the therapeutic agent of activation.The example of this type of medicament comprises antibody in conjunction with polypeptide of interest and micromolecule.In some embodiments, described target therapeutic agent is TKI.In some embodiments, described TKI is RTKI.

" tyrosine kinase inhibitor " or " TKI " refers to the activation disturbing tyrosine kinase or the function mediated by the tyrosine kinase activity of tyrosine kinase, such as, partially or completely block, suppress or neutralize the medicament of the biologic activity mediated by the tyrosine kinase activity of tyrosine kinase.

" receptor tyrosine kinase inhibitors " or " RTKI " refers to the activation disturbing receptor tyrosine kinase or the function mediated by the tyrosine kinase activity of receptor tyrosine kinase, such as, partially or completely block, suppress or neutralize the medicament of the biologic activity mediated by the tyrosine kinase activity of receptor tyrosine kinase.

Except as otherwise noted, as used in this article, term " polypeptide " refers to any natural polypeptide of interest from any vertebrate origin (comprising mammal, such as primate (such as people) and rodent (such as Mouse and rat)).This term is contained " total length ", undressed polypeptide and is derived from any type of polypeptide processed in cell.The natural generation variant of polypeptide also contained in this term, such as splice variant or allelic variant.

" polynucleotide " or " nucleic acid " are used interchangeably in this article, refer to the nucleotide polymer of any length, comprise DNA and RNA.Nucleotide can be deoxyribonucleotide, ribonucleotide, the nucleotide passing through modification or base and/or its analog, or by DNA or RNA polymerase, or any substrate in polymer is mixed by synthetic reaction.Polynucleotide can comprise the nucleotide through modifying, such as methylated nucleotide and analog thereof.If exist, can carry out before or after assembling polymer the modification of nucleotide structure.Nucleotide sequence can be interrupted by non-nucleotide component.Polynucleotide can be modified in post synthesis further, such as by puting together with label.The modification of other type comprises such as " cap ", one or more naturally occurring nucleotide analog is substituted, modify between nucleotide and such as such as there is neutral connection (such as methyl phosphonate, phosphotriester, phosphoramidate (phosphoamidate), carbamate etc.) and there is electrically charged connection (such as thiophosphate, phosphorodithioate etc.) modification, containing pendency module (pendant moiety) such as such as protein (such as nuclease, toxin, antibody, signal peptide, polylysine etc.) modification, there is intercalator (such as acridine, psoralen etc.) modification, containing chelating agen (such as metal, radioactive metal, boron, oxidisability metal etc.) modification, modification containing alkylating agent, there is the modification of modified connection (such as α anomeric nucleic acid (anomeric nucleic acid) etc.), and the polynucleotide of unmodified form.In addition; usually any oh group be present in saccharide can be replaced with such as phosphonic acids (phosphonate) group, phosphoric acid (phosphate) group; protect with standard protecting group; or activation is connected with other of other nucleotide with preparation, or solid or semi-solid support can be conjugated to.Can phosphorylation or add cap group module with amine or 1-20 carbon atom organic and replace 5 ' and 3 ' end OH.Other hydroxyl also can be derivatized to standard protecting group.The analog form of the ribose that polynucleotide also generally can be known containing this area or deoxyribose saccharide, comprise such as 2 '-oxygen-methyl-, 2 '-oxygen-pi-allyl-, 2 '-fluoro-or 2 '-nitrine-ribose, carba sugars, α-anomeric sugar, epimerism sugar is arabinose, xylose or lyxose, pyranose, furanose, sedoheptulose such as, acyclic analog and dealkalize yl nucleosides analog such as methylribonucleotide.Available alternative linking group is replaced one or more di-phosphate ester and is connected.These alternative linking groups include but not limited to following embodiment, wherein phosphate ester P (O) S (" thioester " (thioate)), P (S) S (" dithioester " (dithioate)), (O) NR ₂(" carboxylic acid amide esters " (amidate)), P (O) R, P (O) OR ', CO or CH ₂(" dimethoxym ethane " (formacetal)) substitutes, wherein R or R ' is independent is separately H or substituted or unsubstituted alkyl (1-20 C), optionally containing ether (-O-) connection, aryl, thiazolinyl, cyclic hydrocarbon radical, cycloalkenyl group or aryl (araldyl).All connections not in polynucleotide are all necessarily identical.Aforementioned description is applicable to all polynucleotide mentioned in this article, comprises RNA and DNA.

Term " micromolecule " refers to have about 2000 dalton or less, preferably any molecule of about 500 dalton or less molecular weight.

" separation " antibody refers to the antibody separated with the component of its natural surroundings.In some embodiments, antibody purification, to the purity being greater than 95% or 99%, measures as passed through such as electrophoresis (such as SDS-PAGE, isoelectrofocusing (IEF), capillary electrophoresis) or chromatography (such as ion exchange or reversed-phase HPLC).About the summary of the method for assessment of antibody purity, see the people such as such as Flatman, J.Chromatogr.B 848:79-87 (2007).

Term " antibody " herein uses with most broad sense, and contain various antibody structure, include but not limited to monoclonal antibody, polyclonal antibody, multi-specificity antibody (such as bi-specific antibody) and antibody fragment, as long as they show the antigen-binding activity of expectation.

Term " anti-polypeptide of interest antibody " and " antibody in conjunction with polypeptide of interest " refer to can with enough affinitys in conjunction with polypeptide of interest, makes this antibody can be used as diagnostic agent and/or the therapeutic agent antibody for targeting polypeptide of interest.In one embodiment, according to the measurement such as by radioimmunoassay (RIA), the degree of protein that anti-polypeptide of interest antibodies has nothing to do, non-polypeptide of interest is less than this antibody to about 10% of the combination of polypeptide of interest.In certain embodiments, the antibody in conjunction with polypeptide of interest has≤1 μM ,≤100nM ,≤10nM ,≤1nM ,≤0.1nM ,≤0.01nM or≤0.001nM (such as 10 ^-8m or less, such as 10 ^-8m to 10 ^-13m, such as 10 ^-9m to 10 ^-13m) dissociation constant (Kd).In certain embodiments, the polypeptide of interest epi-position that anti-polypeptide of interest antibodies is conservative in from the polypeptide of interest of different plant species.

" barrier " antibody or " Antagonism " antibody are the antibody of the biologic activity suppressing or reduce the antigen that it combines.Preferred blocking antibody or antagonistic antibodies are substantive or suppress the biologic activity of antigen completely.

" affinity " refers to single binding site and its intensity in conjunction with noncovalent interaction summation whole between spouse's (such as antigen) of molecule (such as antibody).Unless otherwise directed, as used in this article, " binding affinity " refers to reflect in conjunction with the interactional inherent binding affinity of 1:1 between right member's (such as antibody and antigen).Molecule X can state with dissociation constant (Kd) usually to the affinity of its spouse Y.The common method that affinity can be known by this area is measured, and comprises method described herein.Described below is the illustrated specifically for measuring binding affinity and exemplary embodiment.

" antibody fragment " refers to the molecule different from complete antibody, and it comprises the part in conjunction with the antigen of complete antibody combination in complete antibody.The example of antibody fragment includes but not limited to Fv, Fab, Fab ', Fab '-SH, F (ab ') ₂; Double antibody; Linear antibodies; Single-chain antibody molecules (such as scFv); With the multi-specificity antibody formed by antibody fragment.

With refer to the antibody with reference to antibody, the combination of its antigen being blocked to 50% or more in competition assay with reference to antibody " antibody in conjunction with identical epi-position ", and contrary, in competition assay, this antibody is blocked 50% or more to the combination of its antigen with reference to antibody.

Term " is fitted together to " antibody and refers to that a part for weight wherein and/or light chain derives from specific source or species, and the remainder of heavy and/or light chain is from separate sources or the derivative antibody of species.

Term " full length antibody ", " complete antibody " and " whole antibody " are used interchangeably in this article, refer to the antibody having substantially similar structure with native antibody structure or have the heavy chain containing Fc district.

As used in this article, term " monoclonal antibody " refers to the antibody obtained from the antibody of a group homogeneity substantially, namely each antibody forming colony is identical and/or in conjunction with identical epi-position, except such as containing except naturally occurring sudden change or the possible variant antibodies that occurs between the generation of monoclonal antibody preparations, this type of variant is generally with indivisible existence.Different from the polyclonal antibody preparations usually comprised for the different antibodies of different determinant (epi-position), each monoclonal antibody of monoclonal antibody preparations is for the single determinant on antigen.So, modifier " monoclonal " indicate antibody from a group substantially homogeneity antibody obtain characteristic, and should not be construed as require generate antibody by any ad hoc approach.Such as, the monoclonal antibody that will use according to the present invention can be generated by multiple technologies, include but not limited to that hybridoma method, recombinant DNA method, phage display method and utilization contain the method for the transgenic animal of all or part human immunoglobulin gene seat, for generating these class methods and other exemplary methods of monoclonal antibody.

" people's antibody " refer to have with by people or people's Hemapoiesis or the antibody of aminoacid sequence that the aminoacid sequence of the antibody that utilizes people's antibody repertoire or other people's antibody coding sequence to derive from inhuman source is corresponding.This definition clear-cut of people's antibody gets rid of the humanized antibody comprising inhuman antigen binding residues.

" humanization " antibody refers to the chimeric antibody comprised from the amino acid residue of inhuman HVR and the amino acid residue from people FR.In certain embodiments, humanized antibody can comprise at least one, usual two whole variable domains substantially, wherein all or substantially all HVR (such as CDR) corresponding to those of non-human antibody, and all or substantially all FR correspond to those of people's antibody.Optionally, humanized antibody at least can comprise a part for the antibody constant region derived from people's antibody.Antibody, " the humanization form " of such as non-human antibody refers to experience humanized antibody.

" immunoconjugates " refers to and one or more heterologous molecule, includes but not limited to the antibody that cytotoxic agent is puted together.

" individual response " or " response " can use any end-point assessment of instruction to individual benefit, includes but not limited to: (1) suppresses progression of disease (such as cancer progression) to a certain extent, comprises and slows down and block completely; (2) tumor size is reduced; (3) (namely alleviate, slow down or stop completely) cancer cell infiltration is suppressed to enter to close on peripheral organs and/or tissue; (4) (namely alleviate, slow down or stop completely) transfer is suppressed; (5) one or more symptoms relevant with disease or disease (such as cancer) are alleviated to a certain extent; (6) extension of progresson free survival; And/or the mortality rate of (8) treatment point rear preset time reduces.

Term " substantially the same " is for representing sufficiently high similarity degree between two numerical value during this paper, so that those skilled in the art will think having by the difference in the biological characteristics background measured by described numerical value (such as Kd value or express) between two numerical value very little or do not have biology and/or significance,statistical.As the function of reference/fiducial value, the difference between described two numerical value is such as less than about 50%, is less than about 40%, is less than about 30%, be less than about 20%, and/or be less than about 10%.

Phrase " substantive different " is for representing sufficiently high difference degree between two numerical value during this paper, so that those skilled in the art will think having significance,statistical by the difference in the biological characteristics background measured by described numerical value (such as Kd value) between two numerical value.As with reference to/compare the function of numerator value, the difference between described two numerical value is such as greater than about 10%, is greater than about 20%, is greater than about 30%, be greater than about 40%, and/or be greater than about 50%.

" effective dose " of substances/molecules (such as pharmaceutical composition) refers at required dosage and effectively realizes the amount for the treatment of or the prevention result expected the time period.

" the treatment effective dose " of substances/molecules can cause the factors such as the ability expecting response according to such as individual morbid state, age, sex and weight and this substances/molecules and change in individuality.Treatment effective dose also refers to that the treatment beneficial effect of substances/molecules surpasses the amount of any poisonous or deleterious effects." prevention effective dose " refers in required dosage and the amount effectively realizing the prevention result expected the time period.Typically but not necessarily, because preventive dose is before disease or at disease early stage for experimenter, therefore prevent effective dose will lower than treatment effective dose.

Term " pharmaceutical formulation " refers to that its form allows that the biologic activity of the active component wherein contained is effective, and does not contain the preparation of other composition experimenter that can use this preparaton being produced to unacceptable toxicity.

" pharmaceutical acceptable carrier " refers to composition nontoxic to experimenter except active component in pharmaceutical formulation.Pharmaceutical acceptable carrier includes but not limited to buffer agent, excipient, stabilizing agent or antiseptic.

As used in this article, " pharmaceutically acceptable salt " refers to that the pharmacy of compound can accept organic or inorganic salt.

As used in this article, " treatment/process " (and grammatical variants) refers to attempt to change the clinical intervention that the nature process of individuality is treated by institute, can be to prevent or carrying out in the process of clinical pathology.The desired effects for the treatment of include but not limited to prophylactic generation or recurrence, relief of symptoms, any direct or indirect pathological consequences of weakening disease, prevention transfer, slow down progression of disease speed, improve or the state and exempt or improve prognosis of palliating a disease.In some embodiments, use antibody of the present invention to postpone the formation of disease or to slow down the progress of disease.

Term " anti-cancer therapies " refers to therapy useful in Therapeutic cancer.The example of anticancer therapeutic agent includes but not limited to the medicament of medicament, antiangiogenic agent, apoptosis agent, antitublin and other Therapeutic cancer used in such as chemotherapeutics, growth inhibitor, cytotoxic agent, X-ray therapy, anti-CD 20 antibodies, platelet derived growth factor inhibitor (such as Gleevec ^tM(Imatinib Mesylate)), cox 2 inhibitor (such as celecoxib), interferon, cytokine, in conjunction with the antagonist (such as neutrality antibody) (PDGFR-β, BlyS, APRIL, BCMA receptor, TRAIL/Apo2) of one or more following target things and other biological activity and organic chemistry agent, etc.The present invention also comprises their combination.

Term " cytotoxic agent " is suppressing for referring to during this paper or prevent cell function and/or cause the material of cell death or destruction.This term intention comprises radiosiotope (such as At ²¹¹, I ¹³¹, I ¹²⁵, Y ⁹⁰, Re ¹⁸⁶, Re ¹⁸⁸, Sm ¹⁵³, Bi ²¹², P ³², Pb ²¹², with the radiosiotope of Lu), chemotherapeutics or chemotherapeutics (such as methotrexate (methotrexate), amycin (adriamicin), vinca alkaloids (vinca alkaloids) (vincristine (vincristine), vinblastine (vinblastine), etoposide (etoposide)), doxorubicin (doxorubicin), melphalan (melphalan), mitomycin (mitomycin) C, chlorambucil (chlorambucil), daunorubicin (daunorubicin) or other intercalator), growth inhibitor, enzyme and fragment thereof, such as nucleolytic enzyme, antibiotic, and toxin, such as small molecule toxins or antibacterial, fungus, the enzyme toxin alive of plant or animal origin, comprise its fragment and/or variant, and the various antineoplastic agent hereafter disclosed or anticarcinogen.Hereafter describe other cytotoxic agent.Kill the destruction that tumor guiding drug plays tumor cell.

" chemotherapeutics " refers to the chemical compound that can be used for Therapeutic cancer.The example of chemotherapeutics comprises alkylating agent class (alkylating agents), such as phosphinothioylidynetrisaziridine (thiotepa) and endoxan (cyclosphosphamide) Alkyl sulfonate esters class (alkyl sulfonates), such as busulfan (busulfan), Improsulfan (improsulfan) and piposulfan (piposulfan); Aziridines (aziridines), such as Benzodepa (benzodopa), card ripple quinone (carboquone), U.S. appropriate in sending (meturedopa) and uredepa (uredopa); Ethylenimine class (ethylenimines) and methylmelamine class (methylamelamines), comprise hemel (altretamine), triethylenemelamine (triethylenemelamine), APO (triethylenephosphoramide), TESPA (triethylenethiophosphoramide) and trimethylolmelamine (trimethylomelamine); Annonaceousacetogenicompounds (acetogenin) (especially bullatacin (bullatacin) and bullatacin ketone (bullatacinone)); Delta-9-Tetrahydrocannabinol (tetrahydrocannabinol) (Dronabinol (dronabinol), ); β-lapachol (lapachone); Lapachol (lapachol); Colchicine class (colchicines); Betulic acid (betulinicacid);Camptothecine (camptothecin) (comprises synthetic analogues Hycamtin (topotecan) CPT-11 (Irinotecan (irinotecan), ), acetyl camptothecine, scopoletin (scopolectin) and 9-aminocamptothecin), bryostatin (bryostatin), callystatin, CC-1065 (comprising its Adozelesin (adozelesin), Carzelesin (carzelesin) and Bizelesin (bizelesin) synthetic analogues), podophyllotoxin (podophyllotoxin), podophyllic acid (podophyllinic acid), Teniposide (teniposide), hidden algae element class (cryptophycins) (particularly hidden algae element 1 and hidden algae element 8), dolastatin (dolastatin), duocarmycin (comprising synthetic analogues, KW-2189 and CB1-TM1), Eleutherobin (eleutherobin), pancratistatin, sarcodictyin, sponge chalone (spongistatin), nitrogen mustards (nitrogen mustards), such as Chlorambucil (chlorambucil), Chlornaphazine (chlornaphazine), courage phosphamide (chlorophosphamide), Estramustine (estramustine), ifosfamide (ifosfamide), chlormethine (mechlorethamine), mustron (mechlorethamine oxide hydrochloride), melphalan (melphalan), novoembichin (novembichin), phenesterin (phenesterine), prednimustine (prednimustine), Trofosfamide (trofosfamide),Uracil mastard (uracil mustard), nitrosourea (nitrosoureas), such as BCNU (carmustine), chlorozotocin (chlorozotocin), Fotemustine (fotemustine), lomustine (lomustine), Nimustine (nimustine) and Ranimustine (ranimnustine), antibiotics, such as Enediyne Antibiotic (enediyne) (as Calicheamicin (calicheamicin), especially Calicheamicin γ 1I and Calicheamicin ω I1 are (see people such as such as Nicolaou, Angew.Chem Intl.Ed.Engl., 33:183-186 (1994)), CDP323, a kind of oral administration of alpha-4 integrin inhibitor, anthracycline antibiotic (dynemicin), comprises dynemicin A, Ai Sibo mycin (esperamicin), and Neocarzinostatin (neocarzinostatin) chromophore and related colour albumen Enediyne Antibiotic chromophore), aclacinomycin (aclacinomysin), D actinomycin D (actinomycin), anthramycin (authramycin), azaserine (azaserine), bleomycin (bleomycin), act-C (cactinomycin), carabicin, carminomycin (carminomycin), cardinophyllin (carzinophilin), chromomycin (chromomycin), actinomycin D (dactinomycin), daunorubicin (daunorubicin), Detorubicin (detorubicin), 6-phenodiazine-5-oxygen-L-nor-leucine, Doxorubicin (doxorubicin) (comprises morpholino Doxorubicin, Cyanomorpholino Doxorubicin, 2-pyrroles are for Doxorubicin, doxorubicin hydrochloride liposome injection Liposomal doxorubicin TLC D-99 PEGization liposomal doxorubicin and deoxydoxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marcellomycin (marcellomycin), mitomycin (mitomycins) is such as mitomycin C, mycophenolic acid (mycophenolic acid), nogalamycin (nogalamycin), olivomycin (olivomycin), Peplomycin (peplomycin), porfiromycin (porfiromycin), puromycin (puromycin), triferricdoxorubicin (quelamycin), rodorubicin (rodorubicin), streptonigrin (streptonigrin), streptozotocin (streptozocin), tubercidin (tubercidin), ubenimex (ubenimex), Zinostatin (zinostatin), zorubicin (zorubicin), antimetabolite class, such as methotrexate (MTX), gemcitabine (gemcitabine) Tegafur (tegafur) Capecitabine (capecitabine) Epothilones (epothilone) and 5 FU 5 fluorouracil (5-FU); Folacin, such as denopterin (denopterin), methotrexate (MTX), pteroyltriglutamic acid (pteropterin), Trimetrexate (trimetrexate); Purine analogue, such as fludarabine (fludarabine), Ismipur (mercaptopurine), ITG (thiamiprine), thioguanine (thioguanine); Pyrimidine analogue, such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine, Carmofur (carmofur), cytarabine (cytarabine), two BrdU (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine); Androgens, such as calusterone (calusterone), dromostanolone propionate (dromostanolone propionate), epitiostanol (epitiostanol), Mepitiostane (mepitiostane), Testolactone (testolactone); Anti-adrenal gland class, such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), Trilostane (trilostane); Folic acid supplement, such as folinic acid (frolinic acid); Aceglatone (aceglatone); Aldophosphamide glucosides (aldophosphamide glycoside); Amino-laevulic acid (aminolevulinic acid); Eniluracil (eniluracil); Amsacrine (amsacrine); Bestrabucil;Bisantrene (bisantrene); Edatrexate (edatraxate); Defosfamide (defofamine); Demecolcine (demecolcine); Diaziquone (diaziquone); Elfornithine; Elliptinium Acetate (elliptinium acetate); Epothilone; Ethoglucid (etoglucid); Gallium nitrate; Hydroxyl urea (hydroxyurea); Lentinan (lentinan); Lonidamine (lonidainine); Maytansinoid class (maytansinoids), such as maytansine (maytansine) and ansamitocin (ansamitocin); Mitoguazone (mitoguazone); Mitoxantrone (mitoxantrone); Mopidamol (mopidanmol); C-283 (nitraerine); Pentostatin (pentostatin); Phenamet (phenamet); THP (pirarubicin); Losoxantrone (losoxantrone); 2-ethyl hydrazides (ethylhydrazide); Procarbazine (procarbazine); Polysaccharide compound (JHS NaturalProducts, Eugene, OR); Razoxane (razoxane); Rhizomycin (rhizoxin); Sizofiran (sizofiran); Spirogermanium (spirogermanium); Tenuazonic acid (tenuazonic acid); Triethyleneiminobenzoquinone (triaziquone); 2,2 ', 2 "-RA3; Trichothecin class (trichothecenes) (especially T-2 toxin, verrucarine (verracurin) A, roridin (roridin) A and the rhzomorph (anguidine) that crawls); Urethane (urethan);Eldisine (vindesine) Dacarbazine (dacarbazine); Mannomustin (mannomustine); Dibromannitol (mitobronitol); Mitolactol (mitolactol); Pipobroman (pipobroman); Gacytosine; Cytarabine (arabinoside) (" Ara-C "); Phosphinothioylidynetrisaziridine (thiotepa); Taxoid (taxoid), for example Taxol (paclitaxel) The nano particle formulation Taxol (ABRAXANE of albumin transformation ^TM) and Taxotere (docetaxel) Chlorambucil (chloranbucil); 6-thioguanine (thioguanine); Purinethol (mercaptopurine); Methotrexate (MTX) (methotrexate); Platinum agent, such as cis-platinum (cisplatin),Oxaliplatin (oxaliplatin) (for example ) and carboplatin (carboplatin); Changchun medicine class (vincas), it stops tubulin polymerization to form microtubule, comprises vincaleukoblastinum (vinblastine) Vincristine (vincristine) Eldisine (vindesine) And vinorelbine (vinorelbine) Etoposide (etoposide) (VP-16); Ifosfamide (ifosfamide); Mitoxantrone (mitoxantrone); Folinic acid (leucovorin); NSC-279836 (novantrone); Edatrexate (edatrexate); Daunomycin (daunomycin);Aminopterin (aminopterin); Ibandronate (ibandronate); Topoisomerase enzyme inhibitor RFS 2000; DFMO (DMFO); Class Tretinoin (retinoids), such as Tretinoin (retinoic acid), comprises bexarotene (bexarotene) Diphosphonates (bisphosphonates), such as clodronate (clodronate) (for example Or ), etidronate (etidronate) NE-58095, zoledronic acid/zoledronate (zoledronicacid/zoledronate) Alendronate (alendronate) Pamidronate (pamidronate) Tiludronate (tiludronate) Or Risedronate (risedronate) And troxacitabine (troxacitabine) (DOX nucleosides analogue of cytosine); ASON, the signal that particularly suppresses to involve abnormal cell proliferation by way of in the ASON of gene expression, such as example PKC-α, Raf, H-Ras and EGF-R ELISA (EGF-R); Vaccine, such as Vaccine and gene therapy vaccine,For example Vaccine, Vaccine and Vaccine; Topoisomerase 1 inhibitor (for example ); RmRH (for example ); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, Pfizer); Perifosine (perifosine),Cox 2 inhibitor (as celecoxib (celecoxib) or etoricoxib (etoricoxib)), proteasome inhibitor (for example PS341); Bortezomib CCI-779; Tipifarnib (R11577); Orafenib, ABT510; Bcl-2 inhibitor, such as oblimersensodium Pixantrone; EGFR inhibitor (definition sees below); Tyrosine kinase inhibitor (definition sees below); Serine-threonine kinase inhibitor, such as rapamycin (rapamycin) (sirolimus, ); Farnesyl transferase inhibitor, such as lonafarnib (SCH 6636, SARASAR ^TM); And any above-mentioned every pharmaceutically acceptable salt, acid or derivative; And two or more above-mentioned every combinations, such as CHOP (abbreviation of endoxan, Doxorubicin, vincristine and prednisolone conjoint therapy) and FOLFOX (oxaliplatin (ELOXATIN ^TM) abbreviation of therapeutic scheme of associating 5-FU and folinic acid).

Chemotherapeutics as defined herein has comprised adjustment, reduction, blocking-up or has suppressed " antihormone agent " or " endocrine therapy agent " class of the hormone effect effect that cancer can be promoted to grow.They self can be hormones, include but not limited to: the anti-estrogens with the agonist/antagonist characteristic of mixing, comprise tamoxifen (tamoxifen) 4-hydroxytamoxifen, toremifene (toremifene) idoxifene (idoxifene), droloxifene (droloxifene), raloxifene (raloxifene) trioxifene (trioxifene), that Lip river former times sweet smell (keoxifene), and selective estrogen receptor modulators class (SERM), such as SERM3; There is no the pure anti-estrogens of agonist properties, such as fulvestrant (fulvestrant) with EM800 (this type of medicament estrogen receptor capable of blocking (ER) dimerization, suppression DNA combine, improve ER turnover and/or containment ER level); Aromatase inhibitor class, comprises steroidal aromatase inhibitor class, such as formestane (formestane) and exemestane (exemestane) with on-steroidal aromatase inhibitor class, such as Anastrozole (anastrazole) letrozole (letrozole) with aminoglutethimide (aminoglutethimide), and other aromatase inhibitor class, comprise vorozole (vorozole) megestrol acetate (megestrol acetate) fadrozole (fadrozole) and 4 (5)-imidazoles; Gonadotropin-releasing hormone agonist class, comprise leuprorelin (leuprolide) ( with ), goserelin (goserelin), buserelin (buserelin) and triptorelin (tripterelin); Sex steroid class (sexsteroids), comprise ethisterone class (progestine), such as megestrol acetate (megestrol acetate) and medroxyprogesterone acetate (medroxyprogesterone acetate), estrogens, such as diethylstilbestrol (diethylstilbestrol) and premarin (premarin), with androgens/retinoic acid-like class, such as fluoxymesterone (fluoxymesterone), all trans retinoic acids (transretionic acid) and fenretinide (fenretinide); Onapristone (onapristone); Progesterone antagonist class; Estrogen receptor down-regulation agent class (ERD); Anti-androgens, such as Drogenil (flutamide), nilutamide (nilutamide) and than Ka meter Te (bicalutamide); And the acceptable salt of the pharmaceutics of any above-mentioned substance, acid or derivant; And the combination of two or more above-mentioned substances.

Term " prodrug " is less and can enzymatic activation or change precursor or the derivative form of the active medicinal matter having more active parent form at the cytotoxicity for referring to compare with female medicine (parent drug) tumor cell during the application.See such as Wilman, " Prodrugs in Cancer Chemotherapy " Biochemical Society Transactions, 14, the people such as pp.375-382,615th Meeting Belfast (1986) and Stella, " Prodrugs:A Chemical Approach to Targeted Drug Delivery; " Directed Drug Delivery, the people such as Borchardt compile, pp.247-267, Humana Press (1985).Prodrug of the present invention include but not limited to phosphate-containing/ester prodrugs, containing sulfo-phosphate/ester prodrug, containing sulfate/ester prodrugs, containing peptide prodrug, D-amino acid-modified prodrugs, glycosylated prodrugs, containing beta-lactam prodrug, the prodrug containing optional substituted benzene oxygen yl acetamide or the prodrug containing optional substituted benzene acetamide, the 5-flurocytosine that can be converted into the medicine having more activity and no cytotoxicity and other 5-FUD prodrug.The example that can derive the cytotoxic drug of the prodrug forms for the present invention's use includes but not limited to those chemotherapeutics above-described.

" growth inhibitor " is for referring to the compound that T suppression cell (such as in vitro or in vivo its growth depends on the cell of polypeptide of interest activity) grows or compositions during this paper.The example of growth inhibitor comprises and blocks cell cycle and to advance the medicament of (being in the position beyond the S phase), such as induces the medicament that G1 stagnates and the M phase stagnates.Classical M phase blocker comprises Changchun medicine class (vincas) (vincristine (vincristine) and vinblastine (vinblastine)), taxanes (taxanes) and Topoisomerase II inhibitors such as doxorubicin (doxorubicin), epirubicin (epirubicin), daunorubicin (daunorubicin), etoposide (etoposide) and bleomycin (bleomycin).Medicaments of those retardances G1 also overflow and enter the S phase and stagnate, such as DNA alkylating agent class such as tamoxifen (tamoxifen), prednisone (prednisone), dacarbazine (dacarbazine), chlormethine (mechlorethamine), cisplatin (cisplatin), methotrexate (methotrexate), 5-fluorouracil (5-fluorouracil) and ara-C.More information can be see the molecular Basis of Cancer, Mendelsohn and Israel, eds., the people (WBSaunders:Philadelphia, 1995) such as Chapter 1, entitled " Cellcycle regulation, oncogenes; and antineoplastic drugs " by Murakami, especially the 13rd page.Taxanes (Pa Litasai (paclitaxel) and docetaxel (docetaxel)) is the anticarcinogen derived from yew tree.Derived from European yew docetaxel ( rhone-Poulenc Rorer) be Pa Litasai ( bristol-Myers Squibb) semi-synthetic analog.Pa Litasai and docetaxel promote be assembled into microtubule by tubulin dimer and by preventing depolymerization from making microtubule stabilization, cause suppression mitotic in cell.

" X-ray therapy " refers to use directed gamma ray or beta ray to bring out enough damages to cell, the ability worked orderly with restrictive cell or destroy cell completely.Will be appreciated that many modes are known to determine dosage and the persistent period for the treatment of in this area.Typical treatment gives as applied once, and typical dosage range is 10-200 unit every day (gray(Gy) (Gray)).

" individuality " or " experimenter " refers to mammal.Mammal includes but not limited to performing animal (such as cattle, sheep, cat, dog and horse), primates (such as people and non-human primates such as monkey), rabbit and Rodents (such as Mouse and rat).In certain embodiments, individual or experimenter is people.

Term " adjoint " is used in reference in this article uses two or more therapeutic agents, and the time of administration is enough close, and wherein the effect of various therapeutic agent is overlapping in time.Thus, walk abreast the dosage regimen used and be included in and continue to use one or more other medicaments after one or more medicaments are used in termination.In some embodiments, concomitant administration is concurrently, sequentially, and/or side by side.

" reduce or suppress " ability having guided the overall reduction of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or larger.Reduce or suppress to refer to the size of the symptom of treated disease, the existence of transfer or size or primary tumor.

Term " package insert " is used in reference to the description in the commercial packing being usually included in treatment product, they include close relate to the indication of this type for the treatment of products application, usage, dosage, use, the information of combination treatment, contraindication and/or warning.

" goods " comprise at least one reagent, such as, be used for the treatment of any manufacture thing (such as packaging or container) or the test kit of the medicine of disease or disease (such as cancer) or the probe for specific detection biomarker described herein.In certain embodiments, manufacture thing or test kit are promoted for the unit implementing methods described herein, distribute or are sold.

As understood by a person skilled in the art, carry herein and state " about " certain value or parameter comprises (and describe) embodiment for this value or parameter itself.Such as, propose the description stating " about X " and comprise description to " X ".

Should be appreciated that aspect of the present invention described herein and embodiment comprise by and/or substantially " be made up of " each side and embodiment.As used in this article, singulative " ", " one " and " described/to be somebody's turn to do " comprise plural number and mention thing, unless otherwise instructed.

II. method and purposes

The method utilizing ALDH inhibitor (such as disulfiram and/or its derivant) and target therapeutic agent (such as TKI) to carry out Therapeutic cancer is provided herein.

Especially, be provided in the method for Therapeutic cancer in individuality herein, it comprises this individual concomitant administration ALDH inhibitor (such as disulfiram and/or its derivant) and target therapeutic agent (such as TKI).In some embodiments, described ALDH inhibitor (such as disulfiram and/or its derivant) and the respective amount of described target therapeutic agent (such as TKI) effectively extend the period of cancer sensitivity and/or postpone cell and form resistance to described target therapeutic agent (such as TKI).In some embodiments, described ALDH inhibitor (such as disulfiram and/or its derivant) and the respective amount of described target therapeutic agent (such as TKI) effectively improve the effect of the treatment of cancer comprising target therapeutic agent (such as TKI).Such as, in some embodiments, with be included in there is no described ALDH inhibitor (such as disulfiram and/or its derivant) when (under described ALDH inhibitor (such as disulfiram and/or its derivant) lacks) use compared with the standard care of the described target therapeutic agent (such as TKI) of effective dose, described ALDH inhibitor (such as disulfiram and/or its derivant) and the respective amount of described target therapeutic agent (such as TKI) effectively improve effect.In some embodiments, with be included in there is no described ALDH inhibitor (such as disulfiram and/or its derivant) when (under described ALDH inhibitor (such as disulfiram and/or its derivant) lacks) use compared with the standard care of the described target therapeutic agent (such as TKI) of effective dose, described ALDH inhibitor (such as disulfiram and/or its derivant) and the respective amount of described target therapeutic agent (such as TKI) effectively improve response (such as totally linearization).In some embodiments, described target therapeutic agent is tyrosine kinase inhibitor (TKI).In some embodiments, described TKI is EGFR inhibitor, HER2 inhibitor, MET/HGF inhibitor, ALK inhibitor, BRAF inhibitor, ROS1 inhibitor and/or mek inhibitor.In some embodiments, described TKI is receptor tyrosine kinase inhibitors (RTKI).In some embodiments, described RTKI is EGFR inhibitor, HER2 inhibitor, MET inhibitor and/or ALK inhibitor.

In addition, be provided in herein in individuality to improve and comprise the method for effect of the treatment of cancer of target therapeutic agent (such as TKI), it comprises the ALDH inhibitor (such as disulfiram and/or its derivant) of described target therapeutic agent (such as TKI) to this individual concomitant administration effective dose and effective dose.Also be provided in the method for Therapeutic cancer in individuality herein, wherein treatment of cancer comprises the ALDH inhibitor (such as disulfiram and/or its derivant) of target therapeutic agent (such as TKI) to this individual concomitant administration effective dose and effective dose, wherein said treatment of cancer have be included in there is no described ALDH inhibitor (such as disulfiram and/or its derivant) when (under described ALDH inhibitor (such as disulfiram and/or its derivant) lacks) use the described target therapeutic agent (such as TKI) of effective dose standard care compared with effect of rising.In some embodiments, described target therapeutic agent is TKI.In some embodiments, described TKI is EGFR inhibitor, HER2 inhibitor, MET/HGF inhibitor, ALK inhibitor, BRAF inhibitor, ROS1 inhibitor and/or mek inhibitor.In some embodiments, described TKI is RTKI.In some embodiments, described RTKI is EGFR inhibitor, HER2 inhibitor, MET/HGF inhibitor and/or ALK inhibitor.

In addition, be provided in herein in individuality and postpone and/or stop formation of cancer to the method for the resistance of target therapeutic agent (such as TKI), it comprises the described target therapeutic agent (such as TKI) of ALDH inhibitor (such as disulfiram and/or its derivant) to this individual concomitant administration effective dose and effective dose.Also be provided in individuality the method for the sensitivity improved target therapeutic agent (such as TKI) herein, it comprises the described target therapeutic agent (such as TKI) of ALDH inhibitor (such as disulfiram and/or its derivant) to this individual concomitant administration effective dose and effective dose.In some embodiments, described target therapeutic agent is TKI.In some embodiments, described TKI is EGFR inhibitor, HER2 inhibitor, MET/HGF inhibitor, ALK inhibitor, BRAF inhibitor, ROS1 inhibitor and/or mek inhibitor.In some embodiments, described TKI is RTKI.In some embodiments, described RTKI is EGFR inhibitor, HER2 inhibitor, MET/HGF inhibitor and/or ALK inhibitor.

In addition, be provided in the individuality with cancer the method for the period extending target therapeutic agent (such as TKI) sensitivity herein, it comprises the described target therapeutic agent (such as TKI) of ALDH inhibitor (such as disulfiram and/or its derivant) to this individual concomitant administration effective dose and effective dose.Also be provided in the individuality with cancer the method for the persistent period of the response extended target therapeutic agent (such as TKI) herein, it comprises the described target therapeutic agent (such as TKI) of ALDH inhibitor (such as disulfiram and/or its derivant) to this individual concomitant administration effective dose and effective dose.In some embodiments, described target therapeutic agent is TKI.In some embodiments, described TKI is EGFR inhibitor, HER2 inhibitor, MET/HGF inhibitor, ALK inhibitor, BRAF inhibitor, ROS1 inhibitor and/or mek inhibitor.In some embodiments, described TKI is RTKI.In some embodiments, described RTKI is EGFR inhibitor, HER2 inhibitor, MET/HGF inhibitor and/or ALK inhibitor.

In some embodiments of any described method, described ALDH inhibitor and/or target therapeutic agent (such as TKI) be antibody, Binding peptide, in conjunction with micromolecule or polynucleotide, all as described in this article those.In some embodiments, described ALDH inhibitor is disulfiram and/or its derivant.In some embodiments, described ALDH inhibitor is disulfiram.In some embodiments of any described method, described ALDH inhibitor is gossypol and/or its ALDH inhibition derivant or metabolite.In some embodiments, described ALDH inhibitor is gossypol.In some embodiments, described ALDH inhibitor is 2,2 '-two-(formoxyl-1,6,7-trihydroxy-5-isopropyl-3-methyl naphthalene) or its pharmaceutical acceptable salts.In some embodiments, described ALDH inhibitor is 2,2 '-two-(formoxyl-1,6,7-trihydroxy-5-isopropyl-3-methyl naphthalene).

The cancer having a resistance to therapy used herein comprises the cancer that the ability that not respond therapy and/or produce remarkable response (such as partial response and/or totally linearization) reduces.Resistance can be the acquired resistance produced in Therapeutic Method process.In some embodiments, described acquired drug resistance is instantaneous and/or reversible pharmacological tolerance.Comprise wherein said drug resistance to the instantaneous of therapy and/or reversible pharmacological resistance to reentry to the sensitivity of therapy after described Therapeutic Method suspends.In some embodiments, described acquired resistance is permanent resistance.The permanent resistance of therapy is comprised to the heredity change of giving drug resistance.Permanent resistance can occur as the result of the treatment of general chemotherapy-cyclophosphamide, platinum agent and/or paclitaxel.

Cancer therapy used herein to sensitivity comprises response and/or can produce the cancer of remarkable response (such as partial response and/or totally linearization).

The assay method that therapy assessment resistance obtains and/or sensitivity maintains is known in the art and is described in an embodiment.The change of resistance acquisition and/or sensitivity maintenance (such as drug resistance) can be assessed by the growth such as measuring drug resistance supporter described in the people such as embodiment and Sharma.The change of resistance acquisition and/or sensitivity maintenance (such as permanent resistance and/or expansion resistant) is assessed in the growth that can expand supporter by such as measuring drug resistance described in the people such as embodiment and Sharma.In some embodiments, IC can be passed through ₅₀, EC ₅₀change or the reduction that expands tumor growth in supporter of drug resistance supporter and/or drug resistance indicate resistance.In some embodiments, described change be greater than about 50%, 100% and/or 200% arbitrary.In addition, the change that resistance obtains and/or sensitivity maintains can be assessed in vivo, such as by assessment to the time before the response of therapy, duration of response and/or progress, such as partial response and totally linearization.The change that resistance obtains and/or sensitivity maintains can based in a group individuality to the change of time before the response of therapy, duration of response and/or progress, the number of such as partial response and totally linearization.

In some embodiments of any described method, described cancer is solid tumor cancer.In some embodiments, described cancer is gastric cancer.In some embodiments, described cancer is pulmonary carcinoma (such as nonsmall-cell lung cancer (NSCL)).In some embodiments, described cancer is breast carcinoma.In some embodiments, described cancer is colorectal carcinoma (such as colon cancer and/or rectal cancer).In some embodiments, described cancer is basal cell carcinoma.In some embodiments of any described cancer, described cancer is adenocarcinoma.Cancer when starting the Therapeutic Method comprising described ALDH inhibitor (such as disulfiram and/or its derivant) and described target therapeutic agent (such as TKI) in any combination treatment method described herein can be (example of sensitivity includes but not limited to response and/or can produce remarkable response (such as partial response and/or totally linearization)) to the Therapeutic Method sensitivity comprising independent described target therapeutic agent.Start to comprise described ALDH inhibitor, (such as disulfiram and/or its derivant) and described target therapeutic agent, the cancer during Therapeutic Method of (such as TKI) in any combination treatment method described herein can not be the Therapeutic Method resistance to comprising independent described target therapeutic agent, (example of resistance includes but not limited to not respond and/or can produce remarkable response, the ability of (such as partial response and/or totally linearization) reduces and/or can not produce remarkable response, (such as partial response and/or totally linearization)).

In some embodiments of any described method, the individuality according to any above-mentioned embodiment can be people.

In some embodiments of where method in office, the combination treatment recorded above contains combined administration (wherein two or more therapeutic agents are included in identical or point other preparaton), and separate administration (in this case antagonist of the present invention can before other therapeutic agent and/or adjuvant are used, simultaneously, sequential, parallel and/or use afterwards).In some embodiments, combination treatment comprises X-ray therapy and/or other therapeutic agent further.

Can by any suitable means, comprise in oral, parenteral, lung and intranasal, if and be expected to be useful in topical therapeutic, use ALDH inhibitor described herein (such as disulfiram and/or its derivant) and/or target therapeutic agent (such as TKI) in damage.Parenteral infusions comprises intramuscular, intravenous, intra-arterial, intraperitoneal or subcutaneous administration.Part is of short duration or long-term according to using, and administration can pass through any suitable path, and such as, by injection, such as intravenous or subcutaneous injection carry out.Contain various administration schedule herein, include but not limited to single administration or repeatedly using in multiple time point, inject and use and pulse infusion.

The mode that ALDH inhibitor described herein (such as disulfiram and/or its derivant) and/or target therapeutic agent (such as TKI) a kind ofly can meet good medical practice is prepared, is determined dosage and use.The factor considered in this context is included in the particular condition for the treatment of, specific mammal in treatment, the clinical state of individual patient, disease reason, drug delivery position, application process, administration schedules and other factor known to medical practitioner.ALDH inhibitor (such as disulfiram and/or its derivant) and/or target therapeutic agent (such as TKI) without the need to but optionally prepare together with the medicament that prevents or treat described disease at present with one or more.The effective dose of other medicament this kind of depends on the amount of ALDH inhibitor (such as disulfiram and/or its derivant) existing in formula and/or target therapeutic agent (such as TKI), disease or the type for the treatment of and the factor of other above-mentioned discussion.These medicaments usually use with identical dosage and have route of administration described herein, or use with the dosage described herein of about 1-99%, or used by any approach with any dosage, described dosage and approach be to determine by rule of thumb/suitable through clinical assays.

In order to prevent or disease therapy, the suitable dose of ALDH inhibitor described herein (such as disulfiram and/or its derivant) and/or target therapeutic agent (such as TKI) (when separately or when using with one or more other other therapeutic agent) should depend on the type of the disease that will treat, the seriousness of disease and the course of disease, using ALDH inhibitor (such as disulfiram and/or its derivant) and/or target therapeutic agent (such as TKI) is for prevention or therapeutic purposes, treatment before, the clinical history of patient and the response to ALDH inhibitor (such as disulfiram and/or its derivant), and the consideration of attending doctor determines.ALDH inhibitor (such as disulfiram and/or its derivant) is suitable for once or in a series for the treatment of giving patient.For the repetitive administration within a couple of days or longer time, according to situation, treatment generally will continue until there is the suppression to disease symptoms expected.This kind of dosage can intermittently be used, and as weekly or use for every three weeks, such as, makes patient accept about 2-about 20 doses, or the ALDH inhibitor of such as about 6 doses (such as disulfiram and/or its derivant) and/or target therapeutic agent (such as TKI).Initial higher loading dose can be used, then use one or more lower dosage.Exemplary dosage regimen comprises to be used.But, other dosage regimen can be used.The progress of monitoring this treatment is easy to by routine techniques and algoscopy.

Understanding can use immunoconjugates to implement any above-mentioned preparaton or Therapeutic Method as ALDH inhibitor and/or target therapeutic agent (such as TKI).

III. therapeutic combination

Providing package contains the combination of ALDH inhibitor (such as disulfiram and/or its derivant) and target therapeutic agent (such as TKI) herein.On the one hand, provide pharmaceutical products, it comprises a) as the ALDH inhibitor of the effective dose of the first composition, and b) as the targeting agent (target therapeutic agent) of the effective dose of the second composition, its for or sequentially to make for Therapeutic cancer.In certain embodiments, this combination improves the effect of this target therapeutic agent used separately.In certain embodiments, this combinatorial delays and/or prevention formation of cancer are to the resistance of this target therapeutic agent.In certain embodiments, this is combined in the period extending this target therapeutic agent sensitivity in the individuality with cancer.

The ALDH inhibitor and/or target therapeutic agent that can be used for combination treatment method described herein are also provided herein.In some embodiments, this ALDH inhibitor and/or target therapeutic agent be antibody, Binding peptide, in conjunction with micromolecule and/or polynucleotide.

In some embodiments of any combination treatment method described herein, this ALDH inhibitor suppress in ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, ALDH1L1, ALDH1L2, ALDH2, ALDH3A1, ALDH3A2, ALDH3B1, ALDH3B2, ALDH4A1, ALDH5A1, ALDH6A1, ALDH7A1, ALDH8A1, ALDH9A1, ALDH16A1 and/or ALDH18A1 one or more.In some embodiments, according to ALDH inhibitor of the present invention be the compound of one or more activity in several isozymes that can suppress ALDH.In some embodiments, described ALDH inhibitor is general ALDH inhibitor.ALDH inhibitor includes but not limited to disulfiram, coprinin (coprine), cyanamide (cyanamide), amino ring propanol (ACP) of 1-, daidzin (daidzin) (i.e. 4', the 7-glucoside of 7-dihydroxy isoflavone), cephalosporin, anti-diabetic sulfonylureas, metronidazole, diethyldithiocarbamate, phenethyl isothiocyanate (PEITC), prunusetin. (prunetin) (4', 5-dihydroxy-7-methoxy isoflavone), 5-hydroxyl daidzin (genistin (genistin)), and represent any metabolite or the analog of ALDH inhibitory activity.In another embodiment, described ALDH inhibitor is disulfiram or its ALDH inhibition metabolite.This metabolite comprises such as S-methyl N, N-diethyldithiocarbamate, S-methyl N, N-diethyldithiocarbamate sulfoxide and S-methyl N, N-diethyl thiocarbamate sulfoxide.In some embodiments, described ALDH inhibitor is disulfiram.In some embodiments of any described method, described ALDH inhibitor is gossypol and/or its ALDH inhibition derivant or metabolite.In some embodiments, described ALDH inhibitor is gossypol.In some embodiments, described ALDH inhibitor is 2,2 '-two-(formoxyl-1,6,7-trihydroxy-5-isopropyl-3-methyl naphthalene) or its pharmaceutical acceptable salts.In some embodiments, described ALDH inhibitor is 2,2 '-two-(formoxyl-1,6,7-trihydroxy-5-isopropyl-3-methyl naphthalene).

ALDH inhibitor also comprises the compound of following formula:

Wherein:

R ₁be selected from lower group: hydrogen, carboxyl, halogen, branch or unbranched (C ₁-C ₆) halo alkyl, (C ₃-C ₆) ring oxyl, (C ₁-C ₆) halo oxyl, (C ₃-C ₆) ring halo oxyl, (C ₃-C ₆) ring Alkyloxyalkyl, (C ₁-C ₆) oxyl (C ₃-C ₆) cyclic hydrocarbon radical, (C ₃-C ₆) cyclic hydrocarbon radical carbonyl, replacement or unsubstituted phenyl, phenyl (C ₁-C ₆) alkyl, heterocyclic radical, and heterocyclyloxy base, Heterocyclylcarbonyl, wherein substituent group is 1 to 4 and is selected from lower group: halogen, amino carbonyl, aminothiocarbonyl, carboxyl, formoxyl, hydroxyl, amino, carbamoyl, (C ₁-C ₃) alkyl, (C ₁-C ₃) halo alkyl, (C ₁-C ₃) oxyl, (C ₁-C ₃) halo oxyl, (C ₁-C ₃) hydrocarbylamino, two (C ₁-C ₃) hydrocarbylamino, (C ₁-C ₂) oxyl (C ₁-C ₂) alkyl, (C ₁-C ₂) hydrocarbylamino (C ₁-C ₂) alkyl, two (C ₁-C ₂) hydrocarbylamino (C ₁-C ₂) alkyl, (C ₁-C ₃) alkyl carbonyl, (C ₁-C ₃) alkyloxycarbonyl group, (C ₁-C ₃) hydrocarbylamino carbonyl, and two (C ₁-C ₃) hydrocarbylamino carbonyl;

R ₂be selected from lower group: hydrogen and oxyl;

R ₃be selected from lower group: hydrogen, C ₁-C ₆alkyloxycarbonyl group, carboxyl and sugar;

R ₄be selected from lower group: hydrogen and hydroxide;

R ₅be selected from lower group: hydrogen, carboxyl, hydroxyl, halogen, branch or unbranched (C ₁-C ₆) alkyl, (C ₁-C ₆) halo alkyl, (C ₂-C ₆) alkenyl, (C ₃-C ₆) alkadienyl, (C ₁-C ₆) oxyl, (C ₃-C ₆) ring oxyl, (C ₁-C ₆) halo oxyl, (C ₃-C ₆) ring halo oxyl, (C ₂-C ₆) alkynyl group oxygen base, (C ₁-C ₆) oxyl (C ₁-C ₆) alkyl, (C ₃-C ₆) ring Alkyloxyalkyl, (C ₁-C ₆) oxyl (C ₃-C ₆) cyclic hydrocarbon radical, (C ₁-C ₆) alkyl carbonyl, (C ₃-C ₆) cyclic hydrocarbon radical carbonyl, (C ₁-C ₆) alkyloxycarbonyl group, (C ₄-C ₆) alkyloxycarbonyl group alkyl, (C ₁-C ₆) hydroxy alkylene, replacement or unsubstituted phenyl, phenyl (C ₁-C ₆) alkyl, heterocyclic radical, heterocyclyloxy base, Heterocyclylcarbonyl, wherein substituent group is 1 to 4 and is selected from lower group: halogen, amino carbonyl, aminothiocarbonyl, carboxyl, formoxyl, hydroxyl, amino, carbamoyl, (C ₁-C ₃) alkyl, (C ₁-C ₃) halo alkyl, (C ₁-C ₃) oxyl, (C ₁-C ₃) halo oxyl, (C ₁-C ₃) hydrocarbylamino, two (C ₁-C ₃) hydrocarbylamino, (C ₁-C ₂) oxyl (C ₁-C ₂) alkyl, (C ₁-C ₂) hydrocarbylamino (C ₁-C ₂) alkyl, two (C ₁-C ₂) hydrocarbylamino (C ₁-C ₂) alkyl, (C ₁-C ₃) alkyl carbonyl, (C ₁-C ₃) alkyloxycarbonyl group, (C ₁-C ₃) hydrocarbylamino carbonyl, and two (C ₁-C ₃) hydrocarbylamino carbonyl;

R ₆be selected from lower group: hydrogen and hydroxide; And

R ₇be selected from lower group: hydrogen, halogen, and C ₁-C ₆oxyl.

ALDH inhibitor also comprises the compound with following CAS registration number: 1069117-57-2,1069117-56-1,1069117-55-0,1055417-23-6,1055417-22-5,1055417-21-4,1055417-20-3,1055417-19-0,1055417-18-9,1055417-17-8,1055417-16-7,1055417-15-6 and 1055417-13-4, and salt.

ALDH inhibitor also comprises the compound of following formula:

Wherein R ₁, R ₂the C of saturated or undersaturated linear or branch is independently represented with R3 ₁-C ₆alkyl, or its salt.

ALDH inhibitor also comprises 4-amino-4-methyl-valerylene thio-acid (S)-methyl ester and salt thereof.

In some embodiments of any combination treatment method described herein, described target therapeutic agent is TKI.In some embodiments, described TKI is EGFR inhibitor, HER2 inhibitor, MET/HGF inhibitor, ALK inhibitor, BRAF inhibitor, ROS1 inhibitor and/or mek inhibitor.In some embodiments, described TKI is RTKI.In some embodiments, described RTKI is EGFR inhibitor, HER2 inhibitor, MET/HGF inhibitor and/or ALK inhibitor.

In some embodiments of any combination treatment method described herein, described target therapeutic agent is EGFR inhibitor.Exemplary EGFR inhibitor (anti-egfr antibodies) comprises antibody, such as be called the Humanized monoclonal antibodies of nimotuzumab (YM Biosciences), completely people ABX-EGF (panitumumab, Abgenix Inc.) and be called E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 and E7.6.3 and be recorded in US 6, the fully human antibodies of 235,883; MDX-447 (Medarex Inc).Pertuzumab (2C4) is a kind of directly in conjunction with HER2 but interference HER2-EGFR dimerization suppresses the humanized antibody of EGFR intracellular signaling thus.Other example in conjunction with the antibody of EGFR comprises GA201 (RG7160; Roche Glycart AG), MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL8509) is (see U.S. Patent No. 4,943,533, the people such as Mendelsohn) and variant, such as chimerization 225 (C225 or Cetuximab; ) and reset pattern people 225 (H225) (see WO 96/40210, Imclone Systems Inc.); IMC-11F8, the EGFR target antibody (Imclone) of a kind of complete people; In conjunction with the antibody (U.S. Patent No. 5,212,290) of II class mutant egf R; In conjunction with EGFR, be recorded in U.S. Patent No. 5,891, the humanization of 996 and chimeric antibody; With the people's antibody in conjunction with EGFR, such as ABX-EGF (see WO98/50433, Abgenix); EMD 55900 (people such as Stragliotto, Eur.J.Cancer 32A:636-640 (1996)); EMD7200 (matuzumab), a kind of for EGFR, compete with both EGF and TGF-α the humanization EGFR antibody that EGFR is combined; With mAb 806 or humanization mAb 806 (people such as Johns, J.Biol.Chem.279 (29): 30375-30384 (2004)).Anti-egfr antibodies can be conjugated with cytotoxic agent, so generates immunoconjugates (see such as EP659,439A2, Merck Patent GmbH).In some embodiments, this anti-egfr antibodies is cetuximab.In some embodiments, this anti-egfr antibodies is panitumumab.In some embodiments, this anti-egfr antibodies is zalutumumab, nimotuzumab and/or matuzumab.

The anti-egfr antibodies that can be used for the method comprises with enough affinitys and specific binding EGFR and can reduce or suppress any antibody of activity of EGFR.The antibody selected can have the enough strong binding affinity to EGFR usually, and such as antibody can with the Kd value between 100nM-1pM in conjunction with people c-met.Can by the algoscopy (the BIAcore algoscopy recorded in such as PCT application publication number WO2005/012359) such as resonated based on surperficial plasmon; Enzyme-linked immunosorbent assay (ELISA); Affinity of antibody is measured with competition assay (such as RIA).Preferably, anti-egfr antibodies of the present invention can be used as therapeutic agent involves EGFR/EGFR ligand activity disease or situation for targeting and interference.Further, this antibody can carry out other biological activity assavs, such as, in order to assess its effectiveness as therapeutic agent.This type of algoscopy is known in the art and depends on the intended purpose of target antigen and antibody.In some embodiments, can by EGFR arm and in conjunction with molecule triggering on leukocyte such as T-cell receptors molecule (such as CD2 or CD3), or the Fc receptor of IgG (Fc γ R), the arm combination of such as Fc γ RI (CD64), Fc γ RII (CD32) and Fc γ RIII (CD16), thus cellular defence mechanisms is focused on the cell of expressing EGFR.Bi-specific antibody also can be used for cytotoxic agent being positioned to the cell of expressing EGFR.These antibody have EGFR brachium conjunctivum and the arm in conjunction with cytotoxic agent (such as sapotoxin albumen, anti-interferon-α, vinca alkaloids, ricin A chain, methotrexate or radioactive isotope hapten).Can as full length antibody or antibody fragment (such as F (ab') ₂bi-specific antibody) prepare bi-specific antibody.

Exemplary EGFR inhibitor also comprises micromolecule, such as US5616582, US5457105, US5475001, US5654307, US5679683, US6084095, US6265410, US6455534, US6521620, US6596726, US6713484, US5770599, US6140332, US5866572, US6399602, US6344459, US6602863, US6391874, WO9814451, WO9850038, WO9909016, WO9924037, WO9935146, WO0132651, US6344455, US5760041, US6002008, and/or the compound recorded in US5747498.Concrete micromolecule EGFR antagonist comprises OSI-774 (CP-358774, Erlotinib, OSI Pharmaceuticals); PD 183805 (CI 1033,2-acrylamide, N-[4-[(the chloro-4-fluorophenyl of 3-) is amino]-7-[3-(4-morpholinyl) propoxyl group]-6-quinazolyl]-, dihydrochloride, Pfizer Inc.); (ZD1839, gefitinib, AstraZeneca); ZM 105180 (6-amino-4-(3-aminomethyl phenyl-amino)-quinazoline, Zeneca); BIBX-1382 (N8-(the fluoro-phenyl of the chloro-4-of 3-)-N2-(1-methyl-pi-4-base)-pyrimido [5,4-d] pyrimidine-2,8-diamidogen, Boehringer Ingelheim); PKI-166 ((R)-4-[4-[(1-phenylethyl) is amino]-1H-pyrrolo-[2,3-d] pyrimidine-6-base]-phenol); (R)-6-(4-hydroxy phenyl)-4-[(1-phenylethyl) is amino]-7H-pyrrolo-[2,3-d] pyrimidine; CL-387785 (N-[4-[(3-bromophenyl) is amino]-6-quinazolyl]-2-butyne amide); EKB-569 (N-[4-[(the chloro-4-fluorophenyl of 3-) is amino]-3-cyanogen-7-ethyoxyl-6-quinolyl]-4-(dimethylamino)-2-butylene amide); Lapatinib (Tykerb, GlaxoSmithKline); ZD6474 (Zactima, AstraZeneca); CUDC-101 (Curis); Canertinib (CI-1033); AEE788 (6-[4-[(4-ethyl-1-piperazinyl) methyl] phenyl]-N-[(1R)-1-phenylethyl]-7H-pyrrolo-[2,3-d] pyrimidine-4-amine, WO2003013541, and PKI166 (4-[4-[[(1R)-1-phenylethyl] amino]-7H-pyrrolo-[2 Novartis), 3-d] pyrimidine-6-base]-phenol, WO9702266, Novartis).In some embodiments, this EGFR antagonist is N-(3-ethynyl phenyl)-6,7-bis-(2-methoxy ethoxy)-4-quinazoline amine and/or its pharmaceutical acceptable salts (such as N-(3-ethynyl phenyl)-6,7-bis-(2-methoxy ethoxy)-4-quinazoline amine-HCl).In some embodiments, this EGFR antagonist is gefitinib and/or its pharmaceutical acceptable salts.In some embodiments, this EGFR antagonist is Lapatinib and/or its pharmaceutical acceptable salts.In some embodiments, this EGFR antagonist is gefitinib and/or Erlotinib.

In some embodiments of any combination treatment method described herein, described target therapeutic agent is HGF/MET inhibitor.Exemplary HGF/MET inhibitor (anti-HGF and/or anti-MET antibody) comprises antibody, the anti-MET antibody (including but not limited to antibody 13.3.2,9.1.2,8.70.2,8.90.3) disclosed in such as WO05/016382; The anti-met antibody generated by the hybridoma cell line being preserved in Genoa CBA with ICLC PD 03001 or identify the anti-met antibody of epi-position identical with the epi-position of this monoclonal antibody identification on HGF receptor β chain extracellular domain; The anti-met antibody (including but not limited to 04536,05087,05088,05091,05092,04687,05097,05098,05100,05101,04541,05093,05094,04537,05102,05105,04696,04682) disclosed in WO2007/126799; The anti-met antibody disclosed in WO2009/007427 (includes but not limited at CNCM (InstitutPasteur, Paris, France) on March 14th, 2007 with numbering I-3731, on March 14th, 2007 with numbering I-3732, on July 6th, 2007 with numbering I-3786, on March 14th, 2007 with the antibody of numbering I-3724 preservation); The anti-met antibody disclosed in US20110129481; The anti-met antibody disclosed in US20110104176; The anti-met antibody disclosed in WO2009/134776; The anti-met antibody disclosed in WO2010/059654; The anti-met antibody disclosed in WO2011020925 (includes but not limited to comfortable CNCM (Institut Pasteur, Paris, France) on March 12nd, 2008 with the hybridoma of numbering I-3949 preservation and the antibody secreted with the hybridoma of numbering I-4273 preservation on January 14th, 2010); And/or MetMAb (onartuzumab) or its biofacies seemingly pattern (WO2006/015371; The people such as Jin, CancerRes (2008) 68:4360).In some embodiments, this MET/HGF inhibitor is onartuzumab.

In some embodiments of any combination treatment method described herein, this MET/HGF inhibitor is anti-hepatocyte growth factor (HGF) antibody, the humanized antibody 2B8 such as, recorded in humanization anti-HGF antibody TAK701, rilotumumab, Ficlatuzumab and/or WO2007/143090.In some embodiments, this anti-HGF antibody is the anti-HGF antibody recorded in US7718174B2.

In some embodiment of any combination treatment method described herein, this MET/HGF inhibitor be following any one: SGX-523, gram azoles is for Buddhist nun (PF-02341066; 3-[(1R)-1-(the chloro-3-fluorophenyl of 2,6-bis-) ethyoxyl]-5-(1-piperidin-4-yl pyrazoles-4-base) pyridine-2-amine; CAS 877399-52-5); JNJ-38877605 (CAS 943540-75-8), BMS-698769, PHA-665752 (Pfizer), SU5416, INC-280 (Incyte); SU11274 (Sugen; [(3Z)-N-(3-chlorphenyl)-3-({ 3,5-dimethyl-4-[(4-methylpiperazine-1-yl) carbonyl]-1H-pyrroles-2-base } methylene)-N-methyl-2-oxygen indoline-5-sulfonamide; CAS 658084-23-2]), Foretinib (GSK1363089), XL880 (CAS 849217-64-7; XL880 is the inhibitor of MET/HGF and VEGFR2 and KDR); MGCD-265 (MethylGene; MGCD-265 targeting met, VEGFR1, VEGFR2, VEGFR3, Ron and Tie-2 receptor; CAS 875337-44-3), Tivantinib (ARQ 197; (-)-(3R, 4R)-3-(5,6-dihydro-4H-pyrrolo-[3,2,1-ij] quinoline-1-base)-4-(1H-indol-3-yl) pyrrolidine-2,5-diketone; See people such as Munchi, Mol Cancer Ther June 20109; 1544; CAS 905854-02-6), LY-2801653 (Lilly), LY2875358 (Lilly), MP-470, Rilotumumab (AMG 102, anti-HGF monoclonal antibody), antibody 223C4 or humanized antibody 223C4 (WO2009/007427), humanization L2G7 (humanization TAK701; The anti-HGF monoclonal antibody of humanization); EMD 1214063 (Merck Sorono), EMD 1204831 (Merck Serono), NK4, Cabozantinib (XL-184, CAS 849217-68-1; Carbozantinib is the double inhibitor of MET/HGF and VEGFR2), MP-470 (SuperGen; The new inhibitor of c-KIT, MET, PDGFR, Flt3 and AXL), Comp-1, Ficlatuzumab (AV-299; Anti-HGF monoclonal antibody), E7050 (CAS 1196681-49-8; E7050 is dual MET/HGF and VEGFR2 inhibitor; Esai); MK-2461 (Merck; N-((2R)-Isosorbide-5-Nitrae-dioxa hexamethylene-2-ylmethyl)-N-methyl-N'-[3-(1-methyl isophthalic acid H-pyrazoles-4-base)-5-oxygen-5H-benzo [4,5] ring also [1,2-b] pyridin-7-yl in heptan] sulfonamide; CAS 917879-39-1); MK8066 (Merck), PF4217903 (Pfizer), AMG208 (Amgen), SGX-126, RP1040, LY2801653, AMG458, EMD637830, BAY-853474, DP-3590.In certain embodiments, this met inhibitor is that gram azoles is for any one or more in Buddhist nun, tivantinib, carbozantinib, MGCD-265, ficlatuzumab, humanization TAK-701, rilotumumab, foretinib, h224G11, DN-30, GDC-0712, MK-2461, E7050, MK-8033, PF-4217903, AMG208, JNJ-38877605, EMD1204831, INC-280, LY-2801653, SGX-126, RP1040, LY2801653, BAY-853474 and/or LA480.In certain embodiments, this met inhibitor gram azoles is for any one or more in Buddhist nun, tivantinib, carbozantinib, MGCD-265, ficlatuzumab, humanization TAK-701, rilotumumab and/or foretinib.In some embodiments, this met inhibitor is that gram azoles is for Buddhist nun.

In some embodiments of any combination treatment method described herein, described target therapeutic agent is BRAF inhibitor.Exemplary BRAF inhibitor is known in the art and comprises such as sorafenib, PLX4720, PLX-3603, dabrafenib (GSK2118436), GDC-0879, RAF265 (Novartis), XL281, ARQ736, BAY73-4506, Wei Luofeini and WO2007/002325, WO2007/002433, WO2009111278, WO2009111279, WO2009111277, WO2009111280 and U.S. Patent No. 7,491, in 829 record those.In some embodiments, this BRAF inhibitor is selectivity BRAF inhibitor.In some embodiments, this BRAF inhibitor is the selective depressant of BRAF V600.In some embodiments, BRAF V600 is BRAFV600E, BRAF V600K and/or V600D.In some embodiments, BRAF V600 is BRAFV600R.In some embodiments, this BRAF inhibitor is Wei Luofeini.In some embodiments, this BRAF inhibitor is Wei Luofeini.

Wei Luofeini (RG7204, PLX-4032, CAS registration number 1029872-55-5) has demonstrated and caused programmed cell death in multiple cancerous cell line (such as melanoma cell series).If the Wei Luofeini BRAF/MEK interrupted in BRAF/MEK/ERK approach walks rapid – BRAF have common V600E sudden change.Wei Luofeini its cancer have V600E BRAF suddenly change (namely at amino acid position number 600 place of BRAF protein, normal valine is replaced by glutamic acid) patient in work, such as FDA approval melanoma patient.The melanoma of about 60% has V600E BRAF and suddenlys change.V600E sudden change is present in other cancer multiple, comprises lymphoma, colon cancer, melanoma, thyroid carcinoma and pulmonary carcinoma.Wei Luofeini has following structure:

(Wei Luofeini) (Genentech, Inc.) is given the ratification in the U.S. and indicates a kind of drug products of patient being used for the treatment of and having to suddenly change with BRAF V600E can not excising of (as the test ratified by FDA is detected) or metastatic melanoma. (Wei Luofeini) melanoma patient for lacking BRAF V600E sudden change (wild type BRAF melanoma) is not recommended.

In some embodiments of any combination treatment method described herein, described target therapeutic agent is ALK inhibitor.In some embodiments, this ALK inhibitor is that gram azoles is for Buddhist nun.Gram azoles is that Met and ALK (anaplastic lymphoma kinase ALK Alk receptor tyrosine kinase) inhibitor of the aminopyridine chemical series developed by Pfizer Incorporated is (see people such as Zou for Buddhist nun (also referred to as PF-02341066 or 1066), Cancer Research67:4408-4417,2007 and supplementary data).Other exemplary ALK inhibitor comprises such as TAE-684 (Novartis; See people such as Galkin, Proc.National Acad.Sci.104 (1) 270-275,2007), AP26113 (Ariad Pharmaceuticals, Inc.), and CEP-14083, CEP-14513, and CEP-11988 (Cephalon; See people such as Wan, Blood 107:1617-1623,2006); With WHI-P131 and WHI-P154 (EMD Biosciences), the chloro-N of 5- ⁴-[2-(isopropelsulfonyl) phenyl]-N ₂-{ 2-methoxyl group-4-[4-(4--methylpiperazine-1-yl) piperidin-1-yl] phenyl } pyrimidine-2,4-diamidogen and 2-[(the bromo-2-{ of 5-[2-methoxyl group-4-(4-methylpiperazine-1-yl) phenyl] is amino } pyrimidine--n-4-yl) amino]-N-methyl benzenesulfonamide (see people such as Mosse, Clin Cancer Res.2009Sep.15; 15 (18): 5609-14,2009; J.of Med.Chem.49:1006-1015,2006; Cancer Research, (US), 2004,64:8919-8923,2004; Proc.Natl.Acad.Sci.101:13306-13311,2004; Annual Review of Medicine, (US) 54:73,2003; Science 278:1309-1312,1997; Oncogene 14 (4): 439-449,1997; Oncogene 9:1567-1574,1994; Am J Pathol 160:1487-1494,2002; Am J Pathol 157:377-384,2000; Blood90:2901-2910,1997; Am J.Pathol.156 (3): 781-9,2000; J Comb Chem.8:401-409,2006 and US publication 20100152182; 20100099658; 20100048576; 20090286778; 20090221555; 20090186801; 20090118216; 20090099193; 20080176881; 20080090776; 2008/0300273; WO 2005/097765; WO2005/009389; WO 2005/016894; WO 2004/080980; And WO2004079326).

In some embodiments of any combination treatment method described herein, described target therapeutic agent is mek inhibitor.In some embodiments, this mek inhibitor is MEK1 inhibitor, MEK2 inhibitor and/or MEK1/2 inhibitor.Exemplary mek inhibitor includes but not limited to trametinib (GSK1120212), MEK162, selumetinib (AZD 6244, ARRY-142886), pimasertib (MSC1936369B, AS-703026, AS703026), GDC-0973, GDC-0623, PD-325901, GDC-0973, CI-1040, PD035901.In some embodiments, this Mek inhibitor is selumetinib, pimasertib, GDC-0973, GDC-0623 or trametinib.In certain embodiments, this Mek inhibitor is GDC-0973.

GDC-0973 (XL518) is a kind of selective depressant of MEK, is also called inhibition of mitogen-activated protein kinase kinases (MAPKK), and it is a kind of key component of the frequent RAS/RAF/MEK/ERK approach activated in people's tumor.Improper activation Promote cell's growth under exogenous growth factors disappearance of MEK/ERK approach.GDC-0973 is carrying out the clinical trial assessing solid tumor.GDC-0973 can be prepared as described in International Patent Application Publication No. WO2007044515 (A1).GDC-0973 title is (S)-(the fluoro-2-of 3,4-bis-(the fluoro-4-idodophenylamino of 2-) phenyl) (3-hydroxyl-3-(piperidin-2-yl) azetidine-1-base) ketone, and has following structure:

Trametinib (GSK 1120212, CAS registration number 871700-17-3) title is N-(3-{3-cyclopropyl-5-[(the fluoro-4-iodophenyl of 2-) amino]-6,8-dimethyl-2,4,7-tri-oxygen-3,4,6,7-tetrahydropyridine is [4,3-d] pyrimidine-1 (2H)-Ji also } phenyl) acetamide, and there is following structure:

In some embodiments of any TKI and/or RTKI, this inhibitor can be to the specific inhibitor of polypeptide of interest, such as, to the specific inhibitor of EGFR, HER2, MET/HGF, ALK, BRAF, ROS1 and/or MEK.In some embodiments of any TKI and/or RTKI, this inhibitor can be double inhibitor or general inhibitor, wherein this TKI and/or RTKI suppresses one or more polypeptide of interest, such as to EGFR, HER2, MET/HGF, ALK, BRAF, ROS1 and/or MEK, and the inhibitor of one or more other target polypeptid specificities.

A. antibody

There is provided the antibody of the separation in conjunction with polypeptide of interest (such as ALDH and/or tyrosine kinase (such as receptor tyrosine kinase)) herein, for methods described herein.In any above-mentioned embodiment, antibody is humanized.In addition, the antibody according to any above-mentioned embodiment is monoclonal antibody, comprises chimeric, humanization or people's antibody.In one embodiment, antibody is antibody fragment, such as Fv, Fab, Fab ', scFv, double antibody or F (ab ') ₂fragment.In another embodiment, antibody is full length antibody, such as " complete IgG1 " antibody or other antibody class defined herein or isotype.

In still another embodiment, the antibody according to above-mentioned any embodiment can mix any feature, alone or in combination as described in following part:

1. affinity of antibody

In certain embodiments, the antibody provided herein has≤1 μM ,≤100nM ,≤10nM ,≤1nM ,≤0.1nM ,≤0.01nM or≤0.001nM (such as 10 ^-8m or less, such as 10 ^-8m to 10 ^-13m, such as 10 ^-9m to 10 ^-13m) dissociation constant (Kd).In one embodiment, Kd is measured by radio-labelled antigen binding assay (RIA).In one embodiment, with antibody interested and the antigen enforcement RIA thereof of Fab pattern.Such as, by the Cmin when there is the titration series of unlabelled antigen ( ¹²⁵i) labelled antigen balance Fab, is then caught by plate the antigen combined with anti-Fab antibody bag and measures the solution binding affinity (see people such as such as Chen, J.Mol.Biol.293:865-881 (1999)) of Fab to antigen.In order to set up the condition of algoscopy, will porous plate (Thermo Scientific) catches with 5 μ g/ml in 50mM sodium carbonate (pH 9.6) and is spent the night with anti-Fab antibody (Cappel Labs) bag, uses 2% in PBS (w/v) bovine serum albumin to close 2-5 hour in room temperature (about 23 DEG C) subsequently.In non-adsorbed plate (Nunc#269620), by 100pM or 26pM [ ¹²⁵i] Fab interested (such as with the people such as Presta, VEGF antibody in Cancer Res.57:4593-4599 (1997), the assessment of Fab-12 the is consistent) mixing of-antigen and serial dilution.Then interested Fab is incubated overnight; But the incubation sustainable longer time, (such as about 65 hours) were to guarantee to reach balance.After this, mixture is transferred to seizure plate, in incubation at room temperature (such as 1 hour).Then solution is removed, and with 0.1% polysorbate 20 in PBS wash plate 8 times.After dull and stereotyped drying, add 150 μ l/ hole scintillation solution (MICROSCINT-20 ^tM; Packard), then at TOPCOUNT ^tMto plate count 10 minutes in gamma counter (Packard).The concentration selecting each Fab to provide to be less than or equal to maximum combined 20% is for competitive binding assay method.

According to another embodiment, Kd uses surface plasmon resonance assays is measured.Such as, use or (BIAcore, Inc., Piscataway, NJ) uses immobilized antigen CM5 chip to implement algoscopy about 10 response units (RU) in 25 DEG C.In one embodiment, carboxy methylation dextran biosensor matrix chip (CM5 is activated according to instructions hydrochloric acid N-ethyl-N '-(3-dimethylamino-propyl)-carbodiimide (EDC) of supplier and N-hydroxy-succinamide (NHS), BIACORE, Inc.).Antigen 10mM sodium acetate pH 4.8 is diluted to 5 μ g/ml (about 0.2 μM), then injects with the coupling protein matter obtaining about 10 response units (RU) with the flow velocity of 5 μ l/ minutes.After injecting antigen, inject 1M ethanolamine with closed unreacted group.In order to carry out kinetic measurement, be infused in containing 0.05% polysorbate 20 (TWEEN-20 in 25 DEG C with the flow velocity of about 25 μ l/ minutes ^tM) surfactant PBS (PBST) in the Fab (0.78nM to 500nM) of twice serial dilution.Use simple Lang Gemiaoer (Langmuir) combination model one to one ( evaluation Software version 3.2) combined and the sensing figure calculations incorporated speed (k that dissociates by matching simultaneously _on) and dissociation rate (k _off).Equilibrium dissociation constant (Kd) is with ratio k _off/ k _oncalculate.See the people such as such as Chen, J.Mol.Biol.293:865-881 (1999).If according to surperficial plasmon resonance assays above, association rate is more than 10 ⁶m ^-1s ^-1, so association rate can use fluorescent quenching technology to measure, and is namely such as equipped with spectrophotometer (Aviv Instruments) or the 8000 serial SLM-AMINCO of cut-off device according to spectrometer ^tMwith the measurement of stirring cuvette in spectrophotometer (ThermoSpectronic), when there is the antigen of increasing concentration, measuring the anti-antigen-antibody of 20nM (Fab form) in PBS pH 7.2 and (exciting=295nm in the fluorescent emission intensity of 25 DEG C; Transmitting=340nm, 16nm band is logical) rising or reduction.

2. antibody fragment

In certain embodiments, the antibody provided herein is antibody fragment.Antibody fragment includes but not limited to Fab, Fab ', Fab '-SH, F (ab ') ₂, Fv and scFv fragment, and other hereafter described fragment.About the summary of some antibody fragment, see the people such as Hudson, Nat.Med.9:129-134 (2003).About the summary of scFv fragment, see such as Pluckth ü n, in The Pharmacology of MonoclonalAntibodies, 113rd volume, Rosenburg and Moore compiles, (Springer-Verlag, New York), 269-315 page (1994); Be also shown in WO 93/16185; And U.S. Patent No. 5,571,894 and 5,587,458.About comprising salvage receptor binding epitope residue, and there is Fab and F (ab ') of the Half-life in vivo of prolongation ₂the discussion of fragment, is shown in U.S. Patent No. 5,869,046.

Double antibody is the antibody fragment with two antigen binding sites, and it can be bivalence or bispecific.See such as EP 404,097; WO 1993/01161; The people such as Hudson, Nat.Med.9:129-134 (2003); And the people such as Hollinger, Proc.Natl.Acad.Sci.USA 90:6444-6448 (1993).Three antibody and four antibody are also recorded in the people such as Hudson, Nat.Med.9:129-134 (2003).

Single domain antibody comprises the heavy chain variable domain all or in part of antibody or the antibody fragment of light-chain variable domain all or in part.In certain embodiments, single domain antibody is people's single domain antibody (Domantis, Inc., Waltham, MA; See such as U.S. Patent No. 6,248,516B1).

Can multiple technologies be passed through, include but not limited to the proteolytic digestion of complete antibody and the generation of recombinant host cell (such as escherichia coli or phage) to generate antibody fragment, as described in this article.

3. chimeric with humanized antibody

In certain embodiments, the antibody provided herein is chimeric antibody.Some chimeric antibody is recorded in such as U.S. Patent No. 4,816,567; And the people such as Morrison, Proc.Natl.Acad.Sci.USA, 81:6851-6855 (1984)).In one example in which, chimeric antibody comprises non-human variable domains (such as, from mice, rat, hamster, rabbit or non-human primates, the variable region that such as monkey is derivative) and human constant region.In another example, chimeric antibody is " class conversion " antibody, and wherein class or subclass change from the class of parental antibody or subclass.Chimeric antibody comprises its Fab.

In certain embodiments, chimeric antibody is humanized antibody.Usually, by non-human antibody's humanization to reduce the immunogenicity to people, retain specificity and the affinity of parent non-human antibody simultaneously.Usually, humanized antibody comprises one or more variable domain, wherein HVR, and such as CDR (or its part) derives from non-human antibody, and FR (or its part) derives from human antibody sequence.Optionally, humanized antibody also at least can comprise a part for human constant region.In some embodiments, the corresponding residue of some the FR residues in humanized antibody from non-human antibody's (such as antibody of derivative HVR residue) is substituted, such as, to recover or to improve antibody specificity or affinity.

Humanized antibody and generation method survey thereof are in such as Almagro and Fransson, Front.Biosci.13:1619-1633 (2008), and be recorded in the people such as such as Riechmann further, Nature 332:323-329 (1988); The people such as Queen, Proc.Nat ' l Acad.Sci.USA 86:10029-10033 (1989); U.S. Patent No. 5,821,337,7,527,791,6,982,321 and 7,087,409; The people such as Kashmiri, Methods 36:25-34 (2005) (describing specificity determining area (SDR) grafting); Padlan, Mol.Immunol.28:489-498 (1991) (describing " resurfacing "); The people such as Dall ' Acqua, Methods 36:43-60 (2005) (describing " FR reorganization "); And the people such as Osbourn, the people such as Methods 36:61-68 (2005) and Klimka, Br.J.Cancer, 83:252-260 (2000) (describing " pathfinder selection " method of FR reorganization).

May be used for humanized people framework region to include but not limited to: the framework region (see people such as such as Sims, J.Immunol.151:2296 (1993)) using " best fit (best-fit) " method choice; The derivative framework region of the consensus sequence of people's antibody of the specific subgroup from light or variable region of heavy chain (see people such as such as Carter, Proc.Natl.Acad.Sci.USA, 89:4285 (1992); And the people such as Presta, J.Immunol., 151:2623 (1993)); (somatic mutation) framework region of people's maturation or people's germline framework region (see such as Almagro and Fransson, Front.Biosci.13:1619-1633 (2008)); With the framework region (see people such as such as Baca, the people such as J.Biol.Chem.272:10678-10684 (1997) and Rosok, J.Biol.Chem.271:22611-22618 (1996)) derived by screening FR library.

4. people's antibody

In certain embodiments, the antibody provided herein is people's antibody.Multiple technologies human antibodies in next life as known in the art can be used.Usually, people's antibody is recorded in van Dijk and van de Winkel, Curr.Opin.Pharmacol.5:368-74 (2001) and Lonberg, Curr.Opin.Immunol.20:450-459 (2008).

Can by using immunity original preparation people antibody to transgenic animal, described transgenic animal have been modified to response antigenic challenge and have generated complete human antibody or have the complete antibody of people variable region.This type of animal is usually containing all or part human immunoglobulin gene seat, and it replaces endogenous immunoglobulin locus, or it to exist outward or random integration enters in the chromosome of animal at chromosome.In this type of transgenic mice, generally by the deactivation of endogenous immunoglobulin locus.Obtain the summary of the method for people's antibody about transgenic animal, see Lonberg, Nat.Biotech.23:1117-1125 (2005).Be also shown in such as U.S. Patent No. 6,075,181 and 6,150,584, which depict XENOMOUSE ^tMtechnology; U.S. Patent No. 5,770,429, which depict technology; U.S. Patent No. 7,041,870, which depict K-M technology, and U.S. Patent Application Publication text No.US 2007/0061900, which depict technology).Can such as modify from the people variable region by the zoogenic complete antibody of this class further by combining with different people constant region.

Also can by the raw human antibodies of method based on hybridoma.Human myeloma for generating human monoclonal antibodies and mouse-human heteromyeloma's cell line are described (see such as Kozbor J.Immunol., 133:3001 (1984); The people such as Brodeur, Monoclonal Antibody Production Techniquesand Applications, 51-63 page (Marcel Dekker, Inc., New York, 1987); And the people such as Boerner, J.Immunol., 147:86 (1991)).The people's antibody generated via human B-lymphocyte hybridoma technology is also recorded in the people such as Li, Proc.Natl.Acad.Sci.USA, 103:3557-3562 (2006).Other method comprises those and is such as recorded in U.S. Patent No. 7,189,826 (which depict and generate monoclonal human IgM antibody from hybridoma cell line) and Ni, Xiandai Mianyixue, 26 (4): 265-268 (2006) (which depict people-people's hybridoma).People's hybridoma technology (Trioma technology) is also recorded in Vollmers and Brandlein, Hist. & Histopath., 20 (3): 927-937 (2005) and Vollmers and Brandlein, Methods Find.Exp.Clin.Pharmacol., 27 (3): 185-91 (2005).

Also the raw human antibodies of variable domain sequence can be cloned by the Fv being separated the phage display library selection derived from people.Then, people's constant domain of this type of variable domain sequence and expectation can be combined.Described below is the technology selecting people's antibody from antibody library.

5. the antibody that library is derivative

Separation antibody can be carried out by antibody combinatorial library screening to one or more activity of expectation.Such as, expect that the multiple method in conjunction with the antibody of feature is as known in the art for generating phage display library and having this type of library screening.This type of method survey in people such as such as Hoogenboom, Methods Mol.Biol.178:1-37 (O ' people such as Brien compiles, Human Press, Totowa, NJ, 2001), and be recorded in the people such as such as McCafferty further, Nature 348:552-554; The people such as Clackson, Nature 352:624-628 (1991); The people such as Marks, J.Mol.Biol.222:581-597 (1992); Marks and Bradbury, Methods Mol.Biol.248:161-175 (Lo compiles, HumanPress, Totowa, NJ, 2003); The people such as Sidhu, J.Mol.Biol.338 (2): 299-310 (2004); The people such as Lee, J.Mol.Biol.340 (5): 1073-1093 (2004); Fellouse, Proc.Natl.Acad.Sci.USA 101 (34): 12467-12472 (2004); And the people such as Lee, J.Immunol.Methods 284 (1-2): 119-132 (2004).

In some phage display method, the complete or collected works of VH and VL gene are cloned respectively by polymerase chain reaction (PCR), and recombinate at random in phage library, then can to described phage library screening antigen in conjunction with phage, as being recorded in the people such as Winter, Ann.Rev.Immunol., 12:433-455 (1994).Phage is usually with scFv (scFv) fragment or with Fab fragment display antibody fragment.The hang oneself library in source of immunity provides for immunogenic high-affinity antibody, and does not need to build hybridoma.Or, (such as from people) natural complete or collected works can be cloned with when without any providing for large quantities of non-self and also have the single source of antibody of autoantigen when immunity, as by people such as Griffiths, EMBO J, 12:725-734 (1993) describe.Finally, also can by the V constant gene segment C do not reset from stem cell clone, and use the variable CDR3 district of the PCR primer code level containing random sequence and realize rearrangement in vitro to synthesize generating non-non-immune libraries, as by Hoogenboom and Winter, J.Mol.Biol., described by 227:381-388 (1992).The open text of patent describing people's antibody phage libraries comprises such as: U.S. Patent No. 5,750,373 and U.S. Patent Publication text No.2005/0079574,2005/0119455,2005/0266000,2007/0117126,2007/0160598,2007/0237764,2007/0292936 and 2009/0002360.

Think that the antibody that is separated from people's antibody library or antibody fragment are people's antibody herein or people's antibody fragment.

6. multi-specificity antibody

In certain embodiments, the antibody provided herein is multi-specificity antibody, such as bi-specific antibody.Multi-specificity antibody is the monoclonal antibody at least two different loci to binding specificity.In certain embodiments, one of binding specificity is for polypeptide of interest (such as ALDH and/or tyrosine kinase (such as receptor tyrosine kinase)), and another kind is for other antigen any.In certain embodiments, bi-specific antibody can in conjunction with two different epi-positions of polypeptide of interest (such as ALDH and/or tyrosine kinase (such as receptor tyrosine kinase)).Also bi-specific antibody can be used to be positioned cytotoxic agent to express the cell of polypeptide of interest (such as ALDH and/or tyrosine kinase (such as receptor tyrosine kinase)).Bi-specific antibody can with full length antibody or antibody fragment preparation.

Technology for generating multi-specificity antibody includes but not limited to have the right recombinant co-expression of not homospecific two pairs of heavy chain immunoglobulin-light chains (see Milstein and Cuello, Nature 305:537 (1983)), the people such as WO 93/08829 and Traunecker, EMBO is (1991) J.10:3655) and " swell-enter-hole " through engineering approaches (see such as U.S. Patent No. 5,731,168).Also can by the through engineering approaches electrostatic manipulation effects (WO 2009/089004A1) for generating antibody Fc-heterodimeric molecule; Two or more antibody crosslinked or fragment (see such as U.S. Patent No. 4,676,980, and the people such as Brennan, Science, 229:81 (1985)); Leucine zipper is used to generate bi-specific antibody (see people such as such as Kostelny, J.Immunol., 148 (5): 1547-1553 (1992)); Use " double antibody " technology (see people such as such as Hollinger, Proc.Natl.Acad.Sci.USA, 90:6444-6448 (1993)) for generating bispecific antibody fragment; And use scFv (sFv) dimer (see people such as such as Gruber, J.Immunol., 152:5368 (1994)); And as people such as such as Tutt, described in J.Immunol.147:60 (1991), prepare three-specific antibody to generate multi-specificity antibody.

Also comprise the engineered antibody with three or more functional antigen binding sites herein, comprise " Octopus antibody " (see such as US 2006/0025576A1).

Antibody herein or fragment also comprise " dual function FAb " or " DAF " (see the such as US 2008/0069820) comprised in conjunction with polypeptide of interest (such as ALDH and/or tyrosine kinase (such as receptor tyrosine kinase)) and the another kind not antigen binding site of synantigen.

7. antibody variants

A) glycosylation variants

In certain embodiments, the antibody that provides is changed herein to improve or to reduce the degree of antibody glycosylation.Can, by changing aminoacid sequence, make create or eliminate interpolation or the deletion that one or more glycosylation site realizes the glycosylation site of antagonist easily.

When antibody comprises Fc district, the carbohydrate of its attachment can be changed.The natural antibody generated by mammalian cell comprises branch, two antennary oligosaccharide usually, and it generally connects the Asn297 being attached to the CH2 territory in Fc district by N.See the people such as such as Wright, TIBTECH 15:26-32 (1997).Oligosaccharide can comprise various carbohydrate, such as, and mannose, N-acetyl-glucosamine (GlcNAc), galactose and sialic acid, and the fucose being attached to the GlcNAc in two antennary oligosaccharide structure " trunk ".In some embodiments, can modify to create the antibody variants with the characteristic that some improves to the oligosaccharide in antibody of the present invention.

In one embodiment, provide antibody variants, it has shortage attachment (directly or indirectly) in the carbohydrate structure of the fucose in Fc district.Such as, the fucose amount in this antibody-like can be 1% to 80%, 1% to 65%, 5% to 65% or 20% to 40%.By relative to being attached to all sugared structure of Asn297 (such as, compound, heterozygosis with the structure of high mannose) summation, the average magnitude calculating fucose in Asn297 place sugar chain measures fucose amount, as measured by MALDI-TOF mass spectrometry, such as, as being recorded in WO 2008/077546.Asn297 refers to the asparagine residue of the about the 297th (the Eu numbering of Fc district residue) being arranged in Fc district; But Asn297 also can be positioned at the 297th upstream or about ± 3, downstream aminoacid, namely between the 294th and the 300th due to the minor sequence variation in antibody.This type of fucosylation variant can have the ADCC function of improvement.See such as U.S. Patent Publication text No.US 2003/0157108 (Presta, L.); US 2004/0093621 (KyowaHakko Kogyo Co., Ltd).The example relating to the publication of " de-fucosylation " or " fucose lacks " antibody variants comprises: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; The people such as Okazaki, J.Mol.Biol.336:1239-1249 (2004); The people such as Yamane-Ohnuki, Biotech.Bioeng.87:614 (2004).The example that can generate the cell line of de-defucosylated antibody comprises the Lec13CHO cell of protein fucosylation defect (people such as Ripka, Arch.Biochem.Biophys.249:533-545 (1986); U.S. Patent application No US2003/0157108 A1, Presta, L; And WO 2004/056312 A1, the people such as Adams, especially in embodiment 11), with knock out cell line, such as α-1,6-fucosyl transferase gene FUT8 knock out Chinese hamster ovary celI (see people such as such as Yamane-Ohnuki, Biotech.Bioeng.87:614 (2004); The people such as Kanda, Y., Biotechnol.Bioeng., 94 (4): 680-688 (2006); And WO2003/085107).

Further provide the antibody variants with two typing oligosaccharide, such as, be wherein attached to two antennary oligosaccharide in antibody Fc district by GlcNAc two points.This type of antibody variants can have the fucosylation of reduction and/or the ADCC function of improvement.The example of this type of antibody variants is recorded in such as WO 2003/011878 (people such as Jean-Mairet); U.S. Patent No. 6,602,684 (people such as Umana); And US2005/0123546 (people such as Umana).Additionally provide the antibody variants in the oligosaccharide being attached to Fc district with at least one galactose residue.This type of antibody variants can have the CDC function of improvement.This type of antibody variants is recorded in such as WO 1997/30087 (people such as Patel); WO 1998/58964 (Raju, S.); And WO 1999/22764 (Raju, S.).

B) Fc region variants

In certain embodiments, can by a place or many places are amino acid modified be incorporated herein in the Fc district of antibody that provides, generate Fc region variants thus.Fc region variants can be included in the people Fc region sequence (such as, human IgG1, IgG2, IgG3 or IgG4Fc district) that one or more amino acid position comprises amino acid modified (such as substituting).

In certain embodiments, the present invention is contained and is had some but the antibody variants of not all effector functions, described effector functions becomes the expectation material standed for of following application, wherein the Half-life in vivo of antibody is important, and some effector functions (such as complement and ADCC) is unnecessary or harmful.External and/or in vivo cytotoxicity algoscopy can be carried out to confirm the reduction/abatement of CDC and/or ADCC activity.Such as, Fc receptor (FcR) binding assay can be carried out to guarantee that antibody deficiency Fc γ R combines (therefore likely lacking ADCC active), but retain FcRn binding ability.The main cell NK cell of mediation ADCC only expresses Fc γ RIII, and monocytes Fc γ RI, Fc γ RII and Fc γ RIII.The FcR summarized in table 3 on the 464th page of Ravetch and Kinet, Annu.Rev.Immunol.9:457-492 (1991) on hematopoietic cell expresses.The non-limitative example of the vitro assay of the ADCC activity of assessment molecules of interest is recorded in U.S. Patent No. 5,500,362 (see such as Hellstrom, I. people is waited, Proc.Nat ' l Acad.Sci.USA 83:7059-7063 (1986)) and Hellstrom, I. people is waited, Proc.Nat ' lAcad.Sci.USA 82:1499-1502 (1985); 5,821,337 (see people such as Bruggemann, M., J.Exp.Med.166:1351-1361 (1987)).Or, on-radiation assay method can be adopted (see the ACTI such as flow cytometry ^tMnon-radioactive cell toxicity assay (CellTechnology, Inc.Mountain View, CA; And CytoTox non-radioactive cell toxicity assay (Promega, Madison, WI)).PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC) and NK cell (NK) cell are comprised for the effector lymphocyte that this type of algoscopy is useful.Or/in addition, the ADCC that can assess molecules of interest is in vivo active, such as, in animal model, is such as disclosed in the people such as Clynes, Proc.Nat ' l Acad.Sci.USA's 95:652-656 (1998).Also C1q binding assay can be implemented to confirm that therefore antibody in conjunction with C1q, and can not lack CDC activity.See that C1q and C3c in such as WO 2006/029879 and WO 2005/100402 is in conjunction with ELISA.In order to assess complement activation, can implement CDC algoscopy (see people such as such as Gazzano-Santoro, J.Immunol.Methods 202:163 (1996); The people such as Cragg, M.S., Blood 101:1045-1052 (2003); And Cragg, M.S. and M.J.Glennie, Blood 103:2738-2743 (2004)).Also method as known in the art can be used to combine and removing/half-life mensuration (see people such as such as Petkova, S.B., Int ' l.Immunol.18 (12): 1759-1769 (2006)) in body to implement FcRn.

The antibody with the effector functions of reduction comprises those and has one or more (U.S. Patent No. 6,737,056) that substitutes in Fc district residue 238,265,269,270,297,327 and 329.This type of Fc mutant is included in two places in amino acid position 265,269,270,297 and 327 or more place and has alternative Fc mutant, comprise so-called " DANA " Fc mutant (U.S. Patent No. 7 that residue 265 and 297 is replaced into alanine, 332,581).

Describe some antibody variants of the combination to FcR that is that there is improvement or that reduce (see such as U.S. Patent No. 6,737,056; WO 2004/056312, and the people such as Shields, J.Biol.Chem.9 (2): 6591-6604 (2001)).In certain embodiments, antibody variants comprises the place or many places amino acid replacement that have and improve ADCC, the Fc district substituted of the position 298,333 and/or 334 (the EU numbering of residue) in such as Fc district.In some embodiments, Fc district is made a change, its cause change (that is, improvement or reduce) C1q combine and/or CDC (CDC), such as, as being recorded in U.S. Patent No. 6,194,551; WO 99/51642; And the people such as Idusogie, J.Immunol.164:4178-4184's (2000).

The antibody with the half-life of prolongation and the combination to neonatal Fc receptor (FcRn) of improvement is recorded in US2005/0014934A1 (people such as Hinton), neonatal Fc receptor (FcRn) is responsible for Maternal immunoglobulin G to be transferred to the fetus (people such as Guyer, the people such as J.Immunol.117:587 (1976) and Kim, J.Immunol.24:249 (1994)).Those antibody comprise wherein to have and improve a place that Fc district combines FcRn or the Fc district that many places substitute.This type of Fc variant comprises those at Fc district residue 238,256,265,272,286,303,305,307,311,312,317,340,356,360,362,376,378, a place in 380,382,413,424 or 434 or many places have alternative, such as, Fc district residue 434 substitute (U.S. Patent No. 7,371,826).Be also shown in Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; And WO 94/29351, it pays close attention to other example of Fc region variants.

C) through antibody variants that cysteine is engineered

In certain embodiments, can expect to create through the engineered antibody of cysteine, such as, " thioMAb ", wherein one or more residue cysteine residues of antibody substitute.In particular embodiments, what the residue substituted was present in antibody can close to site.By substituting those residues with cysteine, what reactive thiol group was positioned antibody thus can close to site, and may be used for antibody and other module, and such as drug moiety or linker-drug module are puted together, to create immunoconjugates, as further described herein.In certain embodiments, can with cysteine substitute following residue any one or multiple: the V205 (Kabat numbering) of light chain; The A118 (EU numbering) of heavy chain; With the S400 (EU numbering) in heavy chain Fc district.As such as U.S. Patent No. 7,521, can generate described in 541 through the engineered antibody of cysteine.

B. immunoconjugates

Go back providing package herein containing the immunoconjugates of the antibody in conjunction with polypeptide of interest (such as ALDH and/or tyrosine kinase (such as receptor tyrosine kinase)) or the immunoconjugates comprising the antibody in conjunction with polypeptide of interest (such as ALDH and/or tyrosine kinase (such as receptor tyrosine kinase)), this antibody and one or more cytotoxic agents such as chemotherapeutics or medicine, growth inhibitor, toxin (such as archon, antibacterial, fungus, plant, or the enzyme activity toxin of animal origin, or its fragment), or radiosiotope is puted together, for methods described herein.

In one embodiment, immunoconjugates is antibody-drug conjugates (ADC), wherein antibody and one or more drug conjugate, include but not limited to that Folium Mayteni hookeri alkaloid (maytansinoid) is (see U.S. Patent No. 5,208,020, No.5,416,064 and European patent EP 0425235B1); Auristatin is monomethyl auristatin drug moiety DE and DF (MMAE and MMAF) (see U.S. Patent No. 5,635,483 and No.5,780,588, and No.7,498,298) such as; Dolastatin (dolastatin); Calicheamicin (calicheamicin) or derivatives thereof (see U.S. Patent No. 5,712,374, No.5,714,586, No.5,739,116, No.5,767,285, No.5,770,701, No.5,770,710, No.5,773,001 and No.5,877,296; The people such as Hinman, Cancer Res.53:3336-3342 (1993); And the people such as Lode, Cancer Res.58:2925-2928 (1998)); Anthracycline antibiotics such as daunomycin (daunomycin) or doxorubicin (doxorubicin) (see people such as Kratz, Current Med.Chem.13:477-523 (2006); The people such as Jeffrey, Bioorganic & Med.Chem.Letters 16:358-362 (2006); The people such as Torgov, Bioconj.Chem.16:717-721 (2005); The people such as Nagy, Proc.Natl.Acad.Sci.USA 97:829-834 (2000); The people such as Dubowchik, Bioorg. & Med.Chem.Letters 12:1529-1532 (2002); The people such as King, J.Med.Chem.45:4336-4343 (2002); And U.S. Patent No. 6,630,579); Methotrexate; Vindesine (vindesine); Taxane is docetaxel (docetaxel), Pa Litasai (paclitaxel), larotaxel, tesetaxel and ortataxel such as; Trichothecin (trichothecene); And CC1065.

In another embodiment, immunoconjugates comprises antibody described herein, and this antibody is puted together with enzyme activity toxin or its fragment, includes but not limited to diphtheria toxin, diphtherotoxin A chain, the nonbinding active fragments of diphtheria toxin, diphtherotoxin, exotoxin A chain (from Pseudomonas aeruginosa Pseudomonas aeruginosa), ricin (ricin) A chain, Agglutinin (abrin) A chain, Adenia heterophylla (Bl.) Koord.[A.che-ualieri Gagnep. root toxalbumin (modeccin) A chain, α-broom aspergillin (sarcin), Aleurites fordii Hemsl. (Aleurites fordii) toxalbumin, carnation (dianthin) toxalbumin, phytolacca american (Phytolaca americana) toxalbumin (PAPI, PAPII and PAP-S), Fructus Momordicae charantiae (Momordicacharantia) mortifier, curcin (curcin), crotin (crotin), Saponaria officinalis (sapaonaria officinalis) mortifier, gelonin (gelonin), NSC-69529 (mitogellin), restrictocin (restrictocin), phenomycin (phenomycin), enomycin (enomycin) and trichothecin (tricothecenes).

In another embodiment, immunoconjugates comprises antibody described herein, and this antibody and radioactive atom are puted together to form radiation conjugate.Multiple radiosiotope can be used for generating radiation conjugate.Example comprises At ²¹¹, I ¹³¹, I ¹²⁵, Y ⁹⁰, Re ¹⁸⁶, Re ¹⁸⁸, Sm ¹⁵³, Bi ²¹², P ³², Pb ²¹²with the radiosiotope of Lu.When being used for detecting by radiation conjugate, it can comprise radioactive atom and study for scitiphotograph, such as Tc ^99mor I ¹²³, or spin label is used for nuclear magnetic resonance, NMR (NMR) imaging (also referred to as nuclear magnetic resonance, mri), such as iodo-123, iodine-131, indium-111, fluoro-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or ferrum.

Multiple bifunctional protein coupling agent can be used to carry out the conjugate of Dispersal risk and cytotoxic agent, such as N-succinimido-3-(2-pyridine radicals dithio) propionic ester (SPDP), succinimido-4-(N-Maleimidomethyl) cyclohexane extraction-1-carboxylate (SMCC), iminothiolane (IT), imino-ester (all example hydrochloric acid adipyl imidic acid dimethyl esters), active esters (such as suberic acid two succinimido ester), aldehydes (such as glutaraldehyde), double azido compound (such as two (p-azido benzoyl base) hexamethylene diamine), dual azepine derivatives (such as two (p-diazoniumbenzoyl) ethylenediamine), vulcabond (such as toluene 2, 6-vulcabond), with double activated fluorine compounds (such as 1, 5-bis-fluoro-2, 4-dinitro benzene) dual-function derivative.Such as, can as people such as Vitetta, Science 238:1098 prepares ricin immunotoxin described in (1987).The 1-isothiocyanic acid benzyl-3-methyl diethylene-triamine pentaacetic acid (MX-DTPA) of carbon-14 labelling is for the exemplary chelating agen by radioactive nucleotides and antibody conjugate.See WO94/11026.Joint can be convenient to release cells cytotoxic drug in cell " can cut joint ".Such as, sour unstable joint, peptidase-sensitive linker, photo-labile joint, dimethyl linker or containing disulfde linker (people such as Chari, Cancer Res.52:127-131 (1992) can be used; U.S. Patent No. 5,208,020).

Immunoconjugates herein or ADC are clearly contained but are not limited to this type of conjugate of preparing with following cross-linking agent, include but not limited to: commercialization is (as purchased from Pierce Biotechnology Inc., Rockford, IL, U.S.A) BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, with SVSB (succinimido-(4-vinyl sulfone) benzoate).

C. Binding peptide

Also provide Binding peptide herein, for methods described herein, Binding peptide is following polypeptide, and it combines, preferred specific binding polypeptide of interest (such as ALDH and/or tyrosine kinase (such as receptor tyrosine kinase)).In some embodiments, Binding peptide is the antagonist of polypeptide of interest (such as ALDH and/or tyrosine kinase (such as receptor tyrosine kinase)).

Binding peptide can use known polypeptide synthesis method chemosynthesis, or recombinant technique can be used to prepare and purification.The length of Binding peptide is normally at least about 5 aminoacid, or length is at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 aminoacid or longer, wherein this type of Binding peptide can be in conjunction with, preferred specific binding polypeptide of interest (such as ALDH and/or tyrosine kinase (such as receptor tyrosine kinase)).

Binding peptide just can use known technology to identify without the need to too much testing.In this, notice for can the technology of Binding peptide of specific binding polypeptide target thing be well known in the art (see such as United States Patent (USP) 5,556,762,5 to polypeptide libraries screening, 750,373,4,708,871,4,833,092,5,223,409,5,403,484,5,571,689,5,663,143; PCT publication number WO 84/03506 and WO 84/03564; The people such as Geysen, Proc.Natl.Acad.Sci.U.S.A.81:3998-4002 (1984); The people such as Geysen, Proc.Natl.Acad.Sci.U.S.A.82:178-182 (1985); The people such as Geysen, in SyntheticPeptides as Antigens, 130-149 (1986); The people such as Geysen, J.Immunol.Meth.102:259-274 (1987); The people such as Schoofs, J.Immunol.140:611-616 (1988); The people such as Cwirla, S.E., (1990) Proc.Natl.Acad.Sci.USA 87:6378; The people such as Lowman, H.B., (1991) Biochemistry 30:10832; The people such as Clackson, T., (1991) Nature 352:624; The people such as Marks, J.D., (1991), J.Mol.Biol.222:581; The people such as Kang, A.S., (1991) Proc.Natl.Acad.Sci.USA 88:8363; Smith, G.P., (1991) Current Opin.Biotechnol.2:668).The method producing peptide library and these libraries of screening is also disclosed in United States Patent (USP) 5,723,286,5,432,018,5,580,717,5,427,908,5,498,530,5,770,434,5,734,018,5,698,426,5,763,192, and 5,723,323.

D. in conjunction with micromolecule

Provide herein as micromolecule ALDH inhibitor (such as disulfiram and/or its derivant) and/or micromolecule target therapeutic agent (such as micromolecule TKI (such as micromolecule RTIK)) use in conjunction with micromolecule, for method mentioned above.

Preferably, refer to beyond Binding peptide defined herein or antibody in conjunction with micromolecule, in conjunction with the organic molecule of, preferred specific binding polypeptide of interest (such as ALDH and/or tyrosine kinase (such as receptor tyrosine kinase)).The known formula science of law can be used identify and chemosynthesis (see such as PCT publication number WO 00/00823 and WO 00/39585) in conjunction with organic molecule.Usually about 2000 dalton are less than in conjunction with organic micromolecular size, or its size is less than about 1500,750,500,250 or 200 dalton, wherein this type of can in conjunction with, preferably the organic molecule of specific binding polypeptide described herein just can use known technology to identify without the need to too much experiment.In this, notice for can be (see such as PCT publication number WO 00/00823 and WO 00/39585) well known in the art in conjunction with the technology of the molecule of polypeptide of interest to organic molecule library screening.Can be such as aldehyde in conjunction with organic molecule, ketone, oxime, hydrazone, semicarbazones (semicarbazone), carbonohydrazides (carbazide), primary amine, secondary amine, tertiary amine, the hydrazine that N-replaces, hydrazides, alcohol, ether, sulfur alcohol, sulfur ether, disulphide, carboxylic acid, ester, amide, urea, carbamate (carbamate), carbonic ester (carbonate), ketal, thio ketal ization (thioketal), acetal, mercaptal, aryl halide, aromatic yl sulphonate (aryl sulfonate), alkyl halogen, hydrocarbyl sulfonic ester (alkylsulfonate), aromatic compound, heterocyclic compound, aniline, alkene, alkynes, glycol, amino alcohol, mouth oxazolidine, mouth oxazoline, Thiazolidine, thiazoline, enamine, sulfonamide (sulfonamide), epoxide, ethylene imine (aziridine), isocyanates (isocyanate), sulfonic acid chloride, diazonium compound, acid chloride (acidchloride) etc.

E. antagonist polynucleotide

Provide polynucleotide antagonist herein, for methods described herein.Polynucleotide can be antisensenucleic acids and/or ribozyme.Antisensenucleic acids comprise at least with the sequence of the part complementation of the rna transcription thing of gene of interest (such as ALDH and/or tyrosine kinase (such as receptor tyrosine kinase)).But although preferred Absolute complementarity, it is dispensable.

" at least with the part complementation of RNA " mentioned in this article sequence means to have enough complementarity can hybridize with RNA, forms the sequence of stable duplex; When double stranded antisense nucleic acid, so can test the strand of duplex DNA, or triplex formation can be measured.Hybridization ability can depend on the length of complementary degree and antisensenucleic acids.Usually, the nucleic acid of hybridization is larger, more with the base mispairing of RNA, it can containing and still form stable duplex (or triplex, situation also can so).Those skilled in the art can confirm permissible extent of mismatch by the fusing point using standard schedule to measure hybridization complex.

Hold (such as 5 ' non-translated sequence until AUG start codon and comprise AUG start codon) complementary polynucleotide suppressing should the most effectively work in translation with information 5 '.But, show the translation also effectively suppressing mRNA with the sequence of 3 ' the non-translated sequence complementation of mRNA.Generally see Wagner, R., 1994, Nature 372:333-335.So, can use with 5 '-or 3 '-untranslated of gene, the oligonucleotide of noncoding region complementation the translation suppressing endogenous mRNA in antisense approach.The complement of AUG start codon should be comprised with the polynucleotide of 5 ' the untranslated region complementation of mRNA.Be not too effective translational inhibitor with the antisense polynucleotides of mRNA coding region complementation, but can use according to the present invention.No matter be designed to 5 ' of mRNA, 3 ' or coding region hybridize, the length of antisensenucleic acids should be at least 6 nucleotide, and preferably length range is the oligonucleotide of 6 to about 50 nucleotide.In concrete, oligonucleotide is at least 10 nucleotide, at least 17 nucleotide, at least 25 nucleotide or at least 50 nucleotide.

F. antibody and Binding peptide variant

In certain embodiments, the amino acid sequence variation of antibody and/or the Binding peptide provided herein is provided.Such as, the binding affinity and/or other biological characteristics that improve antibody and/or Binding peptide can be expected.By suitable modification being introduced in the nucleotide sequence of encoding antibody and/or Binding peptide, or the amino acid sequence variation of Dispersal risk and/or Binding peptide can be carried out by peptide symthesis.This type of is modified the deletion of the residue comprised in the aminoacid sequence of such as antagonist and/or Binding peptide and/or inserts and/or substitute.Any combination can carrying out deleting, insert and substituting is to obtain final construct, as long as final construct has the feature of expectation, such as, antigen combines.

In certain embodiments, the antibody variants with a place or many places amino acid replacement and/or Binding peptide variant is provided.Substitute the interested site of mutation and comprise HVR and FR.Conservative substituting shows in Table 1 under the title of " preferably substituting ".More the change of essence provides in Table 1 under the title of " exemplary alternative ", and further describes referring below to amino acid side chain classification.Amino acid replacement can be introduced in interested antibody and/or Binding peptide, and to the activity that product screening is expected, the antigen such as retaining/improve combination, the immunogenicity reduced or ADCC or CDC improved.

table 1

According to common side chain properties, aminoacid can divide into groups as follows:

(1) hydrophobic: nor-leucine, Met, Ala, Val, Leu, Ile;

(2) neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acid: Asp, Glu;

(4) alkalescence: His, Lys, Arg;

(5) residue of chain orientation is affected: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative alternative meeting needs to replace another classification with the member of one of these classifications.

G. antibody and Binding peptide derivant

In certain embodiments, the antibody provided and/or Binding peptide can be modified further herein to comprise other non-proteinaceous module that is as known in the art and that easily obtain.The module being suitable for antibody and/or Binding peptide derivatization includes but not limited to water-soluble polymer.The non-limitative example of water-soluble polymer includes but not limited to Polyethylene Glycol (PEG), the copolymer of ethylene glycol/propylene glycol, carboxymethyl cellulose, dextran (dextran), polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxa Pentamethylene., ethylene/copolymer-maleic anhydride, polyamino acid (or homopolymer or randomcopolymer), dextran or poly-(n-VP) Polyethylene Glycol, polypropylene glycol (propropylene glycol) homopolymer, poly(propylene oxide) (prolypropylene oxide)/ethylene oxide copolymer, polyoxyethylated polyols (such as glycerol), polyvinyl alcohol, and composition thereof.Methoxy PEG-propionaldehyde is conducive to preparation due to its stability in water.Polymer can be any molecular weight, and can be branch or unbranched.The polymer number being attached to antibody and/or Binding peptide is change, and if attachment more than a polymer, then they can be identical or different molecules.Usually, number and/or the type of the polymer of derivatization can be determined based on following consideration, described consideration includes but not limited to, antibody to be modified and/or the concrete property of Binding peptide or function, whether antibody derivatives and/or Binding peptide derivant can be used for the treatment of under qualifications, etc.

In another embodiment, antibody and/or Binding peptide is provided and the conjugate of the non-proteinaceous module that selectivity heats by being exposed to radiation.In one embodiment, non-proteinaceous module is CNT (people such as Kam, Proc.Natl.Acad.Sci.USA 102:11600-11605 (2005)).Radiation can be any wavelength, and includes but not limited to injure ordinary cells but non-proteinaceous module be heated to the wavelength of the killed temperature of cell of contiguous antibody and/or Binding peptide-non-proteinaceous module.

IV. screen and/or identify and there is the ALDH inhibitor of desired function and/or the method for target therapeutic agent

Other antagonist for polypeptide of interest (such as ALDH and/or tyrosine kinase (such as receptor tyrosine kinase)) (comprises the antibody provided herein, Binding peptide, and/or in conjunction with micromolecule), can be identified it by various algoscopy as known in the art, screen or characterize its physical/chemical properties and/or biologic activity, in methods described herein.

The aminoacid sequence of various human ALDH family member (such as " isozyme ") is known in the art and is that the public is available.See such as GenBank accession number NP.sub.--000680 (ALDH 1, member A1); GenBank accession number NP_000684 (ALDH 1, member A3); GenBank accession number AAH02967 and NP.sub.--000681 (ALDH 2); GenBank accession number NP.sub.--001026976 (ALDH 3, member A2, isoform 1); GenBank accession number CA139494 (ALDH 4, member A1); GenBank accession number CAA20248 (ALDH 5, member A1); GenBank accession number EAW81160 (ALDH 6, member A1, isoform CRA_b); GenBank accession number AAH02515 (ALDH 7, member A1); GenBank accession number NP.sub.--072090 (ALDH 8, member A1, isoform 1); GenBank accession number NP.sub.--000687 (ALDH 9, member A1); GenBank accession number AAG42417 (ALDH 12); GenBank accession number AAG42417 (ALDH 12); GenBank accession number NP.sub.--699160 (ALDH 16); With GenBank accession number CAI16766 (ALDH 18, member A1).

The crystal structure of wild type ALDH2 and ALDH2 mutant C302S is (U.S. Patent No. 8,124,389) known in the art, and can be used for design and preparation ALDH inhibitor, in methods described herein and compositions.

In certain embodiments, the computer system that the memorizer comprised comprises the atomic coordinates of ALDH polypeptide can be used as model for the compound of rationality qualification in conjunction with the ligand binding site on ALDH polypeptide.Such as, from the beginning or by improvement known compound this compounds can be designed.In other situation, binding compounds can be identified by test known compound with the molecular model of " stop " ALDH polypeptide of whether determining it.This type of parking scheme is that this area is generally known.

ALDH crystal structural data can use with microcomputer modelling technical tie-up, thus is developed the model of the combination of various ALDH-binding compounds by analyzing crystal structured data.Site model characterizes the three-dimensional topology of site surface, and comprises Van der Waals contact, electrostatic interaction and become the factors such as hydrogen bond chance.Then use computer modeling technique come Position Design become with the interactional functional group in model site (include but not limited to proton, oh group, amine groups, bivalent cation, aromatic series and aliphatic functionality, amide group, alcohol groups, etc.) interaction position.These groups can be designed into pharmacophore or candidate compound, expect that this candidate compound can this site of specific binding.So, pharmacophore design involves to be considered to fall into chemical interaction (comprise into hydrogen bond, Van der Waals, electrostatic and covalent interaction) and the site interactional ability of the candidate compound in pharmacophore via any or all available types, although generally speaking pharmacophore interacts via non-covalent mechanism and site.

Beyond actual analysis, microcomputer modelling technical Analysis pharmacophore or candidate compound can be used in conjunction with the ability of ALDH polypeptide.Only can synthesize those to indicate enough to combine energy (in one example in which, in conjunction with corresponding to 10 by microcomputer modelling ^-2the M order of magnitude or more closely to the dissociation constant of target thing) in conjunction with the candidate of target thing (such as ALDH polypeptide binding site), and use enzyme assay test that those skilled in the art will know that and/or as herein described they they in conjunction with ALDH polypeptide and the ability suppressing ALDH enzyme function.So, calculate appraisal procedure to avoid unlikely with the unnecessary synthesis of suitable affinity in conjunction with the compound of ALDH polypeptide.

Series of steps can be relied on to calculate assessment and design ALDH pharmacophore or candidate compound, wherein to chemical entities or fragment Selection and screening they with each ability of combining in conjunction with target site on ALDH polypeptide.Those skilled in the art can use one of several methods to carry out the ability of combining chemical entities or fragment screening they and ALDH polypeptide (target site more particularly, on ALDH polypeptide).This process can with a subset based on ALDH polypeptide coordinate or those coordinates known in the art, and visual detection such as target site starts on the computer screen.

In order to the ALDH inhibitor of selective enhancement induced cancer cell death, can relative to the forfeiture of film integrality in the combination with reference to assessment and target therapeutic agent (such as TKI), it is indicated by such as propidium iodide (PI), trypan blue or 7AAD picked-up.PI can be implemented under complement and immune effector cell disappearance and absorb algoscopy.By incubation together with the culture medium of tumor cell and independent culture medium or the proper combination containing ALDH and/or target therapeutic agent (TKI).By 3 day time period of cell culture.After each process, cleaning cell and etc. be divided into 35mm and be with the 12x 75 of strainer cover to manage (often pipe 1ml, each processed group 3 is managed) to remove cell lump.Then pipe accepts PI (10 μ g/ml).Can use flow cytometer and sample analyzed by CellQuest software (Becton Dickinson).The ALDH inhibitor combined with target therapeutic agent (such as TKI) that those can be selected compared with independent culture medium and/or independent target therapeutic agent (such as TKI) to induce the cell death of statistical significant level (measuring as absorbed by PI) is as cell death induced antibody, Binding peptide or in conjunction with micromolecule.

In some embodiments of any screening and/or authentication method, described candidate ALDH inhibitor is antibody, Binding peptide, in conjunction with micromolecule, or polynucleotide.In some embodiments, described ALDH inhibitor (such as disulfiram and/or its derivant) is micromolecule.

V. pharmaceutical formulation

By by have expect this antibody-like of purity and one or more optional pharmaceutical acceptable carriers (Remington ' s Pharmaceutical Sciences the 16th edition, Osol, A. compile (1980)) mixing to prepare the pharmaceutical formulation of the antagonist of ALDH inhibitor as described in this article (such as disulfiram and/or its derivant) and/or target therapeutic agent (such as TKI) with freeze-dried formulation or aqueous solution form.In some embodiments, the antagonist of ALDH inhibitor (such as disulfiram and/or its derivant) and/or target therapeutic agent (such as TKI) is in conjunction with micromolecule, antibody, Binding peptide and/or polynucleotide.Usually, pharmaceutical acceptable carrier is nontoxic in adopted dosage and concentration to receiver, and includes but not limited to buffer agent, such as phosphate, citrate and other organic acid; Antioxidant, comprises ascorbic acid and methionine; Antiseptic (such as octadecyl dimethyl benzyl ammonium chloride; Hexamethonium chloride; Benzalkonium chloride, benzethonium chloride; Phenol, butanols or benzyl alcohol; P-hydroxybenzoic acid hydrocarbyl carbonate, such as methyl parahydroxybenzoate or propyl ester; Catechol; Resorcinol; Hexalin; 3-amylalcohol; And metacresol); Low-molecular-weight (being less than about 10 residues) polypeptide; Protein, such as serum albumin, gelatin or immunoglobulin; Hydrophilic polymer, such as polyvinylpyrrolidone; Aminoacid, such as glycine, glutamine, agedoite, histidine, arginine or lysine; Monosaccharide, disaccharide and other carbohydrate, comprise glucose, mannose or dextrin; Chelating agen, such as EDTA; Saccharide, such as sucrose, mannitol, trehalose or sorbitol; Salify counter ion, such as sodium; Metal composite (such as Zn-protein complex); And/or non-ionic surface active agent, such as Polyethylene Glycol (PEG).Exemplary pharmaceutical acceptable carrier herein comprise further interstitial drug dispersant such as soluble neutral reactive transparent matter acid enzyme glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( baxter International, Inc.).Some exemplary sHASEGP and using method, comprise rHuPH20 and be recorded in U.S. Patent Publication text No.2005/0260186 and 2006/0104968.In one aspect, sHASEGP and one or more other glycosaminoglycans enzymes such as chondroitinase are combined.

Exemplary freeze-dried formulation is recorded in U.S. Patent No. 6, and 267,958.Aqueous antibody preparaton comprises those and is recorded in U.S. Patent No. 6,171, and 586 and WO2006/044908, rear a kind of preparaton comprises histidine-acetate buffer.

Preparaton herein also a kind of can treat the necessary active component of concrete indication containing exceeding, preferably those complementary activities and do not have the component of adverse effect each other.This type of active component is suitable for being effective to the amount of required object and combines existence.

Active component can be wrapped and be loaded in such as by (being such as hydroxy methocel or gelatin-microcapsule and poly-(methyl methacrylate) microcapsule respectively) in condensation technique or the microcapsule prepared by interfacial polymerization, in colloidal drug delivery system (such as liposome, albumin microspheres, microemulsion, nano-particle and Nano capsule), or in macro emulsion.This type of technology is disclosed in Remington ' s PharmaceuticalSciences, the 16th edition, and Osol, A. compile (1980).

Extended release preparation can be prepared.The suitable example of extended release preparation comprises the semipermeable matrices of the solid hydrophobic polymers of the antagonist containing ALDH inhibitor (such as disulfiram and/or its derivant) and/or target therapeutic agent (such as TKI), this substrate is shaping commercial form, such as film, or microcapsule.

Preparaton for using in body is generally aseptic.Aseptic can easily realize, such as, by filtering through sterilised membrane filter.

VI. goods

In another aspect of the present invention, provide containing mentioned abovely can be used for treating, the goods of material of prevention and/or diagnose medical conditions.Described goods comprise the label or package insert that container is connected with on described container or with described container.Suitable container comprise such as medicine bottle, pencil, syringe, IV solution bag, etc.Described container can be made with multiple material, such as glass or plastics.Described container is equipped with alone or effectively treats, prevents and/or diagnose the compositions of described illness with another combination of compositions, and can have sterile access port (phial of stopper that such as described container can be intravenous solution bag or can pierce through with hypodermic needle).At least one active agents in described compositions is the antagonist of ALDH inhibitor described herein (such as disulfiram and/or its derivant) and/or target therapeutic agent (such as TKI).Described label or package insert indicate the illness that said composition is used for the treatment of selection.In addition, goods can comprise the first container that (a) is wherein equipped with compositions, and wherein said compositions comprises the antagonist of ALDH inhibitor (such as disulfiram and/or its derivant) and/or target therapeutic agent (such as TKI); (b) second container of compositions is wherein housed, and wherein said compositions comprises other cytotoxic agent or other therapeutic agent.

In some embodiments, described goods comprise the compositions contained in label on container, described container and described container; Wherein said compositions comprises one or more reagent (such as in conjunction with the primary antibodie of one or more biomarkers, or for the probe of one or more biomarkers described herein and/or primer), instruction said composition can be used for the label on the container of the existence assessing one or more biomarkers in sample, and for using described reagent to assess the description of the existence of one or more biomarkers in sample.Described goods can comprise a set of for the preparation of sample and the description and the material that utilize reagent further.In some embodiments, described goods can comprise reagent such as primary antibodie and two anti-both, wherein two anti-ly put together such as, in a kind of label, enzyme marker.In some embodiments, described goods comprise one or more probes for one or more biomarkers described herein and/or primer.

In some embodiments of any goods, the antagonist of described ALDH inhibitor (such as disulfiram and/or its derivant) and/or target therapeutic agent (such as TKI) be antibody, Binding peptide, in conjunction with micromolecule or polynucleotide.In some embodiments, described ALDH inhibitor (such as disulfiram and/or its derivant) and/or the antagonist of target therapeutic agent (such as TKI) are micromolecule.In some embodiments, described ALDH inhibitor (such as disulfiram and/or its derivant) and/or the antagonist of target therapeutic agent (such as TKI) are antibody.In some embodiments, described antibody is monoclonal antibody.In some embodiments, described antibody is people, humanized or chimeric antibody.In some embodiments, described antibody be antibody fragment and this antibody fragment in conjunction with ALDH inhibitor (such as disulfiram and/or its derivant) and/or target therapeutic agent (such as TKI).

Goods in this embodiment of the present invention also can comprise the package insert that the described compositions of instruction can be used for treating specific illness.Or/in addition, described goods can comprise second (or 3rd) container further, the acceptable buffer of pharmacy is wherein housed, such as injection bacteriostatic water (BWFI), phosphate buffered saline (PBS), Lin Ge (Ringer) family name's solution and dextrose solution.It can comprise other material from business and user's position needs further, comprises other buffer, diluent, filter, syringe needle and syringe.

Other optional member in goods comprises one or more buffer (such as Block buffer, cleaning buffer solution, substrate buffer solution etc.), other reagent such as by the substrate of enzyme marker chemical modification (such as chromogen), epi-position repair liquid, control sample (positive and/or negative control), contrast microscope slide etc.

Be appreciated that any said products can comprise immunoconjugates described herein replacement or supplement the antagonist of ALDH inhibitor (such as disulfiram and/or its derivant) and/or target therapeutic agent (such as TKI).

Embodiment

It is below the embodiment of method and composition of the present invention.Should be appreciated that in view of general description provided above, other embodiment various can be implemented.

Embodiment 1

Materials and methods

Human carcinoma cell line and reagent

In the RPMI culture medium being supplemented with Sodium Pyruvate, 10% hyclone and antibiotics penicillin and streptomycin in 37 DEG C at 5%CO ₂human carcinoma cell line is cultivated under existence.

ALDH activation measurement

The ALDH substrate (Aldefluor Kit, Stem Cell Technology) of the bodipy labelling using the scheme according to supplier to rebuild detects ALDH activity.In RPMI culture medium, dilute substrate (5ul substrate/ml culture medium), and be added into attached cell.In 37 DEG C at CO ₂in incubator, incubation is after 30 minutes, with RPMI culture medium cleaning cell twice, and uses IncuCyte HD System (Essen BioScience) and 10 times of object lens to gather image.

Flow cytometry and RNA extract

The ALDH using Aldefluor algoscopy to detect in Kato II parental cell is active.Flow cytometry sorting is used to represent the ALDH of about 5% parental cell with the highest and minimum ALDH activity respectively ^highand ALDH ^lowcell.Be used in DEAB (a kind of cold competitive substrate) exist under together with the substrate of bodipy labelling the Kato II cell of incubation as negative control.The total serum IgE that use RNAEasy post (Qiagen) extracts carries out the gene expression analysis based on microarray.

Cell Viability Assay

With 4% paraformaldehyde fixed cell at the end of the mensuration period, and the nucleic acid staining agent Syto60 (Life Technologies) being used in 1:5000 dilution in water measures viability.SpectraMax M5 is used (to excite 635nm and launch 695nm; Molecular Device) measure fluorescence intensity.Viability states the % of non-processor contrast as.

The generation of drug resistance cell

By processing parental cell 30 days with 1uM gram of azoles for Buddhist nun, produce Kato II and GTL-16DTP.With 2uM Erlotinib process PC9 parental cell 9 days, produce DTP.In all situations, every three days replaced medium.

Immunoblotting

The NP-40 lysis buffer containing protease and inhibitors of phosphatases is used to extract protein from cell granule.Use SDS-PAGE gel (BioRad) separated by protein and implement immune detection, this uses standard scheme to carry out.For the antibody of ALDH1A1 purchased from R & D Systems, PARP and the phospho-ATM/ATR substrate antibody of GAPDH, cutting is purchased from Cell Signaling Technology, and phospho-γ H2A.x antibody is purchased from Millipore.

ROS algoscopy

Use the carboxy derivatives CM-H of fluorescein ₂dCFDA (Molecular Probes) implements ROS algoscopy.The ROS indicator of reconstruction is added into containing the growth medium of DTP in plate, and incubation 30 minutes.Use trypsin-EDTA that DTP is departed from from plate, and use Flow cytometry ROS level, use untreated parental cell in contrast.

Xenograft tumor is studied

At growth medium (RPMI 1640,10% heat-inactivated hyclone, 2mM L-glutaminate) in cultivate PC-9, PC-9-GFP, EBC-1 and GTL-16 cell to 80% and converge, then trypsin treatment is used, with PBS cleaning once, and HankShi balanced salt solution (HBSS) or HBSS and Matrigel [somatomedin minimizing; Catalogue #356231 (BD Biosciences, West Grove, PA)] 1:1 mixture in resuspension to 5x 10 ⁷the final concentration of individual cell/ml.The 5x10 that the right back side subcutaneous (s.c.) being used in immunocompromised host mice is inoculated ⁶individual cell (100 μ L) sets up often kind of xenograft tumor model.GTL-16 cell is implanted in naked (nu/nu) mice (Charles River Laboratories, Hollister, CA) in HBSS when there is no Matrigel.PC-9 and PC-9-GFP cell is implanted in naked (nu/nu) mice (Charles RiverLaboratories, Hollister, CA) in HBSS when there being Matrigel.EBC-1 cell is implanted in naked (nu/nu) mice (Charles River Laboratories, Hollister, CA) in HBSS when there is no Matrigel.When gross tumor volume reaches about 100-200mm ³time, mice is divided into the group of 10-15 the animal with size similar tumor, and start treatment that day after grouping.GDC-0712 (Genentech is taken to mice through every day (QD) oral gavage (PO), Inc., a kind of MET micromolecular inhibitor, in water, be mixed with 100mg/kg), Erlotinib (50mg/kg, in 7.5%Captisol) and/or disulfiram (Sigma, Thiuram, catalogue #86720,200mg/kg administration is mixed with in Oleum Helianthi 95%, benzylalcohol 5%), or only corresponding medium.Digital caliper (Fred V.FowlerCompany, Inc.) is used to use formula (L x W x W)/2 to measure gross tumor volume.As area (AUC) under each dosage group matched curve every day relative to the percentage ratio of medium, calculate Tumor growth inhibition (%TGI), make %TGI=100x 1 – (AUC process/sky)/(AUC medium/sky).Use R packagenlme, version 3.1 – 97in R v2.12.0 uses linear hybrid effect (LME) model curve fitting to be applied to each tumor volume data converted through Log2.

Hippocampus algoscopy

Cultivate in micro plate (SeahorseBioscience) at XF 96 porocyte and distribute each hole about 5,000 parental cell and 15,000 DTP cell, and in 37 DEG C at 5%CO ₂incubation 24 hours in incubator.Disulfiram and NAC process 48 hours is implemented under TKI exists.Oxygen consumption rate (OCR) and extracellular acidification speed (ECAR) measurement is implemented in without bicarbonate, serum-free, 37 DEG C of pre-warm culture medium.After analysis completes, with 4% paraformaldehyde fixed cell, with Hoechst dyeing, use the average core enumerate of Molecular DevicesImageXpress HCS to 4 quadrant/borescopic imagings and to each quadrant.Rod figure presents the average +/-SEM that standardized (cell number) OCR and ECAR from six holes measures.

Result

Cancer stem cell marker gene ALDH1A1 differential expression in drug resistance cell

Implement to identify at the gene of gram azoles for differential expression in Buddhist nun's toleration Kato II stomach cancer cell based on the gene expression analysis of microarray.In the gene of many rises, in drug resistance supporter (DTP) colony, identify cancer stem cell marker gene ALDH1A1.The ALDH using Aldefluor algoscopy (StemCell Technology) to measure in the Kato II cell of living is active, after gram azoles processes one month for Buddhist nun, in groupuscule (about 5%) Kato II parental cell (Figure 1A) and detect that in nearly all Kato II DTP high ALDH is active.To from ALDH ^highthe RNA of cell separation implement based on the gene expression analysis of microarray identify ALDH1A1 be in ALDH gene family with ALDH ^lowcell compares the unique member (Fig. 1 b) of rise about 8 times.Consistent with rna level, at ALDH ^highin cell and the ALDH1A1 protein level (Fig. 1 c) in Kato II detected in Kato II and the derivative DTP of GTL-16 cell (gastric cancer).Gram azoles process for Buddhist nun the ALDH1A1 protein level (Fig. 1 c) improved in 24 hours in Kato II parental cell, is detecting that any drug-induced apoptosis (the appearance measurement as the PARP protein by cutting) is not before (data show).The ALDH1A1 struck in low Kato II and GTL-16 cell expresses and has no significant effect drug susceptibility or DTP formation.

ALDH inhibitor disulfiram kills drug resistance cell

In order to understand the effect of ALDH in drug resistance, with the irreversible ALDH inhibitor process Kato II and the GTL-16 parental cell that are called disulfiram (DS).DS and metabolite thereof suppress the enzymatic activity (people such as Koppaka, 2012) of multiple ALDH family member.The growth of independent disulfiram to these cancerous cell has no significant effect, but when combining for Buddhist nun with gram azoles, DS eliminates Drug tolerance Kato II and GTL-16 cell (Fig. 2 A, B).To not expressing ALDH1A1 but expressing the similar effect (Fig. 2 C) of nonsmall-cell lung cancer PC9 cell observation to DS of other ALDH family member.When maintaining more than 10 days in Erlotinib, about 20%PC9 DTP starts division as parent PC9 cell, maintains their drug resistance characteristic simultaneously.This growth group of Erlotinib toleration PC9 DTP is called drug resistance and expands supporter or DTEP people such as (, 2010) Sharma, and different with DTP, they are much lower to the sensitivity of DS.Excellent figure in Fig. 2 presents the data from triplicate hole, DS and TKI is to the combined effect of the viability of the DTP from all three kinds of cell lines mentioned above for diagram.TKI does not eliminate DTP with independent DS pretreatment PC9 and GTL-16 cell in 3-6 days before exposing, and instruction lasting blockade ALDH is active is vital for killing DTP.

To mammary gland, colon and pulmonary carcinoma origin and 8 kinds of other cancerous cell lines depending on various oncogene test the effectiveness that multiple TKI-DS combines.Although independent DS has no significant effect viability, all TKI-DS are combined in and eliminate or significantly reduce corresponding DTP number aspect height effectively (Fig. 3).These results emphasize that the survival of drug resistance cell is generally to the dependency of ALDH activity further, and hint uses DS eliminating/postponing the potential beneficial effect in polytype cancer return in TKI combination.

Drug resistance cell has high ROS level

Compared with their normal homologue, cancerous cell has higher ROS level, thinks that this promotes cell proliferation (people such as Szatrowski, 1991; The people such as Boonstra, 2004).Be exposed to chemotherapy and X-ray therapy and improve ROS level, even higher in cancerous cell, this can cause and generates multiple aldehyde product via membrane lipid peroxidatio.Some (as malonaldehyde and 4-hydroxyl-aldehyde C-9s (4-HNE)) in these aldehyde products have the longer half-life and can cause DNA damage and cell death subsequently (people such as Chiu, 2012; The people such as Casares, 2012; The people such as Li, 2009).Respond DNA that ROS level raises in CD133+ glioblastoma stem cell and repair approach acute activation for they provide basis people such as (, 2006) Bao to the resistance of radiation.In order to determine whether that the similar mechanism involving ROS plays a role in drug resistance, in DTP, measure bioenergetics, ROS level, the degree of DNA damage and the activity of DNA reparation approach.The ROS level all observed in the DTP that PC9 with GTL-16 derives more than 6 times compared with their parental cell raises (Fig. 4 A).DS process causes the ROS level in DTP to raise further for 48 hours, and this can by being added into culture medium to reverse by NAC.The ROS level raised causes oxygen consumption rate to raise (OCR) (Fig. 4 B) and drug resistance cell double center chain DNA break increases and DNA repair mechanism activation (Fig. 4 C).These results prompting ALDH family member plays ROS street cleaner's effect, and this survival for DTP is vital.

NAC saves disulfiram to the lethal effect of DTP

Next, we want to ask whether NAC process is enough to stop TKI+DS process killing and wounding DTP.PC9 and GTL-16 cell is processed for Buddhist nun respectively with Erlotinib and gram azoles, or separately or combine with DS and NAC.As expected, DS and TKI is combined in 14 days and kills all PC9 DTP, and this is almost saved (Fig. 5 A) completely when adding NAC together with DS with TKI.Obtain similar results with GTL-16 DTP, wherein avoid the death of DS induction with the GTL-16 DTP comprising NAC (Fig. 5 B) redemption about 80% during TKI+DS process.

In order to understand the mechanism of action of DS, the DTP derivative with DS and NAC process GTL-16 reaches 48 hours, and implements immunoblot experiment with the protein extracted.DS process causes ALDH1A1 and NF κ B level to reduce, and causes γ H2A.x to raise several times, the activation (the PARP level as passed through cutting significantly raises announcement) of the extensive DNA damage in the DTP of this prompting DS process and subsequently apoptosis pathway.The existence of ROS street cleaner NAC recovers ALDH1A1 and NF κ B level, and stops γ H2A.x and DTP apoptosis to raise.

In xenograft mouse model, disulfiram postpones tumor recurrence

Use xenograft mouse model to investigate DS and eliminate/postponing the effect in tumor recurrence in vivo.First in vitro the processing scheme of PC9 In vivo study is tested in experiment, wherein with separately or the Erlotinib process PC9 cell that combines with DS 6 days.Allow that PC9 DTP in Erlotinib, a DS and Erlotinib+DS group is when growing without any when medicine, another Erlotinib+DS group continues to accept DS simultaneously.As shown in FIG, compared with Erlotinib group, observe the remarkable delay of PC9 DTP growth from the Erlotinib+DS group of two kinds of medicines of stopping using.As expected, the Erlotinib+DS subgroup continuing to accept DS is survived without PC9 DTP.Rod figure presents the data from triplicate hole, the effect of diagram DS.

For the research of PC9 xenograft, to mouse inoculation PC9 cell, and allow that tumor growth is to 100-200mm ³size, is then divided into four processed group, i.e. vehicle control, DS contrast, independent TKI and TKI+DS group.Stop process after 11 days, the animal exception of TKI+DS group, they continue to accept DS until research terminates.As depicted in figure 6b, after Erlotinib process, PC9 tumor regression is completely observed almost.For Erlotinib processed group, be 60 days as the time (TTP) before the tumour progression that 5xTTP measures, have 10 routine PR and 1 routine CR, and for Erlotinib+DS group, average tumor size, lower than primary tumor volume, has 9 routine PR and 6 routine CR (P=0.0007).With cell line data consistent, xenografts data display TKI and DS combination significantly can postpone tumor recurrence.

List of references

Koppaka,V et al.(2012).Pharmacol Rev 64,520-539.

Szatrowski,T.P.and Nathan,C.F.(1991)Cancer Research,51,794-798.

Boonstra,J.and Post,J.A.(2004)Gene,337,1-13.

Chiu,W.H.et al.(2012).Biochemical Pharmacology,83,1159-1171.

Li,Y.et al.(2009).Anti-Cancer Drugs,20,770-778.

Bao,S.et al.(2006)Nature 444:756-760.

Although in order to understand clearly object, foregoing invention is described in more detail by illustration and embodiment, and description and embodiment should not be construed as and limit the scope of the invention.By mentioning clearly complete disclosure of including all patents and the scientific literature quoted herein.

Claims

1. the method for Therapeutic cancer in individuality, it comprises the target therapeutic agent of ALDH inhibitor to this individual concomitant administration effective dose and effective dose.

2. the process of claim 1 wherein that described ALDH inhibitor and described target therapeutic agent amount separately effectively extend the cancer sensitivity period and/or postpone cell and form resistance to described target therapeutic agent.

3. improve in individuality and comprise the method for effect of the treatment of cancer of target therapeutic agent, it comprises the ALDH inhibitor of this target therapeutic agent to this individual concomitant administration effective dose and effective dose.

4. the method for a Therapeutic cancer in individuality, wherein treatment of cancer comprises the ALDH inhibitor of target therapeutic agent to this individual concomitant administration effective dose and effective dose, wherein this treatment of cancer have use this target therapeutic agent of effective dose with (under this target therapeutic agent lacks) when being included in not this target therapeutic agent standard care compared with effect of rising.

5. postpone in individuality and/or stop formation of cancer to a method for the resistance of target therapeutic agent, it comprises this target therapeutic agent of ALDH inhibitor to this individual concomitant administration effective dose and effective dose.

6. treat and form the method with the individuality of cancer that raises the probability of the resistance of target therapeutic agent, it comprises this target therapeutic agent of ALDH inhibitor to this individual concomitant administration effective dose and effective dose.

7. in the individuality with cancer, improve a method for the sensitivity to target therapeutic agent, it comprises this target therapeutic agent of ALDH inhibitor to this individual concomitant administration effective dose and effective dose.

8. in the individuality with cancer, extend the method for the period of target therapeutic agent sensitivity, it comprises the target therapeutic agent of ALDH inhibitor to this individual concomitant administration effective dose and effective dose.

9. in the individuality with cancer, extend the method for the persistent period of the response to target therapeutic agent, it comprises this target therapeutic agent of ALDH inhibitor to this individual concomitant administration effective dose and effective dose.

10. the method for any one of claim 1-9, wherein said ALDH inhibitor is micromolecule ALDH inhibitor.

11. the method for claim 10, wherein said micromolecule ALDH inhibitor is disulfiram (disulfiram) or its ALDH inhibition derivant or metabolite.

12. the method for claim 10, wherein said ALDH inhibitor is N, N-diethyl [(diethyl thiocarbamoyl) disulphanes base] thioformamide or its pharmaceutical acceptable salts.

13. the method for claim 10, wherein said ALDH inhibitor is N, N-diethyl [(diethyl thiocarbamoyl) disulphanes base] thioformamide.

The method of 14. claim 10, wherein said ALDH inhibitor is 2,2 '-two-(formoxyl-1,6,7-trihydroxy-5-isopropyl-3-methyl naphthalene) or its pharmaceutical acceptable salts.

15. the method for claim 10, wherein said ALDH inhibitor is 2,2 '-two-(formoxyl-1,6,7-trihydroxy-5-isopropyl-3-methyl naphthalene).

The method of 16. any one of claim 1-15, wherein said target therapeutic agent is tyrosine kinase inhibitor (TKI).

The method of 17. claim 16, wherein said TKI is EGFR inhibitor, HER2 inhibitor, MET inhibitor, ALK inhibitor, BRAF inhibitor, ROS1 inhibitor and/or mek inhibitor.

The method of 18. claim 16, wherein said TKI is receptor tyrosine kinase inhibitors (RTKI).

19. the method for claim 18, wherein said RTKI is EGFR inhibitor, HER2 inhibitor, MET inhibitor and/or ALK inhibitor.

20. the method for any one of claim 16-19, wherein said inhibitor is antibody inhibition, micromolecular inhibitor, Binding peptide inhibitor and/or polynucleotide antagonist.

The method of 21. claim 16, wherein said TKI is N-(3-ethynyl phenyl)-6,7-bis-(2-methoxy ethoxy) quinazoline-4-amine or its pharmaceutical acceptable salts (such as Erlotinib (erlotinib)).

The method of 22. claim 16; wherein said TKI is N-(4-(3-fluorine benzyloxy)-3-chlorphenyl)-6-(5-((2-(methyl sulphonyl) ethylamino) methyl) furan-2-base) quinazoline-4-amine, two 4-toluene sulfonic acide esters or its pharmaceutical acceptable salts (such as Lapatinib (lapatinib)).

The method of 23. claim 16, wherein said TKI is (S)-N-(2,3-dihydroxypropyl)-3-(the fluoro-4-idodophenylamino of 2-) Pyrazinamide or its pharmaceutical acceptable salts (such as AS703026).

The method of 24. claim 16, wherein said TKI is Wei Luofeini (vemurafenib).

The method of 25. claim 16, wherein said TKI is 3-((R)-1-(the chloro-3-fluorophenyl of 2,6-bis-) ethyoxyl)-5-(1-(piperidin-4-yl)-1H-pyrazoles-4-base) pyridine-2-amine or its pharmaceutical acceptable salts (such as gram azoles is for Buddhist nun (crizotinib)).

26. the method for any one of claim 1-25, wherein said cancer is gastric cancer, pulmonary carcinoma (such as nonsmall-cell lung cancer (NSCL)), colorectal carcinoma (such as colon cancer and/or rectal cancer) or basal cell carcinoma.

27. 1 kinds of pharmaceutical products, it comprises a) as the ALDH inhibitor of the effective dose of the first composition, and b) as the targeting agent of the effective dose of the second composition, for or sequentially to make for Therapeutic cancer.

The pharmaceutical products of 28. claim 27, wherein said ALDH inhibitor is micromolecule ALDH inhibitor.

29. the pharmaceutical products of claim 27 or 28, wherein said ALDH inhibitor is disulfiram or its ALDH inhibition derivant or metabolite.

The pharmaceutical products of 30. claim 27 or 28, wherein said ALDH inhibitor is N, N-diethyl [(diethyl thiocarbamoyl) disulphanes base] thioformamide or its pharmaceutical acceptable salts.

31. the pharmaceutical products of claim 27 or 28, wherein said ALDH inhibitor is N, N-diethyl [(diethyl thiocarbamoyl) disulphanes base] thioformamide.

The pharmaceutical products of 32. claim 27 or 28, wherein said ALDH inhibitor is 2,2 '-two-(formoxyl-1,6,7-trihydroxy-5-isopropyl-3-methyl naphthalene) or its pharmaceutical acceptable salts.

The pharmaceutical products of 33. claim 27 or 28, wherein said ALDH inhibitor is 2,2 '-two-(formoxyl-1,6,7-trihydroxy-5-isopropyl-3-methyl naphthalene).

The pharmaceutical products of 34. any one of claim 27 to 33, wherein said target therapeutic agent is tyrosine kinase inhibitor (TKI).

The pharmaceutical products of 35. claim 34, wherein said TKI is EGFR inhibitor, HER2 inhibitor, MET inhibitor, ALK inhibitor, BRAF inhibitor, ROS1 inhibitor and/or mek inhibitor.

The pharmaceutical products of 36. claim 34, wherein said TKI is receptor tyrosine kinase inhibitors (RTKI).

37. the pharmaceutical products of claim 36, wherein said RTKI is EGFR inhibitor, HER2 inhibitor, MET inhibitor and/or ALK inhibitor.

38. the pharmaceutical products of claim 34, wherein said inhibitor is antibody inhibition, micromolecular inhibitor, Binding peptide inhibitor and/or polynucleotide antagonist.

39. the pharmaceutical products of claim 34, wherein said TKI is N-(3-ethynyl phenyl)-6,7-bis-(2-methoxy ethoxy) quinazoline-4-amine or its pharmaceutical acceptable salts, particularly Erlotinib.

The pharmaceutical products of 40. claim 34; wherein said TKI is N-(4-(3-fluorine benzyloxy)-3-chlorphenyl)-6-(5-((2-(methyl sulphonyl) ethylamino) methyl) furan-2-base) quinazoline-4-amine; two 4-toluene sulfonic acide esters or its pharmaceutical acceptable salts, particularly Lapatinib.

The pharmaceutical products of 41. claim 34, wherein said TKI is (S)-N-(2,3-dihydroxypropyl)-3-(the fluoro-4-idodophenylamino of 2-) Pyrazinamide or its pharmaceutical acceptable salts, particularly AS703026.

The pharmaceutical products of 42. claim 34, wherein said TKI is Wei Luofeini.

The pharmaceutical products of 43. claim 34, wherein said TKI is 3-((R)-1-(2, the chloro-3-fluorophenyl of 6-bis-) ethyoxyl)-5-(1-(piperidin-4-yl)-1H-pyrazoles-4-base) pyridine-2-amine or its pharmaceutical acceptable salts, particularly gram azoles be for Buddhist nun.

The pharmaceutical products of 44. any one of claim 27 to 43, wherein said cancer is gastric cancer, pulmonary carcinoma, nonsmall-cell lung cancer (NSCL), colon cancer and/or rectal cancer or basal cell carcinoma.