CN103721255A - Application of co-blocking of PD-1 and TIM-3 signal paths to anti-stomach-cancer treatment - Google Patents

Application of co-blocking of PD-1 and TIM-3 signal paths to anti-stomach-cancer treatment Download PDF

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CN103721255A
CN103721255A CN201410006645.6A CN201410006645A CN103721255A CN 103721255 A CN103721255 A CN 103721255A CN 201410006645 A CN201410006645 A CN 201410006645A CN 103721255 A CN103721255 A CN 103721255A
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tim
cell
gastric cancer
signal path
blocker
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杨林
宗云辉
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Suzhou University
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Suzhou University
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Abstract

The invention relates to application of co-blocking of PD-1 and TIM-3 signal paths to anti-stomach-cancer treatment, and in particular relates to a blocking agent of the PD-1 signal path, a blocking agent of the TIM-3 signal path and application of the blocking agents together with the conventional anti-stomach-cancer treatment medicaments to preparation of a medicinal composition for resisting stomach cancer. The blocking agents or a combination of the blocking agents and the conventional anti-stomach-cancer medicaments treats stomach cancer by enhancing the stomach cancer cell killing effect of T cells. The composition has a remarkable treatment effect, and has the advantages of small toxic and side effects and cooperation with the conventional stomach cancer treatment medicaments.

Description

Common blocking-up PD-1 and the purposes of TIM-3 signal path in anti-curing gastric cancer
Technical field
The invention belongs to biotechnology and field of medicaments, be specifically related to a kind of new method for the treatment of gastric cancer, jointly block PD-1 and TIM-3 signal path; Proved that the method and existing anti-curing gastric cancer drug combination have synergy simultaneously.Accordingly, the application of the coupling that the invention provides PD-1 signal path blocker and TIM-3 signal path blocker or itself and existing anti-curing gastric cancer medicine in curing gastric cancer.
Background technology
Tumor-infiltrated lymphocyte (the Tumor Infiltration Lymphocytes that has about half in multiple mouse tumor model, TILs) coexpression TIM-3 and PD-1, and show the exhaustion of function, lose the ability of propagation and secretion IL-2, TNF-α and these cytokines of IFN-γ.Common blocking-up PD-1 and TIM-3 signal path, be found to compare with independent blocking-up PD-1 or TIM-3 signal path, can more effectively suppress tumor growth.In human cancer, TIM-3 is at PD-1 +the specific CD8 of NY-ESO-1 +high expressed on T cell, TIM-3 +/ PD-1 +the NY-ESO-1 specific C D8 of two positives +t cell compares TIM-3 -/ PD-1 +and TIM-3 -/ PD-1 -nY-ESO-1 specific C D8 +t cell function is more disorderly, secretion IL-2, TNF and IFN-γ still less, and blocking-up TIM-3 signal path can strengthen NY-ESO-1 specific C D8 +the ability of the increment of T cell and secrete cytokines, jointly blocks PD-1 signal path and has significant synergism.
Some previous researchs are just studied colon sarcoadenoma (MC38 and CT26) and these 3 kinds of mouse tumor models of fibrosarcoma (WTMCA2); Research in human tumor is also the research for the specific CD8+T cell of NY-ESO-1 in melanoma patients.Shortage is to the especially global sickness rate the 4th of other cancers of the mankind, the research of the cancer of mortality rate second---gastric cancer, and it is global 42% that Chinese incidence gastric cancer rate accounts for, and average every 3 minutes Jiu You Chineses die from gastric cancer.In the face of the severe gastric cancer situation of China, we wish to treat gastric cancer by the immunization therapy approach of common blocking immunity Inhibitory signal path.
In cancer human body.Immune system is a whole existence.By to whole CD3 +, CD4 +and CD8 +can the result that the research of T cell draws will embody really more comprehensively patient and accept to obtain effective curative effect after PD-1 and the blocking-up of TIM-3 signal path.Research in melanoma patient, has just studied PD-1 and TIM-3 at the specific CD8 of NY-ESO-1 +expression on T cell, and after blocking-up PD-1 and TIM-3 signal path to the specific CD8 of NY-ESO-1 +the impact of T cytoactive.Previous research lacks research PD-1 and TIM-3 CD3 in cancer patient periphery PBMC and TIL of globality +, CD4 +and CD8 +the research of T cells level, and lack after common blocking-up PD-1 and TIM-3 signal path CD4 +and CD8 +the impact assessment of T cytoactive.
Summary of the invention
The object of the invention is for above-mentioned the deficiencies in the prior art, a kind of common blocking-up PD-1 and the purposes of TIM-3 signal path in anti-curing gastric cancer are provided.
For achieving the above object, the technical solution used in the present invention is as follows.
The purposes of the blocker of the blocker of PD-1 signal path and TIM-3 signal path in the pharmaceutical composition of the anti-gastric cancer of preparation.
Further, described pharmaceutical composition comprises:
1) blocker of the blocker of PD-1 signal path and TIM-3 signal path;
2) acceptable carrier or excipient pharmaceutically or in immunology;
With 3) optional, other anti-gastric cancer medicine.
Further, described pharmaceutical composition and other anti-gastric cancer Drug combination.
Further, described pharmaceutical composition comprises other anti-gastric cancer medicine.
Further, the blocker of described PD-1 signal path is anti-PD-1 monoclonal antibody, and the blocker of described TIM-3 signal path is anti-TIM-3 monoclonal antibody.
Further, described other anti-gastric cancer medicine is one or more in alkylating agent, antimetabolite, antitumor antibiotics, plant anticarcinogen, hormone.
Further, described other anti-gastric cancer medicine is one or more in cisplatin, epirubicin, fluorouracil.
The present invention further provides a kind of pharmaceutical composition, having comprised: the 1) blocker of the blocker of PD-1 signal path and TIM-3 signal path; 2) acceptable carrier or excipient pharmaceutically or in immunology; With 3) optional, other anti-gastric cancer medicine.
The present invention further provides a kind of medicine box, comprising: the container that i) contains the blocker of PD-1 signal path and the blocker of TIM-3 signal path; Ii) container that contains other anti-gastric cancer medicine; With optional iii) operation instructions.
The present invention also provides a kind of method for the treatment of gastric cancer, described method comprises the object that pharmaceutical composition of the present invention or medicine box is needed to this treatment, or by the blocker of PD-1 and TIM-3 signal path and the medication combined object that needs this treatment of other anti-gastric cancer.
The invention has the beneficial effects as follows: jointly block the effect that PD-1 and TIM-3 signal path can effectively strengthen T cell killing stomach cancer cell, thereby for the clinical immunization therapy of gastric cancer provides strong foundation, and for providing new effective way for the control of clinical application and gastric cancer.
Accompanying drawing explanation
Fig. 1 is the operational flowchart of embodiments of the invention 1.
Fig. 2 A, Fig. 2 B, Fig. 2 C, Fig. 2 D are the peripheral blood in gastric cancer patients mononuclear cell (PBMC) of embodiments of the invention 1, the representative schematic diagram of adjacent mucosa (AGM) and tumor-infiltrated lymphocyte (TILs) and integrate schematic diagram.
Wherein, the data display CTLA-4(C of integration) with TIGIT(D) at CD3 +, CD4 +and CD8 +expression in T cell mass.
Fig. 3 is normal person's peripheral blood lymphocytes (NDMC) of embodiments of the invention 1, peripheral blood in gastric cancer patients mononuclear cell (PBMC), the representative schematic diagram of adjacent mucosa (AGM) and tumor-infiltrated lymphocyte (TIL).
Wherein, data display PD-1 and TIM-3 are at CD3 +, CD4 +and CD8 +expression in T cell mass.
Fig. 4 shown in embodiment 1, respectively from the tumor-infiltrated lymphocyte of 15 Patients with Gastric Cancer, and adjacent mucosa and peripheral blood lymphocytes (TIL, AGM and PBMC), and 9 normal person's peripheral blood lymphocytes (NDMC) PD-1 +, TIM-3 +, PD-1 +/ TIM-3 +, PD-1 +/ TIM-3 -, PD-1 -/ TIM-3 +and PD-1 -/ TIM-3 -shared CD3 +the percentage rate of T cell.
Fig. 5 shown in embodiment 1, respectively from the tumor-infiltrated lymphocyte of 15 Patients with Gastric Cancer, and adjacent mucosa and peripheral blood lymphocytes (TIL, AGM and PBMC), and the PD-1 of 9 normal person's peripheral blood lymphocytes (NDMC) +, TIM-3 +, PD-1 +/ TIM-3 +, PD-1 +/ TIM-3 -, PD-1 -/ TIM-3 +and PD-1 -/ TIM-3 -shared CD4 +the percentage rate of T cell.
Fig. 6 shown in embodiment 1, respectively from the tumor-infiltrated lymphocyte of 15 Patients with Gastric Cancer, and adjacent mucosa and peripheral blood lymphocytes (TIL, AGM and PBMC), and 9 normal person's peripheral blood lymphocytes (NDMC) PD-1 +, TIM-3 +, PD-1 +tIM-3 +, PD-1 +tIM-3 -, PD-1 -tIM-3 +and PD-1 -tIM-3 -shared CD8 +the percentage rate of T cell.
Fig. 7 shown in embodiment 1, at the TIL of 15 patients with gastric cancer, and in AGM and PBMC, CD3 +(A and B), CD4 +(C and D) and CD8 +the average fluorescent strength (MFI) of (E and F) T cells PD-1 and TIM-3.P value calculates with Wilcoxon signed rank sum test.
Fig. 8 A, Fig. 8 B, Fig. 8 C, Fig. 8 D shown in embodiment 1, with anti-Tim-3(α TIM-3) and/or anti-PD-1(α PD-1) after monoclonal antibody or homotype contrast (IgG) blocking-up, CD4 +(A and C) and CD8 +the expression of cytokine in (B and D) TIL.
Fig. 9 has shown in embodiment 1, with anti-Tim-3(α TIM-3) and/or anti-PD-1(α PD-1) cytotoxicity of MAbs blocking enhancing TIL to AGS.AGS-GFP and TIL cultivate 4h altogether with the form of 1:6, and AGS mortality rate detects by the cell streaming method of 7-AAD dyeing.
The specific embodiment
In order to make object of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is described in further detail.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
The present inventor after research and testing, find that common blocking-up PD-1 and TIM-3 signal path can effectively strengthen the effect of T cell killing stomach cancer cell, the blocker of the blocker of PD-1 signal path and TIM-3 signal path is used simultaneously can obviously suppress proliferation of human gastric cancer cell, promote apoptosis in gastric cancer, thereby reach the effect for the treatment of gastric cancer, and find that such medicine and existing anti-curing gastric cancer drug combination have the effect of significant Synergistic treatment gastric cancer.
the blocker of the blocker of PD-1 signal path and TIM-3 signal path
Herein, term " PD-1 signal path " refers to PD-1 receptor and its part PDL1(B7-H1, the CD274 that is expressed in activated T cell surface) or PDL2 (B7-DC, CD273) contact after, by signal path suppressor T cell such as phosphorylation SHP2, activate; Term " TIM-3 signal path " refers to that TIM-3 can raise at the T cell surface expression of IFN-γ secretion, after being combined with its part galectin-9, Bat-3 is dissociated from TIM-3 receptor, thereby secretion and the propagation of suppressor T cell IFN-γ even cause T cell death.
Herein, term " blocker of PD-1 signal path " refers to the active substance that can be used for blocking PD-1 signal path, and term " blocker of TIM-3 signal path " refers to the active substance that can be used for blocking TIM-3 signal path.Term " blocker " refers to part or all of prevention or suppresses the material of a certain effect.
Blocker of the present invention can be active substance as known in the art, comprising: PD-1 monoclonal antibody, TIM-3 monoclonal antibody, CTLA-4 monoclonal antibody, LAG3 monoclonal antibody, but be not limited to above-mentioned active substance.
In a preferred embodiment of the present invention, described blocker is preferably antibody, more preferably monoclonal antibody.Described antibody can obtain by method as known in the art.
The effect of the inhibition proliferation of human gastric cancer cell that blocker of the present invention can obviously be brought into play, promotion apoptosis in gastric cancer, has significant synergy with the drug combination of existing anti-curing gastric cancer.
other anti-gastric cancer medicine
The effect of the inhibition proliferation of human gastric cancer cell that blocker of the present invention not only itself can obviously be brought into play, promotion apoptosis in gastric cancer, it also can produce significant synergy with existing anti-gastric cancer drug combination.
Described other anti-gastric cancer medicine comprises: alkylating agent, antimetabolite, antitumor antibiotics, plant anticarcinogen, hormone.Be preferably cisplatin, epirubicin, fluorouracil.
pharmaceutical composition and medicine box
Pharmaceutical composition provided by the invention comprises: the blocker of the blocker of PD-1 signal path and TIM-3 signal path, acceptable carrier or excipient, other anti-gastric cancer medicine pharmaceutically or in immunology.
This pharmaceutical composition can be effective to the treatment of gastric cancer.As there is other anti-gastric cancer medicine in compositions, itself and the blocker of PD-1 signal path are, the blocker three's of TIM-3 signal path mol ratio is (8 – 40): 1:1.
Herein, term " pharmaceutically or in immunology acceptable carrier " refers to the carrier for health product or pharmaceutical preparation, comprises various excipient and diluent.Applicable carrier is that those skilled in the art is known, preferred diluent of the present invention, concrete optional normal saline (0.9% sodium chloride solution).
Compositions of the present invention is liquid solution.
The present invention also provides a kind of medicine box for curing gastric cancer, comprising: accommodate the blocker of PD-1 signal path and the blocker of TIM-3 signal path container, accommodate the container of other anti-gastric cancer medicine, optional operation instructions.
In embodiments of the present invention, before the blocker that gives the blocker of PD-1 signal path, TIM-3 signal path, give other anti-gastric cancer medicine afterwards or simultaneously, to produce synergistic therapeutic effect.
method of application
Medicine of the present invention is by following which kind of administering mode: in parenteral, subcutaneous, intraperitoneal, lung, in intranasal, tumor, topical, or other administering mode, preferably intravenous injection or intravenous drip.The amount of application of the present composition, by active substance weighing scale, is generally about 0.001-5mg blocker/kg body weight every day, preferably about 0.01-1/kg body weight, preferably 0.01-0.5mg/kg body weight.
advantage of the present invention
The present invention has advantages of as follows: jointly block the effect that PD-1 and TIM-3 signal path can effectively strengthen T cell killing stomach cancer cell, thereby for the clinical immunization therapy of gastric cancer provides strong foundation, and for providing new effective way for the control of clinical application and gastric cancer.
embodiment: jointly block PD-1 and TIM-3 signal path to In vitro culture Stomach Carcinomas cell lethal effect
(1) material
1. specimen and cell line
1) stomach organization, cancer beside organism and peripheral blood in gastric cancer patients (by the attached First People's Hospital of University Of Suzhou, being provided);
2) normal person's peripheral blood (being provided by attached the second the People's Hospital of University Of Suzhou);
3) gastric carcinoma cell lines (AGS is from the Sino-British hematology of Tang of University Of Suzhou research center).
2. main agents and material
1) RPMI1640, DMEM high glucose medium (HyClone company);
2) hyclone (FBS), 0.25% pancreatin (HyClone company);
3) penicillin-streptomycin (HyClone company) for cell culture;
4) Ficoll-paque plus lymphocyte separation medium (GE healthcare company);
5) 7-AAD dyestuff (BD company);
6) the fixing dead cell dye reagent box (invitrogen company) of LIVE/DEAD;
7) fixing and rupture of membranes solution and rupture of membranes/cleaning buffer solution (BD company);
8) Golgi body blocking-up reagent (BD company);
9) Trizol lysate (invitrogen company);
10) viral packaging plasmid Δ R, REV and peplos plasmid VSV-G;
11) activation of PersonGen human T-lymphocyte and amplification kit (Bo Shengji company).
3. key instrument and equipment
1) FACS Aria III and FACS Caliber flow cytometer (BD company);
2) superclean bench (Thermo company);
3) CO 2cell culture incubator (Thermo company);
4) low speed refrigerated centrifuge (Thermo company);
5) high speed centrifuge (Beckman Coulter company);
4. main antibody
1) mouse-anti people CD3-FITC, mouse-anti people CD4-FITC, mouse-anti people CD8-FITC, mouse-anti people CD4-APC, mouse-anti people PD-1-APC, mouse-anti people PD-1-FITC, mouse-anti-human T IM-3-PE, mouse-anti-human T IGIT-PerCP Cy5.5, mouse-anti people CTLA-4-PerCP Cy5.5, mouse-anti people CD8-PE Cy7, mouse-anti human IL-2-PE, mouse-anti people IFN-γ-PerCP Cy5.5, mouse-anti-human T NF-α-APC, mouse-anti people CD3-PerCP Cy5.5, mouse-anti people CD8-PerCP Cy5.5(BD company);
2) mouse-anti people PD-1 monoclonal antibody, mouse-anti people CD3 monoclonal antibody (Bo Shengji company);
3) mouse-anti-human T IM-3 monoclonal antibody clone2E2(Millipore company);
4) normal mouse IgG antibody (invitrogen company).
(2) method
According to conventional cell culture processes, at 37 ℃, 5%CO 2environment cultivate people's cell.Comprise gastric carcinoma cell lines: AGS, AGS-GFP; The tumor-infiltrated lymphocyte (TIL) extracting from the stomach organization of Patients with Gastric Cancer; The T lymphocyte extracting from Patients with Gastric Cancer or normal person's peripheral blood.AGS, AGS-GFP, TIL, T cell are with containing 10%FBS DMEM culture medium culturing.
Fig. 1 is the operational flowchart of embodiments of the invention 1, and as shown in Figure 1, the present embodiment specifically comprises the steps:
1. set up the positive ags cell of GFP-luc system
The preparation of 1.1GFP-luc slow virus
1.1.1 plasmid transforms
1) get 100 μ l competent cells and be placed in ice bath;
2) in competent cell suspension, add 1ng object plasmid (GFP-luc, Δ R, REV or VSV-G), rotating centrifugal pipe to be to mix content gently, standing 30min in ice bath;
3) centrifuge tube is placed in to 42 ℃ of water-baths and places 60s, then fast pipe is transferred in ice bath, make the cooling 2min of cell;
4) in centrifuge tube, add the aseptic SOC culture medium of 900 μ l (not containing antibiotic), mix and be placed on 37 ℃ of shaking table shaken cultivation 45min(150rpm/min);
5) centrifuge tube content is mixed, the competent cell that absorption 100 μ l have transformed is coated on the LB solid medium that contains ammonia benzyl antibiotic (Ampicillin, Amp), is inverted flat board, cultivates 12h for 37 ℃.
1.1.2 plasmid extraction
1) monoclonal of selecting on above-mentioned LB flat board is placed in 3ml LB culture medium (Amp), 37 ℃ of shaking table shaken cultivation 12h(250rpm/min);
2) after 12h, draw 1ml bacterium liquid to 200ml LB culture medium (Amp), 37 ℃ of shaking table shaken cultivation 18h-20h(250rpm/min);
3) according to Axygen plasmid, extract in a large number test kit description and extract plasmid, by UV spectrophotometer measuring plasmid concentration and purity, be placed in-20 ℃ of preservations.
1.1.3 slow virus is packed
1) cell culture: get 6 * 10 6individual 293T cell is inoculated in 100mm Tissue Culture Dish, spends the night, and when cell degree of converging reaches 90%, changes the fresh DMEM culture medium of 6ml;
2) after 3h, carry out packaging virus, in EP pipe, add 450 μ l sterilized water, add following plasmid, mix;
Plasmid DNA content (μ g)
Object plasmid (GFP-PDL1, GFP-luc) 10
ΔR 6.5
REV 3.5
VSV-G 2.5
3) dropwise add 50 μ l CaCl 2, mix;
4) separately prepare new 1.5ml EP pipe, add 500 μ l2 * HBS;
5) by 2) 3) add 4), mix, the standing 15min of room temperature is muddy as seen;
6) above-mentioned mixed liquor is added in 293T cell, cross method mixes, and incubator is cultivated 24h;
7) after 24h, change 6ml fresh culture, continue to cultivate;
8) after 48h, collect first virus, and add 6ml fresh culture;
9) after 72h, collect second batch virus, now available fluorescence microscope GFP fluorescence intensity detects transfection efficiency;
10) the viral centrifuging and taking supernatant of collecting, with the membrane filtration of 0.22 μ m ,-80 ℃ of preservations after subpackage.
1.2 supercentrifugation concentrating virus
1) in superclean bench, 6 metal canulas and lid are sprayed to 75% ethanol, tip upside down on filter paper, air-dry standby.
2) culture supernatant of collecting, 1200rpm, 5min(is in order to remove 293T cell).
3) by the supernatant 0.22um membrane filtration degerming of centrifugal acquisition.
4) virus (filtering) is transferred in 6 ultracentrifugation pipes to 35ml/tube.
5) centrifuge tube is put into metal tube, tighten lid, 25000rpm, 90min.
6) centrifuge tube is carefully taken out, outwell supernatant, tip upside down on the filter paper disinfecting in alcohol, blot residual media.
7) to centrifuge tube bottom, directly add the virolysis liquid of preparation in advance.
8) centrifuge tube is put into the common centrifuge tube of 50ml, tightened lid, sealed membrane is sealed, vortex vibration 40-60min
9) the resuspended liquid of the virus at the bottom of collecting pipe, 10000rpm, 1min(removes cell debris).
10) supernatant of centrifugal acquisition is divided and installed in cryopreservation tube, 100ul/tube ,-80 ℃ of preservations.
The titer determination of 1.3 viruses
1) first day: 24 orifice plate pavings 3 * 10 5individual 293T cell, every hole adds 1ml culture medium.
2) second day: get cell counting in two holes and (should obtain 6-8 * 10 5individual), average.Viral with ten times of gradient dilutions, cumulative volume 250ul, 2-3 hole of each dilution factor transfection.
3) the 3rd day: in every hole, add 1ml culture medium.
4) the 5th day: flow cytometer showed 293T cell GFP positive rate, calculates virus titer by the positive rate of 5-10%.
1.4GFP-luc slow-virus transfection ags cell
5 * 10 5ags cell is suspended in 250 μ l DMEM culture medium, and bed board in 24 orifice plates adds GFP-luc virus by ratio (1:10), mixes.According to GFP fluorescence, sorting obtains AGS-GFP cell two days later.
2. separated and prepare lymphocyte single cell suspension
2.1 histiocytes are separated
1) Jiang Yi block organization is placed on 40-50 object rustless steel and organizes on filter screen, guarantees that tissue is immersed in PBS;
2) with scalpel, cut out the about 2mm penetrability of spacing otch;
3) rotate the piston portion of syringe, make tissue through filter screen;
4) cell suspension is filtered with 200 μ m nylon mesh screens, and collect in centrifuge tube;
5) 1000RPM, centrifugal 10min;
6) PBS re-suspended cell.
2.2T cell or PBMC preparation
1) blood or the resuspended histiocyte of PBS after dilution are slowly superimposed on Ficoll with pipet along tube wall;
2) centrifugal: 400g, 30min;
3) collect the mononuclearcell of upper plasma layer and Ficoll interface, move in a new centrifuge tube;
4) PBS washed cell is twice;
5) with culture medium or PBS re-suspended cell.
3. phenotype analytical (flow cytometer showed)
Use the PBMC from patient or normal person of Ficoll separation or from the patient patient TILHuo T of cancer beside organism cell and CD3-FITC or CD4-FITC or CD8-FITC, PD-1-APC, TIM-3-PE antibody is hatched jointly, finally add 7-AAD to distinguish living cells, or use TIGIT-PerCP Cy5.5 or CTLA-4-PerCP Cy5.5 antibody incubation.On FACS Aria III flow cytometer, detect, then use FlowJo software (Tree Star company) analysis result.
4.T cells in vitro activates
Use the PBMC from patient or normal person or patient's til cell counting 1 * 10 of Ficoll separation 6, add once in a week 100 activation of μ l PersonGen human T-lymphocyte and amplifing reagents, every other day add 30U/ml IL-2, after two weeks, T cell is in exponential phase, and then collecting cell carries out follow-up test.
5. intracellular cytokine dyeing
1) 2 * 10 6the TIL of Activation In Vitro and 2 * 10 5aGS stomach cancer cell mixes, and at 10 μ g/ml normal mouse IgG, anti-TIM-3 is or/and jointly hatch under the existence of anti-PD-1 monoclonal antibody;
2) after 2 hours, 1:1000 adds Golgi body blocking-up reagent;
3) collecting cell after 5 hours, PBS cleans;
4) use
Figure BDA0000454128620000121
fixing dead cell stain reagent dyeing;
5) with CD4-FITC or CD8-PE Cy7, cell is carried out to padding;
6) fixing and rupture of membranes solution is hatched 20min;
7) Perm/Wash buffer solution for cleaning cell is twice;
8) with IFN-γ-PerCP Cy5.5, IL-2-PE and TNF-α-APC carry out dyeing in born of the same parents;
9) on FACS Aria III flow cytometer, detect, then use FlowJo software (Tree Star company) analysis result.
6. cell killing experiment
1) get 4 * 10 5aGS-GFP cell and 2.4 * 10 6tIL cultivates altogether, adds 10 μ g/ml normal mouse IgG simultaneously, and anti-TIM-3 is or/and anti-PD-1 monoclonal antibody is hatched 4-6 hour;
2) cell mixture digestion is taken out, PBS washes twice;
3) add 1 μ l7-AAD, use on FACS Caliber flow cytometer and detect.
7. statistical analysis
Use paired data t check and Wilcoxon signed rank sum test method to carry out statistical significance analysis.GraphPad Prism software (GraphPad software company) is for all statistical analysis.Two tails detect, and p < 0.05 represents that result difference is remarkable, and p < 0.01 represents that result difference is extremely remarkable.
8. result of the test
8.1PD-1 and TIM-3 raise at Patients with Gastric Cancer T cells
In order to determine which co-suppression molecule plays an important role in gastric cancer TILs process tired out, TILs in separated stomach organization, T cell in adjacent mucosa, and Patients with Gastric Cancer and normal person's peripheral blood lymphocytes (PBMC), then analyze CTLA-4, TIGIT, PD-1 and TIM-3 at CD3 +, CD4 +and CD8 +expression in T cell.
CTLA-4 is at patient P BMC CD3 +/ CD4 +in T cell, substantially do not express, but at CD8 +t cellular expression is higher; CD3 +/ CD4 +/ CD8 +cTLA-4 high expressed in TIL (Fig. 2 A and C).But from average fluorescent strength (mean fluorescence intensity, MFI) analysis result, expression and the CTLA-4 expression basic zero difference (Fig. 2 C) in PBMC T cell of CTLA-4 in TIL.
TIGIT is at Patients with Gastric Cancer CD3 +/ CD4 +expression ratio in TIL is at normal person CD3 +/ CD4 +expression in T cell raises, and TIGIT is at Patients with Gastric Cancer CD8 +expression ratio in TIL is at normal person CD8 +expression in T cell reduces.The ratio of TIGIT positive cell accounts for from the CD3 in Patients with Gastric Cancer cancer beside organism (AGM) +and CD4 +in T cell, be the highest (Fig. 2 B and 2D).With MFI, analyze TIGIT and obtain analog result (Fig. 2 D).
PD-1 +cell is at CD3 +in TIL, shared ratio is that (60.18 ± 23.57%) are apparently higher than PD-1 +the shared patient P BMC CD3 of cell +ratio (35.85 ± 19.34% in T cell; P=0.0126); While PD-1 +cell accounts for patient P BMC CD3 +the ratio of T cell is apparently higher than normal pbmc CD3 +pD-1 in T cell +ratio (11.70 ± 6.436%, the p=0.0022 of cell; Fig. 3 and 4A).PD-1 is at CD4 +/ CD8 +expression in T cell and PD-1 are at CD3 +expression of results in T cell is consistent, at CD4 +/ CD8 +in TIL, express the highest, at Patients with Gastric Cancer periphery CD4 +/ CD8 +t cells significantly reduces, at normal person's periphery CD4 +/ CD8 +t cells significantly reduces (Fig. 3,5A and 6A).Use MFI to analyze PD-1 at Patients with Gastric Cancer CD3 +, CD4 +and CD8 +expression in TIL and PBMCs obtains identical result (Fig. 4 A, 5A and 6A).In the other mucosal tissue of Patients with Gastric Cancer cancer and in TILs, express the CD3 of PD-1 +, CD4 +and CD8 +the ratio similar (Fig. 4 A, 5A and 6A) of T cell.
TIM-3 +cell is at CD3 +in TIL, shared ratio (44.89 ± 29.42%) is apparently higher than TIM-3 +the shared patient P BMC CD3 of cell +ratio (26.77 ± 17.25% in T cell; P=0.0119); TIM-3 +the shared patient P BMC CD3 of cell +the ratio of T cell is apparently higher than TIM-3 +the shared normal pbmc CD3 of cell +ratio (10.78 ± 11.54%, p=0.0314 in T cell; Fig. 2 and 3B).TIM-3 is at CD4 +expression in T cell and TIM-3 are at CD3 +expression of results consistent (Fig. 3 and 5B) in T cell.TIM-3 is at normal pbmc, patient P BMC, adjacent mucosa and TILs CD8 +between T cell, there is not differential expression, although express (34.29 ± 14.31%, 46.51 ± 18.70%, 56.16 ± 24.77%, 60.08 ± 24.07%) in rising trend (Fig. 3 and 6B).Clearly, patient P BMC CD3 +/ CD4 +the expression of T cell TIM-3 is 2.5 times of normal level, CD3 +/ CD4 +the expression of TILs TIM-3 is 4.5 times of normal level.Use MFI to analyze TIM-3 at Patients with Gastric Cancer CD3 +/ CD4 +expression in TILs and PBMCs obtains identical result (as Fig. 7 B, C, D).In the other mucosal tissue of Patients with Gastric Cancer cancer and in TILs, express the CD3 of TIM-3 +, CD4 +and CD8 +the ratio similar (Fig. 4 B, 5B and 6B) of T cell.
There are some researches show PD-1 in Patients with Gastric Cancer stomach organization +cD4 +/ PD-1 +cD8 +the number of T cell is all apparently higher than PD-1 in Patients with Gastric Cancer normal gastric mucosa +cD4 +/ PD-1 +cD8 +the number of T cell.But our result show the PD-1 of T cellular expression in adjacent mucosa and TIL and TIM-3 without significant difference (as Fig. 4 A, B; 5A, B and 6A, B).And we also observe TIGIT +cell accounts for the CD3 from AGM +and CD4 +t cells ratio is higher than CD3 +and CD4 +tILs(Fig. 2 D).These results suggest adjacent mucosa tissues are similar to stomach organization in immunosuppressive condition, thereby have caused the tired out of T cell.
8.2PD-1 +tIM-3 +t cells ratio increases in gastric cancer PBMCs
Compare normal pbmc, in patient P BMC T cell, the expression of PD-1 and TIM-3 is all obviously risen, and in most of TIL, PD-1 and TIM-3 further express rising.Then we analyze PD-1 and TIM-3 expresses in identical or different T cell subsets.
Compare normal pbmc, PD-1 in patient P BMC +/ TIM-3 +shared CD3 +t cell proportion is obvious (2.228 ± 4.196%vs11.70 ± 11.70%, the p=0.0348 of raising respectively; Fig. 4 C); Compare normal pbmc, PD-1 in patient P BMC +/ TIM-3 -the shared CD3 of T cell +t cell proportion is obvious (9.467 ± 4.177%vs22.06 ± 11.13%, the p=0.0053 of raising respectively; Fig. 4 D).But, compare PD-1 in patient and normal pbmc -/ TIM3 +shared CD3 +t cell proportion does not have notable difference (as Fig. 3 and 4E).PD-1 +/ TIM-3 +, PD-1 +/ TIM-3 -and PD-1 -/ TIM3 +cell accounts for Patients with Gastric Cancer and normal person CD4 +the ratio of T cell with at CD3 +ratio in T cell is consistent, does not just repeat here (Fig. 5 C, 5D and 5E).Compare normal pbmc, PD-1 +/ TIM-3 +/ CD8 +t cell is expressed obviously rise (2.225 ± 3.957%vs15.09 ± 9.652%, p=0.0017 in patient P BMC; Fig. 6 C); And PD-1 +/ TIM-3 -/ CD8 +and PD-1 -/ TIM-3 +/ CD8 +the expression of T cell in patient P BMC be no significant difference (p=0.0795; P=0.9263; Fig. 3,6D and 6E).In other words, PD-1 in patient P BMC +/ TIM-3 +the CD3 of two positives +, CD4 +, CD8 +t cell has increased respectively 5 times, 5 times, 7 times than normal level; PD-1 +/ TIM-3 -cD3 +, CD4 +t cell has also on average increased by 2 times than normal water; PD-1 in patient P BMC -/ TIM-3 +cD3 +, CD4 +, CD8 +t cell is not compared with normal PBMC and is significantly raise.These results demonstrations, in Patients with Gastric Cancer, the rise that TIM-3 expresses occurs on the T cell of expressing PD-1 specially; Yet the rise that PD-1 expresses can occur on the T cell of not expressing TIM-3.
8.3PD-1 +tIM-3 +t cells ratio further increases in gastric cancer TILs
PD-1 +/ TIM-3 +the shared CD3 of cell +the ratio of TILs is apparently higher than patient P BMC CD3 +ratio in T cell (33.68 ± 27.20%vs11.70 ± 11.70%, p=0.0082; Fig. 4 C)., compare TIL and patient P BMC CD3 +pD-1 in T cell -/ TIM-3 +or PD-1 +/ TIM-3 -shared ratio separately, result zero difference (p=0.4721 and p=0.3994; Fig. 4 D and 4E).PD-1 +/ TIM-3 +, PD-1 +/ TIM-3 -and PD-1 -/ TIM3 +cell accounts for CD4 +the ratio of T cell with at CD3 +ratio in T cell is consistent, does not just repeat here (Fig. 3,5C, 5D and 5E).Compare patient P BMC, PD-1 in TIL +/ TIM-3 +/ CD8 +cell proportion obviously rise (15.09 ± 9.652%vs33.52 ± 15.80%, p=0.0058; Fig. 6 C), PD-1 -/ TIM-3 +/ CD8 +t cell proportion decline (31.42 ± 15.83%vs26.56 ± 17.55%, p=0.0094; And PD-1 Fig. 6 E), +/ TIM-3 -/ CD8 +the ratio of cell is zero difference (17.16 ± 14.04%vs18.00 ± 15.18%, p=0.0887; Fig. 6 D).Our result shows, PD-1 +/ TIM-3 +tIL has represented that the major part of TIL (accounts for CD3 +, CD4 +, CD8 +the ratio of TIL has reached respectively 33.68 ± 27.20%, 40.47 ± 24.25%, 33.52 ± 15.80%).In Patients with Gastric Cancer TILs, PD-1 +/ TIM-3 +cells ratio is higher than PD-1 +/ TIM-3 -and PD-1 -/ TIM-3 +cell.Result of study from other tumor model also shows all TIM-3 +tIL coexpression PD-1, and TIM-3 +/ PD-1 +tILs has represented the main part of T cellular infiltration tumor.In Patients with Gastric Cancer or normal pbmc s, PD-1 +/ TIM-3 +cells ratio is all lower than PD-1 +/ TIM-3 -and PD-1 -/ TIM-3 +cell.Therefore, the TILs from Patients with Gastric Cancer recently shows more serious function exhaustion from patient's T cell.
8.4 blocking-up PD-1 and TIM-3 strengthen TIL secrete cytokines
In order to prove that PD-1 and TIM-3 are the target spots of immunotherapy for gastric cancer, be necessary whether the cytokine secretion that detects TILs by blocking-up PD-1 and TIM-3 is enhanced.Due to the TILs limited amount directly obtaining from stomach organization separation, we have carried out amplification in vitro to TILs.After two weeks, the quantity of TILs is from 1X10 6be increased to 5X10 7.Subsequently by 2X10 6tILs and 2X10 5stomach cancer cell mixes, with anti-TIM-3 or/and the monoclonal antibody of anti-PD-1 or IgG block after 5h, flow cytometry analysis CD4 +and CD8 +the change of TIL secrete cytokines.
Blocking-up TIM-3 signal path, only has CD4 separately +iFN-γ and IL-2 that TILs expresses increase to some extent, CD8 +tILs does not change.Block separately PD-1 signal path, CD4 +/ CD8 +the IFN-γ that TILs expresses and IL-2 all increase to some extent, but TNF-α does not increase (Fig. 8).Combine and use the monoclonal antibody of anti-TIM-3 and anti-PD-1 jointly to block, observe CD4 +and CD8 +tILs secretion of gamma-IFN and TNF-α obviously increase, and CD8 +tILs secretion IL-2 obviously increases.Block separately a wherein signal paths CD8 +tILs secretes the TNF-α less than 5%, and these two paths of combined occlusion have 32% CD8 +the TNF-α of TILs secretion, the inhibitory action of PD-TIM-3 signal path can be broken by this two signal paths of common blocking-up thus.
8.5 blocking-up PD-1 and TIM-3 strengthen TILs cytotoxicity
In order to determine the cytotoxicity that whether can strengthen TILs by blocking-up PD-1 and TIM-3 signal path.2.4x10 6tIL and 4x10 5aGS-GFP cell, at IgG, is cultivated 4h under the existence of anti-TIM-3 and/or anti-PD-1 monoclonal antibody altogether, detects the mortality rate of AGS-GFP cell after 4h by the method for cell streaming, and the cell of the 7-AAD positive is dead cell.
Blocking-up PD-1 or TIM-3 signal path TIL strengthen than IgG antibody blocking to some extent to the cytotoxicity of ags cell separately.And, jointly blocking PD-1 and TIM-3 signal path, TIL is obviously better than independent blocking-up PD-1 or TIM-3 signal path (as Fig. 9) to the fragmentation effect of tumor cell.
9. discuss
We observe TIGIT +cD8 +the ratio of TILs is lower than patient's peripheral t IGIT +cD8 +the ratio of T cell.Previous research shows that T cell can high expressed co-suppression factor TIGIT after Long contact time tumor antigen.But the result of contradiction is that co-suppression factor TIGIT is but at CD8 +in TILs, express and reduced, this phenomenon needs further to be studied.
PD-1 has confirmed in melanoma, high expressed in the TILS of renal cell carcinoma and Patients with Non-small-cell Lung.Up-to-date achievement in research proves, PD-1 is at the CD4 of Patients with Gastric Cancer +and CD8 +high expressed in T cell, and the expression positive correlation of the expression of PD-1 and PD-L1.The polymorphism of TIM-3 studies show that TIM-3 gene promoter region exists a dangerous genetic mutation, is the risk factor that in Chinese population, gastric cancer occurs.But do not have positive evidence to show whether high expressed in Patients with Gastric Cancer of TIM-3, whether exist and contact with the high expressed of PD-1.Our result shows PD-1 and TIM-3 high expressed all in Patients with Gastric Cancer PBMC T cell and TILs, and PD-1 +/ TIM-3 +tILS accounts for the ratio of total TILS more than 1/3, simultaneously PD-1 +/ TIM-3 +tILs is than normal pbmc PD-1 +/ TIM-3 +t cell has increased 10-14 doubly (Fig. 4 A, 4B, 5A, 5B, 6A and 6B).Patients with Gastric Cancer periphery PD-1 +/ TIM-3 +the level of T cell is than the PD-1 of normal person periphery +/ TIM-3 +t cell has increased by 5 to 7 times (Fig. 4 C, 5C and 6C).The expression of PD-1 and/or TIM-3 is exhausted with the function of T cell or is disorderly relevant.The TILs of our results suggest Patients with Gastric Cancer shows more serious function than periphery T cell and exhausts, Patients with Gastric Cancer periphery T cell shows more serious function than normal person periphery T cell and exhausts.
Compare normal person's peripheral blood cells, PD-1 +/ TIM-3 +cell and PD-1 +/ TIM-3 -the ratio that cell accounts for TILs and Patients with Gastric Cancer periphery T cell all obviously raises (except PD-1 +/ TIM-3 -cell accounts for CD8 +tILs and Patients with Gastric Cancer periphery CD8 +the ratio of T cell), but PD-1 -/ TIM-3 +cells ratio does not but raise, and the prompting up-regulated expression of TIM-3 and the up-regulated expression of PD-1 are proportionate.And, we find Patients with Gastric Cancer PBMC/TILs and are slightly higher than Patients with Gastric Cancer PBMC/TILs than the TIM-3 expression raising in normal pbmc s (having increased 2.5-4.5 doubly) than the PD-1 expression raising in normal pbmc s (having increased 3-5 doubly), hint PD-1 is singly at the up-regulated of Patients with Gastric Cancer periphery T cell, and the up-regulated in TILs may be slightly early than the up-regulated of TIM-3.
Compare patient P BMC, PD-1 -/ TIM-3 +/ CD8 +when TILs ratio declines to some extent, PD-1 +/ TIM-3 +/ CD8 +tILs ratio rises to some extent.It should be noted that CD8 +pD-1 in TILs -/ TIM-3 -t cells ratio and patient P BMC CD8 +pD-1 in T cell -/ TIM-3 -between T cells ratio, difference does not reach statistical significance (Fig. 6 C, 6D and 6E).This is explanation just, PD-1 in TIL +/ TIM-3 +/ CD8 +t cell is probably directed to PD-1 in PBMC -/ TIM-3 +/ CD8 +t cell.Consistent result studies show that mouse tumor model, TIM-3 in tumor microenvironment +/ CD8 +t cell is because the hint of response environment can produce the phenomenon of PD-1 up-regulated.Simultaneously because TIM-3 is at normal person's peripheral blood CD8 +t cells very high (34.29 ± 14.31%, Fig. 5 B).It is believed that CD8 +t cell just starts to express TIM-3, TIM-3 in periphery +/ CD8+T cell dead or exhaust is to be subject to the expression of the part galectin-9 of TIM-3 to regulate, this time TIM-3 +/ CD8 +t cell does not touch galectin-9, so show serious function, does not exhaust.But work as TIM-3 +/ CD8 +t cell migration to high expressed galectin-9 tumor tissues has just formed PD-1 around +/ TIM-3 +/ CD8 +t cell, compares PD-1 -/ TIM-3 +/ CD8 +t cell just shows more serious function and exhausts.With PD-1 +/ TIM-3 +/ CD8 +tIL derives from PD-1 -/ TIM-3 +/ CD8 +t cell is different, due to PD-1 +/ TIM-3 -or PD-1 -/ TIM-3 +cD3 +/ CD4 +til cell group ratio does not have significant change, the PD-1 therefore newly increasing +/ TIM-3 +/ CD3 +/ CD4 +tIL may be mainly derived from PBMC PD-1 -/ TIM-3 -/ CD3 +/ CD4 +t cell.We get rid of PD-1 +/ TIM-3 +the increase of cells ratio and PD-1 -/ TIM-3 -and PD-1 -/ TIM-3 +the reduction of cells ratio is to pass through PD-1 +/ TIM-3 +cell self propagation causes.Because PD-1 +/ TIM-3 +cell shows more serious function exhaustion than PD-1 or the mono-positive cell of TIM-3, and the ability that loses multiplication capacity and secrete cytokines is considered to T cell function and exhausts.
PD-L1 high expressed in gastric cancer is proved, and PD-L1 +cancer patient compare PD-L1 -patient's survival probability lower.Recently, PD-1 high expressed in gastric cancer is also in the news, and PD-1 in gastric cancer is also thought in this research +tIL compares PD-1 -tILs secretion IFN-γ still less.Anti-PD-1 and anti-PD-L1 antibody have very deep research in mice and the mankind.But up to the present, not yet find to use the monoclonal antibody of anti-PD-1 or anti-PD-L1 to carry out blocking test, assess PD-1 in gastric cancer +the report whether TIL function can be restored.Our experimental result shows to use the antibody of anti-PD-1 can strengthen CD8 +and CD4 +the ability of TIL secretion of gamma-IFN and IL-2, the antibody of anti-PD-1 has the ability (Fig. 8 and Fig. 9) that TIL kills and wounds stomach cancer cell that strengthens simultaneously.Our result demonstrates anti-PD-1 antibody for the wide prospect of immunotherapy for gastric cancer.
While assessing anti-TIM-3 antibody antitumous effect at present, be mainly to analyze PD-1 in mice +/ TIM-3 +cD8 +t cell, or NY-ESO-1 tumour-specific PD-1 in the mankind +/ TIM-3 +cD8 +the function of T cell disappearance cytokine secretion.The research to colon sarcoadenoma (MC38 and CT26) and these 3 kinds of mouse tumor models of fibrosarcoma (WTMCA2) of report has recently been compared anti-TIM-3 monoclonal antibody to CD8 +and CD4 +the impact of T cell viability, found that effective anti-TIM-3 treatment is mainly to depend on the CD8 that can produce IFN-γ +t cell.In our research, we have analyzed CD8 in anti-TIM-3 antibody on human class gastric cancer +and CD4 +the impact of TILs function, result proof is used separately anti-TIM-3 antibody can promote CD4 +tILs secretion of gamma-IFN and IL-2.But the antibody of not seeing anti-TIM-3 can further promote CD8 +the phenomenon of TIL secretion of gamma-IFN.This may be amplified 2 Zhou Houcai in vitro with our TILs, and to carry out blocking test relevant, because TILs has obtained certain activation in amplification procedure, thereby cause using separately TIM-3 antibody to carry out blocking test, fails further to strengthen its secretion.When combining while using the antibody of anti-PD-1 and anti-TIM-3 to block, use separately the antibody blocking CD8 of anti-TIM-3 or anti-PD-1 +tILs secretes higher levels of IFN-γ, IL-2 and TNF-α (Fig. 8 D), and similar result also shows to combine while using the antibody of anti-PD-1 and anti-TIM-3 to block, and uses separately the antibody blocking CD4 of anti-TIM-3 or anti-PD-1 +tIL secretes higher levels of IFN-γ and TNF-α (Fig. 8 C).Our anti-TIM-3 antibody of presentation of results can promote CD4 under the synergism of PD-1 antibody +and CD8 +tILs secrete cytokines.
The secretion situation of the til cell factor from the antibody blocking of the anti-TIM-3 of independent use or anti-PD-1, anti-PD-1 antibody makes the ability of TIL functional rehabilitation be better than anti-TIM-3 antibody (Fig. 8 D).This may be because TIM-3 signal path than PD-1 signal path in T Cellular immunity suppression a little less than role.PD-1 +/ TIM-3 +t cell or PD-1 -/ TIM-3 +t cell will be likely all the target of anti-TIM-3 antibody.And PD-1 +/ TIM-3 +cell is the major part of TIL, PD-1 tired out +/ TIM-3 +t is difficult to by independent blocking-up TIM-3 signal path, function is restored.In tumor model, the mechanism that anti-TIM-3 antibody plays a role is to make TIM-3 +leukopenia, evidence suggests PD-1 -/ TIM-3 +the minimizing of cell is accompanied by PD-1 +/ TIM-3 -the increase of cell number.But this mechanism is impossible to CD4 in our 4-5h extracorporeal blocking process +and CD8 +tILs restore funcitons plays important effect.So, use separately anti-TIM-3 antibody blocking, the ability of TIL secrete cytokines does not significantly strengthen (Fig. 8), and the ability of TIL killing tumor cell also just improves (Fig. 9) slightly.
Although we observe, block separately PD-1 the secretion of the til cell factor is increased, be not that all previous research all thinks that independent blocking-up PD-1/PD-L1 can make T cell function be replied.Externally by of short duration stimulation, block separately PD-1/PD-L1 signal path and can not recover equally NY-ESO-1 tumour-specific CD8 +the function of T cell.Research shows about 50%PD-1 +tILs is TIM-3 -/ PD-1 +tILs, representative be the T cell that a group activates, extracorporeal blocking PD-1/PD-L1 signal path can recover its function, needs in vivo combined vaccine to use and just can make tumor regression, blocks separately PD-1/PD-L1 signal path and can not make tumor disappear.Similar result of study is to block TIM-3 and PD-1 signal path simultaneously, and the ability of observing TIL secretion of gamma-IFN is obviously better than independent blocking-up TIM-3 or PD-1 signal path.Separately the effectiveness of blocking-up PD-1 needs the experiment of further inside and outside and verifies, our result shows that in conjunction with forefathers' achievement in research it is apparent that common blocking-up PD-1 and TIM-3 show TIL secrete cytokines and the enhancing that suppresses tumor growth ability.Therefore, combined occlusion PD-1 and TIM-3 signal path are the effective ways of immunotherapy for gastric cancer.
The foregoing is only preferred embodiment of the present invention, be not used for limiting practical range of the present invention; If do not depart from the spirit and scope of the present invention, the present invention is modified or is equal to replacement, all should be encompassed in the middle of the protection domain of the claims in the present invention.

Claims (9)

  1. The purposes of the blocker of the blocker of 1.PD-1 signal path and TIM-3 signal path in the pharmaceutical composition of the anti-gastric cancer of preparation.
  2. 2. purposes according to claim 1, is characterized in that, described pharmaceutical composition comprises:
    1) blocker of the blocker of PD-1 signal path and TIM-3 signal path;
    2) acceptable carrier or excipient pharmaceutically or in immunology;
    With 3) optional, other anti-gastric cancer medicine.
  3. 3. purposes according to claim 1, is characterized in that, described pharmaceutical composition and other anti-gastric cancer Drug combination.
  4. 4. purposes according to claim 1, is characterized in that, described pharmaceutical composition comprises other anti-gastric cancer medicine.
  5. 5. according to the purposes described in claim 1-4 any one, it is characterized in that, the blocker of described PD-1 signal path is anti-PD-1 monoclonal antibody, and the blocker of described TIM-3 signal path is anti-TIM-3 monoclonal antibody.
  6. 6. according to the purposes described in claim 2-4 any one, it is characterized in that, described other anti-gastric cancer medicine is one or more in alkylating agent, antimetabolite, antitumor antibiotics, plant anticarcinogen, hormone.
  7. 7. according to the purposes described in claim 2-4 any one, it is characterized in that, described other anti-gastric cancer medicine is one or more in cisplatin, epirubicin, fluorouracil.
  8. 8. a pharmaceutical composition, comprising:
    1) blocker of the blocker of PD-1 signal path and TIM-3 signal path;
    2) acceptable carrier or excipient pharmaceutically or in immunology;
    With 3) optional, other anti-gastric cancer medicine.
  9. 9. a medicine box, comprising:
    The container of the blocker of the blocker that i) contains PD-1 signal path and TIM-3 signal path;
    Ii) container that contains other anti-gastric cancer medicine;
    With optional iii) operation instructions.
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