CN104630145A

CN104630145A - Anti-tumor T cell as well as preparation method and anti-tumor drug thereof

Info

Publication number: CN104630145A
Application number: CN201510013987.5A
Authority: CN
Inventors: 杨世成
Original assignee: 杨世成
Current assignee: ZMKS International Cancer Therapy Biotechnologies Co., Ltd.
Priority date: 2015-01-12
Filing date: 2015-01-12
Publication date: 2015-05-20

Abstract

The invention provides a method for preparing an anti-tumor T cell in vitro through DC (dendritic cell, namely antigen presenting cell), wherein the anti-tumor T cell is a tumor total RNA-loaded DC amplification and enrichment anti-tumor T cell. The invention also provides a preparation method of the anti-tumor T cell and an anti-tumor drug or inoculation vaccine taking the anti-tumor T cell as an active ingredient. According to the invention, a specific anti-tumor T cell is amplified in vitro by taking the tumor total RNA-loaded DC as a platform, a small amount of tumor total RNA is amplified to an enough amount in vitro so as to realize clinical application, and the in-vivo anti-tumor effect in an IC (intracranial) tumor-bearing model is evaluated, including how the mature state of DC and the cell factor combination adjust the differentiation of the anti-tumor T cell as well as the synergistic anti-tumor effect thereof in a preclinical experiment model.

Description

A kind of antitumor T cell, its preparation method and antitumor drug

Technical field

The present invention relates to a kind of antitumor T cell and preparation method thereof, and be activeconstituents antitumor drug or the vaccination of this antitumor T cell.

Background technology

The antitumor T cell of direct use amplification in vitro is based on two facts: 1) have the patient of malignant brain tumor to have very strong systemic immune and suppress, seriously hinder the effect of inoculation anti-tumor vaccine method, 2) compared with vaccination method, use comprises tumor infiltrating lymphocyte (tumor infiltrated lymphocytes, or genetically engineered peripheral blood lymphocyte (peripheral blood lymphocytes TIL), PBL) (adoptive cell transfer is shifted at the adoptive cellular of the antitumor T cell of interior amplification in vitro, ACT) tumour cell (Dudley ME remaining in melanoma patient can more effectively be eradicated, Yang JC, Sherry R, Hughes MS, Royal R, et al. (2008) Adoptive cell therapy for patients with metastatic melanoma:evaluation of intensive myeloablative chemoradiation preparative regimens.J Clin Oncol 26:5233-5239, Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, et al. (2006) Cancer regression in patients after transfer of genetically engineered lymphocytes.Science 314:126-129), and thoroughly remove in blood and tumour cell (the Porter DL of transfer, Levine BL, Kalos M, Bagg A, June CH (2011) Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia.The New England journal of medicine 365:725-733).But the method for these two kinds of antitumor T cell of external generation is inevitably in the face of some significantly limits.The T cell of modifying through the genetically engineered of TCR or CAR can only a kind of tumour antigen of target killing, and the Endodontic failure that this single target killing finally can bring due to the change of tumour antigen.And TIL represents multi-functional antitumor T cell, its successful Application is only limitted to Melanoma.Due to tumor sample small-sized of excision, the TIL that can obtain is limited, so do not apply the clinical data that TIL treats malignant brain tumor.What is interesting is, Finding case in the 519 routine glioblastomas reported recently, the infiltration of cd8 t cell (Yang I relevant to the long-term surviving of patient, Tihan T, Han SJ, Wrensch MR, Wiencke J, et al. (2010) CD8+T-cell infiltrate in newly diagnosed glioblastoma is associated with long-term survival.J Clin Neurosci 17:1381-1385).Owing to there is the difficulty obtaining TIL in cerebral tumor in actual clinical treatment, the method being necessary to seek other obtains the antitumor T cell of similar TIL.

Summary of the invention

The present invention solves the aforementioned problems in the prior to propose.The present invention uses dendritic cell (the dendritic cells of ttRNA (tumour total serum IgE) load, DC) as the antitumor T cell of Mutiple Targets of the similar TIL of platform amplification in vitro, realize a small amount of ttRNA of amplification in vitro to enough amounts and for clinical application, and evaluate its synergistic antitumor effect in encephalic (IC) bearing mouse model.

The invention provides a kind of antitumor T cell and preparation method thereof.

Present invention also offers more than one and state the antitumor drug that antitumor T cell is activeconstituents.

For achieving the above object, the present invention is by the following technical solutions:

First aspect of the present invention is to provide a kind of antitumor T cell, it is characterized in that, described antitumor T cell is with the dendritic cell DC specific amplification of tumour total serum IgE load and the antitumor T cell of enrichment.

In above-mentioned antitumor T cell, preferably, also cytokine is comprised in described antitumor T cell, described cytokine preferably from but any one or the multiple combination that are not limited in IL-4, IL-7, IL-12, IL-15, IL-21, GM-CSF, CD40L, be more preferably the combination of IL-7, IL-12, IL-21.

In above-mentioned antitumor T cell, preferably, described dendritic cell are from the myelocyte of the mankind or mouse or lymphocyte.

Second aspect of the present invention is to provide the preparation method as above-mentioned antitumor T cell, comprises the steps:

The preparation of step 1, tumour total serum IgE: the tumour cell of cell cultures of hanging oneself synthesizes tumour total serum IgE through external;

The preparation of step 2, ripe tumour total serum IgE-DC: the mode that tumour total serum IgE step 1 obtained and the dendritic cell DC be separated transduces by electricity imports in DC, subsequently overnight incubation in the medium, obtain the tumour total serum IgE-DC of maturation;

Step 3, the ripe tumour total serum IgE-DC and the T cell Dual culture that step 2 are obtained, obtain antitumor T cell as claimed in claim 1.

In above-mentioned preparation method, preferably, the tumor cell line of described step 1 from the tumour cell of the mankind or mouse, more preferably from but be not limited to astrocytoma cell system KR158 and the Melanoma cells B16 F10OVA of the mouse with height invasiveness.

In above-mentioned preparation method, preferably, the dendritic cell of described step 2 are from the myelocyte of the mankind or mouse or lymphocyte.

In above-mentioned preparation method, preferably, described step 2 adds CD40L or lipopolysaccharides LPS in the medium to realize the maturation of DC.

In above-mentioned preparation method, preferably, the Dual culture of described step 3 carries out under the existence of cytokine; More preferably, described cytokine is selected from any one or the multiple combination in IL-4, IL-7, IL-12, IL-15, IL-21, GM-CSF, CD40L; Most preferably, described cytokine is selected from the combination of IL-7, IL-12, IL-21.

In above-mentioned preparation method, preferably, the T cell of described step 3 from the spleen of the mankind or mouse, more preferably from the spleen of the mankind or mouse.

In above-mentioned preparation method, preferably, the T cell of described step 3 is antigen responsive cell, comprises CD4+T cell, CD8+T cell, effector T cell, memory T cell.

In above-mentioned preparation method, preferably, the ripe ttRNA-DC of described step 3 and the number ratio of T cell are 1:(15-25), such as 1:20.

3rd aspect of the present invention is to provide a kind of antitumor drug, described antitumor drug with antitumor T cell according to claim 1 for activeconstituents.

In above-mentioned antitumor drug, preferably, described antitumor drug is medicament or vaccination.

In above-mentioned antitumor drug, preferably, described tumour comprises the tumour of the such as cancer of the brain.

Load has the DC of total tumour antigen to provide all needs to meet ex vivo enrichment and the antitumor T cell of amplification.DC is strong irritation cell, constantly supervise host antigenic environment and specifically activating T cell and B cell response (Steinman RM (2001) Dendritic cells and the control of immunity:enhancing the efficiency of antigen presentation.The Mount Sinai journal of medicine, New York 68:160-166; Banchereau J, Steinman RM (1998) Dendritic cells and the control of immunity.Nature 392:245-252).Use and be loaded with that the immunotherapy of the DC of polypeptide or tumour antigen extract is verified has strong anti-tumor activity (Iwami K in cerebral tumor, Shimato S, Ohno M, Okada H, Nakahara N, et al. (2012) Peptide-pulsed dendritic cell vaccination targeting interleukin-13receptor alpha2chain in recurrent malignant glioma patients with HLA-A*24/A*02allele.Cytotherapy 14:733-742; Yu JS, Wheeler CJ, Zeltzer PM, Ying H, Finger DN, et al. (2001) Vaccination of malignant glioma patients with peptide-pulsed dendritic cells elicits systemic cytotoxicity and intracranial T-cell infiltration.Cancer research 61:842-847; Liau LM, Black KL, Prins RM, Sykes SN, DiPatre PL, et al. (1999) Treatment of intracranial gliomas with bone marrow-derived dendritic cells pulsed with tumor antigens.Journal of neurosurgery 90:1115-1124; Labeur MS, Roters B, Pers B, Mehling A, Luger TA, et al. (1999) Generation of tumor immunity by bone marrow-derived dendritic cells correlates with dendritic cell maturation stage.Journal of immunology 162:168-175).What is interesting is, contriver finds, in synergistic tumour lotus knurl model, the DC of load tumor lysis product has demonstrated strong protection and therapeutic action as vaccination, and finds that the maturity state of DC is directly connected to the effect of antineoplastic immune; Data suggestion Clinical practice maturation instead of jejune tumour antigen load DC as effective tumor inoculation vaccine.Because representative easily uses with the ttRNA of total antigen of the tumour cell of rna form, the invention provides an innovative approach and use ttRNA-DC as the multi-functional antitumor T cell of the external generation of main platform.In the present invention, replace tumor lysis product as the source of tumour antigen, the tumor sample from cerebral tumor based on excision is very little and be not enough to produce this fact of tumor lysis cell, so contriver uses ttRNA load DC.And the operating process (SOP) of contriver's Criterion realizes a small amount of ttRNA of amplification in vitro and reaches enough for the scheme of clinical application.Therefore, in the present invention, based on ttRNA-DC as treatment vaccination, contriver improves its treatment potentiality, the immunotherapy of tumors of the antitumor T cell of direct external generation for innovating.

For using adoptive cellular therapy (ACT) for cancer therapy, one actual considers is the progressively eventually end differentiation of T cell in vitro in culturing process, this differentiation produces strong vitro effect anti-tumor activity, but antitumous effect (the Gattinoni L greatly reduced on the contrary in body, Klebanoff CA, Palmer DC, Wrzesinski C, Kerstann K, et al. (2005) Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8+ T cells.The Journal of clinical investigation 115:1616-1626).The differentiation state of T cell can directly be adjusted by following factors: the process of 1) cultivating; 2) the adding of medicine; And 3) body interior use combination cytokine (Gattinoni L, Klebanoff CA, Palmer DC, Wrzesinski C, Kerstann K, et al. (2005) Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8+T cells.The Journal of clinical investigation 115:1616-1626; Gattinoni L, Klebanoff CA, Restifo NP (2009) Pharmacologic induction of CD8+T cell memory:better living through chemistry.Science translational medicine; Hinrichs CS, Spolski R, Paulos CM, Gattinoni L, Kerstann KW, et al. (2008) IL-2and IL-21confer opposing differentiation programs to CD8+T cells for adoptive immunotherapy.Blood 111:5326-5333; Klebanoff CA, Gattinoni L, Torabi-Parizi P, Kerstann K, Cardones AR, et al. (2005) Central memory self/tumor-reactive CD8+T cells confer superior antitumor immunity compared with effector memory T cells.Proceedings of the National Academy of Sciences of the United States of America 102:9571-9576).The cytokine sharing γ c chain acceptor comprises IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, plays a key effect in vivo in immunne response process, and contributes to forming long lasting T cell memory.At present, some clinical ACT flow processs use IL-2 to be used for the amplification in vitro of antitumor T cell, although IL-2 increases in large quantities and activates have immunosuppressant CD4+CD25+FoxP3+T _regs.By contrast, IL-7 directly suppresses T _regsfunction by being in T that is spontaneous and memory _regson the affine IL-7R of height send signal to discharge spontaneous control to T cell.Except the cytokine of shared γ c chain acceptor, IL-12 is another important cytokine, and contribute to T cell differentiation and memory formation, and optionally can improve the survival of CD8+CD62Lhigh cell, such cell has superpower anti-tumor activity.In addition, compared with IL-2, the end eventually that IL-7 and IL-21 can reduce effector T cell breaks up and contributes to the enrichment of the CD8+T cell for tumor response.At present, the antitumor T cell being derived from ttRNA-DC-T system uses different cytokines or their combination are not also systematically studied, belong to a brand-new field.

The advantage using ACT to treat cerebral tumor is that T cell can by hemato encephalic barrier (the Hong JJ stoping humoral immunization composition to enter brain, Rosenberg SA, Dudley ME, Yang JC, White DE, et al. (2010) Successful treatment of melanoma brain metastases with adoptive cell therapy.Clinical cancer research:an official journal of the American Association for Cancer Research 16:4892-4898; Blankenhorn EP, Stranford SA, Smith PD, Hickey WF (1991) Genetic differences in the T cell receptor alleles of LEW rats and their encephalomyelitis-resistant derivative, LER, and their impact on the inheritance of EAE resistance.European journal of immunology 21:2033-2041).Therefore, in the present invention, contriver sets up innovative approach, uses the external generation of ttRNA-DC antitumor T cell, and these methods comprise the maturity state of DC and combination of cytokines and how to regulate antitumor T cell to break up and assess their antitumous effects in clinical front IC tumor model.

Accompanying drawing explanation

Fig. 1 is phenotype and the function of the mouse DC of external generation.

A. the height of ripe mouse DC surface marker is expressed.Mouse DC is prepared according to standard method.At the 7th day, in the substratum of DC, add CD40L (1 μ g/ml) or add LPS (0.5 μ g/ml) and spend the night, promote DC differentiation and maturation.Use one group of antibody by flow cytometry analysis phenotype.Upper hurdle: the expression showing the DC surface marker from a representative experiment in histogram, and from the data conclusion collection of illustrative plates as described below of the DC of three kinds of different experiments: represented by dotted arrows DC is from IL-4/GM-CSF nutrient solution group; It is adding from CD40L that half solid line represents DC; Solid line represents DC and adds group from lipopolysaccharides (LPS).Lower hurdle: the mean value ± standard deviation representing the average fluorescent strength (MFI) of the surface marker from 5 independent experiments.Asterisk shows compared with the DC from IL-4/GM-CSF group, p<0.001.

B. ripe DC spontaneously discharges high-level cytokine.After adding CD40L or LPS, with every hole 2*10 ⁶individual DC is laid in 24 orifice plates, spends the night, and collects supernatant, and by Duke University's core laboratory what test platform completed detects cytokine with mouse 10-Plex kit (Invitrogen company).The data representing 5 independent experiments are marked and drawed as mean value ± standard deviation.Asterisk shows compared with the DC from IL-4/GM-CSF condition, p<0.001.

Fig. 2 is that ripe ttRNA-DC regulates the vaccination of platform can promote that T cell is bred.

A. the multiplication capacity of the T cell of vitro culture is promoted.With KR158-ttRNA (ttRNA-DC), the DC electroporation prepared by above-mentioned condition is used for subcutaneous (ID) vaccination.After one week, obtain from spleen T cell and with Fluoresceincarboxylic acid etheric acid (CFSE) mark, single culture or IL-2 exist under cultivate 7 days.Analyze CD3 colony, be y-axis from the representative data often organizing 1 mouse with CD3, be that x-axis is marked and drawed with CFSE.Upper hurdle: T cell is cultivated when not adding cytokine; Lower hurdle: T cell IL-2 cultivates.

B. ripe ttRNA-DC promotes the propagation of cd8 t cell.Marked and drawed the data from the representative mean value ± standard deviation often organizing 3 mouse, asterisk shows compared with the DC from IL-4/GM-CSF condition, p<0.001.

Fig. 3 is that ripe ttRNA-DC adds that cytokine-cocktail produces the T cell with low phenotypic differentiation.

A. from the phenotype often organizing 1 representative mouse.As described in Figure 2, the T cell from the vaccinated mouse of different ttRNA-DC uses corresponding ttRNA-DC amplification in vitro 6 days further, and T cell phenotype uses one group of fluorescent-labeled antibody analysis by flow cytometer.The use of ttRNA-DC is marked on the top of every pictures, and cultivation carries out under the existence of IL-2.

B. dissimilar ttRNA-DC produces the T cell with similar phenotype.Analysis is limited to CD8 colony, and the subset of reflection CD44 height CD62L low (Tem), CD44 height CD62L high (Tcm) and the low CD62L of CD44 high (Tn) is determined by T cell marker CD44 and CD62L.The per-cent comprised from each subset of CD8, Tem and Tcm of often organizing 3 mouse is marked and drawed as mean value ± standard deviation.

C. from the representative phenotype of the T cell of 1 mouse.Under IL-2 or cytokine-cocktail exists, use LPS-DC amplification in vitro after 6 days, the phenotype from 1 representative mouse is marked and drawed.The use of IL-2 or cytokine-cocktail is marked on the top of every pictures.

D.ttRNA-DC adds that cytokine-cocktail causes PD phenotype.Based on as the LPS-DC as described in C figure, reflect that the per-cent from CD8, Tem, Tcm and Tn of often organizing 5 mouse is marked and drawed as mean value ± standard deviation.Asterisk shows compared with IL-2 group, p<0.001.

Fig. 4 is that the antitumor T cell of LPS-DC amplification adds that cytokine-cocktail causes tumor-bearing mice prolonged survival period.

The T cell of A.DC amplification specifically identifies antigen associated target.Based on LPS-DC, dissimilar RNA-DC amplification T cell is used as effector cell, and antigen associated target is from RNA-DC target as an alternative, or from KR158 tumour system.Except directly using KR158 tumour system, contrast target is ctrl-DC or with OVA and KR158ttRNA; The T cell increased with these RNA-DC is called after ctrl-T, OVA-T and KR158-T respectively.The concentration that cytokine uses is as described below.Increase after 6-7 days, T cell (every hole 2.5*10 ⁵individual) with target with ratio 10:1 Dual culture in round bottom 96-orifice plate, IFN γ detects horizontally through ELISA test kit.Data are marked and drawed from the same form three hole as mean value ± standard deviation.

B.LPS-DC demonstrates superior anti-tumor activity.Female C57bl/6 mouse (often organizing 7 mouse) uses the implanted IC tumour of KR158 cell 5 days, the front body irradiation accepting 5Gy for 1 day of ACT.At the 0th day, at ttRNA-DC vaccination posterior vein injection (IV) 1*10 ⁶individual from different ttRNA-DC and cytokine-cocktail exist amplification T cell.After ACT, inject twice extra ttRNA-DC vaccination by abdominal cavity (IP) weekly.Show to use different DC to add each group of cytokine-cocktail the right being marked on figure.

The antitumor T cell of C.LPS-DC amplification can not extend the survival time under the culture condition of IL-2.Mouse (often organize 7 mouse) implanted IC tumour, shows only to use DC or is combined the right that each group of dissimilar T cell is marked on figure.

D.LPS-DC adds that cytokine-cocktail shows superior anti-tumor activity.In brief, as B and C experiment, and in these IC bearing mouse model, cytokine or they combinationally use is detected.Show each group of the T cell being combined DC and different cultivation the right being marked on figure.DC+T w/o DC enrichment:T cell uses cytokine-cocktail to cultivate but does not add the amplification of LPS-DC; DC+IL-7/IL-21+IL-12T or IL-15T: use the T cell of LPS-DC, IL-7 and IL-21 amplification first to cultivate 3 days, supplement the cultivation that IL-12 or IL-15 is used for section remaining time.Except IL-2 is supplemented to 30IU/ml, the concentration of other cytokine is 20ng/ml.Asterisk represents p<0.001.

The per-cent of the positive subset of Fig. 5 CD62L that has been cytokine-cocktail culture and improvement, but do not improve the per-cent of OVAtetra+T cell.

A. be called as OVA RNA-DC with the LPS-DC of OVA-RNA electroporation, the spleen t-cell that the 1:1 from WT or OT-1 mouse mixes and OVA RNA-DC with T cell and DC ratio for 10:1 Dual culture.Under the existence of different cytokines or their combination, vitro culture is after 6 days, uses per-cent and the phenotype of flow cytometry analysis OVA Tetra+T cell.Different cytokines or their composite marking are at the top of every pictures; Cytokine above line illustrates first 3 days that use cytokine, and the cytokine below line shows the in the end 3 days cytokines used.

B. the OVA Tetra+CD8+CD62L+T cell combinationally using the higher per-cent of generation of cytokine.In figure numerical value be based on flow cytometry analysis from the phenotype of Tetra+CD8+T cell often organizing 3 mouse, and to mark and draw as mean value ± standard deviation.Asterisk shows compared with IL-2 group, p<0.001.

Fig. 6 is the long-term surviving that cytokine amplification in vitro T cell causes tumor-bearing mice.

A. the ACT of the T cell of cytokine amplification is used to cause prolonging survival.Female C57bl/6 mouse (often organizing 7 mouse) uses B16F10OVA cell to implant IC tumour 5 days, and ACT accepts 5Gy body irradiation in first 1 day.Spleen t-cell from the 1:1 mixing of WT mouse and OT-1 mouse uses OVA RNA-DC amplification under IL-2 or cytokine-cocktail exists; After 6 days, at OVA RNA-DC abdominal cavity (IP) vaccination posterior vein injection (IV) 10*10 ⁶individual T cell.The each group profile showing to be combined DC and T cell is shown in the right mark.Asterisk represents p<0.001.

The powerful amplification of the specific cd8 t cell of B.OVA antigen.Spleen t-cell from the 1:1 mixing of WT mouse and OT-1 mouse used the tetrameric marker analysis of CD8 and OVA at the 0th day, and used OVA RNA-DC amplification 6 days under IL-2 or cytokine-cocktail exists.Be marked in each picture after part CD8+Tetra+T cell flow cytometer showed.

The gene expression atlas of the CD8+OVA Tetra+T cell after C.OVA RNA-DC amplification in vitro.From the CD8+OVA Tetra+T cell extraction total serum IgE of sorting, and use mouse Affymetrix GeneChip (mouse 430A 2.0 array) analyzing gene expression map.Upper hurdle: based on the gene expression dose by Partek Genomics Suit 6.6 software analysis, representing 601 genes, being greater than the loose than marking and drawing of 2 times of changes from the having of T cell of difference cultivation.Lower hurdle: one group of representational gene is plotted in histogram.Their effects in T cell activity, cytokine network and the interaction (see Fig. 7,8,9) between cytokine and acceptor is summarised by the gene selected.

Fig. 7 is the gene expression atlas of change and the dependency of T cell activity.As described in figure 6, represent that reflection is greater than one group of gene of 2 times of changes with yellow star in the graph, to summarize their effects in T cell activity.This analysis be based on web analytics (http://david.abcc.ncifcrf.gov) after amendment, the active routing information of T cell is produced by BIOCARTA software.

Fig. 8 is the correlation analysis of the gene expression atlas cytokine network of change.This chart is produced by BIOCARTA software, and the different genes from 2 times of cut-off lists represents with arrow.

Fig. 9 is the interaction of different genes expression map and cytokine-cocktail acceptor.This chart is produced by KEGG, and the different genes from 2 times of cut-off lists represents with arrow.

Embodiment

Carry out detailed and concrete introduction below by specific embodiment to the present invention, to make better to understand the present invention, but following embodiment does not limit the scope of the invention.

embodiment

One, materials and methods:

1, from the cell cultures of mouse tumor cell system and the preparation of ttRNA:

Mouse cytokine IL-4, IL-7, IL-12, IL-15, IL-21, GM-CSF and CD40L are purchased from PEPROTECH company (Los Angeles, New Jersey), and IL-2 (Aldesleukin) is purchased from PROLEUKIN company (San Diego, Jia Nifuniya).Mouse T cell is cultivated in RPMI, comprising being supplemented with the L-glutaminate of 5%FBS, 1mM of penicillin/streptomycin of 100U/ml, 55uM β-ME, 1mM Sodium.alpha.-ketopropionate, 0.1mM NEAA, 10mM HEPES (Invitrogen company).The present embodiment preferably has mouse astrocytoma model and the melanoma IC tumor model of height invasiveness, namely from NF1 ^-/-: p53 ^-/-astrocytoma cell system KR158 in C57BL/6-mouse, and carry out the comfortable mouse melanin tumor cell line B16F10OVA containing cultivating in the DMEM of 10%FBS.All cells are cultivated in 5%CO2 humidified incubator at 37 DEG C.Lipopolysaccharides (LPS) available from Sigma (St. Louis, the Missouri State).Tumour total serum IgE (total tumor RNA, ttRNA) from KR158, B16F10OVA clone is prepared according to product manual by using RNeasy Maxi test kit (Qiagen company).OVA RNA synthesizes in vitro by using T7mMESSAGE mMACHINE RNA test kit (Ambion company).The integrity of ttRNA and quality are evaluated by use 1% agarose gel electrophoresis, and quantitative at 260nm wavelength place by NanaDrop.

2, the preparation of the dendritic cell DC of mouse:

Producing the program of mouse dcs DC based on disclosed experiment flow has carried out revising (Inaba K at cerebral tumor immunotherapy center, Duke University, Inaba M, Romani N, Aya H, Deguchi M, et al. (1992) Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.The Journal of experimental medicine 176:1693-1702).The medullary cell that the present embodiment preferably comprises shin bone, femur and breastbone from two rear feet is processed according to standard sterile procedures and is extracted.Collect these medullary cells and by 70 μm of cell filters (BD Biosciences company) filter after, by the cell of every 5 mouse of results with 500g at room temperature centrifugal 5min, and settling flux is in 3ml ammonium chloride, then at room temperature hatches 3-4 minute.Lysis for red corpuscle (RBC) is stopped by the 30ml RPMI adding the precooling comprising 10%FBS.Then these cells are filtered by 70 μm of cell filters again, centrifugal, and be resuspended in the RPMI comprising 5%FBS, GM-CSF and IL-4 (20ng/ml) (Peprotech company), other supplement are described above.Cell is laid in every hole 3*10 ⁵6 orifice plates in.At the 3rd day, slowly remove substratum in a circulating manner with suction pipe assistor, add substratum fresh as above.At the 7th day, with suction pipe assistor promptly harvested cell.After centrifugal, by these cell washings and with 2.5*10 ⁷/ ml settling flux is in OptiMEM, 200 μ l aliquots are divided to transfer in the cuvette filling 25 μ g KR158ttRNA or 10 μ g OVA RNA, and put into Electro Square Porator ECM 830 (BTX Harvard Apparatus company), then in 300V pulse 500 microsecond (Mode, LV).Collecting cell is also laid in again with every hole 5*10 ⁶the RPMI substratum as above of 6 orifice plates and 0.5 extra μ g/ml LPS in spend the night.Within second day, collect ripe ttRNA-DC be used for immunization or with mouse T cell Dual culture.

3, the Dual culture of dendritic cell DC and T cell:

According to standard operating procedure (SOPs), take out fresh for mouse T cell from spleen, and with ttRNA-or OVA RNA-DC with the ratio of 10:1 (T cell: DC cell) Dual culture.Dual culture uses with every hole 2*10 ⁶carry out in 24 orifice plates of T cell.As shown in each accompanying drawing explanation, add cytokine or their combination.In some experiments, with 0.5 μM of CFSE (Invitrogen company) according to product manual mark T cell.

4, flow cytometer and analysis:

FACSCalibur (BD Biosciences company) is used to carry out flow cytometry.The viability of cell is determined by the combination of the 7-AAD dyeing of forward angle light scatter and dead cell.The antibody (BD Biosciences company) using fluorescence to combine detects the cell surface expression of CD3, CD4, CD8, CD40, CD44, CD70, CD80, CD86, CCR7, PD-L1, I-A, CD62L and CD45RO.FlowJo 8.1.1 software (FlowJo company, Ya Shilan, Oregon) is used to analyze all FACS data.For the CD8+OVATetra+T cell for microarray analysis, vitro culture after 6 days, by being selected CD8+OVATetra+T cell divide by the FACSAria II of FACSDiva software operation at Duke University's flow cytometer core laboratory (http://shared-resources.dhvi.duke.edu/research-flow-cytometry).

5, cytokines measurement and microarray analysis:

Preparation described above also cultivates mouse dcs DC, at the 7th day, collects DC and is again laid in every hole 2*10 with different concns CD40L (maximum 1 μ g/ml) and LPS 0.5 μ g/ml ⁶spend the night in 24 orifice plates of individual DC.Second day, collect supernatant, and be used in the Cytokine Mouse 10-Plex Panel (Invitrogen company) that Duke University's core laboratory (http://shared-resources.dhvi.duke.edu/home) operates by many times of Luminex micro-sphere array analysis and detect cytokine.Carried out altogether the independent experiment of all five times, collect supernatant and be stored in-20 DEG C, cytokine is detected simultaneously.For microarray analysis, from WT mouse and OT-1 mouse and with the T cell of 1:1 mixing and OVA RNA-DC Dual culture 6 days.CD8+OVATetra+T cell is selected by classification described above.Total serum IgE uses the mini test kit of RNAeasy (Qiagen company) to be purified from cell, and the genetic expression spectrogram gene chip (mouse 430A 2.0 array) of mouse Affymetrix company is analyzed.Genetic expression spectrogram, by Partek Genomics Suit 6.6 software analysis, selects the T cell of cultivating from difference to have 601 genes being greater than twice change.Analyzed further by web analytics instrument (http://david.abcc.ncifcrf.gov/summary.jsp) by the gene selected, their T cell under Biocarta subclass be active to summarize, effect in the interphase interaction of cytokine network and cytokine and acceptor.

6, animal model:

Female C57BL/6 mouse (6-8 week is large) is used to implant IC tumour.With 0.25% trypsinase results KR158 and the B16F10OVA tumour cell containing 0.02%EDTA.Cell is mixed with isopyknic methylcellulose gum in zinc option medium, and loads in the syringe (Hamilton company, Reno, the state of Nevada) of 250 μ l with No. 25 additional syringe needles.30000 cells (KR158) and 5000 cells (B16F10OVA) are used Quitessential Stereotaxic Injector system (Stoelting company with the volume of 5 μ l, Wood Dale, IL) implant in the RCN of brain.After IC tumour implants five days, mouse accepts the body irradiation of 5Gy, uses Shepherd irradiator to carry out in Duke University's medical centre.Second day, inject 0.5-1*10 to mouse intracutaneous (ID) ⁶individual ttRNA-DC, and intravenous injection (IV) 1*10 ⁶individual T cell is used for KR158 model, and 10*10 ⁷individual T cell is used for B16F10OVA model.The 7th day after ACT and the 14th day, mouse peritoneal (IP) vaccination twice 0.5-1*10 ⁶individual ttRNA-DC is used for KR158 tumor model.Every day checks mouse, and human endpoint procedure mouse as required, and experimentally need to retain sample.

Two, interpretation of result:

1, the ripe costimulatory molecules of DC and the high level expression of cytokine:

Contriver compares the impact of LPS and CD40L on vitro culture DC, with determine implant phenotype after this maturing step with the change of function.Generally, supplement CD40L and LPS to spend the night the increase trend of costimulatory molecules on mouse DC surface demonstrating such as CD40, CD80 and CD86.But, detected by MFI, these molecules statistically significance be only embodied in the ripe DC of LPS surface on (see Figure 1A).When measuring the cytokine discharged from the supernatant liquor of collected overnight, contriver finds, the interpolation of CD40L and LPS considerably improves the secretion (see Figure 1B) of cytokine IL-1 β, IL-6, IL-12, IFN γ and TNF α.The level of IL-2 and IL-5 remains unchanged, the level of IL-10 is in the limit of error of background value, only a small amount of IL-10 detects in the DC culture supernatant of LPS maturation, this and previous report (Labeur MS, Roters B, Pers B, Mehling A, Luger TA, et al. (1999) Generation of tumor immunity by bone marrow-derived dendritic cells correlates with dendritic cell maturation stage.Journal of immunology 162:168-175) be consistent (see Figure 1B).In addition, the low-level of IL-10 is also proved to be due to the existence of IL-4 in DC Process of in vitro, inhibit generation (the Jiang HR of IL-10, Muckersie E, Robertson M, Xu H, Liversidge J, et al. (2002) Secretion of interleukin-10or interleukin-12by LPS-activated dendritic cells is critically dependent on time of stimulus relative to initiation of purified DC culture.J Leukoc Biol 72:978-985).

2, ripe DC promote cd8 t cell propagation and cytokine-cocktail collaborative under maintain the low differentiation of cd8 t cell:

For determining the biological function of cylinder mature DC, import the DC of ttRNA to mouse inoculation vaccine with the external preparation of difference by electroporation.Obtain its spleen after one week of inoculation, and mark T cell with CFSE.Contriver finds, demonstrates the profound influence (see Figure 1A) to T cell in-vitro multiplication through the DC of CD40L and LPS maturation.Compare with control group and immature DC group, T cell a large amount of as seen under classification, and CD4 and cd8 t cell enter the multiplicative stage (see Fig. 1).What is interesting is, in fact strengthen the adding of IL-2 the differentiation of T cell in control group and immature DC group, and demonstrate the minimum chemiluminescence (see Figure 1A) with T cell in ripe DC group.Except imagery these for vaccinated DC biological function in vivo, final purpose be utilize the ttRNA-DC of these external preparations to carry out enrichment as a platform and expansion for the antitumor precursor cell of ACT.But, under the existence of IL-2, these ttRNA-DC when the T cell with spleen in vitro Dual culture time create cd8 t cell, Tem and Tcm (see Fig. 3 A and Fig. 3 B) of similar per-cent.For directly regulating the phenotype of the T cell of vitro culture, be applied to validity and the persistence of oncotherapy, contriver optimizes the culture condition of the application cell factor-cocktail.As shown in Figure 3 C, on the basis of the ripe DC of LPS, relative to interpolation IL-2, supplement the phenotype that cytokine-cocktail have significantly changed the T cell of vitro culture, show as PD phenotype, such as, (CD44 is low to significantly increase Tcm and Tn, CD62 is high) ratio of subset, and the per-cent of cd8 t cell remains unchanged.

3, it is active that the antitumor T cell come from ripe DC change adds that cytokine-cocktail demonstrates noticeable anti-tumor in vivo:

Have been reported display, the T cell of PD vitro culture has the advantage of long term growth in xenograft mouse model (Yang S, Ji Y, Gattinoni L, Zhang L, Yu Z, et al. (2013) Modulating the differentiation status of ex vivo-cultured anti-tumor T cells using cytokine cocktails.Cancer Immunol Immunother 62:727-736; Yang S, Gattinoni L, Liu F, Ji Y, Yu Z, et al. (2011) In vitro generated anti-tumor T lymphocytes exhibit distinct subsets mimicking in vivo antigen-experienced cells.Cancer immunology, immunotherapy:CII 60:739-749).And the T cell of these types has demonstrated has stronger anti-tumor activity (Hinrichs CS in lotus knurl model, Borman ZA, Gattinoni L, Yu Z, Burns WR, et al. (2011) Human effector CD8+T cells derived from naive rather than memory subsets possess superior traits for adoptive immunotherapy.Blood 117:808-814; Gattinoni L, Lugli E, Ji Y, Pos Z, Paulos CM, et al. (2011) A human memory T cell subset with stem cell-like properties.Nature medicine 17:1290-1297).But, this PD T cell hang oneself CAR genetically engineered modify or from transgenic mice; At present, resist tumor T cells and also do not have systematic evaluation with their anti-tumor activity; Therefore, in the present invention, contriver systematically assess anti-tumor activity and from the T cell of external ttRNA-DC platform differentiation state between relation.Consistent with report, people and mouse through the T cell of ttRNA-DC amplification and enrichment demonstrate tumor associated target to specific recognition.Contriver finds, the antitumor T cell of ttRNA-DC amplification can identify that tumor associated antigen and ttRNA prepare applied tumour cell (see Fig. 4 A) specifically.

Owing to inoculating the remarkable promoter action to T cell after ripe DC vaccine in vivo, anti-tumor in vivo effect also finds, shows different antitumous effects (see Fig. 4 B) by using the T cell from the different ttRNA-DC cultivated through cytokine-cocktail.For evaluating anti-tumor in vivo effect, intracranial tumors is after implanted 5 days, and in ACT the day before yesterday, mouse accepts the body irradiation of 5Gy; Second day, mouse was respectively by the antitumor T cell that external intravenous injection (IV) is increased, and abdominal cavity (IP) inoculates ttRNA-DC vaccine.Compare with untreated fish group and IL-4/GM-CSF DC group, CD40L and LPS-DC significantly improves the median survival of tumor-bearing mice, and LPS-DC group reaches statistical significance (see Fig. 4 B).Due to the Affording acquisition in LPS-DC body and superiority (see Fig. 1, Fig. 2, Fig. 4 B), next attempt the interpolation of comparing different cytokines, and under the synergy of LPS-DC, observe its antitumous effect in IC tumor model.Compared with use IL-2, the T cell that cytokine-cocktail cultivates significantly improves the median survival (see Fig. 4 C) of tumor-bearing mice.In the present embodiment, cytokine-cocktail is combined IL-7, IL-12, IL-21, and does not use IL-2, and contriver has found at the low yield (not shown) cultivating terminal T cell; And compare other antitumous effect (see Fig. 4 D) comprising the combination of cytokines using IL-15.But the T cell that the cytokine-cocktail based on the ripe DC of LPS cultivates continues the median survival extending mouse, and other combination does not demonstrate significant antitumous effect (see Fig. 4 D).For proving that ttRNA-DC is a steps necessary for the antitumor T cell of external generation, add the T cell group that one group of similar training method not having ttRNA-DC to increase is cultivated, as desired, these T cell can not extend the median survival (see Fig. 4 D) of tumor-bearing mice.

4, the cultivation of cytokine-cocktail or other combinations can promote the ratio of memory T cell:

Based on the ripe DC of LPS, contriver finds, the T cell that cytokine-cocktail cultivates can extend the median survival of tumor-bearing mice.The more important thing is, the anti-tumor activity demonstrating its (see Fig. 4) is in vivo and in vitro subject to the impact of ttRNA-DC platform, such as, antigen is correlated with the specific recognition of target and the median survival of prolongation, infers that biology effect results from the amplification of antitumor precursor cell and enrichment.Although the DC of ttRNA load provides the source of an innovation, ttRNA represents all tumor associated antigens of the tumour antigen comprising Tumor mutations antigen, and from this point, most of tumor associated antigen still needs research further to determine.

When the holographic antigen of weight that ttRNA comprises be not also understand completely when, contriver attempts answer very important problem: in ttRNA-DC-T system, adding cytokine-cocktail is the antitumor precursor cell being enriched T cell, still the phenotype of antitumor T cell is changed? for addressing this problem, have selected the response of the well-known OVA antigen from OT-1 mouse T cell, i.e. T cell identifiable design OVA antigen.In Figure 5, use the T cell from OT-1 mouse, and utilize OVA-DC to increase in vitro, contriver finds, compared with IL-2 group, the combination of other cytokines all is used to create the CD8OVAtetra+T cell of similar per-cent, except demonstrating the CD8OVAtetra+T cell (see Fig. 5 A and Fig. 5 B, upper hurdle) of reduction with IL7+IL21/IL12+IL15 group.When analyzing the per-cent of this group CD8OVAtetra+CD62L+T cell, contriver finds, compared with IL-2 group, other combination of cytokines all significantly increase the ratio (see Fig. 5 A and Fig. 5 B, lower hurdle) of the T cell of this type.Seem to seem, except using IL-2, the phenotype combinationally using the T cell that can change vitro culture of cytokine.About from data in the body of Fig. 4, contriver finds, combinationally uses IL-7, IL-12 and IL-21 (a kind of cytokine-cocktail) and within three days, plays crucial effect at Dual culture.

5, in OVA-RNA DC body, the antitumor T cell of enrichment extends the median survival of tumor-bearing mice significantly:

In experiment above, the antitumor T cell from ttRNA-DC-T platform having demonstrated external generation extends the median survival (participation Fig. 4) of tumor-bearing mice significantly.But the median survival in the therapeutic regimen that cytokine-cocktail cultivates extends two weeks usually, at present in this system, does not reach long-term survival by once injecting adoptive transfer T cell.Inquisitive, by ttRNA-DC ex vivo enrichment and amplification, shown by low-level IFN γ induction, the per-cent of antitumor precursor cell is low (participating in Fig. 4 A).For cultivating frequency and the per-cent of the antitumor T cell of terminal enrichment, attempting to reuse ttRNA-DC is stimulating T cell; But the mouse T cell of vitro culture became fragile and is easy to apoptosis after 7 days of first round ttRNA-DC Dual culture; And even if carry out second to take turns ttRNA-DC under cytokine-cocktail culture environment, PD CD62LHigh phenotype can not be raised.

For proving that adoptive transfer T cell can not only improve the median survival of IC tumor-bearing mice significantly, and can long-term surviving be reached, contriver selects to have more invasive melanocyte tumour system B16F10OVA, and be designed to express OVA antigen to implant IC, this kind of tumor-bearing mice generally can in 3 weeks dead (see Fig. 6 A).Consistent with OVA antigen, utilize the T cell from OT-1 mouse spleen to mix with wild-type (WT) T cell with 1:1 ratio.Upon mixing the 0th day, be typically about 5% (see Fig. 6 B) by the per-cent of flow cytomery CD8OVAtetra+T cell.But after 6 days of OVA RNA-DC amplification in vitro, the finished product are primarily of CD8OVAtetra+T cell composition, and its use with IL-2 or cytokine-cocktail has nothing to do (see Fig. 6 B).Although use the T cell of the vitro culture obtained to create the CD8OVAtetra+T cell of similar level by cytokine difference, and the T cell significant prolongation of two types IC tumour stands the median survival of mouse, but result in the long-term surviving (median survival of 51 days from the T cell that cytokine-cocktail organizes, by contrast, IL-2 group is 28 days, untreated fish group is 21 days) (participating in Fig. 6 A).

For determining the molecular biosciences basis under the T cell that IL-2 and cocktail cultivates, pick out the CD8OVAtetra+T cell of amplification in vitro after 6 days, its purity is determined (see Fig. 6 B) by flow cytometry analysis.Based on the genetic expression through Partek Genomics Suit 6.6 software analysis, the T cell of cultivating from difference has the scatter diagram drawn (see Fig. 6 C, upper hurdle) being greater than representative 601 genes that 2 times change.And have selected one group of gene, this group gene summarises their effects in T cell activity, cytokine network and the mutual relationship (see Fig. 6 C, lower hurdle and Fig. 7,8,9) between cytokine and acceptor.Be consistent with phenotype analytical, CD27, CD62L level is raised, and what is interesting is, finds that the expression level being used for 4-1BB has also been increased (see Fig. 6 C, lower hurdle); But the IFN γ level obviously increased and PD phenotype inconsistent (see Fig. 5).What is interesting is, contriver finds, is lowered, comprise CXCR3, CXCL10, CCL5, CCR9 etc. (see Fig. 6 C, lower hurdle) in the T cell that several chemokine is cultivated at cocktail compared with IL-2.

6, the dependency of molecular marked compound and anti-tumor in vivo effect:

Different between IL-2 and cocktail amplification in vitro T cell are defined as molecular marked compound.But contriver analyzes 601 genes that a group has 2 times of changes, and concentrate several candidate genes to summarize their effects (see Fig. 7) in T cell activity.The active path of T cell is produced by BIOCARTA software, reflects that 2 times of key molecules changed are represented by asterisk.Contriver finds in the T cell that external cocktail increases, and the 4-1BB on T cell surface raises, and this may facilitate the interaction between DC and T cell; IL-2R in T cell too increases, and infers this interaction that may help itself and cd4 t cell subclass; But, the implication of low-level TCR ζ chain and IL-18 is fallen still unknown (see Fig. 7).

In order to the curative effect of clear and definite ACT, once inject the mutual relationship between the T cell of tumor-bearing mice and scavenger cell, neutrophilic leukocyte, basophilic leukocyte, eosinophil, mastocyte, B cell, the cytokines of 2 times of changes that accurately find some keys, that obtained by microarray analysis, and with arrows to summarize their effects (see Fig. 8 and Fig. 9) in cytokine network.As mentioned above, IFN γ and IL-10 is raised, and IL-18 is lowered.IL-10 can suppress the synthesis of pro-inflammatory cytokine IFN-γ, IL-2, IL-3, TNF α and GM-CSF, and these pro-inflammatory cytokines are secreted by scavenger cell and regulatory T cells.In addition, IL-10 also has the potential HLA-II antigen that can suppress antigen presenting cell, also can stimulate some T cell (Th2) and mastocyte, and stimulates B cell maturation and antibody to produce.Although the main purpose of the present embodiment is the molecular marked compound of clearly antitumor T cell, and then clearly utilize the anti-tumor-stroma of antitumor T cell of cocktail amplification in vitro, but the data deficiencies that the present embodiment provides, with clear and definite and antitumor relevant molecular marked compound, requires further study to determine.

Three, discuss:

In the present invention, inventors have demonstrated that ripe DC and combination cytokine are how the outer ttRNA-DC of support increases the propagation of antitumor T cell and differentiation.Contriver finds, ripe DC can secrete one group of cytokine, and extends the active condition of T cell in vitro in culturing process in vivo after vaccination significantly.Based on ripe DC, do not use the cytokine cocktail of IL-2 to create CD62L High phenotype, this successfully extends has the height mouse astrocytoma model of invasiveness and the median survival of melanoma IC tumor model.Contriver advises, the high-caliber CD62L in these antitumor T cell can contribute to optimizing anti-tumor in vivo effect, or can be used as marking reliably with the phenotype redefining best antitumor T cell and being applied to clinical.And contriver has attempted with the molecule marker of microarray analysis identification based on superior antitumous effect, and accurately find some to T cell activity have Main Function and and the interactional key molecule of cytokine network.But the different phenotypes relevant from result for the treatment of need to be further explained.

A large amount of evidence shows, RNA transfection is a kind of good solution be loaded into by antigen on DC.TtRNA representative take RNA as the full spectrum antigen of tumour antigen, they are ideal targets (Robbins PF of application cell immunotherapy, Lu YC, El-Gamil M, Li YF, Gross C, et al. (2013) Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells.Nat Med 19:747-752).Inventors have demonstrated that the T cell of ttRNA-DC amplification in vitro can specific recognition associated target antigen, comprise ttRNA and be extracted applied tumour cell (see Fig. 4).But, demonstrate, only in IC tumor model, can not result for the treatment of be implemented as vaccinated ttRNA-DC, although it shows the effect of T cell in potential activation body.The present embodiment adopts the tumour antigen of tumor model and load DC.First, unlike subcutaneous injection of tumor and by measuring gross tumor volume evaluation tumor growth, in the present invention, contriver adopts the tumor-bearing mice that grown 5 days to be the model evaluating oncotherapy effect; Therefore, contriver reaches a conclusion, and ttRNA-DC has certain result for the treatment of as Inoculation vaccine, and this effect may be not enough to the median survival extending animal, and needs to act synergistically with the antitumor T cell in ACT.The second, contriver does not concentrate on and studies the preferably source what is tumour antigen, such as ttRNA or total tumor lysis product; Due in brain tumor, cut tumor sample is very little, therefore can not obtain enough tumour cells to prepare total tumor lysis product, can be applied to clinical application by a small amount of ttRNA of amplification in vitro on the contrary to enough amounts.At present, it is feasible that existing positive clinical data confirms that the ttRNA increased from tumor stem cell (taking CD33 as marker) is applied to clinical application.

With existing gene engineering method (Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, et al. (2006) Cancer regression in patients after transfer of genetically engineered lymphocytes.Science 314:126-129; Kalos M, Levine BL, Porter DL, Katz S, Grupp SA, et al. (2011) T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia.Science translational medicine 3:95ra73) different, the invention provides the ttRNA-DC-T platform of innovation, successfully extend the survival of two kinds of mouse IC tumor models.Contriver infers, vaccination subsequently provides and is conducive to external adoptive transfer T cell and survives in vivo lastingly required microenvironment, consistent with the result in Fig. 2, and in Fig. 2, ripe DC can regulate the proliferation in vivo of T cell after amplification.In this case, adoptive transfer T cell can finally be transferred to knub position and kill tumour cell.With the model reported (Jiang HR, Muckersie E, Robertson M, Xu H, Liversidge J, et al. (2002) Secretion of interleukin-10or interleukin-12by LPS-activated dendritic cells is critically dependent on time of stimulus relative to initiation of purified DC culture.J Leukoc Biol 72:978-985) be consistent, the anti-tumor in vivo effect of adoptive transfer T cell seriously relies on three factors: lymph serum is except (lymphodepletion), injection (the Gattinoni L of vaccination subsequently and IL-2, Klebanoff CA, Palmer DC, Wrzesinski C, Kerstann K, et al. (2005) Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8+T cells.The Journal of clinical investigation 115:1616-1626).The ultimate aim of ACT eliminates tumour cell and sets up the memory of lasting antineoplastic immune.Or, compare as vaccine with use ttRNA-DC, utilize its potentiality directly to produce the antitumor T cell of Multifunctional external, do not need to rely on the compromised immune system in cancer patients.Experiment proves, T cell very accurately can eliminate tumour (Brenner MK, Heslop HE (2010) Adoptive T cell therapy of cancer.Current opinion in immunology 22:251-257 large, ripe in mouse and the mankind, Rosenberg SA (2011) Cell transfer immunotherapy for metastatic solid cancer-what clinicians need to know.Nature reviews Clinical oncology 8:577-585, Johnson LA, Morgan RA, Dudley ME, Cassard L, Yang JC, et al. (2009) Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen.Blood 114:535-546, Robbins PF, Morgan RA, Feldman SA, Yang JC, Sherry RM, et al. (2011) Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1.J Clin Oncol 29:917-924), or even tumour is in (Hong JJ in " Immune privilege " brain, Rosenberg SA, Dudley ME, Yang JC, White DE, et al. (2010) Successful treatment of melanoma brain metastases with adoptive cell therapy.Clinical cancer research:an official journal of the American Association for Cancer Research 16:4892-4898, Rosenberg SA, Yang JC, Sherry RM, Kammula US, Hughes MS, et al. (2011) Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy.Clinical cancer research:an official journal of the American Association for Cancer Research 17:4550-4557).In fact, the immunotherapy that the hemato encephalic barrier stoping humoral immunization composition to enter brain can not hinder T cell to mediate; As the T cell of activation, research confirmation can pass through hemato encephalic barrier (Hong JJ, Rosenberg SA, Dudley ME, Yang JC, White DE, et al. (2010) Successful treatment of melanoma brain metastases with adoptive cell therapy.Clinical cancer research:an official journal of the American Association for Cancer Research 16:4892-4898).

Contriver advises service-strong CD62L as molecular marked compound to predict the anti-tumor in vivo effect of the antitumor T cell of vitro culture, and redefines the concept of the antitumor T cell of the vitro culture of the best of the ACT for cancer.

Be described in detail specific embodiments of the invention above, but it is just as example, the present invention is not restricted to specific embodiment described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and substituting also all among category of the present invention.Therefore, equalization conversion done without departing from the spirit and scope of the invention and amendment, all should contain within the scope of the invention.

Claims

1. an antitumor T cell, is characterized in that, described antitumor T cell is with the dendritic cell DC specific amplification of tumour total serum IgE load and the antitumor T cell of enrichment.

2. antitumor T cell according to claim 1, is characterized in that, also comprises cytokine in described antitumor T cell.

3. antitumor T cell according to claim 2, is characterized in that, described cytokine is selected from any one or multiple combination in IL-4, IL-7, IL-12, IL-15, IL-21, GM-CSF, CD40L.

4. antitumor T cell according to claim 2, is characterized in that, described cytokine is selected from the combination of IL-7, IL-12, IL-21.

5. the preparation method of antitumor T cell as claimed in claim 1, is characterized in that, comprise the steps:

6. preparation method according to claim 5, is characterized in that, the tumour cell of described step 1 is from the tumour cell of the mankind or mouse.

7. preparation method according to claim 5, is characterized in that, the dendritic cell of described step 2 are from the myelocyte of the mankind or mouse or lymphocyte.

8. preparation method according to claim 5, is characterized in that, described step 2 adds CD40L or lipopolysaccharides LPS in the medium to realize the maturation of DC.

9. preparation method according to claim 5, is characterized in that, the Dual culture of described step 3 carries out under the existence of cytokine.

10. preparation method according to claim 9, is characterized in that, described cytokine is selected from any one or multiple combination in IL-4, IL-7, IL-12, IL-15, IL-21, GM-CSF, CD40L.

11. preparation methods according to claim 9, is characterized in that, described cytokine is selected from the combination of IL-7, IL-12, IL-21.

12. preparation methods according to claim 5, is characterized in that, the T cell of described step 3 is from the spleen of the mankind or mouse.

13. preparation methods according to claim 5, is characterized in that, the T cell of described step 3 is antigen responsive cell, comprise CD4+T cell, CD8+T cell, effector T cell, memory T cell.

14. preparation methods according to claim 5, is characterized in that, the ripe tumour total serum IgE-DC of described step 3 and the number ratio of T cell are 1:(15-25).

15. 1 kinds of antitumor drugs, is characterized in that, described antitumor drug with antitumor T cell according to claim 1 for activeconstituents.

16. antitumor drugs according to claim 15, is characterized in that, described antitumor drug is medicament or vaccination.