CN104630146A - Preparation method and application of tumour cell specific polyclonal T cells - Google Patents
Preparation method and application of tumour cell specific polyclonal T cells Download PDFInfo
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Abstract
The invention relates to the technical fields of molecular biology and cellular immunology and in particular relates to a preparation method of tumour cell specific polyclonal T cells. The preparation method of the tumour cell specific polyclonal T cells comprises the following steps: (1) obtaining peripheral blood mononuclear cells; (2) mixing the obtained peripheral blood mononuclear cells with gene modifying tumour cells; and (3) resuspending the obtained mixed cells in a culture medium containing cell factors which can maintain cell growth, proliferation and differentiation. The prepared tumour cell specific polyclonal T cells are mainly applied to medicines which are sued for preventing or treating cancers. The preparation method of the tumour cell specific polyclonal T cells has the advantages that the prepared tumour cell specific polyclonal T cells have the characteristics of large quantity, high specificity and strong killing capability; besides, a technology is simple, and the preparation cost is obviously reduced compared with the prior art.
Description
Technical field
The present invention relates to molecular biology and technical field of cellular immunology, particularly relate to tumor cell specific polyclone T cell preparation method, the tumor cell specific polyclone T cell prepared by the method prevention or Therapeutic cancer medicine in application.
Background technology
At present, adoptive immunity cell therapy achieves breakthrough in the tumour for the treatment of melanoma and blood system; Therefore, the focus of immunotherapy of tumors research field is again become.In the world, the effector cell selected mainly comprises tumor infiltrating lymphocyte (tumor infiltrating lymphocyte, TIL), tumour antigen or virus antigen specific cytotoxic t lymphocytes (cytotoxic T lymphocyte, CTL) and Chimeric antigen receptor modify T cell (chimeric antigen receptor modified T lymphocyte, CART); Domestic, the cytokine induced kill cell (cytokine induced killer cell, CIK) of the also mainly invention eighties in 20th century of clinical application and the cytokine induced kill cell (DC-CIK) of activated dendritic cell improved on this basis.CIK and DC-CIK cell preparation technology is relatively simple, be easy to carry out large-scale promotion clinical, but because it is mainly through non-specific mechanism killing tumor cell, current clinical effectiveness display, curative effect is very limited.TIL, CTL and CART achieve good clinical therapeutic efficacy clinically, but TIL but exists difficulty of drawing materials, and need the cultivation through 1 ~ 2 month long period, therefore, are difficult to apply at clinical-scale; The preparation of CTL cell, first tumour or viral-specific antigens is isolated, then separating monocytic cell induction DC, add specific antigens in the process, make ripe DC finally can by major histocompatibility complex (major histocompatibility complex, the form submission of I, II quasi-molecule of MHC) encoding is to DC surface, then with the generation of T cell co-culturing, inducing CTL cell, relate to antigen preparation, DC and T cell separation and Culture in whole process, therefore technique is comparatively complicated; The current major way of preparation of CART carries out genetic modification by lentiviral vectors, and technical process is more complicated, and with high costs, therefore, is also in clinical experimental stage at present, is difficult to apply in clinical-scaleization.Recent studies have shown that, chronic lymphocytic leukemia (chronic lymphocytic leukemia, CLL) peripheral blood mononuclear cell (the Peripheral blood mononuclear cell in patient source, PBMC), a kind of two targeting specific antibody (Bispecific Antibody is added in cultivating in vitro, BsAb) medicine blinatumomab(CD3 × CD19), add IL-2 simultaneously, the leukemia cell in PBMC can be eliminated, the polyclone T cell of simultaneously inducing can demonstrate stronger Tumor-cytotoxic efiect (Golay etc., 2014).But two targeting specific antibody that this technology uses relate to carry out in vitro producing, purifying, packaging, transport, the link such as preservation, therefore with high costs and waste time and energy; In addition, polyclone T cell prepared by this technology is only for B cell system tumour, and range of application is very limited.Therefore, how to overcome the restricted of above-mentioned series technique, develop a kind of can for most of tumour even all tumor type, tumor specific T cells that technique is simple, with low cost prepares current techique, be then the key issue that faces of current immunotherapy of tumors and a difficult problem.
Summary of the invention
For solving the problem, the invention provides a kind of tumor cell specific polyclone T cell (tumor specific polyclonal T lymphocyte, TSPT) preparation method and prevention or Therapeutic cancer medicine in application.
First aspect, the invention provides the preparation method of TSPT cell, and described TSPT cell then prepares gained with the PBMC Dual culture of genetic modification tumour cell and acquisition by obtaining PBMC.Although obtain PBMC and the external method preparing specific T-cells is a lot, but be not also described through at present and dual genetic modification is carried out to tumour cell, the tumour cell energy modified expression-secretion type genetic engineering antibody (Genetic engineering Antibody simultaneously, and film mating type target antigen (Target Antigen GeAb), TAg), the combined method of TSPT cell is then obtained with above-mentioned genetic modification tumour cell and PBMC Dual culture.In aforesaid combination, the principle of preparation TSPT cell is: genetic modification tumour cell can secrete GeAb can express film mating type TAg again simultaneously, with the process of PBMC mixed culture, GeAb is by identifying TAg and T cell, by the two hinge together, thus being conducive to the antigen presentation of genetic modification tumour cell to T cell, the T cell that then stimulates further, activates and increase forms TSPT cell, and in culturing process, genetic modification tumour cell is killed; The tumour cell clear and definite concerning tumour antigen, carry out genetic modification and express TAg, serve the effect of strengthening antigen and antibody specific identification, and concerning the indefinite tumour cell of tumour antigen, by selecting a known TAg to modify, then serve the effect of erecting antigen and antibody specific identification.Therefore this technical scheme, solves the technical barrier that current most of tumour antigen is indefinite or expression amount is low tumour cell cannot produce with T cell identification and then inducing specific T cell.The method comprises the steps: first to gather peripheric venous blood, then be separated with ficoll-general shadow glycosamine density gradient centrifugation and obtain PBMC, above-mentioned PBMC is mixed with genetic modification tumour cell, then Dual culture in the substratum containing RhIL-2 (Recombinant Human Interleukin-2, rhIL-2).Every 2 ~ 3 days subsequently, count cell, then continue to cultivate with after the fresh culture adjustment cell to Suitable Density containing rhIL-2, cultivate after 9 ~ 20 days, collected cell is TSPT cell.
Preferably, the density of initial incubation PBMC and genetic modification tumour cell is (0.5 ~ 2) × 10 respectively
6/ ml, both volume ratios of mixing are 0.1 ~ 10.
Further preferably, the density of initial incubation PBMC and genetic modification tumour cell is 1 × 10 respectively
6/ ml, both volume ratios of mixing are 1.
Preferably, the final concentration using rhIL-2 is 50 ~ 1000 IU/ml.
Further preferably, the final concentration using rhIL-2 is 300 IU/ml.
Preferably, every 2 ~ 3 days subsequently, the Suitable Density of cell cultures was (0.5 ~ 3) × 10
6/ ml.
Further preferably, every 2 ~ 3 days subsequently, the Suitable Density of cell cultures was 1 × 10
6/ ml.
Second aspect, the invention provides a kind of preparation method of genetic modification tumour cell, and described genetic modification tumour cell can by have coding GeAb and TAg(GeAb.TAg simultaneously) the carrier transfection tumor cell of gene obtains.
Preferably, described GeAb gene has the base sequence of coding GeAb, and described GeAb is BsAb, and described BsAb has the binding site of targeting T-cells epitope and the secretor type BsAb of TCSA epitope binding site.
Preferably, described TAg gene is the base sequence with coding TAg, the one in the tissue-specific differentiation antigen of described TAg choosing, the tumour antigen of oncovirus expression, mutator gene or the expression product of oncogene, the cell protein of unconventionality expression and embryonal antigen.
Preferably, the one in the Lentiviral of described carrier choosing, retroviral vector, gland relevant viral vector, adenovirus carrier, non-replicating viral vector, duplicating virus carrier and sleeping beauty transposon stand etc.
Preferably, the one in the liver cancer of described tumour cell choosing, lung cancer, cancer of the stomach, the esophageal carcinoma, intestinal cancer, cervical cancer, the tumour cell such as mammary cancer and nasopharyngeal carcinoma.
The third aspect, the invention provides a kind of construction process of GeAb.TAg genophore of encoding, the described carrier with coding GeAb.TAg gene in the multiple clone site (MCS) of carrier, inserts GeAb.TAg gene order combine, and described GeAb.TAg gene order comprises the base sequence of the encoding immune sphaeroprotein κ chain signal peptide connected successively, the base sequence of coding Flag label, the base sequence of GeAb, the base sequence of encoding histidine His6 label, the base sequence of P2A, TAg gene and terminator codon.
Preferably, the one in the Lentiviral of described carrier choosing, retroviral vector, gland relevant viral vector, adenovirus carrier, non-replicating viral vector, duplicating virus carrier and sleeping beauty transposon stand etc.
Further preferably, described carrier is Lentiviral.
Preferably, the base sequence of described immunoglobulin kappa chain signal peptide is as shown in SEQ ID NO:1.
Preferably, the base sequence of described Flag label is as shown in SEQ ID NO:2.
Preferably, described GeAb gene is BsAb gene.
Further preferably, the gene of described BsAb is formed (CD20 × CD3) by short chain (GGGGS) link by single-chain antibody (single-chain antibody fragment, the scFv) gene of anti-CD20 antibodies of encoding and the scFv gene of coding AntiCD3 McAb.
Further preferably, the base sequence of described BsAb is as shown in SEQ ID NO:3.
Preferably, the base sequence of described histidine 6 label is as shown in SEQ ID NO:4.
Preferably, the base sequence of described P2A is as shown in SEQ ID NO:5.
Preferably, the tissue-specific differentiation antigen gene of described TAg gene choosing, oncovirus express tumor antigen gene, mutator gene or oncogene, one in the cell protein gene of unconventionality expression and embryonal antigen gene.
Further preferably, the tissue-specific differentiation antigen gene of described TAg gene choosing.
Further preferably, described TAg gene is CD20 gene.
Further preferably, the base sequence of described TAg is as shown in SEQ ID NO:6.
Preferably, the base sequence of described terminator codon is TTA.
Fourth aspect, the invention provides the construction process of the preparation method of the TSPT cell of tumor cell specific as described in relation to the first aspect, the preparation method of the genetic modification tumour cell as described in second aspect, the coding GeAb.TAg genophore as described in the third aspect.
Preferably, the cell of TSPT as described in relation to the first aspect provided by the invention, genetic modification tumour cell as described in second aspect, GeAb.TAg genophore of encoding as described in the third aspect are for the preparation of the application in the medicine of prevention or Therapeutic cancer.
Further preferably, the cell of TSPT as described in relation to the first aspect provided by the invention, genetic modification tumour cell as described in second aspect, GeAb.TAg genophore of encoding as described in the third aspect are for the preparation of the application in the medicine of prevention or Hepatoma therapy.
Method provided by the invention has following beneficial effect:
(1) the TSPT cell that prepared by the present invention has obvious tumor cell specific, and the killing-efficiency to the effector T cell that the killing-efficiency of tumour cell is cultivated apparently higher than current classical way (AntiCD3 McAb and anti-CD28 antibody associating rhIL-2 induce PBMC).From current data, through HepG2-BsAb.TAg inducing culture TSPT cell to liver cancer cell killing-efficiency comparatively classical way improve 3.78 times, namely killing-efficiency has mentioned 61.72% from original 12.91%; And liver cancer-specific TSPT cell is not significantly improved compared with classical way to the killing-efficiency of K562 cell, namely classical way is 12.18%, and the killing-efficiency of liver cancer cell specificity TSPT cell is 13.47%.
(2) genetic modification tumour cell provided by the invention, can either express film mating type TAg, simultaneously again can secretion expression BsAb.At the beneficial effect of genetic modification tumour cell Membrane surface expression TAg be: the tumour antigen of current most of tumour cell is also indefinite or expression amount is low, so then solve the difficult problem to tumour cell targets identification by this method, thus make the even whole tumour of most of tumour cell all can carry out high level expression specific targeted antigen by the method, become the target of two targeting specific antibody recognition.The beneficial effect that genetic modification tumour cell carries out secretion expression BsAb is: avoid the links such as produced in vitro, purifying, packaging, transport and preservation, thus greatly reduce production cost.Simultaneously by the two united application, as the combination of CD20 and CD20 × CD3, more easily form current techique, be applied to the application in the even whole prevention of tumor type of major part or the medicine of Therapeutic cancer.
(3) GeAb.TAg genophore provided by the invention, avoids and carries out transfection and expression at two carriers, optimizes production technique greatly like this, makes it simpler.
(4) tumor cell specific TSPT cell provided by the invention can be applied separately, or with one or more in chemotherapy, radiotherapy, operation, interventional therapy, biotherapy, immunotherapy be united and applied in prevent or Therapeutic cancer medicine in application.
Accompanying drawing explanation
The physical map of the pUC57 carrier that Fig. 1 provides for the embodiment of the present invention 1.
Fig. 2 is the physical map of the recombinant plasmid pUC57.BsAb containing CD20 × CD3 gene that the embodiment of the present invention 1 builds.
The physical map of the pCDH carrier that Fig. 3 provides for the embodiment of the present invention 1.
Fig. 4 is the physical map of the recombinant slow virus expression vector pCDH-BsAb.TAg containing CD20 × CD3.CD20 gene that the embodiment of the present invention 1 builds.
The HepG2-BsAb.TAg process screening film mating type antigens c D20 expression flow cytometry analysis figure that Fig. 5 provides for the embodiment of the present invention 2.This figure shows, through the positive rate of HepG2-BsAb.TAg of screening up to 99.8%; Therefore, from the expression of film mating type CD20, technical scheme provided by the invention is successful.
The flow cytometry analysis figure that CD20 × CD3 that Fig. 6 provides for the embodiment of the present invention 2 is combined with Raji cell and Jurkat cell respectively.Through the curve of the experimental group of CD20 × CD3 process when in this figure, the curve of arrow (→) indication is dyeing, therefore, result display from figure, CD20 × CD3 can combine respectively at the Jurkat cell of the Raji cell of the CD20 positive and the CD3 positive; Therefore, from the expression of secretor type CD20 × CD3, technical scheme provided by the invention is successful.Composition graphs 5, genetic modification tumour cell preparation method provided by the invention and coding GeAb.TAg genophore construction process are all successful.
The trend map (n=8) that in the PBMC inducing culture process that Fig. 7 provides for the embodiment of the present invention 4, CD3 positive cell and CD20 positive cell change along with incubation time change in the ratio of total cell.This figure shows, and in the process of mixed cell culture, the ratio of genetic modification tumour cell sharply declines, and within the 6th day, drops to about 5%, can't detect genetic modification tumour cell after 9 days; Meanwhile, the ratio of CD3 positive cell raises fast in the process of cultivating.Can more than 96% be reached after 6 days.Consider from safety perspective, the present invention can remove the genetic modification tumour in culture system completely, and the TSPT cell that therefore prepared by the present invention is safe.
In the PBMC inducing culture process that Fig. 8 provides for the embodiment of the present invention 4, CD3 positive cell and CD20 positive cell are along with incubation time change cell absolute number changing trend diagram (n=8).This figure shows, and in the process of mixed cell culture, the absolute number of genetic modification tumour cell was increase at first 3 days, reduces gradually, can't detect genetic modification tumour cell after 9 days after 3 days; Meanwhile, the absolute number of CD3 positive cell continues to raise in the process of cultivating always.Therefore, no matter from the angle of safety or consider to the angle of T cell amplification efficiency, method provided by the invention is all successful.
The cultivation TSPT Immunophenotyping analysis chart (n=8) of the 14th day that Fig. 9 provides for the embodiment of the present invention 5.In this figure, pre represents the PBMC group of fresh separated, and post represents the cultivation TSPT groups of cells of the 14th day, therefore shows in figure, and the method can significantly increase the T cell of the CD3 positive; Meanwhile, the positive CTL cell of advantage pcr CD8.Therefore, TSPT cell prepared by the present invention is a kind of based on the heterogeneous cell of a group of CTL cell, thus is conducive to Efficient killing effect tumour cell.
Cultivation TSPT cell-specific tumor cytotoxicity effect analysis figure (n=8) of the 14th day that Figure 10 provides for the embodiment of the present invention 6.This figure CD3-28 represents the effector T cell that classical way is cultivated, therefore result display in figure, through HepG2-BsAb.TAg inducing culture TSPT cell to liver cancer cell killing-efficiency comparatively classical way be significantly increased, and demonstrate good specificity.Therefore, TSPT cell have precisely, the ability of Efficient killing effect tumour cell.
Embodiment
The following stated is the preferred implementation of the embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from embodiment of the present invention principle; can also make some improvements and modifications, these improvements and modifications are also considered as the protection domain of the embodiment of the present invention.Unreceipted concrete technology or condition person in embodiment, (such as show with reference to J. Pehanorm Brooker etc. according to the technology described by the document in this area or condition, " Molecular Cloning: A Laboratory guide " that Huang Peitang etc. translate, the third edition, Science Press) or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
Embodiment 1: present embodiments provide one can secrete again the gene recombination Lentiviral of anti-CD20 × CD3 construction process at surface of cell membrane stably express CD20, comprise the steps:
(1) a kind of construction process of the pUC57 recombinant plasmid vector containing CD20 × CD3 gene is present embodiments provided
(1a) mode adopting complete genome sequence to synthesize synthesizes base sequence (SEQ ID NO:4)-P2A base sequence (SEQ ID NO:5)-Nhe I restriction enzyme site of base sequence (SEQ ID the NO:3)-encoding histidine His6 label of base sequence (SEQ ID the NO:2)-BsAb of base sequence (SEQ ID the NO:1)-coding Flag label of the Xba I restriction enzyme site-encoding immune sphaeroprotein κ chain signal peptide connected successively, the each base sequence connected successively described in the present embodiment is only one in gene (i.e. DNA fragmentation) double-strand of full genome synthesis, another chain and each base sequence complementary be connected successively.
(1b) with the base sequence double digestion that Xba I and Nhe I pair of pUC57 carrier and embodiment 1 step (1a) are synthesized, adopt agarose gel electrophoresis qualification enzyme to cut result, and reclaim respectively linear pUC57 carrier and enzyme cut after nucleotide fragments.
(1c) nucleotide fragments after adopting T4 DNA ligase enzyme to be cut by embodiment 1 step (1b) gained enzyme carries out connection with linear pUC57 carrier by the mol ratio of 3:1 and spends the night or be connected 2 hours under room temperature at 16 DEG C, then connection product is added in E.coli DH5 α bacterial strain competent cell suspension, transform, and the dull and stereotyped upper 37 DEG C of overnight incubation of LB be seeded in containing penbritin, picking list bacterium colony carries out double digestion (Xba I and the Nhe I) checking of recombinant plasmid vector; Choose enzyme and cut result positive colony, check order, the base sequence on all four carrier called after pUC57.BsAb(4309 bp that nucleotide fragments and embodiment 1 step (1a) are synthesized will be inserted), this pUC57. BsAb(4309 bp) plasmid map as shown in Figure 2.
(2) method of a kind of CD20 gene order clone is present embodiments provided
(2a) extraction of cell total rna: according to TRIZOL
tM(Invitrogen) test kit process specifications, from people Burkitts lymphoma cell line---extract total serum IgE Daudi cell (Chinese Academy of Sciences's cell bank), extract ultraviolet spectrophotometer is quantitative, agarose gel electrophoresis qualification RNA quality, residue RNA product saves backup in-80 ° of C.
(2b) cDNA first chain synthesis: utilize Reverse Transcription System (Fermentas) reverse transcription synthesis cDNA first chain in grads PCR instrument (Biometra), concrete operations are: get 1 μ g total serum IgE, Oligo (dT) 18 primer(0.2 μ g/ μ l) 1 μ l, be supplemented to cumulative volume 12 μ l with the water of nuclease free.Soft mixing, proceeds as follows after simply centrifugal 3 ~ 5 s in PCR instrument: 70 ° of C 5 min, 4 ° of C Pause.Take out PCR pipe, add AMV 5 × reaction buffer 4 μ l successively on ice, 10mM dNTP Mix 2 μ l, proceeds as follows after Ribonuclease inhibitor 1 μ l in PCR instrument again: 37 ° of C 5 min, 4 ° of C Pause.Take out PCR pipe, add M-Mulv Reverse Transcriptase 1 μ l on ice, PCR pipe is mixed gently and simply centrifugal after, with 42 ° of C incubation 60 min in PCR instrument, last 70 ° of C 10 min, 4 ° of C Pause.Reverse transcription product saves backup in-80 ° of C.
(2c) PCR reaction: get the above-mentioned cDNA product of 2 μ l and carry out pcr amplification as template.Reaction conditions is: after 95 DEG C of denaturation 3 min, and with 1. 95 DEG C of sex change 45 s 2. 60 DEG C of annealing 45 s 3. 72 DEG C, 1 min 30 s extends, and react 34 circulations, last takes turns extension 10 min.The primer sequence of PCR, the base sequence of forward primer is as SEQ ID NO:7, and the base sequence of reverse primer is as SEQ ID NO:8.And introduce BamH I restriction enzyme site at reverse primer tail end.The size of PCR primer is 922bp, and agarose gel electrophoresis is separated, cut glue recovery reacts for over-lap PCR.
(3) a kind of method that CD20 × CD3 gene that 3 ' end is contained P2A base sequence is connected with CD20 gene is present embodiments provided
(3a) PCR reaction: get the above-mentioned pUC57.BsAb plasmid of 2 μ l and carry out pcr amplification as template.Reaction conditions is: after 95 DEG C of denaturation 3 min, and with 1. 95 DEG C of sex change 45 s 2. 59.5 DEG C of annealing 45 s 3. 72 DEG C, 1 min 30 s extends, and react 34 circulations, last takes turns extension 10 min.The primer sequence of PCR, the base sequence of forward primer is as SEQ ID NO:9, and the base sequence of reverse primer is as SEQ ID NO:10.Wherein, Xba I restriction enzyme site is introduced before forward primer.The size of PCR primer is 1669bp, and agarose gel electrophoresis is separated, cut glue recovery reacts for over-lap PCR.
(3b) over-lap PCR reaction: with SEQ ID NO:8 and SEQ ID NO:9 for primer, cut CD20 gene that glue reclaims and the PCR primer of CD20 × CD3 gene is template, carry out PCR reaction, reaction conditions is: after 95 DEG C of denaturation 3 min, with 1. 95 DEG C of sex change 45 s 2. 59.5 DEG C of annealing 45 s 3. 72 DEG C, 1 min 30 s extends, and react 34 circulations, last takes turns extension 10 min.The size of PCR primer is 2551bp, called after CD20 × CD3.CD20 gene, and agarose gel electrophoresis is separated, cut the structure of glue recovery for subsequent recombination carrier.
(4) structure and the authentication method of CD20 × CD3.CD20 gene recombination Lentiviral of a kind of Lentiviral mediation is present embodiments provided
(4a) with Xba I and BamH I, CD20 × CD3.CD20 gene double digestion is reclaimed to pCDH carrier (System Bioscience) and embodiment 1 step (3b) glue, adopt agarose gel electrophoresis qualification enzyme to cut result, and reclaim CD20 × CD3.CD20 gene fragment that linear pCDH carrier and enzyme cut respectively.
(4b) CD20 × CD3.CD20 gene fragment after adopting T4 DNA ligase enzyme to be cut by embodiment 1 step (4a) gained enzyme is carried out connection with linear pCDH carrier in 3:1 ratio and is spent the night or be connected 2 hours under room temperature at 16 DEG C, then connection product is added in E.coli DH5 α bacterial strain competent cell suspension, transform, and the dull and stereotyped upper 37 DEG C of overnight incubation of LB be seeded in containing penbritin, picking list bacterium colony carries out double digestion (Xba I and the BamH I) checking of recombinant expression vector; Choose enzyme and cut result positive colony, check order, will the correct carrier called after pCDH-BsAb.TAg(9894bp of nucleotide fragments base sequence be inserted), the plasmid map of this pCDH-BsAb.TAg recombinant expression vector is as shown in Figure 4.The remaining bacterium liquid glycerine of 30% is preserved, and makes the final concentration of glycerine be 15%.
(4c) connect bacterium LB substratum with the glycerol stock liquid preserved, carry out a large amount of plasmid extraction (Qiagen), obtain the recombinant plasmid for virus packaging of q.s.
(5) a kind of production method of the slow sick particle containing CD20 × CD3.CD20 gene is present embodiments provided
(5a) cultivation of 293T cell before transfection: 24 h before transfection, with the 293T cell (purchased from Chinese Academy of Sciences's cell bank) of 0.25% tryptic digestion logarithmic phase, be 4 × 10 with the DMEM substratum adjustment cell density containing 10% foetal calf serum (Mediatech)
5/ ml, is then inoculated in the culture dish of 10 cm, about 10ml.2h before transfection, when cell reaches the density of 70 ~ 80%, removes substratum, changes the DMEM substratum of serum-free.
(5b) preparation of transfection composite: the recombinant plasmid pCDH-BsAb.TAg and the 10 μ g pPACKH1 that 2 μ g are contained CD20 × CD3.CD20 gene
tMpackaging plasmid Mix adds one containing in the aseptic 15 ml centrifuge tubes of 750 μ l opti-MEM substratum, fully mixes, incubated at room 5 min.Meanwhile, by the Lipofectamine 2000 of 60 μ l
tMtransfection reagent is diluted in another the 15 ml centrifuge tube containing 750 μ l opti-MEM substratum, mixes lightly, incubated at room 5 min.Then, by the Lipofectamine 2000 of dilution
tMreagent dropwise joins containing in plasmid opti-MEM mixed solution, mixing of turning upside down lightly, incubated at room 20 min.
(5c) transfection 293T cell: wash 293T cells once with 10 ml opti-MEM substratum before transfection, then adds 8.5 ml containing 2% serum not containing antibiotic DMEM substratum.Join in culture dish by the transfection composite of incubated at room 20 min, mixing gently makes transfection composite be evenly distributed in substratum, 37 DEG C, cultivate in 5% CO2 incubator.After 6 min, replace the substratum containing transfection composite with the fresh DMEM substratum containing microbiotic and 2% foetal calf serum.
(5d) collection of virion and concentrated: after transfection, 24 h and 48h collect vial supernatant (24h collects the fresh training liquid of rear substitution) respectively for twice, by centrifugal 5 min of supernatant liquor 3000 rpm room temperature containing virion, remove cell debris, then by the membrane filtration of the supernatant liquor after centrifugal with 0.45 μm, in 40 mL ultracentrifugation pipes, 4 DEG C, centrifugal 120 min of 72000g/min.Then use the resuspended virion of 500 μ l PBS, and be dispensed into the interior 20 μ l/pipes of EP pipe of 0.25ml ,-80 DEG C save backup for a long time.
Embodiment 2: present embodiments providing one can secrete again the construction process of the HepG2 cell (called after: HepG2-BsAb.TAg) of CD20 × CD3 by surface of cell membrane stably express CD20 simultaneously, comprises the steps:
(1) recombinant slow virus particle transfection HepG2
Cultivate HepG2 cell according to a conventional method, with the DMEM/F12 perfect medium containing 100 mL/L foetal calf serums, adjustment cell density is 5 × 10
5/ mL, transfection first 1 day tryptic digestion HepG2, is re-seeded in culturing bottle; Treat that Growth of Cells degree of converging reaches 70%-80%, the cultivation more renewed, add the recombinant slow virus particle containing 20 μ L.After infecting 4 h, replaced medium continues to cultivate, not contain the empty carrier of goal gene for contrast.
(2) stably expressing cell line is screened
Adding final concentration after infecting 48 h is that the tetracycline of 2 g/L carries out stably expressing cell line screening.Picking resistant cell colonies is in 96 well culture plates, 24 well culture plates, 6 well culture plates enlarged culturing step by step.Stable HepG2-BsAb.TAg is set up through about 14 days.
(3) flow cytomery HepG2-BsAb.TAg surface of cell membrane CD20 expresses
(3a) the film surface-stable of cultivation expressed CD20 and secrete the HepG2-BsAb.TAg collected by centrifugation of CD20 × CD3, supernatant liquor saves backup; Use PBS buffer solution cell 2 times simultaneously, finally adjust cell density 1 × 10
6/ ml, gets 100 μ l; Then add mouse-anti h CD20 antibody that fluorescein isothiocyanate (FITC) marks and hatch 30 min on ice.The HepG2 cell simultaneously arranging unmodified does negative control.
(3b) Incubating Solution of step (3a) is carried out centrifugal after, the cell bovine serum albumin (BSA) of 1% and the PBS(dye solution of 0.02% sodium azide) after dye solution washs 2 times, use 200 μ l dye solution re-suspended cells again, finally carry out detecting and analyzing with flow cytometer, result such as Fig. 5 shows, the HepG2 cell that the present invention modifies can specific adsorption anti-CD20 antibodies, illustrates that surface of cell membrane can stably express CD20 antigen.
(4) can flow cytomery HepG2-BsAb.TAg secrete CD20 × CD3
(4a) by the supernatant liquor that embodiment 2 step (3a) is collected, 10000 rpm, after centrifugal 5min, then get 50 μ l respectively with Jurkat cell and Raji cytomixis in dye solution, final volume 100 μ l, cell density is 1 × 10
6/ ml, to hatching 30 min on ice.
(4b) Incubating Solution of embodiment 2 step (4a) is carried out centrifugal after, gained cell PBS buffer solution 2 times, then adds the anti-His Tag antibody that fluorescein isothiocyanate (FITC) marks and hatches 30min on ice.
(4c) Incubating Solution of embodiment 2 step (4b) is carried out centrifugal after, after gained cell dye solution washs 2 times, use 200 μ l dye solution re-suspended cells again, then carry out detecting and analyzing with flow cytometer, result such as Fig. 6 shows, and recombinant antibodies of the present invention can be combined with the Raji cell of the CD20 positive and the Jurkat cell of the CD3 positive respectively.Therefore, the present invention can secrete CD20 × CD3 through the HepG2-BsAb.TAg modified.
Embodiment 3: the preparation method present embodiments providing the TSPT cell that a kind of HepG2-BsAb.TAg mediates, comprises the steps:
(1) gather detection in peripheral blood of patients underwent, obtain mononuclearcell through ficoll-general shadow glycosamine density gradient centrifugation.Concrete steps are: with the hemocyte of physiological saline two-fold dilution precipitation, human lymphocyte parting liquid and dilute blood add in centrifuge tube in the ratio of 1:2,2000 rpm, centrifugal 20 min, careful draw tunica albuginea layer, with brine 2 times, rotating speed is respectively 1600 rpm, 1300 rpm, all centrifugal 7 min, namely obtain PBMC.
(2) above-mentioned PBMC is resuspended in the commercialization serum free mediums such as PAA or GT-T551, adjustment cell density 1 × 10
6about/ml, mixes with equal-volume, isopycnic HepG2-BsAb.TAg, adds rhIL-2 after mixing, and final concentration is 300 IU/ml, in 37 DEG C, cultivates in the incubator of 5% CO2; After 48 h, cell is counted; Then adjusting cell density with the fresh culture of the rhIL-2 containing 300 IU/ml is 1 × 10
6/ ml; Every 2 ~ 3 days later, to cell count rear use contain the rhIL-2 of 300 IU/ml fresh culture adjustment cell density be 1 × 10
6after/ml, in 37 DEG C, cultivate in the incubator of 5% CO2, cultivate after 9 ~ 20 days, collected cell is TSPT cell.Do positive control with effector T cell prepared by classical cultural method simultaneously.
Embodiment 4: the detection method present embodiments providing TSPT morphocytology and multiplication capacity thereof, comprises the steps:
(1) cell cultures 24 h and visible cell are sunken to the bottom of culturing bottle, and in comparatively intensive colony state, after cultivating 48 h, colony becomes large.Auspicious Ji's Albert'stain Albert is carried out to the cultivation cell of 12 ~ 14 days, finds that most cells volume increases, ovalize or irregular shape, nucleus is mostly oval or circular, and nuclear staining is loosened, and nuclear membrane is irregular, have projection, visible 1 the little kernel of each cell, in mazarine; Kytoplasm enriches, and dye dusty blue, form is irregular, has pseudopodium, and there is light dye phenomenon at nearly core place, also has in irregular shape.Get the cultivation cell of 12 ~ 14 days 100 μ l, add 100 μ l 0.4% Trypan Blue liquid, viable cell does not dye, and dead cell dyes blueness, counted under microscope.Cell viability prepared by the present invention is greater than 97%.
(2) counted with cell counting count board and cell counter respectively at the 0th, 3,6,9,12,15,18,21 day, simultaneously by the method for following embodiment 5, AntiCD3 McAb-FITC and CD20-APC is selected to dye to cell, then flow cytomery is carried out, the ratio of the ultimate analysis CD3 positive and CD20 positive cell, and calculate proportion in conjunction with cell counting, as Fig. 7, shown in table 1 and absolute number as shown in Fig. 8, table 2.In the TSPT cell that result display is cultivated, can't detect the existence of genetic modification tumour cell after 9 days, illustrated that TSPT cell prepared by the present invention is safe; The present invention simultaneously is also the method for efficient amplification T cell.
In table 1:PBMC inducing culture process, CD3 positive cell and CD20 positive colonies are in the ratio of total cell
The absolute number of CD3 positive cell and CD20 positive colonies in table 2:PBMC inducing culture process
Embodiment 5: present embodiments provide a kind of TSPT Immunophenotyping detection method, comprise the steps:
Get the cell of the 14th day, wash 2 times with dye solution, adjustment cell density is 1 × 10
6/ ml, 100 μ l cell suspensions are added respectively in each detector tube, experiment tube is divided into 2 pipes, wherein a pipe adds fluorescent-labeled antibody is AntiCD3 McAb-FITC, CD4-PE, CD8-PECY5.5 and CD20-APC, another pipe adds AntiCD3 McAb-FITC, CD4-PE, CD25-PECY5.5 and CD56-APC dyes, 4 DEG C of lucifuges hatch 30 min, after washing 2 times with dye solution, use 200 μ l dye solution re-suspended cells again, finally carry out detecting and analyzing with flow cytometer, result is as Fig. 9, shown in table 3, illustrate that TSPT cell prepared by the present invention is the heterogeneous population body that is main cell with the positive CTL of CD8.
Table 3: the TSPT Immunophenotyping of the 14th day
Embodiment 6: present embodiments provide a kind of TSPT cells against tumor cells kill capability detection method, comprise the steps:
(1) get the cultivation TSPT cell of the 14th day, detect the killing activity to HepG2, K562 tumour cell.
(2) cell density adjusting HepG2, K562 is 1 × 10
5/ ml, every hole 100 μ l, 96 orifice plates, in ratio and the TSPT cytomixis of 1:10, arrange 6 multiple holes, arrange independent target cell (tumour cell) contrast, separately TSPT cell (effector cell) contrast and single culture base simultaneously and do blank.
(3) be placed in 37 DEG C, cultivate after 24 hours in the incubator of 5% CO2 and saturated humidity, every hole adds CCK-8 solution of 10 μ l, then cultivates 4h, then measures absorbancy (OD) value by microplate reader in 450nm wavelength place.
(4) according to formula: kill ratio of outflow (%)=［ 1-(experimental group OD value-effector cell organizes OD value) ］/(effector cell organizes OD value) × 100%, calculate and kill knurl efficiency.Wherein, in formula, each OD value is the value after deducting blank group OD value, and result is as shown in Figure 10, table 4, and TSPT cell has efficiently, the ability of specific killing tumour.
Table 4: the killing-efficiency of the TSPT cells against tumor cells of the 14th day
Reference:
Golay J, D'Amico A, Borleri G, et al. A Novel Method Using Blinatumomab for Efficient, Clinical-Grade Expansion of Polyclonal T Cells for Adoptive Immunotherapy. J Immunol, 2014, 193(9): 4739-4747.
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Claims (10)
1. tumor cell specific polyclone T cell preparation method, is characterized in that, adopts following preparation process: (1) obtains peripheral blood mononuclear cell; (2) peripheral blood mononuclear cell that step (1) obtains is mixed with genetic modification tumour cell; (3) cell mixing that step (2) obtains is resuspended in containing can maintain Growth of Cells, propagation and differentiation cytokine substratum in.
2. tumor cell specific polyclone T cell preparation method according to claim 1, it is characterized in that, described is collection peripheric venous blood from peripheral blood acquisition mononuclearcell, is then separated acquisition peripheral blood mononuclear cell with ficoll-general shadow glycosamine density gradient centrifugation.
3. tumor cell specific polyclone T cell preparation method according to claim 1, is characterized in that, described genetic modification tumour cell is genetic engineering antibody gene association target antigen Gene Double rebuilding decorations tumour cells.
4. tumor cell specific polyclone T cell preparation method according to claim 3, it is characterized in that, described genetic engineering antibody gene has the base sequence of encoding gene engineered antibody, described genetic engineering antibody is two targeting specific antibody, the two targeting specific antibody of secretor type that described pair of targeting specific antibody has the binding site of targeting T-cells epitope and the binding site of TCSA epi-position.
5. tumor cell specific polyclone T cell preparation method according to claim 3, it is characterized in that, described target antigen gene is the base sequence with coding film mating type target antigen, the one in the tumour antigens such as the tissue-specific differentiation antigen of described film mating type target antigen choosing, the tumour antigen of oncovirus expression, mutator gene or the expression product of oncogene, the cell protein of unconventionality expression and embryonal antigen.
6. tumor cell specific polyclone T cell preparation method according to claim 1, is characterized in that, the cytokine of described maintenance Growth of Cells, propagation and differentiation is rhIL-2.
7. the tumor cell specific polyclone T cell of the specific killing tumour cell prepared by claim 1.
8. the application of tumor cell specific polyclone T cell in the medicine for the preparation of immunotherapy or vaccine inoculation of specific killing tumour cell according to claim 7, the tumor cell specific polyclone T cell of described specific killing tumour cell is obtained by the method comprised the following steps: (1) obtains peripheral blood mononuclear cell; (2) peripheral blood mononuclear cell that step (1) obtains is mixed with genetic modification tumour cell; (3) cell mixing that step (2) obtains is resuspended in containing can maintain Growth of Cells, propagation and differentiation cytokine substratum in.
9. the application of tumor cell specific polyclone T cell in the medicine for the preparation of immunotherapy or vaccine inoculation of specific killing tumour cell according to claim 8, the vaccine of the tumor cell specific polyclone T cell of described specific killing tumour cell is obtained by the method comprised the following steps: (1) obtains peripheral blood mononuclear cell; (2) peripheral blood mononuclear cell that step (1) obtains is mixed with genetic modification tumour cell; (3) cell mixing that step (2) obtains is resuspended in containing can maintain Growth of Cells, propagation and differentiation cytokine substratum in.
10. the application of tumor cell specific polyclone T cell in the medicine for the preparation of immunotherapy or vaccine inoculation of specific killing tumour cell described in claim 8 or 9, the application in the medicine of described immunotherapy or vaccine inoculation refer to for prevent or Therapeutic cancer medicine in application.
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