CN1609196A - High-yield T lymphocyte cloning technology for finding tumor specific antigen and tumor specific antigen determinant - Google Patents
High-yield T lymphocyte cloning technology for finding tumor specific antigen and tumor specific antigen determinant Download PDFInfo
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Abstract
The present invention discloses the method of finding antigen determinant with tumor specificity, shown by HLA acceptor and identified by CD4 positive lymphocyte or CD8 positive lymph cell from the tumor sample and tumor penetrating lymphocyte gathered from patient body. The present invention discloses also the method of screening the latent curative effect of treating vaccine or immunogen in cell immunizing therapy on patient of specific race. The present invention discloses also the method of extracorporeal amplifying to generate T-lymphocyte with specific reaction on antigen and antigen determinant for cell transferring therapy or developing treating vaccine to activate cell immunity system.
Description
Technical field
The present invention relates to the tumor immunology field, further, the present invention relates to a kind of high yield T lymphocyte clone technology that is used to find tumour specific antigen and tumour specific antigen determinant.
Background technology
Many studies show that, can utilize the operation human immune system to reach the purpose of control and treatment cancer, such as the mucus immunity reaction that utilizes body, can be at various growth factor receptorses and cell surface marker molecule exploitation monoclonal antibody, to reach the effect of treatment cancer; On the other hand, utilize the immune cell immune response of human activin, the kill tumor cell reaches the treatment method for cancer and more and more obtains paying attention to specifically.
Human immune system's cell immune response mainly is to realize by T lymphocyte (Tlymphocytes).The T lymphocyte can be divided into CD8 male cytotoxic T lymphocyte (CD8+cytotoxic T lymphocyte, CTL or Tc) and CD4 male helper T cell (CD4+T-helper lymphocytes).CD8 male cytotoxic T lymphocyte (CD8+cytotoxic T lymphocyte, CTL or Tc) and CD4 male helper T cell are all discerned human leukocyte antigens (Human Leukocyte Antigen, HLA; Human main consistency complex body Major Histocompatibility Complex (MHC)) polypeptide antigen of surperficial ditch.These antigens are positioned at cell surface.CD8 male T lymphocyte can be discerned the polypeptide portion of the antigenic 8-10 of a MHC I class HLA amino acid long, and these polypeptide are that cytoplasm protein produces by degrading, the MHC I class HLA antigen of respectfully presenting at cell surface by endoplasmic reticulum.And the polypeptide antigen of CD4 male helper T cell identification is through different cell passages: extracellular protein enters cell through the cell endocytosis, be degraded to short polypeptide antigen at intracellular endocytosis body (endosome), the MHC II class HLA antigen of respectfully presenting at cell surface, the complex body that CD4 male helper T cell identification MHC II class HLA antigen and short polypeptide antigen form.T lymphocyte identification antigen is by discerning polypeptide antigen simultaneously and special HLA antigen forms.
The method that is used to evoke cell immune response comprises passive (post and comfort) transfer method, utilizes and shifts immunocyte with tumour resistance and the active immunization that uses vaccine, and utilizing stimulates patient's immunity system to produce the immunocyte with tumour resistance in a large number.
Post and comfort the clinical study that transfer method starts from the eighties, these studies show that the lymphocyte that penetrates solid tumor (being called for short the tumour penetration cell) can obtain and carry out amplification in vitro from the single-cell suspension liquid of the tumor sample of excision, then the cell of these amplification in vitros is added that the IL-2 of high dosage refills in the melanoma patient body, can obtain certain clinical therapeutic efficacy.But early stage research only uses CD8 male tumour penetration cell thereby curative effect limited.The CD8 positive tumor penetration cell and the CD4 positive tumor penetration cell of amplification in vitro used in a research of finishing recently simultaneously.Two kinds of cells of mixing use are obtained the significant curative effect to the melanoma patient, and the potential use of the tumour antigen that the tumour penetration cell is discerned is obviously pointed out in this class research.
The active immunity rule requires to produce in vivo the lymphocyte that in a large number tumour is had high resistance.These cells must not limited by the tolerant mechanism of general tumour, and solid tumor is had the long-term immune resistance that continues.The tumor vaccine that current active immunization mainly relies on two classes researching and developing is based on the vaccine of cell or based on the vaccine of tumour antigen.
Use homology or allogenic intact tumor cells or tumour cell extract to come immunity at first based on the vaccine of cell, but generally on intact cell tumour antigen very a small amount of, seriously limited the effect of this method.Diverse ways is attempted in the immunity that strengthens whole tumour cell, vaccine based on cell comprises intact tumor cells (homology or allos) now, the tumour cell that gene was revised (allow hormone or co-activation molecular gene between its express cell), the syzygy of tumour cell extract (lysate, cytolemma and heat shock protein(HSP)) and tumour cell and antigen performance cell.
The discovery of human tumor antigen makes development become possibility based on the vaccine of tumour antigen.This class antigen can use or suddenly change with enhancing immunity in a large number, thereby cause tumour resistance stronger and that more can regulate and control, the tumour antigen (natural or artificial recombination) that comprises purification based on the vaccine of tumour antigen synthesizes polypeptide, " exposing " DNA, the virus of artificial recombination and bacterium.
Be used to clone the existing quite development of technology of the tumour antigen of respectfully presenting on MHC/HCA I receptoroid (such antigenic antigenic determinant is that CD8 male T lymphocyte is discerned).Many these class antigens are by with tumour cell cDNA expression library, transform the target cell of expressing suitable HLA molecule, then with there being the T lymphocyte of tumour resistance to find out suitable transformant.In addition, from the polypeptide of the tumor cell surface wash-out polypeptide of wash-out (or on the HLA molecule that is purified into from tumour cell) can be pulsed onto on the antigen performance cell and test to the lymphocytic reactivity of specificity anti-tumor is arranged, can find its tumor antigen gene to these peptide purifications and order-checking at last.The third technology often is called as " converse immunization ", and this technology successfully has been used for confirming at tumour cell excess or unique expressed proteins tumour antigen whether.In addition, external sensitization technology also is used to produce the T lymphocyte that particular candidate antigen is had resistance, discerns complete tumour cell specifically if these T lymphocytes can have specially, and this candidate albumen then can be thought a tumour antigen.The 4th kind of technology is commonly referred to as SEREX (the blood sample analysis technology of recombinant cDNA expression library).This technology is based on a specific protein is produced on the prerequisite of the essential helper T lymphocyte of antibody.Be used for after tumour patient serum on one's body is diluted detecting in the tumour cDNA library of prokaryotic organism (as bacterium etc.) expression, this SEREX method generally be used to find can antibody recognition gene.
Under comparing, the technology of cloning the tumour antigen of being respectfully presented for MHC/HLA II receptoroid (being generally the identification of CD4 positive t lymphocytes) then develops limited because of technical difficulty.It is not to be exactly the loading and unloading department that the cell intrinsic protein is transported to special MHC two receptoroids from extracellular environment that technical difficulty comes from this class tumour antigen, and processed then back is transported to cell surface with MHC two receptoroids on bonding again.A nearest technology based on biochemical protein purification and mass spectrum order-checking is developed out.This technology successfully is used to find penetrated by homology CD4 positive tumor the unique tumour antigen on 1558 K-1735s of being expressed in of lymphocyte identification.This method may other highly expresses to cloning, and can be respectfully presented cell effectively from the external environment picked-up and to be the proteantigen of dedicating the CD4 positive t lymphocytes to also useful by antigen.The library that the method for another latest developments then is to use a lI-cDNA translation to merge.This method is based on the heredity decision, uses 293 clones.This clone is expressed the important component part gene of invariant chain (lI) DMA, DMB and processing of other MHC two receptoroids and performance after by genetic modification.This method also might be for being widely used in cloning MHC II class antigen.
Existing method difficulty has technically seriously limited its application in the treatment of Chinese population tumour is controlled.The technology of existing tumour penetration cell and be applicable to melanoma based on the technology of tumour penetration cell, and melanoma is not general in Chinese population.Other cancer such as lung cancer, nasopharyngeal carcinoma and liver cancer are bigger threats to the health of Chinese population, yet existing tumour through-transmission technique but can't be used for these cancers, and is main because following two reasons:
1, now uses in the method in the overwhelming majority, in case behind the tumor resection, the tumour penetration cell generally obtains by the limited dilution cloning method.This method is by CD 3-resisting monoclonal antibody (OKT-3), feeding cell (FEEDER CELL, the for example illuminated allos ring week blood mononuclear cell that kills) existence stimulates the T lymphocyte growth down, this method successfully is used to the to increase a large amount of T of collective lymphocyte and from melanomatosis person clone T lymphocyte.But often cause it to lose to the melanoma specific immune of tumour in addition by the be situated between T lymphocyte amplification connect and clone technology of OKT-3.Same problem also is present in the T lymphocyte that T lymphocyte that " converse immunization " obtain and external polypeptide stimulate gained.A kind of hypothesis of current trend is thought, in vivo tumour there is the compound framework of t lymphocyte receptor on the precursor T lymphocyte of special resistance and CD3 by the tumour cell repetitive stimulation, and these tumour cells itself and non-professional antigen show cell and lack the co-activation factor, for example CD80 and CD86.Therefore, the TCR/CD3 signal transduction path is by with exhausting in vivo, and existing limited dilution cloning rule is to rely on to repeat OKT-3 and stimulate with the TCR/CD3 signal transduction path that exhausts.In addition, generally the converse immunization of Shi Yonging leans on all blood mononuclear cell co-cultivation of ring of external and polypeptide pulse to obtain the T lymphocyte, also be by TCR/CD3 approach conducted signal, as a result, amplification of the T lymphocyte of existing dependence OKT-3 and clone technology cause tumour is had the lymphocytic positive guide death of T of special resistance probably.
2, since the technical restriction in the limiting dilution assay now still do not have effective high yield (high-throughput) method up to now and find tumour specific antigen on a large scale.These class methods are particularly useful with the vaccine as future for seeking effective antigens.
Summary of the invention
Technical problem to be solved by this invention be to provide a kind of use the tumor sample collected on one's body from patient and tumour penetrate lymphocyte seek tumour-specific, respectfully presented by the HLA receptoroid and be the method for the antigenic determinant of CD4 positive lymphocyte or the identification of CD8 positive lymphocyte, this method may further comprise the steps:
A. obtain tumor sample on one's body and make single-cell suspension liquid from patient;
B. extract CD4 positive lymphocyte or CD8 positive lymphocyte from single-cell suspension liquid, and prepare single clone and these clones that increase with limiting dilution;
C. the T lymphocyte and the tumour cell co-cultivation of amplification, and inspection analyses special iuntercellular hormone in the supernatant liquor, thereby finds out the T lymphocyte clone of tumor response;
D. from patient's tumour is cut the part of sample, extract mRNA, construction cDNA library, and be used to transform the target cell;
E. tumor response T lymphocyte and the sub-co-cultivation of target cell transformation are also examined the special iuntercellular hormone of analysing in the supernatant liquor, finally find out the tumour specific antigen gene;
F. find out the tumour specific antigen determinant from the tumour specific antigen gene.
Further, in embodiments of the present invention, the above-mentioned clone that is used to set up the target cell is 293 clones or bhk cell system or Cos clone.
In the present invention, the HLA acceptor of expressing on the above-mentioned target cell is specific including, but not limited to Chinese population, for example: HLA-A24 or HLA-A2 or HLA-DP5 or HLA-DR4.
In the present invention, the lymphocytic nutrient solution of T that is used to increase comprises the antibody of interleukine-2, PHA-L and anti-CD28.
In the present invention, tumor response T lymphocyte is to find by GM-CSF or IFN-g that inspection is analysed in supernatant liquor.
Technical problem to be solved by this invention also is to provide a kind of and is used in treatment vaccine that the cellular immunization therapy uses or immunogen and penetrates lymphocyte from tumor sample and tumour that patient collects on one's body, screen the method for cellular immunization therapy, may further comprise the steps the patient's of particular race potential curative effect:
A. obtain tumor sample on one's body and make single-cell suspension liquid from patient;
B. extract CD4 positive lymphocyte or CD8 positive lymphocyte from single-cell suspension liquid, and prepare single clone and these clones that increase with limiting dilution;
C. the T lymphocyte and the tumour cell co-cultivation of amplification, and inspection analyses special iuntercellular hormone in the supernatant liquor, thereby finds out the T lymphocyte clone of tumor response;
D. the T lymphocyte of amplification and the treatment vaccine that in the cellular immunization therapy, uses or immunogen co-cultivation to find out to treatment vaccine or the original atopic T lymphocyte of immunity;
E. get data by contrast from step c and steps d, can make treatment vaccine or the lymphocytic relative frequency of the original atopic T of immunity.
Further, in embodiments of the present invention, the above-mentioned clone that is used to set up the target cell is 293 clones, or bhk cell system, or Cos clone.
In the present invention, the HLA acceptor of expressing on the above-mentioned target cell is specific including, but not limited to Chinese population, as HLA-A24 or HLA-A or HLA-DP5 or HLA-DR4.
In the present invention, the above-mentioned lymphocytic nutrient solution of T that is used to increase comprises the antibody of interleukine-2, PHA-L and anti-CD28.
In the present invention, above-mentioned tumor response T lymphocyte is to find by the content that GM-CSF in the supernatant liquor or IFN-g are analysed in inspection.
Technical problem to be solved by this invention also is to provide a kind of and creates antagonism former or antigenic determinant has atopic T lymphocyte with amplification in vitro, comprise CD4 positive lymphocyte and CD8 positive lymphocyte, carrying out cell posts and comforts transfer therapy, or the development therapeutic vaccine may further comprise the steps with the immune method of activating cells:
A. one kind is used for cultivating the lymphocytic high yield pattern of T, particularly with 96 holes, 192 holes, 384 holes or other culture plates to stimulate the lymphocytic growth of T; Or
B. one kind is used for examining the high yield pattern of analysing the T lymphocyte activity, particularly analyses iuntercellular hormone or cell life and death situation with 96 holes, 192 holes, 384 holes or the inspection of other culture plates.
The present invention is the T lymphocyte technological system that is used to find to clone MHC I type and MHC II type tumour specific antigen and tumour specific antigen determinant of a high yield, and this invention comprises melanoma applicable to kinds of tumors.This invention is used to find new tumour specific antigen and tumour specific antigen determinant, so that utilize the immunization method of tumour penetration cell to treat tumour except that melanoma and kidney.
The lymphocytic positive guide death of T that the T lymphocyte amplification of existing dependence OKT-3 and clone technology cause tumour is had special resistance probably, in order to address this problem, clone T lymphocyte from the plain knurl patient's tumor of non-black cell, the inventor succeeds in developing the lymphocytic growth hormone of a kind of new stimulation, this growth hormone does not rely on the TCR/CD3 signal transduction path, and can substitute OKT-3 and be used for limiting dilution assay and the lymphocytic rapid amplifying of gained T.This growth hormone mixed solution is by non-TCR/CD3 approach conduction T lymphocyte growth signal, and therefore, what it can not cause obtaining from the tumour penetration cell has the lymphocytic positive guide death of T of special resistance to tumour.When this mixed solution directly contrasts when being used to clone tumour-specific CD4 positive t lymphocytes and CD8 positive t lymphocytes with OKT-3, this mixed solution has shown higher cloning efficiency and has obtained more tumour-specific T lymphocyte that this type of effect is quite consistent in repeated experiments.
Based on this new T lymphocyte growth element, the inventor has invented a kind of high yield T lymphocyte clone system, and the tumour that is used for cloning effectively tumour-specific penetrates lymphocyte, and finds out the positive and CD8 positive t lymphocytes specific antigens of CD4.This system can be used for high productivity and finds tumour specific antigen and antigenic determinant, and uses tumor sample and the hemocyte sample that obtains from patient on one's body, rather than essential at the external cell that is of building in the existing method of the overwhelming majority.This method has overcome the technical restriction in the limiting dilution assay of prior art.
Description of drawings
Fig. 1 has shown the method flow diagram of finding out tumour specific antigen and tumour specific antigen determinant.
Embodiment
Embodiment 1
Shown in 1, an exemplary of HITC technology can be described below as shown:
One. find out and the T lymphocyte of the tumor response that increases
1, tumor resection sample: obtain tumour on one's body from patient and cut sample.
2, single-cell suspension liquid: tumour is cut the part of sample and is handled via plurality of enzymes or handled by automation strength, makes it to become single-cell suspension liquid.
3, cellular segregation: with the positive and CD8 positive cell of magnetic bead separation of C D4 that antibody is covered, remaining cell (mainly being tumour cell) is then pressed attached cell and is cultivated.
4, carry out limiting dilution with STIMULEUKIN (new growth hormone mixed solution): based on needs to the T lymphocyte clone, isolated CD4 is positive and the CD8 positive t lymphocytes is diluted, put then on 96 orifice plates of illuminated culturing cell covering of killing, can only there be maximum cells in each hole, annotates STIMULEUKIN with these single cell clones that increase in each hole.
5, find out the T lymphocyte clone of tumor response: the tumour cell that gets from the 3rd step is changed in the 96 new orifice plates, and the T lymphocyte that gets from the 4th step is then added these by commentaries on classics then has in the hole of tumour cell.Because there is the reactive T lymphocyte of tomour specific can be, analyze the T lymphocyte clone that iuntercellular hormone in each hole can find to have tumor response at last by the special iuntercellular hormone of justacrine that tumour cell activates (GM-CSF or IFN-g).Use existing reagent system,, analyze the iuntercellular hormone in the supernatant liquor in each hole as granulocyte-macrophage colony stimulating factor (GM-CSF) ELISA kit.
6, amplification has the T lymphocyte of tumor response: this class cell is placed into and continues amplification in the big incubator.
Two. find out tumour specific antigen
7, set up the cDNA library: cut from patient's tumour and extract mRNA the part of sample and be used for the construction cDNA library, the selection of expression vector can determine that this library specifically is to be used to look for MHC I class or mhc class ii receptor-specific antigen.
8, transform: the cDNA library generally is an incubation growth in bacterium, and this library then is amplified and is divided into subgroup, and each subgroup comprises the first source cDNA of some amount.The cDNA that spreads cultivation from these subgroups is used to transform and expresses correct HLA kind and be grown in 293 cells on 96 orifice plates in advance.Like this, the transformant in each hole can have the cDNA kind that is no more than number in the initial subgroup.
9, add tumor response T lymphocyte: going on foot the tumor response sexual cell that obtains and increase from the 6th is had 96 orifice plates of transformant by a minute adding.
10, the release of hormone and find out the lymphocytic hole of the T that is activated between analysis of cells: because there is the T lymphocyte of tumor response can be activated and discharge special iuntercellular hormone by 293 cells of the correct antigenic determinant of performance, by analysis between the specific cell in porose hormone can find out by correct 293 cells that tumor antigen gene transformed.
11, subclone and repeat the T lymphocyte and detect: 293 cells of finding out in the 10th step are actually a group cell, are transformed and get by the first source cDNA of a subgroup, and this subgroup is branch groupuscule and be used to transform 293 same cells again, repeats the 9th and 10 and goes on foot.
12, clone cDNA: repeated for the 11st step till obtaining to activate the lymphocytic single clone of T of tumor response, from this cell, clone cDNA, Here it is tumour specific antigen.
13, find out antigenic determinant: use with batch tumor response T lymphocyte, a plurality of methods of Cai Yonging all can be used for finding out special antigenic determinant in the prior art.
In this application, some technology well known to those skilled in the art are not described in detail.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read foregoing description content of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (11)
1. a use penetrates lymphocyte from tumor sample and the tumour that patient collects on one's body, that searching has is tumour-specific, that respectfully presented by the HLA receptoroid, be the method for the antigenic determinant of CD4 positive lymphocyte or the identification of CD8 positive lymphocyte, and this method comprises:
A. obtain tumor sample on one's body and make single-cell suspension liquid from patient;
B. extract CD4 positive lymphocyte or CD8 positive lymphocyte from single-cell suspension liquid, prepare single clone with limiting dilution, these clones increase;
C. the T lymphocyte and the tumour cell co-cultivation of amplification, the special iuntercellular hormone in the supernatant liquor is analysed in inspection, finds out the T lymphocyte clone of tumor response;
D. extract mRNA from patient's tumour is cut the part of sample, the construction cDNA library is used to transform the target cell;
E. with tumor response T lymphocyte and the sub-co-cultivation of target cell transformation, the special iuntercellular hormone in the supernatant liquor is analysed in inspection, finds out the tumour specific antigen gene;
F. find out the tumour specific antigen determinant from the tumour specific antigen gene.
2. method according to claim 1, the clone that wherein is used to set up the target cell are 293 clones or bhk cell system or Cos clone.
3. method according to claim 1, wherein the HLA acceptor of expressing on the target cell is specific including, but not limited to Chinese population, is: HLA-A24 or HLA-A2 or HLA-DP5 or HLA-DR4.
4. method according to claim 1, the lymphocytic nutrient solution of T that wherein is used to increase comprises interleukine-2, the antibody of PHA-L and anti-CD28.
5. method according to claim 1, wherein tumor response T lymphocyte is to determine by the content that GM-CSF in the supernatant liquor or IFN-g are analysed in inspection.
6. one kind is used in treatment vaccine that the cellular immunization therapy uses or immunogen and penetrates lymphocyte from tumor sample and tumour that patient collects on one's body, and screening cellular immunization therapy is to the method for the patient's of particular race potential curative effect, and this method comprises:
A. obtain tumor sample on one's body from patient, make single-cell suspension liquid;
B. extract CD4 positive lymphocyte or CD8 positive lymphocyte from single-cell suspension liquid, prepare single clone with limiting dilution, these clones increase;
C. the T lymphocyte and the tumour cell co-cultivation of amplification, the special iuntercellular hormone in the supernatant liquor is analysed in inspection, finds out the T lymphocyte clone of tumor response;
D. T lymphocyte and the treatment vaccine that in the cellular immunization therapy, uses or the immunogen co-cultivation of amplification, find out treatment vaccine or the original atopic T lymphocyte of immunity;
E. by contrasting, make treatment vaccine or the lymphocytic relative frequency of the original atopic T of immunity from step c and steps d gained data.
7. method according to claim 6, the clone that wherein is used to set up the target cell is 293 clones, or bhk cell system or Cos clone.
8. method according to claim 6, wherein the HLA acceptor of expressing on the target cell is specific including, but not limited to Chinese population, is HLA-A24 or HLA-A or HLA-DP5 or HLA-DR4.
9. method according to claim 6, the lymphocytic nutrient solution of T that wherein is used to increase comprises the antibody of interleukine-2, PHA-L and anti-CD28.
10. method according to claim 6, wherein tumor response T lymphocyte is to determine by the content that GM-CSF in the supernatant liquor or IFN-g are analysed in inspection.
11. one kind creates antagonism former with amplification in vitro or antigenic determinant has atopic T lymphocyte, carry out cell and post and comfort transfer therapy, or the development therapeutic vaccine is with the immune method of activating cells, this method comprises:
A. one kind is used for cultivating the lymphocytic high yield pattern of T, selects 96 holes, 192 holes, 384 holes or porous culture plate more for use, stimulates the lymphocytic growth of T; Or
B. one kind is used for examining the high yield pattern of analysing the T lymphocyte activity, selects 96 holes, 192 holes, 384 holes or porous culture plate more for use, and iuntercellular hormone or cell life and death situation are analysed in inspection.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628621A (en) * | 2019-10-28 | 2019-12-31 | 合肥中科干细胞再生医学有限公司 | Equipment and method for obtaining tumor specific T cells |
| CN111733131A (en) * | 2013-07-15 | 2020-10-02 | 美国卫生和人力服务部 | Method for preparing T cells against human papilloma virus antigens |
| CN113214383A (en) * | 2020-05-23 | 2021-08-06 | 湖南源品细胞生物科技有限公司 | TCR enrichment clone type and acquisition method and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111733131A (en) * | 2013-07-15 | 2020-10-02 | 美国卫生和人力服务部 | Method for preparing T cells against human papilloma virus antigens |
| US11918640B2 (en) | 2013-07-15 | 2024-03-05 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Methods of preparing anti-human papillomavirus antigen T cells |
| CN110628621A (en) * | 2019-10-28 | 2019-12-31 | 合肥中科干细胞再生医学有限公司 | Equipment and method for obtaining tumor specific T cells |
| CN110628621B (en) * | 2019-10-28 | 2023-12-22 | 合肥中科干细胞再生医学有限公司 | Device and method for obtaining tumor-specific T cells |
| CN113214383A (en) * | 2020-05-23 | 2021-08-06 | 湖南源品细胞生物科技有限公司 | TCR enrichment clone type and acquisition method and application thereof |
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