CN102719400A - Preparation method of HLA-A0201 restrictive anti-carcinoembryonic antigen (CEA) antigenic specificity cytotoxic T lymphocyte (CTL) - Google Patents
Preparation method of HLA-A0201 restrictive anti-carcinoembryonic antigen (CEA) antigenic specificity cytotoxic T lymphocyte (CTL) Download PDFInfo
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Abstract
The invention discloses a preparation method of an HLA-A0201 restrictive anti-CEA antigenic specificity CTL. The preparation method includes collecting peripheral blood mononuclear cells (PBMCs) through hemapheresis, and enriching and purifying CD8+T lymphocytes; stimulating the CD8+T lymphocytes by utilizing mature dendritic cells loaded with the HLA-A0201 restrictive CEA antigen polypeptides and promoting T cells to grow by using rhIL-2 and rhIL-7; purifying a target CTL by utilizing a tetramer marking method and a fluorescene-activated cell sorting; stimulating the target CTL to grow with an anti-human CD3 monoclonal antibody coated by solid phase and IL-2; adding autologous PBMCs after radiation of gamma rays to strengthen activation of the target CTL; and adding rhIL-15 for breeding and amplification, collecting and identifying. The prepared target CTL has high purity, multiplication capacity, killing activity and proportion of CTL-CM, and can be applied to immunotherapy such as tumors.
Description
Technical field
The invention belongs to biotechnology exploitation and applied research field, relate to the restricted anti-CEA antigen-specific CTL preparation method of HLA-A0201.
Background technology
One, the predicament of oncotherapy and chance
Malignant tumour has become human number one " killer ", and operation, radiotherapy and chemotherapy are three big ordinary methods of treatment tumour, can load by effective in a short time ameliorate tumor, but still can not effectively remove tumour cell.MRD, be the root of tumor recurrence and transfer to the part sensitivity and the resistance of radiotherapy, chemotherapy, and recurrence with shift tumour patient main causes of death just.Conventional treatments is unable to do what one wishes, and the oncotherapy technology of development of new is extremely urgent with product.
Two, the birth of cellular immunization treatment technology and development
Play developing rapidly of immunology and oncology the eighties in last century; Research group headed by the nineteen eighty-two America NI H Rosenberg professor develops killer cell (LAK) immunotherapy of lymphokineactivation, and deep discussion has been carried out in the clinical application to the LAK cell in follow-up study.But LAK cell killing power is strong inadequately, and clinical application needs a large amount of infusions (3 * 10
10-11), it is limited in one's ability to increase on the other hand, need be in the infusion cell heavy dose ofly uses interleukin II (IL-2) (100,000 IU/kg q8h), thereby have produced relevant untoward reaction.Then Rosenberg has proposed tumor infiltrating lymphocyte (TIL) again, and it kills the knurl ability and is significantly improved than LAK, and need not heavy dose of IL-2 combined utilization, but cell need separate acquisition from tumor tissues, and this greatly limits its clinical application widely.On above working foundation; The employing interferon-gammas such as Schmidt-Wolf (IFN-γ) of Stanford university in 1991, IL-2 and mouse-anti people CD3 monoclonal antibody (Mouse anti-HumanCD3mAb) have induced the cell mass with powerful anti-tumor activity jointly, called after cytokine induced kill cell (CIK).
BMDC (DCs) is the most powerful full-time antigen presenting cell (APC) of function in the known up to now body; There is abundant antigen capture molecule in its surface; Antigen presentation molecule (MHC I class and MHC class), immune costimulatory molecules (CD80, CD83, CD86, CD40 etc.).DC cell after antigenic stimulation moves to lymphoglandula, and its antigenic information of carrying is passed to corresponding T lymphocyte, starts, excites the CD4+/CD8+T cellullar immunologic response, kills tumour cell specifically.But simultaneously the DCs secreting leukocytes mesonium-8 (IL-8) after antigenic stimulation, interferon alpha (IFN-α), interleukin 12 (IL-12), tumor necrosis factor alpha (TNF-α) etc.; Enhancing body nonspecific immune response (natural immunity); So DC has the laudatory title of " natural immunity adjuvant ", its discoverer Ralph professor Steinman obtains Nobel Prize in medicine in 2011.In view of DC residing middle cardiac status in the immunity of organism system of defense, the anti-tumor vaccine of developing based on DC has made it to become one of immunotherapy of tumors technology most advanced, the most likely, and Chinese scholars has carried out exploring widely to it in recent years.2 professors of Stanford medical college in 1992 have set up in the world, and first hand is that (Dendreon, CA), the product P rovenge of the said firm obtained FDA approval listing on April 29th, 2010 in basic tumor vaccine company with DC.But DC function in vivo receives the immunological status of patient own, the influence of tumor microenvironment, and have a large amount of supressors in the tumour patient body, so effect is also desirable not as in vitro effects in the body of DC.
It is the immunotherapy technology of rising in the recent period that cytotoxic T lymphocyte (CTL) makes up, and because of it has ability special, direct killing tumour target cell, therefore becomes the focus of research.
Three, the effect of CEACAMS (CEA) in immunotherapy of tumors
(Carcinoembryonic antigen is that nineteen sixty-five is found in intestinal canal tumour and embryo's digestive tube first CEA) to CEACAMS, is made up of 686 amino acid, and molecular weight is 18～20kD.Recently research confirms that CEA is one of member of ig supergene family, possibly participate in cell recognition and iuntercellular and react, and CEA still is a kind of adhesion factor, can promote tumour cell to combine with Normocellular, and the transfer of tumour is played an important role.Therefore CEA is not only a kind of simple tumor markers, and is the molecule relevant with malignancy of tumorization, in kinds of tumors, expresses.Surpass 90% primary colorectal cancer expression CEA, especially in the hepatic metastases patient, change of serum C EA concentration raises in 80% hepatic metastases patient.The positive rate of patients with gastric cancer change of serum C EA is about 40%, and is and relevant with by stages, raises gradually with course of disease progress.The patients with lung cancer of 40%-80% CEA occurs and raises, and the reactivity of change of serum C EA level and treatment and prognosis have dependency preferably.CEA also can produce from mammary tissue.Therefore CEA can be used as effective target antigen of immunotherapy of tumors.
The M & M of the lung cancer of CEA antigen high expression level, cancer of the stomach, colorectal cancer, mammary cancer all is tumours of rank front three, and the antigenic medicine listing to CEA is not also arranged at present, so development of new is imperative to the medicine of CEA target spot.The exploitation of anti-CEA specific CTL is a kind of developing direction.
Four, the CTL mechanism of action
1, body t cell immune response process
There is huge T cell bank in body; Through the different TXi Baoshouti of its surface expression (TCR) specific recognition different external antigen molecule (bacterium, virus and tumour antigen polypeptide); Be activated to having the CTL of kill target cell; Remove the infection and the tumour cell of bacterium, virus, and can produce immunological memory property CTL, prevent the invasion once more of bacterium, virus and the recurrence of tumour.
The CTL immunne response is roughly divided four-stage:
(1) antigen recognition: APC is through surface receptor identification and capture antigen (bacterium, virus, tumour cell);
(2) antigen processing and submission: antigen digests, is cracked into peptide molecule through APC, and the latter combines to form respectively MHC-I-polypeptide or MHC-II-polypeptide complex with APC born of the same parents interior abundant MHC-I class or class, and is transported to the APC surface;
(3) t cell activation (dual signal theory): APC and T cell meet; The T cell is through the MHC-I/II-polypeptide complex of self TCR identification APC submission; CD8+T cell recognition MHC-I-polypeptide complex wherein; CD4+T cell recognition MHC-II-polypeptide complex, T cell have been accepted antigenic stimulus signal (first signal) and have been added immune costimulatory signal (second signal) beginning activation, and having cytotoxic effect is CTL;
(4) target cell is killed: activation CTL is circulated to position, antigen place, kills and wounds and removes behind the TCR specific recognition antigen presentation cell through the CTL surface.
But, found to have a large amount of immunosuppressive factors in the tumour patient body, influence effective activation and the propagation of CTL, quantity is very few, is not enough to contend with the tumour of breeding rapidly.If can and feed back give the patient at a large amount of antigen-specific CTL of external preparation, can be directly, quick killing tumor cell plays therapeutic action, and the residual tumor cell that conventional treatment can't be removed is killed, and can prevent the recurrence and the transfer of tumour.
2, CTL phenotype and function difference
Expression kind according to the CTL surface molecular can be divided into amphitypy: central memory-type CTL (CTL
-CM) and effect memory-type CTL (CTL
-EM).
Klebanoff etc. discover: phenotype analytical is the CTL of CD3+CD45RO+CD62L+CCR7+
-CMHas stronger anti-tumor capacity; Johnson etc. have compared the CTL that the MART-1 tcr gene is modified
-CMAnd CTL
-EM, the result finds that the former the disappear ability of malignant melanoma cell is 10 times of the latter.Results of study such as Berger also show CTL
-CMBe the CTL of CD3+CD45RO+CD62L-CCR7-than phenotype behind the infusion
-EMCan survive the longer time in vivo.
3, CTL technological development present situation
The external technology of preparing of comprehensively domestic, external CTL can classify as three kinds of methods and source:
(1) tumor infiltrating lymphocyte (TIL) amplification in vitro method
From the tumour patient tumor tissues, separate TIL, add interleukin II (IL-2) and cultivate, clone's dilution method screening tumour antigen male T cell clone adds mouse-anti people CD3 monoclonal antibody and IL-2 again and carries out the amplification cultivation acquisition.
Li etc. select HLA-A201 male IV phase malignant melanoma patient; After its tumor tissues is prepared into single cell suspension, adds IL-2 and cultivate, the positive T cell clone of CD8+T+MART-1+ in the 5 weeks back screening amplifying cells; Add the PBMC feeder cell amplification cultivation behind mouse-anti people CD3 monoclonal antibody and the irradiation again; Add IL-2 next day, cultivate again and can obtain CTLs in 12 days, can to the T2 cell strain specific recognition of load MART-1 polypeptide with kill and wound.
(2) tcr gene is modified method
TCR is the key molecule of T cell recognition antigen, mediation immunne response; Comprise α, β, γ, four chains of δ; Connect into α/β and two kinds of heterodimers of gamma/delta by disulfide linkage, wherein α/β+T cell accounts for the 90-95% of periphery blood T cell, is main immune effector cell.Tcr gene is modified and is promptly passed through the exogenous plasmid transfection from body T lymphocyte, imports the tcr gene of ability identification specificity antigenic peptide, selects positive colony, cultivates at amplification in vitro and obtains CTL.Plasmid comparatively commonly used has retroviral plasmid or slow virus plasmid.
Morgan etc. adopt anti-CD3+CD28+ magnetic bead to activate the CD8+T cell, use the slow virus plasmid transfection and modify MART-1 or the specific tcr gene of gp100, adopt mouse-anti people's CD3 monoclonal antibody and IL-2 culture system to prepare CTLs, 12 days ability amplification cultivation to 10
9Individual cell is used for the treatment of the melanoma patients of 15 routine HLA-A2+, and the result shows 2 routine patients and tangible clinical efficacy occurred, and 52 years old male patient's of 1 example armpit metastatic lesion disappears, and the hepatic metastases focus has dwindled 89%, and the DFS phase is 21 months; 30 years old male patient of 1 example, lung's metastatic lesion disappears, and the DFS phase is 20 months.
Perro etc. adopt IL-15 (IL-15) and interleukin II 1 (IL-21) combination of cytokines to promote the T cell of the non-propagation of tcr gene transfection, can keep T cell high expression level CD62L and CD28.
(3) exo-antigen sensitization method
With artificial APC (Artificial APC), or the external repetitious stimulation T cell of antigen (protein, polypeptide, or full cell), antigen-specific CTL obtained.
(patent No.: CN102168066A) method of external evoked hepatitis b virus specificity cell toxicity T lymphocyte promptly adopts this method to the patent of invention of Zhejiang University.MHC-I-Ig and anti-CD28mAb are encapsulated magnetic bead make up artificial APC, behind the epitope peptide load APC, external sensitization 3-4 week, again with magnetic bead fractionation by adsorption antigen-specific CTL, the Tetramer evaluation of dyeing.
The defective of above-mentioned three kinds of methods:
(1) TIL amplification in vitro method
The fresh tumor tissues that 1. need obtain the patient is originated as CTL, only limits to the patient that can perform the operation, and the tumor tissues source is limited;
2. TIL separates, and obtaining with authentication method of ctl clone is complicated, and various cellular constituents are more in the tumor tissues, and complicated, the time of isolated cell is long, generally all needs more than 1 month;
3. be mixed with the CD4+CD25+Treg subgroup among the TIL, it can suppress the CTL amplification efficiency;
4. because the in-vitro separation proliferation time is long, increased the chance of polluting.
(2) tcr gene is modified method
The introducing of 1. exogenous plasmid (retrovirus or slow virus etc.) brings certain risk to clinical application;
2. endogenous TCR disturbs, and the tcr gene of importing possibly be able to not import the site of expection, and with endogenic TCR wrong row is taken place, and it can discern antigen is not desired design, and is unknown, has very big risk.
(3) external artificial APC load antigen sensibilization method
1. introduce allogenic material: whether artificial APC, the CTL of preparation have that artificial APC is residual to be still query;
2. the cycle of the external sensitization CTL of artificial APC is longer, needs 3-4 week, obtain the needs that CTL also needs could satisfy after the amplification cultivation clinical treatment, so the cycle of vitro culture is long, and the probability of pollution is bigger.
The preparation method of the restricted antigen-specific CTL of HLA-A201 that do not have also at present that reported in literature crosses that preparation time is short, amplification times is high, CTL purity height and fragmentation effect is good.
Summary of the invention
The objective of the invention is preparation method to the restricted anti-CEA antigen-specific CTL of HLA-A201 that above-mentioned technical problem provides that a kind of preparation time is short, amplification times is high, CTL purity height and fragmentation effect are good.
The objective of the invention is to realize through following technical proposal:
The preparation method of the restricted antigen-specific CTL of HLA-A0201, this method comprises the following steps:
A. the enrichment and the purification process of the first step CTL precursor cell derived cell:
Gathering periphery mononuclearcell (PBMC) with automatic mononuclearcell Acquisition Instrument (Dan Caiyi) originates as cell; Adopt the negative magnetic bead partition method of clinical grade, enrichment from PBMC, purifying CTL precursor cell are the CD8+T lymphocyte.
B. target CTL induces with the first round and increases:
Stimulate the CD8+T lymphocyte with the restricted CEA antigenic peptide of load HLA-A0201 from body maturation BMDC (mDC); Not only offer first signal (antigen) of T cell activation, DC offers the necessary second signal of T cell activation (immune costimulatory molecules CD40, CD80, CD83 etc.) simultaneously; Add two kinds of cytokines of rhIL-2 and rhIL-7 simultaneously and unite the growth of promotion T cell, suppress activation inductive apoptosis reaction (AICD), prolong the life cycle of Kiwi.
C. target CTL purification process:
The first round with load antigenic mDC stimulate and amplification target CTL quantity after; Adopt Tetramer marking method and fluidic cell sorting art again purification of target CTL (target CTL is meant and expresses the CD8+T that discerns CEA antigenic peptide TCR; Be the Tetramer+CD8+T lymphocyte); Promptly select Tetramer+CD8+T, reject the T cell of non-specific amplification, to reduce its influence target CTL propagation.
D. second of target CTL take turns amplification:
Adopt the anti-human-CD3mAb of solid-phase coating and growth and the propagation of IL-2 stimulation target CTL; Add again and strengthen activation from body PBMC target CTL through gamma-ray irradiation; Adding rhIL-15 can promote the propagation of memory t cell, apoptosis capable of inhibiting cell on the other hand on the one hand.
E. second take turns amplification back target CTL phenotypic evaluation:
Adopt flow cytometry that the target CTL that has prepared is carried out phenotype analytical, it is divided into center memory CTL (CTL according to the molecular spectra difference of its expression
-CM: CD8+CD45RO+CD62L+CCR7+) with responsiveness CTL (CTL
-EM: CD8+CD45RO+CD62L-CCR7-).
Described preparation method, the target antigen among the step b is the preferred 9-15 of a CEA antigenic peptide length amino acid, further the restricted CEA antigenic peptide of the HLA-A0201 of preferred sequence shown in SEQ ID NO.1.
Described preparation method; Load targets is antigenic among the step b prepares through following method from body maturation DC: the adherent back of PBMC adds mixing normal people AB serum and the RPMI1640 culture medium culturing three days of rhGM-CSF, rhIFN α, deactivation, and the DC of results adds target antigen again and hatches promptly and get.
Described preparation method is used for the preparing through following method from body PBMC of gamma-ray irradiation of target CTL amplification: leave and take the PBMC that part is singly adopted acquisition, behind the gamma-ray irradiation of 30-50GY, preserve among the step c; Preserve liquid: contain the RPMI1640 nutrient solution of 20% (V/V) DMSO and 20% (V/V) normal people AB serum, storage temperature :-80 ℃ of very low temperature are preserved.
Described preparation method; The CD3 monoclonal antibody clonal antibody (anti-human-CD3mAb that is used for target CTL amplification in the steps d; Be called for short the CD3 monoclonal antibody) be following the operation: in the vessel surface that is used for amplifying cells, the CD3 monoclonal antibody can combine with the CD3 molecule on a plurality of CTL surface, promotes the formation of CTL cell clone with CD3 monoclonal antibody solid-phase coating; Promote cells contacting, get into proliferating cycle fast.
Described preparation method, the consumption of each step moderate stimulation molecule or cell is respectively:
A. the target CTL first round each add-on that increases: the rhIL-2 final concentration is 500-750IU/ml, and the rhIL-7 final concentration is 50-75ng/ml, and DC and initial T cell quantity are than being 1:5.
B. target CTL second takes turns amplification: the rhIL-2 final concentration is 200-500IU/ml, and the rhIL-15 final concentration is 100-250ng/ml, and it is 1.0-2.5 μ g/ml that the CD3 monoclonal antibody encapsulates concentration, and the PBMC of gamma-ray irradiation is 2:1 with initial CTL quantity ratio.
Described preparation method, cell quantity and phenotypic characteristic are seen table 1 after each procedure.
Table 1
Below be more detailed explanation to preparing method's of the present invention key step:
1, CTL precursor cell purifying and enriching method:
Usually adopting periphery PBMC is the lymphocytic source of CD8+T as the CTL precursor cell; Contain CD4 positive t lymphocytes (CD4+T), CD8 positive t lymphocytes (CD8+T), bone-marrow-derived lymphocyte, monocyte (Mo), nk cell various kinds of cell compositions such as (NK) among the PBMC, wherein the CD8+T starting quantity directly influences the ultimate capacity of target CTL.
Adopt that the method for singly adopting is disposable gathers a large amount of periphery PBMC, obtain a large amount of initiator cells, but unnecessary PBMC irradiation or frozen be used for repeatedly the CTL preparation and use, and method is easy and simple to handle.
Adopt the negative back-and-forth method of clinical grade magnetic bead purifying CD8+T from PBMC, reject CD4+T, B, NK cell, reduce of the interference of its rapid amplifying CD8+T amplification growth.Can the purity of CD8+T be improved 2-4 doubly (being increased to average more than 90%) through the negative back-and-forth method of magnetic bead, see accompanying drawing 1.
2, target CTL induces with the first round and increases:
Patent of the present invention adopts three days ripe DC preparing methods, and that DC prepares is simple and direct, the time short, and its form, phenotype and conventional 7-10 days DC comparison no significant differences are seen accompanying drawing 2.
Patent of the present invention is antigenic from body maturation DC with load targets, gives the CD8+T activatory first signal; DC is rich in immune costimulatory molecules simultaneously, gives CD8+T activatory second signal, and assurance CD8+T is induced to be divided into target CTL under the dual signal effect efficiently.Associating rhIL-2 and rhIL-7 increase to CD8+T and are superior to the effect of monofactor (IL-2), and IL-7 promotes memory t cell clone's propagation, suppress the AICD reaction because of causing behind the long-time IL-2 activating T cell, prolong the T cells survival cycle.
3, target CTL purifying and enriching method:
After inducing amplification in the first round; The CD8+T that target CTL promptly expresses recognition objective antigen (being the restricted CEA antigenic peptide of HLA-A0201) TCR in the CD8+T cell only accounts for the part ratio; And ratio is not high, is the CD8+T of non-specific amplification in a large number, does not have the effect of identification, removing target cell.The present invention adopts that the Tetramer marking method is separated with fluidic cell sorting art, purification of target CTL, and enrichment can be discerned the CTL of target antigen again, and these CTL just possess the ability of killing and wounding target cell.
The purity of target CTL reaches more than 95% behind the purifying.And the several method of conventional report adopts limiting dilution assay mostly, methods such as electroinjection, and the time cost is longer, complicated operation, specificity is relatively low.See accompanying drawing 3.
4, target CTL second takes turns amplification:
Adopt the mouse-anti people CD3/CD28 monoclonal antibody of liquid phase and IL-2 combination to increase to the T cell amplification more.The present invention adopts solid phase mouse-anti people CD3 monoclonal antibody (anti-human-CD3mAb) coating technique, unite through gamma-ray irradiation from body PBMC and rhIL-15 as the multiple signal that stimulates T cell proliferation.Solid phase CD3-mAb can promote the T cell clone to form, and accelerates the cell growth; IL-15 makes CTL have anti-apoptosis, promotes the ability of memory T cell growth; The a large amount of costimulatory molecules justacrine of the PBMC surface expression various kinds of cell factor provides CTL good growing environment, and behind gamma-ray irradiation, can not breed and disturb the growth of CTL, thereby guarantees the efficient amplification of CTL.
Compare with conventional amplification system: the target CTL output of this patent method preparation is high, CTL
CMRatio is high, and it is stronger to kill and wound the target cell ability.See accompanying drawing 4-5.
Beneficial effect of the present invention:
The present invention unites and adopts efficient, simple and direct singly adopting with the magnetic bead sorting method to obtain CD8+T than ordinary method a greater number as target CTL precursor cell; As sensitinogen, associating rhIL-2 and rhIL-7 induce CTL precursor clone to increase to the target CTL differentiation and the first round with the BMDC of the restricted CEA antigenic peptide of HLA-A0201 load; From the thin crowd of mixed C D8+T lymph of sensitization, isolate target CTL through the tetramer (tetramer) mark and fluidic cell sorting technology again; Through adopting solid phase anti-human-CD3-mAb; The PBMC of gamma-ray irradiation is as feeder cell, and associating IL-2 and IL-15 carry out second to target CTL and take turns efficient amplification.The target CTL that adopts the inventive method to prepare possesses high purity, the high proliferation ability, and High Fragmentation is active, at high proportion CTL
-CMCharacteristics can be used for the immunotherapy of CEA associated malignancies and communicable disease.
The present invention adopts different methods to pass through the secondarily purified and enrichment to the restricted anti-CEA antigen-specific CTL of HLA-A0201; Through different amplification systems the target CTL of enrichment is carried out two and take turns inducing of amplification and function; Make target CTL on the purity, quantitatively with on the function all be superior to several kinds of CTL preparation methods of the prior art, be embodied in the following aspects especially:
1, the invention provides natural, activated T cell dual signal efficiently
1. antigen-specific signal: DCs is the strongest full-time antigen presenting cell of function in the body of generally acknowledging; Body series imitates, duplicates antigen is caught submission in vivo by DC process; Select dominance antigen peptide load live body DC as sensitinogen; Provide near the MHC-polypeptide complex space structure in the human immunity answering, for the T cell activation provides specificity first signal.
2. costimulatory signal: the DC costimulatory molecules of expressed in abundance simultaneously is for the T cell activation provides natural second signal.
2, the invention provides efficient, simple and direct CTL precursor and target CTL enrichment and purification process
1. the enrichment of CTL precursor cell: adopt singly adopt with the negative magnetic bead sorting CTL of clinical grade precursor cell be the CD8+T lymphocyte; Easy and simple to handle; Starting quantity is sufficient, and the CD8+T cell purity of acquisition reaches more than 90%, for preparation q.s, enough purity target CTL provide basic assurance.
2. the purifying of target CTL cell: adopting Tetramer mark and airflow classification technological, is that Tetramer+CD8+T carries out separation and purification to target CTL, simple and direct, efficient, save time (4-5 hour), and purity is up to more than 95%.
3, the invention provides two take turns, efficient target CTL amplification system, to guarantee to provide sufficient amount target CTL
1. the first round induces and increases: adopt the DC of polypeptide load, unite two kinds of cytokine IL-2 and IL-7 amplification system, amplification times reaches 30-50 doubly in two weeks.
2. second take turns target CTL amplification: adopt solid phase mouse-anti people CD3 monoclonal antibody, associational cells factor IL-15 and from body PBMC amplification system, left and right sides amplification times was average 40 times in 10 days, was up to 60 times.Can obtain 10
9Individual target CTL cell reaches the needs of clinical application, and ordinary method (background technology is mentioned three kinds of external evoked CTL that reach same quantity of method needs 3-4 week.
4, the external vigor that kills and wounds of target CTL of the present invention's preparation is higher
The restricted anti-CEA antigen-specific CTL of HLA-A0201 of the present invention preparation external can specific recognition with kill and wound the target cell of expressing corresponding antigens, and the high day ordinary method of kill rate is imitated target kill rate when identical and is on average exceeded about 15%.See accompanying drawing 5.
M1 is an ordinary method one: the target CTL first round increase and the sorting purifying with this patent method, target CTL second takes turns when amplification, with containing the RPMI1640 perfect medium re-suspended cell that 10% deactivation mixes normal people AB serum, cell density is 2 * 10
6/ ml is seeded to 75cm
2In the Tissue Culture Flask, every bottle of 30ml, adding final concentration are that mouse-anti people CD3 monoclonal antibody (liquid phase) and the final concentration of 50ng/ml is the rhIL-2 of 500IU/ml, and mixing places 37 ℃ gently, cultivates in the 5%CO2 cell culture incubator.Every day observation of cell growth conditions, replenish fresh culture in good time and contain the RPMI1640 perfect medium that 10% deactivation mixes normal people AB serum, 200IU/ml rhIL-2, cell proliferation is to a certain amount of expansions bottle.Be cultured to the 10th day harvested cell.With saline water washing 2 times and resuspended, calculate the cell quantity that obtains.Get 3 * 10
5Individual left and right sides cell adds anti-people CD8, CD45RO, CD62L, CCR7 monoclonal antibody and carries out labeling CT L, carries out the ratio of check and analysis CD8+CD45RO+CD62L+CCR7+T with flow cytometer.And to the kill capability of target cell.Average acquisition CTL cell total amount is (1.0 ± 0.3) * 10
9, CTL
CMThe ratio average out to: 31.9 ± 6.5%, CTL lethality average out to 23.7 ± 4.3% when imitating the target ratio for 30:1.
M2 is an ordinary method two: the target CTL first round increase and the sorting purifying with this patent method, target CTL second takes turns when amplification, with containing the RPMI1640 perfect medium re-suspended cell that 10% deactivation mixes normal people AB serum, cell density is 2 * 10
6/ ml is seeded to 75cm
2In the Tissue Culture Flask, every bottle of 30ml adds 1.5 * 10
7Anti-CD3/28 magnetic bead of/ml and final concentration are the rhIL-2 of 500IU/ml, and mixing places 37 ℃ gently, cultivate in the 5%CO2 cell culture incubator.Every day observation of cell growth conditions, replenish fresh culture in good time and contain the RPMI1640 perfect medium that 10% deactivation mixes normal people AB serum, 200IU/ml rhIL-2, cell proliferation is to a certain amount of expansions bottle.Be cultured to the 10th day harvested cell.With saline water washing 2 times and resuspended, calculate the cell quantity that obtains.Get 3 * 10
5Individual left and right sides cell adds anti-people CD8, CD45RO, CD62L, CCR7 monoclonal antibody and carries out labeling CT L, carries out the ratio of check and analysis CD8+CD45RO+CD62L+CCR7+T with flow cytometer, and to the kill capability of target cell.Average acquisition CTL cell total amount is (1.2 ± 0.3) * 10
9, CTL
CMThe ratio average out to: 33.4 ± 3.2%, CTL lethality average out to 30.2 ± 4.1% when imitating the target ratio for 30:1.
M3 is this patent method, on average obtains CTL cell total amount and is (3.7 ± 0.3) * 10
9, CTL
CMThe ratio average out to: 75.3 ± 4.9%, CTL lethality average out to 41.3 ± 3.3% when imitating the target ratio for 30:1.
5, CTL in the target CTL cell of the present invention's preparation
CMRatio is high
The centre type memory CTL ratio of expressing the CD3+CD45RO+CD62L+CCR7+ phenotype among the CTLs of the present invention's preparation is higher than ordinary method, sees accompanying drawing 4.
Compare amplification ability, phenotype and the lethality of the target CTL of three kinds of amplification system preparations, the result shows: this patent induces system inductive CTL amplification ability the strongest, and the cell total amount can reach (3.7 ± 0.3) * 10
9, CTL wherein
CMRatio can reach 75.3%, is imitating target than for 30:1 the time, on average can reach 41.3% for the kill capability of target cell (the T2 cell strain of load HLA-A2-CEA polypeptide).
Description of drawings
Fig. 1: the comparison of purity before and after the CTL precursor cell purifying.
Figure A: singly adopt the PBMC fluidic cell and detect collection of illustrative plates (before the purifying)
Method: get 50 μ l PBMC (about 3 * 10 respectively
5Individual cell) adds in 3 streaming pipes, add the anti-people CD4 antibody of 5 μ l FITC marks in first pipe again, the anti-people CD8 antibody of 5 μ l PE marks, the anti-people CD56 antibody of the anti-people CD3 antibody of 5 μ l PerCP marks and 5 μ lAPC marks; The anti-people CD14 antibody that adds 5 μ l APC marks in second pipe; The 3rd pipe adds the anti human CD 19 antibody of 5 μ l APC marks; Abundant mixing; 4 ℃ of lucifuges were hatched 30 minutes; Take out the every pipe in back add the 1ml precooling twice of the saline water washing that contains 2% (V/V) NBCS and resuspended after, detect employing Cellquest software analysis CD3+CD4+T%, CD3+CD8+T%, CD14+%, CD19+%, CD3-CD56+% ratio with FACS Calibur flow cytometer.
The result:
CD14+%（Mo）：9.99% CD19+%（B）：6.15%
CD3+CD4+T%：10.91% CD3+CD8+T%：45.01%
CD3-CD56+%（NK）：0.45%
The negative back CTL precursor cell fluidic cell of selecting of figure B magnetic bead detects collection of illustrative plates (behind the purifying)
Method: singly adopt PBMC and select with CD4+CD19+CD16/56+T sorting magnetic bead is negative; After rejecting CD4+T cell, NK cell and B cell; The streaming dyeing process of collecting before not combining the magnetic bead cell according to purifying dyes, and analyzes the variation of CD3+CD4+T%, CD3+CD8+T%, CD14+%, CD19+%, CD3+CD56+%, CD3-CD56+% ratio.The result shows that after the negative selection of magnetic bead, the CD3+CD8+T% ratio reaches 90.97%, and purity has improved two times.
The result:
CD3+CD4+T%：2.65% CD3+CD8+T%：97.13%
CD14+%（Mo）：0.04% CD19+%（B）：1.01%
CD3-CD56+%（NK）：0.00%
Fig. 2: two kinds of different methods inductive DC phenotypes relatively
Method: get 50 μ l rhGM-CSF+rhIFN-α respectively and induce 3 days DC (about 3 * 10
5Individual cell) adds in 4 streaming pipes; First pipe adds CD40, the HLA-DR of 5 μ l PerCP marks and the anti-people CD11c antibody of 5 μ l APC marks of 5 μ lPE marks; Second pipe adds anti-people CD80 antibody, the anti-people HLA-DR antibody of 5 μ l PerCP marks and the anti-people CD11c antibody of 5 μ lAPC marks of 5 μ l PE marks; The 3rd pipe adds anti-people CD83 antibody, the anti-people HLA-DR antibody of 5 μ l PerCP marks and the anti-people CD11c antibody of 5 μ l APC marks of 5 μ l PE marks; The 4th pipe adds anti-people CD86 antibody, the anti-people HLA-DR antibody of 5 μ l PerCP marks and the anti-people CD11c antibody of 5 μ l APC marks of 5 μ l PE marks; Abundant mixing; 4 ℃ of lucifuges were hatched 30 minutes; Take out the every pipe in back add the 1ml precooling twice of the saline water washing that contains 2% (V/V) NBCS and resuspended after, detect employing Cellquest software analysis HLA-DR+CD11c+%, CD40+HLA-DR+CD11c+%, CD80+HLA-DR+CD11c+%, CD83+HLA-DR+CD11c+%, CD86+HLA-DR+CD11c+% ratio with FACS Calibur flow cytometer.RhGM-CSF+rhIL-4 induces 7 days DC to use with quadrat method and detects.The result shows that the DC purity phenotype (HLA-DR+CD11c+) that the rhGM-CSF+rhIFN-α that this patent adopts induced 3 days is 90.52%, and ripening degree phenotype (CD83+) is 77.23%.The DC purity phenotype that rhGM-CSF+rhIL-4 induced 7 days is 90.95%, and the ripening degree phenotype is 77.29%, both no significant differences.
The result:
Figure A:rhGM-CSF+rhIFN α induces 3 days DC phenotype streamings to detect collection of illustrative plates:
HLA-DR+CD11c+%：90.52% CD40+HLA-DR+CD11c+%：87.15%
CD80+HLA-DR+CD11c+%：82.27%?CD83+HLA-DR+CD11c+%：77.23%
CD86+HLA-DR+CD11c+%：87.47%
Figure B:rhGM-CSF+rhIL-4 induces 7 days DC phenotype streamings to detect collection of illustrative plates:
HLA-DR+CD11c+%：90.95% CD40+HLA-DR+CD11c+%：86.85%
CD80+HLA-DR+CD11c+%：88.76% CD83+HLA-DR+CD11c+%：77.29%
CD86+HLA-DR+CD11c+%：88.21%
Fig. 3: the target CTL first round back streaming that increases detects collection of illustrative plates
Figure A: the target CTL first round back cell streaming detection collection of illustrative plates (before the purifying) that increase
Figure B: the target CTL first round back cell streaming detection collection of illustrative plates (behind the purifying) that increase
Method: the CTL first round increases and afterwards with the Tetramer of PE mark HLA-A2+CEA+ and the anti-people CD8 antibody of FITC mark the CTL that increases is carried out mark; Adopt selected by flow cytometry apoptosis Tetramer+CD8+CTL cell; Tetramer+CD8+T purity is 3.13% before the sorting, reaches 95.14% after the sorting.
The comparison of the CTL phenotype of three kinds of different methods preparations of Fig. 4
Figure A (M1): the CTL phenotype streaming of liquid phase mouse-anti people CD3 monoclonal antibody+IL-2 amplification system preparation detects collection of illustrative plates
Figure B (M2): liquid phase CD3/28 magnetic bead, the CTL phenotype streaming of IL-2 amplification system preparation detects collection of illustrative plates
Figure C (M3): solid phase mouse-anti people CD3 monoclonal antibody, IL-2, IL-7, IL-15 is from the CTL phenotype streaming detection collection of illustrative plates of body PBMC amplification system preparation
Method: the target CTL behind the purifying is divided into 3 parts; First part adds final concentration 50ng/ml mouse-anti people's CD3 monoclonal antibody and 500IU/ml IL-2 carries out amplification cultivation; Second part adds final concentration 10 μ g/ml CD3/28 magnetic beads and 250IU/mlIL-2 carries out amplification cultivation; The 3rd part adds mouse-anti people CD3 monoclonal antibody 2 μ g/ml and encapsulates the culturing bottle that spends the night in advance; Ratio according to target CTL:PBMC=1:2 adds the PBMC through gamma-ray irradiation, and IL-2 and the final concentration 25ng/ml IL-15 of adding final concentration 500IU/ml carry out amplification cultivation.Be cultured to the 7th day results CTL.Get 50 μ lCTL (about 3 * 10
5Individual cell) add in the 2 fluid-guiding type pipes, first pipe adds the anti-people CD62L antibody of 5 μ l FITC marks, the anti-people CCR7 antibody of 5 μ lPE marks; The anti-people CD45RO antibody of 5 μ lAPC marks; Second pipe adds the anti-people CD3 antibody of 5 μ lPerCP marks and the anti-people CD8 antibody of 5 μ lPE marks, abundant mixing, and 4 ℃ of lucifuges were hatched 30 minutes; Take out the every pipe in back add the 1ml precooling twice of the saline water washing that contains 2% (V/V) NBCS and resuspended after; Detect with FACS Calibur flow cytometer, adopt Cellquest software analysis CD3+CD8+T%, CD45RO+CD62L+% and CCR7+CD62L+% ratio, obtain the phenotype streaming figure of CTL; The CD3+CD8+T% of the CTL of this patent method preparation is 97.24%; Wherein CD45RO+CD62L+% reaches 74.76%, and CCR7+CD62L+% reaches 77.52%, apparently higher than method 1 and 2.
The comparison of the CTL kill capability of Fig. 5 different methods preparation is (with CEA
691-699CTL is an example).
The CTL kill capability of figure A (M1) liquid phase mouse-anti people CD3 monoclonal antibody+IL-2 amplification system preparation
Figure B (M2) liquid phase CD3/28 magnetic bead, the CTL kill capability of IL-2 amplification system preparation
Figure C (M3) solid phase mouse-anti people CD3 monoclonal antibody, IL-2, IL-7, IL-15 is from the CTL kill capability of body PBMC amplification system preparation
Method: target cell 1: be load C EA
691-699The HLA-A2+T2 cell strain of polypeptide, target cell 2: be the unloaded cell strain of HLA-A2+T2, target cell 3:K562 (strain of NK sensitive cells).
The CTL of embodiment 1 preparation respectively with above-mentioned 3 kinds of target cells according to imitating the mixed of target than 3:1,10:1 and 30:1, mtt assay detects the CTL killing activity.The result shows, the CTL of the inventive method preparation imitating target than for 10:1 the time, reaches 20.5%, apparently higher than method 1 and 2 to the specific killing activity of target cell.
The practical implementation method
Below through embodiment the present invention is done further elaboration.
Embodiment 1CEA
691-699The preparation of antigenic peptide specific CTL
1, the HLA-A201+CEA+ colorectal cancer patients is prepared
Extract patient's periphery anticoagulation 1ml, add the HLA-A2-mAb of FITC mark, detect HLA-A2 (abbreviation of HLA-A201, down together) expression through streaming; Detect the CEA antigenic expression.HLA-A2 and CEA express simultaneously, and positive person is selected in.
2, the restricted CEA antigenic peptide of HLA-A201 is synthetic
The site is 691-699, and sequence is IMIGVLVGV (SEQ ID NO.1) 9 peptides, hereinafter to be referred as CEA
691-699Polypeptide through chemosynthesis (the biochemical ltd of Shanghai gill), fully dissolves with aseptic double-distilled water, and peptide concentration is 5mg/ml, and packing is stored in-80 ℃.
3, PBMC gathers
With singly adopting appearance collection patient periphery PBMCs, separate PBMCs through the Ficoll density gradient centrifugation, part is used for DC induces, and part is used for enrichment of CTL precursor cell and purifying, and part is through gammairradiation and frozen in-80 ℃.
4, DC induces and antigen load
Adherent method prepares DC: PBMCs is suspended in the RPMI1640 substratum, and cell density is 3 * 10
6/ ml, inoculating cell is in 75cm
2In the culturing bottle, in 5%CO
2, hatched 90 minutes in 37 ℃ of incubators, wash gently with preparatory warm saline and wash 2-3 time.Attached cell is used for DC and induces.
DC induces: in culturing bottle, add mixing normal people AB serum, the final concentration 200ng/ml rhGM-CSF of 30ml DC inducing culture (volume ratio, the down with) deactivation that contains 10%, the RPMI1640 perfect medium of final concentration 500ng/ml rhIFN-α.Induce after 3 days and gather in the crops DCs.
Polypeptide DC load: the DC of results is suspended in the RPMI1640 substratum, and cell density is 1 * 10
6/ ml also is inoculated in cell cultures 6 orifice plates, and every hole 3ml adds CEA in the hole
691-699Polypeptide to final concentration is 10 μ g/ml, is positioned over 37 ℃, 5%CO
2Educate results after 2 hours in the incubator altogether, with RPMI1640 substratum washing DC secondary, use the RPMI1640 substratum resuspended again, cell density is 1 * 10
6/ ml is used for the stimulation of target CTL (being the restricted anti-CEA antigen-specific CTL of HLA-A0201).
The preparing through following method of gamma-ray irradiation that is used for target CTL amplification: leave and take the PBMC that part is singly adopted acquisition, behind the gamma-ray irradiation of 30-50GY (average 40GY), preserve from body PBMC; Preserve liquid: contain the RPMI1640 nutrient solution of 20% (V/V) DMSO and 20% (V/V) normal people AB serum, storage temperature :-80 ℃ of very low temperature are preserved.
5, the isolation and purification of CTL precursor cell
In the frozen PBMC of DC results recovery on the same day, with the PBS solution washing cell 2 times that 2% deactivation mixes normal people AB serum that contains of precooling, and re-suspended cell, adjusting cell density is 2 * 10
7/ ml; In cell, add clinical grade CD4+CD19+CD16/56+T sorting magnetic bead (Miltenyi CliniMACS); Educate altogether after 30 minutes and to cross post, collect effusive CD8+T lymphocyte, wash 2 times with RPMI 1640 substratum of precooling; Cell is resuspended in contains 10% deactivation and mix in the normal people AB serum RPMI1640 perfect medium, cell density is 1 * 10
6/ ml.
6, the target CTL first round increases
The CD8+T of purifying is inoculated in cell cultures 6 orifice plates, and every hole adds 3ml, adds the 0.6ml DC of load polypeptide (step 4 preparation) again, adds IL-2 and IL-7 and makes final concentration reach 500IU/ml and 50ng/ml respectively, puts into 5%CO behind the mixing gently
2, cultivate in 37 ℃ of cell culture incubators; Every day observation of cell growth conditions, replenish fresh culture (contain 10% deactivation and mix normal people AB serum RPMI1640 perfect medium, 500IU/ml IL-2 and 50ng/ml IL-7) in good time.The 7th day, stimulate once more in every Kong Zhongzai adding 0.6ml DC of load polypeptide (ditto).Every day observation of cell growth conditions, replenish fresh culture (ditto) in good time, expand bottle when cell density is excessive, cell proliferation can be moved to 75cm after a certain amount of
2Continuing amplification in the culturing bottle cultivates.
7, target CTL sorting purifying
The DC of load polypeptide and CD8+T educated 12-14 days altogether, harvested cell, and with the PBS solution washing cell 2 times that 2% deactivation mixes normal people AB serum that contains of precooling, and re-suspended cell, the adjustment cell density is 1 * 10
7/ ml, the HLA-A201+CEA of adding PE mark in cell suspension
691-699The CD8mAb of Tetramer and FITC mark behind the mixing, educated 20 minutes in 4 ℃ of refrigerators gently altogether.With the PBS solution washing cell 2 times that 2% deactivation mixes normal people AB serum that contains of precooling, re-suspended cell, the adjustment cell density is 1 * 10
7/ ml carries out airflow classification with cell, and results CD8+Tetramer+T lymphocyte is abandoned supernatant after centrifugal, and with containing the RPMI1640 perfect medium re-suspended cell that 10% deactivation mixes normal people AB serum, cell density is 2 * 10
6/ ml.
8, target CTL second takes turns amplification
Tissue Culture Flask encapsulates: in step 7 previous day, get 75cm
2Tissue Culture Flask, in wherein add 2ml mouse-anti people CD3 monoclonal antibody (concentration 2 μ g/ml, producer: U.S. R&D company, catalog number (Cat.No.): 59-MAB100, clone number: C1UCHT1, down with), the rearmounted 4 ℃ of refrigerators of sealing encapsulate and spend the night;
Before inoculating cell; Taking-up encapsulates culturing bottle, inhales and abandons unnecessary CD3-mAb in the culturing bottle, and every bottle adds 30ml sorting CD8+Tetramer+T lymphocyte; Adding is through the PBMC of gamma-ray irradiation (quantity 2 times to target CTL); Add final concentration 500IU/ml IL-2 and final concentration 250ng/ml IL-15, mixing places 37 ℃ gently, 5%CO
2Cultivate in the cell culture incubator.Every day observation of cell growth conditions, replenish fresh culture (contain 10% people's deactivation and mix normal people AB serum, 200IU/ml IL-2, RPMI 1640 perfect mediums of 100ng/ml IL-15) control cell density in good time; Cell proliferation is to a certain amount of expansion bottle.
9, target CTL results detect with phenotype
Second takes turns target CTL amplification about 10 days, harvested cell.With saline water washing 2 times, be resuspended in the 100ml saline water, cell density is 10
7/ ml is used for immunotherapy.
Get 3 * 10
5Individual left and right sides cell; Add anti-people CD8, CD45RO, CD62L, CCR7 monoclonal antibody and carry out labeling CT L; Carry out the ratio of check and analysis CD8+CD45RO+CD62L+CCR7+T with flow cytometer, the result sees Fig. 4 C, and the CD3+CD8+T% of the CTL of present method preparation is 97.24%; Wherein CD45RO+CD62L+% reaches 74.76%, and CCR7+CD62L+% reaches 77.52%.
Cell quantity and phenotypic characteristic are seen table 1 after each procedure.
10, target CTL kill capability
With the preparation target CTL respectively with last load C EA
691-699The HLA-A2+T2 cell strain of polypeptide, the unloaded cell strain of HLA-A2+T2 and three kinds of target cells of K562 (strain of NK sensitive cells) are according to imitating the mixed of target than 3:1,10:1 and 30:1, and mtt assay detects the CTL killing activity.The result shows, the CTL of the inventive method preparation imitating target than for 10:1 the time, reaches 20.5% to the specific killing activity of target cell, sees Fig. 5 C.
Claims (9)
1.HLA-A0201 restricted anti-CEA antigen-specific CTL preparation method is characterized in that this method comprises the following steps:
The enrichment and the purifying of a.CTL precursor cell derived cell:
The collection periphery of singly gathering mononuclearcell is originated as the CTL precursor cell; Adopt the negative magnetic bead partition method of clinical grade, enrichment from collect the periphery mononuclearcell, purifying CTL precursor cell are the CD8+T lymphocyte;
B. target CTL induces with the first round and increases:
Mature dendritic cell with the restricted CEA antigenic peptide of load HLA-A0201 stimulates the CD8+T lymphocyte, adds two kinds of cytokines of rhIL-2 and rhIL-7 simultaneously and unites the growth of promotion T cell; Second week again repetitive stimulation once accomplish first round amplification;
C. target CTL purification process:
Adopt Tetramer marking method and fluidic cell sorting art purification of target CTL, promptly select the Tetramer+CD8+T lymphocyte;
D. target CTL second takes turns amplification:
Adopt the anti-human-CD3mAb of solid-phase coating and the growth of IL-2 stimulation target CTL; Adding strengthens the activation to target CTL from external all mononuclearcell behind gamma-ray irradiation; Add rhIL-15 and continue to cultivate, accomplish second and take turns amplification, collect and identify.
2. preparation method according to claim 1 is characterized in that the restricted CEA antigenic peptide of HLA-A0201 length is 9-15 amino acid among the step b.
3. preparation method according to claim 2 is characterized in that the restricted CEA antigenic peptide of described HLA-A0201 sequence is shown in SEQ ID NO.1.
4. preparation method according to claim 1; The mature dendritic cell that it is characterized in that the restricted CEA antigenic peptide of load HLA-A0201 among the step b prepares through following method: the adherent back of periphery mononuclearcell was added the mixing normal people AB serum that contains rhGM-CSF, rhIFN α, deactivation and RPMI1640 culture medium culturing three days, and results DC adds target antigen again and hatches promptly and get.
5. preparation method according to claim 1; It is characterized in that being used among the step c the preparing through following method of gamma-ray irradiation of target CTL amplification: leave and take part claim 1 and singly adopt and obtain the periphery mononuclearcell, behind the gamma-ray irradiation of 30-50GY, preserve from external all mononuclearcells; Preserve liquid: contain the RPMI1640 nutrient solution of 20% (V/V) DMSO and 20% (V/V) normal people AB serum, storage temperature :-80 ℃ of very low temperature are preserved.
6. preparation method according to claim 1; The anti-human-CD3mAb that it is characterized in that being used in the steps d target CTL amplification is following operation: with the anti-human-CD3mAb solid-phase coating in the vessel surface that is used for amplifying cells; Anti-human-CD3mAb combines with the CD3 molecule on a plurality of CTL surface; Promote the formation of CTL cell clone, promote cells contacting, get into proliferating cycle fast.
7. preparation method according to claim 1 is characterized in that target CTL is meant the CD8+T that expresses identification CEA antigenic peptide TCR, i.e. Tetramer+CD8+T lymphocyte.
8. preparation method according to claim 1 is characterized in that the consumption of each step moderate stimulation molecule or cell is respectively:
A. the target CTL first round each add-on that increases: the rhIL-2 final concentration is 500-750IU/ml, and the rhIL-7 final concentration is 50-75ng/ml, and DC is 1: 5 with initial CD8+T cell quantity ratio;
B. target CTL second takes turns amplification: the rhIL-2 final concentration is 200-500IU/ml; The rhIL-15 final concentration is 100-250ng/ml; It is 1.0-2.5 μ g/ml that anti-human-CD3mAb encapsulates concentration, and the periphery mononuclearcell of gamma-ray irradiation is 2: 1 with initial CTL quantity ratio.
9. preparation method according to claim 1 is characterized in that cell quantity and phenotypic characteristic are respectively after each procedure:
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