CN105505873A - Preparation method of HLA-A0201 restriction anti-Her2-neu antigen-specific CTL (cytotoxic lymphocyte) - Google Patents

Abstract

The invention belongs to the field of biotechnology development and application and discloses a preparation method of HLA-A0201 restriction anti-Her2-neu antigen-specific CTL (cytotoxic lymphocyte). The method includes: collecting peripheral mononuclear cells by single collecting, and enriching and purifying CD8+T lymphocyte; stimulating the CD8+T lymphocyte with mature dendritic cells that support HLA-A0201 restriction Her2-neu antigen polypeptides, and promoting T cell growth with rhIL-2 and rhIL-7; purifying a target CTL by Tetramer labeling and flow activated cell sorting; stimulating the target CTL to grow with antihuman CD3 monoclonal antibodies and IL-2; enhancing activation of the target CTL through autologous PBMC (peripheral blood mononuclear cells) after irradiation of gamma ray; adding rhIL-15 for culturing and amplification, and collecting and identifying. The target CTL prepared by the method is high in purity, multiplication capacity, killing activity and CTL-CM ratio and is used in immunotherapy for tumors and the like.

Description

The preparation method of the restricted anti-Her2-neu Peptide-specific CTL of HLA-A0201

Technical field

The invention belongs to biotechnology development & application research field, relate to the restricted anti-Her2-neu Peptide-specific CTL preparation method of HLA-A0201.

Background technology

One, the predicament of oncotherapy and chance

Malignant tumour has become mankind's number one " killer ", and operation, radiotherapy and chemotherapy are the three large ordinary methods for the treatment of tumour, can effective ameliorate tumor load in a short time, but still effectively can not remove tumour cell.MRD, to the part of radiotherapy, chemotherapy, responsive and resistance is the root of tumor recurrence and transfer, and recurrence with shift tumour patient main causes of death just.Conventional treatments is unable to do what one wishes, the oncotherapy technology of development of new and product extremely urgent.

Two, the birth of cellular immunotherapy technology and development

Play developing rapidly of immunology and oncology the eighties in last century, research group headed by nineteen eighty-two America NI HRosenberg teaches develops Lymphatic circulation (LAK) immunotherapy, and has carried out deep discussion to the clinical application of LAK cell in follow-up study.But LAK Execution is strong not, and clinical application needs a large amount of infusion (3 × 10 ^10-11), increase on the other hand limited in one's ability, need heavy dose of application interleukin II (IL-2) (100,000 IU/kg, q8h) while infused cells, thus create relevant untoward reaction.Then Rosenberg also been proposed tumor infiltrating lymphocyte (TIL), its kill knurl ability comparatively LAK be significantly improved, and without the need to heavy dose of IL-2 combined utilization, but cell needs to be separated from tumor tissues to obtain, and this greatly limits its clinical application widely.On above working foundation, the Schmidt-Wolf of Stanford university in 1991 etc. adopt interferon-gamma (IFN-γ), IL-2 and mouse-anti people CD3 monoclonal antibody (Mouseanti-HumanCD3mAb) has induced the cell mass with powerful anti-tumor activity, called after cytokine induced kill cell (CIK) jointly.

Dendritic cell (DCs) is the professional antigen presenting cell (APC) that in known up to now body, function is the most powerful, there is abundant antigen capture molecule in its surface, antigen presentation molecule (MHCI class and MHC class Ⅱmolecule), co-stimulators (CD80, CD83, CD86, CD40 etc.).DC cell after antigenic stimulation moves to lymphoglandula, and the antigenic information of being carried passes to corresponding T lymphocyte, starts, excites CD4+/CD8+T cellullar immunologic response, killing off tumor cells specifically.DCs simultaneously after antigenic stimulation can secreting leukocytes mesonium-8 (IL-8), interferon alpha (IFN-α), interleukin 12 (IL-12), tumor necrosis factor alpha (TNF-α) etc., enhancing body nonspecific immune response (natural immunity), therefore DC has the laudatory title of " natural immunity adjuvant ", its discoverer professor RalphSteinman obtains Nobel Prize in medicine in 2011.In view of the Central Position that DC is residing in immunity of organism system of defense, the anti-tumor vaccine based on DC exploitation has made it to become one of most advanced, most promising immunotherapy of tumors technology, and recent domestic scholar has carried out exploring widely to it.2 professors of Stanford medical college in 1992 have set up the tumor vaccine company (Dendreon, CA) of first hand based on DC in the world, and the product P rovenge of the said firm obtains FDA approval listing on April 29th, 2010.But DC function is in vivo subject to the immunological status of patient own, the impact of tumor microenvironment, and there is a large amount of supressor in tumour patient body, effect in the body of therefore DC is also desirable not as in vitro effects.

It is the immunotherapy techniques risen in the recent period that cytotoxic T lymphocyte (CTL) builds, and because it has ability that is special, direct killing tumour target cell, therefore becomes the focus of research.

Three, the effect of Her2-neu in immunotherapy of tumors

Her2-neu is a kind of proto-oncogene, and coded product is the transmembrane glycoprotein p185 of 185kD, and mainly comprise extracellular region, cross-film district and intracellular region three part, wherein halfcystine is rich in extracellular region, containing 2 ligand binding domains; Weigh anchor to have made to order and use in cross-film district; Intracellular region has inherent tyrosine kinase activity, and containing self Tyr phosphorylation site.Her2-neu proto-oncogene is expressed by increasing or transcribe exception, is also activated by transgenation, and the tumour originated from multiple epithelial cell is related closely.The high expression level state of amplification and p185 is there is in Several Kinds of Malignancy tissue.

Within 1998, the humanized restructuring Her2 monoclonal antibody Herceptin of U.S. FDA approval is applied to clinical, and treatment has the patient with breast cancer of Her2-neu gene amplification and process LAN; Within 2010, European Union ratifies again the treatment of Herceptin for Her2 positive gastric carcinoma patient, and in October in the same year, U.S. FDA is also ratified Herceptin and is used for the treatment of late gastric cancer.

But Herceptin only has about 20-30% for the clinical effective rate of Her2-neu+++ patient, use initial response is good, life-time service will produce resistance phenomenon (antibody neutralization), and price comparison is expensive, and the medical expense of a year is about 300,000.Within 2003, U.S. FDA ratifies the Patients with Non-small-cell Lung of Her2-neu signal transduction inhibitor Iressa (Gefitinib) for Her2 high expression level.But tumour cell exists the abnormal activation of many signal paths, because which limit the clinical efficacy of molecular targeted agents.

Therefore, development of new is imperative for the medicine of Her2/neu target spot.The exploitation of anti-Her2/neu 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 storehouse in body, by the extracellular antigens molecule (bacterium, virus and tumor-antigen peptide) that φt cell receptor (TCR) specific recognition that its surface expression is different is different, by the CTL of activation for having kill target cell, the infection of bacteria removal, virus and tumour cell, and can immunological memory CTL be produced, prevent the recurrence of bacterium, viral invasion and tumour again.

CTL immunne response roughly divides four-stage:

(1) antigen recognition: APC is by surface receptor identification and capture antigen (bacterium, virus, tumour cell);

(2) antigen processing and submission: antigen digests through APC, is cracked into peptide molecule, MHC-I class that the latter is abundant in APC born of the same parents or class Ⅱmolecule are combined and form MHC-I-polypeptide or MHC-II-polypeptide complex respectively, and are transported to APC surface;

(3) t cell activation (dual signal theory): APC and T cell are met, T cell identifies the MHC-I/II-polypeptide complex of APC submission by self TCR, wherein CD8+T cell recognition MHC-I-polypeptide complex, CD4+T cell recognition MHC-II-polypeptide complex, the stimulus signal (the first signal) that T cell receives antigen adds that immune costimulatory signal (second signal) starts activation, has cytotoxic effect and CTL;

(4) target cell is killed: activation CTL is circulated to position, antigen place, by carrying out killing and wounding and removing after the TCR specific recognition antigen-expressing cells on CTL surface.

But found to there is a large amount of immunosuppressive factor in tumour patient body, affect effective activation and the propagation of CTL, quantity is very few, be not enough to contend with the tumour of breeding rapidly.If a large amount of Peptide-specific CTL can be prepared in vitro and feed back to patient, can directly, quick killing tumor cell plays therapeutic action, the remaining tumor cells that conventional treatment cannot be removed is killed, recurrence and the transfer of tumour can be prevented.

2, CTL phenotype and function difference

Expression kind according to CTL surface molecular can be divided into amphitypy: central memory-type CTL (CTL _-CM) and effect memory-type CTL (CTL _-EM).

Klebanoff etc. study discovery: phenotype analytical is the CTL of CD3+CD45RO+CD62L+CCR7+ _-CMthere is stronger anti-tumor capacity; Johnson etc. compare the CTL of MART-1TCR genetic modification _-CMand CTL _-EM, found that the former the disappear ability of malignant melanoma cell is 10 times of the latter.The results of study such as Berger also show CTL _-CMbe the CTL of CD3+CD45RO+CD62L-CCR7-compared with phenotype after infusion _-EMcan survive the longer time in vivo.

3, CTL technological development present situation

Comprehensively the external technology of preparing of domestic, external CTL can classify as three kinds of methods and source:

(1) tumor infiltrating lymphocyte (TIL) amplification in vitro method

From tumour patient tumor tissues, be separated TIL, add interleukin II (IL-2) and cultivate, the T cell clone of the clonal dilutions method screening tumour antigen positive, then add mouse-anti people CD3 monoclonal antibody and IL-2 carries out amplification cultivation acquisition.

Li etc. select IV phase melanoma cancer patients of the HLA-A201 positive, after its tumor tissues is prepared into single cell suspension, add IL-2 to cultivate, after 5 weeks, in screening amplifying cells, the positive T cell of CD8+T+MART-1+ is cloned, add the PBMC feeder cell amplification cultivation after mouse-anti people CD3 monoclonal antibody and irradiation again, next day adds IL-2, then cultivates and can obtain CTLs in 12 days, to the T2 cell strain specific recognition of load MART-1 polypeptide and can kill and wound.

(2) tcr gene modifies method

TCR is the key molecule of T cell identification antigen, mediated immunity response, comprise α, β, γ, δ tetra-chains, connect into α/β and gamma/delta two kinds of heterodimers 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 namely by exogenous plasmid transfection into autologous T lymphocyte, and import the tcr gene of energy identification specificity antigenic peptide, select positive colony, amplification cultivation obtains CTL in vitro.Comparatively conventional plasmid has retroviral plasmid or slow virus plasmid.

Morgan etc. adopt AntiCD3 McAb+CD28+ magnetic bead to activate CD8+T cell, and application slow virus plasmid transfection modifies the specific tcr gene of MART-1 or gp100, adopts mouse-anti people's CD3 monoclonal antibody and IL-2 culture system to prepare CTLs, 12 days energy amplification cultivation to 10 ⁹individual cell, for the treatment of the melanoma patients of 15 routine HLA-A2+, result shows 2 routine patients and has occurred obvious clinical efficacy, and the armpit metastatic lesion of 1 example 52 years old male patient disappears, and hepatic metastases focus reduces 89%, and the disease free survival phase is 21 months; 1 example 30 years old male patient, lung's metastatic lesion disappears, and the disease free survival phase is 20 months.

Perro etc. adopt Interleukin-15 (IL-15) and IL-21 (IL-21) combination of cytokines to promote the T cell of tcr gene transfection non-proliferative, can maintain T cell high expression level CD62L and CD28.

(3) exo-antigen sensitization method

With artificial APC (ArtificialAPC), or antigen (protein, polypeptide, or full cell) external repetitious stimulation T cell, obtain Peptide-specific CTL.

Namely the method for patent of invention (patent No.: CN102168066A) the external evoked hepatitis b virus specificity cell toxicity T lymphocyte of Zhejiang University adopts the method.MHC-I-Ig and anti-CD28mAb bag is built artificial APC by magnetic bead, and after epitope peptide load APC, external sensitization 3-4 week, then with magnetic bead fractionation by adsorption Peptide-specific CTL, Tetramer dyes qualification.

The defect of above-mentioned three kinds of methods:

(1) TIL amplification in vitro method

1. need the fresh tumor tissue obtaining patient to originate as CTL, be only limitted to the patient that can perform the operation, and tumor tissues limited source;

2. TIL is separated, the acquisition of ctl clone and authentication method complexity, and in tumor tissues, various cellular constituent is more, more complicated, and the time of isolated cell is long, generally all needs more than 1 month;

3. be mixed with CD4+CD25+Treg subgroup in TIL, it can suppress CTL amplification efficiency;

4. because in-vitro separation proliferation time is long, the chance of pollution is added.

(2) tcr gene modifies method

1. the introducing of exogenous plasmid (retrovirus or slow virus etc.), brings certain risk to clinical application;

2. endogenous TCR disturbs, and the tcr gene of importing may can not import the site of expection, and with endogenic TCR, wrong row occurs, and it can identify that antigen is not desired design, and is unknown, there is very large risk.

(3) external artificial APC Antigen sensitization method

1. introduce allogenic material: artificial APC, whether the CTL of preparation has artificial APC to remain is still query;

2. the cycle of the artificial external sensitization CTL of APC is longer, needs 3-4 week, and obtain the needs that could meet clinical treatment after CTL also needs amplification cultivation, therefore the cycle of vitro culture is long, and the probability of pollution is larger.

At present also not having reported in literature to cross, preparation time is short, amplification times is high, CTL purity is high and the preparation method of the restricted Peptide-specific CTL of fragmentation effect is good HLA-A0201.

Summary of the invention

The object of the invention is to provide that a kind of preparation time is short, amplification times is high, CTL purity is high and the preparation method of the restricted anti-Her2-neu Peptide-specific CTL of fragmentation effect is good HLA-A0201 for above-mentioned technical problem.

The object of the invention is to be realized by following technical proposal:

The preparation method of the restricted anti-Her2-neu Peptide-specific CTL of HLA-A0201, the method comprises the following steps:

A. the enrichment of the first step CTL precursor cell derived cell and purification process:

Peripheral blood mononuclear cells (PBMC) is gathered as cell derived with automatic mononuclearcell Acquisition Instrument (Dan Caiyi); Adopt the negative Beads enrichment method of clinical grade, enrichment from PBMC, purifying CTL precursor cell and CD8+T lymphocyte.

B. target CTL induction was increased with the first round:

CD8+T lymphocyte is stimulated with the autologous mature dendritic cell (mDC) of load HLA-A0201 restricted Her2-neu antigenic peptide, not only be supplied to first signal (antigen) of T cell activation, DC is supplied to the necessary second signal of T cell activation (co-stimulators CD40, CD80, CD83 etc.) simultaneously; Add rhIL-2 and rhIL-7 two kinds of cytokines simultaneously and combine the growth of promotion T cell, suppress the apoptosis of activation induction to react (AICD), extend the life cycle of active cells.Second week again repetitive stimulation once, completes first round amplification.

C. target CTL purification process:

To stimulate and after the target CTL quantity that increases at the mDC of first round Antigen, adopt Tetramer marking method and Flow cytometry again purification of target CTL (target CTL refers to the CD8+T expressing and identify Her2-neu antigenic peptide TCR, i.e. Tetramer+CD8+T lymphocyte), namely Tetramer+CD8+T is selected, reject the T cell of non-specific amplification, to reduce the impact that it is bred target CTL.

D. second of target CTL amplification is taken turns:

Adopt growth and the propagation of anti-human-CD3mAb and the rhIL-2 stimulation target CTL of solid-phase coating; Add the activation of autologous PBMC enhancing to target CTL through gamma-ray irradiation again; Add the propagation that rhIL-15 mono-aspect can promote memory t cell, apoptosis capable of inhibiting cell on the other hand.

E. second the rear target CTL phenotypic evaluation of amplification is taken turns:

Flow cytometry is adopted to carry out phenotype analytical to the target CTL prepared, different from Memory CTL (CTL centered by its point according to its molecular spectra of expressing _-CM: CD8+CD45RO+CD62L+CCR7+) and responsiveness CTL (CTL _-EM: CD8+CD45RO+CD62L-CCR7-).

Described preparation method, the target antigen in step b is the preferred 9-15 of a Her2-neu antigenic peptide length amino acid, the restricted Her2-neu antigenic peptide of the HLA-A0201 of further preferred sequence as shown in SEQIDNO.1.

Described preparation method, being prepared by following method from body maturation DC of load target antigen in step b: add rhGM-CSF, rhIFN α, the mixing normal human AB serum of deactivation and RPMI1640 culture medium culturing three days after PBMC is adherent, the DC of results adds target antigen again and hatches and get final product.

Described preparation method, is prepared by following method for the autologous PBMC of the gamma-ray irradiation of target CTL amplification in steps d: leave and take part in step a and singly adopt the PBMC of acquisition, preserve after the gamma-ray irradiation of 30-50GY; Conserving liquid: containing the RPMI1640 nutrient solution of 20% (V/V) DMSO and 20% (V/V) normal human AB serum, storage temperature :-80 DEG C of Excised Embryos.

Described preparation method, for the CD3 monoclonal antibody clonal antibody (anti-human-CD3mAb of target CTL amplification in steps d, be called for short CD3 monoclonal antibody) be following operation: by CD3 monoclonal antibody solid-phase coating in the vessel surface being used for amplifying cells, CD3 monoclonal antibody can be combined with the CD3 molecule on multiple CTL surface, promote the formation of CTL cell clone, promote cells contacting, enter proliferating cycle fast.

Described preparation method, the consumption of each step moderate stimulation molecule or cell is respectively:

A. the target CTL first round increases each add-on: rhIL-2 final concentration is 500-750IU/ml, rhIL-7 final concentration be 50-75ng/ml, DC and initial T cell number ratio is 1:5.

B. target CTL second takes turns amplification: rhIL-2 final concentration is 200-500IU/ml, rhIL-15 final concentration be 100-250ng/ml, CD3 monoclonal antibody bag is 1.0-2.5 μ g/ml by concentration, and PBMC and the initial CTL number ratio of gamma-ray irradiation are 2:1.

Described preparation method, after each step operation, cell quantity and phenotypic characteristic are in table 1.

Table 1

Below the explanation specifically of the key step to preparation method of the present invention:

1, CTL precursor cell purifying and enriching method:

Usual employing periphery PBMC is as CTL precursor cell and the lymphocytic source of CD8+T, containing various kinds of cell compositions such as CD4 positive t lymphocytes (CD4+T), CD8 positive t lymphocytes (CD8+T), bone-marrow-derived lymphocyte, monocyte (Mo), natural killer cells (NK) in PBMC, wherein CD8+T starting quantity directly affects the ultimate capacity of target CTL.

Adopt and singly adopt that method is disposable gathers a large amount of periphery PBMC, obtain a large amount of initiator cell, unnecessary PBMC can irradiation or frozen, and for repeatedly CTL Synthesis and applications, and method is easy and simple to handle.

Adopt clinical grade magnetic bead Solid phase method purifying CD8+T from PBMC, reject CD4+T, B, NK cell, reduce the interference of its rapid amplifying to CD8+T amplification growth.The purity of CD8+T can be improved 2-4 doubly (being increased to average more than 90%) by magnetic bead Solid phase method, see accompanying drawing 1.

2, target CTL induction was increased with the first round:

The present invention adopts three days ripe DC preparation methods, DC prepare simple and direct, the time is short, its form, phenotype compare no significant difference with a conventional 7-10 days DC, see accompanying drawing 2.

Patent load target antigen of the present invention from body maturation DC, give CD8+T activation the first signal; DC is rich in co-stimulators simultaneously, gives the CD8+T second signal of activation, ensures that CD8+T is induced to differentiate into target CTL efficiently under dual signal effect.Associating rhIL-2 and rhIL-7 carries out to CD8+T the effect that amplification is better than monofactor (IL-2), and IL-7 promotes the propagation of memory t cell clone, suppresses the AICD because causing after long-time IL-2 activating T cell react, prolongation T cell life cycle.

3, target CTL purifying and enriching method:

After first round induced amplification, in CD8+T cell, namely target CTL expresses and identifies that the CD8+T of target antigen (i.e. the restricted Her2-neu antigenic peptide of HLA-A0201) TCR only accounts for fraction, and ratio is not high, the CD8+T of non-specific amplification in a large number, without the effect identifying, remove target cell.The present invention adopts that Tetramer marking method is separated with Flow cytometry, purification of target CTL, and enrichment can identify the CTL of target antigen again, and these CTL just possess the ability of killing and wounding target cell.

After purifying, the purity of target CTL reaches more than 95%.And the several method of conventional report adopts limiting dilution assay, the methods such as electroinjection mostly, time cost is longer, and complicated operation, specificity is relatively low.See accompanying drawing 3.

4, target CTL second takes turns amplification:

The mouse-anti people CD3/CD28 monoclonal antibody of liquid phase and IL-2 combination is adopted to increase to T cell amplification.The present invention adopts solid phase mouse-anti people CD3 monoclonal antibody (anti-human-CD3mAb) coating technique, combines the multiple signal of breeding as stimulation T cell through autologous PBMC and rhIL-15 of gamma-ray irradiation.Solid phase CD3-mAb can promote T cell Clone formation, accelerates Growth of Cells; IL-15 makes CTL have anti-apoptotic, promotes the ability of memory T cell growth; The a large amount of costimulatory molecules of PBMC surface expression also secretes the growing environment that cytokine profiles provides CTL good, and can not breed after gamma-ray irradiation and disturb the growth of CTL, thus ensures the efficient amplification of CTL.

Compare with conventional amplification system: target CTL output prepared by present method is high, CTL _cMratio is high, and it is stronger to kill and wound target cell ability.See accompanying drawing 4-5.

Beneficial effect of the present invention:

The present invention combines and adopts efficient, the simple and direct CD8+T that to obtain with magnetic bead sorting method than ordinary method a greater number of singly adopting as target CTL precursor cell; By the dendritic cell of HLA-A0201 restricted Her2-neu antigenic peptide load as sensitinogen, associating rhIL-2 and rhIL-7 induces CTL precursor clone to increase to target CTL differentiation and the first round; From the thin group of mixed C D8+T lymph of sensitization, target CTL is isolated again through the tetramer (tetramer) mark and fluidic cell sorting technology, by adopting solid phase anti-human-CD3-mAb, the PBMC of gamma-ray irradiation is as feeder cell, and associating IL-2 and IL-15 carries out second to target CTL and take turns efficient amplification.The target CTL adopting the inventive method to prepare possesses high purity, high proliferation ability, and High Fragmentation is active, CTL at high proportion _-CMfeature, can be used for the immunotherapy of Her2-neu associated malignancies.

Secondarily purified and the enrichment that the present invention adopts different methods to pass through the restricted anti-Her2-neu Peptide-specific CTL of HLA-A0201, by different amplification system, the induction that two take turns amplification and function is carried out to the target CTL of enrichment, make target CTL in purity, quantitatively and be functionally all better than several CTL preparation method of the prior art, be embodied in the following aspects especially:

1, the invention provides natural, efficient activated T cell dual signal

1. antigen-specific signal: DCs is the professional antigen presenting cell that in generally acknowledged body, function is the strongest, body series imitates, copies antigen is caught submission in vivo process by DC, select dominant antigen peptide load live body DC as sensitinogen, there is provided closest to the MHC-polypeptide complex space structure in human immunity answering, for T cell activation provides specificity first signal.

2. the costimulatory molecules of expressed in abundance while of costimulatory signal: DC, for 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 and singly adopt negative magnetic bead sorting CTL precursor cell and CD8+T lymphocyte with clinical grade, easy and simple to handle, starting quantity is sufficient, and the CD8+T cell purity of acquisition reaches more than 90%, and for preparing q.s, enough purity target CTL provide basic guarantee.

2. the purifying of target CTL cell: adopt Tetramer mark and flow sorting techniques, separation and purification is carried out to target CTL and Tetramer+CD8+T, simple and direct, efficient, save time (4-5 hour), high purity more than 95%.

3, the invention provides two to take turns, efficient target CTL amplification system, to ensure to provide sufficient amount target CTL

1. first round induction and amplification: the DC adopting polypeptide load, combine two kinds of cytokine IL-2 and IL-7 amplification system, in two weeks, amplification times reaches 30-50 doubly.

2. second target CTL amplification is taken turns: adopt solid phase mouse-anti people CD3 monoclonal antibody, associational cells factor IL-15 and autologous PBMC amplification system, about 10 days average 40 times of amplification times, are up to 60 times.Can 10 be obtained ⁹individual target CTL cell, reaches the needs of clinical application, and ordinary method (background technology mention three kinds of external evoked CTL reaching same quantity of method need 3-4 week.

4, the target CTL Cytotoxicity in vitro vigor prepared of the present invention is higher

The restricted anti-Her2-neu Peptide-specific CTL of HLA-A0201 prepared by the present invention external energy specific recognition and the target cell killing and wounding expression corresponding antigens, and kill rate height sky ordinary method, effect target on average exceeds about 15% than kill rate time identical.See accompanying drawing 5.

M1 is ordinary method one: the target CTL first round increase and sorting purifying with this patent method, when target CTL second takes turns amplification, with containing 10% deactivation mixing normal human AB serum RPMI1640 perfect medium re-suspended cell, cell density is 2 × 10 ⁶/ ml, is seeded to 75cm ²in Tissue Culture Flask, every bottle of 30ml, adds mouse-anti people CD3 monoclonal antibody (liquid phase) that final concentration is 50ng/ml and final concentration is the rhIL-2 of 500IU/ml, and mixing is placed in 37 DEG C gently, cultivates in 5%CO2 cell culture incubator.Every day observation of cell growth conditions, supplement the RPMI1640 perfect medium of fresh culture containing 10% deactivation mixing normal human AB serum, 200IU/mlrhIL-2, cell proliferation is to a certain amount of expansion bottle in good time.Be cultured to the 10th day harvested cell.With brine 2 times and resuspended, calculate the cell quantity obtained.Get 3 × 10 ⁵individual left and right cell, adds anti-human CD8, CD45RO, CD62L, CCR7 monoclonal antibody and carries out labeling CT L, carries out detecting the ratio analyzing CD8+CD45RO+CD62L+CCR7+T with flow cytometer.And the kill capability to target cell.Average acquisition CTL cell total amount is (0.9 ± 0.4) × 10 ⁹, CTL _cMratio average out to: 30.5 ± 4.1%, effect target is than being CTL lethality average out to 25.6 ± 4.8% during 30:1.

M2 is ordinary method two: the target CTL first round increase and sorting purifying with this patent method, when target CTL second takes turns amplification, with containing 10% deactivation mixing normal human AB serum RPMI1640 perfect medium re-suspended cell, cell density is 2 × 10 ⁶/ ml, is seeded to 75cm ²in Tissue Culture Flask, every bottle of 30ml, adds 1.5 × 10 ⁷/ ml AntiCD3 McAb/28 magnetic bead and final concentration are the rhIL-2 of 500IU/ml, and mixing is placed in 37 DEG C gently, cultivates in 5%CO2 cell culture incubator.Every day observation of cell growth conditions, supplement the RPMI1640 perfect medium of fresh culture containing 10% deactivation mixing normal human AB serum, 200IU/mlrhIL-2, cell proliferation is to a certain amount of expansion bottle in good time.Be cultured to the 10th day harvested cell.With brine 2 times and resuspended, calculate the cell quantity obtained.Get 3 × 10 ⁵individual left and right cell, adds anti-human CD8, CD45RO, CD62L, CCR7 monoclonal antibody and carries out labeling CT L, carries out detecting the ratio analyzing CD8+CD45RO+CD62L+CCR7+T with flow cytometer, and the kill capability to target cell.Average acquisition CTL cell total amount is (1.0 ± 0.3) × 10 ⁹, CTL _cMratio average out to: 32.1 ± 3.6%, effect target is than being CTL lethality average out to 23.2 ± 4.4% during 30:1.

M3 is this patent method, and average acquisition CTL cell total amount is (2.9 ± 0.4) × 10 ⁹, CTL _cMratio average out to: 71.4 ± 5.3%, effect target is than being CTL lethality average out to 40.6 ± 3.9% during 30:1.

5, CTL in the target CTL cell prepared of the present invention _cMratio is high

The centre type memory CTL ratio expressing CD3+CD45RO+CD62L+CCR7+ phenotype in CTLs prepared by the present invention, higher than ordinary method, is shown in accompanying drawing 4.

Compare the amplification ability of target CTL prepared by three kinds of amplification systems, phenotype and lethality, result shows: the CTL of this patent induction system induction increases ability the most by force, and cell total amount can reach (2.9 ± 0.4) × 10 ⁹, wherein CTL _cMratio can reach 71.4%, and when imitating target ratio for 30:1, the kill capability for target cell (the T2 cell strain of load HLA-A2-Her2-neu polypeptide) on average can reach 40.6%.

Accompanying drawing explanation

The comparison of purity before and after Fig. 1: CTL precursor cell purifying.

Figure A: singly adopt PBMC FCM analysis collection of illustrative plates (before purifying)

Method: get 50 μ lPBMC (about 3 × 10 respectively ⁵individual cell) add in 3 streaming pipes, add the anti-human CD4 antibody that 5 μ lFITC mark in the first pipe again, the anti-human CD8 antibody that 5 μ lPE mark, the anti-CD3antibody that 5 μ lPerCP mark and the anti-human CD56 antibody that 5 μ lAPC mark; The anti-human CD14 antibody that 5 μ lAPC mark is added in second pipe; 3rd pipe adds the anti human CD 19 antibody that 5 μ lAPC mark, abundant mixing, 4 DEG C of lucifuges hatch 30 minutes, after taking-up often pipe add 1ml precooling containing 2% (V/V) new-born calf serum brine twice and resuspended after, detect with FACSCalibur flow cytometer, adopt Cellquest software analysis CD3+CD4+T%, CD3+CD8+T%, CD14+%, CD19+%, CD3-CD56+% ratio.

Result:

CD14+％(Mo)：8.32％CD19+％(B)：0.16％

CD3+CD4+T％：7.81％CD3+CD8+T％：46.97％

CD3-CD56+％(NK)：0.20％

CTL precursor cell FCM analysis collection of illustrative plates (after purifying) after figure B magnetic bead Solid phase

Method: singly adopt PBMC CD4+CD19+CD16/56+T sorting magnetic bead Solid phase, after rejecting CD4+T cell, NK cell and B cell, collect and dye according to the streaming dyeing process before purifying in conjunction with magnetic cell, analyze the change of CD3+CD4+T%, CD3+CD8+T%, CD14+%, CD19+%, CD3+CD56+%, CD3-CD56+% ratio.Result shows, and after magnetic bead Solid phase, CD3+CD8+T% ratio reaches 90.55%, and purity improves two times.

Result:

CD3+CD4+T％：1.80％CD3+CD8+T％：90.55％

CD14+％(Mo)：0.14％CD19+％(B)：0.35％

CD3-CD56+％(NK)：0.89％

The DC phenotype of Fig. 2: two kinds of different methods inductions compares

Method: get the DC (about 3 × 10 that 50 μ lrhGM-CSF+rhIFN-α induce 3 days respectively ⁵individual cell) add in 4 streaming pipes, first pipe adds the CD40 that 5 μ lPE mark, the anti-human CD11c antibody that the HLA-DR that 5 μ lPerCP mark and 5 μ lAPC marks, second pipe adds the anti-human CD80 antibody that 5 μ lPE mark, the anti-human HLA-DR antibodies that 5 μ lPerCP mark and the anti-human CD11c antibody that 5 μ lAPC mark, 3rd pipe adds the anti-human CD83 antibody that 5 μ lPE mark, the anti-human HLA-DR antibodies that 5 μ lPerCP mark and the anti-human CD11c antibody that 5 μ lAPC mark, 4th pipe adds the anti-human CD86 antibody that 5 μ lPE mark, the anti-human HLA-DR antibodies that 5 μ lPerCP mark and the anti-human CD11c antibody that 5 μ lAPC mark, abundant mixing, 4 DEG C of lucifuges hatch 30 minutes, after taking-up often pipe add 1ml precooling containing 2% (V/V) new-born calf serum brine twice and resuspended after, detect with FACSCalibur flow cytometer, adopt Cellquest software analysis HLA-DR+CD11c+%, CD40+HLA-DR+CD11c+%, CD80+HLA-DR+CD11c+%, CD83+HLA-DR+CD11c+%, CD86+HLA-DR+CD11c+% ratio.The DC same method that rhGM-CSF+rhIL-4 induces 7 days detects.Result shows, and the DC purity phenotype (HLA-DR+CD11c+) that the rhGM-CSF+rhIFN-α that this patent adopts induces 3 days is 95.66%, and ripening degree phenotype (CD83+) is 82.94%.The DC purity phenotype that rhGM-CSF+rhIL-4 induces 7 days is 95.36%, and ripening degree phenotype is 90.16%, both no significant differences.

Result:

Figure A:rhGM-CSF+rhIFN α induces 3 days DC phenotype flow cytometer detection collection of illustrative plates:

HLA-DR+CD11c+％：95.66％CD40+HLA-DR+CD11c+％：93.60％

CD80+HLA-DR+CD11c+％：81.97％CD83+HLA-DR+CD11c+％：82.94％

CD86+HLA-DR+CD11c+％：84.94％

Figure B:rhGM-CSF+rhIL-4 induces 7 days DC phenotype flow cytometer detection collection of illustrative plates:

HLA-DR+CD11c+％：95.36％CD40+HLA-DR+CD11c+％：92.24％

CD80+HLA-DR+CD11c+％：82.36％CD83+HLA-DR+CD11c+％：90.16％

CD86+HLA-DR+CD11c+％：91.88％

Fig. 3 target CTL first round increase after flow cytometer detection collection of illustrative plates

A: the target CTL first round of figure increase afterwards cell flow cytometer detection collection of illustrative plates (before purifying)

B: the target CTL first round of figure increase afterwards cell flow cytometer detection collection of illustrative plates (after purifying)

Method: the CTL first round increase after with PE mark HLA-A2+Her2-neu+ Tetramer and FITC mark anti-human CD8 antibody to amplification CTL mark, adopt selected by flow cytometry apoptosis Tetramer+CD8+CTL cell, before sorting, Tetramer+CD8+T purity is 1.25%, reaches 98.41% after sorting.

The comparison of CTL phenotype prepared by Fig. 4 tri-kinds of different methods

Figure A (M1): CTL phenotype flow cytometer detection collection of illustrative plates prepared by liquid phase mouse-anti people CD3 monoclonal antibody+IL-2 amplification system

Figure B (M2): liquid phase CD3/28 magnetic bead, CTL phenotype flow cytometer detection collection of illustrative plates prepared by IL-2 amplification system

Figure C (M3): solid phase mouse-anti people CD3 monoclonal antibody, IL-2, IL-7, IL-15, CTL phenotype flow cytometer detection collection of illustrative plates prepared by autologous PBMC amplification system

Method: the target CTL after purifying is divided into 3 parts, first part adds final concentration 50ng/ml mouse-anti people's CD3 monoclonal antibody and 500IU/mlIL-2 carries out amplification cultivation, second part adds final concentration 10 μ g/mlCD3/28 magnetic bead and 250IU/mlIL-2 carries out amplification cultivation, 3rd part adds the pre-coated culturing bottle spent the night of mouse-anti people CD3 monoclonal antibody 2 μ g/ml, add the PBMC through gamma-ray irradiation according to the ratio of target CTL:PBMC=1:2, and add the IL-2 of final concentration 500IU/ml and final concentration 25ng/mlIL-15 and carry out amplification cultivation.Be cultured to the 7th day results CTL.Get 50 μ lCTL (about 3 × 10 ⁵individual cell) add in 2 fluid-guiding type pipes, first pipe adds the anti-human CD62L antibody that 5 μ lFITC mark, the anti-human CCR7 antibody that 5 μ lPE mark, the anti-human CD45RO antibody that 5 μ lAPC mark, second pipe adds the anti-CD3antibody of 5 μ lPerCP marks and the anti-human CD8 antibody of 5 μ lPE mark, abundant mixing, 4 DEG C of lucifuges hatch 30 minutes, after taking-up often pipe add 1ml precooling containing 2% (V/V) new-born calf serum brine twice and resuspended after, detect with FACSCalibur 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 CTL prepared by this patent method is 97.87%, wherein CD45RO+CD62L+% reaches 73.10%, CCR7+CD62L+% reaches 71.81%, apparently higher than method 1 and 2.

The comparison of CTL kill capability prepared by Fig. 5 different methods is (with Her2-neu _650-658cTL is example).

CTL kill capability prepared by figure A (M1) liquid phase mouse-anti people CD3 monoclonal antibody+IL-2 amplification system

Figure B (M2) liquid phase CD3/28 magnetic bead, CTL kill capability prepared by IL-2 amplification system

Figure C (M3) solid phase mouse-anti people CD3 monoclonal antibody, IL-2, IL-7, IL-15, CTL kill capability prepared by autologous PBMC amplification system

Method: target cell 1: be load Her2-neu _650-658the HLA-A2+T2 cell strain of polypeptide, target cell 2: be the strain of HLA-A2+T2 non-loaded cells, target cell 3:K562 (strain of NK sensitive cells).

CTL prepared by embodiment 1 mixes according to imitating the ratio of target than 3:1,10:1 and 30:1 with above-mentioned 3 kinds of target cells respectively, and mtt assay detects CTL killing activity.Result shows, and CTL prepared by the inventive method imitating target than during for 10:1, reaches 23.64%, apparently higher than method 1 and 2 to the specific killing activity of target cell.

Specific implementation method

The invention will be further elaborated by the following examples.

Embodiment 1Her2-neu _650-658prepared by antigenic peptide specific CTL

1, HLA-A201+Her2-neu+ patient with breast cancer prepares

Extract patient peripheral's anticoagulation 1ml, add the HLA-A2-mAb of FITC mark, through flow cytometer detection HLA-A2 (abbreviation of HLA-A201, lower same) expression; Detect Her2-neu antigenic expression.HLA-A2 and Her2-neu expresses positive simultaneously and is selected in.

2, the restricted Her2-neu antigenic peptide synthesis of HLA-A201

Site is 650-658, and sequence is PLTSIISAV (SEQIDNO.1) 9 peptide, hereinafter referred to as Her2-neu _650-658polypeptide, by chemosynthesis (the biochemical company limited of Shanghai gill), fully dissolve with aseptic double-distilled water, peptide concentration is 5mg/ml, and packing is stored in-80 DEG C.

3, PBMC gathers

With singly adopting instrument collection patient peripheral PBMCs, be separated PBMCs through Ficoll density gradient centrifugation, part is used for DC induction, and part is used for the enrichment of CTL precursor cell and purifying, and part is frozen in-80 DEG C through gammairradiation.

4, DC induction and antigen load

Adherent method prepares DC: be suspended in by PBMCs in RPMI1640 substratum, and cell density is 3 × 10 ⁶/ ml, inoculating cell is in 75cm ²in culturing bottle, in 5%CO ₂, hatch 90 minutes in 37 DEG C of incubators, wash with pre-warm saline and wash 2-3 time gently.Attached cell is used for DC induction.

DC induces: the RPMI1640 perfect medium adding the mixing normal human AB serum of 30mlDC inducing culture (volume ratio, lower with) deactivation containing 10%, final concentration 200ng/mlrhGM-CSF, final concentration 500ng/mlrhIFN-α in culturing bottle.Induce and gather in the crops DCs after 3 days.

Polypeptide DC load: be suspended in by the DC of results in RPMI1640 substratum, cell density is 1 × 10 ⁶/ ml is also inoculated in cell cultures 6 orifice plate, and every hole 3ml, adds Her2-neu in hole _650-658polypeptide to final concentration is 10 μ g/ml, is positioned over 37 DEG C, 5%CO ₂educate altogether in incubator after 2 hours and gather in the crops, wash DC bis-times with RPMI1640 substratum, then use RPMI1640 substratum resuspended, cell density is 1 × 10 ⁶/ ml, for the stimulation of target CTL.

Autologous PBMC for the gamma-ray irradiation of target CTL amplification is prepared by following method: leave and take the PBMC that acquisition adopted by part list, preserve after the gamma-ray irradiation of 30-50GY (average 40GY); Conserving liquid: containing the RPMI1640 nutrient solution of 20% (V/V) DMSO and 20% (V/V) normal human AB serum, storage temperature :-80 DEG C of Excised Embryos.

5, the isolation and purification of CTL precursor cell

Gather in the crops in DC and to recover frozen PBMC the same day, with the PBS solution washed cell 2 times containing 2% deactivation mixing normal human AB serum of precooling, and re-suspended cell, adjustment cell density is 2 × 10 ⁷/ ml, clinical grade CD4+CD19+CD16/56+T sorting magnetic bead (MiltenyiCliniMACS) is added in cell, educate altogether after 30 minutes and cross post, collect the CD8+T lymphocyte flowed out, 2 times are washed with the RPMI1640 substratum of precooling, be resuspended in by cell containing in 10% deactivation mixing normal human 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 plate, every hole adds 3ml, add the DC (prepared by step 4) of 0.6ml load polypeptide again, add IL-2 and IL-7 and make final concentration reach 500IU/ml and 50ng/ml respectively, put into 5%CO after mixing gently ₂, cultivate in 37 DEG C of cell culture incubators; Every day observation of cell growth conditions, supplement fresh culture (containing 10% deactivation mixing normal human AB serum RPMI1640 perfect medium, 500IU/mlIL-2 and 50ng/mlIL-7) in good time.7th day, the DC (ditto) adding 0.6ml load polypeptide in every Kong Zhongzai stimulated again.Every day observation of cell growth conditions, in good time supplement fresh culture (ditto), carry out expansion bottle when cell density is excessive, cell proliferation can be moved to 75cm after a certain amount of ²continue amplification in culturing bottle to cultivate.

7, target CTL sorting purifying

Load Her2-neu _650-658dC and the CD8+T of polypeptide educates 12-14 days altogether, harvested cell, with the PBS solution washed cell 2 times containing 2% deactivation mixing normal human AB serum of precooling, and re-suspended cell, adjustment cell density is 1 × 10 ⁷/ ml, adds the HLA-A201+Her2-neu of PE mark in cell suspension _650-658the CD8mAb of Tetramer and FITC mark, gently after mixing, educates 20 minutes altogether in 4 DEG C of refrigerators.With the PBS solution washed cell 2 times containing 2% deactivation mixing normal human AB serum of precooling, re-suspended cell, adjustment cell density is 1 × 10 ⁷/ ml, carries out airflow classification by cell, and results CD8+Tetramer+T lymphocyte, abandons supernatant after centrifugal, and with the RPMI1640 perfect medium re-suspended cell containing 10% deactivation mixing normal human AB serum, cell density is 2 × 10 ⁶/ ml.The target CTL first round increase after flow cytometer detection collection of illustrative plates see Fig. 3.

8, target CTL second takes turns amplification

Tissue Culture Flask bag quilt: in step 7 the day before yesterday, get 75cm ²tissue Culture Flask, in wherein adding 2ml mouse-anti people CD3 monoclonal antibody (concentration 2 μ g/ml, producer: R & D company of the U.S., catalog number (Cat.No.): 59-MAB100, clone number: C1UCHT1, lower same), seal rearmounted 4 DEG C of refrigerator bags and spent the night.

Before inoculating cell, take out bag by culturing bottle, unnecessary CD3-mAb in culturing bottle is abandoned in suction, every bottle adds 30ml sorting CD8+Tetramer+T lymphocyte, add the PBMC (quantity 2 is doubly to target CTL) through gamma-ray irradiation, add final concentration 500IU/mlIL-2 and final concentration 250ng/mlIL-15, mixing is placed in 37 DEG C, 5%CO gently ₂cultivate in cell culture incubator.Every day observation of cell growth conditions, in good time supplement fresh culture (containing 10% people's deactivation mixing normal human AB serum, the RPMI1640 perfect medium of 200IU/mlIL-2,100ng/mlIL-15) control cell density; Cell proliferation is to a certain amount of expansion bottle.

9, target CTL results and Phenotypic examination

Second takes turns target CTL increases about 10 days, harvested cell.With brine 2 times, be resuspended in 100ml physiological saline, cell density is 10 ⁷/ ml, for immunotherapy.

Get 3 × 10 ⁵individual left and right cell, add anti-human CD8, CD45RO, CD62L, CCR7 monoclonal antibody and carry out labeling CT L, carry out with flow cytometer detecting the ratio analyzing CD8+CD45RO+CD62L+CCR7+T, the results are shown in Figure 4C, the CD3+CD8+T% of CTL prepared by present method is 97.87%, wherein CD45RO+CD62L+% reaches 73.10%, CCR7+CD62L+% and reaches 71.81%.

After each step operation, cell quantity and phenotypic characteristic are in table 1.

10, target CTL kill capability

By preparation target CTL respectively with load Her2-neu _650-658the HLA-A2+T2 cell strain of polypeptide, the strain of HLA-A2+T2 non-loaded cells and K562 (strain of NK sensitive cells) three kinds of target cells mixs than the ratio of 3:1,10:1 and 30:1 according to effect target, mtt assay detection CTL killing activity.Result shows, and CTL prepared by the inventive method imitating target than during for 10:1 to the specific killing activity of target cell, reaches 23.64%, sees Fig. 5 C.

Claims (9)

1.HLA-A0201 restricted anti-Her2-neu Peptide-specific CTL preparation method, is characterized in that the method comprises the following steps:

The enrichment of a.CTL precursor cell derived cell and purifying:

Collection peripheral blood mononuclear cells of singly gathering is originated as CTL precursor cell; Adopt the negative Beads enrichment method of clinical grade, enrichment from collection peripheral blood mononuclear cells, purifying CTL precursor cell and CD8+T lymphocyte;

B. target CTL induction was increased with the first round:

Stimulate CD8+T lymphocyte with the mature dendritic cell of the restricted MUC1 antigenic peptide of load HLA-A0201, add rhIL-2 and rhIL-7 two kinds of cytokines simultaneously and combine the growth of promotion T cell; Second week again repetitive stimulation once, completes first round amplification;

C. target CTL purification process:

Adopt Tetramer marking method and Flow cytometry purification of target CTL, namely select Tetramer+CD8+T lymphocyte;

D. target CTL second takes turns amplification:

Adopt the growth of anti-human-CD3mAb and the rhIL-2 stimulation target CTL of solid-phase coating; Add the activation of the enhancing of the autologous peripheral mononuclearcell after gamma-ray irradiation to target CTL; Add rhIL-15 to continue to cultivate, complete second and take turns amplification, collect qualification.

2. preparation method according to claim 1, is characterized in that HLA-A0201 restricted MUC1 antigenic peptide length 9-15 amino acid in step b.

3. preparation method according to claim 2, is characterized in that the restricted MUC1 antigen polypeptide sequence of described HLA-A0201 is as shown in SEQIDNo.1.

4. preparation method according to claim 1, it is characterized in that the mature dendritic cell of load HLA-A0201 restricted MUC1 antigenic peptide in step b is prepared by following method: add after peripheral blood mononuclear cells is adherent containing the mixing normal people AB of rhGM-CSF, rhIFN α, deactivation and RPMI1640 culture medium culturing three days, results DC adds target antigen again and hatches and get final product.

5. preparation method according to claim 1, it is characterized in that being prepared by following method for the autologous peripheral mononuclearcell of the gamma-ray irradiation of target CTL amplification in steps d: leave and take in Partial rights requirement 1 step a and singly adopt acquisition peripheral blood mononuclear cells, preserve after the gamma-ray irradiation of 30-50GY; Conserving liquid: containing the RPMI1640 nutrient solution of 20% (V/V) DMSO and 20% (V/V) normal human AB serum, storage temperature :-80 DEG C of Excised Embryos.

6. preparation method according to claim 1, it is characterized in that the anti-human-CD3mAb increased for target CTL in steps d is following operation: by anti-human-CD3mAb solid-phase coating in the vessel surface being used for amplifying cells, anti-human-CD3mAb is combined with the CD3 molecule on multiple CTL surface, promote the formation of CTL cell clone, promote cells contacting, enter proliferating cycle fast.

7. preparation method according to claim 1, is characterized in that target CTL refers to the CD8+T expressing and identify MUC1 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 increases each add-on: rhIL-2 final concentration to be 500-750IU/ml, rhIL-7 final concentration be 50-75ng/ml, DC with initial CD8+T cell quantity than being 1:5;

B. target CTL second takes turns amplification: rhIL-2 final concentration is 200-500IU/ml, rhIL-15 final concentration is 100-250ng/ml, anti-human-CD3mAb bag is 1.0-2.5ug/ml by concentration, and peripheral blood mononuclear cells and the initial CTL number ratio of gamma-ray irradiation are 2:1.

9. preparation method according to claim 1, after it is characterized in that the operation of each step, cell quantity and phenotypic characteristic are respectively: