CN1844372A - Amplification in vitro method for T lymphocyte specific for tumour antigen - Google Patents

Abstract

This invention relates to a cytotoxic T lymphocyte and its application in antineoplastic immunity. Concretely, the invention provides a method to induce T cells into cytotoxic T lymphocyte by combined use of interleukins 2, interleukins 15, activated anti-CD28 monocolonal antibodies and tumor antigen loaded antigen-presenting cells. The application of cytotoxic T lymphocyte in the antineoplastic immunity is also provided.

Description

The amplification in vitro method of T lymphocyte specific for tumour antigen

FIELD OF THE INVENTION

The present invention relates to cytotoxic T lymphocyte and the application in anti tumor immune response thereof.Specifically, the present invention relates to unite the antigen presenting cell that uses interleukin-22, interleukin 15 and anti-CD28 monoclonal antibody of excitated type and tumour antigen load becomes cytotoxic T lymphocyte with inducing T cell method.The invention further relates to the application of cytotoxic T lymphocyte in anti tumor immune response according to said method preparation.

The background of invention

In the antitumor immunity of organism answering, cellular immunization is in middle cardiac status.The specific cellular immune function of in-vivo tumour lowly be one of tumour major reason of escaping the unrestricted growth of immune supervision.How can supply the cytotoxic T lymphocyte (CTL) of the specific for tumour antigen of clinical application at external acquisition q.s is the problem that at first will face in the tumour adoptive immunotherapy.

The T cell is CD8 particularly ⁺And CD4 ⁺The T cell plays irreplaceable effect in the mediation anti-tumor immune response.The activation of T cell and amplification need the participation of a series of immunocytes and cytokine.Dendritic cell (DC) is given the T cell as the most powerful antigen presenting cell of function (APC) with the antigen presentation after picked-up, processing and the processing, gives first signal of T cell activation.Simultaneously, behind costimulatory molecules such as the CD80/CD86 on DC surface (B7-1/B7-2), CD40 and corresponding acceptor of T cell surface or the ligand interaction, the second signal of mediation T cell activation.In addition, the activation of CTL also needs CD4 ⁺The participation of cytokines such as the IL-2 of T emiocytosis and IFN-γ.

For tumour patient particularly for the middle and advanced stage tumour patient, because the interior tumor load of body is big and tumor cell proliferation is rapid, so when carrying out the autoimmunity treatment, require and to feed back then in patient's body to reach the purpose that suppresses or kill tumour cell at a large amount of self antigen specific CTL of amplification in vitro.Cytokine such as IL-2, IL-7, IL-15 and IFN-γ and the anti-CD28 of excitated type and CD3 monoclonal antibody studies show that, though all can be used for amplification in vitro CTL.Yet; the efficient of existing amplification method still remains further to be improved; particularly use increase regular meeting often of these cytokines to cause the apoptosis (AICD) of the T cell that is activated merely, thereby this area is wished to set up and a kind ofly can be avoided that AICD takes place and can be with the method for higher efficient amplification in vitro CTL.

The purpose of invention

The method that the purpose of this invention is to provide a kind of amplification in vitro cytotoxic T lymphocyte, this method comprises:

(1) by separating the monocyte that obtains adherent growth in the tumour patient peripheral blood;

(2) in the presence of proper concn granulocyte-macrophage colony stimutaing factor and interleukin-4, the monocyte that culturing step (1) obtains forms immature dendritic cell to induce;

(3) exist down in the excitated type anti-CD 40 monoclonal antibody, immature dendritic cell and tumour cell that step (2) obtains are educated altogether, make said immature dendritic cell become the sophisticated dendritic cell of specific tumour antigen load;

(4) antigen presenting cell that obtains with step (3) and the isolating in advance T cell of mixture process of the anti-CD28 monoclonal antibody of interleukin-2, interleukin-15 and excitated type are to promote the amplification of said T cell;

(5) cytotoxic T lymphocyte that obtains of separating step (4) amplification.

According to the preferred embodiments of the invention, patient's autologous tumor histocyte that wherein said tumour cell is an apoptosis after treatment.

According to the preferred embodiments of the invention, wherein said tumour cell comprises hematological system tumor and solid tumor cell.

According to the preferred embodiments of the invention, wherein said cytotoxic T lymphocyte is a specific for tumour antigen.

Another object of the present invention provides the application of cytotoxic T lymphocyte in the adoptive immunotherapy of tumour that as above obtains.

Brief Description Of Drawings

Fig. 1 shows the phenotypic alternation of different biotic factor combination inductive T cells in different time points.Wherein F is a RPMI-1640 blank group; A is the IL-2+IL-15+CD28 group; B is the IL-7+IL-2 group; C is the IL-15+IL-2 group; D is the CD28+IL-2 group; E is independent IL-2 group.X-coordinate provides three different somes detection time: 1 for before DC excites; 2 excite back 7 days for DC; 3 excite back 14 days for DC.Ordinate zou provides the percentage expression rate of each phenotype on the cell that flow cytometer (FCM) detects.

Fig. 2 shows that different biotic factor combinations influences T cell proliferating number purpose.Wherein the F group is RPMI-1640 blank group; A is the IL-2+IL-15+CD28 group; B is the IL-7+IL-2 group; C is the IL-15+IL-2 group; D is the CD28+IL-2 group; E uses the IL-2 group separately.X-coordinate is the fate after inducing; Ordinate zou is a cell count (unit 10 ⁶/ ml).

Fig. 3 shows that different biotic factor combinations is to the influence from body T cell proliferation.Wherein the F group is RPMI-1640 blank group; A is the IL-2+IL-15+CD28 group; B is the IL-7+IL-2 group; C is the IL-15+IL-2 group; D is the CD28+IL-2 group; E uses the IL-2 group separately.Ordinate zou is stimulation index (SI).

Fig. 4 shows the influence of different biotic factor combinations to the cytotoxic activity of the CTL of amplification.Wherein the F group is RPMI-1640 blank group; A is the IL-2+IL-15+CD28 group; B is the IL-7+IL-2 group; C is the IL-15+IL-2 group; D is the CD28+IL-2 group; E uses the IL-2 group separately.Raji is the B lymphoma cell strain; XG-2 is a multiple myeloma cell line; K562 is killer cell (NK) sensitive strain.

Fig. 5 shows the change in concentration of IFN-γ in the T cell conditioned medium of different biotic factor shootings on group.Wherein the F group is RPMI-1640 blank group; A is the IL-2+IL-15+CD28 group; B is the IL-7+IL-2 group; C is the IL-15+IL-2 group; D is the CD28+IL-2 group; E is independent IL-2 group.

The particular content of invention

The present invention relates to cytotoxic T lymphocyte and the application in anti-tumor immune response thereof. Particularly Say, the present invention relates to unite use interleukin-22, interleukin 15 and the anti-CD28 monoclonal antibody of excitated type with And the antigen presenting cell of tumour antigen load, the inducing T cell amplification is the side of cytotoxic T lymphocyte Method. The invention further relates to by the cytotoxic T lymphocyte of the method preparation and exempt from that adopting property is antitumor Application in the epidemic disease.

In people and mammiferous immune system, the cell-mediated delayed allergy of T, graft rejection With a series of cell immune responses such as cytotoxic activities. Some T cell and special cell cortex protein are namely The MHC interaction of molecules is with submission antigen on cell surface. The antigen presenting cell of specialization such as huge have a liking for thin Born of the same parents breed with helper cell with the Dendritic Cells Induced cytotoxicity and identify on the target cell surface and express mutually Answer antigen. And then T cell (CTL) kills and wounds these target cells, perhaps induces these targets of other cell killings Cell.

It is to manage in a large amount of tumour of external acquisition that use CTL carries out one of key technology in the oncotherapy Peptide-specific CTL. Traditional amplification in vitro CTL method is to adopt heavy dose of IL-2, although like this can With the T cell that is activated in a large number in a short time, but be easy to cause activating the cell death of inducing (AICD), it is required finally to be difficult to obtain long-term and stably q.s tumour-specific adoptive immunotherapy Cytotoxic T lymphocyte.

The effect of IL-2 mediation can be divided into two stages, and the IL-2 of early stage low dosage can mediate the T cell Propagation; But in the later stage, along with the gradually rising with IL-2 concentration of increasing of activating T cell quantity, T Cell just enters cell death (AICD) process of being induced by the activation of Fas/FasL mediation soon. Therefore, The simple IL-2 that uses can not make the amplification of T cell in the external long period.

As a kind of cell factor of disactivation T cell derived, IL-15 is mainly by the adhesiveness peripheral blood mononuclear Cell produces, and is by the α chain of uniqueness and β and the γ (being γ c) that shares with the IL-2 acceptor on the structure Chain forms. IL-15 can sting the T cell proliferation of activation, induces killing and wounding of CTL and Lymphokine Cell (LAK) produces, and promotes B emiocytosis IgM, IgG, IgA. Show on evidence IL-15 Promoting non-antigen dependence CD8⁺Play an important role in the differentiation of T cell and the long-term surviving. IL-15 and its The interaction of acceptor can cause CD8⁺The differentiation of T cell. In the T cell activation process, IL-15 and TCR Two approach have common ground in some aspects. Therefore, IL-15 has similar biology effect to IL-2 Should, namely both can both promote the differentiation and proliferation of T cell. Yet different from IL-2 is, IL-15 and its After the receptors bind, can stablize γ c chain, and raise apoptosis-related genes bcl-2, thereby performance there is anti-AICD Effect with the time-to-live that prolongs cell.

CD28 can provide the T cell activation required costimulatory signal, promotes T cell activation propagation. CD28 Molecule claims again Tp44, is a kind of 202 amino acid whose I type transmembrane glycoproteins that comprise. As immunoglobulin (Ig) The member of superfamily (IGSF), CD28 molecule and CTLA-4 are all costimulatory molecules CD80 and CD86 Natural receptor. After receiving the antigentic specificity signal, on the CD28 antigen on the T cell and the B cell Interaction between B7 antigen is further to provide activated T cell, to cause high-level cytokine secretion Secondary signal. In addition, the membrane CD28 molecule is reducing T cell activation threshold value, is inducing the anti-apoptotic genes expression table Reaching, increase the aspects such as cytokine secretion, Promote immunity cynapse formation and prevention T cell anergy also has heavily Act on. Therefore, no matter in vivo still external, the growth of T cell and propagation need that not only IL-2, IL-15 are arranged Deng the participation of SCIF, and the participation of the costimulating factor such as CD28 also is indispensable. This Be used for the mixture of inducing T cell activation and proliferation in the inventive method except comprising proleulzin, interleukin-15, Also be added with the anti-CD28 monoclonal antibody of excitated type (CD28mAb), purpose namely is to excite and provide CD28 The above-mentioned intrinsic activity of molecule.

Known BMDC is to swash by submission antigen on cell surface I and II class MHC molecule The most strong antigen presenting cell (the Banchereau and of T cell alive, NK cell and other immunocytes Schmit, Advances in Experimental Medicine and Biology (Back et al., eds) volume 378, Plenum Press, NY). Except to T cell and NK cell submission antigen, BMDC also can Originally stimulated the mitosis of T cell by producing the T cell mitogen. Traditional amplification in vitro DC's Method be in the PMBC culture of in vitro culture, add granulocyte-macrophage colony stimulate because of The CD34 that son (GM-CSF) and TNF (TNF-α) stimulate⁺Hemopoietic forebody cell is to lure Lead the DC Hemapoiesis; Perhaps in the PMBC culture of in vitro culture, add GM-CSF, white The cell factors such as cytokine 4 (IL-4) and TNF-α are to obtain GM-CSF dependence DC cell.

In order further to improve the amplification efficiency of CTL cell, satisfy the needs of clinical tumor immunization therapy, and For the foundation of the inventive method provides sufficient basic research data, our further investigated has also compared various thin Intracellular cytokine and various combination thereof are to the impact of T cell activation and propagation.

We studies show that, excitated type CD28mAb, IL-2, IL-7 and IL-15 make alone or in combination Time spent is to the amplification effect difference of T cell, and the existing obvious complementation of these SCIF tables The property. For example, we find, use separately the increase of the front 7 days T cell quantities of excitated type CD28mAb Obviously, by initial 0.2 * 10⁶Rapidly increase to 4.1 * 10⁶, but the increase of T cell quantity obviously slows down (4.1 * 10 in after this 7 days⁶Be increased to 6.0 * 10⁶). The costimulatory signal of this phenomenon prompting CD28 mediation Mainly the T cell excite and breed play a role in early days. Show the T cell institute that CD28mAb excites The endogenous IL-2 that produces is not sufficient to keep its growth and propagation, and must be by means of ectogenic IL-2. In addition, our experiment shows, uses separately IL-2, or unites and use IL-2+IL-7 or IL-2+IL-15 Though but the inducing T cell amplification, efficient is not high, and the CTL of the generation of inducing is to the kill rate of target cell Also on the low side.

On the contrary, use in conjunction excitated type CD28mAb, IL-2 and IL-15, not only the most obviously (the T cell quantity is by 0.2 * 10 for T cell amplification effect⁶Continue to be expanded to 2.4 * 10⁷), and the T cell of amplification is shown The Fas antigen that reaches reduces, the CTL that induces to the kill rate of target cell up to about 35%. Excitated type The possible cause of CD28mAb, IL-2 and IL-15 use in conjunction best results is that this combination has not only strengthened First and second signals that the T cell activation is required by the anti-AICD effect of IL-15, have prolonged T simultaneously The life cycle of cell, thereby in quality with quantitatively guaranteed the killing-efficiency of CTL to target cell.

On the other hand, consider that the Th0 cell can be divided into the Th1 cell of main secretion of gamma-IFN and mediated cell immunity under different condition, or secrete IL-10 and mediate the Th2 cell of humoral immunization.。Therefore we have also measured the content of IFN-γ and IL-10 in the T cells and supernatant that different amplification modes produce.The result shows, contains IFN-γ in various different cytokines and the CD28 monoclonal antibody combination inductive T cell conditioned medium, and does not almost detect IL-10.Show that the Th0 cell mainly is divided into the Th1 cell of secretion of gamma-IFN and mediated cell immunity.Uniting under the situation of using CD28mAb, IL-2 and IL-15, the IFN-γ level in the T cell culture supernatant that amplification obtains is up to 6.4ng/ml.

Moreover, in order to make dendritic cell (DC) load tumour specific antigen, we adopt the tumor cell line cell as basic substance, choosing tumor-cell antigen and to its processing, submission from whole cell antigen by DC, may be a kind of simple and effective method that can remove loaded down with trivial details tumor associated antigen screening step from.

Based on above-mentioned fundamental research, the inventor has successfully set up a kind of method of new amplification in vitro cytotoxic T lymphocyte.In the method for the present invention, we are obtained the monocyte of adherent growth at first basically by separation in the tumour patient peripheral blood according to traditional method, in the presence of proper concn granulocyte-macrophage colony stimutaing factor and interleukin-4, cultivate resulting monocyte then, form immature dendritic cell to induce.At last, use comprises the anti-CD28 monoclonal antibody of interleukin-2, interleukin-15 and excitated type and the isolating T cell of mixture process of the antigen presenting cell that obtains as stated above, obtains the special cytotoxic T lymphocyte of required tumour antigen with amplification.

Therefore, the method for amplification in vitro cytotoxic T lymphocyte of the present invention consists essentially of following steps:

(1) by separating the monocyte that obtains adherent growth in the tumour patient peripheral blood;

(2) in the presence of proper concn granulocyte-macrophage colony stimutaing factor and interleukin-4, the monocyte that culturing step (1) obtains forms immature dendritic cell to induce;

(3) exist down in the excitated type anti-CD 40 monoclonal antibody, immature dendritic cell and same patient's tumor cell that step (2) obtains are educated altogether, make said immature dendritic cell become the sophisticated dendritic cell of specific tumour antigen load;

(4) antigen presenting cell that obtains with step (3) and the isolating in advance T cell of mixture process of the anti-CD28 monoclonal antibody of interleukin-2, interleukin-15 and excitated type are to promote the amplification of said T cell;

(5) cytotoxic T lymphocyte that obtains of separating step (4) amplification.

According to a preferred embodiment of the invention, wherein said tumour cell patient's autologous tumor histocyte that is apoptosis after treatment.

According to another preferred embodiment of the present invention, wherein said cytotoxic T lymphocyte is a specific for tumour antigen.

According to another preferred embodiment of the present invention, wherein said tumour cell comprises hematological system tumor and solid tumor cell.

The present invention further provides the application of CTL in immunotherapy of tumors that obtains as stated above.Our cell in vitro is learned experiment and is shown, isolating T cell can be the special cytotoxic T lymphocyte of tumour antigen (CTL) with higher speed differentiation and propagation under antigen presenting cell, IL-2, IL-15 and the monoclonal combined action of anti-D28 of the tumour cell load of apoptosis.And these have very strong tumor cytotoxicity activity through the CTL of external evoked amplification performance.In addition, we show indivedual middle and advanced stage tumour patient volunteers' test of cure result, and the CTL that obtains according to the inventive method amplification can effectively delay the progress of the state of an illness, alleviate patient's symptom.

Embodiment 1: the preparation of the antigen presenting cell of tumour antigen load

Present embodiment is described by the tumour cell of the apoptosis method at dendritic cell (DC) area load tumour antigen.

1, inducing of apoptosis of tumor cells: in the substratum (RPMI-1640+10%FCS) of B lymphoma cell strain Daudi, add CD40 antibody (CD40mAb) (5 μ g/ml), induce 24 hours (h) for 37 ℃.Behind the washed cell, usefulness radionuclide cobalt ( ⁶⁰Co) (50Gy, dose rate 10Gy/min) irradiation.37 ℃ of (5%CO then ₂) continue to cultivate 48 hours standby.

2, the external evoked and amplification of DC: adopt the fresh peripheral blood of healthy people's anticoagulant heparin (available from blood station, center, Suzhou City), the Ficoll density gradient separation obtains peripheral blood mononuclear cell, is adjusted to 3～5 * 10 with the RPMI-1640+10%FCS substratum ⁶/ ml adds 37 ℃ of adherent culture 2h in 24 well culture plates.Collect the cell cultures of adherent growth and be added with rhGM-CSF (1000IU/ml), rhIL-4 (500IU/ml), L-glutaminate (0.02mmol/L), 2 mercapto ethanol (5 * 10 ^-5Mol/L), 37 ℃, 5%CO in the RPMI-1640 substratum of penicillin (100IU/ml), Streptomycin sulphate (100 μ g/ml) and 10% foetal calf serum ₂Cultivate.Half amount was changed liquid once in every 2-3 days.

3, after the 7th day that the Daudi cell loading dendritic cell of apoptosis: DC cultivates, in apoptosis Daudi: DC=3: 1 ratio co-cultivation 16h.The washing back adds CD40mAb (5 μ g/ml), continues to cultivate 2 days to making maturing dendritic cell.

Embodiment 2: the amplification in vitro of antigen-specific CTL and application

The method of the dendritic cell of present embodiment description use antigen load and the mixture amplification CTL of multiple biotic factor.Wherein use flow cytometry (FCM) to detect cell phenotype, use the existence and the level thereof of the ELISA kit detection cell factor, with ³H-TdR mixes the propagation of measuring cell, and with ⁵¹The Cr relief test method is measured cytotoxic activity.

(1) purifying of T cell: the PBMC that obtains by peripheral blood centrifugation (Ficoll).The PBMC cell uses flow cytometry to detect cell purity＞85% behind nylon hair column chromatography purification.

(2) load the DC of Daudi cell of apoptosis to the activation that excites of T cell: under the existence of the cytokine of various combination, with the DC of sophisticated apoptosis Daudi load and purifying from the ratio co-cultivation of body T cell in E/T=1/10.Be divided into is six groups: A.CD28mAb (5 μ g/ml)+IL-15 (5ng/ml)+IL-2; B.IL-7 (5ng/ml)+IL-2; C.IL-15 (5ng/ml)+IL-2; D.CD28mAb (5 μ g/ml)+IL-2; E.IL-2; F. blank.With the sophisticated DC+ that do not add cytokine from body T cell as negative control.The concentration of the first week IL-2 that experiment adds is 30IU/ml, and the concentration of the IL-2 that will use separately after the week is increased to 100IU/ml.

(3) FCM of T cell phenotype analyzes: the T cell was handled with CD3, CD4, CD8, CD25, CD28 and the CD95 monoclonal antibody of PE mark respectively in 1 week and activation two weeks of back before the DC of load tumour antigen activation, after the activation.As seen the result on the same group in cytokine and the excitated type monoclonal antibody combined induction T cell activation process, CD3 all do not occur ⁺, CD4 ⁺And CD8 ⁺The percentage of T cell improves, and CD25 ⁺And CD28 ⁺T cell showed increased.CD95 (Fas) expression level of CD28mAb+IL-15+IL-2 group T cell is lower than other each groups, and IL-15+IL-2 organizes the (see figure 1) of taking second place.These results show that the signal of CD28mAb and IL-15 mediation has the effect of anti-T cell AICD.

(4) detection of IFN-γ, IL-10 level in the T cells and supernatant: the DC that collects apoptosis Daudi cell loading respectively excites the 7th day T cells and supernatant with the mixture of different biotic factors, detects IFN-γ, IL-10 concentration according to the described method of test kit specification sheets.The ELISA detected result shows all have IFN-γ to exist in each experimental group supernatant, and wherein the IFN-γ concentration with the CD28mAb+IL-15+IL-2 group is the highest (see figure 5).All do not detect IL-10 in the cell culture of each group.

(5) DC of the Daudi cell loading of apoptosis to from the influence of body T ability of cell proliferation ( ³H-TdR mixes method): the aperture (2 * 10 that will place 96 orifice plates through the ripe DC and the T cell (1: 10) of apoptosis Daudi cell loading ⁴/ hole), 37 ℃ of (5%CO ₂) cultivate.Cultivate after 56 hours, mix in every hole ³H-TdR3.7 * 10 ⁴Bq continues to be cultured to 72h and stops.Adopt liquid scintillation instrument to measure the cpm value, and calculate stimulation index (SI): SI=(experimental group cpm-background cpm)/(control group cpm-background cpm) by following formula.Experimental result shows, the mixture of the cytokine of variant combination and excitated type monoclonal antibody all can stimulate CTL propagation, but wherein with the CD28mAb+IL-15+IL-2 group to T cells in vitro proliferation function significantly (referring to Fig. 3).

(6) DC of the Daudi cell loading of apoptosis activate specific for tumour antigen CTL cytotoxicity ( ⁵¹The Cr release test): by per 2 * 10 ⁵The Daudi cell mixes 3.7 * 10 ⁶Bq ⁵¹Cr is hatched 2h for 37 ℃.After washing 3 times, will and cultivate 4h, measure the cpm value with gamma counter then through T cell and Daudi cytomixis (E/T=50/1) after the DC of the antigen load activation.Calculate kill rate by following formula: kill rate (%)=100 * [(experimental group cpm-nature release group cpm)/(maximum release group cpm-nature release group cpm)].Use without antigen load DC inductive CTL/Daudi, Ag-DC inductive CTL/Raji, Ag-DC inductive CTL/XG-2, Ag-DC inductive CTL/K562 and organize in contrast.Test-results shows that DC, the CD28, IL-15 and the IL-2 that unite the tumour cell load of using apoptosis excite the CTL of amplification to Daudi kill rate the highest (39.7 ± 8.2%); Unite other each groups of using IL-2/IL-7+IL-2/IL-15+IL-2 the Daudi cell is also showed in various degree killing activity, but Raji and XG-2 cell are not then had tangible cytotoxicity (referring to Fig. 4).

Embodiment 3: the CTL that obtains by the inventive method uses in oncotherapy

Present embodiment illustrates the cytotoxic T lymphocyte that obtains according to the inventive method amplification and uses in the clinical malignant neoplastic disease people of treatment.

Ovarian cancer patient, woman, 75 years old.Before the ovarian tumor excision, gather patient's anticoagulation 1ml, the flow cytometry method detects the T cell phenotype and is: CD ₃55%, CD ₄29.7%, CD ₈35.7%CD ₂₅4.4%, CD ₂₈22%.Retained part tumor tissues in the art is made single cell suspension with 400 eye mesh screens, and-80 ℃ of freezing preservations are standby.The postoperative conventional chemotherapy, after finishing first course of treatment (3 week), it is normal to detect routine blood test.Give the continuous intramuscular injection GM-CSF of patient (75 μ g/ days) two days, to improve the quantity of DC precursor cell in patient's peripheral blood.Got periphery anticoagulation 50ml on the 3rd day, separate acquisition PBMC and routine and induce DC.At the 6th day of DC growth, recovery part tumour cell, cis-platinum (DDP) is apoptosis-induced and carry out the DC load.After treating the DC maturation, extract patient's peripheral blood 50ml once more, obtain the T cell and educate altogether with antigen load DC.The quantity of the CTL that obtains by the inventive method reaches 10 ⁸The time, the part cell continues amplification, and all the other cells mix with 100ml injection Portugal salt solution with physiological saline washing 3 times, and vein is fed back in patient's body.Add during this and use dexamethasone 5mg, the prevention transfusion reaction.Feed back once more after the CTL amplification that continues to cultivate, feed back altogether 3 times.There is no any side reaction in the feedback process and after feeding back.Treatment is checked T cell phenotype: CD after January ₃68.8%, CD ₄45.5%, CD ₈26.6%, CD ₂₅14.9%, CD ₂₈51.4%; Check once more after 1 year: CD ₃46.6%, CD ₄30%, CD ₈24.8%, CD ₂₅20%, CD ₂₈33.1%.

Claims (5)

1, a kind of method of amplification in vitro cytotoxic T lymphocyte, this method comprises:

(1) by separating the mononuclearcell that obtains adherent growth in the tumour patient peripheral blood;

(2) in the presence of proper concn granulocyte-macrophage colony stimutaing factor and interleukin-4, the monocyte that culturing step (1) obtains forms immature antigen presenting cell to induce;

(3) exist down in anti-CD 40 monoclonal antibody, immature antigen presenting cell and tumour cell that step (2) obtains are educated altogether, make said prematurity antigen presenting cell become the sophisticated antigen presenting cell of specific tumour antigen load;

(4) antigen presenting cell that obtains with step (3) and the isolating in advance T cell of mixture process of the anti-CD28 monoclonal antibody of interleukin-2, interleukin-15 and excitated type are to promote the amplification of said T cell;

(5) cytotoxic T lymphocyte that obtains of separating step (4) amplification.

2, according to the process of claim 1 wherein that said tumour cell is patient's autologous tumor histocyte of apoptosis after treatment.

3, according to the process of claim 1 wherein that said tumour cell comprises hematological system tumor and solid tumor cell.

4, according to the process of claim 1 wherein that said cytotoxic T lymphocyte is a specific for tumour antigen.

5, the application of cytotoxic T lymphocyte in the adoptive immunotherapy of tumour that obtains according to the method for claim 1.

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