CN102719399A - In vitro amplification method of self-specific T cell, prepared T cell system, pharmaceutical use of cell system and component monitoring method of cell system - Google Patents
In vitro amplification method of self-specific T cell, prepared T cell system, pharmaceutical use of cell system and component monitoring method of cell system Download PDFInfo
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Abstract
The invention relates to an in vitro amplification method of self-specific T cells, a T cell system prepared by the method, pharmaceutical use of the cell system, and a component monitoring method of the cell system. The in vitro amplification method comprises the steps of: (1) separating peripheral blood mononuclear cells from peripheral blood of a patient; (2) adding corresponding T cell stimulating factors as needed, adding or not adding corresponding cell growth factors, and amplifying T cells by cultivating the peripheral blood mononuclear cells separated in the step (1) in a corresponding culture medium. According to the invention, polyclonal immunocompetent cells with various functionalities can be prepared according to different clinical needs by adopting the amplification method of the invention in a GMP environment, and the prepared polyclonal immunocompetent cells can be used in immune system regeneration and immunotherapy of patients.
Description
Technical field
The present invention relates to a kind of amplification in vitro method from the body specific T-cells, also relate to adopt obtain after this method amplification from body specific T-cells system, the application of this cell system in medication preparation and to the component monitoring method of this cell system.
Background technology
Tumor treatment adopts three kinds of traditional therapies, i.e. radiotherapy, chemotherapy and operative treatment always.Conventional surgical treatment can only solve local problem, and radiotherapy and chemotherapy also can make human normal tissue and cell sustain damage when killing and wounding cancer cells, and is especially very big to the Immune Function of the T cell of leading cellular immunization, is prone to formation secondary tumour.Therefore, radiotherapy, chemotherapy and the three great tradition therapeutic modalities of performing the operation such as exist thoroughly always, are prone to shift, spinoff is big at drawback.
In recent years, new tumour autogenous cell immunotherapy method (also claiming tumor biotherapy) has become the 4th kind of pattern of modern oncotherapy.Comprise CIK (Cytokine Induced Killer; Cytokine induced kill cell) cellular immunization therapy, LAK cell (Lymphokine Activated Killer Cells; Lymphokine activated killer cell) immunotherapy, til cell (Tumor Infiltrating Lymphocyte, tumor infiltrating lymphocyte) immunotherapy and DC cell (BMDC) immunotherapy etc.
Wherein, the CIK cell is with CD3 monoclonal antibody activated killer cell (claiming the CD3AK cell again), is a kind of more novel anti-tumour effect cell that LAK and TIL occur afterwards, uses more at present at home.The CIK cell has the strong and certain characteristics such as anti-knurl ability of amplification in vitro ability; LAK and the til cell some shortcomings in oncotherapy have been overcome; But the main limitation of CIK therapy is this method cells in vitro activation has broad spectrum, lacks the lethal effect special to some tumour cell.
In a word, at present, the bottleneck that tumour autogenous cell immunotherapy runs into is mainly aspect following two:
1. to carry out the system that cell in vitro cultivates perfect inadequately for autogenous cell, has a lot of uncontrollable factors, thereby cause cell amplification quantity not sufficient or amplification failure, and the cell that more crucial is after cultivating lacks the specificity of killing tumor cells;
2. lack the Quality Control monitoring and evaluation method that cell in vitro is cultivated preparation, feed back its curative effect of the interior back of patient's body and can not accurately with timely be estimated again, hindered this new technology and standardized and standardized enforcement.
Summary of the invention
In order to overcome the above-mentioned defective of prior art; The object of the present invention is to provide a kind of product that obtains from the amplification in vitro method and the cultivation of body specific T-cells (from body specific T-cells system), this product in pharmacy application and to the monitoring method of this product fraction; Adopt this amplification in vitro method can be under the environment of GMP to prepare the polyclone immunologically competent cell of different functionalities according to clinical different needs; Can be used for tumour, virus infection or put, the immunologic reconstitution after the immunologic injury that chemotherapy causes; Also can prepare the cloning T cell of high degree of specificity with the virus of killing and wounding or function of tumor; Be called CTC (The Cloned T cells; CTC), be used for antitumor and antiviral cellular immunization treatment, corresponding component monitoring method can be used for prepared quality monitoring and evaluation from body specific T-cells system or medicine; Guaranteeing the standardization and the stdn from body specific T-cells system of this amplification in vitro method and preparation thereof, and can carry out effectively evaluating to clinical efficacy.
The main technical schemes that the present invention adopts is:
A kind of amplification in vitro method from the body specific T-cells comprises the steps:
Step (1) is separated PMNC (being PBMCs) from peripheral blood of patients;
Step (2) adds corresponding T LSF as required, adds or do not add corresponding ESC, in corresponding substratum, the isolated PMNC of step (1) is carried out the vitro culture T cell that increases.
In said step (2); Can also produce also and can not produce the DC cell vaccine; The mode of said production DC cell vaccine is: when said PMNC vitro culture, add the corresponding D C cell maturation factor to obtain sophisticated DC cell, in the sophisticated DC cell that obtains, add corresponding tumour or virus-specific antigen and carry out antigenic stimulation to produce the DC cell vaccine.
Said promotion DC maturation factor preferably added before vitro culture.
Preferably; Require and the cultivation needs according to specificity; Said T LSF can comprise in following any one, any several kinds or all cytokine: SUPF-1, SUPF-2, SUPF-3, SUPF-4, SUPF-5, SUPF-6, SUPF-7, SUPF-8, SUPF-9, SUPF-10 and SUPF-31; When adding multiple T LSF; The ratio of various T LSFs can require confirm according to specificity, can or under experiment instruction, confirm in the add-on of following T LSF kind that will add of different situations and the T LSF that added and the proportionlity of various T LSFs according to experimental verification, theoretical derivation.
Said substratum can adopt or comprise serum free medium usually.
Said serum free medium preferably adopts or comprises substratum MEDO-A.
Preferably, said step (2) comprising:
Step (2.1), with substratum MEDO-A said step (1) being separated the PMNC obtain, to process cell density be 1x10
6/ ml-2x10
6The cell suspension of/ml;
Step (2.2); Said step (2.1) adds SUPF-31, SUPF-5, SUPF-6 and SUPF-7 and supplies to cultivate afterwards, and incubation time was generally about 48-72 hour; Afterwards; Remove or do not remove the etcetera of T LSF, the mode of said adding SUPF-31, SUPF-5, SUPF-6 and SUPF-7 is preferably processes the mixture of SUPF – 31 and SUPF-5 and the mixture of SUPF-6 and SUPF-7 respectively earlier, and then adds respectively;
Step (2.3), said step (2.2) add SUPF-3 and SUPF-10 afterwards again and in substratum MEDO-A, continue to cultivate, and the mode of said adding SUPF-3 and SUPF-10 is preferably the two and adds respectively.
Usually, said step (2.3) also comprises afterwards: step (2.4), results T cell.
Can confirm incubation time according to cell quantity, for example, usually, the condition of said results T cell can be following arbitrary condition: the incubation time of said step (2.3) reaches 10-12 days (when being used for immunologic reconstitution) or 10-14 days (when being used for immunotherapy); Perhaps, the T cell quantity reaches the cultivation requirement; Perhaps, the omnidistance vitro culture time reaches 10-12 days (when being used for immunologic reconstitution) or 10-14 days (when being used for immunotherapy).
Preferably, the peripheral blood in the said step (1) can adopt density gradient method to separate PMNC with EDTA or anticoagulant heparin.
Be preferably ground; For above-mentioned any one described amplification in vitro method from the body specific T-cells; In the said step (2), also stimulate so that increase in the T cell after increasing to have anti-tumor capacity and immunocompetent CTC cell precursors through the interpolation tumour antigen.
Said carry out mode that tumour antigen stimulates can for: adding tumour or virus-specific antigen directly stimulates, and/or, carry said specific anti primary stimuli by the self antigen presenting cell.
Be preferably, said tumour antigen comprise in following any one, any several kinds or all: TAG-1, TAG-2, TAG-3, TAG-4, TAG-5, TAG-6, TAG-7, TAG-9, TAG-10, TAG-11, TAG-12, TAG-13, TAG-14, TAG-15, TAG-16, TAG-17, TAG-18, TAG19 and TAG-20.
Can adopt following manner that said step (2) is improved; The cell suspension that said step (2.1) is made at first carries out adherent culture; Again cell suspension after the adherent culture and attached cell are cultivated separately respectively, then, the cell suspension of cell suspension single culture gained and the attached cell of attached cell single culture gained are mixed; Add SUPF-6 and SUPF-7 after the mixing, carry out said step (2.3) afterwards.
The condition of said adherent culture can be 37 ℃ of 5% CO
2 Adherent culture 2 hours-3 hours.
The single culture of attached cell after the said adherent culture can be carried out as follows:
(i) any one among adding TAG-1 or the TAG-2 and TAG-3, TAG-4, TAG-5, TAG-6, TAG-7, TAG-9, TAG-10, TAG-11, TAG-12, TAG-13, TAG-14, TAG-15, TAG-16, TAG-17, TAG-18, TAG19 and TAG-20; Said TAG19 and TAG-20 mix the back and add; In substratum MEDO-A, cultivate, incubation time can be about 48 hours;
(ii), step (i) adds TAG-4 and TAG-5 afterwards again and continues to cultivate, and incubation time can be about 24 hours, for example, can be cultured to next day;
(iii), step (ii) adds corresponding tumour more afterwards or virus antigen polypeptide is cultivated altogether, and incubation time can be 3 hours.
The single culture of the cell suspension after the said adherent culture can be undertaken by said step (2.2).
A kind of from body specific T-cells system, all or part of T cell wherein makes through amplification in vitro for adopting above-mentioned any one amplification in vitro method from the body specific T-cells (or any method among the claim 1-15).
Preferably, wherein T cell total amount (CD3
+The T cell) is higher than 98%.
When said system is used for immunologic reconstitution, CD4 T cell subsets (CD3 wherein
+CD4
+The T cell) content is higher than 60%; When said system is used for immunotherapy, CD4 T cell subsets (CD3 wherein
+CD4
+The T cell) content is not less than 15%.
When said system is used for immunologic reconstitution, CD8 T cell subsets (CD3 wherein
+CD8
+The T cell) content is not higher than 40% and be not less than 20%; When said system is used for immunotherapy, CD8 T cell subsets (CD3 wherein
+CD8
+The T cell) content is higher than 80%, is preferably to be higher than 90%.
Wherein the T cell content is higher than 90%, is preferably to be higher than 95%.Said activated T cell subsets should comprise CD4 usually
+CD38
+HLADR
+And CD8
+CD38
+HLADR
+Two T cell subsets, preferably, CD4
+CD38
+HLADR
+And CD8
+CD38
+HLADR
+Content separately all is higher than 45%.
Anti-tumor activity T cell subsets (CD8 wherein
+CD45RO
+CD62L
+, Tcm) content is higher than 70%, is preferably to be higher than 90%.
Said anti-tumor activity T cell subsets (CD8
+CD45RO
+CD62L
+, Tcm) be no more than 5% and do not express CD62L, be preferably and be no more than 1% and do not express CD62L, more preferably be no more than 0.1% and do not express CD62L.
Memory T cell subgroup content wherein is higher than 95%.Said memory T cell subgroup should be CD8 usually
+CD45RO
+, preferably, CD8
+CD45RO
+Content be higher than 95%.
Be preferably, above-mentioned any from body specific T-cells system, regulatory T cells subgroup (CD4 wherein
+CD25
+FoxP3
+, Treg) content is lower than 5%.
A kind ofly be used in preparation from body specific T-cells system that immunity system is rebuild and the application of the medicine of immunotherapy, saidly constitute from body specific T-cells system (or any claim is described from body specific T-cells system in by claim 16-28) by above-mentioned any one from body specific T-cells system.
Usually, said immunity system is rebuild and is mainly referred to rebuild for the immunity system behind the immune system injury that causes because of tumour, virus infection or radiotherapy, chemotherapy, and said immunotherapy mainly refers to the cellular immunization biotherapy for tumour, virus infection.
Above-mentioned any said from body specific T-cells system in the application of preparation in the medicine; Effective constituent in its medicine can all be said from body specific T-cells system (i.e. the independent utility of this system), also can partly be said from body specific T-cells system (i.e. the mixing application of this system and other composition).
A kind of component monitoring method from body specific T-cells system; Can be used for above-mentioned any component monitoring, comprise cell phenotype analysis and/or TCR cloning analysis from body specific T-cells system (or the described system of each claim of claim 16-31).
Can adopt flow cytometry to carry out described cell phenotype analysis.For example, can at first adopt the cell in fluorescent mark monoclonal antibody and the said system to make up, carry out the quantitative analysis of cell phenotype then.
Said monoclonal antibody can comprise in following any one, any several kinds or all: anti-CD3
+, CD4
+, CD8
+, CD45RA
+, CD45RO
+, CD38
+, CD62L
+, HLADR
+, CD25
+, FoxP3
+, CD28
+And CD57
+
The concrete monitoring project of said cell phenotype analysis comprises: T cell total amount (CD3
+The T cell), CD4 T cell subsets (CD3
+CD4
+The T cell), CD8 T cell subsets (CD3
+CD8
+The T cell), activated T cell subsets (CD4
+CD38
+HLADR
+And/or CD8
+CD38
+HLADR
+), anti-tumor activity T cell subsets (CD8
+CD45RO
+CD62L
+, Tcm), regulatory T cells subgroup (CD4
+CD25
+FoxP3
+, Treg, and/or CD8
+CD28
-/ CD8
+CD57
+), memory T cell subgroup (CD4
+CD45RO
+And/or CD8
+CD45RO
+).
Said TCR cloning is analyzed can comprise that 22 V β gene families to TXi Baoshouti carry out visual and/or quantitative analysis.
The concrete grammar that said TCR cloning is analyzed can be the functional status of the antiviral or tumour that reflects the antigen specific T cells of frequency, length and the distribution quantification that occurs through the cloning of measuring TCR CDR3 gene regions.
The invention has the beneficial effects as follows:
The invention provides the standard method that autogenous cell carries out vitro culture; The cell expansion ex vivo effect stability, success ratio is high, can prepare the immunologically competent cell of corresponding function according to clinical various demands; As for the patient who causes immunologic injury because of tumour, virus infection or radiotherapy, chemotherapy; Can its disorderly T cell system be returned to normal through vitro culture, and can reach the amplification of hundred times polycloneization, can be used for patient's immune system after being fed back in patient's body to rebuild; In addition; Also can turn out cloning T cell, can be used for antitumor and antiviral cellular immunization and treat, especially can in culture system, produce a large amount of have anti-tumor capacity and immunocompetent CTC cell precursorss with the virus of killing and wounding and high degree of specificity of function of tumor; Make the T cell of cloning retain more lastingly in that the patient is intravital, help improving curative effect;
Through experimental verification, for the homologous parallel samples, adopt method of the present invention to cultivate after, CD3
+CD8
+The T cell expressing far above 49.25% of prior art, and almost all is the CD8 of high expression level up to 92.46%
+The T cell is especially almost all expressed the CD3CD8 cell, after cultivating with the method for prior art a lot of low CD8 that express is arranged still
+It is obvious that T cell and the CD3CD8 cell of not expressing are compared beneficial effect; What is more important adopts in the sample after method of the present invention is cultivated, phenotype be CD8CD45ROCD62L have the effect of height tumor-killing central type memory cell (Tcm) up to 92.85%; And wherein only 0.09% do not express CD62L; And in the sample after cultivating with the method for prior art, its Tcm only accounts for 39.64%, and wherein has 47.77% memory cell (CD45RO) not express CD62L;
Component monitoring method of the present invention can effectively be analyzed and estimate the cell system after cultivating; Not only can forward and backwardly analyze respectively and estimate using; Especially cloning that can track of the whole process T cell changes, and the active constituent content in can the accurate response cell system is for the curative effect of estimating cell system and patient's recovery provide effective monitoring; Help the adjustment of regimen, help the curative effect that reaches best.
Description of drawings
Fig. 1 is the application of antineoplastic specificity T cells in vitro amplification method of the present invention, antineoplastic specificity T cell system and the schematic diagram of antineoplastic specificity T cell killing tumour;
Fig. 2 is the schematic flow sheet (from body specific T-cells system can being used for immunologic reconstitution after the preparation) of an embodiment of the amplification in vitro method from the body specific T-cells of the present invention;
Fig. 3 is the schematic flow sheet (from body specific T-cells system can being used for cellular immunization treatment after the preparation) of another embodiment of the amplification in vitro method from the body specific T-cells of the present invention;
Fig. 4 adopts amplification in vitro method and the prior art from the body specific T-cells of the present invention that same increment is originally cultivated, and adopts monitoring method of the present invention that the system that obtains is carried out cell phenotype and analyze resulting result and contrast synoptic diagram and (mainly show and have the CD8 that kills and wounds viral and tumour
+The T cell subsets, wherein, A1, A2 cultivate the CD3 in the T cell system that obtains afterwards respectively for adopting prior art and method of the present invention
+CD8
+T cell subsets content analysis, B1, B2 cultivate the CD8 in the T cell system that obtains afterwards respectively for adopting prior art and method of the present invention
+CD45RO
+CD62L
+T cell subsets content analysis);
Fig. 5 is the schematic flow sheet that TXi Baoshouti of the present invention (TCR) cloning is analyzed;
Fig. 6 adopts monitoring method of the present invention to the sample analysis before and after the body specific T-cells is being cultivated of the present invention synoptic diagram (A is the analysis to the PCMCs sample the cultivation before, and B is the analysis from body specific T-cells system to obtaining after cultivating from the step shown in same this employing of increment Fig. 3 of A) as a result.
Embodiment
Referring to the embodiment shown in Fig. 2, at first, gather patient 50mlEDTA anticoagulation; Then, adopt density gradient method to separate PMNC (PBMCs), leave and take a certain amount of PMNC as required; So that carry out cell phenotype flow cytometer showed and TCR polymorphism analysis, again the PMNC of remainder is prepared cell suspension 40ml with substratum MEDO-A, add stimulating factor SUPF-31 and SUPF-5 then; SUPF-6; In substratum MEDO-A, cultivated altogether 48-72 hour after the SUPF-7, add SUPF-3 and SUPF-10 stimulating factor again, continue to cultivate at substratum MEDO-A; Harvested cell after 10-14 days (concrete incubation time can be confirmed according to cell quantity) carries out cell phenotype flow cytometer showed and TCR cloning afterwards and analyzes.
, through embodiment the present invention is described in more detail the present invention in order better to explain below in conjunction with accompanying drawing so that better understand.
Referring to Fig. 1 to Fig. 6; The invention provides a kind of from the amplification in vitro method of body specific T-cells and thus method make from body specific T-cells system, said monitoring method of monitoring this quality from body specific T-cells system (promptly wherein component) from the application and being used to of body specific T-cells system in immunologic reconstitution and immunotherapy medicaments also is provided.
Said amplification in vitro method from the body specific T-cells comprises the steps:
(1) from peripheral blood of patients, separates PMNC (being PBMCs);
(2) add corresponding T LSF as required; Add or do not add corresponding ESC; In corresponding substratum, the isolated PMNC of step (1) is carried out the vitro culture T cell that increases; Also adding or not adding promotes the DC maturation factor to produce sophisticated DC cell; Said promotion DC maturation factor preferably adds before the vitro culture, and adding corresponding ESC can increase T cell quantity and TXi Baoshouti polymorphum to be used for the reconstruction of T cellular immunization.
Be preferably, said T LSF comprise in following any, appoint several kinds or all: SUPF-1, SUPF-2, SUPF-3, SUPF-4, SUPF-5, SUPF-6, SUPF-7, SUPF-8, SUPF-9, SUPF-10, SUPF-31.The kind of the T LSF of being selected for use can be selected according to patient's tumour situation; Be preferably and comprise at least and can produce the T LSF that the tumour that the patient is suffered from has the T cell of specific killing effect in the T cell system after cultivation; When selecting more than one T LSF for use; Various T LSFs can mix according to arbitrary proportion; The principle of selecting for use can for: according to experience, can spread usually or shift the new tumour that forms and select the T LSF for use, or improve and can generate the accounting of T LSF that this new tumour is had the T cell of specific killing effect according to the tumour that the patient suffered from.
Wherein, SUPF-1, SUPF-2, SUPF-3, SUPF-4, SUPF-5, SUPF-6, SUPF-7, SUPF-8, SUPF-9, SUPF-10 and SUPF-31 are corresponding cytokines, like IL2, and IL4, IL12, GMC-SF etc.
Said substratum comprises serum free medium, and being preferably all is serum free medium.
Said serum free medium comprises substratum MEDO-A, and being preferably all is substratum MEDO-A.
Be preferably, step (2) comprising:
(2.1), it is 1x10 that the PMNC that with substratum MEDO-A step (1) separation is obtained is processed cell density
6/ ml-2x10
6The cell suspension of/ml;
(2.2); Step (2.1) was cultivated 48-72 hour after adding SUPF-31, SUPF-5, SUPF-6 and SUPF-7 afterwards altogether; Afterwards; Remove or do not remove the etcetera of T LSF, be preferably and process SUPF – 31 and the mixture of SUPF-5 and the mixture of SUPF-6 and SUPF-7 at first respectively, add respectively more afterwards;
(2.3), step (2.2) adds SUPF-3 and SUPF-10 afterwards again and in substratum MEDO-A, continues to cultivate, and is preferably the two and adds respectively.
Step (2.3) can also comprise afterwards: (2.4), results T cell.
The condition of results T cell can for: the incubation time of step (2.3) reaches 10-12 days or 10-14 days; Perhaps, the T cell quantity reaches the cultivation requirement; Perhaps, omnidistance incubation time reaches 10-12 days or 10-14 days.
Peripheral blood in the step (1) can adopt density gradient method to separate PMNC with EDTA or anticoagulant heparin.
Referring to the embodiment shown in Fig. 2, at first, gather patient 50mlEDTA anticoagulation; Then, adopt density gradient method to separate PMNC (PBMCs), leave and take a certain amount of PMNC as required; Analyze so that carry out cell phenotype flow cytometer showed and TCR cloning, in the PMNC of remainder, add stimulating factor SUPF-31 and SUPF-5, SUPF-6 and SUPF7 again, in substratum MEDO-A, cultivated altogether 72 hours afterwards; Cultivate the etcetera that stimulating factor is removed in the back; Add SUPF-3 and SUPF-10 stimulating factor afterwards again, continue to cultivate, harvested cell after 10-12 days (concrete incubation time can be confirmed according to cell quantity) at substratum MEDO-A; Carry out cell phenotype flow cytometer showed and TCR cloning afterwards and analyze, the cell phenotype flow cytometer showed is the result see the following form.
Be preferably; Above-mentioned any one described amplification in vitro method from the body specific T-cells; Also comprise in the step (2) through adding tumour antigen and stimulate so that increase and have the central type memory T cell of anti-tumor capacity in the T cell after the amplification and/or be fed back to the step of CTC cell precursors (promptly having anti-tumor capacity and immunocompetent CTC cell precursors) that back generation in the body has the cloning T cell of killing tumor cells ability; The stimulation of said tumour antigen comprises: adopt tumour or virus-specific antigen directly to stimulate; And/or, carry said specific anti primary stimuli by the self antigen presenting cell.
The said tumour antigen that adds comprises or does not comprise the specific antigens that can make sophisticated DC cell generate the DC cell vaccine.
Be preferably, said tumour antigen comprise in following any, appoint several kinds or all: TAG-1, TAG-2, TAG-3, TAG-4, TAG-5, TAG-6, TAG-7, TAG-9, TAG-10, TAG-11, TAG-12, TAG-13, TAG-14, TAG-15, TAG-16, TAG-17, TAG-18, TAG19 and TAG-20.
Increase said CTC cell precursors step particular content can for: the cell suspension that step (2.1) is made at first carries out adherent culture; Again cell suspension after the adherent culture and attached cell are cultivated separately respectively; Then; The cell suspension of cell suspension single culture gained and the attached cell of attached cell single culture gained are mixed, add SUPF-6 and SUPF-7 after the mixing, afterwards performing step (2.3).Usually, be preferably attached cell and more help obtaining sophisticated DC cell, can be used to cultivate the DC cell vaccine, the T cell and the non-adherent cell in the cell suspension more helps increasing can be used for the T cell amplification.
The condition of adherent culture can be 37 ℃ of 5% CO2 adherent culture, 2 h ~ 3 hours.
The cell suspension of adherent culture gained (2.2) is set by step cultivated.
Attached cell after the adherent culture can be cultivated as follows:
(i); Attached cell adds kinds of tumors antigen and in substratum MEDO-A, cultivates; Said kinds of tumors antigen comprises a kind of, TAG-3 among TAG-1 or the TAG-2, TAG-4, TAG-5, TAG-6, TAG-7, TAG-9, TAG-10, TAG-11, TAG-12, TAG-13, TAG-14, TAG-15, TAG-16, TAG-17, TAG-18 and by arbitrary proportion blended TAG19 and TAG-20; For example attached cell adds SUPF-1, SUPF-2 and SUPF-3 and in substratum MEDO-A, cultivates, and incubation time can be 48 hours;
(ii), step (i) adds SUPF-4 and SUPF-5 afterwards again and continues to cultivate, and incubation time can be 24 hours, for example, can be cultured to next day;
(iii), step adds corresponding tumour again after (ii) or virus antigen polypeptide is cultivated altogether, and what corresponding tumour or virus antigen polypeptide can be in the said kinds of tumors antigens is a kind of, and incubation time can be 3 hours.
Referring to the embodiment shown in Fig. 3, at first, gather patient 50mlEDTA anticoagulation; Then; Adopt density gradient method to separate PMNC (PBMCs), leave and take a certain amount of PMNC as required, so that carry out cell phenotype flow cytometer showed and TCR cloning analysis; Again the PMNC of remainder is prepared cell suspension 40ml, 37 ℃ of 5% CO then with substratum MEDO-A
2Adherent culture 3 hours is cultivated the back and is separated attached cell and suspension cell and cultivation respectively, promptly; Add stimulating factor SUPF-31 and SUPF-5 in the suspension cell after separation, in substratum MEDO-A, cultivated altogether 72 hours afterwards, cultivate the etcetera that stimulating factor is removed in the back; Attached cell after will separating simultaneously adds SUPF-1, SUPF-2 and SUPF-3, cultivates to add SUPF-4 and SUPF-5 after 48 hours, adds tumour or virus antigen polypeptide next day and cultivates altogether 3 hours; Attached cell and suspension cell after cultivating are respectively mixed; Add SUPF-6 and SUPF7 again, add SUPF-3 and SUPF-10 stimulating factor again, continue to cultivate at substratum MEDO-A; Harvested cell after 10-14 days (concrete incubation time can be confirmed according to cell quantity); Carry out cell phenotype flow cytometer showed and TCR cloning afterwards and analyze, the partial results of cell phenotype flow cytometer showed is seen Fig. 4, and the partial results that the TCR cloning is analyzed is seen Fig. 6.
Can know by Fig. 4, adopt the method for prior art to cultivate in the sample CD3 afterwards
+CD8
+T cell expressing 49.25%, and a lot of low CD8 that express are arranged
+T cell and the CD3CD8 cell (referring to Fig. 4 A1) of not expressing are compared, and adopt method of the present invention to cultivate in the sample CD3 afterwards
+CD8
+T cell expressing 92.46% especially almost all is the CD8 of high expression level
+The T cell, and almost all express CD3CD8 cell (referring to Fig. 4 A2).What is more important; Its phenotype of central type memory cell (Tcm) with the effect of height tumor-killing is CD8CD45ROCD62L, and in the sample after cultivating with the method for prior art, its Tcm only accounts for 39.64%; And there is 47.77% memory cell (CD45RO) not express CD62L (referring to Fig. 4 B1); And adopt in the sample after method of the present invention is cultivated, its Tcm can be up to 92.85%, and only 0.09% memory cell is not expressed CD62L.
Can be known that by Fig. 6 method of the present invention can go out CTC cloning precursor in vitro culture, these cloning precursors can produce a large amount of cloning T cells (CTC) after in being fed back to patient's body, and these cells are killing tumor cells or the special T cell of virus.
Method of the present invention can go out to have the T cell of polymorphum in vitro culture.These polyclones T cell can reach the purpose of immunologic reconstitution after in being fed back to chemotherapy patients's body.
Said from body specific T-cells system, all or part of T cell wherein makes through amplification in vitro for adopting above-mentioned any amplification in vitro method from the body specific T-cells (or the arbitrary method among the claim 1-15).
Be preferably T cell total amount (CD3 wherein
+The T cell) is higher than 98%.
When said system is used for immunologic reconstitution, CD4 T cell subsets (CD3 wherein
+CD4
+The T cell) content is higher than 60%; When said system is used for immunotherapy, CD4 T cell subsets (CD3 wherein
+CD4
+The T cell) content is not less than 15%.
When said system is used for immunologic reconstitution, CD8 T cell subsets (CD3 wherein
+CD8
+The T cell) content is not higher than 40% and be not less than 20%; When said system is used for immunotherapy, CD8 T cell subsets (CD3 wherein
+CD8
+The T cell) content is higher than 80%, is preferably to be higher than 90%.
Wherein activated T cell subsets content is higher than 90%, is preferably to be higher than 95%.Be preferably, said activated T cell subsets comprises CD4
+CD38
+HLADR
+And CD8
+CD38
+HLADR
+Be preferably CD4
+CD38
+HLADR
+And CD8
+CD38
+HLADR
+Content separately all is higher than 45%.
Anti-tumor activity T cell subsets (CD8 wherein
+CD45RO
+CD62L
+, Tcm) content is higher than 70%, is preferably to be higher than 90%.Said anti-tumor activity T cell subsets (CD8
+CD45RO
+CD62L
+, Tcm) be no more than 5% and do not express CD62L, be preferably and be no more than 1% and do not express CD62L, more preferably be no more than 0.1% and do not express CD62L.
Memory T cell subgroup content wherein is higher than 95%.Be preferably, said memory T cell subgroup is CD8
+CD45RO
+Be preferably CD8
+CD45RO
+Content be higher than 95%.
Be preferably, above-mentioned any from body specific T-cells system, regulatory T cells subgroup (CD4 wherein
+CD25
+FoxP3
+, Treg) content is lower than 5%.。
Saidly be used in preparation from body specific T-cells system that immunity system is rebuild and the application of the medicine of immunotherapy, said all or part of in body specific T-cells system come from above-mentioned any from body specific T-cells system (or coming from the described system of each claim among the claim 16-28).
Be preferably, said immunity system is rebuild and is mainly referred to rebuild for the immunity system behind the immune system injury that causes because of tumour, virus infection or radiotherapy, chemotherapy, and said immunotherapy mainly refers to the cellular immunization biotherapy for tumour, virus infection.The case that success is treated.Case 1. patient Zhao, the women, 38 years old, mammary cancer, through two the course of treatment chemotherapy, the TXi Baoshouti polymorphum of its t lymphocyte subset crowd CD4 and CD8 all changes, 11 days vitro culture of warp, the 95% T cell subsets that changes has recovered polymorphum.Case 2. patient kings so-and-so, the male sex, 74 years old, small cell lung cancer.Before the treatment, tumour is 5 x, 4 cm, and its tumour is reduced to 1x2cm after the immunotherapy of 3 courses of treatment.Other state of patient also all improves accordingly.
It is above-mentioned that any is said from the application of body specific T-cells system in pharmacy; Can all be said from body specific T-cells system (promptly using separately), also can partly be said from body specific T-cells system (promptly use or title mixing are used separately).
Said monitoring method from body specific T-cells system; Can be used for the monitoring of above-mentioned any corresponding component content in body specific T-cells system (or the described system of each claim of claim 16-31), comprise cell phenotype analysis and/or TCR cloning analysis.
Comprise before use and the monitoring of using the back to corresponding component content in the said system.Import before the human body after referring to before the said use cultivate completion, refer to import after the said use after the human body, the corresponding component content that is collected in the peripheral blood of patients is monitored.
Can adopt flow cytometry to carry out the cell phenotype analysis.
At first adopt the cell in fluorescent mark monoclonal antibody and the said system to make up, carry out the quantitative analysis of cell phenotype then.
Said monoclonal antibody comprise in following any, appoint several kinds or all: anti-CD3
+, CD4
+, CD8
+, CD45RA
+, CD45RO
+, CD38
+, CD62L
+, HLADR
+, CD25
+, FoxP3
+, CD28
+And CD57
+Said monoclonal antibody refers to monoclonal antibody and some etcetera, like anti-CD3
+Refer to anti-CD3 monoclonal antibody and some etcetera, anti-CD28
+Refer to anti-CD28 monoclonal antibody and some etcetera.
The concrete monitoring project of said cell phenotype analysis comprises: T cell total amount (CD3
+The T cell), CD4 T cell subsets (CD3
+CD4
+The T cell), CD8 T cell subsets (CD3
+CD8
+The T cell), activated T cell subsets (CD4
+CD38
+HLADR
+And/or CD8
+CD38
+HLADR
+), anti-tumor activity T cell subsets (CD8
+CD45RO
+CD62L
+, Tcm), regulatory T cells subgroup (CD4
+CD25
+FoxP3
+, Treg, and/or CD8
+CD28
-/ CD8
+CD57
+), memory T cell subgroup (CD4
+CD45RO
+And/or CD8
+CD45RO
+).
Be preferably, above-mentioned any monitoring method from body specific T-cells system, said TCR cloning is analyzed and is comprised that 22 V β gene families to TXi Baoshouti carry out visual and/or quantitative analysis.
The concrete grammar that said TCR cloning is analyzed can be the functional status of the antiviral or tumour that reflects the antigen specific T cells of frequency, length and the distribution quantification that occurs through the cloning of measuring TCR CDR3 gene regions.Because each T cell all has an antigen receptor; The CDR3 district (being made up of V-D-J) of this acceptor is and the main site of epitope bonded; In identification antigen process gene reset, edit, correction or advantage pcr; Formation has the different T lymphocyte clone of specific CDR3 gene regions; Therefore represent that this antigen has caused special T cellullar immunologic response, measure the functional status that frequency, length and distribution (polymorphum) that the cloning of specific T CR CDR3 gene regions occurs can reflect the lymphocytic antiviral or tumour of antigen specific T.
For accuracy, safety and the repeatability that guarantees that the TXi Baoshouti cloning is analyzed, can select any suitable method for use, can measure the grand method of microgram as adopting.For example, TXi Baoshouti is qualitative can carry out with reference to the record of patent US007375211 with quantitative analysis.This patented technology is to design for biological immunotherapy comprises the evaluation of clinical curative effect of the treatment of adopting property T cellular immunization, therapeutic tumor vaccine specially.This patent comprises technology, method and is used for the analysis software of statistics quantitative T cell cloneization and polymorphum.Application should technology can detect 67 V (Variable) district gene, and 2 D (Diversity) distinguish gene, different 24 the TXi Baoshouti V β gene families being formed of arranging with 2 C (Constant) district gene of 13 J (Joining) district gene.Wherein the Statistics Application quantitation software quantitative degree of cloning not only also can be calculated simultaneously the relation between these cloning degree and the curative effect.
Fig. 5 is a TXi Baoshouti cloning testing process.Use the PCR Sptting plate that encapsulates all ingredients in advance and make its term harmonization, measure the result comparability is arranged to guarantee it.Concrete detection method is: extract the total RNA in the cell; Then it is joined 24 hole PCR Sptting plates of the special primer that is coated with the TCR acceptor in advance and various RT and PCR reaction; After adding PCR reaction buffer and enzyme again; On the pcr amplification appearance, increase, it is quantitative with scanning that the PCR product after the amplification can carry out the high resolving power gel separation.This method adopts the monoclonal cell strain as positive control, and application of high resolution glue makes that 3 to 5 base differences are promptly separable, and polyclone, few clone or mono-clonal can both clearly be observed (referring to Fig. 5).
When relating to the technology contents of several kinds of materials mixing uses among the present invention; Unless otherwise specified; Can mix according to arbitrary proportion otherwise all refer to, and, to those skilled in the art; Its mixed every kind of material effect separately all can derive out according to the kind of the compounding substances of being selected for use, so repeat no more in the present invention.
Claims (38)
1. the amplification in vitro method from the body specific T-cells is characterized in that comprising the steps:
Step (1) is separated PMNC from peripheral blood of patients;
Step (2); Add corresponding T LSF as required; Add or do not add corresponding ESC; In corresponding substratum, the isolated PMNC of step (1) is carried out the vitro culture T cell that increases; Produce or do not produce the DC cell vaccine, the mode of said production DC cell vaccine is: when said PMNC vitro culture, add the corresponding D C cell maturation factor to obtain sophisticated DC cell, in the sophisticated DC cell that obtains, add corresponding tumour or virus-specific antigen and carry out antigenic stimulation to produce the DC cell vaccine.
2. method according to claim 1, it is characterized in that said T LSF comprise in following any, any several kinds or all SUPF-1, SUPF-2, SUPF-3, SUPF-4, SUPF-5, SUPF-6, SUPF-7, SUPF-8, SUPF-9, SUPF-10 and SUPF-31.
3. method according to claim 2 is characterized in that said substratum comprises serum free medium.
4. method according to claim 3 is characterized in that said serum free medium comprises substratum MEDO-A.
5. method according to claim 4 is characterized in that said step (2) comprising:
Step (2.1), with substratum MEDO-A said step (1) being separated the PMNC obtain, to process cell density be 1x10
6/ ml-2x10
6The cell suspension of/ml;
Step (2.2), said step (2.1) were cultivated 48-72 hour after adding SUPF – 31, SUPF-5, SUPF-6 and SUPF-7 afterwards altogether, afterwards, removed or do not remove the etcetera of T LSF;
Step (2.3), said step (2.2) add SUPF-3 and SUPF-10 afterwards again and in substratum MEDO-A, continue to cultivate.
6. method according to claim 5 is characterized in that said step (2.3) also comprises afterwards:
Step (2.4), results T cell.
7. method according to claim 6, the condition that it is characterized in that gathering in the crops the T cell is following arbitrary condition:
The T cell quantity reaches the cultivation requirement;
The incubation time of step (2.3) reaches 10-12 days (when being used for immunologic reconstitution) or 10-14 days (when being used for immunotherapy);
The omnidistance vitro culture time reaches 10-12 days (when being used for immunologic reconstitution) or or 10-14 days (when being used for immunotherapy).
8. method according to claim 1 is characterized in that peripheral blood in the step (1) with EDTA or anticoagulant heparin, adopts density gradient method to separate PMNC.
9. according to the described method of each claim among the claim 1-8; It is characterized in that in the said step (2); Also stimulate so that increase in the T cell after increasing and have anti-tumor capacity and immunocompetent CTC cell precursors through the interpolation tumour antigen; The mode that said interpolation tumour antigen stimulates is: adopt tumour or virus-specific antigen directly to stimulate, and/or, carry said specific anti primary stimuli by the self antigen presenting cell.
10. method according to claim 9, it is characterized in that said tumour antigen comprise in following any one, any several kinds or all: TAG-1, TAG-2, TAG-3, TAG-4, TAG-5, TAG-6, TAG-7, TAG-9, TAG-10, TAG-11, TAG-12, TAG-13, TAG-14, TAG-15, TAG-16, TAG-17, TAG-18, TAG19 and TAG-20.
11. method according to claim 10; It is characterized in that the cell suspension that step (2.1) is made at first carries out adherent culture; Again cell suspension after the adherent culture and attached cell are cultivated separately respectively, then, with the cell suspension and the attached cell mixing of single culture gained; Add SUPF-6 and SUPF-7 after the mixing, afterwards performing step (2.3).
12. method according to claim 11, the condition that it is characterized in that said adherent culture are 37 ℃ of 5% CO
2Adherent culture 2 hours-3 hours.
13. method according to claim 12 is characterized in that the single culture of the attached cell after the said adherent culture is carried out as follows:
(i) any one among adding TAG-1 or the TAG-2 and TAG-3, TAG-4, TAG-5, TAG-6, TAG-7, TAG-9, TAG-10, TAG-11, TAG-12, TAG-13, TAG-14, TAG-15, TAG-16, TAG-17, TAG-18, TAG19 and TAG-20; Said TAG19 and TAG-20 mix the back and add, and in substratum MEDO-A, cultivate;
(ii) step (i) adds TAG-4 and TAG-5 continuation cultivation afterwards again;
(iii), step adds corresponding tumour after (ii) or virus antigen polypeptide is cultivated altogether again.
14. method according to claim 13, it is characterized in that said step (i), (ii), incubation time (iii) was respectively 48 hours, 24 hours and 3 hours.
15. method according to claim 11 is characterized in that the single culture of the cell suspension after the said adherent culture is undertaken by said step (2.2).
16. one kind from body specific T-cells system, it is characterized in that wherein all or part of for adopting arbitrary method among the claim 1-15 to make through amplification in vitro.
17. system according to claim 16 is characterized in that T cell total amount (CD3 wherein
+The T cell) is higher than 98%.
18. system according to claim 16 is characterized in that when said system is used for immunologic reconstitution, CD4 T cell subsets (CD3 wherein
+CD4
+The T cell) content is higher than 60%; When said system is used for immunotherapy, CD4 T cell subsets (CD3 wherein
+CD4
+The T cell) content is not less than 15%.
19. system according to claim 16 is characterized in that when said system is used for immunologic reconstitution, CD8 T cell subsets (CD3 wherein
+CD8
+The T cell) content is not higher than 40% and be not less than 20%; When said system is used for immunotherapy, CD8 T cell subsets (CD3 wherein
+CD8
+The T cell) content is higher than 80%, is preferably to be higher than 90%.
20. system according to claim 16 is characterized in that wherein the T cell content is higher than 90%, is preferably to be higher than 95%.
21. system according to claim 20 is characterized in that said activated T cell subsets comprises CD4
+CD38
+HLADR
+And CD8
+CD38
+HLADR
+Two T cell subsets.
22. system according to claim 21 is characterized in that CD4
+CD38
+HLADR
+And CD8
+CD38
+HLADR
+Content separately all is higher than 45%.
23. system according to claim 16 is characterized in that anti-tumor activity T cell subsets (CD8 wherein
+CD45RO
+CD62L
+, Tcm) content is higher than 70%, is preferably to be higher than 90%.
24. system according to claim 23 is characterized in that said anti-tumor activity T cell subsets (CD8
+CD45RO
+CD62L
+, Tcm) be no more than 5% and do not express CD62L, be preferably and be no more than 1% and do not express CD62L, more preferably be no more than 0.1% and do not express CD62L.
25. system according to claim 16 is characterized in that memory T cell subgroup content wherein is higher than 95%.
26. system according to claim 25 is characterized in that said memory T cell subgroup is CD8
+CD45RO
+
27. system according to claim 26 is characterized in that CD8
+CD45RO
+Content be higher than 95%.
28., it is characterized in that regulatory T cells subgroup (CD4 wherein according to the described system of each claim among the claim 16-27
+CD25
+FoxP3
+, Treg) content is lower than 5%.
29. one kind is used in preparation from body specific T-cells system that immunity system is rebuild and the application of the medicine of immunotherapy, it is characterized in that said any claim is described in by claim 16-28 constitutes from body specific T-cells system from body specific T-cells system.
30. application according to claim 29; It is characterized in that said immunity system reconstruction mainly refers to rebuild for the immunity system behind the immune system injury that causes because of tumour, virus infection or radiotherapy, chemotherapy, said immunotherapy mainly refers to the cellular immunization biotherapy for tumour, virus infection.
31. according to claim 29 or 30 described application, it is characterized in that said from body specific T-cells system be independent utility or with other composition mixing application.
32. one kind can be used for the described component monitoring method from body specific T-cells system from body specific T-cells system of each claim among the claim 16-31, it is characterized in that comprising cell phenotype analysis and TCR cloning analysis.
33. monitoring method according to claim 32 is characterized in that adopting flow cytometry to carry out described cell phenotype analysis.
34. monitoring method according to claim 33 is characterized in that at first adopting the cell in fluorescent mark monoclonal antibody and the said system to make up, and carries out the quantitative analysis of cell phenotype then.
35. monitoring method according to claim 34, it is characterized in that said monoclonal antibody comprise in following any, appoint several kinds or all: anti-CD3
+, CD4
+, CD8
+, CD45RA
+, CD45RO
+, CD38
+, CD62L
+, HLADR
+, CD25
+, FoxP3
+, CD28
+And CD57
+
36. monitoring method according to claim 35 is characterized in that the concrete monitoring project of said cell phenotype analysis comprises: T cell total amount (CD3
+The T cell), CD4 T cell subsets (CD3
+CD4
+The T cell), CD8 T cell subsets (CD3
+CD8
+The T cell), activated T cell subsets (CD4
+CD38
+HLADR
+And/or CD8
+CD38
+HLADR
+), anti-tumor activity T cell subsets (CD8
+CD45RO
+CD62L
+, Tcm), regulatory T cells subgroup (CD4
+CD25
+FoxP3
+, Treg, and/or CD8
+CD28
-/ CD8
+CD57
+), memory T cell subgroup (CD4
+CD45RO
+And/or CD8
+CD45RO
+).
37., it is characterized in that said TCR cloning analysis comprises that 22 V β gene families to TXi Baoshouti carry out visual and/or quantitative analysis according to claim 32,33,34,35 or 36 described monitoring methods.
38., it is characterized in that the concrete grammar that said TCR cloning is analyzed is: the functional status of the antiviral or tumour that reflects the antigen specific T cells of frequency, length and the distribution quantification that occurs through the cloning of measuring TCR CDR3 gene regions according to the described monitoring method of claim 37.
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| CN105349488A (en) * | 2015-12-04 | 2016-02-24 | 深圳源正细胞医疗技术有限公司 | Combined factor for inducing tumor specific T cells and method for obtaining tumor specific T cells |
| CN108603171A (en) * | 2015-12-23 | 2018-09-28 | 基因医疗免疫疗法有限责任公司 | Antigen-specificity TCR of new generation |
| CN110241086A (en) * | 2012-06-11 | 2019-09-17 | 威尔逊沃夫制造公司 | Improved cell culture processes for adoptive cellular therapy |
| CN112304851A (en) * | 2020-10-28 | 2021-02-02 | 上海睿钰生物科技有限公司 | Evaluation method of in vitro natural killer cell immunocompetence and application thereof |
| JP2023551819A (en) * | 2020-11-25 | 2023-12-13 | ジーニアス・バイオテクノロジー・インコーポレイテッド | Antigen-specific T cells and methods for their production and use |
-
2012
- 2012-04-28 CN CN201210130553XA patent/CN102719399A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110241086A (en) * | 2012-06-11 | 2019-09-17 | 威尔逊沃夫制造公司 | Improved cell culture processes for adoptive cellular therapy |
| CN105349488A (en) * | 2015-12-04 | 2016-02-24 | 深圳源正细胞医疗技术有限公司 | Combined factor for inducing tumor specific T cells and method for obtaining tumor specific T cells |
| CN108603171A (en) * | 2015-12-23 | 2018-09-28 | 基因医疗免疫疗法有限责任公司 | Antigen-specificity TCR of new generation |
| CN112304851A (en) * | 2020-10-28 | 2021-02-02 | 上海睿钰生物科技有限公司 | Evaluation method of in vitro natural killer cell immunocompetence and application thereof |
| JP2023551819A (en) * | 2020-11-25 | 2023-12-13 | ジーニアス・バイオテクノロジー・インコーポレイテッド | Antigen-specific T cells and methods for their production and use |
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