CN110452870A - A kind of isolated culture method of tumor specific T cells and the product obtained by it - Google Patents

A kind of isolated culture method of tumor specific T cells and the product obtained by it Download PDF

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CN110452870A
CN110452870A CN201910420697.0A CN201910420697A CN110452870A CN 110452870 A CN110452870 A CN 110452870A CN 201910420697 A CN201910420697 A CN 201910420697A CN 110452870 A CN110452870 A CN 110452870A
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mononuclearcell
cells
isolated
positive
tumor
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李铁鹏
高全立
王瑶
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HENAN PROV TUMOUR HOSPITAL
Henan Cancer Hospital
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HENAN PROV TUMOUR HOSPITAL
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Abstract

The present invention relates to a kind of isolated culture method of tumor specific T cells and the products obtained by it, the isolated culture method are as follows: the peripheral blood of tumor patient is pre-processed first, the mononuclearcell in peripheral blood is separated again, then the mononuclearcell of the PD-1 positive is separated, finally the mononuclearcell of the PD-1 positive is expanded, obtains the tumor specific T cells.The method original material according to the present invention for being separately cultured tumor specific T cells is the peripheral blood of tumor patient, tumor tissues or pernicious Pleural effusions without using patient are as material, it is more convenient to draw materials, and almost all of tumor patient can facilitate blood sampling, and without compared with major trauma；The T cell tumour-specific finally obtained is high, and simultaneously includes the T cell of the CD4 positive and the CD8 positive；The operating process of this method is simple, required time is shorter.

Description

A kind of isolated culture method of tumor specific T cells and the product obtained by it
Technical field
The invention belongs to technical field of cell culture, and in particular to a kind of isolated culture method of tumor specific T cells And the product obtained by it.
Background technique
Malignant tumour seriously endangers the life of the mankind and health, the death rate occupy first of various diseases.Tumour existing three Big conventional treatments (i.e. operative treatment, radiotherapy and chemotherapy) in the past few decades in improve tumor patient Survival rate is made that major contribution, but can not solve transfer and the Problems Concerning Their Recurrence of tumour always, and the survival rate of tumor patient is in recent years Also it is no longer significantly improved, three big conventional therapies have suffered from curative effect bottleneck, and there is an urgent need to find new to break through tumour The method for treating curative effect bottleneck.
Tumor specific cytotoxic T lymphocyte (CTL) i.e. cytotoxic T lymphocyte, be received from antigen presenting cell it is anti- Prime information, and by clonal expansion can specific recognition and killing antigentic specificity target cell effector T cell, CTL It is the main mechanism that body removes cancerous tumor cell, plays main function during antineoplastic immune.CTL can not only by The substances direct killing tumour cell such as granzyme and perforin, can also be by secreting some cell factor such as IFN γs and TNF α etc. Killing tumor cell indirectly.With biomedical development, tumor specific cytotoxic T lymphocyte treatment is increasingly subject to weight in recent years Depending on showing that good application prospect, a variety of specific CTL Fiber differentiation schemes are come into being.Tumor-specific CTL treatment is swollen Tumor disease has the characteristics that safety, targets, is efficient.
But the Fiber differentiation of tumor-specific CTL needs to consume a large amount of time, and separation and incubation step are complicated, Requirement to technology is also very high, therefore significantly limits extensive utilization of this treatment method with cancer patient.Have Person has found that the cell with cytotoxicity can be expanded after IL-2 is added in cultivating system in animal experiment, but studies hair Existing this method inducing cell lethality is not strong enough, inducing specific CTL inefficiency.The TIL technology of the exploitations such as Rosenberg is deposited It is difficult to the problem of obtaining in cell, also limits its clinical application.And it is thin that tumour-specific T is separately cultured in currently available technology Cell origin needed for born of the same parents is mainly autologous patient tumor tissues or pernicious Pleural effusions, and acquiring way is inconvenient, and can give patient Bring wound.
CN107043749A discloses a kind of separation method of tumor-infiltrated T lymphocyte, and the invention is provided from reality The method of the tumor-infiltrated T lymphocyte of separant induction culture includes the following steps: (1) by size 2-3mm in body tumour3Tumour Tissue block is added in the culture hole containing separant induction culture solution and is cultivated；The separant induction culture solution is only to contain IL-7 With the RPMI-1640 complete medium of IL-15；(2) it carries out within every 3-4 days primary half amount and changes liquid；(3) it after 2-3 weeks, removes described swollen Tumor tissue block, the cell in culture hole is tumor-infiltrated T lymphocyte, using the isolated TIL purity of the method for the present invention It is high and active with stronger tumor cytotoxicity.But this method needs to obtain autologous patient tumor tissues and is more troublesome, and It is time-consuming long.
CN109402054A discloses a kind of CD4+The amplification cultivation method of memory T-lymphocyte, by culture medium In add a certain amount of cell factor IL-2, IL-7 and IL-21 simultaneously, and make to inactivate self blood in incubation in culture medium Slurry, cell factor IL-2, IL-7 and IL-21 are maintained in certain concentration range, to promote CD4+Memory T-lymphocyte Amplification cultivation effect, improve CD4+The purity and functionality of memory T-lymphocyte.The amplification cultivation method of the invention can be very Good is suitable for CD4+The amplification cultivation of memory T-lymphocyte, can induced amplification go out required enough cell concentration, satisfaction pair The demand of such T lymphocyte amplification in vitro culture.But this method can only get CD4+T lymphocyte.
To sum up, if the simple tumour extensive, operation is convenient, required time is shorter of a source material acquisition modes can be developed The isolated culture method of specific T-cells is significantly.
Summary of the invention
In view of the deficiencies of the prior art, being separately cultured the purpose of the present invention is to provide a kind of tumor specific T cells Method and the product obtained by it, such isolated culture method operating process is simple, and elapsed time is short, and raw material facilitate acquisition, And the tumour-specific for cultivating obtained T lymphocyte is higher, while including the T lymphocyte of the CD4 positive and the CD8 positive.
In order to achieve that object of the invention, the invention adopts the following technical scheme:
On the one hand, the present invention provides a kind of isolated culture method of tumor specific T cells, the isolated culture method Are as follows: the peripheral blood of tumor patient is pre-processed first, then separates the mononuclearcell in peripheral blood, finally to the PD-1 positive Mononuclearcell expanded, obtain the tumor specific T cells.
Most tumor patients not can be carried out operation at the beginning of making a definite diagnosis, and have no chance to obtain tumor tissues, simultaneously only The patient of part tumor kind (such as lung cancer, liver cancer) has pernicious Pleural effusions, and ratio is lower, while and not all Pleural effusions in all Containing greater number of T lymphocyte, T lymphocyte specific quantity is less, and according to the present invention is separately cultured tumour The method original material of specific T-cells is the peripheral blood of tumor patient, tumor tissues or pernicious chest and abdomen without using patient For water as material, it is more convenient to draw materials, and almost all of tumor patient can facilitate blood sampling, and without compared with major trauma；Isolated T Cell tumour specificity is high, and simultaneously includes the T cell of the CD4 positive and the CD8 positive；The operating process of this method is simple, institute It takes time shorter.
In the present invention, the pretreated method can be with are as follows: tumor patient is made to receive PD-1 Antybody therapy within 3 weeks.
The mode for receiving PD-1 Antybody therapy are as follows: tumor patient according to granted clinic anti-human PD-1 antibody medicine Object specification carries out the treatment (such as: receiving Wu Liyou monoclonal antibody injection by the intravenous injection of 3mg/kg weight) of a cycle.
In the present invention, the pretreated method may be: take out after the peripheral blood of tumor patient in peripheral blood PD-1 antibody is added, is then incubated for.
If patient received PD-1 Antybody therapy within 3 weeks, subsequent extraction operation can be directly entered；If patient exists Do not received PD-1 Antybody therapy in 3 weeks, then need first to carry out above-mentioned pretreatment operation, was flowed subsequently into subsequent extraction Journey.
It is had the advantage that using the first above-mentioned pretreatment mode one is tumour original in peripheral blood can be improved The ratio of specific T-cells；The second is the ability of PD-1 positive cell activation and proliferation can be improved.
Preferably, final concentration of 0.2-0.8 μ g/mL of the PD-1 antibody in peripheral blood, such as 0.2 μ g/mL, 0.3 μ G/mL, 0.4 μ g/mL, 0.5 μ g/mL, 0.6 μ g/mL, 0.7 μ g/mL or 0.8 μ g/mL etc..
Preferably, the temperature of the incubation is 0-6 DEG C, such as 0 DEG C, 1 DEG C, 2 DEG C, 3 DEG C, 4 DEG C, 5 DEG C or 6 DEG C.
Preferably, the time of the incubation be 0.5-24h, such as 0.5h, 1h, 2h, 4h, 5h, 8h, 10h, 12h, 15h, 20h or for 24 hours etc..
In the present invention, the blood of cancer patients include lung cancer, kidney, intestinal cancer, the cancer of the esophagus, malignant mela noma or The peripheral blood of cholangiocarcinoma patients.
The peripheral blood can be derived from including lung cancer, kidney, intestinal cancer, the cancer of the esophagus, malignant mela noma or cholangiocarcinoma patients Peripheral blood, the source of original material is very extensive, and the scope of application is also relatively wide.
Preferably, the method for the mononuclearcell in the separation blood of cancer patients are as follows: blood of cancer patients is used The isolated mononuclearcell of Ficoll density gradient method.
Density gradient can directly be used by carrying out cleaning without human peripheral blood salting liquid etc. before the separation mononuclearcell Method is separated, and operation is enormously simplified.
Preferably, the method for the mononuclearcell of separation PD-1 positive are as follows: by isolated mononuclearcell and 4 antibody of anti-human igg of biotin modification carries out primary incubation altogether, adds anti-biotin antibodies or the coated magnetic of Streptavidin Pearl carries out secondary total incubation, and magnetic separation obtains the mononuclearcell of the PD-1 positive.
Herein 4 antibody of anti-human igg of biotin modification can also with biotinylated LAG-3 antibody, TIM-3 antibody etc. into Row replacement, magnetic separating mode can also be substituted with selected by flow cytometry apoptosis mode herein.
As application of the PD-1 antibody in clinic is more extensive, most of tumor patient can receive controlling for such drug It treats, the PD-1 molecule on PD-1 positive cell surface has been selectively bound by the antibody in these medication peripheral blood in patients, at this time cannot be straight It connects and is separated using PD-1 antibody, separated here with IgG4 antibody and cleverly evaded this problem, and existing granted face The PD-1 antibody of bed application is IgG4 hypotype.
Preferably, the temperature being once incubated for altogether is 20-30 DEG C, such as 20 DEG C, 22 DEG C, 24 DEG C, 25 DEG C, 26 DEG C, 28 DEG C or 30 DEG C etc..
Preferably, once time for being incubated for altogether is 10-30min, for example, 10min, 15min, 20min, 25min, 28min or 30min etc..
Preferably, the use concentration of 4 antibody of anti-human igg of the biotin modification is every 107A mononuclearcell uses 1-4 μ g, such as 1 μ g, 1.5 μ g, 2 μ g, 2.5 μ g, 3 μ g or 4 μ g etc..
Preferably, the secondary temperature being incubated for altogether is 20-30 DEG C, such as 20 DEG C, 22 DEG C, 24 DEG C, 25 DEG C, 26 DEG C, 28 DEG C or 30 DEG C etc..
Preferably, the secondary time being incubated for altogether is 5-20min, such as 5min, 8min, 10min, 12min, 15min Or 20min etc..
Preferably, the method that the mononuclearcell to the PD-1 positive is expanded are as follows: by isolated PD-1 sun Property mononuclearcell be resuspended with serum-free medium, plant and expanded into pre-coated culture bottle, it is described swollen after being expanded Tumor specific T-cells.
Preferably, the pre-coated culture bottle is by recombinant human fibronectin polypeptide segment (such as trade name RetroNectinTM) and anti-human CD3 activating antibodies (such as clone number: OKT3) carry out coated culture bottle.
Culture bottle recombinant human fibronectin polypeptide segment and anti-human CD3 activating antibodies are subjected to coating processing, energy simultaneously Significantly improve the expanding effect of tumor specific T cells.
Preferably, the final concentration of 4-8 μ g/mL of the fibronectin, such as 4 μ g/mL, 4.5 μ g/mL, 5 μ g/mL, 5.5 μ G/mL, 6 μ g/mL, 7 μ g/mL or 8 μ g/mL etc..
Preferably, the clone position OKT3 of the anti-human CD3 activating antibodies, final concentration of 1-2 μ g/mL, such as 1 μ g/mL, 1.2 μ g/mL, 1.4 μ g/mL, 1.6 μ g/mL, 1.8 μ g/mL or 2 μ g/mL etc..
Preferably, the serum-free medium includes IL-2 and protein kinase B inhibitor (AKTi).
Protein kinase B inhibitor (AKTi) is added in serum-free medium can to expand Memorability T in finished product Percentage of lymphocyte significantly improves, and concentration selects 0.5-2 μM.
Preferably, the serum-free medium further includes any one in IL-7, IL-15 or IL-21 or at least two Combination, combination, IL-7 and IL-15 and combination of IL-21 etc. of described at least two combination including IL-7 and IL-15.
The use concentration of IL-2, IL-7, IL-15 and IL-21 be respectively 800-1200IU, 8-12ng/mL, 8-12ng/mL, 8-12ng/mL。
As the preferred technical solution of the present invention, the isolated culture method specifically comprises the following steps:
(1) take tumor patient peripheral blood be added PD-1 antibody make its final concentration of 0.2-0.8 μ g/mL, at 0-6 DEG C into Row is incubated for 0.5-24h；Or tumor patient is made to receive PD-1 Antybody therapy within 3 weeks；
(2) product for obtaining step (1) the isolated mononuclearcell of Ficoll density gradient method；
(3) the isolated mononuclearcell of step (2) and biotinylated 4 antibody of anti-human igg is total at 20-30 DEG C It is incubated for 10-30min, anti-biotin antibodies is added or the coated magnetic bead of Streptavidin is incubated for 5- at 20-30 DEG C altogether 20min, magnetic separation obtain the mononuclearcell of the PD-1 positive；
(4) by the mononuclearcell of the isolated PD-1 positive of step (3) with including IL-2, IL-7, IL-15, IL- 21 and protein kinase B inhibitor serum-free medium be resuspended, plant by 4-8 μ g/mL fibronectin and the anti-human CD3 of 1-2 μ g/mL Treated that pre-coated culture bottle is expanded for antibody, the tumor specific T cells after being expanded.
On the other hand, the present invention provides a kind of tumour-specific isolated using isolated culture method as described above T cell.
Compared with the existing technology, the invention has the following advantages:
The method original material according to the present invention for being separately cultured tumor specific T cells is the periphery of tumor patient Blood, the tumor tissues or pernicious Pleural effusions without using patient are as material, and it is more convenient to draw materials, and almost all of tumor patient is equal Blood sampling can be facilitated, and without compared with major trauma；Isolated T cell tumour-specific is high, and simultaneously includes the CD4 positive and CD8 Positive T cell；The operating process of this method is simple, required time is shorter.
Detailed description of the invention
Fig. 1 is that (CD4 is positive thin for the finished product internal effect memory t cell ration statistics figure that obtains of five kinds of training methods Born of the same parents)；
Fig. 2 is that (CD8 is positive thin for the finished product internal effect memory t cell ration statistics figure that obtains of five kinds of training methods Born of the same parents)；
Fig. 3 is tumor specific T cells proliferation times of the sample 1 under two different pretreatment modes in embodiment 2 As a result statistical chart；
Fig. 4 is tumor specific T cells proliferation times of the sample 2 under two different pretreatment modes in embodiment 2 As a result statistical chart.
Specific embodiment
The technical scheme of the invention is further explained by means of specific implementation.Those skilled in the art should be bright , the described embodiments are merely helpful in understanding the present invention, should not be regarded as a specific limitation of the invention.
Embodiment 1
Influence of the factor pattern to effect memory T-lymphocyte ratio in cell finished product in culture medium
The present embodiment probes into five kinds of different training methods to effect memory t cell (specific table in cell finished product Type are as follows: CD3+CD8+CD45RA-CCR7-And CD3+CD4+CD45RA-CCR7-) ratio influence, concrete operations include following step It is rapid:
(1) it takes in 3 weeks by the peripheral blood of the patients with renal cell carcinoma of PD-1 Antybody therapy；
(2) peripheral blood for obtaining step (1) the isolated mononuclearcell of Ficoll density gradient method；
(3) the isolated mononuclearcell of step (2) and biotinylated 4 antibody of anti-human igg are incubated altogether at 25 DEG C 20min is educated, the coated magnetic bead of anti-biotin antibodies is added and is incubated for 10min altogether at 25 DEG C, magnetic separation obtains the PD-1 positive Mononuclearcell；
(4) mononuclearcell of the isolated PD-1 positive of step (3) used containing IL-2 respectively, contain IL-2, IL- 7 and IL-15, contain IL-2 and IL-21, contain IL-2 and AKTi and contain IL-2, IL-7, IL-15, IL-21, AKTi simultaneously Serum-free medium be resuspended, plant by treated the pre-coated training of 6 μ g/mL fibronectins and 1.5 μ g/mL anti-CD3antibodies Feeding bottle is expanded, tumor specific T cells product (IL-2, IL-7, IL-15, IL-21, AKTi after being expanded after 10 days Usage amount be respectively 1000IU, 10ng/mL, 10ng/mL, 10ng/mL, 1 μM).
Effect memory t cell (the T for being included to CD4 positive cell in finished product and CD8 positive cell respectivelyEM, i.e., CD3+CD8+CD45RA-CCR7-And CD3+CD4+CD45RA-CCR7-) ratio counted, value is higher to represent tumour-specific The expanding effect of T cell is better, as a result (CD45RA in ordinate in figure as depicted in figs. 1 and 2-CCR7-I.e. characterization CD45RA and The Effector memory T cell subgroup of the double-negative expression of CCR7, abscissa IL-2, IL-2/7/15, IL-2/21, IL-2/AKTi, IL- 2/7/15/21/AKTi respectively indicate with containing IL-2, containing IL-2, IL-7 and IL-15, containing IL-2 and IL-21, contain IL- 2 and AKTi and simultaneously containing the serum-free medium of IL-2, IL-7, IL-15, IL-21, AKTi): with containing IL-2 respectively Culture solution in be added IL-7, IL-15, IL-21 compare, in the culture solution containing IL-2 be added protein kinase B inhibitor can To significantly improve the expanding effect of T cell, there is statistical difference.
Embodiment 2
Influence of two different PD-1 antibody pretreatment modes to PD-1 positive cell proliferative capacity
The present embodiment is probed into two different PD-1 antibody pretreatment modes and is expanded isolated tumor specific T cells The influence of speedup degree, the present embodiment carry out the experiment of two samples altogether, one is the peripheral blood (sample 1) of cervical cancer patient, Second is that the peripheral blood (sample 2) of melanoma cancer patients, concrete operations include the following steps:
(1) cervical cancer patient and melanoma cancer patients are taken (in 3 weeks) PD-1 antibody drug treatment is forward and backward respectively Peripheral blood.PD-1 antibody is added in peripheral blood in patients without the treatment of PD-1 antibody drug makes its final concentration of 0.5 μ g/mL, 4 Incubation 0.5h is carried out at DEG C, in this, as pretreated group 1；Peripheral blood in patients after the treatment of PD-1 antibody drug is directly used in Step (2), as pretreated group 2；
(2) peripheral blood for obtaining step (1) the isolated mononuclearcell of Ficoll density gradient method；
(3) the isolated mononuclearcell of step (2) and biotinylated 4 antibody of anti-human igg are incubated altogether at 25 DEG C 20min is educated, the coated magnetic bead of anti-biotin antibodies is added and is incubated for 10min altogether at 25 DEG C, magnetic separation obtains the PD-1 positive Mononuclearcell；
(4) serum-free of the mononuclearcell of the isolated PD-1 positive of step (3) containing IL-2 and AKTi is trained Nutrient solution is resuspended, and plants by 6 μ g/mL fibronectins and 1.5 μ g/mL anti-CD3antibodies, treated that pre-coated culture bottle is expanded Increase, counts the quantity of PD-1 positive T cell after 0 day, 6 days and 14 days respectively, as a result as shown in Figure 3 and Figure 4: with pretreated group 1 compares, and the amplification ability of two in pretreated group 2 sample tumor specific T cells significantly improves.
Embodiment 3
Evaluation test to the tumour-specific of isolated T cell (sample is patients with renal cell carcinoma peripheral blood)
The present embodiment evaluates the ability of isolated T cell identification autologous tumour cell, thin with secretion of gamma-IFN Born of the same parents account for CD8+And CD4+Ratio (the i.e. CD8 of cell+IFN-γ+%, CD4+IFN-γ+It %) is characterized, ratio is higher illustrates T The tumour-specific of cell is higher.
Concrete operations include the following steps:
(1) test is divided into three groups, does not sort group, PD-1 feminine gender group and PD-1 positive group respectively；
(2) peripheral blood of patients with renal cell carcinoma is taken, is added PD-1 antibody incubation (pretreatment)；
(3) peripheral blood for obtaining step (2) the isolated mononuclearcell of Ficoll density gradient method；
(4) the isolated mononuclearcell of step (3) and biotinylated 4 antibody of anti-human igg are incubated altogether at 25 DEG C 20min is educated, the coated magnetic bead of anti-biotin antibodies is added and is incubated for 10min altogether at 25 DEG C, magnetic separation obtains the PD-1 positive Mononuclearcell, i.e. PD-1 positive group；Sorting remainder is PD-1 feminine gender group；It is not sort without this step operation Group；
(5) above-mentioned three groups of cells are resuspended with the serum-free medium containing IL-2 and AKTi respectively, are planted by 6 μ g/mL Treated that pre-coated culture bottle is expanded for fibronectin and 1.5 μ g/mL anti-CD3antibodies, the T after being expanded after 14 days Cell makes T cell respectively in the case where having tumour cell stimulation and negative for tumor cells stimulation, and statistics secretion of gamma-IFN cell accounts for CD8+With CD4+The ratio of cell, method particularly includes:
Autologous patient tumour cell (deriving from tumor tissues) is obtained first: the renal carcinoma tissue that operation obtains being shredded, is added Add clostridiopetidase A, hyaluronidase, DNA enzymatic to digest 0.5-2h in 37 DEG C of constant incubators, is washed twice with phosphate buffer later. Then tumour cell is obtained with Ficoll Percoll gradient centrifugation, is frozen in liquid nitrogen container spare (thin in this, as specific T lymphocytes The target cell of born of the same parents).
Carry out T lymphocyte specific evaluation (being incubated for method altogether) again: take above-mentioned three groups of cultures T cell (do not sort group, PD-1 feminine gender group and PD-1 positive group), with addition IFN-γ secretion blocking agent Brefeldin after serum-free medium washes twice A (concentration 5ng/ml) but the serum-free medium resuspension for being free of any cell factor, with autologous tumor cell in the ratio of 1:1 Mixing, 37 DEG C of constant incubators receive cell after being incubated for 6h.Fixer is added after cell surface marker CD3, CD4, CD8 fluorescence antibody It is fixed, with staining kit rupture of membranes intracellular and IFN-γ fluorescence antibody is marked later, is finally secreted using flow cytomery IFN-γ cell accounts for CD8+And CD4+Ratio (the CD8 of cell+IFN-γ+%, CD4+IFN-γ+%).Wherein, by IFN-γ antibody Isotype control (isotype) antibody mark group be used as " Isotype control group ", autologous tumor cell group will be not added with as " not piercing Swash group " (embodying T lymphocyte Autocrine IFN-γ ability, as background value), it (is embodied co-cultivation group as " stimulation group " Respond the T lymphocyte ratio of autologous tumor cell stimulation).
According to experimental design, " stimulation group " subtracts " not stimulating group " and obtains this kind of intracellular specific T-cells ratio, with this Evaluate the ratio height of contained tumor specific T cells.The value is higher, illustrates that this kind of training method can obtain more tumours Specific T-cells.
The results are shown in Table 1: in CD4+In cell subsets, not sorting group, PD-1 feminine gender group and PD-1 positive component is not 0.62%, 0.8%, 1.19%, wherein PD-1 positive group numerical value highest, shows that contained tumor specific T cells are most；Equally , in CD8+In cell subsets, do not sort group, PD-1 feminine gender group and PD-1 positive component be not 0.63%, 0.68%, 1.66%, wherein PD-1 positive group numerical value highest, shows that contained tumor specific T cells are most.
Table 1
Embodiment 4
Evaluation test to the tumour-specific of isolated T cell (sample is peripheral blood from patients with lung cancer)
Concrete operations include the following steps:
Obtain autologous patient tumour cell (deriving from Pleural effusions): the pernicious Pleural effusions centrifugation that will acquire, phosphate-buffered Liquid is resuspended, and obtains tumour cell with Ficoll Percoll gradient centrifugation, freezes in liquid nitrogen container spare (thin as specific T lymphocytes The target cell of born of the same parents).
Concrete operations include the following steps:
(1) test is divided into three groups, does not sort group, PD-1 feminine gender group and PD-1 positive group respectively；
(2) peripheral blood of patients with lung cancer is taken, is added PD-1 antibody incubation (pretreatment)；
(3) peripheral blood for obtaining step (2) the isolated mononuclearcell of Ficoll density gradient method；
(4) the isolated mononuclearcell of step (3) and biotinylated 4 antibody of anti-human igg are incubated altogether at 25 DEG C 20min is educated, the coated magnetic bead of anti-biotin antibodies is added and is incubated for 10min altogether at 25 DEG C, magnetic separation obtains the PD-1 positive Mononuclearcell, i.e. PD-1 positive group；Sorting remainder is PD-1 feminine gender group；It is not sort without this step operation Group；
(5) above-mentioned three groups of cells are resuspended with the serum-free medium containing IL-2 and AKTi respectively, are planted by 6 μ g/mL Treated that pre-coated culture bottle is expanded for fibronectin and 1.5 μ g/mL anti-CD3antibodies, the T after being expanded after 14 days Cell makes T cell respectively in the case where having tumour cell stimulation and negative for tumor cells stimulation, and statistics secretion of gamma-IFN cell accounts for CD8+With CD4+The ratio of cell, method particularly includes:
Autologous patient tumour cell (deriving from Pleural effusions) is obtained first: the pernicious Pleural effusions centrifugation that will acquire, phosphate Buffer is resuspended, and obtains tumour cell with Ficoll Percoll gradient centrifugation, freezes in liquid nitrogen container spare (in this, as specificity The target cell of T lymphocyte).
Carry out T lymphocyte specific evaluation (being incubated for method altogether) again: take above-mentioned three groups of cultures T cell (do not sort group, PD-1 feminine gender group and PD-1 positive group), with addition IFN-γ secretion blocking agent Brefeldin after serum-free medium washes twice A (concentration 5ng/ml) but the serum-free medium resuspension for being free of any cell factor, with autologous tumor cell in the ratio of 1:1 Mixing, 37 DEG C of constant incubators receive cell after being incubated for 6h.Fixer is added after cell surface marker CD3, CD4, CD8 fluorescence antibody It is fixed, with staining kit rupture of membranes intracellular and IFN-γ fluorescence antibody is marked later, is finally secreted using flow cytomery IFN-γ cell accounts for CD8+And CD4+Ratio (the CD8 of cell+IFN-γ+%, CD4+IFN-γ+%).
The results are shown in Table 2: in CD4+In cell subsets, not sorting group, PD-1 feminine gender group and PD-1 positive component is not 0.09%, -0.03%, 0.98%, wherein PD-1 positive group numerical value highest, shows that contained tumor specific T cells are most；Equally , in CD8+In cell subsets, do not sort group, PD-1 feminine gender group and PD-1 positive component be not 0.61%, 0.97%, 3.25%, wherein PD-1 positive group numerical value highest, shows that contained tumor specific T cells are most.
Table 2
Embodiment 5
Evaluation test to the tumour-specific of isolated T cell (sample is melanoma cancer patients' peripheral blood)
Concrete operations include the following steps:
(1) test is divided into three groups, does not sort group, PD-1 feminine gender group and PD-1 positive group respectively；
(2) peripheral blood of melanoma cancer patients is taken, is added PD-1 antibody incubation (pretreatment)；
(3) peripheral blood for obtaining step (2) the isolated mononuclearcell of Ficoll density gradient method；
(4) the isolated mononuclearcell of step (3) and biotinylated 4 antibody of anti-human igg are incubated altogether at 25 DEG C 20min is educated, the coated magnetic bead of anti-biotin antibodies is added and is incubated for 10min altogether at 25 DEG C, magnetic separation obtains the PD-1 positive Mononuclearcell, i.e. PD-1 positive group；Sorting remainder is PD-1 feminine gender group；It is not sort without this step operation Group；
(5) above-mentioned three groups of cells are resuspended with the serum-free medium containing IL-2 and AKTi respectively, are planted by 6 μ g/mL Treated that pre-coated culture bottle is expanded for fibronectin and 1.5 μ g/mL anti-CD3antibodies, the T after being expanded after 14 days Cell makes T cell respectively in the case where having tumour cell stimulation and negative for tumor cells stimulation, and statistics secretion of gamma-IFN cell accounts for CD8+With CD4+The ratio of cell, method particularly includes:
Autologous patient tumour cell (deriving from tumor tissues): the melanoma cancer tissue that operation is obtained is obtained first It shreds, addition clostridiopetidase A, hyaluronidase, DNA enzymatic digest 0.5-2h in 37 DEG C of constant incubators, use phosphate buffer later It washes twice.Then obtain tumour cell with Ficoll Percoll gradient centrifugation, freeze in liquid nitrogen container it is spare, in this, as specificity The target cell of T lymphocyte).
T lymphocyte specific evaluation (being incubated for method altogether) is carried out again: same as Example 3.
The results are shown in Table 3: in CD4+In cell subsets, do not sort group, PD-1 feminine gender group and PD-1 positive component be not - 0.13%, 0.07%, 0.51%, wherein PD-1 positive group numerical value highest, shows that contained tumor specific T cells are most；Equally , in CD8+In cell subsets, do not sort group, PD-1 feminine gender group and PD-1 positive component be not -0.15%, 0.07%, 0.5%, wherein PD-1 positive group numerical value highest, shows that contained tumor specific T cells are most.
Table 3
Embodiment 6
Evaluation test to the tumour-specific of isolated T cell (sample is peripheral blood from patients with esophageal cancer)
Concrete operations include the following steps:
(1) test is divided into three groups, does not sort group, PD-1 feminine gender group and PD-1 positive group respectively；
(2) peripheral blood of patient with esophageal carcinoma is taken, is added PD-1 antibody incubation (pretreatment)；
(3) peripheral blood for obtaining step (2) the isolated mononuclearcell of Ficoll density gradient method；
(4) the isolated mononuclearcell of step (3) and biotinylated 4 antibody of anti-human igg are incubated altogether at 25 DEG C 20min is educated, the coated magnetic bead of anti-biotin antibodies is added and is incubated for 10min altogether at 25 DEG C, magnetic separation obtains the PD-1 positive Mononuclearcell, i.e. PD-1 positive group；Sorting remainder is PD-1 feminine gender group；It is not sort without this step operation Group；
(5) above-mentioned three groups of cells are resuspended with the serum-free medium containing IL-2 and AKTi respectively, are planted by 6 μ g/mL Treated that pre-coated culture bottle is expanded for fibronectin and 1.5 μ g/mL anti-CD3antibodies, the T after being expanded after 14 days Cell makes T cell respectively in the case where having tumour cell stimulation and negative for tumor cells stimulation, and statistics secretion of gamma-IFN cell accounts for CD8+With CD4+The ratio of cell, method particularly includes:
Autologous patient tumour cell (deriving from tumor tissues) is obtained first: the human esophageal carcinoma that operation obtains is shredded, It adds clostridiopetidase A, hyaluronidase, DNA enzymatic and digests 0.5-2h in 37 DEG C of constant incubators, wash two with phosphate buffer later Time.Then tumour cell is obtained with Ficoll Percoll gradient centrifugation, freezes in liquid nitrogen container spare, drenched in this, as specificity T The target cell of bar cell).
T lymphocyte specific evaluation (being incubated for method altogether) is carried out again: same as Example 3.
The results are shown in Table 4: in CD4+In cell subsets, not sorting group, PD-1 feminine gender group and PD-1 positive component is not 0.15%, 0.54%, 1.36%, wherein PD-1 positive group numerical value highest, shows that contained tumor specific T cells are most；Equally , in CD8+In cell subsets, not sorting group, PD-1 feminine gender group and PD-1 positive component is not 1.44%, 2.1%, 6.15%, Wherein PD-1 positive group numerical value highest shows that contained tumor specific T cells are most.
Table 4
Embodiment 7
Evaluation test to the tumour-specific of isolated T cell (sample is cholangiocarcinoma patients peripheral blood)
Concrete operations include the following steps:
(1) test is divided into three groups, does not sort group, PD-1 feminine gender group and PD-1 positive group respectively；
(2) peripheral blood of cholangiocarcinoma patients is taken, is added PD-1 antibody incubation (pretreatment)；
(3) peripheral blood for obtaining step (2) the isolated mononuclearcell of Ficoll density gradient method；
(4) the isolated mononuclearcell of step (3) and biotinylated 4 antibody of anti-human igg are incubated altogether at 25 DEG C 20min is educated, the coated magnetic bead of anti-biotin antibodies is added and is incubated for 10min altogether at 25 DEG C, magnetic separation obtains the PD-1 positive Mononuclearcell, i.e. PD-1 positive group；Sorting remainder is PD-1 feminine gender group；It is not sort without this step operation Group；
(5) above-mentioned three groups of cells are resuspended with the serum-free medium containing IL-2 and AKTi respectively, are planted by 6 μ g/mL Treated that pre-coated culture bottle is expanded for fibronectin and 1.5 μ g/mL anti-CD3antibodies, the T after being expanded after 14 days Cell makes T cell respectively in the case where having tumour cell stimulation and negative for tumor cells stimulation, and statistics secretion of gamma-IFN cell accounts for CD8+With CD4+The ratio of cell, method particularly includes:
Autologous patient tumour cell (deriving from Pleural effusions) is obtained first: the pernicious Pleural effusions centrifugation that will acquire, phosphate Buffer is resuspended, and obtains tumour cell with Ficoll Percoll gradient centrifugation, freezes in liquid nitrogen container spare (in this, as specificity The target cell of T lymphocyte).
T lymphocyte specific evaluation (being incubated for method altogether) is carried out again: same as Example 3.
The results are shown in Table 5: in CD4+In cell subsets, PD-1 feminine gender group and PD-1 positive component be not 0.46%, 0.3%, wherein PD-1 positive group numerical value highest, shows that contained tumor specific T cells are most；Likewise, in CD8+Cell is sub- In group, PD-1 feminine gender group and PD-1 positive component are not -0.19%, 1.02%, and wherein PD-1 positive group numerical value highest, shows institute It is most containing tumor specific T cells.
Table 5
Embodiment 8
Evaluation test to the tumour-specific of isolated T cell (sample is patients with bowel cancer peripheral blood)
Concrete operations include the following steps:
(1) test is divided into three groups, does not sort group, PD-1 feminine gender group and PD-1 positive group respectively；
(2) peripheral blood of patients with bowel cancer is taken, is added PD-1 antibody incubation (pretreatment)；
(3) peripheral blood for obtaining step (2) the isolated mononuclearcell of Ficoll density gradient method；
(4) the isolated mononuclearcell of step (3) and biotinylated 4 antibody of anti-human igg are incubated altogether at 25 DEG C 20min is educated, the coated magnetic bead of anti-biotin antibodies is added and is incubated for 10min altogether at 25 DEG C, magnetic separation obtains the PD-1 positive Mononuclearcell, i.e. PD-1 positive group；Sorting remainder is PD-1 feminine gender group；It is not sort without this step operation Group；
(5) above-mentioned three groups of cells are resuspended with the serum-free medium containing IL-2 and AKTi respectively, are planted by 6 μ g/mL Treated that pre-coated culture bottle is expanded for fibronectin and 1.5 μ g/mL anti-CD3antibodies, the T after being expanded after 14 days Cell makes T cell respectively in the case where having tumour cell stimulation and negative for tumor cells stimulation, and statistics secretion of gamma-IFN cell accounts for CD8+With CD4+The ratio of cell, method particularly includes:
Autologous patient tumour cell (deriving from Pleural effusions) is obtained first: the pernicious Pleural effusions centrifugation that will acquire, phosphate Buffer is resuspended, and obtains tumour cell with Ficoll Percoll gradient centrifugation, freezes in liquid nitrogen container spare (in this, as specificity The target cell of T lymphocyte).
T lymphocyte specific evaluation (being incubated for method altogether) is carried out again: same as Example 3.
The results are shown in Table 6: in CD4+In cell subsets, PD-1 feminine gender group and PD-1 positive component be not 5.37%, 8.43%, wherein PD-1 positive group numerical value highest, shows that contained tumor specific T cells are most；Likewise, in CD8+Cell is sub- In group, PD-1 feminine gender group and PD-1 positive component are not 13.54%, 20.3%, and wherein PD-1 positive group numerical value highest, shows institute It is most containing tumor specific T cells.
Table 6
Embodiment 9
Evaluation test to the tumour-specific of isolated T cell (sample is another patients with renal cell carcinoma peripheral blood)
Concrete operation step is consistent with embodiment 3.
The results are shown in Table 7, from 7 data of table: in CD4+In cell subsets, group, PD-1 feminine gender group and PD- are not sorted 1 positive component is not 0.42%, 7%, wherein PD-1 positive group numerical value highest, shows that contained tumor specific T cells are most；Together Sample, in CD8+In cell subsets, not sorting group, PD-1 feminine gender group and PD-1 positive component is not 0.58%, 8.64%, wherein PD-1 positive group numerical value highest shows that contained tumor specific T cells are most.
Table 7
Embodiment 10
To the evaluation test of the tumour-specific of isolated T cell, (sample is another melanoma cancer patients periphery Blood)
Concrete operation step is consistent with embodiment 5.
The results are shown in Table 8, from 8 data of table: in CD4+In cell subsets, group, PD-1 feminine gender group and PD- are not sorted 1 positive component is not 0.086%, -0.34%, 0.38%, wherein PD-1 positive group numerical value highest, shows contained tumour-specific T cell is most；Likewise, in CD8+In cell subsets, not sorting group, PD-1 feminine gender group and PD-1 positive component is not 0.005%, -0.15%, 0.71%, wherein PD-1 positive group numerical value highest, shows that contained tumor specific T cells are most.
Table 8
The Applicant declares that a kind of point of the present invention is explained by the above embodiments tumor specific T cells of the invention From cultural method and the product obtained by it, but the present invention is not limited to the above embodiments, that is, does not mean that the present invention is necessary Relying on above-described embodiment could implement.It should be clear to those skilled in the art, any improvement in the present invention, to this The equivalence replacement of each raw material of invention product and addition, the selection of concrete mode of auxiliary element etc., all fall within protection of the invention Within range and the open scope.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above Detail within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, this A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can No further explanation will be given for the combination of energy.

Claims (10)

1. a kind of isolated culture method of tumor specific T cells, which is characterized in that the isolated culture method are as follows: right first The peripheral blood of tumor patient is pre-processed, then separates the mononuclearcell in peripheral blood, then separates the single of the PD-1 positive Nucleus finally expands the mononuclearcell of the PD-1 positive, obtains the tumor specific T cells.
2. the isolated culture method of tumor specific T cells as described in claim 1, which is characterized in that described pretreated Method are as follows: tumor patient is made to receive PD-1 Antybody therapy within 3 weeks.
3. the isolated culture method of tumor specific T cells as described in claim 1, which is characterized in that described pretreated Method are as follows: PD-1 antibody is added in peripheral blood after taking out the peripheral blood of tumor patient, is then incubated for；
Preferably, final concentration of 0.2-0.8 μ g/mL of the PD-1 antibody in peripheral blood；
Preferably, the temperature of the incubation is 0-6 DEG C；
Preferably, the time of the incubation is 0.5-24h.
4. the isolated culture method of tumor specific T cells as claimed in any one of claims 1-3, which is characterized in that institute State the peripheral blood that blood of cancer patients includes lung cancer, kidney, intestinal cancer, the cancer of the esophagus, malignant mela noma or cholangiocarcinoma patients.
5. such as the isolated culture method of tumor specific T cells of any of claims 1-4, which is characterized in that institute The method for stating the mononuclearcell in separation blood of cancer patients are as follows: blood of cancer patients Ficoll density gradient method point From obtaining mononuclearcell.
6. the isolated culture method of tumor specific T cells according to any one of claims 1 to 5, which is characterized in that institute The method for stating the mononuclearcell of the separation PD-1 positive are as follows: by isolated mononuclearcell and biotinylated anti-human igg 4 Antibody carries out primary incubation altogether, adds anti-biotin antibodies or the coated magnetic bead of Streptavidin carries out secondary total incubation, magnetic The mononuclearcell of the isolated PD-1 positive of power；
Preferably, the temperature being once incubated for altogether is 20-30 DEG C；
Preferably, the time being once incubated for altogether is 10-30min；
Preferably, the use concentration of 4 antibody of biotinylated anti-human igg is every 107A mononuclearcell uses 1-4 μ g；
Preferably, the secondary temperature being incubated for altogether is 20-30 DEG C；
Preferably, the secondary time being incubated for altogether is 5-20min.
7. such as the isolated culture method of tumor specific T cells of any of claims 1-6, which is characterized in that institute State the method expanded to the mononuclearcell of the PD-1 positive are as follows: by the mononuclearcell nothing of the isolated PD-1 positive Serum free culture system liquid is resuspended, and plants and is expanded into pre-coated culture bottle, the tumor specific T cells after being expanded.
8. the isolated culture method of tumor specific T cells as claimed in claim 7, which is characterized in that the pre-coated training Feeding bottle is to carry out coated culture bottle by recombinant human fibronectin polypeptide segment and anti-human CD3 activating antibodies；
Preferably, the final concentration of 4-8 μ g/mL of the fibronectin；
Preferably, the final concentration of 1-2 μ g/mL of the anti-CD3antibody；
Preferably, the serum-free medium includes interleukin 2 and protein kinase B inhibitor；
Preferably, the serum-free medium further includes appointing in interleukin-17, interleukin 15 or interleukin 21 It anticipates a kind of or at least two combinations.
9. such as the isolated culture method of tumor specific T cells of any of claims 1-6, which is characterized in that institute Isolated culture method is stated to specifically comprise the following steps:
(1) taking the peripheral blood of tumor patient that PD-1 antibody is added makes its final concentration of 0.2-0.8 μ g/mL, is incubated at 0-6 DEG C Educate 0.5-24h；Or tumor patient is made to receive PD-1 Antybody therapy within 3 weeks；
(2) product for obtaining step (1) the isolated mononuclearcell of Ficoll density gradient method；
(3) 4 antibody of anti-human igg of the isolated mononuclearcell of step (2) and biotinylation modification is total at 20-30 DEG C It is incubated for 10-30min, anti-biotin antibodies is added or the coated magnetic bead of Streptavidin is incubated for 5- at 20-30 DEG C altogether 20min, magnetic separation obtain the mononuclearcell of the PD-1 positive；
(4) by the mononuclearcell of the isolated PD-1 positive of step (3) with including interleukin 2, interleukins 7, the serum-free medium of interleukin 15, interleukin 21 and protein kinase B inhibitor is resuspended, and plants by 4-8 μ g/mL Treated that pre-coated culture bottle is expanded for recombinant human fibronectin polypeptide segment and the anti-human CD3 activating antibodies of 1-2 μ g/mL, obtains Tumor specific T cells after amplification.
10. a kind of tumour-specific T isolated using the isolated culture method of any of claims 1-9 is thin Born of the same parents.
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