CN110713978A

CN110713978A - Separation method of tumor antigen specific tumor infiltrating T cells

Info

Publication number: CN110713978A
Application number: CN201911122814.1A
Authority: CN
Inventors: 谢云青; 郑秋红
Original assignee: Fujian Cancer Hospital (fujian Cancer Institute Fujian Cancer Prevention And Treatment Center)
Current assignee: Fujian Cancer Hospital (fujian Cancer Institute Fujian Cancer Prevention And Treatment Center)
Priority date: 2019-11-16
Filing date: 2019-11-16
Publication date: 2020-01-21
Anticipated expiration: 2039-11-16
Also published as: CN110713978B

Abstract

The invention provides a separation culture method of Tumor specific Tumor Infiltrating T cell (TIL) cells from Tumor tissues or hydrothorax and ascites Tumor of a Tumor patient, belonging to the technical field of cell engineering. The invention utilizes CD8 and PD-1 antibodies as markers for separating tumor specific T lymphocytes in TIL cells, separates the tumor antigen specific T lymphocytes from TIL cell populations by combining with a flow sorting technology, and obtains the tumor antigen specific TIL cells with high specific killing tumor activity by combining with an in vitro high-efficiency amplification technology. The technical scheme of the invention can obviously shorten the time of TIL cell in vitro separation culture, the preparation process is easy to operate, the cost is low, the popularization and the use are convenient, and TIL cells with tumor antigen specificity and more specific and stronger activity for killing tumors can be separated and obtained.

Description

Separation method of tumor antigen specific tumor infiltrating T cells

Technical Field

The invention belongs to the technical field of cell culture, and particularly relates to a separation culture method of tumor antigen specific infiltrating lymphocytes.

Background

At present, the treatment of tumor is in a bottleneck stage, so we should actively seek new tumor treatment means while continuously innovating the traditional tumor treatment method. The tumor immunotherapy is judged as the most important scientific breakthrough by the journal of science in 2013, and has become the fourth major tumor treatment mode in parallel with operations, radiotherapy and chemotherapy, and the tumor immunotherapy has great potential for resisting tumors. With the proposal of an accurate medical plan, Tumor precise cellular immunotherapy also comes forward, and is expected to become an important breakthrough for Tumor precise therapy, and Tumor-infiltrating lymphocytes (TILs) also become one of research hotspots for Tumor precise cellular immunotherapy due to the strong specific killing effect of the Tumor-infiltrating lymphocytes on Tumor cells.

TIL is tumor antigen-induced, a heterogeneous lymphocyte population mainly present in the tumor stroma, invented by NIH's Rosenberg in the mid 90 s, who isolated expanded lymphocytes directly from surgically excised tumor tissue for reinfusion in melanoma patients, in combination with chemotherapy and high concentrations of IL-2, and was able to achieve complete remission in 20% of melanoma patients for more than 3 years (Rosenberg SA, et al, Clin Cancer Res, 2011;17(13): 4550-7). The clinical use of TIL is subsequently becoming more widespread. In 2015, the total remission rate of 120 patients with advanced renal Cancer treated by Baldan V and the like by TIL combined chemotherapy was 38%, wherein 13 patients (11%) with complete remission, 32 patients (27%) with partial remission, the tumor of 18 patients with partial remission was 90% reduced, and the tumor-free period of patients with complete remission was 7-8 months (Br J Cancer, 2015,112: 1510-) (1518). The DENIGER et al study in 2017 included 11 patients with advanced malignant melanoma and given TIL in combination with trametinib after non-myeloablative treatment resulted in partial clinical remission in 7 of 11 patients and complete clinical remission in 2 patients within 3 years (Clin Cancer Res, 2017, 23: 351-.

The TIL cells currently in clinical use are mainly derived from two aspects: (1) surgically resected tumor tissue or lymph nodes; (2) cancerous hydrothorax and ascites. Generally, after mechanical shearing or protease digestion and other steps are needed for separating TIL cells from a surgical sample, the TIL cells from cancerous ascites are separated by using Ficoll to obtain mononuclear cells, and the TIL cells from the cancerous ascites and hydrothorax are relatively simple to separate and can be directly separated by using the Ficoll. The conventional isolated culture of TIL cells comprises the following steps:

(1) obtaining mononuclear cells by utilizing Ficoll separation from sheared or digested tumor tissues of a tumor patient or cancerous breast and abdominal water;

(2) adding the obtained mononuclear cells into TIL cell culture medium, adding amplification factor, and standing at 37 deg.C and 5% CO₂Culturing in an incubator, and performing expansion culture of TIL cells.

In 2014, Rosenberg continuously published articles in Top-level journal Science and JCI demonstrating that TIL technology is effective because of the presence of specific T-cell clones in these TIL cells against tumor antigens, mainly CD8⁺Effector T cells. This population of CD8⁺TIL cell surface TCR comprising a set of specific endogenous polyclonal repertoires against tumor-associated antigens and unique neoantigens, these CD8 infiltrated intratumorally under appropriate circumstances⁺T cells are the effector of a targeted anti-tumor response. Takashi et al found that this population of activated CD8⁺TIL cells in tumors continuously express PD-1 inhibitory receptors on the surface due to the increase of the secretion of self IFN-gamma and the influence of tumor immunosuppressive microenvironment. This population PD-1 positively expressed CD8⁺TIL cells represent immunoreactive cell clones in tumors (gross A, et al.J ClinInvestig. 2014;124(5): 2246-59). And Rosenberg et al again verified CD8 in vitro⁺PD-1⁺The TIL cell population of (2) and CD8⁺PD-1^-The TIL cell population of (a) has a stronger tumor-specific killing activity than the TIL cell population of (a). MacLean Hall et al found that the expression level of PD-1 in this population of immunosuppressive TIL cells with high PD-1 expression in the tumor microenvironment was reduced to about 6.1% after PD-1 signaling disruption by the addition of anti-PD-1 antibody during in vitro culture (MacLeanHall, Journal for ImmunoTherapy of Cancer (2016) 4: 61). Thus, the population of CD8 was obtained from tumor tissue or thoraco-abdominal water⁺PD-1⁺The TIL cells are subjected to in vitro massive amplification and activation, and the obtained cells are applicable to clinical tumor-specific immunotherapy or combined with other tumor treatment schemes, and have low immune score and tumor swelling improvementThe curative effect of cold tumor immunotherapy with less number of infiltrating lymphocytes in the tumor microenvironment and the prevention of tumor postoperative recurrence and metastasis have great clinical significance.

However, TIL cells isolated and cultured by conventional methods were CD4⁺And CD8⁺T cells are a heterogeneous mixture of cells. Therefore, the invention provides a method for separating and culturing tumor-specific TIL cells aiming at the heterogeneity of the TIL cells obtained in vitro.

Disclosure of Invention

In the peritumoral infiltrated cells, CD8, as described above⁺PD-1⁺The TIL cell population has a stronger tumor-specific killing activity. The invention takes CD8 and PD-1 as sorting markers for obtaining TIL cell subsets aiming at tumor antigens, and uses a flow sorting method to sort CD8⁺PD-1⁺TIL cells are selected from TIL cell population obtained from tumor tissue or cancerous hydrothorax and ascites by favoring CD8 in vitro⁺The cytokine combined preparation for high-efficiency amplification and activation of T cells greatly expands the group of cells to obtain a large amount of tumor-specific CD8⁺Effector T cells.

The invention is realized by the following technical scheme:

a method for isolating and culturing Tumor antigen-specific Tumor Infiltrating T cells (TILs), comprising the following steps:

the method comprises the following steps: preparing single cell suspension of tumor tissue, namely obtaining the single cell suspension containing tumor-specific TIL cells from tumor tissue blocks or cancerous pleural effusion;

step two: adding the obtained single cell suspension into an efficient amplification agent 1 for culturing for 1-4 d;

step three: separating the cell suspension in the second step by using a flow sorting cell technology to obtain a tumor antigen specific TIL cell;

step four: and adding the tumor antigen specific TIL cells obtained by sorting into a high-efficiency amplification agent 2 to perform amplification culture in vitro continuously to obtain a large number of TIL cells with tumor specific killing activity.

The first step comprises the following sub-steps:

collecting tumor tissue block, removing necrotic tissue and blood vessel, coating, and shearing into 1cm with sterile scissors²Slowly shearing the left and right small tissues into minced meat for short, adding 5ml of 1640 cell culture medium containing a tissue digestive enzyme composition agent, slowly shaking for 4-6 hours to ensure that the tumor tissues are digested into minced meat, and filtering by using a 100um cell filter screen to remove cell masses to obtain single cell suspension; adding 50mL GT-T551H3 culture medium for heavy suspension; performing density gradient separation on single cell suspension obtained from the tumor tissue by using Ficoll lymphocyte separation fluid to obtain mononuclear cells;

or directly taking cancerous hydrothorax and ascites, and performing density gradient separation by using Ficoll lymphocyte separation liquid to obtain mononuclear cell sediment.

The second step specifically comprises the following operations: the obtained mononuclear cell pellet was resuspended in GT-T551H3 cell culture medium to adjust the cell concentration to 5X 10⁵-1×10⁶Adding TIL cell high-efficiency amplification agent 1 into the mixture per ml, placing the mixture in a 6-well cell culture plate at the temperature of 37 ℃ and 2ml per well with 5% CO₂Culturing for 1-4 days under the condition for rapid amplification.

The third step is specifically operated as follows: centrifuging and collecting the mononuclear cells obtained in the step two, and then performing centrifugation according to the ratio of 1 × 10⁶Cell pellets were resuspended in PBS buffer at a concentration of/ml and then 10⁶Adding 1ug/ul antibody into cells, adding 5-100ul CD8-ECD antibody, 5-100ul PD-1-PE antibody and 5-100ul CD4-FITC antibody for labeling for 30 minutes, adding PBS buffer solution for washing, resuspending, detecting on machine, sorting according to the operation steps of Beckmann MoFlo XDP flow sorter, and collecting CD8⁺PD-1⁺TIL cells specific for tumor antigens.

The fourth step is specifically operated as follows:

substep 1: the CD8 obtained in the third step⁺PD-1⁺After centrifugation of the TIL cell subsets, cell pellets were adjusted to 1X 10 cell concentration by adding GT-T551H3 cell culture medium⁶Adding TIL cell high-efficiency amplification agent 2 into the mixture per ml, and placing the mixture at 75cm²In a cell culture flask, 5% CO at 37 ℃₂The culture was continued under the conditions.

Substep 2: performing amplification flask culture according to the amplification condition of the cells, wherein the culture medium added for amplification is GT-T551H3 culture medium containing IL-2 with the final concentration of 300 u/mL;

substep 3: culturing for 15-20 days, collecting the final tumor specific TIL cells when the cells are not amplified exponentially any more, and detecting the tumor killing activity by using a real-time unmarked cell function analyzer.

The tissue digesting enzyme composition comprises the following components in final concentration in 1640 cell culture medium: 2-10mg/ml of collagenase,

0.1-5mg/ml hyaluronidase, 10-500U/ml DNase I.

The TIL cell high-efficiency expansion agent 1 comprises one or more of IL-2, IFN-gamma and CD3 antibodies; the IL-2 concentration in the GT-T551H3 cell culture medium is 1000-5000U/ml, the IFN-gamma concentration is 500-2000U/ml, and the CD3 monoclonal antibody concentration is 0.1-10 ug/ml.

The CD4 antibody used for the flow detection of the tumor specific TIL cell marker is a FITC fluorescent dye marker, the CD8 antibody is an ECD fluorescent dye marker, and the PD-1 is a PE fluorescent dye marker; as a further improvement of the invention, the labeled antibody for flow sorting the tumor-specific TIL cells is two antibodies of CD8 and PD-1. The purity of the tumor specific TIL cells sorted by the scheme can reach 85-95%.

As a further optimization of the technical scheme, in order to improve the cell expansion speed and optimize the cell function after the expansion, the TIL cell efficient expansion promoting agent 2 is a GT-T551H3 cell culture medium containing 500-5000U/ml IL-2, 0.5-300ug/ml IL-15, 0.5-300ug/ml IL-7 and 0.5-30ug/ml anti-PD-1 monoclonal antibody, and the preferable technical scheme is adopted to ensure that the tumor specificity CD8 is subjected to sorting⁺TIL cells are efficiently amplified in vitro, the amplification multiple can reach 100-1000 times, and the cell amplification culture time with the maximum multiple is about 15-20 days.

Furthermore, the method is applied to the preparation of tumor antigen specific TIL cells.

Compared with the prior art, the invention has the beneficial effects that:

the presence of a T cell clone specific for the tumor antigen, mainly CD8, in TIL cells⁺Effector T cells. Aiming at the problem of heterogeneity of TIL cells obtained by in vitro separation at present, in order to improve the tumor specificity of the TIL cells after separation, the technology of the invention uses two antibodies of CD8 and PD-1 as markers for sorting TIL cell clones aiming at tumor antigens, and obtains CD8 by flow cytometry⁺PD-1⁺TIL cell subset, and efficiently amplifying the cell subset by using cell factor mixture in vitro, thereby obtaining the TIL cell subset with more tumor specificity, and facilitating clinical popularization and application.

Flow Cytometry (FCM) is a highly developed and comprehensively utilized high-tech product of monoclonal antibodies, immunocytochemistry techniques, fluorescent labeling, laser techniques, electronic computer science, and the like. The advantage of using flow cytometry for cell sorting is that the measuring speed is fast, tens of thousands of cells can be measured in 1 second at the fastest, pollution is reduced, cell activity is kept, meanwhile, nearly 100 parameters of physical and chemical characteristics can be measured for the same cell by flow cytometry, loss is reduced, and the purity of the sorted cell can reach 85-95%. In order to improve the cell yield and activity of flow sorting and facilitate later expansion culture, TIL cells are separated from tumors, and then are subjected to cell sorting after being rapidly expanded for 1-4 days in vitro by using high-dose IL-2, IFN-gamma and CD3 antibodies. The CD3 antibody can make T cell proliferate and promote its secretion of various cytokines such as IL-2, TNF-alpha, CSF, IFN-gamma, etc., and IL-2 cytokine is important for the durability and proliferation of T cell activity.

The invention adopts the combination agent 2 of the high-efficiency amplification cell factors, which comprises IL-2, IL-15, IL-7 cell factors and anti-PD-1 monoclonal antibody, to sort CD8⁺PD-1⁺TIL cell subsets were amplified rapidly and efficiently. IL-15 has some advantages over IL-2, such as the ability of IL-15 to activate and amplify CD8⁺T cells, do not activate regulatory T lymphocytes (Tregs), and release the T cell suppressive effect of Tregs (Alenzi FQ, et al. J Med Life,2011, (04): 399-.IL-7 is CD8⁺Another potent growth, activation and survival factor for T cells can increase CD8⁺The anti-tumor capacity of T cells, whereas inhibition of PD-1 signaling enhances expansion of tumor-specific TIL cells (Mazzucchelli R, et al. Nat. Rev. Immunol. 2007,7: 144-154). Therefore, the invention adds two beneficial CD8 types of IL-15 and IL-7 on the basis of the traditional expansion of TIL cells by using high-dose IL-2⁺Cytokine efficiently expanded by T cells and increased CD8 by blocking PD-1 signaling via anti-PD-1 antibody⁺Yield of TIL cell subpopulations.

By improving the technology of the invention, the tumor specific CD8 with higher purity is obtained⁺The TIL cell subset has the advantages of greatly improved cell activity, large cell number, simple and easy operation and obviously shortened cell culture period, is expected to become a means for realizing precise cell immunotherapy of tumors, and is convenient for clinical popularization and use. In vitro experiments prove that the TIL cell prepared by the invention has stronger specific tumor killing activity, and if the TIL cell is applied to clinic or is combined with an anti-PD-1/PD-L1 monoclonal antibody, the TIL cell has great significance for improving the curative effect of cold tumor with lower immune score, prolonging the postoperative recurrence and metastasis time of tumor patients and improving the life cycle and life quality of the patients.

Drawings

FIG. 1 TIL cell phenotype isolated from tumor tissue of a melanoma patient in example 1; wherein a is CD4-FITC/CD8-ECD, and b is CD 8-ECD/PD-1-PE.

FIG. 2 CD8 isolated from example 1⁺PD-1⁺And (3) performing in-vitro amplification culture on the TIL cells for 20 days by using IL-2, IL-15, IL-7 and an anti-PD-1 antibody, and observing a cell morphology graph under a microscope.

FIG. 3 flow-assay of the cell phenotype results of example 1 and its comparative example; wherein a is CD8 in example 1⁺PD-1⁺TIL-IL-2+ IL-15+ IL-7+ PD-1 cells, b is TIL-IL-2+ IL-15+ IL-7+ PD-1 cells; c is TIL-IL-2 cells.

FIG. 4 flow-assay results of intracellular cytokine secretion ability of example 1 and its comparative example; wherein a is CD8 in example 1⁺PD-1⁺TIL-IL-2+IL-15+ IL-7+ PD-1 cells; b is TIL-IL-2+ IL-15+ IL-7+ PD-1 cell; c is TIL-IL-2 cells.

FIG. 5 TIL cell phenotype isolated from ascites of a patient with bladder cancer in example 2; wherein a is CD4-FITC/CD8-ECD, and b is CD 8-ECD/PD-1-PE.

FIG. 6 flow-assay of the cell phenotype results of example 2 and its comparative examples; wherein a is CD8 in example 2⁺PD-1⁺TIL-IL-2+ IL-15+ IL-7+ PD-1 cells; b is TIL-IL-2+ IL-15+ IL-7+ PD-1 cell; c is TIL-IL-2 cells.

FIG. 7 flow-assay results of intracellular cytokine secretion ability of example 2 and its comparative example; wherein a is CD8 in example 2⁺PD-1⁺TIL-IL-2+ IL-15+ IL-7+ PD-1; b is TIL-IL-2+ IL-15+ IL-7+ PD-1 cell; c is TIL-IL-2 cells.

FIG. 8 cell proliferation curves;

FIG. 9 RTCA measures the killing activity of example 1 and its comparative examples on syngeneic tumor cells.

The specific embodiments are as follows

The following describes the embodiments of the present invention in further detail with reference to the accompanying drawings.

The method for flow detection of TIL cell phenotype comprises the following specific operation steps:

taking TIL cell suspension, adjusting cell concentration to 1 × 10 with PBS⁶Each per ml, at 1ug antibody/10⁶And respectively adding CD4-FITC, CD8-ECD and PD-1-PE flow-labeled antibodies into the cells according to the proportion, incubating for 30min in a dark place, adding 1ml PBS to wash the cells twice, discarding the supernatant, suspending the cell precipitate in 1ml PBS, and detecting on a computer.

The method for flow detection of the secretion capacity of the cell factors in the TIL cells comprises the following specific operation steps:

the method comprises the following steps: TIL cells were collected and PBS was used to adjust the cell concentration to 2X 10⁶Cells per ml, stimulated with PMA (10ng/ml) for 6 hours;

step two: resuspending the cells in PBS containing 10% inactivated serum, blocking the Fc antibody;

step three: staining the cell surface by using a CD8-ECD antibody for 20min, centrifuging for 5min at 500g, and discarding the supernatant;

step four: adding fixative (dosage refer to specification PN IM2716U and PN IM3279U, Beckman company), incubating at room temperature in dark for 15min, adding 2-3ml PBS, centrifuging for 500g, 5min, and discarding supernatant;

step five: adding membrane breaking agent (dosage is referred to the instruction), incubating at room temperature in dark for 10min, adding 2-3ml PBS, centrifuging for 500g for 5min, and discarding the supernatant; adding IFN-gamma-PC 7 and TNF-alpha-PC 5 antibody for staining for 30min, adding 2-3ml PBS, centrifuging for 500g for 5min, discarding supernatant, resuspending fixed cells in 500ul PBS containing 2% paraformaldehyde, and performing flow detection.

Example 1:

the isolated culture of tumor antigen specific TIL cells in tumor tissues of melanoma patients comprises the following steps:

(1) taking tumor tissues of melanoma patients (Wangxx, female, 81 years old), removing normal tissues and necrotic tissues, shearing the tumor tissues by using surgical scissors, adding 5ml of serum-free RPMI1640 (containing 2mg/ml collagenase, 5mg/ml hyaluronidase and 100u/ml DNase I), slowly shaking and incubating for about 6 hours to see that the tumor tissues are digested into chylous, and removing cell masses by using a 100um cell filter screen to obtain a single cell suspension;

(2) after the obtained single cell suspension is centrifugally precipitated, adding 50ml of GT-T551H3 culture medium into the cell precipitate for heavy suspension;

(3) preparing 2-level density gradient separation liquid (the lower layer is 100% Ficoll, and the upper layer is a mixed liquid of 75% Ficoll and 25% RPMI 1640) by taking a 15ml centrifugal tube;

(4) adding the single cell suspension obtained in the step (2) into the uppermost layer of the 2-level separation liquid prepared in the step (3), centrifuging at 2000rpm/min for 30min, and taking cells between the upper layer and the middle layer, wherein the cells are enriched tumor cells; taking the cells between the middle layer and the lower layer, this is a mononuclear cell suspension, and the total number of obtained mononuclear cells is about 3 × 10⁶；

(5) The obtained mononuclear cell pellet was resuspended in GT-T551H3 cell culture medium to adjust the cell concentration to 5X 10⁵Adding IL-2 with final concentration of 5000U/ml, CD3 monoclonal antibody with final concentration of 10ug/ml and IFN-gamma with final concentration of 2000U/ml, placing on 6-well cell plateMedium culture, 2 ml/well, 37 ℃, 5% CO₂Culturing for 2d under the condition for rapid amplification;

(6) the cells in (5) were collected by centrifugation at 1X 10⁶Cell pellets were resuspended in PBS at a concentration of about 15 ml. According to 10⁶Adding 1ug of antibody into cells, adding 15ul of each of CD4-FITC, CD8-ECD and PD-1-PE antibody, labeling for 30 minutes, adding 35ml of PBS, mixing uniformly, centrifuging to collect cell precipitate, and adding 15ml of PBS to resuspend cells;

(7) performing cell phenotype analysis according to the operation steps of the Beckmann MoFlo XDP flow sorter, and sorting and collecting tumor specific CD8⁺PD-1⁺TIL cells; the results are shown in FIG. 1, CD8 in TIL cells after 2d in vitro culture⁺The cell proportion is 39.2%, CD4⁺The cell proportion was 41.7%, while CD8⁺PD-1⁺The proportion of TIL cells was about 33.4%. (8) Collecting CD8⁺PD-1⁺After centrifugation of the TIL cell subsets, cell pellets were adjusted to 1X 10 cell concentration by adding GT-T551H3 cell culture medium⁶Adding GT-T551H3 cell culture medium containing IL-2 of 5000U/ml, IL-15 of 0.5ug/ml, IL-7 of 0.5ug/ml and anti-PD-1 monoclonal antibody of 1ug/ml, and placing at 75cm²In a cell culture flask, 5% CO at 37 ℃₂Continuing culturing under the condition;

(9) performing amplification flask culture according to the amplification condition of the cells, wherein the culture medium added for amplification is GT-T551H3 culture medium containing IL-2 with the final concentration of 300 u/ml;

(10) the cells obtained in example 1 (hereinafter referred to as CD 8)⁺PD-1⁺TIL-IL-2+ IL-15+ IL-7+ PD-1) on day 20 of culture, cells were observed under a microscope and photographed (as shown in FIG. 2). The cells were collected, subjected to a cytophenotypic assay by flow, and the cytokine secretion ability level thereof was measured, and compared with that of the comparative example unsorted TIL cells (hereinafter referred to as TIL-IL-2+ IL-15+ IL-7+ PD-1) and the comparative example TIL cells which were expanded in vitro with only 5000U/ml of IL-2 cytokine (hereinafter referred to as TIL-IL-2), the results are shown in FIGS. 3 and 4.

FIG. 1 shows CD8 in TIL cells isolated from tumor tissue of melanoma patients⁺TIL cell proportion 392% (as shown in FIG. 1 a), wherein CD8⁺PD-1⁺The proportion of TIL is as high as 33.4% (as shown in FIG. 1 b). Sorted CD8 improved by the present technique⁺PD-1⁺The TIL cells are basically purified CD8 after being amplified and cultured for 20 days in vitro by IL-2, IL-15, IL-7 cytokines and anti-PD-1 monoclonal antibody⁺T cells of which CD8⁺PD-1⁺The ratio of (A) was reduced to 10.2% (as shown in FIG. 3 a) while the ability to endocrinically secrete IFN-. gamma.was relatively high, about 34% (as shown in FIG. 4 a) compared to the comparative example, indicating CD8 after sorting⁺TIL cells gradually recover their activation function from their original "depleted" state during in vitro culture.

FIG. 3a is the CD8 of example 1⁺PD-1⁺TIL-IL-2+ IL-15+ IL-7+ PD-1 cells were cultured in vitro for 20 days, followed by flow-identified cell phenotypes. Substantially purified CD8⁺TIL cells of which CD8⁺PD-1⁺The proportion of TIL cells decreased to 10.2%. FIGS. 3b and 3c show the cell phenotypes of the control examples of example 1, namely, TIL-IL-2+ IL-15+ IL-7+ PD-1 and TIL-IL-2, respectively, and from FIG. 3b, CD8 in the TIL-IL-2+ IL-15+ IL-7+ PD-1 group⁺PD-1⁺The proportion of TIL cells decreased to 15.5%; as can be seen in FIG. 3c, CD8 was obtained after culturing cells of the TIL-IL-2 group of the control example⁺PD-1⁺The proportion of (A) is up to 30.9%. Explanation of post-flow sort CD8⁺PD-1⁺The TIL cells are cultured for a long time to obtain substantially purified CD8⁺TIL cells; the combination of IL-2, IL-15, IL-7 cytokine and anti-PD-1 monoclonal antibody is beneficial to CD8⁺Expansion of T cells; TIL cells were cultured in vitro for prolonged periods of time under the action of PD-1 blockers, CD8⁺PD-1⁺The proportion of TIL will decrease.

As shown in FIG. 4a, CD8 cultured in example 1⁺PD-1⁺The level of IFN-gamma in cells with TIL-IL-2+ IL-15+ IL-7+ PD-1 is up to 34.0%, and the level of secreted TNF-alpha is lower, about 4.5%. As shown in FIGS. 4b-4c, the level of IFN- γ secretion by TIL-IL-2+ IL-15+ IL-7+ PD-1 cells in the comparative example was 20.2%, and the level of TNF- α secretion was also lower, about 6.9%, whereas the level of IFN- γ secretion by the comparative example TIL-IL-2 was lower, about 19.3%; the level of secreted TNF-alpha is about8.0 percent. The combination of IL-2, IL-15, IL-7 cytokine and anti-PD-1 monoclonal antibody is more beneficial to CD8⁺TIL in vitro cell activation.

Example 2:

the separation culture of the tumor antigen specific TIL cells in ascites of patients with bladder cancer comprises the following steps:

(1) preparing 2-level density gradient separation liquid (the lower layer is 100% Ficoll, and the upper layer is a mixed liquid of 75% Ficoll and 25% RPMI 1640) by taking a 15ml centrifugal tube;

(2) taking 100ml of cancerous ascites of a patient with bladder cancer (Huangxxx, female, 76 years old), adding the uppermost layer of the grade-2 separation solution prepared in the step (1), centrifuging at 2000rpm/min for 30min, and taking cells between the upper layer and the middle layer, wherein the cells are enriched tumor cells; collecting cells between the middle layer and the lower layer as mononuclear cell suspension, and centrifuging to collect the obtained mononuclear cells with total cell number of about 1 × 10⁷；

(3) Resuspending the cell pellet in GT-T551H3 cell culture medium to adjust the cell concentration to 1X 10⁶Adding IL-2 of 5000U/ml, CD3 monoclonal antibody of 0.1ug/ml and IFN-gamma of 2000U/ml, culturing in 6-well cell plate at 37 deg.C and 5% CO of 2 ml/well₂Culturing for 4d under the condition for rapid amplification;

(4) the cells in (3) were collected by centrifugation at 1X 10⁶The cell pellet was resuspended in PBS at a concentration of about 100 ml. According to 10⁶Adding 1ug of antibody into cells, adding 100ul of each of CD4-FITC, CD8-ECD and PD-1-PE antibody, labeling for 30 minutes, adding 370ml of PBS, mixing uniformly, centrifuging to collect cell precipitate, and adding 100ml of PBS to resuspend cells;

(5) performing phenotype analysis according to the operation procedure of Beckmann MoFlo XDP flow sorter (the result is shown in FIG. 5), sorting and collecting tumor antigen specific CD8⁺PD-1⁺TIL cells; the results showed CD8 in TIL cells after 4 days in vitro culture⁺The cell proportion was 52.3%, CD4⁺The proportion of cells was 19.3%, while CD8⁺PD-1⁺The proportion of TIL cells was about 49.4%.

(6) Collecting CD8⁺PD-1⁺TIL cell lineAfter centrifugation of the population, the cell pellet was adjusted to 1X 10 cell concentration by adding GT-T551H3 cell culture medium⁶Adding GT-T551H3 cell culture medium containing 500U/ml IL-2, 3ug/ml IL-15, 3ug/ml IL-7, and 3ug/ml anti-PD-1 monoclonal antibody, and placing at 75cm²In a cell culture flask, 5% CO at 37 ℃₂The culture was continued under the conditions.

(7) According to the condition of cell amplification, the flask amplification culture is carried out, and the culture medium added for amplification is GT-T551H3 culture medium containing 300u/ml IL-2.

(8) When the cells are no longer exponentially expanded after approximately 15 days of culture, the cultured CD8 is collected⁺PD-1⁺TIL-IL-2+ IL-15+ IL-7+ PD-1 cells were phenotyped by flow and tested for their cytokine secretion capacity level and compared with the comparative TIL cells, and the results are shown in FIGS. 6 and 7.

FIG. 5a and FIG. 5b show CD8 in TIL cells isolated from ascites of patients with bladder cancer⁺TIL cells accounted for 52.3%, of which CD8⁺PD-1⁺The proportion of TIL is as high as 49.4%. Sorted CD8 improved by the present technique⁺PD-1⁺The TIL cells are subjected to in vitro amplification culture for 15 days by cytokines such as IL-2, IL-15, IL-7 and the like and an anti-PD-1 antibody, and then are basically purified CD8⁺T cells, and wherein CD8⁺PD-1⁺The ratio of (A) was reduced to 12.7% (as shown in FIG. 6 a) while the ability to endocrinically secrete IFN-. gamma.was relatively higher, about 44.5% (as shown in FIG. 7 a) compared to the comparative example, indicating CD8 after sorting⁺TIL cells gradually recover their activation function from their original "depleted" state during in vitro culture.

As shown in FIG. 6a, CD8 in example 2⁺PD-1⁺After the TIL-IL-2+ IL-15+ IL-7+ PD-1 cells were cultured, they were essentially purified CD8⁺TIL cells of which CD8⁺PD-1⁺The proportion in TIL cells decreased to 12.7%. FIGS. 6b and 6c show the cell phenotype of the control TIL-IL-2+ IL-15+ IL-7+ PD-1, TIL-IL-2 groups, as shown in FIG. 6b, CD8 after 15 days of culture of TIL-IL-2+ IL-15+ IL-7+ PD-1 cells⁺PD-1⁺The proportion of TIL cells was reduced to 15.8%; FIG. 6c is a photograph of CD8 in TIL-IL-2 cells of control example⁺PD-1⁺The proportion of TIL cells was as high as 42.3%. Further demonstrating post-flow sorting CD8⁺PD-1⁺The TIL cells are cultured for a long time to obtain substantially purified CD8⁺TIL cells; IL-2, IL-15, IL-7 cytokines and PD-1 blockers in combination are beneficial for CD8⁺Expansion of T cells; the PD-1 blocker combination is more favorable for CD8⁺TIL in vitro cell activation, prolonged culture in vitro, CD8⁺PD-1⁺The proportion of TIL will decrease.

As shown in FIG. 7, CD8 in example 2⁺PD-1⁺The level of IFN-gamma in cells with TIL-IL-2+ IL-15+ IL-7+ PD-1 reached 44.5%, and the level of secreted TNF-alpha was lower, about 1.3% (FIG. 7 a); compared to the examples, the comparative TIL-IL-2+ IL-15+ IL-7+ PD-1 cells secreted IFN-. gamma.at a lower level of about 29.6% and secreted TNF-. alpha.at a lower level of about 0.8% (FIG. 7 b), whereas the comparative TIL-IL-2 cells secreted IFN-. gamma.at a lower level of 20.8% and secreted TNF-. alpha.at a lower level of 5.6%, respectively (FIG. 7 c), than the examples. Further illustrates that the combination of IL-2, IL-15, IL-7 cytokines and PD-1 blocking agent is more beneficial to CD8⁺TIL cells are activated in vitro.

Example 3

A Real-Time unlabeled cell function analyzer (RTCA) detects the tumor killing activity, and the specific operation steps are as follows:

the method comprises the following steps: autologous tumor cells

Adding the cell suspension obtained from the tumor tissue in the example 1 into the uppermost layer of the prepared 2-stage density gradient separation liquid (the lower layer is 100% Ficoll, and the upper layer is a mixed solution of 75% Ficoll and 25% RPMI 1640), centrifuging at 2000rpm/min for 30min, and collecting cells between the uppermost layer and the middle layer, wherein the cells are enriched tumor cells; after washing the cells twice with PBS, the cells were resuspended in DMEM containing 10% FBS and adjusted to a cell concentration of 2X 10⁴Per ml, added to 25cm²In a cell culture flask, 5% CO at 37 ℃₂Carrying out adherent culture under the condition, and carrying out amplification of adherent cells according to the growth condition of the cells;

step two: culture of unsorted TIL cells in comparative example (TIL-IL-2+ IL-15+ IL-7+ PD-1)

The unsorted TIL cells cultured in the 6-well cell plate of example 1 were subjected to flask expansion culture according to the cell expansion conditions, and the medium thus expanded was mixed with the CD8 of example 1⁺PD-1⁺The TIL cell culture medium was the same.

Step three: in the comparative example, TIL cells were cultured with IL-2 cells alone (TIL-IL-2)

The unsorted TIL cells of example 1 were expanded and cultured by adding IL-2 only at 5000 u/ml.

FIG. 8 is a cell growth curve with the number of days in cell culture on the abscissa and the number of cells on the ordinate. As shown in the figure, under the action of IL-2, IL-15 and IL-7 cytokines and PD-1 blocking agent, CD8⁺TIL cells proliferate at the highest rate, while unsorted TIL cells under the same culture conditions were located next. However, in the case of IL-2 cytokine alone, the proliferation rate of TIL cells was relatively slow, suggesting that the combination of IL-2, IL-15 and IL-7 cytokines and PD-1 blocking agents may promote TIL cell proliferation, particularly in favor of CD8⁺Expansion of T cells.

Step four: RTCA detection of CD8 in example 1⁺PD-1⁺Tumor specific TIL cells (CD 8)⁺PD-1⁺TIL-IL2+ IL-15+ IL-7+ PD-1), comparative example TIL-IL-2+ IL-15+ IL-7+ PD-1 and comparative example TIL-IL-2 tumoricidal activity, the procedure was as follows:

the first substep: adding a culture medium into an E-Plate detection Plate and measuring a background impedance value;

and a second substep: collecting the logarithmic phase autologous tumor cells obtained in the first step as target cells, counting, adjusting the concentration of the cell suspension,

to E-Plate assay Plate was added a volume of cells, 1X 10⁵Placing in a room temperature ultra-clean bench for 30 min;

and a third substep: the E-Plate detection Plate with the added cells is placed on a detection platform (the detection platform is placed in an incubator in advance), and real-time dynamic cell proliferation detection is carried out.

And a fourth substep: after overnight detection, example CD8 was added to each well⁺PD-1⁺TIL-IL2+ IL-15+ IL-7+ PD-1, comparative example TIL-IL-2+ IL-15+ IL-7+ PD-1 and comparative example TIL-IL-2 as effector cells, the ratio of effect to target was 20: 1, and continuing the detection, a real-time cell effect curve can be obtained (the result is shown in figure 9).

As can be seen from FIG. 9, example 1CD8⁺PD-1⁺TIL-IL2+ IL-15+ IL-7+ PD-1 and comparative example TIL-IL-2+ IL-15+ IL-7+ PD-1 and TIL-IL-2 effector cells obtained by amplification by the method of the invention compared to comparative examples in the case of equivalent target ratios to target cells, tumor antigen-specific CD8⁺TIL effector cells have a more specific tumor killing activity. Demonstrating that the combination of cytokines used in the protocol of the present invention enhances CD8⁺The tumor-specific killing activity of TIL was named tumor antigen-specific TIL cells.

And (4) conclusion: from the above analysis results, it can be seen that the tumor-specific CD8 isolated by the technique of the present invention⁺PD-1⁺After the TIL cells are subjected to in vitro amplification culture, the cells are fully activated and rapidly amplified, and the method has the advantages of short in vitro amplification culture time, simplicity and easiness in operation and suitability for clinical popularization.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the present invention pertains, the technical solutions in the embodiments may be appropriately combined without departing from the spirit of the present invention, and all of the technical solutions should be considered as belonging to the protection scope of the present invention.

Claims

1. The isolated culture method of tumor antigen specific tumor infiltrating T cells is characterized by comprising the following steps:

2. The method of claim 1, wherein the first step comprises the following substeps:

3. The method for culturing TIL cells specific for tumor antigen as claimed in claim 1, wherein the second step is performed by: the obtained mononuclear cell pellet was resuspended in GT-T551H3 cell culture medium to adjust the cell concentration to 5X 10⁵-1×10⁶Adding TIL cell high-efficiency amplification agent 1 into the mixture per ml, placing the mixture in a 6-well cell culture plate at the temperature of 37 ℃ and 2ml per well with 5% CO₂Culturing for 1-4 days under the condition for rapid amplification.

4. The method for culturing TIL cells specific for tumor antigens as claimed in claim 1, wherein said step three is specifically performed as follows: centrifuging and collecting the mononuclear cells obtained in the step two, and then performing centrifugation according to the ratio of 1 × 10⁶Resuspending the cell pellet in P at a concentration of/mlBS buffer, then according to 10⁶Adding 1ug of antibody into cells, adding CD4, CD8, PD-1 and flow detection antibody for labeling for 30min, adding PBS buffer solution for washing, resuspending, mechanically sorting, sorting according to the operation steps of Beckmann MoFloXDP flow sorter, collecting CD8⁺PD-1⁺TIL cells specific for tumor antigens.

5. The method for culturing TIL cells specific for tumor antigens as claimed in claim 1, wherein said step four comprises the following steps:

substep 1: the CD8 obtained in the third step⁺PD-1⁺After centrifugation of the TIL cell subsets, cell pellets were adjusted to 1X 10 cell concentration by adding GT-T551H3 cell culture medium⁶Adding TIL cell high-efficiency amplification agent 2 into the mixture per ml, and placing the mixture at 75cm²In a cell culture flask, 5% CO at 37 ℃₂Continuing culturing under the condition;

6. The method for culturing and isolating tumor antigen-antigen specific TIL cells according to claim 2, wherein the tissue digesting enzyme composition comprises the following final concentration components in 1640 cell culture medium: 2-10mg/ml collagenase, 0.1-5mg/ml hyaluronidase, 10-500U/ml DNase I.

7. The method of claim 3, wherein the TIL cell expansion agent 1 comprises one or more of IL-2, IFN- γ, and CD3 antibodies; the IL-2 concentration in the GT-T551H3 cell culture medium is 1000-5000U/ml, the IFN-gamma concentration is 500-2000U/ml, and the CD3 monoclonal antibody concentration is 0.1-10 ug/ml.

8. The isolated culture method of TIL cell specific for tumor antigen as claimed in claim 4, wherein the CD4 antibody for flow detection of the marker of TIL cell specific for tumor is labeled with FITC fluorochrome, the CD8 antibody is labeled with ECD fluorochrome, and the PD-1 is labeled with PE fluorochrome, and the CD8 is collected by sorting⁺PD-1⁺TIL cells specific for tumor antigens.

9. The method of claim 5, wherein the TIL cell expansion promoter 2 is GT-T551H3 cell culture medium containing 500-5000U/ml IL-2, 0.5-300ug/ml IL-15, 0.5-300ug/ml IL-7, 0.5-30ug/ml anti-PD-1 mab.

10. Use of the isolated culture method of tumor antigen specific tumor infiltrating T cell of claim 1 in the preparation of tumor antigen specific TIL cell.