CN110713978B

CN110713978B - Separation method of tumor antigen specific tumor invasive T cells

Info

Publication number: CN110713978B
Application number: CN201911122814.1A
Authority: CN
Inventors: 谢云青; 郑秋红
Original assignee: Fujian Tumour Hospital (fujian Tumour Institute Fujian Cancer Control And Prevention Center)
Current assignee: Fujian Tumour Hospital (fujian Tumour Institute Fujian Cancer Control And Prevention Center)
Priority date: 2019-11-16
Filing date: 2019-11-16
Publication date: 2023-08-18
Anticipated expiration: 2039-11-16
Also published as: CN110713978A

Abstract

The invention provides a separation culture method of tumor-specific tumor-infiltrating T cells (Tumor Infiltrating Lymphocytes, TIL) cells derived from tumor tissues or hydrothorax and ascites of a tumor patient, belonging to the technical field of cell engineering. The invention uses CD8 and PD-1 antibodies as the markers for separating tumor-specific T lymphocytes in TIL cells, separates the tumor-antigen-specific T lymphocytes from a TIL cell population by combining a flow separation technology, and combines an in-vitro high-efficiency amplification technology to obtain the tumor-antigen-specific TIL cells with higher specific killing tumor activity. The technical scheme of the invention can obviously shorten the time of in vitro separation and culture of TIL cells, has the advantages of easy operation of the preparation process, low cost and convenient popularization and use, and can separate and obtain the TIL cells with more specific tumor killing tumor and stronger tumor antigen specificity.

Description

Separation method of tumor antigen specific tumor invasive 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 tumors enters a bottleneck stage, so that when the traditional tumor treatment method is continuously innovated, new tumor treatment means are actively sought. Tumor immunotherapy was evaluated by the journal of science as the most significant scientific breakthrough in 2013, and has become the fourth largest treatment modality for tumors in parallel with surgery, radiotherapy and chemotherapy, and tumor immunotherapy is showing great potential against tumors. Along with the proposal of the 'accurate medical plan', accurate cell immunotherapy of tumor is also generated, and is expected to become an important break of accurate therapy of tumor, and tumor-infiltrated lymphocyte (Tumor infiltrating lymphocytes, TIL) has stronger specific killing effect on tumor cells and also becomes one of research hot spots of accurate cell immunotherapy of tumor.

TIL is a tumor antigen-induced heterogeneous lymphocyte population mainly existing in the tumor stroma, is invented by Rosenberg of NIH in the middle of 90 s, directly separates amplified lymphocytes from tumor tissues excised by surgery for feedback of melanoma patients, and can achieve a state of complete remission of 20% of the melanoma patients by combining chemotherapy and high-concentration IL-2 for more than 3 years (Rosenberg SA, et al Clin Cancer Res, 2011;17 (13): 4550-7). The clinical use of TIL subsequently becomes increasingly widespread. In 2015, baldan V et al treated 120 patients with advanced renal carcinoma with TIL combined chemotherapy, the total remission rate was 38%, 13 cases (11%) were completely remitted, 32 cases (27%) were partially remitted, the tumor of 18 cases of partially remitted patients was 90% smaller, and the tumor-free period of completely remitted patients was 7-8 months (Br J Cancer, 2015, 112:1510-1518). DENIGER et al 2017 study included 11 patients with advanced malignant melanoma, and TIL and trametinib combined therapy administered after non-myeloablative treatment, resulting in a partial relief of clinical symptoms in 7 of 11 patients and a complete relief of clinical symptoms in 2 patients within 3 years (Clin Cancer Res, 2017, 23:351-362).

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. The isolation of TIL cells from surgical samples generally requires mechanical shearing or protease digestion, and the like, and then the Ficoll is used for isolation to obtain mononuclear cells, while the isolation of the TIL cells from cancerous hydrothorax and ascites is relatively simple, and can be directly carried out through the Ficoll. The traditional isolated culture of TIL cells includes the following steps:

(1) Separating mononuclear cells from tumor tissues of a tumor patient after shearing or digestion or from cancerous hydrothorax and ascites by using Ficoll;

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

In 2014 Rosenberg was continuously published in top journal Science and JCI, demonstrating that the reason for the effectiveness of the TIL technology was due to the presence of specific T cell clones, mainly CD8, against tumor antigens in these TIL cells ⁺ Effector T cells. This isGroup CD8 ⁺ The TIL cell surface TCR comprises a set of specific endogenous polyclonal libraries for tumor-associated antigens and unique neoantigens, under appropriate circumstances, these CD 8's are infiltrated intratumorally ⁺ T cells are effectors of targeted anti-tumor responses. Takashi et al have found that this population of activated CD8 ⁺ TIL cells express PD-1 inhibitory receptors on the surface continuously in tumors due to the increase of self IFN-gamma secretion and the influence of tumor immunosuppression microenvironment. CD8 positive expressed by the group of PD-1 ⁺ TIL cells represent immunoreactive cell clones in tumors (Gros A, et al J Clin invest g.2014; 124 (5): 2246-59). And Rosenberg et al again validated CD8 in vitro ⁺ PD-1 ⁺ TIL cell populations and CD8 of (a) ⁺ PD-1 ^- Has stronger tumor-specific killing activity than the TIL cell population of (C). MacLean Hall et al found that the expression level of PD-1 was reduced to about 6.1% after the addition of anti-PD-1 antibodies to block PD-1 signals during in vitro culture in TIL cells in which PD-1 was highly expressed in tumor microenvironment (MacLean Hall, journal for ImmunoTherapy of Cancer (2016) 4:61). Thus, the group of CD8 is obtained from tumor tissue or hydrothorax and ascites ⁺ PD-1 ⁺ The obtained cells are applicable to clinical tumor specific immunotherapy or combined with other tumor treatment schemes, and the TIL cells have great clinical significance for improving the curative effect of 'cold' tumor immunotherapy with lower immune scores and smaller number of infiltrating lymphocytes in tumor microenvironment and preventing postoperative recurrence and metastasis of tumors.

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

Disclosure of Invention

As described above, CD8 in peri-tumor infiltrating cells ⁺ PD-1 ⁺ Has stronger tumor specific killing activity. The invention uses CD8. PD-1 as a sorting marker for obtaining TIL cell subsets against tumor antigens CD8 was sorted by flow-sorting ⁺ PD-1 ⁺ TIL cells are selected from TIL cell population obtained from tumor tissue or cancerous hydrothorax and ascites, and pass through in vitro cell culture medium for CD8 ⁺ The cytokine composition agent for efficient amplification and activation of T cells can be used for amplifying a large number of cells in the population to obtain a large number of tumor-specific CD8 ⁺ Effector T cells.

The invention is realized by the following technical scheme:

a method of isolated culture of tumor antigen specific tumor infiltrating T cells (Tumor Infiltrating Lymphocytes, TIL) comprising the steps of:

step one: preparing a tumor tissue single cell suspension, namely obtaining a single cell suspension containing tumor specific TIL cells from a tumor tissue block or cancerous hydrothorax and ascites;

step two: adding the single cell suspension into the efficient amplicon 1 to culture for 1-4d;

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

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

The first step comprises the following sub-steps:

taking tumor tissue block, removing necrotic tissue and blood vessel, coating, cutting into 1cm pieces with sterile scissors ² Slowly cutting left and right small tissues into meat paste, adding 5ml of 1640 cell culture medium containing tissue digestive enzyme composition, slowly shaking for 4-6 hours to obtain tumor tissues, and filtering with 100um cell filter screen to remove cell mass to obtain single cell suspension; adding 50mL of GT-T551H 3 culture medium to resuspend; carrying out density gradient separation on single cell suspension obtained from tumor tissues by using Ficoll lymphocyte separation liquid to obtain mononuclear cells;

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

The specific operation of the second step is as follows: adding the obtained mononuclear cell precipitate into GT-T551H 3 cell culture medium to resuspend, and adjusting cell concentration to 5×10 ⁵ -1×10 ⁶ Per ml, and adding TIL promoting cell high efficiency amplification agent 1, placing in 6-well cell culture plate, 2 ml/well, 37deg.C, 5% CO ₂ Culturing for 1-4d under the condition for rapid amplification.

The third specific operation is as follows: centrifuging and collecting the mononuclear cells obtained in the step two, and then mixing the mononuclear cells according to the ratio of 1 multiplied by 10 ⁶ The cell pellet was resuspended in PBS buffer at a concentration of/ml and then taken up at 10 ⁶ The ratio of 1ug/ul antibody to cells is added, CD8-ECD antibody 5-100ul, PD-1-PE antibody 5-100ul and CD4-FITC antibody 5-100ul are added for labeling for 30 minutes, then PBS buffer solution is added for washing, the suspension is carried out after being re-suspended, the detection is carried out on the cells, the cells are separated according to the operation steps of a Beckmann MoFlo XDP flow separator, and CD8 is collected ⁺ PD-1 ⁺ Tumor antigen specific TIL cells.

The specific operation of the step four is as follows:

sub-step 1: CD8 obtained in the step three ⁺ PD-1 ⁺ Centrifuging TIL cell subpopulation, adding GT-T551H 3 cell culture medium into cell sediment to adjust cell concentration to 1×10 ⁶ Per ml, adding TIL-promoting cell high-efficiency amplification agent 2, and standing at 75cm ² In a cell culture flask, 37 ℃,5% CO ₂ Culturing was continued under the conditions.

Sub-step 2: performing bottle expansion culture according to the cell expansion condition, wherein the expansion culture medium is GT-T551H 3 culture medium containing IL-2 with the final concentration of 300 u/mL;

sub-step 3: and culturing until 15-20 days, collecting and obtaining final tumor specific TIL cells when the cells are not amplified exponentially, and detecting the tumor killing activity by using a real-time label-free cell function analyzer.

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

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

The components of the TIL cell high-efficiency amplification agent 1 comprise one or more of IL-2, IFN-gamma and CD3 antibodies; the concentration of IL-2 in the GT-T551H 3 cell culture medium is 1000-5000U/ml, the concentration of IFN-gamma is 500-2000U/ml, and the concentration of CD3 monoclonal antibody is 0.1-10 ug/ml.

The CD4 antibody used for detecting the tumor specific TIL cell markers is FITC fluorescent dye marker, the CD8 antibody is ECD fluorescent dye marker and the PD-1 is PE fluorescent dye marker; as a further improvement of the invention, the labeled antibodies used for flow sorting tumor-specific TIL cells are 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 promote the cell expansion speed, the cell function after expansion is optimized, the TIL-promoting cell high-efficiency expansion agent 2 is a GT-T551H 3 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 monoclonal antibody, and by adopting the preferred technical scheme, the tumor-specific CD8 after separation is caused ⁺ The TIL cells are efficiently amplified in vitro, the amplification factor can reach 100-1000 times, and the maximum factor cell amplification culture time is about 15-20 days.

Further, the above 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 specific T cell clones, predominantly CD8, against tumor antigens 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 utilizes two antibodies of CD8 and PD-1 as markers for sorting TIL cell clones aiming at tumor antigens, and obtains CD8 by a flow cytometry technology ⁺ PD-1 ⁺ And the TIL cell subgroup is efficiently amplified by utilizing a cytokine composition in vitro, so that the TIL cell subgroup with more tumor specificity is obtained, and the TIL cell subgroup is convenient for clinical popularization and application.

Flow Cytometry (FCM) is a highly developed and comprehensive high-technology product of monoclonal antibodies, immunocytochemistry technology, fluorescent labeling, laser technology, and electronic computer science. The advantage of using flow cytometry to conduct cell sorting is that the measurement speed is fast, the ten thousand cells can be measured within 1 second at maximum, the pollution is reduced, the cell activity is maintained, meanwhile, the flow cytometry can simultaneously conduct nearly 100 parameter measurement on physical and chemical characteristics of the same cell, the loss is reduced, and the purity of the sorted cells can reach 85-95%. In order to improve the yield and activity of flow-type sorted cells and facilitate the later amplification culture, the invention firstly adopts large-dose IL-2, IFN-gamma and CD3 antibodies to carry out rapid amplification for 1-4d in vitro after separating TIL cells from tumors, and then carries out cell sorting. Wherein the CD3 antibody can proliferate T cells and promote the secretion of various cytokines such as IL-2, TNF-alpha, CSF, IFN-gamma, etc., and the IL-2 cytokine is critical for the persistence and proliferation of T cell activity.

The invention adopts the combination of the high-efficiency amplification cytokine combination agent 2 comprising IL-2, IL-15, IL-7 cytokine and anti-PD-1 monoclonal antibody to separate CD8 ⁺ PD-1 ⁺ The TIL cell subgroup is subjected to high-efficiency and rapid expansion. IL-15 has some advantages not possessed by IL-2, such as IL-15 being able to activate and amplify CD8 ⁺ T cells, do not activate regulatory T lymphocytes (Tregs), and relieve the inhibition of T cells by Tregs (Alenzi FQ, et al J Med Life,2011, (04): 399-406). IL-7 is CD8 ⁺ Another potent growth, activation and survival factor for T cells increases CD8 ⁺ The anti-tumor capacity of T cells, while inhibition of PD-1 signaling enhances the expansion of tumor-specific TIL cells (Mazzuchelli R, et al Nat. Rev. Immunol. 2007,7:144-154). Therefore, the invention increases IL-15 and IL-7 which are beneficial to CD8 based on the traditional method of using high dose IL-2 to amplify TIL cells ⁺ Cytokines that efficiently expand T cells and block PD-1 signaling by anti-PD-1 antibodies to increase CD8 ⁺ Yield of TIL cell subpopulations.

By improving the technology of the invention, the tumor specific CD8 with higher purity is obtained ⁺ Til cell subpopulations, cell activityGreatly improves the cell quantity, has simple and easy operation and obviously shortens the cell culture period, is hopeful to become a means for realizing accurate cell immunotherapy of tumors, and is convenient for clinical popularization and use. In vitro experiments prove that the TIL cells prepared by the invention have stronger specific tumor killing activity, if the TIL cells are applied to clinic or combined with anti-PD-1/PD-L1 monoclonal antibody, the TIL cells have great significance in improving the curative effect of 'cold' tumors with lower immune scores, prolonging the postoperative recurrence and metastasis time of tumor patients and improving the life time and life quality of the patients.

Drawings

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

FIG. 2 CD8 isolated from example 1 ⁺ PD-1 ⁺ In vitro, TIL cells were cultured for 20 days with IL-2, IL-15, IL-7 and anti-PD-1 antibodies, and then observed under a microscope.

FIG. 3 flow-through detection 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 is a flow chart showing the results of the intracellular cytokine secretion capacity 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. 5 TIL cell phenotype isolated from ascites in bladder cancer patients in example 2; wherein a is CD4-FITC/CD8-ECD, and b is CD8-ECD/PD-1-PE.

FIG. 6 flow-through detection of cell phenotype results for example 2 and its comparative example; 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 cells; c is TIL-IL-2 cells.

FIG. 7 is a flow chart showing the results of the intracellular cytokine secretion capacity 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 cells; c is TIL-IL-2 cells.

FIG. 8 cell proliferation curve;

FIG. 9 RTCA detects the killing activity of example 1 and its comparative example against homologous tumor cells.

The detailed description is as follows

The invention will be further described in detail with reference to the accompanying drawings.

The method for detecting the phenotype of the TIL cells in a flow mode comprises the following specific operation steps:

taking TIL cell suspension, PBS adjusting the cell concentration to 1×10 ⁶ Each ml, 1ug antibody/10 ⁶ The ratio of individual cells was added with CD4-FITC, CD8-ECD and PD-1-PE flow-labeled antibody, incubated for 30min in the absence of light, washed twice with 1ml PBS, the supernatant was discarded, and the cell pellet was resuspended in 1ml PBS and checked on the fly.

The flow assay for the cytokine secretion capacity in TIL cells comprises the following steps:

step one: TIL cells were collected and PBS was used to adjust the cell concentration to 2X 10 ⁶ Cells were stimulated with PMA (10 ng/ml) for 6 hours at each ml;

step two: the cells were resuspended in PBS containing 10% inactivated serum, blocking Fc antibodies;

step three: cell surface staining with CD8-ECD antibody for 20min, centrifuging for 500g and 5min, and discarding supernatant;

step four: adding fixative (dosage reference 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 a membrane breaker (dosage reference instruction), incubating for 10min at room temperature in dark, adding 2-3ml PBS, centrifuging for 500g and 5min, and discarding the supernatant; IFN-gamma-PC 7 and TNF-alpha-PC 5 antibodies were added and stained for 30min, 2-3ml PBS was added, 500g,5min were centrifuged, the supernatant was discarded, and 500ul of PBS containing 2% paraformaldehyde was used to resuspend the fixed cells for flow detection.

Example 1:

isolated culture of tumor antigen specific TIL cells in tumor tissue of a patient with melanoma, comprising the steps of:

(1) Taking tumor tissue of a melanoma patient (Wang XX, female, 81 years old), removing normal tissue and necrotic tissue, shearing the tumor tissue 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, wherein the tumor tissue is digested into a chyme shape, and removing cell masses by using a 100um cell filter screen to obtain single cell suspension;

(2) After the obtained single cell suspension is centrifugally precipitated, adding 50ml of GT-T551H 3 culture medium into the cell precipitate for resuspension;

(3) Preparing a 2-grade density gradient separating liquid (the lower layer is 100% Ficoll, and the upper layer is a mixed liquid of 75% Ficoll and 25% RPM 1640) by using a 15ml centrifuge tube;

(4) Adding the single cell suspension obtained in the step (2) into the uppermost layer of the 2-stage 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 cells between the middle layer and the lower layer, which are single nuclear cell suspensions, and obtaining single nuclear cells with total number of about 3×10 ⁶ ；

(5) Resuspending the obtained mononuclear cell pellet in adding GT-T551H 3 cell culture medium to adjust the cell concentration to 5×10 ⁵ Per ml, simultaneously adding IL-2 with a final concentration of 5000U/ml, CD3 monoclonal antibody with a final concentration of 10ug/ml, IFN-gamma with a final concentration of 2000U/ml, placing into 6-well cell plates for culture, 2 ml/well, 37 ℃ and 5% CO ₂ Culturing for 2d under the condition to carry out rapid amplification;

(6) The cells in (5) were collected by centrifugation at 1X 10 ⁶ The cell pellet was resuspended in PBS at a concentration of/ml, about 15ml. According to 10 ⁶ The proportion of 1ug antibody added to the cells, 15ul of each of CD4-FITC, CD8-ECD and PD-1-PE antibodies was added to the cells and labeled for 30 minutes, 35ml of PBS was added to the cells, and after mixing the cells uniformly, the cell pellet was collected by centrifugation and 15ml of PBS was added to resuspend the cells;

(7) Cell phenotype analysis was performed according to the procedure of the beckman MoFlo XDP flow sorter, and tumor-specific CD8 was collected by sorting ⁺ PD-1 ⁺ TIL cells; the results are shown in FIG. 1, cultured in vitroAfter 2d, CD8 in TIL cells ⁺ Cell proportion is 39.2%, CD4 ⁺ Cell proportion was 41.7%, while CD8 ⁺ PD-1 ⁺ The proportion of TIL cells was about 33.4%. (8) Collected CD8 ⁺ PD-1 ⁺ Centrifuging TIL cell subpopulation, adding GT-T551H 3 cell culture medium into cell sediment to adjust cell concentration to 1×10 ⁶ Per ml, adding GT-T551H 3 cell culture medium containing 5000U/ml IL-2, 0.5ug/ml IL-15, 0.5ug/ml IL-7, 1ug/ml anti-PD-1 monoclonal antibody, and standing at 75cm ² In a cell culture flask, 37 ℃,5% CO ₂ Continuing to culture under the condition;

(9) Performing bottle expansion culture according to the cell expansion condition, wherein the expansion culture medium is GT-T551H 3 culture medium containing IL-2 with the final concentration of 300 u/ml;

(10) In the cells of example 1 (hereinafter referred to as CD 8) ⁺ PD-1 ⁺ On day 20 of TIL-IL-2+IL-15+IL-7+PD-1 culture, cells were observed under a microscope and photographed (as shown in FIG. 2). Cells were collected, flow-through for cell phenotype identification and cytokine secretion capacity levels were examined, and compared with unsorted TIL cells of comparative example (hereinafter abbreviated as TIL-IL-2+IL-15+IL-7+PD-1) and TIL cells of comparative example were subjected to in vitro expansion with only 5000U/ml IL-2 cytokine (hereinafter abbreviated as TIL-IL-2), and the results are shown in FIGS. 3 and 4.

FIG. 1 shows CD8 in TIL cells isolated from tumor tissue of melanoma patients ⁺ TIL cells account for 39.2% (as shown in FIG. 1 a), in which CD8 ⁺ PD-1 ⁺ The proportion of TIL is as high as 33.4% (as shown in fig. 1 b). CD8 selected by improving the technology ⁺ PD-1 ⁺ TIL cells were cultured in vitro for 20 days with IL-2, IL-15, IL-7 cytokine and anti-PD-1 mAb, and then substantially purified CD8 ⁺ T cells, wherein CD8 ⁺ PD-1 ⁺ The ratio of (a) was reduced to 10.2% (as shown in FIG. 3 a) while the ability of the cell to endocrine IFN-gamma was relatively high compared to the comparative example, about 34% (as shown in FIG. 4 a), indicating that CD8 was sorted ⁺ TIL cells gradually recover the activation function of the cells from their original "depleted" state during in vitro culture.

FIG. 3a is example 1Middle CD8 ⁺ PD-1 ⁺ The cell phenotype was confirmed by flow assay after culturing TIL-IL-2+IL-15+IL-7+PD-1 cells in vitro for 20 days. Substantially purified CD8 ⁺ TIL cells, wherein CD8 ⁺ PD-1 ⁺ The proportion of TIL cells was reduced to 10.2%. FIGS. 3b and 3c show the cell phenotypes of the control TIL-IL-2+IL-15+IL-7+PD-1 and TIL-IL-2, respectively, of example 1, after culturing, as can be seen in FIG. 3b, CD8 in the TIL-IL-2+IL-15+IL-7+PD-1 group ⁺ PD-1 ⁺ The proportion of TIL cells was reduced to 15.5%; as can be seen in FIG. 3c, the control TIL-IL-2 group cells were cultured for CD8 ⁺ PD-1 ⁺ Up to 30.9%. Description of CD8 after flow sorting ⁺ PD-1 ⁺ Cell substantially purified CD8 obtained by culturing TIL cells for a long period of time ⁺ TIL cells; IL-2, IL-15, IL-7 cytokines and anti-PD-1 monoclonal antibodies in combination favor 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, example 1 shows cultured CD8 ⁺ PD-1 ⁺ TIL-IL-2+IL-15+IL-7+PD-1 cells secreted IFN-gamma levels up to 34.0% with lower levels of TNF-alpha secretion of about 4.5%. As shown in FIGS. 4b-4c, the comparative example had a level of IFN-gamma secretion from TIL-2+IL-15+IL-7+PD-1 cells of 20.2%, a lower level of TNF-alpha secretion of about 6.9%, and the comparative example had a lower level of IFN-gamma secretion from TIL-IL-2 of about 19.3%; levels of secreted TNF- α were about 8.0%. Illustrating that IL-2, IL-15, IL-7 cytokines and anti-PD-1 mAbs in combination are more beneficial to CD8 ⁺ TIL cell activation in vitro.

Example 2:

isolated culture of tumor antigen specific TIL cells in ascites of a patient with bladder cancer comprising the steps of:

(1) Preparing a 2-grade density gradient separating liquid (the lower layer is 100% Ficoll, and the upper layer is a mixed liquid of 75% Ficoll and 25% RPM 1640) by using a 15ml centrifuge tube;

(2) Taking 100ml of cancerous ascites of bladder cancer patient (yellow X, female, 76 years old), adding the 2-stage separating liquid prepared in (1) into the uppermost layer, centrifuging at 2000rpm/min for 30min, and taking the upper layer and the middle layerCells between layers, which are enriched tumor cells; taking cells between the middle layer and the lower layer, which are mononuclear cell suspensions, centrifuging and collecting the obtained mononuclear cells, wherein the total number of cells is about 1×10 ⁷ ；

(3) The cell pellet was resuspended in 1X 10 cells by addition of GT-T551H 3 cell culture medium ⁶ Per ml, 5000U/ml IL-2, 0.1ug/ml CD3 mab, 2000U/ml IFN-gamma were added simultaneously, and cultured in 6 well cell plates, 2 ml/well, 37℃at 5% CO ₂ Culturing for 4d under the condition to carry out rapid amplification;

(4) The cells in (3) were collected by centrifugation at 1X 10 ⁶ The cell pellet was resuspended in PBS at a concentration of/ml, about 100ml. According to 10 ⁶ The proportion of 1ug antibody added to the cells, 100ul of each of CD4-FITC, CD8-ECD and PD-1-PE antibodies was added to the cells and labeled for 30 minutes, then 370ml of PBS was added, after mixing, the cell pellet was collected by centrifugation, and 100ml of PBS was added to resuspend the cells;

(5) Phenotypic analysis (results as in FIG. 5) was performed according to the procedure of Beckmann MoFlo XDP flow sorter, sorting and collecting tumor antigen specific CD8 ⁺ PD-1 ⁺ TIL cells; the results indicate that CD8 in TIL cells after 4d in vitro culture ⁺ Cell proportion is 52.3%, CD4 ⁺ Cell proportion was 19.3%, while CD8 ⁺ PD-1 ⁺ The proportion of TIL cells was about 49.4%.

(6) Collected CD8 ⁺ PD-1 ⁺ Centrifuging TIL cell subpopulation, adding GT-T551H 3 cell culture medium into cell sediment to adjust cell concentration to 1×10 ⁶ Per ml, adding GT-T551H 3 cell culture medium containing 500U/ml IL-2, 3ug/ml IL-15, 3ug/ml IL-7, 3ug/ml anti-PD-1 monoclonal antibody, and placing at 75cm ² In a cell culture flask, 37 ℃,5% CO ₂ Culturing was continued under the conditions.

(7) According to the cell expansion condition, the bottle expansion culture is carried out, and the expansion culture medium is GT-T551H 3 culture medium containing 300u/ml IL-2.

(8) Culturing for about 15 days, collecting cultured CD8 when the cells no longer exponentially expand ⁺ PD-1 ⁺ TIL-IL-2+IL-15+IL-7+PD-1 cells, flow-throughCytophenotype was identified and its level of cytokine secretion capacity was measured and compared to the comparative TIL cells, and the results are shown in fig. 6 and 7.

FIGS. 5a and 5b show CD8 in TIL cells isolated from ascites of a patient with bladder cancer ⁺ TIL cells account for 52.3% of CD8 ⁺ PD-1 ⁺ The proportion of TIL is as high as 49.4%. CD8 selected by improving the technology ⁺ PD-1 ⁺ The TIL cells are subjected to amplification culture for 15 days by cytokines such as IL-2, IL-15, IL-7 and the like and anti-PD-1 antibody in vitro, and then are basically purified to obtain CD8 ⁺ T cells, and CD8 therein ⁺ PD-1 ⁺ The ratio of (a) was reduced to 12.7% (as shown in FIG. 6 a) while the ability of the cell to endocrine IFN-gamma was relatively high compared to the comparative example, about 44.5% (as shown in FIG. 7 a), indicating that CD8 was sorted ⁺ TIL cells gradually recover the activation function of the cells from their original "depleted" state during in vitro culture.

As shown in FIG. 6a, CD8 in example 2 ⁺ PD-1 ⁺ Substantially purified CD8 after TIL-IL-2+IL-15+IL-7+PD-1 cell culture ⁺ TIL cells, wherein CD8 ⁺ PD-1 ⁺ The proportion in TIL cells was reduced to 12.7%. FIG. 6b and FIG. 6c are cell phenotypes of the TIL-IL-2+IL-15+IL-7+PD-1 and TIL-IL-2 groups of the control examples, as shown in FIG. 6b, after 15 days of culture of TIL-IL-2+IL-15+IL-7+PD-1 cells, CD8 ⁺ PD-1 ⁺ The proportion of TIL cells was reduced to 15.8%; FIG. 6c is a graph showing CD8 in control TIL-IL-2 cells ⁺ PD-1 ⁺ The proportion of TIL cells is up to 42.3%. Further demonstrating flow post-sorting CD8 ⁺ PD-1 ⁺ Cell substantially purified CD8 obtained by culturing TIL cells for a long period of time ⁺ TIL cells; combinations of IL-2, IL-15, IL-7 cytokines and PD-1 blockers facilitate CD8 ⁺ Expansion of T cells; the PD-1 blocker combination is more beneficial to CD8 ⁺ TIL in vitro cell activation, long-term culture in vitro, CD8 ⁺ PD-1 ⁺ The proportion of TIL will decrease.

As shown in FIG. 7, CD8 in example 2 ⁺ PD-1 ⁺ TIL-IL-2+IL-15+IL-7+PD-1 cell endocrine IFN-gamma level reaches 44.5%, TNF-alpha secretion level is lowerAbout 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%, TNF-alpha at a level of about 0.8% (FIG. 7 b), whereas the comparative TIL-IL-2 cells secreted IFN-gamma at a lower level than the examples of 20.8%, respectively 5.6% (FIG. 7 c). Further elucidation of the favor of CD8 by IL-2, IL-15, IL-7 cytokine and PD-1 blocker combinations ⁺ TIL cells are activated in vitro.

Example 3

The real-time label-free cell function analyzer (Real Time Cellular Analysis, RTCA) detects the tumor killing activity, and the specific operation steps are as follows:

step one: autologous tumor cells

Adding the cell suspension obtained in the tumor tissue of the above example 1 into the prepared 2-grade density gradient separating liquid (the lower layer is 100% Ficoll, the upper layer is a mixed liquid of 75% Ficoll and 25% RPM 1640), centrifuging at 2000rpm/min for 30min, and collecting cells between the uppermost layer and the middle layer, which are the enriched tumor cells; after washing the cells twice with PBS, the cells were resuspended in DMEM medium containing 10% FBS and the cell concentration was adjusted to 2X 10 ⁴ Per ml, added to 25cm ² In a cell culture flask, 37 ℃,5% CO ₂ Performing adherent culture under the condition, and performing adherent cell expansion according to the growth condition of cells;

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

Taking unsorted TIL cells cultured in 6-well cell plates in example 1, performing flask expansion culture according to cell expansion conditions, and expanding the added culture medium with example 1CD8 ⁺ PD-1 ⁺ The TIL cell culture medium was identical.

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

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

FIG. 8 is a graph showing cell proliferation, in which the abscissa indicates the number of days of cell culture and the ordinate indicates the number of cells. As shown in the figure, IL-2, IL-15 and IL-7 cytokinesCD8 under the action of son and PD-1 blocker ⁺ TIL cells proliferate at the fastest rate, while unsorted TIL cells of the same culture conditions are located next. However, the proliferation rate of TIL cells was relatively slow with only IL-2 cytokine action, suggesting that IL-2, IL-15 and IL-7 cytokine combinations and PD-1 blockers may promote TIL cell proliferation, particularly for CD8 ⁺ Expansion of T cells.

Step four: RTCA detection of CD8 in example 1 ⁺ PD-1 ⁺ Tumor-specific TIL cells (CD 8 ⁺ PD-1 ⁺ The tumor killing activities of TIL-IL2+ IL-15+ IL-7+ PD-1), comparative TIL-IL-2+ IL-15+ IL-7+ PD-1 and comparative TIL-IL-2 are as follows:

the method comprises the following substeps: adding a culture medium to the E-Plate assay Plate and measuring a background impedance value;

sub-step two: collecting the logarithmic phase autologous tumor cells obtained in the first step as target cells, counting, adjusting the concentration of cell suspension,

a volume of cells, 1X 10, was added to the E-Plate assay Plate ⁵ Hole, placing in a room temperature super clean bench for 30min;

and a sub-step three: the E-Plate assay Plate with the added cells was placed on an assay table (the assay table was pre-placed in an incubator) for real-time dynamic cell proliferation assay.

And a sub-step four: after overnight detection, example CD8 was added to each culture well separately ⁺ PD-1 ⁺ TIL-IL2+ IL-15+ IL-7+ PD-1, comparative TIL-IL-2+ IL-15+ IL-7+ PD-1 and comparative TIL-IL-2 were used as effector cells with an effective target ratio of 20:1 and continuing the detection, a real-time cell effect curve is obtained (see FIG. 9 for results).

As can be seen from FIG. 9, example 1CD8 ⁺ PD-1 ⁺ TIL-IL2+ IL-15+ IL-7+ PD-1 and comparative examples TIL-IL-2+ IL-15+ IL-7+ PD-1 and TIL-IL-2 effector cells in the same equivalent target ratio to target cells, compared with comparative examples, the tumor antigen specific CD8 amplified by the method of the present invention ⁺ TIL effector cells have more specific tumor killing activity. Demonstrating that the combination of cytokines used in the present protocol enhances CD8 ⁺ TIL (tungsten inert gas)Tumor-specific killing activity, which is designated as tumor antigen-specific TIL cells.

Conclusion: from the above analysis results, it can be seen that tumor-specific CD8 isolated by the technique of the present invention ⁺ PD-1 ⁺ After the TIL cells are amplified and cultured in vitro, the cells are fully activated and rapidly amplified, and the method has short in-vitro amplification and culture time, is simple and feasible to operate, and can be suitable for clinical popularization.

The foregoing is a further detailed description of the invention in connection with the preferred embodiments, and it is not intended that the invention be limited to the specific embodiments described. It should be understood by those skilled in the art that the technical solutions of the embodiments may be appropriately combined without departing from the scope of the present invention.

Claims

1. The method for separating and culturing tumor-antigen-specific tumor-infiltrating T cells is characterized by comprising the following steps of:

step two: adding the single cell suspension into the efficient amplification agent 1 to culture for 1-4d;

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

the first step comprises the following sub-steps:

taking tumor tissue block, removing necrotic tissue and blood vessel, coating, cutting into 1cm pieces with sterile scissors ² Slowly cutting left and right small tissues into minced meat, adding 5ml of 1640 cell culture medium containing tissue digestive enzyme composition, slowly shaking for 4-6 hours to obtain the tumor tissue digested into minced meatFiltering to remove cell mass by using a 100um cell filter screen to obtain single cell suspension; adding 50mL of GT-T551H 3 culture medium to resuspend; carrying out density gradient separation on single cell suspension obtained from tumor tissues by using Ficoll lymphocyte separation liquid to obtain mononuclear cells;

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

the specific operation of the second step is as follows:

adding the obtained mononuclear cell precipitate into GT-T551H 3 cell culture medium to resuspend, and adjusting cell concentration to 5×10 ⁵ ~1×10 ⁶ Adding TIL promoting cell high efficiency amplification agent 1 into each ml, placing into 6-well cell culture plate, 2 ml/well, 37 deg.C, 5% CO ₂ Culturing for 1-4d under the condition to rapidly amplify;

the third specific operation is as follows: centrifuging and collecting the mononuclear cells obtained in the step two, and then mixing the mononuclear cells according to the ratio of 1 multiplied by 10 ⁶ The cell pellet was resuspended in PBS buffer at a concentration of individual/ml and then taken up at 10 ⁶ Adding 1ug antibody into each cell, adding CD4, CD8 and PD-1 flow detection antibody for marking for 30min, adding PBS buffer solution for washing, re-suspending, sorting by a machine, sorting according to the operation steps of a Beckmann MoFlo XDP flow sorter, and collecting CD8 ⁺ PD-1 ⁺ Tumor antigen specific TIL cells;

the specific operation of the step four is as follows:

sub-step 1: CD8 obtained in the step three ⁺ PD-1 ⁺ Centrifuging TIL cell subpopulation, adding GT-T551H 3 cell culture medium into cell sediment to adjust cell concentration to 1×10 ⁶ Adding TIL-promoting cell high-efficiency amplification agent 2 per ml, and standing at 75cm ² In a cell culture flask, 37 ℃,5% CO ₂ Continuing to culture under the condition;

sub-step 3: culturing until 15-20 days, collecting and obtaining final tumor specific TIL cells when the cells are not amplified in an index way, and detecting the tumor killing activity by using a real-time label-free cell function analyzer;

the 1640 cell culture medium containing the tissue digestive enzyme composition is prepared by adding collagenase with a final concentration of 2-10mg/ml, hyaluronidase with a final concentration of 0.1-5 mg/ml and DNase I with a final concentration of 10-500U/ml into the 1640 cell culture medium;

the TIL-promoting cell high-efficiency amplification agent 1 is prepared by adding IL-2 with a final concentration of 1000-5000U/ml, IFN-gamma with a final concentration of 500-2000U/ml and CD3 monoclonal antibody with a final concentration of 0.1-10ug/ml into a GT-T551H 3 cell culture medium;

CD4 antibody used for flow detection of tumor specific TIL cell markers is FITC fluorescent dye marker, CD8 antibody is ECD fluorescent dye marker, PD-1 is PE fluorescent dye marker, and CD8 is collected by separation ⁺ PD-1 ⁺ Tumor antigen specific TIL cells;

the TIL-promoting cell high-efficiency amplification agent 2 is prepared by adding IL-2 with a final concentration of 500-5000U/ml, IL-15 with a final concentration of 0.5-3 ug/ml, IL-7 with a final concentration of 0.5-3 ug/ml and anti-PD-1 monoclonal antibody with a final concentration of 1-3 ug/ml into a GT-T551H 3 cell culture medium.

2. Use of the isolated culture method of tumor antigen specific tumor infiltrating T cells according to claim 1 in the preparation of tumor antigen specific TIL cells.