CN115944650B - Application of tumor infiltration cells in preparation of antitumor drugs and model construction method - Google Patents
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Abstract
The invention belongs to the technical field of cell biology, and discloses an application of tumor infiltration cells in preparing anti-tumor drugs and a model construction method, and an application of autologous tumor side injection CCR2 high-expression tumor infiltration CD8+T cells combined with anti-PD-1 in preparing the anti-tumor drugs; wherein the tumor is lung cancer and glioma of mice. The purification of the tumor infiltration T cells provided by the invention is up to 99%; activation of T cells by tumor infiltrating CD8+ T cells with high expression of CCR 2; intratumoral injection of cd8+ T cells in combination with subcutaneous injection of anti-PD-1; the treatment effects of the combined treatment of the single CD8+ T cells, the anti-PD-1 pretreated tumor-infiltrating CD8+ T cells, the CCR 2-transfected tumor-infiltrating CD8+ T cells, the anti-PD-1 tumor-infiltrating CD8+ T cells and the transfected CCR2 tumor-infiltrating CD8+ T cells are different, the effect of the immunotherapy is reasonable and effective, and good curative effects can be expected to be obtained.
Description
Technical Field
The invention belongs to the technical field of cell biology, and particularly relates to application of tumor infiltration cells in preparation of antitumor drugs and a model construction method.
Background
Lung cancer is a very heterogeneous malignancy, resulting in nearly one-fourth of cancer-related deaths. World health organization surveys report that lung cancer incidence is the leading malignancy in many countries and regions. Lung cancer usually occurs over 40 years old, the incidence age peak is 60-79 years old, and the prevalence rate of men and women is 2.3:1. Lung cancer originates in the bronchial mucosal epithelium, is confined to the basement membrane and forms paraneoplastic cancer, and can grow into the bronchial lumen and/or adjacent lung tissue and can spread through lymphatic circulation or transbronchial metastasis. Lung cancer is mainly of two major basic types, small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC). Of these, non-small cell lung cancer (NSCLC) occurs more, with about 80% of lung cancer patients belonging to this type. Lung adenocarcinoma (LUAD), a member of non-small cell lung cancer, is the most invasive histological type and has a rapidly rising global incidence. At present, common treatment strategies include surgical excision, radio frequency ablation, arterial chemoembolization, chemoradiotherapy and adjuvant immunotherapy, but have certain limitations in improving the survival rate of patients. Gliomas are the most common primary malignant tumors of the central nervous system, and the main clinical treatment schemes at present are traditional surgery and postoperative chemoradiotherapy, so that the gliomas have little effect on improving the life cycle of patients and have poor prognosis. The glioblastoma with the highest malignancy has a very poor prognosis, although the median survival of the patient is only 14 to 16 months after the combination therapy. In recent years, the treatment of glioma is a bottleneck, and immunotherapy of glioma microenvironment is expected to develop a new treatment scheme.
Several studies have demonstrated that tumor infiltrating immune cells are the most promising targets for Tumor Microenvironment (TME) immunotherapy. Tumor Infiltrating Lymphocytes (TILs) are T cell-based heterogeneous lymphocytes that are predominantly present in TMEs, have highly specific cytotoxicity to autologous tumor cells, and have advantages of high efficiency, specificity, and small side effects. Two problems are faced in TILs cell chemotaxis during development of TILs therapy, whether T cells can successfully migrate to tumor sites? Is T cells migrating to the tumor site activated and killed the tumor cells? Early studies showed that T cells highly expressing CXCR2 genes could migrate directionally to tumor sites secreting chemokines, but specific killing effects were not clear, nor were experiments in animals validated.
In recent years, the tumor immunotherapy has made a great breakthrough, and the PD-1 antibody single drug is utilized to treat various malignant tumors, with the effective rate reaching 10-30%. In fact, the method ultimately works with tumor-specific T cells by reactivating tumor-specific T cells in the tumor microenvironment of the patient. Thus, the key to the success of tumor immunotherapy is how to better function as tumor-specific T cells.
However, related technical schemes for preparing antitumor drugs by combining tumor infiltrating CD8+ T cells with high expression of autologous tumor side injection CCR2 with anti-PD-1 have not been reported in the prior art.
In recent years, scientists have conducted a great deal of clinical research on tumor-infiltrating lymphocyte therapy to observe and evaluate the therapeutic effects of TILs treatment on various tumors such as melanoma, ovarian cancer, lung cancer and the like. Wherein, when TILs are used in melanoma patients, the TILs are separated from the tumor microenvironment, and IL-2 is stimulated and amplified in vitro and has good tumor killing effect after being infused back into the patients. TILs have also been shown to have therapeutic efficacy in non-small cell lung cancer, osteosarcoma and ovarian cancer.
Although TILs treatment has proven effective against solid tumors, challenges are faced in clinical use and there is a need to provide a solution strategy.
Difficulty in separation and purification of TILs
Not all tumor tissue microenvironments have more TILs present. Furthermore, the tumor tissue isolated TILs obtained by surgery carry a large number of tumor cells, and in the purification culture, some tumor cells do not die with the time of culture, and excessive tumor cells easily cause depletion death of TILs, resulting in failure of obtaining TILs. Furthermore, TILs comprise a number of heterogeneous subgroups, cd4+ T, CD8+t, effector cells and regulatory cells, etc. In order to ensure better curative effect in the later treatment, it is important to isolate TILs with anti-tumor effect.
2. High doses of IL-2 are typically used for amplification before the TILs therapeutic product is returned to the tumor patient. Expansion promotes T cell differentiation and changes in phenotype thereof, affecting proliferation capacity and persistence of TILs in vivo after reinfusion.
3. Adverse reactions of second generation TILs therapeutic products
Currently, second generation therapeutic products focus mainly on four aspects: enhancing the ability of TILs to target and recognize tumors; enhancing the persistence of TILs in vivo; enhancing tumor infiltration capacity of TILs; enhancing tumor killing efficacy of TILs. In TILs, chimeric antigen receptors of CD3 zeta and CD28 are constructed, but the in-vivo reaction is low due to the overlong in-vitro treatment time; the retrovirus transfects IL-2 gene, but has low treatment response and high toxicity; lentiviruses transfect CXCR2, but have no known effect.
Our technique first separates TILs, purifying cd8+ T, a primary killer potential cell; the magnetic bead purification and other methods are combined to overcome the problem of tumor cell pollution of the purified cells; the method is characterized in that the TILs are transfected by CCR2 high-expression slow virus without in-vitro IL-2 stimulation, and the TiLs are injected beside tumor and act on tumor in a short distance, so that the chemotactic effect of the TILs is enhanced, the TILs are activated, and the anti-PD-1 therapy is combined, so that the method has remarkable anti-tumor therapeutic effect.
Through the above analysis, the problems and defects existing in the prior art are as follows:
1.TILs are difficult to purify and easily cause pollution to tumor cells, so that the T cells are exhausted and dead in the process of in vitro culture;
2. adding IL-2 and the like to amplify in vitro, changing the cell character, and having long time (4-6 weeks);
3. the toxicity of using the cytokines is high;
4. the study of chemokines is deficient, and there is a need to study new chemokines that play roles in chemotaxis and cell activation.
At present, related technical schemes for preparing anti-tumor drugs by combining tumor infiltrating CD8+ T cells with later anti-PD-1 have not been reported.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides an application of tumor infiltration cells in preparing antitumor drugs.
The invention is realized in such a way that the application of the tumor infiltration cells in preparing the anti-tumor drugs is that: application of autologous tumor side injection CCR2 high-expression tumor infiltration CD8+T cells combined with anti-PD-1 in preparation of anti-tumor drugs; wherein the tumor is lung cancer and glioma of mice.
Another object of the present invention is to provide a method for verifying the application of the tumor-infiltrating cd8+ T cells in preparing an anti-tumor drug, wherein the method for constructing the animal model comprises the following steps:
step one, purifying tumor infiltration T cells;
step two, slow virus transfection and analysis of activation and killing effects of tumor infiltration CD8+ T cells with high CCR2 expression on cells;
step three, determining the interval time between the combined subcutaneous injection of the tumor side injection CD8+T cells and the anti-PD-1;
step four, analyzing the difference of the effect of the combined treatment of single body injection of CD8+ T cells, tumor-infiltrating CD8+ T cells pretreated by anti-PD-1, CCR 2-transfected tumor-infiltrating CD8+ T cells, anti-PD-1 and CCR 2-transfected tumor-infiltrating CD8+ T cells.
Further, the purification of tumor-infiltrating T cells in step one comprises: tumor tissue of the mice is obtained and sheared to a size of 1mm by scissors, and the operation blade is used for cutting, and the samePreparing digestive juice; shaking the tissue fragments at 37 ℃ for 25min, and digesting for 2 times; passing through 40 μm filter, centrifuging the filtrate at 4deg.C for 5min at 300g, and discarding the supernatant; re-suspending and separating out lymphocytes by using tissue diluent in a tumor infiltration lymphocyte separation kit, washing for 2 times by PBS, and re-suspending; cd8+ T cells of single cell suspension were purified using meitian gentle Mouse cd8+ T Cell Isolation Kit, magnetic beads and T cell ratios: 10 μL:10 7 The method comprises the steps of carrying out a first treatment on the surface of the Purified cd8+ T cells were cultured in RPMI1640 medium, and purified again using differential digestion attached every 3h for 2 consecutive times.
Further, the formula of the digestive juice is as follows: the lung cancer is RPMI1640 culture medium contains 0.05mg/mL type I collagenase, 0.05mg/mL type IV collagenase, 0.05mg/mL hyaluronidase and 0.01mg/mL DNase I; glioma is RPMI1640 medium containing 0.05mg/mL type II collagenase and 0.01mg/mL DNase I.
Further, the RPMI1640 medium contained 10% FBS and 1% P/S.
Further, the lentivirus transfection in the second step and analysis of activation and killing of the T cells by the tumor-infiltrating CD8+ T cells with high expression of CCR2 comprise:
lentivirus transfected CD8+ T cells, compared with an empty vector lentivirus transfected control group, the tumor-infiltrated CD8+ T cells of the CCR2 high-expression lentivirus transfected group highly express early activation molecule CD69; after the CD8+ T cells and the tumor cells are co-cultured, the expression of granzyme B and interferon gamma is increased, and the activation and killing effects of the high expression CCR2 on the tumor infiltrating CD8+ T cells are verified.
Further, the determination of the time interval between intratumoral injection of cd8+ T cells in combination with subcutaneous injection of anti-PD-1 in step three comprises:
tumor side injection of cd8+ T cells highly expressing CCR2, tumor size was observed daily; taking the rebound of the tumor gradually becoming smaller as a time node, rapidly injecting the anti-PD-1, and continuing to observe until the tumor becomes smaller or disappears.
Further, the difference in effect of the combined treatment of the single individual injection cd8+ T cells, the anti-PD-1 pretreated tumor-infiltrating cd8+ T cells, the CCR2 transfected tumor-infiltrating cd8+ T cells, the anti-PD-1 and the CCR2 transfected tumor-infiltrating cd8+ T cells in step four comprises:
autologous injection of CCR2 transfected CD8+ T cells, tumor becomes smaller after 2-3 days, tumor rebound on day 5; anti-PD-1 and transfected CCR2 tumors infiltrate CD8+ T cells in a combined way, the rebound tendency is inhibited, and the tumors continue to diminish until disappearing; tumors of the other two groups became smaller but rebound.
Further, the method for constructing the animal model further comprises the following steps: anti-PD-1 is used, and tumor infiltration CD8+ T cells with chemotaxis and activation killing are used.
In combination with the technical scheme and the technical problems to be solved, the technical scheme to be protected has the following advantages and positive effects:
the purification of the tumor infiltration T cells provided by the invention is up to 99%; activation and killing effect of tumor infiltration CD8+ T cells with high expression of CCR2 on T cells; the intratumoral injection of cd8+ T cells in combination with subcutaneous injection of anti-PD-1 for a time interval; untreated group, tumor-infiltrating cd8+ T cell reinfusion against PD-1 pretreatment, CCR 2-transfected tumor-infiltrating cd8+ T cell reinfusion, CCR 2-transfected tumor-infiltrating cd8+ T cell and anti-PD-1 combination treatment effect.
The invention verifies the therapeutic effect of the tumor infiltration CD8+T cells combined with the anti-PD-1 with high expression of autologous tumor side injection CCR2 on the glioma and lung cancer of mice, has reasonable and effective immunotherapy effect, can be used as an auxiliary method combining clinical traditional radiotherapy and chemotherapy, and is expected to obtain good curative effect.
The present invention is directed to clinical transformations. If the invention is used for clinical experiments and transformation, the invention provides key technical indexes and optimization schemes for TILs treatment of one of the most potential cell treatment types. The invention has good treatment effect in lung adenocarcinoma and glioma verification, is possibly suitable for other tumors in the future, and provides thought and guidance for development of multi-tumor general drugs. The invention can be combined with anti-PD-1 treatment, and can also be combined with other radiotherapy and chemotherapy and auxiliary treatment, thereby providing an alternative treatment scheme for tumor treatment.
At present, TILs treatment technology is mature, but the problems of difficult separation and purification, long in-vivo durability, high toxicity, long in-vitro operation time, biological property change and the like are faced to be solved. The research separates main anti-tumor TILs CD8+ T cells as a therapeutic drug, adopts a plurality of methods to jointly purify, eliminates tumor cell pollution, improves the purification rate to 99%, perfects the separation and purification technology at home and abroad, and fills the technical blank in the home and abroad. After the high-expression slow virus of the CCR2 with high-efficiency transfection suspension cell is adopted in vitro, firstly, the expression of a cell activation marker molecule is detected, and the cell activation marker molecule is co-cultured with tumor cells, and the killing effect is detected and verified; after detection, CD8+ T cells of the CCR2 high-expression lentivirus are directly injected beside tumor without being amplified by a stimulator, so that chemotaxis of the CD8+ T cells to tumor tissues is facilitated, character change caused by long-time stimulator culture in vitro is avoided, at present, CCR2 is concentrated in macrophage research, related treatment research of the CD8+ T with high CCR2 expression is not available, and the technical blank in the domestic and foreign industries is filled. The anti-PD-1 treatment is adopted at the later stage of CCR2 high-expression CD8+T injection instead of the anti-PD-1 treatment, so that adverse immune reactions such as excessive cytokine storm and the like caused by the simultaneous combined use of CCR2 high-expression CD8+T and anti-PD-1 are avoided, and the external combined treatment scheme is perfected.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of tumor-infiltrating CD8+ T lymphocyte extraction and reinfusion animal treatment provided by an embodiment of the present invention;
FIG. 2 is a flow chart of tumor-infiltrating CD8+ T lymphocyte extraction and reinfusion animal treatment provided by an embodiment of the invention;
FIG. 3 is a schematic diagram of purity detection after magnetic bead sorting of tumor-infiltrating CD8+ T cells provided by an embodiment of the invention;
FIG. 4 (a) is a schematic representation of CCR2 expression after CCR2 lentivirus transfection of tumor infiltrating CD8+ T cells provided in the examples of the present invention; FIG. 4 (b) is a statistical plot of CCR2 expression after CCR2 lentivirus transfection of tumor infiltrating CD8+ T cells provided by the examples of the invention; FIG. 4 (c) is a schematic and statistical diagram of early activation molecule CD69 expression after CCR2 lentivirus transfection of tumor infiltrating CD8+ T cells provided in the examples of the present invention;
FIG. 4 (d) is a statistical chart showing the expression of CD69 as early activating molecule after CCR2 lentivirus is transfected into tumor infiltrating CD8+ T cells according to the present invention; FIG. 4 (e) is a schematic diagram showing the expression of the killing marker molecule CD107a after the CCR2 lentivirus provided by the embodiment of the invention is transfected into tumor infiltrating CD8+ T cells; FIG. 4 (f) is a statistical graph showing the expression of the killing marker CD107a after CCR2 lentivirus transfection of tumor infiltrating CD8+ T cells, as provided in the examples of the present invention.
FIG. 5 (a) is a schematic diagram showing the detection of the expression of CD69, an early activation molecule of CD8+ T cells by flow cytometry after in vitro co-culturing of glioma cells and tumor infiltrating CD8+ T cells provided by the examples of the present invention; FIG. 5 (b) is a statistical chart showing the detection of the expression of CD69, an early activation molecule of CD8+ T cells by flow cytometry after in vitro co-culture of glioma cells and tumor infiltration of CD8+ T cells provided by the examples of the present invention; FIG. 5 (c) is a schematic diagram showing the detection of the expression of CD107a, a killing marker molecule of CD8+ T cells by flow cytometry after in vitro co-culturing glioma cells and tumor infiltrating CD8+ T cells provided by the examples of the invention; FIG. 5 (d) is a statistical chart showing the detection of the expression of CD107a, a killing marker molecule of CD8+ T cells by flow cytometry after in vitro co-culturing glioma cells and tumor infiltrating CD8+ T cells provided by the examples of the invention.
FIG. 6 (a) is a schematic representation of the expression of granzyme B after flow cytometry detection of the killer effector molecule interferon gamma after in vitro co-culture of glioma cells and tumor infiltration of CD8+ T cells provided by the examples of the present invention; FIG. 6 (B) is a statistical chart showing the expression of interferon gamma, granzyme B, which is a killer effector molecule detected by flow cytometry after in vitro co-culturing glioma cells and tumor infiltrating CD8+ T cells provided by the example of the present invention;
FIG. 7 (a) is a schematic diagram showing the detection of the expression of CD69, an early activation molecule of CD8+ T cells by flow cytometry after in vitro co-culturing lung adenocarcinoma cells and tumor infiltrating CD8+ T cells provided by the examples of the present invention; FIG. 7 (b) is a statistical chart showing the detection of the expression of CD69, an early activation molecule of CD8+ T cells by flow cytometry after in vitro co-culturing lung adenocarcinoma cells and tumor infiltrating CD8+ T cells provided by the examples of the present invention; FIG. 7 (c) is a schematic diagram showing the detection of the expression of CD107a, a killing marker molecule of CD8+ T cells by flow cytometry after co-culturing lung adenocarcinoma cells and tumor infiltrating CD8+ T cells in vitro provided by the examples of the invention; FIG. 7 (d) is a statistical graph showing the expression of CD107a, a killer marker molecule, of CD8+ T cells detected by flow cytometry after co-culturing lung adenocarcinoma cells and tumor infiltrating CD8+ T cells in vitro provided by the examples of the present invention.
FIG. 8 (a) is a schematic representation of the expression of the interferon gamma, granzyme B, of the killing effector molecule detected by flow cytometry after in vitro co-culture of lung adenocarcinoma cells and tumor infiltrating CD8+ T cells provided by the examples of the invention; FIG. 8 (B) is a statistical chart showing the expression of interferon gamma, granzyme B, which is a killer effector molecule detected by flow cytometry after in vitro co-culture of lung adenocarcinoma cells and tumor infiltrating CD8+ T cells provided by the example of the present invention;
FIG. 9 (a) is a graph showing comparison of tumor volumes of a glioma mouse model after autologous feedback provided by the examples of the present invention between different treatment groups, wherein the glioma is a control group without treatment, the glioma is a control group transfected with CD8+ T cells by empty lentivirus, the glioma is a group with anti-PD-1 pretreatment, the glioma is a group with CCR2CD8+ T cells, and the glioma is a combination treatment group with anti-PD-1 and CCR2CD8+ T cells; FIG. 9 (B) is a graph showing comparison of tumor volumes of a lung adenocarcinoma mouse model after autologous feedback provided by the embodiment of the invention among different treatment groups, wherein the lung adenocarcinoma is not treated in the A lung adenocarcinoma untreated group control, the lung adenocarcinoma is empty and slow virus transfected CD8+ T cell in the B lung adenocarcinoma air control group, the lung adenocarcinoma is anti-PD-1 pretreated CD8+ T cell in the C lung adenocarcinoma, the lung adenocarcinoma is transfected CCR2CD8+ T cell in the D lung adenocarcinoma, and the lung adenocarcinoma is anti-PD-1 and CCR2CD8+ T cell transfected in the E lung adenocarcinoma combined treatment group; FIG. 9 (c) is a statistical chart of tumor volume change of the glioma mouse model after autologous feedback provided by the example of the present invention between different treatment groups; FIG. 9 (d) is a statistical chart of tumor volume change of the lung adenocarcinoma mouse model after autologous feedback provided by the embodiment of the invention among different treatment groups; FIG. 9 (e) is a statistical chart of the weight change of the glioma mouse model after autologous feedback provided by the example of the present invention between different treatment groups; FIG. 9 (f) is a statistical chart of weight change of mice in different treatment groups of the lung adenocarcinoma mouse model after autologous feedback provided by the embodiment of the invention;
FIG. 10 shows the therapeutic assays of autologous mice following injection provided by the examples of the present invention, with confocal microscopy, and with greater migration of CCR2 transfected CD8+ T cells to tumor sites than empty vector transfected CD8+ T cells.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Aiming at the problems existing in the prior art, the invention provides application of tumor infiltration cells in preparing antitumor drugs, and the invention is described in detail below with reference to the accompanying drawings.
The embodiment of the invention provides an application of tumor-infiltrating cells in preparing antitumor drugs, which comprises the following steps: application of autologous tumor side injection CCR2 high-expression tumor infiltration CD8+T cells combined with anti-PD-1 in preparation of anti-tumor drugs.
The tumor provided by the embodiment of the invention is a glioma of a mouse and lung cancer.
As shown in fig. 1, the schematic diagram of tumor-infiltrating cd8+ T lymphocyte extraction and feedback animal treatment provided by the embodiment of the invention comprises the following steps:
s101: purifying tumor infiltrating T cells;
s102: lentivirus transfection and analysis of activation of cells by tumor infiltrating CD8+ T cells with high expression of CCR 2;
s103: determining the interval between the combined subcutaneous injection of tumor side injected CD8+ T cells and the anti-PD-1;
s104: the effect differences of the combined treatment of autologous cd8+ T cells, tumor-infiltrating cd8+ T cells pretreated with anti-PD-1, CCR 2-transfected tumor-infiltrating cd8+ T cells, anti-PD-1 and CCR 2-transfected tumor-infiltrating cd8+ T cells were analyzed.
In the embodiment of the present invention, step S101, purification of tumor infiltrating T cells is performed; as shown in FIG. 3, the purification rate of CD8+ T cells reached 99.32%.
In an embodiment of the invention, step S102, the activation of T cells by tumor infiltrating CD8+ T cells with high expression of CCR2 is analyzed. As shown in fig. 4 (a), 4 (b), 4 (c), and 4 (d), CCR 2-highly expressed tumor infiltrating cd8+ T cells up-regulate the expression of early activation molecule CD69 of T cells. As shown in fig. 5 (a), 5 (b), 5 (c), 5 (d), glioma cells and tumor-infiltrating cd8+ T cells were co-cultured in vitro, and CD69 and CD107a expression of tumor-infiltrating cd8+ T cells was detected, with CD69 and CD107a expression in both the cd8+ tccr2+ group, cd8+ tccr2+ group in combination with anti-PD-1 group being higher than in the control group (cd8+ T group or anti-PD-1 pretreated tumor-infiltrating cd8+ T cell group). As shown in fig. 6 (a), 6 (B), glioma cells and tumor-infiltrating cd8+ T cells were co-cultured in vitro, and expression of interferon gamma and granzyme B by killing molecules of tumor-infiltrating cd8+ T cells was detected, and expression of CD69 and CD107a in the cd8+tccr2+ group, cd8+tccr2+ group in combination with anti-PD-1 group was higher than that in the control group (cd8+ T group or anti-PD-1 pretreated tumor-infiltrating cd8+ T cell group). As shown in fig. 7 (a), 7 (b), 7 (c), and 7 (d), lung cancer cells and tumor-infiltrating cd8+ T cells were co-cultured in vitro, and CD69 and CD107a expression of tumor-infiltrating cd8+ T cells was detected, with CD69 and CD107a expression in both the cd8+tccr2+ group, and the anti-PD-1 group in combination being higher than in the control group (cd8+t group or the anti-PD-1 pretreated tumor-infiltrating cd8+ T cell group). As shown in fig. 8 (a), 8 (B), lung cancer cells and tumor infiltrating cd8+ T cells were co-cultured in vitro, and expression of interferon gamma and granzyme B by killing molecules of tumor infiltrating cd8+ T cells was detected, and expression of CD69 and CD107a by cd8+tccr2+ group, cd8+tccr2+ group combined with anti-PD-1 group was higher than that of control group (cd8+ T group or anti-PD-1 pretreated tumor infiltrating cd8+ T cell group).
In an embodiment of the invention, step S103, determining the interval between the combined subcutaneous injection of tumor side injected cd8+ T cells against PD-1; after measuring the tumor volume, it was found that tumor rebound was long starting on day 5 after reinfusion of TIL cells, so we chose reinfusion of anti-PD-1 on day 4, tumor was small, and tumor examination size was taken out after 4 days.
In an embodiment of the present invention, step S104, the difference in the effect of the combination treatment group of single individual injection of CD8+ T cells, anti-PD-1 pretreated tumor-infiltrating CD8+ T cells, CCR2 transfected tumor-infiltrating CD8+ T cells, anti-PD-1 and CCR2 transfected tumor-infiltrating CD8+ T cells is analyzed. In vivo mouse experiments, the anti-tumor effect of using CCR2 high-expression CD8+ T cells and combined injection of anti-PD-1 group is best, and the trend of CCR2 high-expression TILs to tumor tissues is confirmed by observation under a confocal microscope.
The purification of tumor-infiltrating T cells in step S101 provided by the embodiment of the invention includes:
the tumor tissue of the mice is taken out, the scissors are sheared to the size of 1mm, and the surgical blade is sheared. Meanwhile, digestive juice is prepared, and the formula is as follows: RPMI1640 medium contained type I collagenase (0.05 mg/mL), type IV collagenase (0.05 mg/mL), hyaluronidase (0.05 mg/mL), and DNase I (0.01 mg/mL). Shaking the tissue fragments at 37 ℃ for 25min, and digesting for 2 times; the filtrate was centrifuged at 300g for 5min at 4℃through a 40 μm filter and the supernatant was discarded. Lymphocytes were resuspended and isolated using tissue dilutions in tumor-infiltrating lymphocyte isolation kit (Solarbio). The PBS was washed 2 times and resuspended. Cd8+ T cells of the single cell suspension were purified using meitian gentle Mouse cd8+ T Cell Isolation Kit. Purified cd8+ T cells were cultured in RPMI1640 medium containing 10% fbs, and cd8+ T cells were purified again using differential digestion attachment every 3h, and purified 2 times in succession. Inoculating purified CD8+ T cells, infecting CCR2 high-expression lentivirus, centrifuging and concentrating after 72h, injecting glioma entity beside tumor, injecting anti-PD-1 antibody on the 4 th day after inoculating purified CD8+ T cells, and eliminating tumor.
Step S102 provided by the embodiment of the invention analyzes activation and killing effects of tumor-infiltrating CD8+ T cells with high expression of CCR2 on T cells, and comprises the following steps:
after the CD8+ T cells and the tumor cells are co-cultured, compared with an empty lentiviral vector transfected control group, the CCR2 high-expression lentiviral transfected tumor infiltration CD8+ T cell group highly expresses CD69 early activation molecules, and the expression of granzyme B and interferon gamma is increased, so that the activation and killing effect of the tumor infiltration CD8+ T cells on the T cells is determined.
The determination of the inter-time of intratumoral injection of cd8+ T cells in combination with subcutaneous injection of anti-PD-1 in the analysis in step S103 provided by the embodiments of the present invention comprises:
tumor side injection of cd8+ T cells highly expressing CCR2, tumor size was observed daily; taking the rebound of the tumor gradually becoming smaller as a time node, rapidly injecting the anti-PD-1, and continuing to observe until the tumor becomes smaller or disappears.
The difference of the effects of the combination treatment group of the single-alone injection cd8+ T cells, the anti-PD-1 pretreated tumor-infiltrating cd8+ T cells, the CCR2 transfected tumor-infiltrating cd8+ T cells, the anti-PD-1 tumor-infiltrating cd8+ T cells and the CCR2 transfected tumor-infiltrating cd8+ T cells in the step S104 provided by the embodiment of the invention comprises:
single-alone body injection of cd8+ T cells transfected with CCR2, tumor becoming smaller after 2-3 days, tumor becoming smaller trend rebound starting at day 5; the rebound tendency of CD8+ T cells transfected with CCR2 and anti-PD-1 is inhibited by combined injection, and the rebound tendency is continuously reduced until the rebound tendency disappears; the single individual injected cd8+ T cells, anti-PD-1 pretreated tumors infiltrated cd8+ T cells, and tumors became small but rebound.
The method for constructing the animal model provided by the embodiment of the invention further comprises the following steps: cd8+ T cells were highly expressed with anti-PD-1, ccr 2.
TILs therapy has been successfully applied to metastatic melanoma and other solid tumor patients. TILs therapy has unique advantages over other immune cell therapies (e.g., CAR-T and TCR-T). TIL consists of T cells with multiple TCR clones, which respond more effectively to tumor heterogeneity, not only directly to shared tumor antigens, but also to specific tumor antigens. TILs usually contain a large number of effector memory T cells, and have a stronger antitumor effect after activation. In addition, TIL comes from the patient himself, without genetic modification, which means that the method is low toxic. However, TIL therapy also has limitations. First, in order to achieve a durable anti-tumor response, effector T cells with anti-tumor activity must be present in the tumor, which is not the case for many solid tumors. Another potential population of transformed anti-tumor cells are tumor infiltrating γδ T cells that exert their anti-tumor activity, usually by secretion of Interferon (IFN) and Tumor Necrosis Factor (TNF), but which are independent of tumor-associated antigens. Second, despite the great progress made in the selection strategy of TILs, the widespread use of TILs therapy in a variety of cancers remains a challenge due to the barrier to immunosuppressive tumor microenvironment, and no TILs products are currently marketed. Since tumor cells often exhibit different types of genetic mutations that produce multiple neoantigens, it is difficult to design universal CAR-TILs to eliminate cancer cells. Meanwhile, immunosuppressive tumor microenvironments may induce failure of invasive cytotoxic T cells, thereby leading to reduced capacity for elimination of cancer cells. Scientists found that high-grade ER breast tumors infiltrated high levels of PD-1 depleted T cells. In addition, injected TILs survive for a short period in vivo. More importantly, in order to improve survival and tumor homing ability of TILs after re-infusion into patients, modification techniques have been explored, and various chemotactic cytokines are used for homing of TILs, but the efficacy is unknown. The high expression CCR2 TILs combined immune checkpoint inhibitor of the invention is anti-PD-1, which can possibly overcome the problem of homing of the TILs and the problem of short-term depletion of the TILs in vivo. However, the present invention does not allow for the current expansion of IL-2, anti-CD 3 and feeder cells prior to injection, which has the disadvantage of a relatively small number, and has the advantage of a fast process (4-6 weeks for general expansion, 3-4 days for the present invention), which is advantageous in maintaining cellular properties. By adopting the tumor side injection to directly reach the tumor part, the consumption of cells after peripheral blood injection is avoided. In addition, the invention realizes almost no tumor cell pollution in the aspect of separating and purifying TILs. The most common procedure is surgical removal of the tumor, cutting into small pieces, tissue culture, and collection and purification after T cells have roamed out. The research directly carries out tissue digestion, the ficoll separating liquid extracts mononuclear cells, the magnetic beads separate and purify CD8+ T cells, differential adherence and other methods to purify the cells again. The method can extract the high-purity CD8+ T cells in a short time.
The embodiment of the invention has a great advantage in the research and development or use process, and has the following description in combination with data, charts and the like of the test process.
Examples: treatment effect of autologous tumor side injection CCR2 high-expression tumor infiltration CD8+ T cell combined with later-stage anti-PD-1 on glioma and lung cancer of mice
The purification of tumor infiltration T cells provided by the embodiment of the invention is up to 99%; activation of T cells by tumor infiltrating CD8+ T cells with high expression of CCR 2; the intratumoral injection of cd8+ T cells in combination with subcutaneous injection of anti-PD-1 for a time interval; the effects of the combined treatment of single-alone injection of cd8+ T cells, anti-PD-1 pretreated tumor-infiltrating cd8+ T cells, CCR2 transfected tumor-infiltrating cd8+ T cells, anti-PD-1 and CCR2 transfected tumor-infiltrating cd8+ T cells were different.
1. Purification of tumor infiltrating T cells, up to 99%.
The tumor tissue of the mice is taken out, the scissors are sheared to the size of 1mm, and the surgical blade is sheared. Meanwhile, digestive juice is prepared, and the formula is as follows: RPMI1640 medium contained type I collagenase (0.05 mg/mL), type IV collagenase (0.05 mg/mL), hyaluronidase (0.05 mg/mL), and DNase I (0.01 mg/mL). The tissue pieces were shaken at 37℃for 25min, digested 2 times, then passed through a 40 μm filter, the filtrate centrifuged at 300g for 5min at 4℃and the supernatant discarded. Lymphocytes were resuspended and isolated using tissue dilutions in tumor-infiltrating lymphocyte isolation kit (Solarbio). The PBS was washed 2 times and resuspended. Cd8+ T cells of the single cell suspension described above were purified using meitian and gentle Mouse cd8+ T Cell Isolation Kit. The ratio of magnetic beads to total cells is: 10 μL:10 7 . Purified cd8+ T cells were cultured in RPMI1640 medium (containing 10% fbs), purified again using differential digestion attached to the wall every 3h, and purified 2 times in succession.
Activation and killing effect of tumor infiltrating CD8+ T cells with high expression of CCR2 on T cells.
After co-culturing the CD8+ T cells and tumor cells, compared with an empty lentiviral vector transfected control group, the CCR2 high-expression lentiviral transfected tumor infiltrates CD69, CD107a, granzyme B and interferon gamma expression of the CD8+ T cell group to be increased.
3. Intratumoral injection of cd8+ T cells in combination with subcutaneous injection of anti-PD-1 for a time interval.
The tumor is injected with CD8+ T cells beside the tumor, the size of the tumor is observed about 4-5 days, and when the tumor gradually becomes smaller and rebound occurs, the anti-PD-1 is rapidly injected at the time point, and the observation is continued until the tumor disappears.
If both CD8+ T cells which express CCR2 and anti-PD-1 are used, the invention also monitors that the cells have low PD-1 expression at the moment because the CD8+ T cells which express CCR2 and start to inject have strong immunocompetence and killing capacity, and the anti-PD-1 is not used for blocking the PD-1 function of the CD8+ T cells which express CCR2 at the moment, and the excessive immune response and adverse reaction are easily caused. Therefore, the invention uses CD8+ T cells with high expression of CCR2 and anti-PD-1 treatment respectively, and the selection interval is about 5 days through experiments.
4. Treatment effect differences of combined treatment of autologous cd8+ T cells, tumor-infiltrating cd8+ T cells pre-treated with anti-PD-1, CCR 2-transfected tumor-infiltrating cd8+ T cells, anti-PD-1 and CCR 2-transfected tumor-infiltrating cd8+ T cells.
Single individual injection of CCR2 transfected cd8+ T cells, tumor became smaller after 2-3 days and tumor tendencies became rebound on day 5. The combined injection of cd8+ T cells and anti-PD-1 inhibited the rebound tendency, continued to diminish until disappearing. The other two groups of tumors became smaller but rebound.
5. The characteristics are combined, so that the immunotherapy effect of the scheme is ensured to be reasonable and effective, and the scheme can be used as a scheme combining other clinical immunotherapy, and is expected to achieve good curative effects.
The combination of TIL therapy and anti-PD-1/PD-L1 antibody therapy showed preliminary beneficial results in recent trials. In cancer patients, the immune checkpoint receptors on effector T cells, CTLA-4 and PD-1, are upregulated, resulting in inhibition of T cell function, based on which inhibition signals can be blocked using anti-CTLA-4 and anti-PD-1. In addition, in vitro and in vivo experiments show that after long-term exposure to tumor antigens, CD8+ T cells apoptosis or enter into an abnormal differentiation state, and PD-1 is highly expressed, so that no response to specific tumor antigens is caused, and based on the result, inhibition signals are blocked through immune checkpoint inhibitors, and immune responses are started. These existing mechanisms provide a theoretical basis for combining TILs with immune checkpoint inhibitors. Currently, the efficacy of TILs in combination with anti-PD-1 therapy as a first-line therapy is still in clinical trial. Recent researches find that Dendritic Cells (DCs) express high levels of PD-L1 besides tumor cells, weaken the activation of T cells and inhibit the anti-tumor activity, and provide theoretical basis for TILs combined immune checkpoint inhibitor treatment. Based on the above, the TILs treatment combined immune checkpoint inhibitor PD-1 provided by the invention overcomes the limitation of single medicine, is hopeful to improve the efficiency of combined medicine and is hopeful to obtain good curative effect.
The BRAF gene plays an important role in cell growth and differentiation. In some cancers, BRAF mutations are the most common mutations that result in excessive activation of MAPK pathways, promoting cell proliferation. Activated BRAF mutations can induce immune escape mechanisms, escaping T cell immune surveillance. Studies have shown that the BRAF inhibitor, verofenib, can reduce associated immunosuppressive signals, promote lymphocyte infiltration, reduce immunosuppressive cells, and enhance presentation of melanoma antigens. However, the clinical response of BRAF inhibitors is often transiently limited. Clinical trials have found that metastatic melanoma patients receiving combination therapy of TIL, IL-2 and vemurafenib respond significantly or completely. Based on the above, the TILs treatment combined with the BRAF inhibitor can overcome the limitation of single drug, and is expected to achieve good curative effect.
DC vaccines can induce immune responses and can activate and increase the number of TILs and clinical trials are underway to evaluate their combination TILs treatment. Combinations of TILs therapy and oncolytic viruses are also under investigation. The virus may combat tumor immunosuppression by producing cytokines that promote TILs anti-tumor effects. Based on the above, the TILs of the invention can be combined with various immunotherapies in the future, and are expected to achieve good curative effects.
The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.
Claims (1)
1. The application of a composition containing tumor-infiltrating cells in preparing an anti-tumor drug is characterized in that the tumor-infiltrating cells are tumor-infiltrating CD8+ T cells with high CCR2 expression, the composition consists of the tumor-infiltrating CD8+ T cells with high CCR2 expression and anti-PD-1, and the tumor is mouse lung adenocarcinoma or glioma.
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