CN115944650A - Application of tumor infiltrating 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 infiltrating cells in preparing anti-tumor drugs and a model construction method, wherein the application of tumor infiltrating CD8+ T cells with high CCR2 expression in preparing anti-tumor drugs in combination with anti-PD-1 is injected by a patient beside a tumor; wherein the tumor is mouse lung cancer and glioma. The purification of the tumor infiltrating T cells provided by the invention is up to 99 percent; activation of T cells by CCR 2-highly expressed tumor infiltrating CD8+ T cells; the tumor side injection of CD8+ T cells is combined with the subcutaneous injection of anti-PD-1; the treatment effect of the combined treatment of the single CD8+ T cell, the anti-PD-1 pretreated tumor-infiltrating CD8+ T cell, the CCR 2-transfected tumor-infiltrating CD8+ T cell, the anti-PD-1 and the transfected CCR2 tumor-infiltrating CD8+ T cell is different, the immunotherapy effect is reasonable and effective, and the expected good curative effect can be obtained.
Description
Technical Field
The invention belongs to the technical field of cell biology, and particularly relates to application of tumor infiltrating cells in preparation of an anti-tumor drug and a model construction method.
Background
Lung cancer is a highly heterogeneous malignancy, leading to nearly one-fourth of cancer-related deaths. The world health organization surveys that the incidence of lung cancer accounts for the first place of malignancy in many countries and regions. Lung cancer usually occurs in more than 40 years, the peak of onset age is 60-79 years, and the male and female prevalence rates are 2.3. Lung cancer originates in the bronchial mucosal epithelium, is confined to the basement membrane and becomes paraneoplastic, can grow into the bronchial lumen and/or adjacent lung tissue, and can spread by lymphatic circulation or transbronchial metastasis. There are two major basic types of lung cancer, namely Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC). Of which non-small cell lung cancer (NSCLC) occurs more, and about 80% of lung cancer patients belong to this category. Lung adenocarcinoma (LUAD), a member of non-small cell lung cancers, is the most aggressive histological type and rapidly increases in incidence worldwide. At present, the commonly used treatment strategies comprise surgical excision, radio frequency ablation, transcatheter arterial chemoembolization, chemoradiotherapy and adjuvant immunotherapy, but have certain limitations on the aspect of improving the survival rate of patients. Glioma is the most common primary malignant tumor of the central nervous system, and the current clinical main treatment scheme is traditional operation and postoperative chemoradiotherapy, which has little effect on improving the survival period of patients and has poor prognosis. The prognosis of glioblastoma with the highest degree of malignancy is extremely poor, although with combined treatment the median survival of patients is only 14 to 16 months. In recent years, treatment for glioma is becoming a bottleneck, and immunotherapy for glioma microenvironment is expected to develop a new treatment scheme.
Several studies have demonstrated that tumor infiltrating immune cells are the most desirable targets for Tumor Microenvironment (TME) immunotherapy. Tumor Infiltrating Lymphocytes (TILs) are heterogeneous lymphocytes mainly comprising T cells, which are mainly present in TME, have highly specific cytotoxicity against autologous tumor cells, and have the advantages of high efficiency, specificity, and few side effects. Two problems are faced in TILs cell chemotaxis during the development of TILs therapy, is the successful migration of T cells to the tumor site? Is T cells migrating to the tumor site activated and killed? Early studies show that T cells with high expression of CXCR2 gene can directionally migrate to tumor sites secreting chemokines, but the specific killing effect is unknown, and experimental verification in animals and objects is not available.
In recent years, the tumor immunotherapy makes a major breakthrough, and the effective rate of the single drug therapy of various malignant tumors by using the PD-1 antibody reaches 10-30%. Indeed, it is the tumor-specific T cells that ultimately function by reactivating them in the patient's tumor microenvironment. Thus, the key to the success of tumor immunotherapy is how to better function tumor-specific T cells.
However, no report has been found on the technical scheme of preparing an antitumor drug by using autologous tumor-side injection of tumor-infiltrating CD8+ T cells with high CCR2 expression in combination with anti-PD-1.
In recent years, scientists have conducted a great deal of clinical studies on the treatment of tumor-infiltrating lymphocytes to observe and evaluate the therapeutic effects of TILs on various tumors such as melanoma, ovarian cancer, lung cancer, etc. Wherein, when the TILs are used in melanoma patients, the TILs are separated from the tumor microenvironment, and the IL-2 is stimulated and amplified in vitro and has good tumor killing effect after being infused back into the patients. TILs treatment has also been shown to be effective in the treatment of non-small cell lung cancer, osteosarcoma and ovarian cancer.
Although TILs treatment has proven effective in solid tumors, clinical applications face challenges and there is a need to provide a solution strategy.
Difficulty in isolating and purifying TILs
Not all tumor tissue microenvironments have a high number of TILs present. Moreover, TILs isolated from tumor tissues obtained by surgery have a large number of tumor cells, some tumor cells cannot die along with the culture time in purification culture, and the excessive tumor cells easily cause exhaustion and death of TILs, so that the TILs are failed to be obtained. Furthermore, TILs comprise a number of heterogeneous subsets, CD4+ T, CD + T, effector and regulatory cells, etc. In order to ensure a better therapeutic effect in the later treatment, it is important to isolate TILs with an anti-tumor effect.
2. Large doses of IL-2 are typically used for expansion prior to reinfusion of TILs therapeutic products back into the tumor patient. The expansion can promote the differentiation of T cells and the change of phenotypes thereof, and influence the proliferation capacity and the durability of TILs in vivo after the reinfusion.
3. Adverse reactions of second generation TILs therapeutic products
Currently, the second generation of therapeutic products focus primarily on four areas: enhancing the ability of TILs to target and recognize tumors; enhancing the durability of the TILs in vivo; enhancing the tumor infiltration capacity of the TILs; enhancing the tumor killing efficacy of TILs. In TILs, a chimeric antigen receptor consisting of CD3 zeta and CD28 is constructed, but the in vitro treatment time is too long, so the in vivo reaction is low; retrovirus transfects IL-2 gene, but the treatment response is low, the toxicity is big; lentiviruses transfect CXCR2, but the effect is not clear.
Firstly, separating TILs, and purifying a main killing potential cell CD8+ T; the tumor cell pollution problem of purified cells is solved by adopting the combined use of various methods such as magnetic bead purification and the like; the TILs are directly transfected by CCR2 high-expression lentivirus without being stimulated by in vitro IL-2, and are injected beside tumors to act on tumors in a short distance, so that the chemotactic effect of the TILs is enhanced, the TILs are activated, and the anti-PD-1 treatment is combined, so that the anti-tumor treatment effect is obvious.
Through the above analysis, the problems and defects of the prior art are as follows:
1. the TILs are difficult to purify, and are easy to cause tumor cell pollution, so that T cells are exhausted and die in the in-vitro culture process;
2. adding IL-2 and the like for in vitro amplification to change the cell characteristics for a long time (4-6 weeks);
3. the use of cytokines is highly toxic;
4. because of the lack of research on chemokines, there is a need to develop new chemokines that have a role in chemotaxis and cell activation.
At present, no report is found on the relevant technical scheme of preparing the antitumor drug by combining tumor infiltrating CD8+ T cells and late anti-PD-1.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an application of tumor infiltrating cells in preparing antitumor drugs.
The invention is realized in such a way that the application of the tumor infiltrating cells in the preparation of the anti-tumor medicament is as follows: the application of tumor infiltration CD8+ T cells with high CCR2 expression combined with anti-PD-1 in preparing anti-tumor drugs is performed by autologous tumor side injection; wherein the tumor is mouse lung cancer and glioma.
The invention also aims to provide a method for verifying the application of the tumor infiltrating CD8+ T cells in preparing anti-tumor drugs, and the construction method of the animal model comprises the following steps:
step one, purifying tumor infiltrating T cells;
step two, transfecting lentiviruses and analyzing the activation and killing effects of CCR2 high-expression tumor infiltration CD8+ T cells on cells;
determining the interval time of the paraneoplastic injection of the CD8+ T cells and the subcutaneous injection of the anti-PD-1;
and step four, analyzing the effect difference of the combined treatment of the independent self-injection CD8+ T cells, the anti-PD-1 pretreated tumor infiltration CD8+ T cells, the CCR2 transfected tumor infiltration CD8+ T cells, the anti-PD-1 and the CCR2 transfected tumor infiltration CD8+ T cells.
Further, the purification of the tumor infiltrating T cells in the first step comprises: obtaining tumor tissues of a mouse, cutting the tumor tissues into 1mm by using scissors, cutting the tumor tissues by using a surgical blade, and preparing digestive juice; shaking the tissue fragments at 37 deg.C for 25min, and digesting for 2 times; passing through a 40 μm filter, centrifuging the filtrate at 4 deg.C and 300g for 5min, and discarding the supernatant; resuspending and separating the lymphocytes by using tissue diluent in the tumor infiltrating lymphocyte separation kit, washing for 2 times by using PBS, and resuspending; CD8+ T cells from the single Cell suspension were purified using the american whirlpool CD8+ T Cell Isolation Kit, at a ratio of magnetic beads to T cells: 10 μ L of: 10 7 (ii) a The purified CD8+ T cells were cultured in RPMI1640 medium and purified again every 3h using differential digestion anchorage, with 2 consecutive purifications.
Further, the formula of the digestive juice is as follows: the lung cancer is that the 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; the glioma is RPMI1640 culture medium containing 0.05mg/mL type II collagenase and 0.01mg/mL DNase I.
Further, the RPMI1640 culture solution contains 10% FBS and 1%P/S.
Further, the lentivirus transfection and analysis of the T cell activation and killing effects of CCR 2-highly expressed tumor infiltrating CD8+ T cells in the step two comprise:
transfecting CD8+ T cells by lentiviruses, and comparing the empty vector lentivirus transfection control group, wherein the CCR2 high-expression lentivirus transfection group has high expression of early activation molecules CD69 by tumor infiltration CD8+ T cells; 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 high-expression CCR2 on tumor infiltrating CD8+ T cells are verified.
Further, the determination of the interval between the peritumoral injection of CD8+ T cells in combination with the subcutaneous injection of anti-PD-1 in step three comprises:
CD8+ T cells with high expression of CCR2 are injected beside the tumor, and the tumor size is observed every day; taking the gradual reduction and rebound of the tumor as a time node, quickly injecting the anti-PD-1, and continuously observing until the tumor becomes small or disappears.
Further, the differences in the effect of the combined treatment of autologous injected CD8+ T cells alone, anti-PD-1 pretreated tumor-infiltrating CD8+ T cells, CCR 2-transfected tumor-infiltrating CD8+ T cells, anti-PD-1 and CCR 2-transfected tumor-infiltrating CD8+ T cells in step four included:
CD8+ T cells transfected with CCR2 are injected in an autologous manner, the tumor becomes small after 2 to 3 days, and the tumor rebounds on the 5 th day; anti-PD-1 and transfection CCR2 tumors infiltrate CD8+ T cells for combined treatment, the rebound tendency is inhibited, and the tumors continue to diminish until disappear; tumors in the other two groups became smaller but rebound.
Further, the construction method of the animal model further comprises the following steps: tumor-infiltrating CD8+ T cells with both chemotactic and activation killing were used with anti-PD-1.
By combining the technical scheme and the technical problem to be solved, the technical scheme to be protected by the invention has the advantages and positive effects that:
the purification of the tumor infiltrating T cells provided by the invention is up to 99%; activation and killing effects of CCR2 high-expression tumor infiltration CD8+ T cells on T cells; interval time between peritumoral injection of CD8+ T cells in combination with subcutaneous injection of anti-PD-1; differences in the combined therapeutic effects of untreated groups, tumor-infiltrating CD8+ T cell reinfusion, anti-PD-1 pretreated tumor-infiltrating CD8+ T cell reinfusion, CCR 2-transfected tumor-infiltrating CD8+ T cell and anti-PD-1.
The invention verifies the treatment effect of autologous tumor-side injection CCR2 high-expression tumor infiltration CD8+ T cells combined with anti-PD-1 on mouse glioma and lung cancer, has reasonable and effective immunotherapy effect, can be used as an auxiliary method combined with clinical traditional chemoradiotherapy, and is expected to obtain good curative effect.
The present invention is directed to clinical transformation. If the invention is used for clinical experiments and transformation, key technical indexes and optimized schemes are provided for TILs treatment which is one of the most potential cell treatment types. The invention has good treatment effect on lung adenocarcinoma and glioma verification, is possibly suitable for other tumors in the future, and provides ideas and guidance for development of multi-tumor universal medicaments. The invention can be used for combining anti-PD-1 treatment, and can also be used for combining other chemoradiotherapy and adjuvant treatment, thereby providing a selectable treatment scheme for tumor treatment.
At present, the TILs treatment technology is mature day by day, but the problems of difficult separation and purification, in-vivo durability, high toxicity, long in-vitro operation time, change of biological properties and the like are faced with urgent need to be solved. The research separates main anti-tumor TILs CD8+ T cells as therapeutic drugs, adopts a plurality of methods for combined purification, eliminates the tumor cell pollution, has the purification rate as high as 99 percent, perfects the separation and purification technology at home and abroad, and fills up the technical blank in the industry at home and abroad. After the CCR2 high-expression lentivirus with high transfection suspension cell property is adopted in vitro, firstly, the expression of a cell activation marker molecule is detected, the cell activation marker molecule and tumor cells are co-cultured, and the killing effect of the cell activation marker molecule is detected and verified; after detection, CD8+ T cells of CCR2 high-expression lentivirus are directly injected beside tumors without being amplified by a stimulant, so that chemotaxis of the CD8+ T cells to tumor tissues is facilitated, the change of characters caused by in-vitro long-time stimulant culture is avoided, at present, CCR2 is mostly focused on macrophage research, related treatment research of CCR2 high-expression CD8+ T is not available, and the technical blank in the domestic and foreign industries is filled. The late stage of CD8+ T with high CCR2 expression injection is treated by anti-PD-1 instead of simultaneously treated by anti-PD-1, so that the adverse immune reactions such as over-strong cytokine storm and the like caused by simultaneously and jointly using CD8+ T with high CCR2 expression 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 needed to be used 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 it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of the treatment of tumor infiltrating CD8+ T lymphocyte extraction and reinfusion animals provided by embodiments of the present invention;
FIG. 2 is a flow chart of the treatment of tumor infiltrating CD8+ T lymphocyte extraction and reinfusion animals provided by the present invention;
FIG. 3 is a schematic diagram of the detection of the purity of tumor-infiltrating CD8+ T-cell after magnetic bead sorting, according to an embodiment of the present invention;
FIG. 4 (a) is a schematic representation of CCR2 expression following transfection of tumor-infiltrating CD8+ T cells by a CCR2 lentivirus as provided by the examples of the present invention; FIG. 4 (b) is a statistical plot of CCR2 expression following transfection of tumors with CD8+ T cells by CCR2 lentiviruses as provided by the examples of the present invention; FIG. 4 (c) is a schematic and statistical graph of the expression of the early activation molecule CD69 after CCR2 lentivirus transfection of tumor infiltrating CD8+ T cells provided by the present invention;
FIG. 4 (d) is a statistical plot of early activation molecule CD69 expression following CCR2 lentivirus transfection of tumor infiltrating CD8+ T cells provided by the examples of the present invention; FIG. 4 (e) is a schematic representation of the expression of the killer marker molecule CD107a after CCR2 lentivirus transfection of tumor infiltrating CD8+ T cells provided by the present examples; FIG. 4 (f) is a statistical plot of the expression of the killer marker molecule CD107a after CCR2 lentivirus transfection tumor infiltration of CD8+ T cells provided by the examples of the present invention.
FIG. 5 (a) is a schematic diagram of flow cytometry detection of the expression of the early activation molecule CD69 of CD8+ T cells after in vitro co-culture of glioma cells and tumor-infiltrating CD8+ T cells provided by the embodiments of the present invention; FIG. 5 (b) is a statistical graph of flow cytometry detection of the expression of the early activation molecule CD69 of CD8+ T cells following in vitro co-culture of glioma cells and tumor-infiltrating CD8+ T cells as provided by the examples of the present invention; FIG. 5 (c) is a schematic representation of flow cytometry detection of the expression of the killer marker molecule CD107a of CD8+ T cells following in vitro co-culture of glioma cells and tumor-infiltrating CD8+ T cells as provided by the examples of the present invention; fig. 5 (d) is a statistical graph of flow cytometry detection of the expression of the killer marker molecule CD107a of CD8+ T cells after in vitro co-culture of glioma cells and tumor-infiltrating CD8+ T cells provided by the embodiments of the present invention.
FIG. 6 (a) is a schematic diagram showing the detection of the killer effector molecule interferon gamma and the granzyme B expression by flow cytometry after in vitro co-culturing of glioma cells and tumor-infiltrating CD8+ T cells provided in the examples of the present invention; FIG. 6 (B) is a statistical chart of the flow cytometry detection of the expression of killer effector molecules interferon gamma and granzyme B after in vitro co-culture of glioma cells and tumor-infiltrating CD8+ T cells provided in the embodiments of the present invention;
FIG. 7 (a) is a schematic diagram of flow cytometry detection of the expression of the early activation molecule CD69 of CD8+ T cells after in vitro co-culturing of lung adenocarcinoma cells and tumor-infiltrating CD8+ T cells provided by an embodiment of the present invention; FIG. 7 (b) is a statistical graph of flow cytometry detection of the expression of the early activating molecule CD69 of CD8+ T cells after in vitro co-culture of lung adenocarcinoma cells and tumor-infiltrating CD8+ T cells provided by an embodiment of the present invention; FIG. 7 (c) is a schematic diagram of flow cytometry detection of CD107a expression of a killer marker molecule of CD8+ T cells after in vitro co-culturing of lung adenocarcinoma cells and tumor-infiltrating CD8+ T cells provided by an embodiment of the present invention; FIG. 7 (d) is a statistical graph of the expression of the killer marker molecule CD107a of CD8+ T cells detected by flow cytometry after in vitro co-culturing lung adenocarcinoma cells and tumor-infiltrating CD8+ T cells provided by the embodiments of the present invention.
FIG. 8 (a) is a schematic diagram showing the detection of the expression of killer effector interferon gamma, granzyme B by flow cytometry after in vitro co-culturing lung adenocarcinoma cells and tumor-infiltrating CD8+ T cells provided in the examples of the present invention; FIG. 8 (B) is a statistical chart of the expression of killer effector interferon gamma and granzyme B detected by flow cytometry after in vitro co-culturing lung adenocarcinoma cells and tumor-infiltrating CD8+ T cells provided by the embodiment of the present invention;
FIG. 9 (a) is a schematic diagram showing the comparison of tumor volume sizes of a mouse model of glioma after autologous reinfusion among different treatment groups, a glioma untreated group control, a glioma null lentivirus-transfected CD8+ T cell control group B glioma, a glioma anti-PD-1 pretreated CD8+ T cell group C glioma, a glioma-transfected CCR2CD8+ T cell group D glioma, and a combination treatment group of glioma anti-PD-1 and transfected CCR2CD8+ T cell provided in the examples of the present invention; fig. 9 (B) is a schematic diagram of tumor volume size comparison between different treatment groups of a lung adenocarcinoma mouse model after autologous transfusion provided in the present invention, a lung adenocarcinoma untreated group control, a B lung adenocarcinoma empty lentivirus-transfected CD8+ T cell control group, a C lung adenocarcinoma anti-PD-1 pretreated CD8+ T cell group, a D lung adenocarcinoma-transfected CCR2CD8+ T cell group, an E lung adenocarcinoma anti-PD-1 and CCR2CD8+ T cell-transfected combination treatment group; FIG. 9 (c) is a statistical graph of tumor volume changes between different treatment groups in a glioma mouse model after autologous reinfusion provided by an embodiment of the invention; FIG. 9 (d) is a statistical graph of tumor volume changes between different treatment groups in a lung adenocarcinoma mouse model after autologous transfusion provided by an embodiment of the present invention; FIG. 9 (e) is a statistical graph of the body weight changes of mice in a glioma mouse model after autologous transfusion provided by an embodiment of the invention among different treatment groups; FIG. 9 (f) is a statistical graph of the change in body weight of mice in a lung adenocarcinoma mouse model after autologous transfusion provided by an example of the present invention among different treatment groups;
FIG. 10 is a confocal microscope showing that CD8+ T cells transfected with CCR2 migrate to a greater extent to the tumor site than CD8+ T cells transfected with empty vector, as measured by treatment following injection in autologous mice as provided by the examples of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.
Aiming at the problems in the prior art, the invention provides an application of tumor infiltrating cells in preparing antitumor drugs, and the invention is described in detail with reference to the accompanying drawings.
The embodiment of the invention provides an application of tumor infiltrating cells in preparing an anti-tumor medicament, which comprises the following steps: the application of the tumor infiltration CD8+ T cells with high CCR2 expression combined with anti-PD-1 in preparing anti-tumor drugs is performed by autologous tumor side injection.
The tumor provided by the embodiment of the invention is mouse glioma and lung cancer.
As shown in fig. 1, the schematic diagram of the treatment of tumor infiltrating CD8+ T lymphocytes extracted and returned to animals provided by the embodiment of the present invention includes the following steps:
s101: purifying the tumor infiltrating T cells;
s102: lentivirus transfection and analysis of the activation of CCR 2-highly expressed tumor infiltrating CD8+ T cells on cells;
s103: determining the interval time between the paraneoplastic injection of CD8+ T cells and the subcutaneous injection of anti-PD-1;
s104: the differences in the effect of the combination treatment of autologous injected CD8+ T cells, anti-PD-1 pretreated tumor-infiltrating CD8+ T cells, 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, the purification of tumor infiltrating T cells is performed; as shown in fig. 3, the purification rate of CD8+ T cells reached 99.32%.
In the present example, step S102, the activation of T cells by CCR 2-highly expressed tumor-infiltrating CD8+ T cells was analyzed. As shown in fig. 4 (a), 4 (b), 4 (c), 4 (d), CCR 2-highly expressed tumor infiltrating CD8+ T cells upregulate the expression of the early activation molecule CD69 of T cells. As shown in fig. 5 (a), fig. 5 (b), fig. 5 (c) and fig. 5 (d), glioma cells and tumor-infiltrating CD8+ T cells were co-cultured in vitro, and the expression of CD69 and CD107a was detected in the tumor-infiltrating CD8+ T cells, and the expression of CD69 and CD107a was higher in both the CD8+ TCCR2+ group, the CD8+ TCCR2+ group in combination with the anti-PD-1 group than in the control group (the CD8+ T group or the anti-PD-1 pretreated tumor-infiltrating CD8+ T cell group). As shown in fig. 6 (a) and fig. 6 (B), glioma cells and tumor-infiltrating CD8+ T cells were co-cultured in vitro, and the expression of killer interferon gamma and granzyme B of the tumor-infiltrating CD8+ T cells was detected, and the expression of CD69 and CD107a of the CD8+ TCCR2+ group, and the anti-PD-1 group were higher than those of the control group (CD 8+ T group or anti-PD-1 pretreated tumor-infiltrating CD8+ T cell group). As shown in fig. 7 (a), fig. 7 (b), fig. 7 (c) and fig. 7 (d), the lung cancer cells and the tumor infiltrating CD8+ T cells were co-cultured in vitro, and the expression of CD69 and CD107a was detected in the tumor infiltrating CD8+ T cells, and the expression of CD69 and CD107a was higher in the CD8+ TCCR2+ group, the CD8+ TCCR2+ group and the anti-PD-1 group, respectively, than in the control group (the CD8+ T group or the anti-PD-1 pretreated tumor infiltrating CD8+ T cell group). As shown in fig. 8 (a) and fig. 8 (B), lung cancer cells and tumor-infiltrating CD8+ T cells were co-cultured in vitro, and the expression of killer interferon gamma and granzyme B was detected in the tumor-infiltrating CD8+ T cells, and the expression of CD69 and CD107a was higher in both the CD8+ TCCR2+ group, and the anti-PD-1 group, than in the control group (CD 8+ T group or anti-PD-1 pretreated tumor-infiltrating CD8+ T cell group).
In an embodiment of the invention, step S103, the interval between the paraneoplastic injection of CD8+ T cells in combination with the subcutaneous injection of anti-PD-1 is determined; after measuring the tumor volume, the tumor rebounds to grow up from 5 days after the TIL cells are reinfused, so that the anti-PD-1 is reinfused on 4 days, the tumor becomes small, and the tumor is taken out for examination after 4 days.
In the present example, step S104, the effect difference of the combined treatment groups of autologous injected CD8+ T cells alone, anti-PD-1 pretreated tumor-infiltrating CD8+ T cells, CCR 2-transfected tumor-infiltrating CD8+ T cells, anti-PD-1 and CCR 2-transfected tumor-infiltrating CD8+ T cells was analyzed. In vivo mouse experiments, the anti-tumor effect of the group using CCR2 high-expression CD8+ T cells and combined injection anti-PD-1 is the best, and the observation under a confocal microscope confirms that CCR2 high-expression TILs tend to be positioned in tumor tissues.
The purification of the tumor infiltrating T cells in step S101 provided by the embodiment of the present invention includes:
the tumor tissue of the mouse is taken out, cut into 1mm by scissors and cut by a surgical blade. Meanwhile, digestive juice is prepared, and the formula is as follows: the RPMI1640 medium contained collagenase I (0.05 mg/mL), collagenase IV (0.05 mg/mL), hyaluronidase (0.05 mg/mL) and DNase I (0.01 mg/mL). Shaking the tissue fragment at 37 deg.C for 25min, and digesting for 2 times; the filtrate was centrifuged through a 40 μm filter at 300g for 5min at 4 ℃ and the supernatant was discarded. Lymphocytes were resuspended and isolated using tissue dilutions from a tumor infiltrating lymphocyte isolation kit (Solarbio). PBS washing 2 times, heavy suspension. CD8+ T cells from single Cell suspensions were purified using the American whirlpool CD8+ T Cell Isolation Kit. The purified CD8+ T cells were cultured in RPMI1640 medium containing 10% FBS, and the CD8+ T cells were purified again at 3h intervals using differential digestion anchorage and were purified 2 times in succession. Inoculating and purifying CD8+ T cells, infecting lentivirus with high CCR2 expression, centrifuging and concentrating after 72h, injecting glioma solid beside tumors, injecting anti-PD-1 antibody on the 4 th day after inoculating and purifying CD8+ T cells, and eliminating tumors.
The step S102 provided by the embodiment of the invention for analyzing the activation and killing effects of CCR2 high-expression tumor infiltration CD8+ T cells on T cells comprises the following steps:
after the CD8+ T cells and the tumor cells are co-cultured, compared with an empty lentivirus vector transfection control group, the CCR2 high-expression lentivirus transfected tumor infiltration CD8+ T cell group high-expression CD69 early-stage activation molecules are increased in granzyme B and interferon gamma expression, and the activation and killing effects of the tumor infiltration CD8+ T cells on the T cells are determined.
The determination of the interval time between the paraneoplastic injection of CD8+ T cells and the subcutaneous injection of anti-PD-1 in the analysis in step S103 provided in the embodiments of the present invention includes:
CD8+ T cells with high expression of CCR2 are injected beside the tumor, and the tumor size is observed every day; taking the gradual decrease and rebound of the tumor as a time node, quickly injecting the anti-PD-1, and continuously observing until the tumor becomes small or disappears.
The effect differences of the combined treatment groups for analyzing the independent autologous injection of CD8+ T cells, the tumor infiltration of CD8+ T cells pretreated by anti-PD-1, the tumor infiltration of CD8+ T cells transfected with CCR2, the anti-PD-1 and the tumor infiltration of CD8+ T cells transfected with CCR2 provided in the step S104 of the present invention include:
independently and self-injecting CD8+ T cells for transfecting CCR2, wherein the tumor becomes small after 2-3 days, and the trend of the tumor becoming small rebounds from the 5 th day; CD8+ T cells transfected with CCR2 and anti-PD-1 are injected in a combined mode, the rebound tendency is inhibited, and the cells continue to become small until the rebound tendency disappears; CD8+ T cells were injected alone, anti-PD-1 pretreated tumors infiltrated CD8+ T cells, and tumors became small but rebound.
The construction method of the animal model provided by the embodiment of the invention further comprises the following steps: anti-PD-1,CCR2 high-expression CD8+ T cells are used.
TILs therapy has been successfully applied to metastatic melanoma and other solid tumor patients. TILs therapy has its unique advantages over other immune cell therapies, such as CAR-T and TCR-T. TILs, which are composed of T cells with multiple TCR clones, respond more efficiently to tumor heterogeneity, and can act not only directly on common tumor antigens, but also on specific tumor antigens. TILs usually contain a large number of effector memory T cells and are more potent in antitumor effect after activation. Furthermore, TIL is derived from the patient himself without genetic modification, which means that the method is of low toxicity. 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 γ δ T cells, which typically exert their anti-tumor activity by secreting Interferon (IFN) and Tumor Necrosis Factor (TNF), but which are independent of tumor-associated antigens. Secondly, despite great advances in the strategy for TILs selection, the widespread use of TILs therapy in a variety of cancers remains challenging due to the barriers to immunosuppression of the tumor microenvironment, and no TILs products are currently on the market. Since tumor cells often exhibit different types of genetic mutations that generate multiple neoantigens, it is difficult to design universal CAR-TILs to eliminate cancer cells. At the same time, immunosuppressive tumor microenvironments may induce the depletion of infiltrating cytotoxic T cells, leading to a decrease in the 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 have a short survival time in vivo. More importantly, in order to improve the survival rate and tumor homing capacity of patients re-infused with TILs, modification techniques have been explored in which various chemotactic cytokines are used to home TILs, but without significant efficacy. The high expression CCR2 TILs combined with an immune checkpoint inhibitor anti-PD-1 can overcome the problem of homing of the TILs and overcome the problem of short-term exhaustion of the TILs in vivo. However, the present invention does not employ the currently common expansion of IL-2, anti-CD 3 and feeder cells prior to injection, and has the disadvantage of relatively small numbers, the advantage of rapid processing (4-6 weeks for general expansion and 3-4 days for the present invention), and the advantage of maintaining the cell characteristics. The tumor site is directly reached by adopting the paraneoplastic injection, so that the cell consumption after the 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, collection and purification after T cells have been shed. In the research, tissue digestion is directly carried out, mononuclear cells are extracted from ficoll separation solution, CD8+ T cells are separated and purified by magnetic beads, and the cells are purified again by various methods such as differential adherence and the like. The high-purity CD8+ T cells can be extracted in a short time.
The embodiment of the invention has some positive effects in the process of research and development or use, and indeed has great advantages compared with the prior art, and the following contents are described by combining data, charts and the like in the test process.
Example (b): autologous tumor-side injection CCR2 high-expression tumor infiltration CD8+ T cell combined late anti-PD-1 therapeutic effect on mouse glioma and lung cancer
The purification of the tumor infiltrating T cells provided by the embodiment of the invention is up to 99 percent; activation of T cells by CCR 2-highly expressed tumor infiltrating CD8+ T cells; interval time between peritumoral injection of CD8+ T cells in combination with subcutaneous injection of anti-PD-1; the effect of combined treatment of autologous injected CD8+ T cells alone, 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 was different.
1. Purification of tumor infiltrating T cells, up to 99%.
The tumor tissue of the mouse is taken out, cut into 1mm by scissors and cut by a surgical blade. Simultaneously preparing digestive juice, wherein the formula is as follows: the RPMI1640 medium contained collagenase I (0.05 mg/mL), collagenase IV (0.05 mg/mL), hyaluronidase (0.05 mg/mL) and DNase I (0.01 mg/mL). The tissue fragments were digested 2 times by shaking on a shaker at 37 ℃ for 25min, then passed through a 40 μm filter, the filtrate was centrifuged at 300g for 5min at 4 ℃ and the supernatant was discarded. Lymphocytes were resuspended and isolated using tissue dilutions from a tumor infiltrating lymphocyte isolation kit (Solarbio). PBS washing 2 times, heavy suspension. CD8+ T cells from the single Cell suspension were purified using the American whirlpool CD8+ T Cell Isolation Kit. The ratio of magnetic beads to total cells was: 10 μ L of: 10 7 . The purified CD8+ T cells were cultured with RPMI1640 medium (containing 10% FBS), and the CD8+ T cells were purified again every 3h using differential digestion anchorage, and were purified 2 times consecutively.
Activation and killing effects of tumor infiltrating CD8+ T cells with high CCR2 expression on T cells.
After co-culturing the CD8+ T cells and the tumor cells, compared with an empty lentivirus vector transfection control group, the expression of CD69, CD107a, granzyme B and interferon gamma of a CCR2 high-expression lentivirus transfected tumor infiltration CD8+ T cell group is increased.
3. Interval of peritumoral injection of CD8+ T cells combined with subcutaneous injection of anti-PD-1.
Injecting CD8+ T cells by tumor side, observing the size of the tumor for about 4-5 days, taking the time point as the rebound of the tumor gradually becomes smaller, quickly injecting anti-PD-1, and continuing observation until the tumor disappears.
If the CD8+ T cells highly expressing CCR2 and the anti-PD-1 are used at the same time, because the CD8+ T cells highly expressing CCR2 which are injected at the beginning have strong immunocompetence and killing capacity, and the invention also monitors that PD-1 of the cells is low at the moment, the anti-PD-1 has the effect of not blocking the PD-1 of the CD8+ T cells highly expressing CCR2 and is easy to cause over-strong immune response and adverse reaction. Therefore, the invention respectively uses CD8+ T cells with high expression of CCR2 and anti-PD-1 for treatment, and experiments show that the interval is selected to be about 5 days.
4. Differences in therapeutic effect of combination treatments of autologous injection of CD8+ T cells, anti-PD-1 pretreated tumor-infiltrating CD8+ T cells, CCR 2-transfected tumor-infiltrating CD8+ T cells, anti-PD-1 and CCR 2-transfected tumor-infiltrating CD8+ T cells.
Independent autologous injection of CD8+ T cells transfected with CCR2 results in a smaller tumor after 2-3 days and a rebound trend of smaller tumors at day 5. Combined injection of CD8+ T cells and anti-PD-1, rebound tendency was suppressed and continued to diminish until they disappeared. The other two groups of tumors became smaller but rebound.
5. By combining the characteristics, the scheme ensures reasonable and effective immunotherapy effect, and can be used as a scheme combined with other clinical immune adjuvant therapies, and expected to obtain good curative effect.
Combination of TIL therapy and anti-PD-1/PD-L1 antibody therapy showed preliminary favorable results in recent trials. In cancer patients, the immune checkpoint receptors CTLA-4 and PD-1 on effector T cells are up-regulated in levels resulting in suppression of T cell function, based on which the use of anti-CTLA-4 and anti-PD-1 can block the inhibitory signal. In addition, in vivo and in vitro experiments show that after long-term contact with tumor antigens, CD8+ T cells are apoptotic or enter 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, an immune response is started by blocking an inhibitory signal through an immune checkpoint inhibitor. These existing mechanisms provide a theoretical basis for combining TILs and immune checkpoint inhibitors. At present, the effect of TILs in combination with anti-PD-1 therapy as first-line therapy is still in clinical trials. Recent studies found that Dendritic Cells (DC) express high levels of PD-L1 in addition to tumor cells, attenuate T cell activation, and inhibit anti-tumor activity, which also provides a theoretical basis for TILs in combination with immune checkpoint inhibitors. Based on the above, the anti-PD-1 inhibitor for treating combined immune checkpoint by using the TILs overcomes the limitation of single medicine, is expected to improve the efficiency of combined medicine, and is expected to obtain good curative effect.
The BRAF gene plays an important role in cell growth and differentiation. In certain cancers, BRAF mutations are the most common mutations that lead to overactivation of the MAPK pathway, promoting cell proliferation. Activated BRAF mutations can induce immune escape mechanisms, evading T cell immune surveillance. Research shows that the BRAF inhibitor Wei Luo Feini can reduce related immunosuppressive signals, promote lymphocyte infiltration, reduce immunosuppressive cells and enhance melanoma antigen presentation. However, the clinical response to BRAF inhibitors is often temporally limited. Clinical trials found that patients with metastatic melanoma who received a combination of TIL, IL-2 and wei Luo Feini had a marked improvement or complete response. Based on the above, the combined BRAF inhibitor for TILs treatment provided by the invention can overcome the limitation of single medicine, and is expected to obtain 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 treatment in combination with TILs. Combinations of TILs therapy and oncolytic viruses are also under investigation. The virus can be used for resisting tumor immunosuppression by producing cytokines which promote the anti-tumor effect of TILs. Based on this, in the future, the TILs of the present invention can be combined with various immunotherapies, and a good therapeutic effect is expected to be obtained.
The above description is only for the purpose of illustrating the embodiments of the present invention, and the scope of the present invention should not be limited thereto, and any modifications, equivalents and improvements made by those skilled in the art within the technical scope of the present invention as disclosed in the present invention should be covered by the scope of the present invention.
Claims (10)
1. The application of the tumor infiltrating cells in preparing the antitumor drugs is characterized in that the tumor infiltrating CD8+ T cells with high CCR2 expression are injected by the tumor side of an autologous tumor to be combined with anti-PD-1 in preparing the antitumor drugs.
2. The use of the tumor infiltrating cell of claim 1 in the preparation of an anti-tumor medicament, wherein the tumor is mouse glioma or lung cancer.
3. A method for constructing an animal model for verifying the effect of the use according to any one of claims 1 to 2, comprising:
step one, purifying tumor infiltrating T cells;
step two, lentivirus transfection and analysis of the activation effect of CCR2 high-expression tumor infiltration CD8+ T cells on cells;
determining the interval time of the paraneoplastic injection of the CD8+ T cells and the subcutaneous injection of the anti-PD-1;
and step four, analyzing the combined treatment effect difference of the CD8+ T cells, the anti-PD-1 pretreated tumor infiltration CD8+ T cells, the CCR2 transfected tumor infiltration CD8+ T cells, the anti-PD-1 and CCR2 transfected tumor infiltration CD8+ T cells.
4. The method of constructing an animal model according to claim 3, wherein the purification of tumor infiltrating T cells in step one comprises:
obtaining tumor tissues of a mouse, cutting the tumor tissues into 1mm by using scissors, cutting the tumor tissues by using a surgical blade, and preparing digestive juice; shaking the tissue fragments at 37 deg.C for 25min, and digesting for 2 times; passing through a 40 μm filter, centrifuging the filtrate at 4 deg.C and 300g for 5min, and removing the supernatant; resuspending and separating the lymphocytes by using tissue diluent in the tumor infiltrating lymphocyte separation kit, washing for 2 times by using PBS, and resuspending; CD8+ T cells from the single Cell suspension were purified using the american whirlpool CD8+ T Cell Isolation Kit, at a ratio of magnetic beads to T cells: 10 μ L of: 10 7 (ii) a The purified CD8+ T cells were cultured in RPMI1640 medium and used at 3h intervalsCD8+ T cells were purified again by rapid digestion adherent, 2 consecutive purifications.
5. The method for constructing an animal model according to claim 4, wherein the digestive fluid is formulated as: the 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; the glioma is RPMI1640 culture medium containing 0.05mg/mL type II collagenase and 0.01mg/mL DNase I.
6. The method of constructing an animal model according to claim 4, wherein the RPMI1640 medium contains 10% FBS and 1%P/S.
7. The method for constructing an animal model according to claim 3, wherein the step two of lentivirus transfection and analysis of the effect of tumor-infiltrating CD8+ T cells with high CCR2 expression on T cells comprises:
transfecting CD8+ T by lentivirus, and comparing with an empty vector lentivirus transfection control group, wherein the CCR2 high-expression lentivirus transfection group has tumor infiltration CD8+ T cells high-expression 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 high-expression CCR2 on tumor infiltration CD8+ T cells are confirmed.
8. The method of constructing an animal model according to claim 3, wherein the determination of the time interval between the paraneoplastic injection of CD8+ T cells in combination with the subcutaneous injection of anti-PD-1 in step three comprises:
injecting CD8+ T cells with high expression of CCR2 in a tumor side way, and observing the size of the tumor every day; taking the gradual decrease and rebound of the tumor as a time node, quickly injecting the anti-PD-1, and continuously observing until the tumor becomes small or disappears.
9. The method for constructing an animal model according to claim 3, wherein the step four of analyzing the differences in the combined treatment effects of CD8+ T cells, anti-PD-1 pretreated tumor-infiltrating CD8+ T cells, CCR 2-transfected tumor-infiltrating CD8+ T cells, anti-PD-1 and CCR 2-transfected tumor-infiltrating CD8+ T cells comprises:
independently and self-injecting CCR2 high expression CD8+ T cells, wherein the tumor becomes small after 2 to 3 days, and the tumor rebounds on the 5 th day; the CCR2 high-expression CD8+ T cells and anti-PD-1 are used in a combined mode, so that the rebound tendency is inhibited, and the tumor is continuously reduced or disappeared; the other two groups of tumors became smaller but rebound.
10. The method of constructing an animal model according to claim 3, further comprising: tumor-infiltrating CD8+ T cells with both chemotactic and activation killing were used with anti-PD-1.
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