WO2023115459A1 - Tumor antigen/mhc-i complex, preparation method therefor and application thereof - Google Patents

Abstract

The present invention relates to a tumor antigen/MHC-I complex, a preparation method therefor and an application thereof. According to the present invention, on the basis of the design principle of the shotgun sequencing method, a complex of a tumor antigen expressed by a tumor cell, and MHC-I is subjected to undifferentiated pull-down at one time from a tumor cell lysate, an immunogenic tumor antigen does not need to be identified in advance, the application range is wider, the steps are simpler, and the cost is lower. The tumor antigen/MHC-I complex provided by the present invention can specifically enrich CD8+ T cells in a tumor-infiltrating lymphocyte, can induce and stimulate the activation and proliferation of the tumor-infiltrating lymphocyte and the CD8+ T cells therein, solves the problem of accurate targeting of tumor antigen-specific T cells, enhances the killing function of the tumor-infiltrating lymphocyte on tumor cells, and can be used for treating tumor diseases and optimizing the ACT method.

Description

A kind of tumor antigen/MHC-I complex and its preparation method and application technical field

The present invention relates to the field of immunology, in particular to a tumor antigen/MHC-I complex and its preparation method, enriching CD8 ⁺ T cells in vitro, stimulating the activation and proliferation of tumor-infiltrating lymphocytes from the same source, and/or preparing and treating tumors It also relates to a method for enriching CD8 ⁺ T cells in vitro and the obtained CD8 ⁺ T cells, a method for stimulating the activation and proliferation of tumor-infiltrating lymphocytes from the same source in vitro and the obtained tumor-infiltrating lymphocytes , a corresponding pharmaceutical composition, and a method for treating tumors.

Background technique

The relationship between the immune system and tumors is intricate: on the one hand, the immune system can kill or eliminate tumor cells through a variety of immune effector mechanisms; on the other hand, tumor cells can also resist or evade the immune system’s killing and clear. In the early stage of tumors, the immune system tries to eliminate tumor cells that are considered "exogenous". Immunosuppressive state, escaping the body's immune recognition. Therefore, how to induce the anti-tumor immune response of T lymphocytes through tumor antigens expressed by tumor cells is the key to the research of tumor immunotherapy.

The so-called tumor antigen refers to the neoantigen (neoantigen) that appears in the process of cell carcinogenesis or the antigen substance abnormally expressed or overexpressed by tumor cells. T cells can distinguish tumor cells from normal cells by specifically recognizing unique tumor antigens expressed by tumor cells to trigger an immune response. Although tumor cells express tumor antigens, the immunogenicity of most tumor cells is relatively weak, and it is difficult to induce the body to produce specific immune responses against these antigens. At the same time, tumor cells reduce the ability of MHC to present tumor antigens by down-regulating or shutting down the expression of major histocompatibility complex (MHC), or directly down-regulating the production of tumor antigens, resulting in the proper immunity against tumor antigens in the body. The response lacks specificity or is insufficient to eliminate tumor cells, resulting in the immune response generated by tumor patients often not being able to effectively eliminate tumor cells.

Adoptive Cell Therapy (ACT) is a method of adoptively infusing autologous or allogeneic immune cells (mainly autologous cells) that have been expanded and activated in vitro into tumor patients to enhance the patient's immune function and achieve therapeutic purposes. . In the process of adoptive T cell therapy, T lymphocytes are the ultimate executors of killing tumor cells, among which, cytotoxic T lymphocytes (CTLs) are the main effector cells of anti-tumor immunity, mediating specific anti-tumor immune response. In the body, this process depends on intercellular contact. In the process of antigen presentation, tumor antigens can be presented on MHC class I molecules for T cells to recognize, induce the activation of CD8 ⁺ cytotoxic T cells, and initiate adaptive immune responses. .

Human MHC, also known as human leukocyte antigen (HLA) gene complex, participates in adaptive immune response as an antigen-presenting molecule, and determines the MHC restriction of antigen recognition by T cells. CD8 ⁺ T cells in CTL cells can recognize endogenous antigen peptides presented by MHC class I molecules and be activated, target tumor cells through a rapid and precise control process, and directly kill tumor cells. However, due to the absence or reduced expression of MHC class I molecules on the surface of mutant cells or tumor cells, tumor cells cannot effectively present tumor antigens, so that they cannot effectively activate specific CD8 ⁺ CTLs to kill tumor cells, resulting in tumor immune escape.

The specificity of ACT therapy depends largely on the recognition of specific tumor antigens, especially neoantigens. Accumulating evidence suggests that the success of ACT therapy may be attributed to neoantigen-specific T cells, a strategy that has been shown to successfully induce tumor regression and even complete remission in patients with metastatic cancer. Therefore, ACT employing T cells specific for these neoantigens would be very promising. Among various ACTs, tumor infiltrating lymphocytes (Tumor Infiltrating Lymphocytes, TILs) have been proven to be a very successful approach in tumor therapy. TIL isolated from tumor tissue contains a variety of tumor-specific T cells with different receptors. These tumor-specific T cells mainly recognize neoantigens caused by gene mutations in cancer cells and can respond to a variety of tumor antigens. Addressing tumor heterogeneity. More and more research attention has been shifted to the identification and selection of neoantigen-specific T cells. This "precise targeting" strategy poses a huge challenge to the identification and isolation of neoantigen-specific T cells. How to effectively identify and isolate tumor antigen-specific T cells from TILs and effectively recognize neoantigens caused by cancer cell gene mutations largely determines the ability of TIL cells to target and kill tumor cells.

Researchers have long attempted to isolate tumor antigen-specific T cell subsets from infused TILs and have developed a number of methods for this purpose. Currently known strategies for isolating tumor antigen-specific T cells include: 1) through whole-exome sequencing, tumor cell cDNA library sequencing, and bioinformatics analysis combined with HLA class I molecular typing analysis to speculate on the tumor-specific antigen sequence, Synthesize predicted antigen peptide pools to screen tumor antigen-specific T cells; 2) Through whole-exome sequencing, screen TandemMinigene (TMG), which encodes tumor mutations in patients, to identify tumor neoantigen-specific TILs from therapeutic TILs. 3) Use surface markers or T cell receptor (TCR) sequence analysis and big data analysis methods to predict tumor antigen-specific T cells, etc. However, these attempts have shortcomings to varying degrees. Some methods require the establishment of autologous tumor cell lines, so they need to face the challenge of establishing autologous tumor cell lines; if a single cell surface marker is used, some tumor antigen-specific T Cells will be missed; especially the currently most widely used method of sequencing and bioinformatics analysis requires more tumor tissue, complex equipment, months of time and expensive expenses, and there are also inaccuracies that may be introduced by computer algorithms At the same time, for most patients who have already developed tumor metastasis, this time frame is unacceptable, coupled with the huge cost, the application of these methods faces great limitations.

Furthermore, artificial antigen-presenting cells (aAPCs) are currently being used as a possible alternative in the production of cancer immunotherapies. The currently employed aAPC is an approach limited to identified and selected peptide-MHC complexes and cannot be applied in the case of unknown tumor neoantigens. Some researchers also combine known, genetically engineered peptide-MHC complexes on magnetic beads to enrich/stimulate T cells, but this process requires pre-identification of immunogenic tumor antigens and synthesis of corresponding patient-specific MHC in vitro -Class I molecular structure also limits the wide application of this method in clinical tumor immunotherapy.

Contents of the invention

The invention provides a tumor antigen/MHC-I complex derived from tumor cells and a preparation method thereof, so as to capture specific tumor antigens in tumor cells to the greatest extent, and can be used for enriching tumor antigen-specific CD8 ⁺ T cells, stimulating Activation, proliferation and tumor killing function of tumor infiltrating lymphocytes from the same source to solve the problem of "precise targeting" of tumor antigen-specific T cells.

The invention provides a method for preparing a tumor antigen/MHC-I complex, which comprises:

Lying the tumor cells to obtain a tumor cell lysate, the tumor cell lysate contains a tumor antigen/MHC-I complex;

The carrier loaded with the antibody is mixed with the tumor cell lysate to obtain the tumor antigen/MHC-I complex; wherein the tumor antigen/MHC-I complex is loaded on the carrier, and the antibody Bind specifically to MHC-I in the tumor antigen/MHC-I complex.

In some embodiments, the method for preparing the tumor antigen/MHC-I complex further comprises the step of isolating the tumor antigen/MHC-I complex from the carrier loaded with the tumor antigen/MHC-I complex .

In some embodiments, the tumor cells are lysed with a cell lysate.

In some preferred embodiments, the cell lysate includes 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt ([(3-Cholanidopropyl)dimethylammonio]-1-propanesulfonate, hereinafter referred to as CHAPS ), Tris buffer and NaCl.

In some embodiments, the antibody is an MHC-I beta chain antibody.

In some preferred embodiments, the antibody is labeled with a first label selected from biotin and/or a first chemical conjugate.

In some embodiments, the carrier is magnetic beads and/or quantum dot fluorescent microspheres.

In some preferred embodiments, the carrier is labeled with a second label selected from avidin and/or a second chemical conjugate.

The present invention also provides a tumor antigen/MHC-I complex, which is prepared by the preparation method of the tumor antigen/MHC-I complex provided in the present invention.

The present invention also provides the use of the tumor antigen/MHC-I complex in enriching tumor antigen-specific CD8 ⁺ T cells in vitro, stimulating the activation and proliferation of tumor-infiltrating lymphocytes from the same source, and/or preparing drugs for treating tumors .

The present invention also provides a method for enriching tumor antigen-specific CD8 ⁺ T cells in vitro, which includes:

The tumor antigen/MHC-I complex provided by the present invention is contacted with tumor infiltrating lymphocytes from the same source, and the tumor antigen-specific CD8 ⁺ T cells in the tumor infiltrating lymphocytes are in contact with the tumor antigen/MHC-I The complex binds to obtain a conjugate enriched with said tumor antigen-specific CD8 ⁺ T cells.

In some embodiments, the tumor antigen/MHC-I complex is combined with an antibody labeled with biotin and loaded together on a carrier labeled with avidin, and, in the tumor antigen/MHC-I complex The tumor infiltrating lymphocytes are supplemented with avidin prior to contacting them with tumor infiltrating lymphocytes from the same source.

In some embodiments, prior to contacting the tumor antigen/MHC-I complexes with tumor infiltrating lymphocytes from the same source, the tumor infiltrating lymphocytes are also supplemented with a tyrosine kinase inhibitor; preferably, the tyrosine kinase The amino acid kinase inhibitor is dasatinib.

In some preferred embodiments, the tyrosine kinase inhibitor is dasatinib.

In some embodiments, the method for enriching tumor antigen-specific CD8 ⁺ T cells in vitro further comprises the step of isolating the tumor antigen-specific CD8 ⁺ T cells from the conjugate.

The present invention also provides a tumor antigen-specific CD8 ⁺ T cell, which is prepared by the method for enriching tumor antigen-specific CD8 ⁺ T cells in vitro provided by the present invention.

The present invention also provides a method for stimulating the activation and proliferation of tumor-infiltrating lymphocytes from the same source in vitro, which includes:

The tumor antigen/MHC-I complex provided by the present invention is contacted with tumor infiltrating lymphocytes from the same source.

In some embodiments, in the step of contacting the tumor antigen/MHC-I complex provided by the present invention with tumor infiltrating lymphocytes from the same source, cytokines are also added.

In some preferred embodiments, the cytokine is selected from at least one of 4-1BB, IL-2, and IL-21.

The present invention also provides an activated tumor-infiltrating lymphocyte, which is prepared by stimulating the activation and proliferation of the tumor-infiltrating lymphocyte from the same source in vitro provided by the present invention.

The present invention also provides a pharmaceutical composition, the pharmaceutical composition includes an active ingredient and a pharmaceutically acceptable carrier, the active ingredient includes the tumor antigen-specific CD8 ⁺ T cells provided in the present invention and/or the Activated tumor infiltrating lymphocytes.

The present invention also provides a method for treating tumors, which includes:

contacting an effective amount of the tumor antigen-specific CD8 ⁺ T cells, the activated tumor infiltrating lymphocytes and/or the pharmaceutical composition with the tumor; wherein the tumor antigen-specific CD8 ⁺ T cells, The activated tumor infiltrating lymphocytes and the active ingredient in the pharmaceutical composition are derived from the tumor.

The present invention also provides a method for identifying tumor antigens, which includes:

The tumor antigen-specific CD8 ⁺ T cells provided by the present invention are subjected to TCR sequencing or protein mass spectrometry analysis to identify their sequences.

The present invention has following beneficial effect:

First of all, the preparation method of the tumor antigen/MHC-I complex provided by the present invention is based on the design principle of the shotgun method (Shot gun), and utilizes the characteristics of specific binding of the antibody to MHC-I to obtain a one-time free antigen from the tumor cell lysate. Differential capture (Pull-down) complexes of tumor antigens expressed by tumor cells and MHC-I. This method can capture the tumor antigen/MHC-I complex in the tumor cell lysate to the greatest extent, without the need to identify the immunogenic tumor antigen in advance, has a wider scope of application, simpler steps, and lower cost.

Secondly, the tumor antigen/MHC-I complex provided by the present invention can specifically enrich CD8 ⁺ T cells in tumor infiltrating lymphocytes, and can induce and stimulate the activation and proliferation of tumor infiltrating lymphocytes and CD8 ⁺ T cells in them, For example, increasing the proportion of stem cell-like tumor antigen-specific T cells, increasing the ability of CD8 ⁺ T cells to secrete IFN-γ, etc., solved the problem of "precise targeting" of tumor antigen-specific T cells, and enhanced the ability of tumor-infiltrating lymphocytes to The killing function of tumor cells has great practical significance for the treatment of tumor diseases and the optimization of ACT methods.

Description of drawings

Fig. 1 is a schematic flow diagram of the preparation method of the tumor antigen/MHC-I complex provided by the present invention based on the design principle of the shotgun method, and the use of the obtained tumor antigen/MHC-I complex;

Figure 2 is the Western blot detection result of the tumor antigen/MHC-I complex preparation method provided in Example 1 of the present invention for capturing the effect of the tumor antigen/MHC-I complex; wherein, 1 is a protein gradient; 2 is a tumor Cell lysate suspension; 3 is tumor antigen/MHC-I complex suspension;

Figure 3 is a schematic diagram of the proportion of CD8 ⁺ T cells after co-incubating the tumor antigen/MHC-I complex with TIL cells in Example 2 of the present invention (N=3; **** indicates P<0.0001); where, blank The control group was TIL cells, and the negative control group was a mixture of TIL cells and 20 μl of magnetic beads Dynabeads ^TM coated with HLA class I antibody [W6/32] (antibody labeled with biotin) and labeled with streptavidin; The group is a mixture of TIL cells and pMHC complexes (at this time, pMHC complexes are loaded on magnetic beads Dynabeads ^TM coated with HLA class I antibody [W6/32] (labeled with biotin) and labeled with streptavidin );

Fig. 4 is a flow cytometric fluorescence analysis graph after co-incubating tumor antigen/MHC-I complexes with TIL cells in Example 2 of the present invention, showing the ratio of CD8 ⁺ T cells and CD4 ⁺ T cells; wherein, A TIL cells derived from the same tumor sample in the blank control group; B is the cells in the supernatant of the negative control group; C is the cells combined with the tumor antigen/MHC-I complex in the experimental group;

Figure 5 is the IFN-γ concentration in the supernatant of the blank control group, the negative control group and the experimental group after stimulating TIL cells derived from the same tumor tissue sample with the tumor antigen/MHC-I complex in Example 3 of the present invention for 48 hours Statistical results (N=3; * means P<0.05; ** means P<0.01);

Fig. 6 is in the embodiment of the present invention 3, after the TIL cell that same tumor tissue sample origin is stimulated with tumor antigen/MHC-I complex, the Tscm (memory stem T cell) ratio of each group (N=3; * represents P <0.05);

Fig. 7 is the result of analyzing the ratio of CD-107a/IFN-γ double-positive cells in each group after stimulating TIL cells derived from the same tumor tissue sample with tumor antigen/MHC-I complex in Example 3 of the present invention (N=3; * means P<0.05; ns means P>0.05);

In Figure 5 to Figure 7, the blank control group is TIL cells, without adding Dynabeads ^TM magnetic beads coated with HLA class I antibody [W6/32] (labeled with biotin) and labeled with streptavidin, negative control Group Dynabeads ^TM coated with HLA class I antibody [W6/32] (labeled with biotin) and labeled with streptavidin were added to TIL cells in the same proportion of the experimental group. The experimental group was in TIL cells pMHC complexes loaded on magnetic beads Dynabeads ^TM coated with HLA class I antibody [W6/32] (labeled with biotin) and labeled with streptavidin were added.

Detailed ways

The present invention will be further described below in conjunction with examples, but these examples do not constitute any limitation to the present invention.

The invention provides a method for preparing a tumor antigen/MHC-I complex, which comprises:

(11) lysing the tumor cells to obtain a tumor cell lysate, which contains a tumor antigen/MHC-I complex;

(12) mixing the antibody-loaded carrier with the tumor cell lysate to obtain the tumor antigen/MHC-I complex; wherein the tumor antigen/MHC-I complex is loaded on the carrier, The antibody specifically binds to MHC-I in the tumor antigen/MHC-I complex.

Specifically, in step (11), tumor cells refer to tumor cells obtained from tumor tissues. The present invention does not limit the method for obtaining tumor cells from tumor tissue and the method for lysing tumor cells, and conventional methods in the art can be used.

Tumor antigens and MHC-I are already in a combined state in tumor cells, that is, tumor antigens and MHC-I exist in tumor cell lysates in the state of "tumor antigen/MHC-I complex (pMHC)".

In some embodiments, tumor cells are lysed using a cell lysate. The tumor cell is lysed by the cell lysate, not only the tumor antigen/MHC-I complex formed by the antigen on the surface of the tumor cell and MHC-I can be present in the tumor cell lysate, but also the internal antigen of the tumor cell (i.e., The tumor antigen/MHC-I complex formed by the part of the antigen that does not exist on the surface of the tumor cell) and MHC-I also exists in the tumor cell lysate, so that there are more tumor antigens in the tumor cell lysate for was crawled.

In some preferred embodiments, the cell lysate is a conditional tumor cell lysate (lysis buffer), which includes CHAPS, Tris buffer and NaCl. Among them, the concentration of CHAPS is preferably 1-25 mg/ml, more preferably 2.5 mg/ml; the concentration of Tris buffer is preferably 25 mM; the concentration of NaCl is preferably 150 mM, so as to better maintain the pH and osmotic pressure when tumor cells are lysed. The conditional tumor cell lysate is used to release more tumor antigen/MHC-I complexes formed by tumor antigens and MHC-I for being captured; meanwhile, the conditional tumor cell lysate has mild lysing conditions , can better protect the tumor antigen/MHC-I complex.

Further, preferably, when adding the cell lysate to lyse the tumor cells, a protease inhibitor is also added. The protease inhibitor can protect the tumor antigen/MHC-I complex and avoid the tumor cell's own protease from degrading the tumor antigen/MHC-I complex in the process of tumor cell lysis.

In step (12), in the carrier loaded with the antibody, the antibody can specifically bind to MHC-I in the tumor cell lysate. Therefore, when the antibody-loaded carrier is mixed with the tumor cell lysate, the antibody specifically binds to MHC-I in the tumor cell lysate, thereby targeting the tumor antigen/antigen present in the tumor cell lysate. The MHC-I complex is captured indiscriminately to obtain a carrier loaded with the tumor antigen/MHC-I complex. At this time, the antibody is also supported on the carrier. Therefore, in some embodiments, further comprising the step of isolating the tumor antigen/MHC-I complex from the carrier loaded with the tumor antigen/MHC-I complex. As the separation method, conventional methods in the art can be used.

In some embodiments, the antibody is an MHC-I beta chain antibody. MHC class I molecules are heterodimers composed of heavy chains (alpha chains) and light chains (beta chains). Among them, the amino acid sequence of the heavy chain has large differences among individuals, while the amino acid sequence of the light chain is highly conserved, and the difference between different species is very small. Therefore, the selection of MHC-I light chain antibodies can be applied to prepare corresponding tumor antigen/MHC-I complexes from tumor tissues of different individuals. In some specific embodiments, the antibody is an HLA (human leukocyte antigen) class I antibody [W6/32], which specifically recognizes the MHC-I light chain, and can be used for the detection of human tumor antigen/MHC-I complexes crawl. It can be understood that when the tumor antigen/MHC-I complex to be captured is derived from an organism other than human, the corresponding antibody that can specifically recognize the MHC-I light chain of the organism can be used.

The method for loading the antibody on the carrier can adopt conventional methods in the art. In some preferred embodiments, the antibody is labeled with a first label selected from biotin and/or a first chemical conjugate. Correspondingly, the carrier is marked with a second marker, and the second marker can be combined with the first marker, so that the antibody is loaded on the carrier. The second label is selected from avidin and/or a second chemical conjugate that can bind to the first chemical conjugate. Preferably, the first label is biotin, and the second label is streptavidin. Biotin and streptavidin have a strong binding force, good binding stability, strong specificity, and are not affected by organic solvents such as reagent concentration, pH environment, and protein denaturants.

The carrier of the present invention can be a carrier commonly used in the field, including but not limited to magnetic beads, quantum dot fluorescent microspheres and the like. In some embodiments, magnetic beads are chosen as the carrier. The magnetic bead carrier is not only easy to obtain, but also can simulate the supporting structure of the antigen peptide/MHC-I complex on the cell membrane in the present invention. In some embodiments, magnetic beads Dynabead ^™ labeled with streptavidin are selected.

In some embodiments, since the tumor antigen/MHC-I complex obtained in step (12) is loaded on the carrier, it also includes separating the tumor antigen/MHC-I complex from the carrier. step. Because in the following, it also involves the use of the obtained tumor antigen/MHC-I complex to enrich tumor antigen-specific CD8 ⁺ T cells and stimulate the activation and proliferation of tumor-infiltrating lymphocytes from the same source, in order to avoid the tumor antigen/MHC-I complex The MHC-I complex exists in a free state, and it is convenient to obtain tumor antigen-specific CD8 ⁺ T cells (the free state is not conducive to the recognition and binding of TCR), preferably the tumor antigen/MHC-I complex obtained in step (12) is loaded The carrier is used to enrich tumor antigen-specific CD8 ⁺ T cells without separating the tumor antigen/MHC-I complex from the carrier. However, it can be understood that those skilled in the art may choose to first separate the tumor antigen/MHC-I complex from the carrier according to actual needs, and then use the tumor antigen/MHC-I complex to to enrich tumor antigen-specific CD8 ⁺ T cells; or, firstly separate the tumor antigen/MHC-I complex from the carrier, and then load the tumor antigen/MHC-I complex on another carrier, and then used to enrich and obtain tumor antigen-specific CD8 ⁺ T cells.

The present invention also provides a tumor antigen/MHC-I complex, which is prepared by the preparation method of the tumor antigen/MHC-I complex provided in the present invention.

The present invention also provides the use of the tumor antigen/MHC-I complex in enriching tumor antigen-specific CD8 ⁺ T cells in vitro, stimulating the activation and proliferation of tumor-infiltrating lymphocytes from the same source, and/or preparing drugs for treating tumors .

Tumor-infiltrating lymphocytes isolated from tumor tissue contain a variety of tumor-specific T cells with different receptors. These tumor-specific T cells mainly recognize antigens caused by gene mutations in cancer cells, so they can respond to a variety of tumor antigens. Responses to address tumor heterogeneity. However, in the prior art, it is difficult to realize the effective recognition and isolation of tumor cell antigens, so the generated tumor-specific T cells generally have the problems of poor specificity and poor targeting, which affects the therapeutic effect of tumors. In view of this problem, in combination with Figure 1, the preparation method of the tumor antigen/MHC-I complex provided by the present invention is based on the design principle of the "shotgun method", which can make the tumor antigen (including tumor neoantigens) are fully released into the lysate, and the tumor antigen/MHC-I is captured indiscriminately from the tumor cell lysate at one time by utilizing the specific binding of antibodies to MHC-I and the characteristics of MHC-I presenting tumor antigens Complex. The preparation method can capture more tumor antigens in the tumor antigen/MHC-I complex in vitro, and the variety is more abundant, which is more conducive to obtaining CD8 ⁺ T cells specific for tumor antigens (tumor neoantigens), And it can selectively induce the activation and proliferation of these cells, thereby increasing the specific killing function of these cells on tumor cells, and realizing the "precise targeting" of tumor cells.

The present invention also provides a method for enriching tumor antigen-specific CD8 ⁺ T cells in vitro, which includes:

The tumor antigen/MHC-I complex provided by the present invention is contacted with tumor infiltrating lymphocytes from the same source, and the tumor antigen-specific CD8 ⁺ T cells in the tumor infiltrating lymphocytes are in contact with the tumor antigen/MHC-I The complex binds to obtain a conjugate enriched with said tumor antigen-specific CD8 ⁺ T cells.

By this method, tumor antigen-specific T cell subsets (CD8 ⁺ T cells) can be easily isolated from tumor-infiltrating lymphocytes, without the need to analyze the sequence of tumor antigens or tumor antigen-specific T cells. At the same time, this method also avoids the problem of missing some antigen-specific T cells when using a single cell surface marker.

In the present invention, the term "contact" includes, but is not limited to, mixing the tumor antigen/MHC-I complex with the tumor infiltrating lymphocytes. In some specific embodiments, the contacting is achieved by mixing and incubating the tumor antigen/MHC-I complex with the tumor infiltrating lymphocytes.

In the present invention, the tumor antigen/MHC-I complex used for contacting with tumor-infiltrating lymphocytes from the same source can be obtained in step (12) of the preparation method of the above-mentioned tumor antigen/MHC-I complex of the present invention The tumor antigen/MHC-I complex loaded on the carrier may also be a tumor antigen/MHC-I complex isolated from the carrier loaded with the tumor antigen/MHC-I complex. In some embodiments, in order to facilitate the isolation of CD8 ⁺ T cells from tumor infiltrating lymphocytes, the tumor antigen/MHC-I complex loaded on the carrier is selected to be contacted with tumor infiltrating lymphocytes from the same source. At this time, CD8 ⁺ T cells in tumor infiltrating lymphocytes recognize the tumor antigen/MHC-I complex through the T cell receptor (TCR), thereby binding to the tumor antigen/MHC-I complex to form a conjugate, and the binding The drug is loaded on the carrier to realize the separation of CD8 ⁺ T cells and tumor infiltrating lymphocytes.

In the present invention, "tumor infiltrating lymphocytes from the same source" means that the source of the tumor infiltrating lymphocytes and the tumor antigen/MHC-I complex is the same. That is, in the preparation method of the tumor antigen/MHC-I complex, the tumor derived from the tumor cells in step (11) is the same as the tumor derived from the tumor-infiltrating lymphocytes, both from the same The same tumor in an individual with a neoplastic disease.

In some embodiments, further comprising the step of isolating CD8 ⁺ T cells from the combination. As the separation method, conventional methods in the art can be used.

Since tumor cells themselves will express biotin, and there will be a small amount of biotin in the culture medium during tumor cell culture, at this time, when the antibody is labeled with the first marker, and the first marker is biotin, and the carrier When there is a second label on the antibody, and the second label is avidin, the biotin expressed by the cell itself or the biotin in the culture medium will bring about the combination of the biotin on the antibody and the avidin on the carrier. interference. Therefore, in some embodiments, in order to avoid the above-mentioned interference problem, before the tumor antigen/MHC-I complex is contacted with tumor infiltrating lymphocytes of the same source, avidin is added to the tumor infiltrating lymphocytes. , to combine with the biotin expressed by the cell itself or the biotin in the culture medium to avoid interference. In some embodiments, the amount of avidin added is 0.5 μg relative to 1*10 ⁶ tumor infiltrating lymphocytes.

CD8 ⁺ T cells bind to MHC-I in the tumor antigen/MHC-I complex through the T cell receptor (TCR). However, TCR will be triggered to internalize after contact with cognate antigen, TCR internalization will cause CD8 ⁺ T cells to fail to recognize or bind tumor antigen/MHC-I complexes, and in the detection experiment of CD8 ⁺ T cells Also results in failure to stain CD8 ⁺ T cells. To avoid this problem, in some embodiments, a tyrosine kinase inhibitor is also added to the tumor infiltrating lymphocytes prior to contacting the tumor antigen/MHC-I complexes with tumor infiltrating lymphocytes from the same source. Tyrosine kinase inhibitors have the effect of inhibiting the internalization of T cell receptors, ensuring the recognition and binding of CD8 ⁺ T cells to tumor antigen/MHC-I complexes. In addition, by adding tyrosine kinase inhibitors, in the detection experiment of CD8 ⁺ T cell enrichment effect by flow cytometry, the background interference signal can be reduced, showing clear grouping results. In some preferred embodiments, the tyrosine kinase inhibitor is Dasatinib. In some specific embodiments, it is added in the form of a mixture consisting of 50 nM dasatinib, 5 g/L human serum albumin (HSA) and PBS buffer at pH 7.4. Among them, HSA mainly plays the role of maintaining osmotic pressure and pH buffering.

The present invention also provides a tumor antigen-specific CD8 ⁺ T cell, which is prepared by the method for enriching tumor antigen-specific CD8 ⁺ T cells in vitro provided by the present invention. The tumor antigen-specific CD8 ⁺ T cells are specific to multiple tumor antigens (tumor neoantigens) in tumor cells, and have a more effective and specific killing effect on tumors.

The tumor antigen-specific CD8 ⁺ T cells provided by the present invention can also be used to identify tumor antigens. Accordingly, the present invention provides a method for identifying tumor antigens, comprising:

The tumor antigen-specific CD8 ⁺ T cells provided by the present invention are subjected to TCR sequencing or protein mass spectrometry analysis to identify their sequences.

The present invention also provides a method for stimulating the activation and proliferation of tumor-infiltrating lymphocytes from the same source in vitro, which includes:

The tumor antigen/MHC-I complex provided by the present invention is contacted with tumor infiltrating lymphocytes from the same source.

The tumor antigen/MHC-I complex provided by the present invention can activate tumor antigen-specific T cell clones by contacting tumor infiltrating lymphocytes from the same source, promote the activation and proliferation of tumor infiltrating lymphocytes, and make stem cell-like tumor antigens The proportion of specific T cells is increased, and the ability of CD8 ⁺ T cells to secrete IFN-γ is improved, etc., to enhance the killing function of tumor cells.

In the present invention, the tumor antigen/MHC-I complex used for contacting with tumor-infiltrating lymphocytes from the same source can be obtained in step (12) of the preparation method of the above-mentioned tumor antigen/MHC-I complex of the present invention The tumor antigen/MHC-I complex loaded on the carrier may also be a tumor antigen/MHC-I complex isolated from the carrier loaded with the tumor antigen/MHC-I complex.

In some embodiments, during the step of contacting the tumor antigen/MHC-I complex with tumor infiltrating lymphocytes from the same source, cytokines are also added. This is because most of the tumor-infiltrating lymphocytes derived from tumor tissue are in a partially exhausted state, and the number of cells is low, which may lead to potential adverse effects on the activation and proliferation of tumor-infiltrating lymphocytes. By adding cytokines, the normal proliferation and stemness of tumor-infiltrating lymphocytes can be guaranteed.

In some preferred embodiments, the cytokine is selected from at least one of 4-1BB, IL-2, and IL-21.

The present invention also provides an activated tumor-infiltrating lymphocyte, which is prepared by stimulating the activation and proliferation of the tumor-infiltrating lymphocyte from the same source in vitro provided by the present invention. The activated tumor-infiltrating lymphocytes provided by the present invention have the characteristics of increasing the proportion of stem cell-like tumor antigen-specific T cells, increasing the ability of CD8 ⁺ T cells to secrete IFN-γ, etc., and can enhance the killing effect on tumors.

The present invention also provides a pharmaceutical composition, the pharmaceutical composition includes an active ingredient and a pharmaceutically acceptable carrier, the active ingredient includes the tumor antigen-specific CD8 ⁺ T cells provided in the present invention and/or the Activated tumor infiltrating lymphocytes. Wherein, the pharmaceutically acceptable carrier may be a conventionally used carrier in the art.

In the pharmaceutical composition, there is no special requirement on the content of the active ingredient and the pharmaceutically acceptable carrier, which may be the conventional content of each component.

In some embodiments, the pharmaceutical composition may also contain other pharmaceutically acceptable excipients, which may be one or more of various preparations or compounds routinely used in the art. For example, the other pharmaceutically acceptable auxiliary materials may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.

In some embodiments, the pharmaceutical composition can be a liquid preparation, such as an injection. Such liquid formulations include, but are not limited to, subcutaneous, intramuscular or intravenous administration. In some specific embodiments, the pharmaceutical composition is for intravenous administration.

The present invention also provides a method for treating tumors, which includes:

contacting an effective amount of the tumor antigen-specific CD8 ⁺ T cells, the activated tumor infiltrating lymphocytes and/or the pharmaceutical composition with the tumor; wherein the tumor antigen-specific CD8 ⁺ T cells, The activated tumor infiltrating lymphocytes and the active ingredient in the pharmaceutical composition are derived from the tumor.

In the present invention, the term "contact" includes, but is not limited to, an effective amount of the tumor antigen-specific CD8 ⁺ T cells, the activated tumor infiltrating lymphocytes and/or the pharmaceutical composition mixed with the tumor, or An effective amount of the tumor antigen-specific CD8 ⁺ T cells, the activated tumor infiltrating lymphocytes and/or the pharmaceutical composition is contacted with the tumor by intravenous injection or the like. In some specific embodiments, an effective amount of the tumor antigen-specific CD8 ⁺ T cells, the activated tumor infiltrating lymphocytes and/or the pharmaceutical composition is mixed with the tumor (tumor cells) in vitro Contact is achieved by means of incubation. In other specific embodiments, an effective amount of the tumor antigen-specific CD8 ⁺ T cells, the activated tumor infiltrating lymphocytes and/or the pharmaceutical composition and the tumor infiltrating lymphocytes are intravenously injected Administered, in vivo circulation achieves contact with the tumor.

In order to make the above-mentioned implementation details and operations of the present invention clearly understood by those skilled in the art, as well as the remarkable embodiment of the progress of the tumor antigen/MHC-I complex and its preparation method and use in the embodiments of the present invention, the following is implemented through multiple An example is used to illustrate the above-mentioned technical solution.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1

1. Cell Lysis of Tumor Tissue

1.1 Carry out verification experiments with lung cancer tumor tissue samples, cut the tumor tissue into 1mm ³ tissue fragments through aseptic operation, add physiological saline, and collect tissue cells through a 70 μm filter with a Pasteur pipette;

1.2 The collected tissue cells were centrifuged at 400g at room temperature for 5min;

1.3 After centrifugation, collect the cells and resuspend them in normal saline for cell counting, and resuspend the remaining cells in complete medium for later use;

1.4 Place the cells in test tubes so that there are 4* ¹⁰⁶ cells in each tube and lyse them; wash with 1mL pre-cooled PBS (pH7.4) before lysing, wash twice in total, and wash with 400g, Centrifuge at 4°C for 5 minutes, then collect the cells;

1.5 Add 300 μl of conditional cell lysate (Lysis buffer: 2.5mg/ml CHAPS, 25mM Tris buffer and 150mM NaCl) to the collected cells, mix thoroughly, place on ice for 10min to lyse, and mix gently several times during the process; Centrifuge at 14000g, 4°C for 15min, transfer the supernatant to a new test tube, which is the tumor cell lysate;

1.6 Determination of concentration: BCA kit was used to measure the total protein concentration in the tumor cell lysate collected in each tube, so as to calculate the total amount of tumor cell protein obtained.

2. Pull-down tumor antigen/MHC-I complex (pMHC complex)

Separation and extraction of tumor antigen/MHC-I complexes by biotin-streptavidin method:

2.1 Take 25 μl of magnetic beads Dynabeads ^TM (Dynabeads ^TM MyOne ^TM Streptavidin T1, purchased from Invitrogen) labeled with streptavidin, after equilibrium preparation: divided into two parts, wherein:

Part 1: Put 20 μl of magnetic beads Dynabeads ^TM (0.2 mg) labeled with streptavidin into a test tube, add 8 μl of HLA class I antibody [W6/32] (labeled with biotin) (ab110665, purchased from Abcam ) antibody, the test tube was placed on a sample mixer (Hula Mixer) and gently rotated, and incubated at room temperature for 30 minutes; the test tube was removed, placed on a magnetic stand, and after standing for 2 minutes, 100 μl PBS (1g/L HSA+2.5mg /mlCHAPS, pH 7.4) to wash off the unstable antibody, place it on the magnetic stand, let it stand for 2min, remove the supernatant, and repeat 3 times in total to obtain the HLA class I antibody [W6/32] (antibody labeled with biotin) and magnetic beads Dynabeads ^™ labeled with streptavidin.

The second part: take 5 μl of magnetic beads Dynabeads ^TM labeled with streptavidin and tumor cell lysate (the volume is calculated according to the concentration determined by BCA, the total protein concentration needs to reach 0.15 μg) and mix well, and the test tube is placed in the sample mixer Incubate at room temperature for 30 minutes, remove the test tube, place it on a magnetic stand, let it stand for 2 minutes, and collect the supernatant.

2.2 Add the second part of the collected supernatant to the first part of the test tube and mix evenly, place it on a sample mixer and rotate gently, and incubate overnight at 4°C.

2.3 The next morning, put the test tube on the magnetic stand for 10 minutes, transfer the supernatant to a new test tube to retain the sample, and detect the grabbing effect by western blotting.

2.4 Resuspend the pMHC complex with 100 μl of PBS (1g/L HSA+2.5mg/ml CHAPS, pH 7.4), place the test tube on a magnetic stand, let it stand for 2min, remove the supernatant, and then resuspend the pMHC complex with 100μl of PBS (1g/LHSA +2.5mg/ml CHAPS, pH 7.4) Gently resuspend the pMHC complex (i.e. magnetic beads Dynabeads ^TM coated with HLA class I antibody [W6/32] (labeled with biotin) and labeled with streptavidin and pMHC-bound product), avoid co-elution of proteins bound to the tube wall during resuspension, and repeat twice in total.

2.5 Take 1/10 of the resuspended pMHC complex suspension and place it in another test tube, gently resuspend with 2× loading buffer, heat at 50°C for 10 min; place the test tube on a magnetic stand, Take the supernatant and transfer it to a new tube for detection by western blot.

2.6 Western blot detection:

In order to verify the capture effect, the tumor cell lysate and the pMHC complex suspension were resuspended in the sample buffer, heated in a water bath at 50°C, and loaded with equal weight for Western blot detection, and the verification marker was streptavidin The situation of binding tumor antigen/HLA I complex on the magnetic beads of the protein.

After SDS-PAGE electrophoresis, use primary anti-MHC class I antibody+HLA A+HLA B antibody (ab134189, purchased from Abcam Company) (rabbit monoclonal antibody [EPR1394Y]) to detect MHC class I molecule+HLA A+HLA B. The fragment size is about 41kDa); goat anti-rabbit IgG H&L (labeled with horseradish peroxidase HRP) was used as the secondary antibody for western blot detection. The result is shown in Figure 2.

It can be seen from Figure 2 that the tumor antigen/HLA I complex (pMHC) in tumor cell lysates can be effectively captured by using streptavidin-labeled magnetic beads as a carrier combined with biotin-labeled antibodies.

Example 2

Validation experiment of the specific enrichment effect of pMHC complex on CD8 ⁺ T cells in TIL cells derived from the same tumor sample:

1. After cell counting, TIL cells from the same lung cancer tumor tissue were centrifuged at 1*10 ⁶ /tube to remove the supernatant; washed once with 1mL PBS (10g/L HSA, pH7.4); centrifuged at 400g, 4°C Remove the supernatant for 5 minutes and resuspend in 100 μl PBS (5 g/L HSA, pH 7.4).

2. In order to remove the interference background, add 0.5U avidin to each tube of the resuspended cells, incubate at room temperature for 10 minutes; add 1mL PBS (5g/L HSA, pH7.4), 400g, centrifuge at 4°C for 5 minutes to remove the supernatant; Suspend the cells in 1ml PBS (5g/L HSA, pH7.4) and centrifuge twice.

3. In order to enhance the binding of pMHC complexes to TIL cells from the same source and reduce the interference background during flow staining and detection analysis, add dasatinib (50nM, 5g/L HSA, PBS pH7.4) and incubate at 37°C for 30min , 400g, 4°C for 5min to remove the supernatant, wash once and resuspend to 100μl with PBS/5g/L HSA.

4. Add Fc receptor blocking agent according to the number of cells, react at room temperature for 10 minutes, add 900 μl PBS/5g/L HSA to wash the cells once. CD8 ⁺ T cell enrichment was carried out after resuspending in 100 μl PBS/5g/L HSA.

5. Using TIL cells as the blank control group (blank), mix TIL cells with 20 μl of magnetic beads Dynabeads ^TM coated with HLA class I antibody [W6/32] (antibody labeled with biotin) and labeled with streptavidin Mix well as a negative control group (negative), TIL cells and pMHC complex (at this time, the pMHC complex is loaded on the surface coated with HLA class I antibody [W6/32] (labeled with biotin) and labeled with streptavidin The magnetic beads Dynabeads ^TM ) were mixed uniformly as the experimental group, and the test tube was placed on the sample mixer and rotated gently, and incubated at room temperature for 1 h.

6. After the incubation is completed, remove the test tube and place it on the magnetic stand, let it stand for 10 minutes, and collect the supernatant and pMHC/cells respectively (in the previous step, the loaded HLA class I antibody [W6/32] (labeled with biotin) and pMHC complexes on magnetic beads Dynabeads ^TM labeled with streptavidin and the product of cell binding) to be detected.

7. The supernatant and pMHC/cells were washed once with 100 μl PBS (5g/L HSA/50nM Dasatinib/2mM EDTA, pH7.4), and 100μl PBS (5g/L HSA/50nM Dasatinib/2mM EDTA, pH7.4) resuspended, flow cytometric analysis of CD45, CD4, CD8 expression. The changes in the proportion of CD45 ⁺ CD8 ⁺ cell populations in each group were evaluated, and the specific enrichment effect of the pMHC complex on CD8 ⁺ T cells was evaluated. The results are shown in Figure 3 and Figure 4 .

It can be seen from Figure 3 and Figure 4 that the proportion of CD8 ⁺ T cells in the experimental group was significantly higher than that of the negative control group and the blank control group, indicating that the pMHC complex can effectively and selectively bind to CD8 ⁺ T cells and achieve the goal of targeting CD8 ⁺ T cells. enrichment effect.

Example 3

1. Cells (including tumor tissue cells and TIL) isolated from the same tumor sample were sorted with CD45 magnetic beads (human CD45 MicroBeads, purchased from Mitenyi Biotec), and CD45 ⁺ cells (ie, TIL) were collected and separated with complete medium (RPMI 1640+10% AB serum+penicillin and streptomycin) were resuspended, and the cell density was adjusted to 1*10 ⁶ /mL, placed in 100 μl of cell suspension per well of a 96-well plate, and supplemented with 3000U/mL IL-2.

2. After the cells were plated and stabilized overnight, the pMHC complex loaded on magnetic beads Dynabeads ^TM coated with HLA class I antibody [W6/32] (labeled with biotin) and labeled with streptavidin was added in proportion as In the experimental group, the number of magnetic beads Dynabeads ^TM particles: cells was 0.5:1 (calculated according to the number of magnetic beads Dynabeads ^TM particles), and the control group was set as a blank control group (blank, without adding HLA class I antibody coated [W6/32] ( Labeled with biotin) and magnetic beads Dynabeads ^TM labeled with streptavidin) and negative control group (negative, add HLA class I antibody coated [W6/32] (labeled with biotin) and labeled in the same proportion Magnetic beads Dynabeads ^TM with streptavidin); supplement IL-2 (3000U/mL), IL-21 (25ng/mL) and 4-1BB (5μg /mL); all samples containing Dynabeads ^TM particles coated with HLA class I antibody [W6/32] (labeled with biotin) and magnetic beads labeled with streptavidin need to be completely washed before adding to the co-culture of cells Supplementary medium was added after resuspension to ensure that the final volume of each well was 200 μl.

3. After the cells have been cultured for 48 hours, perform the first half change of medium (only 3000U/mL IL-2 is added to the complete medium), absorb 100μl of supernatant, centrifuge at 400g for 5min, keep the supernatant for ELISA detection, and analyze IFN in each group - Gamma secretion status.

4. Expand the well after the cell expansion is doubled; pay attention to take a supernatant sample each time the well is expanded, and analyze the secretion of IFN-γ in each group by ELISA.

5. Observe the state of the cells, double the cell expansion, and count the cells, take 7.5*10 ⁵ cells for flow cytometry, and analyze the proportion of the memory phenotype cell population, the results are shown in Figure 6.

6. Observe the state of the cells, count the cells, transfer to a 24-well plate at a density of 1*10 ⁶ /mL to continue to expand and culture, and maintain the cell density below 2*10 ⁶ /mL.

7. Add 3000U/mL IL-2 to the complete medium to maintain the cell culture, take 2*10 ⁶ cells in each group for killing double staining experiment, and analyze the change of CD 107a/IFN-γ double positive cell population ratio by flow cytometry, The results are shown in Figure 5 and Figure 7.

It can be seen from Figure 6 that the pMHC complex can stimulate tumor antigen-specific T cells and increase the proportion of memory stem T cells.

It can be seen from Fig. 5 and Fig. 7 that upon pMHC complex stimulation, the release of IFN-γ is increased, and the cytotoxic function is enhanced, suggesting a better activation effect of cytotoxic T cells.

The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (17)

1. The preparation method of tumor antigen/MHC-I complex, is characterized in that, described preparation method comprises:

   Lying the tumor cells to obtain a tumor cell lysate, the tumor cell lysate contains a tumor antigen/MHC-I complex;

   The carrier loaded with the antibody is mixed with the tumor cell lysate to obtain the tumor antigen/MHC-I complex; wherein the tumor antigen/MHC-I complex is loaded on the carrier, and the antibody Bind specifically to MHC-I in the tumor antigen/MHC-I complex.
2. The preparation method according to claim 1, further comprising the step of separating the tumor antigen/MHC-I complex from the carrier loaded with the tumor antigen/MHC-I complex.
3. The preparation method according to claim 1 or 2, characterized in that, the tumor cells are lysed with a cell lysate; preferably, the cell lysate includes 3-[3-(cholamidopropyl)dimethyl Amino]propanesulfonic acid inner salt, Tris buffer and NaCl.
4. The preparation method according to any one of claims 1-3, wherein the antibody is an MHC-Iβ chain antibody; preferably, the antibody is labeled with a first marker, and the first marker is selected from Biotin and/or first chemical conjugate.
5. The preparation method according to any one of claims 1-4, wherein the carrier is magnetic beads and/or quantum dot fluorescent microspheres; preferably, the carrier is marked with a second marker, and the second The secondary label is selected from avidin and/or a second chemical conjugate.
6. A tumor antigen/MHC-I complex, characterized in that it is prepared by the preparation method described in any one of claims 1-5.
7. The use of the tumor antigen/MHC-I complex according to claim 6 in enriching tumor antigen-specific CD8 ⁺ T cells in vitro, stimulating the activation and proliferation of tumor-infiltrating lymphocytes from the same source, and/or preparing drugs for treating tumors.
8. A method for enriching tumor antigen-specific CD8 ⁺ T cells in vitro, characterized in that the method comprises:

   contacting the tumor antigen/MHC-I complex of claim 6 with tumor infiltrating lymphocytes from the same source, the tumor antigen specific CD8 ⁺ T cells in the tumor infiltrating lymphocytes complexed with the tumor antigen/MHC-I Combining with the tumor antigen specific CD8 ⁺ T cells to obtain the conjugate.
9. The method according to claim 8, wherein the tumor antigen/MHC-I complex is combined with an antibody labeled with biotin and loaded together on a carrier labeled with avidin, and, in the tumor Tumor infiltrating lymphocytes of the same origin were spiked with avidin prior to contacting the antigen/MHC-I complexes with the tumor infiltrating lymphocytes.
10. The method according to claim 8 or 9, characterized in that, before the tumor antigen/MHC-I complex is contacted with tumor infiltrating lymphocytes from the same source, tyrosine is added to the tumor infiltrating lymphocytes Kinase inhibitor; preferably, the tyrosine kinase inhibitor is dasatinib.
11. The method according to any one of claims 8-10, characterized in that the method further comprises the step of isolating the tumor antigen-specific CD8 ⁺ T cells from the conjugate.
12. A tumor antigen-specific CD8 ⁺ T cell, characterized in that it is prepared by the method according to any one of claims 8-11.
13. A method for stimulating the activation and proliferation of tumor-infiltrating lymphocytes from the same source in vitro, characterized in that the method comprises:

    The tumor antigen/MHC-I complex of claim 6 is contacted with tumor infiltrating lymphocytes from the same source.
14. The method according to claim 13, characterized in that, in the step of contacting the tumor antigen/MHC-I complex described in claim 6 with tumor infiltrating lymphocytes from the same source, cytokines are also added; preferably, the Said cytokine is selected from at least one of 4-1BB, IL-2 and IL-21.
15. An activated tumor infiltrating lymphocyte, characterized in that it is prepared by the method according to claim 13 or 14.
16. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises an active ingredient and a pharmaceutically acceptable carrier, and the active ingredient includes the tumor antigen-specific CD8 ⁺ T cells according to claim 12 and/or claim 15 The activated tumor infiltrating lymphocytes.
17. A method for treating tumors, characterized in that the method comprises:

    Contacting an effective amount of the tumor antigen-specific CD8 ⁺ T cells of claim 12, the activated tumor infiltrating lymphocytes of claim 15 and/or the pharmaceutical composition of claim 16 with the tumor; wherein, the The tumor antigen-specific CD8 ⁺ T cells, the activated tumor infiltrating lymphocytes and the active ingredients in the pharmaceutical composition are derived from the tumor.

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