CN115678845A

CN115678845A - Method for culturing tumor-specific CTL cells and cell therapy product

Info

Publication number: CN115678845A
Application number: CN202110852058.9A
Authority: CN
Inventors: 黄英; 李波; 李娜; 胡远; 安婷; 余冰琼; 董碘
Original assignee: Shenzhen Jinuoyin Biotechnology Co ltd; Genoimmune Therapeutics Co Ltd
Current assignee: Shenzhen Jinuoyin Biotechnology Co ltd; Genoimmune Therapeutics Co Ltd
Priority date: 2021-07-27
Filing date: 2021-07-27
Publication date: 2023-02-03

Abstract

The invention provides a culture method of tumor specific CTL cells and a cell therapy product. Wherein, the culture method comprises the following steps: inducing and differentiating the PBMC in vitro into mature DCs loaded with tumor neoantigen polypeptides; co-culturing the mature DCs with CD8+ cells, thereby stimulating activation of the CD8+ cells into CTL cells; the CTL cells were expanded. By adding DC loaded with tumor specific antigen peptide fragment to stimulate PBMC for secondary stimulation before CD3/CD28 loaded magnetic beads are used for stimulating CTL cell to perform undifferentiated cloning, and further adjusting and controlling the time required by each culture stage, the number and the proportion of tumor specific CTL cells in the final T cell product of the method are remarkably increased, and the CTL cell culture period is shortened.

Description

Method for culturing tumor-specific CTL cell and cell therapy product

Technical Field

The invention relates to the field of cell culture, in particular to a culture method of tumor specific CTL cells and a cell therapy product.

Background

In recent years, immunotherapy of tumors has received great attention because it has a significant effect on advanced tumors that are ineffective for conventional therapies. Cancer immunotherapy (Cancer immunotherapy) is a method of specifically killing malignant tumor cells by using the autoimmune system and reducing side effects on normal tissues as much as possible. Tumor immunotherapy is one of the most promising approaches to tumor therapy following chemotherapy and targeted therapy. T cells are currently considered to be the only cells capable of specifically killing tumor cells. DCs present antigens to T cells, inducing activation and proliferation of T cells, including CD4+ helper T cells and CD8+ killer T cells. Tumor antigen-specific T cells have been an important target for tumor therapy.

Tumor neoantigens and tumor associated antigens have received considerable attention from researchers as targets for tumor immunotherapy. Tumor-associated antigens (TAA) are antigen molecules that are expressed in Tumor cells in an elevated manner, and are also expressed in normal cells to a certain extent. Also referred to as "autoantigens," e.g., a family of cancer testis antigens. Thus, tumor-associated antigens are centrally tolerated in the body, i.e. specific T cells against the tumor-associated antigens are cleared in the thymus.

The tumor neoantigen is produced by somatic gene mutation, is mutant protein produced by somatic gene mutation in a patient body, and then stimulates T cell response and kills tumor cells through antigen presentation to achieve the effect of accurate treatment. Non-synonymous point mutations, indel mutations, frameshift mutations, gene fusions, etc. can all produce tumor neoantigens. Tumor neoantigens are also called Tumor Specific Antigens (TSA) or "non-self antigens. The main advantages of the tumor neoantigen as a target of immunotherapy include no central tolerance, strong affinity with Major Histocompatibility Complex (MHC) molecules, reduction of off-target side reactions of normal tissues, high safety and the like. Research shows that compared with a patient inoculated with tumor-associated antigen, the immune response caused by the neoepitope is stronger. The number of novel tumor antigens identified in the past is limited, but with the development of second-generation sequencing and bioinformatics technologies, sequence comparison can be rapidly completed from human normal tissue samples and tumor samples, and finding individual tumor specific antigens is easier and easier.

T-cell Adoptive therapy (ACT) is a very effective method for eliminating malignant tumors in patients, and a large population of tumor-infiltrating lymphocytes (TILs) highly enriched in tumor-specific T-cells often exists around the tumor. As early as 1985, rosenberg et al found that in vivo injection of IL-2 and LAK cells maintained long-lasting regression of certain metastatic melanomas, suggesting specific T cell expansion. However, due to systemic toxicity and expansion of tregs, the treatment effect is poor, and finally, IL-2 cannot be used systemically for tumor treatment. Further in vitro experiments revealed that lymphocytes extracted from melanoma contain specific cells that recognize the own tumor. Subsequent clinical trials have shown that autologous tumor-infiltrating lymphocytes can mediate objective regression of metastatic melanoma. There is a definite advantage to separate, expand and activate T cells in vitro, and then to return them to the patient.

However, in vitro expansion of tumor infiltrating lymphocytes is not always feasible. Especially for some malignancies that have metastasized, such as breast or colorectal cancer, the initial T cell population is difficult to obtain and has limited physical contact with the tumor. There are other reasons that have prevented widespread use of TIL therapy, such as: early isolation procedures require fresh tumor tissue, TILs expansion is difficult, expanded TIL cell function is impaired, and negative regulation from the tumor microenvironment (such as PD-L1 expression and Treg activation) is required.

Therefore, in actual clinical treatment, as an alternative treatment strategy for such cancer patient population, obtaining specific T lymphocytes from Peripheral blood is also a promising method that can easily access a huge number of T cells, i.e., the original T cells are derived from Peripheral Blood Mononuclear Cells (PBMCs) collected from Peripheral blood. However, because a series of treatments such as drug therapy, radiotherapy, chemotherapy, etc. may be performed before ACT therapy is performed on a tumor patient, immune cells in the patient are substantially fully activated to generate an immune tolerance phenomenon, the patient has weak immune cells relative to a healthy person, the capacity of T cells in the patient to differentiate and expand after being stimulated is weak, and particularly in a late-stage tumor patient, the T cell activity is low, and it is difficult to expand T cells to a target amount required in the clinical application process. Therefore, how to enhance the capability of proliferation and activation of T cells into tumor-specific T cells by optimizing the in vitro T cell culture mode is a promising technical improvement scheme.

In the T cell adoptive therapy, T cells in a patient are transferred to the outside of the body for culture and amplification, and a large amount of killer T cells are obtained and then are returned to the patient. There are two main categories of approaches that can be divided in subjects based on the source of the T cells: one is in vitro T cell culture expansion based on TILs (tumor infiltrating lymphocytes); another class is the culture expansion of T cells in vitro based on Peripheral Blood Mononuclear Cells (PBMCs) isolated from peripheral blood.

In view of the wide application prospect of adoptive T cell therapy, there are now a number of methods that have been extensively studied to optimize the production of ex vivo expanded T cells. The existing mature preparation process comprises the following steps:

scheme 1: a large number of naive T cells were expanded from PBMC (peripheral blood mononuclear cells) directly using anti-CD3/CD28 coated magnetic beads as a stimulus. The method for directly expanding T cells in PBMC or TILs by using polyclonal stimuli has the advantages of simplicity, rapidness and availability of a large number of T cells in a short time, and is applied to ACT therapy (T cell adoptive therapy) of various cancers and used as subculture of a large number of autologous T cells derived from peripheral blood. For example, patent applications CN107090433A and CN107254440A disclose a method for culturing T cells in vitro, which comprises directly adding a protective solution coated with CD3 monoclonal antibody or magnetic beads coated with anti-CD3/CD28 antibody to stimulate and amplify primary T cells.

Scheme 2: patent application publication No. CN108220234A discloses an in vitro amplification method of anti-tumor T cells derived from non-sentinel lymph nodes. The method directly uses artificially synthesized tumor neogenetic antigen peptide to pre-stimulate PBMC, then uses immune magnetic beads of CD3/CD28 to perform undifferentiated stimulation and amplification on T cells, so that the T cells are rapidly proliferated and differentiated in a large quantity, and then uses the tumor neogenetic antigen peptide to re-stimulate the T cells.

Scheme 3: patent application publication No. CN108289910A discloses an in vitro amplification method of HER2 specific TH1 cells. The method loads mature DCs with tumor neoantigen peptides to obtain HER 2-peptide pulse type I dendritic cells, and then carries out coculture stimulation on the HER 2-peptide pulse type I dendritic cells to amplify Th1 cells. This stimulation process can be repeated multiple times, resulting in a greater number of proportionally higher HER 2-specific Th1 cells.

Scheme 4: the DC and tumor lysate or the DC is directly mixed with tumor neogenetic antigen peptide for culture, so that the DC is loaded with the tumor neogenetic antigen peptide, DC vaccine is prepared according to the method, then the vaccine is inoculated into PBMC or TILs initial T cells, the PBMC or TILS can be partially differentiated into tumor specific T cells through stimulation of the DC vaccine, and then stimulators such as magnetic beads coupled with anti-CD3/CD28 antibodies are added for indiscriminate amplification of the existing T cells. The DC loaded with tumor neoantigen is used as DC vaccine, and a vaccine stimulation step is added before the magnetic bead nonspecific amplification step. In the final T cell product, the proportion of the tumor specific T cells is obviously improved, and the specificity is enhanced.

Scheme 5: using the TILs method, firstly taking out tumor tissues from a patient body, culturing the TILs and the tumor tissues together, adding cytokines such as IL-2 and the like to activate T cells and kill the tumor cells by the T cells, finally leading the tumor cells to die gradually, culturing the remaining T cells with the tumor specific killing effect, and then returning the patient. This approach increases the number of tumor-specific T cells and removes them from the tumor-suppressed environment. The method is applied to melanoma patients at first, T cells derived from TILs are amplified, and the final result shows that the objective response rate is about 50%.

However, the above culture methods still have to be improved in terms of the number and/or proportion of tumor-specific CTL cells (i.e., cytotoxic T lymphocytes) obtained.

Disclosure of Invention

The main object of the present invention is to provide a method for culturing tumor-specific T cells and a cell therapy product, which can increase the number and/or the proportion of tumor-specific CTL cells in a T cell culture.

In order to achieve the above object, according to one aspect of the present invention, there is provided a method for culturing tumor-specific CTL cells, the method comprising: inducing and differentiating the PBMC in vitro into mature DCs loaded with tumor neoantigen polypeptides; co-culturing the mature DCs with CD8+ cells, thereby stimulating activation of the CD8+ cells into CTL cells; the CTL cells were expanded.

Further, the in vitro induced differentiation of PBMCs into mature DCs loaded with tumor neoantigen polypeptides comprises: isolating the PMBC and sorting CD14+ cells and CD8+ cells from the PBMCs; performing directional differentiation on the CD14+ cells to obtain differentiated DCs; and co-incubating the tumor neoantigen polypeptide and the differentiated DC to obtain the mature DC loaded with the tumor neoantigen polypeptide.

Further, subjecting the CD14+ cells to directed differentiation to obtain differentiated DCs comprises: adopting a DC culture medium to carry out heavy suspension on the CD14+ cells to obtain heavy suspension cells; adding rh GM-CSF and rh IL-4 into the resuspended cells to make directional differentiation so as to obtain first-stage differentiated cells; adding a maturation factor into the differentiated cells at the first stage to continue culturing to obtain differentiated DCs; preferably, the DC medium is a human serum albumin-containing medium; more preferably, the DC medium is obtained by adding human serum albumin to any one of the following media: AIM-V culture medium, X-VIVO 15 culture medium and dendritic Cell culture medium of Cell Genix, preferably, the volume concentration of human serum albumin in DC culture medium is 2.5%, the working concentration of rh GM-CSF is 10 ng/mL-120 ng/mL, and the working concentration of rh IL-4 is 10 ng/mL-120 ng/mL; preferably, the maturation factor is selected from any one or more of: rh GM-CSF, rh IL-4, rh IL-1 beta, rh IL-6, TNF-alpha, PGE-2, and Poly (I: C); more preferably, the final concentrations of the respective maturation factors are as follows: the concentration of rh GM-CSF is 50 ng/mL-200 ng/mL, the concentration of rh IL-4 is 50 ng/mL-120 ng/mL, the concentration of rh IL-1 beta is 10 ng/mL-120 ng/mL, the concentration of rh IL-6 is 10 ng/mL-120 ng/mL, the concentration of TNF-alpha is 10 ng/mL-120 ng/mL, the concentration of PEG-2 is 10 ng/mL-1000 ng/mL, and the concentration of Poly (I: C) is 2 mu g/mL-40 mu g/mL; preferably, the directional differentiation is carried out on days 1-2, the culture is continued on days 3-5, and the tumor neoantigen polypeptide and the differentiated DC are co-incubated on days 6-9 to obtain the mature DC loaded with the tumor neoantigen polypeptide; more preferably, the final concentration of the tumor neoantigen polypeptide is 0.1-40. Mu.g/mL.

Further, co-culturing mature DCs with CD8+ cells, thereby stimulating activation of CD8+ cells into CTL cells, comprises: performing segmented stimulation on the CD8+ cells by adopting mature DCs, so that the CD8+ cells are activated into CTL cells; preferably, the CD8+ cells are pre-stimulated and co-cultured for 7 to 10 days by adopting mature DCs to obtain pre-activated cells; and (3) performing re-stimulation co-culture on the preactivated cells for 1-2 days by adopting the mature DCs to obtain CTL cells.

Further, expanding the CTL cell comprises: amplifying CTL cells by adding magnetic beads or soluble monoclonal antibodies of the monoclonal antibodies coupled with CD3/CD28 into the CTL cells to obtain tumor specific CTL cells; preferably, CTL cells are co-cultured, preferably for 1 to 5 days, with magnetic beads or soluble anti-CD3/CD28 monoclonal antibodies coupled to CD3/CD28 monoclonal antibodies, and then transferred to the cellsCarrying out expansion culture on the culture bag or the G-REX bottle for 5-12 days preferably to obtain tumor specific CTL cells; preferably, the density of the scale-up culture is maintained at 1X 10 ⁶ ～2×10 ⁶ one/mL.

Further, the volume ratio of the CTL cells to the magnetic beads is 100 to 400, preferably 200; preferably, CTL cells and magnetic beads coupled with CD3/CD28 monoclonal antibodies or soluble anti-CD3/CD28 monoclonal antibodies are subjected to co-culture, a co-culture system comprises autologous plasma, rh IL-21, rh IL-7 and rh IL-15, the volume content of the autologous plasma is 0-10%, preferably 2-3%, the concentration of the rh IL-21 is 5-30 ng/mL, preferably 20ng/mL, the concentration of the rh IL-7 is 5-120 ng/mL, preferably 20ng/mL, and the concentration of the rh IL-15 is 5-120 ng/mL, preferably 20ng/mL; preferably, the concentration of the soluble anti-CD3/CD28 monoclonal antibody in the co-culture system is 30-50 ng/mL; preferably, during co-culture, it is determined whether the co-cultured cells require medium addition or rotation of wells based on any one or more of the following observations: cell morphology, cell number, or culture medium color.

Further, isolating PMBC and sorting CD14+ cells and CD8+ cells from PBMCs comprises: mixing peripheral blood or single blood, centrifuging and collecting lower layer blood cells; diluting blood cells by DPBS, placing the blood cells in Ficoll separating medium for centrifugal separation, and collecting intermediate leucocyte cells to obtain PBMC; incubating and sorting PBMCs by adopting CD14 magnetic beads, and collecting CD14+ cells; adding CD8 magnetic beads into the CD 14-cells for incubation, and sorting out CD8+ cells; more preferably, prior to the incubation sorting of PBMCs with CD14 magnetic beads, the culturing method further comprises the step of subjecting the suspension comprising PBMCs to a screen filtration; further preferably, peripheral blood or single blood is mixed evenly and centrifuged for 3-6 min at the rotating speed of 650-750 g to collect lower layer blood cells; diluting blood cells with DPBS containing 2.5% HSA (V/V), centrifuging in Ficoll separating medium, and collecting middle tunica albuginea cells to obtain PBMC; after filtering PBMC with a screen, the filter volume is increased to 10 ⁹ Adding 5-30 mL of MACS buffer solution into each PBMC for resuspension to obtain PBMC resuspension solution; adding 100-1000 mul of CD14 magnetic beads into the heavy suspension of the PBMC for forward sorting, and respectively collecting CD14+ cells and CD 14-cells; and adding 100-1000 mu L of CD8 magnetic beads into the CD 14-cells for forward sorting, and collecting the CD8+ cells.

Further, the tumor neoantigen polypeptide is selected from any one or more of the following: KLMGIVYKV, SLDWWAFGV, VTFHIPFEV, AVGSYVYSV, and KLASYDMRL.

Further, in pre-stimulation co-culture, the ratio of cell number of mature DCs to CD8+ cells was 1; preferably, in the process of pre-stimulation co-culture and/or re-stimulation co-culture, a co-stimulation culture system is adopted for culture, wherein the co-stimulation culture system contains autologous plasma, co-stimulation factors and serum-free culture medium; preferably, the volume content of the autologous plasma in the co-stimulation culture system is 0-10%, more preferably 2% -3%; preferably, the co-stimulation factor is rh IL-21, and the concentration of the rh IL-21 in the co-stimulation culture system is 5-30 ng/ml; more preferably 20ng/ml.

Further, in the process of pre-stimulation co-culture and/or re-stimulation co-culture, the method also comprises a step of supplementing cell growth factors or performing hole transfer on the co-cultured cells, preferably, the supplemented cell growth factors are rh IL-7 with the final concentration of 5-120 ng/mL and rh IL-15 with the final concentration of 5-120 ng/mL; more preferably, the final concentration of rh IL-7 supplemented is 20ng/mL and the final concentration of rh IL-15 is 20ng/mL.

According to another aspect of the present invention, there is provided a cell therapy product comprising tumor-specific CTL cells cultured in vitro, wherein the tumor-specific CTL cells in the cell therapy product are cultured by any one of the above-mentioned culturing methods.

By applying the technical scheme of the invention, DC loaded with tumor specific antigen peptide fragments is added to perform secondary stimulation on PBMC before magnetic beads loaded with CD3/CD28 are used for stimulating T cells to perform undifferentiated cloning, and the time required by each culture stage is further adjusted and controlled. This method allows a significant increase in the number and proportion of tumor-specific CTL cells in the final T cell product, while shortening the CTL cell culture cycle.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a graph showing the effect of different ratios of autologous plasma on the expansion of CTL cells and the secretion of IFN-. Gamma.in example 1 of the present invention;

FIG. 2 shows the effect of different DC to CD8+ co-culture ratios on specific CTL cell culture in example 2 of the present invention;

FIG. 3 shows the effect of different cell growth factors on CTL cell culture in example 3 of the present invention;

FIG. 4 shows the cell expansion fold and the content of specific CTL cells obtained in examples 4 to 7 according to the present invention;

fig. 5 shows the cell killing ability of the T cells obtained in examples 4 to 7 with different effective target ratios.

FIG. 6 shows the IFN-. Gamma.secretion amounts of tumor-specific CTL cells obtained in examples 4 to 7.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.

Interpretation of terms:

PBMC: peripheral Blood Mononuclear cells, peripheral Blood mononary Cell.

DC: dendritic Cell, dendritic Cell. Is the Antigen Presenting Cell (APC) with the strongest function found at present.

CTL: cytotoxic T lymphocytes, also known as killer T lymphocytes. Various cytokines are secreted exclusively to participate in the immune function. It has killing effect on some virus, tumor cell and other antigen matter, and forms important defense line with natural killer cell (NK cell) for resisting virus and tumor immunity.

DPBS: ducheng's Phosphate buffer, fully known as Dulbecco's Phosphate Buffered Salin, contains no calcium or magnesium ions as compared to the commonly used standard PBS. The ionic concentration and osmotic pressure of the solution are isotonic and compatible with the human body. The buffer helps to provide and maintain a stable pH between 7.2 and 7.6. Are often used to dilute or wash the cells.

rh GM-CSF: recombinant human granulocyte-macrophage colony stimulating factor.

rh IL-4: recombinant human interleukin-4.

Poly (I: C): polysarcosine, polyinosinic-polycytidylic acid, is an analog of double-stranded RNA, one is Poly (I) and the other is Poly (C), is an interferon inducer, and is mainly used for activating immune lymphocytes.

As mentioned in the background, although there are some methods for culturing tumor-specific CTL cells, these methods still do not satisfy the requirement of use, and in order to improve the current situation, the present application attempts to provide a novel culture method. Firstly, the inventor carries out research and analysis on the existing method, and finds that the existing schemes only consider the requirements of a certain aspect and ignore other result indexes, or the final product cannot meet the actual requirements due to incomplete process design. In particular, each of the solutions mentioned in the background art has its own unique drawbacks, as detailed below.

Scheme 1: the PBMC (peripheral blood mononuclear cells) is directly subjected to undifferentiated amplification by using the polyclonal stimulant, although the operation is simple, quick and effective, only a few spontaneously induced tumor specific T cells can be amplified by using the polyclonal stimulant similar to anti-CD3/CD28 coated magnetic beads, and meanwhile, a large number of non-tumor related T cells can be obtained, so that the targeting property is weak, and the toxic and side effects are strong.

Scheme 2: PBMC were stimulated directly with neoantigenic peptides, followed by undifferentiated expansion and restimulation of stimulated T cells. The main problem with this approach is that the direct use of antigenic peptides deactivates specific cells in PBMCs, and the presentation effect is much lower than that of professional antigen presenting cells (e.g. DCs).

Scheme 3: the mature DC is loaded with tumor neoantigen peptide to obtain HER 2-peptide pulse type I dendritic cells, and then the HER 2-peptide pulse type I dendritic cells are co-cultured to amplify the Th1 cells. This stimulation process may be repeated multiple times, resulting in a greater number and proportion of HER 2-specific Th1 cells. The main disadvantages of this method are the long culture period and the complicated operation, and the increase of the proportion of tumor-specific T cells, but the increase of the number of the final specific T cells is limited.

Scheme 4: primary T cells were stimulated with DCs loaded with tumor neoantigens, followed by indiscriminate amplification using CD3/CD28 coated magnetic beads. Although the number of tumor-specific T cells in the final cell product is increased, the percentage of final tumor-specific T cells is increased only to a limited extent.

Scheme 5: tumor infiltrating lymphocytes are obtained from tumor tissue directly using the TILs method, although this method has certain advantages. However, the operation is complicated, and the method requires the acquisition of fresh tumor tissue in an early separation step, which is not easily met for some malignant tumors; in the meantime, the subsequent treatments may damage the TILs, resulting in problems such as loss of function of the finally generated T cells.

In view of the above-mentioned drawbacks of the prior art methods, the present application provides an improved method for efficiently culturing tumor-specific CTL cells, and tests prove that the method not only can shorten the culture time, but also can obtain a greater number of tumor-specific CTL cells with a higher ratio. Based on the test results, the applicant proposed the technical means of the present application, which is described in detail below.

In an exemplary embodiment of the present application, there is provided a method for culturing tumor-specific CTL cells, the method comprising: inducing PBMC in vitro to differentiate into mature DC; co-culturing mature DCs with CD8+ cells, thereby stimulating activation of CD8+ cells into CTL cells; the CTL cells were expanded. Before undifferentiated cloning of CTL cells, the culture method firstly adopts mature DCs loaded with tumor specific antigen peptide fragments to stimulate and activate CD8+ cells separated from PBMCs, so that the number and proportion of tumor specific CTL cells in a final T cell product are obviously improved, and correspondingly, the proportion of non-tumor related T cells is reduced, thereby improving targeting property, weakening toxic and side effects and shortening the culture period of the CTL cells.

DC cells need to be induced to differentiate from other cell types (including PBMC, cord blood CD34+ cells, bone marrow, etc., where PBMC are the most accessible and abundant and are widely used clinically as the source cells for DCs) and can be divided into different stages of maturation, usually in 2 steps: 1) Inducing differentiation from DC progenitors (e.g., PBMCs) into Immature DCs (iDC); 2) Differentiation from iDCs was induced into mature DCs (mature DCs). The above procedure for inducing and differentiating PBMCs into mature DCs in vitro can be implemented by using the existing DC cell differentiation method or by modifying the existing DC cell differentiation method on the basis of the existing DC cell differentiation method. In a preferred embodiment, the in vitro induced differentiation of PBMCs into mature DCs comprises: isolating PMBC and sorting CD14+ cells and CD8+ cells from PBMCs; performing directional differentiation on the CD14+ cells to obtain differentiated DCs; and co-incubating the tumor neoantigen polypeptide and the differentiated DC to obtain the mature DC loaded with the tumor neoantigen polypeptide.

Compared with the weak function of directly activating T cells to expand into antigen-specific CTL cells by adopting the antigen polypeptide, the DC is selected as the antigen presenting cells in the application, and the DC loaded with the tumor neoantigen polypeptide and the CTL cells are co-cultured, so that the presenting capacity of the polypeptide-MHC is improved, and the function of activating the CTL cells is improved.

In addition, in the present application, the starting T cells are derived from peripheral blood and are not obtained from a fresh tumor sample. Since peripheral blood is readily available and can be exposed to a large number of T cells, there are no problems in the prior TILs methods due to the material extraction and experimental procedures, such as the difficulty in applying the TILs method to metastatic malignancies, the difficulty in obtaining initial T cell populations and limited contact with the tumor parenchyma, the need for fresh tumor tissue in the early isolation procedure, the difficulty in expanding TILs, the impairment of expanded TIL cell function, and negative regulation from the tumor microenvironment (e.g., PD-L1 expression and Treg activation, etc.).

In the present application, although PBMC are used as an initial T cell source, it is considered that PBMC include various cell types such as T, B, NK cells, monocytes, dendritic cells, and the like, in which PBMC are first induced to differentiate into mature DCs in vitro, then the mature DCs are co-cultured with CD8+ cells to stimulate activation of CD8+ cells into CTL cells, and finally CTL cells are specifically amplified, rather than PBMC being directly activated and amplified. While CD8+ cytotoxic T lymphocytes (i.e., CD8+ CTLs) play a killing role. If PBMC cells are directly used for expansion, not only CD8+ cells but also other lymphocytes are activated and expanded, thereby causing a reduction in the specific proportional number of the resulting CD8+ CTL cells. Therefore, the anti-tumor effect of CTL obtained by the method is better than that of CTL obtained by stimulating PBMC (peripheral blood mononuclear cell).

In a preferred embodiment, committed differentiation of CD14+ cells to obtain differentiated DCs comprises: resuspending the CD14+ cells by using a culture medium to obtain resuspended cells; adding rh GM-CSF and rh IL-4 into the resuspended cells to make directional differentiation so as to obtain first-stage differentiated cells; and adding a maturation factor into the differentiated cells in the first stage to continue culturing to obtain the differentiated DC.

In a preferred embodiment, the culture medium is a culture medium containing human serum albumin; more preferably, the medium is obtained by adding human serum albumin to any one of the following media: AIM-V medium, X-VIVO 15 medium and Cell Genix dendritic Cell medium; preferably, the volume concentration of human serum albumin in the DC medium is 2.5%, the concentration of rh GM-CSF is 10-120 ng/mL (such as 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL, 110ng/mL or 120 ng/mL), more preferably 80-100 ng/mL, the concentration of rh IL-4 is 10-120 ng/mL (such as 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL, 110ng/mL or 120 ng/mL), more preferably 80-100 ng/mL.

In a preferred embodiment, the maturation factor is selected from any one or more of: rh GM-CSF, rh IL-4, rh IL-1 beta, rh IL-6, TNF-alpha, PGE-2, and Poly (I: C); more preferably, the final concentrations of the respective maturation factors are as follows: the concentration of rh GM-CSF is 50 ng/mL-200 ng/mL (for example, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL, 110ng/mL, 120ng/mL, 130ng/mL, 140ng/mL, 150ng/mL, 160ng/mL, 170ng/mL, 180ng/mL, 190ng/mL or 200 ng/mL), and more preferably 100ng/mL; the concentration of rh IL-4 is 50 ng/mL-120 ng/mL (for example, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL, 110ng/mL or 120 ng/mL), and more preferably 100ng/mL; the concentration of rh IL-1 β is 10ng/mL to 120ng/mL (for example, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL, 110ng/mL or 120 ng/mL), and more preferably 40ng/mL; the concentration of rh IL-6 is 10 ng/mL-120 ng/mL (for example, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL, 110ng/mL or 120 ng/mL), and more preferably 40ng/mL; the concentration of TNF-alpha is 10 ng/mL-120 ng/mL (for example, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL, 110ng/mL or 120 ng/mL), and more preferably 20ng/mL; PEG-2 has a concentration of 10ng/mL to 1000ng/mL, more preferably 150 to 250ng/mL (e.g., 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL, 110ng/mL or 120ng/mL, 200ng/mL, 300ng/mL, 400ng/mL, 500ng/mL, 600ng/mL, 700ng/mL, 800ng/mL, 900ng/mL or 1000 ng/mL), and Poly (I: C) has a concentration of 2. Mu.g/mL to 40. Mu.g/mL, more preferably 15. Mu.g/mL to 25. Mu.g/mL (e.g/mL, 25ng/mL, 30ng/mL, 35ng/mL or 40 ng/mL).

In a preferred embodiment, the directed differentiation is performed on days 1-2, the culture is continued on days 3-5, and the tumor neoantigen polypeptide is co-incubated with the differentiated DCs on days 6-9 to obtain mature DCs.

In a preferred embodiment, the final concentration of the tumor neoantigen polypeptide is 0.1-40. Mu.g/mL, more preferably 5-20. Mu.g/mL, and most preferably 10. Mu.g/mL. The tumor neoantigen polypeptide can be reasonably selected according to different tumor types.

In the preferred embodiment, rh GM-CSF is a hematopoietic growth factor, which stimulates the formation of neutrophil and macrophage colonies in vitro, promotes the differentiation of monocytes into macrophagocyte-like cells, and increases the expression of MHC class II molecules on the cell surface, thereby enhancing the antigen presentation function of the cells. In addition, rh GM-CSF can also promote survival of DCs. The rh IL-4 plays a role in the process of inducing monocytes into DCs by inhibiting the overgrowth of macrophages, thereby guiding the monocytes to differentiate toward DCs. If rh IL-4 is not added to the culture system, the monocyte will differentiate into macrophage. Meanwhile, rh IL-4 also has the capability of reducing the expression level of CD14 molecules on the cell surface, and the reduction of the expression level of CD14 is an important mark for differentiating PBMC into DC. The rhGM-CSF and the rhIL-4 act together to directionally differentiate PBMC into Immature DC (iDC), and the DC has strong antigen uptake and processing capacity but weak antigen presenting capacity. The cell surface moderately expresses MHC class I, class II and B7 family molecules (CD 80, CD86, etc.), but not or under-expresses CD14. In the preferred embodiment, the first stage differentiated cells are obtained by adding rh GM-CSF and rh IL-4 in the above concentration ranges to the resuspended cells for directed differentiation, and the DC culture medium supplemented with human blood albumin is used, wherein the DC culture medium has a certain ability to induce differentiation into iDC when the added volume content of human blood albumin in the DC culture medium is 2.5%.

The rh IL-1 beta, rh IL-6 and TNF-alpha are proinflammatory factors, the 3 cytokines can reduce the megakaryocytic action of immature DC and the expression of surface Fc receptor, and up-regulate the expression of MHC (major histocompatibility complex) I-class molecules, II-class molecules and B7 family molecules on the cell surface, and can differentiate the immature DC into the mature DC in an in vitro culture system. The three-factor combined antigen induces complete maturation of DCs under the condition of no calf serum culture, so that DCs which can be applied to clinic are prepared. The addition of PGE2 (Prostaglandin E2 ) to the above three-factor combination further improves the yield, maturation, migration ability and immune activation ability of DCs. The migration ability of rh IL-1 beta, rh IL-6 and TNF-alpha to induce mature DCs is weaker, and the mature DCs cannot reach lymph nodes well to activate T cells, and the mature DCs can be induced to migrate to the lymph nodes more easily due to the high expression of surface chemokine receptors after PGE2 is added, so that the immune response of an organism to the tumor is caused. The combination of these four factors is widely used in the clinic and is considered as the "gold standard" for the preparation of mature DCs, typical working concentrations are rh IL-1 β 10ng/ml, rh IL-6 1000U/ml, TNF- α 10ng/ml and PGE2 mg/ml, respectively.

It should be noted that PGE2 could not be added to the first step of DC induced differentiation, and if it was added in the first step, DC differentiation would be inhibited. The differences in the nature of the serum-free media used in culture systems may also lead to differences in the nature of the mature DCs induced by PGE2 addition, and thus it is desirable to prepare DCs clinically in sufficient preliminary studies and then identify the best preparation for treating a tumor. In the preferred embodiment of the present application, the maturation factor includes rh GM-CSF, rh IL-4 and Poly (I: C), poly (I: C) is Poly-sarcosine, poly-inosinic acid-Poly-cytidylic acid is double-stranded RNA analog, one is Poly (I) and the other is Poly (C), is an interferon inducer, and is mainly used for activating immune lymphocytes. The three factors and the four factors are adopted to induce the iDC to be differentiated into the mDC, so that the DC with directional induction can highly express HLA-DR, CD86, CD83 and the like, and the DC can still maintain the stable maturity after being cultured in a culture medium without the cytokines for 24-48h. When the working concentration of each maturation factor is controlled within the above preferred range, the method has the beneficial effects of less induced DC adherence, high yield and high expression of surface marker molecules. In a more preferred embodiment, the concentration of rh GM-CSF is 50 ng/mL-200 ng/mL, the concentration of rh IL-4 is 50 ng/mL-120 ng/mL, the concentration of rh IL-1 β is 10 ng/mL-120 ng/mL, the concentration of rh IL-6 is 10 ng/mL-120 ng/mL, the concentration of TNF- α is 10 ng/mL-120 ng/mL, the concentration of PEG-2 is 10 ng/mL-1000 ng/mL, and the concentration of Poly (I: C) is 2 g/mL-40 μ g/mL. Most preferably, the working concentrations of each maturation factor are: the concentration of rh GM-CSF is 100ng/mL, the concentration of rh IL-4 is 80ng/mL, the concentration of rh IL-1 beta is 40ng/mL, the concentration of rh IL-6 is 40ng/mL, the concentration of TNF-alpha is 20ng/mL, the concentration of PEG-2 is 200ng/mL, and the concentration of Poly (I: C) is 20 mug/mL.

In the process of inducing iDC to differentiate into mDC, the tumor neogenesis antigen peptide and the differentiated DC are co-cultured to stimulate the mature DC surface to present tumor antigen polypeptide, so that CD8+ cells can be more effectively stimulated and activated, and the method is favorable for obtaining tumor specific CTL cells with higher proportion.

In a preferred embodiment, co-culturing mature DCs with CD8+ cells, thereby stimulating activation of CD8+ cells into CTL cells, comprises: performing segmented stimulation on the CD8+ cells by adopting mature DCs, so that the CD8+ cells are activated into CTL cells; preferably, the CD8+ cells are pre-stimulated and co-cultured for 7 to 10 days by adopting mature DCs to obtain pre-activated cells; and (3) performing re-stimulation co-culture on the pre-activated cells for 1-2 days by adopting mature DCs to obtain CTL cells, and performing large-scale amplification on the CTL cells.

By using mature DC cells loaded with tumor neoantigen polypeptides to pre-stimulate and re-stimulate CD8+ cells, the proportion of tumor specific CTL cells in the finally prepared T cell product is increased, the proportion of non-tumor related T cells is reduced, the targeting property is improved, and the toxic and side effects are weakened. By controlling the time of the pre-stimulation co-culture to be 7-10 days and the time of the re-stimulation co-culture to be 1-2 days, a large amount of tumor-specific CTL cells can be effectively activated and generated in a short time, so that the content of the tumor-specific CTL cells is improved, and the cell culture time is shortened.

The above-mentioned step of amplifying CTL cells is a step of amplifying a large amount of CTL cells to increase the total amount of cultured CTL cells, and the specific steps are similar to those of the conventional undifferentiated amplification step. In a preferred embodiment, expanding CTL cells comprises: amplifying the CTL cells by adding magnetic beads or soluble monoclonal antibodies of the monoclonal antibodies coupled with the CD3/CD28 to the CTL cells; preferably, the CTL cells and the magnetic beads coupled with the CD3/CD28 monoclonal antibody or the soluble anti-CD3/CD28 monoclonal antibody are subjected to co-culture, preferably for 1 to 5 days, and then transferred to a cell culture bag or a G-REX bottle for expansion culture, preferably for 5 to 12 days; preferably, it is expandedThe density of the large culture was maintained at 1X 10 ⁶ ～2×10 ⁶ one/mL.

In the bulk expansion step of the CTL cells, the magnetic beads coupled with the CD3/CD28 monoclonal antibody or the soluble anti-CD3/CD28 monoclonal antibody is used for co-culture, so that main and synergistic stimulation signals required by activation and expansion of T cells can be provided, and thus the large-scale expansion can be realized in a short time by adopting the mode for the amplification. Magnetic beads coupled with monoclonal antibodies of CD3/CD28 can provide primary and co-stimulation signals required by T cell activation and expansion, and activated T cells generate rh IL-2, rh GM-CSF, INF gamma and TNF-alpha and play the role and function of T cells. Magnetic bead-activated T cells coupled with monoclonal antibodies to CD3/CD28 can expand 100-1000 times within a 9-14 day culture period.

In the preferred embodiment, the cells are co-cultured for 1 to 5 days and then transferred to a cell culture bag or a G-REX flask for 5 to 12 days of expansion culture, depending on the culture time and the conditions required for activating T cells with a tumor neoantigen. In the stage of scale-up culture, the density of cultured cells is controlled to 1 × 10 ⁶ ～2×10 ⁶ Within the range of one cell/mL, the amplification speed is high, and the cell quality is good.

In order to further increase the expansion rate of CTL cells, in a preferred embodiment, the volume ratio of CTL cells to magnetic beads is 100 to 400, preferably 200. More preferably, the CTL cell is co-cultured with the CD3/CD28 monoclonal antibody-coupled magnetic bead or the soluble anti-CD3/CD28 monoclonal antibody, and the co-culture system comprises 0 to 10% by volume of autologous plasma (preferably 1 to 5%, more preferably 1 to 3%, further preferably 2 to 3%), rh IL-21 (preferably 10 to 30ng/mL, more preferably 20 ng/mL) at a concentration of 5 to 30ng/mL, rh IL-7 (preferably 15 to 25ng/mL, more preferably 20 ng/mL) at a concentration of 5 to 120ng/mL, and rh IL-15 (preferably 15 to 25ng/mL, more preferably 20 ng/mL) at a concentration of 5 to 120 ng/mL), and the autologous plasma, rh IL-7, and rh IL-15 are added to the co-culture system to facilitate the growth and maintenance of the CTL cell, and the content is controlled within the above range, so that the CTL cell can be significantly proliferated in a short period of time and the aging thereof can be inhibited.

More preferably, the concentration of the soluble anti-CD3/CD28 monoclonal antibody in the co-culture system is 30-50 ng/mL, and the effect of promoting CTL cell proliferation is more remarkable at the concentration; more preferably, during co-culture, to achieve higher expansion, the state of the cell culture is observed during culture and it is determined whether the co-cultured cells require medium addition or rotation of wells based on any one or more of the following observations: cell morphology, cell number, or culture medium color. If cell colonies appear, the number of cells increases, and the color of the medium turns yellow, it is necessary to add the medium or to transfer the wells.

In the preferred embodiment described above, following restimulation of T cells with mature DC cells loaded with neoantigenic peptides, indiscriminate stimulation of expansion of T cells is performed using anti-CD3/CD28 coupled immunomagnetic beads. In the final T cell product prepared, the total number of tumor-specific CTL cells was significantly increased. Meanwhile, the production cycle of preparing tumor specific CTL cells is further shortened by improving the culture process conditions.

In a preferred embodiment, isolating PMBC and sorting CD14+ cells and CD8+ cells from PBMCs comprises: mixing peripheral blood or single blood, centrifuging and collecting lower layer blood cells; diluting blood cells by DPBS, placing the blood cells in Ficoll separation liquid for centrifugal separation, and collecting intermediate white membrane layer cells, namely PBMC; incubating and sorting PBMCs by adopting CD14 magnetic beads, and collecting CD14+ cells; adding CD8 magnetic beads into the CD 14-cells for incubation, and sorting out CD8+ cells; more preferably, the culturing method further comprises a step of filtering the suspension containing the PBMCs by a screen, and further preferably, the screen has a pore size of 20 to 60 μm, before the PBMCs are subjected to incubation sorting by using CD14 magnetic beads.

In a preferred embodiment, peripheral blood or single blood is mixed evenly and centrifuged at 650-750 g for 3-6 min to collect the lower layer blood cells; diluting blood cells with DPBS containing 2.5% HSA (v/v), centrifuging in Ficoll separating medium, and collecting intermediate buffy coat cells to obtain PBMC; after filtering PBMC with a screen, the filter volume is increased to 10 ⁹ Adding 5-30 mL of MA into PBMCRe-suspending the CS buffer solution to obtain a PBMC re-suspension solution; adding 100-1000 mul of CD14 magnetic beads into the PBMC heavy suspension for forward sorting, and respectively collecting CD14+ cells and CD 14-cells; and adding 100-1000 mu L of CD8 magnetic beads into the CD 14-cells for forward sorting, and collecting the CD8+ cells.

The tumor neoantigen polypeptide in the present application varies according to the type of a specific tumor. The polypeptide sequence can be obtained through the existing report, or can be obtained through the comparison of a normal sample and a tumor sample. If the comparison result is obtained, the specific comparison method or analysis software is not particularly limited. For example, tumor neoantigen polypeptides screened by bioinformatic software developed by warfarin can be adopted. In a preferred embodiment, the tumor neoantigen polypeptide is selected from any one or more of: SEQ ID NO:1 (amino acid sequence: KLMGIVYKV), SEQ ID NO:2 (amino acid sequence: SLDWWAFGV), SEQ ID NO:3 (amino acid sequence: VTFHIPEV), SEQ ID NO:4 (amino acid sequence: AVGSYVYSV) and SEQ ID NO:5 (amino acid sequence: KLASYDMRL).

In a preferred embodiment, the ratio of the cell number of mature DC to CD8+ cells in the pre-stimulation co-culture is 1. Within the range of the ratio, the stimulation and activation effect on the T cells is stronger, thereby being beneficial to generating higher ratio of tumor-specific CTL cells. In order to further increase the stimulation activation ratio of the tumor-specific CTL cells, preferably, a co-stimulation culture system is adopted for pre-stimulation co-culture and/or re-stimulation co-culture, wherein the co-stimulation culture system contains autologous plasma, co-stimulation factors and serum-free culture medium; preferably, the volume content of autologous plasma in the co-stimulatory culture system is 0-10%, more preferably 2% -3%; preferably, the co-stimulatory factor is rh IL-21 and the concentration of rh IL-21 in the co-stimulatory culture system is from 5 to 30ng/ml, more preferably 20ng/ml.

On the basis of the co-stimulation culture, in order to further reduce or avoid the influence of nutrition or environment in the cell proliferation process, preferably, in the process of pre-stimulation co-culture and/or re-stimulation co-culture, the method further comprises the step of supplementing cell growth factors or performing hole transfer on the co-cultured cells, preferably, the supplemented cell growth factors are rh IL-7 and rh IL-15, the final concentration of the supplemented rh IL-7 is 5-120 ng/mL, the final concentration of the supplemented rh IL-15 is 5-120 ng/mL, further preferably, the rh IL-7 with the final concentration of 15-20 ng/mL and the rh IL-15 with the final concentration of 15-20 ng/mL are supplemented in the culture medium, and most preferably, the rh IL-7 with the final concentration of 20ng/mL and the rh IL-15 with the final concentration of 20ng/mL are supplemented in the culture medium; by supplementing the final concentrations of the cell growth factors rh IL-7 and rh IL-15, the proliferation of T cells can be further promoted.

According to a second aspect of the present application, there is provided a cell therapy product comprising tumour specific T cells cultured in vitro, wherein the tumour specific T cells have been cultured using any one of the culture methods described above. In the T cell culture obtained by the culture method, the total cell number and the proportion of tumor-specific CTL cells are obviously improved, so that the method is suitable for preparing corresponding cells in cell therapy including TCR-T, CAR-T, DC-CIK or DC-CTL.

The beneficial effects of the cultivation process according to the invention are further illustrated by the following examples.

It should be noted that those who do not indicate conditions in the following examples are performed according to conventional conditions or conditions recommended by the manufacturer. The apparatus or materials used in the examples are all conventional commercial products which are commercially available.

First part, optimization of culture conditions

Example 1: effect of autologous plasma on CTL cell culture

Step 1: separating and sorting PBMC;

mixing peripheral blood or single blood, centrifuging and collecting lower layer blood cells. Blood cells were diluted with DPBS, added to Ficoll separation medium, and centrifuged. Collecting the intermediate leucocyte layer cells, namely the Peripheral Blood Mononuclear Cells (PBMC). The centrifugation conditions were 700g,5min, 2.5% of HSA (v/v) was contained in DPBS (Gibco).

The PBMC suspension was screened, centrifuged, incubated with CD14 magnetic beads (Miltenyi Biotec), column sorted, and CD14+ cells collected. CD8+ cells were then sorted by incubation with CD8 magnetic beads (Miltenyi Biotec) in CD 14-cells.

PBMC after sieving, 10 times ⁹ Each was resuspended and mixed by adding 10mL of MACS buffer, followed by addition of 800. Mu.L of CD14 magnetic beads (Miltenyi Biotec for forward sorting. The collected CD 14-cells were forward sorted by addition of 200. Mu.L of CD8 magnetic beads (Miltenyi Biotec).

Step 2: inoculating CD14+ cells, and inducing and differentiating in vitro into mature DCs;

the sorted CD14+ cells were resuspended in DC medium containing Human Serum Albumin (HSA), rh GM-CSF and rh IL-4 were added for directed differentiation. On the 4 th day, the maturation factors rh GM-CSF, rh IL-4, rh IL-1 beta, rh IL-6, TNF-alpha, PGE-2, poly I: C were added and the culture continued. Tumor neoantigen polypeptides were incubated with DCs on day 5, and then mature DCs were collected.

The medium for culturing DC cells was obtained by adding HSA to AIM V medium at a concentration of 2% (V/V) in HSA, 100ng/mL in rh GM-CSF, and 100ng/mL in rh IL-4.

The maturation inducing factor combination is added on day 4, the concentration of rh GM-CSF is 100ng/mL, the concentration of rh IL-4 is 100ng/mL, the concentration of rh IL-6 is 40ng/mL, the concentration of TNF-alpha is 20ng/mL, the concentration of rh IL-1 beta is 40ng/mL, the concentration of PEG-2 is 200ng/mL, the concentration of Poly (I: C) is 20 mu g/mL, the tumor neoantigen polypeptide is loaded on day 5, and the final concentration of the polypeptide is 10 mu g/mL.

The following tumor neoantigen polypeptides were used, numbered SEQ ID NOs: 1 (sequence: KLMGIVYKV), SEQ ID NO:2 (sequence: SLDWWAFGV), SEQ ID NO:3 (sequence: VTFHIPFEV), SEQ ID NO:4 (sequence: AVGSYVYSV) and SEQ ID NO:5 (sequence: KLASYDMRL).

And step 3: carrying out co-culture on the mature DC loaded with the polypeptide and CD8+ cells, and activating the mature DC and the CD8+ cells into CTL cells;

mature DC and CD8+ cells loaded with polypeptide are added into a serum-free culture medium of HIPP-T009 lymphocytes together, autologous plasma and a costimulatory factor rh IL-21 are added for pre-stimulation, the addition amounts of the autologous plasma are 0%, 1%, 3% and 5% respectively, and the final concentration of the rh IL-21 is 20ng/ml. After three days of culture, cell growth factors rh IL-7 and rh IL-15 are added, cells are collected after 7 days of co-culture, and T cell expansion multiple and IFN-gamma secretion are detected.

FIG. 1 shows the effect of different ratios of autologous plasma (0%, 1%, 3%, 5%) on the fold expansion of specific T cells and the amount of Elispot IFN-. Gamma.secretion. From 0% to 3%, the T cell expansion fold is obviously improved along with the improvement of the autologous plasma proportion, and when the plasma proportion is increased from 3% to 5%, the expansion fold is slowly increased and is basically in a plateau phase.

The secretion of IFN-gamma of specific T cells cultured with the plasma proportion of 5% is the highest, the secretion of IFN-gamma of T cells cultured with the plasma proportion of 1% is 95000, which is higher than that of T cells cultured with 3% autologous plasma, and the secretion of IFN-gamma of T cells cultured without plasma is the lowest, which is 66000.

Considering that the collection amount of autologous plasma is limited, 1% -3% of autologous plasma can meet the requirement of T cell culture by combining the T cell amplification multiple and IFN-gamma secretion amount, and 2% of autologous plasma is preferentially selected for CTL cell amplification culture.

Example 2: effect of the Co-culture ratio of DC and CD8+ on the expansion of specific T cells

Steps

1 and 2 are the same as in example 1.

mature DCs and CD8+ cells loaded with polypeptide were added together to the serum-free culture medium of HIPP-T009 lymphocytes and pre-stimulated by adding co-stimulation factor rh IL-21 at a final concentration of 20ng/mL and autologous plasma at a volume concentration of 2%, the ratio of the number of co-cultured mature DCs to CD8+ cells was 1.

The results are shown in FIG. 2. Figure 2 shows the effect of different culture ratios of DC to CD8+ (1, 4, 1, 8, 1, 10, 1. The co-culture ratios 1.

When the DC: CD8+ coculture ratio 1, specific T cell IFN-gamma secretion is highest, higher than 1: t ratio 1.

And (3) pre-stimulating the CD8+ cells by the mature DC in combination with the amplification multiple and the IFN-gamma secretion amount, wherein the ratio of the number of co-cultured cells is 1.

Example 3: effect of cell growth factors on T cell culture

Steps

1 and 2 are the same as in example 1.

And 3, step 3: co-culturing the DC and the CD8+ cells, and activating the DC and the CD8+ cells into CTL cells;

mature DC and CD8+ cells loaded with polypeptide are added into a serum-free culture medium of HIPP-T009 lymphocytes together for co-culture, and rh IL-21 and 2% (v/v) autologous plasma are added into the culture system at a final concentration of 20ng/ml, the ratio of the number of co-cultured cells is DC: CD8+ =1, and the co-culture time is 7 days. Wherein the co-culture medium is also added with the following cell growth factors:

group A: rh IL-2 final concentration of 5ng/ml;

group B: the final concentration of rh IL-7 is 20ng/ml, and the final concentration of rh IL-15 is 20ng/ml;

group C: the final concentration of rh IL-7 is 10ng/ml, and the final concentration of rh IL-15 is 10ng/ml;

and comparing the expansion times and IFN-gamma secretion amounts of the T cells cultured by different cytokines. The results are shown in FIG. 3.

FIG. 3 shows the effect of three different sets A, B, C of cell growth factors on T cell culture. Wherein group A: the final concentration of rh IL-2 is 5ng/ml; group B: the final concentration of rh IL-7 is 20ng/ml, and the final concentration of rh IL-15 is 20ng/ml; group C: the final concentration of rh IL-7 is 10ng/ml, and the final concentration of rh IL-15 is 10ng/ml. When the DC and the CD8+ are cultured together, the DC is stimulated to be CTL cells, the final concentration of rh IL-7 is 20ng/ml, the final concentration of rh IL-15 is 20ng/ml, the cultured CTL cells are expanded by 16.1 times, the IFN-gamma secretion amount of the cultured CTL cells is 36000 and is higher than that of the group A, the expansion multiple of the CTL cells is 14.6 times, the IFN-gamma secretion amount of the CTL cells is 5200, the expansion multiple of the CTL cells is 13.9 times, and the IFN-gamma secretion amount of the CTL cells is 17200.

Therefore, the final concentration of the cell growth factor rh IL-7 was 20ng/ml and the final concentration of rh IL-15 was 20ng/ml, and specific CTL cell culture was performed.

In the second part, the number, the proportion and the antitumor ability of the tumor neoantigen-specific CTL cells obtained by amplification in example 7 using the improved concept of the present application are compared with those of the tumor neoantigen-specific CTL cells obtained by conventional methods. The culture conditions used in each example were optimized as described above.

Example 4

In this example, PBMC was directly non-specifically amplified, and the specific procedure included the following steps:

step 1: isolation of PBMC；

Mixing peripheral blood or single blood, centrifuging and collecting lower layer blood cells. Blood cells were diluted with DPBS, added to Ficoll separation medium, and centrifuged. Collecting the intermediate leucocyte layer cells, namely the Peripheral Blood Mononuclear Cells (PBMC). Wherein the centrifugation was 700g,5min, and the content of HSA (V/V) in DPBS (Gibco) was 2.5%.

Step 2: carrying out rapid mass amplification on the PBMC and magnetic beads coupled with the CD3/CD28 monoclonal antibody;

PBMC were co-cultured with CD3/CD 28-coupled immunomagnetic beads (Miltenyi Biotec) at a PBMC: beads =200 (V/V) to CD3/CD28 addition ratio.

The basal medium in this co-culture step was HIPP-T009 lymphocyte serum-free medium (purchased from Kyoto Biotech Co., ltd.) to which autologous plasma, co-stimulatory factors and cytokines were further added to give a co-culture system, wherein the co-culture system contained 2% autologous plasma, 20ng/ml rh IL-21, 20ng/ml rh IL-7, 20ng/ml rh IL-15. Observing the cell culture state during the culture period, wherein the observation indexes comprise: cell morphology, cell number, media color. If cell colonies appear, the number of cells increases, and the color of the culture medium turns yellow, the culture medium needs to be added or the wells need to be rotated.

After 2 days of co-culture in the rapid amplification stage, washing off residual magnetic beads, transferring cells to a cell culture bag or a G-REX bottle for large-scale amplification culture, wherein the cell transfer density is (2 multiplied by 10) ⁶ one/mL), cultured for 7 days.

Example 5

In this example, PBMC are stimulated with a tumor neoantigen, non-specifically amplified, and re-stimulated with the antigen, which includes the following steps:

step 1: separating PBMC;

Step 2: pre-stimulating PBMC with tumor neoantigen peptide;

the PBMC is pre-stimulated for 3 days by the tumor neoantigen peptide, and whether culture medium is added or not or a transfer hole is determined according to the cell culture state during culture. The content of HSA in the DC medium was 2% (v/v), the concentration of rh GM-CSF was 100ng/mL, and the concentration of rh IL-4 was 100ng/mL.

The following tumor neoantigen polypeptides were used, numbered SEQ ID NOs: 1 (amino acid sequence: KLMGIVYKV), SEQ ID NO:2 (amino acid sequence: SLDWWAFGV), SEQ ID NO:3 (amino acid sequence: VTFHIPFEV), SEQ ID NO:4 (amino acid sequence: AVGSYVYSV) and SEQ ID NO:5 (amino acid sequence: KLASYDMRL).

And 3, step 3: carrying out rapid mass amplification on the PBMC and magnetic beads coupled with the CD3/CD28 monoclonal antibody;

PBMC were co-cultured with CD3/CD 28-coupled immunomagnetic beads (Miltenyi Biotec) at a PBMC: beads =200 (V/V) to CD3/CD28 ratio. The basic culture medium is HIPP-T009 lymph cells are cultured in serum-free medium (substrate-doubled), on the basis, autologous plasma, co-stimulation factors and cell factors are added to obtain a co-culture system. Wherein the co-culture system comprises 2% (v/v) autologous plasma, 20ng/ml rh IL-21. Observing the cell culture state during the culture period, wherein the observation indexes comprise: cell morphology, cell number, media color. If cell colonies appear, the number of cells increases, and the color of the culture medium turns yellow, the culture medium needs to be added or the wells need to be rotated.

After 3 days of co-culture, washing off residual magnetic beads, transferring cells to a cell culture bag or a G-REX bottle for large-scale amplification culture, wherein the cell transfer density is 2 multiplied by 10 ⁶ pieces/mL, cultured for 7 days. Observing the cell culture state during the culture period, wherein the observation indexes comprise: cell morphology, cell number, culture medium color. If cell colonies appear, the number of cells increases, and the color of the culture medium turns yellow, the culture medium needs to be added or the wells need to be changed.

And 4, step 4: co-culturing the amplified PBMC and the tumor neoantigen for re-stimulation;

and co-culturing the amplified PBMC and the tumor neoantigen in a co-culture system of adding rh IL-7 with a final concentration of 20ng/mL and rh IL-15 with a final concentration of 20ng/mL into the serum-free culture medium of the HIPP-T009 lymphocytes. The cells were cultured for 3 days in total and restimulation was performed. During the culture period, whether to supplement the culture medium or to change the wells is determined according to the state of the cells being cultured.

Example 6

In this embodiment, after stimulating T cells with DCs loaded with tumor neoantigens, re-stimulating T cells with DCs again for specific amplification, the specific procedure includes the following steps:

step 1: PBMC separation and sorting；

Mixing peripheral blood or single blood, centrifuging and collecting lower layer blood cells. Blood cells were diluted with DPBS, added to Ficoll separation medium, and centrifuged. Collecting the intermediate leucocyte layer cells, namely the Peripheral Blood Mononuclear Cells (PBMC). The centrifugation conditions were 700g,5min, 2.5% HSA (v/v) contained in DPBS (Gibco).

PBMC after sieving, 10 times ⁹ Each cell was resuspended and mixed by adding 10mL of MACS buffer, followed by addition of 800. Mu.L of CD14 magnetic beads (Miltenyi Biotec for forward sorting. The collected CD 14-cells were subjected to forward sorting by adding 200. Mu.L of CD8 magnetic beads (Miltenyi Biotec)And (4) selecting.

Step 2: inoculating CD14+ cells, inducing in vitro differentiation into mature DCs；

The sorted CD14+ cells were resuspended in Human Serum Albumin (HSA) -containing DC medium, rh GM-CSF and rh IL-4 were added, and directed differentiation was performed. On the 4 th day, the maturation factors rh GM-CSF, rh IL-4, rh IL-1 beta, rh IL-6, TNF-alpha, PGE-2, poly I: C were added and the culture continued. Tumor neoantigen polypeptides were incubated with DCs on day 5, and then mature DCs were collected.

Wherein, the DC culture medium containing human serum albumin is obtained by adding HAS into AIM V culture medium, wherein the concentration of HAS in the DC culture medium is 2% (V/V), the concentration of added rh GM-CSF is 100ng/mL, and the concentration of rh IL-4 is 100ng/mL.

On day 4, a maturation-inducing factor combination was added, with a rh GM-CSF concentration of 100ng/mL, rh IL-4 concentration of 100ng/mL, rh IL-6 concentration of 40ng/mL, TNF- α concentration of 20ng/mL, rh IL-1 β concentration of 40ng/mL, PEG-2 concentration of 200ng/mL, POLYI: the concentration of C is 20ug/mL, the tumor neoantigen polypeptide is loaded on the 5 th day, and the final concentration of the polypeptide is 10 ug/mL.

And 3, step 3: co-culturing DC and CD8+ cells, activating them to CTL cells；

Mature DCs were pre-stimulated for CD8+ cells, with a ratio of numbers of co-cultured cells DC: CD8+ =1, and a co-culture time of 7 days. Meanwhile, the culture was performed using a costimulatory culture system obtained by adding rh IL-21 to a final concentration of 20ng/mL and 2% (v/v) of autologous plasma to the serum-free culture medium of HIPP-T009 lymphocytes.

The polypeptide-loaded DCs re-stimulated CTL cells for 1 day of co-culture, during which time HIPP-T009 lymphocyte serum-free medium was used for culture, and rh IL-7 was added to the culture system at a final concentration of 20ng/mL, rh IL-15 at a final concentration of 20ng/mL, and 2% (v/v) autologous plasma. Observing the cell culture state during the culture period, wherein the observation indexes comprise: cell morphology, cell number, media color. If cell colonies appear, the number of cells increases, and the color of the culture medium turns yellow, the culture medium needs to be added or the wells need to be changed.

Example 7

In this embodiment, after stimulating T cells with DC loaded with tumor neoantigens, re-stimulating T cells with DC, specifically amplifying, and performing undifferentiated amplification with CD3/CD28 immunomagnetic beads, the specific operation includes the following steps:

step 1: PBMC isolation and sorting, as in example 6;

and 2, step: CD14+ cells were seeded and induced to differentiate into mature DCs in vitro, as in example 6;

and 3, step 3: co-culturing the DC and CD8+ cells, activating them into CTL cells, as in example 6;

and 4, step 4: CTL cells were rapidly amplified in large quantities with magnetic beads coupled with CD3/CD28 monoclonal antibodies.

CTL cells were co-cultured with CD3/CD28 coupled immunomagnetic beads (Miltenyi Biotec) on magnetic beads at a ratio of T: beads =200 (v/v) CTL to CD3/CD28 cells. The co-culture system contained 2% (v/v) autologous plasma, rh IL-21 at a concentration of 20ng/mL, and cytokine rh IL-7 at a final concentration of 20ng/mL and cytokine rh IL-15 at a final concentration of 20ng/mL were added during the culture. Observing the cell culture state during the culture period, wherein the observation indexes comprise: cell morphology, cell number, culture medium color. If cell colonies appear, the number of cells increases, and the color of the culture medium turns yellow, the culture medium needs to be added or the wells need to be rotated.

After 3 days of co-culture, washing off residual magnetic beads, transferring cells to a cell culture bag or a G-REX bottle for large-scale amplification culture, wherein the cell transfer density is 2 multiplied by 10 ⁶ pieces/mL, cultured for 7 days.

And (3) detection: the effect of the cells cultured by the culture methods of examples 4 to 7 was examined, as follows:

(1) Specific T cell expansion fold evaluation test, the result is shown in figure 4. The specific operation comprises the following steps:

step 1:10 μ L of the cell suspension and 10 μ L of trypan blue (Biyun day) staining solution were added to a 1.5mL centrifuge tube, mixed well, 10 μ L of the mixed suspension was placed on a hemocytometer, and counted under a microscope for a plurality of times.

Step 2: live cells will not be stained blue and dead cells will be stained blue. The cell density and the cell number are calculated according to the cell count, and the cell activity requirement is not less than 90%.

Total number of cells = cell density x cell suspension volume;

fold expansion = number of T cells ultimately obtained/number of starting cells.

(2) Specific T cell ratio evaluation assay (tetramer assay) the results are shown in FIG. 4. The specific operation comprises the following steps:

step 1: taking 6X 10 ⁵ After adding 300. Mu.L of DPBS (Gibco) to the T cells prepared in examples 4 to 7, washing centrifugation (400g, 5 min) was performed to discard the supernatant, and 200. Mu.L of DPBS was added to resuspend the cells.

Step 2: 50 mu L of cell suspension is put into 2 centrifuge tubes with 1.5mL respectively, the control group is not processed, and 2 mu L of tetramer (APC-labelled) prepared from tumor neoantigen peptide is added into the experimental group.

And step 3: mu.L of PE anti-human CD8 (Biolegend) staining solution was added to each of the 2 tubes, mixed, and incubated at room temperature for 20min in the dark.

And 4, step 4: DPBS was added to the centrifuge tube to dilute the stained cells, mixed and centrifuged (400g, 5 min) to discard the supernatant. The cell pellet was flicked off, 200. Mu.L of DPBS resuspended cells, 5. Mu.L of 7-ADD (Biolegend) staining solution was added to each tube, incubated at room temperature in the dark for 5min, and then detected by flow cytometry (Beckman).

And 5: firstly, selecting a 7-ADD negative cell population, and then selecting APC +/PE + double positive cells from the 7-ADD negative cell population, wherein the proportion is the proportion of the specific T cells.

(3) The results of the T cell killing ability evaluation test (CFSE, 7-AAD staining test) are shown in FIG. 5. The specific operation comprises the following steps:

step 1: taking 1X 10 ⁶ Each placeThe tumor cells A375 in the logarithmic growth phase are placed in a 15mL centrifuge tube and centrifuged (200g, 5 min) to obtain cell precipitates. To the pellet, CFSE (biolegend, 200. Mu.L, final concentration 0.3. Mu.M) staining solution was added, and the mixture was stained in a37 ℃ cell culture chamber. And (3) taking out the cells after 20min, adding 5-10mL of DPBS (platelet-rich plasma-enhanced Raman Spectroscopy) for washing, and centrifuging (200g, 5 min) to obtain cell precipitates. Then, the cells were resuspended and counted in a T cell medium containing 2% FBS, and the cell suspension density was adjusted to 2X 10 ⁵ And (4) obtaining target cell suspension.

And 2, step: resuspending the T cells prepared in examples 4 to 7 with 2% of FBS-containing T cell medium, filtering with a 40 μm mesh, counting the cell suspension, and adjusting the cell density to 2X 10 ⁶ And (4) obtaining T cell suspension.

And step 3: to a 48-well plate, 250 μ L of T cell suspension and 250 μ L of target cell suspension (T cells: target cells = 10) were added for a total of 500 μ L/well. Blank set was 250 μ L of target cell suspension and 250 μ L of HIPP medium, and 250 μ L of DPBS was added to wells that were not loaded on the periphery of the well plate to prevent evaporation.

And 4, step 4: observing under a microscope, ensuring that the cells are uniformly dispersed in the culture well, placing the culture plate at 5% CO ₂ And culturing in a cell culture box at 37 ℃ for 18-24 h.

And 5: all cells in 48-well plates were collected by trypsinization, centrifuged (300g, 5 min) and the supernatant discarded. To the cells were added 150. Mu.L of DPB and 5. Mu.L of 7-AAD (BioLegend) staining solution and stained for 10min at room temperature in the dark.

Step 6: and (4) carrying out flow cytometry (Beckman) detection, wherein the ratio of the number of the CFSE and 7-ADD double positive cells to the number of the CFSE positive cells is the ratio of dead cells in the target cells.

And 7: cell killing experiments for T cells target cells =20 were performed. The steps are the same as the steps above, and the difference is that in the step 2: counting the cell suspension, adjusting the cell density to 4 × 10 ⁶ And (4) obtaining T cell suspension.

The kill rate is calculated by the formula: killing rate (%) = proportion of dead cells in target cells of experimental group-proportion of dead cells in target cells of negative control group.

(4) IFN-gamma secretion evaluation test (ELISPOT test) and the results are shown in figure 6. The specific operation comprises the following steps:

step 1: taking T2 cells in logarithmic growth phase, counting living cells, centrifuging (200g, 5 min), discarding supernatant, suspending cells by IMDM culture medium, adjusting cell density to 2 × 10 ⁵ one/mL. Cells were divided into 3 groups (positive, negative, example 1) and seeded in 6-well plates, with 1mL of T2 cells added to each group. PHA (phytohemagglutinin) with a final concentration of 4. Mu.g/mL was added to the positive group, and none was added to the negative group, and tumor neoantigen (1-40) with a final concentration of 1. Mu.g/mL was added to examples 4 to 7. Subjecting the T2 cells to CO ₂ Culturing in incubator for 2-6h, centrifuging (400g, 5 min) to collect cells, performing elastic precipitation, adding 0.5mL of 2% FBS-containing T cell culture medium per group, washing, and re-suspending, wherein the cell density after re-suspension is 2 × 10 ⁵ one/mL.

Step 2: taking 5X 10 ⁴ In each of examples 4-7, the supernatant was discarded by centrifugation (400g, 5 min), and the pellet was flicked off, and the cells were resuspended in 0.5mL of 2-% FBS-containing T-cell medium to a cell density of 2X 10 ⁵ One per mL.

And step 3: the ELISPOT (Mabtech) plates were washed 5 times with sterile PBS (150. Mu.L/well) and 150. Mu.L of 10% FBS-containing RPMI 1640 medium was added to each well, which was placed in CO ₂ The incubator is sealed for 30min. Discard the medium, then add 50. Mu.L of T2 cell suspension and 50. Mu.L of T cell suspension per well at 37 ℃ C. 5% ₂ The culture in the incubator is 18-24h, and the ELISPOT well plate is wrapped by tinfoil paper. The subsequent operations were performed strictly according to the kit instructions. When the number of spots in the negative group is less than 6/10 ⁴ The number of spots in the positive group is more than 15/10 ⁴ The set of data is counted as valid data.

IFN-gamma secretion (IFN-gamma expression positive cell number/10) ⁶ Viable cells) = (number of experimental group spots-number of negative control group spots)/number of T cell wells × 10 ⁶ 。

In the above examples, the culture process was performed by counting cells and performing related tests according to the cell demand, and the harvest time was monitored. And after the culture is finished, harvesting a final T cell product, washing by using normal saline, sampling, inspecting, and counting cells, detecting the specific cell proportion, the IFN-gamma secretion amount and the in-vitro cell killing capacity by using a tetramer flow.

The detection results of the above four indices are shown in fig. 4 to 6. Wherein,

FIG. 4 shows the fold expansion of specific T cells and the ratio of specific T cells obtained by cell counting and tetramer detection experiments in the present invention (example 7) and 3 other examples. As can be seen from the data, the amplification fold of example 4 (non-specific amplification of PBMC) was 124 fold, but the proportion of specific T cells was 0.18%, which was the lowest in the 4 examples. Example 5 (direct stimulation of PBMC with tumor neoantigen peptide, non-specific expansion with CD3/CD28, and stimulation again with tumor neoantigen) resulted in a lower fold expansion of T cells (53 fold) and a lower proportion of specific T cells (1.32%). Example 6 (tumor neoantigen peptide presented by DC, after co-culture with T cells, re-stimulation with DC) resulted in a higher proportion of specific T cells (6.48%), which were less amplified by a factor of 68 due to lack of non-specific amplification. In example 7 of the present invention (after stimulating T cells with DC carrying tumor neoantigen, re-stimulating T cells with DC, specifically amplifying them, and then differentially amplifying them with CD3/CD28 immunomagnetic beads), the amplification factor of the specific T cells obtained by culturing was 176, and the ratio thereof was 7.91%. Compared with the T cells cultured in other 3 examples, the expansion multiple of the T cells is the highest, and the content of specific T cells is also the highest.

FIG. 5 shows the cell killing ability of the T cells obtained in the 4 examples at different effective target ratios. When the T cell: the killing rate of T cells obtained in example 7 of the present invention was 71.2% when target cells = 10. The killing rate was 18 times that of example 4, 4.5 times that of example 5, and 1.2 times that of example 4. When the T cell: when the target cell =20, the killing rate of the T cell obtained in example 7 of the present invention is 92%, which is 21.5% higher than the killing rate of the effective target cell which is 10. The kill rate of example 4 increased from 3.9% (10. The kill rate of example 5 increased from 15.4% (10. The kill rate of example 6 increased from 63.7% (10. When the effective target ratio is increased, the killing rate of the T cells obtained in the 4 examples is increased to different degrees. This shows that the killing rate of the target cells is significantly higher in example 7 than in other examples, and that the higher the effective target ratio, the higher the killing rate of the specific T cells.

FIG. 6 shows the IFN-. Gamma.secretion by the tumor-specific T cells obtained in 4 examples. The secretion of IFN-gamma is detected by ELISOPT experiment, and the data shows that the secretion of IFN-gamma of T cell cultured in the example 7 of the invention is the highest, which also shows that the specificity of T cell is the best, and is consistent with the experimental result of FIG. 4.

From the above description, it can be seen that the above-described embodiments of the present invention are adapted by integrating the DC re-stimulation step and the magnetic bead amplification step into the original protocol through the improvement and innovation of the process of the previous protocol. Experiments prove that the innovative process is effective, and the number and the proportion of tumor specific T cells in a T cell final product are improved. The following technical effects are realized:

1) Before indiscriminate stimulation amplification, the DC is used for pre-stimulating and re-stimulating the PBMC-derived initial sample, so that the proportion of tumor-specific T cells in the final T cell product is remarkably increased, the targeted killing property of the tumor is improved, and the toxic and side effects on an organism are reduced.

2) After re-stimulation, T cells were indiscriminately stimulated and expanded using CD3/CD28 loaded magnetic beads, resulting in a significant increase in the total number of tumor-specific T cells in the final T cell product.

3) The preparation time of the tumor specific T cells is shortened.

4) Most of the existing problems of the TILs method are circumvented.

It should be noted that, the time from the stage of obtaining peripheral blood mononuclear cells to the final product is 22-25 days, and the time of each process step can be further optimized on the basis of the time to shorten and simplify the process. In addition, the specific T cell ratio in the pre-stimulation stage CTL can be further increased by further improving the DC maturation process, so that the specific T cell ratio is further improved.

In the cell culture obtained by the culture method for preparing the specific T cells, the total number of tumor specific CTL cells and the ratio of the tumor specific CTL cells to the final T cell product are remarkably improved, so that research and development of corresponding cell therapy products can be carried out on the basis of the process, for example, TCR-T, CAR-T, DC-CIK or DC-CTL and the like can be carried out by adopting the process for preparing the cells.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

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Claims

1. A method for culturing tumor-specific CTL cells, comprising:

inducing and differentiating the PBMC in vitro into mature DCs loaded with tumor neoantigen polypeptides;

co-culturing said mature DCs with CD8+ cells, thereby stimulating activation of said CD8+ cells into CTL cells;

expanding the CTL cells.

2. The culture method of claim 1, wherein inducing differentiation of said PBMCs into mature DCs loaded with tumor neoantigen polypeptides in vitro comprises:

isolating PMBC and sorting CD14+ cells and CD8+ cells from said PBMCs;

performing directional differentiation on the CD14+ cells to obtain differentiated DCs;

and co-incubating the tumor neogenesis antigen polypeptide and the differentiated DC to obtain the mature DC loaded with the tumor neogenesis antigen polypeptide.

3. The culture method according to claim 2, wherein the subjecting the CD14+ cells to committed differentiation to obtain differentiated DCs comprises:

adopting a DC culture medium to carry out resuspension on the CD14+ cells to obtain resuspended cells;

adding rh GM-CSF and rh IL-4 into the resuspended cells for directional differentiation to obtain first-stage differentiated cells;

adding a maturation factor into the first-stage differentiated cells to continue culturing to obtain the differentiated DCs;

preferably, the DC medium is a human serum albumin-containing medium; more preferably, the DC medium is obtained by adding the human serum albumin to any one of the following media: AIM-V medium, X-VIVO 15 medium and CellGenix dendritic cell medium; further preferably, the volume concentration of the human serum albumin in the DC culture medium is 2.5%, the working concentration of the rh GM-CSF is 10 ng/mL-120 ng/mL, and the working concentration of the rh IL-4 is 10 ng/mL-120 ng/mL;

preferably, the maturation factor is selected from any one or more of: rh GM-CSF, rh IL-4, rh IL-1 beta, rh IL-6, TNF-alpha, PGE-2, and Poly (I: C); more preferably, the final concentrations of each of said maturation factors are as follows: the concentration of rh GM-CSF is 50 ng/mL-200 ng/mL, the concentration of rh IL-4 is 50 ng/mL-120 ng/mL, the concentration of rh IL-1 beta is 10 ng/mL-120 ng/mL, the concentration of rh IL-6 is 10 ng/mL-120 ng/mL, the concentration of TNF-alpha is 10 ng/mL-120 ng/mL, the concentration of PEG-2 is 10 ng/mL-1000 ng/mL, and the concentration of Poly (I: C) is 2 mu g/mL-40 mu g/mL;

preferably, the directed differentiation is carried out on days 1-2, the continuous culture is carried out on days 3-5, and the tumor neoantigen polypeptide and the differentiated DC are co-incubated on days 6-9 to obtain a mature DC loaded with the tumor neoantigen polypeptide;

more preferably, the final concentration of the tumor neoantigen polypeptide is 0.1-40. Mu.g/mL.

4. The culture method of any one of claims 1 to 3, wherein co-culturing the mature DCs with CD8+ cells, such that stimulating activation of the CD8+ cells into CTL cells comprises:

(ii) subjecting said CD8+ cells to a step-wise stimulation with said mature DCs, thereby activating said CD8+ cells to CTL cells;

preferably, the mature DC is adopted to perform pre-stimulation co-culture on the CD8+ cells for 7-10 days to obtain pre-activated cells;

and (3) performing re-stimulation co-culture on the pre-activated cells for 1-2 days by adopting the mature DCs to obtain the CTL cells.

5. The culture method according to any one of claims 1 to 3, wherein expanding the CTL cells comprises:

amplifying the CTL cells by adding magnetic beads or soluble monoclonal antibodies of the monoclonal antibodies coupled with the CD3/CD28 into the CTL cells to obtain the tumor-specific CTL cells;

preferably, the CTL cells and magnetic beads coupled with CD3/CD28 monoclonal antibodies or soluble anti-CD3/CD28 monoclonal antibodies are subjected to co-culture, preferably the co-culture is carried out for 1 to 5 days, and then the CTL cells are transferred to a cell culture bag or a G-REX bottle for expansion culture, preferably the expansion culture is carried out for 5 to 12 days, so as to obtain the tumor-specific CTL cells;

preferably, the density of the expanded culture is maintained at 1X 10 ⁶ ～2×10 ⁶ one/mL.

6. The culture method according to claim 5, wherein the volume ratio of the CTL cells to the magnetic beads is from 100 to 1, preferably from 200;

preferably, the CTL cells and magnetic beads coupled with CD3/CD28 monoclonal antibodies or soluble anti-CD3/CD28 monoclonal antibodies are subjected to co-culture, wherein a co-culture system comprises autologous plasma, rh IL-21, rh IL-7 and rh IL-15, the volume content of the autologous plasma is 0-10%, preferably 2-3%, the concentration of the rh IL-21 is 5-30 ng/mL, preferably 20ng/mL, the concentration of the rh IL-7 is 5-120 ng/mL, preferably 20ng/mL, and the concentration of the rh IL-15 is 5-120 ng/mL, preferably 20ng/mL;

preferably, the concentration of the soluble anti-CD3/CD28 monoclonal antibody in the co-culture system is 30-50 ng/mL;

preferably, during the co-culturing process, it is determined whether the co-cultured cells require medium addition or rotation of wells based on any one or more of the following observations: cell morphology, cell number, or culture medium color.

7. The culture method of claim 2, wherein isolating PMBC and sorting CD14+ cells and CD8+ cells from the PBMCs comprises:

mixing peripheral blood or single blood, centrifuging and collecting lower layer blood cells;

diluting the blood cells by DPBS, placing the blood cells in a Ficoll separating medium for centrifugal separation, and collecting intermediate leucocyte cells to obtain the PBMC;

performing incubation sorting on the PBMCs by using CD14 magnetic beads, and collecting the CD14+ cells;

adding CD8 magnetic beads into the CD 14-cells for incubation, and sorting out the CD8+ cells;

more preferably, prior to the incubation sorting of the PBMCs with the CD14 magnetic beads, the culturing method further comprises the step of screen filtering the suspension containing PBMCs;

it is further preferred that the first and second liquid crystal display panels,

mixing peripheral blood or single blood, centrifuging at 650-750 g for 3-6 min, and collecting lower layer blood cells;

diluting said blood cells with said DPBS containing 2.5% HSA (V/V), followed by centrifugation in a Ficoll medium, and collecting intermediate buffy coat cells to obtain said PBMCs;

after the PBMC were subjected to screen filtration, the PBMC were added to 10 portions each ⁹ Adding 5-30 mL of MACS buffer solution into the PBMC for resuspension to obtain a PBMC resuspension solution;

adding 100-1000. Mu.L of CD14 magnetic beads into the PBMC heavy suspension for forward sorting, and respectively collecting the CD14+ cells and the CD 14-cells;

and adding 100-1000 mu L of CD8 magnetic beads into the CD 14-cells for forward sorting, and collecting the CD8+ cells.

8. The culture method according to claim 3, wherein the tumor neoantigen polypeptide is selected from any one or more of the following: KLMGIVYKV, SLDWWAFGV, VTFHIPFEV, AVGSYVYSV, and KLASYDMRL.

9. The culture method according to claim 4, wherein the ratio of the number of cells of the mature DCs to the number of cells of the CD8+ cells in the pre-stimulation co-culture is 1;

preferably, said pre-stimulatory co-culture and/or said re-stimulatory co-culture are performed using a co-stimulatory culture system comprising autologous plasma, a co-stimulatory factor and serum-free medium;

preferably, the volume content of the autologous plasma in the co-stimulation culture system is 0-10%; more preferably 2% -3%;

preferably, the co-stimulatory factor is rh IL-21 and the concentration of the rh IL-21 in the co-stimulatory culture system is 5-30 ng/ml, more preferably 20ng/ml;

preferably, during the pre-stimulation co-culture and/or the re-stimulation co-culture, a step of supplementing cell growth factors or performing hole transfer on the co-cultured cells is further included;

preferably, the supplemented cell growth factors are rh IL-7 and rh IL-15, the final concentration of the supplemented rh IL-7 is 5-120 ng/mL, and the final concentration of the rh IL-15 is 5-120 ng/mL;

more preferably, the final concentration of the supplemented rh IL-7 is 20ng/mL and the final concentration of rh IL-15 is 20ng/mL.

10. A cell therapy product comprising tumor-specific CTL cells cultured in vitro, wherein said tumor-specific CTL cells in said cell therapy product are cultured by the culture method according to any one of claims 1 to 9.