A method for amplifying tumor-infiltrating T lymphocytes derived from autologous cancerous pleural effusion and ascites

Abstract

The present invention discloses a method for amplifying tumor-infiltrating T lymphocytes derived from autologous cancerous pleural effusion, which belongs to the field of cell separation and culture technology, and includes the following steps: S1, collection of autologous pleural effusion; S2, preparation of samples to be sorted; S3, addition of sorting magnetic beads; S4, elution and loading; S5, elution of non-target cells and elution and collection of target cells; S6, in vitro activation of tumor-infiltrating T lymphocytes; S7, in vitro amplification of tumor-infiltrating T lymphocytes; S8, identification and harvesting of tumor-infiltrating T lymphocytes. The amplification method provided by the present invention can fully activate TIL cells by using nano-scale particles coupled to CD3 / CD28 antibodies to activate tumor-infiltrating T lymphocytes obtained by sorting and purification, so that the proliferation ability of TIL cells is greatly improved, and sufficient TIL cells can be obtained at the end of culture.

Description

Technical Field

[0001] The invention belongs to the technical field of cell separation and culture, and in particular relates to a method for amplifying tumor-infiltrating T lymphocytes derived from autologous cancerous pleural effusion and ascites. Background Art

[0002] With the aging of the population, the incidence and mortality of cancer in my country will not be optimistic in the future. Therefore, new treatment methods are urgently needed to overcome the global problem of tumor treatment.

[0003] The occurrence of tumors is the result of somatic gene mutations. Usually, the body's immune system will monitor the existence of such abnormal somatic cells and eliminate them through cytotoxicity. Unfortunately, tumor cells can not only escape the recognition and attack of the immune system through various mechanisms, but also inhibit the function of immune cells through the tumor immune microenvironment, providing conditions for their further growth and spread. It can be seen that the body's immune function plays a vital role in the occurrence and development of tumors, so tumor immunotherapy technology came into being.

[0004] Currently, tumor infiltrating lymphocyte (TIL) therapy has become a new star in the field of solid tumor treatment. Its principle is to infiltrate tumor infiltrating lymphocytes (mainly CD8 + T lymphocytes are extracted, amplified in vitro, and then injected back into the patient's body. Then IL-2 injection is needed to maintain the activity of TIL cells. TIL cells entering the patient's body can effectively migrate to the tumor lesions and exert anti-tumor effects after specifically identifying tumor cells. Compared with other cell therapy methods, the process of TIL cells killing cancer cells has great advantages such as strong specificity, high sensitivity, good tumor chemotaxis and good body tolerance. TIL cell therapy has achieved remarkable therapeutic effects in the field of melanoma, with a total effective rate of about 50%, and up to 25% of patients maintain lasting complete remission after treatment.

[0005] The primary key issue for the success of TIL therapy is how to amplify a large number of TIL cells with strong anti-tumor activity in vitro within a certain period of time. However, two major problems are usually encountered when amplifying TIL cells in vitro: one is the source of TIL. If TIL cells are obtained from tumor tissue, it involves the complexity of clinical cooperation and tumor tissue digestion operations. Not only are the patient screening conditions harsh, resulting in a large number of patients being unable to accept TIL therapy, but also a large number of original TIL cells will be lost, resulting in a reduction in the number of TIL cells finally obtained; the second is that because of the long-term immunosuppressive environment created by tumor cells, the proliferation ability of TIL cells is severely inhibited, resulting in a reduction in the number of TIL cells finally obtained. In response to the above two problems, the present invention provides a method for amplifying tumor-infiltrating T lymphocytes derived from autologous cancerous pleural effusions. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for amplifying tumor-infiltrating T lymphocytes derived from autologous cancerous pleural effusion and ascites.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] A method for amplifying tumor-infiltrating T lymphocytes derived from autologous cancerous pleural effusion and ascites, comprising the following steps:

[0009] S1. Collection of autologous pleural and ascites: Aseptically collect the patient's autologous pleural and ascites, centrifuge, and collect the cell precipitate;

[0010] S2. Preparation of samples to be sorted: adding a sorting buffer to wash the cell precipitate collected in step S1, then centrifuging, removing the supernatant, and then adding a sorting buffer to resuspend the cell precipitate to obtain a cell suspension, counting cells in the cell suspension, and treating the cell suspension by adding a sorting buffer according to the counting result to obtain a cell suspension reaction solution;

[0011] S3, adding sorting magnetic beads: adding sorting magnetic beads to the cell suspension reaction solution obtained in step S2, mixing evenly, incubating at 2-4° C. for 15-20 min, and obtaining a mixture after the incubation is completed;

[0012] S4, elution and loading: add sorting buffer to the mixture obtained in step S3 for elution. After elution, discard the supernatant and adjust the total cell concentration to 1.5-2×10^8 / ml with sorting buffer to obtain a cell magnetic bead mixed suspension, and then perform the loading operation;

[0013] S5. Elution of non-target cells and elution and collection of target cells: After the loading operation in step S4 is completed, non-target cells and target cells are eluted in sequence using a sorting buffer, and then the target cell suspension is collected and counted to determine the number and viability of target cells. When the viability and CD3 positivity of the target cells meet the requirements of sorting and purification, a sorted and purified tumor-infiltrating T lymphocyte suspension is obtained;

[0014] S6. In vitro activation of tumor-infiltrating T lymphocytes: centrifuge the tumor-infiltrating T lymphocyte suspension obtained in step S5 and discard the supernatant to obtain a tumor-infiltrating T lymphocyte precipitate, wash the tumor-infiltrating T lymphocyte precipitate by pipetting, then centrifuge and discard the supernatant, resuspend the cell precipitate to obtain a cell suspension, then add an activation reagent, mix well, add to a cell culture plate, and incubate for 2-3 days to obtain activated cells;

[0015] S7. In vitro expansion of tumor-infiltrating T lymphocytes: collect the activated cells obtained in step S6 and count the cells, then centrifuge and discard the supernatant, resuspend the cell pellet, and culture it in a cell culture plate, then count the cells every other day, readjust the live cell concentration to 1-1.5×10^6 / ml based on the counting results, and add cytokine IL-2. During the routine culture process, the cultured cells are transferred from the cell plate to a cell culture flask and a cell culture bag in turn according to the cell culture volume for culture;

[0016] S8. Identification and harvesting of tumor-infiltrating T lymphocytes: After two weeks of culture, the cultured tumor-infiltrating T lymphocytes can be identified and harvested as needed.

[0017] Preferably, in step S2, step S4 and step S5, the sorting buffer is 1×PBS+0.5% FBS+2mMEDTA, and the sorting buffer is used at 4°C.

[0018] Preferably, in step S2, the processing of the cell suspension by adding a sorting buffer according to the counting result is specifically as follows: when the density of cells in the cell suspension is greater than 1×10^8 / ml, adding a sorting buffer to the cell suspension to adjust the total cell concentration to 1×10^8 / ml.

[0019] Preferably, in step S3, the volume ratio of the cell suspension reaction solution obtained in step S2 to the added amount of the sorting magnetic beads is 10:1.

[0020] Preferably, in step S4, the specific steps of the loading operation are: placing the sorting column on the magnetic rack, then adding sorting buffer to the center of the sorting column to activate the sorting column, and immediately adding the cell magnetic bead mixed suspension to the center of the sorting column after the sorting buffer flows out.

[0021] Preferably, in step S5, the elution of the non-target cells and the target cells specifically comprises the following steps:

[0022] S51, adding a sorting buffer to the center of the sorting column loaded with the cell magnetic bead mixed suspension, repeating the above operation multiple times to obtain a target cell precipitate;

[0023] S52, removing the sorting column loaded with the target cell precipitate obtained in step S51 from the magnetic rack, adding sorting buffer to the center of the sorting column, and then transferring it to a centrifuge tube, mixing it evenly, and collecting the target cell suspension.

[0024] Preferably, in step S5, the target cell viability and CD3 positivity meet the requirements of sorting and purification as follows: the target cell viability reaches more than 90% and the CD3 positivity reaches more than 80%.

[0025] Preferably, in step S6, the activation reagent is a nanometer-scale particle coupled to a CD3 / CD28 antibody.

[0026] Preferably, in step S6, the volume ratio of the cell suspension to the activation reagent is 200:1.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The TIL cells in the present invention are TIL cells derived from pleural effusion and ascites. Compared with tumor tissue, cancerous pleural effusion and ascites are easier to obtain clinically and more easily meet the sample standards required for preparing TIL cells, so they can benefit a wider range of patients. In addition, the amplification method provided by the present invention can obtain original cells through a simple centrifugation operation, and the operation at the laboratory level is also very simple, avoiding the loss of original cells. At the same time, as a simulated waste in the treatment process, the acquisition of cancerous pleural effusion and ascites does not increase the cost of TIL cell therapy, thereby reducing the treatment cost of TIL cell therapy.

[0029] (2) The amplification method provided by the present invention uses nano-scale superparamagnetic microbeads coupled with CD3 antibodies to sort and purify tumor-infiltrating T lymphocytes at the initial stage of culture, which can completely cut off the inhibitory effect of tumor cells at the initial stage of amplification at the source, allowing TIL cells to enter the amplification stage more quickly, and the TIL cells obtained after separation and purification have a higher purity and more ideal cell performance;

[0030] (3) The amplification method provided by the present invention can fully activate TIL cells by using nano-scale particles coupled to CD3 / CD28 antibodies to activate tumor-infiltrating T lymphocytes obtained by sorting and purification, so that the proliferation ability of TIL cells is greatly improved, and sufficient TIL cells can be obtained at the end of culture;

[0031] (4) The nanoscale superparamagnetic microbeads coupled to CD3 antibodies and the nanoscale particles coupled to CD3 / CD28 antibodies used in the present invention do not affect downstream experiments, are GMP grade, biodegradable, harmless to cells and humans, and the cells harvested after culture can be used for clinical trials;

[0032] (5) The amplification method provided by the present invention can produce 10^9 TIL cells in a 15-day culture cycle, and the cell viability is as high as 98%, the CD3 positive rate is as high as 89.6%, and the CD8 positive rate, which mainly plays a role in killing cancer cells, is as high as 98%. + The proportion of cytotoxic T cells was as high as 96.6%. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a diagram showing the results of sorting and purifying tumor-infiltrating T lymphocytes in step S5 of an embodiment of the present invention;

[0034] Figure 2 This is a specific fluorescent image of cell nucleus staining when TIL cells are counted on a cell counter using the AO / PI staining method during initial culture;

[0035] in, Figure 2 (a) is a white light image; Figure 2 (b) is the fluorescence image; Figure 2 (c) is the matching graph;

[0036] Figure 3 The before and after morphology comparison diagram of the activated cell and the unactivated cell obtained in step S6 of the embodiment of the present invention;

[0037] in, Figure 3 (a) is the cell morphology before activation; Figure 3 (b) is the cell morphology after 3 days of activation;

[0038] Figure 4 This is a trend line diagram of the in vitro expansion of tumor-infiltrating T lymphocytes during the culture process of step S7 of an embodiment of the present invention;

[0039] Figure 5 This is a statistical graph of the viability of tumor-infiltrating T lymphocytes during the culture process of step S7 of an embodiment of the present invention;

[0040] Figure 6This is a specific fluorescent image of cell nucleus staining when the tumor-infiltrating T lymphocytes are counted on a cell counter using the AO / PI staining method after 15 days of culture in the embodiment of the present invention;

[0041] in, Figure 6 (a) is a white light image; Figure 6 (b) is the fluorescence image; Figure 6 (c) is the matching graph;

[0042] Figure 7 This is a morphological diagram of tumor-infiltrating T lymphocytes after 15 days of culture in the embodiment of the present invention;

[0043] in, Figure 7 (a) is the morphological image at 20x; Figure 7 (b) is the morphological image at 40x;

[0044] Figure 8 This is a diagram showing the phenotypic identification of tumor-infiltrating T lymphocytes after 15 days of culture in an example of the present invention. DETAILED DESCRIPTION

[0045] The exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described above. On the contrary, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0046] Example 1

[0047] The embodiment of the present invention provides a method for amplifying tumor-infiltrating T lymphocytes derived from autologous cancerous pleural effusion and ascites, which specifically comprises the following steps:

[0048] S1. Collection of autologous pleural and ascites: Autologous pleural and ascites are waste materials from the treatment of malignant tumor patients. With the consent of the patients, the autologous pleural and ascites of the patients are collected aseptically for cell preparation. The amount of pleural and ascites collected at a time is at least 200 ml to avoid red blood cell contamination. The pleural and ascites are centrifuged at 300g for 10 minutes, and the supernatant is fully discarded after the centrifugation, and the cell precipitate is collected;

[0049] S2. Preparation of samples to be sorted: Add an appropriate amount of sorting buffer (sorting buffer formula: 1×PBS+0.5% FBS+2mMEDTA, used at 4°C) to fully blow and rinse the cell pellet collected in step S1, then centrifuge at 300g for 10 minutes, discard the supernatant after the end, add an appropriate amount of sorting buffer to fully resuspend the cell pellet to obtain a cell suspension, and then count the cells of the cell suspension (AO / PI dual fluorescence nucleic acid counting method, Countstar Mira FL cell counter), and treat the cell suspension by adding sorting buffer according to the counting results. Specifically, when the cell density in the cell suspension is greater than 1×10^8 / ml, add sorting buffer to the cell suspension to adjust the total cell concentration to 1×10^8 / ml. When the total number of cells is less than 1×10^7, directly resuspend with 100ul of sorting buffer to obtain a cell suspension reaction solution;

[0050] S3, adding sorting magnetic beads: add sorting magnetic beads (GenScript Biotech, CytoSinct Biotech) to the cell suspension reaction solution obtained in step S2 at a ratio of 10ul of sorting magnetic beads per 100ul of cell suspension reaction solution. TM CD3 Nanobeads, human), shake the magnetic beads in the tube thoroughly before aspirating them, shake the mixture gently after adding them, mix them evenly, incubate them at 4°C for 15 minutes, shake them gently every 5 minutes, and get a mixture after the incubation.

[0051] S4, elution and sample loading: 1-2 ml of sorting buffer was added to the mixture obtained in step S3 at a ratio of 100 ul of the mixture of cells and magnetic beads for elution. After the sorting buffer was added, it was gently inverted upside down. After the elution was completed, centrifugation was performed at 300 g for 10 min. After discarding the supernatant, the total cell concentration was adjusted to 2×10^8 / ml with sorting buffer to obtain a cell magnetic bead mixed suspension, and then the sample loading operation was performed. Before loading, the sorting column (GenScript Biotech, CytoSinct TM gM column) was placed on a magnetic rack (GenScript Biotech, CytoSinct TM gStand; GenScript Biotech, M8 magnet), and at the same time, add 500ul of sorting buffer to the center of the sorting column, so that the added buffer flows out of the column to achieve the purpose of activating the sorting column. After the buffer flows out, immediately add the cell magnetic bead mixed suspension slowly to the center of the sorting column. According to the principle of positive screening, non-target cells will flow out of the sorting column, while target cells will remain in the sorting column;

[0052] S5, elution of non-target cells and elution and collection of target cells: After the loading operation in step S4 is completed, the non-target cells and target cells are eluted in sequence by the sorting buffer, and then the target cell suspension is collected and the cell count is performed to determine the number and viability of the target cells, and then 2×10^4 live cells are taken for flow antibody staining of CD3, CD4, and CD8, and then analyzed using the Live / hCD45+ gate of the flow cytometer. When the target cell viability reaches more than 90% and the CD3 positive rate reaches more than 80%, it can be judged that the sorting and purification effect is ideal, and the tumor-infiltrating T lymphocyte suspension after sorting and purification is obtained. The elution of the non-target cells and target cells specifically includes the following steps:

[0053] S51, add 500ul of sorting buffer to the center of the sorting column loaded with the cell magnetic bead mixed suspension, and collect the elution buffer flowing out with a centrifuge tube at the bottom of the sorting column. Note that this operation needs to be repeated three times. Each time the sorting buffer is added, it is necessary to wait until the last sorting buffer has just flowed out to obtain the target cell precipitation;

[0054] S52. Remove the sorting column loaded with the target cell precipitate obtained in step S51 from the magnetic rack, place it on a 15 ml centrifuge tube, add 1 ml of sorting buffer to the center of the sorting column, and then immediately use the plunger of the sorting column to quickly push the sorting buffer into the 15 ml centrifuge tube to collect the target cells, and then mix evenly to collect the target cell suspension.

[0055] S6. In vitro activation of tumor-infiltrating T lymphocytes: The tumor-infiltrating T lymphocyte suspension obtained in step S5 was centrifuged at 300 g for 10 min, and the supernatant was fully discarded to obtain a tumor-infiltrating T lymphocyte precipitate. The tumor-infiltrating T lymphocyte precipitate was washed with 10 ml of normal saline, and then centrifuged at 300 g for 10 min, and the supernatant was discarded. The cell precipitate was resuspended in serum-free T cell complete medium (medium formula: X-VIVO 15 (LONZA) + 10 ng / ml IL-2 (Dakoway)) at a total cell concentration of 1×10^6 / ml to obtain a cell suspension, and then nano-scale particles coupled to CD3 / CD28 antibodies (from commercially available, manufacturer: GenScript GeneScript Biotech, model: EnceedTM T cell Activation, human), after fully mixing, add to 6-well cell culture plates, control the volume of each well to 3ml-5ml, place in a 37°C, 5% CO2 incubator for 2-3 days to obtain activated cells, observe the cells every day during this period, and be careful not to perform any operations on the cells before 2 days. If the cells are observed to be transformed into permeable round cells and grow in clusters after 2d-3d, it indicates that the activation effect is ideal and the conventional in vitro expansion stage can be entered;

[0056] S7. In vitro expansion of tumor-infiltrating T lymphocytes: collect the activated cells obtained in step S6 and count the cells, then centrifuge at 300g for 10 minutes, discard the supernatant, resuspend the cell pellet with serum-free T cell complete medium at a live cell concentration of 1×10^6 / ml, and place it in a cell culture plate for culture, then observe the cells under a microscope every other day and count the cells, readjust the live cell concentration to 1×10^6 / ml with serum-free T cell complete medium according to the counting results, and add cytokine IL-2 at a concentration of 10ng / ml according to the adjusted total volume. During the routine culture process, the cultured cells are transferred from the cell plate to a cell culture flask and a cell culture bag in turn according to the cell culture volume for culture. Specifically, as the cell culture volume continues to increase, when the culture volume reaches 60ml, the cells can be transferred from the cell plate to a T75 cell culture flask, when the culture volume reaches 150ml, they can be transferred to a T175 cell culture flask, and when the culture volume reaches 500ml, they can be transferred to a 2L cell culture bag for culture;

[0057] S8. Identification and harvesting of tumor-infiltrating T lymphocytes: After two weeks of culture, the cultured tumor-infiltrating T lymphocytes can be identified and harvested as needed. The specific operation is to collect the cell culture suspension to be harvested into a centrifuge tube, centrifuge at 300g for 10 minutes, discard the supernatant, and then use physiological saline to wash the cell pellet, then centrifuge at 300g for 10 minutes, discard the supernatant, and then use physiological saline to wash the cell pellet, mix and count the cells, and determine the total number of cells and cell viability obtained after the end of the culture. Then, according to the results of the cell counting, take 1×10^5 total cells and use flow cytometry analysis technology to perform phenotypic detection on the finally harvested tumor-infiltrating T lymphocytes. The specific operation is to select CD3, CD4, and CD8 flow antibodies to stain the identified cells, and analyze the Live / hCD45+ gate.

[0058] The results of sorting and purifying tumor-infiltrating T lymphocytes in step S5 of the present embodiment are as follows: Figure 1 As shown, through Figure 1 It can be seen that before purification and sorting, CD3 + The proportion of cells in the total cell population was 29.3%, and after purification and sorting, CD3 + The proportion of cells in the total cell population is 82.0%, indicating that the separation and purification effect of step S5 is ideal.

[0059] The counting results of tumor infiltrating T lymphocytes before culture in step S7 of the embodiment of the present invention are shown in Table 1. As can be seen from Table 1, the total number of TIL cells at the beginning of culture is only 9.96 × 10^6, but the cell viability is good at 95%, indicating that the experimental operation did not damage the cells. Figure 2 The specific fluorescence image of the cell nucleus staining when TIL cells are counted on a cell counter using the AO / PI staining method at the beginning of culture is Figure 2 The results further illustrate the correctness and authenticity of the counting results in Table 1.

[0060] Table 1 Statistical table of cell counting results before TIL cell culture

[0061]

[0062] Figure 3 The morphological comparison diagram of the activated cell and the unactivated cell obtained in step S6 of the embodiment of the present invention is shown in FIG. Figure 3 It can be seen that the diameter of the activated T cells is larger than that of the resting cells, and the activated cells grow in clusters, indicating that the T cells are successfully activated. The average diameters of the activated cells and the unactivated cells are shown in Table 2 below.

[0063] Table 2 Statistical table of average cell diameter before and after TIL cell activation

[0064] Before activation(μm) After activation(μm) Average cell diameter 9.04 10.75

[0065] From the results in Table 2, it can be seen that the average diameter of the cells before activation was 9.04 μm, while the average diameter of the cells 3 days after activation was 10.75 μm.

[0066] Figure 4 and Figure 5 They are respectively the in vitro amplification trend line and the activity statistical chart of tumor infiltrating T lymphocytes in the culture process of step S7 of the embodiment of the present invention, Figure 4 It can be seen that after two weeks of culture, the TIL cells expanded from only 9.96×10^6 cells at the beginning to 1.35×10^9, and the overall cell expansion rate showed a "slow fast slow" change, which is consistent with the normal cell expansion state. Figure 5 It can be seen that after the cells entered the conventional expansion stage, the cell viability remained at a high level, indicating that the cell activity was good.

[0067] The statistical results of the counting results of tumor-infiltrating T lymphocytes after 15 days of culture in the embodiment of the present invention are shown in Table 3 below.

[0068] Table 3 TIL cell count results at the end of 15 days of culture

[0069]

[0070] It can be seen from the results in Table 3 that, through the in vitro expansion method provided in the embodiment of the present invention, 10^9 TIL cells can be obtained after 15 days of culture, and the cell viability is as high as 98%. The preparation process of the current leading company in the TIL industry, iovance, USA, prepares 10^9 to 10^11 TIL cells in a 22-day culture cycle. Therefore, the number of TIL cells obtained by the in vitro expansion method provided in the embodiment of the present invention is almost the same as the number of cells obtained by the existing leading companies, and the culture cycle of the present invention is significantly shorter than the existing culture cycle, which saves culture time and improves culture efficiency. Figure 6 The specific fluorescence image of the cell nucleus staining is shown in the cell counting method of the tumor infiltrating T lymphocytes after 15 days of culture in the embodiment of the present invention. Figure 6 The results further illustrate the correctness and authenticity of the counting results in Table 3.

[0071] Figure 7 This is a morphological diagram of tumor-infiltrating T lymphocytes after 15 days of culture in the present invention. Figure 7It can be seen that the cells are full in morphology and in good condition at the end of the culture. Table 4 is a statistical table of morphological parameters of tumor-infiltrating T lymphocytes after 15 days of culture in the embodiment of the present invention.

[0072] Table 4 Statistics of TIL cell morphological parameters at the end of 15 days of culture

[0073] Average cell diameter (μm) 10.17μm Cell roundness (standard circle is 1) 0.92

[0074] It can be seen from Table 4 that the average diameter of the cultured tumor-infiltrating T lymphocytes is 10.17 μm, and the cell roundness (the standard circle is 1) is 0.92, which further illustrates that the cell morphology is full and the cell state is good.

[0075] Figure 8 This is a phenotypic identification diagram of tumor-infiltrating T lymphocytes after 15 days of culture in the embodiment of the present invention. Figure 8 It is shown that 96.6% of the TIL cells cultured by the expansion method provided in the embodiment of the present invention are cytotoxic T cells when finally harvested, and the cell phenotype is CD3 + and CD8 + T, indicating that the TIL cells cultured by the amplification method provided in the embodiment of the present invention have stronger killing function against tumor cells. The statistical table of TIL cell phenotype parameters after 15 days of culture is shown in Table 5 below.

[0076] Table 5 Statistics of TIL cell phenotype ratios at the end of 15 days of culture

[0077]

[0078] Since CD8 plays a cytotoxic role in the process of cellular immunity + T cells, so the tumor-infiltrating T lymphocytes harvested were CD8 + The higher the proportion of T cells, the stronger the anti-cancer activity of tumor-infiltrating T lymphocytes. It can be seen from the results in Table 5 that the CD8 + The proportion of cytotoxic T cells is as high as 96.6%, so the tumor-infiltrating T lymphocytes amplified by the in vitro amplification method provided by the embodiment of the present invention have excellent cancer cell killing effect.

[0079] In summary, the method for amplifying tumor-infiltrating T lymphocytes provided in the embodiment of the present invention can obtain a sufficient amount of TIL cells, and the cell viability is as high as 98%, the CD3 positive rate is as high as 89.6%, and the CD8 positive rate that mainly plays a role in killing cancer cells is as high as 98%. + The proportion of cytotoxic T cells is as high as 96.6%, while reducing the cost of TIL cell therapy.

[0080] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for amplifying tumor-infiltrating T lymphocytes derived from autologous cancerous pleural effusion and ascites, characterized in that: The following steps are involved: S1. Collection of autologous pleural and ascites: Aseptically collect the patient's autologous pleural and ascites, centrifuge, and collect the cell precipitate; S2. Preparation of samples to be sorted: adding a sorting buffer to wash the cell precipitate collected in step S1, then centrifuging, removing the supernatant, and then adding a sorting buffer to resuspend the cell precipitate to obtain a cell suspension, counting cells in the cell suspension, and treating the cell suspension by adding a sorting buffer according to the counting result to obtain a cell suspension reaction solution; S3, adding sorting magnetic beads: adding sorting magnetic beads to the cell suspension reaction solution obtained in step S2, mixing evenly, incubating at 2-4°C for 15-20 min, and obtaining a mixture after the incubation is completed; The volume ratio of the cell suspension reaction solution obtained in step S2 to the added amount of the sorting magnetic beads is 10:1; The separation magnetic beads are nano-scale superparamagnetic microbeads coupled with CD3 antibodies; S4, elution and loading: add sorting buffer to the mixture obtained in step S3 for elution. After elution, discard the supernatant and adjust the total cell concentration to 1.5-2×10^8 / ml with sorting buffer to obtain a cell magnetic bead mixed suspension, and then perform the loading operation; S5. Elution of non-target cells and elution and collection of target cells: After the loading operation in step S4 is completed, non-target cells and target cells are eluted in sequence using a sorting buffer, and then the target cell suspension is collected and counted to determine the number and viability of target cells. When the viability and CD3 positivity of the target cells meet the requirements of sorting and purification, a sorted and purified tumor-infiltrating T lymphocyte suspension is obtained; The target cell viability and CD3 positivity meet the requirements of sorting and purification as follows: the target cell viability reaches more than 90% and the CD3 positivity reaches more than 80%; In step S2, step S4 and step S5, the sorting buffer is 1×PBS + 0.5% FBS + 2mM EDTA, and the sorting buffer is used at 4°C; S6. In vitro activation of tumor-infiltrating T lymphocytes: The tumor-infiltrating T lymphocyte suspension obtained in step S5 is centrifuged and the supernatant is discarded to obtain a tumor-infiltrating T lymphocyte precipitate, the tumor-infiltrating T lymphocyte precipitate is washed by pipetting, then centrifuged and the supernatant is discarded, and the cell precipitate is resuspended in serum-free T cell complete medium at a total cell concentration of 1×10^6 / ml to obtain a cell suspension, and then an activation reagent is added, mixed evenly, and added to a cell culture plate, and incubated for 2-3 days to obtain activated cells; The formula of the culture medium is: X-VIVO 15+10ng / ml IL-2; the volume ratio of the cell suspension to the activation reagent is 200:1; the activation reagent is nano-scale particles coupled with CD3 / CD28 antibodies; S7. In vitro expansion of tumor-infiltrating T lymphocytes: collect the activated cells obtained in step S6 and count the cells, then centrifuge and discard the supernatant, resuspend the cell pellet, and culture it in a cell culture plate, then count the cells every other day, readjust the live cell concentration to 1-1.5×10^6 / ml based on the counting results, and add cytokine IL-2. During the routine culture process, transfer the cultured cells from the cell plate to a cell culture flask and a cell culture bag in turn according to the cell culture volume for culture; S8. Identification and harvesting of tumor-infiltrating T lymphocytes: After two weeks of culture, the cultured tumor-infiltrating T lymphocytes can be identified and harvested as needed.

2. The method for amplifying tumor-infiltrating T lymphocytes derived from autologous cancerous pleural effusion and ascites according to claim 1, characterized in that: In step S2, the cell suspension is processed by adding a sorting buffer according to the counting result. Specifically, when the density of cells in the cell suspension is greater than 1×10^8 / ml, a sorting buffer is added to the cell suspension to adjust the total cell concentration to 1×10^8 / ml.

3. The method for amplifying tumor-infiltrating T lymphocytes derived from autologous cancerous pleural effusion and ascites according to claim 1, characterized in that: In step S4, the specific steps of the sample loading operation are: placing the sorting column on the magnetic rack, then adding sorting buffer to the center of the sorting column to activate the sorting column, and immediately adding the cell magnetic bead mixed suspension to the center of the sorting column after the sorting buffer flows out.

4. The method for amplifying tumor-infiltrating T lymphocytes derived from autologous cancerous pleural effusion and ascites according to claim 3, characterized in that: In step S5, the elution of the non-target cells and the target cells specifically includes the following steps: S51, adding a sorting buffer to the center of the sorting column loaded with the cell magnetic bead mixed suspension, repeating the above operation multiple times to obtain a target cell precipitate; S52, removing the sorting column loaded with the target cell precipitate obtained in step S51 from the magnetic rack, adding sorting buffer to the center of the sorting column, and then transferring it to a centrifuge tube, mixing it evenly, and collecting the target cell suspension.

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