Gene modified K562 cell and application thereof in TIL amplification

Abstract

The invention relates to the field of cell preparation, in particular to a gene modified K562 cell and application thereof in TIL amplification. The invention discloses a gene modified K562 cell, wherein the K562 cell contains a polynucleotide sequence for coding IL-15, 4-1BBL and CD86. The invention also discloses a feeder cell-based TIL cell preparation method, the key quality attributes (such as cell amplification and motility, killing function and the like) of the TIL cells after cryopreservation and recovery of the TIL cells prepared by the method are obviously improved, and the method has important significance for improving the clinical curative effect of TIL cell treatment.

Description

Technical Field

[0001] The present invention relates to the field of cell preparation, and in particular to a gene-modified K562 cell and its application in TIL expansion. Background Art

[0002] Cell and Gene Therapy (CGT) products have become the forefront and hot spot of global medical development. Among them, TIL cell immunotherapy (Tumor Infiltrating Lymphocytes) has shown good results in the treatment of solid tumors. Currently, TIL cell drugs have been approved for marketing, and many TIL cell drugs for different solid tumors have been approved for IND and are in different clinical stages. TIL cell immunotherapy is to separate T cells from the patient's tumor tissue, stimulate and amplify them in vitro, and then return them to the patient's body to expand the immune response and achieve the purpose of treating primary or secondary tumors. Its principle is mainly that a large number of T cells are infiltrated in the tumor tissue, among which there are some T cells targeting tumor-specific antigens, which can penetrate deep into the tumor tissue and specifically kill the tumor. TIL cell immunotherapy shows broad application prospects in the treatment of solid tumors.

[0003] The active raw materials of TIL cells are mainly tumor tissues or lymph nodes removed from patients during surgery. The tumor cell suspension obtained after the tumor tissues or lymph nodes are transferred step by step contains some lymphocytes that are in a "sleeping" state and target tumor-specific antigens. These cells are activated and expanded in vitro to become T cells with effector functions and then returned to the patient's body. They can penetrate deep into the tumor tissue and specifically kill the tumor by restarting the T cell immune response signaling pathway, thereby achieving the purpose of treating primary or secondary tumors.

[0004] The main shortcomings and deficiencies of the current TIL cell activation and expansion process are as follows: (1) Tumor tissue processing stage The traditional method of processing tumor tissue is to place the tumor tissue in a culture dish with sterile shearing fluid, and use sterile scissors to cut all the tumor tissue into small pieces without visible tissue fragments to obtain lymphocyte suspension. This processing method may not be able to completely infiltrate and release the lymphocytes in the tumor tissue, resulting in a decrease in the yield of lymphocyte suspension in the tumor tissue.

[0005] (2) Rapid expansion phase (REP) Traditional cell activation mainly uses 3-6 different healthy donor sources mixed and irradiated heterologous PBMC as feeder cells, which are then added in a certain proportion to activate and expand TIL cells. The main disadvantages of heterologous PBMC-derived feeder cells are as follows: ① Feeder cells from heterologous donors will introduce batch-to-batch heterogeneity and variability when activating TIL, which may affect the product stability and clinical treatment effect of TIL cells during commercial production.

[0006] ② When irradiated PBMC from multiple heterologous donors are used as feeder cells, the amount used is generally large (e.g., TIL: feeder cells = 1:50-1:100), which increases the cost of feeder cells used in the production of a single batch of TILs and increases the risk of residual feeder cells in the product; ③ A large number of allogeneic healthy donor PBMCs need to be separated from single blood samples, which has a high procurement cost. Single blood samples from donors need to be transported through a cold chain and are generally required to be processed within 48 hours, so the logistics and transportation requirements are relatively high. Summary of the invention

[0007] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for preparing TIL cells based on K562 feeder cells and its application. In the rapid expansion phase (REP), feeder cells obtained by irradiating monoclonal genetically modified K562 cells are used to activate and expand TIL cells.

[0008] The present application provides a genetically modified K562 cell, wherein the K562 cell contains polynucleotides encoding IL-15, 4-1BBL and CD86.

[0009] The present application provides a method for amplifying tumor-infiltrating lymphocytes, using the above-mentioned K562 cell culture to amplify tumor-infiltrating lymphocytes.

[0010] The present application provides the use of the above-mentioned K562 cells or the above-mentioned method in the preparation of tumor infiltrating lymphocytes.

[0011] The beneficial effects brought by this application include but are not limited to: (1) The yield of TIL cells obtained by pre-amplification culture of tumor tissue by step-by-step shear infiltration is increased by 1.5 times, and the cell viability is above 90%. (2) Since the monoclonal genetically modified K562 feeder cells are uniform and stable in batches, the stability and efficacy of TILs cell products after activation and expansion of K562 feeder cells can be guaranteed. In addition, genetically modified K562 can be used as needed by establishing a batch cell bank, which can greatly reduce the cost of using allogeneic PBMC-derived feeder cells when producing a single batch of TIL cells, while reducing the risk of residual feeder cells in TIL products. In addition, with the same TIL:feeder cell ratio of 1:25, the expansion multiple of TIL cells stimulated and expanded by genetically modified K562 feeder cells for 14 days reached more than 1300 times (2.14 times the expansion multiple of TIL cells stimulated and cultured by PBMC feeder cells), the cell viability was more than 90%, the TIL cell phenotype (CD3 positive rate) reached more than 95%, the feeder cell residues were negative in the whole process stage, the cell killing efficiency was 25.4%, 46.8% and 55.9% at the effector-target ratio of 1:1, 1:3 and 1:10 respectively (no significant difference from the killing efficiency of TIL cells stimulated and cultured by PBMC feeder cells), and the IFN-γ secretion reached 689pg / ml at the effector-target ratio of 1:10 (slightly higher than the IFN-γ secretion of TIL cells stimulated and cultured by PBMC feeder cells). This preparation method is simple, efficient and conducive to the large-scale production of TIL cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present application will be further described in the form of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not restrictive, and include: Figure 1 This is a cooling program for freezing cells according to some embodiments of the present application.

[0013] Figure 2 The present invention is a flowchart of the TIL cell preparation process according to some embodiments of the present application.

[0014] Figure 3 It is a bar graph of cell viability in the Pre-REP stage according to some embodiments of the present application.

[0015] Figure 4 It is a bar graph showing the number of live cells in the Pre-REP stage according to some embodiments of the present application.

[0016] Figure 5 This is a TIL cell expansion curve diagram according to some embodiments of the present application.

[0017] Figure 6 It is a TIL cell viability curve diagram according to some embodiments of the present application.

[0018] Figure 7 This is the TIL cell phenotype shown in some embodiments of the present application.

[0019] Figure 8 is the yield of TIL cells after recovery as shown in some embodiments of the present application.

[0020] Fig. 9 is the viability of TIL cells after recovery as shown in some embodiments of the present application.

[0021] Fig.10 This is the killing efficiency of TIL cells after recovery as shown in some embodiments of the present application.

[0022] Fig.11 is the amount of IFN-γ secretion of TIL cells after recovery as shown in some embodiments of the present application. DETAILED DESCRIPTION

[0023] In order to more clearly illustrate the technical solutions of the embodiments of this specification, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some examples or embodiments of this specification. For ordinary technicians in this field, this specification can also be applied to other similar scenarios based on these drawings without creative work. Unless it is obvious from the language environment or otherwise explained, the same reference numerals in the figures represent the same structure or operation.

[0024] As shown in this specification and claims, unless the context clearly indicates an exception, the words "a", "an", "an" and / or "the" do not refer to the singular and may also include the plural. Generally speaking, the terms "comprise" and "include" only indicate the inclusion of the steps and elements that have been clearly identified, and these steps and elements do not constitute an exclusive list. The method or device may also include other steps or elements.

[0025] Flowcharts are used in this specification to illustrate the operations performed by the system according to the embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed precisely in order. Instead, the steps may be processed in reverse order or simultaneously. At the same time, other operations may also be added to these processes, or one or more operations may be removed from these processes.

[0026] The present application provides a genetically modified K562 cell, wherein the K562 cell contains polynucleotides encoding IL-15, 4-1BBL and CD86.

[0027] In some embodiments, the K562 cells may express IL-15, 4-1BBL and CD86.

[0028] In some embodiments, the K562 cells may express membrane-bound IL-15, 4-1BBL and CD86.

[0029] The present application also provides a method for amplifying tumor-infiltrating lymphocytes, using the above-mentioned K562 cell culture to amplify tumor-infiltrating lymphocytes.

[0030] In some embodiments, the method may specifically include the following steps: 1) isolating tumor infiltrating lymphocytes from tumor tissue, culturing them in a culture medium containing IL-2 for 7 to 15 days to obtain first cells; 2) irradiating the above-mentioned K562 cells to obtain second cells; 3) mixing the first cells and the second cells at a ratio of (0.5 to 1.5): (20 to 30), culturing, and obtaining CD3-positive tumor infiltrating lymphocytes. In some embodiments, preferably, in step 3), the first cells and the second cells may be mixed at a ratio of 1:25.

[0031] In some embodiments, in the step 1), tumor infiltrating lymphocytes are isolated from tumor tissue and cultured in a culture medium containing IL-2 for 8 to 14 days to obtain a first cell. In some embodiments, in the step 1), tumor infiltrating lymphocytes are isolated from tumor tissue and cultured in a culture medium containing IL-2 for 9 to 13 days to obtain a first cell. In some embodiments, in the step 1), tumor infiltrating lymphocytes are isolated from tumor tissue and cultured in a culture medium containing IL-2 for 10 to 12 days to obtain a first cell. In some embodiments, in the step 1), tumor infiltrating lymphocytes are isolated from tumor tissue and cultured in a culture medium containing IL-2 for 11 to 12 days to obtain a first cell.

[0032] In some embodiments, the first cell and the second cell may be mixed in a ratio of (0.75-1.25):(22-28). In some embodiments, the first cell and the second cell may be mixed in a ratio of (1-1.25):(24-26).

[0033] In some embodiments, in step 1), the concentration of IL-2 in the culture medium may be 2000-4000 IU / ml. For example, the concentration of IL-2 in the culture medium may be 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900 or 4000 IU / ml. Any range characterized by a combination of the above end values ​​is also included, which will not be repeated here.

[0034] In some embodiments, in step 1), the tumor may be melanoma.

[0035] In some embodiments, in step 3), the culture medium used for culturing may contain IL-2 and CD3 antibodies. In some embodiments, preferably, in step 3), the concentration of IL-2 in the culture medium may be 2000-4000 IU / ml. For example, the concentration of IL-2 in the culture medium may be 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900 or 4000 IU / ml. It also includes any range characterized by a combination of the above end values, which will not be repeated here.

[0036] In some embodiments, preferably, in step 3), the concentration of the CD3 antibody in the culture medium can be 20 to 40 ng / ml. For example, the concentration of the CD3 antibody in the culture medium can be 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 ng / ml. Any range characterized by a combination of the above end values ​​is also included, which will not be repeated here.

[0037] In some embodiments, in step 3), the culture time can be 7 to 15 days. For example, the culture time can be 7, 8, 9, 10, 11, 12, 13, 14 or 15 days. Any range characterized by a combination of the above end values ​​is also included, which will not be repeated here.

[0038] In some embodiments, preferably, in step 3), the culturing time can be 14 days.

[0039] In some embodiments, in step 3), the first living cell density may be 1 to 1.5 x 10 6 / ml. For example, the first viable cell density can be 1 x10 6 / ml, 1.1 x10 6 / ml, 1.2 x10 6 / ml, 1.3 x10 6 / ml, 1.4 x10 6 / ml or 1.5x10 6 Any range characterized by a combination of the above end values ​​is also included, which will not be described in detail here.

[0040] In some embodiments, in step 3), after mixed culture, the solution may be replenished every 2 to 4 days. In some embodiments, in step 3), the first living cell culture density may be 5 to 10 x 10 5 / ml.

[0041] In some embodiments, in the steps 1) and 3), the culture medium used in the culture may be a serum-free culture medium. In some embodiments, in the steps 1) and 3), preferably, the serum-free culture medium may be an AIM V serum-free culture medium.

[0042] The present application also provides the use of the above-mentioned K562 cells or the above-mentioned method in the preparation of tumor infiltrating lymphocytes.

[0043] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged. Example 1

[0044] Based on the unique characteristics of TIL cells, K562 cells were precisely genetically modified, mainly by introducing membrane-bound interleukin factors, optimizing immune stimulatory molecules, and modifying antigen-specific genes. The specific modifications are as follows: 1) Induced activation function: Select interleukin factors that can maintain memory T cells and will not cause T cell apoptosis, and design membrane-bound interleukin factors to overcome the short serum half-life of secreted factors, so as to more effectively induce and maintain T cell proliferation and differentiation.

[0045] 2) Selection of co-stimulatory signals: Prefer immunostimulatory molecules that bind to receptors highly expressed by activated CD4+ and CD8+ T cells. By providing co-stimulatory signals, they significantly promote the in vitro expansion of T cells and provide a high-quality cell source for the preparation of cell drugs.

[0046] 3) Antigen-specific modification: Gene modification of markers on the surface of K562 cells that may cause the dominant growth of immune cells avoids the impact on cell consistency, thereby improving the quality controllability of the CMC part of the cell drug and ensuring stable efficacy in the clinical stage. This modification also highlights the advantages of K562 cells as feeder cells compared to PBMC.

[0047] The successfully constructed IL-15, 4-1BBL and CD86 gene-modified K562 cells have undergone monoclonal screening, library construction testing, functional verification and safety evaluation method development and verification. The K562 feeder cells obtained after irradiation can specifically enhance the proliferation ability, cell killing function and sustained survival ability of T cells in vivo, significantly enhancing its efficacy in anti-tumor treatment.

[0048] The steps for constructing the genetically modified K562 monoclonal cell line are as follows: (1) The IL-15, 4-1BBL and CD86 gene sequences were constructed into the Pre-Lenti-EF1-CAR-V2-WPmut vector, and the constructed expression vector was used to transform the IL-15, 4-1BBL and CD86 genes of the K562 wild-type cell line (purchased from ATCC) by lentiviral transduction to obtain IL-15, 4-1BBL and CD86 gene-modified K562 polyclonal cells. Then, the K562 polyclonal cells were sorted by flow cytometry to obtain multiple IL-15, 4-1BBL and CD86 triple-positive monoclonal clones, and each monoclonal cell was traced by photographic origin and expanded and cultured. Finally, 10 IL-15, 4-1BBL and CD86 gene-modified K562 monoclonal cell lines with IL-15, 4-1BBL and CD86 triple-positive rates of more than 99% were successfully screened by flow cytometry. Example 2

[0049] This patent embodiment uses melanoma tissue from melanoma patients to prepare TIL cells, specifically involving the following steps: tumor tissue acceptance and processing, TIL cell rapid expansion preparation, TIL cell activation and rapid expansion culture, and TIL cell harvesting, preparation, and cryopreservation. (1) Tumor tissue acceptance Two melanoma tissues from melanoma patients obtained by surgical resection were stored in isotonic nutrient tissue receiving fluid with antibiotics added [AIM V serum-free medium (Gibco, catalog number: A3021002) + 100mg / ml streptomycin, penicillin (hyclone, catalog number: SV30010) + 50mg / ml gentamicin (Shanghai Shangyao Zhongxi Pharmaceutical Co., Ltd., catalog number: National Medicine Standard H31022243)] and transported to the cell preparation room through a 2-8°C cold chain. At the same time, the tissue receiving fluid was sampled for sterility testing, and the sterility test was negative.

[0050] (2) Tumor tissue processing Melanoma tissue was divided into two equal parts according to weight and processed as follows: ①In the biosafety cabinet, add 5-8 ml of sterile shearing solution [AIM V serum-free medium (Gibco, catalog number: A3021002) + 3000 IU / ml IL-2 (Beijing Shuanglu Pharmaceutical Co., Ltd., catalog number: S19991007)] to 5 culture dishes in turn, and mark them as A, B, C, D, and E. Place one of the melanoma tissues in the tissue receiving solution in culture dish A, and cut the single tumor tissue into 3-5 pieces. Use new sterile tweezers to transfer all tissues in culture dish A to culture dish B and let it stand for 2-3 minutes. Use new sterile tweezers to transfer all tissues in culture dish B to culture dish C, and cut all tumor tissues into 2 pieces. Use new sterile tweezers to transfer all tissues in culture dish C to culture dish D and let it stand for 2-3 minutes. Use new sterile forceps to transfer all tissues in the D dish to the E dish, and cut all tumor tissues into pieces of about 27 cubic millimeters (3×3×3mm blocks). Transfer the tissues in the E dish and the obtained lymphocyte suspension to a T75 cell culture flask containing 15-25ml of sterile shearing fluid, blow 6-12 times and let it stand for 4-6 minutes. If fat floating tissue is found, suck it out and discard it. The sampled cell suspension was tested for sterility, and the sterility test was negative.

[0051] ②In the biosafety cabinet, add 5-8 ml of sterile shearing fluid [AIM V serum-free medium (Gibco, catalog number: A3021002) + 3000 IU / ml IL-2 (Beijing Shuanglu Pharmaceutical Co., Ltd., catalog number: S19991007)] to a culture dish. Place another melanoma tissue in the tissue receiving fluid in a culture dish, and cut all tumor tissues into pieces of about 27 cubic millimeters (3×3×3mm blocks). Then transfer the tissue in the culture dish and the obtained lymphocyte suspension to a T75 cell culture flask containing 15-25 ml of sterile shearing fluid, blow 6-12 times, and let it stand for 4-6 minutes. If fat floating tissue is found, absorb and discard it. The sampled cell suspension was tested for sterility, and the sterility test was negative.

[0052] (3) TIL cell rapid expansion preparation (Pre-REP) Transfer the prepared 1000ml Pre-REP complete medium [AIM V serum-free medium (Gibco, catalog number: A3021002) + 3000IU / ml IL-2 (Beijing Shuanglu Pharmaceutical Co., Ltd., catalog number: S19991007)] to two corresponding G-Rex 100Ms, and then transfer all the cell suspensions and tissues obtained from the cell culture flasks in ① and ② above to G-Rex 100Ms respectively, and perform TIL cell pre-expansion culture in a 37°C, 5% CO2 incubator. The same day is D1, and the expansion does not exceed 15 days. Sampling and counting begin on D12. Pre-REP cells are harvested when the total number of viable cells is ≥2E7. The REP stage is entered on the same day. If the total number of viable cells is less than 2E7, continue to culture and count on D13 and D14 in sequence. If the total number of viable cells counted on D14 is still less than 2E7, harvest directly on D15. The two cell suspensions were filtered through a 70 µm cell strainer into a 50 ml centrifuge tube to remove large cell clumps and tissues. The two cell suspensions were collected and then entered the next step of rapid expansion culture. In addition, the D7 culture supernatant and the cell suspension collected on the last day of Pre-REP were taken for sterility testing, and the sterility test was negative.

[0053] (4) Rapid expansion of TIL cells (REP) ①Activation culture (D1) The two cell suspensions harvested in the Pre-REP stage were combined and mixed evenly, and samples were counted. According to the counting results, they were divided into two parts, centrifuged at 400g for 10min, and the supernatant was discarded. Then, the cells were respectively cultured with REP expansion medium [AIM V serum-free medium (Gibco, catalog number: A3021002) + 3000IU / ml At the same time, add CD3 monoclonal antibody (Takara, Catalog No.: T210) at a final concentration of 30 ng / ml, and then add IL-15, CD86 and 4-1BBL genetically modified and irradiated K562 feeder cells (Puxin Biotech) that have been revived in a 37°C water bath and washed and replaced with new medium, and 3 kinds of allogeneic donors (Heyousheng, batch number: EZ4016 / EZ4131 / 013k058) mixed PBMC irradiated feeder cells in a ratio of 1:25. Finally, the two cell suspensions were transferred to the corresponding cell culture bags (OriGen Biomedical Inc., Catalog No.: PL30-2G) and activated and cultured in a 37°C, 5% CO2 incubator for 3 days.

[0054] ② Fluid culture (D4) After activation and culture of the two cell lines for 3 days, samples were taken and counted. According to the counting results, REP expansion medium [AIM V serum-free medium (Gibco, catalog number: A3021002) + 3000IU / ml IL-2 (Beijing Shuanglu Pharmaceutical Co., Ltd., catalog number: S19991007)] was supplemented to adjust the live cell density to 5-10E5 / ml. At the same time, samples were taken to detect TIL phenotype and residual feeder cells. The two groups of remaining cells in the culture bags were cultured in a 37°C, 5% CO2 incubator for 3 days.

[0055] ③Xuri culture (D7) After culturing the two cell lines for 3 days, samples were taken and counted. The two cell suspensions were washed by centrifugation at 400g for 10 min, and the supernatant was discarded. REP expansion medium [AIM V serum-free medium (Gibco, catalog number: A3021002) + 3000IU / ml IL-2 (Beijing Shuanglu Pharmaceutical Co., Ltd., catalog number: S19991007)] was added to adjust the live cell density to the same 5-10E5 / ml. At the same time, samples were taken to detect the TIL phenotype and residual feeder cells. The remaining two groups of cells were transferred to Cellbag 10 L culture bags (Cytiva, catalog number: 29-1054-99), and the Cellbag 10 L culture bags were assembled into two Xuri-W25 reactors (Cytiva, model: 29064568) respectively. The Xuri reactors were adjusted to the same speed (4-6rpm) and angle (2-4°), and Xuri dynamic culture was started. Thereafter, samples were taken and counted every 2 days, and REP expansion medium was pumped in through sterile welding to adjust to the same viable cell density of 5-10E5 / ml. After the culture volume reached 5000mL, samples were taken and counted every day, and the perfusion rate and Xuri reactor speed and angle were adjusted to the same according to the cell growth density, and dynamic culture was carried out at 37°C and 5% CO2.

[0056] ④TIL cell harvesting, preparation and cryopreservation The total number of viable cells required was determined according to the reinfusion dose of the TIL cell preparation. Generally, the total number of cells can reach the harvest standard within 14 days of culture. At D14, the two groups of TIL cells were sampled and counted, and TIL phenotype, feeder cell residue, sterility and mycoplasma detection were performed based on the counting results. Then, the two groups of cells dynamically cultured by Xuri were harvested and washed and concentrated by LOVO fully automatic cell processing and purification system (Fresenius Kabi, model: 6R4900), and finally replaced with 100ml cell suspension respectively. Then, according to the platform preparation formula [20% HSA: compound electrolyte: CS10 = 2:2:6 (V / V)], the viable cell density was adjusted to 1E7 / ml-1E8 / ml, and they were divided into several cryopreservation bags (Miltenyi, catalog number: 200-074-400) according to a certain volume, and then vented, vacuumed and vacuum-sealed respectively. Finally, a program cooling instrument (Thermo Fisher, model: 7453) was used to preset the cryopreservation program (attached Figure 1 ) The TIL cell preparation was programmed to cool down, and after the end, the TIL cell preparation was transferred to a gas phase liquid nitrogen tank for long-term storage. After 2-7 days of cryopreservation, the two groups of TIL cells were taken out and revived for cell counting, TIL phenotype, feeder cell residues, cell killing and γ-IFN secretion detection.

[0057] The data show that: (1) The yield of TIL cells obtained by pre-amplification culture of tumor tissue by step-by-step shear infiltration increased by 1.5 times (see Figure 4 ), and the cell viability is above 90% (see Figure 3 ).

[0058] (2) With the same TIL:feeder cell ratio of 1:25, the expansion fold of TIL cells stimulated and expanded by genetically modified K562 feeder cells for 14 days reached more than 1300 times (2.14 times the expansion fold of TIL cells stimulated and expanded by PBMC feeder cells) (see Figure 5 ), the cell viability is above 90% (see Figure 6 ), the TIL cell phenotype (CD3 positive rate) of the finished products harvested and frozen during the REP culture stage reached more than 95% after cryopreservation and resuscitation (see Figure 7 ), the feeder cell residues in the entire REP stage and after cryopreservation and resuscitation of the finished product were negative (see Table 1 for details), and the TIL cell yield after cryopreservation and resuscitation of the finished product reached 104% (slightly higher than the yield of TIL cells stimulated by PBMC feeder cells after cryopreservation and resuscitation of the finished product) (see Figure 8 ), the viability of TIL cells after cryopreservation and resuscitation of the finished product reached 92.5% (slightly higher than the viability of TIL cells after cryopreservation and resuscitation stimulated by PBMC feeder cells) (see Fig. 9), the killing efficiency of primary tumor cells of patients at effector-target ratios of 1:1, 1:3 and 1:10 were 25.4%, 46.8% and 55.9%, respectively (with no significant difference in killing efficiency of TIL cells stimulated by PBMC feeder cells) (see Fig.10 ), when the effector-target ratio was 1:10, IFN-γ secretion reached 689pg / ml (slightly higher than IFN-γ secretion of TIL cells cultured by PBMC feeder cells) (see Fig.11 ).

[0059] Table 1

[0060] The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only for example and does not constitute a limitation of this specification. Although not explicitly stated here, those skilled in the art may make various modifications, improvements and corrections to this specification. Such modifications, improvements and corrections are suggested in this specification, so such modifications, improvements and corrections still belong to the spirit and scope of the exemplary embodiments of this specification.

[0061] At the same time, this specification uses specific words to describe the embodiments of this specification. For example, "one embodiment", "an embodiment", and / or "some embodiments" refer to a certain feature, structure or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more in different positions in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures or characteristics in one or more embodiments of this specification can be appropriately combined.

[0062] In some embodiments, numbers describing the number of components and attributes are used. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise specified, "about", "approximately" or "substantially" indicate that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may change according to the required features of individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and adopt the general method of retaining the digits. Although the numerical domains and parameters used to confirm the breadth of the range in some embodiments of this specification are approximate values, in specific embodiments, the setting of such numerical values ​​is as accurate as possible within the feasible range.

[0063] Finally, it should be understood that the embodiments described in this specification are only used to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, as an example and not a limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly introduced and described in this specification.

Claims

1. A genetically modified K562 cell, characterized in that: The K562 cells contain polynucleotides encoding IL-15, 4-1BBL and CD86.

2. The K562 cell according to claim 1, wherein The K562 cells express IL-15, 4-1BBL and CD86.

3. The K562 cell according to claim 1, characterized in that The K562 cells express membrane-bound IL-15, 4-1BBL and CD86.

4. A method for amplifying tumor infiltrating lymphocytes, characterized in that: The K562 cells as described in any one of claims 1 to 3 are used to culture and expand tumor infiltrating lymphocytes.

5. The method according to claim 4, characterized in that The specific steps include: 1) Isolating tumor infiltrating lymphocytes from tumor tissue and culturing them in a medium containing IL-2 for 7 to 15 days to obtain the first cells; 2) irradiating the K562 cells according to any one of claims 1 to 3 to obtain a second cell; 3) The first cell and the second cell are mixed in a ratio of (0.5-1.5): (20-30), and cultured to obtain CD3-positive tumor-infiltrating lymphocytes.

6. The method according to claim 5, characterized in that In the step 1), the concentration of IL-2 in the culture medium is 2000-4000 IU / ml; And / or, in step 1), the tumor is melanoma.

7. The method according to claim 5, characterized in that In the step 3), the culture medium used for culturing contains IL-2 and CD3 antibody; And / or, in step 3), the culturing time is 7 to 15 days; And / or, in step 3), the first living cell density is 1 to 1.5 x 10 6 / ml; And / or, in step 3), the first cells and the second cells are mixed in a ratio of 1:25; And / or, in step 3), after mixed culture, the solution is replenished every 2 to 4 days; And / or, in steps 1) and 3), the culture medium used during culturing is a serum-free culture medium.

8. The method according to claim 7, characterized in that The concentration of IL-2 in the culture medium is 2000-4000 IU / ml, and the concentration of CD3 antibody is 20-40 ng / ml.

9. The method according to claim 7, characterized in that The serum-free medium is AIM V serum-free medium.

10. Use of the K562 cell according to any one of claims 1 to 3 or the method according to any one of claims 4 to 9 in the preparation of tumor infiltrating lymphocytes.

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