Method for preparing and expanding stem-like car-t cell in vitro

Abstract

The present application relates to a method for preparing and expanding a stem-like CAR-T cell in vitro. Specifically, the method comprises: 1) obtaining an isolated CD8+ T cell; 2) activating the CD8+ T cell; 3) introducing a CAR gene into the activated CD8+ T cell to obtain a CD8+ T cell into which the CAR gene is introduced; 4) mixing the CD8+ T cell into which the CAR gene is introduced, a fibrinogen solution and a thrombin solution, and culturing same under conditions suitable for cell growth, so as to obtain a stem-like CAR-T cell culture; and 5) bringing the culture into contact with a dispase so as to obtain the stem-like CAR-T cell. The present application also relates to a kit for preparing and expanding a stem-like CAR-T cell in vitro, the use of fibrinogen and thrombin in the preparation of a kit for preparing and expanding a stem-like CAR-T cell in vitro, and a stem-like CAR-T cell obtained by means of the method or kit of the present application.

Description

Method for preparing and expanding stemness CAR-T cells in vitro TECHNICAL FIELD

[0001] The present application relates to the field of tumor prevention and treatment. Specifically, it relates to a method for preparing and expanding stemness CAR-T cells in vitro. The present application also relates to a kit for preparing and expanding stemness CAR-T cells in vitro, the use of fibrinogen and thrombin in the preparation of the kit, and stemness CAR-T cells obtained by the method or kit of the present application. BACKGROUND

[0002] Chimeric antigen receptor-T cell (CAR-T) technology is a newly emerging adoptive cell therapy, which activates, modifies and proliferates patient T cells in vitro, and then returns them to the patient's body. Through the specific receptors expressed by T cells, tumor cells are specifically recognized and show strong killing activity. At present, several CAR-T cell therapies have been marketed at home and abroad, which are used to treat leukemia, lymphoma and myeloma, etc. However, in solid tumors which account for a larger proportion, the effect of CAR-T cell therapy is very limited. This is mainly because the CAR-T cells returned are prepared based on terminally differentiated effector T cells, which are difficult to survive in the body for a long time, greatly affecting the clinical effect of adoptive cell therapy. The complex microenvironment of solid tumors makes it difficult for CAR-T cells to infiltrate into the tumor. Even if a small amount of CAR-T cells infiltrate into the tumor, the immunosuppressive environment inside the tumor will induce CAR-T cells to enter an exhausted state, which cannot exert an anti-tumor effect.

[0003] Stemness T cells are a rare subset of memory T lymphocytes, which have stem cell-like self-renewal ability and multipotent potential to reconstruct the entire memory and effector T cell subsets. Stemness T cells can specifically recognize a variety of viruses and self-tumor antigens. Compared with other known memory cells, stemness T cells have higher proliferation capacity, can maintain in the body for a long time, and can rapidly produce a large amount of cytokines under the stimulation of antigens, thereby exerting stronger anti-tumor and anti-inflammatory immune functions.

[0004] In view of the strong self-renewal and in vivo survival ability of stemness T cells, stemness CAR-T cells prepared based on stemness T cells will make up for the defects of current CAR-T cell therapy, and are expected to become the most effective cell type for adoptive cell transfer (ACT) therapy of tumors. Because the proportion of stemness T cells in the human body is very low, it is particularly important and urgent to explore a method for preparing and expanding stemness CAR-T cells in vitro on a large scale. SUMMARY

[0005] Human fibrinogen is a glycoprotein synthesized and secreted by hepatocytes. It is one of the highest content coagulation factors in plasma, which can be converted into fibrin under the action of thrombin, and forms a stable, porous fibrin gel after coagulation. As an insoluble protein, fibrin can be digested and degraded by dispase. The fibrin gel culture system can provide a 3D culture environment for cells, better simulate the in vivo environment, and thus provide experimental conditions closer to the physiological state, which is of great significance for studying cell behavior, cell-cell interaction and cell-microenvironment interaction.

[0006] Without being bound to a specific theory, the technical solution of the present application is at least partially based on the finding that the application of the aforementioned 3D culture system to CAR-T cell culture helps to better understand cell behavior and develop more effective cancer treatment strategies.

[0007] The present application establishes a 3D fibrin gel culture system by using fibrinogen and thrombin, which can amplify stem T cells in vitro and complete CAR assembly of stem T cells at the same time. The stem CAR-T cells prepared by this method show stronger proliferation and killing capacity in vitro and in vivo, and have more tumor infiltrating cells and longer survival time.

[0008] In order to solve the problem that stem CAR-T cells cannot be prepared and amplified in vitro, the present application is proposed.

[0009] In a first aspect, the present application provides a method for preparing and amplifying stem CAR-T cells in vitro, which comprises or consists of the following steps:

[0010] 1) obtaining isolated CD8 + T cells;

[0011] 2) activating the CD8 + T cells;

[0012] 3) introducing a CAR gene into the activated CD8 + T cells to obtain CD8 + T cells with CAR gene introduction;

[0013] 4) mixing the CD8 + T cells with CAR gene introduction, fibrinogen solution and thrombin solution, and culturing under suitable cell growth conditions to obtain stem CAR-T cell culture;

[0014] Preferably, the method further comprises:

[0015] 5) contacting said culture with dispersinase, obtaining dry CAR-T cells.

[0016] In some embodiments, in step 1) of the method of the application, said CD8 + T cells are isolated from the peripheral blood, umbilical cord blood, cancer tissue, pleural fluid or ascites fluid of an animal; preferably, said animal is a mammal, said mammal is a non-human mammal or a human, more preferably said mammal is a human.

[0017] In some embodiments, in step 2) of the method of the application, said CD8 + T cells are activated by incubating said CD8 + T cells with CD3 monoclonal antibody, CD28 monoclonal antibody and IL-2 under conditions suitable for cell growth.

[0018] In further embodiments, the final concentration of said CD3 monoclonal antibody is 0.1-10 pg / ml, such as but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 pg / ml, more preferably 1-5 pg / ml, further preferably 2.5 pg / ml.

[0019] In further embodiments, the final concentration of said CD28 monoclonal antibody is 0.1-10 pg / ml, such as but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 pg / ml, more preferably 0.1-2 pg / ml, further preferably 1 pg / ml.

[0020] In further embodiments, the final concentration of said IL-2 is 10-1000 IU / ml, such as but not limited to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 IU / ml, more preferably 50-200 IU / ml, further preferably 100 IU / ml.

[0021] In further embodiments, said CD8 + T cells are incubated with CD3 monoclonal antibody, CD28 monoclonal antibody and IL-2 for 24-72 hours, preferably 24-48 hours, such as but not limited to 24, 26, 28, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 44, 46, 48 hours, further preferably 36 hours.

[0022] In some embodiments, in step 3) of the method of the application, the CAR gene is introduced into the activated CD8 + T cells by a lentivirus expressing the CAR, the lentivirus transfection MOI = 0.5-10, for example but not limited to 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.4, 1.6, 1.8, 2.0, preferably MOI = 1.

[0023] In further embodiments, polybrene is added at a final concentration of 10 pg / ml at lentivirus transfection.

[0024] In some embodiments, in step 4) of the method of the application, the CD8 + T cells into which the CAR gene was introduced are mixed with a fibrinogen solution, seeded into a culture plate pre-incubated with a thrombin solution, and after 30 minutes of incubation medium is added, wherein the fibrinogen is human fibrinogen and the thrombin is human thrombin.

[0025] In some embodiments, the final concentration of the fibrinogen is 1-20 mg / ml, for example but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 pg / ml, more preferably 5-10 mg / ml, further preferably 8 mg / ml.

[0026] In some embodiments, the concentration of the thrombin is 0.01-1 U / pl, for example but not limited to 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 U / pl, more preferably 0.05-0.2 U / pl, further preferably 0.1 U / pl.

[0027] In further embodiments, in step 4) of the method of the application, the CD8 + T cells into which the CAR gene was introduced are incubated with a medium containing beta-mercaptoethanol and human IL-2.

[0028] In some embodiments, in step 5) of the method of the application, the culture obtained in step 4) is contacted with a dispersion enzyme after 2-3 days of incubation, obtaining the dry CAR-T cells; the concentration of the dispersion enzyme is 1-10 mg / ml, for example but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / ml, preferably 2-5 mg / ml, more preferably 4 mg / ml.

[0029] In a second aspect, the present application provides a kit for preparing and expanding stemness CAR-T cells in vitro, and comprising:

[0030] a component for activating T cells;

[0031] a component for introducing a CAR gene;

[0032] fibrinogen;

[0033] thrombin; and

[0034] optionally, dispersin.

[0035] In some embodiments, in the kit of the present application, the component for activating T cells is a combination of CD3 monoclonal antibody, CD28 monoclonal antibody and IL-2.

[0036] In further embodiments, the final concentration of the CD3 monoclonal antibody is 0.1-10 μg / ml, more preferably 1-5 μg / ml, further preferably 2.5 μg / ml.

[0037] In further embodiments, the final concentration of the CD28 monoclonal antibody is 0.1-10 μg / ml, more preferably 0.1-2 μg / ml, further preferably 1 μg / ml.

[0038] In further embodiments, the final concentration of the IL-2 is 10-1000 IU / ml, more preferably 50-200 IU / ml, further preferably 100 IU / ml.

[0039] In some embodiments, in the kit of the present application, the component for introducing a CAR gene is a CAR-expressing lentivirus, which is transfected at MOI = 0.5-10, preferably MOI = 1.

[0040] In further embodiments, the kit further comprises polybrene for lentivirus transfection, at a final concentration of 10 μg / ml.

[0041] In some embodiments, in the kit of the present application, the fibrinogen is human fibrinogen, and the thrombin is human thrombin.

[0042] In further embodiments, the final concentration of the fibrinogen is 1-20 mg / ml, more preferably 5-10 mg / ml, further preferably 8 mg / ml; and the concentration of the thrombin is 0.01-1 U / μl, more preferably 0.05-0.2 U / μl, further preferably 0.1 U / μl.

[0043] In a further embodiment, the final concentration of the dispersin is 1-10 mg / ml, preferably 2-5 mg / ml, more preferably 4 mg / ml ± 10%

[0044] In a third aspect, the present application provides uses of fibrinogen and thrombin in the preparation of a kit for the in vitro preparation and expansion of stemness CAR-T cells.

[0045] In some embodiments, the fibrinogen is human fibrinogen and the thrombin is human thrombin.

[0046] In a further embodiment, the final concentration of the fibrinogen is 1-20 mg / ml, more preferably 5-10 mg / ml, further preferably 8 mg / ml; and the concentration of the thrombin is 0.01-1 U / μl, more preferably 0.05-0.2 U / μl, further preferably 0.1 U / μl.

[0047] In a fourth aspect, the present application provides stemness CAR-T cells obtained by the method or the kit of the present application.

[0048] In some embodiments, the stemness CAR-T cells have one or more of the following characteristics:

[0049] 1) significantly increased CD62L + CD45RO - CD45RA + CCR7 + CD95 + T cell proportion;

[0050] 2) significantly up-regulated expression levels of stemness-related genes SOX2, NANOG and TCF7;

[0051] 3) enhanced tumor killing ability;

[0052] 4) enhanced in vitro expansion ability;

[0053] 5) enhanced in vivo survival ability.

[0054] The present applicant has found through extensive experimental research that, compared with ordinary CAR-T cells, the stemness CAR-T cells prepared and expanded by using fibrinogen and thrombin to form a 3D fibrin gel have the characteristics of high stemness gene expression and self-renewal ability, and the prepared and expanded CAR-T cells have a higher proportion of stemness phenotype (CD62L + CD45RO - CD45RA + CCR7 + CD95 +). In vitro studies show that the method prepared and expanded stem CAR-T cells have stronger expansion ability and killing ability. In addition, in vivo studies show that the method prepared and expanded stem CAR-T cells have stronger tumor infiltration ability and more durable survival ability, thereby showing better anti-tumor effect.

[0055] In the present application, when referring to a numerical value, it is understood to include a reasonable error range, for example ±1%, ±1.5%, ±2%, ±2.5%, ±3%, ±3.5%, ±4%, ±4.5%, ±5%, ±5.5%, ±6%, ±6.5%, ±7%, ±8%, ±9%, ±10%. This is mainly due to the reasonable error introduced by the measurement method, personnel operation, raw materials themselves, operation environment, instrument equipment, etc. For example, due to the accuracy of the measurement method of concentration, enzyme activity, and the proficiency of personnel, sampling and other factors, a concentration of 100 IU / ml can refer to 100 IU / ml within the error range, and the skilled person can determine the specific error range according to the context. When there is no explicit limitation, the error range is ±10%, i.e. "100 IU / ml" should be understood as 90-110 IU / ml.

[0056] When referring to time, it should not be understood as second by second, but there can be an error of ±1%, ±1.5%, ±2%, ±2.5%, ±3%, ±3.5%, ±4%, ±4.5%, ±5%, ±5.5%, ±6%, ±6.5%, ±7%, ±8%, ±9%, ±10%. For example, due to the influence of personnel operation and timer error, 36 hours can have an error of ±10%.

[0057] The method of the present application does not involve the diagnosis or treatment of a disease. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 shows that 3D fibrin gel culture system can be used to prepare and expand CAR-T cells in vitro.

[0059] Figure 2 shows that 3D fibrin gel culture system prepared and expanded CAR-T cells have a higher proportion of stem T cell phenotype.

[0060] Figure 3 shows that 3D fibrin gel culture system prepared and expanded stem CAR-T cells highly express stem-related genes (SOX2, NANOG and TCF7).

[0061] Figures 4A and 4B show that 3D fibrin gel culture system prepared and expanded stem CAR-T cells have stronger tumor cell killing ability and expansion ability in in vitro experiments.

[0062] Fig. 5A, Fig. 5B show that the 3D fibrin gel culture system prepared and expanded dry CAR-T cells have stronger anti-tumor ability and more durable survival ability in in vivo experiments. DETAILED DESCRIPTION

[0063] Unless otherwise defined, all technical and scientific terms used herein have the meanings that are commonly understood by one of ordinary skill in the art in the field of the application.

[0064] Various cell lines, animals used in the examples:

[0065] Human peripheral blood was derived from volunteers; human colorectal cancer cell line HCT116 was purchased from Cell Resource Center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences; immunodeficient NSG mice were purchased from Jiangsu Jicui Yekang Biotechnology Co., Ltd.

[0066] The raw materials and reagents used in the present application are commercially available unless otherwise specified.

[0067] Preparation of lentivirus expressing CAR:

[0068] The three plasmids used in the experiment are: CAR-HER2 plasmid overexpressing human Her2, CAR-B7-H3 plasmid overexpressing human B7-H3, and CAR-MSLN plasmid overexpressing human mesothelin (MSLN). The lentivirus packaging plasmids used in the experiment are pSPAX2 and PMD2G. The 293T cell line was transfected with the above three plasmids and pSPAX2 and PMD2G packaging plasmids, respectively, to obtain lentivirus expressing CAR-HER2, CAR-B7-H3, and CAR-MSLN.

[0069] Taking the CAR-HER2 plasmid overexpressing Her2 as an example, specifically, the plasmid is generated by fusing the human Her2 gene fragment into the lentivirus backbone plasmid containing the EF1a promoter. The plasmid is composed of antigen (Her2) specific scFv, human CD8 signal peptide, human CD8 hinge and transmembrane domain, CD28 and 4-1BB costimulatory domain, and CD3 zeta intracellular domain.

[0070] The specific preparation steps of the lentivirus are as follows:

[0071] 1. Preparation of culture medium

[0072] 293T cell complete culture medium: DMEM basic culture medium + 10% FBS + 100x Glutamax (Gibco, Cat: 35050-061) + 100x double antibody (Solarbio, Cat: P1400);

[0073] Virus packaging medium (ready to use): 293T cell complete medium + 500x sodium butyrate (final concentration 5mM).

[0074] 2. Plasmid transfection

[0075] Transfect plasmid into 293T cells when the cells are 80-90% confluence.

[0076] Transfection system for 10cm dish: Add 250ul Jet buffer into two 1.5ml EP tubes, gently add 40ul Jet liposome into one tube and mix gently, add target plasmid: PSPAX2:PMD2G=10ug:7ug:3ug into another tube and mix.

[0077] 3. Virus concentration

[0078] Pre-treatment of virus spin column (30kD, Merck): Add 6ml PBS to wash the column, centrifuge at 5000g for 5-7min, discard the liquid.

[0079] Collect the supernatant of 10cm dish into 50ml centrifuge tube 48h after transfection, centrifuge at 1000g for 5min, and use 20ml syringe to suck the supernatant after centrifugation, filter through 0.45um filter membrane. Add 15ml virus-containing filtrate after filtration into the pre-treated virus concentration column, centrifuge at 4℃, 5000g for 25min, collect the virus concentrate on the concentration column and store at -80℃. The titer of concentrated virus is greater than 5x10 7 TU / ml, usually between 1x10 8 TU / ml-5x10 8 TU / ml.

[0080] Example 1: Large-scale preparation and expansion of CAR-T cells in 3D fibrin gel

[0081] 1. Experimental procedure

[0082] 1) Collect human peripheral blood and isolate CD8 + T cells;

[0083] 2) Seed T cells into 24-well plates at an initial density of 1x10 6 cells / well, activate T cells with CD3 (2.5ug / ml), CD28 (1ug / ml) monoclonal antibodies and cytokine IL-2 (100IU / ml), and incubate in a 37℃, 5% CO2 and saturated humidity incubator for 36 hours;

[0084] 3) Collect activated T cells, seed 2x10 5 CD8+ T cells were mixed with 100 μl of concentrated lentivirus expressing CAR (MOI = 0.5-10, preferably MOI = 1) and polybrene (final concentration 10 μg / ml) and incubated at 37°C for 2 hours; activated T cells were collected, 2 x 10 5 CD8 + T cells were added with 100 μl of concentrated lentivirus expressing CAR, centrifuged at 1200g for 2 hours, and cultured in a 48-well rigid plate to prepare control CAR-T cells (Con-CAR T), after 6-8 hours of infection with virus, the cells were centrifuged and replaced with fresh culture medium, and the Con-CAR T cells were replaced every other day thereafter, inoculated at 5 x 10 5 / ml into a 24-well plate for culture;

[0085] 4) 3.5 μl of thrombin solution (0.1 U / μl, Sea Run Holdings, Catalog No. SEA-135) was pre-added to a 24-well plate; the incubated T cells were mixed with different concentrations of human fibrinogen solution (preferably 8 mg / ml, equivalent to 1050 Pa; RiaSTAP, Catalog No. NDC 63833-891-90) in a total volume of 250 μl / well, inoculated into a 24-well plate immediately, and incubated in a 37°C, 5% CO2 incubator for 30 min to completely convert fibrinogen into fibrin gel and wrap T cells to form a 3D network structure; 1 ml of culture medium containing β-mercaptoethanol (1000x, Thermo, final concentration 55 μM) and human IL-2 (100 IU / ml) was added to each well for continuous expansion culture to prepare 3D-CAR T cells;

[0086] 5) After 3 days of culture, the 3D fibrin gel formed by fibrinogen and thrombin was digested with dispersin (4 mg / ml), centrifuged at 500g for 5 min, and the expanded 3D-CAR T cells and Con-CAR T cells were harvested and counted. The proportion of CAR + T cells was detected by flow cytometry.

[0087] 2. Experimental results

[0088] The initial total amount of T cells was 5 x 10 6 After 3 days of expansion in the 3D fibrin gel culture system, a total of 4.2 x 10 7 CAR-T cells were prepared; while the T cells cultured in a 24-well rigid plate produced a total of 7.6 x 10 6 CAR-T cells after 3 days of expansion (Figure 1).

[0089] Example 2: 3D fibrin gel culture system produces CAR-T cells with higher proportion of stemness T cell phenotype

[0090] 1. Experimental procedure

[0091] 3D-CAR T and Con-CAR T cells were collected and stained with Live / Dead dye for 10 min at room temperature, then stained with phenotype-related antibodies (CD45RA, CD45RO, CCR7, CD95, CD62L) for 30 min at 4°C. Data collection was performed using Invitrogen Attune NxT flow cytometer, and data analysis was performed using FlowJo software.

[0092] 2. Experimental results

[0093] Flow cytometry analysis found that only 7% of CAR-T cells in Con-CAR T cells cultured in rigid flat plates were CD45RA + CCR7 + CD45RO - CD95 + CD62L + (stemness T cell phenotype); while about 56% of 3D-CAR T cells were CD45RA + CCR7 + CD45RO - CD95 + CD62L + (Figure 2). In summary, CAR-T cells prepared by 3D fibrin gel have a higher proportion of stemness T cell phenotype.

[0094] Example 3: 3D fibrin gel culture system produces and expands stemness CAR-T cells with high expression of stemness-related genes (SOX2, NANOG and TCF7)

[0095] 1. Experimental procedure

[0096] 3D-CAR T cells prepared by 3D fibrin gel culture system and Con-CAR T cells cultured in rigid flat plates were collected, total RNA was extracted after Trizol lysis, and reverse transcribed into cDNA, and the expression levels of SOX2, NANOG and TCF7 genes were detected by qPCR.

[0097] 2. Experimental results

[0098] qPCR results showed that the expression levels of stemness-related genes (SOX2, NANOG and TCF7) of 3D-CAR T cells prepared by 3D fibrin gel culture system were significantly up-regulated compared with Con-CAR T cells cultured in rigid flat plates (Figure 3).

[0099] Example 4: The dry CAR-T cells prepared and expanded by 3D fibrin gel culture system have stronger tumor cell killing ability and expansion ability in in vitro experiments

[0100] 1. Experimental steps

[0101] HCT116 cells were plated in 24-well plates at 5x10 4 cells / well one day in advance, and the dry CAR-T cells prepared by 3D fibrin gel culture system and the Con-CAR T cells cultured in rigid flat plates were collected the next day after they adhered to the wall. The T cells and tumor cells were co-cultured at an effector-to-target ratio of 1:1. After two days, the tumor cell lysis rate was detected, and the supernatant was centrifuged to collect the suspended cells. The CAR + T cells were sorted by flow cytometry, and co-cultured with tumor cells at the same effector-to-target ratio for two days for the second round of killing. In this way, a total of 7 rounds of killing were completed. The killing rate and expansion rate of CAR-T cells on tumor cells were detected in each round.

[0102] 2. Experimental results

[0103] The results showed that, except for the first round of co-culture, the dry CAR-T cells prepared by 3D fibrin gel culture system were significantly better than the Con-CAR T cells cultured in rigid flat plates in the second to seventh rounds of killing on tumor cells (Figure 4A), showing stronger killing ability. After analyzing the number of CAR-T cells after each round of co-culture, it was found that the number of dry CAR-T cells prepared by 3D fibrin gel culture system after each round of co-culture was higher than that of ordinary Con-CAR T cells, showing stronger expansion ability (Figure 4B).

[0104] Example 5: The dry CAR-T cells prepared and expanded by 3D fibrin gel culture system have stronger anti-tumor ability in in vivo experiments

[0105] 1. Experimental steps

[0106] 1x10 6 HCT116 cells were inoculated into NSG mice, and when the tumor grew to about 5x5mm in size, 1x10 6 dry CAR-T cells prepared by 3D fibrin gel culture system, Con-CAR T cells cultured in rigid flat plates, or negative control CD19-CAR T cells against an irrelevant target were adoptively transferred via the tail vein. The tumor size was measured every two days, and the animals were sacrificed when the tumor grew to 2cm 3 in size. The tumor volume and death time of tumor-bearing mice were recorded at each measurement.

[0107] 2. Experimental results

[0108] The experimental results show that, compared with Con-CAR T cells and negative controls in rigid flat plate culture, the stem CAR-T cells prepared by the 3D fibrin gel culture system can significantly inhibit the growth of HCT116 tumors (Figure 5A), and prolong the survival time of tumor-bearing mice (Figure 5B).

[0109] In summary, the scheme of the present application has the following effects:

[0110] 1. The present application can mass-produce and expand stem CAR-T cells by using a 3D fibrin gel culture system formed by fibrinogen and thrombin, and has the characteristics of high expansion efficiency, simple production process, short preparation period, etc.

[0111] 2. The stem CAR-T cells prepared and expanded by the method and kit of the present application have high expression levels of stemness genes such as SOX2, NANOG and TCF7.

[0112] 3. The stem CAR-T cells prepared and expanded by the method and kit of the present application show stronger tumor cell killing ability, expansion ability, infiltration ability and more persistent survival ability in in vivo and in vitro studies, so it is expected to solve many problems such as short in vivo survival time and difficulty in infiltrating into the tumor interior of the current CAR-T cell therapy, and has great clinical application prospect.

Claims

1. A method for in vitro preparation and expansion of stemness CAR-T cells, comprising: 1) obtaining an isolated CD8 + T cell; 2) activating the CD8 + T cells; 3) introducing the CAR gene into the activated CD8 + T cells, obtaining CD8 + T cells with the CAR gene introduced; and 4) the CD8 + T cells, fibrinogen solution and thrombin solution are mixed and cultured under conditions suitable for cell growth to obtain a dry CAR-T cell culture; Preferably, the method further comprises: 5) contacting the culture with dispersin, obtaining stemness CAR-T cells.

2. The method of claim 1, wherein: In step 1), the CD8 + T cells are isolated from the peripheral blood, umbilical cord blood, cancer tissue, pleural fluid or ascites of an animal, Preferably, the animal is a mammal, the mammal is a non-human mammal or a human, more preferably the mammal is a human.

3. The method of claim 1, wherein: In step 2), the CD8 + T cells are activated by incubating them with CD3 monoclonal antibodies, CD28 monoclonal antibodies and IL-2 under conditions appropriate for cell growth + T cells, Preferably, the final concentration of the CD3 monoclonal antibody is 0.1-10 pg / ml, more preferably 1-5 pg / ml, further preferably 2.5 pg / ml ± 10%; Preferably, the final concentration of the CD28 monoclonal antibody is 0.1-10 pg / ml, more preferably 0.1-2 pg / ml, further preferably 1 pg / ml ± 10%; Preferably, the final concentration of the IL-2 is 10-1000 IU / ml, more preferably 50-200 IU / ml, further preferably 100 IU / ml ± 10%; Preferably, the CD8+ T cells are incubated with CD3 monoclonal antibody, CD28 monoclonal antibody and IL-2 + T cells 24-72 hours, more preferably 24-48 hours, further preferably 36 hours ± 10%.

4. The method of claim 1, wherein: In step 3), the CAR gene is introduced into the activated CD8 T cells by lentivirus expressing the CAR + T cells, The MOI of the lentivirus transfection expressing CAR is 0.5-10, preferably MOI = 1 ± 10%; Preferably, polybrene is added at the time of transfection, with a final concentration of 10 pg / ml ± 10%.

5. The method of claim 1, wherein: In step 4) CD8 + T cells were mixed with fibrinogen solution, seeded into culture plates previously incubated with a thrombin solution, and after 30 minutes of incubation the culture medium was added, in which The fibrinogen is human fibrinogen, and the thrombin is human thrombin; Preferably, the final concentration of the fibrinogen is 1-20 mg / ml, more preferably 5-10 mg / ml, further preferably 8 mg / ml ± 10%; Preferably, the final concentration of the thrombin is 0.01-1 U / ul, more preferably 0.05-0.2 U / ul, further preferably 0.1 U / ul ± 10%; Preferably, in step 4), the CD8 + T cells introduced with the CAR gene are cultured with a medium containing beta- mercaptoethanol and human IL-2 T cells.

6. The method of claim 1, wherein: In step 5), the culture is contacted with dispersin after 2-3 days of culture, with a final concentration of 1-10 mg / ml, preferably 2-5 mg / ml, more preferably 4 mg / ml ± 10%.

7. A kit for in vitro preparation and expansion of stemness CAR-T cells, and comprising: a component for activating T cells; a component for introducing a CAR gene; fibrinogen; thrombin; and optionally, dispersin.

8. The kit of claim 7, wherein: Preferably, the component for activating T cells is a combination of CD3 monoclonal antibody, CD28 monoclonal antibody, and IL-2, Preferably, the final concentration of the CD3 monoclonal antibody is 0.1-10 pg / ml, more preferably 1-5 pg / ml, further preferably 2.5 pg / ml ± 10%; Preferably, the final concentration of the CD28 monoclonal antibody is 0.1-10 pg / ml, more preferably 0.1-2 pg / ml, further preferably 1 pg / ml ± 10%; Preferably, the final concentration of the IL-2 is 10-1000 IU / ml, more preferably 50-200 IU / ml, further preferably 100 IU / ml ± 10%. Preferably, the component for introducing the CAR gene is a lentivirus expressing the CAR, the lentivirus expressing the CAR transfecting at an MOI = 0.5-10, preferably MOI = 1 ± 10%; preferably the kit further comprises polybrene for lentivirus transfection at a final concentration of 10 μg / ml ± 10%; Preferably, the fibrinogen is human fibrinogen, the thrombin is human thrombin; Preferably, the fibrinogen has a final concentration of 1-20 mg / ml, more preferably 5-10 mg / ml, further preferably 8 mg / ml ± 10%; Preferably, the thrombin has a concentration of 0.01-1 U / μl, more preferably 0.05-0.2 U / μl, further preferably 0.1 U / μl ± 10%. Preferably, the dispersin has a final concentration of 1-10 mg / ml, preferably 2-5 mg / ml, more preferably 4 mg / ml ± 10%.

9. Use of fibrinogen and thrombin for the preparation of a kit for the in vitro preparation and expansion of stemness CAR-T cells, wherein: Preferably, the fibrinogen is human fibrinogen, the thrombin is human thrombin; Preferably, the fibrinogen has a final concentration of 1-20 mg / ml, more preferably 5-10 mg / ml, further preferably 8 mg / ml ± 10%; Preferably, the thrombin has a concentration of 0.01-1 U / μl, more preferably 0.05-0.2 U / μl, further preferably 0.1 U / μl ± 10%.

10. Stemness CAR-T cells obtained by the method of any one of claims 1 to 6 or the kit of claim 7 or 8, having one or more of the following characteristics: 1) significantly increased CD62L + CD45RO - CD45RA + CCR7 + CD95 + T cell proportion; 2) significantly upregulated expression levels of stemness-related genes SOX2, NANOG and TCF7; 3) enhanced tumor killing capacity; 4) enhanced in vitro expansion capacity; 5) enhanced in vivo survival capacity.

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