CN114645022A - CD 5-targeted CAR-gamma delta T cell and preparation method and application thereof - Google Patents

CD 5-targeted CAR-gamma delta T cell and preparation method and application thereof Download PDF

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CN114645022A
CN114645022A CN202210517330.2A CN202210517330A CN114645022A CN 114645022 A CN114645022 A CN 114645022A CN 202210517330 A CN202210517330 A CN 202210517330A CN 114645022 A CN114645022 A CN 114645022A
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陈志国
吴焕童
赵宇
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Xuanwu Hospital
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Abstract

The invention provides a CD 5-targeted CAR-gamma delta T cell and a preparation method and application thereof. The invention firstly provides a CD 5-targeted CAR-gamma delta T cell preparation, wherein the expression ratio of functional receptors in gamma delta T cells is as follows: CD16 greater than 40%, NKp30 greater than 40%, NKp44 greater than 50%; greater than 40% CXCR4, greater than 35% CCR7, greater than 50% CXCR5, and greater than 55% CXCR 3. The invention also provides a method for preparing CD 5-targeted CAR-gamma delta T cells, which comprises adding AB serum, interleukin-2, interleukin-15, TGF-beta and zoledronic acid in culture. The CD 5-targeted CAR-gamma delta T cells express a plurality of NK activating receptors and chemotactic receptors, show good migration activity in vivo, and can be used for downstream scientific research and clinical transformation.

Description

CD 5-targeted CAR-gamma delta T cell and preparation method and application thereof
Technical Field
The invention relates to a preparation technology and application of a sorting-free chimeric antigen receptor T cell which takes a gamma delta T cell as a main component and targets CD 5.
Background
Chimeric antigen receptor-modified T cells (CAR-T) are a class of genetically modified genetically engineered T cell preparations with targeted recognition of tumor-specific antigens. The CAR-T can rely on the specificity of the CAR receptor to recognize tumor cells on one hand, and can kill the tumor cells through an MHC independent path on the other hand, and has high-efficiency targeted anti-tumor activity. At present, CD19 specific CAR-T has been proved to have good clinical curative effect on various refractory/recurrent B cell malignant tumors, the complete remission rate after treatment is close to 90%, and a brand new solution is provided for the treatment of hematological tumors. Therefore, researchers are also trying to use CAR-T formulations for the treatment of other hematologic and solid tumors, including T cell malignancies.
In T cell malignancies, approximately 80% express CD5, and thus CD5 is currently a potential target for the development of novel therapeutic approaches/drugs for T cell malignancies. However, the effector component of the prior CAR-T preparations is mainly α β T cells. Although studies have shown that autologous CAR- α β T targeting CD5 can effectively kill tumor cells, prolonging the survival time of tumor-bearing mice, and the clinical safety thereof is preliminarily verified. However, in clinical treatment, a considerable number of patients cannot use autologous cells for CAR-T production due to disease conditions and physical causes; when the allogeneic CD 5-specific CAR-alpha beta T is used for treatment, the proportion of graft-versus-host disease (GVHD) generated after infusion is high, and the patient has a high safety risk, so that the clinical application of the traditional CAR-alpha beta T cell in the treatment of T cell malignant tumor is limited. Therefore, there is a need to develop a more safe and effective CAR-T formulation.
The gamma delta T cells are a subset of T cells present in the periphery and account for 1% -5% of circulating T cells. Unlike α β T cells, γ δ T cells expressing V γ 9V δ 2 can, on the one hand, proliferate by recognizing endogenous isopentenyl pyrophosphate (IPP) produced by tumor cells, and, on the other hand, can also kill tumor cells via MHC-independent pathways. It has now been demonstrated that γ δ T cells have a significant killing effect on a variety of tumor cell lines. In addition, γ δ T has a very low risk of inducing GVHD when allogeneic reinfusion is performed, and has better clinical safety and applicability when allogeneic cellular immunotherapy is performed, compared to α β T cells. Therefore, the gamma delta T cells have great clinical transformation potential in the field of anti-tumor cell immunotherapy.
However, because the proportion of endogenous γ δ T cells in total lymphocytes is low, how to prepare effector cells in vitro on a large scale to obtain sufficient numbers has long been a major technical challenge in the field. The currently common method is to enrich the gamma delta T cells in peripheral blood by using a sorting technology and then culture the cells in vitro. The process is long in time consumption on one hand, and high in preparation cost on the other hand, and limits clinical transformation and commercial popularization of the technology.
Disclosure of Invention
It is an object of the present invention to provide CD 5-targeted CAR- γ δ T cells of high purity.
It is another object of the invention to provide a method of making CD 5-targeted CAR- γ δ T cells.
It is another object of the invention to provide the use of the CD 5-targeted CAR- γ δ T cells.
In one aspect, the invention provides a CD 5-targeted CAR- γ δ T cell (CD 5CAR- γ δ T), in particular, a high purity CD 5-targeted CAR- γ δ T cell preparation.
The CD 5-targeted CAR- γ δ T cell preparation of the present invention, wherein the ratio of expression of functional receptors in γ δ T cells is: CD16 greater than 40%, NKp30 greater than 40%, NKp44 greater than 50%; greater than 40% CXCR4, greater than 35% CCR7, greater than 50% CXCR5, and greater than 55% CXCR 3.
According to a particular embodiment of the invention, the CD 5-targeted CAR- γ δ T cell preparation of the invention, wherein γ δ T cells comprise more than 80%.
According to a particular embodiment of the invention, the CD 5-targeted CAR- γ δ T cell preparation of the invention has greater than 55% CAR in γ δ T cells.
According to a particular embodiment of the invention, in the CD 5-targeted CAR- γ δ T cell preparation of the invention, wherein the ratio of expression of functional receptors in γ δ T cells is: CD16 greater than 60%, NKp30 greater than 60%, NKp44 greater than 60%; greater than 60% CXCR4, greater than 44% CCR7, greater than 80% CXCR5, and greater than 80% CXCR 3.
According to a particular embodiment of the invention, in the CD 5-targeted CAR- γ δ T cell preparation of the invention, wherein the ratio of expression of functional receptors in γ δ T cells is: CD16 greater than 80%, NKp30 greater than 75%, NKp44 greater than 65%; greater than 60% CXCR4, greater than 44% CCR7, greater than 85% CXCR5, and greater than 85% CXCR 3.
In another aspect, the invention also provides a method of making a CD 5-targeted CAR- γ δ T cell, the method comprising:
peripheral blood PBMCs were cultured in complete media and infected with lentiviruses expressing CD 5-targeted CARs, and culture continued until cells were harvested (i.e., the CD 5-targeted CAR- γ δ T cells).
According to a specific embodiment of the invention, in the method for preparing the CD 5-targeted CAR- γ δ T cells of the invention, the composition of the complete medium comprises a basal medium and an additive factor. Wherein the basic culture medium is selected from one or more of serum-free RPMI1640, DMEM, alpha-MEM, X-VIVO15 and AIM-V culture medium. The additive factors include AB serum, interleukin-2, interleukin-15, TGF-beta, and Zoledronic Acid (ZA). Preferably, the content of the additive factors in the complete medium is: the volume content of the AB serum is 5-10%, the interleukin-2 is 100-1000 IU/mL, the interleukin-15 is 5-50 ng/mL, the TGF-beta is 1-20 ng/mL, and the zoledronic acid is 0.1-10 mu M.
According to a specific embodiment of the invention, in the method for preparing the CD 5-targeted CAR- γ δ T cells, inactivated aAPC cells are added to a culture system 3-7 days after infection with lentivirus expressing CD 5-targeted CAR, and then culture is continued until the cells are harvested. Preferably, the aAPC is added at a ratio of 1: 2 to 10T cells to aAPC in the culture system.
According to a particular embodiment of the invention, the method of making the CD 5-targeted CAR- γ δ T-cell of the invention comprises:
PBMC were adjusted to a cell density of 1X 10 with complete medium6~5×106Inoculating the strain/mL into a culture container which is pre-coated with 0.1-40 mu g/mL retronic for culture;
half amount of liquid change or complete culture medium supplement is carried out every 2-3 days in the culture process;
culturing for 5-7 days, harvesting cells, adjusting cell density to 1 × 106~5×106mL, infection with lentivirus expressing CD 5-targeted CAR, continued to culture in complete medium;
adding inactivated aAPC cells at a ratio of T cells to aAPC cells =1 to 2-10 for further culture on days 2-3 after infection; in the continuous culture process, supplementing a fresh complete culture medium into the culture system every 2-3 days, and adding inactivated aAPC cells into the culture system according to the ratio of T cells to aAPC cells = 1: 2-10 every 5-7 days;
harvesting cells after culturing for 21-25 days; alternatively, cells were harvested on an initial cell basis when the fold expansion was greater than 300 fold or when the CAR-positive γ δ T cells were more than 10% pure.
On the other hand, the invention also provides application of the CD 5-targeted CAR-gamma delta T cell (the CD 5-targeted CAR-gamma delta T cell product or the CD 5-targeted CAR-gamma delta T cell prepared by the method disclosed by the invention) in preparation of a therapeutic product for resisting tumor cells.
According to a specific embodiment of the invention, the CD 5-targeted CAR- γ δ T cells of the invention are useful for killing CD5 positive T cell tumors, including human acute T lymphocyte leukemia cells, human T cell lymphoma cells, and the like.
In another aspect, the invention also provides an application of the CD 5-targeted CAR- γ δ T cell (the CD 5-targeted CAR- γ δ T cell preparation, or the CD 5-targeted CAR- γ δ T cell prepared by the method of the invention) in preparation of a reagent for evaluation research of immune function of peripheral γ δ T cells and/or research of anti-tumor mechanism of gene-modified γ δ T cells.
The specific experimental result of the invention shows that the CAR-gamma delta T (CD 5 CAR-gamma delta T) targeting CD5 obtained by the preparation method has good tumor cell killing activity; the cell factor has stronger cytokine release capacity in the killing process; in addition, the CD5 CAR-gamma delta T can kill tumor cells of CD5+, can effectively kill tumor cells of CD5-, and has better anti-tumor activity compared with the traditional CD5 CAR-alpha beta T. In vivo anti-tumor activity experiments show that the CD5 CAR-gamma delta T of the invention has better in vivo anti-tumor activity than CD5 CAR-alpha beta T prepared using conventional CD3/CD28 activation methods. In vivo homing ability evaluation experiments show that the CD5 CAR-gamma delta T of the invention has better bone marrow migration ability than conventional CAR-alpha beta T. The CD5 CAR-gamma delta T has better anti-tumor activity, and can induce effector cells to express higher-level chemotactic receptors, so that the effector cells have better in-vivo migration activity.
In conclusion, the invention provides a sorting-free CD 5-targeted CAR-gamma delta T cell and a preparation method thereof, a sufficient amount of high-purity CD 5-targeted CAR-gamma delta T cells can be obtained without sorting, and the CD 5-targeted CAR-gamma delta T cell expresses multiple NK activating receptors and chemotactic receptors, shows better migration activity in vivo, and can be used for downstream scientific research and clinical transformation.
Drawings
Figure 1 shows the results of experiments with serial cell counts during the culture of CD 5-targeted CAR- γ δ T cells of the invention.
Figure 2 shows the results of the γ δ T assay experiments in CAR- γ δ T cell products targeted to CD5 of the present invention.
Figure 3 shows the results of CAR analysis experiments in CD 5-targeted CAR- γ δ T cell products of the invention.
Figure 4 shows the results of CD 5-targeted CAR- γ δ T cell functional receptor expression level analysis experiments of the present invention.
Figure 5 shows the results of the CD 5-targeted CAR- γ δ T tumor cell killing activity assay of the present invention.
Figure 6 shows the results of CD 5-targeted CAR- γ δ T killing-associated cytokine release level assay of the present invention.
Figure 7 shows experimental results of killing activity of CD 5-targeted CAR- γ δ T on CD5 negative tumor cells at different effect-to-target ratios in accordance with the present invention.
Figure 8 shows experimental results of killing activity of CD 5-targeted CAR- γ δ T on CD5 monoclonal antibody before and after blocking target cells.
Figure 9 shows imaging data of CD 5-targeted CAR- γ δ T anti-tumor activity in vivo of the present invention.
Figure 10 shows the results of a survival curve analysis experiment of CD 5-targeted CAR- γ δ T anti-tumor activity in vivo.
Figure 11 shows the results of the expression level test of anti-tumor associated cytokines in mice receiving CD 5-targeted CAR- γ δ T cell therapy of the present invention.
Figure 12 shows the results of an experimental assessment of CD 5-targeted CAR- γ δ T homing ability in vivo.
Figure 13 shows the experimental results of CD 5-targeted CAR- γ δ T induction of effector cell expression of chemotactic receptors in accordance with the present invention.
Detailed Description
For a more clear understanding of the technical features, objects and advantages of the present invention, reference is now made to the following detailed description taken in conjunction with the accompanying specific embodiments, and the technical solutions of the present invention are described, it being understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.
Unless specifically defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art.
Example 1 formulation of culture Medium and preparation of CD 5-Targeted CAR-Gamma Delta T cells (CD 5 CAR-Gamma Delta T)
1) Preparation of complete Medium
The complete medium used in this example was prepared by adding 10% by volume of one or more of human AB serum, 200IU/mL recombinant human interleukin-2 (rhIL-2), 30ng/mL recombinant human interleukin-15 (rhIL-15), 5ng/mL recombinant human TGF-beta (rhTGF-beta), 4. mu.M Zoledronic Acid (ZA) to a commercial serum-free basal medium including, but not limited to, X-VIVO15, according to each experimental group shown in Table 1.
In addition, in experimental group 1, experimental group 3, experimental group 5, and experimental group 7, inactivated Artificial antigen-presenting cell (aAPC) was added during the culture process, respectively. The aAPC cell line is a K562 cell line that overexpresses membrane-bound IL-21, CD86, CD83, and CD 137L. Specifically, the aAPC cell line can be prepared by using a human myeloid leukemia K562 cell line (purchased from Wuhanplosai Biotechnology Co., Ltd.) and expressing four molecules of membrane-bound recombinant human IL-21, CD86, CD83 and CD137L by lentiviral infection.
TABLE 1 complete medium composition and culture method used in different experimental groups and control groups
Figure 494747DEST_PATH_IMAGE001
Note: + represents the addition of the component, -represents the absence of the addition of the component.
2) Preparation of CD 5-targeted CAR- γ δ T cells (CD 5CAR- γ δ T)
Collecting 30mL of peripheral blood of a healthy subject, and slowly spreading the peripheral blood on the upper layer of the human lymphocyte separation liquid to keep a clear layered interface. Using a swing-type centrifuge, PBMC in fresh peripheral blood was separated by centrifugation at a rotation speed of 800g, a centrifugation temperature of 25 ℃, an ascending speed of 0, and a descending speed of 0 for 15 minutes. After centrifugation, the middle white membrane layer was carefully aspirated, and PBS was supplemented to 30mL, and the mixture was centrifuged at 1500rpm, 25 ℃ and 9 ℃ for 10 minutes in an orbital centrifuge. PBS was discarded and suspended in a complete medium (related art documents: CN 108017717A; CN 110358734A; Clinical Cancer Res)Research, 2019, 25 (18): 5595-5607). After counting PBMC were adjusted to 3X 10 cell density with complete medium6Perml inoculated in 1. mu.g/mL Retronetic precoated culture vessel at 37 ℃ with 5% CO2And culturing in a saturated humidity environment. And according to the growth condition of the cells, half of the culture solution is changed or the complete culture medium is supplemented every 2-3 days. Culturing to 7 days according to the growth state of cells, harvesting and counting the cells, and adjusting the cell density to 5 × 10 according to the counting result6mL, lentiviruses infected with CD5CAR expressing a CD 5-targeting gene (lentiviruses prepared by the methods of Clinical Cancer Research, 2019, 25 (18): 5595-5607 and Front Immunol, 2020, 23 (11): 581116). On day 3 post infection, i.e. 10 of culture, radiation inactivated aAPC cells were added at a ratio of T cells to aAPC cells =1 to 5 for further culture. The culture system was supplemented with an equal volume of fresh complete medium every 2-3 days, and the radiation inactivated aAPC cells were added to the culture system every 7 days at a ratio of T cells to aAPC cells =1 to 5 (absolute number of cells added subsequently is the reference T cell number, excluding the aAPC cells that had been added previously). The cells are harvested after 21-25 days of culture (for specific methods for harvesting cells, reference is made to technical literature: Clinical Cancer Research, 2019, 25 (18): 5595-.
The γ δ T cell counts during culture in each experimental group are shown in figure 1.
The flow analysis method is used for detecting the cell subsets of the final products of different experimental groups, and the detection results are shown in fig. 2 and fig. 3. The results show that the proportion of gamma delta T cells in the CD5CAR-T obtained by the culture method of the experimental group 1 and the experimental group 2 can reach more than 80 percent, and is obviously higher than that of the final products obtained by other experimental group cultures. See table 2 below for details.
TABLE 2 purity of γ δ T cells in final product and Positive proportion of CAR in γ δ T cells (mean. + -. standard deviation)
Figure 631462DEST_PATH_IMAGE002
The results of continuous cell counting and final product counting analysis in the culture process show that the complete culture medium added with AB serum, interleukin-2, interleukin-15, TGF-beta and zoledronic acid can obviously improve the in vitro proliferation capacity of the gamma delta T cells, and the number of the gamma delta T cells obtained by the culture method added with aAPC can be further obviously improved.
The results show that the preparation method can effectively induce the in-vitro expansion of the gamma delta T cells, and high-purity CD 5-targeted CAR-gamma delta T cells are obtained.
Example 2 expression of gamma delta T cell-associated functional receptors
The flow analysis detection technology is used for analyzing the CD 5-targeted CAR-gamma delta T cell functional receptor expression level of the final product obtained from different experimental groups, and the result is shown in figure 4. The results show that the CAR- γ δ T cells targeting CD5 prepared by culture in experimental group 1 express higher levels of CD16, NKp30 and NKp44 receptors in addition to the high expression of CD3 and γ δ TCR receptors, and the receptors have positive correlation with the antitumor activity in γ δ T cells, suggesting that the CAR- γ δ T cells targeting CD5 prepared by the culture method of the present invention may have better antitumor activity. See table 3 below for details.
TABLE 3 expression levels (mean. + -. standard deviation) of CAR- γ. delta. T cell functional receptors targeting CD5
Figure 473516DEST_PATH_IMAGE003
Example 3 evaluation of CAR- γ δ T cell killing Activity targeting CD5
CD 5-targeting CAR- γ δ T killing in vitro was evaluated using a CD5 positive human acute T-lymphoblastic leukemia CCRF-CEM cell line (purchased from wuhan punuosai biotechnology limited). Evaluation was carried out by the LDH release method (related art documents: CN 108017717A; CN 110358734A; Clinical Cancer Research, 2019, 25 (18): 5595-.
The results show that after effector cells and target cells are grouped according to different effective target ratios and are subjected to coculture for 12 hours, the killing efficiency of CD5 CAR-gamma delta T cultured in each experimental group on the target cells is enhanced along with the increase of the effective target ratio; and under the condition of the same effective target ratio, the CD5 CAR-gamma delta T cells obtained by using the culture method of the experimental group 1 obviously better kill target cells than other experimental groups (Table 4).
Further, the in vitro continuous killing of CD5CAR- γ δ T cells obtained by detecting different experimental groups by repeatedly adding tumor cells to the co-culture system showed that CD5CAR- γ δ T cells obtained by the culture method of experimental group 1 have better continuous killing activity in vitro. See table 4 below for details.
TABLE 4 evaluation of the killing activity in vitro of the final products of the different experimental groups (mean. + -. standard deviation)
Figure 337567DEST_PATH_IMAGE004
The results show that the CD 5-targeted CAR-gamma delta T obtained by the preparation method has good tumor cell killing activity in vitro.
Example 4 measurement of the level of Release of a killer-related cytokine
The level of the killing-related factor in the co-culture killing system was measured by ELISA method according to the methods described in the published technical literature (CN 108017717A; CN 110358734A; Clinical Cancer Research, 2019, 25 (18): 5595-5607). The results are shown in FIG. 6.
The detection result shows that the effective target ratio is 1: 1, under the condition of co-culture for 12 hours, the CAR-gamma delta T targeting CD5 cultured and prepared in the experimental group 1 kills related factors in the process of killing target cells, wherein the release levels of IL-2, IFN-gamma, TNF-alpha and GM-CSF are obviously higher than those of other experimental groups, so that the CAR-gamma delta T targeting CD5 obtained by the preparation method disclosed by the invention can release higher-level proinflammatory factors in the in-vitro killing process, and the fact that the CAR-gamma delta T has better anti-tumor activity is suggested.
Example 5 killing Activity of CAR- γ δ T against CD 5-negative tumor cells
This example further evaluated the killing activity of CD 5-targeted CAR- γ δ T cells prepared in accordance with the present invention against CD5 negative (CD 5-) tumor cells. First, using human B-cell lymphoma cell line Raji as a target cell, LHD release method compared the killing activity of CD5CAR- γ δ T (product prepared by experimental group 1) and CD5CAR- α β T. The preparation of CD5CAR- α β T is referred to the published technical literature (CN 108017717A; CN 110358734A; Clinical Cancer Research, 2019, 25 (18): 5595-. Results of the experiment referring to fig. 7, the results show that the target ratio between the effective targets is 1: 1-8: 1 range, CD5CAR- α β T has little killing activity on the Raji tumor cell line of CD5-, while CD5CAR- γ δ T shows good killing activity.
On the other hand, the human T acute lymphoblastic leukemia cell line CCRF-CEM using CD5+ as the target cell, the mouse anti-human CD5 monoclonal antibody (beijing yi warz, biotechnology limited) at 1 μ g/mL was used to block the CD5 receptor on the surface of the target cell before killing, and then the ratio of effective target ratio 1: 1 CD5 CAR-gamma delta T (product prepared by experiment group 1) and CD5 CAR-alpha beta T are respectively co-cultured with the pretreated target cells, and the killing activity is detected by an LDH release method after 12 hours. The results of the experiment are shown in FIG. 8 (in the figure mAb + represents treatment with antibody blocking and mAb-represents no blocking treatment with antibody). The results show that CD5CAR- γ δ T can still kill target cells effectively after blocking with monoclonal antibody, while the killing activity of CD5CAR- α β T is significantly inhibited.
The results show that the CD5 CAR-gamma delta T can not only kill tumor cells of CD5+, but also effectively kill tumor cells of CD5-, has better targeted killing activity compared with the traditional CD5 CAR-alpha beta T, and has obvious killing activity on malignant tumors with negative target change, thereby improving the durability of killing.
Example 6 identification of in vivo antitumor Activity
To evaluate the in vivo anti-tumor activity of CD5CAR- γ δ T cells prepared according to the present invention, 6-8 week old NOD/SCID severe immunodeficiency mice were used and injected into the tail vein with 1X 106Human acute T lymph stably expressing luciferaseCell leukemia cell line CCRF-CEM (Luci-CCRF-CEM) establishes mouse acute T lymphocyte leukemia model. After 3 days of modeling, the model was expressed by 1X 107The CD5 CAR-gamma delta T cells (product from experiment 1) prepared according to the culture method of experiment 1 were administered for treatment at a dose of 200. mu.L/mouse. The treatment is carried out by tail vein injection, and the injection is respectively carried out once on the 3 rd day and the 6 th day after the tumor cells are injected. The animals in the treatment control group were injected with the same dose of CD5CAR- α β T prepared by the conventional CD3/CD28 activation method (for detailed information reference: CN 108017717A; CN 110358734A; Clinical Cancer Research 2019, 25 (18): 5595-5607), and the animals in the negative control group were injected with the same dose of normal T cells, and the number of the animals in each treatment group was 3. After the injection treatment, each experimental group was imaged in vivo every 7 days, and the tumor progression in vivo was monitored until all negative control group animals died.
Comparing the data from live imaging of the groups of animals (figure 9) shows that CD5CAR- γ δ T can inhibit the growth of tumor cells in vivo more effectively.
Further survival curve analysis (figure 10) showed that the median survival time of tumor bearing mice could be effectively extended after treatment with CD5CAR- γ δ T cells prepared according to the present invention, indicating that CD5CAR- γ δ T cells have better anti-tumor activity in vivo than CD5CAR- α β T and normal T cells.
In addition, the results of examining anti-tumor associated cytokines in mice from different treatment groups showed that the levels of IL-2, IFN- γ, TNF- γ were significantly higher in mice treated with CD5CAR- γ δ T cells than in the other two treatment groups (FIG. 11).
The above results indicate that the CD5CAR- γ δ T obtained by the preparation method of the present invention has better in vivo anti-tumor activity than the CD5CAR- α β T prepared using the conventional CD3/CD28 activation method.
Example 7 evaluation of in vivo homing ability
To evaluate the in vivo homing activity of cells prepared according to the invention, the treated mice of example 6 above were sacrificed, bone marrow was removed, leukocytes were isolated, and CD5CAR- γ δ T and CD5CAR- α β T (CD 45+/CAR +) in bone marrow were labeled with mouse anti-human CD45, goat anti-mouse Fab antibodies. The results of the test are shown in FIG. 12. The results show that the CAR- γ δ T ratio in bone marrow of mice receiving CD5CAR- γ δ T treatment is significantly higher than that of the CD5CAR- α β T treatment group and the normal T cell treatment group, suggesting that the CD5CAR- γ δ T prepared by the present invention has better bone marrow migration ability than the conventional CAR- α β T.
On the basis of the results, the expression levels of CD5 CAR-gamma delta T prepared by the invention (the product prepared by the experimental group 1), CD5 CAR-alpha beta T prepared by the conventional CD3/CD28 activation method and various chemotaxis receptors on the surface of normal somatic cells are compared by using a flow cytometry detection technology. Will be 1 × 106The cells of the different treatment groups for animal experiments were collected in a flow sample tube, washed once with PBS containing 1% BSA, centrifuged at 1500rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended by adding PBS containing 1% BSA. FITIC-labeled mouse anti-human CXCR4, PE-labeled mouse anti-human CCR7, PerCP-labeled mouse anti-human CXCR5 and APC-labeled mouse anti-human CXCR3 are added to each tube of sample to be tested, and the samples are incubated for 25 minutes at room temperature in a dark place. After the incubation, the sample was washed once with PBS containing 1% BSA, and then added to 200 μ L of the fixation solution for machine detection. The results are shown in FIG. 13 and Table 5.
TABLE 5 expression of normal T cells, CD5CAR- γ δ T and CD5CAR- α β T different chemokines (mean. + -. standard deviation)
Figure 886360DEST_PATH_IMAGE005
The result shows that the CD5 CAR-gamma delta T prepared by the invention can express higher levels of CXCR4, CCR7, CXCR5 and CXCR3, and the CD5 CAR-gamma delta T prepared by the process not only has better anti-tumor activity, but also can induce effector cells to express higher levels of chemotactic receptors, so that the effector cells have better in-vivo migration activity.

Claims (10)

1. A CD 5-targeted CAR- γ δ T cell preparation, wherein the functional receptors are expressed in γ δ T cells in a ratio of: CD16 greater than 40%, NKp30 greater than 40%, NKp44 greater than 50%; greater than 40% CXCR4, greater than 35% CCR7, greater than 50% CXCR5, and greater than 55% CXCR 3.
2. A CD 5-targeted CAR- γ δ T-cell preparation according to claim 1, wherein γ δ T-cells comprise more than 80% and wherein the CAR in γ δ T-cells comprises more than 55%.
3. The CD 5-targeted CAR- γ δ T-cell preparation according to claim 1, wherein the ratio of functional receptor expression in γ δ T-cells is: CD16 greater than 60%, NKp30 greater than 60%, NKp44 greater than 60%; greater than 60% CXCR4, greater than 44% CCR7, greater than 80% CXCR5, and greater than 80% CXCR 3.
4. A method of making a CD 5-targeted CAR- γ δ T cell, comprising:
culturing peripheral blood PBMCs in complete medium and infecting with a lentivirus expressing a CD 5-targeted CAR, continuing the culture until the cells are harvested;
the complete culture medium comprises a basic culture medium and additional factors, wherein the basic culture medium is selected from one or more of serum-free RPMI1640, DMEM, alpha-MEM, X-VIVO15 and AIM-V culture medium, and the additional factors comprise AB serum, interleukin-2, interleukin-15, TGF-beta and zoledronic acid.
5. The method according to claim 4, wherein the content of the additive factors in the complete medium is: the volume content of the AB serum is 5-10%, the interleukin-2 is 100-1000 IU/mL, the interleukin-15 is 5-50 ng/mL, the TGF-beta is 1-20 ng/mL, and the zoledronic acid is 0.1-10 mu M.
6. The method of claim 4, wherein the inactivated aAPC cells are added to the culture system 3-7 days after infection with a lentivirus expressing a CD 5-targeted CAR, and then the culture is continued until the cells are harvested; the addition amount of the aAPC cells is 1: 2-10 according to the number of the T cells to the aAPC cells in the culture system.
7. The method of claim 4, characterized in that it comprises:
PBMC were adjusted to a cell density of 1X 10 with complete medium6~5×106Inoculating the strain/mL into a culture container which is pre-coated with 0.1-40 mu g/mL retronic for culture;
half-amount liquid change or complete culture medium supplement is carried out every 2-3 days in the culture process;
culturing for 5-7 days, harvesting cells, adjusting cell density to 1 × 106~5×106mL, infection with lentivirus expressing CD 5-targeted CAR, continued to culture in complete medium;
adding inactivated aAPC cells at a ratio of T cells to aAPC cells =1 to 2-10 for further culture on days 2-3 after infection; in the continuous culture process, supplementing a fresh complete culture medium into the culture system every 2-3 days, and adding inactivated aAPC cells into the culture system according to the ratio of T cells to aAPC cells = 1: 2-10 every 5-7 days;
culturing for 21-25 days to harvest cells; alternatively, cells were harvested on an initial cell basis when the fold expansion was greater than 300 fold or when the CAR-positive γ δ T cells were more than 10% pure.
8. Use of a CD 5-targeted CAR- γ δ T-cell preparation according to any one of claims 1 to 3 or a CD 5-targeted CAR- γ δ T-cell prepared according to the method of any one of claims 4 to 7 for the preparation of a therapeutic product for use against tumor cells.
9. Use of a CD 5-targeted CAR- γ δ T-cell preparation according to any one of claims 1 to 3 or a CD 5-targeted CAR- γ δ T-cell prepared according to any one of claims 4 to 7 for the preparation of a reagent for the assessment of peripheral γ δ T-cell immune function and/or the study of gene-modified γ δ T-cell anti-tumor mechanisms.
10. A kit, comprising:
a CD 5-targeted CAR- γ δ T-cell preparation according to any one of claims 1 to 3 or a CD 5-targeted CAR- γ δ T-cell prepared according to the method of any one of claims 4 to 7; or
Preparing a reagent composition targeting CD5CAR- γ δ T cells, the reagent composition comprising a complete media composition and aAPC cells; wherein the complete culture medium composition comprises a basic culture medium and additional factors, and the additional factors comprise AB serum, interleukin-2, interleukin-15, TGF-beta and zoledronic acid.
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