Preparation method and application of multi-type CAR-immune cell mixed product containing CAR-macrophages and T cells

Abstract

The invention provides a preparation method and application of a multi-type CAR-immune cell mixed product containing CAR-macrophages and T cells, and relates to the technical field of biology. The preparation method of the mixed product containing the CAR-macrophages and the T cells, provided by the invention, comprises the following steps: culturing peripheral blood mononuclear cells obtained by separation in a culture medium, introducing a gene for coding a chimeric antigen receptor into the macrophages, and continuously culturing to obtain the multi-type CAR-immune cell mixed product containing the CAR-macrophages and the T cells. According to the preparation method, the mixed multi-type CAR-immune cell (CAR-mix) containing CAR-macrophages and T cells is prepared by adopting a one-step method, the cell yield is high, the proportion of the CAR-macrophages to the T cells in the prepared immune cell is proper, the synergistic effect of the CAR-macrophages and the T cells can be fully exerted, the generation of polyclonal T cells is stimulated, and the CAR-mix is used for preparing the CAR-macrophages and the T cells. A stronger solid tumor resisting function is realized.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing a multi-type CAR-immune cell hybrid product comprising CAR-macrophages and T cells, and its application. Background Technology

[0002] Solid tumors are complex in composition. Due to the limited number of effective targets, one of the challenges of CAR-T therapy is that a single target (or dual targets) cannot address the problem of tumor heterogeneity. CAR-macrophage technologies and products composed solely of macrophage components cannot directly kill the large number of tumor cells in solid tumors because macrophages themselves, as effector cells, are not as potent as T cells. Furthermore, whether there are sufficient endogenous T cells (especially in cold tumors) within the tumor to receive antigen presentation and stimulation from macrophages after macrophages are injected into the body is also a challenge, thus failing to guarantee the effective immunomodulatory role of macrophages in stimulating T cells.

[0003] Mounting evidence suggests that treating complex and highly heterogeneous malignant tumors like solid tumors requires leveraging the synergistic effects of lymphocytes' cytotoxic abilities and myeloid cells' immunomodulatory strengths, as well as the systemic roles of the innate immune system (such as macrophage phagocytosis and antigen presentation) and the adaptive immune system's targeted killing. Therefore, the combined use of macrophages and T cells represents a promising future direction.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The primary objective of this invention is to provide a method for preparing a polytypic CAR-immune cell (CAR-mix) product containing CAR-macrophages and T cells, in order to solve the aforementioned technical problems.

[0006] A second objective of the present invention is to provide an immune cell mixture.

[0007] A third objective of this invention is to provide the application of the multi-type CAR-immune cell mixture product containing CAR-macrophages and T cells prepared by the above preparation method in the preparation of drugs for treating tumors.

[0008] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides a method for preparing a multi-type CAR-immune cell hybrid product comprising CAR-macrophages and T cells, comprising the following steps: The isolated peripheral blood mononuclear cells were cultured in animal cell culture medium containing 20-50 ng / ml human M-CSF and 20-50 ng / ml human GM-CSF. Then, the gene encoding the chimeric antigen receptor was introduced into macrophages, and after further culture, a multi-type CAR-immune cell hybrid product containing CAR-macrophages and T cells was obtained.

[0009] As a further technical solution, methods for isolating peripheral blood mononuclear cells include density gradient centrifugation.

[0010] As a further technical solution, the animal cell culture medium includes 20ng-50ng / ml human M-CSF, 20ng-50ng / ml human GM-CSF, and basic culture medium components; The basic components of the culture medium, by volume percentage, include 5%-10% human serum substitute, 0.5%-2% GlutaMAX, and the balance being X-VIVO15.

[0011] As a further technical solution, the chimeric antigen receptor comprises, from the N-terminus to the C-terminus, a single-chain antibody against mesothelin, a CD8α hinge region, a CD8α transmembrane region, a 4-1BB co-stimulatory signal transduction region, and a CD3ζ signal transduction region. The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.1.

[0012] As a further technical solution, the method of introducing the gene encoding the chimeric antigen receptor into macrophages includes Ad5f35 adenovirus transfection.

[0013] As a further technical solution, the MOI of the adenovirus transfection is 20-70.

[0014] As a further technical solution, the preparation method includes the following steps: The isolated peripheral blood mononuclear cells were cultured in animal cell culture medium containing 20-50 ng / ml human M-CSF and 20-50 ng / ml human GM-CSF. After 2-4 days of culture, the gene encoding the chimeric antigen receptor was introduced into macrophages. After 2-3 more days of culture, a multi-type CAR-immune cell mixture product containing CAR-macrophages and T cells was obtained.

[0015] Secondly, the present invention provides an immune cell mixture prepared by the above-described preparation method.

[0016] As a further technical solution, the number of CAR-macrophages in the immune cell mixture accounts for 50%-70%, and the number of T cells accounts for 10%-30%.

[0017] Thirdly, the present invention provides the application of the multi-type CAR-immune cell mixture product containing CAR-macrophages and T cells prepared by the above preparation method in the preparation of drugs for treating tumors.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for preparing a multi-type CAR-immune cell mixture product containing CAR-macrophages and T cells. The method uses a one-step process to prepare a mixed immune cell CAR-mix containing CAR-macrophages and T cells. This preparation method has a high cell yield. The ratio of CAR-macrophages to T cells in the prepared immune cell CAR-mix has been optimized and tested to prove that it is appropriate, which can give full play to the synergistic effect of the two, stimulate the production of polyclonal T cells, and achieve stronger anti-solid tumor function. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 A. Microscopic image of the product prepared by the process of this invention; B. Microscopic image of the product prepared by the prior art solution; C. Immunofluorescence staining observation of the distribution of macrophages and T cells in the product prepared by the process of this invention and the product prepared by the prior art solution; Figure 2 The main components of the cell products prepared by this invention; Figure 3 A. CAR positivity rate of macrophages in cell products prepared by the process of this invention was detected by flow cytometry; B. CAR positivity rate of macrophages in cell products prepared by the background technique was detected by flow cytometry; C. Statistical analysis of the total number of cells in the final product prepared by the process of this invention and the background technique, with the same starting cell amount. Figure 4A. The average fluorescence intensity of CD80 expressed by WT-mix and CAR-mix was detected by flow cytometry; B. The average fluorescence intensity of CD86 expressed by WT-mix and CAR-mix was detected by flow cytometry; C. After WT-mix and CAR-mix were co-cultured with SKOV3 tumor cells at three effector-to-target ratios of 5:1, 10:1, and 20:1 for 48 hours or 72 hours, the killing efficiency against tumor cells was detected by luciferase assay; D. After co-culturing CD3+ T cells, CD14+ CAR-Mac prepared using the prior art, and CAR-mix prepared by the present invention at an effector-to-target ratio of 10:1 with SKOV3 tumor cells for 48 hours, the killing efficiency against tumor cells was detected by luciferase assay. Figure 5 A. The killing ability of T cells, CD14+ WT-Mac, WT-mix, CD14+ CAR-Mac, and CAR-mix against MSLN+ SKOV3 tumor cells was detected by luciferase assay; B. The killing ability of T cells, CD14+ WT-Mac, WT-mix, CD14+ CAR-Mac, and CAR-mix against MSLN knockout SKOV3 tumor cells was detected by luciferase assay; C. The killing ability of T cells, CD14+ WT-Mac, WT-mix, CD14+ CAR-Mac, and CAR-mix against a 1:1 mixture of MSLN+ and MSLN knockout SKOV3 tumor cells was detected by luciferase assay. Figure 6 A. Tumor-bearing mice modeled by SKOV3 intraperitoneal injection were treated with the same number of cells intraperitoneally, and the changes in tumor burden at different time points after treatment were observed by in vivo imaging, including the PBS control group, CD14+ CAR-Mac prepared by existing technology, and CAR-mix cells containing different proportions (15%, 30%, 60%) of T cells; B. Tumor burden change curves at different time points after treatment; C. Survival curves of mice in different treatment groups; Figure 7 A. Tumor-bearing mice induced by intraperitoneal injection of SKOV3 were observed for changes in tumor burden after intraperitoneal injection of different cell therapies using in vivo imaging, including a PBS control group, a CAR-mix group (8e6 cells / mouse), and groups with different numbers of T cells: 15% (1.2×10⁻⁶). 6 (pieces / animal), 30% (2.4×10) 6 (pieces / animal), 60% (4.8×10) 6 (each / piece), 100% (8×10) 6 B. Tumor burden changes at different times after treatment; Figure 8A. The proportion of CD69+ activated T cells after co-culturing CAR-mix and AsPC1 pancreatic cancer cells at a 10:1 effector-target ratio for 72 hours by flow cytometry; B. The proportion of CD69+ activated T cells after co-culturing CAR-mix and SKOV3 ovarian cancer cells at a 10:1 effector-target ratio for 72 hours by flow cytometry; C. The proportion of CD45RO+CD45RA- memory T cells after co-culturing CAR-mix and SKOV3 ovarian cancer cells at a 10:1 effector-target ratio for 72 hours by flow cytometry. Figure 9 After co-culturing CAR-mix and SKOV3 ovarian cancer cells at an effector-target ratio of 10:1 for 72 hours, cells were collected. T-cell clonal diversity indices in individual CAR-mix cells and co-cultured CAR-mix cells were analyzed by TCR-seq sequencing. Figure 10 After co-culturing CAR-mix and SKOV3 ovarian cancer cells at an effector-target ratio of 10:1 for 72 hours, cells were collected. T cell clonal distribution heatmaps in individual CAR-mix cells (A) and co-cultured CAR-mix cells (B) were analyzed by TCR-seq sequencing. Detailed Implementation

[0021] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0022] In a first aspect, the present invention provides a method for preparing a multi-type CAR-immune cell hybrid product comprising CAR-macrophages and T cells, comprising the following steps: The isolated peripheral blood mononuclear cells were cultured in animal cell culture medium containing 20-50 ng / ml human M-CSF and 20-50 ng / ml human GM-CSF. Then, the gene encoding the chimeric antigen receptor was introduced into macrophages, and after further culture, a multi-type CAR-immune cell hybrid product containing CAR-macrophages and T cells was obtained.

[0023] The present invention provides a method for preparing a multi-type CAR-immune cell mixture product containing CAR-macrophages and T cells. The method uses a one-step process to prepare a mixed immune cell CAR-mix containing CAR-macrophages and T cells. This preparation method has a high cell yield. The ratio of CAR-macrophages to T cells in the prepared immune cell CAR-mix has been optimized and verified to be appropriate, which can give full play to the synergistic effect of the two, stimulate the production of polyclonal T cells, and achieve stronger anti-solid tumor function.

[0024] In some alternative embodiments, the animal cell culture medium comprises 20 ng-50 ng / ml human M-CSF, 20 ng-50 ng / ml human GM-CSF, and a basic culture medium component; The basic components of the culture medium, by volume percentage, include 5%-10% human serum substitute, 0.5%-2% GlutaMAX, and the balance being X-VIVO15.

[0025] This invention does not impose specific limitations on the method for isolating and obtaining peripheral blood mononuclear cells; any method well-known to those skilled in the art can be used. In some optional embodiments, the method for isolating and obtaining peripheral blood mononuclear cells includes density gradient centrifugation.

[0026] In some alternative implementations, the step of obtaining peripheral blood mononuclear cells using density gradient centrifugation includes: 1. PBMC Isolation: Dilute the apheresis blood 2-fold with physiological saline (1:1 dilution). Prepare several sterile 50ml centrifuge tubes, add 20ml of Ficoll separation buffer to each, then slowly add 20ml of the diluted and mixed cell suspension along the wall of the centrifuge tube onto the surface of the Ficoll separation buffer. Set the centrifuge speed to 1 for ascending and 0 for descending, the centrifugation force to 400g, 25℃, and centrifuge for 30 minutes. Aspirate the white membrane layer into a 50ml centrifuge tube, and add physiological saline buffer to each tube up to the 45ml mark. Invert to mix and centrifuge: set the speed to 400g, 25℃, time to 10 minutes, and the speed to 5 for ascending and 4 for descending. Discard the supernatant after centrifugation. Then, wash twice with physiological saline buffer. Collect the obtained PBMCs into one tube, dilute the sample, and count them.

[0027] 2. Dilute the cell density to 5.56 × 10⁻⁶ based on the cell count results. 6 / mL, and then placed in T225 culture flasks, 45ml per flask. The culture medium composition is: basic components of culture medium + cytokines. The basic components of culture medium are: 94% X-VIVO15 + 5% human serum substitute + 1% GlutaMAX; cytokines are: 25% human M-CSF + 25% human GM-CSF.

[0028] 3. After 72 hours, discard the supernatant and add fresh complete culture medium. Add adenovirus at MOI=50. Return to culture for further incubation.

[0029] 4. After 24 hours, centrifuge to collect the cells from the supernatant and replace with fresh culture medium.

[0030] 5. After 48 hours, scrape off all the cells with a cell scraper, collect, count, and fill into containers for cryopreservation.

[0031] In some optional embodiments, the chimeric antigen receptor comprises, from the N-terminus to the C-terminus, a single-chain antibody against mesothelin, a CD8α hinge region, a CD8α transmembrane region, a 4-1BB co-stimulatory signal transduction region, and a CD3ζ signal transduction region. The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.1: EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSG GGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK.

[0032] In some alternative implementations, the method of introducing the gene encoding the chimeric antigen receptor into macrophages includes Ad5f35 adenovirus transfection, which can bind to CD46 on the surface of macrophages, thereby targeting macrophages with minimal infection of other cells.

[0033] In some alternative implementations, the MOI of the adenovirus transfection is 20-70.

[0034] In some optional embodiments, the preparation method includes the following steps: The isolated peripheral blood mononuclear cells were cultured in animal cell culture medium containing 20-50 ng / ml human M-CSF and 20-50 ng / ml human GM-CSF. After 2-4 days of culture, the gene encoding the chimeric antigen receptor was introduced into macrophages. After 2-3 more days of culture, a multi-type CAR-immune cell mixture product containing CAR-macrophages and T cells was obtained.

[0035] Secondly, the present invention provides an immune cell mixture prepared by the above-described preparation method.

[0036] In some alternative embodiments, the immune cell mixture contains 50%-70% CAR-macrophages and 10%-30% T cells.

[0037] The ratio of CAR-macrophages to T cells in this immune cell mixture is appropriate, which can give full play to the synergistic effect of the two, stimulate the production of polyclonal T cells, and achieve stronger anti-solid tumor function.

[0038] Thirdly, the present invention provides the application of the multi-type CAR-immune cell mixture product containing CAR-macrophages and T cells prepared by the above preparation method in the preparation of drugs for treating tumors.

[0039] The immune cells prepared by the method of this invention contain an appropriate ratio of CAR-macrophages and T cells, which can give full play to the synergistic effect of the two and can be used to prepare drugs for treating tumors.

[0040] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0041] Example 1 The preparation method of CAR-macrophages and T cells (CAR-mix) includes the following steps: 1. PBMC Isolation: Dilute the apheresis blood 2-fold with physiological saline (1:1 dilution). Prepare several sterile 50ml centrifuge tubes, add 20ml of Ficoll separation buffer to each, then slowly add 20ml of the diluted and mixed cell suspension along the wall of the centrifuge tube onto the surface of the Ficoll separation buffer. Set the centrifuge speed to 1 for ascending and 0 for descending, the centrifugation force to 400g, 25℃, and centrifuge for 30 minutes. Aspirate the white membrane layer into a 50ml centrifuge tube, and add physiological saline buffer to each tube up to the 45ml mark. Invert to mix and centrifuge: set the speed to 400g, 25℃, time to 10 minutes, and the speed to 5 for ascending and 4 for descending. Discard the supernatant after centrifugation. Then, wash twice with physiological saline buffer. Collect the obtained PBMCs into one tube, dilute the sample, and count them.

[0042] 2. Dilute the cell density to 5.56 × 10⁻⁶ based on the cell count results. 6 / mL, and then placed in T225 culture flasks, 45ml per flask. The culture medium composition is: basic components of culture medium + cytokines. The basic components of culture medium are: 94% X-VIVO15 + 5% human serum substitute + 1% GlutaMAX; cytokines are: 25% human M-CSF + 25% human GM-CSF.

[0043] 3. After 72 hours, discard the supernatant and add fresh complete culture medium. Add adenovirus at MOI=50. Return to culture for further incubation.

[0044] 4. After 24 hours, centrifuge to collect the cells from the supernatant and replace with fresh culture medium.

[0045] 5. After 48 hours, scrape off all the cells with a cell scraper, collect, count, and fill into containers for cryopreservation.

[0046] Comparative Example 1 The preparation method of CAR-Mac cells includes the following steps: 1. Mononuclear cells in apheresis blood were separated using density gradient centrifugation.

[0047] 2. Incubate mononuclear cells with CD14 magnetic beads for 15-30 minutes (per 10 mg / L). 7 Add 10 μL of magnetic beads to the cells.

[0048] 3. Add 10 times the volume of buffer solution and centrifuge at 300g for 10 minutes. Discard the supernatant and resuspend in buffer solution.

[0049] 4. Place the sorting column in the magnetic rack and pre-wash with 500 μL of buffer (to equilibrate the matrix inside the column).

[0050] 5. Slowly add the cell suspension to the sorting column and wash three times with buffer. Remove the sorting column, add a certain volume of buffer, and quickly push with the plunger to collect CD14-positive cells.

[0051] 6. Transfer the harvested cells at 1e 6 The culture medium was resuspended at a density of / ml in the culture medium and placed in a T225 flask for culture. The culture medium composition was: basic components of the culture medium + cytokines. The basic components of the culture medium were: 94% X-VIVO15 + 5% human serum substitute + 1% GlutaMAX; the cytokines were: 25% human M-CSF + 25% human GM-CSF.

[0052] 7. Add adenovirus on day 3 of culture at MOI=20-70, and change the medium after 24 hours.

[0053] 8. Collect cells after culturing for another 2 days.

[0054] Experimental Example 1 The CAR-mix cells prepared in Example 1 and the CAR-Mac cells prepared in Comparative Example 1 were seeded onto 24-well cell culture slides. After 12 hours, cell morphology was observed using an optical microscope. Cells were then fixed with 4% paraformaldehyde for 30 minutes. After fixation, anti-human CD11b-FITC and anti-human CD3-PE antibodies were diluted 1:100 (v / v) and stained at room temperature for 30 minutes. After washing, cells were stained with 1 μg / ml DAPI for 5 minutes. The cell culture slides were removed from the culture dishes, placed on glass slides, mounted, and observed and photographed under a confocal microscope.

[0055] The results are as follows Figure 1 As shown, the product prepared by the process of this invention is mainly composed of CAR-macrophages, and T cells can also be clearly seen attached around the macrophages. Figure 1 (A) Comparative Example 1 yielded macrophages with higher purity ( Figure 1 (B in the text). Immunofluorescence staining can produce the same results (see appendix). Figure 1 (C in the middle).

[0056] Experimental Example 2 Take 1×10 6 CAR-mix cells were treated with anti-human CD11b-FITC and anti-human CD3-APC antibodies and stained at room temperature for 15 minutes. After washing once with 1 ml of PBS, the proportion of positive cells was detected by flow cytometry.

[0057] The results are as follows Figure 2 As shown. The cell product prepared by the process of this invention consists of 50%-70% macrophages and 10%-30% T cells (as shown). Figure 2 ).

[0058] Experimental Example 3 Take 1×10 6 WT-mix cells (prepared differently from Comparative Example 1 in that adenovirus-infected cells were not added), CAR-mix cells (cells prepared in Example 1), CD14+ WT-Mac cells (prepared differently from Comparative Example 1 in that adenovirus-infected cells were not added), and CD14+ CAR-Mac cells (cells prepared in Comparative Example 1) were stained with anti-human CD11b-FITC and anti-human MSLN-PE antibodies for 15 minutes at room temperature. After washing once with 1 ml of PBS, the proportion of positive cells was detected by flow cytometry. The results are as follows: Figure 3 As shown in A and B in the diagram.

[0059] PBMCs were isolated from apheresis blood. A portion of the PBMCs was then used to separate CD14-positive cells using CD14 magnetic beads. After counting, cells were analyzed at a concentration of 1×10⁻⁶. 6Cells were seeded at a density of 1 / ml and cultured in 10cm dishes with 10ml of cell suspension. Another portion of PBMCs was collected, and the proportion of CD14-positive cells was determined by flow cytometry. The calculated percentage was 1×10⁻⁶ CD14-positive cells. 6 Cells were seeded at a density of 1 / ml and cultured in 10cm dishes with 10ml of cell suspension per dish. Subsequent culture was performed according to the preparation processes of Comparative Example 1 and Example 1, respectively. Cells were digested and collected on day 6 of culture, and the yields of both processes were calculated using a cell counter. Results are as follows... Figure 3 As shown in C.

[0060] The results are as follows Figure 3 As shown. Compared with existing technologies, the macrophage CAR positivity rate of the cell products prepared by this invention is comparable (see attached diagram). Figure 3 (A and B in the text), but production can be increased to about 3 times ( Figure 3 (C in the middle).

[0061] Test Example 4 Take 1×10 6 WT-mix cells (prepared differently from those in Example 1, except without the addition of adenovirus-infected cells) and CAR-mix cells were stained with anti-human CD11b-FITC, anti-human CD80-APC, and anti-human CD86-PE antibodies for 15 minutes at room temperature. After washing once with 1 ml of PBS, the fluorescence intensity was detected by flow cytometry. The results are as follows: Figure 4 As shown in A and B in the diagram.

[0062] Add 2×10 to each well of the 96-well plate 3 SKOV3-luci tumor cells were cultured at effector-to-target ratios of 5:1, 10:1, and 20:1, with the addition of specific amounts of WT-mix or CAR-mix cells. After 48 or 72 hours of incubation, luciferase substrate was added, and the amount of remaining tumor cells in each well was measured to calculate the killing efficiency. Results are as follows: Figure 4 As shown in C.

[0063] Add 2×10 to each well of the 96-well plate 3 SKOV3-luci tumor cells were treated with a certain amount of CD3 at an effector-target ratio of 20:1. + T cells (i.e., pure T cells), CD14 + CAR-Mac (CAR-macrophages prepared in Comparative Example 1) or CAR-mix cells were placed back into the incubator and cultured for 72 hours. Then, luciferase substrate was added, the amount of remaining tumor cells in each well was detected, and the killing efficiency was calculated.

[0064] The results are as follows Figure 4As shown. Compared with WT-mix, a product prepared using the same process but without viral transfection, the cell product CAR-mix prepared by the present invention showed a significant increase in the surface markers CD80 and CD86 of M1 macrophages. Figure 4 (A and B in the text). CAR-mix exhibits significantly higher tumor cell killing ability in vitro than WT-mix (see attached text). Figure 4 (C) Compared with CD3+ T cells alone and CAR-Mac prepared in Comparative Example 1, the cell product prepared in this invention has a significantly enhanced tumor-killing ability (see appendix). Figure 4 (D in the middle).

[0065] Experimental Example 5 Add 2×10 to each well of the 96-well plate 3 SKOV3-luci tumor cells were cultured at an effector-to-target ratio of 20:1, with the addition of a certain number of CD3+ T cells (i.e., pure T cells), CD14+ WT-Mac (prepared in a manner different from Comparative Example 1, except that adenovirus-infected cells were not added), WT-mix (prepared in a manner different from Example 1, except that adenovirus-infected cells were not added), CD14+ CAR-Mac (CAR-macrophages prepared in Comparative Example 1), or CAR-mix cells (cells prepared in Example 1). After 72 hours of incubation in a culture incubator (containing tumor cells), luciferase substrate was added, and the amount of remaining tumor cells in each well was measured to calculate the killing efficiency. Two types of tumor cells were used: SKOV3 cells expressing MSLN antigen and SKOV3 cells not expressing MSLN antigen. The experiment was divided into three groups: one group used tumor cells expressing MSLN antigen as target cells (SKOV3-MSLN+), one group used tumor cells not expressing MSLN antigen as target cells (SKOV3-MSLN-KO), and one group used a 1:1 mixture of the two types of cells as target cells (SKOV3-MSLN+:KO 1:1).

[0066] The results are as follows Figure 5 (Based on the total number of cells in CAR-mix, T30% refers to T cells that make up 30% of the total number of cells in CAR-mix, and T100% refers to T cells whose number is the same as the total number of cells in CAR-mix.) The CAR-Mac in the cell product prepared by the process of this invention synergistically exerts a better anti-tumor effect with T cells. On tumor cells expressing MSLN antigen, both CAR-mix and CD14+ CAR-Mac have strong tumor cell killing ability, and CAR-mix is ​​significantly stronger than CD14+ CAR-Mac. T cells alone and WT-mix without CAR expression have weaker tumor killing ability (see appendix). Figure 5(A) Compared to tumor cells expressing MSLN antigen, the tumor-killing ability of both CAR-mix and CD14+ CAR-Mac is reduced against tumor cells that do not express MSLN antigen, with the reduction being more pronounced for CD14+ CAR-Mac (see Appendix). Figure 5 (A and B in the text). For tumor cells with a 1:1 mixture of expressed and unexpressed antigens, the killing ability of CAR-mix remained almost unchanged at around 60%, but the CD14+ CAR-Mac decreased significantly, from 40% to less than 20% (see appendix). Figure 5 (C in the text). These results suggest that CAR-mix has a stronger killing effect on highly heterogeneous tumor cells. This may be a result of the synergistic effect of CAR-Mac and T cells.

[0067] Experimental Example 6 Dozens of healthy NSG mice aged 6-8 weeks were selected, and each mouse was injected intraperitoneally with 2×10 5 SKOV3-luci cells were used. Three days later (day D-1), tumor burden in the model mice was assessed using small animal in vivo imaging. Mice with similar tumor burdens were randomly divided into five groups: a PBS control group, a CD14+ CAR-Mac prepared in Comparative Example 1, and a CAR-mix group containing different proportions of T cells (the T cell proportions were adjusted to 15%, 30%, and 60% by adding a certain amount of the same in vivo T cells). On day D0, mice in the corresponding groups were injected with either PBS or the corresponding cell therapy. The volume of both PBS and cell suspension was 200 μL / mouse, and the cell count was 8 × 10⁶ cells / mouse. 6 / mouse. Subsequent monitoring of tumor burden changes in mice was conducted using small animal in vivo imaging, and the differences were statistically analyzed. Mortality rates for each group of mice were recorded, and survival curves were plotted.

[0068] The results are as follows Figure 6 As shown. Animal experiments have demonstrated that the CAR-mix prepared using the process of this invention exhibits superior antitumor effects in mice compared to the CD14+ CAR-Mac prepared in Comparative Example 1.

[0069] Experimental Example 7 Dozens of healthy NSG mice aged 6-8 weeks were selected, and each mouse was injected intraperitoneally with 2×10 5 SKOV3-luci cells were collected. Three days later (day D-1), tumor burden in the model mice was assessed using small animal in vivo imaging. Mice with similar tumor burdens were randomly divided into 6 groups, including a PBS control group, a CAR-mix group (8 × 10⁻⁶ cells), and a CAR-mix group (8 × 10⁻⁶ cells). 6 (number / animal), and different numbers of T cells: 15% (1.2 × 10⁻⁶ cells / animal), and groups with different numbers of T cells: 15% (1.2 × 10⁻⁶ cells / animal). 6 (pieces / animal), 30% (2.4×10) 6(pieces / animal), 60% (4.8×10) 6 (each / piece), 100% (8×10) 6 (Number of mice per animal). Subsequent monitoring of tumor burden changes in mice was conducted using small animal in vivo imaging, and the differences were statistically analyzed.

[0070] The results are as follows Figure 7 As shown. Animal experiments have demonstrated that the CAR-mix prepared using the process of this invention has a better anti-tumor effect than T cells alone in mice, and different doses of T cells did not show significant tumor-suppressive effects.

[0071] Experimental Example 8 The activation of T cells in the cells prepared according to Example 1 of this invention is crucial for enhancing anti-tumor function. We co-cultured the prepared CAR-mix with AsPC1 pancreatic cancer cells and SKOV3 ovarian cancer cells at an effector-target ratio of 10:1 for 72 hours, with 5 × 10⁶ tumor cells included. 5 5 × 10 CAR-mix cells / well 6 Cells / well. Subsequently, flow cytometry antibodies against human CD8-PE and anti-human CD69-APC were added for staining, and T cell activation in the mixture was detected by flow cytometry analysis. Compared with CAR-mix cells without co-culture, the proportion of CD69+ activated T cells in CD8+ T cells after co-culture was significantly increased (see attached image). Figure 8 (A and B in the text). Additionally, a significant increase in the proportion of memory T cells among the T cells was observed after co-culture (see attached text). Figure 8 (C in the middle).

[0072] Experimental Example 9 To further investigate changes in T cells, 2×10⁶ cells were cultured separately. 7 One CAR-mix, or 2×10 7 One CAR-mix with 2×10 6 SKOV3 cells were co-cultured. After 72 hours, cell samples were collected, RNA was extracted, and TCR sequencing was performed. The sequencing results indicated that the diversity of T cell clones decreased significantly after co-culture. Figure 9 ), certain specific clones were amplified in large quantities ( Figure 10 This is an effective response to tumor cells.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a multi-type CAR-immune cell hybrid product comprising CAR-macrophages and T cells, characterized in that, Includes the following steps: The isolated peripheral blood mononuclear cells were cultured in animal cell culture medium containing 20-50 ng / ml human M-CSF and 20-50 ng / ml human GM-CSF. Then, the gene encoding the chimeric antigen receptor was introduced into macrophages, and after further culture, a multi-type CAR-immune cell hybrid product containing CAR-macrophages and T cells was obtained.

2. The preparation method according to claim 1, characterized in that, Methods for isolating peripheral blood mononuclear cells include density gradient centrifugation.

3. The preparation method according to claim 1, characterized in that, The animal cell culture medium includes 20 ng-50 ng / ml human M-CSF, 20 ng-50 ng / ml human GM-CSF, and basic culture medium components; The basic components of the culture medium, by volume percentage, include 5%-10% human serum substitute, 0.5%-2% GlutaMAX, and the balance being X-VIVO15.

4. The preparation method according to claim 1, characterized in that, The chimeric antigen receptor, from N-terminus to C-terminus, comprises an anti-mesothelin single-chain antibody, a CD8α hinge region, a CD8α transmembrane region, a 4-1BB co-stimulatory signal transduction region, and a CD3ζ signal transduction region. The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.

1.

5. The preparation method according to claim 1, characterized in that, The method of introducing the gene encoding the chimeric antigen receptor into macrophages includes Ad5f35 adenovirus transfection.

6. The preparation method according to claim 5, characterized in that, The MOI of the adenovirus transfection was 20-70.

7. The preparation method according to claim 1, characterized in that, The preparation method includes the following steps: The isolated peripheral blood mononuclear cells were cultured in animal cell culture medium containing 20-50 ng / ml human M-CSF and 20-50 ng / ml human GM-CSF. After 2-4 days of culture, the gene encoding the chimeric antigen receptor was introduced into macrophages. After 2-3 more days of culture, a multi-type CAR-immune cell mixture product containing CAR-macrophages and T cells was obtained.

8. A mixture of immune cells, characterized in that, It is prepared by the preparation method according to any one of claims 1-7.

9. The immune cell mixture according to claim 8, characterized in that, The immune cell mixture contains 50%-70% CAR-macrophages and 10%-30% T cells.

10. The use of a multi-type CAR-immune cell mixture product containing CAR-macrophages and T cells prepared by the preparation method according to any one of claims 1-7 in the preparation of a drug for treating tumors.

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