Construction method of CAR-T (chimeric antigen receptor T) cells for enhancing chemotaxis and tumor suppression effects of solid tumors in lung

Abstract

The invention provides a CAR-T cell construction method for enhancing chemotactic and tumor inhibition effects of lung solid tumors, HER2-CAR-T cells co-expressing high-specificity paired double receptors CXCR5 and CCR6 are developed on the basis of high-expression chemotactic factors CXCL13 and CCL20 in a lung cancer tumor microenvironment, and finally chemotactic and homing of the CAR-T cells to the lung solid tumors are enhanced. The construction and the application of the tumor inhibition effect are improved.

Description

Technical Field

[0001] The present invention relates to the field of immune cell technology, and in particular to a method for constructing CAR-T cells that enhance the chemotaxis and tumor inhibition effects of solid lung tumors. Background Art

[0002] Lung cancer is the most common malignant tumor in the world, and its incidence and mortality rate rank first in cancer statistics. Although traditional treatment and targeted therapy have prolonged survival and improved quality of life for many patients, the effectiveness of treatment is still limited by gene mutation targets and drug selection. In addition, the recurrence of lung cancer, the inefficiency of drugs, the increased resistance of tumors to drugs, and the serious adverse reactions that may occur during treatment all pose major challenges to the overall quality of life of patients.

[0003] As a cutting-edge technology in the era of precision medicine, CAR-T therapy is hailed as a ray of hope for conquering tumors because of its potential in solving the difficult problems of tumor treatment. In the treatment of hematological tumors, CAR-T therapy has achieved remarkable clinical results with its high specificity and selectivity. However, in the treatment of solid tumors, due to obstacles such as the lack of tumor-specific antigens, the immunosuppressive effects of the tumor microenvironment (TME), the low infiltration and homing ability of T cells, and T cell exhaustion, although CAR-T therapy has shown great potential, it also faces many challenges.

[0004] Chemokines are important molecules that promote T cell tumor infiltration and homing. The highly specific pairing between chemokines and chemokine receptors is the core factor that determines the tumor homing of CAR-T cells and exerts therapeutic effects. In view of the differences in the expression of chemokines in the TME of solid tumors, the CAR-T optimization strategy of engineering CAR-T cells to express specific chemokine receptors can improve the homing ability of CAR-T cells to tumor tissues, and is expected to achieve precise and effective tumor control. Summary of the invention

[0005] The present invention provides a method for constructing CAR-T cells that enhance the chemotaxis and tumor inhibition effects of solid lung tumors. The method is based on the high expression of chemokines CXCL13 and CCL20 in the tumor microenvironment of lung cancer, and develops HER2-CAR-T cells that co-express highly specific paired dual receptors CXCR5 and CCR6, which ultimately enhance the chemotaxis and homing of CAR-T cells to solid lung tumors and improve the construction and application of tumor inhibition effects.

[0006] The first purpose of the present invention is to determine the chemokine receptors and ligands with high affinity and unique pairing at this stage, so as to avoid unexpected effects or adverse reactions caused by redundancy of chemokine receptors and ligands, so as to further define the chemokines in the lung cancer tumor microenvironment, and analyze the expression spectrum of the above-defined chemokines in the lung cancer tumor microenvironment.

[0007] The present invention provides a method for constructing CAR-T cells that enhance the chemotaxis and tumor inhibition effects of lung solid tumors, comprising the following steps: 1) Identify the chemokine receptors and ligands that have high affinity and are the only pair at this stage, and choose the chemokine receptors and ligands that are avoided as mentioned above; 2) Retrieve the above chemokine expression profiles of lung adenocarcinoma and normal tissues from the TCGA database to identify chemokines whose expression in cancer tissues is significantly higher than that in adjacent tissues; 3) Detect the expression of transcriptional and protein levels in lung adenocarcinoma clinical lung cancer and adjacent tissues to confirm that the chemokines expressed in cancer tissues are significantly higher than those in adjacent tissues; 4) The chemokine protein expression levels of normal lung cell line BEAS-2B and lung cancer cell lines A549, H1734, H1975, H1299, and HCC827 were detected to clarify the expression of chemokines in the cell lines and identify the target cells.

[0008] The second purpose of the present invention is to design CAR-T cells with high-pairing receptors that highly express chemokines in the lung cancer tumor microenvironment, and to verify the expression of each gene in the above-mentioned high-pairing receptor CAR-T cells.

[0009] The present invention uses pCDH-CMV-LucSh(CpG-free)-EF1-coGFP-T2A-Puro as a backbone plasmid, performs full gene synthesis on the gene sequence corresponding to HER2-CAR, and then uses enzyme cutting and ligation to construct it between the XbaI and NotI restriction sites of the pCDH-CMV-LucSh(CpG-free)-EF1-coGFP-T2A-Puro vector, and deletes the redundant EF1-coGFP-T2A-Puro sequence in the plasmid backbone to obtain the pCDH-CMV-HER2-CAR target plasmid. The nucleotide sequence of HER2 is shown in SEQ ID NO.2.

[0010] Furthermore, the intermediate plasmid pCDH-CMV-HER2-CAR-EF1-coGFP-T2A-Puro was used as the plasmid backbone, the CXCR5 and CCR6 genes were fully synthesized, and the coGFP and Puro in the backbone were replaced by homologous recombination to obtain the pCDH-CMV- HER2-CAR-EF1-CXCR5-T2A-CCR6 target plasmid. The nucleotide sequence of CXCR5 is shown in SEQ ID NO.7, and the nucleotide sequence of CCR6 is shown in SEQ ID NO.8.

[0011] The third purpose of the present invention is to detect the proliferation of CAR-T cells in in vitro lung cancer cell lines, and the chemotaxis and killing effects on lung cancer cells.

[0012] HER2-CXCR5-CCR6-CAR-Jurkat / T and HER2-CAR-Jurkat / T were co-incubated with HCC827 target cells that highly expressed chemokines CXCL13 and CCL20.

[0013] 1. After target cell stimulation, detect the CD69 activation index of HER2-CXCR5-CCR6-CAR-Jurkat and HER2-CAR-Jurkat cells. After target cell stimulation, detect the killing efficiency of HER2-CXCR5-CCR6-CAR-T and HER2-CAR-T on target cells.

[0014] 2. After target cell stimulation, detect the proliferation of HER2-CXCR5-CCR6-CAR-T and HER2-CAR-T cells.

[0015] 3. After target cell stimulation, the chemotactic effect of HER2-CXCR5-CCR6-CAR-T and HER2-CAR-T on tumor cells.

[0016] The fourth objective of the present invention is to verify the in vivo tumor killing effect of the above-mentioned high-paired receptor CAR-T cells in an in vivo orthotopic lung cancer mouse model.

[0017] HER2-CXCR5-CCR6-CAR-T group and HER2-CAR-T group, lung orthotopic HCC827-Luciferase target cell tumor-bearing mice were constructed in the treatment, and the tumor-bearing mice were divided into PBS group, HER2-CXCR5-CCR6-CAR-T group and HER2-CAR-T group for treatment.

[0018] 1. Luciferase fluorescence was measured by small animal in vivo imaging every day for three days to determine tumor size and tumor control.

[0019] 2. On the 14th and 28th days, the peripheral blood was measured to show the ratio of CD4+ and CD8+ T cells to determine the in vivo proliferation of CAR-T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a redundancy diagram of chemokine receptor and ligand pairs.

[0021] Figure 2 It is the expression profile of chemokines CX3CL1, CXCL16, CXCL13, CXCL12, CCL25, and CCL20 in 483 lung adenocarcinomas and 347 normal tissues in the TCGA database.

[0022] Figure 3 The above graphs show the expression of chemokines CXCL13 and CCL20 proteins in 6 pairs of lung adenocarcinoma clinical lung cancer and adjacent tissues. The results show that the expression of CXCL13 and CCL20 in cancer tissues is significantly higher than that in adjacent tissues.

[0023] Figure 4 The above is a statistical chart of the protein expression of chemokines CXCL13 and CCL20 in 6 pairs of lung adenocarcinoma clinical lung cancer and adjacent tissues. The results show that the expression of CXCL13 and CCL20 in cancer tissues is significantly higher than that in adjacent tissues.

[0024] Figure 5 Western Blot was used to identify the protein expression of chemokines CXCL13 and CCL20 in BEAS-2B (normal lung cell line) and lung cancer cell lines A549, H1734, H1975, H1299, and HCC827. The results showed that H1975 and HCC827 both highly expressed chemokines CXCL13 and CCL20. HCC827 was used as the target cell for subsequent experiments.

[0025] Figure 6 Real-time quantitative PCR was used to identify the RNA expression of chemokines CXCL13 and CCL20 in BEAS-2B (normal lung cell line) and lung cancer cell lines A549, H1734, H1975, H1299, and HCC827. The results showed that both HCC827 cell lines highly expressed chemokines CXCL13 and CCL20. HCC827 was used as the target cell for subsequent experiments.

[0026] Figure 7HER2-CAR-CXCR5-CCR6 and HER2-CAR were designed and constructed. Based on the pCDH-CMV-MCS-EF1-coGFP-T2A-Puro plasmid (generation 0), the second-generation CAR with the specific scFv-CD8 hinge region, transmembrane region + co-stimulatory signal (CD28 or 4-1BB) of the single target HER2 was constructed in the multiple cloning site (MCS) NheⅠ and EcoRⅠ through enzyme ligation. At the same time, the receptor CXCR5 and CCR6S genes were replaced with the coGFP and Puro genes in the plasmid by homologous recombination, respectively, to obtain the target gene plasmid: pCDH-CMV-HER2CAR-EF1-CXCR5-T2A-CCR6.

[0027] Figure 8 It is the expression detection of each gene in HER2-CXCR5-CCR6-CAR-T and HER2-CAR-T. After the plasmid was packaged by lentivirus and transduced with T cells, the expression levels of HER2-CAR, CXCR5 and CCR6 gene proteins were detected by flow cytometry. HER2-CAR, CXCR5 and CCR6 were all expressed in HER2-CAR-CXCR5-CCR6-T.

[0028] Fig. 9 It is the activation effect of HER2-CXCR5-CCR6-CAR-Jurkat and HER2-CAR-Jurkat. After HER2-CXCR5-CCR6-CAR-Jurkat and HER2-CAR-Jurkat were co-incubated with HCC827 target cells that highly expressed chemokines CXCL13 and CCL20 at different ratios, the activation effect of HER2-CXCR5-CCR6-CAR-Jurkat and HER2-CAR-Jurkat was significantly better than that of HER2-CAR-Jurkat cells.

[0029] Fig.10 It is the killing effect of HER2-CXCR5-CCR6-CAR-T and HER2-CAR-T. After HER2-CXCR5-CCR6-CAR-T and HER2-CAR-T were co-incubated with HCC827 target cells that highly expressed chemokines CXCL13 and CCL20 in different proportions, the killing effect of HER2-CXCR5-CCR6-CAR-T and HER2-CAR-T was significantly better than that of HER2-CAR-T cells.

[0030] Fig.11 It is the proliferation effect of HER2-CXCR5-CCR6-CAR-T and HER2-CAR-T. After HER2-CXCR5-CCR6-CAR-T and HER2-CAR-T were co-incubated with HCC827 target cells that highly expressed chemokines CXCL13 and CCL20 at a ratio of 1:1, the proliferation effect of HER2-CXCR5-CCR6-CAR-T was significantly stronger than that of HER2-CAR-T.

[0031] Fig.12 It is the tumor chemotaxis effect of HER2-CXCR5-CCR6-CAR-T and HER2-CAR-T cells. HER2-CXCR5-CCR6-CAR-T and HER2-CAR-T were co-incubated with HCC827 target cells that highly expressed chemokines CXCL13 and CCL20 in different proportions. The experiment showed that after 24 hours, as the number of target cells increased or decreased, the chemotaxis effect of HER2-CXCR5-CCR6-CAR-T on tumor cells was significantly stronger than that of HER2-CAR-T. Under the same proportion conditions, as time went on, the chemotaxis effect of HER2-CXCR5-CCR6-CAR-T on tumor cells was significantly stronger than that of HER2-CAR-T.

[0032] Fig.13 The tumor control effect of HER2-CXCR5-CCR6-CAR-T group and HER2-CAR-T group in the treatment of lung orthotopic HCC827-Lucferace target cell tumor-bearing mice. In the experiment of treating lung orthotopic HCC827-Luciferase target cell tumor-bearing mice, the tumor control effect of HER2-CXCR5-CCR6-CAR-T group was significantly better than that of HER2-CAR-T, and its tumor size and tumor Luciferase fluorescence value were the lowest. After 32 days of observation, there were 3 mice surviving in the HER2-CXCR5-CCR6-CAR-T group, 2 mice in the HER2-CAR-T group, and 1 mouse in the untreated group. In the treatment of HCC827-Luciferase target cell tumor-bearing mice in the lung, the tumor control effect of the HER2-CXCR5-CCR6-CAR-T group was significantly better than that of the HER2-CAR-T group.

[0033] Fig.14This is a statistical analysis chart of biosignalin signals from in vivo imaging of tumor-bearing mice in the HER2-CXCR5-CCR6-CAR-T group and the HER2-CAR-T group. As time goes by, the biosignalin signals of tumors in the HER2-CXCR5-CCR6-CAR-T group are significantly lower than those in the HER2-CAR-T group.

[0034] Fig.15 The ratio of CD4+ and CD8+ T cells in peripheral blood of HER2-CXCR5-CCR6-CAR-T group and HER2-CAR-T group on the 14th and 28th days after injection of HCC827-Luciferase target cell-bearing mice with orthotopic lung tumors. The ratio of CD4+ and CD8+ T cells in peripheral blood of HER2-CXCR5-CCR6-CAR-T group and HER2-CAR-T group on the 14th and 28th days after injection of HCC827-Luciferase target cell-bearing mice with orthotopic lung tumors was significantly better than that of HER2-CAR-T.

[0035] Fig.16 This is a statistical chart of the ratio of CD4+ and CD8+ T cells in the peripheral blood of tumor-bearing mice in the HER2-CXCR5-CCR6-CAR-T group and the HER2-CAR-T group on the 14th and 28th days after treatment. The results show that the ratio of CD4+ and CD8+ T cells in the HER2-CXCR5-CCR6-CAR-T group was significantly higher than that in the HER2-CAR-T group.

[0036] Fig.17 The HER2-CXCR5-CCR6-CAR-T group and the HER2-CAR-T group were euthanized on the 32nd day after the injection of HCC827-Luciferase target cells in the lung orthotopic tumor-bearing mice, and the tumor tissues were taken for qPCR experimental detection, and the infiltration effect of HER2-CXCR5-CCR6-CAR-T and HER2-CAR-T cells in the tumor tissue. The HER2-CXCR5-CCR6-CAR-T group and the HER2-CAR-T group were euthanized on the 32nd day after the injection of HCC827-Luciferase target cells in the lung orthotopic tumor-bearing mice, and the tumor tissues were taken for qPCR experimental detection, and the infiltration effect of HER2-CXCR5-CCR6-CAR-T and HER2-CAR-T cells in the tumor tissue. The results showed that the proportion of infiltrating HER2-CXCR5-CCR6-CAR-T cells in the tumor tissue was significantly higher than that in the HER2-CAR-T group.

[0037] Fig.18This is a diagram of the homing and killing mechanism of HER2 CAR-CXCR5-CCR6-T cells and HER-CAR-T cells. HER2 CAR-CXCR5-CCR6-T cells are better able to chemotaxis into lung solid tumor cells expressing chemokines CXCL13 and CCL20, and proliferate extensively there, thereby exerting a stronger anti-tumor effect. DETAILED DESCRIPTION

[0038] According to multiple review searches, CX3CL1-CXCR1, CXCL16-CXCR6, CXCL13-CXCR5, CXCL12-CXCR4, CCL25-CCR9, and CCL20-CCR6 are currently the only high-affinity paired chemokine receptors and ligands. Selecting the above chemokine receptors and ligands for subsequent design can avoid unexpected effects or adverse reactions caused by redundancy in receptor and ligand affinity ( Figure 1 ).

[0039] The inventors searched the TCGA database for 483 lung adenocarcinomas and 347 normal tissues for the above chemokines CX3CL1, CXCL16, CXCL13, CXCL12, CCL25, and CCL20 expression profiles and found that only CXCL13 and CCL20 were significantly higher in cancer tissues than in adjacent tissues, while other chemokines did not show this or had no significant expression differences ( Figure 2 ).

[0040] The following is a further detailed description of a method for constructing CAR-T cells that enhances the chemotaxis and tumor inhibition effects of solid lung tumors according to the present invention in conjunction with the accompanying drawings and specific embodiments: Example 1

[0041] Using the pCDH-CMV-LucSh(CpG-free)-EF1-coGFP-T2A-Puro stored in the laboratory as the backbone plasmid, the gene sequence corresponding to HER2-CAR was fully synthesized, and then constructed between the XbaI and NotI restriction sites of the pCDH-CMV-LucSh(CpG-free)-EF1-coGFP-T2A-Puro vector by restriction digestion and ligation. At the same time, the redundant EF1-coGFP-T2A-Puro sequence in the plasmid backbone was deleted to obtain the pCDH-CMV-HER2-CAR target plasmid.

[0042] The intermediate plasmid pCDH-CMV-HER2-CAR-EF1-coGFP-T2A-Puro obtained above was used as the plasmid backbone, the CXCR5 and CCR6 genes were fully synthesized, and the coGFP and Puro in the backbone were replaced by homologous recombination to obtain the pCDH-CMV-HER2-CAR-EF1-CXCR5-T2A-CCR6 target plasmid. Example 2

[0043] Lentivirus packaging process - calcium phosphate method 1. Preparation before the experiment: (1) Virus packaging plasmid: pSPAX2 / pMD2G, (2) Transfection reagent: 1000X chloroquine solution (100mmol / L): 52mg of chloroquine diphosphate (Shanghai Shenggong Cat: C6569) was dissolved in 1mL of deionized water, sterilized by 0.22μm filtration, and stored at -20℃ away from light.

[0044] CaCl2: 2.5 M 2x BBS: NaCl (Sigma Cat: S7653): 1.636 g; BES (Sigma Cat: B4554): 1.065 g; Na2HPO4 (Sigma Cat: S5136): 0.021 g; H2O to 90 mL, adjust pH to about 6.95 (1 M NaOH), make up to 100 mL, and store at 4°C.

[0045] (3) Complete culture medium: DMEM + 10% FBS-PS.

[0046] 2. Packaging process: (1) Spread 10cm, the number of 293T cells is: 4x106, DMEM + 10% FBS-PS complete medium per dish, culture until the next day, and control the cell density to about 80-90%.

[0047] (2) Before preparing the transfection reagent, change the medium (1000X chloroquine solution with a final concentration of 100 μmol / L, 7 ml, DMEM + 2% FBS culture medium). Chloroquine can inhibit cell growth, causing it to produce more viruses, while also inhibiting cell autophagy and plasmid degradation.

[0048] (3) Prepare packaging plasmid and transfection reagent (Ca-DNA), mix well and let stand for 3 minutes 1. Nuclease-free water to 500μl (total volume, water for injection) CaCl2 (2.5M) 50μl Core plasmid 12 μg Pspax2 9 μg PMD2.0G 3μg 2. Slowly vortex and add an equal volume (500ul) of 2x BBS to the Ca-DNA mixture. Vortex the round-bottom centrifuge tube for 10 seconds, mix thoroughly and let it stand for 15 minutes.

[0049] (4) Evenly drip the mixture of packaging plasmid and transfection reagent into a dish containing complete culture medium. Shake the 10 cm dish crosswise 6-8 times and place in an incubator at 37°C and 5% CO2 for incubation.

[0050] 3. Medium change: After incubation for 6 hours, change the medium to 10 ml / well DMEM + 2% FBS.

[0051] 4. Collect the virus supernatant: Take the time point after changing the medium to 12ml of DMEM + 2% FBS as the reference time, collect the virus supernatant twice at 36h and 60h, filter the supernatant with a 0.45um filter, divide into 500ul / tube, and freeze at -80℃.

[0052] 5. Ultracentrifugation and concentration (20°C): Ultracentrifugation, 80,000 x g for 2 h.

[0053] 6. Virus resuspending: DPBS, resuspend, and control the concentration rate between 100x-200x.

[0054] 7. Virus packaging: 20-50ul / tube. Example 3

[0055] CAR-Jurkat activation experiment: A high-throughput method reported by Darin Bloemberg to identify a novel chimeric antigen receptor in Jurkat cells was used to evaluate the sensitivity of CAR and the specific recognition efficiency of target cells. Co-culture of CAR-Jurkat cells and target cells: CAR-Jurkat = 1x10 5 Target cells are 0, 1x10 3 , 5x10 3 , 1x10 4 , 5x10 4 , 1x10 5 The cells were plated in 96-well plates. After 24 hours, the expression of CD69 in CAR-Jurkat cells was detected by flow cytometry and compared with the original NC-Jurkat cells to analyze the activation efficiency of CAR-Jurkat cells. Example 4

[0056] CAR-T cell killing experiment: 96-well plate with opaque flat bottom, 5 x10 luciferase stably transfected target cells 3 cells / well, 4 replicate wells. Effector cells were cultured at four ratios of 1:1, 5:1, 10:1, and 20:1. After 24 hours of culture, the supernatant was removed by centrifugation, and then the cells were added with lysis buffer and firefly luciferin substrate. After incubation for 15-20 minutes, the chemiluminescence value was detected in a chemiluminometer or an ELISA reader with a chemiluminescence detection module (380-780nm can be measured). The obtained values ​​were obtained using the formula (1-(experimental group / maximum value of target cells)) x 100% to obtain the effector cell killing efficiency values ​​at four ratios of 1:1, 5:1, 10:1, and 20:1. Example 5

[0057] HER2-CXCR5-CCR6-CAR-T group and HER2-CAR-T group were used to treat NDG lung orthotopic HCC827-Luciferase target cell tumor-bearing mice, and the proliferation, survival and lymph node homing of CAR-T cells in mice were detected (1) Packaging viruses and preparing HER2-CXCR5-CCR6-CAR-T and HER2-CAR-T.

[0058] (2) NDG mice were randomly divided into 5 groups, with 5 mice in each group. One group was the control group, which was injected with an equal volume of PBS. The following 3 groups were injected with PBS, HER2-CXCR5-CCR6-CAR-T cells, and HER2-CAR-T cells, respectively. Each mouse was injected with 2×10 6 CAR-T cells, volume 100ul.

[0059] (3) Changes in the percentage of CAR-T cells in the peripheral blood of mice. After inoculation of CAR-T cells, 200ul of whole blood was collected from the orbital venous plexus every week, and the CAR-T cell ratio in the peripheral blood of mice was detected by q-PCR technology and flow cytometry technology to determine the proliferation and survival of CAR-T cells in vivo.

[0060] (4) Homing of CAR-T cells in mouse spleen tissue: The mice were euthanized 30 days after inoculation of CAR-T cells, and then the spleen, thymus, bone marrow and other tissues and organs were dissected. The proportion of CAR in these tissues and organs was detected by immunohistochemical staining to determine the homing of CAR-T cells. Example 6

[0061] Antitumor effects of HER2-CXCR5-CCR6-CAR-T group and HER2-CAR-T group in treating NDG lung orthotopic HCC827-Luciferase target cell tumor-bearing mice (1) Preparation of inoculated cells: Take HER2-positive target cells in the logarithmic growth phase, resuspend the tumor cell suspension with an appropriate volume of complete culture medium, take 20ul of the cell suspension for counting, ensure that its viability is above 95%, take out the required volume according to the cell concentration, and centrifuge at 2000rpm for 3min; (2) Discard the supernatant, resuspend the cell pellet in culture medium without antibiotics, gently pipette to mix, and centrifuge at 2000 rpm for 3 min; (3) Discard the supernatant and resuspend a certain volume according to the total amount of cells to ensure that each mouse has a cell inoculation volume of 100ul. The number of cells inoculated is: 2×10 5 , transfer the cell suspension to a 1.5 ml EP tube, mark it, and place it in the transfer chamber of the animal room for inoculation; (4) Intraperitoneal injection to construct a CDX model of malignant lung cancer cells: Use the correct posture to grab NDG mice, wipe the mouse abdomen with a wet cotton ball, and use a syringe to inject the lung cancer target cell suspension with biotin luciferase in the logarithmic growth phase into the mouse subcutaneously and through the tail vein, avoiding puncturing blood vessels and bleeding, and construct subcutaneous tumor-bearing mice. The day of inoculation is recorded as Day 0; (5) On Day 2 of the NDG mice inoculated with tumors, the mice were imaged in vivo to observe the tumor formation in the mice. Some mice without tumor formation were eliminated based on the size of the tumors in the images. The remaining mice were evenly distributed into different groups based on the size of the tumors, and each mouse was ear-tagged for identification. For the mice injected with tail vein, when the tumor fluorescence intensity reached about 1.5x106, the animals were divided into the control group and the treatment group (5 mice in each group).

[0062] (6) The experimental groups were as follows: The mice were weighed and recorded before inoculation, and the survival status of the mice was evaluated; (7) On Day 6, the target cell group was randomly divided into 2 groups (experimental group and control group, 5 mice in each group). CAR-T cell suspension was prepared, with 2×10 per mouse in the experimental group. 6 Each mouse was injected with 100ul of CAR-T cells, and the control group was injected with an equal volume of culture medium or PBS. After that, live imaging was performed every other week, the weight of the mice was recorded, the size of the tumors of the mice was observed, and the length and width of the tumors were measured with a vernier caliper every 5 days, and the tumor volume was calculated using the formula (length × width2) / 2.

[0063] (8) During each in vivo imaging, the total flux of bioluminescence of each mouse was recorded and a growth curve was drawn.

[0064] (9) After 30 days of CAR-T therapy, the animals were euthanized and the tumor tissues and spleens were dissected for fluorescent immunohistochemistry (using mouse anti-human mitochondrial membrane protein antibody as the primary antibody), and photographed.

[0065] The above is only a preferred embodiment of the construction and application of the HER2-CAR-T cells that co-express highly specific paired dual receptors CXCR5 and CCR6 developed by the present invention for the high expression of chemokines XCL13 and CCL20 in the tumor microenvironment of lung cancer, which ultimately enhances the chemotaxis and homing of CAR-T cells to lung solid tumors and improves the tumor inhibition effect. It should be pointed out that for ordinary technicians in this technical field, without departing from the precursor of the method of the present invention, corresponding improvements and supplements to chemokines highly expressed in other tumor microenvironments should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing CAR-T cells that enhance the chemotaxis and tumor inhibition effects of solid lung tumors, characterized in that: The following steps are involved: 1) Identify the chemokine receptors and ligands that have high affinity and are the only pair at this stage, and choose the chemokine receptors and ligands that are avoided as mentioned above; 2) Retrieve the above chemokine expression profiles of lung adenocarcinoma and normal tissues from the TCGA database to identify chemokines whose expression in cancer tissues is significantly higher than that in adjacent tissues; 3) Detect the expression of transcriptional and protein levels in lung adenocarcinoma clinical lung cancer and adjacent tissues to confirm that the chemokines expressed in cancer tissues are significantly higher than those in adjacent tissues; 4) Detect the expression of chemokine protein levels in normal lung cell line BEAS-2B and lung cancer cell lines A549, H1734, H1975, H1299, and HCC827 to clarify the expression of chemokines in the cell lines and identify target cells; The specific operation of the construction method is: using pCDH-CMV-LucSh(CpG-free)-EF1-coGFP-T2A-Puro as the backbone plasmid, the gene sequence corresponding to HER2-CAR is fully synthesized, and then constructed between the XbaI and NotI restriction sites of the pCDH-CMV-LucSh(CpG-free)-EF1-coGFP-T2A-Puro vector by enzyme cutting and ligation, and at the same time, the redundant EF1-coGFP-T2A-Puro sequence in the plasmid backbone is deleted to obtain the pCDH-CMV-HER2-CAR target plasmid, and the nucleotide sequence of HER2 is shown in SEQ ID NO.

2.

2. The method for constructing CAR-T cells for enhancing chemotaxis and tumor inhibition of lung solid tumors according to claim 1, characterized in that: Using the intermediate plasmid pCDH-CMV-HER2-CAR-EF1-coGFP-T2A-Puro as the plasmid backbone, the CXCR5 and CCR6 genes were fully synthesized, and the coGFP and Puro in the backbone were replaced by homologous recombination to obtain the pCDH-CMV-HER2-CAR-EF1-CXCR5-T2A-CCR6 target plasmid. The nucleotide sequence of CXCR5 is shown in SEQ ID NO.7, and the nucleotide sequence of CCR6 is shown in SEQ ID NO.8.

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