CART cell with high expression of CCL5 and CXCL9 and targeting EGFR as well as preparation method and application of CART cell

Abstract

The invention provides a CCL5 and CXCL9 high-expression targeting EGFR (epidermal growth factor receptor) CART (chimeric antigen receptor T) cell as well as a preparation method and application thereof, a strain of targeting EGFR monoclonal antibody with relatively strong affinity is firstly obtained, and a strain of targeting EGFR CART cell with high-expression CCL5 and CXCL9 is obtained on the basis of the monoclonal antibody. The cell can recognize and combine EGFR protein highly expressed on the surface of a tumor cell by expressing scFv of EGFR, and transmits a signal to a T cell through an intracellular signal transduction region, so that the T cell is activated, the T cell is promoted to secrete cytokines, the cytokines can kill tumor cells highly expressing EGFR protein, the T cell can be continuously activated and proliferated, and the tumor cells with high expression of EGFR protein can be killed. Cytokines are continuously secreted, so that the effect of killing tumor cells is enhanced.

Description

Technical Field

[0001] The present invention belongs to the biomedical technology field of cell therapy, and particularly relates to a CART cell highly expressing CCL5 and CXCL9 and targeting EGFR, and a preparation method and application thereof. Background Art

[0002] Epidermal growth factor (EGF). Since its discovery in 1962, great progress has been made in the research on the relationship between growth factors and EGFR. These receptors have been proven to be overexpressed, amplified or rearranged in human tumors. The prior art has confirmed that EGFR plays an important role in the occurrence and development of various malignant tumors, including non-small cell lung cancer (NSCLC), glioblastoma, colorectal cancer, squamous cell carcinoma of the head and neck (SCCHN), etc., suggesting that EGFR may be a potential and mature target for tumors. At present, some projects of tumor treatment methods targeting EGF / EGFR are conducting clinical trials, but the clinical efficacy is limited, and there are often defects such as complex preparation processes, high costs, and difficulty in large-scale application.

[0003] The CAR-T cell therapy has a relatively promising development prospect in tumor immunotherapy. Its mechanism of action is to isolate T lymphocytes from the patient's own body and express chimeric antigen receptors by means of genetic engineering, mediating MHC to kill tumors in a non-restrictive manner. This method has achieved good clinical effects in hematological malignancies, and preclinical studies on solid tumors are also in full swing, bringing new hope to patients. However, problems such as the targeting ability of CART cells, the level of cytokine release, and even the viability of in vitro amplification are important factors restricting their effective killing of tumor cells. Summary of the Invention

[0004] In order to solve the above problems, the present application proposes the following technical solutions:

[0005] First, a monoclonal antibody specifically targeting EGFR is provided. The amino acid sequence of the light chain variable region of the EGFR monoclonal antibody is:

[0006] DIQLQSPVILSLPVTPERVAFSCRASSLLDASDDNIHWAQQRPGASRLAIKYAASYRISGI PSAFSGSGSGTDFGTDFTAKISRVEADIADAYCQQAIEFPTTFGAATKLEIK (SEQ ID NO.1);

[0007] The amino acid sequence of the heavy chain variable region is: QVQLQESGPGPGASLRLSCACTVSGFSLTNYAMVHWARQSPKGLWLGVIWSGANTDYANS VKSRLSINKDNSKSKNTAYLQMSLQSNDTAIAYCARALTYSASWYHNFAYWGQQGVTASS (SEQ ID NO.2);

[0008] Furthermore, the present invention provides a chimeric antigen receptor, which comprises: an antigen-specific recognition region, a transmembrane region, an intracellular signaling region, a CCL5-CXCL9 region,

[0009] wherein, the antigen-specific recognition region is the scFv of the monoclonal antibody against the above-mentioned EGFR;

[0010] The amino acid sequence of the transmembrane domain is: VGVVGGLLGSLVLLVWVLAVI (SEQ ID No.3);

[0011] The amino acid sequence of the intracellular signaling region is: PDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTLNSDGYTPEPARITSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAEAACA (SEQ ID No. 4);

[0012] The CCL5-CXCL9 region is a fusion protein of CCL5 and CXCL9. After truncation and substitution optimization of the above two fragments, the optimized sequences of each are connected by a linker to form a positively charged amphiphilic structure, which is convenient for fixation and insertion into the negatively charged phospholipid bilayer. The specific sequence is SPYSDTTPACFAYIARPLPRHIKEYFYTSGKCSNAVVFVTARKNRQVCANPEKKWVAREYIN SLEMSRRKKTPVAVRKGRCCISTNQGTIHALQSLKDLKQFAPSPSCAEKIEIIATLAKNGV QTCLNAPDSADAV (SEQ ID NO. 5).

[0013] Furthermore, the present invention provides a nucleic acid molecule that encodes the above chimeric antigen receptor.

[0014] Furthermore, the present invention provides a vector, which comprises the above nucleic acid molecule. Further, the vector is at least one of a viral vector, a modified mRNA vector or a transposon-mediated gene transfer vector.

[0015] Furthermore, the present invention provides a host cell, which comprises at least one of the above chimeric antigen receptor, nucleic acid molecule and vector.

[0016] Furthermore, the present invention provides a CART cell that highly expresses CCL5 and CXCL9 and targets EGFR. The CART cell is obtained by the following steps, and its preparation method comprises the following steps:

[0017] 1) Extract RNA from EGFR antibody hybridoma cells, obtain cDNA through reverse transcription PCR, and purify the product by nucleotide precipitation method;

[0018] 2) Use the cDNA as a template to amplify the VH and VL genes of the EGFR monoclonal antibody;

[0019] 3) Use PCR amplification to obtain gene fragments of the transmembrane region, intracellular signal region, and CCL5-CXCL9 region;

[0020] 4) Perform gene fusion on the above fragments to form an EGFR antibody VH-VL-transmembrane region-intracellular signal region-CCL5-CXCL9 region fragment;

[0021] 5) Connect the above fragment into a vector to obtain a CAR expression vector;

[0022] 6) Transform and amplify the target plasmid, perform virus packaging through a lentiviral packaging system to prepare a virus solution, and concentrate and purify the prepared lentiviral liquid to ensure a certain virus titer;

[0023] 5) Use the immunomagnetic bead method to sort T cells from human peripheral blood mononuclear cells, and then activate, amplify and transfect the T cells with lentivirus;

[0024] 6) Culture and amplify the infected T cells to obtain CAR-T cells.

[0025] Furthermore, the present invention provides a pharmaceutical composition, which is characterized in that the pharmaceutical composition comprises at least one of the above chimeric antigen receptor, nucleic acid molecule, vector, host cell and CART cell.

[0026] Furthermore, the pharmaceutical composition provided by the present invention further comprises a cytokine, and the cytokine is selected from at least one of interferon-γ and interleukin.

[0027] Furthermore, the pharmaceutical composition further comprises a monoclonal antibody selected from at least one of cetuximab, alemtuzumab, ipilimumab, and ofatumumab.

[0028] Furthermore, the present invention provides the use of at least one of the above-mentioned monoclonal antibody, chimeric antigen receptor, nucleic acid molecule, vector, host cell, CART cell, and pharmaceutical composition in the preparation of a drug for treating solid tumors.

[0029] Furthermore, the cancers include breast cancer, rectal cancer, skin cancer, colon cancer, pancreatic cancer, liver cancer, ovarian cancer, prostate cancer, brain cancer, kidney cancer, lung cancer, lymphoma, and melanoma.

[0030] Beneficial effects

[0031] In the technical solution of the present invention, first, a monoclonal antibody targeting EGFR with strong affinity was obtained, and on this basis, a CART cell targeting EGFR and highly expressing CCL5 and CXCL9 was obtained. The cell can recognize and bind to the EGFR protein highly expressed on the surface of tumor cells by expressing the scFv of EGFR, and transmit the signal into the T cell through the intracellular signal transduction region, thereby activating the T cell and promoting the T cell to secrete cytokines, which can further kill the tumor cells highly expressing the EGFR protein. Moreover, the intracellular signal transduction region integrates the CCL5-CXCL9 fragment, and in the applicant's previous research, the CCL5-CXCL9 fragment can be used as an effective mRNA fragment with good tumor treatment effects, enabling the continuous activation and proliferation of T cells, continuous secretion of cytokines, and enhancing the effect of killing tumor cells. Brief description of the drawings

[0032] Figure 1 It is a structural diagram of a CAR vector plasmid;

[0033] Figure 2 It is the activated T cells and subsets of CAR-T cells. Among them, A: activated T cells. B: c-Met CAR-T (2G). C: CD19 CAR-T (2G). D: c-Met CAR-T (3G). E: CD19 CAR-T (3G);

[0034] Figure 3 It is the Western blot verification of the protein expressed by EGFR CAR-T cells using CCL5 antibody and CXCL9 antibody;

[0035] Figure 4 It is that CAR-T cells specifically kill MDA-MB-231 triple-negative breast cancer cells in vivo, significantly inhibiting tumor growth. Among them, a is the tumor volume curve, and b is the change curve of mouse body weight;

[0036] Figure 5 For CAR-T cells to specifically kill H1975 lung cancer cells in vivo and significantly inhibit tumor growth. Among them, a is the tumor volume curve and b is the curve of the change in mouse body weight;

[0037] Figure 6 For CAR-T cells to specifically kill SW480 colorectal cancer cells in vivo and significantly inhibit tumor growth. Among them, a is the tumor volume curve and b is the curve of the change in mouse body weight. Specific implementation manners

[0038] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0039] Materials and preparations

[0040] The materials and preparations in this application all belong to the conventional selections in the art and do not belong to the invention and innovation of this application. The following is only an illustrative description.

[0041] Primary human lymphocytes. Primary human CD4+ T cells and CD8+ T cells were isolated from healthy volunteers. T cells were stimulated with anti-CD3 / CD28 Dynabead magnetic beads (Life Technologies, Grand Island, NY) and cultured in R10 medium supplemented with 100 IU / mL IL-2 (10% fetal bovine serum, 1% HEPES, 1% GutaMAX, 1% penicillin and streptomycin, 1% MEM NEAA, and 1% sodium pyruvate were added to RPMI-1640 medium).

[0042] Production and transduction of lentivirus. The lentiviral vector was produced by HEK293T cells, and the HEK293T cells were transfected with the transfer plasmid and packaging plasmids pRSV.REV, pMD2.G, and pMDLg.pRRE. The lentiviral vector was harvested after 24 and 48 hours and concentrated by ultracentrifugation.

[0043] Example 1 Screening and preparation of monoclonal antibodies against EGFR

[0044] (1) Expression and purification of the phage display library: Infect the logarithmic-phase TG1 library culture with freshly thawed M13K07 helper phage at a multiplicity of infection of 20:1 (phage-to-cell ratio), and induce overnight with IPTG; Purify the phage library by PEG / NaCl precipitation, and then determine the phage titer. The phage is stored at 4 °C and the scFv selection is carried out later.

[0045] (2) Selection of EGFR-specific scFv-phage: In the first round of selection, coat the Maxisorp plate with 20 μg / ml EGFR-6His protein dissolved in 1×PBS (2 μg / ml in the second round and 0.5 μg / ml in the third round), and incubate overnight at 4 °C. Then, after washing the plate three times with PBS, add the blocking buffer (containing 5% milk and 1% BSA in 1×PBS) to each well. After incubating for 2 hours at room temperature, discard the blocking buffer, add the phage solution, seal the plate with plastic wrap, gently shake and incubate for 2 hours, and then wash the plate 10 times with PBST (20 cycles in the second round and 30 cycles in the third round). Then use 1 mL of acid elution buffer (pH 2.2) to elute the antigen-binding scFv-phage, neutralize it, inoculate it into 15 mL of logarithmic-phase TG1 culture (OD600 = 0.5), incubate statically for 30 min and shake for 30 min at 37 °C, inoculate it on a 2xYT-GA agar plate, and culture overnight at 30 °C for subsequent selection.

[0046] (3) mpELISA screening: After three rounds of screening, 480 phage-infected colony clones are selected for monoclonal phage ELISA (mpELISA) screening. Generate phage supernatants from individual colony clones and test their binding to EGFR-Fc protein. The supernatant is incubated with a pre-blocked Maxisorp plate coated with 2 μg / ml EGFR-6His protein. After three washes, add 100 μl / well of HRP-conjugated anti-M13 antibody, which is diluted 1:5000 in the blocking buffer (containing 5% milk and 1% BSA in 1×PBS), and then incubate for 60 min at room temperature. After washing the plate 5 times with PBST, add 100 μl / well of TMB substrate solution and incubate for 10 - 30 minutes until a blue color appears. Stop the reaction by adding 50 μl / well of stop solution (2N H2SO4). As determined, among the selected positive colonies, the absorbance value of clone No. 3 is 1.72, and positive colonies with an absorbance ≥ 0.5 at 450 nm are identified as capable of producing anti-EGFR antibodies that bind to EGFR-6His protein.

[0047] Cloning and sequence analysis: Positive clones were screened according to the ELISA results and used as templates for PCR cloning of scFv sequences (forward primer sequence: 5'-tgcagctggcacgacaggtttc-3', reverse primer sequence: 5'-cgtcagactgtagcacgtt-3'). Then, the PCR products were sequenced by the Sanger sequencing method (forward primer sequence: 5'-aacaattgaattcaggagga-3', reverse primer sequence: 5'-cctCCTAAGAAGCGTAGTC-3'). The variable region sequences of the antibody were obtained. The amino acid sequence of the light chain variable region was:

[0048] DIQLQSPVILSLPVTPERVAFSCRASSLLDASDDNIHWAQQRPGASRLAIKYAASYRISGI PSAFSGSGSGTDFGTDFTAKISRVEADIADAYCQQAIEFPTTFGAATKLEIK (SEQ ID NO.1);

[0049] The amino acid sequence of the heavy chain variable region was: QVQLQESGPGPGASLRLSCACTVSGFSLTNYAMVHWARQSPKGLWLGVIWSGANTDYANS VKSRLSINKDNSKSKNTAYLQMSLQSNDTAIAYCARALTYSASWYHNFAYWGQQGVTASS (SEQ ID NO.2). The CDR regions of the scFv were analyzed through the ABYSIS website (HTTP: / / ABYSIS.ORG / ), and the results are shown in Table 1.

[0050] Table 1 Amino acid sequences of scFv light chain CDR and heavy chain CDR

[0051]

[0052] Example 2 Preparation, identification and detection of EGFR CART cells

[0053] First, the scFv sequence was amplified by PCR and concatenated with the transmembrane domain (SEQ ID No.3), intracellular signaling region (SEQ ID No.4), and CCL5-CXCL9 region (SEQ ID NO.5) to obtain the CAR element. The schematic diagram is shown in Figure 1 ,

[0054] The above sequence was digested with two enzymes and inserted into a CAR expression vector. After digestion identification, it was co-transfected with the lentiviral packaging plasmids pSPAX2 and pMD2.G into 293T cells. The viral supernatant was collected 48 hours after infection. Taking a cell culture dish with a diameter of 10 cm as an example, the CAR expression vector to be transfected was 20 μg, pSPAX2 was 15 μg, pMD2.G was 5 μg, and Lip2000 was 30 μL. The prepared lentiviral liquid was concentrated and purified to ensure a certain virus titer.

[0055] According to the conventional operations in the art, CART cells were constructed, which were outlined as follows: 1) 10 mL of peripheral blood from healthy volunteers was collected in a heparin anticoagulation tube, diluted with PBS and added to human lymphocyte separation medium at a ratio of 1:1, and centrifuged at 800 g for 30 min at low temperature; 2) After centrifugation, the middle buffy coat layer was aspirated and washed 3 times in PBS; 3) Resuspended and counted with RPMI1640 complete medium containing 200 U / mL IL-2, and the concentration was adjusted to 1×10 6 / mL; placed in a 48-well plate coated with CD3 / CD28 antibody and activated for 24 h; 4) On the same day, the pro-infection reagent RetroNectin was diluted in PBS at a ratio of 1:40, and 150 μL per well was used to coat the 48-well plate and incubated overnight at 4°C for lentiviral infection. 5) The next day, the T cells activated for 24 h were collected and washed 3 times with PBS (1000 r / min, 5 min); 6) Resuspended the cells with TransB and counted, and the concentration was 1×10 6 cells / mL; 7) The cells were placed in a 48-well plate pre-coated with the pro-infection reagent RetroNectin, 150 μL of cells was added to each well, and lentivirus (2G: MOI = 5, 3G: MOI = 6) and the pro-infection reagent TransA (1:25) were added according to the infection multiplicity value, and cultured in an incubator at 37°C and 5% CO2; 8) After 8 h, 200 μL of RPMI1640 complete medium containing 200 U / mL IL-2 was supplemented; 9) Fresh medium was supplemented at 24 h, 48 h, and 72 h after lentiviral infection, and the cell growth status, density, and fluorescence intensity were observed under a fluorescence microscope. When the cell density ≥ 80%, passage was carried out according to the operation steps for suspension cells. Cultured in an incubator at 37°C and 5% CO2 for more than 10 days.

[0056] On the 4th day after lentiviral infection, 1×10 6 CAR-T and activated T cells were collected respectively. After centrifugation and washing, they were resuspended with 200 μL of PBS respectively, and the positive rate of CAR-T cells was detected by flow cytometry. 2×10 6 CAR-T and 5×10 6Activated T cells were centrifuged and washed, and then 7 μL of APC-labeled anti-human CD3 antibody, PE-labeled anti-human CD4 antibody, and PerCY5.5-labeled anti-human CD8 antibody were added to each tube. After incubating in the dark at 4°C for 40 min, the cells were washed twice with PBS to remove non-specific binding, and then resuspended in 200 μL of PBS per tube. The proportions of CD4+ and CD8+ positive cells in the CAR-T cell subsets were detected by flow cytometry. The results showed that the proportion of CD4+ in activated T cells was approximately 65.8%, and the proportion of CD8+ was approximately 31.5%; the proportion of CD4+ in EGFRCAR-T(2G) cells was approximately 45.4%, and the proportion of CD8+ was approximately 44.3%; the proportion of CD4+ in CD19CAR-T(2G) cells was approximately 63.1%, and the proportion of CD8+ was approximately 27.4%; the proportion of CD4+ in EGFRCAR-T(3G) cells was approximately 65.3%, and the proportion of CD8+ was approximately 32.2%; the proportion of CD4+ in CD19CAR-T(3G) cells was approximately 83%, and the proportion of CD8+ was approximately 15%. In summary, compared with activated T cells, the proportion of the CD8+ cell subset in EGFRtCAR-T(2G) cells was significantly up-regulated (P<0.01, Figure 2 ); there was no obvious change in the EGFRCAR-T(3G) cell subset.

[0057] Furthermore, CCL5 antibody and CXCL9 antibody were used to further verify the proteins expressed by EGFR CAR-T cells. After the CART cells were purified and passaged, the supernatant was collected and the expression of the target protein was detected by Western Blot. After reducing electrophoresis, the membrane was transferred, and a His rabbit polyclonal antibody (commercially available) was used for incubation. The secondary antibody used for detection was an HRP-conjugated goat anti-rabbit antibody. The steps are briefly described as follows:

[0058] Take 80 μl of cell supernatant, add 20 μl of 5×SDS-PAGE protein loading buffer, mix the cell supernatant and 5×SDS-PAGE protein loading buffer, and heat at 100°C for 5 minutes to fully denature the protein.

[0059] Centrifuge at 5000 rpm for 5 minutes. After cooling to room temperature, load 10 μl into the loading wells of the SDS-PAGE gel and start electrophoresis.

[0060] Stop electrophoresis when the blue dye reaches near the bottom of the gel.

[0061] Take out the gel for membrane transfer. After membrane transfer, place the PVDF membrane in a 5% skim milk solution and block for 2 hours.

[0062] Wash the PVDF membrane 3 times with TBST, 10 minutes each time, apply the primary antibody, and incubate overnight at 4°C.

[0063] Wash the PVDF membrane 3 times with TBST, 10 minutes each time, apply the secondary antibody, and incubate for 1 hour.

[0064] Wash the PVDF membrane three times with TBST for 10 minutes each time, and then wash it with TBS for 10 minutes.

[0065] Add the chromogenic solution for color development. The results of Western Blot detection are as Figure 3 shown, where M: Marker (10 - 245 kd), and the truncated CCL5 fragment and CXCL9 fragment are both approximately 8 kd. The results confirm the correct expression of the above two chemokine ligands in CART cells.

[0066] Example 3 In Vivo Experiment of EGFR CART in Animals

[0067] Preparation of tumor-forming cells: MDA-MB-231 (triple-negative breast cancer cell line), H1975 (non-small cell lung cancer cell line), SW480 (colorectal cancer cell line) are from the Cell Bank of the Chinese Academy of Sciences, Shanghai;

[0068] Animal preparation: Order 6-week-old female NCG mice weighing 20 - 25 g one week before inoculation (NOD / ShiLtJGpt-Prkdc em26Il2rg em26 / Gpt, from Jiangsu Jicui Yakang Biotechnology Co., Ltd., strain type KnoCL out, strain number T001475, background NOD / ShiltJGpt). Animal feeding and experimental operations are carried out strictly according to the SPF level standard. Animal experiments are carried out strictly in accordance with the working guidelines of the Animal Research Committee. They are raised in a specific pathogen-free SPF environment at a room temperature of 26°C, with sterile feed and sterile water. The mice are allowed to adapt to the environment for 7 days before starting the relevant experiments.

[0069] Steps for subcutaneous xenograft tumor modeling and in vivo experiment in NCG mice:

[0070] 1) Treat and collect adherent target cells: Take Bxpc3 with 80% - 90% cell confluence, add 3 ml of trypsin to each T75 flask, digest for 6 - 8 minutes, add 6 ml of RPMI1640 complete medium to each flask to terminate digestion, centrifuge at 1500 rpm, accelerate to 9 and decelerate to 9, centrifuge for 5 min, discard the supernatant, then resuspend and mix well with RPMI1640 basal medium, centrifuge at 1500 rpm, accelerate to 9 and decelerate to 9, centrifuge for 5 min, discard the supernatant, and repeat the washing once.

[0071] 2) Inoculation: After rinsing twice, stain with trypan blue for counting, measure the cell viability (above 95%), adjust the cell density to 5.0×107 / ml with basal medium, disinfect the skin at the axilla of the forelimb of NCG mice with 75% alcohol, mix the cell suspension well, and subcutaneously inject 100 ul of the cell suspension with an insulin needle, then slowly pull out the needle and continue to raise at the SPF level.

[0072] 3) Grouping of mice: When the tumor in the mice reached 100 mm 3 random grouping was carried out. The bedding of the mice was changed regularly, and the general condition and feeding activity ability of the mice were observed. The body weight and tumor volume of the mice were measured once every two days.

[0073] 4) Preparation of CAR-T cells: Use AIMV complete medium (5% FBS + 1000 IU / ml IL-2), culture in an incubator at 37 °C and 5% carbon dioxide, observe the cell growth status every 2 days, take microscope photos, count, and replenish the medium. T cells were expanded in vitro to the required cell amount for reinfusion, and sterility, mycoplasma, and CAR transduction efficiency were detected before reinfusion. Treatment of CAR-T cells: Take the CAR-T cells to be reinfused, centrifuge at 1500 rpm, raise to 9 and lower to 9, centrifuge for 5 min, discard the supernatant, wash twice with AIMV basal medium, then stain with trypan blue for counting, determine the cell viability, and then adjust the T cell density to 1.0×108 / ml with basal medium for reinfusion.

[0074] 5) Reinjection of CAR-T cells: When the tumor volume of the mice reached 100 mm 3 the reinjection of CAR-T cells was started. The reinjection protocol of CAR-T cells was 1×10 7 cells per time, and a total of 4 reinjections were performed, with CAR-T reinjection once every 4 days. Reinjection operation: Disinfect the tail of NCG mice with 75% alcohol. After mixing the cell suspension, inject 100 μl of CAR-T cell suspension into the tail vein of the mice using an insulin needle, then slowly pull out the needle, disinfect and stop bleeding the wound for 20 s, and continue to raise in the SPF level environment.

[0075] 5×10 6 target cells MDA-MB-231 (triple-negative breast cancer cell line), H1975 (non-small cell lung cancer cell line), and SW480 (colorectal cancer cell line) were injected into 6-week-old NCG mice through the tail vein respectively. After 4 - 6 days of inoculation, when the tumor volume was about 100 mm 3 the tumor-bearing mice were randomly divided into 3 groups. The experimental mice were given 4 reinjections of CAR-T cell therapy (1×10 7 / mouse / time), with an interval of 3 days in between.

[0076] During the treatment process, we continuously observed and recorded the tumor size, body weight, and activity status of the mice, and statistically analyzed the tumor size of the mice. The experimental results showed that the anti-Trop2 CAR-T cell group could significantly inhibit tumor growth compared with the anti-CD19 CAR-T cell group and the non-treatment group. Figure 4CAR-T cells specifically kill MDA-MB-231 triple-negative breast cancer cells in vivo and significantly inhibit tumor growth. Among them, a is the tumor volume curve and b is the curve of mouse body weight change. Compared with the control group and the CD19 CART treatment group of mice, the tumor volume of the mice treated with CAR-T cells was significantly reduced, indicating that CAR-T cells can significantly inhibit the growth of MDA-MB-231 triple-negative breast cancer xenograft tumors. Figure 5 CAR-T cells specifically kill H1975 lung cancer cells in vivo and significantly inhibit tumor growth. Among them, a is the tumor volume curve and b is the curve of mouse body weight change. Compared with the control group and the CD19 CART treatment group of mice, the tumor volume of the mice treated with CAR-T cells was significantly reduced, indicating that CAR-T cells can significantly inhibit the growth of H1975 lung cancer xenograft tumors. The body weight of the mice treated with CAR-T cells did not decrease significantly, indicating that CAR-T cells are tolerated in mice. Figure 6 CAR-T cells specifically kill SW480 colorectal cancer cells in vivo and significantly inhibit tumor growth. Among them, a is the tumor volume curve and b is the curve of mouse body weight change. Compared with the control group and the CD19 CART treatment group of mice, the tumor volume of the mice treated with CAR-T cells was significantly reduced, indicating that CAR-T cells can significantly inhibit the growth of SW480 colorectal cancer cell xenograft tumors. The body weight of the mice treated with CAR-T cells even increased significantly, indicating that the CART treatment effect is significant and can be the focus of future research in the next step of the experiment.

[0077] The results showed that significant anti-tumor effects were achieved in mouse models of lung cancer cell line H1975, triple-negative breast cancer cell line MDA-MB-231, and colorectal cancer cell line SW480. In summary, the EGFR CART cells provided by the present invention can precisely target EGFR on the surface of tumor cells, enabling them to more accurately recognize and kill various tumor cells expressing the above-mentioned tumor-associated antigens; in addition, the CAR structure mentioned in the present invention endows T cells with stronger proliferative ability and lasting vitality, making them show stronger tumor cell killing ability.

[0078] The above description of the embodiments is for the convenience of those of ordinary skill in the art to understand and use the present invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments. Modifications and improvements made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A monoclonal antibody specifically targeting EGFR, characterized in that The amino acid sequence of the light chain variable region of the anti-EGFR monoclonal antibody is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.

2.

2. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises: an antigen-specific recognition region, a transmembrane region, an intracellular signaling region, a CCL5-CXCL9 region, wherein the antigen-specific recognition region is the scFv of the anti-EGFR monoclonal antibody described in claim 1; the amino acid sequence of the transmembrane domain is shown as SEQ ID No.3; the amino acid sequence of the intracellular signaling region is shown as SEQ ID No.4; the CCL5-CXCL9 region is a fusion protein of CCL5 and CXCL9, and its amino acid sequence is shown as SEQ ID No.

5.

3. A nucleic acid molecule encoding the chimeric antigen receptor according to claim 2.

4. A vector comprising the nucleic acid molecule according to claim 3.

5. The vector according to claim 4, which is at least one of a viral vector, a modified mRNA vector or a transposon-mediated gene transfer vector.

6. A cell comprising at least one of the chimeric antigen receptor according to claim 2, the nucleic acid molecule according to claim 3, and the vector according to claim 4 or 5.

7. The cell according to claim 6, which is a CART cell.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises at least one of the chimeric antigen receptor according to claim 2, the nucleic acid molecule according to claim 3, the vector according to claim 4 or 5, and the cell according to claim 6 or 7.

9. Use of at least one of the monoclonal antibody according to claim 1, the chimeric antigen receptor according to claim 2, the nucleic acid molecule according to claim 3, the vector according to claim 4 or 5, the cell according to claim 6 or 7, and the pharmaceutical composition according to claim 8 in the preparation of a medicament for treating solid tumors.

10. The use according to claim 9, wherein the cancers include breast cancer, rectal cancer, skin cancer, colon cancer, pancreatic cancer, liver cancer, ovarian cancer, prostate cancer, brain cancer, kidney cancer, lung cancer, lymphoma, melanoma.

Images