An antibody for CAR-T cell regulation and its application

Abstract

The present invention relates to an antibody for CAR-T cell regulation and its application, which can recognize CAR receptor molecules containing specific artificial antigen epitopes. In the process of preparing the corresponding CAR-T, by adding the antibody of the present invention, the directional amplification of CAR-T cells can be achieved, and the proportion of CAR-positive cells in the final product can be increased; at the same time, CAR-positive cells can be induced to differentiate into Tcm subpopulations, and the anti-tumor durability of the final product can be improved. Moreover, the CAR-T cells prepared by the antibody have higher in vivo anti-tumor activity; in addition, after the administration of CAR-T, the number of CAR-T cells in the body can also be regulated by the antibody.

Description

Technical Field

[0001] The present invention relates to the field of gene therapy and cell therapy, and in particular to an antibody for CAR-T cell regulation and application thereof. Background Art

[0002] Chimeric antigen receptor modified T cells (CAR-T) have achieved remarkable clinical efficacy in the treatment of B-cell tumors, and two CAR-T products have been approved by the US FDA for marketing. However, there are still many technical bottlenecks in the clinical application of CAR-T, which hinders its further promotion. Among them, how to achieve stable and high-purity CAR-T preparation, reduce the large variation caused by different batches of T cells to lentivirus batches; and how to avoid serious clinical side effects caused by uncontrolled proliferation in the body after CAR-T is reinfused, etc. These are the main technical problems that have not yet been solved.

[0003] Although studies have reported that inactive EGFR receptors can be used as screening marker molecules for CAR-T after co-expression with CAR; the use of small molecule compounds combined with iCas9 protein components to construct fusion CAR molecules can achieve in vivo regulation after CAR-T infusion. However, the realization of the above technical effects must be achieved when constructing the CAR expression vector, and different protein functional components must be co-expressed with the CAR receptor. On the one hand, such a technical solution increases the difficulty of CAR vector construction, and on the other hand, the introduction of too many macromolecular components also increases the uncertainty of the function of modified T cells. Therefore, how to use a simpler method to achieve the preparation of high-purity CAR-positive T cells and in vivo control after CAR-T infusion is still a technical problem in this field. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides an antibody for CAR-T cell regulation and its application. The antibody can recognize CAR receptor molecules containing specific artificial antigen epitopes. In the process of preparing the corresponding CAR-T, by adding the antibody of the present invention, the directional amplification of CAR-T cells can be achieved, and the proportion of CAR-positive cells in the final product can be increased; at the same time, CAR-positive cells can be induced to differentiate into Tcm subpopulations, thereby improving the anti-tumor durability of the final product.

[0005] To this end, in a first aspect, the present invention provides an antibody comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3;

[0006] Wherein, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are selected from the following group:

[0007] HCDR1: TYAMN (SEQ ID NO: 5), HCDR2: RIRSKSNNYATFYGESVRG (SEQ ID NO: 6), HCDR3: DYFGSYADY (SEQ ID NO: 7);

[0008] Or, HCDR1: TFAMN (SEQ ID NO: 15), HCDR2: RMRSKNNYYTTFYADSVKD (SEQ ID NO: 16), HCDR3: DYFGFNNAVDY (SEQ ID NO: 17);

[0009] The amino acid sequences of LCDR1, LCDR2 and LCDR3 are selected from the group consisting of:

[0010] LCDR1: RSSSKSLLHSNGNTYLY (SEQ ID NO: 8), LCDR2: RMSNLAS (SEQ ID NO: 9), LCDR3: MQYLEYPYT (SEQ ID NO: 10);

[0011] Alternatively, LCDR1: RASKSVSTSGYSYMH (SEQ ID NO: 18), LCDR2: LASNLES (SEQ ID NO: 19), LCDR3: QHSRELPWT (SEQ ID NO: 20).

[0012] In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are selected from the group consisting of:

[0013] HCDR1: TYAMN (SEQ ID NO: 5), HCDR2: RIRSKSNNYATFYGESVRG (SEQ ID NO: 6), HCDR3: DYFGSYADY (SEQ ID NO: 7); LCDR1: RSSSKSLLHSNGNTYLY (SEQ ID NO: 8), LCDR2: RMSNLAS (SEQ ID NO: 9), LCDR3: MQYLEYPYT (SEQ ID NO: 7) NO: 10);

[0014] Or, HCDR1: TFAMN (SEQ ID NO: 15), HCDR2: RMRSKNNYYTTFYADSVKD (SEQ ID NO: 16), HCDR3: DYFGFNNAVDY (SEQ ID NO: 17); LCDR1: RASKSVSTSGYSYMH (SEQ ID NO: 18), LCDR2: LASNLES (SEQ ID NO: 19), LCDR3: QHSRELPWT (SEQ ID NO: 17) ID NO: 20).

[0015] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1 or SEQ ID NO: 11.

[0016] In some embodiments, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2 or SEQ ID NO: 12.

[0017] In one embodiment, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is SEQ ID NO: 2.

[0018] In one embodiment, the amino acid sequence of the heavy chain variable region is SEQ ID NO:11, and the amino acid sequence of the light chain variable region is SEQ ID NO:12.

[0019] In some embodiments, the antibody is a full-length antibody, a Fab fragment, a F(ab)2 fragment, a double-chain Fv fragment, or a single-chain Fv fragment.

[0020] In some embodiments, the antibody is a monoclonal antibody.

[0021] In some embodiments, the antibody further includes a heavy chain constant region and a light chain constant region; the heavy chain constant region includes the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 13; the light chain constant region includes the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 14.

[0022] In one embodiment, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:3, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:4.

[0023] In one embodiment, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:13, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:14.

[0024] In some embodiments, the carboxyl terminus and / or amino terminus of the antibody is fused with a tag protein and / or a signal peptide.

[0025] In some embodiments, the signal peptide comprises the sequence shown in SEQ ID NO:21 or SEQ ID NO:22.

[0026] In a second aspect, the present invention provides a nucleic acid molecule encoding the antibody of the present invention.

[0027] In a third aspect, the present invention provides a vector comprising the nucleic acid molecule of the present invention.

[0028] In a fourth aspect, the present invention provides a host cell, which expresses the antibody of the present invention, or comprises the nucleic acid molecule of the present invention, or comprises the vector of the present invention.

[0029] In a fifth aspect, the present invention provides a method for producing the antibody, comprising the following steps: culturing the host cell of the present invention under conditions allowing expression of the antibody; and isolating and purifying the antibody from the obtained culture.

[0030] In a sixth aspect, the present invention also provides the use of the antibody in the preparation of chimeric antigen receptor modified T (CAR-T) cells, wherein the chimeric antigen receptor has a sorting domain, and the amino acid sequence of the sorting domain is SEQ ID NO: 27.

[0031] In some embodiments, the method for preparing the chimeric antigen receptor modified T cells comprises: infecting the activated T cells with a virus encoding the chimeric antigen receptor, and then adding the antibody described in the first aspect of the present invention to the culture system.

[0032] In a seventh aspect, the present invention provides the use of the antibody in the preparation of A) or B) medicine:

[0033] A) Drugs that regulate the number of chimeric antigen receptor modified T (CAR-T) cells in vivo;

[0034] B) Drugs that regulate the anti-tumor activity of chimeric antigen receptor modified T (CAR-T) cells in vivo.

[0035] In previous studies, the inventors of the present invention reported a CAR molecule with an artificial antigen epitope, which can efficiently sort the target cells expressing CAR positive by a secondary sorting method to increase the proportion of positive CAR-T cells in the final product (CN108017717A). Based on this study, the inventors unexpectedly found that during the preparation process, the use of antibodies against artificial antigen epitopes to specifically activate T cells introduced into CAR can not only increase the proportion of CAR positive T cells in the final product, but also significantly increase the proportion of Tcm in CAR positive T cells, bringing unexpected technical effects. In this research technology, the present invention further developed antibodies that can specifically recognize artificial antigen epitopes E-tag and can effectively activate corresponding CAR-T cells. It was further found that the antibody can not only play the above role in the preparation stage of CAR-T cells, but also the CAR-T cells prepared by the antibody have higher in vivo anti-tumor activity; in addition, after the administration of CAR-T, the number of CAR-T cells in the body can also be regulated by the antibody.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1. The antibodies provided by the present invention can be used in the preparation of CAR-T cells, and can effectively increase the proportion of CAR-positive T cells in the product, and can achieve in vitro specific amplification of the Tcm subpopulation in CAR-positive T cells, significantly increasing the proportion of Tcm in the final product.

[0038] 2. The antibodies provided by the present invention are used in the preparation of CAR-T cells, which effectively overcomes the technical problem that Tcm cannot be cultured for a long time in vitro, realizes the long-term culture of Tcm in vitro, and effectively increases the yield of Tcm; the Tcm ratio begins to decrease around the 6th day of culture in the prior art, while the Tcm ratio can still be maintained at a high level when the technical solution of the present invention is cultured to the 12th day.

[0039] 3. The following problems have always existed in the clinical application of CAR-T technology: how to regulate CAR-T cells after they are reinfused into the body to improve their safety; how to effectively regulate the activity of CAR-T in the body to further improve its clinical anti-tumor activity. The antibodies provided by the present invention have been experimentally verified to be able to be used for in vivo regulation after CAR-T is reinfused, thereby improving the safety and controllability of CAR-T technology in clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only used for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. In the accompanying drawings:

[0041] Figure 1 : SDS-PAGE identification results of the antibodies prepared by the present invention;

[0042] Figure 2 : In the process of preparing CAR-T cells, the antibodies provided by the present invention are used for specific activation, and the results of analyzing the proliferation capacity of each group of cells;

[0043] Figure 3 : In the process of preparing CAR-T cells, the antibody provided by the present invention is used for specific activation, and the proportion of CAR-positive T cells in the prepared cell product;

[0044] Figure 4 : In the process of preparing CAR-T cells, the antibody provided by the present invention is used for specific activation, and the ratio of Tcm in the CAR-positive T cells in the prepared cell product;

[0045] Figure 5 : In the process of preparing CAR-T cells, the antibodies provided by the present invention are used for specific activation, and the test results of the in vitro killing activity of the prepared cells on target cells;

[0046] Figure 6 : Evaluation results of the in vivo anti-tumor activity of CAR-T cells prepared by specific activation of the antibodies provided by the present invention;

[0047] Figure 7 : The antibodies provided by the present invention are used to regulate the number of CAR-T cells in the body, and the relative multiples of CAR-T cells in peripheral blood change over time. DETAILED DESCRIPTION

[0048] The exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0049] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field (for example, refer to "Molecular Cloning Experiment Guide" by J. Sambrook et al., translated by He Fuchu et al., 4th edition, Science Press; "Concise Immunology Experiment Guide" by JE Colligan et al., translated by Cao Xuetao et al., Science Press, etc.) or according to the product instructions.

[0050] Example 1 Preparation of monoclonal antibodies targeting chimeric antigen receptor sorting peptides

[0051] (1) The artificial antigen epitope polypeptide and ovalbumin are coupled by the EDC method to prepare a fusion antigen:

[0052] Take 5 mg of the target antigen polypeptide (amino acid sequence is SEQ ID NO: 27, KPLPEVTDEY) and 2.5 mg of ovalbumin (purchased from Solebao, item number A8040) and dissolve them in 250 μl TE buffer (pH 8.0), shake them thoroughly to dissolve them, and then take 79 mg of EDC·HCl and dissolve them thoroughly in 250 μl TE buffer (pH 8.0). Add the EDC solution dropwise to the mixed solution of the target antigen peptide and ovalbumin while shaking, and react in a shaker at 37°C for more than 2 hours. First, use a Sephadex column to separate the coupling product and the unbound target antigen peptide small molecules, and then dialyze and purify them. Dialyze 0.5 ml of the solution in PBS for 3 days, change the solution twice a day, and change 200 ml each time. The dialyzed product is the prepared fusion antigen, which is packaged in small doses and stored at -20°C for use.

[0053] (2) According to the conventional technical method for preparing monoclonal antibodies, the fusion antigen prepared in step (1) was used to immunize 5 Bal b / c mice. After the immunization cycle, the antigen polypeptide (amino acid sequence: KPLPEVTDEY) was used as the antigen and ovalbumin was used as the negative control to detect the mouse serum antibody titer. The test results are shown in Table 1. Mouse No. 4 with the highest serum titer was killed, and spleen cells were isolated from the spleen and fused with the S / P 20 myeloma cell line to prepare hybridoma cells. After subclone screening, two positive cell lines were obtained, namely: MM02H (SmAb-1) and MM04H (SmAb-2).

[0054] Table 1 Serum titer detection of immune mice

[0055]

[0056] Example 2 Identification of monoclonal antibodies

[0057] After the two positive cell lines obtained in Example 1 were amplified, they were injected into the peritoneal cavity of Bal b / c mice respectively. Ten days after the injection, the mice were killed and the ascites was collected. After the ascites was centrifuged at 1200 rpm, the supernatant was purified using a Protein-A gel column to obtain monoclonal antibodies. After the antibodies were precipitated, they were redissolved in PBS and stored at -80°C for later experiments. The obtained antibodies were identified by denaturing polyacrylamide gel electrophoresis (SDS-PAGE). The test results are shown in Figure 1As shown in Table 2, the band with a larger molecular weight is the antibody heavy chain, and the band with a smaller molecular weight is the antibody light chain. The purity of the whole body can reach more than 95%. The titer was determined by ELISA, and the results are shown in Table 2. The binding titer of the two antibodies to the antigen can reach 1:1000.

[0058] Table 2 Antibody titer detection

[0059] Antibody clone number Dilution ratio OD450 detection value -1 OD450 detection value -2 OD450 average MM02H 1∶1000 2.734 2.632 2.683 MM04H 1∶1000 2.911 2.764 2.836

[0060] The genomes of two hybridoma cells expressing monoclonal antibodies were extracted and the antibody coding sequences and protein sequences were sequenced to obtain the sequence information of the two monoclonal antibodies.

[0061] According to the test results, the heavy chain amino acid sequence of MM02H (SmAb-1) is shown in SEQ ID NO: 23, wherein positions 1-20 are signal peptides, positions 21-140 are heavy chain variable regions, and positions 141-466 are heavy chain constant regions; the light chain amino acid sequence is shown in SEQ ID NO: 24, wherein positions 1-19 are signal peptides, positions 20-131 are light chain variable regions, and positions 132-237 are light chain constant regions. Specifically, the heavy chain variable region amino acid sequence of MM02H (SmAb-1) is shown in SEQ ID NO: 1, and the light chain variable region amino acid sequence is shown in SEQ ID NO: 2; the heavy chain constant region amino acid sequence is shown in SEQ ID NO: 3, and the light chain constant region amino acid sequence is shown in SEQ ID NO: 4. The variable region CDR region sequence is shown in Table 3.

[0062] Table 3 MM02H (SmAb-1) variable region CDR region sequence

[0063]

[0064] According to the test results, the heavy chain amino acid sequence of MM04H (SmAb-2) is shown in SEQ ID NO: 25, wherein positions 1-20 are signal peptides, positions 21-142 are heavy chain variable regions, and positions 143-468 are heavy chain constant regions; the light chain amino acid sequence is shown in SEQ ID NO: 26, wherein positions 1-19 are signal peptides, positions 20-129 are light chain variable regions, and positions 130-236 are light chain constant regions. Specifically, the heavy chain variable region amino acid sequence of MM04H (SmAb-2) is shown in SEQ ID NO: 11, and the light chain variable region amino acid sequence is shown in SEQ ID NO: 12; the heavy chain constant region amino acid sequence is shown in SEQ ID NO: 13, and the light chain constant region amino acid sequence is shown in SEQ ID NO: 14. The variable region CDR region sequence is shown in Table 4.

[0065] Table 4 MM04H (SmAb-2) variable region CDR region sequence

[0066]

[0067]

[0068] Example 3 Preparation of Chimeric Antigen Receptor Encoding Virus

[0069] In this example, lentivirus encoding CD19sCAR, CD19CAR and EGFP was prepared. The specific steps are as follows:

[0070] (1) Construction of chimeric antigen receptor expression vector:

[0071] The chimeric antigen receptor CD19sCAR can target the CD19 antigen and comprises an artificial antigen epitope E-tag (SEQ ID NO: 27). The amino acid sequence of CD19sCAR is shown in SEQ ID NO: 28, and the nucleotide sequence is shown in SEQ ID NO: 29.

[0072] At the same time, a control chimeric antigen receptor CD19CAR that does not contain an artificial antigen epitope is set up, and its amino acid sequence is shown in SEQ ID NO: 30, and its nucleotide sequence is shown in SEQ ID NO: 31.

[0073] The CD19sCAR and CD19CAR nucleotide sequences were synthesized in their entirety by chemical synthesis, restriction enzyme sites were added to both ends, and the synthesized sequences were inserted into the downstream of the CMV promoter of the lentiviral expression vector pLenti6.4 containing the CMV promoter by gene cloning to obtain the CD19sCAR lentiviral expression vector and the CD19CAR lentiviral expression vector.

[0074] (2) Preparation of chimeric antigen receptor encoding virus

[0075] HEK293T cells were used as packaging cells to prepare chimeric antigen receptor encoding viruses. HEK293T cells in the logarithmic growth phase were digested and centrifuged at 800 rpm for 5 min. The culture medium was discarded and resuspended in DMEM medium containing 10% FBS. After cell counting, the density of the cell suspension was adjusted to 3.6×10 6 / ml and placed in a 37°C cell culture incubator until use.

[0076] The transfection of the viral packaging plasmid used the Lipofectamine 3000 kit (Thermo Fisher) and was performed according to the instructions of the kit. The three plasmids required for lentiviral packaging, including the lentiviral expression vector (CD19sCAR lentiviral expression vector prepared in step 1, CD19CAR lentiviral expression vector and lentiviral vector pLenti6.4-CMV-EGFP containing the EGFP encoding gene), the plasmid psPAX2 encoding the viral nucleocapsid protein Gag / Pol and Rev, and the plasmid pVSVG encoding the viral envelope protein, were mixed with Lipofectamine 3000 according to the recommended ratio in the instructions to prepare a DNA liposome complex, and allowed to stand at room temperature for 15 minutes. After the standing period, a 6-well culture plate was taken, and the DNA-liposome complex was added to the 6-well plate, 1 ml per well, and the HEK293T cell suspension prepared previously was gently mixed, added to the 6-well plate, and mixed with the liposome complex. The culture plate was placed in an incubator for continued culture, and the culture supernatant containing the virus was collected at 24h and 48h. After the last supernatant was collected, the supernatant was centrifuged at 2000g for 10min and filtered with a 0.45μm filter membrane to prepare lentivirus encoding CD19sCAR, CD19CAR and EGFP, respectively, which were aliquoted and frozen at -80°C for storage.

[0077] Example 4 Preparation of chimeric antigen receptor modified T cells

[0078] The antibody with the highest detection titer in Example 2 (MM04H, SmAb-2) was selected to detect its ability to selectively amplify CD19sCAR-modified T cells in vitro.

[0079] Peripheral blood mononuclear cells (PBMCs) were isolated by Ficol density gradient centrifugation, and the PBMCs were resuspended in D-PBS and the density was adjusted to 0.5×10 6 / mL. According to the ratio of 1:1 between the absolute number of T cells and CD3 / CD28 sorting magnetic beads, add the sorting magnetic beads and mix gently at room temperature for 20 minutes. The mixed cell-magnetic bead suspension is used to sort CD3+T cells using a sorting magnetic rack. The sorted T cells are activated by using X-VIVO15 medium containing 50ng / mL OKT-3 and 500IU / mL IL-2 at a rate of 1×10 6 / mL resuspended T cells, inoculated into the culture container for culture, cultured in a 37°C, 5% CO2 saturated humidity incubator for 24 h. After T cell activation, the cells were collected into a 50 mL centrifuge tube, counted, and the cell density was adjusted to 3×10 6 / mL.

[0080] The activated T cells were grouped according to Table 5, wherein SmAb was the antibody MM04H (SmAb-2) prepared in Example 2, and the negative control group was a treatment group in which T cells were infected with a lentivirus encoding EGFP.

[0081] Table 5 Experimental group grouping

[0082] Serial number Group No. Treatment and culture methods 1 Experimental group a1 Specific activation using SmAb after infection with CD19sCAR 2 Experimental group a2 No specific activation using SmAb after infection with CD19sCAR 3 Experimental group a3 Specific activation using SmAb after infection with CD19CAR 4 Experimental group a4 No specific activation using SmAb after infection with CD19CAR 5 Experimental group a5 Negative control group

[0083] After the activated T cells were grouped according to Table 5, they were infected with the lentivirus encoding CD19sCAR, CD19CAR and EGFP prepared in Example 3. 24h after infection, the cells of each treatment group were transferred to X-VIVO15 medium containing 80ng / mL IL-7, 500IU / mL IL-2 and 20ng / mL IL-15 for culture, and the culture conditions were 37°C and a saturated humidity incubator containing 5% CO2. On the 6th day of culture, the experimental groups a1 and a3 were activated according to the treatment method of the experimental grouping. The treatment method was as follows: the cells of the experimental groups a1 and a3 were transferred to a culture container coated with 20μg / mL SmAb antibodies, and continued to be cultured with X-VIVO15 medium containing 80ng / mL IL-7, 500IU / mL IL-2 and 20ng / mL IL-15 under the same conditions as before.

[0084] During the culture period, the cells were replenished with fluid every 2-3 days according to the cell growth conditions. After that, each group of cells continued to be cultured until the 12th day, and the cell products were harvested respectively.

[0085] Three independent batches of experiments were counted, and the final products of each experimental group were subjected to the following analysis and testing:

[0086] The proliferation ability of cells in each experimental group was analyzed. The results are shown in Figure 2 As shown by Figure 2 It can be seen that after the cells in the experimental group a1 were stimulated and activated by SmAb, the proliferation level was significantly better than that of the cells in other experimental groups; the final products of each experimental group were analyzed by flow cytometry, and the test results are shown in Figure 3 As shown by Figure 3 It can be seen that the proportion of CAR-positive T cells in the final product of experimental group a1 was significantly higher than that of other experimental groups, indicating that the application of SmAb in the preparation of sCAR-T can promote the expansion of CAR-positive T cells and increase their proportion in the final product.

[0087] Example 5 Effect of SmAb on the in vitro differentiation ability of sCAR-modified T cells

[0088] The T cells of each group obtained by culturing for 12 days in Example 4 were subjected to flow cytometry analysis to detect the proportions of different T cell subsets in the final product, including central memory T cells (Tcm), effector memory T cells (Tem), naive T cells ( T) and terminally differentiated T cells (Tte). Molecular markers for sorting different T cells include CD3, CD45RO, CD197 and sCAR.

[0089] Test results are shown in Figure 4 As shown in the figure, the proportion of Tcm in the final product harvested from experimental group a1 was significantly higher than that in other experimental groups, indicating that the application of SmAb in the preparation of sCAR-T can effectively promote the differentiation of CAR-positive T cells into central memory T cells and increase the proportion of Tcm in the final product.

[0090] Example 6 Effect of SmAb on the in vitro cytotoxic activity of sCAR-modified T cells

[0091] The human lymphoma cell line Raji was used as the target cell, and 5×10 4 / mL was inoculated into a U-bottom 96-well plate, and the CAR-T cells of each experimental group obtained by culturing for 12 days in Example 4 were co-cultured at an effector-target ratio (E / T) of 25:1, 12.5:1, 6.25:1, and 1:1. The culture medium was serum-free RPMI1640 culture medium, and the culture conditions were 37°C, 5% CO2, and a saturated humidity incubator. After culturing for 12 hours, the lactate dehydrogenase release method (LDH method) was used to detect the killing activity of CAR-T cells of different experimental groups against target cells. The test results are shown in Figure 5 .Depend on Figure 5 The experimental results show that the killing activity of the cell product obtained by applying SmAb to the preparation of sCAR-T is significantly better than that of other experimental groups.

[0092] Example 7 Regulation of the anti-tumor activity of sCAR-modified T cells by SmAb

[0093] 6-8 week old NOD / SCID IL2Rγc- / - immunodeficient mice were selected and human Raji cell line (Raji-Luci) stably expressing luciferase protein was inoculated with 10 6 The tumor-bearing mouse model was established by tail vein injection at a dose of 100 μL / mouse. Three days after the injection of tumor cells, the experimental animals in each group were divided into groups according to the experimental grouping of Example 4. 6 / 100 μL / animal was injected with the CAR-T cell product prepared in Example 4. After injection, the tumor progression in the experimental animals was observed using a small animal imaging system every week until all experimental animals died. The statistical analysis results of the experimental data are shown in Figure 6As shown, compared with other experimental groups, the CAR-T cells obtained in experimental group a1 can effectively prolong the median survival time of tumor-bearing mice, delay the progression of tumors in vivo, and have more lasting in vivo anti-tumor activity, indicating that the application of SmAb in the preparation of sCAR-T can effectively improve the in vivo anti-tumor activity of the prepared CAR-T cell products.

[0094] Example 8 In vivo regulation of sCAR-modified T cells by SmAb

[0095] (1) SmAb antibody was coupled with anti-tubulin small molecule drug Maytansinoid (DM1) to prepare SmAb-DM1. The specific steps are as follows:

[0096] The MM04H (SmAb-2) antibody was dissolved in PBS containing 1.8N DTT and 1mM DTPA at 1mg / mL, and the pH of the buffer was adjusted to 8.0 with 50mM boric acid. The antibody was incubated at 37°C for 1h to reduce and denature the antibody. The reduced antibody solution was balanced to 4°C and purified using a G25 desalting column. The purified denatured antibody and thioether linker-DM1 were mixed in a ratio of 2.4:4.6, reacted at 4°C for 1 hour, and 20 times the volume of cysteine ​​was added after the reaction to terminate the reaction. The reaction mixture was purified by a G25 desalting column, and the final product was SmAb coupled to DM1. The SmAb-DM1 antibody was stored at 4°C at a concentration of 5mg / mL for subsequent experiments.

[0097] (2) NOD / SCID IL2Rγc- / - immunodeficient mice were grouped according to Table 6. Mice in different experimental groups were divided into 1×10 7 / 200μL / mouse was injected with different CAR-T cell products through the tail vein. 2h after injection, peripheral blood of mice was collected as baseline data, and then SmAb 1mg / kg, SmAb-DM1 1mg / kg or PBS were injected through the tail vein according to the experimental groups. 24h after injection, peripheral blood of mice was collected, and CAR-T cells in the samples were detected by flow cytometry. The molecular markers used in the detection were CD45, CD3 and CAR. The test results are shown in Figure 7 As shown, the proportion of CAR-T cells in mice infused with CD19sCAR-T and given SmAb-DM1 decreased significantly over the detection time point, indicating that SmAb-DM1 can effectively eliminate sCAR-T cells in the body.

[0098] Table 6 Animal experiment groups

[0099] Serial number Group No. Treatment and culture methods 1 Experimental group b1 Injection of SmAb-DM1 after CD19sCAR-T transfusion 2 Experimental group b2 Injection of SmAb after CD19sCAR-T transfusion 3 Experimental group b3 PBS injection after CD19sCAR-T transfusion 4 Experimental group b4 Injection of SmAb-DM1 after CD19CAR-T transfusion 5 Experimental group b5 Inject SmAb after CD19CAR-T transfusion 6 Experimental group b6 PBS injection after CD19CAR-T transfusion 7 Experimental group b7 Negative control group

[0100] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. An antibody, characterized in that The antibody comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3; Wherein, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are respectively: HCDR1: TFAMN (SEQ ID NO: 15), HCDR2: RMRSKNNYYTTFYADSVKD (SEQ ID NO: 16), HCDR3: DYFGFNNAVDY (SEQ ID NO: 17); The amino acid sequences of LCDR1, LCDR2 and LCDR3 are: LCDR1: RASKSVSTSGYSYMH (SEQ ID NO: 18), LCDR2: LASNLES (SEQ ID NO: 19), LCDR3: QHSRELPWT (SEQ ID NO: 20); The amino acid sequence of the antigen to which the antibody is directed is SEQ ID NO:

27.

2. The antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

11.

3. The antibody according to claim 1 or 2, characterized in that The amino acid sequence of the light chain variable region is shown in SEQ ID NO:

12.

4. The antibody according to claim 1, characterized in that The antibody is a full-length antibody, a Fab fragment, a F(ab)2 fragment or a single-chain Fv fragment.

5. The antibody according to claim 1, characterized in that The antibody is a monoclonal antibody.

6. The antibody according to claim 1, characterized in that The antibody also includes a heavy chain constant region and a light chain constant region.

7. The antibody according to claim 6, characterized in that The heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:

13.

8. The antibody according to claim 6 or 7, characterized in that The light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

14.

9. The antibody according to claim 1, characterized in that The carboxyl terminus and / or amino terminus of the antibody is fused with a tag protein and / or a signal peptide.

10. The antibody according to claim 9, characterized in that The signal peptide includes the sequence shown as SEQ ID NO: 21 or SEQ ID NO:

22.

11. A product, characterized in that The product is i), ii) or iii) below, i) a nucleic acid molecule encoding the antibody according to any one of claims 1 to 10; ii) a vector comprising the nucleic acid molecule described in i); iii) a host cell, which expresses the antibody according to any one of claims 1 to 10, or comprises the nucleic acid molecule according to i), or comprises the vector according to ii).

12. The method for preparing the antibody according to any one of claims 1 to 10, characterized in that: The following steps are involved: Cultivating the host cell of claim 11 under conditions that allow expression of the antibody; and isolating and purifying the antibody from the resulting culture.

13. Use of the antibody according to any one of claims 1 to 10 in preparing chimeric antigen receptor modified T cells, wherein the chimeric antigen receptor has a sorting domain, and the amino acid sequence of the chimeric antigen receptor is SEQ ID NO: 28; The method for preparing the chimeric antigen receptor modified T cells comprises: The activated T cells are infected with the virus encoding the chimeric antigen receptor, and then the antibody is added to the culture system.

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