| Objective:Multiple myeloma(MM)is the second most common hematological malignancy globally,and its incidence in China has exceeded that of acute leukemia.In recent years,with the introduction of various new treatments,patients’ rate of clinical remission has improved.However,relapses caused by drug resistance still occur sporadically,and high-risk patients fail to respond to existing treatments.Therefore,currently,multiple myeloma is still incurable.Chimeric antigen receptor T cells(CAR-T)therapy is an emerging immunotherapy,and current clinical studies have shown that it has great potential in treating multiple myeloma.The development of CD38-targeted CAR-T cells(anti-CD38 CAR-T)began with the success of CD38 monoclonal antibody drugs in treating multiple myeloma.Since then,various anti-CD38 CAR-T cells have been developed and evaluated in clinical trials,but so far,no safe and effective anti-CD38 CAR-T cells have been approved for marketing.Therefore,in this study,we prepared two novel anti-CD38 CAR-T cells and evaluated the anti-tumor activities of these CAR-T cells in vivo and in vitro.We expected to develop novel anti-CD3 8 CAR-T cells that can effectively treat multiple myeloma in preclinical studies.Currently,the only CAR-T cell targeting BCMA(B-cell maturation antigen),which is clinically approved for treating multiple myeloma,has the problem of tumor recurrence caused by the escape of BCMA antigen.To solve this problem,based on novel anti-CD38 CAR-T cells,we designed tandem dual CAR-T cells(anti-BCMA/CD38 Tan CAR-T)that can simultaneously target BCMA and CD38.By verifying in vivo and in vitro activities of these CAR-T cells,we expected to develop novel BCMA/CD38 tandem dual-targeting CAR-T cells that can effectively treat multiple myeloma and inhibit the escape of BCMA or CD38 antigens.Methods:Plasmid construction,retroviral vector packaging,and CAR-T cell preparation:(1)The target fragments were obtained by patent search and gene synthesis.The MFG anti-CD38 CAR and MFG CD38/BCMA Tan CAR retroviral vector expression plasmids were constructed using molecular cloning techniques.(2)Phoenix-ECO and PG13 cells were used for packaging the retroviral vector,the supernatant of the retroviral vector was harvested,and the copy number of vectors was detected by RT-q-PCR.(3)Peripheral blood mononuclear cells(PBMC)was isolated from healthy donors,the human primary T cells were obtained after stimulating these PBMCs with anti-CD3 monoclonal antibody(OKT-3)and interleukin-2(IL-2).We transduced these T cells with retroviral vectors to obtain anti-CD38 CAR-T and anti-CD38/BCMATan CAR-T cells.In vitro activity detection of CAR-T cells:(1)Detection of the killing ability of CAR-T cells to target cells.After CAR-T cells were co-cultured with target cells,three methods were used to detect CAR-T cells’ killing efficiency on target cells:① the two co-cultured cells were stained with Annexin V apoptosis reagent,the apoptosis ratio of target cells was detected by flow cytometry.We distinguished the effector cells and target cells by fluorescent-labeled antibodies staining.② After co-culturing,the chemiluminescence signal produced by the reaction between the luciferase expressed by the target cells and the substrate was detected using a multifunctional microplate reader.The survival rate of the target cells was analyzed.③ After co-culturing,the level of pro-inflammatory cytokines secreted by CAR-T cells were detected by the CBA flow cytometry multi-factor detection method or double antibody sandwich ELISA method.(2)CAR-T cell proliferation detection.CAR-T cells were stained with CFSE and co-cultured with target cells.Flow cytometry was used to detect changes in CFSE signal and analyzed the proliferation ability of CAR-T cells.CAR-T cells were counted every 48 hours to plot the CAR-T cells proliferation curve and viability curve.In vivo activity detection of CAR-T cells:(1)The therapeutic effect of CAR-T cells on tumor-bearing mice.The RPMI-Luc cell line was injected into the tail vein of NPG immunodeficient mice to construct a xenograft mouse model.After injecting CAR-T cells into tumor-bearing mice,using MIIS small animal in vivo imaging system to monitor the chemiluminescence signal produced by the reaction of substrate luciferin and the luciferase expressed by tumor cell analyzed the effect of CAR-T cells on tumor clearance.After the in vivo imaging,the mice were sacrificed,and the peripheral blood,bone marrow,liver,spleen,or tumor tissues of the mice were collected.After labeling the tumor cells with fluorescent antibodies,the residual tumor cells in each tissue were detected by flow cytometry.At the same time,the ELISA method was used for detecting the secretion level of interferon-gamma(IFN-gamma)in the serum of mice.(2)In vivo proliferation detection of CAR-T cells.To analyze the proliferation of CAR-T cells in vivo,the percentage of human CD3+T cells in peripheral blood leukocytes of mice was evaluated by flow cytometry.In addition,the Kaplan-Meier survival curve of tumor-bearing mice was plotted by recording the survival time and survival rate of each group of mice.Results:First,two anti-CD38 CAR(CAR021,CAR022)retroviral vectors were successfully constructed,efficiently transduced human primary T cells to prepare CAR-T cells.The in vitro activity test showed that the two novel anti-CD38 CAR-T cells had significant killing effects on the human-derived lymphoma cells Raji,Daudi,and multiple myeloma cells RPMI-Luc expressing CD38.The killing efficiency was higher than 90%.And the secretion level of IFN-y increased significantly after CAR-T cells were incubated with target cells.In vivo activity test showed that it has a significant therapeutic effect on RPMI-Luc tumor-bearing mice.At the same time,the comparison of the anti-tumor activity between the novel anti-CD38 CAR-T cells and the other four positive control anti-CD38 CAR-T cells with known anti-CD38 scFv structure showed that there is no apparent difference in their killing effects on different target cells.Secondly,we successfully constructed novel anti-CD38(21)/BCMA Tan CAR-T cells via connecting the bb2121 anti-BCMA scFv in tandem with the anti-CD38 CAR021 scFv,which can efficiently transduce human primary T cells to prepare CAR-T cells,the transduction efficiency is not significantly different from single-targeted CAR-T cells,indicating that the dual-antigen CAR modification did not affect the transduction efficiency of these CARs.And the integrated copy number of Tan CAR molecules in T cell is less than 5,indicating that the retroviral vector is safe.In vitro anti-tumor activity detection showed that anti-BCMA-OR-CD38(21)Tan CAR-T had significant cytotoxicity toward single-targeted CAR-T that only responded to tumor cells with the expression of CD38 or BCMA and tumor cells expressing both CD38 and BCMA.The cytotoxicity was significantly higher than single-targeted CD38(21)or BCMA CAR-T cells and anti CD38(21)-OR-BCMA Tan CAR-T cells.At the same time,the pro-inflammatory cytokine released by Tan CAR T cells significantly increased after target cell stimulation to levels comparable to that generated by BCMA-CAR or CD38 CAR T cells.The above in vitro activity test results showed that anti CD38(21)/BCMA Tan CAR-T could effectively prevent the escape of BCMA or CD38 antigen.The detection of in vitro proliferation ability showed that the anti BCMA-OR-CD38(21)-Tan CAR-T cells increased significantly more than single-targeted CAR-T cells and anti CD38-OR-BCMA Tan-CAR T cells in response to activation by target cells.The T cell proliferation curve showed that the proliferation of anti BCMA-OR-CD38(21)Tan CAR-T cells were not significantly different from single-targeted CAR-T cells,indicating that Tan CAR expression did not affect T cell proliferation.The detection of in vivo anti-tumor activity of anti BCMA-OR-CD38(21)Tan CAR-T showed that four days after CAR-T cell injection,the anti BCMA-OR-CD38(21)Tan CAR-T cell treatment group had no tumor signals were detected.At the same time,no tumor cells were detected in the peripheral blood,bone marrow,liver,and spleen tissues of the mice,indicating that the tumor cells were eliminated.After 28 days of continuous observation,no tumor recurrence was observed.Compared with the single-targeted CD38(21)or BCMA CAR-T treatment group,Tan CAR-T cells showed no significant difference in tumor clearance.The secretion of IFN-γ in the serum of mice in the Tan CAR-T cell treatment group increased and was significantly higher than that in the single-targeted CAR-T cell treatment group.The proliferation detection of CAR-T cells in vivo showed that Tan CAR-T cells were significantly higher than anti-CD38 CAR-T cells on the 7th day after infusion.The CD3+T could still be detected on the 22nd day after infusion.This indicated that Tan CAR-T cells could increase for a long time in tumor-bearing mice.The survival curve of tumor-bearing mice showed that compared with the model group and Pan-T group,the survival time of the Tan CAR-T cell-treated mice was prolonged.Still,there was no significant difference compared with the single-targeted CAR-T cell treatment.Finally,we successfully constructed novel anti-CD38(22)/BCMA Tan CAR-T cells via connecting the bb2121 anti-BCMA scFv in tandem with the anti-CD38 CAR022 scFv,which can efficiently transduce human primary T cells,preparation of CAR-T cells.The integrated copy number of Tan CAR molecules in T cell is less than 5,indicating that the retroviral vector has high activity and exemplary safety.In vitro anti-tumor activity testing showed that anti BCMA-OR-CD38(22)Tan CAR-T had significant cytotoxicity toward tumor cells with the expression of CD38 or BCMA and tumor cells expressing both CD38 and BCMA.The cytotoxicity was significantly higher than single-targeted CD38(22)or BCMA CAR-T cells and anti CD38(22)-OR-BCMA Tan CAR-T cells.The pro-inflammatory cytokine released by Tan CAR T cells significantly increased after target cell stimulation to levels comparable to that generated by BCMA-CAR or CD38 CAR T cells.These indicated that anti CD38(22)/BCMA Tan CAR-T could effectively prevent the escape of BCMA or CD38 antigen.The detection of in vitro proliferation ability showed that the anti BCMA-OR-CD38(22)Tan CAR-T cells increased significantly more than single-targeted CAR-T cells and anti CD38-OR-BCMA Tan-CAR T cells in response to activation by target cells.The T cell proliferation curve showed that anti BCMA-OR-CD38(21)Tan CAR-T cells were not significantly different from single-targeted CAR-T cells.Conclusion:In this study,we successfully constructed two novel anti-CD38 CAR-T cells based on the structure of human anti-CD3 8 scFvs,which had significant cytotoxicity toward a variety of hematological malignancies with CD3 8 expression and had significant therapeutic effects on multiple myeloma-bearing mice.The cytotoxicity on tumor cells is not significantly different from the existing four positive control anti-CD38 CAR-T cells.We successfully constructed four CD38/BCMA tandem dual CAR-T cells via connecting the anti-BCMA scFv in tandem with the anti-CD38 CAR021 scFv or anti-CD38 CAR022 scFv.The anti BCMA-OR-CD38(21)/CD38(22)Tan CAR-T cells had a significant in vitro anti-multiple myeloma effect,which was higher than anti CD38(21)/CD38(22)-OR-BCMA Tan CAR-T cells and single-targeted CAR-T cells.These Tan CAR-T cells can also effectively prevent the escape of BCMA or CD38 antigen.Notably,the anti BCMA-ORCD38(21)Tan CAR-T cells had a noticeable tumor clearance effect on multiple myeloma tumor-bearing mice and can significantly increase the survival time of mice.In summary,we successfully developed two novels anti-CD38 CAR-T cells and four novels BCMA/CD38 tandem dual-targeting CAR-T cells,which have apparent preclinical multiple myeloma treatment effects,and CD38/BCMA tandem dual-targeting CAR-T cells can effectively avoid the escape of BCMA and CD38 antigen in vitro,providing an effective new strategy for the treatment of multiple myeloma. |
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