Study On The Efficacy And Toxicity Of BCMA-CAR-T Cells

Objective:To construct a mouse model of multiple myeloma,design a reasonable experimental group,select the appropriate method to evaluate the pharmacodynamic effect of human BCMA-CAR-T cells on human multiple myeloma and determine the appropriate dose,while in tumor mice The corresponding toxicity indicators were evaluated in the model,and the toxic effects of BCMA-CAR-T cells were initially observed.The new generation in situ hybridization RNAscope was used to detect the distribution of BCMA-CAR-T,and the expression of BCMA was detected by immunohistochemistry.The feasibility of this method to detect the distribution of CAR-T cells was preliminarily determinedMethods:1.The bioluminescence imaging method was used to identify the luminescence efficiency of luciferase-labeled human multiple myeloma cells,and the human multiple myeloma model was established by transplanting human tumor cells into the tail vein of severe immunodeficiency mice.Bioluminescence imaging was used to identify whether the transplantation was successful2.After injection of CAR-T cells into the tail vein,the therapeutic effect of CAR-T cells on multiple myeloma was detected by bioluminescence imaging,and the corresponding cytokines(IFN-y)in peripheral blood were detected to indirectly evaluate CAR-T therapeutic effect.During the experiment,the body temperature and body weight of the mice were measured,and the general state of the mice was observed.After theexperiment,the mice were dissected,and the anatomical observation was performed,and the pathological sections were taken to obtain the pathological sections of the mice,and the therapeutic effects and toxic effects of the mice were evaluated.3 Tissue paraffin sections were prepared,RNAscope was used to detect the distribution of BCMA-CAR-T in tissues,and positively-derived tissues were immunohistochemically tested for the distribution of BCMA in tissues.Results 1.Fluorescent D-luciferin substrate was added to MM.1S-Luc cells,and Luciferin luminescence imaging was taken.The results and data were read to confirm that the in vitro bioluminescence efficiency of MM.1S-Luc cells was 99%.MM.1S-Luc cells were transplanted into the tail vein of NSG mice.On the 18th day after transplantation,the bioluminescence imaging identification model was successfully constructed.2.Bioluminescence in vivo imaging showed that the BCMA-CAR-T cells showed obvious therapeutic effects on the third day after injection,and the middle dose and high dose CAR-T were better than the low dose group.On the third day after cell treatment,IFN-? was released in a large amount and then immediately decreased.The profile reflected the effect of BCMA-CAR-T.The mice in the non-treated group lost weight,and showed signs of quadriplegia,weight loss,and sparse hair.However,the weight of the mice in the CAR-T treatment group increased naturally,and the clinical signs did not appear abnormal.Gross anatomical observation showed multiple myeloma cells and tumor metastasis in the treatment group,and no abnormalities in the CAR-T treatment group.Therefore,CAR-T cells can rapidly alleviate multiple myeloma without obvious toxic effects.3.RNAscope test results showed that the positive signal of BCMA-CAR-T cells was expressed on the surface of plasma cells and B lymphocytes.The RNAscope positive signal was sliced for immunohistochemistry to detect BCMA antigen.The results showed that the positive signal of BCMA antigen was also expressed in Plasma cell surface and B lymphocyte surface.It is initially demonstrated that RNAscope can be used to detect the distribution of CAR-T cells,and RNAscope can be used to detect one of the methods of CAR-T metabolism and targeted distribution.Conclusion:BCMA-CAR-T cells have significant anti-multiple myeloma effects and safety can be monitored.