Study On The Effect Of CD305 Inhibiting BCMA CAR-T On Multiple Myeloma Cells

【Background】Multiple myeloma(MM)is a heterogeneous disease mainly characterized by malignant proliferation of bone marrow plasma cells,which accounts for about 10% of blood tumors and has become the second largest hematologic malignancy in the world,the median age of onset was 75 years.Currently,the main treatments include proteasome inhibitors,immunomodulators,monoclonal antibodies,hematopoietic stem cell transplantation and CAR-T cell infusion therapy.Most patients will eventually develop a relapse or refractory MM that is difficult to treat.Adoptive immunotherapy targeting BCMA CAR-T cells has made great progress in relapse-refractory MM,with an overall response rate of 81%.However,it has been reported in the literature and our previous studies that BCMA CAR-T has a weak or no killing effect on some highly expressed BCMA cells.Therefore,this study intends to clarify the mechanism of the insensitive killing effect of BCMA CAR-T on MM cells and provide a new idea for the therapeutic effect of BCMA CAR-T by studying the difference of the killing effect of BCMA CART on different MM cell lines and its possible reasons.【Methods】1.BCMA CAR-T and MM cell lines with high expression of BCMA were cocultured.The apoptosis of MM cells was detected by flow cytometry.MM cells were divided into two groups: kill sensitive group and kill insensitive group;2.After co-culture of effector cells(BCMA CAR-T cells)and target cells(MM cells),the binding of effector target cells,the expression of CD69 in effector cells,and the secretion of granzyme in effector cells were detected by flow cytometry,and the differences between the two groups were compared;3.MM cell membrane proteins were extracted and silver-stained.Protein spectrum analysis was used to study the difference proteins on the surface of cell membrane between the two groups;4.The interaction between CD305 and BCMA was detected by Co-IP test;5.CD305 knockdown plasmid was constructed and transferred into insensitive MM cell lines by lentivirus transfection.The CD305 knockdown MM cells were co-cultured with BCMA CAR-T cells,and the binding of effector target cells,the expression of CD69 in effector cells and apoptosis of effector cells were detected by flow cytometry;6.CD305 overexpressed plasmid was constructed and transfected into the kill sensitive group MM cells.【Results】1.According to the apoptosis of MM cells and BCMA CAR-T cells after co-culture,MM cells were divided into two groups: MM.1S,KMS-11 and ARD,and AMO-1 and LAMA-84,respectively,in the insensitive group;2.No binding between effector cells and target cells was found in the killing insensitive group during co-culture,no expression of CD69 was found in effector cells,and no release of granzyme was detected in effector cells;However,in the killing sensitive group,the binding of effector cells and target cells was found during co-culture,CD69 expression was found in effector cells,and granzyme release was detected in effector cells;3.The differential protein analysis indicated that CD305 was highly expressed in AMO-1 and LAMA-84 cells of the insensitive group,while CD305 was not detected in the three cell lines of the sensitive group;4.After CD305 knockdown of MM cells in the insensitive group,sh CD305-AMO-1 and sh CD305-LAMA-84 cells were co-cultured with BCMA CAR-T,the expression of CD69 in effector cells was 20.60% and 25.00%,respectively,but the apoptosis rate was only 6.70% and 0.00%,respectively.【Conclusion】1.BCMA CAR-T cells have different killing effects on different MM cell lines.2.The insensitivity to killing effect by BCMA CAR-T cells was related to the high expression of CD305 in MM cells.3.Inhibition of CD305 expression can improve the activation ability of BCMA CAR-T cells,which is a potential target for MM therapy.