Clinical And Mechanistic Study Of CD123-Targeted CAR-T In The Treatment Of AML

Purpose: The present study aimed to verify the anti-leukemia effect and controllability of CD123 CAR-T,conduct a clinical trial of donor-derived CD123 CAR-T serving as preconditioning for haploidentical transplantation in AML patient who relapses after transplantation,and explore the mechanism of side effects after CD123 CAR-T treatment.Methods: 1.The expression of CD123,cytotoxicity,and depletion of CAR-T was assessed by flow cytometry.Detected CAR expression,cytotoxicity,and depletion of CAR-T by flow cytometry.Cytokine release was assessed by ELISA or CBA.2.A FUS-ERG+ AML patient who relapse after transplantation was enrolled and using donor-derived CD123 CAR-T as preconditioning for haploidentical transplantation.3.An in vitro co-culture model was designed to mimic the status,wherein CART123 was stimulated and cytokines were released,and its influence to different types of cells.4.Cytotoxicity to HUVEC was assessed by in vitro live cell imaging system.5.The differentiation potential of MPB CD34+ cells after co-incubated with CD123 CAR-T was assessed by colony-forming assays.Results: Ⅰ.1.We generated CD123 CAR-T and exhibited specific killing and increased intracellular IFN-γ of CD123 CAR-T to CD123+ AML cells.2.Pretreated with low-dose DAC enhanced the killing sensitivity of CD123 CAR-T to MOLM-13 and THP-1.3.CD123 CAR-T was depleted specifically by cetuximab and non-specifically by rATG in vitro.4.CD123 CAR-T significantly prolonged the overall survival of NOG mice bearing MOLM-13 compared with the NT group.Ⅱ.1.Donor-derived CD123 CAR-T exhibited specific killing to patient-derived AML cells.2.The BM blast cells decreased after CAR-T reinfusion,and the patient achieved full donor chimerism on day 18,CRi on day32,and complete myeloid implantation on day 42 after transplantation.3.The proliferation of CAR-T after the infusion was detected by qPCR,and the trend was consistent with the decrease of tumor burden and the rapid development of CRS.Ⅲ.1.The expression level of CD123 on HUVEC was varied in vitro.2.IFN-γ and TNF-α upregulate the expression of CD123 on HUVEC and HDMEC while IL-4 downregulates it in vitro.3.CD123 CAR-T exhibits specific cytotoxicity and cytokines production on HVUEC,while IFN-γ and TNF-α aggravate endothelial damage caused by CD123 CAR-T.4.Co-incubation of CD123 CAR-T and myeloid leukemia cells in in vitro co-culture model upregulated the expression of CD123 on HVUEC,and IFN-γ and TNF-α neutralizing antibodies were able to antagonize it specifically.Moreover,IFN-γ and TNF-α neutralizing antibodies did not affect the cytotoxicity of CD123 CAR-T to CD123+ AML cell lines in vitro.5.The expression of CD123 on K562 cells was up-regulated after incubated with CD123 CAR-T or treated by TNF-α,and the expression of CD123 on MOLM-13 cells was up-regulated after treated by IFN-γ in vitro.6.The expression of CD123 on CD123 CAR-T could be up-regulated.Moreover,compared to control group,CD123 CAR-T had a higher level of CD123,CD25,PD-1,and TIM-3,and more terminally differentiated immunophenotype,lower expansion fold and a higher level of apoptosis in vitro.7.CD123 CAR-T impairs erythroid colony forming ability of nHSPC cells;Although CD123 on nHSPC could be upregulated by IFN-γ and TNF-α or in co-culture with CD123 CAR-T and AML cells,pretreated with IFN-γ and TNF-α did not enhance the damage of CD123 CAR-T to nHSPC.Conclusion:1.The anti-leukemia activity and controllability of CD123 CAR-T in vitro and in vivo.2.CD123 CAR-T reduces the chemotherapy-resistant AML blasts for FUS-ERG+ AML without affecting the full donor chimerism and myeloid implantation.Also,the patient achieved CRi.3.In summary,this study identified that the expression of CD123 on endothelial cells could be upregulated when co-cultured with CD123 CAR-T.Furthermore,IFN-γ and TNF-α could aggravate endothelial damage caused by CD123 CAR-T in vitro.Moreover,blocking IFN-γ and TNF-α does not affect the cytotoxicity of CD123 CAR-T to AML cells.4.CD123 expresses in CART123 and would be upregulated after activation,putatively causing an overactivated and fratricide effect.5.CD123 CAR-T affects the erythroid colony forming ability of nHSPC.Up-regulation of CD123 on CD34+ cells does not aggravate the hematopoietic toxicity of CD123 CAR-T.6.The expression of CD123 on a variety of normal hematopoietic cells,myeloid leukemia cells,endothelial cells could be upregulated,which is closely related to safety in the CD123-targeted therapy,and needs attention.