靶向Siglec-6 CAR-T细胞的体外优化及其抗急性髓系白血病的临床前评价OA

Objective:To construct and optimize chimeric antigen receptor T(CAR-T)cells targeting sialic acid-binding immunoglobulin-like lectin 6(Siglec-6),and to evaluate their targeted cytotoxicity against acute myeloid leukemia(AML)cells,preliminary in vivo efficacy and short-term safety,thereby providing experimental evidence for Siglec-6 as an immunotherapeutic target for AML.Methods:A recombinant CAR gene targeting Siglec-6 was designed.Three humanized single-chain variable fragments(scFvs)were screened,and the effects of different co-stimulatory domains,including 4-1BB,CD28,ICOS,HVEM,TMIGD2 and OX40,on CAR-T cell function were compared within a second-generation CAR framework.Siglec-6 CAR-T cells were generated by lentiviral transduction of primary human T cells,and CAR positivity and cellular phenotypes were detected by flow cytometry.Surface expression of Siglec-6 on U937,MV4-11 and K562 cells was examined,and 293T-Siglec-6 overexpression(OE)cells,U937-Siglec-6 knockout(KO)cells,and U937-Luc/U937-Siglec-6 KO-Luc cell models were established.Flow cytometry-based cytotoxicity assays,ELISA,real-time cell analysis(RTCA)and luciferase bioluminescence assays were used to evaluate the in vitro cytotoxic activity and target specificity of CAR-T cells.A U937-Luc xenograft leukemia model was established to assess in vivo antitumor efficacy,and short-term safety was evaluated based on body weight,bioluminescence imaging,peripheral blood T-cell levels,hematoxylin-eosin staining of major organs,organ indices and serum biochemical parameters.Results:Among the three candidate CAR-T cells,CAR-T-1 showed superior performance in terms of CAR expression level,short-term cytotoxic activity and feasibility for subsequent construct development.Comparison of different co-stimulatory domains showed that 4-1BB-CAR-T cells exhibited relatively stable CAR positivity and stronger in vitro cytotoxic activity under multiple effector-to-target ratios.Siglec-6 CAR-T cells effectively killed Siglec-6-positive U937 and MV4-11 cells and significantly promoted the release of IFN-γ and TNF-α(P<0.000 1),whereas no obvious cytotoxicity was observed against Siglec-6-negative K562 cells.The 293T-Siglec-6 overexpression model and U937-Siglec-6 KO model further confirmed that this cytotoxic effect was clearly dependent on Siglec-6 targeting.After antigen stimulation,Siglec-6 CAR-T cells differentiated into effector phenotypes and were significantly activated(P<0.000 1),accompanied by increased expression of some exhaustion-related markers(P<0.01).In vivo experiments showed that Siglec-6 CAR-T treatment reduced tumor burden and prolonged survival in U937-Luc tumor-bearing mice.During short-term observation,no obvious difference in body weight change was observed between the two groups,and hematoxylin-eosin staining of major organs revealed no apparent morphological injury,although some organ indices and serum biochemical parameters differed between groups(P<0.01).Conclusion:Siglec-6 CAR-T cells specifically killed Siglec-6-positive AML cells and exerted preliminary anti-leukemic activity in an AML xenograft model,and the underlying mechanism may be associated with the target-dependent recognition of Siglec-6,the activation of CAR-T cells and the increased secretion level of IFN-γ/TNF-α.