B7-H3低免疫原性嵌合抗原受体T细胞可克服免疫排斥并保持针对胆管癌的抗肿瘤活性

Background. Intrahepatic cholangiocarcinoma (ICC) is a rare, aggressive liver cancer, with limited treatment options and poor survival. Because surgery is the only curative option, but most patients present with advanced disease, novel therapies are urgently needed. CAR-T cell therapy has revolutionized hematologic cancer treatment, but its application in solid tumors is limited.We investigated an allogeneic “off-the-shelf” hypoimmune (HIP) CAR-T platform designed to overcome barriers that hinder CAR-T efficacy in solid tumors. Targeting B7-H3, aberrantly expressed in ICC, the HIP CAR T cells demonstrated promising activity in vitro and in vivo studies. These findings support the potential of allogeneic HIP B7H3 CAR-T cells to improve antitumor responses in advanced ICC. Methods: Immunohistochemical staining of an ICC tumor vs normal tissue. Generation of HIP T cells with disrupted B2M, CIITA, and TRAC genes using CRISPR-Cas9 editing. In addition, CD47 and anti-B7H3 CAR were expressed using lentiviral transduction. These allogeneic HIP B7H3 CAR T cells were compared to allogeneic WT B7H3 CAR T cells (TCR-/-) that only expressed the anti-B7H3 CAR. HIP phenotype characterization was analyzed by flow cytometry. Killing assay performed with xCELLicence real-time system. BLI for tumor growth. Results: Immunohistochemistry of human ICC tumors showed strong membrane and cytoplasmic B7-H3 expression compared to normal liver tissue. The B7-H3 epitope recognized by mAb 376.96 was highly expressed in ICC2, ICC3 and hCAF cell lines. B7-H3 CAR T specifically recognized and eliminated ICC targets in vitro. Lentiviral constructs engineering produced both WT and HIP allogeneic CAR T cells with high editing efficiency: TCR knockout in 91% (WT) and 89.8% (HIP) and HLA-I and HLA-II knocked out in 93.3% and 98.7% of HIP B7-H3 cells. CD47 overexpression was restricted to the HIP B7-H3 cells (47.3%). In co-culture assay T cells, NK cells, and macrophages, HIP cells showed protection from adaptive and innate immune clearance. Impedance cytotoxicity assays confirmed that HIP B7-H3 CAR T cells maintained full antitumor activity, matching WT B7-H3 CAR T cell killing rate. HIP CAR-T cells are now validated in in vivo ICC model. Conclusion: Our findings showed that allogeneic HIP B7-H3 CAR T cells can be efficiently engineered, maintain protection from adaptive and innate immune rejection, and retain full cytotoxic activity against ICC tumors. By combining the tumor-specific activity of B7-H3 CAR-T cells with the immune-evasive properties of HIP cells, this approach offers a promising and widely applicable allogeneic “off-the shelf” therapy for ICC, providing a strong rationale for further preclinical translational studies.