CAR-T/NK细胞在实体肿瘤（如结直肠癌）治疗中取得了有限的成功，部分原因是肿瘤相关抗原的异质性导致初始反应后出现抗原阴性复发。通过增强内源性免疫细胞的招募和持久性可以克服这一障碍。采用免疫组织化学和流式细胞术分别评估组织微阵列和细胞系中CD133抗原的表达。采用逆转录病毒载体转导来产生分泌CBLB502的CAR133-NK92细胞（CAR133-i502-NK92）。通过LDH释放、RTCA测定、脱颗粒试验以及小鼠异种移植瘤中测量肿瘤生物发光信号强度来量化CAR133-NK92细胞的体外和体内肿瘤杀伤能力。我们对CAR133-i502-NK92细胞进行了工程改造，并证明这些细胞在体外和异种移植小鼠模型中显示出增强的增殖（9.0×104细胞 vs. 7.0×104细胞）和特异性抗肿瘤活性，并且耐受性良好。值得注意的是，CAR133-i502-NK92细胞分泌的CBLB502能够有效激活内源性免疫细胞。此外，在hCD133+/hCD133-混合癌异种移植模型中，CAR133-i502-NK92细胞比对照组更好地抑制了癌症的生长（n = 5, P = 0.0297）。T细胞浸润与抗肿瘤效力的增加有关（P < 0.0001）。通过携带CBLB502 TLR5激动剂，CAR133-NK92细胞能够以CAR133依赖的方式特异性消除CD133阳性结肠癌细胞，并以CBLB502特异性内源性免疫反应的方式间接根除CD133阴性结肠癌细胞。本研究描述了一种用于优化CAR-T/NK细胞治疗抗原多样的实体肿瘤的新技术。版权所有 © 2023年癌症生物学与医学。

CAR-T/NK cells have had limited success in the treatment of solid tumors, such as colorectal cancer (CRC), in part because of the heterogeneous nature of tumor-associated antigens that lead to antigen-negative relapse after the initial response. This barrier might be overcome by enhancing the recruitment and durability of endogenous immune cells.Immunohistochemistry and flow cytometry were used to assess the expression of CD133 antigen in tissue microarrays and cell lines, respectively. Retroviral vector transduction was used to generate CBLB502-secreting CAR133-NK92 cells (CAR133-i502-NK92). The tumor killing capacity of CAR133-NK92 cells in vitro and in vivo were quantified via LDH release, the RTCA assay, and the degranulation test, as well as measuring tumor bioluminescence signal intensity in mice xenografts.We engineered CAR133-i502-NK92 cells and demonstrated that those cells displayed enhanced proliferation (9.0 × 104 cells vs. 7.0 × 104 cells) and specific anti-tumor activities in vitro and in a xenogeneic mouse model, and were well-tolerated. Notably, CBLB502 secreted by CAR133-i502-NK92 cells effectively activated endogenous immune cells. Furthermore, in hCD133+/hCD133- mixed cancer xenograft models, CAR133-i502-NK92 cells suppressed cancer growth better than the counterparts (n = 5, P = 0.0297). Greater T-cell infiltration was associated with greater anti-tumor potency (P < 0.0001).Armed with a CBLB502 TLR5 agonist, CAR133-NK92 cells were shown to be capable of specifically eliminating CD133-positive colon cancer cells in a CAR133-dependent manner and indirectly eradicating CD133-negative colon cancer cells in a CBLB502-specific endogenous immune response manner. This study describes a novel technique for optimizing CAR-T/NK cells for the treatment of antigenically-diverse solid tumors.Copyright © 2023 Cancer Biology & Medicine.