源自信号调节蛋白 α Ig 样结构域的新型模块化 T 细胞嵌合抗原受体间隔区

Background: T cells equipped with chimeric antigen receptors (CAR) have shown remarkable efficacy in targeting B lineage malignancies. Improvement of the CAR structure is needed, however, with a view to developing flexibly modifiable spacers that are inert in interactions with unwanted cells. Specifically, binding to cells carrying receptors for IgG's crystallizable fragment (FcR), that recognize IgG-derived domains in CARs is to be avoided. Methods: Two novel CARs targeting the CD19 antigen where the IgG1-CH2 and -CH3 domains were replaced with Ig-like domains from signal-regulatory protein (SIRP ) were designed in silico . An IgG1-based CAR and a CAR lacking both SIRP and IgG1 domains were used as comparators. The phenotype and memory phenotype of the expanded cells were analyzed by flow cytometry, and CAR T cell activation and cytotoxic efficacy were assessed in co-culture experiments in response to CD19 + target cells. Unwanted interactions with FcR-expressing myeloid cells were interrogated in co-culture assays with THP-1 monocytic cells. Results: T cells carrying the novel SIRP -based CARs enacted potent in vitro cytotoxicity against CD19 positive B-lineage leukemia cells, comparable to traditional IgG1-based CAR T cells.

Co-culture of IgG1-based CAR T cells with FcR-expressing THP-1 monocytic cells led to prominent cell surface expression of CD69 on T cells together with production of Interleukin (IL)-2 and Interferon- , and production of IL-1 , indicating activation of the T cells and monocytes, respectively. Longer co-culture led to killing of the monocytes. No signs of T cell nor monocyte activation were detected in co-cultures of SIRP -based CAR T cells with THP-1 cells.

Arming T cells with the SIRP -based CARs favored differentiation towards CD4 + phenotype during expansion, while the effects on memory phenotype of the T cells were equivalent between the SIRP - and IgG1-based CARs. In a pilot experiment, T cells modified with one of the SIRP -based CARs showed dose dependent leukemia cell control.

Conclusion: The novel SIRP based spacers offer a suitable backbone for developing chimeric antigen receptors that evade the off-target binding to FcR while the cells retain a favorable memory phenotype and efficient cytotoxicity, establishing a promising candidate for future in vivo and clinical testing.