铰链/跨膜区与信号转导区的改造提高隐球菌特异性 GXMR-CAR 的表达与信号阈值

Chimeric antigen receptors (CARs) redirect T cells to recognize a specific target. CAR components play a pivotal role in antigen specificity, structure stability, expression on cell surface, and induction of cellular activation, which together determine the success of CAR T-cell therapy. CAR products targeting B-cell lymphoma encouraged the development of new CAR applications beyond cancer.

For example, our group developed a CAR to specifically target glucuronoxylomannan (GXM) in the capsule of Cryptococcus species, called GXMR-CAR or GXMR-IgG4-28 . Cryptococcus are fungi that cause the life-threatening disease cryptococcosis, and GXMR-IgG4-28 redirected T cells to target yeast and titan cell forms of Cryptococcus spp.

Here, we replaced the IgG4-hinge and CD28-transmembrane domains from GXMR-CAR with a CD8 molecule as the hinge/transmembrane and used CD28 or 4-1BB molecules as co-stimulatory domains, creating GXMR-8-28 and GXMR-8-BB , respectively. Jurkat cells expressing GXMR-CAR containing CD8 as the hinge/transmembrane improved the CAR expression and induced a tonic signaling. GXMR-8-28 and GXMR-8-BB induced high levels of IL-2 and up-regulation of CD69 expression in the presence of reference strains of C. neoformans and C. gattii .

Moreover, GXMR-8-28 and GXMR-8-BB showed increased strength in response to incubation with clinical isolates of Cryptococcuss spp. , and 4-1BB co-stimulatory domain triggered a more pronounced cellular activation. Dasatinib, a tyrosine kinase inhibitor, attenuated the GXMR-CAR signaling cascade's engagement in the presence or absence of its ligand.

This study optimized novel second-generation GXMR-CARs containing the CD8-hinge/transmembrane domain that improved CAR expression, antigen recognition, and signal strength in T-cell activation.