Modification of Hinge/Transmembrane and Signal Transduction Domains Improves the Expression and Signaling Threshold of GXMR-CAR Specific to Cryptococcus spp.

Author(s)

dos Santos, Matheus Henrique ; Machado, Michele Procopio ; Kumaresan, Pappanaicken R. ; da Silva, Thiago Aparecido

• Document type: Journal article
• Source: CELLS; v. 11, n. 21, p. 20-pg., 2022-11-01.

Abstract

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 zeta. Cryptococcus are fungi that cause the life-threatening disease cryptococcosis, and GXMR-IgG4-28 zeta 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 alpha molecule as the hinge/transmembrane and used CD28 or 4-1BB molecules as co-stimulatory domains, creating GXMR-8-28 zeta and GXMR-8-BB zeta, respectively. Jurkat cells expressing GXMR-CAR containing CD8 alpha as the hinge/transmembrane improved the CAR expression and induced a tonic signaling. GXMR-8-28 zeta and GXMR-8-BB zeta 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 zeta and GXMR-8-BB zeta 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. (AU)

• FAPESP's process: 18/18538-0 - Bioengineered of T- and NK-cells by CAR against invasive fungal infections
• Grantee: Thiago Aparecido da Silva
• Support Opportunities: Research Grants - Young Investigators Grants
• FAPESP's process: 20/09113-6 - Functional activity of T cells modified with GXMR-CAR expressing the intracellular domain of CD28 or CD137, to control of experimental cryptococcosis
• Grantee: Matheus Henrique dos Santos
• Support Opportunities: Scholarships in Brazil - Master
• FAPESP's process: 20/11307-3 - Impact of different scFv in GXMR-CAR expressed by T lymphocytes during T-cell activation against Cryptococcus spp
• Grantee: Michele Procópio Machado
• Support Opportunities: Scholarships in Brazil - Master
• FAPESP's process: 19/26074-7 - Bioengineered of T- and NK-cells by CAR against invasive fungal infections
• Grantee: Thiago Aparecido da Silva
• Support Opportunities: Scholarships in Brazil - Young Researchers