PT  - JOURNAL ARTICLE
AU  - Zenere, Giorgio
AU  - Wu, Chengxiang
AU  - Midkiff, Cecily C.
AU  - Johnson, Nathan M.
AU  - Grice, Christopher P.
AU  - Wimley, William C.
AU  - Kaur, Amitinder
AU  - Braun, Stephen E.
TI  - Extracellular domain, hinge, and transmembrane determinants affecting surface CD4 expression of a novel anti-HIV chimeric antigen receptor (CAR) construct
AID  - 10.1101/2023.10.25.563930
DP  - 2023 Jan 01
TA  - bioRxiv
PG  - 2023.10.25.563930
4099  - http://biorxiv.org/content/early/2023/10/26/2023.10.25.563930.short
4100  - http://biorxiv.org/content/early/2023/10/26/2023.10.25.563930.full
AB  - Chimeric antigen receptor (CAR)-T cells have demonstrated clinical potential, but current receptors still need improvements to be successful against chronic HIV infection. In this study, we address some requirements of CAR motifs for strong surface expression of a novel anti-HIV CAR by evaluating important elements in the extracellular, hinge, and transmembrane (TM) domains. When combining a truncated CD4 extracellular domain and CD8α hinge/TM, the novel CAR did not express extracellularly but was detectable intracellularly. By shortening the CD8α hinge, CD4-CAR surface expression was partially recovered and addition of the LYC motif at the end of the CD8α TM fully recovered both intracellular and extracellular CAR expression. Mutation of LYC to TTA or TTC showed severe abrogation of CAR expression by flow cytometry and confocal microscopy. Additionally, we determined that CD4-CAR surface expression could be maximized by the removal of FQKAS motif at the junction of the extracellular domain and the hinge region. CD4-CAR surface expression also resulted in cytotoxic CAR T cell killing of HIV Env+ target cells. In this study, we identified elements that are crucial for optimal CAR surface expression, highlighting the need for structural analysis studies to establish fundamental guidelines of CAR designs.Competing Interest StatementThe authors have declared no competing interest.