Arrangement of VL and VH Domains in VRC01-Based Chimeric Antigen Receptor (CAR) Affects Functions and Exhaustion Status of CAR-T Cells

Abstract

Background: Immunotherapy using engineered T cells expressing chimeric antigen receptor (CAR) is used in an increasing number of cases. The CAR molecule has an antigen-binding domain usually consisting of an antibody-derived VL and VH domains. The arrangement of VL and VH domains has been shown to affect CAR characteristics and functions of the CAR-transduced T (CAR-T) cells. Objectives: This study aims to clarify whether the relative arrangement of VL and VH domains in CAR affects the exhaustion status of CAR-T cells especially bearing herpes virus entry mediator (HVEM)-derived co-stimulatory signal domain (CSSD). Method: We generated CARs bearing the HVEM-derived CSSD and the VL and VH domains derived from HIV-1 broadly neutralizing antibody VRC01 in different arrangements (VL-VH or VH-VL) as a model system, and compared effector functions and characteristics of the CAR-T cells. Results: In the transduced T cells, the VL-VH CAR was expressed at higher levels and led to more potent effector functions than the VH-VL CAR. Phenotypic analysis showed that the VH-VL CAR-T cells contained two populations: one with higher CAR expression and CAR-mediated tonic signaling associated with more exhausted phenotype, and the other with the opposite characteristics. Interestingly, the VL-VH CAR-T cells, although expressing higher levels of CAR, exhibited lower CAR-mediated tonic signaling associated with lower percentage of exhausted population, which led to higher expansion upon cognate antigen simulation. These results suggested that the VL-VH CAR-T cells appeared to escape exhaustion induced by CAR-mediated tonic signaling despite the high CAR expression. Conclusions: The arrangement of VL and VH domains in VRC01-based CAR appears to play important roles in not only functions but also exhaustion status of CAR-T cells with HVEM-derived CSSD. Therefore, preclinical validation of the relative position of VL and VH domains combined with various CSSDs in CAR could assist in developing effective CAR-T cell-mediated immunotherapy.

Keywords

• Chimeric antigen receptor
• single chain antibody
• variable region