Generation and functional analysis of spacer-modified HBV-specific chimeric antigen receptors

T-cell therapy using chimeric-antigen-receptors (CARs) is an established immunotherapeutic strategy for treating cancers, but also interesting for chronic viral infections. Our study aimed to optimize CAR constructs to exclude target-independent tonic signaling and realize recognition of HBV envelope protein (HBVenv) merely on HBV-positive cells, but not as soluble antigens.

We constructed S-CARs containing the HBV S-specific single-chain antibody C8 as the binding domain, CD28-CD3 intracellular signaling domains, and various spacers. We transduced human T cells with single S-CARs or logic-gated HER2-synNotch-inducible S-CARs and characterized the function of S-CAR-T cells on HBVenv-positive hepatoma cells.

S-CARs containing IgG1-CH2CH3, IgG4-CH3Hinge, IgG2-CH3, or zEGF spacers of 119-225 amino acids in length specifically activated T-cells to secrete cytokines and eliminate HBVenv transgenic hepatoma cell lines. Moreover, the S-CAR-T cells showed antiviral activity and significantly decreased the level of viral antigens, intracellular HBV DNA, and HBV cccDNA in HBV-infected HepG2-NTCP cells. HER2-synNotch-inducible CAR-T cells expressed the S-CAR upon HER2-synNotch activation only when HER2 was recognized on target cells. Unlike S-CAR transduced T cells, they were not activated when incubated with soluble HBsAg and selectively killed dual antigen (HER2+HBVenv+) HepG2 cells but not cells only positive for HBVenv or HER2.

Taken together, our study demonstrates that after spacer-modification of the S-CAR unwanted, ligand-independent tonic signaling is eliminated, while the antiviral function of S-CAR-T cells is maintained. Inducing the S-CAR by the synNotch strategy improves CAR T-cell efficacy and specificity. Thus, our optimized CARs are interesting therapeutic candidates for treating chronic hepatitis B and HBV-associated hepatocellular carcinoma.

Publication History

Article published online:
20 January 2025