Human single domain antibody-based CAR-T cells targeting BAFF-R demonstrate promising preclinical activity in B-cell malignancies.

BACKGROUND

Single-chain antibody-based CAR-T cells targeting BAFF-R have demonstrated antitumor effects against human B-cell malignancies and can overcome CD19 antigen loss. However, CARs built on antigen-specific single-chain antibody variable fragments may have limitations, e.g., large CAR binding domain size. To improve the function of BAFF-R CAR-T cells, we designed CARs with only a fully human heavy-chain variable domain.

METHODS

A fully human antibody phage display library was used to select anti- BAFF- R clones using protein/cell alternate panning. The screened clones were grafted into a second-generation CAR to generate CAR-T cells. CD107a degranulation, luciferase-based cytolysis, repeat antigen stimulation, activation markers, and exhaustion markers were used to detect the function of CAR-T cells in vitro. Xenografts were established in NPG mice following intravenous injection of Jeko-1 cells. Tumor burden and survival of mice were recorded weekly.

RESULTS

Three single-domain heavy chain antibodies, namely Clone #5, Clone #77, and Clone #80, were selected for further evaluation. CAR-T cells constructed with these three candidate clones demonstrated specific target cell activation and effective cytotoxicity in vitro. Clone #5 and Clone #77 CAR-T cells exhibited superior cytotoxic capacity and expansion ability. No significant difference was observed in the expression levels of exhaustion markers on the surface of the three CAR-T cell types. In vivo studies revealed that Clone #5 CAR-T cells displayed enhanced antitumor efficacy compared to other groups, resulting in prolonged survival.

CONCLUSION

We developed a novel BAFF-R CAR-T cell product structured on a fully human single-domain antibody. This product demonstrated promising preclinical activity and may provide a potential alternative treatment for patients with B-cell malignancies.