Impact of Glycine-Serine Linker on Target Antigen Binding and Subsequent CD37CAR-T Performance

CD19-chimeric antigen receptor (CAR) has shown promising outcomes in B-cell malignancies. However, relapses due to poor CAR-T persistence and antigen escape have occurred. CD37 is a potential alternative immunotherapy for CD19^- tumors. Inferior CAR-T cytotoxicity was observed in CD37CAR-T compared to CD19CAR-T model that was possibly due to lower CD37CAR affinity. To alleviate CD37CAR functions, we optimized the most prevalent linkers, Whitlow (18aaL) and glycine-serine (GS4L). CD37.GS4L CAR-T showed higher transduction efficiency and T-cell expansion contributed by minimizing T-cell fratricide. In chronic antigen stimulation, CD37.GS4L CAR demonstrated robust T-cell proliferation while preserving stemness and a decrease in induced exhaustion phenotype, which resulted in greater tumoricidal activity among various CD37⁺ malignancies. In silico analysis showed that CD37.GS4L scFv altered structural dynamic behaviors by facilitating variable heavy-chain region closer to CD37 receptor with higher binding affinity and less aggregation of negatively charged protein, which contributed to lower tonic signaling during CAR activation and diminished exhaustion. Ultimately, effective anti-tumor control with notable memory T-cell persistence was exhibited in Burkitt lymphoma mice treated with CD37.GS4L CAR-T. Additionally, CD37.GS4L CAR-T illustrated the potential in vivo cytotoxicity in myeloma-inoculated mice. In summary, the flexibility and affinity of glycine-serine linker of CD37CAR can potentiate CAR-T functionality.