Fine-Tuned Anti-CD19 CAR-T Cells to Improve Anti-Tumor Activity By Smash Platform

Chimeric Antigen Receptor T-cell (CAR-T) therapy has emerged as a groundbreaking advancement in the field of immunotherapy, offering new hope for patients with certain types of hematologic malignancies such as B-cell lymphomas and acute lymphoblastic leukemia. CAR-T therapy has demonstrated significant clinical efficacy, leading to remarkable remission rates in patients with otherwise refractory cancers. Despite its promising outcomes, CAR-T therapy faces several challenges that need further improvements. Current CAR designs can result in severe adverse effects such as cytokine release syndrome and neurotoxicity, and they often lack efficacy against solid tumors due to the immunosuppressive tumor microenvironment and antigen escape.

One promising biological approach for improving CAR-T therapy involves optimizing the binding kinetics of the antigen-binding domain (ABD) in CAR constructs. By fine-tuning the binding affinity and association/dissociation rates (K_on/K_off) of ABDs to target antigens, several studies have been conducted to improve the specificity and efficacy of CAR-T cells while reducing off-target effects and toxicity.

Single-molecule Protein Interaction Detection (SPID) technology developed by PROTEINA can provide the rapid and precise protein-protein interaction (PPI) data through analyzing high resolution single-molecule image. Here, we demonstrate Single-Molecule Antibody Screening with High-throughput imaging system (SMASH) platform utilizing SPID technology that not only compiles hit finding and validation processes, but also provides ultra-sensitive binding kinetics using just 10 pg of crude antibody.

We selected single chain variable region (ScFv) of anti-CD19 (clone FMC63) to develop a fine-tuned novel CAR variant using SMASH platform. In this study, we assessed the binding affinity (K_D) of a site-mutagenesis library consisting of 760 variants and identified 6 novel key residues that were not previously reported in the literature, which may modulate binding. To investigate whether K_D value affects functional activity, we generated CD19 CAR-Jurkat stable cell (CAR-J) using a lentiviral system. CAR-J tended to show an increase in CD69 activation marker levels as binding affinity inreased when stimulated with CD19-expressing cells, specifically the Raji B and TMD8 cell lines. Next, to understand the effect of binding kinetics on anti-tumor activity, we stratified variants with similar K_D values. Candidates that exhibited significant changes in binding kinetics were selected, and CAR-T cells were generated using PBMCs through the lentivirus system. The cytotoxic activity, and the resulting cytokine release effects of CAR-T cells appeared to be enhanced at lower K_D values, while a fast dissociation rate was crucial for determining their functional activity.

Although further research on off-target effects is required, this study demonstrates that binding kinetics are closely associated with the improvement of CAR-T's anti-tumor activity. Thus, our SMASH platform serves as a novel and attractive tool that not only constructs ABD-mutated libraries to rapidly screen the binding affinity and kinetics of interested CARs, but also provides optimal CAR candidates to enhance anti-tumor activity.