Fine-Tuning Signal Strength in CD5 CAR-NK Cells for Targeted T Cell Cancer Therapy

Seona Jo^1,2Yu Bin Lee^3,4Seok Min Kim¹Soo Yun Lee¹Myeongjin Choi^3,4Mi-Lang Kyun³Seo Yule Jeong³Sunyoung Lee¹Ji Hyun Kim¹Yoonji Kim⁵Yu Jung Kim⁵Sora Park⁵Kyoung- Sik Moon^3,4Tae-Don Kim^1,2*

• ¹Center for Gene and Cell Therapy, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea
• ²KRIBB School of Advanced Bioconvergence, University of Science and Technology (UST), Daejeon, Republic of Korea
• ³Center for Global Biopharmaceutical Research, Korea Institute of Toxicology (KIT), Daejeon, Republic of Korea
• ⁴Human and Environmental Toxicology, University of Science and Technology (UST), Daejeon, Republic of Korea
• ⁵New Drug Development Center, Osong Medical Innovation Foundation, Cheongju-si, Republic of Korea

T cell hematological malignancies are aggressive blood cancers that remain challenging despite various treatments. Current chimeric antigen receptor (CAR)-T and natural killer (NK) therapies show potential but struggle with nonselective elimination during tumor targeting. This study aims to develop a safe and effective therapy targeting T cell malignancies using CD5 CAR-NK cells. The strength of CAR signaling, determined by the single-chain variable fragment (scFv) and CAR expression level, enables selective antigen recognition while protecting normal T cells. We aimed at optimizing CAR-NK (OptiCAR-NK) to enhance anti-cancer effects against CD5+ tumors and mitigate on-target off-tumor toxicity in vitro and in vivo. By modulating scFv and CAR expression levels through single-cell isolation and mRNA transfection, we identified variations in the activity of CAR-NK cells against CD5+ cells. In vivo studies using xenograft and humanized mouse models confirmed the selective anti-tumor effects and safety of OptiCAR-NK cells. OptiCAR-NK with optimal scFv and CAR expression showed strong anti-tumor activity with minimal toxicity to normal cells. We identified that fine-tuning CAR is important to harnessing NK cells' innate discriminatory ability, activated by endogenous ligands on target cells. Optimized modulation of scFv and CAR expression is crucial for designing a CAR that achieves high anti-cancer efficacy and is safe in normal cells. Our results suggest a promising avenue for optimized CD5 CAR-NK cell therapy to manage T cell malignancies while minimizing off-tumor effects.