Intrinsic Functional Potential of NK-Cell Subsets Constrains Retargeting Driven by Chimeric Antigen Receptors

Area of Science:

• Immunology
• Cell Therapy
• Cancer Research

Background:

• Natural killer (NK) cells are promising allogeneic cytotoxic effector cells for chimeric antigen receptor (CAR)-mediated therapies.
• Understanding NK cell subset potential is crucial for optimizing CAR-mediated retargeting efficiency.

Purpose of the Study:

• To explore the feasibility of transfecting CAR-encoding mRNA into primary NK cells.
• To investigate how intrinsic NK cell subset properties affect CAR retargeting efficiency.

Main Methods:

• Screened five anti-CD19 CAR constructs, selecting a third-generation CAR with a removed CH2-domain.
• Optimized CAR expression via IL15 stimulation prior to mRNA transfection.
• Assessed CAR-engineered NK cell degranulation and activation thresholds in response to CD19+ and CD19- targets.

Main Results:

• Achieved over 80% CAR expression in NK cells after 3 days of IL15 stimulation and transfection.
• CAR-engineered NK cells showed enhanced degranulation toward CD19+ targets while retaining responses to CD19- targets.
• NK cell subset responses to CAR targets depended on intrinsic activation thresholds influenced by KIR and CD94/NKG2A interactions.
• Adaptive NK cells demonstrated superior killing of CD19+, HLA low, or mismatched tumor cells.

Conclusions:

• Primary allogeneic NK cells are feasible for CAR engineering.
• NK cell diversity and intrinsic properties must be considered to optimize CAR-NK cell therapy efficacy in cancer immunotherapy.