Single-chain variable fragment affinity tuning can optimize anti-AML CAR-NK cell functionality.

Area of Science:

• Immunology
• Cell Therapy
• Oncology

Background:

• Natural Killer (NK) cells possess inherent anticancer properties.
• Chimeric antigen receptor (CAR) engineering redirects NK cell activity against cancer, including acute myeloid leukemia (AML).
• The impact of CAR binding affinity and epitope targeting on CAR-NK cell function requires further investigation.

Purpose of the Study:

• To investigate the functional consequences of varying CAR binding affinities and targeted epitopes on CAR-NK cell activation.
• To analyze CAR-NK cell cytolytic synapse formation and antitumor efficacy against AML.
• To understand how CAR affinity influences CAR-NK cell responses in different timeframes and models.

Main Methods:

• Generated NK-92 and primary NK cells expressing AML-specific CARs (26292 or 7G3 targeting CD123) with variant affinities.
• Conducted in vitro binding, activation, and cytotoxicity assays.
• Utilized high-resolution imaging and mouse xenograft models for in vivo evaluation.

Main Results:

• CARs were highly expressed and mediated antigen-specific activation.
• High-affinity CAR-NK cells showed rapid AML cell killing, while low-affinity CAR-NK cells demonstrated superior antigen density discrimination and sustained cytotoxicity.
• Low-affinity CAR-NK cells exhibited enhanced expansion in vivo.

Conclusions:

• CAR-NK cell expression with varying CD123 binding affinities induces antigen-specific activation and cytotoxicity against AML.
• Affinity-dependent functional differences are time-course and scFv/epitope specific.
• Low-affinity CARs may offer advantages for sustained antitumor activity and in vivo expansion.