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Here, we study the functional consequences of CAR binding affinity and targeted epitope on CAR-NK cell activation, cytolytic synapse formation, and antitumor activity.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>We characterized NK-92 and primary NK cell populations expressing variant affinity AML-specific CARs containing single-chain variable fragments (scFvs, 26292 or 7G3) targeting two epitopes on CD123. 26292 affinity variants were discovered through directed evolution of an error-prone mutagenic library, while 7G3 affinity variants were previously reported. The resulting CAR-NK cell panel was studied with in vitro binding, activation, and cytotoxicity studies and in mouse xenograft models.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>26292 and 7G3 CARs of variable CD123 binding affinities were highly expressed in NK cells and conferred antigen-specific activation in vitro. High-resolution imaging demonstrated greater clustering of high-affinity 7G3 CAR-NK cells and consequent AML target cell death in a short-term time lapse. Low-affinity 7G3 CAR-NK cells exhibited enhanced antigen density discrimination with greater membrane-proximal signaling, cytokine production, and cytotoxicity. In longer-term assays, low-affinity 7G3 CAR-NK cells demonstrated more sustained killing of AML cells. In vivo testing highlighted greater expansion of low-affinity 7G3 CAR-NK cells in two xenograft models.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Conclusions<\/jats:title>\n                  <jats:p>Expression of 26292 and 7G3 CARs with a range of CD123 binding affinities in NK cells leads to antigen-specific activation and cytotoxicity against AML. Affinity-based differences in functional activation and antitumor activity are dependent on time course and are scFv\/epitope specific.<\/jats:p>\n               <\/jats:sec>","DOI":"10.1136\/jitc-2024-010763","type":"journal-article","created":{"date-parts":[[2025,2,6]],"date-time":"2025-02-06T05:35:38Z","timestamp":1738820138000},"page":"e010763","update-policy":"https:\/\/doi.org\/10.1136\/crossmarkpolicy","source":"Crossref","is-referenced-by-count":17,"title":["Single-chain variable fragment affinity tuning can optimize anti-AML CAR-NK cell functionality"],"prefix":"10.1136","volume":"13","author":[{"given":"Ruyan","family":"Rahnama","sequence":"first","affiliation":[{"name":"Oncology, Johns Hopkins Medicine Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Monika","family":"Kizerwetter","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, Johns Hopkins University 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