414 Leveraging modular TriKE-PACC molecules capable of dual-antigen targeting to enhance NK-cell immunotherapy in AML.

Objectives/Goals: Acute myeloid leukemia (AML) is an aggressive blood cancer that hides from the immune system, which led to ~140,000 deaths worldwide in 2024. AML often recurs or resists therapy; therefore, our goal is to develop and test TriKE-PACCs, which combine dual tumor antigen targeting with NK cell activation, to overcome antigen escape and heterogeneity in AML. Methods/Study Population: TriKE-PACC molecules were engineered by combining a TriKE [anti-CD16 single-domain antibody (sdAb), an IL-15 moiety, and an antitumor antibody domain single-chain variable fragment (scFv) or an sdAb] with a PACC arm containing a second antigen-binding scFv/sdAb linked to IL-15Rα. TriKE-PACC constructs were produced in Expi293 cells. To identify clinically relevant AML tumor targets, we analyzed the expression of tumor antigens in AML bone marrow samples obtained from 20 patients using flow cytometry. We modeled antigen escape and heterogeneity using CRISPR/Cas9-generated knockout AML lines. TriKE-PACC-mediated NK cell activity was measured via degranulation, cytokine production, and Incucyte-based cytotoxicity assays against AML cell lines and CRISPR knockout models. Results/Anticipated Results: Our examination of AML patient bone marrow samples revealed diverse antigen profiles, highlighting the need for modular TriKE-PACC designs. The dual-targeting TriKE-PACCs enhanced NK cell activation via CD107a expression and the production of IFNγ and TNFα in co-culture assays with AML cell lines. Notably, TriKE-PACCs retained activity against AML models lacking a targeted antigen, demonstrating the ability of the second tumor targeting arm to overcome antigen escape. Furthermore, NK cell-mediated killing was improved against antigen-deficient AML cells when treated with the TriKE-PACCs compared to standard TriKEs. Discussion/Significance of Impact: TriKE-PACCs offer a modular and customizable NK-engaging immunotherapy to address AML heterogeneity and resistance. Our data, collected utilizing primary patient samples, support the use of this platform for the future development of personalized, dual-targeted therapies to enhance the efficacy of AML treatments.