Leveraging modular TriKE-PACC molecules capable of dual-antigen targeting to enhance NK-cell immunotherapy in AML 2257266

Abstract Introduction Natural Killer (NK) cells are potent effectors of the innate immune system that kill tumor cells via natural cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC). In acute myeloid leukemia (AML), tumor heterogeneity and antigen escape challenge current therapies, resulting in a 5-year survival rate of < 40%. To overcome these barriers and build on our ongoing clinical trial of a CD33-targeted Tri-Specific Killer Engager (TriKE), we engineered TriKEs with Poly Antigen Cytokine Complexes (TriKE-PACCs), which combine dual AML-specific targeting with NK activation and persistence. Methods TriKE and TriKE-PACC constructs were expressed in Expi293 cells and purified by affinity chromatography. TriKE-PACCs consist of an anti-CD16 sdAb, an IL-15 moiety, and an anti-AML scFv or sdAb with a PACC arm containing IL-15Rα linked to an additional anti-AML scFv or sdAb. AML antigens were evaluated on cell lines and 20 primary samples by flow cytometry. Antigen escape was modeled using CRISPR knockout AML lines or primary patient samples. NK activation (CD107a, IFNγ, TNFα) and cytotoxicity were measured using flow cytometry and Incucyte assays. Results Our examination of AML patient samples revealed diverse antigen profiles, highlighting the need for modular TriKE-PACC designs. The TriKE-PACCs enhanced CD107a, IFNγ, and TNFα production and exhibited improved cytotoxicity compared to single-antigen TriKEs, demonstrating resistance to antigen escape. Conclusion TriKE-PACCs offer a customizable, modular approach to treating AML heterogeneity and antigen escape with NK cell-based immunotherapies. By combining IL-15 and CD16 signaling with dual tumor-antigen targeting, we enhance NK cell proliferation and antitumor activity compared to TriKEs or IL-15 therapies alone. These data support further preclinical development and testing of modular and personalized AML immunotherapies to improve upon our current CD33 TriKE clinical trial. Funding Source NIH/NCATS: 1T32TR004376 and 1UM1TR004405 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)