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.
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.
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.
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.
NIH/NCATS: 1T32TR004376 and 1UM1TR004405
Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
Anders W. Matson, Caden Caligiuri, Katherine M. Koehler et al.· Journal of Immunology· 0 citations
Lung cancer is the leading cause of cancer-related death worldwide, with ∼230,000 new U.S. cases projected in 2026. Non-small cell lung cancer (NSCLC), the most common subtype, is treated with surgery, chemotherapy, radiation, targeted therapy, and immunotherapy. While checkpoint blockade therapies have improved outcomes, only ∼15% of advanced NSCLC patients achieve long-term survival, leaving most patients with limited options after relapse. This underscores the need for more effective treatments. Natural Killer (NK) cells play a key role in tumor surveillance, and higher NK cell levels correlate with better patient outcomes. To harness them, we developed Tri-specific Killer Engager (TriKE) molecules to enhance NK cell function through CD16 engagement, IL-15 signaling, and tumor antigen targeting. However, current TriKEs face limitations including rapid clearance (through the glomerulus) and restriction to single-antigen targeting. To overcome these challenges, we engineered TriKE-PACCs (Poly-Antigen Cytokine Complexes), a next-generation platform designed to extend NK cell persistence and enable dual-antigen targeting. TriKE-PACCs combine an anti-CD16 single domain antibody (sdAb), an IL-15 moiety, and an anti-mesothelin single chain variable fragment (scFv), along with a PACC module consisting of IL-15 receptor alpha linked to an anti-B7H3 sdAb. This bifunctional design enables dual targeting of mesothelin and B7H3, addressing both tumor heterogeneity and antigen escape, as seen in NSCLC. Using H322 NSCLC cells with B7-H3 and/or mesothelin CRISPR knockouts to model tumor heterogeneity and antigen loss, we evaluated NK cell degranulation (CD107a), cytokine production (IFNγ, TNFα), and cytotoxicity (Xcelligence and Incucyte live imaging assays). Treatment with TriKE-PACCs significantly enhanced NK cell degranulation, cytokine production, and tumor killing compared to single-antigen TriKEs and IL-15 alone, particularly in heterogeneous antigen settings. Next steps include validating our findings in NSCLC 3D organoid and spheroid models, as well as confirming the efficacy of TriKE-PACCs in vivo. To summarize, our findings show that TriKE-PACCs are a promising immunotherapy approach for NSCLC, with potential to overcome antigen escape, improve NK cell persistence, and enhance clinical outcomes for lung cancer patients.
Rachel N. Steinmetz, Shee Kwan Phung, Joshua T. Walker, Tumpa Dasgupta, Yvette Soignier, Philippa R. Kennedy, Martin Felices. Enhancing NK cell immunotherapy in non-small cell lung cancer using dual-antigen targeting Tri-Specific Killer Engagers (TriKEs/TriKE PACCs) [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr A021.
R. N. Steinmetz, S. Phung, Joshua T Walker et al.· Clinical Cancer Research· 0 citations
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