Abstract 3516: TriNKETs®: trispecific immune cell engager therapies exhibit potent preclinical in vivo anti-tumor activity and broad combinability in diverse cancer models
Authors: Colin LaCroix, Adam Stockmann, Mitchell Bigelow, Cornelis van der Poel, Jolanta Dubauskaite, Jeremy Beech, Samia Khan, Eva Gutierrez, Nathan Felix, Stuart W. Hicks, Nicolai Wagtmann, Ann F Cheung
Journal: Cancer Research (2025)

Abstract The trispecific immune cell engager TriNKET® platform is designed to direct the activity of cytotoxic immune cells against cancer cells through simultaneous engagement of a tumor-associated antigen and the activating receptors CD16a and NKG2D (natural killer group 2 member D), a receptor expressed on natural killer (NK) cells, γδ T cells, and CD8+ T cells. To evaluate the preclinical efficacy of the TriNKET platform, a TriNKET targeting the murine Tyrosinase-related protein 1 (TYRP1), a melanocyte protein expressed on melanomas and normal melanocytes, was developed and evaluated using TYRP1-expressing immunocompetent syngeneic models. The TYRP1-TriNKET model system bridges the gap between in vitro studies and potential clinical activity, providing a robust system to validate the immune-based activities of TriNKETs, and their ability to be combined with standard-of-care therapies, such as checkpoint blockade or chemotherapy. The anti-tumor activity of the TYRP1-targeting TriNKET was evaluated in the B16F10 melanoma model, which endogenously expresses TYRP1, as well as in MC38 and CT26 colon tumor models that were engineered to express TYPR1. Immunological changes within the tumor microenvironment were characterized using flow cytometry. In addition, the potential for enhanced activity was explored by testing the TYRP1-TriNKET in combination with anti-PD1, Abraxane, and various chemotherapy regimens. The TYRP1-TriNKET demonstrated strong binding to TYRP1+ cell lines and robustly activated NK cells in vitro when co-cultured with tumor cells. TYRP1-TriNKET monotherapy showed superior in vivo anti-tumor efficacy compared to a TYRP1-targeting monoclonal antibody in all tumor models tested. TYRP1-TriNKET increased infiltration of NK cells and CD8+ T cells, promoted immune cell activation, and increased levels of pro-inflammatory cytokines and chemokine within the tumor microenvironment without inducing peripheral immune activation or cytokine release. Depletion of immune cells prior to treatment confirmed that the observed anti-tumor responses are mediated through both NK cells and CD8+ T cells. Furthermore, TriNKET efficacy was enhanced when combined with standard-of-care therapies, including anti-PD-1, Abraxane, cyclophosphamide, gemcitabine, and 5-FU, with no evidence of antagonism in combination treatments. TriNKETs display potent preclinical in vivo anti-tumor responses driven by NK cell and CD8+ T cell effector functions. These findings highlight the potential of TriNKETs as promising immunotherapeutic agents, demonstrating their potential both as standalone treatments and as components of combination strategies with standard-of-care therapies. Citation Format: Colin LaCroix, Adam Stockmann, Mitchell Bigelow, Cornelis van der Poel, Jolanta Dubauskaite, Jeremy Beech, Samia Khan, Eva Gutierrez, Nathan Felix, Stuart W. Hicks, Nicolai Wagtmann, Ann F Cheung. TriNKETs®: trispecific immune cell engager therapies exhibit potent preclinical in vivo anti-tumor activity and broad combinability in diverse cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3516.