Abstract 1826: In vivo evaluation of NK-TICA®, a novel Bicycle® tumor-targeted immune cell agonist (TICA) designed to engage NK cells
Authors: Stanczuk Lukas , Lahdenranta Johanna , Dufort Fay J. , Leitheiser Christopher J. , Upadhyaya Punit , Brandish Philip E. , Keen Nicholas 
Published: April 2023

Abstract
Natural Killer (NK) cells are cytotoxic cells of the innate immune system with well characterized anti-tumor properties. Their ability to directly kill malignant cells and elicit an adaptive immune response makes them a promising candidate for a precision-guided immunotherapy for cancer patients.
Bicycle® peptides are small (~1.5-2 kDa), chemically synthetic, structurally constrained bicyclic peptides discovered using phage display. We have developed a Bicycle® that specifically binds to NKp46, a target expressed on NK cells which acts as a key activating receptor contributing to cytolytic function of NK cells. The NKp46-binding Bicycle® conjugated to the tumor antigen, EphA2-binding Bicycle® creates a NK tumor-targeted immune cell agonist (NK-TICA®). EphA2 is a well-characterized tumor-associated antigen overexpressed in many cancer types including prostate, lung, esophageal, urothelial, cervical, head and neck and colorectal cancers. Thus, NK-TICA® is designed to engage and activate NK cells locally in the tumor to induce tumor cell killing and stimulate the adaptive immune system.
To evaluate the effects of NK-TICA® administration in vivo, we used a human NKp46 knock-in mouse model subcutaneously engrafted with a syngeneic MC38 tumor. Characterization of NK populations in blood and tumor samples was conducted by multiplex flow cytometry combined with immunohistochemistry. Plasma samples were collected at various timepoints to measure a number of relevant cytokines.
All tested doses of NK-TICA® were tolerated well with no record of body weight loss or clinical signs. A single dose of NK-TICA® led to a significant upregulation of activation markers in the NK cells isolated from blood and tumor tissue with no increase over baseline in plasma levels of proinflammatory cytokines. We show that NK cell activation in the tumor is dependent on binding of the TICA® to a tumor antigen highlighting the importance of the precision-guided approach.
In vivo proof-of-concept data shown here, in combination with extensive in vitro preclinical characterization of NK-TICA® reported elsewhere, validate this novel class of NK cell engagers as a prospective therapeutic modality.
These studies were approved by the Institutional Animal Care and Use Committee (IACUC). The care and use of animals was conducted in accordance with regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).
Citation Format: Lukas Stanczuk, Johanna Lahdenranta, Fay J. Dufort, Christopher J. Leitheiser, Punit Upadhyaya, Philip E. Brandish, Nicholas Keen. In vivo evaluation of NK-TICA®, a novel Bicycle® tumor-targeted immune cell agonist (TICA) designed to engage NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1826.