The tumor specific activation of natural killer (NK) cells with Bicycles is an area of active investigation in immune oncology. NK cells are highly responsive immune cells that can detect and eliminate tumor cells and bridge innate to adaptive immune responses. Bicycles are small (ca.1.5kDa), chemically synthetic, structurally constrained peptides discovered via phage display and optimized using structure-driven design and medicinal chemistry approaches. We have applied the Bicycle platform technology to discover and evaluate a new class of fully synthetic molecules termed NK tumor immune cell agonists (NK-TICA®). The NK-TICA® consists of chemically coupled Bicycles® that bind specifically to the key activating receptor, NKp46, and to tumor antigens, that results in highly potent, antigen-dependent receptor activation and NK cell activation. We demonstrate potent, selective binding of our Bicycles to receptor-expressing cells and the capability of the bifunctional molecule to induce NK cell function in vitro. With Bicycle’s novel NK-TICA® compound, we demonstrate the engagement of NK cells, the specific activation and function of NK cells, and enhanced tumor cytotoxicity in a tumor target- and dose-dependent manner. In conclusion, NK-TICAs drive NK cell-mediated tumor cell killing and cytokine production in vitro and as such have the potential to catalyze the development of durable anti-tumor immunity in tumor types not well served by current therapies. We hypothesize that utilization of Bicycle NK-TICA® as a multifunctional immune cell engager will promote the modulation of NK cells, as well as the infiltration and anti-tumor activity of NK cells in solid tumors. Citation Format: Fay Dufort, Christopher J. Leitheiser, Kathleen Ho, Tucker Ezell, Alexandra Rezvaya, Peter Brown, Liuhong Chen, Philip Brandish, Kevin McDonnell, Michael Skynner, Nicholas Keen. Modulation of natural killer cell immune response to tumor with novel synthetic tumor -immune cell agonist, NK-TICA(r) [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 1806.

Background Natural killer (NK) cells are immune cells that can detect and eliminate tumor cells and bridge innate to adaptive immune responses. Bicycles are small (ca. 1.5 kDa), chemically synthetic, structurally constrained peptides discovered via phage display and optimized using structure-driven design and medicinal chemistry approaches. We have now applied this technology to identify Bicycles that bind specifically to a key activating receptor, NKp46. We term this new class of fully synthetic molecules Natural Killer tumor-targeted immune cell agonists (NK-TICA™) and herein we will describe their in vitro evaluation. Methods Using our unique phage display screening platform, we have identified high affinity, selective binders to NKp46. By conjugating the Bicycle® NK cell-engaging binders to model tumor antigen EphA2-binding Bicycle®, we have developed a bifunctional Bicycle® NK-TICA™ molecule. In in vitro functional assays, we evaluated the ability of the Bicycle® NK-TICA™ to induce NK cell activation and proliferation as well as cell-mediated cytotoxicity and cytokine production in NK-tumor co-culture assays. We similarly evaluated changes in EphA2 tumor binding affinity of the Bicycle® NK-TICAs in their ability to enhance NK immune response. Results We have developed novel modular compounds with high affinity and selectivity to NK cell receptors with specific tumor targeting capability. We demonstrate potent, selective binding of our Bicycles to receptor-expressing cells and the capability of the bifunctional molecule to induce NK cell function. With Bicycle's novel NK-TICA™ compounds, we demonstrate engagement of NK cells, specific activation and function of NK cells, and enhanced tumor cytotoxicity across multiple tumor antigens, in a dose dependent manner. With changes in tumor binding affinity, we found changes in the activation of the NK cells in vitro. Conclusions We have shown that when chemically coupled to a tumor antigen binding Bicycle®, the result is a bifunctional molecule (NK-TICA™) capable of directing potent and specific NK cell activation and kill in vitro.

Background The use of humanized animal models in preclinical evaluation of immunotherapeutics is often dictated by lack of homology between mouse and human target or divergence in the biology of studied immune cell subtypes. A target expressed on NK cells, NKp46 is a key activating receptor contributing to cytolytic function of NK cells. Bicycle® peptides are small (~1.5 kDa), chemically synthetic, structurally constrained bicyclic peptides discovered using phage display. NKp46-binding Bicycles conjugated to a tumor antigen-binding Bicycle® directed human NK cells to kill the tumor cells expressing the target antigen, and we term these molecules NK tumor-targeted immune cell agonists (NK-TICATM). Due to low homology of NKp46 ectodomain between mouse and human (~63% by NCBI BLAST), we evaluated different approaches to animal humanization to establish the most suitable model for in vivo evaluation of NK-TICATM. The main selection criteria included the number of circulating NKp46+ human NK cells as well as expression of activating and inhibitory receptors. Methods Distinct approaches to animal humanization were employed: 1) reconstitution of human immune system (HIS) with CD34+ hematopoietic stem cells (HSCs) in NCG mice with hIL-15 hydrodynamic injection (HDI); 2) reconstitution of HIS with CD34+ HSCs in NCG.hIL-15 transgenic mice; 3) infusion of human PBMC-derived NK cells in NCG.hIL-15 transgenic mice. Characterization of NK populations in blood was conducted by multiplex flow cytometry. Results Immunophenotyping analysis revealed striking differences in numbers of circulating NKp46+ NK cells between different humanized models. The lowest number was observed in mice engrafted with HSCs with transient expression of hIL-15 and the highest in mice infused with PBMC-derived NK cells in a transgenic model constitutively expressing hIL-15. We also observed expansion of NK cells over time in mice with transgenic expression of hIL-15 but not in mice treated with single hIL-15 HDI. Despite these differences, the proportion of NKp46+ NK cells expressing activation receptor NKG2D or activation marker CD69 was similar between all three study designs. In contrast, expression of NKp30 and NKG2A was different across tested models. Conclusions Transgenic mice with constitutive expression of hIL-15 showed the highest numbers of NKp46+ NK cells in blood and a better expression profile of NK activating and inhibitory receptors making it a more suitable model for in vivo evaluation of NK-TICAsTM. Ethics Approval 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).

Natural killer (NK) cells are immune cells that can detect and eliminate tumor cells and bridge innate to adaptive immune responses. Tumor specific activation of NK cells is thus an area of active investigation in immune oncology, but to date has relied on complex biologic modalities (e.g., antibodies, fusion proteins, or cell therapies), each of which has inherent disadvantages in this application. Thus, alternative approaches are warranted. Bicycle® are small (ca. 1.5 kDa), chemically synthetic, structurally constrained peptides discovered via phage display and optimized using structure-driven design and medicinal chemistry approaches. We have now applied this technology to identify Bicycles that bind specifically to the key activating receptors, NKp46 and CD16a. When chemically coupled to tumor antigen binding Bicycles this results in highly potent, antigen-dependent receptor activation and NK cell activation. We term this new class of fully synthetic molecules Bicycle® natural killer- tumor-targeted immune cell agonists (NK-TICAs™) and we will describe their discovery and evaluation in this presentation.Using our unique phage display screening platform, we have identified high affinity, selective binders to NKp46 and CD16a. By conjugating the Bicycle® NK cell-engaging binders to a model tumor antigen EphA2-binding Bicycle®, we have developed a bifunctional Bicycle NK-TICA™ molecule. In in vitro functional assays, we evaluated the ability of the Bicycle NK-TICAs™ to induce NK cell activation as well as cell-mediated cytotoxicity and cytokine production in NK-tumor co-culture assays.We have developed a novel modular compound with high affinity and selectivity to NK cell receptors with specific tumor targeting capability. We demonstrate potent, selective binding of our Bicycles to receptor-expressing cells and the capability of the bifunctional molecule to induce NK cell function. With Bicycle's novel NK-TICA™ compound, we demonstrate engagement of NK cells, specific activation and function of NK cells, and enhanced EphA2-expressing tumor cytotoxicity, in a dose dependent manner.Bicycle NK-TICAs™ are novel therapeutic agents capable of enhancing the landscape of immune oncology. We hypothesize that utilization of Bicycle NK-TICA™ as a multifunctional immune cell engager will promote modulation of NK cells, and infiltration and anti-tumor activity of NK cells in solid tumors. The data presented here provide initial proof of concept for application of the Bicycle technology to drive NK cell-mediated tumor immunity.