Abstract 1250: Fibronectin domains engineered for specificity to individual murine Fc gamma receptors modulate tumoricidal activity of immune cells.

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Abstract Activating and inhibitory Fc gamma receptors (FcγRs) can play a large role in the efficacy of certain antibody therapeutics. The Fc regions of the antibody bind to the FcγRs expressed on immune cells and modulate responses against antibody-coated tumor cells. Previous studies with FcγR knock-out mice have shown that antibody-mediated tumor clearance is improved when activating FcγRs are bound preferentially over inhibitory FcγRs. The ectodomains of FcγRs are highly homologous, making it challenging to engineer Fc regions to specifically bind individual FcγRs with high affinity and lack binding to the other FcγRs. To address this challenge, we engineered the human tenth type III fibronectin (Fn3) domain scaffold to bind individual murine FcγRs at epitopes that are distinct from the Fc binding region for testing in a mouse model. The most established pre-clinical models of cancer are mouse models; therefore studying the effect of modulating individual murine FcγRs should provide insight into engaging specific FcγRs as an anti-cancer therapeutic for humans. Using directed evolution with yeast surface display, we isolated Fn3 clones specific for murine FcγRI, FcγRII, FcγRIII, and FcγRIV through a combination of depletion, enrichment, and mutagenesis steps. Cell titrations measured with flow cytometry confirmed the binding specificity of the Fn3s. Binding epitopes were determined by fluorescence activated cell sorting of a single-point mutant yeast library of FcγR and competition studies. To confirm the biological activity of the Fn3 clones, a tumor-targeting single-chain variable fragment (scFv) was fused to each Fn3 clone. Each scFv-Fn3 construct was antigen specific and bound specifically to the FcγR that it was designed to target. The murine colon carcinoma cell line MC38 stably transfected with carcinoembryonic antigen (CEA) was used for our studies. Tumor cells were pre-incubated with scFv-Fn3 constructs targeting CEA and then combined with thioglycollate- induced peritoneal macrophages pre-treated with interferon gamma. The phagocytic activity of the macrophages was measured using flow cytometry. Pre-incubation with scFv-Fn3 constructs specific for FcγRI or FcγRIV significantly increased phagocytosis compared with pre-incubations with the control scFv-Fn3 construct. Pre-incubation with a combination of both the FcγRI- and FcγRIV-specific constructs resulted in a phagocytosis level close to that of murine IgG2a, which can interact with all of the FcγRs. These Fn3 tools will allow us to conduct future studies on the immune response that is generated by triggering individual FcγRs in other in vitro assays and in vivo models of cancer. This work was supported by a Sanofi Aventis Biomedical Innovation Award and the NIH/NIGMS Biotechnology Training Program. Citation Format: Tiffany F. Chen, Kevin Li, K. Dane Wittrup. Fibronectin domains engineered for specificity to individual murine Fc gamma receptors modulate tumoricidal activity of immune cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1250. doi:10.1158/1538-7445.AM2013-1250