Engineering therapeutic proteins for immune modulation

Abstract

Antibody-dependent cell-mediated cytotoxicity (ADCC) is implicated in the efficacy, to some degree, of most anti-cancer monoclonal antibodies. This interaction is mediated through Fc gamma receptor (Fc[gamma]R) binding to the antibody Fc. Activating and inhibitory Fc[gamma]Rs are expressed on immune cells and bind to the Fc regions of IgGs, which either promote or hinder responses against the tumor cell. Since mouse models of cancer are currently the most established in this field, studying the effect of modulating individual murine Fc[gamma]Rs would provide insight into this type of therapeutic for humans. The ectodomains of murine Fc[gamma]Rs are highly homologous and therefore it has been difficult to engineer antibody Fc regions to specifically bind only one Fc[gamma]R. To address this challenge, we engineered the human tenth type III fibronectin (Fn3) domain scaffold to bind individual murine FcγRs. The Fn3 scaffold has the advantage of binding at epitopes on the Fc[gamma]R that are distinct from the Fc binding region. Fn3 clones have been isolated with specificity to each known murine Fc[gamma]R: Fc[gamma]RI, Fc[gamma]RIIB, Fc[gamma]RIII, and Fc[gamma]RIV. Measured KDs of Fn3 binding to Fc[gamma]R range on the order of 1-100nM, which fall within the range of normal Fc-Fc[gamma]R binding affinity or even higher affinity. Candidate Fn3 clones are fused to tumor antigen specific scFvs and a murine serum albumin (MSA) to maintain in vivo half-life. Each scFv- MSA-Fn3 construct was antigen specific and bound specifically to the Fc[gamma]R that it was designed to target. To confirm the biological activity of the Fn3 clones, phagocytosis assays with peritoneal macrophages were conducted. Pharmacokinetic studies have shown all scFv-MSA-Fn3 constructs to have approximate beta half-lives of 25 hours in C56BL/6 mice. Biodistribution of scFv-MSA-Fn3 constructs demonstrate preferential accumulation in antigen positive subcutaneous tumors. Multiple in vivo models were optimized to detect antitumor efficacy of our engineered constructs. In a subcutaneous tumor model with aggressive prophylactic dosing, our binders to the activating Fc[gamma]RI, Fc[gamma]RIII, and Fc[gamma]RIV demonstrate similar control to the mIgG2a antibody. These tools will allow us to conduct future studies on the immune response of triggering individual FcγR in models of cancer. The binding of human IgG1 to human Fc gamma receptors (hFcγR) is highly sensitive to the presence of a single N-linked glycosylation site at asparagine 297 (N297) of the Fc, with deglycosylation resulting in a complete loss of hFc[gamma]R binding. Thus, aglycosylated variants that can bind to hFc[gamma]Rs have the potential to allow therapeutic antibodies to be produced in virtually any expression system. Previously, we demonstrated that aglycosylated human IgG1 Fc variants are capable of engaging the human Fc gamma RII subset of the low-affinity hFc[gamma]Rs, demonstrating that N-linked glycosylation of the Fc is not a strict requirement for hFc[gamma]R engagement. In the present study, we demonstrate that aglycosylated IgG variants can be engineered to productively engage with Fc gamma RIIIA, and that these variants can also bind the human Fc gamma RII subset. In this study, we also assess the biophysical properties and serum half-life of the aglycosylated IgG variants. Phagocytosis assays with monocytes and macrophages were performed to determine which constructs optimally drove tumor cell killing. A mathematical model of phagocytosis suggests that hFc[gamma]R dimers of hFc[gamma]RI were the main drivers of phagocytosis.