Study on the development of novel nonlytic human Fc and its application

Abstract

Although extracellular signals such as hormones, cytokines, and growth factors are of therapeutic interest in treating various diseases, they have inherent shortcomings

one such weakness is the short circulating half-lives of these therapeutic agents. Thus, extending the longevity of therapeutic agents by increasing their half-lives has become highly desirable, carrying benefits such as reduced frequency of administration. However, although currently developed methods such as PEGylation, hyperglycosylation, CTP-, albumin- or Fc-fusion efficiently improved in vivo potency, they still has drawbacks such as decreased in vitro bioactivity of target proteins. In the case of IgG1 Fc, different with other strategies, because the region involving in in vitro bioactivity (hinge region) is differed from that for FcRn-mediated recycling (CH3 region), IgG1 Fc is able to be refined for better in vitro bioactivity with retaining FcRn-binding ability. However, IgG1 Fc induces Fc-mediated effector functions that potentially detrimental to the host. As a solution to the aforementioned problem, the first part of this study proposes the use of IgD and IgG4 hybridization, creating a natural nonimmunogenic, noncytolytic and flexible human Fc (hyFc). To gauge its level of efficacy, an erythropoietin (EPO)-hyFc conjugate was prepared and its function was analyzed. Despite low amino acid homology (20.5%) between IgD Fc and IgG4 Fc, EPO-hyFc retained a stable “Y-shaped” structure and repeated intravenous administrations of EPO-hyFc into monkeys did not generate EPO-hyFc-specific antibody responses. Importantly, EPO-hyFc exhibited better in vitro and in vivo bioactivity in rats than EPO-IgG1 Fc, presumably owing to high hinge flexibility of IgD. Furthermore, the mean serum half-life of EPO-hyFc(H), a high sialic acid content form of EPO-hyFc, was approximately 2-fold longer than that of darbepoetin alfa, a heavily glycosylated EPO, in rats. Subcutaneous injection of EPO-hyFc(H) also induced a significantly greater elevation of serum hemoglobin levels than darbepoetin alfa in both normal rats and cisplatin-induced anemic rats in addition to displaying a delayed time to maximal serum level. Taken together, hyFc may be a more attractive Fc conjugate for agonistic proteins/peptides than IgG1 Fc due to its ability to elongate half-lives without inducing host effector functions and hindering the bioactivity of fused molecules. Additionally, a head-to-head comparison demonstrated that hyFc-fusion strategy more effectively improved the in vivo bioactivity of EPO than the traditional hyperglycosylation approach. In conclusion, the hyFc fusion strategy is the first trial for extending a serum half-life without a detriment of in vitro bioactivity followed by better in vivo potency than previous methods such as IgG1 Fc fusion and hyperglycosylation. Despite the effectiveness of influenza vaccine in protecting against homologous influenza infection, it is unable to provide cross-protective efficacy. As such, if a new influenza pandemic was to occur, creating a new vaccine would involve identifying the amino sequences of the pandemic virus and a six month time span in order to produce sizable stock of vaccine. An attractive approach to overcome these limitations found in conventional influenza vaccines is the incorporation of adjuvants. In the second part of this study, we find another application for nonlytic Fc and examine the role of nonlytic Fc-fused IL-7 as an adaptive adjuvant for influenza vaccine. We firstly demonstrate that recombinant IL-7 protein significantly increased influenza vaccine-induced IgG titers, which were bolstered further by mouse nonlytic Fc fusion to IL-7 protein (IL-7-mFc). Compared to squalene emulsion, a clinically available adjuvant for influenza vaccine, IL-7-mFc protein induced 3-fold higher IgG responses as well. Interestingly, a one-tenth dose of influenza vaccine co-delivered with IL-7-mFc exhibited higher IgG titers than a single dose of influenza vaccine alone in mice with this result being confirmed in monkeys using IL-7 fused to hyFc as well. In addition, co-delivery of IL-7-mFc provided superior protection (88 % vs. 13 % by influenza vaccine alone) against lethal challenge with an antigenically distant PR8 influenza virus due to the induction of cross-reactive IgG responses. IL-7-mFc increased not only the number of vaccine-induced CD4+ cells but also ICOS expression and secretion of cytokines including, IL-4 and IL-17. Interestingly, IL-7-mFc markedly compensated for any deficit in IFN-γ, IL-4 or IL-17-mediated signaling as well as blockade of ICOS for induction of IgG responses

presumably this occurs due to the pleiotropic effect of IL-7 despite the capacity of IL-7-mFc for complementing the defect of CD40L being relatively weak. In conclusion, nonlytic Fc-fused IL-7 protein holds promise as a novel adaptive adjuvant for enhancing humoral immunity, serving both to overcome limited influenza vaccine supply and to induce protective efficacy against future pandemics of influenza viruses with unmatched sequences of HA protein and other highly variable viruses. From these two studies, it is possible to conclude that fusion of agonistic molecules, such as EPO and IL-7, with nonlytic Fc may be a prominent platform for significantly potentiating the activity of fused molecules and applying them to a variety of applications.