The Development of a Modified Human IFN-α2b Linked to the Fc Portion of Human IgG1 as a Novel Potential Therapeutic for the Treatment of Hepatitis C Virus Infection

Abstract

Interferon-alpha (IFN-alpha), in conjunction with ribavirin, is the current standard for the treatment of chronic hepatitis C virus (HCV) infection. This treatment requires frequent dosing, with a significant risk of the development of anti-IFN-alpha neutralizing antibodies that correlates with lack of efficacy or relapse. We have developed an IFN-alpha linked to the Fc region of human IgG1 for improved half-life and less frequent dosing. We have also identified, using a human T cell proliferation assay, three regions of IFN-alpha2b that are potentially immunogenic, and a variant containing a total of six mutations within these regions was made. This variant was made as a fusion to Fc either with or without a flexible linker between the fusion partners. Both configurations of the variant were less active than native IFN-alpha alone, although the variant containing the flexible linker had in vitro antiviral activity within the range of other modified IFN-alphas currently in clinical use. Peptides spanning the modified regions were tested in T cell proliferation assays and found to be less immunogenic than native controls when using peripheral blood mononuclear cells (PBMCs) from both healthy individuals and HCV-infected patients who had been treated previously with IFN-alpha2b.

CEA/CD3 bispecific antibody MEDI-565/AMG 211 activation of T cells and subsequent killing of human tumors is independent of mutations commonly found in colorectal adenocarcinomas

“…63 Briefly, a humanized CEACAM5-specific scFv fragment of A5B7 65 was fused via a short peptide linker to a de-immunized human CD3-specific scFv 66 derived from the murine monoclonal antibody L2K 67 in the orientation anti-human CEACAM5 VH-VL-glycine/serine linker-anti-human CD3 VH-VL. MEDI-565 was selected from a panel of these BiTE Ò antibody constructs, produced in CHO cells and affinity purified via a 6X histidine tag at the C-terminal end of the antibody.…”

A deimmunised form of the ribotoxin, α-sarcin, lacking CD4+T cell epitopes and its use as an immunotoxin warhead

“…Peptides were dissolved in DMSO to a concentration of 10 mM and peptide culture stocks prepared by diluting into AIM-V ® culture medium (Gibco, Paisley, UK) to a final concentration of 5 µM. Peptide assays were performed as previously described (Jones et al ., 2004) using a cohort of 52 donor PBMC. Briefly, for each peptide and each donor, sextuplicate cultures were established in a flat bottomed 96-well plate.…”

“…To this end, we sought to deimmunise α-sarcin using a preclinical ex vivo human T cell assay (EpiScreen TM ) (Jones et al ., 2004) to identify linear T cell epitopes present within the α-sarcin sequence. Overlapping peptides spanning the entire sequence were tested for the capacity to induce T cell proliferation against a cohort of community blood donors carefully selected based on expression of a broad range of major histocompatibility complex (MHC) class II haplotypes.…”

Elimination of N-glycosylation by site mutation further prolongs the half-life of IFN-α/Fc fusion proteins expressed in Pichia pastoris

“…IFN-α is commonly used to treat chronic hepatitis B and C virus (HBV and HCV) infections and certain cancers [2, 3]. The conventional IFN-α with a small molecular size of approximately 18 kDa has a short half-life of approximately 2–3 h because of its high sensitivity to serum proteolytic enzymes and rapid clearance by the kidneys [4].…”

“…Unfortunately, Albuferon has not yet been approved because of safety concerns at high doses [5]. Several forms of human IgG Fc-fused IFN-α fusion proteins expressed in mammalian cell lines have produced promising results in preclinical studies [2, 5, 6]. In fact, more than ten therapeutic Fc fusion proteins have been approved for clinical use by the FDA, and some of these proteins have achieved great success on the market, such as Enbrel (TNFR-Fc) [7].…”