Short peptide type I interferon mimetics: therapeutics for experimental allergic encephalomyelitis, melanoma, and viral infections.

The classical canonical model of interferon (IFN) signaling focuses solely on the activation of STAT transcription factors, which limits the model in terms of specific gene activation, associated epigenetic events, and IFN mimetic development. Accordingly, we have developed a noncanonical model of IFN signaling and report the development of short type I IFN peptide mimetic peptides based on the model. The mimetics, human IFNα1(152-189), human IFNß(150-187), and ovine IFNτ(156-195) are derived from the C-terminus of the parent IFNs and function intracellularly based on the noncanonical model. Vaccinia virus produces a decoy IFN receptor (B18R) that inhibits type I IFN, but the IFN mimetics bypass B18R for effective antiviral activity. By contrast, both parent IFNs and mimetics inhibited vesicular stomatitis virus. The mimetics also possessed anti-tumor activity against murine melanoma B16 tumor cells in culture and in mice, including synergizing with suppressor of cytokine signaling 1 antagonist. Finally, the mimetics were potent therapeutics against experimental allergic encephalomyelitis, a mouse model of multiple sclerosis. The mimetics lack toxic side effects of the parent IFNs and, thus, are a potent therapeutic replacement of IFNs as therapeutics.

Biological activities of synthetic peptides of the sequence of human interferon-alpha.

Theoretical and experimental data were obtained indicating a possible role of the amino acid sequence 69-80 of HuIFN-alpha for antiviral activity of interferon. In this paper, synthetic peptides (penta-, octa- and dodecapeptides), representing the sequence region of positions 69-80 of the primary structure of HuIFN-alpha-2 were investigated with regard to their ability to inhibit the cytopathic effect of VSV on MDBK cells and on embryonic human fibroblasts. The peptides themselves exhibit only a very weak antiviral activity; however, it was found that these peptides even increase the antiviral activity of HuIFN-alpha-2.

[Synthesis and biological activity of peptides from interferon alpha A region 125-131]. / Sintez i biologicheskaia aktivnost' peptidov raiona 125-131 interferona alpha A.

Two peptides, IFN-(125-129) (RITLY-I) and [Arg7]IFN-(125-131) (RITLYLR-II), belonging to the putative immunologically active region of interferon alpha A (IFN) were synthesised by the solid-phase method. Both peptides suppress the delayed-type hypersensitivity reaction in vivo as assayed in mice. The peptide (II) either suppresses (0.01-0.1 mg/kg) or stimulates (approximately 1.0 mg/kg) antibody production in mice in response to sheep red blood cells.

A crystalline synthetic peptide representing the epitope of a monoclonal antibody raised against synthetic interferon-alpha 1 fragment 111-166.

The antigenic determinant recognized by the monoclonal antibody that had been raised against synthetic human interferon-alpha 1 (IFN-alpha 1) fragment 111-166 [Arnheiter, H., Thomas, R.M., Leist, T., Fountoulakis, M., and Gutte, B. (1981) Nature (Lond.) 294, 278-280] and that cross-reacted with human IFN-alpha 1, IFN-alpha 2, and IFN-alpha A made in Escherichia coli, was localized to the region between residues 151 and 166 using synthetic COOH-terminal interferon fragments. In solid-phase radioimmunoassays neither the strongly hydrophilic COOH-terminal nonapeptide IFN 158-166 nor its mixtures with IFN 151-162 or IFN 149-158 showed any measurable interaction with the antigen binding site of the monoclonal antibody. For antibody binding, the full covalent structure of IFN 151-166 was required. Quantitatively very similar results were obtained with IFN 149-166 and IFN 143-166. The synthetic COOH-terminal hexadecapeptide of human IFN-alpha 1 (IFN 151-166) could be crystallized.

Antibodies to synthetic peptides of human interferon-beta. Use in biosynthetic studies.

Two peptides from the amino terminus of human interferon-beta were synthesized corresponding to amino acids 1-21 and 18-45. The peptides were conjugated to bovine serum albumin, and rabbits were immunized with either the (1-21)- or the (18-45)-peptide conjugate. Antibodies to the synthetic peptides were detected in the sera using a radioimmunoassay with 125I-labeled peptide. Two of the antisera, one against peptide 1-21 and one against peptide 18-45, immunoprecipitated [35S]interferon-beta. The former was used to study the biosynthesis of interferon-beta in human diploid fibroblasts. In cells induced with double-stranded RNA (poly(I:C] to synthesize interferon-beta, two intracellular proteins with estimated molecular weights of 23,000 and 18,000 were precipitated with the antiserum. Three exocellular proteins from the same induced cells were precipitated with molecular weights of 23,000, 18,000, and 10,000. Reduced amounts of the intra- and exocellular Mr = 23,000 component and enhanced amounts of the Mr = 18,000 component were observed when induced cells were treated with the glycosylation inhibitor tunicamycin. Neither the antibody to peptide 1-21, the antibody to peptide 18-45, nor a combination of both antibodies neutralized the interferon-beta antiviral activity. We conclude that the amino terminus of interferon-beta may not be involved in the binding of interferon-beta to its receptor.

Synthesis and physicochemical characterization of major fragments of human leucocyte interferon alpha 1.

Four oligopeptides corresponding to overlapping regions of the human leucocyte interferon alpha 1 (IFN) amino acid sequence have been synthesized by the solid-phase method. The physicochemical properties of these peptides were investigated by analytical ultracentrifugation and circular dichroism, absorbance, and fluorescence spectroscopies. IFNs 24-81 and 111-166 were shown to possess considerable ordered structure in solution. IFN 111-166 appeared to adopt a beta-stranded conformation, which could be reversibly unfolded by the addition of urea. Equimolar mixtures of IFNs 24-81 and 111-166 formed a noncovalent complex that was resistant to the formation of the Cys29-Cys139 disulfide bond found in native interferon alpha 1. Predictions of the secondary structure of interferon alpha 1 were made and the results compared to the measured properties of the fragments. In general, it was found that the prediction methods favored a highly alpha-helical conformation.

Chemical synthesis of a human interferon-alpha 2 gene and its expression in Escherichia coli.

A 511-base pair DNA fragment encoding human interferon-alpha 2 has been chemically synthesised and expressed from a lac UV5 and a synthetic trp promoter in Escherichia coli. The synthesis involved preparation of 68 oligodeoxyribonucleotides and their enzymic ligation. The product expressed from the trp promoter system had high antiviral activity and displayed biological effects similar to those of Namalwa interferon on natural killer cell activity and in a Daudi cell growth inhibition assay. E.coli minicells containing plasmid DNA with the synthetic IFN-alpha 2 gene under trp promoter control produce a protein with the same electrophoretic mobility as a sample of authentic IFN-alpha 2. The protein from E.coli cross-reacts with the monoclonal antibody NK-2 and was readily purified, close to homogeneity, by immunoadsorption chromatography on NK-2 sepharose.