Novel Modified β-Interferons: Gene Cloning, Expression, and Biological Activity in Bacterial Extracts

Abstract

A series of novel, modified interferons based on the structure of human beta-interferon have been expressed in Escherichia coli. Modified interferon genes were constructed from sequences derived from the natural beta-interferon gene, a synthetic beta-interferon gene, or a specific combination of the two. A total of 23 out of the 25 novel interferons exhibited antiviral (AV) and antiproliferative (AP) activity which varied from 3 to 230% and 8 to 490% of the values for beta-interferon, respectively. None of the novel interferons had only AV or AP activity, although one had a much reduced ratio of AV/AP activity compared with beta-interferon. Substitution of beta-interferon amino acids 2-7 or 28-46 resulted in interferons with significantly increased AP activity on Daudi lymphoblastoid cells (four- to fivefold). All the novel interferons except two with modifications in the 82-105 region reacted with a neutralizing beta-interferon monoclonal antibody.

Antibodies that neutralize human beta interferon biologic activity recognize a linear epitope: analysis by synthetic peptide mapping.

“…In addition, the immunoreactive profile of neutralizing polyclonal antisera demonstrated a peak of reactivity with octapeptides that contained these essential residues. Our study supports the work of others emphasizing the importance of amino-terminal regions in the biologic activity of hIFN-,B (7,8). Creation of IFN-f3 variants, genetically engineered such that the amino-terminal sequences of hIFN-,l (residues 1-81) were replaced with corresponding sequences from hIFN-al, resulted in significant effects on biologic activity in nearly all of the IFNs evaluated, while carboxyl-terminal replacements had no significant effects (7).…”

“…Previous structure-function studies have utilized genetically altered IFN gene products (2)(3)(4)(5)(6)(7)(8), peptide mapping of large fragments (9)(10)(11)(12)(13)(14), and immunochemical mapping with monoclonal antibodies (mAbs) (15)(16)(17)(18). Such analyses of human a and p interferon (hIFN-a and -4) molecules have yielded inconclusive results, implicating large regions near the amino terminus (2,3,7,9,15), carboxyl terminus (11,16), or both (4-6, 8, 10), as domains responsible for biologic activity. The screening of short, sequential overlapping peptides for antibody reactivity as described by Geysen et al (19,20), also known as "pepscan" (21), permits the simultaneous scanning of entire molecules for linear immunoreactive epitopes.…”