Immunochemical mapping of .alpha.-2 interferon

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Abstract
A panel of 5 monoclonal antibodies, designated U1-U5, produced by murine hybridoma clones has been raised to recombinant interferon (IFN) .alpha.-2, and 1 monoclonal antibody, designated U6, has been raised to a mixture of cyanogen bromide fragments of IFN .alpha.-2. These antibodies have been characterized with respect to neutralization of IFN antiviral and antiproliferative activities, binding to 4 cloned IFN .alpha. subtypes (.alpha.-1, .alpha.-2, .alpha.-4, and .alpha.-7) that are naturally occurring and to 2 novel products of recombinant DNA technology (.delta.-4 .alpha.-1 and .delta.-4 .alpha.-2/.alpha.-1 hybrid), and binding to 3 cyanogen bromide fragments of IFN .alpha.-2. Four of the 6 monoclonal antibodies inhibited IFN antiviral activity. In conjunction with the previously reported monoclonal antibodies III/21 [Arnheiter, H., Thomas, R.M., Leist, T., Fountoulakis, M., and Gutte, B. (1981) Nature (London) 294, 278-280] and NK-2 [Secher, D.S., and Burke, D.C. (1980) Nature (London) 285, 446-450], 8 unique epitopes have been described. Analysis of cross-reactivity patterns with IFN .alpha. fragments and subtypes indicated that monoclonal antibodies U1 and NK-2, which neutralized both antiviral and antiproliferative activities, and U2, which was nonneutralizing in these assays, were directed to distinct epitopes located in a polypeptide consisting of the amino-terminal 15 amino acid residues linked to residues 60-110 by a disulfide bond. The epitope recognized by U1 was determined to reside, at least in part, between residues 5 and 15. Competitive binding studies indicated that neutralizing monoclonal antibody U3, which did not bind to any of the cyanogen bromide fragments, was directed to an epitope partially overlapping that of NK-2. Epitopes to which neutralizing monoclonal antibodies U3, U4, and U5 and nonneutralizing antibody U6 were directed were readily distinguished by cross-reactivity with IFN .alpha. subtypes. The nonneutralizing monoclonal antibody U6 was determined to be active in an antiviral assay indicated a lack of direct functional significance for the first 4 amino-terminal amino acid residues and the Cys1-Cys918 disulfide bond. However, reduction with 2-mercaptoethanol of IFN .alpha.-2 altered the integrity of 4 of the 8 epitopes. These data support a critical role for disulfide linkages in maintaining the native conformation of IFN .alpha.-2 and provide a potential basis for predicting the location of functionally important domains.