A potent neutralizing monoclonal antibody can discriminate amongst IFNgamma from various primates with greater specificity than can the human IFNgamma receptor complex.
A monoclonal antibody (AF2) generated against recombinant human interferongamma (IFNgamma) exhibited potent IFNgamma neutralizing activity and prevented human IFNgamma from binding to the cell surface IFNgamma receptor complex. The AF2 antibody also neutralized IFNgamma from higher primates (superfamily Hominoidea) but did not react with IFNgamma from rhesus or other primates in the suborder Anthropoidea IFNgamma from all primates tested, however, could signal via the human IFNgamma receptor complex, as indicated by the ability to upregulate the level of MHC class II molecule expression on the surface of a responsive human cell line. We cloned and sequenced the IFNgamma gene from chimpanzee, gorilla, orangutan, and gibbon, and compared those with the previously reported IFNgamma sequences of human, rhesus, baboon and marmoset. This comparison revealed that, of the primate IFNgammas that were not reactive with AF2, rhesus IFNgamma was most homologous to human IFNgamma, differing at only nine amino acids and containing a one amino acid deletion. Comparing the sequence of human IFNgamma with that of rhesus IFNgamma suggested residues of the human IFNgamma molecule that were involved in the formation of the epitope recognized by the AF2 antibody. Constructing human/rhesus chimeric IFNgamma molecules, combined with site-directed mutagenesis of both human and rhesus IFNgamma revealed that this epitope was dependent upon two non-contiguous amino acids that are juxtaposed in the tertiary structure of IFNgamma. The determinant recognized by AF2 antibody resides in a portion of IFNgamma that is proximal to, but distinct from the surface that interacts with the IFNgamma receptor. Therefore, this neutralizing monoclonal antibody reacts with a conformational determinant that distinguishes primate IFNgammas serologically, but not functionally.
Thakur
A B
AB
Protein Design Labs Inc., Fremont, CA 94555, USA.
Landolfi
N F
NF
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018604:Epitope Mapping; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D011323:Primates / Q000235:genetics / Q000276:immunology*; D017471:Receptors, Interferon / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D000096743:Interferon gamma Receptor
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D007371:Interferon-gamma; D017471:Receptors, Interferon; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000096743:Interferon gamma Receptor