Variable region cDNA sequences and characterization of murine anti-human interferon gamma receptor monoclonal antibodies that inhibit receptor binding by interferon gamma.
Murine monoclonal antibodies (mAbs) are described that recognize the extracellular human interferon gamma receptor alpha-chain (IFN gamma R) and inhibit the binding to it of interferon gamma. The inhibitory activities (IC50s) of these mAbs, quantified by radioimmunoassay using native receptor on human Raji cells, lie in the range 0.5-24 nM, whereas their relative affinities for the immobilised recombinant extracellular receptor, determined using surface plasmon resonance technology, are in the range 0.6-40.9 nM. Nine mAbs derived from one immunization, were shown by variable region cDNA sequencing to be clonally related, with mAb A6 from this group showing the highest affinity for the receptor. Another two mAbs, gamma R38 and gamma R99, derived from a separate immunization, are clonally unrelated to each other and to those in the A6 family. From the V-region sequences, the L-chains of mAbs A6, gamma R38 and gamma R99 were shown to belong to the V kappa 34C, V kappa 34C and V kappa 1 families, whereas the H-chains belong to the 3069, J606 and J558 families, respectively. The mAbs A6 and gamma R38 recognize overlapping epitopes on the N-terminal Ig-like domain of the IFN gamma R, whereas the gamma R99 epitope is located largely in the membrane proximal Ig-like domain. Sequence comparisons with Ig structures solved by X-ray diffraction allowed deductions concerning likely CDR canonical conformations. These studies provide essential information for crystallographic and mutagenesis experiments aimed at understanding the molecular basis of the interactions of these mAbs with the extracellular IFN gamma R.
Bridges
A
A
Institute of Organic Chemistry, University of Zurich, Switzerland.
Birch
A
A
Williams
G
G
Aguet
M
M
Schlatter
D
D
Huber
W
W
Garotta
G
G
Robinson
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D015703:Antigens, CD / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D001666:Binding Sites, Antibody; D001667:Binding, Competitive / Q000276:immunology; D018076:DNA, Complementary / Q000302:isolation & purification; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D007371:Interferon-gamma / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D017471:Receptors, Interferon / Q000235:genetics / Q000276:immunology*; D013329:Structure-Activity Relationship; D000096743:Interferon gamma Receptor
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018076:DNA, Complementary; D007135:Immunoglobulin Variable Region; D007371:Interferon-gamma; D017471:Receptors, Interferon; D000096743:Interferon gamma Receptor