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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.
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