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Identification of functionally distinct domains of human granulocyte-
macrophage colony-stimulating factor using monoclonal antibodies
Y Kanakura, SA Cannistra, CB Brown, M Nakamura, GF Seelig, WW Prosise, JC Hawkins, K Kaushansky and JD Griffin
Division of Tumor Immunology, Dana-Farber Cancer Institute, Boston, MA
02115.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein
that is required for the survival, growth, and differentiation of
hematopoietic progenitor cells. Although the primary structure of GM-CSF is
known from cDNA cloning, the relationship between structure and function of
GM-CSF is not fully understood. Fifteen different monoclonal antibodies
(MoAbs) to human GM-CSF were generated to map immunologically distinct
areas of the molecule. Each of the MoAbs was biotinylated and shown by
enzyme-linked immunosorbent assay to bind to recombinant GM-CSF that had
been affixed to a solid phase. Each of the 15 unconjugated MoAbs was then
used to compete with each biotinylated MoAb for binding to GM-CSF. These
cross-blocking studies identified eight distinct epitopes of native GM-CSF.
Seven of these epitopes were also present in denatured GM-CSF by Western
blotting, and four of the epitopes were at least partially conserved on
GM-CSF that was reduced in beta-mercaptoethanol. MoAbs to four of eight
epitopes neutralized both recombinant (glycosylated and nonglycosylated)
and natural human GM-CSF in a GM colony-forming unit (CFU-GM) assay and
blocked GM-CSF-induced activation of neutrophils. For most of the
antibodies there was a good correlation between neutralizing activity and
the capacity to block binding of 125I-GM-CSF to neutrophils or blasts.
Non-neutralizing antibodies to one epitope partially blocked binding of
125I-GM-CSF to neutrophils. None of the MoAbs neutralized interleukin-3,
G-CSF, or M-CSF. The locations of seven of the epitopes could be partially
mapped with regard to the amino acid structure by determining reactivity to
GM-CSF synthetic peptides or to human-mouse chimeric GM-CSFs. The
neutralizing antibodies were found to map to amino acids 40-77, 78-94, or
110-127. Thus, these MoAbs are useful to identify functional domains of
GM-CSF and in identifying regions that are likely to be involved in
receptor interaction.
Volume 77,
Issue 5,
pp. 1033-1043,
03/01/1991
Copyright © 1991 by The American Society of Hematology
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