Identification of a functional domain of human granulocyte colony-stimulating factor using neutralizing monoclonal antibodies.
Human granulocyte colony-stimulating factor (G-CSF) is a hemopoietic growth factor that is being used successfully to treat various forms of neutropenia. To define functionally important regions of G-CSF, we have prepared 37 monoclonal anti-G-CSF antibodies and mapped the regions of G-CSF recognized by different antibody groups. Antibodies recognizing similar epitopes were identified by competition assays, neutralization assays, conformation dependence and cross-reactivity with canine G-CSF. Seven of eight neutralizing antibodies fell into two related epitope groups and were conformation-dependent. The eighth was unrelated and conformation-independent. Peptides of G-CSF were generated by chemical or enzymatic digestion and tested for antibody reactivity. One of the neutralizing antibodies (LMM351) recognized a small, disulfide-bonded peptide from the V8 protease digest (residues 34-46). A synthetic peptide (residues 20-58) was recognized by all the neutralizing antibodies, implicating this disulfide-bonded loop in receptor binding. The epitopes recognized by nonneutralizing antibodies were found throughout G-CSF. Thus, regions of G-CSF that are not involved in receptor binding have also been defined. A CNBr peptide (residues 1-121) had greatly reduced biological activity, indicating that the COOH terminus is required for receptor binding. We predict that residues 20-46 and the COOH terminus bind to the G-CSF receptor.
Layton
J E
JE
Melbourne Tumour Biology Branch, Ludwig Institute for Cancer Research, Australia.
Morstyn
G
G
Fabri
L J
LJ
Reid
G E
GE
Burgess
A W
AW
Simpson
R J
RJ
Nice
E C
EC
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001666:Binding Sites, Antibody; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D016179:Granulocyte Colony-Stimulating Factor / Q000276:immunology / Q000494:pharmacology / Q000502:physiology*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010446:Peptide Fragments / Q000276:immunology / Q000494:pharmacology*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D012984:Software
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D010450:Endopeptidases