RATIONAL DESIGN, ANALYSIS, AND POTENTIAL UTILITY OF GM-CSF ANTAGONISTS

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an import ant cytokine involved in many immune and inflammatory processes and is believed to act in the early stages of immune responses. GM-CSF stimu lates antigen-presenting cells, enhancing antigen presentation and ind ucing macrophage tumoricidal activity. GM-CSF binds to specific cellul ar receptors that are potential targets for pharmacological design. Ra tional de sign of small-molecule mimics is an important approach to ph armacophore design. One of the strategies in the development of small- molecular mimics of larger polypeptyde ligands is analysis of alternat ive ligands that bind the same site as does the native ligand. Molecul ar studies of GM-CSF-receptor interactions have led to the development of interaction site analogs and the development of an ''anti-anti-GM- CSF'' recombinant antibody (rAb) analog of a site on GM-CSF important for biological activity and receptor binding. This rAb and a peptide d erived from the rAb first complementarity determining region (CDR) seq uence bind to a monoclonal anti-GM-CSF antibody that mimics the GM-CSF R alpha chain, compete with GM-CSF for binding to GM-CSF receptor alph a chain (GM-CSFR alpha), and are specific biological antagonists. Mole cular modeling of the rAb suggests structural similarity with a site p reviously implicated in GM-CSF binding to the GM-CSFR alpha. Two cycli c peptides, 1785 and 1786, also were developed on the basis of structu ral analysis of the GM-CSF region mimicked by anti-anti-GM-CSF recombi nant antibody (rAb) 23.2. These peptides were designed to mimic struct urally the positions of specific residues on the B and C helicies of h uman GM-CSF implicated in receptor binding and bioactivity. Both 1785 and 1786 were recognized specifically by polyclonal anti-GM-CSF antibo dy. 1786 also competitively inhibited binding of GM-CSF to the GM-CSF receptor and demonstrated antagonist bioactivity, as shown by its reve rsal of GM-CSF's ability to inhibit apoptosis of the GM-CSF-dependent cell line MO7E. These studies support the role of residues on the GM-C SF B and C helicies in receptor binding and bioactivity and suggest st rategies for mimicking binding sites on four-helix bundle proteins wit h cyclic peptides.