Development and characterization of high affinity leptins and leptin antagonists.

Leptin is a pleiotropic hormone acting both centrally and peripherally. It participates in a variety of biological processes, including energy metabolism, reproduction, and modulation of the immune response. So far, structural elements affecting leptin binding to its receptor remain unknown. We employed random mutagenesis of leptin, followed by selection of high affinity mutants by yeast surface display and discovered that replacing residue Asp-23 with a non-negatively charged amino acid leads to dramatically enhanced affinity of leptin for its soluble receptor. Rational mutagenesis of Asp-23 revealed the D23L substitution to be most effective. Coupling the Asp-23 mutation with alanine mutagenesis of three amino acids (L39A/D40A/F41A) previously reported to convert leptin into antagonist resulted in potent antagonistic activity. These novel superactive mouse and human leptin antagonists (D23L/L39A/D40A/F41A), termed SMLA and SHLA, respectively, exhibited over 60-fold increased binding to leptin receptor and 14-fold higher antagonistic activity in vitro relative to the L39A/D40A/F41A mutants. To prolong and enhance in vivo activity, SMLA and SHLA were monopegylated mainly at the N terminus. Administration of the pegylated SMLA to mice resulted in a remarkably rapid, significant, and reversible 27-fold more potent increase in body weight (as compared with pegylated mouse leptin antagonist), because of increased food consumption. Thus, recognition and mutagenesis of Asp-23 enabled construction of novel compounds that induce potent and reversible central and peripheral leptin deficiency. In addition to enhancing our understanding of leptin interactions with its receptor, these antagonists enable in vivo study of the role of leptin in metabolic and immune processes and hold potential for future therapeutic use in disease pathologies involving leptin.

A novel recombinant fusion protein encoding a 20-amino acid residue of the third extracellular (E3) domain of CCR2 neutralizes the biological activity of CCL2.

CCL2 is a key CC chemokine that has been implicated in a variety of inflammatory autoimmune diseases and in tumor progression and it is therefore an important target for therapeutic intervention in these diseases. Soluble receptor-based therapy is a known approach for neutralizing the in vivo functions of soluble mediators. Owing to the complexity of seven-transmembrane G protein-coupled receptors, efforts to generate neutralizing soluble chemokine receptors have so far failed. We developed a strategy that is based on the generation of short recombinant proteins encoding different segments of a G protein-coupled receptor, and tested the ability of each of them to bind and neutralize its target chemokine. We show that a fusion protein comprised of as few as 20 aa of the third extracellular (E3) domain of the CCL2 receptor, stabilized by the IgG H chain Fc domain (E3-IgG or BL-2030), selectively binds CCL2 and CCL16 and effectively neutralizes their biological activities. More importantly, E3-IgG (BL-2030) could effectively suppress the in vivo biological activity of CCL2, attenuating ongoing experimental autoimmune encephalomyelitis, as well as the development of human prostate tumor in SCID mice.

A novel recombinant soluble splice variant of Met is a potent antagonist of the hepatocyte growth factor/scatter factor-Met pathway.

PURPOSE: The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), are involved in a wide range of biological activities, including cell proliferation, motility, invasion, and angiogenesis. The HGF/SF-Met signaling pathway is frequently activated in a variety of cancers, and uncontrolled Met activation correlates with highly invasive tumors and poor prognosis. In this study, we investigated the inhibitory effect of a novel soluble splice variant of Met on the HGF/SF-Met pathway. EXPERIMENTAL DESIGN: Using our alternative splicing modeling platform LEADS, we have identified a novel splice variant of the Met receptor, which encodes a truncated soluble form of the receptor. This variant was produced as a recombinant Fc-fused protein named Cgen-241A and was tested in various cell-based assays representing different outcomes of the HGF/SF-Met pathway. RESULTS: Cgen-241A significantly inhibited HGF/SF-induced Met phosphorylation as well as cell proliferation and survival. In addition, Cgen-241A showed a profound inhibitory effect on cell scattering, invasion, and urokinase up-regulation. The inhibitory effects of Cgen-241A were shown in multiple human and nonhuman cell types, representing different modes of Met activation. Furthermore, Cgen-241A showed direct binding to HGF/SF. CONCLUSIONS: Taken together, our results indicate that Cgen-241A is a potent antagonist of the HGF/SF-Met pathway, underlining its potential as a therapeutic agent for the treatment of a wide variety of human malignancies that are dependent on this pathway.