CNTF VARIANTS WITH INCREASED BIOLOGICAL POTENCY AND RECEPTOR SELECTIVITY DEFINE A FUNCTIONAL SITE OF RECEPTOR INTERACTION

Human CNTF is a neurocytokine that elicits potent neurotrophic effects by activating a receptor complex composed of the ligand-specific alph a-receptor subunit (CNTFR alpha) and two signal transducing proteins, which together constitute a receptor for leukemia inhibitory factor (L IFR). At high concentrations, CNTF can also activate the LIFR and poss ibly other cross-reactive cytokine receptors in the absence of CNTFR a lpha. To gain a better understanding of its structure-function relatio nships and to develop analogs with increased receptor specificity, the cytokine was submitted to affinity maturation using phage display tec hnology. Variants with greatly increased CNTFR alpha affinity were sel ected from a phage-displayed library of CNTF variants carrying random amino acid substitutions in the putative D helix, Selected variants co ntained substitutions of the wild-type Gln167 residue, either alone or in combination with neighboring mutations. These results provide evid ence for an important functional role of the mutagenized region in CNT FR alpha binding. Affinity enhancing mutations conferred to CNTF incre ased potency to trigger biological effects mediated by CNTFR alpha and enhanced neurotrophic activity on chicken ciliary neurons. In contras t, the same mutations did not potentiate the CNTFR alpha-independent r eceptor actions of CNTF, These CNTF analogs thus represent receptor-sp ecific superagonists, which should help to elucidate the mechanisms un derlying the pleiotropic actions of the neurocytokine.