258 : Bone-targeting properties of the IL-27/IL-30 poly-glutamic acid motif

IL-27 is composed of the IL-30 cytokine subunit associated to the soluble receptor EBI3. IL-30 comprises an unusual poly-glutamic (poly E) motif in the loop connecting α helix C and D. The binding of sialoprotein to hydroxyapatite, the main mineral component of bone, is mediated by a very similar motif. We analysed the functions of the poly E motif using alanine substitutions. We observed that IL-27 binds hydroxyapatite and bone matrix and that this binding was lost in the alanine substitution mutant (poly A). Hydroxyapatite-bound IL-27 retained the capacity to activate the IL-27 receptor, indicating that it does not represent a latent form requiring release for biological activity. To investigate the relevance of these observations, we compared the localisation of WT and poly A mutant IL-27 in vivo . Mice were subjected to hydrodynamic transfection with plasmids coding for the BirA biotin ligase and WT or mutant IL-27 tagged with an AviTag biotin ligase substrate. Plasmid coding for BirA alone was used as control. Western blot analysis of the transfected mice livers and femurs indicated that WT and mutant IL-27 were expressed similarly and that WT IL-27 was targeted preferentially to bone. This indicates that the poly E motif confers a bone tropism to IL-27/IL-30. As IL-27 is known to have potent anti-tumor activities, this could be beneficial for therapeutic applications targeting bone metastasis. The bone marrow endosteal niche represents a preferential site for the localisation of hematopoietic stem, Treg, memory T and long lived plasma cells as well as for B cell development. The poly E motif could therefore contribute to the immunoregulatory effects of IL-27. Acid motifs are used to target therapeutic proteins such as asfotase alfa to bone. Their insertion in loops connecting type I cytokine α helixes could be useful in generating derivatives targeting bone resident cells.