When proteins bind their cognate receptors on the cell surface, signalling cascades are set in motion. The cytokine granulocyte colony-stimulating factor (GCSF) stimulates the proliferation of certain immune cells and is used to treat cancer patients who have low levels of white blood cells as a result of cytotoxic drugs. Unfortunately, the value of such therapeutic proteins is limited because of rapid clearance by receptor-mediated endocytosis and consequent protein degradation. In the September issue of Nature Biotechnology, scientists from the Massachussetts Institute of Technology (MIT) and Amgen used computer modelling and protein chemistry to engineer GCSF variants with lower receptor affinity that are as biologically potent as wild-type GCSF, but remain active for longer.

Activation of a cytokine receptor causes internalization of the receptor–ligand complex by the cell and results in delivery of the complex to acidified endosomal compartments. At this point, the cytokine ligand is either sent to lysosomal organelles, where the protein is degraded, or it is routed to recycling vesicles, which transport the cytokine out of the cell. What determines the fate of the cytokine–receptor complex in the endosome? Evidence indicates that when the receptor–ligand complex dissociates in the endosome, both the ligand and receptor are more likely to be recycled to the cell surface. Recycling preserves the structural integrity and biological activity of the ligand, and leads to an increase in the amplitude of the signal from a given concentration of ligand.

Sarkar et al. postulated that if they could increase the recycling of GCSF and its receptor without disrupting the ligand–receptor complex at the cell surface, the efficacy and potency of GCSF could be improved. Exploiting the pH difference in the endosome compared with the cell surface, the authors set out to create a cytokine that might have lower receptor affinity at a more acidic pH.

Using the solved crystal structure of the interactions between GCSF and its receptor, a series of sites were identified at the ligand–receptor interface where the presence of a positive charge would destabilize the complex. Introducing histidine residues at these sites by site-directed mutagenesis resulted in a neutral charge at the cell surface and a positive charge in the endosome. This allowed pH-dependent modulation of the affinity of the complex, so that the GCSF variants bound to the receptor at the cell surface with equal affinity to that of the wild type, but with much lower affinity at the lower endosomal pH. As the authors had predicted, in vitro assays showed that the variants were depleted less quickly from the growth medium and were more effective in promoting cell proliferation.

It is not yet clear whether the surface histidine mutations alter the antigenic effects of the protein, or its stability, and these engineered GCSF variants still need to be tested in an animal model. However, these variants show the validity of the approach, which could be widely applied to other protein ligands, whose efficacy is enhanced by increasing recycling back to the cell surface.