Agonistic activity of a CD40-specific single-chain Fv constructed from the variable regions of mAb G28-5

A single-chain Fv (sFv) was expressed from the variable regions of the CD40-specific mAb G28-5. The molecule bound CD40 with a high affinity (2.2 nM) and was a monomer in solution. Surprisingly, G28-5 sFv was a potent CD40 agonist that rapidly crosslinked CD40 on the cell surface but did not crosslink CD40-Ig in solution. G28-5 sFv was a more potent agonist than G28-5 IgG and was able to stimulate CD40 responses by B cells and monocytes. G28-5 IgG partially blocked, whereas G28-5 sFv augmented CD40 responses during stimulation with natural ligand (gp39-CD8 fusion protein). These results indicate that the functional activity of ligands built from the binding site of G28-5 is highly dependent upon the size and physical properties of the molecule both in solution and on the cell surfaces.