Differential inhibition of bispecific GPVI and CLEC-2 nanobody ligands by a monovalent nanobody

We have previously characterised a series of monospecific multivalent nanobodies to the platelet receptors GPVI (Nb2) and CLEC-2 (Nb4) showing that tri- and tetravalent nanobodies induce potent platelet aggregation. In the present study we generated a series of di-, tri- and tetravalent bispecific nanobodies for GPVI and CLEC-2 to mimic ligands such as diesel exhaust particles and polysulphated glycopolymers which crosslink both receptors. In contrast to results with the divalent monospecific nanobodies, the bispecific divalent nanobody, Nb2:Nb4, stimulated weak aggregation in most donors with some donors reaching maximal aggregation over a course of ten minutes. The trivalent nanobodies, Nb2-2:Nb4 (two copies of GPVI nanobody) and Nb2:Nb4–2 (two copies of CLEC-2 nanobody), and the tetravalent nanobody, Nb2-2:Nb4-2, stimulated full aggregation in all donors. Activation by the trivalent nanobodies was blocked by Src and Syk tyrosine kinase inhibitors. Nb2 (100 nM) is a competitive antagonist against the monospecific tetravalent nanobody, Nb2-4. At the same concentration, Nb2 had a negligible effect on the response to Nb2:Nb4-2 but converted the concentration-response curve for Nb2-2:Nb4 to a bell-shape. Modelling the interaction of trivalent bispecific ligands, composed of two different epitopes, using equilibrium mass action also predicted a differential effect. The results have important implications for inhibiting ligands that crosslink more than one type of receptor.