Genetically engineered IgG1 and nanobody oligomers acquire strong intrinsic CD40 agonism

Most anti-CD40 antibodies show robust agonism only upon binding to Fc gamma R+ cells, such as B cells, macrophages, or DCs, but a few anti-CD40 antibodies display also strong intrinsic agonism dependent on the recognized epitope and/or isotype. It is worth mentioning, however, that also the anti-CD40 antibodies with intrinsic agonism can show a further increase in agonistic activity when bound by Fc gamma R-expressing cells. Thus, conventional antibodies appear not to be sufficient to trigger the maximum possible CD40 activation independent from Fc gamma R-binding. We proved here the hypothesis that oligomeric and oligovalent anti-CD40 antibody variants generated by genetic engineering display high intrinsic, thus Fc gamma R-independent, agonistic activity. We generated tetra-, hexa- and dodecavalent variants of six anti-CD40 antibodies and a CD40-specific nanobody. All these oligovalent variants, even when derived of bivalent antagonistic anti-CD40 antibodies, showed strongly enhanced CD40 agonism compared to their conventional counterparts. In most cases, the CD40 agonism reached the maximum response induced by Fc gamma R-bound anti-CD40 antibodies or membrane CD40L, the natural engager of CD40. In sum, our data show that increasing the valency of anti-CD40 antibody constructs by genetic engineering regularly results in molecules with high intrinsic agonism and level out the specific limitations of the parental antibodies.