Genetically engineered IgG1 and nanobody oligomers acquire strong intrinsic CD40 agonism

ABSTRACT

Most anti-CD40 antibodies show robust agonism only upon binding to FcγR⁺ 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 FcγR-expressing cells. Thus, conventional antibodies appear not to be sufficient to trigger the maximum possible CD40 activation independent from FcγR-binding. We proved here the hypothesis that oligomeric and oligovalent anti-CD40 antibody variants generated by genetic engineering display high intrinsic, thus FcγR-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 FcγR-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.

KEYWORDS:

• Antibody fusion protein
• CD40
• nanobody
• valency
• TNFRSF

Disclosure statement

The University of Würzburg has filed a patent application for the use of anti-CD40 antibody fusion proteins with intrinsic agonistic activity with I.L., D.S., and H.W. as co-inventors.

Author contribution statement

N.H., M.A., A.F., M.B., D.S., O.Z., and I.L. performed the experiments. N.H., M.A., I.L., and H.W. designed and interpreted the data and also wrote the manuscript. All authors agree to be accountable for all aspects of the work.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request or are already available within the article or its supplementary materials.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21655979.2024.2302246.

Additional information

Funding

This work has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No [813871] and Deutsche Krebshilfe-Projekt [70114009] to H.W.