Pyroglutamation of cell surface proteins CD47 and TRP1 by glutaminyl cyclase modulates therapeutic antibody binding

Plas-van Duijn, A. van der; Winkelman, L.M.T.; Lensen, C.; Duivendijk, M. van; Derksen, I.; Veenendaal, J.M.F. van; Schaap, G.; Sluijter, M.; Groningen, T.J.B. van; Doorn, R. van; Hall, T. van; Slieker, R.C.; Sharp, T.H.; Beltman, J.B.; Bresser, K.; Scheeren, F.A.

doi:doi:10.1038/s42003-025-08938-4

Monoclonal antibodies are important modalities in the treatment of cancer. Post-translational modifications of proteins, such as glycosylation, can affect the binding affinity of therapeutic antibodies. Whether other PTMs modulate therapeutic antibody binding to different surface proteins is currently underexplored. Pyroglutamation is the post-translational cyclization of an N-terminal glutamine or glutamic acid residue into a pyroglutamate by glutaminyl cyclase. In this study, we investigated the impact of pyroglutamation on the binding affinity of three therapeutic antibodies targeting CD47 and TRP1. Here, we show that pyroglutamation on CD47 and TRP1 modulates the binding of anti(alpha)-CD47 magrolimab and alpha TRP1 TA99 and flanvotumab. Furthermore, the N-terminal glutamine on CD47 is crucial for effective antibody recognition, while pyroglutamation of TRP1 is involved in trafficking to the cell surface. These findings highlight that the pyroglutamation by glutaminyl cyclase...Show moreMonoclonal antibodies are important modalities in the treatment of cancer. Post-translational modifications of proteins, such as glycosylation, can affect the binding affinity of therapeutic antibodies. Whether other PTMs modulate therapeutic antibody binding to different surface proteins is currently underexplored. Pyroglutamation is the post-translational cyclization of an N-terminal glutamine or glutamic acid residue into a pyroglutamate by glutaminyl cyclase. In this study, we investigated the impact of pyroglutamation on the binding affinity of three therapeutic antibodies targeting CD47 and TRP1. Here, we show that pyroglutamation on CD47 and TRP1 modulates the binding of anti(alpha)-CD47 magrolimab and alpha TRP1 TA99 and flanvotumab. Furthermore, the N-terminal glutamine on CD47 is crucial for effective antibody recognition, while pyroglutamation of TRP1 is involved in trafficking to the cell surface. These findings highlight that the pyroglutamation by glutaminyl cyclase can modulate the binding affinity of antibodies with therapeutic potential.Show less

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Pyroglutamation of cell surface proteins CD47 and TRP1 by glutaminyl cyclase modulates therapeutic antibody binding