Biochemical characterization of forimtamig and synapse formation. (A) The molecular model of forimtamig was generated and visualized with discovery studio 2021; residues involved in the P329G LALA silent Fc mutations are shown as spheres colored in cyan. (B) Binding kinetics was measured using horseradish peroxidase (HRP)-based colorimetric analysis in Chinese hamster ovary (CHO) cells transfected with mouse, cynomolgus, or human GPRC5D or human GPRC5A and incubated with increasing concentrations of forimtamig. (C) The likely epitope of forimtamig could be mapped to the unstructured N terminus of GPRC5D and is shown in the context of 2 recently published cryo-electro magnetic (EM) structures of GPRC5D homodimer, in complex with 2 scFvs (protein data bank (PDB) ID 8yzk) and in complex with a single talquetamab Fab anti-GPRC5D (PDB ID 9ima); the core epitope (yellow ball and stick representation) consists of GPRC5D residues 5 to 10, but an additional involvement of residues 11 to 16 (gray stick representation) cannot be ruled out; as the unstructured N terminus is not or only partially resolved in the published structures, the missing N-terminal segments (purple ribbon representation) were remodeled from the AlphaFold model and the shown conformations represent only a placeholder for a whole ensemble of possible conformations; the actual conformation of GPRC5D’s N-terminal segment in complex with forimtamig anti-GPRC5D has yet to be determined. (D) Grating-coupled interferometry (GCI) was used to determine affinities (equilibrium dissociation constant, K_D) of forimtamig and forimtamig monovalent Fab to human GPRC5D and of forimtamig to human CD3ε; black lines indicate fit according to a 1:1 Langmuir model. (E) Correlation of contact duration and speed of fluorescently labeled T cells as measured by confocal live cell imaging to determine the stability of immunological synapses; all treatments are at 200 ng/mL. scFv, single-chain variable fragment.