Residue-Level Characterization of Antibody Binding Epitopes Using Carbene Chemical Footprinting.

Characterization of antibody binding epitopes is an important factor in therapeutic drug discovery, as the binding site determines and drives antibody pharmacology and pharmacokinetics. Here, we present a novel application of carbene chemical footprinting with mass spectrometry for identification of antibody binding epitopes at the single-residue level. Two different photoactivated diazirine reagents provide complementary labeling information allowing structural refinement of the antibody binding interface.

Humanization of a strategic CD3 epitope enables evaluation of clinical T-cell engagers in a fully immunocompetent in vivo model.

T-cell engagers (TCEs) are a growing class of biotherapeutics being investigated in the clinic for treatment of a variety of hematological and solid tumor indications. However, preclinical evaluation of TCEs in vivo has been mostly limited to xenograft tumor models in human T-cell reconstituted immunodeficient mice, which have a number of limitations. To explore the efficacy of human TCEs in fully immunocompetent hosts, we developed a knock-in mouse model (hCD3E-epi) in which a 5-residue N-terminal fragment of murine CD3-epsilon was replaced with an 11-residue stretch from the human sequence that encodes for a common epitope recognized by anti-human CD3E antibodies in the clinic.

Structures of mouse and human GITR-GITRL complexes reveal unique TNF superfamily interactions.

Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) and GITR ligand (GITRL) are members of the tumor necrosis superfamily that play a role in immune cell signaling, activation, and survival. GITR is a therapeutic target for directly activating effector CD4 and CD8 T cells, or depleting GITR-expressing regulatory T cells (Tregs), thereby promoting anti-tumor immune responses. GITR activation through its native ligand is important for understanding immune signaling, but GITR structure has not been reported.

Integrated Approach for Characterizing Bispecific Antibody/Antigens Complexes and Mapping Binding Epitopes with SEC/MALS, Native Mass Spectrometry, and Protein Footprinting.

Bispecific antibodies (BsAbs), with a unique mechanism of recognizing two different epitopes or antigens, have shown potential in various therapeutic areas. Molecular characterization of BsAbs' epitopes not only allows for detailed understanding of their mechanism of actions but also guides the design and selection of drug candidate molecules. In this study, we illustrate the practical utility of an integrated approach, including size exclusion chromatography with multiangle light scattering and native mass spectrometry (MS) for the biophysical characterization of complex formation of a BsAb with two target antigens, cluster of differentiation 3 (CD3) and B-cell maturation antigen (BCMA).