Multispecific antibodies are a recent breakthrough in therapeutic interventions for many complex diseases. Multispecific antibodies recognize two or more epitopes located on the same or distinct targets. Enhancing the stability and improving the binding affinity for multiple targets without cross-reactivity remains a significant challenge in the rational design and engineering of multispecific antibodies.

This work explores the molecular interactions between bispecific antibodies and respiratory syncytial virus F (RSV F) antigen using tandem-trapped ion mobility spectrometry (Tandem-TIMS). Utilizing the ability of Tandem-TIMS to conduct non-ensemble measurements, we characterize the impact of sequence mutations and distinct protein conformations on the stability of various multispecific antibodies and antigen binding. Solutions of bispecific antibodies (provided by Johnson & Johnson Innovative Medicine, designed for research purposes) in the apo and holo forms at a concentration of 0.5 µM were infused into the electrospray source. Structural analysis of the experimental data was conducted using molecular dynamics-based structure relaxation approximation (SRA) method. Our results indicate that sequence permutations in the Fc region play a role in the glycoform formation and the intact protein conformation. Further, our data suggest that the RSV F peptide binds to the bispecific antibodies in a conformation-dependent manner. In contrast, distinct glycoforms of bispecific antibodies do not affect the binding to the RSV F antigen.

Harsha Gunawardena

Janssen Global Services LLC

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