Temperature promotes Fab-arm exchange in Pembrolizumab

Area of Science:

• Immunology
• Biochemistry
• Protein Engineering

Background:

• Immunoglobulin G4 (IgG4) antibodies are dynamic molecules capable of Fab domain swapping.
• This process generates bispecific antibodies with reduced pro-inflammatory activity under specific in vitro conditions.

Purpose of the Study:

• To investigate the effect of physiological and non-physiological temperatures on the intramolecular rearrangements of the immunotherapeutic IgG4 antibody, Pembrolizumab.
• To understand how temperature influences the structural dynamics and antigen-binding properties of Pembrolizumab.

Main Methods:

• Comparative analysis of Pembrolizumab structure and function at different temperatures (25°C, 37°C, 38.5°C, and 40°C).
• Assessment of conformational changes related to Fab-arm exchange.
• Measurement of antibody-antigen binding affinity to the Programmed Death-1 (PD-1) receptor.

Main Results:

• Pembrolizumab undergoes conformational changes supporting Fab-arm exchange at elevated temperatures (38.5°C and 40°C) compared to lower temperatures (25°C and 37°C).
• Higher temperatures lead to reduced binding affinity of Pembrolizumab to its target, the Programmed Death-1 receptor.
• Elevated temperatures promote Fc domain binding to Fc/complement receptors and decrease on-target residence time.

Conclusions:

• Temperature significantly influences the structural integrity and functional activity of Pembrolizumab.
• Fab-arm exchange, while affected by temperature, is not abrogated by the canonical S228P mutation.
• The observed changes in binding affinity and Fc receptor interactions at higher temperatures have implications for the therapeutic use of Pembrolizumab.