The functionality of a therapeutic antibody candidate restored by a single mutation from proline to threonine in the variable region

ABSTRACT

mAbs play an essential role in the therapeutic arsenal. Our laboratory has patented the Rendomab-B49 mAb targeting the endothelin B receptor (ET_B). This G protein-coupled receptor plays a driving role in the progression of numerous cancers. We chimerized our mAb (xiRB49) to evaluate its preclinical therapeutic efficacy in different ET_B⁺ tumor models with an antibody drug conjugate approach. As previously reported, the chimerization process of an antibody can alter its functionality. In this article, we present the chimerization of RB49. xiRB49 purified by Protein A remained perfectly soluble and did not aggregate, but it lost all its ability to recognize ET_B. A detailed analysis of its variable region using IMGT tools allowed us to identify an unusual proline at position 125. In silico mAb modeling and in vitro experiments were performed for a better understanding of xiRB49 structure-function relationships. Our results show that the proline in position 125 on the heavy chain alters the xiRB49 CDR3 light chain conformation and its mutation to threonine allows complete functional recovery.

KEYWORDS:

• Monoclonal antibody
• chimerization
• proline
• in silico modelling

Author contributions

Conceptualization – DB, IM, SPL, SE, Investigation – MH, IM, NC, AH; Original draft preparation – MH, IM; Figures and visualizations – MH, IM; Review and editing –. DB, SPL, MH, IM.

Disclosure statement

DB and AH are scientific cofounders and hold equity in Skymab Biotherapeutics.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website at https://doi.org/10.1080/21645515.2023.2279867.

Additional information

Funding

This work was funded by the CEA, the ANR (ANR-19-CE18-0013, DualMab) and HPC/AI resources from GENCI-IDRIS [Grant 2022-AD010713707].