Inference of molecular structure for characterization and improvement of clinical grade immunocytokines.

Abstract

The use of immunomodulatory agents for the treatment of cancer is gaining a growing biopharmaceutical interest. Antibody-cytokine fusion proteins, namely immunocytokines, represent a promising solution for the regulation of the immune system at the site of disease. The three-dimensional arrangement of these molecules can profoundly influence their biological activity and pharmacokinetic properties. Structural techniques might provide important insight in the 3D arrangement of immunocytokines. Here, we performed structure investigations on clinical grade fusion proteins L19-IL2, IL12-L19L19 and L19L19-IL2 to elucidate their quaternary organization. Crystallographic characterization of the common L19 antibody fragment at a resolution of 2.0-Å was combined with low-resolution studies of the full-length chimeric molecules using small-angle synchrotron X-ray scattering (SAXS) and negative stain electron microscopy. Characterization of the full-length quaternary structures of the immunocytokines in solution by SAXS consistently supported the diabody structure in the L19-IL2 immunocytokine and allowed generation of low-resolution models of the chimeric proteins L19L19-IL2 and IL12-L19L19. Comparison with 3D reconstructions obtained from negative-stain electron microscopy revealed marked flexibility associated to the linker regions connecting the cytokine and the antibody components of the chimeric proteins. Collectively, our results indicate that low-resolution molecular structure characterizations provide useful complementary insights for the quality control of immunocytokines, constituting a powerful tool to guide the design and the subsequent optimization steps towards clinical enhancement of these chimeric protein reagents.

• Authors: Tiziano Ongaro, Salvatore R Guarino, Luigi Scietti, Martina Palamini, Sarah Wulhfard, Dario Neri, Alessandra Villa, Federico Forneris
• Journal: Journal of structural biology (J Struct Biol) Vol. 213 Issue 1 Pg. 107696 (03 2021) ISSN: 1095-8657 [Electronic] United States
• PMID: 33493635 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2021 Elsevier Inc. All rights reserved.

Chemical References

• Cytokines
• Recombinant Fusion Proteins

Topics

• Animals
• Cell Line, Tumor
• Cytokines (chemistry)
• Humans
• Mice
• Molecular Structure
• Recombinant Fusion Proteins (chemistry)
• Scattering, Small Angle
• X-Ray Diffraction (methods)