Analysis of biomolecules by negative staining is a common technique used in electron microscopy (EM) due to its speed, ease of use, higher image contrast, and efficient visualization of small or flexible molecules. Combining negative staining with image classification techniques makes it possible to work with heterogeneous molecule populations, which are difficult or even impossible to analyze using vitrified specimens. In this study, high-resolution structural techniques such as X-ray crystallography and NMR were considered, but not prioritized due to technical limitations. This paper explores how Negative staining EM helped the team at Maverick Therapeutics, Inc., a wholly owned subsidiary of Takeda, to characterize novel COBRA T-cell engagers to support the development of efficient biotherapeutics.
The paper, published in Antibody Therapeutics concludes:
- COBRA™ (Conditional Bispecific Redirected Activation) T-cell engagers are designed to target solid tumors as a single polypeptide chain prodrug that becomes activated by proteolysis in the tumor microenvironment.
- To understand the interdomain interactions in COBRA molecules, we studied the domain organization of EGFR COBRA in solution by structural and biochemical methods. While high-resolution structural techniques such as X-ray crystallography and NMR were considered, they were not prioritized due to either inability to produce crystals of EGFR COBRA in an extensive screening campaign, or due to the large size of the molecule.
- …the structural information was obtained using low-resolution techniques: negative stain electron microscopy (EM) and small-angle X-ray scattering (SAXS).
- Imaging was done by NanoImaging Services, Inc. (4940 Carroll Canyon Road, Suite 115 San Diego, CA 92121).
- To understand the structure of EGFR COBRA, we applied negative stain EM followed by single particle analysis that allowed us to visualize domains in this molecule at low resolution.
- EGFR COBRA in solution is typically a monomer (>97.5% by analytical SEC) with MW around 93 kDa as determined by SEC-MALS, close to the predicted 95 kDa MW . We subjected a sample of EGFR COBRA of similar quality, with very little aggregation observed by SEC-MALS, to EM imaging. EM images showed that the protein particles of EGFR COBRA were well-dispersed, with only a few instances of clumping observed, confirming little aggregation in the sample.
- Visible particles showed variation in size and morphology (Fig. 2A). The elongated particles varied from ~ 11 to ~ 16 nm in length and from ~ 2.3 to ~ 6 nm in width.
- The 2D class averages obtained for EGFR COBRA (Fig. 2B) were well-defined and consistent with initial imaging observations…. The arrangement of the domains relative to one another varied widely across the classes.
- We found that EGFR COBRA is a very flexible molecule, and some domains are likely changing their position relative to each other by considerable distance without any preferred conformational state.
- our studies of EGFR COBRA by SEC-MALS-SAXS and negative stain EM allow us to propose a three-dimensional model for this molecule where instead of a conventional diabody with VH-VLi and VL-VHi Fvs, we have only one Fv between VH and VLi.
- The model is consistent with analysis of the post-translational modifications (PTMs) in EGFR COBRA and assessment of the exposure of MMP9 cleavable linker. This EGFR COBRA forms stable monomers with a very dynamic interdomain arrangement.
- Findings provide insights for design of the novel protein therapeutics.
Jessica Krakow, Michal Hammel, Ying Zhu, Brian J Hillier, Bryce Paolella, Austin Desmarais, Rusty Wall, Tseng-Hui T Chen, Rex Pei, Chulani Karunatilake. Antibody Therapeutics, Volume 5, Issue 1, January 2022, Pages 1–10, doi: 10.1093/abt/tbab028