Stability engineering of anti-EGFR scFv antibodies by rational design of a lambda-to-kappa swap of the V_L framework using a structure-guided approach

Abstract

Phage-display technology facilitates rapid selection of antigen-specific single-chain variable fragment (scFv) antibodies from large recombinant libraries. ScFv antibodies, composed of a V_H and V_L domain, are readily engineered into multimeric formats for the development of diagnostics and targeted therapies. However, the recombinant nature of the selection strategy can result in V_H and V_L domains with sub-optimal biophysical properties, such as reduced thermodynamic stability and enhanced aggregation propensity, which lead to poor production and limited application. We found that the C10 anti-epidermal growth factor receptor (EGFR) scFv, and its affinity mutant, P2224, exhibit weak production from E. coli. Interestingly, these scFv contain a fusion of lambda3 and lambda1 V-region (LV3 and LV1) genes, most likely the result of a PCR aberration during library construction. To enhance the biophysical properties of these scFvs, we utilized a structure-based approach to replace and redesign the pre-existing framework of the V_L domain to one that best pairs with the existing V_H. We describe a method to exchange lambda sequences with a more stable kappa3 framework (KV3) within the V_L domain that incorporates the original lambda DE-loop. The resulting scFvs, C10KV3_LV1DE and P2224KV3_LV1DE, are more thermodynamically stable and easier to produce from bacterial culture. Additionally, C10KV3_LV1DE and P2224KV3_LV1DE retain binding affinity to EGFR, suggesting that such a dramatic framework swap does not significantly affect scFv binding. We provide here a novel strategy for redesigning the light chain of problematic scFvs to enhance their stability and therapeutic applicability.

Keywords:

• antibody engineering
• EGFR
• scFv antibody
• stability engineering
• thermostability

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors would like to thank Dr. Mark Andrake for aid in dynamic light scattering acquisition and to the Spectroscopy Support Facility for providing access to CD spectroscopy.

Funding

This work was supported in part by the NIH Cancer Center Support Grant CA06927 to Fox Chase Cancer Center, as well as NIH grants T32 CA009035 to J.F.W., R01 GM084453 and R01 GM111819 to R.L.D., and R21 CA181868 to M.K.R. was partially supported by a grant from the Rosetta Commons (rosettacommons.org).

Supplemental Material

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