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

MAbs
Andreas LehmannMatthew K Robinson

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 VH and VL 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 VH and VL 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 VL domain to one that best pairs with the existing VH. We describe a method to exchange lambda sequences with a more stable kappa3 framework (KV3) within the VL domain that incorporates the original lambda DE-lo...Continue Reading

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Citations

Apr 28, 2018·PLoS Computational Biology·Jared Adolf-BryfogleRoland L Dunbrack
Dec 8, 2017·Frontiers in Immunology·Peter BannasFriedrich Koch-Nolte
Dec 11, 2019·Antibodies·Mark L ChiuGary L Gilliland
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Aug 23, 2018·BioDrugs : Clinical Immunotherapeutics, Biopharmaceuticals and Gene Therapy·Bushra Husain, Diego Ellerman
Feb 27, 2021·Science·Rogelio A Hernandez-LopezWendell A Lim
Oct 16, 2020·International Journal of Molecular Sciences·Manali S SawantPeter M Tessier
Mar 12, 2021·Biochemistry·Clara T SchoederRocco Moretti

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Methods Mentioned

BETA
PCR
circular dichroism
light scattering
Dynamic light scattering
size-exclusion chromatography
Sizing
FACS

Software Mentioned

FlowJo
THESEUS
Dynamics V6
SCWRL4
UCSF Chimera
Rosetta
Modeler
RosettaAntibody
RosettaAntibody webserver
PyIgClassify

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