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.
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-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.
a Molecular Therapeutics Program, Fox Chase Cancer Center , Philadelphia , PA.|b Current address: Biogen , Cambridge MA.
Andreas
A
Lehmann
a Molecular Therapeutics Program, Fox Chase Cancer Center , Philadelphia , PA.
Josephine H F
JH
Wixted
a Molecular Therapeutics Program, Fox Chase Cancer Center , Philadelphia , PA.
Maxim V
MV
Shapovalov
a Molecular Therapeutics Program, Fox Chase Cancer Center , Philadelphia , PA.
Heinrich
H
Roder
a Molecular Therapeutics Program, Fox Chase Cancer Center , Philadelphia , PA.
Roland L
RL
Dunbrack
a Molecular Therapeutics Program, Fox Chase Cancer Center , Philadelphia , PA.
Matthew K
MK
Robinson
D000595:Amino Acid Sequence; D000915:Antibody Affinity / Q000276:immunology; D015195:Drug Design; D066246:ErbB Receptors / Q000276:immunology*; D006801:Humans; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007146:Immunoglobulin lambda-Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019151:Peptide Library; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D017386:Sequence Homology, Amino Acid; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D013816:Thermodynamics
D007147:Immunoglobulin Light Chains; D007145:Immunoglobulin kappa-Chains; D007146:Immunoglobulin lambda-Chains; D019151:Peptide Library; D057127:Single-Chain Antibodies; C512478:EGFR protein, human; D066246:ErbB Receptors
EGFR; antibody engineering; scFv antibody; stability engineering; thermostability