Functional aspects of co‐variant surface charges in an antibody fragment

Abstract

A mutational analysis of three co-variant pairs of residues, located at the surface of a single-chain fragment, variable (scFv), remote from the antigen-binding site, was performed to investigate the tolerance of these positions to amino acid changes. The replacements consisted of the elimination or addition of charges, or in their replacement by a charge of opposite sign. As measured by Biacore, antigen-binding kinetics and specificity were essentially unaffected by the mutations. The purified scFvs remained mostly 100% active for 14 h, and their sensitivity to guanidinium-chloride denaturation was similar. These observations indicate that the mutations did not affect antigen-binding properties and that protein folding was conserved. However, the various scFvs differed greatly in half-life in periplasmic extracts (<4 h to >16 h at 25°C). The deleterious effect on half-life produced by single mutations could be reversed by introducing a second mutation that restores the natural combination of amino acids in the co-variant pair, indicating that the consequence of charge modifications at these locations depends on the sequence context. We propose that the differences in half-life result from differences in aggregation propensities with other periplasmic proteins, related to the presence of charged patches at the surface of the scFvs. The practical implication is that changes in surface charge may drastically affect the level of active molecules in complex protein mixtures, a potentially important consideration in engineering scFvs for biotechnological or medical purposes.Key words: Co-variance; antibody surface charges; Biacore; antigen-binding properties; half-life; recombinant protein yield A mutational analysis of a recombinant antibody fragment was performed with two aims: (1) to gain a better understanding of the relationship between antibody sequence and function and (2) to identify candidate positions in engineering antibodies for biotechnological or medical applications.We are searching for protein engineering rules, applicable to at least a subgroup of antibodies, and have selected for mutagenesis residues located within the structurally conserved framework region, rather than in the highly variable complementarity determining regions (CDRs) that determine antigen-binding specificity. The residues in question are co-variant positions that were identified in an analysis of antibody germline sequence alignments : Two positions are co-variant if the nature of amino acids at position A is not independent of the nature of amino acids at position B, indicating the existence of functional constraints linking these two residues (Altschuh et al. 1987). The study by Choulier et al. (2000) indicated the existence of a co-variance signal for framework positions located at the surface of the variable region. In particular, for two co-variant pairs identified in mouse antibody germline sequences (L18-L74, H46-H62, Kabat numbering), alternative amino acid types presented large differences in solvation free energ...

Conserved amino acid networks involved in antibody variable domain interactions

“…Despite an enormous amount of research involving antibodies and antibody‐like therapeutics, very little use has been made of covariation analyses to investigate functional features of antibody domains. A study by Altschuh and coworkers59, 60 investigated covariations across murine and human germline V H or V L sequences, with the goal of defining positions within each germline subclass that use mutually exclusive framework amino acid pairs to influence the structural conformations of CDR loops. Our study had a different goal: to use covariation analyses for determining naturally occurring amino acid networks, in antibody variable domains, that are generally important for antibody structure and function.…”

Probing the local conformational change of α1‐antitrypsin

“…The disulfide engineering of K168C–F189C mutant α 1 ‐AT turned out to be a very useful outcome in practical respects. Protein engineering through mutational approaches, such as single amino acid substitutions and disulfide bridges, has been effectively applied to improve protein properties for industrial or medical purposes (Reiter et al 1996; Hugo et al 2002; Eijsink et al 2004; McHugh et al 2004). Patients with α 1 ‐AT deficiency require replacement therapy with available protein (Abusriwil and Stockley 2006).…”

Antibody Binding Selectivity: Alternative Sets of Antigen Residues Entail High-Affinity Recognition

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