Arginine mutations in antibody complementarity-determining regions display context-dependent affinity/specificity trade-offs

Abstract

Antibodies commonly accumulate charged mutations in their complementarity-determining regions (CDRs) during affinity maturation to enhance electrostatic interactions. However, charged mutations can mediate non-specific interactions, and it is unclear to what extent CDRs can accumulate charged residues to increase antibody affinity without compromising specificity. This is especially concerning for positively charged CDR mutations that are linked to antibody polyspecificity. To better understand antibody affinity/specificity trade-offs, we have selected single-chain antibody fragments specific for the negatively charged and hydrophobic Alzheimer's amyloid β peptide using weak and stringent selections for antibody specificity. Antibody variants isolated using weak selections for specificity were enriched in arginine CDR mutations and displayed low specificity. Alanine-scanning mutagenesis revealed that the affinities of these antibodies were strongly dependent on their arginine mutations. Antibody variants isolated using stringent selections for specificity were also enriched in arginine CDR mutations, but these antibodies possessed significant improvements in specificity. Importantly, the affinities of the most specific antibodies were much less dependent on their arginine mutations, suggesting that over-reliance on arginine for affinity leads to reduced specificity. Structural modeling and molecular simulations reveal unique hydrophobic environments near the arginine CDR mutations. The more specific antibodies contained arginine mutations in the most hydrophobic portions of the CDRs, whereas the less specific antibodies contained arginine mutations in more hydrophilic regions. These findings demonstrate that arginine mutations in antibody CDRs display context-dependent impacts on specificity and that affinity/specificity trade-offs are governed by the relative contribution of arginine CDR residues to the overall antibody affinity.

Understanding and overcoming trade-offs between antibody affinity, specificity, stability and solubility

“…Complex mixtures such as serum or milk are particularly effective at blocking non-specific interactions. The use of complex mixtures of non-target molecules during positive selections was recently evaluated during the affinity maturation of single-chain (scFv) antibodies against the Alzheimer’s Aβ42 peptide [41]. The investigators generated antibody libraries with sequence diversity restricted to heavy chain CDR3, displayed the libraries on the surface of yeast, and selected for antibody variants with increased affinity.…”

“…A strikingly different result was obtained when the antibody selections were repeated against the Aβ42 peptide in the presence of high concentrations of non-target molecules (1% milk) [41]. In this case, the selected antibodies had increased specificity despite that some of the antibody variants had several arginine CDR mutations.…”

“…The fact that measurements of antibody specificity (non-specific binding and self-association) are the best biophysical descriptors of the likelihood of antibody success in the clinic [67] highlights the importance of developing computational and bioinformatics methods for predicting antibody specificity. Some of the key factors that determine antibody specificity are becoming clearer, including the numbers of charged, hydrophobic and hydrophilic residues in CDRs [26, 27, 39, 41, 42, 44, 68–70] as well as the net charge of the variable regions [46, 47, 49]. However, methods are needed to collectively describe these disparate findings and provide guidelines for identifying antibody variants with high specificity based only on their amino acid sequences or their combined sequences and structures.…”

“…Future work in antibody library design will need to address many outstanding challenges, especially those related to developing constraints for focusing amino acid diversity on mutations that are most likely to lead to high antibody specificity, solubility and stability in addition to high affinity. This is non-trivial because some mutations (e.g., positively charged mutations in the CDRs) that are helpful for one biophysical property (e.g., solubility) can be detrimental for other properties (e.g., specificity) [5, 26, 41]. Therefore, it will be especially important to generate guidelines for predicting antibody CDR sequences that maximize several biophysical properties (e.g., specificity and solubility) without eliminating key interactive residues that contribute to antibody affinity.…”

Nature-inspired design and evolution of anti-amyloid antibodies

“…Therefore, we have sought to combine our motif-grafting (16,18,19,22) and natural diversity mutagenesis (17) methods to enable robust and systematic isolation of conformational antibodies against Alzheimer's A␤42 fibrils with high affinity and specificity ( Fig. 1).…”