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Affinity maturation of antibodies by combinatorial codon mutagenesis versus error-prone PCR.

ABSTRACT:

In vitro

affinity maturation of therapeutic monoclonal antibodies is commonly applied to achieve desired properties, such as improved binding kinetics and affinity. Currently there are no universally accepted protocols for generation of variegated antibody libraries or selection thereof. Here, we performed affinity maturation using a yeast-based single-chain variable fragment (scFv) expression system to compare two mutagenesis methods: random mutagenesis across the entire V(D)J region by error-prone PCR, and a novel combinatorial mutagenesis process limited to the complementarity-determining regions (CDRs). We applied both methods of mutagenesis to four human antibodies against well-known immuno-oncology target proteins. Detailed sequence analysis showed an even mutational distribution across the entire length of the scFv for the error-prone PCR method and an almost exclusive targeting of the CDRs for the combinatorial method. Though there were distinct mutagenesis profiles for each target antibody and mutagenesis method, we found that both methods improved scFv affinity with similar efficiency. When a subset of the affinity-matured antibodies was expressed as full-length immunoglobulin, the measured affinity constants were mostly comparable to those of the respective scFv, but the full-length antibodies were inferior to their scFv counterparts for one of the targets. Furthermore, we found that improved affinity for the full-length antibody did not always translate into enhanced binding to cell-surface expressed antigen or improved immune checkpoint blocking ability, suggesting that screening with full-length antibody or antigen-binding fragment formats might be advantageous and the subject of a future study.

SUBMITTER: Simons JF

PROVIDER: S-EPMC7531523 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The identification of mutations that enhance antibody affinity while maintaining high antibody specificity and stability is a time-consuming and laborious process. Here, we report an efficient methodology for systematically and rapidly enhancing the affinity of antibody variable domains while maximizing specificity and stability using novel synthetic antibody libraries. Our approach first uses computational and experimental alanine scanning mutagenesis to identify sites in the complementarity-determining regions (CDRs) that are permissive to mutagenesis while maintaining antigen binding. Next, we mutagenize the most permissive CDR positions using degenerate codons to encode wild-type residues and a small number of the most frequently occurring residues at each CDR position based on natural antibody diversity. This mutagenesis approach results in antibody libraries with variants that have a wide range of numbers of CDR mutations, including antibody domains with single mutations and others with tens of mutations. Finally, we sort the modest size libraries (~10 million variants) displayed on the surface of yeast to identify CDR mutations with the greatest increases in affinity. Importantly, we find that single-domain (VHH) antibodies specific for the α-synuclein protein (whose aggregation is associated with Parkinson's disease) with the greatest gains in affinity (>5-fold) have several (four to six) CDR mutations. This finding highlights the importance of sampling combinations of CDR mutations during the first step of affinity maturation to maximize the efficiency of the process. Interestingly, we find that some natural diversity mutations simultaneously enhance all three key antibody properties (affinity, specificity, and stability) while other mutations enhance some of these properties (e.g., increased specificity) and display trade-offs in others (e.g., reduced affinity and/or stability). Computational modeling reveals that improvements in affinity are generally not due to direct interactions involving CDR mutations but rather due to indirect effects that enhance existing interactions and/or promote new interactions between the antigen and wild-type CDR residues. We expect that natural diversity mutagenesis will be useful for efficient affinity maturation of a wide range of antibody fragments and full-length antibodies.

Dataset Information

Affinity maturation of antibodies by combinatorial codon mutagenesis versus error-prone PCR.

In vitro

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