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General strategy for the generation of human antibody variable domains with increased aggregation resistance.

ABSTRACT: The availability of stable human antibody reagents would be of considerable advantage for research, diagnostic, and therapeutic applications. Unfortunately, antibody variable heavy and light domains (V(H) and V(L)) that mediate the interaction with antigen have the propensity to aggregate. Increasing their aggregation resistance in a general manner has proven to be a difficult and persistent problem, due to the high level of sequence diversity observed in human variable domains and the requirement to maintain antigen binding. Here we outline such an approach. By using phage display we identified specific positions that clustered in the antigen binding site (28, 30-33, 35 in V(H) and 24, 49-53, 56 in V(L)). Introduction of aspartate or glutamate at these positions endowed superior biophysical properties (non-aggregating, well-expressed, and heat-refoldable) onto domains derived from common human germline families (V(H)3 and V(?)1). The effects of the mutations were highly positional and independent of sequence diversity at other positions. Moreover, crystal structures of mutant V(H) and V(L) domains revealed a surprising degree of structural conservation, indicating compatibility with V(H)/V(L) pairing and antigen binding. This allowed the retrofitting of existing binders, as highlighted by the development of robust high affinity antibody fragments derived from the breast cancer therapeutic Herceptin. Our results provide a general strategy for the generation of human antibody variable domains with increased aggregation resistance.

SUBMITTER: Dudgeon K

PROVIDER: S-EPMC3390889 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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General strategy for the generation of human antibody variable domains with increased aggregation resistance.

Dudgeon Kip K Rouet Romain R Kokmeijer Iris I Schofield Peter P Stolp Jessica J Langley David D Stock Daniela D Christ Daniel D

Proceedings of the National Academy of Sciences of the United States of America 20120627 27

The availability of stable human antibody reagents would be of considerable advantage for research, diagnostic, and therapeutic applications. Unfortunately, antibody variable heavy and light domains (V(H) and V(L)) that mediate the interaction with antigen have the propensity to aggregate. Increasing their aggregation resistance in a general manner has proven to be a difficult and persistent problem, due to the high level of sequence diversity observed in human variable domains and the requireme ...[more]

PMID: 22745168

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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 ?-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

General strategy for the generation of human antibody variable domains with increased aggregation resistance.

Publications

General strategy for the generation of human antibody variable domains with increased aggregation resistance.

Similar Datasets

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