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Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries

Abstract

We constructed a single-chain Fv antibody library that permits human complementarity-determining region (CDR) gene fragments of any germline to be incorporated combinatorially into the appropriate positions of the variable-region frameworks VH-DP47 and VL-DPL3. A library of 2 × 109 independent transformants was screened against haptens, peptides, carbohydrates, and proteins, and the selected antibody fragments exhibited dissociation constants in the subnanomolar range. The antibody genes in this library were built on a single master framework into which diverse CDRs were allowed to recombine. These CDRs were sampled from in vivo-processed gene sequences, thus potentially optimizing the levels of correctly folded and functional molecules, and resulting in a molecule exhibiting a lower computed immunogenicity compared to naive immunoglobulins. Using the modularized assembly process to incorporate foreign sequences into an immunoglobulin scaffold, it is possible to vary as many as six CDRs at the same time, creating genetic and functional variation in antibody molecules.

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Figure 1: Schematic diagram for the construction of n-CoDeR, using in vivo-proofread gene segments.
Figure 2: Top view of the scFv antibody fragments specific for fluorescein isothiocyanate (FITC; clone FITC8), MUC1 peptide (Muc159), and cholera toxin (&β-subunit) (clone CT17) showing the molecular surface of the individual combining sites.

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Acknowledgements

This investigation was supported by grants from The National Research Council for Engineering Sciences, the National Board for Laboratory Animals, and in part by the BIOSAM program of NUTEK and the European Commission DGXII.

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Correspondence to Eskil Söderlind.

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Söderlind, E., Strandberg, L., Jirholt, P. et al. Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries. Nat Biotechnol 18, 852–856 (2000). https://doi.org/10.1038/78458

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