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 x 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.

Epitope‐specificity of recombinant antibodies reveals promiscuous peptide‐binding properties

“…In this study, we used human recombinant scFv antibodies selected from a large phage display library 32 as binding probes. In order to make sure that the phenomenon of degenerate peptide-binding specificity was biologically relevant and not a bias introduced by the library design, we examined the reactivity pattern of two conventional antibodies raised in rabbits against similar tryptic peptide motifs.…”

“…Eight human recombinant CIMS scFv antibody fragments directed against five short C-terminal amino acid peptide motifs were selected from the n-CoDeR library, 32 and kindly provided by Bioinvent International AB, Sweden (Table I). CIMS antibodies selected against the same selection peptide, but displaying different amino acid sequences in their antigen-binding regions, were denoted sister clones.…”

Preclinical Antitumor Efficacy of BAY 1129980—a Novel Auristatin-Based Anti-C4.4A (LYPD3) Antibody–Drug Conjugate for the Treatment of Non–Small Cell Lung Cancer

“…The C4.4A-Ab (BAY 1112623) was generated by phage display panning using the n-CoDeR library of BioInvent International AB (24) as described in the Supplementary Methods and previously (24)(25)(26)(27). All positions in the complementaritydetermining regions (CDR) of the C4.4A-Ab not required for antigen binding were reverted to human germline sequences as previously described (27).…”

Perspectives for systematic in vitro antibody generation

“…The diversity is introduced by PCRs with DNA -oligonucleotides having degenerated codons at desired positions. This procedure has the advantage that variations are only allowed in positions essential for antigen binding and by choosing wellexpressed and well-folding frameworks (Pini et al, 1998;Knappik et al, 2000;Sö derlind et al, 2000). Both naïve and semi-synthetic antibody libraries have yielded antibodies with KD's down to the sub-nanomolar range, so both types of libraries -or combinations thereof -can be considered as sources for the selection of antibodies for functional genomics.…”