Synthetic single-framework antibody library integrated with rapid affinity maturation by VL shuffling

Abstract

Affinity maturation is often applied to improve the properties of antibodies isolated from universal antibody libraries in vitro. A synthetic human scFv antibody library was constructed in single immunoglobulin framework to enable rapid affinity maturation by updated Kunkel's mutagenesis. The initial diversity was generated predominantly in the V(H) domain combined with only 36 V(L) domain variants yielding 3 × 10(10) unique members in the phage-displayed library. After three rounds of panning the enriched V(H) genes from the primary library selections against lysozyme were incorporated into a ready-made circular single-stranded affinity maturation library containing 7 × 10(8) V(L) gene variants. Several unique antibodies with 0.8-10 nM (K(d), dissociation constant) affinities against lysozyme were found after panning from the affinity maturation library, contrasted by only one anti-lysozyme scFv clone with K(d) <20 nM among the clones panned from the primary universal library. The presented single-framework strategy provides a way to convey significant amount of functional V(H) domain diversity to affinity maturation without bimolecular ligation leading to a diverse set of antibodies with binding affinities in the low nanomolar range.

Lactobacillus helveticus MIMLh5-Specific Antibodies for Detection of S-Layer Protein in Grana Padano Protected-Designation-of-Origin Cheese

“…The deduced amino acid sequences were aligned with the library template. The framework gene and anti-S-layer scFvs differed in CDR1 and CDR3 of both heavy and light chains and CDR2 of the heavy chain (Table 2), which corresponds to the phage library design rules (19). Only one clone, PolyF5, originated from the ScFvP repertoire based on the presence of a tryptophan (W) as the last amino acid in the CDR-L3 loop (26).…”

“…These antibodies carry only the variable domains, the minimal fragments needed for antigen recognition by a full-length IgG, linked to each other with a flexible glycine-serine linker (18). Their relatively small size allows easy genetic manipulation and construction of large libraries in the range of 10 10 members (19). The antibody phage display technique for monoclonal antibody generation is much faster than the conventional hybridoma method and does not require any sophisticated equipment (17).…”

Fast conversion of scFv to Fab antibodies using type IIs restriction enzymes

“…The scFv clones specific to glial cell line-derived neurotrophic factor (GDNF) or Journal of Immunological Methods xxx (2015) xxx-xxx glutathione S-transferase (GST) were in alkaline phosphatase vector pAK600 (Krebber et al, 1997). In high-throughput cloning the first 21 scFv clones were from the primary anti-lysozyme phage screening and were in the phagemid vector pEB91 (Brockmann et al, 2011). The 12 anti-streptavidin (STR) clones were in the alkaline phosphatase (AP) vector pLK06 and the 17 anti-prostate specific antigen (PSA) clones The method involves combination of four DNA fragments in one digestion/ligation reaction.…”

Degradation of C-terminal tag sequences on domain antibodies purified fromE. colisupernatant

“…2 Numerous antibody fragment libraries for phage display have been created, and the creation of new libraries continues as knowledge and technology expands the possibilities. [3][4][5][6][7][8][9][10] Improvements concern stability and functionality of the various antibody fragments themselves, regulation of the expression, the fusion partner utilized in the display system and not least the peptide tag sequences applied (Fig. 1).…”