Cognizance of Molecular Methods for the Generation of Mutagenic Phage Display Antibody Libraries for Affinity Maturation

Abstract

Antibodies leverage on their unique architecture to bind with an array of antigens. The strength of interaction has a direct relation to the affinity of the antibodies towards the antigen. In vivo affinity maturation is performed through multiple rounds of somatic hypermutation and selection in the germinal centre. This unique process involves intricate sequence rearrangements at the gene level via molecular mechanisms. The emergence of in vitro display technologies, mainly phage display and recombinant DNA technology, has helped revolutionize the way antibody improvements are being carried out in the laboratory. The adaptation of molecular approaches in vitro to replicate the in vivo processes has allowed for improvements in the way recombinant antibodies are designed and tuned. Combinatorial libraries, consisting of a myriad of possible antibodies, are capable of replicating the diversity of the natural human antibody repertoire. The isolation of target-specific antibodies with specific affinity characteristics can also be accomplished through modification of stringent protocols. Despite the ability to screen and select for high-affinity binders, some ‘fine tuning’ may be required to enhance antibody binding in terms of its affinity. This review will provide a brief account of phage display technology used for antibody generation followed by a summary of different combinatorial library characteristics. The review will focus on available strategies, which include molecular approaches, next generation sequencing, and in silico approaches used for antibody affinity maturation in both therapeutic and diagnostic applications.

Protein design and variant prediction using autoregressive generative models

“…This experiment is a proof-of-concept that this small library contains antigen-binding sequences that can be starting points for affinity maturation to identify strong binders. Though not explicitly designed to minimize poly-reactive nanobody sequences, training on a naïve llama repertoire, which presumably contains a moderate proportion of poly-reactive sequences [81][82][83][84][85][86][87] , the designed library shows similar levels of poly-reactivity to the synthetic library, which had been designed according to a small set of highly specific nanobodies (Supplementary Fig. 11).…”

Peptides, Antibodies, Peptide Antibodies and More

“…For phage display, engineered single chain fragment variable (scFv) antibodies are a preferred format due to the reduced size, which makes expression and production easier. Affinity maturation of such scFvs is feasible by various methods, as described by Lim et al in this issue, but may require substantial work before Abs with the desired properties are obtained [114].…”

How repertoire data are changing antibody science

“…The "humanness" of the repertoire was validated through sequencing of the mouse BCR repertoire (114). Sequencing techniques have been used to characterize phage display libraries, to monitor their diversity and hence evaluate their capability of isolating antibodies that bind to different antigens (115). Screening libraries can also be designed using BCR repertoire data-Zhai et al (116) and Prassler et al (117) have shown how this is possible, by reproducing the observed amino acid usages at each sequence position.…”