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.

Keywords: affinity maturation; combinatorial libraries; human monoclonal antibodies; phage display.

Figure 1

Construction of antibody libraries can be done either using a phage or phagemid vector. An antibody sequence (AS) is located between a leader sequence (LP) and a pIII coding sequence (pIII). Both vectors consist of a promoter (P), an antibiotic resistance (AR) gene for selection, a tag (T) for detection or purification, and an f1 origin (f1 ori) for replication of single-stranded DNA and generation of antibody-displayed phage particles. Phagemid vectors have in addition a plasmid origin (plasmid ori) for propagation in Escherichia coli. An amber stop codon (AC) is essential for both phage and phagemid vectors and located in between the antibody and pIII coding sequences. Phage vectors will have the genes of the other coat proteins (pX, pV, pVII, pIX, pVIII, pVI, pI, and pIV), whereas phagemid vectors only have the pIII gene.

Figure 2

The biopanning process utilizing an antibody phage library against various target antigens. The process encompasses a few steps, including (1) preparation and (2) purification of the antibody phage library, and (3) repetitive binding, washing, (4) elution, rescue, and (5) amplification of the bound phages. The process is usually repeated three to five times to obtain an enrichment population.