The onset of advanced molecular biology techniques created new possibilities to leverage the natural ingenuity of biological systems. In the late 1980s, this led to the construction of the first antibody libraries. These libraries were created by amplifying antibody encoding genes from lymphocytes, cloning them, and expressing them in bacterial systems.
These early attempts at transferring immune and naïve repertoires to the lab preceded the creation of antibody display techniques. At that time, the libraries would be either expressed as single-chain variable fragments (scFv) or as antibody combinatorial libraries of Fab fragments in bacteria. However, screening these libraries was quite labor-intensive and involved the use of radio-labeled antigens to identify the bacterial clones expressing antibodies with the highest affinity and specificity.
Shortly after these initial breakthroughs, the phage display technology was born. It created not only the possibility to screen libraries efficiently but also the ability to engineer antibodies by random and site-directed mutagenesis.
Currently, antibody libraries can be classified as:
Genomic studies led to the discovery of conserved regions in antibody-encoding genes across different species. This discovery further prompted the development of primers targeting these regions, allowing the amplification of variable light and heavy chains (VL and VH, respectively) for many different host species, and subsequent cloning antibody-encoding genes using standard molecular biology techniques.
These VL and VH repertoires are commonly generated from naïve or immune hosts by harvesting either B lymphocytes (B cells) or peripheral blood mononuclear cells (PBMC) which consist of a mixture of T cells, B cells, and NK cells (natural killer cells), isolated from peripheral blood samples by simple density gradient centrifugation.
This initial harvesting step is followed by mRNA isolation, cDNA synthesis, and antibody gene amplification using a mixture of primers that ensures the highest antibody diversity is captured at this stage. These genes are subsequently cloned into the proper vector.
In the case of phage display, vector systems based on Escherichia coli filamentous M13 phage are the most popular choice. M13 is composed of a circular single-stranded DNA (ssDNA) genome enveloped by capsid proteins.
For phage display, the antibody fragment is typically fused to the gp3 (pIII) protein, one of the minor capsid proteins in phage M13. But due to its vital role in adhesion to E. coli cells via the F factor, researchers developed a complex system to preserve M13’s infectivity during antibody display. This system involves the use of a phagemid, a simplified version of M13, containing only:
Moreover, phagemids are complemented using a helper phage (similar to the native M13 phage) with a weakened version of the origin of replication. The phagemid and phage compete in E. coli during replication, assembly, and packaging of the phage particles resulting in a hybrid phage containing a mixture of native gp3 and gp3-antibody fusion complexes.
The phagemid system also ensures that only a single antibody fragment is displayed per phage particle which, in turn, ensures only the binders with the highest affinity towards a specific antigen are enriched during the multiple rounds of biopanning.
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