Phage display technology is associated with the genetic engineering of bacteriophages and the repetition of multiple rounds of antigen-guided selection and phage reproduction. Generally, the tool facilitates the production and the selection of monoclonal antibodies in vitro.
The process of antibody phage display starts with the preparation of an antibody-library and the ligation of antibody variable heavy (VH) and light chain (VL) PCR products into a phage display vector (phagemid). In order to express a fusion protein containing VH and VL in combination with the pIII minor capsid protein derived from the M13 bacteriophage, the phage display vector is constructed. Moreover, the vector does not contain all genes necessary to form a full bacteriophage. Therefore, a helperphage, which is also called hyperphage, is used that contains the phage genome including a pIII minor capsid deletion. Furthermore, the helperphage is cotransformed with the phage display vector library to E.coli resulting in a library of phages that carry a functional pIII on their surface but lack the pIII gene in their genome. Each phage expresses a mAb displayed on the phage surface and harbors the respective nucleotide sequence.
In order to find the phage displaying the target mAb, the phage library is screened by a process called panning. In this process, the phage library expressing different kinds of mAbs on its surface is introduced to immobilized antigens. This technique allows the selection of rare antigen-binding clones and includes of multiple rounds of phage binding to the antigen, washing steps, elution and reamplification steps via E.coli. After several rounds (most commonly 3-4 rounds for non-eukaryotic cells) of selecting specific binders and washing away of non-binders, highly specific phage clones remain.
Afterwards, testing of the phage pool is performed by phage ELISA. The identified clones of interest can easily be produced by E.coli by transforming the bacteria with the phage display vector of interest. Moreover, the monoclonal antibodies can then be purified by metal chelation and affinity purification since the phagemid encodes a protease cleavage site between pIII and the antibody fragment (scFv).
The antibody phage display technology has proven to be a robust platform to discover human antibodies and it is a powerful tool do improve the properties of antibodies.
