Figure 4

Developing a fully human monoclonal antibody (mAb) using (a) phage display technology and (b) transgenic mouse technology. (a) Step 1: A suitable source of starting material (for example, human blood) is subjected to polymerase chain reaction using appropriate primers, providing 'libraries' of heavy chain V domain (VH) and light chain V domain (VL) sequences. Step 2: Randomly combined VH and VL sequences, connected via a short linker, are incorporated into the genome of a bacteriophage such that they will be expressed at the phage surface. The combination marked with an asterisk encodes the desired specificity. Step 3: The phage library is used to infect a bacterial culture, and the resulting supernatant, containing single-chain Fv-expressing phage particles, is incubated with an appropriate source of target antigen (panning). This can be on a column, Petri dish, and so on. Phage with appropriate specificity adheres to the antigen source. Step 4: Adherent phage is eluted and enriched for the appropriate specificity by further rounds of panning. Step 5: After several rounds of panning, adherent phage is sequenced. A successful procedure should lead to the presence of just one or a few Fv specificities, which can be individually cloned and their specificity checked. At this stage, in vitro affinity maturation procedures can be performed if required (see 'Human antibodies' section for details). Ultimately, the desired specificity is recloned into an appropriate vector containing full-length mAb sequence for expression in a mammalian cell line. (b) Step 1: A transgenic mouse that produces human antibodies is created by targeted disruption of the endogenous murine immunoglobulin heavy- and light-chain genetic loci and their replacement by the equivalent human sequences. Step 2: The mouse, now containing human immunoglobulin genes, is immunised in a conventional manner using the target antigen. Step 3: Splenocytes from the immunised mouse are used to generate hybridomas via conventional fusion technology. Step 4: Resulting hybridomas are screened, leading to isolation and cloning of a hybridoma-secreting high-affinity mAb against the target antigen. Note: In theory, phage display rather than fusion technology can be applied from stage 3 onwards.