Many studies have explored the efficacy of phage display in isolating the autoantibody repertoires of autoantibodies against human epitopes.
For example, Payne and colleagues isolated the repertoires of human anti-Dsg antibodies from patients with active mucocutaneous pemphigus vulgaris using phage display (16). All isolated scFvs bind to Dsg, but only some of them were pathogenic. The pathogenicity was measured by injecting the mAbs into the culture of normal human skin or by passive transfer to neonatal mice. A consensus amino acid sequence (D/E-x-x-x-W) was found in the heavy chain complementarity-determining region 3 (H-CDR3) in six of nine pathogenic antibodies. Also, site-directed mutagenesis revealed that the replacement of amino acid residues in this sequence, especially the tryptophan, could block pathogenicity but not necessarily the binding (17). The pathogenicity is due to direct antibody interference with Dsg adhesion, probably by binding to the W2 acceptor pocket of Dsg, important for its adhesion to cadherin (18,19). In summary, phage display was useful to isolate the repertoires of antibodies against Dsg to find sequence homology between them. Common epitopes in Dsg and hotspot residues can be identified which could be potential therapeutic targets to treat autoimmune diseases.
Another example is the study carried out by Wang et al. where they successfully generated antibodies against the main pathogenic epitope (COL17 NC16A) for autoantibodies in bullous pemphigoid (BP) (20). They combined the heavy- and light-chain genes amplified from antibody repertoires of two BP patients to construct phage display libraries which were subsequently screened against NC16A. Epitope mapping studies indicated that the isolated recombinant Fabs bind different but close or overlapping epitopes on the NC16A domain. Only Fab-B4 and Fab-19 could inhibit the binding of autoantibodies from patients with BP disease to COL17 in vitro, both in an inhibitory ELISA and on skin sections. Also, they inhibited the deposition of BP antibody-activated C1q and C3 at the DEJ in human skin. None of the three Fabs were able to induce clinical signs of BP or histopathological manifestations of BP in a mouse model, however, BP autoantibodies prepared from BP patients induced the disease. Also, these results showed that the selected Fabs were not pathogenic to the mice. Only Fab-B4 and Fab-19 seem to completely block the clinical signs and stimulation of the complement C1q and C3 mediated by BP autoantibodies (20). In conclusion, it was possible with phage display to amplify the repertoire of antibodies against the COL17 NC16A domain from patients with active BP. It was identified Fabs that block the binding of autoantibodies and reduce the clinical signs of BP. This has a therapeutic potential that makes it likely to apply a more specialized treatment for BP or similar diseases.
The effectiveness of the phage display technique has been exemplified in a report by Vugmeyster et al. They selected a group of blocking antibodies against the human and mouse receptors for interleukin 21 (IL-21), a cytokine associated with the pathogenesis of many kinds of autoimmune diseases (21). They constructed a scFv library based on the variable (V) genes isolated from human B-cells from 160 donors (22) and screened it against purified recombinant IL-21 receptor (IL-21R) or expressed by BaF3 cells. A batch of scFvs that recognized human IL-21R and inhibited its binding to IL-21 was selected from this library. From this, 18A5 was the scFv with the strongest blocking activity of IL-21-dependent proliferation of human IL-21R transfected BaF3-, TF1-, B- and T-cells. Further, phage display libraries of variants of 18A5 were constructed by blocking six adjacent codons in the CDR3s in both VH and VL, substituted with NNS randomized codons. Of these libraries, two scFvs fragments (Ab-01 and Ab-02) with only four amino acids differences in VL CDR3 (MSRSIWGNPHVL vs. VARSNKGNPHVL) had the strongest potency of neutralization in cell-based assays. However, Ab-01 and Ab-02, regardless of their similarities, showed distinct pharmacokinetics and pharmacological properties. Also, Ab-01, but not Ab-02, decreased anti-dsDNA antibody titers and the amount of anti-IL-21R antibodies deposited in the kidneys of MRL-Faslpr mice. Untreated mouse strain developed an autoimmune disease that resembled the systemic lupus erythematosus. The effect of Ab-01 was even more remarkable in rodents and cynomolgus monkeys. The reduced blocking effect of Ab-02 was probably due to the consequence of its fast elimination in rodents and monkeys (21). In summary, neutralizing scFvs of the IL-21 and IL-21R interaction were selected by phage display. These scFvs can prevent IgG deposition in the kidney in animal models; therefore, they can be considered potential therapeutic molecules to treat systemic lupus erythematosus.