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The drive to exploit novel targets and biological pathways has lead to the expansion of classical antibody research into innovative fragment adaptations and novel scaffolds. The hope being that alternative or cryptic epitopes may be targeted, tissue inaccessibility may be overcome, and easier engineering options will facilitate multivalent, multi-targeting approaches. To this end, we have been isolating shark single domains to gain a greater understanding of their potential as therapeutic agents. Their unique shape, small size, inherent stability, and simple molecular architecture make them attractive candidates from a drug discovery perspective. Here we describe protocols to capture the immune repertoire of an immunized shark species and to build and select via phage-display target-specific IgNAR variable domains (VNARs).

The PD-1/PD-L1 pathway is a key immune checkpoint that regulates T cell activation. There is strong rationale to develop PD-1 agonists as therapeutics against autoimmunity, but progress in this area has been limited. Here, we generated TCR targeting, PD-1 agonist bispecifics called ImmTAAI molecules that mimic the ability of PD-L1 to facilitate the co-localization of PD-1 with the T cell receptor (TCR) complex at the target cell-T cell interface. PD-1 agonist ImmTAAI molecules specifically bound to target cells and were highly effective in activating the PD-1 receptor on interacting T cells to achieve immune suppression. Potent PD-1 antibody ImmTAAI molecules closely mimicked the mechanism of action of endogenously expressed PD-L1 in their localization to the target cell-T cell interface, inhibition of proximal TCR signalling events and suppression of T cell function. At picomolar concentrations, these bispecifics suppressed cytokine production and inhibited CD8 T cell-mediated cytotoxicity in vitro. Crucially, in soluble form the PD-1 ImmTAAI molecules were inactive and hence could avoid systemic immunosuppression. This study outlines a promising new route to generate more effective, potent, tissue-targeted PD-1 agonists that can inhibit T cell function locally with the potential to treat autoimmune and chronic inflammatory diseases of high unmet need.

Low yields, poor folding efficiencies and improper disulfide bridge formation limit large-scale production of cysteine-rich proteins in Escherichia coli. Human renal dipeptidase (MDP), the only human beta-lactamase known to date, is a homodimeric enzyme, which contains six cysteine residues per monomer. It hydrolyses penem and carbapenem beta-lactam antibiotics and can cleave dipeptides containing amino acids in both D: - and L: -configurations. In this study, MDP accumulated in inactive form in high molecular weight, disulfide-linked aggregates when produced in the E. coli periplasm. Mutagenesis of Cys361 that mediates dimer formation and Cys93 that is unpaired in the native MDP led to production of soluble recombinant enzyme, with no change in activity compared with the wild-type enzyme. The removal of unpaired or structurally inessential cysteine residues in this manner may allow functional production of many multiply disulfide-linked recombinant proteins in E. coli.