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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 T cell receptor (TCR) targeting, PD-1 agonist bispecifics called ImmTAAI molecules that mimic the ability of PD-L1 to facilitate the colocalization of PD-1 with the 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 signaling 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.

In a recombinant antibody scFv format, the presence of an unpaired cysteine (Cys) is implicated in reduced soluble expression and inefficient presentation in phage display. Compared to other species, antibodies derived from rabbits are more likely to contain this unpaired Cys residue at position 80 (Cys80), when generated in a scFv format. In a screening campaign to isolate rabbit scFv against cardiac troponin I (cTnI), it was found that, a large proportion of isolated cTnI-specific clones contained unpaired Cys80. To analyze the factors that led to the selection of anti-cTnI Cys80 scFv, after five rounds of biopanning, the biopanning experiments were repeated with a Cys80 scFv (MG4 Cys ), its alanine variant (MG4 Ala ), and an irrelevant high expressing scFv control. It was found that the selection and subsequent enrichment of MG4 Cys scFv was ousted by the superior expressing variant MG4 Ala , indicating that the Cys80 scFv was selected primarily due to its affinity. It is evident that phage-based selection is influenced by specific sequence characteristics affecting the expression as well as the binding specificity and this needs to be taken into account for selection of optimal antibody derivatives.

Surface plasmon resonance (SPR) is now widely embraced as a technology for monitoring a diverse range of protein-protein interactions and is considered almost de rigueur for characterizing antibody-antigen interactions. The technique obviates the need to label either of the interacting species, and the binding event is visualized in real time. Thus, it is ideally suited for screening crude, unpurified antibody samples that dominate early candidate panels following antibody selection campaigns. SPR returns not only concentration and affinity data but when used correctly can resolve the discrete component kinetic parameters (association and dissociation rate constants) of the affinity interaction. Herein, we outline some SPR-based generic antibody screening configurations and methodologies in the context of expediting data-rich ranking of candidate antibody panels and ensuring that antibodies with the optimal kinetic binding characteristics are reliably identified.

The use of optical biosensors for studying macromolecular interactions is gaining increasing popularity. In one study, 1514 papers that involved the application of biosensor data were identified for the year 2009 alone (Rich and Myszka, J Mol Recognit 24:892-914, 2011), the sheer volume and variety of which present a daunting task for the burgeoning biosensor user to accumulate and decipher. This chapter is designed to provide the reader with the tools necessary to prepare, design, and efficiently execute a kinetic experiment on Biacore. It is written to guide the Biacore user through basic theory, system maintenance, and assay setup while also offering some practical tips that we find useful for Biacore-based studies. Many kinetic-based screening assays require rigorous sample preparation and purification prior to analysis. To highlight these procedures, this protocol describes the kinetic characterization of single chain Fv (scFv) antibody fragments from crude bacterial lysates using an antibody affinity capture approach. Even though we specifically describe the capture of HA-tagged scFv antibody fragments to an anti-HA tag monoclonal antibody-immobilized surface prior to kinetic analysis, the same methodologies are universally applicable and can be used for practically any affinity pair and most Biacore systems.

Rabbit-derived recombinant antibodies have traditionally been viewed as intractable molecules due to the presence of a cysteine in position 80 of the VL domain that becomes rendered 'aberrant' when present in the 'unpaired' context of a single chain Fv (scFv) and chimeric Fab formats. This aberrant Cys80 can severely impinge on the achievable expression levels when rabbit recombinant antibodies are produced in prokaryote systems. The unpaired Cys residue also renders purification problematic. Consequently, researchers often disregard rabbit antibody libraries due to perceived limitations in accessible repertoire diversity. We have shown that by switching the orientation of the VH and VL domains in an aberrant-Cys-containing rabbit scFv isolated in a bona fide screening campaign, it was possible to substantially increase the expression and purification yields of this clone. Furthermore, by incorporating a novel rabbit C-kappa constant fusion domain, we were able to potentiate a further increase in expression level and purify this antibody to a high degree of homogeneity, hitherto impossible to achieve using the aberrant-Cys-containing wild-type scFv. Cumulatively, these findings demonstrate that facile re-formatting can help make the rabbit antibody repertoire, a very valuable resource, more accessible to researchers in the field.