The inhibitory mechanism of these mAbs does not appear to be linked to cytotoxicity (Supplementary Figure 3). only partially competed with Nbs for binding to hVISTA. All mAbs and one Nb (hVISTANb7) were able to strongly detect VISTA expression on primary human monocytes. Importantly, the murine anti-hVISTA mAbs 7E12 and 7G5 displayed strong agonistic activity in human peripheral blood mononuclear cell cultures, while Nb7 and rat anti-hmVISTA mAbs 3C3, 7C6, 7C7, and 7G1 also behaved as hVISTA agonists, albeit to a lesser extent. Cross-reactive mAbs 7C7 and 7G1 further displayed agonistic potential in murine splenocyte assays. Importantly, mAb 7G1 significantly reduced inflammation associated with the murine model of imiquimod-induced psoriasis. These agonistic VISTA mAbs may represent therapeutic leads to treat inflammatory disorders. use of the cross-species anti-human/murine VISTA (anti-hmVISTA) mAb clone 7G1 in an IMQ-induced murine model of psoriasis exemplifies the therapeutic potential of agonizing VISTA for the treatment of inflammatory disorders. Results Characterization of anti-VISTA mAbs and Nbs Following ELISA screenings against human and mouse VISTA (hVISTA and mVISTA), five hybridoma clones generating murine anti-hVISTA mAbs (7G5, 7E12, 10B5, 5F2, and 8G10) and five hybridoma clones generating rat anti-hmVISTA mAbs (3C3, 7C6, 7C7, 7G1, and 11A1) were selected and further expanded based on their production levels. All of the selected murine clones generated mouse IgG1 anti-hVISTA mAbs. The variable regions of the five chosen clones were sequenced and pooled into three different families based on similarities in their heavy chain complementarity-determining region 3 (CDR3) (Physique 1a), a region typically associated with mAb specificity.14 The presence of heavy and light chains as well as the purity of mAbs were confirmed by SDS-PAGE and by Western blot detected using an anti-mouse IgG heavy and light chain antibody (Determine 1b). Physique 1. Sequence alignment and production of anti-VISTA mAbs and Nbs. (a) Sequence alignments of the variable regions of heavy chains of mouse anti-hVISTA mAbs, anti-hVISTA Nbs, and rat anti-hmVISTA mAbs, clustered based on sequence similarity of CDR3. (b) SDS-PAGE gel and Western blot of anti-VISTA mAbs and Nbs under reducing conditions. Bands on Western blots were detected using an antibody conjugated to HRP that recognizes heavy and light chains of mouse or rat IgGs Representative mAbs from each of the three families, namely 7E12, 7G5, and 8G10, were chosen for further characterization based on their sub-nanomolar binding (equilibrium dissociation constant; KD) to human VISTA (KD values of 0.44?nM, 0.14?nM, and 0.98?nM, respectively) as measured by surface plasmon resonance (SPR) (Figure 2a). As expected, none of these murine antibodies recognized recombinant mouse VISTA (Supplementary Figure s1). In addition to the murine anti-hVISTA clones, five rat hybridoma clones were selected based on their production of mAbs that bind to both human and murine VISTA. All five clones produce mAbs (Figure 1b) Aldosterone D8 of the rat IgG2a subtype. As sequencing shows that two of the rat mAbs (7C6 and 11A1) have the same heavy chain sequence, four hybridoma Aldosterone D8 clones were selected for further characterization (3C3, 7C6, 7C7, and 7G1) (Figure 1a). All of the selected clones bound to both hVISTA (KD values of 0.24?nM, 840?nM, 49?nM, and 44?nM, respectively; Figure 2b) and mVISTA (KD values of 1 1.3 pM, 3?nM, 51?nM, and 6.7?nM, respectively; Figure 2c). Figure 2. Binding of mAbs and Nbs to VISTA. (a-c) SPR single-cycle kinetics sensorgrams (in red) and fitted Rabbit Polyclonal to Tyrosine Hydroxylase curves (in black) with equilibrium (KD), association (kon) and dissociation (koff) rate constants depicting the binding of (a) mouse anti-hVISTA mAbs (7E12, Aldosterone D8 7G5, 8G10) and anti-hVISTA Nbs (Nb1, Nb5, Nb7) to human VISTA, (b) rat anti-hmVISTA mAbs (3C3, 7C6, 7C7, 7G1) to human VISTA, and (c) rat anti-hmVISTA mAbs to mouse VISTA. (d) Competition ELISA of anti-hVISTA Nbs, or an irrelevant control Nb19, with representative anti-hVISTA (7E12) or anti-hmVISTA (7C6) mAbs (n?=?3) In addition to the mAbs, seven positive anti-hVISTA Nbs phage clones (hVISTANb1 to 7; Nb1 to Nb7) were identified from bio-panning against hVISTA. The seven anti-hVISTA Nbs phage clones identified were regrouped into three families based Aldosterone D8 on amino acid sequence differences within their CDR domains (Figure 1a). One clone corresponding to each of the families, termed Nb1, Nb5, and Nb7, was chosen for production and characterization. All three Nbs were successfully purified as evidenced by the expected molecular weight band (~15 kDa).