Purpose Human interleukin-21 (IL-21) is a class I cytokine previously reported in clinical studies on immune responsive cancers. Here we report the eVects of systemic IL-21 therapy on the immune system in two phase 1 trials with this novel cytokine. Experimental design Recombinant IL-21 was administered by intravenous bolus injection at dose levels from 1 to 100 g/kg using two planned treatment regimens: thrice weekly for 6 weeks (3/week); or once daily for Wve consecutive days followed by nine dose-free days (5 + 9). The following biomarkers were studied in peripheral blood mononuclear cells (PBMC) during treatment: phosphorylation of STAT3, alterations in the composition of leukocyte subsets, ex vivo cytotoxicity, expression of eVector molecules in enriched CD8 + T cells and CD56 + NK cells by quantitative RT-PCR, and gene array proWling of CD8 + T cells. Results EVects of IL-21 were observed at all dose levels. In the 5 + 9 regimen IL-21 induced a dose dependent decrease in circulating NK cells and T cells followed by a return to baseline in resting periods. In both CD8 + T cells and CD56+ NK cells we found up-regulation of perforin and granzyme B mRNA. In addition, full transcriptome analysis of CD8 + T cells displayed changes in several transcripts associated with increased cell cycle progression, cellular motility, and immune activation. Finally, cytotoxicity assays showed that IL-21 enhanced the ability of NK cells to kill sensitive targets ex vivo. Conclusions IL-21 was biologically active at all dose levels administered with evidence of in vivo NK cell and CD8 + T cell activation.

IBD and experimental murine colitis have a high degree of similarity in the colonic transcriptional profile, probably secondary to non-specific inflammatory processes. However, differences do exist between models, emphasising the need for careful selection and interpretation of qualified animal models in preclinical research.

Hemophilia A (HA) is a bleeding disorder resulting from deficient Factor VIII (FVIII), which normally functions as a cofactor to activated Factor IX (FIXa) that facilitates activation of Factor X (FX). To mimic this property in a bispecific antibody (biAb) format, a screening was conducted to identify functional pairs of anti-FIXa and anti-FX antibodies, followed by optimization of functional and biophysical properties. The resulting biAb (Mim8) assembled efficiently with FIXa and FX on membranes, and supported activation with an apparent equilibrium dissociation constant (KD) of 16 nM. Binding affinity with FIXa and FX in solution was much lower, with KD-values for FIXa and FX of 2.3 and 1.5 µM, respectively. In addition, the activity of Mim8 was dependent on stimulatory activity contributed by the anti-FIXa arm, which enhanced the proteolytic activity of FIXa by four orders of magnitude. In hemophilia A plasma and whole blood, Mim8 normalized thrombin generation and clot formation with potencies 13 and 18 times higher than a sequence-identical analog of emicizumab, respectively. A similar potency difference was observed in a tail-vein transection model in hemophilia A mice, while reduction of bleeding in a severe tail-clip model was observed only for Mim8. Furthermore, the pharmacokinetics of Mim8 were investigated and a half-life of 14 days demonstrated in cynomolgus monkey. In conclusion, Mim8 is a FVIIIa-mimetic with a potent and efficacious hemostatic effect based on preclinical data.

We demonstrate that IL-33, GLP-1R, and CCL20 are deregulated in human IBD, and that prophylactic treatment with 0.6 mg/kg liraglutide improves disease in AdTr colitis. In addition, GLP-1 receptor agonists upregulate IL-33, mucin 5b, and CCL20 in murine Brunner's glands. Taken together, our data indicate that GLP-1 receptor agonists affect gut homeostasis in both proximal and distal parts of the gut.

To understand the ability of regulatory T-cells to control diabetes development in clinically relevant situations, we established a new model of accelerated diabetes in young DP-BB rats by transferring purified T-cells from DR-BB rats made acutely diabetic. Transfer of 3, 5, 10, or 23 million pure in vitro؊activated T-cells accelerated diabetes onset in >90% of the recipients, with the degree of acceleration being dosage dependent. Cotransfer of unfractionated leukocytes from healthy donors prevented diabetes. Full protection was achieved when protective cells were transferred 3-4 days before diabetogenic cells, whereas transfer 2 days before conferred only partial protection. Protection resided in the CD4 ؉ fraction, as purified CD4 ؉ T-cells prevented the accelerated diabetes. When CD25 ؉ cells were depleted from these cells before they were transferred, their ability to prevent diabetes was impaired. CD25ϩ cells prevent Th1-and Th2-induced colitis as well as Leishmania major infection (10), control active experimental autoimmune encephalomyelitis (11), and ameliorate established colitis (12).In the NOD mouse model of human type 1 diabetes, regulatory T-cells were first defined as a subpopulation of cells expressing CD4 ϩ CD45RB low or CD4 ϩ CD62L ϩ (13-15). Later, these regulatory T-cells were shown to coexpress CD62L and CD25 (16).In the other spontaneous model of type 1 diabetes, the DP-BB, regulatory cells have not been characterized with regard to their expression of CD25. Previously, spontaneous disease was prevented by a single transfer of spleen cells (17) and the prevention was enhanced by enrichment for CD4 ϩ cells (18). However, transfer of leukocytes from normal rats has so far been successful only if the transfer takes place before the initiation of insulitis (i.e., at about age 1 month) (17,19); transfers conducted at 2 months accelerate diabetes (19). Protection is dependent on the engraftment of the ART2 ϩ (formerly RT6) cells (20); in the control strain, the DR-BB, depletion of these induces diabetes (21). We recently showed that accelerated diabetes induced by the transfer of in vitro phorbol myristate acetate (PMA) plus ionomycinϪactivated unfractionated splenocytes from acutely diabetic donors is delayed by transfer of DR-BBϪderived leukocytes (22).In this study, we proposed to prevent diabetes using a new rat model where diabetes onset was accelerated by transfer of pure, in vitro PMA plus ionomycinϪactivated T-cells; that is, without activated antigen-presenting cells as were used in our previous study (22). Using preactivated diabetogenic T-cells in the current study allowed us to explore a situation that is relevant to understanding how such already activated T-cells can be controlled in patients with ongoing ␤-cell destruction. Establishing the conditions required for preventing diabetes by cotransferring various leukocytes from healthy DR-BB rats allowed us to narrow a powerful regulatory potential to CD4 ϩ CD25 ϩ T-cells. This new model in rats is well suited to testing preventive s...