Dominic C. Borie

A new modality for immunosuppression: targeting the JAK/STAT pathway

Thousands of organs are transplanted each year and millions of people suffer from autoimmune diseases, which creates a need for an armamentarium of immunosuppressive drugs. Unfortunately, immunosuppressants have unwanted side effects owing, in part, to the fact that they have ubiquitous molecular targets. Cytokines have emerged as important controllers of the immune response, and work during the past decade has identified Janus kinases (JAKs) and signal transducers, and activators of transcription (STATs), as crucial intracellular elements in cytokine signalling. Here, we discuss the potential of the JAK/STAT pathway as a target for new immunosuppressants. In particular, the inhibition of JAK3 seems to be an excellent strategy, because of the selective expression and precise functions of this kinase.

Immunosuppression by the JAK3 inhibitor CP-690,550 delays rejection and significantly prolongs kidney allograft survival in nonhuman primates.

Background. Janus kinase 3 (JAK3) mediates signal transduction from cytokine receptors using the common chain (&ggr;c). Because mutations in genes encoding &ggr;c or JAK3 result in immunodeficiency, we investigated the potential of a rationally designed inhibitor of JAK3, CP-690,550, to prevent renal allograft rejection in nonhuman primates. Methods. Life-supporting kidney transplantations were performed between mixed leukocyte reaction-mismatched, ABO blood group-matched cynomolgus monkeys. Animals were treated with CP-690,550 (n=18) or its vehicle (controls, n=3) and were euthanized at day 90 or earlier if there was allograft rejection. Results. Mean survival time (± standard error of mean) in animals treated with CP-690,550 (53±7 days) was significantly longer than in control animals (7±1 days, P=0.0003) and was positively correlated with exposure to the drug (r=0.79, P<0.01). Four treated animals were euthanized at 90 days with a normal renal function and low-grade rejection at final pathology. Occurrence of rejection was significantly delayed in treated animals (46±7 days from transplantation vs. 7±1 days in controls, P=0.0003). Persistent anemia, polyoma virus-like nephritis (n=2), and urinary calcium carbonate accretions (n=3) were seen in animals with high exposure. Natural killer cell and CD4+ and CD8+ T-cell numbers were significantly reduced in treated animals. Blood glucose, serum lipid levels, and arterial blood pressure were within normal range in treated animals, and no cancers were demonstrated. Conclusions. CP-690,550 is the first reported JAK3 inhibitor combining efficacy and good tolerability in a preclinical model of allotransplantation in nonhuman primates and thus has interesting potential for immunosuppression in humans.

Combined use of the JAK3 inhibitor CP-690,550 with mycophenolate mofetil to prevent kidney allograft rejection in nonhuman primates

Background. Immunosuppression via Janus kinase (JAK) 3 inhibition affords significant prolongation of allograft survival. We investigated the effects of an immunosuppressive regimen combining the JAK3 inhibitor CP-690,550 with mycophenolate mofetil (MMF) in nonhuman primates (NHPs). Methods. Life-supporting kidney transplantations were performed between ABO-compatible, MLR-mismatched NHPs. Animals were treated orally twice a day with CP-690,550 and MMF (n=8) or MMF alone (n=2) and were euthanized at day 90 or earlier due to allograft rejection. Results. Mean survival time (±SEM) in animals treated with MMF alone (23±1 days) was significantly extended in animals that concurrently received CP-690,550 (59.5±9.8 days, P=0.02). Combination animals exposed to higher levels of CP-690,550 had a significantly better survival (75.2±8.7 days) than animals that received less CP-690,550 (33.3±12.6 days, P=0.02). Three combination therapy animals were euthanized at day 90 with a subnormal renal function and early-stage acute graft rejection. Rejection, delayed by treatment, ultimately developed in other animals. Anemia and gastrointestinal intolerance was seen in combination therapy animals that otherwise did not show evidence of viral or bacterial infection besides signs consistent with subclinical pyelonephritis (n=3). One incidental lymphosarcoma was noted. Conclusions. Addition of CP-690,550 to MMF significantly improved allograft survival. The observed side effects appear amenable to improvements upon alteration of dosing strategies. Efficacy of this combination regimen suggests that it could become the backbone of calcineurin inhibitor-free regimens.

Effects of JAK3 inhibition with CP-690,550 on immune cell populations and their functions in nonhuman primate recipients of kidney allografts.

Background. Janus Kinase (JAK) 3 is a tyrosine kinase essential for proper signal transduction downstream of selected cytokine receptors and for robust T-cell and natural killer cells activation and function. JAK3 inhibition with CP-690,550 prevents acute allograft rejection. To provide further insight into the mechanisms of efficacy, we investigated the immunomodulatory effects of CP-690,550 in vitro and in vivo in nonhuman primates. Methods. Pharmacodynamic assessments of lymphocyte activation, function, proliferation and phenotype were performed in three settings: in vitro in whole blood isolated from untransplanted cynomolgus monkeys (cynos), in vivo in blood from untransplanted cynos dosed with CP-690,550 for 8 days, and in vivo in blood from transplanted cynos immunosuppressed with CP-690,550. Cell surface activation markers expression, IL-2- enhanced IFN-γ production, lymphocyte proliferation and immune cell phenotype analyzes were performed with multiparametric flow cytometry. Results. In vitro exposure to CP-690,550 resulted in significant reduction of IL-2-enhanced IFN-γ production by T-cells (maximum inhibition of 55-63%), T-cell surface expression of CD25 (50% inhibitory concentration (IC50); 0.18 &mgr;M) and CD71 (IC50; 1.6 &mgr;M), and T-cell proliferative capacities measured by proliferating cell nuclear antigen expression (IC50; 0.87 &mgr;M). Similar results were observed in animals dosed with CP-690,550. In addition, transplanted animals displayed significant reduction of NK cell (90% from baseline) and T-cell numbers whereas CD8+ effector memory T-cell populations were unaffected. Conclusions. Potent in vitro and in vivo immunomodulatory effects of the JAK3 inhibitor CP-690,550 likely contribute to its efficacy in the prevention of organ allograft rejection.

New strategies for immunosuppression: interfering with cytokines by targeting the Jak/stat pathway

Purpose of reviewNumerous immunosuppressants are available, but their adverse effects related to actions on nonlymphoid cells is problematic. Cytokines are key regulators of immune and inflammatory responses, and blocking their actions has become an important modality in treating autoimmune disorders. This review will discuss strategies to develop novel immunosuppressants that arise from advances in the understanding of cytokine signaling. Recent findingsIt is now recognized that large number of cytokines exert their effect by binding to receptors that activate the Janus kinase/signal transducer and activator of transcription pathway, so targeting intracellular signaling pathways is a logical strategy. A selective inhibitor of Janus kinase 3 has now been generated and is effective for transplant rejection in nonhuman primates and other models. Advances have also been made in understanding the functions of Stat family transcription factors, and approaches to interfering with the action of these DNA binding proteins are being devised. In addition, the identification of negative regulators of cytokine signaling offers additional therapeutic opportunities. SummaryA selective inhibitor of Janus kinase 3 has now been generated and likely represents a new class of effective immunosuppressants. Strategies for targeting signal transducers and activators of transcription pathway are being intensively studied at present and hold potential promise. Multiple endogenous mechanisms exist for negatively regulating cytokine signaling; whether novel therapies can be devised that exploit these mechanisms remains to be determined.

Janus kinase 3 inhibition with CP-690,550 prevents allograft vasculopathy

Janus kinase 3 (JAK3) mediates signal transduction from cytokine receptors using the common γ chain. The rationally designed inhibitor of JAK3, CP‐690,550, prevents acute allograft rejection in rodents and in nonhuman primates. Here we investigated the ability of CP‐690,550, to prevent allograft vasculopathy in a rodent model of aorta transplantation. Aortas from AxC Irish (RT1a) or Lewis (RT1l) rats were heterotopically transplanted into the infra‐renal aorta of Lewis recipients and harvested at 28 or 56 days. Treated recipients received CP‐690,550 by osmotic pumps (mean drug exposure of 110 ± 38 ng/ml). Significant intimal hyperplasia was demonstrated in untreated allografts when compared with isografts at 28 days (2.08 ± 0.85% vs. 0.43 ± 0.2% luminal obliteration, respectively, P = 0.001) and 56 days (5.3 ± 2.4% vs. 0.38 ± 0.3%, P = 0.002). Treatment caused a 51% reduction in intimal hyperplasia at day 56. CP‐690,550‐treated animals also had a significant reduction of donor‐specific IgG production and of the gene expression for suppressor of cytokine signaling‐3 and with unchanged levels of expression of RANTES, IP‐10 and transforming growth factor‐β1. These results are the first to show that JAK3 blockade by CP‐690,550 effectively prevents allograft vasculopathy in this rat model of aorta transplantation.

Microbiological hazards related to xenotransplantation of porcine organs into man.

Pigs are emerging as the most likely providers of genetically engineered organs and cells for the purpose of clinical xenotransplantation. Introduction of clinical trials has been delayed primarily by uncertainties regarding the risk of swine pathogen transmission that could harm the recipient. The concern that xenotransplantation carries the potential for a new epidemic has been highlighted by recent experiences with both bovine spongiform encephalopathy and human immunodeficiency diseases. As clinical trials have been postponed and xenotransplantation teams are working actively to gather data for an estimation of the risk, this review provides the reader with a state-of-the-art estimation of the microbiological hazards related to xenotransplantation of porcine organs to man. Particular emphasis is put on viral and retroviral hazards. Both current diagnostic tools and those under development are described, along with breeding strategies to provide donor animals that would not put the recipient or the general population at risk.

Compared with cyclosporine, ISATX247 significantly prolongs renal-allograft survival in a nonhuman primate model

Background. ISATX247 is a novel calcineurin inhibitor that has shown more potency than cyclosporine in vitro. This is the first study to compare the survival times of renal allografts in nonhuman primates treated with either ISATX247 or cyclosporine. Methods. Adult, male cynomolgus monkeys were divided into blood-group compatible and mixed-lymphocyte, stimulation-mismatched, donor-recipient pairs. Heterotopic renal transplantation and bilateral native nephrectomies were performed. The monkeys were placed into either an ISATX247 or cyclosporine treatment group. Both groups were dosed twice daily to maintain a 12-hour drug-trough level of 150 ng/mL. Whole-blood concentrations of ISATX247 and cyclosporine, complete blood counts, and serum chemistry profiles were performed three times a week. Euthanasia was performed if the serum creatinine concentration became 7 or more mg/dL or a serious complication developed. Results. The group receiving ISATX247 (n=8) survived significantly (P=0.0036) longer than the group receiving cyclosporine (n=7). The mean trough blood concentration of ISATX247 was 120±32 ng/mL and cyclosporine was 189±130 ng/mL. The average area under the curve0–12 for ISATX247 was 6045±1679 ng/mL/hr and for cyclosporine was 4919±823 ng/mL/hr. The average calcineurin inhibition at trough blood concentrations was 80±11% for ISATX247 and 48±12% for cyclosporine. Conclusions. Allografts in monkeys treated with ISATX247 survived significantly longer than those treated with cyclosporine. On the basis of survival times and degree of calcineurin inhibition, ISATX247 is a more potent immunosuppressive agent than cyclosporine in this nonhuman primate model of renal-allograft transplantation.

Functional metabolic characteristics of intact pig livers during prolonged extracorporeal perfusion: potential for a unique biological liver-assist device.

Background. The clinical development of liver-support devices based on perfusion of either pig hepatocytes cartridges or whole pig livers has been hampered by the ability to use sufficient liver cell mass to provide adequate metabolic support, limited perfusion times, and the potential for patient exposure to pig zoonotic diseases. Methods. We designed an original system in which an isolated intact pig liver was perfused extracorporeally under physiological conditions in a closed loop circuit with allogeneic pig blood and constant monitoring of major physiological and functional parameters. The perfusion circuit further included an interface membrane to provide for separation of patient and liver perfusion circulation. Results. Prolonged (6–21 hr) liver perfusion did not produce significant liver damage as reflected by modest rises in the levels of the serum transaminases, stability of main biochemical parameters (including potassium), and the maintenance of normal cellular morphology. Optimal liver function was documented as measured by lactate consumption, control of glycemia, and the results of clotting studies and functional assays. The perfused liver cleared 82% and 79% of peak bilirubin and ammonia concentrations with clearing kinetics identical throughout perfusion. Indocyanine green clearance was identical to that observed in the living donor before explant surgery. Conclusions. In conclusion, the extracorporeal pig liver perfusion apparatus described here allows optimal pig liver function for prolonged periods of time. The microporous membrane to provide separation of donor organ and recipient and the high level of functional activity suggest that this form of liver metabolic support may have important clinical applications.

Similarities in the immunoglobulin response and VH gene usage in rhesus monkeys and humans exposed to porcine hepatocytes

BackgroundThe use of porcine cells and organs as a source of xenografts for human patients would vastly increase the donor pool; however, both humans and Old World primates vigorously reject pig tissues due to xenoantibodies that react with the polysaccharide galactose α (1,3) galactose (αGal) present on the surface of many porcine cells. We previously examined the xenoantibody response in patients exposed to porcine hepatocytes via treatment(s) with bioartficial liver devices (BALs), composed of porcine cells in a support matrix. We determined that xenoantibodies in BAL-treated patients are predominantly directed at porcine αGal carbohydrate epitopes, and are encoded by a small number of germline heavy chain variable region (VH) immunoglobulin genes. The studies described in this manuscript were designed to identify whether the xenoantibody responses and the IgVH genes encoding antibodies to porcine hepatocytes in non-human primates used as preclinical models are similar to those in humans. Adult non-immunosuppressed rhesus monkeys (Macaca mulatta) were injected intra-portally with porcine hepatocytes or heterotopically transplanted with a porcine liver lobe. Peripheral blood leukocytes and serum were obtained prior to and at multiple time points after exposure, and the immune response was characterized, using ELISA to evaluate the levels and specificities of circulating xenoantibodies, and the production of cDNA libraries to determine the genes used by B cells to encode those antibodies.ResultsXenoantibodies produced following exposure to isolated hepatocytes and solid organ liver grafts were predominantly encoded by genes in the VH3 family, with a minor contribution from the VH4 family. Immunoglobulin heavy-chain gene (VH) cDNA library screening and gene sequencing of IgM libraries identified the genes as most closely-related to the IGHV3-11 and IGHV4-59 germline progenitors. One of the genes most similar to IGHV3-11, VH3-11cyno, has not been previously identified, and encodes xenoantibodies at later time points post-transplant. Sequencing of IgG clones revealed increased usage of the monkey germline progenitor most similar to human IGHV3-11 and the onset of mutations.ConclusionThe small number of IGVH genes encoding xenoantibodies to porcine hepatocytes in non-human primates and humans is highly conserved. Rhesus monkeys are an appropriate preclinical model for testing novel reagents such as those developed using structure-based drug design to target and deplete antibodies to porcine xenografts.