Ex vivo delivery of Mirococept: A dose-finding study in pig kidney after showing a low dose is insufficient to reduce delayed graft function in human kidney.

The complement system plays a pivotal role in the pathogenesis of ischemia-reperfusion injury in solid organ transplantation. Mirococept is a potent membrane-localizing complement inhibitor that can be administered ex vivo to the donor kidney prior to transplantation. To evaluate the efficacy of Mirococept in reducing delayed graft function (DGF) in deceased donor renal transplantation, we undertook the efficacy of mirococept (APT070) for preventing ischaemia-reperfusion injury in the kidney allograft (EMPIRIKAL) trial (ISRCTN49958194). A dose range of 5-25 mg would be tested, starting with 10 mg in cohort 1. No significant difference between Mirococept at 10 mg and control was detected; hence the study was stopped to enable a further dose saturation study in a porcine kidney model. The optimal dose of Mirococept in pig kidney was 80 mg. This dose did not induce any additional histological damage compared to controls or after a subsequent 3 hours of normothermic machine perfusion. The amount of unbound Mirococept postperfusion was found to be within the systemic dose range considered safe in the Phase I trial. The ex vivo administration of Mirococept is a safe and feasible approach to treat DGF in deceased donor kidney transplantation. The porcine kidney study identified an optimal dose of 80 mg (equivalent to 120 mg in human kidney) that provides a basis for further clinical development.

Rationale for targeting complement in COVID-19.

A novel coronavirus, SARS-CoV-2, has recently emerged in China and spread internationally, posing a health emergency to the global community. COVID-19 caused by SARS-CoV-2 is associated with an acute respiratory illness that varies from mild to the life-threatening acute respiratory distress syndrome (ARDS). The complement system is part of the innate immune arsenal against pathogens, in which many viruses can evade or employ to mediate cell entry. The immunopathology and acute lung injury orchestrated through the influx of pro-inflammatory macrophages and neutrophils can be directly activated by complement components to prime an overzealous cytokine storm. The manifestations of severe COVID-19 such as the ARDS, sepsis and multiorgan failure have an established relationship with activation of the complement cascade. We have collected evidence from all the current studies we are aware of on SARS-CoV-2 immunopathogenesis and the preceding literature on SARS-CoV-1 and MERS-CoV infection linking severe COVID-19 disease directly with dysfunction of the complement pathways. This information lends support for a therapeutic anti-inflammatory strategy against complement, where a number of clinically ready potential therapeutic agents are available.

Effects of multiple-dose ponesimod, a selective S1P1 receptor modulator, on lymphocyte subsets in healthy humans.

This study investigated the effects of ponesimod, a selective S1P1 receptor modulator, on T lymphocyte subsets in 16 healthy subjects. Lymphocyte subset proportions and absolute numbers were determined at baseline and on Day 10, after once-daily administration of ponesimod (10 mg, 20 mg, and 40 mg each consecutively for 3 days) or placebo (ratio 3:1). The overall change from baseline in lymphocyte count was -1,292±340×106 cells/L and 275±486×106 cells/L in ponesimod- and placebo-treated subjects, respectively. This included a decrease in both T and B lymphocytes following ponesimod treatment. A decrease in naïve CD4+ T cells (CD45RA+CCR7+) from baseline was observed only after ponesimod treatment (-113±98×106 cells/L, placebo: 0±18×106 cells/L). The number of T-cytotoxic (CD3+CD8+) and T-helper (CD3+CD4+) cells was significantly altered following ponesimod treatment compared with placebo. Furthermore, ponesimod treatment resulted in marked decreases in CD4+ T-central memory (CD45RA-CCR7+) cells (-437±164×106 cells/L) and CD4+ T-effector memory (CD45RA-CCR7-) cells (-131±57×106 cells/L). In addition, ponesimod treatment led to a decrease of -228±90×106 cells/L of gut-homing T cells (CLA-integrin ß7+). In contrast, when compared with placebo, CD8+ T-effector memory and natural killer (NK) cells were not significantly reduced following multiple-dose administration of ponesimod. In summary, ponesimod treatment led to a marked reduction in overall T and B cells. Further investigations revealed that the number of CD4+ cells was dramatically reduced, whereas CD8+ and NK cells were less affected, allowing the body to preserve critical viral-clearing functions.

A double-blind randomised controlled investigation into the efficacy of Mirococept (APT070) for preventing ischaemia reperfusion injury in the kidney allograft (EMPIRIKAL): study protocol for a randomised controlled trial.

BACKGROUND: Delayed graft function (DGF) is traditionally defined as the requirement for dialysis during the first week after transplantation. DGF is a common complication of renal transplantation, and it negatively affects short- and long-term graft outcomes. Ischaemia reperfusion injury (IRI) is a prime contributor to the development of DGF. It is well established that complement system activation plays a pivotal role in the pathogenesis of IRI. Mirococept is a highly effective complement inhibitor that can be administered ex vivo to the donor kidney just before transplantation. Preclinical and clinical evidence suggests that Mirococept inhibits inflammatory responses that follow IRI. The EMPIRIKAL trial (REC 12/LO/1334) aims to evaluate the efficacy of Mirococept in reducing the incidence of DGF in cadaveric renal transplantation. METHODS/DESIGN: EMPIRIKAL is a multicentre double-blind randomised case-control trial designed to test the superiority of Mirococept in the prevention of DGF in cadaveric renal allografts, as compared to standard cold perfusion fluid (Soltran®). Patients will be randomised to Mirococept or placebo (Pbo) and will be enrolled in cohorts of N = 80 with a maximum number of 7 cohorts. The first cohort will be randomised to 10 mg of Mirococept or Pbo. After the completion of each cohort, an interim analysis will be carried out in order to evaluate the dose allocation for the next cohort (possible doses: 5-25 mg). Immunosuppression therapy, antibiotic and antiviral prophylaxis will be administered as per local centre protocols. The enrolment will take approximately 24 months, and patients will be followed for 12 months. The primary endpoint is DGF, defined as the requirement for dialysis during the first week after transplantation. Secondary endpoints include duration of DGF, functional DGF, renal function at 12 months, acute rejection episodes at 6 and 12 months, primary non-function and time of hospital stay on first admission and in the first year following transplant. Safety evaluation will include the monitoring of laboratory data and the recording of all adverse events. DISCUSSION: The EMPIRIKAL trial is the first study to evaluate the efficacy of an ex vivo administered complement inhibitor (Mirococept) in preventing DGF in cadaveric human renal transplantation. Mirococept has a unique 'cytotopic' property that permits its retention in the organ microvasculature. TRIAL REGISTRATION: ISRCTN registry, ISRCTN49958194 . Registered on 3 August 2012.

HES1 in immunity and cancer.

Hairy and enhancer of split homolog-1 (HES1) is a part of an extensive family of basic helix-loop-helix (bHLH) proteins and plays a crucial role in the control and regulation of cell cycle, proliferation, cell differentiation, survival and apoptosis in neuronal, endocrine, T-lymphocyte progenitors as well as various cancers. HES1 is a transcription factor which is regulated by the NOTCH, Hedgehog and Wnt signalling pathways. Aberrant expression of these pathways is a common feature of cancerous cells. There appears to be a fine and complicated crosstalk at the molecular level between the various signalling pathways and HES1, which contributes to its effects on the immune response and cancers such as leukaemia. Several mechanisms have been proposed, including an enhanced invasiveness and metastasis by inducing epithelial mesenchymal transition (EMT), in addition to its strict requirement for tumour cell survival. In this review, we summarize the current biology and molecular mechanisms as well as its use as a clinical target in cancer therapeutics.

FRA2 is a STAT5 target gene regulated by IL-2 in human CD4 T cells.

Signal transducers and activators of transcription 5(STAT5) are cytokine induced signaling proteins, which regulate key immunological processes, such as tolerance induction, maintenance of homeostasis, and CD4 T-effector cell differentiation. In this study, transcriptional targets of STAT5 in CD4 T cells were studied by Chromatin Immunoprecipitation (ChIP). Genomic mapping of the sites cloned and identified in this study revealed the striking observation that the majority of STAT5-binding sites mapped to intergenic (>50 kb upstream) or intronic, rather than promoter proximal regions. Of the 105 STAT5 responsive binding sites identified, 94% contained the canonical (IFN-γ activation site) GAS motifs. A number of putative target genes identified here are associated with tumor biology. Here, we identified Fos-related antigen 2 (FRA2) as a transcriptional target of IL-2 regulated STAT5. FRA2 is a basic -leucine zipper (bZIP) motif 'Fos' family transcription factor that is part of the AP-1 transcription factor complex and is also known to play a critical role in the progression of human tumours and more recently as a determinant of T cell plasticity. The binding site mapped to an internal intron within the FRA2 gene. The epigenetic architecture of FRA2, characterizes a transcriptionally active promoter as indicated by enrichment for histone methylation marks H3K4me1, H3K4me2, H3K4me3, and transcription/elongation associated marks H2BK5me1 and H4K20me1. FRA2 is regulated by IL-2 in activated CD4 T cells. Consistently, STAT5 bound to GAS sequence in the internal intron of FRA2 and reporter gene assays confirmed IL-2 induced STAT5 binding and transcriptional activation. Furthermore, addition of JAK3 inhibitor (R333) or Daclizumab inhibited the induction in TCR stimulated cells. Taken together, our data suggest that FRA2 is a novel STAT5 target gene, regulated by IL-2 in activated CD4 T cells.

Role of the Fc region in CD70-specific antibody effects on cardiac transplant survival.

BACKGROUND: The role of the CD70-specific antibody and the mechanisms by which it extends transplant survival are not known. METHODS: Fully major histocompatibility complex-mismatched heterotopic heart transplantation (BALB/c to C57BL/6) was performed. Treated mice received intraperitoneal injections of wild-type (WT) CD70-specific antibody (FR70) or IgG1 or IgG2a chimeric antibodies on days 0, 2, 4, and 6 posttransplantation. RESULTS: WT FR70 antibody significantly extended heart transplant survival to 19 days compared with untreated mice (median survival time [MST]=10 days). Graft survival using the nondepleting IgG1 antibody was significantly shorter (MST=14 days), whereas the survival using depleting IgG2a antibody (MST=18) was similar to that using WT FR70. The FR70 and IgG2a antibodies demonstrated a greater efficiency of fixing mouse complement over the IgG1 variant in vitro. CD4 and CD8 T-cell graft infiltration was reduced with treatment; however, this was most pronounced with WT FR70 and IgG2a antibody therapy compared with the IgG1 chimeric variant. Circulating donor-specific IgG alloantibodies were initially reduced with WT FR70 treatment (day 8 posttransplantation) but increased at days 15 and 20 posttransplantation to the level detected in untreated controls. CONCLUSION: We conclude that WT (FR70) and the IgG2a depleting variant of CD70-specific antibody reduce graft infiltrating CD4 and CD8 T cells, transiently reduce serum alloantibody levels, and extend graft survival. In contrast, the nondepleting IgG1 variant of this antibody showed lower efficacy. These data suggest that a depleting mechanism of action and not merely costimulation blockade plays a substantial role in the therapeutic effects of CD70-specific antibody.

Intermittent antibody-based combination therapy removes alloantibodies and achieves indefinite heart transplant survival in presensitized recipients.

BACKGROUND: It is well established that primed/memory T cells play a critical role in heart transplant rejection. This contributes to the challenges faced in the transplant clinic because current treatments that are efficient in controlling naïve T cell alloresponses have limited efficacy on primed T cell responders. METHODS: Fully MHC-mismatched heart transplantation was performed from BALB/c to C57BL/6 mice presensitized with BALB/c splenocytes 14 days pretransplantation. A combination therapy comprising CD70-, CD154-, and CD8-specific antibodies (Abs) was administered at day 0 and 4 posttransplantation with rapamycin on days 0 to 4. RESULTS: The Ab combination therapy extended heart transplant survival in presensitized recipients from median survival time 8 days (MST) to MST 78 days. A decrease in the number of splenic interferon-gamma-secreting cells measured by ELISpot assay was seen in the treated group compared with the untreated controls. However, graft-infiltrating CD8+ and CD4+ T cells persisted despite treatment and the number of intragraft CD4+ T cells increased at day 30 posttransplantation. When an additional "rescue therapy" comprising the same Abs was readministered at days 30, 60, and 90 posttransplantation, T cell infiltration was reduced and indefinite graft survival was observed. Furthermore, rescue therapy resulted in gradual decrease in titer and, by day 90 posttransplantation, the complete loss of the preexisting, donor-specific Abs. CONCLUSION: We conclude that our Ab combination therapy extends allograft survival in presensitized recipients. When combined with intermittent Ab-mediated rescue therapy, this results in indefinite allograft survival and a loss of the preexisting, donor-specific Abs from the circulation.