Field Study and Correlative Studies of Factor IX Variant FIX-R338L in Participants Treated with Fidanacogene Elaparvovec.

BACKGROUND: Fidanacogene elaparvovec, an adeno-associated virus-based gene therapy vector expressing the high-activity factor IX (FIX) variant FIX-R338L, is in development for hemophilia B. One-stage clotting (OS) assays and chromogenic substrate (CS) assays are commonly used to measure FIX-R338L variant activity. Data from ongoing trials suggest FIX activity varies between different OS and CS assays. MATERIAL AND METHODS: To better understand FIX-R338L activity in clinical samples, an international multisite field study was conducted across a central laboratory and 18 local laboratories, using standard protocols, reagents, and instrumentation, with individual participant samples from a phase 1/2a study of fidanacogene elaparvovec. RESULTS: Unlike the wild-type FIX control, FIX-R338L activity was higher with the OS silica-based assay versus OS ellagic acid-based and CS assays. Variation in FIX activity was greater at the lowest activity levels. Activated FIX (FIXa) in plasma could result in higher OS assay activity or increased thrombin generation, which could overestimate FIX activity. However, FIXa was not detected in the participant samples, indicating that it was not contributing to the OS assay differences. Since individuals on gene therapy may receive exogenous replacement FIX products, replacement products were spiked into patient plasma samples to target a therapeutic concentration. Exogenous FIX was additive to endogenous FIX-R338L, with no interference from FIX-R338L. CONCLUSION: These results demonstrate FIX-R338L activity can be measured with OS and CS assays in clinical laboratories and provide insight into assay variability when measuring FIX with endogenously produced FIX-R338L. The findings may help establish best practices for measuring FIX-R338L activity (Clinicaltrials.gov identifier: NCT02484092).

To Clot or Not to Clot: Deepening Our Understanding of Alterations in the Hemostatic System.

The session on the hemostatic system focused on new developments in coagulation and platelet biology as well as how therapeutic agents may affect hemostasis. The classic cascade model of coagulation was compared with the more recent models of cell-based and vascular-based coagulation, which may provide better insight on how the coagulation cascade works in vivo. A review of platelet biology highlighted that, as platelets age, desialylated platelets form and are recognized by Ashwell-Morell receptor (AMR), leading to hepatic uptake and subsequent increase in thrombopoietin (TPO) production. Administration of therapeutics that induce thrombocytopenia was also discussed, including Mylotarg, which is an antibody-drug conjugate that was shown to decrease human megakaryocyte development but had no effect on platelet aggregation. An acetyl co-A carboxylase inhibitor was shown to cause thrombocytopenia by inhibiting de novo lipogenesis, which is critical for the formation of the megakaryocyte demarcation membrane system responsible for platelet production. It was also illustrated how preclinical translation models have been very helpful in the development of adeno-associated virus (AAV) hemophilia B gene therapy and what old and new preclinical tools we have that can predict the risk of a prothrombotic state in people.

Validation of patient-reported vaso-occlusive crisis day as an endpoint in sickle cell disease studies.

Individuals with sickle cell disease (SCD) experience vaso-occlusive crises (VOC). Historically, VOC episodes have been assessed through medical utilization, thereby excluding events managed at home. In order to validate a daily patient-reported outcome for patients with SCD to accurately report their VOC status and experience of a pain crisis, a SCD Diary was included in Evaluation of Longitudinal Pain Study in Sickle Cell Disease (ELIPSIS), a longitudinal, six-month, non-interventional study. The daily patient-completed diary included a description of SCD pain crisis, followed by questions on: pain crisis in the past 24 h (VOC Day question; respective response yes or no), worst pain, tiredness, and functioning. Thirty-five patients with SCD participated in ELIPSIS. Analyses were performed to validate the patient-reported VOC Day. Mean symptoms and functioning scores on the first or last VOC Day of a VOC Event were compared using t-tests with the mean of the three non-VOC Days before and after the event. Mean severity of symptoms and functioning scores on all VOC Days compared to all non-VOC Days were higher, with statistically significant mean differences between first/last VOC Days and respective three non-VOC Days (p's < .01). A subset of patients (n = 15) and caregivers (n = 9) were interviewed to evaluate their understanding of the SCD Diary questions. Nearly all confirmed that the pain crisis description accurately described the VOC experience, and participants expressed confidence differentiating SCD crisis pain from everyday pain. These results demonstrate patients can reliably report their experiences with VOC-related pain crises using the SCD Diary.

Hemostatic efficacy of marstacimab alone or in combination with bypassing agents in hemophilia plasmas and a mouse bleeding model.

Background: Patients with hemophilia have deficiencies in intrinsic coagulation factors and can develop inhibitors that limit the effectiveness of replacement coagulation factors. Marstacimab, a human monoclonal antibody, binds and inhibits the human tissue factor pathway inhibitor. Marstacimab is currently under development as a potential prophylactic treatment to prevent bleeding episodes in patients with hemophilia A and B. Objective: To assess the effects of marstacimab alone or in combination with the bypassing agent recombinant factor FVIIa (rFVIIa) or activated prothrombin complex concentrate (aPCC) on thrombin generation and bleeding. Methods: Marstacimab and/or rFVIIa or aPCC were added to hemophilic A or B plasma or nonhemophilic plasma in vitro. Hemostatic activity was measured using the thrombin generation assay. In vivo effects were assessed using a mouse acute bleeding model. Male hemophilia A mice were dosed with marstacimab plus aPCC before tail clip; blood loss was quantified by measuring hemoglobin. Results: Marstacimab plus rFVIIa or aPCC slightly increased peak thrombin levels compared with either agent alone. This increase was within the reported range for nonhemophilic plasma and did not exceed levels observed in nonhemophilic plasma treated with marstacimab alone. Hemophilia A mice that received 200 U/kg aPCC had significantly reduced bleeding (62%) compared with vehicle-treated mice (p < 0.05), and marstacimab plus aPCC reduced bleeding by 83.3% compared with vehicle (p= 0.0009). Conclusions: Marstacimab alone or with bypassing agents increased hemostasis in hemophilia plasma without generating excessive thrombin. The hemostatic activity of marstacimab plus aPCC was confirmed in hemophilia A mice.

Evaluation of Longitudinal Pain Study in Sickle Cell Disease (ELIPSIS) by patient-reported outcomes, actigraphy, and biomarkers.

Clinical trials in sickle cell disease (SCD) often focus on health care utilization for painful vaso-occlusive crises (VOCs). However, no objective, quantifiable pain biomarkers exist, pain is not specific to VOCs, health care utilization varies between patients, unreported at-home VOCs likely contribute to long-term outcomes, and patient-reported outcomes are seldom considered. This noninterventional, longitudinal, 6-month study aimed to develop tools to identify VOCs in SCD patients with or without health care utilization. Participants wore an actigraph device, tracking sleep and activity. Patients with SCD used an electronic patient-reported outcome (ePRO) tool to collect data on pain, medication, fatigue, and daily function. Patients self-reported when they experienced VOC pain (VOC day). Biomarkers were collected every 3 weeks (non-VOC). Self-reported VOCs triggered at-home or in-hospital blood collection. The study enrolled 37 participants with SCD; 35 completed the study. Participants reported 114 VOC events and 346 VOC days, of which 62.3% and 78.3%, respectively, were self-treated at home. The ePRO and actigraphy captured end points of pain, functionality, fatigue, activity, and sleep; each was significantly altered on VOC days compared with non-VOC days. Biomarkers collected at home or in the hospital on VOC days were significantly altered compared with non-VOC baseline values, including leukocyte-platelet aggregates, microfluidic-based blood cell adhesion, interleukin-6, C-reactive protein, interleukin-10, tumor necrosis factor-α, and thrombin-antithrombin. The Evaluation of Longitudinal Pain Study in Sickle Cell Disease (ELIPSIS) trial shows the feasibility of accurately monitoring out-of-hospital pain by using patient-reported VOC days as potential end points for clinical trials in SCD; it describes the changes in biomarkers and activity measured by actigraphy that may enable improved identification and assessment of VOCs.

Investigating the clearance of VWF A-domains using site-directed PEGylation and novel N-linked glycosylation.

BACKGROUND: Previous studies have demonstrated that the A1A2A3 domains of von Willebrand factor (VWF) play a key role in regulating macrophage-mediated clearance in vivo. In particular, the A1-domain has been shown to modulate interaction with macrophage low-density lipoprotein receptor-related protein-1 (LRP1) clearance receptor. Furthermore, N-linked glycans within the A2-domain have been shown to protect VWF against premature LRP1-mediated clearance. Importantly, however, the specific regions within A1A2A3 that enable macrophage binding have not been defined. OBJECTIVE AND METHODS: To address this, we utilized site-directed PEGylation and introduced novel targeted N-linked glycosylation within A1A2A3-VWF and subsequently examined VWF clearance. RESULTS: Conjugation with a 40-kDa polyethylene glycol (PEG) moiety significantly extended the half-life of A1A2A3-VWF in VWF-/- mice in a site-specific manner. For example, PEGylation at specific sites within the A1-domain (S1286) and A3-domain (V1803, S1807) attenuated VWF clearance in vivo, compared to wild-type A1A2A3-VWF. Furthermore, PEGylation at these specific sites ablated binding to differentiated THP-1 macrophages and LRP1 cluster II and cluster IV in-vitro. Conversely, PEGylation at other positions (Q1353-A1-domain and M1545-A2-domain) had limited effects on VWF clearance or binding to LRP1.Novel N-linked glycan chains were introduced at N1803 and N1807 in the A3-domain. In contrast to PEGylation at these sites, no significant extension in half-life was observed with these N-glycan variants. CONCLUSIONS: These novel data demonstrate that site specific PEGylation but not site specific N-glycosylation modifies LRP1-dependent uptake of the A1A2A3-VWF by macrophages. This suggests that PEGylation, within the A1- and A3-domains in particular, may be used to attenuate LRP1-mediated clearance of VWF.

Antibodies against the erythroferrone N-terminal domain prevent hepcidin suppression and ameliorate murine thalassemia.

Erythroferrone (ERFE) is produced by erythroblasts in response to erythropoietin (EPO) and acts in the liver to prevent hepcidin stimulation by BMP6. Hepcidin suppression allows for the mobilization of iron to the bone marrow for the production of red blood cells. Aberrantly high circulating ERFE in conditions of stress erythropoiesis, such as in patients with ß-thalassemia, promotes the tissue iron accumulation that substantially contributes to morbidity in these patients. Here we developed antibodies against ERFE to prevent hepcidin suppression and to correct the iron loading phenotype in a mouse model of ß-thalassemia [Hbb(th3/+) mice] and used these antibodies as tools to further characterize ERFE's mechanism of action. We show that ERFE binds to BMP6 with nanomolar affinity and binds BMP2 and BMP4 with somewhat weaker affinities. We found that BMP6 binds the N-terminal domain of ERFE, and a polypeptide derived from the N terminus of ERFE was sufficient to cause hepcidin suppression in Huh7 hepatoma cells and in wild-type mice. Anti-ERFE antibodies targeting the N-terminal domain prevented hepcidin suppression in ERFE-treated Huh7 cells and in EPO-treated mice. Finally, we observed a decrease in splenomegaly and serum and liver iron in anti-ERFE-treated Hbb(th3/+) mice, accompanied by an increase in red blood cells and hemoglobin and a decrease in reticulocyte counts. In summary, we show that ERFE binds BMP6 directly and with high affinity, and that antibodies targeting the N-terminal domain of ERFE that prevent ERFE-BMP6 interactions constitute a potential therapeutic tool for iron loading anemias.

Marstacimab, a tissue factor pathway inhibitor neutralizing antibody, improves coagulation parameters of ex vivo dosed haemophilic blood and plasmas.

INTRODUCTION: Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of the extrinsic pathway that negatively regulates thrombin production during coagulation. Under haemophilic conditions, where the intrinsic coagulation pathway is impaired, inhibition of TFPI may improve clotting. AIM: We investigated the ex vivo effects of a human TFPI neutralizing antibody, marstacimab (previously PF-06741086), in coagulation assays including rotational thromboelastometry (ROTEM), thrombin generation assay (TGA) and the dilute prothrombin time (dPT) assay, performed in haemophilic whole blood and plasmas. We compared the effects of marstacimab to the effects of recombinant coagulation factors and investigated the reproducibility of marstacimab in restoring haemostasis by comparing its effect in whole blood collected from the same study participants on differing days. METHODS: Citrated whole blood and plasmas obtained from haemophilia participants were supplemented ex vivo with vehicle, marstacimab, recombinant FVIII (rFVIII) or recombinant factor IX (rFIX) and analysed in ROTEM, TGA and the dPT assay using low tissue factor concentrations to trigger coagulation. RESULTS: Marstacimab induced pro-coagulant responses in ROTEM parameters including reduction in clotting times and increases in angle. Similarly, participant plasmas supplemented with marstacimab exhibited improvements in TGA parameters, including reduced lag times, increased peak thrombin concentrations and reductions in dPT clotting time. Concentrations of marstacimab tested showed activity comparable to addition of rFVIII or rFIX and were reproducible. CONCLUSIONS: These studies show the ex vivo potency of marstacimab in restoring haemostasis in whole blood and plasmas from haemophilia participants and comparability to ex vivo reconstitution with recombination coagulation factors.

Induction and Impact of Anti-Drug Responses Elicited by a Human Recombinant Coagulation Factor FXaI16L in Preclinical Species.

This paper presents a systemic investigation of ADA development and ADA impact of a human coagulation factor in nonclinical species during drug development and provides insights into potential implications in human if a similar ADA occurs. FXaI16L-induced ADA response was characterized in monkey, mouse, rat, and dog in different studies, and ADA effects on pharmacokinetic and/or pharmacodynamics of FXaI16L were further examined in ADA-negative and ADA-positive animals. After repeated administrations, FXaI16L elicited a dose and exposure day-dependent ADA response which ranged from no response to a transient or persistent response. Increase in exposure day and increase in dose generally enhanced ADA incidence except for a decrease in ADA incidence was observed in monkeys after repeated high-dose administrations. The observable ADA impact on pharmacokinetics was only found in some ADA+ animals and included decrease in clearance and increase in systemic exposure but no increase in half-life. In addition, no or limited effect on pharmacodynamics by ADA was observed. The earliest ADA response was observed after three exposure days, marked elevation of drug exposure was observed in some animals at log titer > 2.0, and the highest antibody titer excited was about 4 (Log10) in all species. A correlation between ADA induction and accumulative exposure after various repeat treatments in different species was found for FXaI16L. In addition, potential immunogenicity risk and mitigation of ADA in clinics are discussed.

PF-04447943, a Phosphodiesterase 9A Inhibitor, in Stable Sickle Cell Disease Patients: A Phase Ib Randomized, Placebo-Controlled Study.

This phase Ib study randomized patients with stable sickle cell disease (SCD) aged 18-65 years to twice-daily PF-04447943 (a phosphodiesterase 9A inhibitor; 5 or 25 mg) or placebo, with/without hydroxyurea coadministration, for up to 29 days. Blood samples were collected at baseline and various posttreatment time points for assessments of PF-04447943 pharmacokinetics (PKs)/pharmacodynamics (PDs). Change from baseline in potential SCD-related biomarkers was evaluated. Of 30 patients, 15 received hydroxyurea and 28 completed the study. PF-04447943, with/without hydroxyurea, was generally well tolerated, with no treatment-related serious adverse events. Plasma PF-04447943 exposure was dose proportional. Twice-daily PF-04447943 25 mg significantly reduced the number and size of circulating monocyte-platelet and neutrophil-platelet aggregates and levels of circulating soluble E-selectin at day 29 vs. baseline (adjusted P < 0.15). PF-04447943 demonstrated PK/PD effects suggestive of inhibiting pathways that may contribute to vaso-occlusion. This study also provides guidance regarding biomarkers for future SCD studies.

Translational Pharmacokinetic/Pharmacodynamic Characterization and Target-Mediated Drug Disposition Modeling of an Anti-Tissue Factor Pathway Inhibitor Antibody, PF-06741086.

Tissue factor pathway inhibitor (TFPI) exhibits multiple isoforms, which are known to present in multiple locations such as plasma, endothelium, and platelets. TFPI is an endogenous negative modulator of the coagulation pathway, and therefore, neutralization of TFPI function can potentially increase coagulation activity. A human monoclonal antibody, PF-06741086, which interacts with all isoforms of TFPI is currently being tested in clinic for treating hemophilia patients with and without inhibitors. To support clinical development of PF-06741086, pharmacokinetics (PK) and pharmacodynamics of PF-06741086 were characterized in monkeys. In addition, a mechanistic model approach was used to estimate PK parameters in monkeys and simulate PK profiles in human. The results show that PF-06741086 exhibited target-mediated drug disposition and had specific effects on various hemostatic markers including diluted prothrombin time, thrombin generation, and thrombin-antithrombin complex in monkeys after administration. The model-predicted and observed human exposures were compared retrospectively, and the result indicates that the exposure prediction was reasonable within less than 2-fold deviation. This study demonstrated in vivo efficacy of PF-06741086 in monkeys and the utility of a rational mechanistic approach to describe PK for a monoclonal antibody with complex target binding.

Preclinical Pharmacokinetics, Pharmacodynamics, Tissue Distribution, and Interspecies Scaling of Recombinant Human Coagulation Factor XaI16L.

FXaI16L is a recombinant human FXa variant which is currently being evaluated in the clinic for treating intracerebral hemorrhage. The aim of our studies is to investigate overall pharmacokinetics, pharmacodynamics, and distribution of FXaI16L in preclinical species, and to understand its potential implication in human. Pharmacokinetics of FXaI16L was examined using active site probes and the results showed that FXaI16L displayed fast clearance, low volume of distribution, and a very short plasma resident time in mice, rats, and monkeys. When pharmacodynamics was examined in monkeys, concentration effects of FXaI16L on shortening of active partial prothrombin time and formation of thrombin-antithrombin complex were observed. Furthermore, biodistribution study was conducted in mice using radiolabeled FXaI16L, and showed that 125I-FXaI16L has high plasma protein binding and significant liver and kidney distribution. Human pharmacokinetic prediction for first-in-human dosing was evaluated using allometric scaling, liver blood flow, and a fixed coefficient method, and single species allometric scaling using monkey data was most predictive for human pharmacokinetics of FXaI16L.

A zymogen-like factor Xa variant corrects the coagulation defect in hemophilia.

Effective therapies are needed to control excessive bleeding in a range of clinical conditions. We improve hemostasis in vivo using a conformationally pliant variant of coagulation factor Xa (FXa(I16L)) rendered partially inactive by a defect in the transition from zymogen to active protease. Using mouse models of hemophilia, we show that FXa(I16L) has a longer half-life than wild-type FXa and does not cause excessive activation of coagulation. Once clotting mechanisms are activated to produce its cofactor FVa, FXa(I16L) is driven to the protease state and restores hemostasis in hemophilic animals upon vascular injury. Moreover, using human or murine analogs, we show that FXa(I16L) is more efficacious than FVIIa, which is used to treat bleeding in hemophilia inhibitor patients. FXa(I16L) may provide an effective strategy to enhance blood clot formation and act as a rapid pan-hemostatic agent for the treatment of bleeding conditions.

Inhibition of the Unfolded Protein Response by metformin in renal proximal tubular epithelial cells.

Metformin (Met), an AMP-activated protein kinase (AMPK) inducer, is primarily transported by organic cation transporters expressed at the surface of renal proximal tubular epithelial cells. However, the implication of Met in renal function remains poorly understood. Interestingly, AICAR, another AMPK inducer, has been shown to inhibit the Unfolded Protein Response (UPR) generated by tunicamycin in cardiomyocytes in an AMPK-kinase dependent fashion suggesting metformin may also block the UPR. In this work, we have examined the effect of metformin on the expression of UPR-related markers (GRP94 and CHOP) induced by glucosamine (GlcN), 2-deoxyglucose (2-DOG) and tunicamycin (TUNI) in renal proximal tubular epithelial cells and in murine mesangial cells. Met attenuated GRP94 and CHOP expression induced by GlcN and 2-DOG, but not TUNI only in renal epithelial cells, even though the AMPK activation was observed in both renal epithelial and mesangial cells. Met did not require the contribution of its AMPK kinase inducing activity to block UPR markers expression. This report has identified a novel inhibitory function of metformin on UPR, which may have a beneficial impact on kidney homeostatic function.

A monoclonal antibody against RAGE alters gene expression and is protective in experimental models of sepsis and pneumococcal pneumonia.

The RAGE (receptor for advanced glycation end products) is believed to play a role in sepsis by perpetuating inflammation. The interaction of RAGE with a variety of host-derived ligands that accumulate during stress and inflammation further induces the expression of RAGE. It was previously shown that a rat anti-RAGE monoclonal antibody protected mice from lethality in a cecal ligation and puncture model. We studied the effects of a humanized anti-RAGE monoclonal antibody in the murine pneumococcal pneumonia model of sepsis. Moreover, a gene expression analysis was performed in lung tissue of animals that underwent cecal ligation and puncture and treated with the rat anti-RAGE monoclonal antibody, compared with controls. Administration of humanized anti-RAGE mAb 6 h after intratracheal infection with Streptococcus pneumoniae improved mortality in BALB/c mice whether a 7.5 mg/kg (P < 0.01) or a 15 mg/kg dose (P < 0.01) was administered in combination with antibiotics. Gene expression analysis showed that many of the genes modulated by treatment with the anti-RAGE antibody were those that play an important role in regulating inflammation. Anti-RAGE monoclonal antibody offered a survival advantage to septic mice. This protective role in treated animals is supported by the observed gene expression profile changes of genes involved in sepsis and inflammation.

Pharmacokinetics and lung distribution of a humanized anti-RAGE antibody in wild-type and RAGE-/- mice.

A neutralizing antibody to the receptor for the advanced glycation end products (anti-RAGE Ab) was developed as a potential treatment of acute and chronic inflammatory conditions. Previous pharmacology studies demonstrated efficacy of the anti-RAGE antibody in the mouse model of sepsis. We examined pharmacokinetics and lung distribution of [125I]anti-RAGE Ab in RAGE-/- and wild-type (129S5) mice following single IV administration. Serum pharmacokinetics of [125I]anti-RAGE Ab was similar in RAGE-/- and 129S5 mice, with the total body clearance of 0.3 mL/hr/kg and the elimination half-life of 11-12 days, suggesting the target expression had limited impact on overall elimination of [125I]anti-RAGE Ab from mice. [125I]Anti-RAGE Ab accumulated in the lung of 129S5 mice, with ~4% of total dose retained in the lung at days 6-27 and the lung AUC0-∞ of ~300% of that in serum. The SDS-PAGE analysis suggested that most of retained lung radioactivity was attributed to intact antibody. No accumulation of radioactivity was observed in the lung of RAGE-/- mice, indicating that lung uptake of [125I]anti-RAGE Ab was target-dependent in wild-type mice. These data suggest that the anti-RAGE Ab was able to localize to the site of RAGE expression, the lung, and support the findings in the previous pharmacology studies.

Complement C3a, CpG oligos, and DNA/C3a complex stimulate IFN-α production in a receptor for advanced glycation end product-dependent manner.

The receptor for advanced glycation end products (RAGE) is a multiligand transmembrane receptor implicated in a number of diseases including autoimmune diseases. To further understand the pathogenic mechanism of RAGE in these diseases, we searched for additional ligands. We discovered that C3a bound to RAGE with an EC(50) of 1.9 nM in an ELISA, and the binding was increased both in magnitude (by >2-fold) and in affinity (EC(50) 70 pM) in the presence of human stimulatory unmethylated cytosine-guanine-rich DNA A (hCpGAs). Surface plasmon resonance and fluorescence anisotropy analyses demonstrated that hCpGAs could bind directly to RAGE and C3a and form a ternary complex. In human PBMCs, C3a increased IFN-α production in response to low levels of hCpGAs, and this synergy was blocked by soluble RAGE or by an Ab directed against RAGE. IFN-α production was reduced in response to mouse CpGAs and C3a in RAGE(-/-) mouse bone marrow cells compared wild-type mice. Taken together, these data demonstrate that RAGE is a receptor for C3a and CpGA. Through direct interaction, C3a and CpGA synergize to increase IFN-α production in a RAGE-dependent manner and stimulate an innate immune response. These findings indicate a potential role of RAGE in autoimmune diseases that show accumulation of immunostimulatory DNA and C3a.

IL-22 induces an acute-phase response.

IL-22 is made by a unique set of innate and adaptive immune cells, including the recently identified noncytolytic NK, lymphoid tissue-inducer, Th17, and Th22 cells. The direct effects of IL-22 are restricted to nonhematopoietic cells, its receptor expressed on the surface of only epithelial cells and some fibroblasts in various organs, including parenchymal tissue of the gut, lung, skin, and liver. Despite this cellular restriction on IL-22 activity, we demonstrate that IL-22 induces effects on systemic biochemical, cellular, and physiological parameters. By utilizing adenoviral-mediated delivery of IL-22 and systemic administration of IL-22 protein, we observed that IL-22 modulates factors involved in coagulation, including fibrinogen levels and platelet numbers, and cellular constituents of blood, such as neutrophil and RBC counts. Furthermore, we observed that IL-22 induces thymic atrophy, body weight loss, and renal proximal tubule metabolic activity. These cellular and physiological parameters are indicative of a systemic inflammatory state. We observed that IL-22 induces biochemical changes in the liver including induction of fibrinogen, CXCL1, and serum amyloid A that likely contribute to the reported cellular and physiological effects of IL-22. Based on these findings, we propose that downstream of its expression and impact in local tissue inflammation, circulating IL-22 can further induce changes in systemic physiology that is indicative of an acute-phase response.

TREM-1 expression is increased in the synovium of rheumatoid arthritis patients and induces the expression of pro-inflammatory cytokines.

OBJECTIVES: To investigate the expression and function of triggering receptor expressed on myeloid cells-1 (TREM-1) in the synovium of human RA patients as well as the level of soluble TREM-1 in the plasma of RA patients. METHODS: Twenty-four RA synovial samples were analysed by gene expression oligonucleotide microarrays. Expression levels of TREM-1 mRNA in murine CIA paws were determined by quantitative PCR (qPCR). TREM-1 protein expression was detected by immunohistochemistry in five RA synovial samples and two OA synovial samples. TREM-1-positive cells from five RA synovial tissues were analysed by FACS staining to determine the cell type. Activation of TREM-1 was tested in five RA synovial samples. Soluble TREM-1 was measured in serum from 32 RA patients. RESULTS: The expression of TREM-1 mRNA was found to increase 6.5-fold in RA synovial samples, whereas it was increased 132-fold in CIA paws. Increased numbers of TREM-1-positive cells were seen in RA synovium sections and these cells co-expressed CD14. Using a TREM-1-activating cross-linking antibody in RA synovial cultures, multiple pro-inflammatory cytokines were induced. The average amount of soluble TREM-1 in plasma from RA patients was found to be higher than that in plasma from healthy volunteers. CONCLUSIONS: These findings suggest that the presence of high levels of functionally active TREM-1 in RA synovium may contribute to the development or maintenance of RA, or both. Inhibiting TREM-1 activity may, therefore, have a therapeutic effect on RA. High levels of soluble TREM-1 in the plasma of RA patients compared with healthy volunteers may indicate disease activity.

Affinity maturation of a humanized rat antibody for anti-RAGE therapy: comprehensive mutagenesis reveals a high level of mutational plasticity both inside and outside the complementarity-determining regions.

Antibodies that neutralize RAGE (receptor for advanced glycation end products)-ligand interactions have potential therapeutic applications in both acute and chronic diseases. We generated XT-M4, a rat anti-RAGE monoclonal antibody that has in vivo efficacy in an acute sepsis model. This antibody was subsequently humanized. To improve the affinity of this antibody for the treatment of chronic indications, we used random and targeted mutagenesis strategies in combination with ribosome and phage-display technologies, respectively, to generate libraries of XT-M4 variants. We identified a panel of single-chain Fv antibody fragments (scFv's) that was improved up to 110-fold in a homogeneous time-resolved fluorescence competition assay against parental XT-M4 immunoglobulin G (IgG). After reformatting to bivalent scFv-Fc fusions and IgGs, we observed similar gains in potency in the same assay. Further analysis of binding kinetics as IgG revealed multiple variants with subnanomolar apparent affinity that was dictated primarily by improvements in the off-rate. All variants also had improved binding to cell surface-expressed human RAGE, and all retained, or had improved, apparent affinity for mouse RAGE. F100bL in V(H) (variable region of the heavy chain) complementarity-determining region 3 (CDR3) was one of a number of key mutations that correlated with affinity improvements and was independently identified by both mutagenesis strategies. Random mutagenesis coupled with ribosome display and high-throughput screening revealed an unexpectedly high level of mutational plasticity across the whole length of the humanized scFv, suggesting greater scope for structural optimization outside of the primary antigen-combining site defined by V(H) CDR3 and V(kappa) CDR3. In summary, our comprehensive mutagenesis approach not only achieved the desired affinity maturation of XT-M4 but also defined multiple mutational hotspots across the antibody sequence, provided an insight into the specificity-determining residues of the antibody paratope, and identified additional sites within the CDR loops where human germ-line amino acids may be introduced without affecting function.