Targeted delivery of anti-inflammatory therapy to rheumatoid tissue by fusion proteins containing an IL-4-linked synovial targeting peptide

We provide first-time evidence that the synovial endothelium-targeting peptide (SyETP) CKSTHDRLC successfully delivers conjugated IL-4 to human rheumatoid synovium transplanted into SCID mice. SyETP, previously isolated by in vivo phage display and shown to preferentially localize to synovial xenografts, was linked by recombinant technology to hIL-4 via an MMP-cleavable sequence. Both IL-4 and the MMP-cleavable sequence were shown to be functional. IL-4-SyETP augmented production of IL-1ra by synoviocytes stimulated with IL-1[beta] in a dose-dependent manner. In vivo imaging confirmed increased retention of SyETP-linked-IL-4 in synovial grafts which was enhanced by increasing number of copies (one to three) in the constructs. Strikingly, SyETP delivered bioactive IL-4 in vivo as demonstrated by increased pSTAT6 in synovial grafts. Thus, this study provides proof of concept for peptide-tissue-specific targeted immunotherapy in rheumatoid arthritis. This technology is potentially applicable to other biological therapies providing enhanced potency to inflammatory sites and reducing systemic toxicity

Targeting of viral interleukin-10 with an antibody fragment specific to damaged arthritic cartilage improves its therapeutic potency

INTRODUCTION: We previously demonstrated that a single-chain fragment variable (scFv) specific to collagen type II (CII) posttranslationally modified by reactive oxygen species (ROS) can be used to target anti-inflammatory therapeutics specifically to inflamed arthritic joints. The objective of the present study was to demonstrate the superior efficacy of anti-inflammatory cytokines when targeted to inflamed arthritic joints by the anti-ROS modified CII (anti-ROS-CII) scFv in a mouse model of arthritis.METHODS: Viral interleukin-10 (vIL-10) was fused to anti-ROS-CII scFv (1-11E) with a matrix-metalloproteinase (MMP) cleavable linker to create 1-11E/vIL-10 fusion. Binding of 1-11E/vIL-10 to ROS-CII was determined by enzyme-linked immunosorbent assay (ELISA), Western blotting, and immune-staining of arthritic cartilage, whereas vIL-10 bioactivity was evaluated in vitro by using an MC-9 cell-proliferation assay. Specific in vivo localization and therapeutic efficacy of 1-11E/vIL-10 was tested in the mouse model of antigen-induced arthritis.RESULTS: 1-11E/vIL-10 bound specifically to ROS-CII and to damaged arthritic cartilage. Interestingly, the in vitro vIL-10 activity in the fusion protein was observed only after cleavage with MMP-1. When systemically administered to arthritic mice, 1-11E/vIL-10 localized specifically to the arthritic knee, with peak accumulation observed after 3 days. Moreover, 1-11E/vIL-10 reduced inflammation significantly quicker than vIL-10 fused to the control anti-hen egg lysozyme scFv (C7/vIL10).CONCLUSIONS: Targeted delivery of anti-inflammatory cytokines potentiates their anti-arthritic action in a mouse model of arthritis. Our results further support the hypothesis that targeting biotherapeutics to arthritic joints may be extended to include anti-inflammatory cytokines that lack efficacy when administered systemically.</p

Targeting of viral interleukin-10 with an antibody fragment specific to damaged arthritic cartilage improves its therapeutic potency

Introduction: We previously demonstrated that a single-chain fragment variable (scFv) specific to collagen type II (CII) posttranslationally modified by reactive oxygen species (ROS) can be used to target anti-inflammatory therapeutics specifically to inflamed arthritic joints. The objective of the present study was to demonstrate the superior efficacy of anti-inflammatory cytokines when targeted to inflamed arthritic joints by the anti-ROS modified CII (anti-ROS-CII) scFv in a mouse model of arthritis.Methods: Viral interleukin-10 (vIL-10) was fused to anti-ROS-CII scFv (1-11E) with a matrix-metalloproteinase (MMP) cleavable linker to create 1-11E/vIL-10 fusion. Binding of 1-11E/vIL-10 to ROS-CII was determined by enzyme-linked immunosorbent assay (ELISA), Western blotting, and immune-staining of arthritic cartilage, whereas vIL-10 bioactivity was evaluated in vitro by using an MC-9 cell-proliferation assay. Specific in vivo localization and therapeutic efficacy of 1-11E/vIL-10 was tested in the mouse model of antigen-induced arthritis.Results: 1-11E/vIL-10 bound specifically to ROS-CII and to damaged arthritic cartilage. Interestingly, the in vitro vIL-10 activity in the fusion protein was observed only after cleavage with MMP-1. When systemically administered to arthritic mice, 1-11E/vIL-10 localized specifically to the arthritic knee, with peak accumulation observed after 3 days. Moreover, 1-11E/vIL-10 reduced inflammation significantly quicker than vIL-10 fused to the control anti-hen egg lysozyme scFv (C7/vIL10).Conclusions: Targeted delivery of anti-inflammatory cytokines potentiates their anti-arthritic action in a mouse model of arthritis. Our results further support the hypothesis that targeting biotherapeutics to arthritic joints may be extended to include anti-inflammatory cytokines that lack efficacy when administered systemically

Identification of autoantigens and their potential post-translational modification in EGPA and severe eosinophilic asthma

BACKGROUND: The chronic airway inflammation in severe eosinophilic asthma (SEA) suggests potential autoimmune aetiology with unidentified autoantibodies analogous to myeloperoxidase (MPO) in ANCA-positive EGPA (eosinophilic granulomatosis with polyangiitis). Previous research has shown that oxidative post-translational modification (oxPTM) of proteins is an important mechanism by which autoantibody responses may escape immune tolerance. Autoantibodies to oxPTM autoantigens in SEA have not previously been studied. METHODS: Patients with EGPA and SEA were recruited as well as healthy control participants. Autoantigen agnostic approach: Participant serum was incubated with slides of unstimulated and PMA-stimulated neutrophils and eosinophils, and autoantibodies to granulocytes were identified by immunofluorescence with anti-human IgG FITC antibody. Target autoantigen approach: Candidate proteins were identified from previous literature and FANTOM5 gene set analysis for eosinophil expressed proteins. Serum IgG autoantibodies to these proteins, in native and oxPTM form, were detected by indirect ELISA. RESULTS: Immunofluorescence studies showed that serum from patients with known ANCA stained for IgG against neutrophils as expected. In addition, serum from 9 of 17 tested SEA patients stained for IgG to PMA-stimulated neutrophils undergoing NETosis. Immunofluorescent staining of eosinophil slides was evident with serum from all participants (healthy and with eosinophilic disease) with diffuse cytoplasmic staining except for one SEA individual in whom subtle nuclear staining was evident. FANTOM5 gene set analysis identified TREM1 (triggering receptor expressed on myeloid cells 1) and IL-1 receptor 2 (IL1R2) as eosinophil-specific targets to test for autoantibody responses in addition to MPO, eosinophil peroxidase (EPX), and Collagen-V identified from previous literature. Indirect ELISAs found high concentrations of serum autoantibodies to Collagen-V, MPO, and TREM1 in a higher proportion of SEA patients than healthy controls. High concentrations of serum autoantibodies to EPX were evident in serum from both healthy and SEA participants. The proportion of patients with positive autoantibody ELISAs was not increased when examining oxPTM compared to native proteins. DISCUSSION: Although none of the target proteins studied showed high sensitivity for SEA, the high proportion of patients positive for at least one serum autoantibody shows the potential of more research on autoantibody serology to improve diagnostic testing for severe asthma. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, identifier, NCT04671446

Rheumatoid arthritis - treatment: 180. Utility of Body Weight Classified Low-Dose Leflunomide in Japanese Rheumatoid Arthritis

Background: In Japan, more than 20 rheumatoid arthritis (RA) patients died of interstitial pneumonia (IP) caused by leflunomide (LEF) were reported, but many of them were considered as the victims of opportunistic infection currently. In this paper, efficacy and safety of low-dose LEF classified by body weight (BW) were studied. Methods: Fifty-nine RA patients were started to administrate LEF from July 2007 to July 2009. Among them, 25 patients were excluded because of the combination with tacrolimus, and medication modification within 3 months before LEF. Remaining 34 RA patients administered 20 to 50 mg/week of LEF were followed up for 1 year and enrolled in this study. Dose of LEF was classified by BW (50 mg/week for over 50 kg, 40 mg/week for 40 to 50 kg and 20 to 30 mg/week for under 40 kg). The average age and RA duration of enrolled patients were 55.5 years old and 10.2 years. Prednisolone (PSL), methotrexate (MTX) and etanercept were used in 23, 28 and 2 patients, respectively. In case of insufficient response or adverse effect, dosage change or discontinuance of LEF were considered. Failure was defined as dosages up of PSL and MTX, or dosages down or discontinuance of LEF. Last observation carried forward method was used for the evaluation of failed patients at 1 year. Results: At 1 year after LEF start, good/ moderate/ no response assessed by the European League Against Rheumatism (EULAR) response criteria using Disease Activity Score, including a 28-joint count (DAS28)-C reactive protein (CRP) were showed in 14/ 10/ 10 patients, respectively. The dosage changes of LEF at 1 year were dosage up: 10, same dosage: 5, dosage down: 8 and discontinuance: 11 patients. The survival rate of patients in this study was 23.5% (24 patients failed) but actual LEF continuous rate was 67.6% (11 patients discontinued) at 1 year. The major reason of failure was liver dysfunction, and pneumocystis pneumonia was occurred in 1 patient resulted in full recovery. One patient died of sepsis caused by decubitus ulcer infection. DAS28-CRP score was decreased from 3.9 to 2.7 significantly. Although CRP was decreased from 1.50 to 0.93 mg/dl, it wasn't significant. Matrix metalloproteinase (MMP)-3 was decreased from 220.0 to 174.2 ng/ml significantly. Glutamate pyruvate transaminase (GPT) was increased from 19 to 35 U/l and number of leukocyte was decreased from 7832 to 6271 significantly. DAS28-CRP, CRP, and MMP-3 were improved significantly with MTX, although they weren't without MTX. Increase of GPT and leukopenia were seen significantly with MTX, although they weren't without MTX. Conclusions: It was reported that the risks of IP caused by LEF in Japanese RA patients were past IP history, loading dose administration and low BW. Addition of low-dose LEF is a potent safe alternative for the patients showing unsatisfactory response to current medicines, but need to pay attention for liver function and infection caused by leukopenia, especially with MTX. Disclosure statement: The authors have declared no conflicts of interes

Identification of autoantigens and their potential post-translational modification in EGPA and severe eosinophilic asthma

BackgroundThe chronic airway inflammation in severe eosinophilic asthma (SEA) suggests potential autoimmune aetiology with unidentified autoantibodies analogous to myeloperoxidase (MPO) in ANCA-positive EGPA (eosinophilic granulomatosis with polyangiitis). Previous research has shown that oxidative post-translational modification (oxPTM) of proteins is an important mechanism by which autoantibody responses may escape immune tolerance. Autoantibodies to oxPTM autoantigens in SEA have not previously been studied.MethodsPatients with EGPA and SEA were recruited as well as healthy control participants. Autoantigen agnostic approach: Participant serum was incubated with slides of unstimulated and PMA-stimulated neutrophils and eosinophils, and autoantibodies to granulocytes were identified by immunofluorescence with anti-human IgG FITC antibody. Target autoantigen approach: Candidate proteins were identified from previous literature and FANTOM5 gene set analysis for eosinophil expressed proteins. Serum IgG autoantibodies to these proteins, in native and oxPTM form, were detected by indirect ELISA.ResultsImmunofluorescence studies showed that serum from patients with known ANCA stained for IgG against neutrophils as expected. In addition, serum from 9 of 17 tested SEA patients stained for IgG to PMA-stimulated neutrophils undergoing NETosis. Immunofluorescent staining of eosinophil slides was evident with serum from all participants (healthy and with eosinophilic disease) with diffuse cytoplasmic staining except for one SEA individual in whom subtle nuclear staining was evident. FANTOM5 gene set analysis identified TREM1 (triggering receptor expressed on myeloid cells 1) and IL-1 receptor 2 (IL1R2) as eosinophil-specific targets to test for autoantibody responses in addition to MPO, eosinophil peroxidase (EPX), and Collagen-V identified from previous literature. Indirect ELISAs found high concentrations of serum autoantibodies to Collagen-V, MPO, and TREM1 in a higher proportion of SEA patients than healthy controls. High concentrations of serum autoantibodies to EPX were evident in serum from both healthy and SEA participants. The proportion of patients with positive autoantibody ELISAs was not increased when examining oxPTM compared to native proteins.DiscussionAlthough none of the target proteins studied showed high sensitivity for SEA, the high proportion of patients positive for at least one serum autoantibody shows the potential of more research on autoantibody serology to improve diagnostic testing for severe asthma.Clinical trial registrationClinicalTrials.gov, identifier, NCT04671446