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A1.39 Modulation of atp:adenosine balance by exosome and rain (Recombinant Anti-Inflammatory Fusion Protein) delivery of CD39 and CD73 is effective in reducing pro-inflammatory cytokine and chemokine production
  1. Jonathan D Finn1,2,
  2. Susanne Snoek1,2,
  3. Jan van Ittersum1,2,
  4. Niels Broekstra1,2,
  5. Carola Feijt 1,2,
  6. Paul P Tak1,2,
  7. Margriet J Vervoordeldonk1,2
  1. 1Arthrogen B. V., Amsterdam, The Netherlands
  2. 2Div. of Clinical Immunology and Rheumatology, Academic Medical Center/University of Amsterdam, Amsterdam, The Netherlands

Abstract

Background and Objectives The conversion of ATP to adenosine is an important mechanism of immune suppression by Tregs, and this is done by expression of CD39 and CD73. CD39 is a membrane bound ATPase that converts extracellular ATP and ADP to AMP, whereas CD73 is a membrane bound ecto-nucleotidase that converts AMP to adenosine. We have previously found evidence that the balance between pro-inflammatory ATP and anti-inflammatory adenosine is skewed in the synovial compartment of rheumatoid arthritis (RA) patients. We have developed two novel systems for the delivery of CD39 and CD73. The first is a Recombinant Anti-Inflammatory fusioN protein (RAIN) that consists of soluble CD73 fused to soluble CD39 by a flexible linker. The second approach uses exosomes containing CD39 and CD73. Here we present data characterising these CD39-CD73 delivery methods and investigate their efficacy in vitro.

Materials and Methods CD39-CD73 samples were produced by transfecting 293T cells with CD39-CD73 expressing plasmids. Exosomes were isolated using ExoQuick TC (System Biosciences) and soluble proteins were concentrated using a spin column. Antigen levels were assayed by quantitative western blot (Odyssey). CD39 and CD73 activity was measured by Malachite Green Phosphate (R&D Systems) assay using ATP or AMP as a substrate. In vitro inflammation assay was performed by incubating LPS activated THP-1 cells with CD39-CD73 containing samples in the presence of ATP. Whole blood inflammasome activation assay was performed by incubating whole blood with CD39-CD73 samples prior to 2 hr LPS stimulation followed by 1 hr ATP stimulation. Pro-inflammatory cytokine/chemokine levels were measured by ELISA.

Results RAIN and exosomes demonstrated high specific activity (RAIN: CD39 = 6660 U/pmol, CD73 = 16100 U/pmol; Exo: CD39 = 10900 U/pmol, CD73 = 2820 U/pmol). Both were potent in reducing pro-inflammatory cytokine and/or chemokine (IL-6, CCL2) expression in a THP-1 based in vitro inflammation assay (CCL2 EC50: RAIN 44 pM ± 1.13, exosomes 12.4 pM ± 1.27; IL-6 EC50: RAIN 29.3 pM ± 1.1, exosomes 5.9 pM ± 1.2). RAIN and CD39-CD73 exosomes were also effective in reducing IL-1β secretion in a whole blood inflammasome activation assay (IL-1β EC50: RAIN ~4000 pM, exosome ~100 pM).

Conclusions We have developed two novel biological therapeutics, RAIN and exosomes expressing CD39 and CD73 that are able to simultaneously decrease a potent pro-inflammatory molecule while increasing an anti-inflammatory molecule. The use of CD39 and CD73 to modulate inflammation should be applicable to broad spectrum of acute and chronic inflammatory disorders.

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