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Abstract: TH-PO008

Modeling Glomerular Complement Activity In-Vitro Using a Novel Human Glomerular Endothelial Cell Assay

Session Information

Category: Glomerular

  • 1005 Clinical Glomerular Disorders


  • Ruan, Qin, Regeneron Pharmaceuticals, Tarrytown, New York, United States
  • Devalaraja-Narashimha, Kishor B., Regeneron Pharmaceuticals, Tarrytown, New York, United States
  • Mao, Shu, Regeneron Pharmaceuticals, Tarrytown, New York, United States
  • Broden, Joshua Reid, Regeneron Pharmaceuticals, Tarrytown, New York, United States
  • Morton, Lori, Regeneron Pharmaceuticals, Tarrytown, New York, United States
  • Macdonnell, Scott, Regeneron Pharmaceuticals, Tarrytown, New York, United States

Reproducible methods for evaluating inhibitory effects of drug candidates on complement activation are essential for preclinical development and potential clinical translation. Due to the complexity of complement activation pathways, an in-vitro assay should use relevant cells and endpoints to the given therapeutic indication. Here, using primary human glomerular endothelial cells (HGECs), we validated a complement C3 & C5 deposition model for use evaluating the blocking activity of C5 monoclonal antibodies (mAb).


Resting or adenosine 5′-diphosphate (ADP)-activated confluent HGECs were incubated for 4 hours with 50% normal human serum, C3 or C5 depleted human serum, or pretreated with anti-C5 mAbs. Thereafter, HGECs were fixed with BD Cytofix, stained with ThermoFisher anti-human C3/C3b or Abcam anti-human C5b-9 antibodies and complement deposits were analyzed (9 sites per well in duplicated wells) by ImageXpress.


C3 and C5b-9 deposition was observed on ADP-activated HGECs exposed to normal human serum but not on non-activated HGECs (C3: 1.5x107±1.0x107; C5: 7.9x106±6.6x106, P<0.05 vs non-ADP-activated HGEC). The deposition of C3 and C5b-9 were significantly reduced on ADP-activated HGEC exposed to C3 or C5 depleted serum (C3: 3.3x105±4.8x104; C5: 1.5x106±6.0x105, P<0.05). Addition of a blocking anti-C5 mAb significantly reduced normal human serum derived C5b-9 deposition onto ADP-activated HGEC, deposition was comparable to C5 depleted sera (C5 mAb: 1.02x106±6.0x105, Control mAb 3.7x106±1.6x106, P<0.05 vs. control mAb).


These data demonstrate utility of an in vitro primary human glomerular endothelial cell assay to model complement C3 & C5 deposition. In addition to in-vitro screening, this assay offers potential as a translational model to evaluate anti-complement strategies in renal disease using patient derived serum samples.


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