Abstract: SA-PO646

Targeted C4 Inhibition by Affinity Purified Immunoglobulins

Session Information

Category: Pharmacokinetics, Pharmacodynamics, and Pharmacogenetics

  • 1601 Pharmacokinetics, Pharmacodynamics, Pharmacogenomics

Authors

  • Volokhina, Elena, Radboud university medical center, Nijmegen, Netherlands
  • Van der velden, Thea J., Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands
  • Michels, Marloes, Radboudumc, Nijmegen, Netherlands
  • Van De Kar, Nicole, UMC St. Radboud Nymegen, Nijmegen, Netherlands
  • Okroj, Marcin, Medical University Gdansk, Gdansk, Poland
  • Van den heuvel, Bert, Radboudumc, Nijmegen, Netherlands
Background

Immunoglobulins (Igs) can activate complement when bound to their antigens. Moreover, they may inhibit complement activation and in clinical practice intravenous Igs are widely used to treat immunodeficiencies as well as inflammatory conditions. Complement inhibitory properties of Igs are poorly understood, which limits their use for targeted complement modulation in renal disorders. In this study we describe immunoglobulin preparations with specific complement inhibiting properties.

Methods

Igs from healthy donors were purified using Protein L or Protein A/G affinity chromatography. Classical (CP) and alternative pathway (AP) activation was assessed using hemolytic assays. Activation of C1q, C4b, C3b and C5b-9 in CP was assessed by ELISA. Purified fractions were analyzed by SDS-PAGE and silver staining.

Results

Igs purified from serum using Protein L, but not Protein A/G inhibited CP in normal human serum (NHS) to below 10% of normal activity when added at 13 mg/mL. Igs from lepirudin, citrate and heparin plasma showed similar inhibiting results, while those isolated from EDTA plasma had no effect. None of the fractions had effect on AP. CP ELISA revealed normal deposition of C1q, and strongly decreased deposition of C4b, C3b and C5b-9. All fractions showed same band pattern in SDS-PAGE. All purified Igs retained ability to activate CP when heat-aggregated. Moreover, Igs isolated from serum spiked with 100 µg/mL eculizumab were still able to fully block AP when added to NHS.

Conclusion

Thus, Igs purified using Protein L chromatography block complement in NHS at the stage of C4. These findings have important therapeutic potential for the inhibition of classical complement pathway in the future in such conditions as antibody-mediated renal graft rejection, lupus nephritis and antiphospholipid syndrome.