Abstract Number: PB0754
Meeting: ISTH 2020 Congress
Background: Plasmin (Plm) which is generated from its zymogen plasminogen (Plg) is a serine protease which plays an important role in fibrin degradation and is a potent modulator of immunological processes and cancer progression. A heavy chain only antibody (VHH), nanobody, which specifically binds to Plg or Plm might render diagnostic and clinical applications.
Aims: To generate VHH(s) against Plg/Plm with high affinity and specificity.
Methods: Plg was isolated and purified from citrated pool plasma using lysine sepharose affinity chromatography (AC) followed by size-exclusion chromatography (SEC). The Plg activation process was optimized in terms of incubation time and temperature, and the activator type. The purified active Plm (200 µg) was injected 6 times subcutaneously to two llamas and peripheral blood lymphocytes were collected and subjected to mRNA extraction and cDNA reverse transcription. The cDNA was used for phage display, panning and ELISA-affinity assays. Selected VHHs were characterized in terms of binding affinity and specificity to both Plg and Plm using bio-layer interferometry (BLI).
Results: Using AC and SEC, 2 mg Plg was isolated from 250 ml citrated pool plasma having 90% purity determined by activity testing and Bradford protein determination. The optimum activation of Plg to Plm was achieved by incubation of Plg with urokinase-type plasminogen activator for 72h at RT. Based on ELISA-affinity results and BLI, 46 hit compounds were found. Among them, seven VHHs bind Plg and Plm with KDs ranging from 1 to 20 nM. One VHH showed selective binding to Plm.
Conclusions: Using purified Plm to immunize llamas, a group of affine nanobodies were isolated against Plg and/or Plm. Further comprehensive functional characterization will reveal the potential of VHHs for the future development of possible drug candidates.
To cite this abstract in AMA style:Klein EM, Shahidi Hamedani N, Müller J, Rühl H, König PA, Oldenburg J, Pötzsch B. Production of Nanobodies Targeting Plasminogen and Plasmin [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/production-of-nanobodies-targeting-plasminogen-and-plasmin/. Accessed January 21, 2022.
« Back to ISTH 2020 Congress
ISTH Congress Abstracts - https://abstracts.isth.org/abstract/production-of-nanobodies-targeting-plasminogen-and-plasmin/