A trypanosome-derived immunotherapeutics platform elicits potent high-affinity antibodies, negating the effects of the synthetic opioid fentanyl

Gianna Triller, Evi P. Vlachou, Hamidreza Hashemi, Monique van Straaten, Johan P. Zeelen, Yosip Kelemen, Carly Baehr, Cheryl L. Marker, Sandra Ruf, Anna Svirina, Monica Chandra, Katharina Urban, Anastasia Gkeka, Sebastian Kruse, Andreas Baumann, Aubry K. Miller, Marc Bartel, Marco Pravetoni, C. Erec Stebbins, F. Nina PapavasiliouJoseph P. Verdi

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

Poorly immunogenic small molecules pose challenges for the production of clinically efficacious vaccines and antibodies. To address this, we generate an immunization platform derived from the immunogenic surface coat of the African trypanosome. Through sortase-based conjugation of the target molecules to the variant surface glycoprotein (VSG) of the trypanosome surface coat, we develop VSG-immunogen array by sortase tagging (VAST). VAST elicits antigen-specific memory B cells and antibodies in a murine model after deploying the poorly immunogenic molecule fentanyl as a proof of concept. We also develop a single-cell RNA sequencing (RNA-seq)-based computational method that synergizes with VAST to specifically identify memory B cell-encoded antibodies. All computationally selected antibodies bind to fentanyl with picomolar affinity. Moreover, these antibodies protect mice from fentanyl effects after passive immunization, demonstrating the ability of these two coupled technologies to elicit therapeutic antibodies to challenging immunogens.

Original languageEnglish (US)
Article number112049
JournalCell reports
Volume42
Issue number2
DOIs
StatePublished - Feb 28 2023

Bibliographical note

Funding Information:
We acknowledge synchrotron time at the Paul Scherrer Institut, Villingen, Switzerland (SLS, beamline PXIII, Vincent Olieric and colleagues). We acknowledge the DKFZ Flow Cytometry core facility and the DKFZ Single-Cell Open Lab (scOpenLab). We acknowledge Dr. Claudia Pitzer and Barbara Kurpiers of the Interdisciplinary Neurobehavioral Core (INBC) facility, Heidelberg, for the use of the LABORAS system and the hot plate and their assistance. We thank Dustin Hicks, Jenny Vigliaturo (University of Minnesota), Elias Amro (DKFZ), and Vanessa Hofmann for key experimental assistance. This project was funded by grants from the NIH (R01AI097127 to F.N.P. and 1R43DA052960-01 to J.P.V.) and funds from the Helmholtz Foundation (to F.N.P. and C.E.S.). We also acknowledge early contributions by P. Stavropoulos and J. Pinger, who helped initiate the work presented here. C.E.S. and F.N.P. conceived and initiated the project. Immunological work: A.B. and A.K.M. generated fen-G4 and fen-sort. H.H. produced the sortaggable trypanosome lines. A.S. G.T. H.H. and J.P.V. conducted sortagging experiments. A.S. quantified sortagging efficiency. G.T. H.H. and K.U. performed ELISA assays. F.N.P. G.T. H.H. M.C. and S.K. conducted immunization experiments and isolated immunological tissues. F.N.P. G.T. and S.R. and conducted fentanyl challenges and behavioral assays. M.P. coordinated and analyzed MS. G.T. and P.V. baited and sorted splenocytes and also generated cDNA libraries for sequencing. Bioinformatic derivation of antibodies: P.V. conceived and constructed the bioinformatic pipeline and analyzed the SmartSeq data with assistance from G.T. and F.N.P. deriving antibody sequences. Antibody characterization: M.v.S. cloned and produced Fabs, and Y.K. cloned and produced mAbs. C.B. performed ELISA and BLI assays. A.S. performed and analyzed ITC. A.G. F.N.P. G.T. and K.U. performed and analyzed passive therapy experiments. M.B. performed MS. crystallography, and M.v.S. crystallized Fab-fentanyl complexes. J.P.Z. and M.v.S. prepared crystals for beamline data collection, collected the data, and solved the structures. C.E.S. J.P.Z. and M.v.S analyzed the structural data. Manuscript: C.E.S. C.L.M. F.N.P. G.T. J.P.V. and M.P. coordinated the project. C.E.S. G.T. and J.P.V. wrote the manuscript. G.T. C.E.S. F.N.P. and J.P.V. report being shareholders of Panosome GmbH and Hepione Therapeutics and having patents planned, pending, or issued broadly relevant to the work. C.E.S. and F.N.P. report being the managing directors of Panosome GmbH. S.K. reports being a shareholder of Panosome GmbH and Hepione Therapeutics. S.R. and K.U. report being employees of Panosome GmbH. P.V. K.U. M.v.S. J.P.Z. Y.K. A.S. A.K.M, A.B. and H.H. report having patents planned, pending, or issued broadly relevant to the work. M.P. and C.B. have patents pending related to fentanyl haptens, conjugates, and mAbs against fentanyl-like compounds.

Funding Information:
We acknowledge synchrotron time at the Paul Scherrer Institut, Villingen, Switzerland (SLS, beamline PXIII, Vincent Olieric and colleagues). We acknowledge the DKFZ Flow Cytometry core facility and the DKFZ Single-Cell Open Lab (scOpenLab). We acknowledge Dr. Claudia Pitzer and Barbara Kurpiers of the Interdisciplinary Neurobehavioral Core (INBC) facility, Heidelberg, for the use of the LABORAS system and the hot plate and their assistance. We thank Dustin Hicks, Jenny Vigliaturo (University of Minnesota), Elias Amro (DKFZ), and Vanessa Hofmann for key experimental assistance. This project was funded by grants from the NIH ( R01AI097127 to F.N.P. and 1R43DA052960-01 to J.P.V.) and funds from the Helmholtz Foundation (to F.N.P. and C.E.S.). We also acknowledge early contributions by P. Stavropoulos and J. Pinger, who helped initiate the work presented here.

Publisher Copyright:
© 2023 The Authors

Keywords

  • CP: Immunology
  • Trypanosoma brucei
  • antibody repertoire analysis
  • fentanyl
  • humoral immunity
  • immunological memory
  • opioid overdose
  • sortase
  • variant surface glycoprotein

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

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