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 Papavasiliou; Joseph P. Verdi

doi:10.1016/j.celrep.2023.112049

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

Pharmacology

Research output: Contribution to journal › Article › peer-review

1 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 language	English (US)
Article number	112049
Journal	Cell reports
Volume	42
Issue number	2
DOIs	https://doi.org/10.1016/j.celrep.2023.112049
State	Published - 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

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.celrep.2023.112049

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Triller, G., Vlachou, E. P., Hashemi, H., van Straaten, M., Zeelen, J. P., Kelemen, Y., Baehr, C., Marker, C. L., Ruf, S., Svirina, A., Chandra, M., Urban, K., Gkeka, A., Kruse, S., Baumann, A., Miller, A. K., Bartel, M., Pravetoni, M., Stebbins, C. E., ... Verdi, J. P. (2023). A trypanosome-derived immunotherapeutics platform elicits potent high-affinity antibodies, negating the effects of the synthetic opioid fentanyl. Cell reports, 42(2), Article 112049. https://doi.org/10.1016/j.celrep.2023.112049

Triller, G, Vlachou, EP, Hashemi, H, van Straaten, M, Zeelen, JP, Kelemen, Y, Baehr, C, Marker, CL, Ruf, S, Svirina, A, Chandra, M, Urban, K, Gkeka, A, Kruse, S, Baumann, A, Miller, AK, Bartel, M, Pravetoni, M, Stebbins, CE, Papavasiliou, FN & Verdi, JP 2023, 'A trypanosome-derived immunotherapeutics platform elicits potent high-affinity antibodies, negating the effects of the synthetic opioid fentanyl', Cell reports, vol. 42, no. 2, 112049. https://doi.org/10.1016/j.celrep.2023.112049

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

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.",

keywords = "CP: Immunology, Trypanosoma brucei, antibody repertoire analysis, fentanyl, humoral immunity, immunological memory, opioid overdose, sortase, variant surface glycoprotein",

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

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: {\textcopyright} 2023 The Authors",

year = "2023",

month = feb,

day = "28",

doi = "10.1016/j.celrep.2023.112049",

language = "English (US)",

volume = "42",

journal = "Cell reports",

issn = "2211-1247",

publisher = "Cell Press",

number = "2",

}

TY - JOUR

T1 - A trypanosome-derived immunotherapeutics platform elicits potent high-affinity antibodies, negating the effects of the synthetic opioid fentanyl

AU - Triller, Gianna

AU - Vlachou, Evi P.

AU - Hashemi, Hamidreza

AU - van Straaten, Monique

AU - Zeelen, Johan P.

AU - Kelemen, Yosip

AU - Baehr, Carly

AU - Marker, Cheryl L.

AU - Ruf, Sandra

AU - Svirina, Anna

AU - Chandra, Monica

AU - Urban, Katharina

AU - Gkeka, Anastasia

AU - Kruse, Sebastian

AU - Baumann, Andreas

AU - Miller, Aubry K.

AU - Bartel, Marc

AU - Pravetoni, Marco

AU - Stebbins, C. Erec

AU - Papavasiliou, F. Nina

AU - Verdi, Joseph P.

N1 - 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

PY - 2023/2/28

Y1 - 2023/2/28

N2 - 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.

AB - 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.

KW - CP: Immunology

KW - Trypanosoma brucei

KW - antibody repertoire analysis

KW - fentanyl

KW - humoral immunity

KW - immunological memory

KW - opioid overdose

KW - sortase

KW - variant surface glycoprotein

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DO - 10.1016/j.celrep.2023.112049

M3 - Article

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VL - 42

JO - Cell reports

JF - Cell reports

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ER -

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

Abstract

Bibliographical note

Keywords

UN SDGs

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