TY - JOUR
T1 - A bispecific, crosslinking lectibody activates cytotoxic T cells and induces cancer cell death
AU - Rosato, Francesca
AU - Pasupuleti, Rajeev
AU - Tomisch, Jana
AU - Meléndez, Ana Valeria
AU - Kolanovic, Dajana
AU - Makshakova, Olga N.
AU - Wiltschi, Birgit
AU - Römer, Winfried
N1 - Funding Information:
Open Access funding enabled and organized by Projekt DEAL. This research was funded by the European Union’s Horizon 2020 Research and Innovation Program under the Marie Skłodowska-Curie grant agreement synBIOcarb (No. 814029). Moreover, WR acknowledges support by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy (GSC-4, EXC-294 and EXC-2189) and the German Research Foundation grant Major Research Instrumentation (project number: 438033605), by the Ministry for Science, Research and Arts of the State of Baden-Württemberg (Az: 33-7532.20; and partial funding of SGBM PhD thesis), and by the Freiburg Institute for Advanced Studies (FRIAS). This publication is partially based upon work from COST Action CA18103 (INNOGLY), supported by COST (European Coorperation in Science and Technology). ONM thanks the financial support from the government assignment for FRC Kazan Scientific Center of RAS. In addition, we would like to express our gratitude to the Life Imaging Center (LIC) of the University of Freiburg for support.
Funding Information:
We thank the Blood Donation Center, Medical Centre of the University of Freiburg for providing LRS chambers. We also express gratitude to Dr. Pavel Salavei of the CIBSS Signaling Factory, University of Freiburg, Germany, for his help and guidance in flow cytometry. Barbara Darnhofer and Ruth Birner-Grünberger as well as the BOKU Core Facility Mass Spectrometry are acknowledged for excellent support with mass spectrometry.
Publisher Copyright:
© 2022, The Author(s).
PY - 2022/12
Y1 - 2022/12
N2 - Background: Aberrant glycosylation patterns play a crucial role in the development of cancer cells as they promote tumor growth and aggressiveness. Lectins recognize carbohydrate antigens attached to proteins and lipids on cell surfaces and represent potential tools for application in cancer diagnostics and therapy. Among the emerging cancer therapies, immunotherapy has become a promising treatment modality for various hematological and solid malignancies. Here we present an approach to redirect the immune system into fighting cancer by targeting altered glycans at the surface of malignant cells. We developed a so-called “lectibody”, a bispecific construct composed of a lectin linked to an antibody fragment. This lectibody is inspired by bispecific T cell engager (BiTEs) antibodies that recruit cytotoxic T lymphocytes (CTLs) while simultaneously binding to tumor-associated antigens (TAAs) on cancer cells. The tumor-related glycosphingolipid globotriaosylceramide (Gb3) represents the target of this proof-of-concept study. It is recognized with high selectivity by the B-subunit of the pathogen-derived Shiga toxin, presenting opportunities for clinical development. Methods: The lectibody was realized by conjugating an anti-CD3 single-chain antibody fragment to the B-subunit of Shiga toxin to target Gb3+ cancer cells. The reactive non-canonical amino acid azidolysine (AzK) was inserted at predefined single positions in both proteins. The azido groups were functionalized by bioorthogonal conjugation with individual linkers that facilitated selective coupling via an alternative bioorthogonal click chemistry reaction. In vitro cell-based assays were conducted to evaluate the antitumoral activity of the lectibody. CTLs, Burkitt´s lymphoma-derived cells and colorectal adenocarcinoma cell lines were screened in flow cytometry and cytotoxicity assays for activation and lysis, respectively. Results: This proof-of-concept study demonstrates that the lectibody activates T cells for their cytotoxic signaling, redirecting CTLs´ cytotoxicity in a highly selective manner and resulting in nearly complete tumor cell lysis—up to 93%—of Gb3+ tumor cells in vitro. Conclusions: This research highlights the potential of lectins in targeting certain tumors, with an opportunity for new cancer treatments. When considering a combinatorial strategy, lectin-based platforms of this type offer the possibility to target glycan epitopes on tumor cells and boost the efficacy of current therapies, providing an additional strategy for tumor eradication and improving patient outcomes.
AB - Background: Aberrant glycosylation patterns play a crucial role in the development of cancer cells as they promote tumor growth and aggressiveness. Lectins recognize carbohydrate antigens attached to proteins and lipids on cell surfaces and represent potential tools for application in cancer diagnostics and therapy. Among the emerging cancer therapies, immunotherapy has become a promising treatment modality for various hematological and solid malignancies. Here we present an approach to redirect the immune system into fighting cancer by targeting altered glycans at the surface of malignant cells. We developed a so-called “lectibody”, a bispecific construct composed of a lectin linked to an antibody fragment. This lectibody is inspired by bispecific T cell engager (BiTEs) antibodies that recruit cytotoxic T lymphocytes (CTLs) while simultaneously binding to tumor-associated antigens (TAAs) on cancer cells. The tumor-related glycosphingolipid globotriaosylceramide (Gb3) represents the target of this proof-of-concept study. It is recognized with high selectivity by the B-subunit of the pathogen-derived Shiga toxin, presenting opportunities for clinical development. Methods: The lectibody was realized by conjugating an anti-CD3 single-chain antibody fragment to the B-subunit of Shiga toxin to target Gb3+ cancer cells. The reactive non-canonical amino acid azidolysine (AzK) was inserted at predefined single positions in both proteins. The azido groups were functionalized by bioorthogonal conjugation with individual linkers that facilitated selective coupling via an alternative bioorthogonal click chemistry reaction. In vitro cell-based assays were conducted to evaluate the antitumoral activity of the lectibody. CTLs, Burkitt´s lymphoma-derived cells and colorectal adenocarcinoma cell lines were screened in flow cytometry and cytotoxicity assays for activation and lysis, respectively. Results: This proof-of-concept study demonstrates that the lectibody activates T cells for their cytotoxic signaling, redirecting CTLs´ cytotoxicity in a highly selective manner and resulting in nearly complete tumor cell lysis—up to 93%—of Gb3+ tumor cells in vitro. Conclusions: This research highlights the potential of lectins in targeting certain tumors, with an opportunity for new cancer treatments. When considering a combinatorial strategy, lectin-based platforms of this type offer the possibility to target glycan epitopes on tumor cells and boost the efficacy of current therapies, providing an additional strategy for tumor eradication and improving patient outcomes.
KW - Bispecific targeting
KW - Cancer immunotherapy
KW - Click chemistry
KW - Globotriaosylceramide
KW - Lectins
KW - Shiga toxin
KW - T cells
KW - Tumor-associated carbohydrate antigens
UR - http://www.scopus.com/inward/record.url?scp=85143656097&partnerID=8YFLogxK
U2 - 10.1186/s12967-022-03794-w
DO - 10.1186/s12967-022-03794-w
M3 - Article
C2 - 36494671
AN - SCOPUS:85143656097
VL - 20
JO - Journal of Translational Medicine
JF - Journal of Translational Medicine
SN - 1479-5876
IS - 1
M1 - 578
ER -