Klick und Link: ein chemieorientierter Ansatz für bispezifische Antikörper
Zusammenfassung der Projektergebnisse
The development of new therapeutic agents which specifically target malignant over healthy tissues has become a paradigm of modern cancer therapy. Bispecific antibodies (bsAbs) combine two monospecific antibodies onto a common scaffold, enabling the exploration of novel functionalities beyond traditional immunomodulators. This dual-targeting concept, which has proven effective in patients in the format of T-Cell engagers, holds great therapeutic potential since it could be extended to any combination of two proteins. Despite their modular nature, the current development of bsAbs is slow due to their tedious and linear production process that mostly relies on genetic approaches. To test N combinations of antibodies as bispecifics, N constructs need to be cloned, expressed, purified, and characterized. As an alternative, this project aimed at establishing a new, chemistry-driven, paradigm for the construction of bispecific antibodies using site-specific biorthogonal chemistry. It was our premise that, a chemistry-driven bsAbs assembly would enable a fast exploration of dissimilar protein conjugations, enabling the generation of a wide variety of protein-protein combinations from a small set of binders. In this endeavour, several chemical tools were attempted, including novel isonitrile-tetrazine multicomponent processes and stepwise Cysteine-specific clickable handles. Some of the approaches that failed at the protein level were valuable for the derivatization of peptides of biomedical relevance. An important result was the development of a new class of stable maleimides which enable ultrafast protein-protein bioconjugation while leading to stable conjugates. These stable maleimides enabled the construction of a small bsAbs library, from the combination of cancerrelated VHH antibodies. It was also proven how the fast exploration of different binders specific to the HER2 antigen can generate dimeric constructs with distinctive binding kinetics as determined by Biolayer interferometry assays. Moreover a chemistry-driven approach enables further derivatization of these bsAbs beyond the protein-protein conjugation. In this direction, prepared fluorescently labeled biparatopic antibodies as well as dimeric antibody-drug conjugates were prepared. Confocal microscopy experiments confirmed the improved membrane binding capabilities of dimeric constructs as compared to their monomeric analogs. Finally, the methodology was extended to the synthesis of higher order constructs. This included a trimeric T-Cell engager and a tetrametric α-HER2/α-PD-L1 bispecific, assembled from the combination of monomeric α-HER2, α-CD3 and α-PD-L1 VHH antibodies.
Projektbezogene Publikationen (Auswahl)
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Merging the Isonitrile‐Tetrazine (4+1) Cycloaddition and the Ugi Four‐Component Reaction into a Single Multicomponent Process. Angewandte Chemie International Edition, 62(44).
Méndez, Yanira; Vasco, Aldrin V.; Ivey, Galway; Dias, Ana Laura; Gierth, Peter; Sousa, Bárbara B.; Navo, Claudio D.; Torres‐Mozas, Angel; Rodrigues, Tiago; Jiménez‐Osés, Gonzalo & Bernardes, Gonçalo J. L.
