Site-specific dual-labelling of proteins with tetrazines – ZINELABEL

Bioconjugates have become essential tools for biomedical research, diagnosis and therapy. A second generation of bioconjugates begins to emerge. It consists in doubly-modified proteins, which have optimized properties and / or open new fields of application such as bimodal medical imaging or theranostics. For instance, these last years have seen the emergence of bimodal imaging agents based on a biovector labeled with both a fluorescent dye and a chelator for a radiometal (for PET or SPECT), and some of them are used nowadays in clinic for surgery assistance. Some tremendous advances have also been made in the field of Antibody Drug Conjugates (ADC), which became a major class of highly potent drugs for targeted therapy. Attaching two different cytotoxic payloads, or both a drug and an imaging reporter, to an antibody (theranostic), will contribute to the development of improved ADC.

The development or theses new bioconjugates is currently hampered by the technical difficulties associated with their construction. Increasing attention has been paid in the last years to the use of site-specific labeling of antibodies, allowing access to chemically defined constructs, more reproducible procedures, a better control of the degree of labeling, which finally may facilitate approval process. However, in many cases, advanced level of protein engineering is skill required to obtain such bioconjugates. Thus, the development of technologies or procedures able to facilitate the production of such biomolecules should enable the explosion of this next generation of bioconjugates and their translation into the market. These innovative technologies must fulfill several criteria: ideally they should be i) easy to perform, ii) site-specific, iii) tolerant/biocompatible, iv) modular.

The ZINELABEL project aims at developing a robust, modular, small, ternary platform, so-called dichloro-s-tetrazine, enabling the direct or indirect, site-specific, double-labeling of proteins in fully biocompatible conditions. The reactivity of dichloro-s-tetrazine will be exploited to prepare a library of mono- and di-substituted derivatives, by successive introduction of different moieties (NIR dye, macrocyclic chelator, PEG chain, targeting agent). The capacity of these modified tetrazines to undergo iEDDA click reaction will be studied and these platforms will be used for i) site-specific labeling of an Affibody, ii) dual modification of a protein aptamer targeting HSP70. These proofs of concept will serve at demonstrating the high potential and versatility of the ZINELABEL technology for the easy and efficient dual-labeling of proteins.