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CF LINK™ – protein bioconjugation and characterization technology opening new frontiers in life science

Dr Václav Matoušek, CF Plus Chemicals s.r.o.; Prof. Antonio Togni, ETH Zürich; Dr Petr Beier, IOCB Prague ; Dr Petr Novák, Institute of Microbiology of the Czech Academy of Sciences (IMIC)

IOCBTech


Challenge

Mild and site-selective protein bioconjugation is a key technology that enables the preparation of homogeneous protein conjugates with applications in clinical diagnostics and targeted antitumour therapy using antibody-drug-conjugates. Bioconjugation methods that operate under mild conditions without the use of toxic transition metals are also particularly advantageous from the manufacturing and regulatory perspective. Detailed understanding of the protein surface map, its solution-dynamics and its interactions with various partners (antigen, complementary protein, etc.) is essential for the development of protein drugs. Robust methods that quickly uncover the delicate nature of protein-protein interactions can therefore dramatically accelerate the discovery of new protein therapeutics.


Technology

Herein we disclose a technology for site-selective bioconjugation of proteins using hypervalent iodine-fluoroalkyl reagents, also known as Togni reagents. Proteins containing free cysteine groups undergo site-selective bioconjugation affording the corresponding S-conjugates that unlike maleimides are not subject to gradual thiol exchange or deconjugation. Tryptophan residues are subject to site-selective bioconjugation when sodium ascorbate, an inexpensive, non-toxic and biocompatible reductant is used in combination with our reagents. When azido fluoroalkyl groups are attached to the protein, various functional groups, such as fluorescent dyes, radionuclides or ADC-toxins can be subsequently ligated via click reaction. This bioconjugation method can also be, under slightly modified reaction conditions, extended to other aromatic amino acids and hence to proteins lacking tryptophan. Thus, it was possible to successfully modify human recombinant insulin, demonstrating the potential for post-translational modification of proteins and protein engineering. The extremely rapid nature of this reaction that targets solvent-accessible aromatic amino acids was turned into a convenient tool for mapping of protein surfaces and study of protein-protein interactions.


Commercial Opportunity

- Reagents for synthesis/modification of small molecule drug candidates

- Kits/technology for protein characterization, stabilization and crosslinking, out-licensing options

- Kits/technology for site-selective bioconjugation of antibodies for ADCs, out-licensing options


Development Status

- scalable, chromatography-free synthesis optimized

- novel reagents with improved performance and new functional groups in the pipeline

- selective thiol bioconjugation showcased on an enzyme, outperforming conventional reagents

- site-selective bioconjugation of tryptophans showcased on myoglobin, work on therapeutically relevant proteins (mAb, other formats) in progress

- kits for protein fingerprinting/surface mapping soon to be launched

- kits for selective bioconjugation of proteins via thiols and tryptophans soon to be launched


Patent Situation

WO2016019475A1, Granted US Patent US 10,040,812 B2, excl. lic. EP 19172756.9, excl. lic. US patent. appl. 15/827,931


Further Reading

1) V. Matoušek, J. Václavík, P. Hájek, J. Charpentier, Z. E. Blastik, E. Pietrasiak, A. Budinská, A. Togni, P. Beier, Chem. Eur. J. 2016, 22, 417–424.,

2) J. Václavík, R. Zschoche, I. Klimánková, V. Matoušek, P. Beier, D. Hilvert, A. Togni, Chem. Eur. J. 2017, 23, 6490–6494.

3) Chem. Eur. J., 2019., accepted manuscript. doi:10.1002/chem.201902944 2) Press release at: www.uochb.cz/en/news/142/new-technology-cf-link-for-protein-bioconjugation-and-structural-proteomics


 

CF LINK™ – protein bioconjugation and characterization technology opening new frontiers in life science