Exploring polymerisation chemistry for synthesising radiolabelled metal-ion polymer bioconjugates for radioimmunotherapy Samy Kichou, Cesare Berton, Patrick A. Cieslik, Jason P. Holland University of Zurich, Department of Chemistry, Switzerland Radioimmunotherapy (RIT) involves the combination of a radionuclide with biologically active vector like a monoclonal antibody (mAb) that delivers the therapeutic payload specifically to a disease lesion. The main challenge in developing radiopharmaceuticals for RIT is to find ways of labelling a protein with sufficiently high levels of radioactivity without compromising the biological properties of the vector. State-of-the-art methods include the combination of enzymatic labelling and click chemistry to achieve site-specific functionalisation and well-defined drug-to-antibody (DAR) ratios in the range of 3 – 5. However, this low stoichiometric DAR ratio imposes limits on the radiochemistry and therapeutic potential of agents for RIT. 1–5 Our objective is to use polymerisation chemistry combined with metal ion selective chelates as monomer units, and photoinitiation water to graft a radiolabelled metal-ion containing polymer (MCP) directly from a bioactive protein. Here, we present our early experiments toward the synthesis of radiolabelled, MCP-proteins using acrylamide-derivatives of metal ion binding chelates. An octadentate, tetra- aza -macrocycle (DOTAGA) was functionalised with an acrylamide moiety using classical amide coupling reactions. Proteins including human serum albumin (HSA) and an IgG 1 monoclonal antibody were successfully functionalized with a photoactivatable benzophenone (BP) unit using N -hydroxylsuccinimide (NHS) to a free lysine or Michael-addition of a maleimide handle to free cysteine residues. Photoinitiation reactions using a 365 nm light-emitting diode confirmed that MCP grafting from the BP-protein occurred in water at room temperature leading radiolabelled MCP-proteins. Radiochemical tests were performed with the positron-emitting radionuclide 68 Ga and the beta-particle emitter 177 Lu. Initial experiments suggest that the successful radiolabelling, as well as polymer chain length and stoichiometry can be controlled by varying the experimental conditions such as protein and monomer concentrations, buffer composition, pH, irradiation time and wavelength. We continue to explore the reactivity and scope of polymerisation chemistry for future applications in the synthesis and testing of novel radiotracers for RIT. References 1. Qi, Y.; Chilkoti, A. Growing Polymers from Peptides and Proteins: A Biomedical Perspective. Polym. Chem. 2013 , 5 (2), 266–276. https://doi.org/10.1039/C3PY01089A. 2. Le, P. J.; Miersch, S.; Forbes, M. W.; Jarvik, N.; Ku, A.; Sidhu, S. S.; Reilly, R. M.; Winnik, M. A. Site-Specific Conjugation of Metal-Chelating Polymers to Anti-Frizzled-2 Antibodies via Microbial Transglutaminase. Biomacromolecules 2021 , 22 (6), 2491–2504. https://doi.org/10.1021/acs.biomac.1c00246. 3. Shatat, R. S.; Niazi, S. K.; Ariffin, A. Synthesis and Characterization of Different Molecular Weights Polyacrylamide. IOSR J. Appl. Chem. 2017 , 10 (04), 67–73. https://doi.org/10.9790/5736-1004016773. 4. Cho, H.; Liu, P.; Boyle, A. J.; Reilly, R. M.; Winnik, M. A. Synthesis of a Metal-Chelating Polymer with NOTA Pendants as a Carrier for 64Cu, Intended for Radioimmunotherapy. Eur. Polym. J. 2020 , 125 , 109501. https://doi.org/10.1016/j. eurpolymj.2020.109501. 5. Shalgunov, V.; Engudar, G.; Bohrmann, L.; Wharton, L.; Maskell, K.; Johann, K.; Barz, M.; Schaffer, P.; Herth, M. M.; Radchenko, V. Radiolabeling of a Polypeptide Polymer for Intratumoral Delivery of Alpha-Particle Emitter, 225Ac, and Beta- Particle Emitter, 177Lu. Nucl. Med. Biol. 2022 , 104–105 , 11–21. https://doi.org/10.1016/j.nucmedbio.2021.11.001.
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