Development of Novel Radiohalogen Based Multifunctional Bioconjugation Reagents for Cancer Imaging

Student thesis: Doctoral ThesisDoctor of Philosophy


This thesis describes the work to develop two novel radiohalogen-based multifunctional bioconjugation reagents, 124I-Sulfo-Cy7-(PhS)2Mal and 18F-AlF-NOTA-(Br)2Mal. The 124I-Sulfo-Cy7-(PhS)2Mal is equipped with iodine-124 for longitudinal PET imaging; a newly developed near-infrared (NIR) reporter, Cy 7.5 cyanine dye for accurate fluorescence imaging with improved in vivo stability; and a dithiophenol maleimide for site-specific protein bioconjugation. 124I-Sulfo-Cy7-(PhS)2Mal was conjugated to both the HER2 targeting antibody, trastuzumab and the α−PD-L1 targeting affibody, NM-01. The corresponding bioconjugates were systematically evaluated in vitro and in vivo for cancer PET imaging and fluorescence-guided surgery. The 18F-AlF-NOTA-(Br)2Mal was developed to harvest the convenient chelation approach of [18F]Al-F complex using a NOTA chelator and, decorated with a dibromo maleimide for bioconjugation of small proteins through the reduced disulphide bridges.

Chapter one focuses on a review of the relevant radioisotopes, imaging techniques, and fluorescent reagents related to this work, this includes (i) an overview of PET imaging with examples of common PET tracers labelled with primary PET radioisotopes; (ii) an introduction to the use of radioiodine isotopes in biomedical imaging and radiotherapy; (iii) a brief overview of the fluorescence mechanisms and a review on most common fluorescent dyes with the focus on biomedical imaging applications of the most important classes of NIR emitting fluorophores; (iv) examples of the dual PET and NIR imaging reagents applied to cancer PET imaging and fluorescence-guided surgery in clinic.

Chapter two reviews the development of radiolabelling of iodine isotopes using the ‘one-pot three components click chemistry approach’; this includes initial radiochemical development, the insight and evidence of the reaction mechanism, and applications of radioiodinated reagents prepared using this method for dual radioluminography and fluorescence imaging, SPECT and fluorescence imaging, and the further implementation of iodine-124 for PET imaging.

Chapter three describes the chemical and radiochemical preparation and characterisation of a novel NIR azide labelling precursor, azido-Sulfo-Cy7 and the non-radioactive reference compound of the dual labelling reagent, 124I-Sulfo-Cy7-(PhS)2Mal as well as the radiochemical synthesis and characterisation of 124I-Sulfo-Cy7-(PhS)2Mal. The newly developed NIR azide was obtained in 46% isolated yield. The non-radioactive reference compound was obtained in a final isolated yield of 23%. This is followed by radiochemical development using iodine-125 to form the 125I-Sulfo-Cy7-(PhS)2Mal in radiochemical yields (RCYs) of 30±5% (n=4). Next, radiolabelling with iodine-124 was conducted using the optimised conditions to produce the desired 124I-Sulfo-Cy7-(PhS)2Mal in RCYs of 23±4% (n=4). The identity of 124I-Sulfo-Cy7-(PhS)2Mal was confirmed by the co-elution with its non-radioactive reference compound. Its molar activity was determined as 20.2 GBq/mol and its lipophilicity was measured as logD 0.96±0.28 (n=3).

Chapter four is focused on the biological evaluation of the newly developed dual PET/NIR bioconjugation reagent, 124I-Sulfo-Cy7-(PhS)2Mal for cancer PET imaging and fluorescence-guided surgery. The 124I-Sulfo-Cy7-(PhS)2Mal was conjugated to two biological entities: the affibody NM-01 targeting α-PD-L1 in cancer, and the antibody Trastuzumab targeting HER-2 expressing cancer in radiolabelling efficiency of 7% and 64%, respectively. The structural integrity of the protein conjugates was assessed using SDS-PAGE gel, fluorescence imaging, and autoradiography. All three methods indicated that both protein conjugates were fully re-bridged post-bioconjugation. Furthermore, the preservation of biological activity for both bioconjugates was assessed using an ELISA assay. The EC50 of the NM-01 towards PD-L1 was 0.52 nM and the NM-01 125I-Sulfo-Cy7-(PhS)2Mal bioconjugate was 1.13 nM. The EC50 of the Trastuzumab towards HER2 was 0.15 nM and the Trastuzumab 124I-Sulfo-Cy7-(PhS)2Mal bioconjugate was 0.17 nM. The in vitro cell uptake study indicated that NM-01 125I-Sulfo-Cy7-(PhS)2Mal bioconjugate had higher uptake (6.9±2.0%, n=3) by the PD-L1 positive Jurkat cells. While significantly lower NM-01 125I-Sulfo-Cy7-(PhS)2Mal bioconjugate uptake (3.1±1.7% , n=3) was observed by the PD-L1 negative SKBR-3 cells. The Trastuzumab 124I-Sulfo-Cy7-(PhS)2Mal bioconjugate was also tested for in vitro cell uptakes. Excellent cell uptake of the conjugate by the two HER2-positive SKBR-3 (53.7±10.0%, n=3) and SKOV3 (56.1±6.4%, n=3) cancer cells was observed. These uptakes could be largely blocked by pre-treating cells with excess of native Trastuzumab. In contrast, the Trastuzumab 124I-Sulfo-Cy7-(PhS)2Mal bioconjugate has negligible uptakes by the HER2 negative MDA-MB-231 (2.4±1.0%, n=3) cancer cells. Next, we further evaluated the Trastuzumab 124I-Sulfo-Cy7-(PhS)2Mal bioconjugate using the NSG mice bearing the HER2-positive SKBR-3 tumour xenograft. The tumours were successfully visualised by both PET imaging and ex vivo NIR fluorescence imaging.

Chapter 5 introduces the advantages of the [18F]Al-F complex radiolabelling chelation method, describes its chemistry, and illustrates the relevant PET tracers prepared using this approach in literature. Next, the radiolabelling precursor, NOTA-(Br)2Mal and the non-radioactive reference compound of a novel bioconjugation reagent, 19F-AlF-NOTA-(Br)2Mal were prepared in 75% and 30% yield, respectively. The radiolabelling of 18F-AlF-NOTA-(Br)2Mal was optimised with RCCs of 51±7% (n=3). The identity of this compound was confirmed by the co-elution with its non-radioactive reference compound.

In conclusion, two novel multifunctional bioconjugation reagents were developed. The dual PET/NIR reporter 124I-Sulfo-Cy7-(PhS)2Mal was successfully conjugated to two cancer targeting proteins NM-01 and Trastuzumab, respectively. The Trastuzumab 124I-Sulfo-Cy7-(PhS)2Mal bioconjugate can visualise tumour xenograft with both PET imaging and ex vivo NIR fluorescence imaging. This demonstrated its potential for clinical applications in cancer PET imaging and fluorescence guided surgery. The precursor and the non-radioactive reference compound of 18F-AlF-NOTA-(Br)2Mal was synthesized and fully characterised, and its 18F-labelling was established and optimised.

Date of Award1 Mar 2024
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorRan Yan (Supervisor) & John Maher (Supervisor)

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