Bifunctional chelators containing dibromomaleimides for the preparation of radioimmunoconjugates for PET imaging

Student thesis: Doctoral ThesisDoctor of Philosophy


Bifunctional chelators (BFCs) can be conjugated to antibodies, and then radiolabelled with metallic radioisotopes, to produce radioimmunoconjugates capable of imaging disease, predicting patient response to therapy, and informing pharmacokinetics of antibody biodistribution. Non-site-specific bioconjugations are simple and reliable but generate heterogeneous conjugates that can show compromised target receptor affinity and stability in vivo. Site-specific bioconjugation techniques are more complex but generate homogeneous bioconjugates, often with high target receptor affinity and stability in vivo. Dibromomaleimides (DBM) enable simple and reliable site-specific conjugation to antibodies via rebridging of reduced disulfide bonds to first give dithiomaleimide conjugates, that rapidly undergo hydrolysis to yield stable dithiomaleamic acid (DTM) conjugates. Here, new DBM-based BFCs have been synthesised and conjugated to the HER2-targeting antibody, trastuzumab, which is used in breast cancer treatment. These new immunoconjugates have been radiolabelled, and then studied in serum, in vitro and in vivo, to assess their stabilities and targeting properties.

Sarcophagine (sar) is a macrobicyclic chelator which has been extensively radiolabelled with 64Cu2+ (t1/2 12.7 h). This chelator-radiometal pair were used to develop a proof-of-concept platform for DBM-based BFCs. Sarcophagine-dibromomaleimide (sar-DBM) was synthesised in 8 % yield following optimisation, and subsequently reacted with trastuzumab, to give sar-DTM-trastuzumab. Near-quantitative radiolabelling of the immunoconjugate with 64Cu2+ was achieved in 5 min at pH 7. The 64Cu-sar-DTM-trastuzumab radioimmunoconjugate was stable in human serum over 40 h. Cell uptake SKOV3 cells (HER2 +ve) demonstrated retained radioimmunoconjugate specificity for the HER2 receptor. Lastly, the stability and HER2 affinity of 64Cu-sar-DTM-trastuzumab were assessed in vivo in healthy female mice using PET imaging and ex vivo biodistribution studies over 48 h. Size-exclusion HPLC and SDS-PAGE analysis of blood serum showed no evidence of bioconjugate degradation or transchelation of radiometallic ions to serum proteins over 48 h.
Additionally, low initial uptake and gradual clearance of radioactivity in the liver, indicated high radioimmunoconjugate stability whilst uptake and retention in HER2-expressing healthy tissues (skin, ovaries, uterine horn) demonstrated HER2 affinity.

To facilitate radiolabelling with the longer-lived PET isotope 89Zr4+ (t1/2 3.3 d), desferrioxamine-DBM (DFO-DBM) was prepared. This enabled comparison between a dibromomaleimide-based BFC and the commercially available maleimide derivative of DFO, which is commonly used to radiolabel antibodies with 89Zr4+. DFO-DBM was synthesised in 22 % yield whilst DFO-maleimide was obtained commercially. The bifunctional chelators were conjugated to trastuzumab to give DFO-DTM-trastuzumab and DFO-mal-trastuzumab. Radiolabelling with 89Zr4+, near-quantitative radiochemical yields were achieved for both species in just 10 min at pH 7, although higher specific activities were achieved with the maleimide conjugate (40 MBq mg-1) than with the DTM conjugate (5 MBq mg-1). Radioimmunoconjugate stability was compared in glutathione solution (10 mM) at pH 5.5 and pH 7, as well as in human serum over 8 days. Both species exhibited high stability in serum and in glutathione solution at pH 7. However, 89Zr-DFO-DTM-trastuzumab was significantly less stable than 89Zr-DFO-mal-trastuzumab in glutathione solution at pH 5.5. Uptake studies in SKOV3 cells showed that HER2 specificity, and the fraction of radioimmunoconjugate with retained affinity for the target receptor (immunoreactive fraction) was similar for both conjugates.

New dibromomaleimide-based BFCs have been developed to prepare site-specific radioimmunoconjugates with retained target receptor affinity and sufficiently high stability for preclinical in vivo use. The use of native unmodified antibodies, and interchain disulfide rebridging in dibromomaleimide conjugations are advantages over existing site-specific and maleimide-based conjugation techniques respectively.
Date of Award1 Aug 2022
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorMichelle Ma (Supervisor) & Nicholas J. Long (Supervisor)

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