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Comparison of (64)Cu-complexing bifunctional chelators for radioimmunoconjugation: labeling efficiency, specific
Maggie S Cooper1, Michelle T Ma, Kavitha Sunassee
1King's College London , Division of Imaging Sciences and Biomedical Engineering, 4th Floor Lambeth Wing, St. Thomas' Hospital, SE1 7EH, London, United Kingdom.
Insights
The study compared eight bifunctional chelators for radiolabeling the anti-CD20 antibody rituximab with copper-64. NOTA and sarcophagine chelators demonstrated superior efficiency and stability for radioimmunoconjugates in molecular imaging applications.
Area of Science:
- Radiochemistry
- Immunoconjugate Development
- Molecular Imaging
Background:
- High radiolabeling efficiency and stability are crucial for successful radioimmunoconjugates used in medical imaging and therapy.
- Copper-64 (64Cu) is a promising radioisotope for positron emission tomography (PET) imaging due to its suitable half-life and decay properties.
Purpose of the Study:
- To compare the radiolabeling efficiency, in vitro stability, and in vivo biodistribution of rituximab-based immunoconjugates utilizing eight different bifunctional chelators labeled with 64Cu.
- To identify optimal chelators for developing high-performance 64Cu-labeled antibody conjugates for molecular imaging.
Main Methods:
- Conjugation of rituximab with eight distinct bifunctional chelators: four macrocyclic (DOTA, Oxo-DO3A, NOTA, PCTA), three DTPA derivatives, and one sarcophagine derivative.
- Radiolabeling with 64Cu under various conditions, assessing efficiency and specific activity.
- In vitro stability studies in human serum at 37°C over 48 hours.
- In vivo biodistribution studies in normal mice over 48 hours to evaluate pharmacokinetic profiles.
Main Results:
- NOTA-rituximab and sar-CO2H-rituximab conjugates exhibited high radiolabeling efficiencies (95% and 98%, respectively), even under dilute conditions.
- All macrocyclic radioimmunoconjugates demonstrated excellent in vitro stability (<6% 64Cu dissociation over 48 hours), unlike DTPA-based conjugates which showed poor stability.
- In vivo biodistribution showed similar profiles for macrocyclic conjugates, with prolonged blood pool retention up to 48 hours.
Conclusions:
- Macrocyclic bifunctional chelators, particularly NOTA and sarcophagine derivatives, are highly suitable for 64Cu-labeled antibody conjugates in molecular imaging.
- NOTA and sar-CO2H offer significant advantages, enabling rapid radiolabeling at room temperature under dilute conditions to achieve high specific activity.
Abstract:
High radiolabeling efficiency, preferably to high specific activity, and good stability of the radioimmunoconjugate are essential features for a successful immunoconjugate for imaging or therapy. In this study, the radiolabeling efficiency, in vitro stability, and biodistribution of immunoconjugates with eight different bifunctional chelators labeled with (64)Cu were compared. The anti-CD20 antibody, rituximab, was conjugated to four macrocyclic bifunctional chelators (p-SCN-Bn-DOTA, p-SCN-Bn-Oxo-DO3A, p-SCN-NOTA, and p-SCN-PCTA), three DTPA derivatives (p-SCN-Bn-DTPA, p-SCN-CHX-A″-DTPA, and ITC-2B3M-DTPA), and a macrobicyclic hexamine (sarcophagine) chelator (sar-CO2H) = (1-NH2-8-NHCO(CH2)3CO2H)sar where sar = sarcophagine = 3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane). Radiolabeling efficiency under various conditions, in vitro stability in serum at 37 °C, and in vivo biodistribution and imaging in normal mice over 48 h were studied. All chelators except sar-CO2H were conjugated to rituximab by thiourea bond formation with an average of 4.9 ± 0.9 chelators per antibody molecule. Sar-CO2H was conjugated to rituximab by amide bond formation with 0.5 chelators per antibody molecule. Efficiencies of (64)Cu radiolabeling were dependent on the concentration of immunoconjugate. Notably, the (64)Cu-NOTA-rituximab conjugate demonstrated the highest radiochemical yield (95%) under very dilute conditions (31 nM NOTA-rituximab conjugate). Similarly, sar-CO-rituximab, containing 1/10th the number of chelators per antibody compared to that of other conjugates, retained high labeling efficiency (98%) at an antibody concentration of 250 nM. In contrast to the radioimmunoconjugates containing DTPA derivatives, which demonstrated poor serum stability, all macrocyclic radioimmunoconjugates were very stable in serum with <6% dissociation of (64)Cu over 48 h. In vivo biodistribution profiles in normal female Balb/C mice were similar for all the macrocyclic radioimmunoconjugates with most of the activity remaining in the blood pool up to 48 h. While all the macrocyclic bifunctional chelators are suitable for molecular imaging using (64)Cu-labeled antibody conjugates, NOTA and sar-CO2H show significant advantages over the others in that they can be radiolabeled rapidly at room temperature, under dilute conditions, resulting in high specific activity.
