Comparison of (64)Cu-complexing bifunctional chelators for radioimmunoconjugation: labeling efficiency, specific activity, and in vitro/in vivo stability

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.

Figure 1

Structures of bifunctional chelators p-SCN-Bn-DOTA, p-SCN-Bn-NOTA, p-SCNBn-oxo-DO3A, p-SCN-Bn-PCTA, sar-CO₂H, p-SCN-Bn-DTPA, CHX-A”-DTPA, 2B3M-ITC-DTPA.

Figure 2

Elution profile for ⁶⁴Cu purification. Initial elution is with 5 mL 9M HCl (fraction 1), followed by 2 fractions of 3 mL and 2 mL 6M HCl respectively (fractions 2-3), then 1 mL 0.1M HCl (fraction 4) with all other fractions (5-12) being eluted with 0.5 mL 0.1M HCl.

Figure 3

Exemplar HPLC radiochromatograms of ⁶⁴Cu labeled Rituximab conjugated with different bifunctional chelators (A) p-SCN-Bn-NOTA (B) p-SCN-Bn-PCTA (C) p-SCN-Bn-DTPA. NOTA- and PCTA-Rituximab show very high radiolabeling efficiency while DTPA-Rituximab shows a significant level of ⁶⁴Cu impurities. The level of antibody aggregates (the peak eluting at 6 min, prior to the antibody peak) was higher for PCTA-Rituximab compared with the other immunoconjugates. Similar data acquired for the other three bifunctional chelator conjugates are shown in supplementary data.

Figure 4

Radiolabeling efficiency of immunoconjugates under increasingly dilute conditions expressed as % labeling efficiency against effective concentration of bifunctional chelator

Figure 5

Serum stability of ⁶⁴Cu-Rituximab immunoconjugates at 37°C over 48 h (mean ± SD, n = 3) determined by size exclusion radiochromatography.

Figure 6

Biodistribution of ⁶⁴Cu-Rituximab-immunoconjugates in normal female Balb/C mice at 48 h post injection. The biodistribution patterns fall into two distinct groups, those containing macrocyclic chelators (A) and those containing DTPA derivatives (B).

Figure 7

PET/CT images (maximum intensity projection) of Balb/C mice 24 h post injection with ⁶⁴Cu-Rituximab-immunoconjugates. (A) ⁶⁴Cu-Sar-CO-Rituximab and (B) ⁶⁴Cu-DTPA-Rituximab given as examples of macrocycle containing immunoconjugates (A, showing predominantly blood pool activity) and ,immunoconjugates containing DTPA derivatives (B, showing predominantly liver and gut activity). Corresponding images for the other chelators tested are presented in supplementary data.

Figure 8

Radioactivity (%ID) in the feces of normal female Balb/C mice at 4, 24 and 48 h post injection with ⁶⁴Cu-Rituximab-immunoconjugates.