Summary

Radiotheranostic agents provide a unique ability to detect, characterize, treat, and monitor sites of disease with exceptional specificity. A persistent challenge for clinical theranostics is the development of suitably matched therapeutic and diagnostic agents that provide correlating pharmacokinetic data to guide therapeutic application. Ideally, this goal is realized in the form of a targeted agent that can be labelled with radionuclides for either imaging or therapy without other chemical changes.

²²⁷Th is a promising radioisotope for targeted α-particle therapy. It produces 5 α-particles through its decay, with the clinically approved ²²³Ra as its first daughter. There is an ample supply of ²²⁷Th, allowing for clinical use; however, the chemical challenges of chelating this large tetravalent f-block cation are considerable. Radiometals must be stably bound to a molecularly specific vector (a small molecule, peptide, or antibody) to achieve localized uptake. The extended biologic residency time and longer radiologic half-life (t½) of isotopes used for antibody-based agents add a requirement for greater stability.

Using the CD20-targeting antibody ofatumumab, evaluation chelation of ²²⁷Th⁴⁺ for α-particle–emitting and radiotheranostic applications was performed. ²²⁷Th-labeled ofatumumab-chelator constructs were synthesized and showed moderate in vitro stability. In this work, 4 antibody-chelator conjugates for in vitro and in vivo stability using ofatumumab, a human anti-CD20 antibody were evaluated. The most stable ²²⁷Th chelator conjugate, L804, was evaluated in vivo for tumour-targeting capability. ²²⁷Th-L804-ofatumumab coordinated ²²⁷Th rapidly and efficiently at high yields, and demonstrated extended stability. An ⁸⁹Zr-L804- theranostic analogue was compared. In vivo tumour targeting confirmed the utility of this chelator, and the diagnostic analogue, ⁸⁹Zr-L804-ofatumumab, showed organ distribution matching that of ²²⁷Th to delineate SU-DHL-6 tumours.

Commercially available and novel chelators for ²²⁷Th showed a range of performances. The L804 chelator can be used with potent radiotheranostic capabilities for ⁸⁹Zr/²²⁷Th quantitative imaging and α-particle therapy.

Results from nanoScan® PET/CT

PET/CT of the SU-DHL-6 tumor–bearing animals was performed on the Nanoscan (Mediso) on day 7. A CT acquisition of 720°, 70 kV/980 μA of 90 ms, and 4× binning was reconstructed by filtered backprojection to produce isotropic 124-μm voxels (122 × 122 × 97 mm). PET data (400–600 keV, 5-ns timing) were reconstructed using the iterative, 3-dimensional TeraTomo algorithm (4 iterations and 6 subsets; Mediso Medical Imaging Systems). Decay, attenuation, and scatter corrections were applied to quantify injected activity.

The ²²⁷Th-L804 conjugate was selected as the lead agent for further evaluation. The tumour-targeting ability of ²²⁷Th-L804-ofatumumab in CD20–positive Raji tumours was investigated. Mice were randomized to receive ²²⁷Th-L804-ofatumumab, control ²²7Th-L804-IgG, or ²²⁷Th-L804-ofatumumab preceded by unmodified ofatumumab. The early blood signal for the unblocked group (21.6 ± 1.9 %IA/g) decreased with time to 7.5 ± 1.8 %IA/g at 7 d. Control IgG uptake was significantly greater in the spleen over the course of the experiment, whereas ²²⁷Th tumor uptake was significantly higher for the targeted construct at all time points (to control IgG, P < 0.01) and to blocked group at 7 d (P < 0.05). Peak tumour uptake at day 3 for ²²⁷Th-L804-ofatumumab achieved 20 ± 1 %IA/g (Fig. 4).

Figure 4. Organ distribution of ²²⁷Th-L804-ofatumamab and ²²⁷Th-L804-IgG and blocking with ofatumumab using Raji tumour–bearing mice (R2G2, female), reporting ²²⁷Th only (%IA/g) at 24 h, 3 d, and 7 d after injection. GI = gastrointestinal tract.

The pharmacokinetics of ²²⁷Th- and ⁸⁹Zr-L804-ofatumumab and conventional ⁸⁹Zr-DFO-ofatumumab was compared (Fig. 5). Small but statistically significant differences were observed between ²²⁷Th- and ⁸⁹Zr-L804-ofatumumab for blood, heart, lung, and cecal tissues at 1 d (Fig. 5A). At 14 d, no differences were detected (Fig. 5B). Together, these data demonstrate that use of different chelators (DFO and L804) alters radiopharmaceutical distribution to a greater degree than does exchange of radiometals.

Figure 5. Pharmacokinetic comparison of ²²⁷Th-L804-, ⁸⁹Zr-L804-, and ⁸⁹Zr-DFO-ofatumumab in naïve female mice at 1 d (A) and 14 d (B) after injection. Significant differences were observed for blood, heart, and lung accumulation of ²²⁷Th-L804- vs. ⁸⁹Zr-L804-ofatumumab at 1 d (P < 0.05). At 14 d, no differences were seen between ²²⁷Th-L804- and ⁸⁹Zr-L804-ofatumumab. ⁸⁹Zr bone uptake was greater for DFO (7.3 ± 1.1 %IA/g) than for L804 (⁸⁹Zr, 3.7 ± 0.5 %IA/g; ²²⁷Th, 3.0 ± 0.4 %IA/g [P < 0.001]). p.i. = postinjection.

The theranostic capability of L804-ofatumumab for ⁸⁹Zr PET was tested in CD20–positive SU-DHL-6 xenografts (Fig. 6). Recapitulating Raji accumulation of ²²⁷Th-L804-ofatumumab, we observed high-contrast delineation with ⁸⁹Zr-L804-ofatumumab and a low skeletal signal.

Figure 6. (A) Representative PET images of SU-DHL-6 tumour–bearing animal with ⁸⁹Zr-L804-ofatumumab, with or without blocking. (B) PET/CT at 7 d, without blocking. On right are cross-sectional images of cervical lymph nodes, brachial lymph nodes, and tumour, from top to bottom.

To confirm specificity, blocking antibody was administered to a representative animal; the result was decreased tumour uptake (Supplemental Fig. 11).

Supplemental Figure 11. Biodistribution of ⁸⁹Zr-L804-Ofatumumab in SU-DHL-6 Tumor bearing Mice. A) Organ distribution at 7 d p.i. of ⁸⁹Zr-L804-ofatumumab for SU-DHL-6 xenografted Nu/Nu mice; B) quantitation of SU-DHL-6 uptake to 7 d.

• Commercially available and novel chelators for ²²⁷Th showed a range of performances.
• L804 chelator is the most stable and versatile chelator of those tested, providing facile coordination of ²²⁷Th and ⁸⁹Zr and can be used with potent radiotheranostic capabilities for ²²⁷Th/⁸⁹Zr quantitative imaging and α-particle therapy.
• Stable chelation of ²²⁷Th was demonstrated and applied for tumour-targeted delivery across 2 lymphoma models.
• Obtained data support the further development of ²²⁷Th/⁸⁹Zr antibody theranostics using this chemically identical precursor.

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