Immuno-PET/CT evaluation of two CAIX VHH-based [89Zr]Zr(IV) radioimmunoconjugates for specific targeting of hypoxic solid tumor areas in colon rectal cancer: a step closer to radioimmunotherapy.

Introduction: Carbonic anhydrase IX (CAIX) is a membrane bound extracellular enzyme that is selectively expressed on hypoxic tumor cells with minimal expression in benign tissues. We report a CAIX-VHH enzyme inhibiting antibody whose serum half-life has been extended by fusing it to human IgG1 Fc domain. To use these constructs for specific imaging and targeted therapy¹ of CAIX expressing tumor cell, CAIX-VHH-Fc was conjugated to two different bifunctional chelators (DFO*-pPhe-NCS and H₄pypa-pPhe-NCS) and followed by labelling with [⁸⁹Zr][Zr(ox)₄] for preclinical PET/CT imaging in mice bearing human HT-29 colorectal cancer cells.

Methods: The high affinity single-domain antibody targeting CAIX presented in this study was recombinantly produced in CHO cells and biophysically and functionally characterized. The antibody was then conjugated to two different chelators (DFO*-pPhe-NCS and H₄pypa-pPhe-NCS) at Ab:chelator ratios of 1:5 and radiolabeled with [⁸⁹Zr][Zr(ox)₄] for 1h at room temperature. Purity and specific activities of purified radioimmunoconjugates were determined by iTLC-SG chromatography and size exclusion chromatography using HPLC. The two radiolabeled Abs (12.7±0.2 μg, 1.6±0.2 MBq for [⁸⁹Zr]Zr(DFO*)-CAIX-VHH-Fc and 13.7±0.2 μg, 1.2±0.1 MBq for [⁸⁹Zr]Zr(pypa)-CAIX-VHH-Fc) were injected intravenously to athymic nude mice (n=4 per group) bearing subcutaneous HT-29 colorectal adenocarcinoma xenografts. PET/CT imaging and biodistribution studies were performed at 1, 3 and 6 days post injection (p.i).

Results: CAIX-VHH-Fc Ab was efficiently conjugated with two different chelators (DFO*-pPhe-NCS and H₄pypa-pPhe-NCS), and radiolabeled with [⁸⁹Zr][Zr(ox)₄]. Radiochemical purities of purified [⁸⁹Zr]Zr(IV) labeled immunoconjugates were >99% and specific activities were 0.44-0.47 MBq/μg. PET/CT imaging with anti-CAIX Abs showed high tumor uptake and tumor-to-background ratios as early as day 1 post-injection (p.i.) for both radioimmunoconjugates, with relatively low uptake in liver and spleen and no significant kidney uptake or bowel accumulation. The H₄pypa-conjugated VHH had faster overall clearance and lower tumor uptake. Low level bone uptake was also observed at early time points, possibly caused by non-specific [⁸⁹Zr]Zr(IV) binding to the protein during radiolabeling. However there was no increase in bone accumulation over time. Biodistribution at day 6 p.i confirmed the high tumor uptake with 16.1±5.0, 6.8±0.9%ID/g for [⁸⁹Zr]Zr(DFO*)-CAIX-VHH-Fc and [⁸⁹Zr]Zr(pypa)-CAIX-VHH-Fc respectively. High and comparable tumor-to-muscle ratios for [⁸⁹Zr]Zr(DFO*)-CAIX-VHH-Fc and [⁸⁹Zr]Zr(pypa)-CAIX-VHH-Fc (21.2 and 21.8, respectively) were also observed, confirming the selectivity of both radioimmunoconjugates.

Conclusions: High affinity Fc fused CAIX VHHs allowed for the clear preclinical imaging of CAIX-positive human HT-29 colorectal tumors. High tumor-to-muscle ratios were obtained with the two radioimmunoconjugates, with faster clearance observed with the [⁸⁹Zr]Zr(pypa)-CAIX-VHH-Fc immunoconjugate. [⁸⁹Zr]Zr(pypa)-CAIX-VHH-Fc shows promise as the H₄pypa chelator has been previously shown to efficiently chelate [¹⁷⁷Lu]Lu(III), and could thus be used for [⁸⁹Zr]Zr(IV)/[¹⁷⁷Lu]Lu(III) labeling as a theranostic pair.

References

(1) Lenferik AE et al.; National Research Council of Canada, assignee. Single domain antibodies targeting CA-IX as well as compositions comprising same. WIPO application WO2022157714A1

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