177Lu-DPI-4452 & 68Ga-DPI-4452, a new radiopeptide tandem targeting Carbonic Anhydrase IX displays strong theranostic potential in CRC and ccRCC tumors

Introduction: Carbonic Anhydrase IX (CAIX) is a transmembrane metalloprotease expressed in many hypoxic tumor types and to a very limited extent in healthy organs. CAIX represents an attractive diagnostic and therapeutic target in hypoxic solid tumors such as Colorectal Cancer (CRC) and clear cell Renal Cell carcinoma (ccRCC) for which an important unmet medical need remains. DPI-4452 is a peptidomimetic targeting CAIX which carries a DOTA cage that can be labelled with several different radionuclides for theranostic purposes. Here we report CAIX expression in CRC and ccRCC human samples and evaluate for the first time therapeutic and diagnostic performance of ¹⁷⁷Lu-DPI-4452 and ⁶⁸Ga-DPI-4452 in vitro and in vivo.

Methods: CAIX protein expression was assessed using a validated immunohistochemistry assay (IHC) with an anti-CAIX antibody (M75) on a panel of 30 ccRCC and 85 CRC tumor specimens.

DPI-4452, ^natLu-DPI-4452, and ^natGa-DPI-4452 affinity and kinetic interaction to CAIX was assessed by Surface Plasmon Resonance (SPR).

For in vivo characterization of the ¹⁷⁷Lu-DPI-4452 and ⁶⁸Ga-DPI-4452, CAIX-positive human cancer cell line HT-29 (CRC) or SK-RC-52 (ccRCC) were subcutaneously implanted into immunodeficient mice. Animals with each tumor type were divided into 4 treatment groups of 10 mice each: a) Single administration (day 1) of vehicle b) Single administration (day 1) of 100MBq of ¹⁷⁷Lu-DPI-4452 c) Single administration (day 1) of 33MBq of ¹⁷⁷Lu-DPI-4452 d) Three administrations (day 1, 8, 15) of 33MBq of ¹⁷⁷Lu-DPI-4452. To assess radioactivity uptake (as % of injected dose /gram tissue) in the tumor, kidney, and liver, 3 animals per treatment group were imaged by SPECT 4h after each ¹⁷⁷Lu-DPI-4452 administration. In both models, tumor volume and body weight were monitored for 42 days from treatment initiation. To correlate the ⁶⁸Ga-DPI-4452 signal with the ¹⁷⁷Lu-DPI-4452 signal in tumors, a satellite group of animals in each model received one injection of ⁶⁸Ga-DPI-4452 (for PET imaging 1h later) followed 7 days later by an injection of ¹⁷⁷Lu-DPI-4452 (for SPECT imaging 4h later).

Results: IHC results demonstrated high-level CAIX expression (H score ≥ 150) in 83% of ccRCC and 29% of CRC tumor cases tested, confirming the relevance of CAIX as a therapeutic target in these 2 indications.

In vitro, DPI-4452 selectively bound to CAIX with sub-nanomolar affinity and showed slow dissociation kinetics. DPI-4452 labeling with lutetium or gallium did not alter its interaction with the target.

In vivo, ¹⁷⁷Lu-DPI-4452 treatments were well tolerated. SPECT imaging of HT-29 and SK-RC-52 xenografted animals at 4h after injection demonstrated rapid and high ¹⁷⁷Lu-DPI-4452 tumor uptake in both models. Tumor uptake remained stable over multiple ¹⁷⁷Lu-DPI-4452 weekly injections suggesting CAIX expression in tumors was not modulated following repeated ¹⁷⁷Lu-DPI-4452 administration. Tumor-to-kidney and tumor-to-liver ratios showed preferential uptake in the tumor (T/K: 1.5 and 7.5 and T/L 60 and 300, for HT-29 and SK-RC-52, respectively). A strong and dose-dependent tumor growth inhibition (maximal T/C<20%) was observed in the 100MBq and the 3x33MBq treatment groups for the HT-29 model and the 100MBq, 3x33MBq, and the 33MBq treatment groups for the SK-RC-52 model. Interestingly, in both models, the dose fractionation (3x33MBq) seemed beneficial in the long run, and in the SK-RC-52 model led to tumor stasis sustained until study end (day 42). ⁶⁸Ga-DPI-4452 tumor uptake evaluated by PET imaging closely matched the ¹⁷⁷Lu-DPI-4452 tumor uptake evaluated by SPECT imaging obtained 1 week later for the same animals, demonstrating the theranostic performance of the ⁶⁸Ga/¹⁷⁷Lu-DPI-4452 pair.

Conclusions: These results demonstrate the promising theranostic potential of DPI-4452 for targeting CAIX-expressing CRC and ccRCC tumors.