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Despite the high interest and concern due to an increasing incidence and death rate, patients who develop muscle invasive bladder cancer (MIBC) have few options available. However, the past decade has produced many candidate bladder tumor-specific markers but further development of these markers is still needed for creating effective targeted medications to solve this urgent need. Interleukin-5 receptor α-subunit (IL-5Rα) has recently been reported to be involved in MIBC progression. Thus, we aimed to validate IL-5Rα as a target for antibody-conjugates to better manage patients with MIBC. Patients were recruited and their tumors were processed for IL-5Rα immunohistochemical analysis. NOD/SCID mice were also heterotopically implanted with the human MIBC HT-1376 and HT-B9 cell lines and established xenografts immunohistochemically evaluated for IL-5Rα and compared against patient tumors. Using the mAb A14, an antibody-drug conjugate (ADC) and a radiolabeled immunoconjugate (RIC) were developed by conjugating to vinblastine and to the positron emitter copper-64 (64Cu), respectively. As a proof-of-concept for ADC and RIC efficacy, in vitro cytotoxicity and in vivo positron emission tomography (PET) imaging in tumor-bearing mice were performed, respectively. In addition, as rapid internalization and accumulation are important components for effective antibody-conjugates, we evaluated these aspects in response to IL-5 and 64Cu-A14 treatments. Our findings suggest that although IL-5Rα protein expression is preferentially increased in MIBC, it is rapid IL-5Rα-mediated internalization allowing vinblastine-A14 to have cytotoxic activity and 64Cu-A14 to detect MIBC tumors in vivo. This is the first report to elucidate the potential of IL-5Rα as an attractive MIBC target for antibody-conjugate applications.

Overexpression of G protein-coupled receptors (GPCRs) in tumours is widely used to develop GPCR-targeting radioligands for solid tumour imaging in the context of diagnosis and even treatment. The human vasoactive neuropeptide urotensin II (hUII), which shares structural analogies with somatostatin, interacts with a single high affinity GPCR named UT. High expression of UT has been reported in several types of human solid tumours from lung, gut, prostate, or breast, suggesting that UT is a valuable novel target to design radiolabelled hUII analogues for cancer diagnosis. In this study, two original urotensinergic analogues were first conjugated to a DOTA chelator via an aminohexanoic acid (Ahx) hydrocarbon linker and then -hUII and DOTA-urantide, complexed to the radioactive metal indium isotope to successfully lead to radiolabelled DOTA-Ahx-hUII and DOTA-Ahx-urantide. The 111In-DOTA-hUII in human plasma revealed that only 30% of the radioligand was degraded after a 3-h period. DOTA-hUII and DOTA-urantide exhibited similar binding affinities as native peptides and relayed calcium mobilization in HEK293 cells expressing recombinant human UT. DOTA-hUII, not DOTA-urantide, was able to promote UT internalization in UT-expressing HEK293 cells, thus indicating that radiolabelled 111In-DOTA-hUII would allow sufficient retention of radioactivity within tumour cells or radiolabelled DOTA-urantide may lead to a persistent binding on UT at the plasma membrane. The potential of these radioligands as candidates to target UT was investigated in adenocarcinoma. We showed that hUII stimulated the migration and proliferation of both human lung A549 and colorectal DLD-1 adenocarcinoma cell lines endogenously expressing UT. In vivo intravenous injection of 111In-DOTA-hUII in C57BL/6 mice revealed modest organ signals, with important retention in kidney. 111In-DOTA-hUII or 111In-DOTA-urantide were also injected in nude mice bearing heterotopic xenografts of lung A549 cells or colorectal DLD-1 cells both expressing UT. The observed significant renal uptake and low tumour/muscle ratio (around 2.5) suggest fast tracer clearance from the organism. Together, DOTA-hUII and DOTA-urantide were successfully radiolabelled with 111Indium, the first one functioning as a UT agonist and the second one as a UT-biased ligand/antagonist. To allow tumour-specific targeting and prolong body distribution in preclinical models bearing some solid tumours, these radiolabelled urotensinergic analogues should be optimized for being used as potential molecular tools for diagnosis imaging or even treatment tools.