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Patients with high-risk non-muscle-invasive bladder cancer (HR-NMIBC) who are unresponsive to or ineligible for intravesical Bacille Calmette-Guérin (BCG) therapy have limited organ-sparing treatment options. Systemic immune checkpoint inhibitors (ICIs) targeting the programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) axis have demonstrated activity in advanced urothelial carcinoma, and pembrolizumab is now approved for BCG-unresponsive NMIBC [1, 2]. However, systemic delivery of ICIs in the NMIBC population raises concerns regarding toxicity, cost, and the consequent risk–benefit balance, with treatment-related adverse events (TRAEs) occurring in two-thirds of cases, and durable responses remaining limited [1, 3]. While early-phase studies have explored intravesical administration of ICIs with minimal observed toxicity, these trials generally failed to show meaningful clinical activity, possibly due to poor urothelial penetration of these large molecules [4, 5]. Therefore, delivery approaches that achieve good local tissue penetration to retain immunotherapeutic benefits while minimising systemic exposure are of growing interest. The sub-urothelial injection technique represents one such novel delivery strategy. By injecting the agent directly into the lamina propria of the bladder wall, this approach likely achieves high local drug concentrations with limited systemic absorption, potentially reducing immune-related AEs while preserving immunological efficacy. Durvalumab is a human monoclonal antibody that inhibits the interaction between PD-L1 and its receptor PD1, restoring T-cell activity and promoting anti-tumour immune responses. The phase Ib SUB-urothelial DUrvalumab InjEction study (SUBDUE-1), was a first-in-human dose-escalation trial, which evaluated sub-urothelial durvalumab in patients undergoing radical cystectomy [6]. The study confirmed the safety and feasibility of this novel approach, with no dose limiting TRAEs reported. Furthermore, early histological data suggested immunomodulatory activity within the tumour microenvironment, providing a rationale for continued investigation in earlier disease stages. Before advancing to a phase II efficacy study in HR-NMIBC, a key translational question remains: to what extent does durvalumab, when administered via sub-urothelial injection, remain confined to the bladder wall, travel to regional lymphatics, or enter the systemic circulation? Understanding the biodistribution of sub-urothelial durvalumab is essential for assessing its therapeutic potential and safety profile in the context of local immunotherapy. The SUBDUE-3 trial will address this question using radiolabelled [89Zr] Zr-DFOSq- durvalumab (89Zr-durvalumab) to evaluate both local retention and systemic biodistribution. By combining advanced molecular imaging techniques with innovative delivery methods, this study seeks to generate foundational pharmacokinetic data to support the ongoing development of sub-urothelial immunotherapy in bladder cancer and may have relevance in other oncological scenarios. Our central hypothesis is that locally administered checkpoint inhibition may provide a complementary or alternative therapeutic pathway to intravesical BCG or chemotherapy by offering a distinct mechanism of anti-cancer activity with potentially reduced systemic toxicity. The SUBDUE-3 is an investigator-initiated, open-label, single-centre phase 0 clinical study being conducted at Fiona Stanley Hospital, Perth, Western Australia (WA). The study is sponsored by the South Metropolitan Health Service (SMHS) and supported by the Australian and New Zealand Urogenital and Prostate Cancer Trials Group (ANZUP 2402). Ethics approval has been granted by the SMHS Human Research Ethics Committee (Research Governance Service [RGS]:7169), and the trial is registered on the Australian New Zealand Clinical Trials Registry (ACTRN12624001245583p). Funding support is provided by the University of Western Australia, AstraZeneca, and Cancer Network WA. Up to three patients with either muscle-invasive bladder cancer (MIBC) or HR-NMIBC who are scheduled for radical cystectomy will be enrolled. Key inclusion and exclusion criteria are shown in Table 1. Participants will receive 89Zr-durvalumab via multiple sub-urothelial injections delivered during a single cystoscopic session ~2 weeks prior to cystectomy. The investigational product will be prepared as 25 mL 89Zr-durvalumab solution, comprising 37 megabecquerels (MBq) in 1 mg of durvalumab diluted in 4 mL of 0.9% saline, combined with 24 mg of unlabelled durvalumab in 21 mL of 0.9% saline for a final durvalumab concentration of 1 mg/mL. In vitro quality control testing will be conducted to quantify potential dissociation of 89Zr from the antibody prior to administration. Adequate organ and marrow function as defined below: Under general anaesthesia, the total 25 mL dose will be administered in 1 mL aliquots distributed throughout the bladder wall using a 5Fr Bonee needle via a 22 Fr rigid cystoscope. Injections will be distributed in a grid-like pattern to achieve bladder-wide distribution. The procedure will be performed by a urologist under the supervision of a trained nuclear medicine physician. Standard radiation safety protocols and theatre precautions for radiopharmaceutical administration, will be observed throughout. Serial positron emission tomography-computed tomography (PET-CT) imaging will be conducted to evaluate local and systemic biodistribution of 89Zr-durvalumab. Imaging time points include: 1–3 h post-injection (bladder-only imaging); 24 h (whole-body and dedicated pelvic scan); 72 h (whole-body); and 5–7 days (whole-body). Concurrent with each imaging session, blood samples will be collected for gamma counting to assess systemic bioavailability of the radiolabelled antibody. At the time of the initial PET scan, the urinary catheter bag will be analysed to calculate urinary radiation excretion, and the catheter will be removed following this scan. The investigational product, 89Zr-durvalumab, will be manufactured and radiolabelled at the Olivia Newton-John Cancer Research Institute (Victoria), shipped to Fiona Stanley Hospital at 2°C - 8°C temperature, and handled under institutional radiation-safety and Good Clinical Practice protocols. A certified radiopharmacist will confirm the radioactive dose and dispense the product into a lead-shielded syringe. The primary endpoint is qualitative and quantitative assessment of local and systemic biodistribution of 89Zr-durvalumab using PET imaging. Secondary endpoints include: (i) safety (CTCAE version 5.0); (ii) distribution patterns within the bladder wall and regional lymphatics; (iii) pharmacokinetic profiling through serial blood sampling; (iv) urinary radioactivity to evaluate excretion; and (v) comparative dosimetry with existing systemic 89Zr-durvalumab datasets. The PET scans will be reviewed by two independent nuclear medicine physicians, and dosimetric analysis performed by medical physicists using Medical Image Merge (MIM; MIM Software Inc., Cleveland, OH, USA) and Organ Level INternal Dose Assessment/EXponential Modelling (OLINDA/EXM) dosimetry software (Hermes Medical Solutions, Stockholm, Sweden). Data will be summarised descriptively. A sample size of three patients is considered adequate for this exploratory phase 0 study. Sub-urothelial delivery of ICIs is a novel concept offering a targeted approach that may overcome the limitations of both systemic and intravesical immunotherapy, namely systemic toxicity and poor tissue penetration respectively. By delivering the agent directly into the lamina propria, a compartment rich in immune effector cells, this method may enhance local immune activity while limiting systemic exposure. If biodistribution demonstrates high local retention with minimal toxicity, sub-urothelial immunotherapy could be explored in earlier disease settings as an adjunct to BCG or alternative for BCG-unresponsive HR-NMIBC, where the therapeutic window for systemic agents can be narrow, particularly in the setting of pre-existing autoimmune conditions. 89Zr-based immuno-PET is an emerging area in uro-oncology. The use of 89Zr enables high-resolution, antibody-specific PET imaging, well-suited to monoclonal antibody kinetics due to its longer half-life (~78 h) [7]. Early studies, including ZIRCON (ClinicalTrials.gov identifier: NCT03849118) in renal carcinoma, have shown its sensitivity and feasibility [8]. In urothelial carcinoma, the ongoing ZiPUP (ClinicalTrials.gov identifier: NCT05046665) trial is exploring 89Zr-girentuximab (TLX250-CDx) imaging in both localised and metastatic disease [9]. Serial PET-CT and blood sampling in SUBDUE-3 will quantify tracer retention and systemic dissemination, generating foundational data to inform dosing, safety, and future efficacy trials. The influence of locoregional tissue expression of the durvalumab target protein PD-L1 on drug distribution, as revealed by PET imaging, will be of additional interest and could lead to future response prediction studies. Beyond NMIBC, sub-urothelial delivery could complement bladder-preserving strategies in MIBC, enabling local immunomodulation with reduced systemic toxicity. More broadly, these findings could inform future development of alternative delivery strategies for immunotherapy in other oncological scenarios. Open access publishing facilitated by The University of Western Australia, as part of the Wiley - The University of Western Australia agreement via the Council of Australian University Librarians. The authors disclose the following potential conflicts of interest: Dr Kevin G. Keane; no conflicts of interest to declare relevant to this study. Dr Richard Gauci; no conflicts of interest to declare relevant to this study. A/Prof. Roslyn J. Francis; Charlies Foundation Research grant; Trial funding to institution. iCare Dust Diseases Board Grant; Trial Funding to institution. Australasian Radiopharmaceutical Trials Network (ARTnet) - Scientific Chair, unpaid. Prof. Andrew M. Scott; EMD Serono (Trial Funding to Institution), ITM (Trial Funding to Institution), Telix Pharmaceuticals (Trial and Research Funding to Institution), AVID Pharmaceuticals Trial (Trial Funding to Institution), Fusion Pharmaceuticals (Trial Funding to Institution), Cyclotek Trial (Trial Funding to Institution), Medimmune (Research Funding to Institution), Antengene (Research Funding to Institution), Humanigen (Research Funding to Institution), National Health and Medical Research Council of Australia (NHMRC; Fellowship Grant 1 177 837). ImmunOs - Scientific Advisory Board (SAB) Member, Immagion Bio - SAB Member. Telix Pharmaceuticals Advisory Board, unpaid. Australian and New Zealand Society of Nuclear Medicine (ANZSNM) - Board member, unpaid. World Federation of Nuclear Medicine and Biology (WFNMB) - Board member, unpaid. Dr Cynthia Hawks; no conflicts of interest to declare relevant to this study. Dr Andrisha-Jade Inderjeeth; Pfizer - 2022, BMS - 2023, Astellas - 2024; Honoraria for teaching and educational events (speaker/preparation fees). Prof. Ian D. Davis; AstraZeneca Advisory Board (no personal payment), Bayer Advisory Board (no personal payment), Roche Advisory Board (no personal payment), Telix Advisory Board (no personal payment), Astellas Advisory Board (no personal payment), Merck Sharp & Dohme (MSD) Advisory Board (no personal payment). ANZUP Cancer Trials Group; Director and Board chair (unpaid). Jayne Lim; no conflicts of interest to declare relevant to this study. A/Prof. Andrew Redfern; no conflicts of interest to declare relevant to this study. MSD and Astellas Advisory Board and guest speaker. Prof. Dickon Hayne; no conflicts of interest to declare relevant to this study. Previous advisory boards; BMS (2024), Urogen (2021), Pacific Edge (2022), AstraZeneca (2025). Travel/Meeting Support; Telix (2022). Speaker Meetings; BMS (2024).