Background: Most head and neck cancers originate in the mucosa of the upper aerodigestive tract, and are largely squamous cell carcinoma (HNSCC). Nectin-4 is an immunoglobulin-like cell adhesion molecule that is overexpressed in a wide variety of cancers, including ~85% of HNSCC cases (1). An antibody-drug conjugate (ADC) against nectin-4, PADCEV (enfortumab vedotin, Astellas), has performed impressively in the management of urothelial carcinoma (2), and is currently being explored for HNSCC (EV-202 trial, NCT04225117). Here we adapt the base antibody of PADCEV into a beta-emitting radioimmunotherapy for the management of HNSCC with promising early results compared to PADCEV itself. 

Methods: Enfortumab IgG1 was conjugated with CHX-A”-DTPA to lysine residues followed by complexation of [¹⁷⁷Lu]LuCl₃ (nca) in a mildly acidic acetate buffer and isolated with a PD-10 desalting column. Instant thin layer chromatography (ITLC) was used to assess radiochemical purity (RCP) and serum stability of the [¹⁷⁷Lu]Lu-CHX-A”-DTPA-enfortumab, while non-reducing SDS-PAGE was used to monitor the structural integrity of the antibody. A plate binding assay was performed to test the ability of ¹⁷⁷Lu-CHX-A”-DTPA-enfortumab to bind to its antigen. Next, [¹⁷⁷Lu]Lu-CHX-A”-DTPA-enfortumab (50 µg, 500 µCi) was administered to female nude mice bearing subcutaneous FaDu xenografts, a HNSCC line of low-to-moderate nectin-4 expression. Biodistribution studies were performed over a 120 h period, including a blocking cohort where a 20-fold excess of unmodified enfortumab was co-administered. The therapeutic response to [¹⁷⁷Lu]Lu-CHX-A”-DTPA-enfortumab, monitored with biweekly tumor measurements and animal weights, was compared against PADCEV (1.25 mg/kg) and a 1x PBS control. ImmunoPET images with zirconium-89 labeled enfortumab ([⁸⁹Zr]Zr-DFO-enfortumab) was also performed to help visualize uptake into the FaDu xenografts.

Results: [¹⁷⁷Lu]Lu-CHX-A”-DTPA-enfortumab was obtained with a specific activity of ~20 mCi/mg with a radiochemical conversion (RCC) of 88.1 ± 3.6% and RCP of 97.9 ± 0.6%. Upon challenge in serum for 7 days, 94.0 ± 1.2% of the radiometal remained antibody bound. No obvious signs of antibody aggregation or degradation were observed with SDS-PAGE during the radiolabeling process. [¹⁷⁷Lu]Lu-CHX-A”-DTPA-enfortumab was found to bind to recombinant human nectin-4, which could be specifically blocked to background levels with the addition of 1000-fold excess enfortumab or PADCEV. Ex vivo biodistribution analysis found [¹⁷⁷Lu]Lu-CHX-A”-DTPA-enfortumab accumulated to into the FaDu xenografts with an activity concentration of ~10% ID/g from 24 h post-injection onward; these results are in line with the PET/CT renderings using [⁸⁹Zr]Zr-DFO-enfortumab. Imaging also highlights the aggressive nature of the FaDu model, which nearly doubled in volume over the 120 h acquisition period. [¹⁷⁷Lu]Lu-CHX-A”-DTPA-enfortumab was able to extend survival of the animals to a median of ~18 days compared to 7 days for the 1x PBS group and 9 days for the PADCEV group.

Conclusion: Enfortumab was successfully used to develop a beta-emitting radioimmunotherapy and extensively characterized to ensure it had an appropriate binding and stability profile for in vivo use. In a highly aggressive FaDu model with low-to-moderate nectin-4 expression, [¹⁷⁷Lu]Lu-CHX-A”-DTPA-enfortumab was able to control the tumors to a greater extent than PADCEV. Additional work is underway exploring [¹⁷⁷Lu]Lu-CHX-A”-DTPA-enfortumab in the FaDu model, including as an orthotopic tongue model, along with other HNSCC and bladder xenografts of varying nectin-4 expression.

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