Fig 2: In vitro specificity of [68Ga]AJ647 for Nectin-4 in bladder cancer cell linesA) [68Ga]AJ647 binding (percent incubated activity, %IA) to various bladder cancer cell lines. Cells were incubated with ~37 kBq (~1 μCi) [68Ga]AJ647 at 4°C for 1 hour. Specificity was confirmed by pre-incubating with 2 μM of non-radioactive AJ647 (blocking dose), which significantly reduced radiotracer uptake, indicating that [68Ga]AJ647 targets Nectin-4. B) Flow cytometry analysis of Nectin-4 receptor expression across different bladder cancer cell lines, providing insight into the variability in receptor density among the cell lines. C) Correlation between in vitro [68Ga]AJ647 uptake and Nectin-4 receptor density, demonstrating that the uptake of [68Ga]AJ647 is dependent on cell surface density of Nectin-4 receptors. Data in figure A and C are represented as mean ± SD. Pearson correlation used in C.

Fig 3: Competition binding assays of [68Ga]AJ647 and EV-FITC with Nectin-4 in HT1376 cell lineA) Schematic representation of competition assays between [68Ga]AJ647 and EV for binding to Nectin-4. On the left, the schematic illustrates the impact of varying EV concentrations on Nectin-4 levels, with accessible Nectin-4 quantified using [68Ga]AJ647. On the right, the schematic depicts how varying concentrations of AJ647 affect Nectin-4 levels, with accessible Nectin-4 determined by EV-FITC binding. B) In vitro binding of [68Ga]AJ647 to HT1376 cells with varying concentrations of EV. The data show concentration-dependent inhibition of [68Ga]AJ647 binding, with an IC50 of 2.7 nM, indicating the competitive nature of EV in blocking [68Ga]AJ647 binding to Nectin-4. C) In vitro binding of EV-FITC to HT1376 cells, assessed by flow cytometry, with varying concentrations of AJ647. The results demonstrate concentration-dependent inhibition of EV-FITC binding, with an IC50 of 35 nM, highlighting that AJ647 competes with EV-FITC for Nectin-4 binding. data represented as mean±SD (n=3–4). D) In vitro binding of [68Ga]AJ647 to HT1376, SCaBER and T24 cells with and without 60 nM EV. E) In vivo PET-CT imaging in mice harboring SCaBER (left) and HT1376 (right) tumors pre- and post- 20 mpk EV treatment (n=2). PET-CT was acquired 60-minutes after 7.4 MBq (~200 μCi) [68Ga]AJ647 injection. E) In vivo PET-CT imaging in mice harboring SCaBER (left) and HT1376 (right) tumors at saturating dose of 20 mpk or saline (n=2). PET-CT was acquired at 60-minutes after 7.4 MBq (~200 μCi) [68Ga]AJ647 injection. F) Ex vivo biodistribution in mice harboring SCaBER (left) and HT1376 (right) tumors treated with 20 mpk or saline control (n=5). Mice were sacrificed at 60-minutes after 1.85 MBq (~50 μCi) [68Ga]AJ647 injection.

Fig 4: Structural design and in vitro affinity assessment of AJ647.A) Molecular structure of AJ647, a bicyclic peptide having NOTA as a bifunctional chelator for 68Ga-labeling. B) and C) Surface Plasmon Resonance (SPR) analysis showing the binding affinity of AJ647 towards Nectin-4, assessed using recombinant human and mouse nectin-4 proteins respectively. Data is presented as mean±SD (n=3).

Fig 5: [68Ga]AJ647 derived Nectin-4 pharmacodynamics and correlation with response in HT1376 tumor xenograft.A) Schematic illustration of the experimental design showing the changes in the [68Ga]AJ647 tumor uptake following different doses of EV at Day 6. B) Tumor growth curve following single dose of EV treatment, showing growth inhibition in both EV treated groups (n=10 per group). C) Fused trans-axial PET-MR images showing the differences in the SUVs of [68Ga]AJ647 pre and post 6 days of EV treatment, indicating that EV can cause heterogenous accessible nectin-4 levels. D) SUVs derived for each mouse from the PET-MR imaging, showing the difference in uptake in before and after EV treatment. E) Correlation of tumor weights with tumor SUV before (left), after (middle) and difference (right) in before and after EV treatment. Student’s t-test used in B (unpaired) and D (paired). Spearman correlation used in E.