Objectives: The integrin α_vβ₆ is an epithelial specific cell surface receptor that is overexpressed on numerous malignancies including pancreatic ductal adenocarcinomas (PDAC) [1]. We previously demonstrated that α_vβ₆-targeted theranostic agents, ⁶⁸Ga-DOTA-5G and ¹⁷⁷Lu-DOTA-ABM-5G show promise in preclinical studies [2] and are currently being evaluated in several Phase 1 clinical trial in patients with pancreas cancer (NCT04665947), metastatic non-small cell lung cancer (NCT06228482) and any metastatic carcinoma (NCT06389123). With the goal to develop a “true theranostic” peptide pair, we designed and synthesized peptides DOTA-ABM-5G (1) and NOTA-ABM-5G (2) radiolabeled with the radionuclide copper-64/copper-67 and evaluated their pharmacokinetic properties and therapeutic efficacy.  

Methods: Peptides 1 and 2 were synthesized using standard Fmoc chemistry. Affinity for α_vβ₆ was assessed by ELISA. The peptides were radiolabeled with copper-64 (t_1/2 = 12.7 h;  β⁺ E_max= 660 keV) for imaging and copper-67 (t_1/2 = 2.6 d; β^– E_max= 562 keV) for treatment. ⁶⁴Cu-1, ⁶⁴Cu-2, ⁶⁷Cu-1 and ⁶⁷Cu-2 were evaluated in vitro for binding and internalization in α_vβ₆ (+) pancreatic Capan-1 cells, and for stability in mouse and human serum. In vivo PET/CT imaging (⁶⁴Cu-1 and ⁶⁴Cu-2 at 4, 24, 48, 72 h) and biodistribution (⁶⁴Cu-1 and ⁶⁴Cu-2 at 4, 24, 48, 72 h) were performed in mice bearing α_vβ₆ (+) Capan-1 tumors.

Results: Peptides 1 and 2 demonstrated high affinity for α_vβ₆ (ELISA; 8.4±0.8 and 10.5±1 nM, respectively). ⁶⁴Cu/⁶⁷Cu-1 and ⁶⁴Cu/⁶⁷Cu-2 were synthesized at 18.5 GBq/μmol molar activity in >98% radiochemical purity (RCP). All radiolabeled peptides showed preferential binding to (18-35% at 1 h) and rapid internalization into (> 50% of bound at 1 h) α_vβ₆ (+) cells (Figure 1A), and > 45% and >95% stability in mouse and human serum respectively, at 48 h. Both ⁶⁴Cu-1 and ⁶⁴Cu-2 clearly delineated α_vβ₆ (+) Capan-1 tumors by PET/CT imaging (Figure 1B).  This was corroborated by the biodistribution study where uptake and slow washout over time of ⁶⁴Cu-1 and ⁶⁴Cu-2 in the Capan-1 tumor was observed (percentage of injected dose per gram, % ID/g: ⁶⁴Cu-1: 4.3±0.5 at 4 h and 2.4±0.2 at 72 h; ⁶⁴Cu-2: 4.2±0.3 at 4 h and 2.0±0.2 at 72 h (Figure 1C). Uptake in the kidneys was significantly lower for ⁶⁴Cu-2 than for ⁶⁴Cu-1 (2.4±0.3 vs. 5.8±1.0 at 72 h; P = 0.006), resulting in improved tumor-to-kidney ratios of 0.85±0.1 (⁶⁴Cu-2) vs 0.43±0.0 (⁶⁴Cu-1) (Figure 1D). ⁶⁴Cu-2 also showed partial clearance through the liver (%ID/g at 24 h and 72 h: 4.6±0.7 and 2.6±0.4; vs ⁶⁴Cu-1: 1.5±0.2 and 1.4±0.2). The tumor uptake for both ⁶⁴Cu-1 and ⁶⁴Cu-2 is comparable to ¹⁷⁷Lu-1, with improved kidney clearance for both ⁶⁴Cu-1 and ⁶⁴Cu-2 (%ID/g at 72 h: ¹⁷⁷Lu-1: 10.6±2.7, ⁶⁴Cu-1: 5.8±1.0, and ⁶⁴Cu-2: 2.4±0.3; Figure 1D).

Conclusions: Imaging peptides ⁶⁴Cu-1 and ⁶⁴Cu-2 showed α_vβ₆– targeted tumor uptake comparable to our current radiotherapeutic peptide ¹⁷⁷Lu-1, and demonstrated significantly decreased kidney uptake. Further preclinical evaluations, including therapeutic efficacy studies and dosimetry estimations, are currently underway for the copper-67 peptides. Since ⁶⁴Cu/⁶⁷Cu is a “true theranostic” radionuclide pair, we anticipate the pharmacokinetics of the copper-67 peptides to match those of the copper-64 peptides, resulting in more favorable dosimetry, especially with respect to the kidneys (current dose limiting organ).  

Acknowledgements: This work was supported by the Stand Up To Cancer and Lustgarten Foundation Pancreatic Cancer Collective (PCC) New Therapies Challenge Grant (SU2C-AACR-PCC-06-18).

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Image/Figure Caption:

Figure 1: (A) Cell-binding and internalization in integrin α_vβ₆ (+) Capan-1 cells (1 h, 37°C, n = 3/compound); bars: SD.  Peptides 1 = DOTA-ABM-5G; 2 = NOTA-ABM-5G. (B) Representative transaxial (top) and coronal (bottom) PET/CT images of mice bearing Capan-1 tumors (arrows) obtained after injection of imaging peptides ⁶⁴Cu-1 and ⁶⁴Cu-2. K- Kidneys, L- Liver. PET scale in color. (C) Biodistribution of imaging peptides ⁶⁴Cu-1 and ⁶⁴Cu-2 in selected organs presented as percentage of injected dose per gram (%ID/g) of tissue in mice bearing α_vβ₆ (+) Capan-1 tumors (n = 3/group/time point). (D) Capan-1 tumor uptake, Kidney uptake and Capan-1-to-Kidney ratios for ⁶⁴Cu-1, ⁶⁴Cu-2, compared to the current radiotherapeutic ¹⁷⁷Lu-DOTA-ABM-5G (¹⁷⁷Lu-1)[2].

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