Theranostic role of 89Zr/177Lu-labeled aflibercept in breast cancer

Introduction: Breast cancer is a heterogeneous disease and it is the most common cancer among women around the world. Although systemic therapy has improved outcomes for patients, the development of new molecularly targeted drugs and treatment regimens is imperative. Vascular endothelial growth factor (VEGF) represents a growth factor with important pro-angiogenic activity, having a mitogenic and an anti-apoptotic effect on endothelial cells, increasing the vascular permeability, promoting cell migration, etc. Targeting and blocking angiogenesis could be helpful for the diagnosis and treatment of breast cancer. Aflibercept (Abe), a chimeric recombinant protein, contains the ligand-binding domains of both VEGFR-1 and VEGFR-2 and can sequester all isoforms of VEGF-A, PlGF, and VEGF-B, therefore affecting pathological and physiological angiogenesis. In this study, the theranostic role of ⁸⁹Zr- and ¹⁷⁷Lu-labeled aflibercept was investigated for PET imaging and treatment in breast cancer murine models.

Methods: Aflibercept was conjugated with desferrioxamine (DFO) for radiolabeling with ⁸⁹Zr (t_1/2 = 78.4 h) and it was conjugated with 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for radiolabeling with ¹⁷⁷Lu (t_1/2 = 6.65 d). After the breast cancer 4T1 tumor-bearing mice model was established, PET imaging and biodistribution studies were performed for 7 days after injection of ⁸⁹Zr-Df-aflibercept. Further, six groups were employed for the treatment study, including PBS, aflibercept, ¹⁷⁷Lu-only, ¹⁷⁷Lu-DOTA-IgG (human non-specific), ¹⁷⁷Lu-DOTA-aflibercept-low dose, and ¹⁷⁷Lu-DOTA-aflibercept-high dose. Tumor sizes and body weight were measured within 16 days post-injection (n = 5-8). Mice injected with CY5.5-aflibercept (n=4) and CY5.5-IgG (n=4) were imaged for optical imaging. Finally, histological analysis was performed to examine VEGF expression in tumors.

Results: For ⁸⁹Zr and ¹⁷⁷Lu, the labeling yields were more than 90% (n=5). PET imaging of ⁸⁹Zr-DFO-aflibercept showed an increased tumor uptake with the maximum SUVmax of 5.61 ± 0.92 at 120 h post-injection for 4T1 tumors (n = 3) (Fig. 1a). After being labeled with CY5.5 in optical imaging, the uptake of tumors in the experimental group was higher obviously than control IgG (Fig. 1b). The SUVmax value of tumor uptake increased from 1.51 ± 0.75 to 5.61 ± 0.92. The tumor-to-blood and tumor-to-muscle ratios increased over time and the highest value was 3.16 ± 0.45 and 19.76±6.42, respectively (Fig. 1c). The above results of imaging suggest the high uptake of tumors from radiolabeled aflibercept. We further labeled ¹⁷⁷Lu with aflibercept. The results showed that for the treatment group of ¹⁷⁷Lu-DOTA-aflibercept-high, significant inhibition of tumor growth was observed. Within 16d, the standard tumor volume of ¹⁷⁷Lu-DOTA-aflibercept-high were significantly less than aflibercept-only, ¹⁷⁷Lu-only, ¹⁷⁷Lu-DOTA-IgG and ¹⁷⁷Lu-DOTA-aflibercept-low, with 3646.52%, 2554.04%, 3794.30%, 1947.46% and 1805.38%, respectively (Fig. 1d). Therefore, the effectiveness of the treatment was demonstrated in our study. Besides, the bodyweight of ¹⁷⁷Lu-DOTA-aflibercept-high and ¹⁷⁷Lu-DOTA-aflibercept-low did not change significantly, indicating the safety of radiolabeled aflibercept in vivo. In the comparison, the weight loss of ¹⁷⁷Lu-only was more than 20%, suggesting that high liver intake may have increased hepatotoxicity (Fig. 1e).

Conclusions: ⁸⁹Zr- and ¹⁷⁷Lu-labeled aflibercept displayed a significant VEGF positive tumor affinity and effective tumor therapy without significant toxicity. Therefore, ⁸⁹Zr- and ¹⁷⁷Lu-labeled aflibercept could be further investigated in the theranostic field of breast cancer in the clinic.

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