A novel peptide-based 68Ga-PET radiotracer for TIGIT expression visualization

Introduction: Accumulating studies have demonstrated that the novel immune checkpoint receptor T cell Ig and ITIM domain (TIGIT) is a promising candidate in cancer immune checkpoint blockade therapy. Elevated TIGIT expression in the tumor microenvironment correlates with improved therapeutic response to anti-TIGIT therapies in preclinical studies. As such, a noninvasive method to quantify TIGIT expression in tumors is crucial to predict the therapeutic effect. In this study, we report a peptide-based positron emission tomography (PET) imaging agent, ⁶⁸Ga-DOTA-^DTBP-3, to visualize TIGIT expression in BALB/c mice bearing 4T1 breast cancer.

Methods: ^DTBP-3, a 12 amino acids D-peptide, is radiolabeled with ⁶⁸Ga through DOTA conjugation. To assess the affinity of ^DTBP-3 to TIGIT, a microscale thermophoresis binding assay was performed. Diagnostic value of ⁶⁸Ga-DOTA-^DTBP-3 was further evaluated via stability assay in vitro, animal microPET/CT imaging, biodistribution study, autoradiography, and immunohistochemical staining using 4T1 tumor-bearing BALB/c mice and tissues of interest. For the blocking group, mice were pretreated with one dose of the 400-fold excessive amount of ^DTBP-3 at 1 h before tracer administration.

Results: ⁶⁸Ga-DOTA-^DTBP-3 was synthesized with a radiochemical purity of 97.29 ± 0.59% after purification, and the retention time was 13.4 min as identified by radio-HPLC. ⁶⁸Ga-DOTA-^DTBP-3 was maintained stably in PBS for 1 h at room temperature with a radiochemical purity of 94.39 ± 0.47%. The binding assay revealed a Kd value of 4.1 μM, indicating a high affinity of the tracer to TIGIT. MicroPET/CT imaging, biodistribution, autoradiography, and immunohistochemical staining studies demonstrated that ⁶⁸Ga-DOTA-^DTBP-3 could specifically target TIGIT and delineate 4T1 tumors. Biodistribution studies showed the tumor uptake of 1.10 ± 0.19 %ID/g at 0.5 h post-injection, whereas the tumor uptake in the blocking group was 0.58 ± 0.11 %ID/g, consistent with the in vivo PET imaging results.

Conclusions: ⁶⁸Ga-DOTA-^DTBP-3 has great potential for detecting the expression of TIGIT, evaluating the therapeutic effect, and optimizing the prescription of TIGIT checkpoint blockade therapy. It may become a candidate for facilitating the screening of patients and may benefit future clinical practice. Acknowledgments: This work was financially supported by the “Fundamental Research Funds for the Central Universities” (Grant NO. 2042018kf0179).

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