A Pretargeted Imaging Strategy for Immune Checkpoint Ligand PD-L1 Expression in Tumor Based on Bioorthogonal Diels-Alder Click Chemistry

The use of antibodies as tracers requires labeling with isotopes with long half-lives due to their slow pharmacokinetics, which creates prohibitively high radiation dose to non-target organs. Pretargeted methodology could avoid the high radiation exposure due to the slow pharmacokinetics of antibodies. In this investigation, we reported the development of a novel pretargeted single photon emission computed tomography (SPECT) imaging strategy (atezolizumab-TCO/[99mTc]HYNIC-PEG11-Tz) for evaluating immune checkpoint ligand PD-L1 expression in tumor based on bioorthogonal Diels-Alder click chemistry.

The radioligand [^99mTc]HYNIC-PEG₁₁-Tz was achieved by the synthesis of a 6-hydrazinonicotinc acid (HYNIC) modified 1,2,4,5-tetrazine (Tz) and subsequently radiolabeled with technetium-99m (Tc-99m). The stability of [^99mTc]HYNIC-PEG₁₁-Tz was evaluated in vitro, and its blood pharmacokinetic test was performed in vivo. Atezolizumab was modified with trans-cyclooctene (TCO). The [^99mTc]HYNIC-PEG₁₁-Tz and atezolizumab-TCO interaction was tested in vitro. Pretargeted H1975 cell immunoreactivity binding and saturation binding assays were evaluated. Pretargeted biodistribution and SPECT imaging experiments were performed in H1975 and A549 tumor-bearing modal mice to evaluate the PD-L1 expression level.

[^99mTc]HYNIC-PEG₁₁-Tz was successfully radiosynthesized with a specific activity of 9.25 MBq/μg and a radiochemical purity above 95 % as confirmed by reversed-phase HPLC (RP-HPLC). [^99mTc]HYNIC-PEG₁₁-Tz showed favorable stability in NS, PBS, and FBS and rapid blood clearance in mice. The atezolizumab was modified with TCO-NHS ester to produce a conjugate with an average 6.4 TCO moieties as confirmed by liquid chromatograph-mass spectrometer (LC-MS). Size exclusion HPLC revealed almost complete reaction between atezolizumab-TCO and [^99mTc]HYNIC-PEG₁₁-Tz in vitro, with the 1:1 Tz-to-mAb reaction providing a conversion yield of 88.65 ± 1.22 %. Pretargeted cell immunoreactivity binding and saturation binding assays showed high affinity to H1975 cells. After allowing 48 h for accumulation of atezolizumab-TCO in H1975 tumor, pretargeted in vivo biodistribution revealed high uptake of the radiotracer in the tumor with a tumor-to-muscle ratio of 27.51 and tumor-to-blood ratio of 1.91. Pretargeted SPECT imaging delineated the H1975 tumor clearly. Pretargeted biodistribution and SPECT imaging in control groups demonstrated a significantly reduced tracer accumulation in the A549 tumor.

We have developed a HYNIC-modified Tz derivative, and the HYNIC-PEG₁₁-Tz was labeled with Tc-99m with a high specific activity and radiochemical purity. [^99mTc]HYNIC-PEG₁₁-Tz reacted rapidly and almost completely towards atezolizumab-TCO in vitro with the 1:1 Tz-to-mAb reaction. SPECT imaging using the pretargeted strategy (atezolizumab-TCO/[^99mTc]HYNIC-PEG₁₁-Tz) demonstrated high-contrast images for high PD-L1 expression H1975 tumor and a low background accumulation of the probe. The pretargeted imaging strategy is a powerful tool for evaluating PD-L1 expression in xenograft mice tumor models and a potential candidate for translational clinical application.