Development And Evaluation Of PD-L1 And TIGIT-Targeted PET Tracers For Immune Checkpoint Imaging In Oncology

Cancer immunotherapy has been boosted by the discovery of CTLA4 and PD1/PD-L1 inhibitors in the past decade,which revolutionize the clinical treatment of solid tumors and open an exciting era of clinical oncology.While blockade of CTLA4 or PD-1/PD-L1 has manifested compelling responses against certain cancer types in the clinic,only a subset of cancer patients could benefit from these inhibitors.The monotherapy was often less effective and combination therapy could be required.The adaptive resistance and immune-related adverse events were often observed.Moreover,the tumor response was more complex and heterogeneous than other therapies,including processes such as hyperprogression or pseudoprogression.All these difficulties could bring challenges to clinical decision-making and efficacy evaluation.Fortunately,PET imaging could provide non-invasive whole-body visualization of tumor and immune cells characteristics and might support patient selection,therapy optimization and combination,and response evaluation for immune checkpoint therapies.Therefore,the development of specific PET tracers that directly target immune checkpoint-ligand interactions is crucial for immunoPET imaging and is also the focus of this paper.Firstly,we reported an 18F-labeled D-peptide antagonist,18F-NOTA-NF12,for specific PET imaging of PD-L1 status.The radiosynthesis of 18F-NOTA-NF12 were achieved with RCY of 41.7 ± 10.2%,RCP of>95%and specific activity of 22.3 ± 2.5 GBq/μmol,respectively.The partition coefficient(Log P)was determined to be-2.02±0.2。In vitro binding assays demonstrated high specificity and affinity with an IC50 of 78.35 nM and KD of 85.08 nM.18F-NOTA-NF12 exhibited renal excretion and rapid clearance from blood and other non-specific organs,contributing to high contrast imaging in the clinical time frame.The tumors were clearly visualized with the optimized tumor-to-muscle ratios of 7.13 ±1.78 at 60 min after injection.Gemcitabine and selumetinib-induced modulation of PD-L1 dynamics were monitored by 18FNOTA-NF12 PET.Subsequently,18F-NOTA-NF12 was translated into the first-in-human trial to evaluate its safety,radiation dosimetry,biodistribution and PD-L1 imaging potential in patients.The large-scale radiosynthesis of 18F-NOTA-NF12 was optimized in a custommade automatic platform with RCY of 70.6 ± 4.2%.All the participants had good tolerability without any adverse and clinically detectable pharmacologic effects.The biodistribution and pharmacokinetics in healthy volunteers were similar to those in preclinical studies.The effective dose was determined to be 9.92E-03 mSv/MBq,which is lower than that of conventional 18F-FDG PET/CT.In NSCLC and esophageal cancer,the patients with high PD-L1 expression had higher tumor uptake of 18F-NOTA-NF12 than patients with low PD-L1 expression.The 18F-NOTA-NF12 PET/CT and 18F-FDG PET/CT had equivalent findings in patients with high PD-L1 expression.In another study,we developed and validated a 68Ga-labeled D-peptide antagonist,68Ga-GP12,for PET imaging of TIGIT expression in cancers.The preparation of 68Galabeled D-peptides was accomplished with RCY of 43.5-83.3%,RCP of>99%,and specific activity of 30.6-57.3 GBq/μmol.The partition coefficient(Log P)was determined to be-3.31--1.56.Among them,68Ga-GP12 has high affinity and specificity for TIGIT with a KD of 37.28 nM.In vivo studies demonstrated the capacity of 68GaGP12 for PET imaging of TIGIT expression with high tumor uptake of 4.22±0.68%ID/g and the tumor-to-muscle ratio of 12.94±2.64 at 60 min post-injection.The in vivo targeting and specificity of 68Ga-GP12 for TIGIT were identified.The capacity of 68GaGP12 for detecting TIGIT expression and evaluating prognosis in course of anti-TIGIT therapy was discovered.As expected,tumor uptake of 68Ga-GP12 was positively associated with TIGIT expression on CD4+T cell,CD8+T cell,NK cell and Treg cell,respectively.The considerable tumor accumulation and rapid blood clearance of 68GaGP12 contributed to the optimized PET imaging with high contrast in a short time.From the perspective of radiation dosimetry,68Ga-GP 12 is safe for use in humans.The optimized time-point for image acquisition is 40 min after injection of 68Ga-GP12.In NSCLC patients,primary and metastatic lesions found in 68Ga-GP12 PET images were comparable to that in 18F-FDG PET images.Moreover,tracer uptake in primary and metastatic lesions and intra-tumoral distribution in large tumor were inhomogenous,indicating the heterogeneity of TIGIT expression.Finally,longitudinal PET imaging with 18F-NOTA-NF12 and 18F-NOTA-GP12 was used to monitor the spatiotemporal dynamics between tumor cell growth and T cell activation after PD-1 blockade,which might explain the heterogeneity of immunotherapy response.The PD-L1 PET and TIGIT PET signals in tumors were negatively correlated with tumor growth rate(TGR),which may and predict the efficacy of immunotherapy.Mass cytometry(Cy-TOF)provided the landscape of tumor immune microenvironment after PD-1 blockade.To some extent,the correlation between PD-L1 and TIGIT expression in host immune cells mediated tumor immune escape(growth)and T cell activation and the efficacy of PD-1 therapy was revealed.During PD-1 therapy,the upregulation of TIGIT expression could provide theoretical support for the combination therapy of anti-PD-1 and anti-TIGIT.The combination of anti-PD-1 and anti-TIGIT therapy significantly inhibited tumor growth and prolonged survival of tumor-bearing mice,demonstrating the powerful efficacy of combined immunotherapy.In conclusion,we developed PD-L1 and TIGIT-targeted PET tracers and systematically evaluated their potentials in tumor immune checkpoint imaging and evaluation and prediction of immunotherapy in preclinical and clinical studies,which may provide a good theoretical and scientific basis for clinical application of accurate immunotherapy.