PD-L1 promotes oncolytic virus infection via a metabolic shift that inhibits the type I IFN pathway.

While conventional wisdom initially postulated that PD-L1 serves as the inert ligand for PD-1, an emerging body of literature suggests that PD-L1 has cell-intrinsic functions in immune and cancer cells. In line with these studies, here we show that engagement of PD-L1 via cellular ligands or agonistic antibodies, including those used in the clinic, potently inhibits the type I interferon pathway in cancer cells. Hampered type I interferon responses in PD-L1-expressing cancer cells resulted in enhanced efficacy of oncolytic viruses in vitro and in vivo. Consistently, PD-L1 expression marked tumor explants from cancer patients that were best infected by oncolytic viruses. Mechanistically, PD-L1 promoted a metabolic shift characterized by enhanced glycolysis rate that resulted in increased lactate production. In turn, lactate inhibited type I IFN responses. In addition to adding mechanistic insight into PD-L1 intrinsic function, our results will also help guide the numerous ongoing efforts to combine PD-L1 antibodies with oncolytic virotherapy in clinical trials.

Quinazoline-2-Carboxamides as Selective PET Radiotracers for Matrix Metalloproteinase-13 Imaging in Atherosclerosis.

Matrix metalloproteinase-13 (MMP-13) plays a critical role in the progression of unstable atherosclerosis. A series of highly potent and selective MMP-13 inhibitors were synthesized around a quinazoline-2-carboxamide scaffold to facilitate radiolabeling with fluorine-18 or carbon-11 positron-emitting nuclides and visualization of atherosclerotic plaques. In vitro enzyme inhibition assays identified three compounds as promising radiotracer candidates. Efficient automated radiosyntheses provided [11C]5b, [11C]5f, and [18F]5j and enabled pharmacokinetic characterization in atherosclerotic mice. The radiotracers displayed substantial differences in their distribution and excretion. Most favorably for vascular imaging, [18F]5j exhibited low uptake in metabolic organs with minimal retention of myocardial radioactivity, substantial renal clearance, and high metabolic stability in plasma. Ex vivo aortic autoradiography and competition studies revealed that [18F]5j specifically binds to MMP-13 within atherosclerotic plaques and localizes to lipid-rich regions. This study demonstrates the utility of the quinazoline-2-carboxamide scaffold for MMP-13 selective positron emission tomography (PET) radiotracer development and identifies [18F]5j for imaging atherosclerosis.

Synthesis and Evaluation of [11C]MCC950 for Imaging NLRP3-Mediated Inflammation in Atherosclerosis.

Overexpression of the NLRP3 inflammasome has been attributed to the progressive worsening of a multitude of cardiovascular inflammatory diseases such as myocardial infarction, pulmonary arterial hypertension, and atherosclerosis. The recently discovered potent and selective NLRP3 inhibitor MCC950 has shown promise in hindering disease progression, but NLRP3-selective cardiovascular positron emission tomography (PET) imaging has not yet been demonstrated. We synthesized [11C]MCC950 with no-carrier-added [11C]CO2 fixation chemistry using an iminophosphorane precursor (RCY 45 ± 4%, >99% RCP, 27 ± 2 GBq/µmol, 23 ± 3 min, n = 6) and determined its distribution both in vivo and ex vivo in C57BL/6 and atherogenic ApoE-/- mice. Small animal PET imaging was performed in both strains following intravenous administration via the lateral tail vein and revealed considerable uptake in the liver that stabilized by 20 min (7-8.5 SUV), coincident with secondary renal excretion. Plasma metabolite analysis uncovered excellent in vivo stability of [11C]MCC950 (94% intact). Ex vivo autoradiography performed on excised aortas revealed heterogeneous uptake in atherosclerotic plaques of ApoE-/- mice in comparison to C57BL/6 controls (48 ± 17 %ID/m2 vs 18 ± 8 %ID/m2, p = 0.002, n = 4-5). Treatment of ApoE-/- mice with nonradioactive MCC950 (5 mg/kg, iv) 10 min prior to radiotracer administration increased uptake in the intestine (5.3 ± 1.8 %ID/g vs 11.0 ± 3.7 %ID/g, p = 0.04, n = 4-6) and in aortic lesions (48 ± 17 %ID/m2 vs 104 ± 15 %ID/m2, p = 0.0002, n = 5) by 108% and 117%, respectively, without significantly increasing plasma free fraction (fp, 1.3 ± 0.4% vs 1.7 ± 0.8%, n = 2). These results suggest that [11C]MCC950 uptake demonstrates specific binding and may prove useful for in vivo NLRP3 imaging in atherosclerosis.

Selective Imaging of Matrix Metalloproteinase-13 to Detect Extracellular Matrix Remodeling in Atherosclerotic Lesions.

PURPOSE: Overexpression and activation of matrix metalloproteinase-13 (MMP-13) within atheroma increases susceptibility to plaque rupture, a major cause of severe cardiovascular complications. In comparison to pan-MMP targeting [18F]BR-351, we evaluated the potential for [18F]FMBP, a selective PET radiotracer for MMP-13, to detect extracellular matrix (ECM) remodeling in vascular plaques possessing markers of inflammation. PROCEDURES: [18F]FMBP and [18F]BR-351 were initially assessed in vitro by incubation with en face aortae from 8 month-old atherogenic ApoE-/- mice. Ex vivo biodistributions, plasma metabolite analyses, and ex vivo autoradiography were analogously performed 30 min after intravenous radiotracer administration in age-matched C57Bl/6 and ApoE-/- mice under baseline or homologous blocking conditions. En face aortae were subsequently stained with Oil Red O (ORO), sectioned, and subject to immunofluorescence staining for Mac-2 and MMP-13. RESULTS: High-resolution autoradiographic image analysis demonstrated target specificity and regional concordance to lipid-rich lesions. Biodistribution studies revealed hepatobiliary excretion, low accumulation of radioactivity in non-excretory organs, and few differences between strains and conditions in non-target organs. Plasma metabolite analyses uncovered that [18F]FMBP exhibited excellent in vivo stability (≥74% intact) while [18F]BR-351 was extensively metabolized (≤37% intact). Ex vivo autoradiography and histology of en face aortae revealed that [18F]FMBP, relative to [18F]BR-351, exhibited 2.9-fold greater lesion uptake, substantial specific binding (68%), and improved sensitivity to atherosclerotic tissue (2.9-fold vs 2.1-fold). Immunofluorescent staining of aortic en face cross sections demonstrated elevated Mac-2 and MMP-13-positive areas within atherosclerotic lesions identified by [18F]FMBP ex vivo autoradiography. CONCLUSIONS: While both radiotracers successfully identified atherosclerotic plaques, [18F]FMBP showed superior specificity and sensitivity for lesions possessing features of destructive plaque remodeling. The detection of ECM remodeling by selective targeting of MMP-13 may enable characterization of high-risk atherosclerosis featuring elevated collagenase activity.

Cardiac Sympathetic Positron Emission Tomography Imaging with Meta-[18F]Fluorobenzylguanidine is Sensitive to Uptake-1 in Rats.

Dysfunction of the cardiac sympathetic nervous system contributes to the development of cardiovascular diseases including ischemia, heart failure, and arrhythmias. Molecular imaging probes such as meta-[123I]iodobenzylguanidine have demonstrated the utility of assessing neuronal integrity by targeting norepinephrine transporter (NET, uptake-1). However, current radiotracers can report only on innervation due to suboptimal kinetics and lack sensitivity to NET in rodents, precluding mechanistic studies in these species. The objective of this work was to characterize myocardial sympathetic neuronal uptake mechanisms and kinetics of the positron emission tomography (PET) radiotracer meta-[18F]fluorobenzylguanidine ([18F]mFBG) in rats. Automated synthesis using spirocyclic iodonium(III) ylide radiofluorination produces [18F]mFBG in 24 ± 1% isolated radiochemical yield and 30-95 GBq/µmol molar activity. PET imaging in healthy rats delineated the left ventricle, with monoexponential washout kinetics (kmono = 0.027 ± 0.0026 min-1, Amono = 3.08 ± 0.33 SUV). Ex vivo biodistribution studies revealed tracer retention in the myocardium, while pharmacological treatment with selective NET inhibitor desipramine, nonselective neuronal and extraneuronal uptake-2 inhibitor phenoxybenzamine, and neuronal ablation with neurotoxin 6-hydroxydopamine reduced myocardial retention by 33, 76, and 36%, respectively. Clearance of [18F]mFBG from the myocardium was unaffected by treatment with uptake-1 and uptake-2 inhibitors following peak myocardial activity. These results suggest that myocardial distribution of [18F]mFBG in rats is dependent on both NET and extraneuronal transporters and that limited reuptake to the myocardium occurs. [18F]mFBG may therefore prove useful for imaging intraneuronal dysfunction in small animals.