Local and distant response to intratumoral immunotherapy assessed by immunoPET in mice.

BACKGROUND: Despite the promising efficacy of immune checkpoint blockers (ICB), tumor resistance and immune-related adverse events hinder their success in cancer treatment. To address these challenges, intratumoral delivery of immunotherapies has emerged as a potential solution, aiming to mitigate side effects through reduced systemic exposure while increasing effectiveness by enhancing local bioavailability. However, a comprehensive understanding of the local and systemic distribution of ICBs following intratumoral administration, as well as their impact on distant tumors, remains crucial for optimizing their therapeutic potential.To comprehensively investigate the distribution patterns following the intratumoral and intravenous administration of radiolabeled anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and to assess its corresponding efficacy in both injected and non-injected tumors, we conducted an immunoPET imaging study. METHODS: CT26 and MC38 syngeneic colorectal tumor cells were implanted subcutaneously on both flanks of Balb/c and C57Bl/6 mice, respectively. Hamster anti-mouse CTLA-4 antibody (9H10) labeled with zirconium-89 ([89Zr]9H10) was intratumorally or intravenously administered. Whole-body distribution of the antibody was monitored by immunoPET imaging (n=12 CT26 Balb/c mice, n=10 MC38 C57Bl/6 mice). Tumorous responses to injected doses (1-10 mg/kg) were correlated with specific uptake of [89Zr]9H10 (n=24). Impacts on the tumor microenvironment were assessed by immunofluorescence and flow cytometry. RESULTS: Half of the dose was cleared into the blood 1 hour after intratumoral administration. Despite this, 7 days post-injection, 6-8% of the dose remained in the intratumoral-injected tumors. CT26 tumors with prolonged ICB exposure demonstrated complete responses. Seven days post-injection, the contralateral non-injected tumor uptake of the ICB was comparable to the one achieved through intravenous administration (7.5±1.7% ID.cm-3 and 7.6±2.1% ID.cm-3, respectively) at the same dose in the CT26 model. This observation was confirmed in the MC38 model. Consistent intratumoral pharmacodynamic effects were observed in both intratumoral and intravenous treatment groups, as evidenced by a notable increase in CD8+T cells within the CT26 tumors following treatment. CONCLUSIONS: ImmunoPET-derived pharmacokinetics supports intratumoral injection of ICBs to decrease systemic exposure while maintaining efficacy compared with intravenous. Intratumoral-ICBs lead to high local drug exposure while maintaining significant therapeutic exposure in non-injected tumors. This immunoPET approach is applicable for clinical practice to support evidence-based drug development.

Efficient PD-L1 imaging of murine glioblastoma with FUS-aided immunoPET by leveraging FcRn-antibody interaction.

Rationale: The passage of antibodies through the blood-brain barrier (BBB) and the blood-tumoral barrier (BTB) is determinant not only to increase the immune checkpoint inhibitors efficacy but also to monitor prognostic and predictive biomarkers such as the programmed death ligand 1 (PD-L1) via immunoPET. Although the involvement of neonatal Fc receptor (FcRn) in antibody distribution has been demonstrated, its function at the BBB remains controversial, while it is unknown at the BTB. In this context, we assessed FcRn's role by pharmacokinetic immunoPET imaging combined with focused ultrasounds (FUS) using unmodified and FcRn low-affinity IgGs targeting PD-L1 in a preclinical orthotopic glioblastoma model. Methods: Transcranial FUS were applied over the whole brain in mice shortly before injecting the anti-PD-L1 IgG 89Zr-DFO-C4 or its FcRn low-affinity mutant 89Zr-DFO-C4Fc-MUT in a syngeneic glioblastoma murine model (GL261-GFP). Brain uptake was measured from PET scans acquired up to 7 days post-injection. Kinetic modeling was performed to compare the brain kinetics of both C4 formats. Results: FUS efficiently enhanced the delivery of both C4 radioligands in the brain with high reproducibility. 89Zr-DFO-C4Fc-MUT mean concentrations in the brain reached a significant uptake of 3.75±0.41%ID/cc with FUS against 1.92±0.45%ID/cc without, at 1h post-injection. A substantial and similar entry of both C4 radioligands was observed at a rate of 0.163±0.071 mL/h/g of tissue during 10.4±4.6min. The impaired interaction with FcRn of 89Zr-DFO-C4Fc-MUT significantly decreased the efflux constant from the healthy brain tissue to plasma compared with non-mutated IgG. Abolishing FcRn interaction allows determining the target engagement related to the specific binding as soon as 12h post-injection. Conclusion: Abolishing Fc-FcRn interaction confers improved kinetic properties to 89Zr-DFO-C4Fc-MUT for immunoPET imaging. FUS-aided BBB/BTB disruption enables quantitative imaging of PD-L1 expression by glioblastoma tumors within the brain.

TSPO PET Imaging as a Potent Non-Invasive Biomarker for Diffuse Intrinsic Pontine Glioma in a Patient-Derived Orthotopic Rat Model.

Diffuse intrinsic pontine gliomas (DIPG), the first cause of cerebral pediatric cancer death, will greatly benefit from specific and non-invasive biomarkers for patient follow-up and monitoring of drug efficacy. Since biopsies are challenging for brain tumors, molecular imaging may be a technique of choice to target and follow tumor evolution. So far, MR remains the imaging technique of reference for DIPG, although it often fails to define the extent of tumors, an essential parameter for therapeutic efficacy assessment. Thanks to its high sensitivity, positron emission tomography (PET) offers a unique way to target specific biomarkers in vivo. We demonstrated in a patient-derived orthotopic xenograft (PDOX) model in the rat that the translocator protein of 18 kDa (TSPO) may be a promising biomarker for monitoring DIPG tumors. We studied the distribution of 18F-DPA-714, a TSPO radioligand, in rats inoculated with HSJD-DIPG-007 cells. The primary DIPG human cell line HSJD-DIPG-007 highly represents this pediatric tumor, displaying the most prevalent DIPG mutations, H3F3A (K27M) and ACVR1 (R206H). Kinetic modeling and parametric imaging using the brain 18F-DPA-714 PET data enabled specific delineation of the DIPG tumor area, which is crucial for radiotherapy dose management.

Impact of Radiolabeling Strategies on the Pharmacokinetics and Distribution of an Anti-PD-L1 PET Ligand.

Molecular imaging with PET offers an alternative method to quantify programmed-death-ligand 1 (PD-L1) to accurately select patients for immunotherapies. More and more clinical and preclinical trials involve radiolabeling of antibody fragments for their desirably fast clearance and high tumor penetration. As the radiolabeling strategy can significantly impact pharmacokinetics and biodistribution, we explored in this work a site-specific radiofluorination strategy on an anti-PD-L1 fragment antigen-binding (Fab) and compared the pharmacokinetic and biodistribution properties with the same Fab labeled using stochastic radiolabeling chemistry. We applied an enzymatic bioconjugation mediated by a variant of the lipoic acid ligase (LplA) that promotes the formation of an amide bond between a short peptide cloned onto the C terminus of the Fab. A synthetic analogue of the enzyme natural substrate, lipoic acid, was radiolabeled with fluorine-18 for site-specific conjugation by LplA. We compared the biodistribution of the site-specifically labeled Fab with a stochastically labeled Fab on lysine side chains in tumor-bearing mice. The two methods of fluorination demonstrate a comparable whole-body biodistribution. The 89Zr-labeled Fab had different biodistribution compared to either 18F-labeled Fab. We attribute the difference to [89Zr] metabolism. Fab-LAP-[18F]FPyOctA therefore reflects better the true pharmacokinetic profile of the Fab.

Optimizing Immuno-PET Imaging of Tumor PD-L1 Expression: Pharmacokinetic, Biodistribution, and Dosimetric Comparisons of 89Zr-Labeled Anti-PD-L1 Antibody Formats.

PET imaging of programmed cell death ligand 1 (PD-L1) may help to noninvasively predict and monitor responses to anti-programmed cell death 1/anti-PD-L1 immunotherapies. In this study, we compared the imaging characteristics of 3 radioligands derived from the anti-PD-L1 IgG1 complement 4 (C4). In addition to the IgG C4, we produced a fragment antigen-binding (Fab) C4, as well as a double-mutant IgG C4 (H310A/H435Q) with minimal affinity for the murine neonatal Fc receptor. Methods: The pharmacokinetics, biodistribution, and dosimetry of the 3 89Zr-labeled C4 ligands were compared by longitudinal PET/CT imaging in nude mice bearing subcutaneous human non-small cell lung cancer xenografts with positive (H1975 model) or negative (A549 model) endogenous PD-L1 expression. Results: The C4 radioligands substantially accumulated in PD-L1-positive tumors but not in PD-L1-negative tumors or in blocked PD-L1-positive tumors, confirming their PD-L1-specific tumor targeting. 89Zr-Fab C4 and 89Zr-IgG C4 (H310A/H435Q) were rapidly eliminated compared with 89Zr-IgG C4. Consequently, maximal tumor-to-muscle ratios were obtained earlier, at 4 h after injection for 89Zr-Fab C4 (ratio, ∼6) and 24 h after injection for 89Zr-IgG C4 (H310A/H435Q) (ratio, ∼9), versus 48 h after injection for 89Zr-IgG C4 (ratio, ∼8). Background activity in nontumor tissues was low, except for high kidney retention of 89Zr-Fab C4 and persistent liver accumulation of 89Zr-IgG C4 (H310A/H435Q) compared with 89Zr-IgG C4. Dosimetry estimates suggested that the C4 radioligands would yield organ-absorbed doses tolerable for repeated clinical PET imaging studies. Conclusion: This study highlights the potential of designing radioligands with shorter pharmacokinetics for PD-L1 immuno-PET imaging in a preclinical model and encourages further clinical translation of such radioligands.

Population pharmacokinetics of oral baclofen at steady-state in alcoholic-dependent adult patients.

Baclofen has been proposed for few years to help treating alcohol dependence at higher doses than those used in neurology. Baclofen pharmacokinetics has been previously well described at low oral or intravenous doses but remains poorly investigated with such high oral doses. We here describe dose regimens of baclofen in 143 alcohol-dependent patients treated with steady-state oral doses of baclofen. Plasma baclofen levels were measured in blood samples using liquid chromatography coupled with tandem mass spectrometry. One hundred and forty-nine baclofen concentrations were sampled 30 min to 15 h after the last dose, and baclofen pharmacokinetics was determined using population pharmacokinetics approach. Our population, whose average age and BMI were 51.5 years and 25.5 kg/m2 , respectively, was composed of two-thirds of men. Daily baclofen doses ranged from 15 to 250 mg and 26% were higher than 120 mg. A one-compartment model with first-order absorption and elimination allowed to determine mean values for clearance (CL/F), volume of distribution (V/F) and absorption rate constant at 8.0 L/h, 44.5 L and 2.23 h-1 , respectively. Inter-individual variability on CL/F and V/F was 27.4 and 86% for the parameters. None of the demographic and biological covariates significantly decreased inter-individual variability. A proportional relationship between oral dose and plasma baclofen exposure indicated a linear pharmacokinetics of baclofen even at doses over 120 mg/day. Our large population study evidenced a linear pharmacokinetics of oral baclofen even at high daily doses with an inter-individual variability of baclofen exposure that could not be explained by demographic and biological data.