Multiparameter Flow Cytometry Assay for Quantification of Immune Cell Subsets, PD-1 Expression Levels and PD-1 Receptor Occupancy by Nivolumab and Pembrolizumab

We report the development and validation of a 12 parameter immunofluorescence flow cytometry method for the sensitive determination of cell concentrations, their expression of PD-1, and PD-1 receptor occupancy. Cell subsets include CD4+ and CD8+ -T-cells, B-cells, natural killer cells, classical-, intermediate- and non-classical monocytes, and myeloid- and plasmacytoid dendritic cells. Cells were isolated from peripheral blood by density gradient centrifugation. The validation parameters included specificity, linearity, limit of quantification, precision, biological within- and between subject variations. The lower limit of quantification was 5.0% of PD-1+ cells. Samples were stable for at least 153 days of storage at -80°C. The clinical applicability of the method was demonstrated in 11 advanced cancer patients by the successful determination of immune cell concentrations, relative number of PD-1+ immune cells, and number of PD-1 molecules per immune cell. Shortly after infusion of nivolumab, receptor occupancy on CD8+ -T-cells was 98%. Similar values were found predose cycle 2, suggesting receptor occupancy remained high throughout the entire cycle. © 2019 International Society for Advancement of Cytometry

Multiparameter Flow Cytometry Assay for Quantification of Immune Cell Subsets, PD‐1 Expression Levels and PD‐1 Receptor Occupancy by Nivolumab and Pembrolizumab

We report the development and validation of a 12 parameter immunofluorescence flow cytometry method for the sensitive determination of cell concentrations, their expression of PD-1, and PD-1 receptor occupancy. Cell subsets include CD4+ and CD8+ -T-cells, B-cells, natural killer cells, classical-, intermediate- and non-classical monocytes, and myeloid- and plasmacytoid dendritic cells. Cells were isolated from peripheral blood by density gradient centrifugation. The validation parameters included specificity, linearity, limit of quantification, precision, biological within- and between subject variations. The lower limit of quantification was 5.0% of PD-1+ cells. Samples were stable for at least 153 days of storage at -80°C. The clinical applicability of the method was demonstrated in 11 advanced cancer patients by the successful determination of immune cell concentrations, relative number of PD-1+ immune cells, and number of PD-1 molecules per immune cell. Shortly after infusion of nivolumab, receptor occupancy on CD8+ -T-cells was 98%. Similar values were found predose cycle 2, suggesting receptor occupancy remained high throughout the entire cycle. © 2019 International Society for Advancement of Cytometry

Multiparameter Flow Cytometry Assay for Quantification of Immune Cell Subsets, PD-1 Expression Levels and PD-1 Receptor Occupancy by Nivolumab and Pembrolizumab

We report the development and validation of a 12 parameter immunofluorescence flow cytometry method for the sensitive determination of cell concentrations, their expression of PD-1, and PD-1 receptor occupancy. Cell subsets include CD4+ and CD8+ -T-cells, B-cells, natural killer cells, classical-, intermediate- and non-classical monocytes, and myeloid- and plasmacytoid dendritic cells. Cells were isolated from peripheral blood by density gradient centrifugation. The validation parameters included specificity, linearity, limit of quantification, precision, biological within- and between subject variations. The lower limit of quantification was 5.0% of PD-1+ cells. Samples were stable for at least 153 days of storage at -80°C. The clinical applicability of the method was demonstrated in 11 advanced cancer patients by the successful determination of immune cell concentrations, relative number of PD-1+ immune cells, and number of PD-1 molecules per immune cell. Shortly after infusion of nivolumab, receptor occupancy on CD8+ -T-cells was 98%. Similar values were found predose cycle 2, suggesting receptor occupancy remained high throughout the entire cycle. © 2019 International Society for Advancement of Cytometry

⁸⁹Zr-Immuno-PET with Immune Checkpoint Inhibitors: Measuring Target Engagement in Healthy Organs

Introduction: 89Zr-immuno-PET (positron emission tomography with zirconium-89-labeled monoclonal antibodies ([89Zr]Zr-mAbs)) can be used to study the biodistribution of mAbs targeting the immune system. The measured uptake consists of target-specific and non-specific components, and it can be influenced by plasma availability of the tracer. To find evidence for target-specific uptake, i.e., target engagement, we studied five immune-checkpoint-targeting [89Zr]Zr-mAbs to (1) compare the uptake with previously reported baseline values for non-specific organ uptake (ns-baseline) and (2) look for saturation effects of increasing mass doses. Method: 89Zr-immuno-PET data from five [89Zr]Zr-mAbs, i.e., nivolumab and pembrolizumab (anti-PD-1), durvalumab (anti-PD-L1), BI 754,111 (anti-LAG-3), and ipilimumab (anti-CTLA-4), were analysed. For each mAb, 2–3 different mass doses were evaluated. PET scans and blood samples from at least two time points 24 h post injection were available. In 35 patients, brain, kidneys, liver, spleen, lungs, and bone marrow were delineated. Patlak analysis was used to account for differences in plasma activity concentration and to quantify irreversible uptake (Ki). To identify target engagement, Ki values were compared to ns-baseline Ki values previously reported, and the effect of increasing mass doses on Ki was investigated. Results: All mAbs, except ipilimumab, showed Ki values in spleen above the ns-baseline for the lowest administered mass dose, in addition to decreasing Ki values with higher mass doses, both indicative of target engagement. For bone marrow, no ns-baseline was established previously, but a similar pattern was observed. For kidneys, most mAbs showed Ki values within the ns-baseline for both low and high mass doses. However, with high mass doses, some saturation effects were seen, suggestive of a lower ns-baseline value. Ki values were near zero in brain tissue for all mass doses of all mAbs. Conclusion: Using Patlak analysis and the established ns-baseline values, evidence for target engagement in (lymphoid) organs for several immune checkpoint inhibitors could be demonstrated. A decrease in the Ki values with increasing mass doses supports the applicability of Patlak analysis for the assessment of target engagement for PET ligands with irreversible uptake behavior

89Zr-immuno-PET using the anti-LAG-3 tracer [89Zr]Zr-BI 754111: demonstrating target specific binding in NSCLC and HNSCC

Purpose: Although lymphocyte activation gene-3 (LAG-3) directed therapies demonstrate promising clinical anti-cancer activity, only a subset of patients seems to benefit and predictive biomarkers are lacking. Here, we explored the potential use of the anti-LAG-3 antibody tracer [89Zr]Zr-BI 754111 as a predictive imaging biomarker and investigated its target specific uptake as well as the correlation of its tumor uptake and the tumor immune infiltration. Methods: Patients with head and neck (N = 2) or lung cancer (N = 4) were included in an imaging substudy of a phase 1 trial with BI 754091 (anti-PD-1) and BI 754111 (anti-LAG-3). After baseline tumor biopsy and [18F]FDG-PET, patients were given 240 mg of BI 754091, followed 8 days later by administration of [89Zr]Zr-BI 754111 (37 MBq, 4 mg). PET scans were performed 2 h, 96 h, and 144 h post-injection. To investigate target specificity, a second tracer administration was given two weeks later, this time with pre-administration of 40 (N = 3) or 600 mg (N = 3) unlabeled BI 754111, followed by PET scans at 96 h and 144 h post-injection. Tumor immune cell infiltration was assessed by immunohistochemistry and RNA sequencing. Results: Tracer uptake in tumors was clearly visible at the 4-mg mass dose (tumor-to-plasma ratio 1.63 [IQR 0.37-2.89]) and could be saturated by increasing mass doses (44 mg: 0.67 [IQR 0.50–0.85]; 604 mg: 0.56 [IQR 0.42–0.75]), demonstrating target specificity. Tumor uptake correlated to immune cell-derived RNA signatures. Conclusions: [89Zr]Zr-BI-754111 PET imaging shows favorable technical and biological characteristics for developing a potential predictive imaging biomarker for LAG-3-directed therapies. Trial registration: ClinicalTrials.gov, NCT03780725. Registered 19 December 2018

89Zr-DFO-durvalumab PET/CT prior to durvalumab treatment in patients with recurrent or metastatic head and neck cancer

Background: In the PINCH study we performed 89Zr-DFO-durvalumab (anti-PD-L1) PET/CT in patients with recurrent or metastatic squamous cell carcinoma of the head and neck (SCCHN) prior to monotherapy durvalumab treatment. The primary aims were to assess safety and feasibility of 89Zr-DFO-durvalumab PET-imaging and predict disease control rate during durvalumab treatment. Secondary aims were to correlate 89Zr-DFO-durvalumab uptake to tumor PD-L1 expression, 18F-FDG uptake, and treatment response of individual lesions. Methods: In this prospective multicenter phase I-II study (NCT03829007), patients with incurable R/M SCCHN underwent baseline [18F]FDG PET and CT or MRI imaging. Subsequently, PD-L1 PET-imaging was performed 5 days after 37MBq [89Zr]Zr-DFO-durvalumab administration. To optimize imaging conditions, dose-finding was performed in the first 14 patients. For all patients, durvalumab treatment (1500mg/4 weeks, IV) was started <1 week after PD-L1 PET imaging and continued until disease progression or unacceptable toxicity (maximum 24 months). CT evaluation was assessed according to RECIST 1.1 every 8 weeks. PD-L1-expression was determined by combined positive score (CPS) on (archival) tumor-tissue. [89Zr]Zr-DFO-durvalumab uptake was measured in [18F]FDG-positive lesions, primary and secondary lymphoid organs, and bloodpool. Results: In total, 33 patients with locoregional recurrent (n = 12) or metastatic SCCHN (n = 21) were enrolled. [89Zr]Zr-DFO-durvalumab injection was safe. A dose of 10mg durvalumab resulted in highest tumor-to-blood-ratios. After a median follow-up of 12.6 months, overall response rate was 26%. The disease control rate at 16 weeks was 48% with a mean duration of 7.8 months (range 1.7-21.1). On a patient level, [89Zr]Zr-DFO-durvalumab-SUVpeak or tumor-to-blood ratio could not predict treatment response (HR 1.4 (95%CI 0.5-3.9, P = 0.54) and (HR 1.3 (95%CI 0.5-3.6, P = 0.61) respectively). Also, on a lesion level, [89Zr]Zr-DFO-durvalumab-SUVpeak showed no substantial correlation to treatment response (Spearman ρ= 0.45, P = 0.051). Lesional [89Zr]Zr-DFO-durvalumab-uptake did not correlate to PD-L1 CPS score, but did correlate to [18F]FDG SUV peak (Spearman ρ= 0.391, P = 0.005). Conclusion: PINCH is the first PD-L1 PET/CT study in patients with R/M SCCHN and has shown the feasibility and safety of [89Zr]Zr-DFO-durvalumab PET/CT in a multi-center trial. [89Zr]Zr-DFO-durvalumab-uptake did not correlate to durvalumab treatment response

Design, development and clinical translation of CriPec®-based core-crosslinked polymeric micelles

Nanomedicines are used to improve the efficacy and safety of pharmacotherapeutic interventions. Unraveling the biological behavior of nanomedicines, including their biodistribution and target site accumulation, is essential to establish design criteria that contribute to superior performance. CriPec® technology is based on amphiphilic methoxy-poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide lactate] (mPEG-b-pHPMAmLacn) block copolymers, which are designed to upon self-assembly covalently entrap active pharmaceutical ingredients (API) in core-crosslinked polymeric micelles (CCPM). Key features of CCPM are a prolonged circulation time, high concentrations at pathological sites, and low levels of accumulation in the majority of healthy tissues. Proprietary hydrolysable linkers allow for tunable and sustained release of entrapped API, including hydrophobic and hydrophilic small molecules, as well as peptides and oligonucleotides. Preclinical imaging experiments provided valuable information on their tumor and tissue accumulation and distribution, as well as on uptake by cancer, healthy and immune cells. The frontrunner formulation CPC634, which refers to 65 nm-sized CCPM entrapping the chemotherapeutic drug docetaxel, showed excellent pharmacokinetic properties, safety, tumor accumulation and antitumor efficacy in multiple animal models. In the clinic, CPC634 also demonstrated favorable pharmacokinetics, good tolerability, signs of efficacy, and enhanced localization in tumor tissue as compared to conventional docetaxel. PET imaging of radiolabeled CPC634 showed quantifiable accumulation in ∼50 % of tumors and metastases in advanced-stage cancer patients, and demonstrated potential for use in a theranostic setting even when applied at a companion diagnostic dose. Altogether, the preclinical and clinical results obtained to date demonstrate that mPEG-b-pHPMAmLacn CCPM based on CriPec® technology are a potent, tunable, broadly applicable and well-tolerable platform for targeted drug delivery and improved anticancer therapy

Circulating T cell status and molecular imaging may predict clinical benefit of neoadjuvant PD-1 blockade in oral cancer

Background Addition of neoadjuvant immune checkpoint inhibition to standard-of-care interventions for locally advanced oral cancer could improve clinical outcome.Methods In this study, 16 evaluable patients with stage III/IV oral cancer were treated with one dose of 480 mg nivolumab 3 weeks prior to surgery. Primary objectives were safety, feasibility, and suitability of programmed death receptor ligand-1 positron emission tomography (PD-L1 PET) as a biomarker for response. Imaging included 18F-BMS-986192 (PD-L1) PET and 18F-fluorodeoxyglucose (FDG) PET before and after nivolumab treatment. Secondary objectives included clinical and pathological response, and immune profiling of peripheral blood mononuclear cells (PBMCs) for response prediction. Baseline tumor biopsies and postnivolumab resection specimens were evaluated by histopathology.Results Grade III or higher adverse events were not observed and treatment was not delayed in relation to nivolumab administration and other study procedures. Six patients (38%) had a pathological response, of whom three (19%) had a major (≥90%) pathological response (MPR). Tumor PD-L1 PET uptake (quantified using standard uptake value) was not statistically different in patients with or without MPR (median 5.3 vs 3.4). All major responders showed a significantly postnivolumab decreased signal on FDG PET. PBMC immune phenotyping showed higher levels of CD8+ T cell activation in MPR patients, evidenced by higher baseline expression levels of PD-1, TIGIT, IFNγ and lower levels of PD-L1.Conclusion Together these data support that neoadjuvant treatment of advanced-stage oral cancers with nivolumab was safe and induced an MPR in a promising 19% of patients. Response was associated with decreased FDG PET uptake as well as activation status of peripheral T cell populations

Praluzatamab ravtansine, a CD166-targeting antibody-drug conjugate, in patients with advanced solid tumors: An Open-Label Phase I/II Trial

Purpose: Praluzatamab ravtansine (CX-2009) is a conditionally activated Probody drug conjugate (PDC) comprising an anti-CD166 mAb conjugated to DM4, with a protease-cleavable linker and a peptide mask that limits target engagement in normal tissue and circulation. The tumor microenvironment is enriched for proteases capable of cleaving the linker, thereby releasing the mask, allowing for localized binding of CX-2009 to CD166. CX-2009 was evaluated in a phase I/II clinical trial for patients with advanced solid tumors. Patients and Methods: Eligible patients had metastatic cancer receiving ≥2 prior treatments. CX-2009 was administered at escalating doses every 3 weeks (0.25–10 mg/kg) or every 2 weeks (4–6 mg/kg). Primary objective was to determine the safety profile and recommended phase II dose (RP2D). Results: Of 99 patients enrolled, the most prevalent subtype was breast cancer (n ¼ 45). Median number of prior therapies was 5 (range, 1–19). Dose-limiting toxicities were observed at 8 mg/kg every 3 weeks and 6 mg/kg every 2 weeks. On the basis of tolerability, the RP2D was 7 mg/kg every 3 weeks. Tumor regressions were observed at doses ≥4 mg/kg. In the hormone receptor–positive/HER2-nonamplified breast cancer subset (n ¼ 22), 2 patients (9%) had confirmed partial responses, and 10 patients (45%) had stable disease. Imaging with zirconium-labeled CX-2009 confirmed uptake in tumor lesions and shielding of major organs. Activated, unmasked CX-2009 was measurable in 18 of 22 posttreatment biopsies. Conclusions: CD166 is a novel, ubiquitously expressed target. CX-2009 is the first conditionally activated antibody–drug conjugatetoCD166todemonstratebothtranslationalandclinicalactivity in a variety of tumor types

Praluzatamab ravtansine, a CD166-targeting antibody-drug conjugate, in patients with advanced solid tumors: an open-label phase 1/2 trial.

PURPOSE: Praluzatamab ravtansine (CX-2009) is a conditionally activated Probody EXPERIMENTAL DESIGN: Eligible patients had metastatic cancer receiving {greater than or equal to}2 prior treatments. CX-2009 was administered at escalating doses every 3 weeks (0.25-10 mg/kg; Q3W) or every 2 weeks (4-6 mg/kg; Q2W). Primary objective was to determine the safety profile and recommended phase 2 dose (RP2D). RESULTS: Of 99 patients enrolled, the most prevalent subtype was breast cancer (BC) (n=45). Median number of prior therapies was 5 (range, 1-19). Dose-limiting toxicities were observed at 8 mg/kg Q3W and 6 mg/kg Q2W. Based on tolerability, the RP2D was 7 mg/kg Q3W. Tumor regressions were observed at doses {greater than or equal to}4 mg/kg. In the HR-positive/HER2-non-amplified BC subset (n=22), two patients (9%) had confirmed partial responses, and 10 patients (45%) had stable disease. Imaging with zirconium-labeled CX-2009 confirmed uptake in tumor lesions and shielding of major organs. Activated, unmasked CX-2009 was measurable in 18/22 post-treatment biopsies. CONCLUSION: CD166 is a novel, ubiquitously expressed target. CX-2009 is the first conditionally activated antibody-drug conjugate to CD166 to demonstrate both translational and clinical activity in a variety of tumor types