A first-in-human phase 1 study of cavrotolimod, a TLR9 agonist spherical nucleic acid, in healthy participants: Evidence of immune activation.

Introduction: Tumor immunotherapy is designed to control malignancies through the host immune response but requires circumventing tumor-dysregulated immunomodulation through immunostimulation, relieving immunorepression, or a combination of both approaches. Here we designed and characterized cavrotolimod (formerly AST-008), an immunostimulatory spherical nucleic acid (SNA) compound targeting Toll-like receptor 9 (TLR9). We assessed the safety and pharmacodynamic (PD) properties of cavrotolimod in healthy participants in a first-in-human Phase 1 study under protocol AST-008-101 (NCT03086278; https://clinicaltrials.gov/ct2/show/NCT03086278). Methods: Healthy participants aged 18 to 40 years were enrolled to evaluate four dose levels of cavrotolimod across four cohorts. Each cohort included four participants, and all received a single subcutaneous dose of cavrotolimod. The dose levels were 5, 10, 12.5 and 18.8 µg/kg. Results and discussion: Cavrotolimod was well tolerated and elicited no serious adverse events or dose limiting toxicities at the doses tested. The results demonstrated that cavrotolimod is a potent innate immune activator, specifically stimulating Th1-type immune responses, and exhibits PD properties that may result in anti-tumor effects in patients with cancer. This study suggests that cavrotolimod is a promising clinical immunotherapy agent.

IMMUNO-COV v2.0: Development and Validation of a High-Throughput Clinical Assay for Measuring SARS-CoV-2-Neutralizing Antibody Titers.

Neutralizing antibodies are key determinants of protection from future infection, yet well-validated high-throughput assays for measuring titers of SARS-CoV-2-neutralizing antibodies are not generally available. Here, we describe the development and validation of IMMUNO-COV v2.0, a scalable surrogate virus assay, which titrates antibodies that block infection of Vero-ACE2 cells by a luciferase-encoding vesicular stomatitis virus displaying SARS-CoV-2 spike glycoproteins (VSV-SARS2-Fluc). Antibody titers, calculated using a standard curve consisting of stepped concentrations of SARS-CoV-2 spike monoclonal antibody, correlated closely (P < 0.0001) with titers obtained from a gold standard 50% plaque-reduction neutralization test (PRNT50%) performed using a clinical isolate of SARS-CoV-2. IMMUNO-COV v2.0 was comprehensively validated using data acquired from 242 assay runs performed over 7 days by five analysts, utilizing two separate virus lots, and 176 blood samples. Assay performance was acceptable for clinical use in human serum and plasma based on parameters including linearity, dynamic range, limit of blank and limit of detection, dilutional linearity and parallelism, precision, clinical agreement, matrix equivalence, clinical specificity and sensitivity, and robustness. Sufficient VSV-SARS2-Fluc virus reagent has been banked to test 5 million clinical samples. Notably, a significant drop in IMMUNO-COV v2.0 neutralizing antibody titers was observed over a 6-month period in people recovered from SARS-CoV-2 infection. Together, our results demonstrate the feasibility and utility of IMMUNO-COV v2.0 for measuring SARS-CoV-2-neutralizing antibodies in vaccinated individuals and those recovering from natural infections. Such monitoring can be used to better understand what levels of neutralizing antibodies are required for protection from SARS-CoV-2 and what booster dosing schedules are needed to sustain vaccine-induced immunity. IMPORTANCE Since its emergence at the end of 2019, SARS-CoV-2, the causative agent of COVID-19, has caused over 100 million infections and 2.4 million deaths worldwide. Recently, countries have begun administering approved COVID-19 vaccines, which elicit strong immune responses and prevent disease in most vaccinated individuals. A key component of the protective immune response is the production of neutralizing antibodies capable of preventing future SARS-CoV-2 infection. Yet, fundamental questions remain regarding the longevity of neutralizing antibody responses following infection or vaccination and the level of neutralizing antibodies required to confer protection. Our work is significant as it describes the development and validation of a scalable clinical assay that measures SARS-CoV-2-neutraling antibody titers. We have critical virus reagent to test over 5 million samples, making our assay well suited for widespread monitoring of SARS-CoV-2-neutralizing antibodies, which can in turn be used to inform vaccine dosing schedules and answer fundamental questions regarding SARS-CoV-2 immunity.

Development and validation of IMMUNO-COV™: a high-throughput clinical assay for detecting antibodies that neutralize SARS-CoV-2.

We here describe the development and validation of IMMUNO-COV™, a high-throughput clinical test to quantitatively measure SARS-CoV-2-neutralizing antibodies, the specific subset of anti-SARS-CoV-2 antibodies that block viral infection. The test measures the capacity of serum or purified antibodies to neutralize a recombinant Vesicular Stomatitis Virus (VSV) encoding the SARS-CoV-2 spike glycoprotein. This recombinant virus (VSV-SARS-CoV-2-S-Δ19CT) induces fusion in Vero cell monolayers, which is detected as luciferase signal using a dual split protein (DSP) reporter system. VSV-SARS-CoV-2-S-Δ19CT infection was blocked by monoclonal α-SARS-CoV-2-spike antibodies and by plasma or serum from SARS-CoV-2 convalescing individuals. The assay exhibited 100% specificity in validation tests, and across all tests zero false positives were detected. In blinded analyses of 230 serum samples, only two unexpected results were observed based on available clinical data. We observed a perfect correlation between results from our assay and 80 samples that were also assayed using a commercially available ELISA. To quantify the magnitude of the anti-viral response, we generated a calibration curve by adding stepped concentrations of α-SARS-CoV-2-spike monoclonal antibody to pooled SARS-CoV-2 seronegative serum. Using the calibration curve and a single optimal 1:100 serum test dilution, we reliably measured neutralizing antibody levels in each test sample. Virus neutralization units (VNUs) calculated from the assay correlated closely (p < 0.0001) with PRNT EC50 values determined by plaque reduction neutralization test against a clinical isolate of SARS-CoV-2. Taken together, these results demonstrate that the IMMUNO-COV™ assay accurately quantitates SARS-CoV-2 neutralizing antibodies in human sera and therefore is a potentially valuable addition to the currently available serological tests. The assay can provide vital information for comparing immune responses to the various SARS-CoV-2 vaccines that are currently in development, or for evaluating donor eligibility in convalescent plasma therapy studies.

Onapristone Extended Release: Safety Evaluation from Phase I-II Studies with an Emphasis on Hepatotoxicity.

INTRODUCTION: Antiprogestins have demonstrated promising activity against breast and gynecological cancers, but liver-related safety concerns limited the advancement of this therapeutic class. Onapristone is a full progesterone receptor antagonist originally developed as an oral contraceptive and later evaluated in phase II studies for metastatic breast cancer. Because of liver enzyme elevations identified during clinical studies, further development was halted. Evaluation of antiprogestin pharmacology and pharmacokinetic data suggested that liver enzyme elevations might be related to off-target or metabolic effects associated with clinical drug exposure. OBJECTIVE: We explored whether the use of a pharmaceutic strategy targeting efficacious systemic dose concentrations, but with diminished peak serum concentrations and/or total drug exposure would mitigate hepatotoxicity. Twice-daily dosing of an extended-release formulation of onapristone was developed and clinically evaluated in light of renewed interest in antiprogestin therapy for treating progesterone receptor-positive breast and gynecologic cancers. The hepatotoxic potential of extended-release onapristone was assessed from two phase I-II studies involving patients with breast, ovarian, endometrial, and prostate cancer. RESULTS: Among the 88 patients in two phase I-II studies in progesterone receptor-positive malignancies treated with extended-release onapristone, elevated alanine aminotransferase/aspartate aminotransferase levels were found in 20% of patients with liver metastases compared with 6.3% without metastases. Of five patients with grade 3 or higher alanine aminotransferase elevations with or without bilirubin elevations (four with breast cancer and one with endometrial cancer), four were assessed as unrelated to extended-release onapristone by the safety data review committee. Furthermore, while the fifth patient's liver enzyme elevations were considered possibly drug related by the study investigator, they were adjudicated as unlikely to be related (< 25% likelihood) by a subsequent independent hepatologist. CONCLUSIONS: These results suggest that the extended-release formulation by reducing drug exposure may be associated with a reduced risk of hepatotoxicity, and supports the continued clinical evaluation of extended-release onapristone for treating progesterone receptor-positive cancers.

Oncolytic Measles Virotherapy and Opposition to Measles Vaccination.

Recent measles epidemics in US and European cities where vaccination coverage has declined are providing a harsh reminder for the need to maintain protective levels of immunity across the entire population. Vaccine uptake rates have been declining in large part because of public misinformation regarding a possible association between measles vaccination and autism for which there is no scientific basis. The purpose of this article is to address a new misinformed antivaccination argument-that measles immunity is undesirable because measles virus is protective against cancer. Having worked for many years to develop engineered measles viruses as anticancer therapies, we have concluded (1) that measles is not protective against cancer and (2) that its potential utility as a cancer therapy will be enhanced, not diminished, by prior vaccination.

Phase I study of onapristone, a type I antiprogestin, in female patients with previously treated recurrent or metastatic progesterone receptor-expressing cancers.

INTRODUCTION: Onapristone is a type I progesterone receptor (PR) antagonist, which prevents PR- mediated DNA transcription. Onapristone is active in multiple preclinical models and two prior studies demonstrated promising activity in patients with breast cancer. We conducted a study of extended release (ER) Onapristone to determine a recommended dose and explore the role of transcriptionally-activated PR (APR), detected as an aggregated subnuclear distribution pattern, as a predictive biomarker. METHODS: An open-label, multicenter, randomized, parallel-group, phase 1 study (target n = 60; NCT02052128) included female patients ≥18 years with PRpos tumors. APR analysis was performed on archival tumor tissue. Patients were randomized to five cohorts of extended release (ER) onapristone tablets 10, 20, 30, 40 or 50 mg BID, or immediate release 100 mg QD until progressive disease or intolerability. Primary endpoint was to identify the recommended phase 2 dose. Secondary endpoints included safety, clinical benefit and pharmacokinetics. RESULTS: The phase 1 dose escalation component of the study is complete (n = 52). Tumor diagnosis included: endometrial carcinoma 12; breast cancer 20; ovarian cancer 13; other 7. Median age was 64 (36-84). No dose limiting toxicity was observed with reported liver function test elevation related only to liver metastases. The RP2D was 50 mg ER BID. Median therapy duration was 8 weeks (range 2-44), and 9 patients had clinical benefit ≥24 weeks, including 2 patients with APRpos endometrial carcinoma. CONCLUSION: Clinical benefit with excellent tolerance was seen in heavily pretreated patients with endometrial, ovarian and breast cancer. The data support the development of Onapristone in endometrial endometrioid cancer. Onapristone should also be evaluated in ovarian and breast cancers along with APR immunohistochemistry validation.

A single-dose PK study of onapristone including the effect of food on absorption.

PURPOSE: Onapristone is an antiprogestin with activity in breast cancer and is under investigation for use in endometrial, ovarian and prostate cancers. Megestrol acetate and abiraterone generally show variability in absorption and, depending on the formulation, food effect. This study was conducted to determine the effect of food on 10 mg oral immediate-release (IR) onapristone and to help identify a formulation to minimize variability. METHODS: This is an open-label, randomized, crossover study to determine the pharmacokinetic profile of onapristone and its main metabolite, N-mono-desmethyl onapristone. Twelve healthy female subjects received 10 mg of oral IR onapristone after an overnight fast, or within 30 min of a high-fat, high-calorie meal with a 2-week washout between dosing periods. RESULTS: Onapristone plasma t1/2 (mean ± SD) was 4.36 ± 0.81 h for the fasted state and 3.76 ± 0.36 h for the fed state. Following food, onapristone tmax was delayed from 1 to 4 h. Food intake was also associated with a small increase in AUC0-∞ of approximately 13 % and a statistically significant decrease in Cmax of approximately 18 %. One subject experienced a 23-day delay in menses after one 10 mg onapristone dose, while another subject experienced transient grade 2 NCI-CTCAE liver enzyme elevation 3 weeks post dose. CONCLUSION: The results are consistent with previous observations, indicating that there is a small increase in onapristone exposure and a significant decrease in Cmax when taken with food. These changes are within acceptable limits set out by the FDA. Thus, our findings indicate that onapristone could be administered without regard to food.

Antitumor activity and safety of combination therapy with the Toll-like receptor 9 agonist IMO-2055, erlotinib, and bevacizumab in advanced or metastatic non-small cell lung cancer patients who have progressed following chemotherapy.

BACKGROUND: IMO-2055 is a Toll-like receptor 9 (TLR9) agonist that potentially enhances the efficacy of antitumor agents through immune stimulation. The objective of this phase Ib dose-escalation trial (3 + 3 design) was to determine the recommended phase II dose (RP2D) of IMO-2055 when combined with erlotinib and bevacizumab in patients with advanced non-small cell lung cancer (NSCLC). METHODS: Patients with stage 3/4 NSCLC and progressive disease (PD) following chemotherapy received IMO-2055 0.08, 0.16, 0.32, or 0.48 mg/kg once weekly plus erlotinib 150 mg daily and bevacizumab 15 mg/kg every 3 weeks. Patients could receive treatment until PD or unacceptable toxicity. RESULTS: Thirty-six patients were enrolled; 35 received at least one treatment dose. Two dose-limiting toxicities were observed across the dose range (Grade 3 dehydration and fatigue) with neither suggestive of a consistent toxicity pattern. IMO-2055 0.32 mg/kg was adopted as RP2D based on clinical and pharmacodynamic data. The most common treatment-emergent adverse events (TEAEs) were diarrhea (74 %), nausea (51 %), fatigue (51 %), rash (51 %), and injection-site reactions (49 %). Four patients experienced serious TEAEs considered to be study drug related. Five patients died, all due to PD. High-grade neutropenia and electrolyte disturbances previously reported with TLR9 agonists combined with platinum-based therapy were not observed in this study. Five of 33 patients evaluable for response (15 %) achieved partial response; another 20 (61 %) had stable disease, including 13 with stable disease ≥4 months. CONCLUSIONS: IMO-2055 demonstrated good tolerability and possible antitumor activity in combination with erlotinib and bevacizumab in heavily pretreated patients with advanced NSCLC.

Impact of liver transplantation on the survival of patients treated for hepatorenal syndrome type 1.

The development of hepatorenal syndrome type 1 (HRS1) is associated with a poor prognosis. Liver transplantation improves this prognosis, but the degree of the improvement is unclear. Most patients receive vasoconstrictors such as terlipressin before transplantation, and this may affect the posttransplant outcomes. We examined a cohort of patients with access to liver transplantation from our previously published study of terlipressin plus albumin versus albumin alone in the treatment of HRS1. The purpose of this analysis was the quantification of the survival benefits of liver transplantation for patients with HRS1. Ninety-nine patients were randomized to terlipressin or placebo. Thirty-five patients (35%) received a liver transplant. Among those receiving terlipressin plus albumin, the 180-day survival rates were 100% for transplant patients and 34% for nontransplant patients; among those receiving only albumin, the rates were 94% for transplant patients and 17% for nontransplant patients. The survival rate was significantly better for those achieving a reversal of hepatorenal syndrome (HRS) versus those not achieving a reversal (47% versus 4%, P < 0.001), but it was significantly lower for the responders versus those undergoing liver transplantation (97%). We conclude that the use of terlipressin plus albumin has no significant impact on posttransplant survival. Liver transplantation offers a clear survival benefit to HRS1 patients regardless of the therapy that they receive or the success or failure of HRS reversal. The most likely benefit of terlipressin in patients undergoing liver transplantation for HRS1 is improved pretransplant renal function, and this should make the posttransplant management of this difficult group of patients easier. For patients not undergoing transplantation, HRS reversal with terlipressin and/or albumin improves survival.

Predictors of response to terlipressin plus albumin in hepatorenal syndrome (HRS) type 1: relationship of serum creatinine to hemodynamics.

BACKGROUND & AIMS: Administration of terlipressin plus albumin is effective in reversing type 1 HRS as compared to albumin alone. However, only about 1/3 of patients respond to treatment, therefore, predictors of response and survival would help identify the patients most likely to benefit from treatment. METHODS: We analyzed our controlled trial of terlipressin vs. placebo (Gastroenterology 2008;134:1360) to define factors predictive of a response and to correlate hemodynamic changes to changes in renal function. RESULTS: Single variant analysis showed treatment with terlipressin, MELD score, and baseline serum creatinine to be predictive of HRS reversal. Alcoholic hepatitis, baseline serum creatinine, and MELD score were predictive of survival. When treatment was not considered as a variable, only baseline serum creatinine predicted HRS reversal. Baseline serum creatinine, presence of alcoholic hepatitis, and Child-Pugh score were also predictive of survival on multivariate analysis. The rise in mean arterial pressure (MAP) following terlipressin administration was not predictive of HRS reversal. However, in those who achieved HRS reversal from terlipressin, there was a significant rise in MAP from beginning to end of treatment. CONCLUSIONS: The most consistent predictor of response to terlipressin and of survival is the baseline serum creatinine. Patients most likely to benefit from terlipressin have earlier onset renal failure (i.e. serum creatinine <5.0mg/dl). A sustained rise in MAP is required for HRS reversal. As MAP is a surrogate marker for the hyperdynamic circulation, it is only with improvement in the hyperdynamic circulation that HRS reversal is observed.

Capecitabine plus 3-weekly irinotecan (XELIRI regimen) as first-line chemotherapy for metastatic colorectal cancer: phase II trial results.

BACKGROUND: Capecitabine results in superior response rate, improved safety, and improved convenience compared with 5-fluorouracil (FU)/leucovorin (LV) in metastatic colorectal cancer (MCRC). Irinotecan in combination with 5-FU/LV has been shown to improve efficacy compared with 5-FU/LV alone in MCRC. Therefore, we evaluated the efficacy and safety of capecitabine plus irinotecan every 3 weeks (XELIRI regimen) as first-line treatment. METHODS: Patients with MCRC who were <65 years of age received irinotecan 250 mg/m i.v. on day 1 + capecitabine 1000 mg/m orally twice daily on days 1 to 14, every 3 weeks. Patients >or=65 years of age and those with impaired renal function or with a history of prior radiotherapy received lower doses of both agents (200 mg/m and 750 mg/m twice daily, respectively). RESULTS: A total of 52 patients (29 men, 23 women) were enrolled between October 2001 and August 2003. Median age was 57.5 years (range, 30-79 years); median Karnofsky performance status was 90 (range, 70-100). Treatment led to a response rate of 50% (ITT population) and a disease control rate of 71%. With a median cohort follow-up of 30.5 months, median time to progression and overall survival are 7.8 months (95% confidence interval, 5.6-10.0) and 16.8 months (95% confidence, 11.9 to not reached), respectively. Most common treatment-related grade 3/4 adverse events were neutropenia (25%), diarrhea (20%), vomiting (16%), dehydration (10%), nausea (6%), abdominal pain (6%), and hand-foot syndrome (6%). CONCLUSION: XELIRI is an active first-line treatment of MCRC. Implementation of upfront dose reductions for both agents in patients with risk factors for toxicity appears to have produced a safer regimen compared with previous studies of XELIRI without such dose reductions.