A vaccine targeting mutant IDH1 in newly diagnosed glioma.

Mutated isocitrate dehydrogenase 1 (IDH1) defines a molecularly distinct subtype of diffuse glioma1-3. The most common IDH1 mutation in gliomas affects codon 132 and encodes IDH1(R132H), which harbours a shared clonal neoepitope that is presented on major histocompatibility complex (MHC) class II4,5. An IDH1(R132H)-specific peptide vaccine (IDH1-vac) induces specific therapeutic T helper cell responses that are effective against IDH1(R132H)+ tumours in syngeneic MHC-humanized mice4,6-8. Here we describe a multicentre, single-arm, open-label, first-in-humans phase I trial that we carried out in 33 patients with newly diagnosed World Health Organization grade 3 and 4 IDH1(R132H)+ astrocytomas (Neurooncology Working Group of the German Cancer Society trial 16 (NOA16), ClinicalTrials.gov identifier NCT02454634). The trial met its primary safety endpoint, with vaccine-related adverse events restricted to grade 1. Vaccine-induced immune responses were observed in 93.3% of patients across multiple MHC alleles. Three-year progression-free and death-free rates were 0.63 and 0.84, respectively. Patients with immune responses showed a two-year progression-free rate of 0.82. Two patients without an immune response showed tumour progression within two years of first diagnosis. A mutation-specificity score that incorporates the duration and level of vaccine-induced IDH1(R132H)-specific T cell responses was associated with intratumoral presentation of the IDH1(R132H) neoantigen in pre-treatment tumour tissue. There was a high frequency of pseudoprogression, which indicates intratumoral inflammatory reactions. Pseudoprogression was associated with increased vaccine-induced peripheral T cell responses. Combined single-cell RNA and T cell receptor sequencing showed that tumour-infiltrating CD40LG+ and CXCL13+ T helper cell clusters in a patient with pseudoprogression were dominated by a single IDH1(R132H)-reactive T cell receptor.

Identification of Miro1 and Miro2 as mitochondrial receptors for myosin XIX.

Mitochondrial distribution in cells is critical for cellular function and proper inheritance during cell division. In mammalian cells, mitochondria are transported predominantly along microtubules by kinesin and dynein motors that bind indirectly via TRAK1 and TRAK2 to outer mitochondrial membrane proteins Miro1 and Miro2 (Miro1/2). Here, using proximity labelling, we identified Miro1/2 as potential binding partners of myosin XIX (Myo19). Interaction studies show that Miro1 binds directly to a C-terminal fragment of the Myo19 tail region and that Miro1/2 recruit the Myo19 tail in vivo This recruitment is regulated by the nucleotide state of the N-terminal Rho-like GTPase domain of Miro1/2. Notably, Myo19 protein stability in cells depends on its association with Miro1/2. Downregulation of Miro1/2 or overexpression of the adaptor proteins TRAK1 and TRAK2 caused a reduction in Myo19 protein levels. Myo19 regulates the subcellular distribution of mitochondria, and downregulation, as well as overexpression, of Myo19 induced perinuclear collapse of mitochondria, phenocopying loss of the kinesin KIF5, dynein or their mitochondrial receptors Miro1/2. These results suggest that Miro1 and Miro2 coordinate microtubule- and actin-based mitochondrial movement.This article has an associated First Person interview with the first author of the paper.

INFORM2 NivEnt: The first trial of the INFORM2 biomarker driven phase I/II trial series: the combination of nivolumab and entinostat in children and adolescents with refractory high-risk malignancies.

BACKGROUND: Pediatric patients with relapsed or refractory disease represent a population with a desperate medical need. The aim of the INFORM (INdividualized Therapy FOr Relapsed Malignancies in Childhood) program is to translate next generation molecular diagnostics into a biomarker driven treatment strategy. The program consists of two major foundations: the INFORM registry providing a molecular screening platform and the INFORM2 series of biomarker driven phase I/II trials. The INFORM2 NivEnt trial aims to determine the recommended phase 2 dose (RP2D) of the combination treatment of nivolumab and entinostat (phase I) and to evaluate activity and safety (phase II). METHODS: This is an exploratory non-randomized, open-label, multinational and multicenter seamless phase I/II trial in children and adolescents with relapsed / refractory or progressive high-risk solid tumors and CNS tumors. The phase I is divided in 2 age cohorts: 12-21 years and 6-11 years and follows a 3 + 3 design with two dose levels for entinostat (2 mg/m2 and 4 mg/m2 once per week) and fixed dose nivolumab (3 mg/kg every 2 weeks). Patients entering the trial on RP2D can seamlessly enter phase II which consists of a biomarker defined four group basket trial: high mutational load (group A), high PD-L1 mRNA expression (group B), focal MYC(N) amplification (group C), low mutational load and low PD-L1 mRNA expression and no MYC(N) amplification (group D). A Bayesian adaptive design will be used to early stop cohorts that fail to show evidence of activity. The maximum number of patients is 128. DISCUSSION: This trial intends to exploit the immune enhancing effects of entinostat on nivolumab using an innovative biomarker driven approach in order to maximize the chance of detecting signs of activity. It prevents exposure to unnecessary risks by applying the Bayesian adaptive design for early stopping for futility. The adaptive biomarker driven design provides an innovative approach accelerating drug development and reducing exposure to investigational treatments in these vulnerable children at the same time. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03838042. Registered on 12 February 2019.

Early metabolic response in sequential FDG-PET/CT under cetuximab is a predictive marker for clinical response in first-line metastatic colorectal cancer patients: results of the phase II REMOTUX trial.

BACKGROUND: To assess the predictive value of early metabolic response (ΔSUV) after short-term treatment with first-line cetuximab in patients (pts) with RAS-wt metastatic colorectal cancer (mCRC). METHODS: In this prospective phase II study, RAS-wt mCRC pts received a single-agent cetuximab run-in therapy of 2 weeks. ΔSUV was assessed with FDG-PET/CT on days 0 and 14. Early clinical response (ECR) was evaluated with CT on day 56 after treatment with FOLFIRI-cetuximab. Primary endpoint was the predictive significance of ΔSUV for ECR. Secondary endpoints were PFS (progression free survival), OS and the influence of ΔSUV on survival. RESULTS: Forty pts were enroled and 33 pts were evaluable for the primary endpoint. The CT response rate was 57.6%. For responders, ΔSUV was significantly higher (p = 0.0092). A significant association of ΔSUV with ECR was found (p = 0.02). Median PFS was 11.7 months and median OS was 33.5 months with a 1-year survival rate of 87.9%. ΔSUV was found to significantly impact the hazard for OS (p = 0.045). CONCLUSIONS: We demonstrate that cetuximab induces metabolic responses in mCRC pts. The study endpoint was met with the ΔSUV discriminating between responders and non-responders. However, these data should be validated in larger patient cohorts.

Investigation of the immunogenicity of different types of aggregates of a murine monoclonal antibody in mice.

PURPOSE: The potential contribution of protein aggregates to the unwanted immunogenicity of protein pharmaceuticals is a major concern. In the present study a murine monoclonal antibody was utilized to study the immunogenicity of different types of aggregates in mice. Samples containing defined types of aggregates were prepared by processes such as stirring, agitation, exposure to ultraviolet (UV) light and exposure to elevated temperatures. METHODS: Aggregates were analyzed by size-exclusion chromatography, light obscuration, turbidimetry, infrared (IR) spectroscopy and UV spectroscopy. Samples were separated into fractions based on aggregate size by asymmetrical flow field-flow fractionation or by centrifugation. Samples containing different types and sizes of aggregates were subsequently administered to C57BL/6 J and BALB/c mice, and serum was analyzed for the presence of anti-IgG1, anti-IgG2a, anti-IgG2b and anti-IgG3 antibodies. In addition, the pharmacokinetic profile of the murine antibody was investigated. RESULTS: In this study, samples containing high numbers of different types of aggregates were administered in order to challenge the in vivo system. The magnitude of immune response depends on the nature of the aggregates. The most immunogenic aggregates were of relatively large and insoluble nature, with perturbed, non-native structures. CONCLUSION: This study shows that not all protein drug aggregates are equally immunogenic.

Comparison of ice fog methods and monitoring of controlled nucleation success after freeze-drying.

Improving freeze-drying processes regarding drying time and batch homogeneity is subject of ongoing research work. In this context, controlled nucleation raised great expectations. However, practically we face some challenges, e.g. how to non-destructively monitor successfully performed controlled nucleation. The question if different controlled nucleation methods lead to comparable products, as not every method can easily be implemented in lab and production scale equipment, is also of high interest. Additionally, the optimal nucleation temperature for controlled nucleation is an open question. In our study, we addressed these challenges. We successfully evaluated frequency modulated spectroscopy as a fast and non-destructive method to monitor controlled nucleation success and batch homogeneity. We found that the better homogeneity generated by controlled nucleation during the freezing step did not sustain in the dried product. Lyophilizates produced by three different ice fog methods for controlled nucleation were characterized by comparable specific surface areas but differed in residual moisture content. To investigate the impact of the ice nucleation temperature (TN) on the resulting specific surface area, we performed controlled nucleation at -3 °C and -10 °C. We concluded that TN is not the only specific surface area determining factor and a high TN does not necessarily lead to larger pores but poses a higher risk of not-nucleating vials.

Phase I/II intra-patient dose escalation study of vorinostat in children with relapsed solid tumor, lymphoma, or leukemia.

BACKGROUND: Until today, adult and pediatric clinical trials investigating single-agent or combinatorial HDAC inhibitors including vorinostat in solid tumors have largely failed to demonstrate efficacy. These results may in part be explained by data from preclinical models showing significant activity only at higher concentrations compared to those achieved with current dosing regimens. In the current pediatric trial, we applied an intra-patient dose escalation design. The purpose of this trial was to determine a safe dose recommendation (SDR) of single-agent vorinostat for intra-patient dose escalation, pharmacokinetic analyses (PK), and activity evaluation in children (3-18 years) with relapsed or therapy-refractory malignancies. RESULTS: A phase I intra-patient dose (de)escalation was performed until individual maximum tolerated dose (MTD). The starting dose was 180 mg/m2/day with weekly dose escalations of 50 mg/m2 until DLT/maximum dose. After MTD determination, patients seamlessly continued in phase II with disease assessments every 3 months. PK and plasma cytokine profiles were determined. Fifty of 52 patients received treatment. n = 27/50 (54%) completed the intra-patient (de)escalation and entered phase II. An SDR of 130 mg/m2/day was determined (maximum, 580 mg/m2/day). n = 46/50 (92%) patients experienced treatment-related AEs which were mostly reversible and included thrombocytopenia, fatigue, nausea, diarrhea, anemia, and vomiting. n = 6/50 (12%) had treatment-related SAEs. No treatment-related deaths occurred. Higher dose levels resulted in higher Cmax. Five patients achieved prolonged disease control (> 12 months) and showed a higher Cmax (> 270 ng/mL) and MTDs. Best overall response (combining PR and SD, no CR observed) rate in phase II was 6/27 (22%) with a median PFS and OS of 5.3 and 22.4 months. Low levels of baseline cytokine expression were significantly correlated with favorable outcome. CONCLUSION: An SDR of 130 mg/m2/day for individual dose escalation was determined. Higher drug exposure was associated with responses and long-term disease stabilization with manageable toxicity. Patients with low expression of plasma cytokine levels at baseline were able to tolerate higher doses of vorinostat and benefited from treatment. Baseline cytokine profile is a promising potential predictive biomarker. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01422499. Registered 24 August 2011.

Does controlled nucleation impact the properties and stability of lyophilized monoclonal antibody formulations?

This study provides the first systematic investigation of the impact of the nucleation protocol during freeze-drying on physico-chemical properties and long-term stability of two IgG1 antibodies in sugar formulations. We hypothesized that the lower specific surface area (SSA) generated by controlled nucleation could be beneficial for the stability of interface sensitive proteins. The study compares controlled nucleated (CN) and randomly nucleated (RN) lyophilizates with high and low antibody concentrations stored at different temperatures. Formulations with and without polysorbate (PS) were included. In the "high concentration" study the formulation without PS showed reduced particle formation for CN samples compared to RN samples. PS containing formulations had an overall lower particle level with no further advantage of CN on stability. Besides the intended comparison of CN and RN samples, we observed that PS promoted sucrose crystallization in both low concentration antibody studies during storage. Additionally, our results indicate that the nucleation temperature (TN) was not the only determining factor for the resulting ice crystal size and consequently the product`s SSA. Overall, the application of CN had neither a positive nor a negative impact on the product's physico-chemical stability. The surfactant had a much higher stabilizing effect than the reduction of the SSA by CN.

Evaluation of Heat Flux Measurement as a New Process Analytical Technology Monitoring Tool in Freeze Drying.

This study investigates the suitability of heat flux measurement as a new technique for monitoring product temperature and critical end points during freeze drying. The heat flux sensor is tightly mounted on the shelf and measures non-invasively (no contact with the product) the heat transferred from shelf to vial. Heat flux data were compared to comparative pressure measurement, thermocouple readings, and Karl Fischer titration as current state of the art monitoring techniques. The whole freeze drying process including freezing (both by ramp freezing and controlled nucleation) and primary and secondary drying was considered. We found that direct measurement of the transferred heat enables more insights into thermodynamics of the freezing process. Furthermore, a vial heat transfer coefficient can be calculated from heat flux data, which ultimately provides a non-invasive method to monitor product temperature throughout primary drying. The end point of primary drying determined by heat flux measurements was in accordance with the one defined by thermocouples. During secondary drying, heat flux measurements could not indicate the progress of drying as monitoring the residual moisture content. In conclusion, heat flux measurements are a promising new non-invasive tool for lyophilization process monitoring and development using energy transfer as a control parameter.

Next-generation personalised medicine for high-risk paediatric cancer patients - The INFORM pilot study.

The 'Individualized Therapy for Relapsed Malignancies in Childhood' (INFORM) precision medicine study is a nationwide German program for children with high-risk relapsed/refractory malignancies, which aims to identify therapeutic targets on an individualised basis. In a pilot phase, reported here, we developed the logistical and analytical pipelines necessary for rapid and comprehensive molecular profiling in a clinical setting. Fifty-seven patients from 20 centers were prospectively recruited. Malignancies investigated included sarcomas (n = 25), brain tumours (n = 23), and others (n = 9). Whole-exome, low-coverage whole-genome, and RNA sequencing were complemented with methylation and expression microarray analyses. Alterations were assessed for potential targetability according to a customised prioritisation algorithm and subsequently discussed in an interdisciplinary molecular tumour board. Next-generation sequencing data were generated for 52 patients, with the full analysis possible in 46 of 52. Turnaround time from sample receipt until first report averaged 28 d. Twenty-six patients (50%) harbored a potentially druggable alteration with a prioritisation score of 'intermediate' or higher (level 4 of 7). Common targets included receptor tyrosine kinases, phosphoinositide 3-kinase-mammalian target of rapamycin pathway, mitogen-activated protein kinase pathway, and cell cycle control. Ten patients received a targeted therapy based on these findings, with responses observed in some previously treatment-refractory tumours. Comparative primary relapse analysis revealed substantial tumour evolution as well as one case of unsuspected secondary malignancy, highlighting the importance of re-biopsy at relapse. This study demonstrates the feasibility of comprehensive, real-time molecular profiling for high-risk paediatric cancer patients. This extended proof-of-concept, with examples of treatment consequences, expands upon previous personalised oncology endeavors, and presents a model with considerable interest and practical relevance in the burgeoning era of personalised medicine.

Dose levels in particulate-containing formulations impact anti-drug antibody responses to murine monoclonal antibody in mice.

Dosage levels and particulate contents of therapeutic protein formulations are potential factors that impact immunogenicity of protein therapeutics. Here, we evaluated the effect of dose levels on the immunogenicity of protein particulates formed by adsorbing a murine monoclonal IgG2c/κ antibody (mAb1) onto silicone oil microdroplets, glass, or aluminum hydroxide (Alhydrogel) microparticles. Immune responses to these particulate-containing preparations were compared against responses to solutions of mAb1 that had been ultracentrifuged to minimize particle levels. Formulations containing 5 or 500 µg of adsorbed mAb1 were administered subcutaneously to C57BL/6J or BALB/c mice. Antidrug antibodies (ADAs) were detected using an isotype-specific enzyme-linked immunosorbent assay (ELISA) method or a chemiluminescence method. Sera from BALB/c mice showed greater ADA responses to administration of particles at the 5-µg dose level than at the 500-µg dose level. In sera from C57BL/6J mice, ADA levels detected by ELISA were independent of the particle dose levels tested. ADAs were not detected in sera from C57BL/6J mice performing the chemiluminescence technique. In conclusion, mice administered formulations of a murine antibody adsorbed onto silicone oil microdroplets, glass microparticles, or Alhydrogel(®) showed greater ADA responses that those that received particle-free mAb1 preparations, and responses were greater for formulations containing lower doses of antibody. .

Antibody responses in mice to particles formed from adsorption of a murine monoclonal antibody onto glass microparticles.

Immunogenicity of therapeutic monoclonal antibodies (mAbs) is a concern because of the effects of anti-drug antibodies (ADAs) on therapeutic efficacy. Particulate matter has been suggested as a potential contributing factor to immunogenicity. In this study, we investigated ADA levels in mice in response to administration of a murine immunoglobulin G (IgG)2c/κ mAb (mAb1) that was generated in C57BL/6J mice. Particles of mAb1 were formed by adsorbing the protein to glass microparticles. Formulations containing microparticles were administered subcutaneously to mice of either the syngeneic strain, C57Bl/6J, or the allogeneic strain, BALB/c. ADA levels were measured using an isotype-specific enzyme-linked immunosorbent assay method. Whereas BALB/c mice showed strong IgG1 and IgG2b responses against both the particulate and native mAb1 samples, adsorption of mAb1 to particles rendered it slightly more immunogenic than its native, soluble form. In BALB/c mice, immunoglobulin M (IgM) was produced after the first week of injections and then faded gradually. In contrast, C57BL/6J mice showed moderate IgM, IgG1, IgG2b, and IgG3 responses to injections of glass particle-adsorbed mAb1. ADA responses were higher in the allogeneic BALB/c mice, which do not produce mAbs of the IgG2c/κ isotype. Thus, the presence of both foreign epitopes and particles may be important in inducing ADA responses.