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Applying Pattern Recognition as a Robust Approach for Silicone Oil Droplet Identification in Flow-Microscopy Images of Protein Formulations
No full textDiscrimination between potentially immunogenic protein aggregates and harmless pharmaceutical components, like silicone oil, is critical for drug development. Flow imaging techniques allow to measure and, in principle, classify subvisible particles in protein therapeutics. However, automated approaches for silicone oil discrimination are still lacking robustness in terms of accuracy and transferability. In this work, we present an image-based filter that can reliably identify silicone oil particles in protein therapeutics across a wide range of parenteral products. A two-step classification approach is designed for automated silicone oil droplet discrimination, based on particle images generated with a flow imaging instrument. Distinct from previously published methods, our novel image-based filter is trained using silicone oil droplet images only and is, thus, independent of the type of protein samples imaged. Benchmarked against alternative approaches, the proposed filter showed best overall performance in categorizing silicone oil and non-oil particles taken from a variety of protein solutions. Excellent accuracy was observed particularly for higher resolution images. The image-based filter can successfully distinguish silicone oil particles with high accuracy in protein solutions not used for creating the filter, showcasing its high transferability and potential for wide applicability in biopharmaceutical studies
Development of anti-CD32b antibodies with enhanced Fc function for the treatment of B and plasma cell malignancies
No full textThe sole inhibitory Fcγ receptor CD32b (FcγR2b) is expressed throughout B and plasma cell development and on their malignant counterparts with the highest expression found on multiple myeloma. Additionally, CD32b expression on tumor cells is known to sequester IgG Fc whereby providing a mechanism of resistance to therapeutic monoclonal antibodies (mAb) with Fc dependent activity. Taken together, CD32b represents an attractive tumor antigen for targeting with a mAb. To this end, two anti-CD32b mAbs, NVS32b1 and NVS32b2, were developed. The complementarity-determining regions (CDRs) of these antibodies bind the CD32b Fc binding domain with high specificity and affinity while the Fc region is afucosylated to enhance activation of FcR on immune effector cells. NVS32b mAbs selectively depletes CD32b+ healthy and malignant B cells but spares myeloid cells and CD32a+ cells. These antibodies mediate potent killing of opsonized cells via antibody dependent cellular cytotoxicity and phagocytosis (ADCC & ADCP), as well as complement dependent cytotoxicity (CDC). Additionally, NVS32b CDRs block the CD32b Fc binding domain, thereby minimizing CD32b mediated resistance to therapeutic mAbs with Fc dependent activity, including rituximab, obinutuzumab, and daratumumab. NVS32b mAbs demonstrate robust antitumor activity against CD32b positive xenografts in vivo and immunomodulatory activity including recruitment of macrophages to the tumor microenvironment and enhancement of DC maturation in response to immune-complexes. The activity of NVS32b mAbs on CD32b+ primary malignant B and plasma cells was confirmed on samples from CLL and MM patients. NVS32b mAbs demonstrated great therapeutic potential, as a single agent or in combination with other mAb therapeutics
Process Development for the Synthesis of a -monobactam antibiotic - LYS228
No full textA scalable process for the novel monobactam antibiotic LYS228 has been developed. This paper covers a novel scalable process for producing β-lactam 10 in the multiple kilogram scale. An alternative approach to compound 7 without protecting-group exchange has been demonstrated to enable a shortened synthesis. A scalable process for the side chain 20, which used an alternative reagent to mitigate a safety risk from the initial med-chem approach, was performed. The final assembling to LYS228, a stable isolated product, was identified and executed in the multiple kilogram scale
Spray 1 drying of API nanosuspensions: Importance of particle size, matrix former content and drying temperature for successful formulation and process development
No full textActive pharmaceutical ingredient (API) nanosuspensions from naproxen (Nap) and itraconazole (Itra) stabilized with Kollidon®VA64 (KVA) and sodium dodecylsulfate (SDS) were produced in two different size classes each by wet media milling. These API nanosuspensions were spray dried with lactose, trehalose and sucrose as matrix formers in different proportions and at different drying temperatures (Toutlet). Toutlet as well as the API content significantly influenced the redispersibility of the API nanoparticles. It could be shown that these two parameters are related to each other, with an increasing API content leading to a decreasing maximum applicable Toutlet (Tcritical). Drying above the Tcritical always led to a change in the nanoparticulate state and thus to a non-redispersible product (with respect to the defined quality criteria). For each proportion of API to matrix former a Tcritical could be found. Tcritical showed a linear relation to the API content. The linear regression of this relation was defined as process boundary. The y-intercept of the process boundary correlated well with the Tg of the pure matrix former used for spray drying. The two API under investigation led to virtually identical behaviour if other parameters were kept constant. The particle size of the initial nanosuspension seems to have an important influence. Nanosuspensions with comparatively small particle sizes led to significantly lower process boundaries compared to the use of larger particle sizes. The maximum API content that leads to a redispersible product is thus determined decisively by the particle size of API nanoparticles, Toulet and the Tg of the matrix former used
Gene-signature-derived IC50s/EC50s reflect the potency of causative upstream targets and downstream phenotypes
No full textMultiplexed gene-signature-based phenotypic assays are increasingly used for the identification and profiling of small molecule-tool compounds and drugs. Here we introduce a method (provided as R-package) for the quantification of the dose-response potency of a gene-signature as EC50 and IC50 values. Two signaling pathways were used as models to validate our methods: beta-adrenergic agonistic activity on cAMP generation (dedicated dataset generated for this study) and EGFR inhibitory effect on cancer cell viability. In both cases, potencies derived from multi-gene expression data were highly correlated with orthogonal potencies derived from cAMP and cell growth readouts, and superior to potencies derived from single individual genes. Based on our results we propose gene-signature potencies as a novel valid alternative for the quantitative prioritization, optimization and development of novel drugs
The roles of human induced pluripotent stem cell-derived cardiomyocytes in drug discovery: managing in vitro safety study expectations
No full textIntroduction: Human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) preparations are increasingly employed in in vitro cardiac safety studies to support candidate drug selection and regulatory submissions. The value of hiPSC-CM-based approaches depends on their ability to recapitulate the cellular mechanisms responsible for cardiotoxicity as well as overall assay characteristics (thus defining model performance). Different expectations at different drug development stages define the utility of these human-derived models. Areas covered: Herein, the authors review the importance of understanding the functional characteristics of the evolving spectrum of simpler (2D) and more complex (co-cultures, 3D constructs, and engineered tissues) human-derived cardiac preparations, and how their performance may be evaluated based on analytical sensitivity, variability, and reproducibility in order to correctly match preparations with expectations of different safety assays. The need for consensus clinical examples of electrophysiologic, contractile, and structural cardiotoxicities essential for benchmarking human-derived models is also discussed. Expert opinion: It is helpful (but not essential) that hiPSC-CMs preparations fully recapitulate pharmacological responses of native adult human ventricular myocytes when evaluating cardiotoxicity in vitro. Further calibration and model standardization (aligning concordance with clinical findings) are necessary to understand the role of hiPSC-CMs in guiding cardiotoxicity assessments in early drug discovery efforts
eLive revised manuscript: A POLYOMAVIRUS PEPTIDE BINDS TO THE CAPSID VP1 PORE AND HAS POTENT ANTIVIRAL ACTIVITY AGAINST BK AND JC POLYOMAVIRUSES
No full textIn pursuit of therapeutics for human polyomaviruses, we identified a peptide derived from the BK polyomavirus (BKV) minor structural proteins VP2/3 that is a potent inhibitor of BKV infection with no observable cellular toxicity. The thirteen-residue peptide binds to major structural protein VP1 with single-digit nanomolar affinity. Alanine scanning of the peptide identified three key residues, substitution of each of which results in ~1000-fold loss of binding affinity with a concomitant reduction in antiviral activity. Structural studies demonstrate specific binding of the peptide to the five-fold pore of pentameric VP1. Cell-based assays demonstrate nanomolar inhibition (EC50) of BKV infection and suggest that the peptide acts early in the viral entry pathway. Homologous peptide exhibits similar binding to JC polyomavirus VP1 and inhibits infection with similar potency to BKV in a model cell line. Lastly, these studies validate targeting the VP1 pore as a novel strategy for the development of anti-polyomavirus agents
A first-in-human phase 1 dose escalation study of spartalizumab (PDR001), an anti-PD-1 antibody, in patients with advanced solid tumors
No full textBackground Spartalizumab is a humanized IgG4κ monoclonal antibody that binds programmed death-1 (PD-1) and blocks its interaction with PD-L1 and PD-L2. This phase 1/2 study was designed to assess the safety, pharmacokinetics, and preliminary efficacy of spartalizumab in patients with advanced or metastatic solid tumors. Methods In the phase 1 part of the study, 58 patients received spartalizumab, intravenously, at doses of 1, 3, or 10 mg/kg, administered every 2 weeks (Q2W), or 3 or 5 mg/kg every 4 weeks (Q4W). Results Patients had a wide range of tumor types, most commonly sarcoma (28%) and metastatic renal cell carcinoma (10%); other tumor types were reported in ≤3 patients each. Most patients (93%) had received prior antineoplastic therapy (median three prior lines) and two-thirds of the population had tumor biopsies negative for PD-L1 expression at baseline. The maximum tolerated dose was not reached. The recommended phase 2 doses were selected as 400 mg Q4W or 300 mg Q3W. No dose-limiting toxicities were observed, and adverse events included those typical of other PD-1 antibodies. The most common treatment-related adverse events of any grade were fatigue (22%), diarrhea (17%), pruritus (14%), hypothyroidism (10%), and nausea (10%). Partial responses occurred in two patients (response rate 3.4%); one with atypical carcinoid tumor of the lung and one with anal cancer. Paired tumor biopsies from patients taken at baseline and on treatment suggested an on-treatment increase in CD8+ lymphocyte infiltration in patients with clinical benefit. Conclusions Spartalizumab was well tolerated at all doses tested in patients with previously treated advanced solid tumors. On-treatment immune activation was seen in tumor biopsies; however, limited clinical activity was reported in this heavily pretreated, heterogeneous population. The phase 2 part of this study is ongoing in select tumor types. Trial registration number NCT02404441
Discovery of WD Repeat-Containing Protein 5 (WDR5)-MYC Inhibitors Using Fragment-Based Methods and Structure-Based Design.
No full textThe frequent deregulation of MYC and its elevated expression via multiple mechanisms drives cells to a tumorigenic state. Indeed, MYC is overexpressed in up to ∼50% of human cancers and is considered a highly validated anticancer target. Recently, we discovered that WD repeat-containing protein 5 (WDR5) binds to MYC and is a critical cofactor required for the recruitment of MYC to its target genes and reported the first small molecule inhibitors of the WDR5-MYC interaction using structure-based design. These compounds display high binding affinity, but have poor physicochemical properties and are hence not suitable for studies. Herein, we conducted an NMR-based fragment screening to identify additional chemical matter and, using a structure-based approach, we merged a fragment hit with the previously reported sulfonamide series. Compounds in this series can disrupt the WDR5-MYC interaction in cells, and as a consequence, we observed a reduction of MYC localization to chromatin
A phenotypic screen identifies calcium overload as a key mechanism of beta cell glucolipotoxicity
No full textType 2 diabetes (T2D) is caused by loss of pancreatic beta cell mass and failure of the remaining beta cells to deliver sufficient insulin to meet demand. Beta cell glucolipotoxicity (GLT), which refers to combined, deleterious effects of elevated glucose and fatty acid levels on beta cell function and survival, contributes to T2D-associated beta cell failure. Drugs and mechanisms that protect beta cells from GLT stress could potentially improve metabolic control in T2D patients. In a phenotypic screen seeking low molecular weight compounds that protected beta cells from GLT we identified compound A that selectively blocked GLT-induced apoptosis in rat insulinoma cells. Compound A and its optimized analogs also improved viability and function in primary rat and human islets under GLT. Further studies revealed that active compound from this series largely reversed GLT-induced global transcriptional changes. We discovered that compound A analogs decreased glucose- and lipid-induced cytosolic calcium influx in islet cells, and all measured beta cell-protective effects correlated with this activity. Our results suggest that taming cytosolic calcium overload in pancreatic islets can improve beta cell survival and function under GLT stress and thus could be an effective strategy for T2D treatment