Immunogenicity Risk Assessment of Process-Related Impurities in An Engineered T Cell Receptor Cellular Product.

Cell therapies such as genetically modified T cells have emerged as a promising and viable treatment for hematologic cancers and are being aggressively pursued for a wide range of diseases and conditions that were previously difficult to treat or had no cure. The process development requires genetic modifications to T cells to express a receptor (engineered T cell receptor (eTCR)) of specific binding qualities to the desired target. Protein reagents utilized during the cell therapy manufacturing process, to facilitate these genetic modifications, are often present as process-related impurities at residual levels in the final drug product and can represent a potential immunogenicity risk upon infusion. This manuscript presents a framework for the qualification of an assay for assessing the immunogenicity risk of AA6 and Cas9 residuals. The same framework applies for other residuals; however, AAV6 and Cas9 were selected as they were residuals from the manufacturing of an engineered T cell receptor cellular product in development. The manuscript: 1) elucidates theoretical risks, 2) summarizes analytical data collected during process development, 3) describes the qualification of an in vitro human PBMC cytokine release assay to assess immunogenicity risk from cellular product associated process residuals; 4) identifies a multiplexed inflammatory innate and adaptive cytokine panel with pre-defined criteria using relevant positive controls; and 5) discusses qualification challenges and potential solutions for establishing meaningful thresholds. The assessment is not only relevant to establishing safe exposure levels of these residuals but also in guiding risk assessment and CMC strategy during the conduct of clinical trials.

A functional three-dimensional microphysiological human model of myeloma bone disease.

Human myeloma bone disease (MBD) occurs when malignant plasma cells migrate to the bone marrow and commence inimical interactions with stromal cells, disrupting the skeletal remodeling process. The myeloma cells simultaneously suppress osteoblastic bone formation while promoting excessive osteoclastic resorption. This bone metabolism imbalance produces osteolytic lesions that cause chronic bone pain and reduce trabecular and cortical bone structural integrity, and often culminate in pathological fractures. Few bone models exist that enable scientists to study MBD and the effect therapies have on restoring the bone metabolism imbalance. The purpose of this research was to develop a well characterized three-dimensional (3D) bone organoid that could be used to study MBD and current or potential treatment options. First, bone marrow stromal cell-derived osteoblasts (OBs) mineralized an endosteal-like extracellular matrix (ECM) over 21 days. Multiple analyses confirmed the generation of hydroxyapatite (HA)-rich bone-like tissue fragments that were abundant in alkaline phosphatase, calcium, and markers of osteoblastic gene expression. On day 22, bone marrow macrophage (BMM)-derived osteoclasts (OCs) were introduced to enhance the resorptive capability of the model and recapitulate the balanced homeostatic nature of skeletal remodeling. Tartrate-resistant acid phosphatase 5b (TRAcP-5b), type I collagen C-telopeptide (CTX-1), and gene expression analysis confirmed OC activity in the normal 3D organoid (3D in vitro model of normal bonelike fragments [3D-NBF]). On day 30, a human multiple myeloma (MM)-derived plasmacytoma cell line was introduced to the 3D-NBF to generate the 3D-myeloma bone disease organoid (3D-MBD). After 12 days, the 3D-MBD had significantly reduced total HA, increased TRAcP-5b levels, increases levels of CTX-1, and decreased expression of osteoblastic genes. Therapeutic intervention with pharmaceutical agents including an immunomodulatory drug, a bisphosphonate, and monoclonal restored HA content and reduced free CTX-1 in a dose-dependent manner. This osteogenically functional model of MBD provides a novel tool to study biological mechanisms guiding the disease and to screen potential therapeutics. © 2021 American Society for Bone and Mineral Research (ASBMR).

Investigation Into the Role of ERK in Tyrosine Kinase Inhibitor-Induced Neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common and debilitating adverse event that can alter patient treatment options and halt candidate drug development. A case study is presented here describing the preclinical and clinical development of CC-90003, a small molecule extracellular signal-regulated kinase (ERK)1/2 inhibitor investigated as an oncology therapy. In a Phase Ia clinical trial, CC-90003 elicited adverse drug-related neuropathy and neurotoxicity that contributed to discontinued development of CC-90003 for oncology therapy. Preclinical evaluation of CC-90003 in dogs revealed clinical signs and electrophysiological changes consistent with peripheral neuropathy that was reversible. Mice did not exhibit signs of neuropathy upon daily dosing with CC-90003, supporting that rodents generally poorly predict CIPN. We sought to investigate the mechanism of CC-90003-induced peripheral neuropathy using a phenotypic in vitro assay. Translating preclinical neuropathy findings to humans proves challenging as no robust in vitro models of CIPN exist. An approach was taken to examine the influence of CIPN-associated drugs on human-induced pluripotent stem cell-derived peripheral neuron (hiPSC-PN) electrophysiology on multielectrode arrays (MEAs). The MEA assay with hiPSC-PNs was sensitive to CIPN-associated drugs cisplatin, sunitinib, colchicine, and importantly, to CC-90003 in concordance with clinical neuropathy incidence. Biochemical data together with in vitro MEA data for CC-90003 and 12 of its structural analogs, all having similar ERK inhibitory activity, revealed that CC-90003 disrupted in vitro neuronal electrophysiology likely via on-target ERK inhibition combined with off-target kinase inhibition and translocator protein inhibition. This approach could prove useful for assessing CIPN risk and interrogating mechanisms of drug-induced neuropathy.

Cardiovascular microphysiological systems (CVMPS) for safety studies - a pharma perspective.

The integrative responses of the cardiovascular (CV) system are essential for maintaining blood flow to provide oxygenation, nutrients, and waste removal for the entire body. Progress has been made in independently developing simple in vitro models of two primary components of the CV system, namely the heart (using induced pluripotent stem-cell derived cardiomyocytes) and the vasculature (using endothelial cells and smooth muscle cells). These two in vitro biomimics are often described as immature and simplistic, and typically lack the structural complexity of native tissues. Despite these limitations, they have proven useful for specific "fit for purpose" applications, including early safety screening. More complex in vitro models offer the tantalizing prospect of greater refinement in risk assessments. To this end, efforts to physically link cardiac and vascular components to mimic a true CV microphysiological system (CVMPS) are ongoing, with the goal of providing a more holistic and integrated CV response model. The challenges of building and implementing CVMPS in future pharmacological safety studies are many, and include a) the need for more complex (and hence mature) cell types and tissues, b) the need for more realistic vasculature (within and across co-modeled tissues), and c) the need to meaningfully couple these two components to allow for integrated CV responses. Initial success will likely come with simple, bioengineered tissue models coupled with fluidics intended to mirror a vascular component. While the development of more complex integrated CVMPS models that are capable of differentiating safe compounds and providing mechanistic evaluations of CV liabilities may be feasible, adoption by pharma will ultimately hinge on model efficiency, experimental reproducibility, and added value above current strategies.

Nonclinical safety assessment of epigenetic modulatory drugs: Current status and industry perspective.

Pharmaceutic products designed to perturb the function of epigenetic modulators have been approved by regulatory authorities for treatment of advanced cancer. While the predominant effort in epigenetic drug development continues to be in oncology, non-oncology indications are also garnering interest. A survey of pharmaceutical companies was conducted to assess the interest and concerns for developing small molecule direct epigenetic effectors (EEs) as medicines. Survey themes addressed (1) general levels of interest and activity with EEs as therapeutic agents, (2) potential safety concerns, and (3) possible future efforts to develop targeted strategies for nonclinical safety assessment of EEs. Thirteen companies contributed data to the survey. Overall, the survey data indicate the consensus opinion that existing ICH guidelines are effective and appropriate for nonclinical safety assessment activities with EEs. Attention in the framework of study design should, on a case by case basis, be considered for delayed or latent toxicities, carcinogenicity, reproductive toxicity, and the theoretical potential for transgenerational effects. While current guidelines have been appropriate for the nonclinical safety assessments of epigenetic targets, broader experience with a wide range of epigenetic targets will provide information to assess the potential need for new or revised risk assessment strategies for EE drugs.

Human ileal organoid model recapitulates clinical incidence of diarrhea associated with small molecule drugs.

Drug-induced gastrointestinal toxicity (GIT) is a common treatment-emergent adverse event that can negatively impact dosing, thereby limiting efficacy and treatment options for patients. An in vitro assay of GIT is needed to address patient variability, mimic the microphysiology of the gut, and accurately predict drug-induced GIT. Primary human ileal organoids (termed 'enteroids') have proven useful for stimulating intestinal stem cell proliferation and differentiation to multiple cell types present in the gut epithelium. Enteroids have enabled characterization of gut biology and the signaling involved in the pathogenesis of disease. Here, enteroids were differentiated from four healthy human donors and assessed for culture duration-dependent differentiation status by immunostaining for gut epithelial markers lysozyme, chromogranin A, mucin, and sucrase isomaltase. Differentiated enteroids were evaluated with a reference set of 31 drugs exhibiting varying degrees of clinical incidence of diarrhea, a common manifestation of GIT that can be caused by drug-induced thinning of the gut epithelium. An assay examining enteroid viability in response to drug treatment demonstrated 90% accuracy for recapitulating the incidence of drug-induced diarrhea. The human enteroid viability assay developed here presents a promising in vitro model for evaluating drug-induced diarrhea.

Developing in vitro assays to transform gastrointestinal safety assessment: potential for microphysiological systems.

Drug-induced gastrointestinal toxicities (DI-GITs) are among the most common adverse events in clinical trials. High prevalence of DI-GIT has persisted among new drugs due in part to the lack of robust experimental tools to allow early detection or to guide optimization of safer molecules. Developing in vitro assays for the leading GI toxicities (nausea, vomiting, diarrhoea, constipation, and abdominal pain) will likely involve recapitulating complex physiological properties that require contributions from diverse cell/tissue types including epithelial, immune, microbiome, nerve, and muscle. While this stipulation may be beyond traditional 2D monocultures of intestinal cell lines, emerging 3D GI microtissues capture interactions between diverse cell and tissue types. These interactions give rise to microphysiologies fundamental to gut biology. For GI microtissues, organoid technology was the breakthrough that introduced intestinal stem cells with the capability of differentiating into each of the epithelial cell types and that self-organize into a multi-cellular tissue proxy with villus- and crypt-like domains. Recently, GI microtissues generated using miniaturized devices with microfluidic flow and cyclic peristaltic strain were shown to induce Caco2 cells to spontaneously differentiate into each of the principle intestinal epithelial cell types. Second generation models comprised of epithelial organoids or microtissues co-cultured with non-epithelial cell types can successfully reproduce cross-'tissue' functional interactions broadening the potential of these models to accurately study drug-induced toxicities. A new paradigm in which in vitro assays become an early part of GI safety assessment could be realized if microphysiological systems (MPS) are developed in alignment with drug-discovery needs. Herein, approaches for assessing GI toxicity of pharmaceuticals are reviewed and gaps are compared with capabilities of emerging GI microtissues (e.g., organoids, organ-on-a-chip, transwell systems) in order to provide perspective on the assay features needed for MPS models to be adopted for DI-GIT assessment.

Thalidomide Inhibits Human iPSC Mesendoderm Differentiation by Modulating CRBN-dependent Degradation of SALL4.

Exposure to thalidomide during a critical window of development results in limb defects in humans and non-human primates while mice and rats are refractory to these effects. Thalidomide-induced teratogenicity is dependent on its binding to cereblon (CRBN), the substrate receptor of the Cul4A-DDB1-CRBN-RBX1 E3 ubiquitin ligase complex. Thalidomide binding to CRBN elicits subsequent ubiquitination and proteasomal degradation of CRBN neosubstrates including SALL4, a transcription factor of which polymorphisms phenocopy thalidomide-induced limb defects in humans. Herein, thalidomide-induced degradation of SALL4 was examined in human induced pluripotent stem cells (hiPSCs) that were differentiated either to lateral plate mesoderm (LPM)-like cells, the developmental ontology of the limb bud, or definitive endoderm. Thalidomide and its immunomodulatory drug (IMiD) analogs, lenalidomide, and pomalidomide, dose-dependently inhibited hiPSC mesendoderm differentiation. Thalidomide- and IMiD-induced SALL4 degradation can be abrogated by CRBN V388I mutation or SALL4 G416A mutation in hiPSCs. Genetically modified hiPSCs expressing CRBN E377V/V388I mutant or SALL4 G416A mutant were insensitive to the inhibitory effects of thalidomide, lenalidomide, and pomalidomide on LPM differentiation while retaining sensitivity to another known limb teratogen, all-trans retinoic acid (atRA). Finally, disruption of LPM differentiation by atRA or thalidomide perturbed subsequent chondrogenic differentiation in vitro. The data here show that thalidomide, lenalidomide, and pomalidomide affect stem cell mesendoderm differentiation through CRBN-mediated degradation of SALL4 and highlight the utility of the LPM differentiation model for studying the teratogenicity of new CRBN modulating agents.

Cardiovascular Toxicity Induced by Kinase Inhibitors: Mechanisms and Preclinical Approaches.

Kinase inhibitors have transformed the treatment of many cancers and are showing the same promise for other indications including inflammatory diseases. This class of drugs is one of the most predominant in the pharmaceutical industry, but development and clinical utility is often limited by a broad spectrum of cardiovascular (CV) toxicities including QT prolongation and arrhythmia, left ventricular dysfunction, congestive heart failure, ischemia and myocardial infarction, and hypertension. In this review article, we provide a broad overview of the spectrum of CV events detected in clinical trials of kinase inhibitors and the known and proposed on- and off-target links between kinase inhibitor targets and these specific cardiotoxicities. We also examine the unique features of kinase inhibitors that have impeded complete mechanistic understanding of kinase inhibitor-associated cardiotoxicities and contributed to the disconnect between preclinical predictions and clinical findings. We then discuss various in vitro models currently in use that are amenable for high-throughput screening as well as lower throughput models that are valuable for mechanistic insight. These physiologically relevant models, together with newer "omic"-wide approaches will help to increase our understanding of the mechanisms underlying kinase inhibitor-associated cardiotoxicity and enable rational design of kinase inhibitors in the future.

Stem Cell Research.

Stem cells have great potential in basic research and are being slowly integrated into toxicological research. This symposium provided an overview of the state of the field, stem cell models, described allogenic stem cell treatments and issues of immunogenicity associated with protein therapeutics, and tehn concentrated on stem cell uses in regenerative medicine focusing on lung and testing strategies on engineered tissues from a pathologist's perspective.

Human stem cell-derived cardiomyocytes in cellular impedance assays: bringing cardiotoxicity screening to the front line.

Cardiovascular (CV) toxicity is a leading cause of drug attrition and withdrawal. Introducing in vitro assays with higher throughput should permit earlier CV hazard identification and enable medicinal chemists to design-out liabilities. Heretofore, development of in vitro CV assays has been limited by the challenge of replicating integrated cardiovascular physiology while achieving the throughput and consistency required for screening. These challenges appear to be met with a combination of human stem cell-derived cardiomyocytes (CM) which beat spontaneously and monitoring the response with technology that can assess drug-induced changes in voltage dependent contraction such as cellular impedance which has been validated with excellent predictivity for drug-induced arrhythmia and contractility. Here, we review advances in cardiomyocyte impedance with emphasis on stem cell-derived cardiomyocyte models for toxicity screening. Key perspectives include: the electrical principles of impedance technology, impedance detection of cardiomyocyte beating, beat parameter selection/analysis, validation in toxicity and drug discovery, and future directions. As a conclusion, an in vitro screening cascade is proffered using the downstream, inclusive detection of CM impedance assays as a primary screen followed by complementary CM assays chosen to enable mechanism-appropriate follow-up. The combined approach will enhance testing for CV liabilities prior to traditional in vivo models.

Disease modeling and phenotypic drug screening for diabetic cardiomyopathy using human induced pluripotent stem cells.

Diabetic cardiomyopathy is a complication of type 2 diabetes, with known contributions of lifestyle and genetics. We develop environmentally and genetically driven in vitro models of the condition using human-induced-pluripotent-stem-cell-derived cardiomyocytes. First, we mimic diabetic clinical chemistry to induce a phenotypic surrogate of diabetic cardiomyopathy, observing structural and functional disarray. Next, we consider genetic effects by deriving cardiomyocytes from two diabetic patients with variable disease progression. The cardiomyopathic phenotype is recapitulated in the patient-specific cells basally, with a severity dependent on their original clinical status. These models are incorporated into successive levels of a screening platform, identifying drugs that preserve cardiomyocyte phenotype in vitro during diabetic stress. In this work, we present a patient-specific induced pluripotent stem cell (iPSC) model of a complex metabolic condition, showing the power of this technique for discovery and testing of therapeutic strategies for a disease with ever-increasing clinical significance.

A high-throughput screen for teratogens using human pluripotent stem cells.

There is need in the pharmaceutical and chemical industries for high-throughput human cell-based assays for identifying hazardous chemicals, thereby reducing the overall reliance on animal studies for predicting the risk of toxic responses in humans. Despite instances of human-specific teratogens such as thalidomide, the use of human cell-teratogenicity assays has just started to be explored. Herein, a human pluripotent stem cell test (hPST) for identifying teratogens is described, benchmarking the in vitro findings to traditional preclinical toxicology teratogenicity studies and when available to teratogenic outcomes in humans. The hPST method employs a 3-day monolayer directed differentiation of human embryonic stem cells. The teratogenic risk of a compound is gauged by measuring the reduction in nuclear translocation of the transcription factor SOX17 in mesendodermal cells. Decreased nuclear SOX17 in the hPST model was strongly correlated with in vivo teratogenicity. Specifically, 71 drug-like compounds with known in vivo effects, including thalidomide, were examined in the hPST. A threshold of 5 µM demonstrated 94% accuracy (97% sensitivity and 92% specificity). Furthermore, 15 environmental toxicants with physicochemical properties distinct from small molecule pharmaceutical agents were examined and a similarly strong concordance with teratogenicity outcomes from in vivo studies was observed. Finally, to assess the suitability of the hPST for high-throughput screens, a small library of 300 kinase inhibitors was tested, demonstrating the hPST platform's utility for interrogating teratogenic mechanisms and drug safety prediction. Thus, the hPST assay is a robust predictor of teratogenicity and appears to be an improvement over existing in vitro models.

Stem cells and stem cell-derived tissues and their use in safety assessment.

Toxicology has long relied on animal models in a tedious approach to understanding risk of exposure to an uncharacterized molecule. Stem cell-derived tissues can be made in high purity, quality, and quantity to enable a new approach to this problem. Currently, stem cell-derived tissues are primarily "generic" genetic backgrounds; the future will see the integration of various genetic backgrounds and complex three-dimensional models to create truly unique in vitro organoids. This minireview focuses on the state of the art of a number of stem cell-derived tissues and details their application in toxicology.

Refining the human iPSC-cardiomyocyte arrhythmic risk assessment model.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are capable of detecting drug-induced clinical arrhythmia, Torsade de Pointes (TdP), and QT prolongation. Efforts herein employ a broad set of structurally diverse drugs to optimize the predictive algorithm for applications in discovery toxicology and cardiac safety screening. The changes in the beat rhythm and rate of a confluent monolayer of hiPS-CMs by 88 marketed and 30 internal discovery compounds were detected with real-time cellular impedance measurement and quantified by measures of arrhythmic beating (IB20, lowest concentration inducing ≥ 20% arrhythmic [irregular, atypical] beats in 3 consecutive 20-s sweeps, and predicted proarrhythmic score [PPS]-IB20) or changes in beat rate (BR20, the lowest concentration inducing a reduction in beat rate of ≥ 20% at 3 consecutive sweeps compared with the time-matched vehicle control group, and PPS-BR20). Drug-induced arrhythmic beats and reductions in beat rates are predictive of clinical arrhythmia and QT prolongation, respectively. A threshold of ≤ 10 µM for class determination results in 82% in vitro-in vivo concordance for TdP prediction and 91% sensitivity for non-TdP arrhythmia detection, or 83% and 91% if clinically efficacious plasma (effective serum therapeutic concentration [C eff]) values are incorporated. This human cardiomyocyte arrhythmic risk (hCAR) model provides greater predictivity for torsadogenicity in humans compared with either human ether-a-go-go-related gene (hERG) inhibition (75%) or QT prolongation (81%). The concordance of beat rate reductions to predict clinical QT prolongation is 86%, or 87% when C eff is considered, which is greater than a hERG signal (80%). Further, arrhythmic beats resulting from cytotoxicity were identified by a distinct arrhythmic beating pattern, which occurred after the onset of cytolethality. This hCAR assay showed increased performance over existing preclinical tools in predicting clinical QT prolongation, arrhythmia, and TdP. Thus, hiPS-CMs are a relevant cell system to improve evaluating cardiac safety liabilities of drug candidates.

Cancer genetics and the cardiotoxicity of the therapeutics.

Cancer genomics has focused on the discovery of mutations and chromosomal structural rearrangements that either increase susceptibility to cancer or support the cancer phenotype. Protein kinases are the most frequently mutated genes in the cancer genome, making them attractive therapeutic targets for drug design. However, the use of some of the kinase inhibitors (KIs) has been associated with toxicities to the heart and vasculature, including acute coronary syndromes and heart failure. Herein we discuss the genetic basis of cancer, focusing on mutations in the kinase genome (kinome) that lead to tumorigenesis. This will allow an understanding of the real and potential power of modern cancer therapeutics. The underlying mechanisms that drive the cardiotoxicity of the KIs are also examined. The preclinical models for predicting cardiotoxicity, including induced pluripotent stem cells and zebrafish, are reviewed, with the hope of eventually being able to identify problematic agents before their use in patients. Finally, the use of biomarkers in the clinic is discussed, and newer strategies (i.e., metabolomics and enhanced imaging strategies) that may allow earlier and more accurate detection of cardiotoxicity are reviewed.

The effects of jaspamide on human cardiomyocyte function and cardiac ion channel activity.

Jaspamide (jasplakinolide; NSC-613009) is a cyclodepsipeptide that has antitumor activity. A narrow margin of safety was observed between doses required for efficacy in mouse tumor models and doses that caused severe acute toxicity in rats and dogs. We explored the hypothesis that the observed toxicity was due to cardiotoxicity. Jaspamide was tested in a patch clamp assay to determine its effect on selected cardiac ion channels. Jaspamide (10 µM) inhibited Kv1.5 activity by 98.5%. Jaspamide also inhibited other channels including Cav1.2, Cav3.2, and HCN2; however, the Kv11.1 (hERG) channel was minimally affected. Using spontaneously contracting human cardiomyocytes derived from induced pluripotent stem cells, effects on cardiomyocyte contraction and viability were also examined. Jaspamide (30 nM to 30 µM) decreased cardiomyocyte cell indices and beat amplitude, putative measurements of cell viability and cardiac contractility, respectively. Concentration-dependent increases in rhythmic beating rate were noted at ≤ 6 h of treatment, followed by dose-dependent decreases after 6 and 72 h exposure. The toxic effects of jaspamide were compared with that of the known cardiotoxicant mitoxantrone, and confirmed by multiparameter fluorescence imaging analysis. These results support the hypothesis that the toxicity observed in rats and dogs is due to toxic effects of jaspamide on cardiomyocytes.