ABSTRACT
Vast quantities of multi-omic data have been produced to characterize the development and diversity of cell types in the cerebral cortex of humans and other mammals. To more fully harness the collective discovery potential of these data, we have assembled gene-level transcriptomic data from 188 published studies of neocortical development, including the transcriptomes of ~30 million single-cells, extensive spatial transcriptomic experiments and RNA sequencing of sorted cells and bulk tissues: nemoanalytics.org/landing/neocortex. Applying joint matrix decomposition (SJD) to mouse, macaque and human data in this collection, we defined transcriptome dynamics that are conserved across mammalian neurogenesis and which elucidate the evolution of outer, or basal, radial glial cells. Decomposition of adult human neocortical data identified layer-specific signatures in mature neurons and, in combination with transfer learning methods in NeMO Analytics, enabled the charting of their early developmental emergence and protracted maturation across years of postnatal life. Interrogation of data from cerebral organoids demonstrated that while broad molecular elements of in vivo development are recapitulated in vitro, many layer-specific transcriptomic programs in neuronal maturation are absent. We invite computational biologists and cell biologists without coding expertise to use NeMO Analytics in their research and to fuel it with emerging data (carlocolantuoni.org).
ABSTRACT
One of the two X chromosomes in female mammals is epigenetically silenced in embryonic stem cells by X chromosome inactivation (XCI). This creates a mosaic of cells expressing either the maternal or the paternal X allele. The XCI ratio, the proportion of inactivated parental alleles, varies widely among individuals, representing the largest instance of epigenetic variability within mammalian populations. While various contributing factors to XCI variability are recognized, namely stochastic and/or genetic effects, their relative contributions are poorly understood. This is due in part to limited cross-species analysis, making it difficult to distinguish between generalizable or species-specific mechanisms for XCI ratio variability. To address this gap, we measured XCI ratios in nine mammalian species (9,143 individual samples), ranging from rodents to primates, and compared the strength of stochastic models or genetic factors for explaining XCI variability. Our results demonstrate the embryonic stochasticity of XCI is a general explanatory model for population XCI variability in mammals, while genetic factors play a minor role.
ABSTRACT
BACKGROUND: Checkpoint inhibitors targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) have demonstrated clinical efficacy in advanced melanoma, but only a subset of patients with inflamed tumors are responsive. Talimogene laherparepvec (T-VEC), a modified herpes simplex virus type 1 (HSV-1) expressing granulocyte-macrophage colony-stimulating factor (GM-CSF), is a first-in-class oncolytic immunotherapy approved for the treatment of melanoma and has been shown to inflame the tumor microenvironment. To evaluate the potential and mechanisms of T-VEC to elicit systemic antitumor immunity and overcome resistance to checkpoint inhibitors in murine tumor models, OncoVEXmGM-CSF was developed similarly to T-VEC, except the human GM-CSF transgene was replaced with murine GM-CSF. Previous work had demonstrated that OncoVEXmGM-CSF generated systemic antitumor immunity dependent on CD8+ T cells in an immune checkpoint-sensitive tumor cell model. METHODS: A novel B16F10 syngeneic tumor model with both HSV-1-permissive subcutaneous tumors and HSV-1-refractory experimental lung metastasis was used to study the local and systemic effects of OncoVEXmGM-CSF treatment alone or in combination with checkpoint inhibitors. RESULTS: Intratumoral injection of OncoVEXmGM-CSF in combination with an anti-CTLA-4 or anti-PD-1 blocking antibody led to increased tumor growth inhibition, a reduction in the number of lung metastases, and prolonged animal survival. OncoVEXmGM-CSF induced both neoantigen-specific and tumor antigen-specific T-cell responses. Furthermore, cured mice from the combination treatment of OncoVEXmGM-CSF and anti-CTLA-4 antibody rejected tumor rechallenges. CONCLUSIONS: These data support the concept that T-VEC and checkpoint inhibition may be an effective combination to treat patients with advanced melanoma.
Subject(s)
Melanoma , Oncolytic Virotherapy , Humans , Animals , Mice , Granulocyte-Macrophage Colony-Stimulating Factor , CD8-Positive T-Lymphocytes/pathology , Antigens, Neoplasm , Tumor MicroenvironmentABSTRACT
Human neural organoid models offer an exciting opportunity for studying often inaccessible human-specific brain development; however, it remains unclear how precisely organoids recapitulate fetal/primary tissue biology. Here, we characterize field-wide replicability and biological fidelity through a meta-analysis of single-cell RNA-sequencing data for first and second trimester human primary brain (2.95 million cells, 51 datasets) and neural organoids (1.63 million cells, 130 datasets). We quantify the degree to which primary tissue cell-type marker expression and co-expression are recapitulated in organoids across 12 different protocol types. By quantifying gene-level preservation of primary tissue co-expression, we show neural organoids lie on a spectrum ranging from virtually no signal to co-expression near indistinguishable from primary tissue data, demonstrating high fidelity is within the scope of current methods. Additionally, we show neural organoids preserve the cell-type specific co-expression of developing rather than adult cells, confirming organoids are an appropriate model for primary tissue development. Overall, quantifying the preservation of primary tissue co-expression is a powerful tool for uncovering unifying axes of variation across heterogeneous neural organoid experiments.
ABSTRACT
The advancement of an investigational new drug in humans is a significant developmental milestone. In first-in-human (FIH)-enabling toxicology studies, the highest dose without a test article-related adverse effect (no-observed-adverse-effect-level [NOAEL]) serves as the basis for deriving a safe FIH starting dose. For anticancer pharmaceuticals, the FIH dose may be calculated using the highest non-severely toxic dose (HNSTD) in nonrodent models or the dose severely toxic to 10% (STD10) in rodents. Given the practice of reporting the NOAEL, but the lack of regulatory requirements to do so for anticancer pharmaceuticals, we conducted an informal survey of 20 companies to answer the question "How is our industry reporting toxic/adverse dose levels in FIH-enabling toxicology studies for anticancer indications?" The data indicated 4 reporting approaches, each providing a path to regulatory acceptance. Within the integrated toxicology study report, 45% of respondents report the HNSTD/STD10, 25% report the NOAEL, 20% report both the HNSTD/STD10 and NOAEL, and 10% do not define either, reserving definitions for regulatory submissions. One reporting approach may be preferred over another for reasons including consistency across indications, repurposing pharmaceuticals, regulatory feedback, or simplicity. The reporting approach should be defined in advance of study initiation, and the pathologist should provide context to support the chosen approach.
Subject(s)
Adverse Drug Reaction Reporting Systems , Antineoplastic Agents , No-Observed-Adverse-Effect Level , Pharmaceutical Preparations , Toxicology , Drugs, Investigational/adverse effects , Antineoplastic Agents/adverse effectsABSTRACT
The potential for effector functions of therapeutic antibodies, including antibody-dependent cell-mediated cytotoxicity (ADCC), is a biological activity of interest for characterization, regardless of if ADCC is an intended primary pharmacological effect. The composition of the conserved antibody Fc glycan can vary as a function of post-translational processing which may affect the binding affinity to Fc receptors, leading to a change of effector activity. Ordesekimab (AMG 714 or PRV-015), a fully human immunoglobulin G1-kappa anti-interleukin (IL)-15 monoclonal antibody, is in clinical development for celiac disease. The binding of ordesekimab to IL-15 inhibits the interaction of IL-15 with the IL-2Rß and common γ chain of the IL-15 receptor complex, but not with the IL-15Rα chain. Therefore, the simultaneous binding of ordesekimab to the Fcγ receptor (R) IIIα expressed on natural killer (NK) cells and to the IL-15/IL-15Rα complex on cells such as monocytes may theoretically enable ADCC toward the IL-15Rα-expressing cells. The high mannose (HM) levels on the Fc glycan were found to vary in different lots of ordesekimab resulting from refinements to the manufacturing process, and the impact on ordesekimab-mediated ADCC activity was evaluated in in vivo and in vitro studies. A review of nonclinical and clinical data found no evidence of ordesekimab-induced depletion of monocytes, or cytotoxicity in organs with wide IL-15Rα expression, suggesting a lack of in vivo ADCC activity. In addition, in vitro peripheral blood mononuclear cells-based ADCC assay did not reveal any cytolytic effect of ordesekimab with various levels of HM content when cocultured with recombinant human IL-15. Taken together, these data demonstrate that ADCC is not a potential liability for ordesekimab and does not contribute to the reduction of IL-15-mediated inflammation, the intended pharmacological effect.
Subject(s)
Interleukin-15 , Leukocytes, Mononuclear , Antibodies, Monoclonal, Humanized , Humans , Interleukin-15/pharmacology , PolysaccharidesABSTRACT
X-chromosome inactivation (XCI) is a random, permanent, and developmentally early epigenetic event that occurs during mammalian embryogenesis. We harness these features to investigate characteristics of early lineage specification events during human development. We initially assess the consistency of X-inactivation and establish a robust set of XCI-escape genes. By analyzing variance in XCI ratios across tissues and individuals, we find that XCI is shared across all tissues, suggesting that XCI is completed in the epiblast (in at least 6-16 cells) prior to specification of the germ layers. Additionally, we exploit tissue-specific variability to characterize the number of cells present during tissue-lineage commitment, ranging from approximately 20 cells in liver and whole blood tissues to 80 cells in brain tissues. By investigating the variability of XCI ratios using adult tissue, we characterize embryonic features of human XCI and lineage specification that are otherwise difficult to ascertain experimentally.
Subject(s)
Embryo, Mammalian , X Chromosome Inactivation , Adult , Animals , Chromosomes, Human, X/genetics , Humans , Mammals/genetics , X Chromosome Inactivation/geneticsABSTRACT
Sexually mature nonhuman primates are often used in nonclinical safety testing when evaluating biopharmaceuticals; however, there is limited information in historical control databases or in the published literature on the spontaneous findings in the male reproductive system. This review evaluated digital slides from the male reproductive tract (testes, epididymides, prostate, and seminal vesicles) in sexually mature cynomolgus macaques (Macaca fascicularis; n = 255) from vehicle control groups in nonclinical toxicology studies and compared the observations with body weight, organ weight, and geographical origin. The most common microscopic findings were hypospermatogenesis and tubular dilatation in the testes; inflammatory cell infiltrate, cellular debris, and decreased sperm in the epididymides; inflammatory cell infiltrate and acinar dilatation in the prostate; and corpora amylacea and atrophy in the seminal vesicles. There were a few correlative observations in animals when grouped by weight or geographical origin: animals with lower terminal body weights (<5 kg) often displayed features of late puberty despite having sperm in the epididymis, while animals originating from Mauritius had a lower incidence of inflammatory cell infiltrates than those from Southeast Asia/China. This review provides incidence, descriptions, and photomicrographs of the common spontaneous microscopic findings in the reproductive system of mature male cynomolgus macaques.
Subject(s)
Epididymis , Semen , Animals , Macaca fascicularis , Male , Organ Size , TestisABSTRACT
Sotorasib is a first-in-class KRASG12C covalent inhibitor in clinical development for the treatment of tumors with the KRAS p.G12C mutation. A comprehensive nonclinical safety assessment package, including secondary/safety pharmacology and toxicology studies, was conducted to support the marketing application for sotorasib. Sotorasib was negative in a battery of genotoxicity assays and negative in an in vitro phototoxicity assay. Based on in vitro assays, sotorasib had no off-target effects against various receptors, enzymes (including numerous kinases), ion channels, or transporters. Consistent with the tumor-specific target distribution (ie, KRASG12C), there were no primary pharmacology-related on-target effects identified. The kidney was identified as a target organ in the rat but not the dog. Renal toxicity in the rat was characterized by tubular degeneration and necrosis restricted to a specific region suggesting that the toxicity was attributed to the local formation of a putative toxic reactive metabolite. In the 3-month dog study, adaptive changes of hepatocellular hypertrophy due to drug metabolizing enzyme induction were observed in the liver that was associated with secondary effects in the pituitary and thyroid gland. Sotorasib was not teratogenic and had no direct effect on embryo-fetal development in the rat or rabbit. Human, dog, and rat circulating metabolites, M24, M10, and M18, raised no clinically relevant safety concerns based on the general toxicology studies, primary/secondary pharmacology screening, an in vitro human ether-à-go-go-related gene assay, or mutagenicity assessment. Overall, the results of the nonclinical safety program support a high benefit/risk ratio of sotorasib for the treatment of patients with KRAS p.G12C-mutated tumors.
Subject(s)
Antineoplastic Agents/toxicity , Piperazines/toxicity , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Pyridines/toxicity , Pyrimidines/toxicity , Animals , Antineoplastic Agents/pharmacology , Drug Evaluation, Preclinical , Humans , Mutation , Neoplasms/drug therapy , Neoplasms/genetics , Piperazines/pharmacology , Pyridines/pharmacology , Pyrimidines/pharmacologyABSTRACT
Sotorasib is a first-in class KRASG12C covalent inhibitor in clinical development for the treatment of tumors with the KRAS p.G12C mutation. In the nonclinical toxicology studies of sotorasib, the kidney was identified as a target organ of toxicity in the rat but not the dog. Renal toxicity was characterized by degeneration and necrosis of the proximal tubular epithelium localized to the outer stripe of the outer medulla (OSOM), which suggested that renal metabolism was involved. Here, we describe an in vivo mechanistic rat study designed to investigate the time course of the renal toxicity and sotorasib metabolites. Renal toxicity was dose- and time-dependent, restricted to the OSOM, and the morphologic features progressed from vacuolation and necrosis to regeneration of tubular epithelium. The renal toxicity correlated with increases in renal biomarkers of tubular injury. Using mass spectrometry and matrix-assisted laser desorption/ionization, a strong temporal and spatial association between renal toxicity and mercapturate pathway metabolites was observed. The rat is reported to be particularly susceptible to the formation of nephrotoxic metabolites via this pathway. Taken together, the data presented here and the literature support the hypothesis that sotorasib-related renal toxicity is mediated by a toxic metabolite derived from the mercapturate and ß-lyase pathway. Our understanding of the etiology of the rat specific renal toxicity informs the translational risk assessment for patients.
Subject(s)
Acetylcysteine/metabolism , Acute Kidney Injury/metabolism , Piperazines/metabolism , Piperazines/toxicity , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Pyridines/metabolism , Pyridines/toxicity , Pyrimidines/metabolism , Pyrimidines/toxicity , Signal Transduction/drug effects , Acute Kidney Injury/chemically induced , Acute Kidney Injury/pathology , Animals , Dose-Response Relationship, Drug , Male , Rats , Rats, Sprague-Dawley , Signal Transduction/physiologyABSTRACT
Phosphatidylinositol 3-kinase (PI3K) δ is a lipid kinase primarily found in leukocytes, which regulates important cell functions. AMG2519493 was a PI3K δ-specific inhibitor in development for treatment of various inflammatory diseases. AMG2519493-related changes in the male and/or female reproductive organs were observed in the 1- and 3-month oral repeat dose toxicology studies in the rat and cynomolgus monkey. Hemorrhagic corpora lutea cysts and increased incidence of corpora lutea cysts without hemorrhage were observed in the ovaries at supra pharmacological doses in the rat. A decrease in seminiferous germ cells in the testis, indicative of spermatogenesis maturation arrest, was observed in both the rat and cynomolgus monkey. Although the characteristics were comparable, the drug systemic exposures associated with the testicular changes were very different between the 2 species. In the rat, the testicular change was only observed at supra pharmacologic exposure. Isotype assessment of PI3K signaling in rat spermatogonia in vitro indicated a role for PI3K ß, but not δ, in the c Kit/PI3K/protein kinase B signaling pathway. Therefore, changes in both the ovary and testis of the rat were considered due to off target effect as they only occurred at suprapharmacologic exposure. In contrast, the testicular changes in the cynomolgus monkey (decrease in seminiferous germ cells) occurred at very low doses associated with PI3K δ-specific inhibition, indicating that the PI3K δ isoform may be important in spermatogenesis maturation in the cynomolgus monkey. Our results suggest species-related differences in PI3K isoform-specific control on reproductive organs.
Subject(s)
Ovary/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Quinolines/pharmacology , Testis/drug effects , Animals , Female , Macaca fascicularis , Male , Mice , Ovary/enzymology , Rats , Rats, Sprague-Dawley , Spermatogonia/enzymology , Testis/enzymologyABSTRACT
The sexual maturity status of animals in nonclinical safety studies can have a significant impact on the microscopic assessment of the reproductive system, the interpretation of potential test article-related findings, and ultimately the assessment of potential risk to humans. However, the assessment and documentation of sexual maturity for animals in nonclinical safety studies is not conducted in a consistent manner across the pharmaceutical and chemical industries. The Scientific and Regulatory Policy Committee of the Society of Toxicologic Pathology convened an international working group of pathologists and nonclinical safety scientists with expertise in the reproductive system, pathology nomenclature, and Standard for Exchange of Nonclinical Data requirements. This article describes the best practices for documentation of the light microscopic assessment of sexual maturity in males and females for both rodent and nonrodent nonclinical safety studies. In addition, a review of the microscopic features of the immature, peripubertal, and mature male and female reproductive system and general considerations for study types and reporting are provided to aid the study pathologist tasked with documentation of sexual maturity.
Subject(s)
Pathologists , Toxicity Tests , Animals , Documentation , Female , Humans , Male , Policy , Research DesignABSTRACT
Toxicologic Pathology is the official journal of the Society of Toxicologic Pathology (STP), the British Society of Toxicological Pathology, and the European STP (ESTP). Toxicologic Pathology publishes articles related to topics in various aspects of toxicologic pathology such as anatomic pathology, clinical pathology, experimental pathology, and biomarker research. Publications include society-endorsed Best Practice/Position and Points to Consider publications and ESTP Expert Workshop articles that are relevant to toxicologic pathology and scientific regulatory processes, Opinion articles under the banner of the STP Toxicologic Pathology Forum, Original Articles, Review Articles (unsolicited/contributed, mini, and invited), Brief Communications, Letters to the Editor, Meeting Reports, and Book Reviews. This article provides details on the various publication categories in Toxicologic Pathology and will serve as a reference for authors and readers.
Subject(s)
Pathology, Clinical , Pathology , Publications/classification , HumansABSTRACT
Primary neurulation is the process by which the neural tube, the central nervous system precursor, is formed from the neural plate. Incomplete neural tube closure occurs frequently, yet underlying causes remain poorly understood. Developmental studies in amniotes and amphibians have identified hingepoint and neural fold formation as key morphogenetic events and hallmarks of primary neurulation, the disruption of which causes neural tube defects. In contrast, the mode of neurulation in teleosts has remained highly debated. Teleosts are thought to have evolved a unique mode of neurulation, whereby the neural plate infolds in absence of hingepoints and neural folds, at least in the hindbrain/trunk where it has been studied. Using high-resolution imaging and time-lapse microscopy, we show here the presence of these morphological landmarks in the zebrafish anterior neural plate. These results reveal similarities between neurulation in teleosts and other vertebrates and hence the suitability of zebrafish to understand human neurulation.
Subject(s)
Epithelial Cells/physiology , Neural Plate/embryology , Neural Tube/embryology , Neurulation , Prosencephalon/embryology , Zebrafish/embryology , Animals , Animals, Genetically Modified , Cell Movement , Cell Shape , Epithelial Cells/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Morphogenesis , Neural Plate/metabolism , Neural Tube/metabolism , Neural Tube Defects/embryology , Prosencephalon/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Time Factors , Time-Lapse Imaging , Transcription Factors/genetics , Transcription Factors/metabolism , Zebrafish/genetics , Zebrafish/metabolismABSTRACT
PURPOSE: Small-cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with a high relapse rate, limited therapeutic options, and poor prognosis. We investigated the antitumor activity of AMG 757, a half-life extended bispecific T-cell engager molecule targeting delta-like ligand 3 (DLL3)-a target that is selectively expressed in SCLC tumors, but with minimal normal tissue expression. EXPERIMENTAL DESIGN: AMG 757 efficacy was evaluated in SCLC cell lines and in orthotopic and patient-derived xenograft (PDX) mouse SCLC models. Following AMG 757 administration, changes in tumor volume, pharmacodynamic changes in tumor-infiltrating T cells (TILs), and the spatial relationship between the appearance of TILs and tumor histology were examined. Tolerability was assessed in nonhuman primates (NHPs). RESULTS: AMG 757 showed potent and specific killing of even those SCLC cell lines with very low DLL3 expression (<1,000 molecules per cell). AMG 757 effectively engaged systemically administered human T cells, induced T-cell activation, and redirected T cells to lyse tumor cells to promote significant tumor regression and complete responses in PDX models of SCLC and in orthotopic models of established primary lung SCLC and metastatic liver lesions. AMG 757 was well tolerated with no AMG 757-related adverse findings up to the highest tested dose (4.5 mg/kg weekly) in NHP. AMG 757 exhibits an extended half-life in NHP, which is projected to enable intermittent administration in patients. CONCLUSIONS: AMG 757 has a compelling safety and efficacy profile in preclinical studies making it a viable option for targeting DLL3-expressing SCLC tumors in the clinical setting.
Subject(s)
Antibodies, Bispecific , Antibodies, Monoclonal , Gene Expression Regulation, Neoplastic , Intracellular Signaling Peptides and Proteins , Lung Neoplasms , Membrane Proteins , Small Cell Lung Carcinoma , T-Lymphocytes , Animals , Female , Humans , Mice , Antibodies, Bispecific/pharmacology , Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Apoptosis , Cell Proliferation , Gene Expression Regulation, Neoplastic/drug effects , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Lung Neoplasms/drug therapy , Lung Neoplasms/immunology , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Membrane Proteins/antagonists & inhibitors , Mice, Inbred NOD , Mice, SCID , Small Cell Lung Carcinoma/drug therapy , Small Cell Lung Carcinoma/immunology , Small Cell Lung Carcinoma/metabolism , Small Cell Lung Carcinoma/pathology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Phosphatidylinositol 3-kinases (PI3Ks) regulate intracellular signaling events for multiple cell surface receptors. Phosphatidylinositol 3-kinase δ, 1 of 4 class I PI3K isoforms, is primarily found in leukocytes and regulates immune cell functions. Here, we report changes in the immune and digestive systems that were associated with AMG2519493, a highly selective small-molecule PI3Kδ inhibitor. Following 1- or 3-month oral repeat dosing in the cynomolgus monkey, changes were observed in circulating B cells, lymphoid tissues (spleen, lymph nodes, gut-associated lymphoid tissue, tonsil), and the digestive tract. Decreased circulating B cells and lymphoid cellularity in B cell-rich zones in lymphoid tissues were attributed to the intended pharmacologic activity of AMG2519493. Dose- and duration-dependent digestive system toxicity was characterized by inflammation in the large intestine and secondary opportunistic infections restricted to the digestive tract. Digestive tract changes were associated with moribundity and mortality at high-dose levels, and the effect level decreased with increased duration of exposure. These observations demonstrate the role of PI3Kδ in regulation of the immune system and of host resistance to opportunistic infections of the digestive tract.
Subject(s)
Opportunistic Infections , Phosphatidylinositol 3-Kinase , Animals , Immunomodulation , Macaca fascicularis , Phosphatidylinositol 3-KinasesABSTRACT
Mast cells play key roles in allergy, anaphylaxis/anaphylactoid reactions, and defense against pathogens/toxins. These cells contain cytoplasmic granules with a wide spectrum of pleotropic mediators that are released upon activation. While mast cell degranulation (MCD) occurs upon clustering of the IgE receptor bound to IgE and antigen, MCD is also triggered through non-IgE-mediated mechanisms, one of which is via Mas-related G protein-coupled receptor X2 (MRGPRX2). MRGPRX2 can be activated by many basic biogenic amines and peptides. Consequently, MRGPRX2-mediated MCD is an important potential safety liability for peptide therapeutics. To facilitate peptide screening for this liability in early preclinical drug development, a rapid, high-throughput engineered CHO-K1 cell-based MRGPRX2 activation assay was evaluated and compared to histamine release in CD34+ stem cell-derived mature human mast cells as a reference assay, using 30 positive control and 29 negative control peptides for MCD. Both G protein-dependent (Ca2+ endpoint) and -independent (ß-arrestin endpoint) pathways were assessed in the MRGPRX2 activation assay. The MRGPRX2 activation assay had a sensitivity of 100% for both Ca2+ and ß-arrestin endpoints and a specificity of 93% (ß-arrestin endpoint) and 83% (Ca2+ endpoint) compared to histamine release in CD34+ stem cell-derived mature human mast cells. These findings suggest that assessing MRGPRX2 activation in an engineered cell model can provide value as a rapid, high-throughput, economical mechanism-based screening tool for early MCD hazard identification during preclinical safety evaluation of peptide-based therapeutics.
Subject(s)
Cell Degranulation/drug effects , High-Throughput Screening Assays/methods , Mast Cells/drug effects , Nerve Tissue Proteins/metabolism , Peptides/adverse effects , Receptors, G-Protein-Coupled/metabolism , Receptors, Neuropeptide/metabolism , Antigens, CD34/metabolism , Cell Degranulation/immunology , Cell Engineering , Cells, Cultured , Cytotoxicity Tests, Immunologic/methods , Drug Evaluation, Preclinical/methods , Hematopoietic Stem Cell Mobilization , Hematopoietic Stem Cells/metabolism , Histamine/analysis , Histamine/metabolism , Humans , Mast Cells/immunology , Mast Cells/metabolism , Primary Cell Culture , Sensitivity and SpecificityABSTRACT
KRAS is the most frequently mutated oncogene in cancer and encodes a key signalling protein in tumours1,2. The KRAS(G12C) mutant has a cysteine residue that has been exploited to design covalent inhibitors that have promising preclinical activity3-5. Here we optimized a series of inhibitors, using novel binding interactions to markedly enhance their potency and selectivity. Our efforts have led to the discovery of AMG 510, which is, to our knowledge, the first KRAS(G12C) inhibitor in clinical development. In preclinical analyses, treatment with AMG 510 led to the regression of KRASG12C tumours and improved the anti-tumour efficacy of chemotherapy and targeted agents. In immune-competent mice, treatment with AMG 510 resulted in a pro-inflammatory tumour microenvironment and produced durable cures alone as well as in combination with immune-checkpoint inhibitors. Cured mice rejected the growth of isogenic KRASG12D tumours, which suggests adaptive immunity against shared antigens. Furthermore, in clinical trials, AMG 510 demonstrated anti-tumour activity in the first dosing cohorts and represents a potentially transformative therapy for patients for whom effective treatments are lacking.
Subject(s)
Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Lung Neoplasms/drug therapy , Lung Neoplasms/immunology , Piperazines/pharmacology , Piperazines/therapeutic use , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Pyridines/pharmacology , Pyridines/therapeutic use , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Drug Synergism , Humans , Immunotherapy , Inflammation/chemically induced , Inflammation/immunology , Inflammation/pathology , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mice , Phosphorylation/drug effects , Piperazines/administration & dosage , Piperazines/chemistry , Proto-Oncogene Proteins p21(ras)/genetics , Pyridines/administration & dosage , Pyridines/chemistry , Pyrimidines/administration & dosage , Pyrimidines/chemistry , Signal Transduction/drug effects , Treatment Outcome , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunologyABSTRACT
Off-target activities of drug candidates observed during in vitro pharmacological profiling frequently do not translate to adverse events (AEs) in human. This could be because off-target activities do not have functional consequences, are not observed at exposures achieved during clinical testing, or may not translate into clinical outcomes. We report clinical consequences of an off-target activity observed during profiling of AMG 337, a selective inhibitor of the mesenchymal-epithelial transition factor being evaluated for treatment of solid tumors. In our screen of 151 potential off-targets, AMG 337 inhibited only adenosine transporter (AT). During clinical trials, headache emerged as the dose-limiting AE in the first-in-human trial. It was thought that headache was caused by extracellular accumulation of adenosine from inhibition of AT by AMG 337 and subsequent adenosine-mediated vasodilation through adenosine receptors (ARs). Further nonclinical studies were performed to evaluate this hypothesis. AMG 337 inhibited AT function in dog and human cells in vitro and dog and human arteries ex vivo. In a dog telemetry study, AMG 337 caused hypotension, which was reduced by pretreatment with theophylline, an AR antagonist. Overall, nonclinical and clinical data suggested that headache was due to cerebral vasorelaxation caused by AMG 337-mediated inhibition of AT. When subjects were advised to drink coffee, an AR antagonist, prior to AMG 337, the severity of headaches was reduced, allowing them to continue treatment. These findings demonstrate the importance of carefully evaluating clinical observations during early drug development and the value of translational nonclinical studies to investigate the mechanism of action driving clinical observations.
ABSTRACT
We assessed the variability of spleen and mesenteric lymph node (MLN) microscopic observations and the correlations of these observations with other study data from 478 control cynomolgus monkeys from 53 routine nonclinical safety studies. Spleen weight parameters (absolute and relative to body or brain weights) were highly variable both within a control group on an individual study (up to 5.11-fold) and among animals with the same light microscopic observation. Grades for microscopic observations were also highly variable. The most frequent microscopic observations for spleen were changes in the size and number of germinal centers (58%), acidophilic (hyaline) material in lymphoid follicles (52%), and compound lymphoid follicles (20%). The most frequent microscopic observations in the MLN were eosinophil infiltrates (90%), changes in size and number of germinal centers (42%), and brown pigment (21%). The only meaningful relationships ( r2 > 0.3) were positive correlations between reticuloendothelial hyperplasia and malarial pigment in the spleen and between each of these observations and spleen weight parameters. We conclude that determination of test article-related effects on the immune system in routine monkey toxicology studies requires careful consideration and a weight-of-evidence approach due to the low numbers of animals/group, the inherent variability in spleen and MLN parameters, and the infrequent correlation among immune system-related end points.