RESUMO
Innate pattern recognition receptor agonists, including Toll-like receptors (TLRs), alter the tumor microenvironment and prime adaptive antitumor immunity. However, TLR agonists present toxicities associated with widespread immune activation after systemic administration. To design a TLR-based therapeutic suitable for systemic delivery and capable of safely eliciting tumor-targeted responses, we developed immune-stimulating antibody conjugates (ISACs) comprising a TLR7/8 dual agonist conjugated to tumor-targeting antibodies. Systemically administered human epidermal growth factor receptor 2 (HER2)-targeted ISACs were well tolerated and triggered a localized immune response in the tumor microenvironment that resulted in tumor clearance and immunological memory. Mechanistically, ISACs required tumor antigen recognition, Fcγ-receptor-dependent phagocytosis and TLR-mediated activation to drive tumor killing by myeloid cells and subsequent T-cell-mediated antitumor immunity. ISAC-mediated immunological memory was not limited to the HER2 ISAC target antigen since ISAC-treated mice were protected from rechallenge with the HER2- parental tumor. These results provide a strong rationale for the clinical development of ISACs.
Assuntos
Imunoterapia , Neoplasias , Imunidade Adaptativa , Animais , Antígenos de Neoplasias , Imunoterapia/métodos , Camundongos , Neoplasias/tratamento farmacológico , Microambiente TumoralRESUMO
We have used a supervised classification approach to systematically mine a large microarray database derived from livers of compound-treated rats. Thirty-four distinct signatures (classifiers) for pharmacological and toxicological end points can be identified. Just 200 genes are sufficient to classify these end points. Signatures were enriched in xenobiotic and immune response genes and contain un-annotated genes, indicating that not all key genes in the liver xenobiotic responses have been characterized. Many signatures with equal classification capabilities but with no gene in common can be derived for the same phenotypic end point. The analysis of the union of all genes present in these signatures can reveal the underlying biology of that end point as illustrated here using liver fibrosis signatures. Our approach using the whole genome and a diverse set of compounds allows a comprehensive view of most pharmacological and toxicological questions and is applicable to other situations such as disease and development.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Xenobióticos/farmacologia , Animais , Bases de Dados Genéticas , Genômica , Fígado/patologia , Cirrose Hepática/genética , Ratos , Reprodutibilidade dos TestesRESUMO
The economic hurdles of drug development and the emergence of genomic technologies such as chemogenomics are combining to shift the existing paradigms in preclinical drug development. Today, the information gleaned from high content molecular data has begun to augment traditional approaches to the assessment of drug safety. The optimal approach is a hybrid strategy employing chemogenomic data and gene expression-based biomarkers of drug efficacy and toxicity to supplement low content and insensitive methods for risk assessment and mechanistic evaluation of drug candidates. Large reference databases of chemogenomic data are essential to the derivation and validation of accurate and predictive gene expression biomarkers. An example of the development of a predictive biomarker for hepatic bile duct hyperplasia is described herein. As gene expression technologies improve, biomarkers will achieve higher throughput, and become more cost effective and increasingly accurate. This will elevate the value of chemogenomics in drug development, shift attrition to earlier in the process, and reduce the overall cost of drug development. Over the past 2 to 3 years, the transition of chemogenomics from a research tool to a decision-making tool has begun and regulatory agencies are anxiously awaiting implementation of this technology to make faster and more informed evaluations of potential drugs.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Fígado/efeitos dos fármacos , Farmacogenética/métodos , Animais , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Biomarcadores , Carcinógenos/toxicidade , Bases de Dados Factuais , Perfilação da Expressão Gênica , Humanos , Hiperplasia , Técnicas In Vitro , Fígado/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Oncogenes/efeitos dos fármacos , Farmacogenética/estatística & dados numéricos , SegurançaRESUMO
Successful drug discovery requires accurate decision making in order to advance the best candidates from initial lead identification to final approval. Chemogenomics, the use of genomic tools in pharmacology and toxicology, offers a promising enhancement to traditional methods of target identification/validation, lead identification, efficacy evaluation, and toxicity assessment. To realize the value of chemogenomics information, a contextual database is needed to relate the physiological outcomes induced by diverse compounds to the gene expression patterns measured in the same animals. Massively parallel gene expression characterization coupled with traditional assessments of drug candidates provides additional, important mechanistic information, and therefore a means to increase the accuracy of critical decisions. A large-scale chemogenomics database developed from in vivo treated rats provides the context and supporting data to enhance and accelerate accurate interpretation of mechanisms of toxicity and pharmacology of chemicals and drugs. To date, approximately 600 different compounds, including more than 400 FDA approved drugs, 60 drugs approved in Europe and Japan, 25 withdrawn drugs, and 100 toxicants, have been profiled in up to 7 different tissues of rats (representing over 3200 different drug-dose-time-tissue combinations). Accomplishing this task required evaluating and improving a number of in vivo and microarray protocols, including over 80 rigorous quality control steps. The utility of pairing clinical pathology assessments with gene expression data is illustrated using three anti-neoplastic drugs: carmustine, methotrexate, and thioguanine, which had similar effects on the blood compartment, but diverse effects on hepatotoxicity. We will demonstrate that gene expression events monitored in the liver can be used to predict pathological events occurring in that tissue as well as in hematopoietic tissues.
Assuntos
Biotecnologia/métodos , Desenho de Fármacos , Indústria Farmacêutica/métodos , 5-Aminolevulinato Sintetase/biossíntese , Animais , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Automação , Ductos Biliares/patologia , Carmustina/toxicidade , Biologia Computacional , Bases de Dados como Assunto , Relação Dose-Resposta a Droga , Regulação para Baixo , Expressão Gênica , Humanos , Hiperplasia/etiologia , Fígado/efeitos dos fármacos , Masculino , Metotrexato/toxicidade , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Tamanho do Órgão , Farmacologia/métodos , RNA/química , RNA Complementar/metabolismo , Ratos , Ratos Sprague-Dawley , Reticulócitos/citologia , Reticulócitos/metabolismo , Tioguanina/toxicidade , Fatores de Tempo , Distribuição Tecidual , Toxicologia/métodosRESUMO
Toxicogenomics using a reference database can provide a better understanding and prediction of toxicity, largely by creating biomarkers that tie gene expression to actual pathology events. During the course of building a toxicogenomic database, an observation was made that a number of non-steroidal anti-inflammatory compounds (NSAIDs) at supra-pharmacologic doses induced an acute phase response (APR) and displayed hepatic gene expression patterns similar to that of intravenous lipopolysaccharide (LPS). Since NSAIDs are known to cause injury along the gastrointestinal tract, it has been suggested that NSAIDs increase intestinal permeability, allowing LPS and/or bacteria into the systemic circulation and stimulating an APR detectable in the liver. A short term study was subsequently conducted examining the effects of aspirin, indomethacin, ibuprofen, and rofecoxib to rats and a variety of endpoints were examined that included serum levels of inflammatory cytokines, histologic evaluation, and hepatic gene expression. Both indomethacin and ibuprofen injured the gastrointestinal tract, induced an APR, and increased serum levels of LPS, while rofecoxib and aspirin did not affect the GI tract or induce an APR. In treatments that eventually showed a systemic inflammatory response, hepatic expression of many inflammatory genes was noted as early as 6 hours after treatment well before alterations in traditional clinical pathology markers were detected. This finding led to the creation of a hepatic gene expression biomarker of APR that was effectively shown to be an early identifier of imminent inflammatory injury. In terms of the relative gastrointestinal safety and the NSAIDs studied, an important safety distinction can be made between the presumptive efficacious dose and the APR-inducing dose for indomethacin (1-2-fold), ibuprofen (5-fold), and rofecoxib (approximately 250-fold). Our data support the notion that NSAID-induced intestinal injury results in leakage of commensural bacteria and/or LPS into the circulation, provoking a systemic inflammatory response and that hepatic gene expression-based biomarkers can be used as early and sensitive biomarkers of APR onset.
Assuntos
Reação de Fase Aguda/induzido quimicamente , Anti-Inflamatórios não Esteroides/toxicidade , Expressão Gênica/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Reação de Fase Aguda/genética , Reação de Fase Aguda/metabolismo , Animais , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Inibidores de Ciclo-Oxigenase 2/toxicidade , Bases de Dados Factuais , Relação Dose-Resposta a Droga , Ibuprofeno/toxicidade , Indometacina/toxicidade , Intestinos/efeitos dos fármacos , Lactonas/farmacologia , Lipopolissacarídeos/sangue , Fígado/efeitos dos fármacos , Masculino , Permeabilidade/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Sulfonas/farmacologia , Fatores de TempoRESUMO
A large gene expression database has been produced that characterizes the gene expression and physiological effects of hundreds of approved and withdrawn drugs, toxicants, and biochemical standards in various organs of live rats. In order to derive useful biological knowledge from this large database, a variety of supervised classification algorithms were compared using a 597-microarray subset of the data. Our studies show that several types of linear classifiers based on Support Vector Machines (SVMs) and Logistic Regression can be used to derive readily interpretable drug signatures with high classification performance. Both methods can be tuned to produce classifiers of drug treatments in the form of short, weighted gene lists which upon analysis reveal that some of the signature genes have a positive contribution (act as "rewards" for the class-of-interest) while others have a negative contribution (act as "penalties") to the classification decision. The combination of reward and penalty genes enhances performance by keeping the number of false positive treatments low. The results of these algorithms are combined with feature selection techniques that further reduce the length of the drug signatures, an important step towards the development of useful diagnostic biomarkers and low-cost assays. Multiple signatures with no genes in common can be generated for the same classification end-point. Comparison of these gene lists identifies biological processes characteristic of a given class.