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ABSTRACT: Chimeric antigen receptor (CAR) T cells hold promise as a therapy for B-cell-derived malignancies, and despite their impressive initial response rates, a significant proportion of patients ultimately experience relapse. Although recent studies have explored the mechanisms of in vivo CAR T-cell function, little is understood about the activation of surrounding CARneg bystander T cells and their potential to enhance tumor responses. We performed single-cell RNA sequencing on nonhuman primate (NHP) and patient-derived T cells to identify the phenotypic and transcriptomic hallmarks of bystander activation of CARneg T cells following B-cell-targeted CAR T-cell therapy. Using a highly translatable CD20 CAR NHP model, we observed a distinct population of activated CD8+ CARneg T cells emerging during CAR T-cell expansion. These bystander CD8+ CARneg T cells exhibited a unique transcriptional signature with upregulation of natural killer-cell markers (KIR3DL2, CD160, and KLRD1), chemokines, and chemokine receptors (CCL5, XCL1, and CCR9), and downregulation of naïve T-cell-associated genes (SELL and CD28). A transcriptionally similar population was identified in patients after a tisagenlecleucel infusion. Mechanistic studies revealed that interleukin-2 (IL-2) and IL-15 exposure induced bystander-like CD8+ T cells in a dose-dependent manner. In vitro activated and patient-derived T cells with a bystander phenotype efficiently killed leukemic cells through a T-cell receptor-independent mechanism. Collectively, to our knowledge, these data provide the first comprehensive identification and profiling of CARneg bystander CD8+ T cells following B-cell-targeting CAR T-cell therapy and suggest a novel mechanism through which CAR T-cell infusion might trigger enhanced antileukemic responses. Patient samples were obtained from the trial #NCT03369353, registered at www.ClinicalTrials.gov.
Assuntos
Efeito Espectador , Linfócitos T CD8-Positivos , Imunoterapia Adotiva , Animais , Humanos , Imunoterapia Adotiva/métodos , Linfócitos T CD8-Positivos/imunologia , Efeito Espectador/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos B/imunologia , Ativação Linfocitária/imunologia , Macaca mulatta , Linfócitos T Citotóxicos/imunologiaRESUMO
Recombinant adeno-associated virus (rAAV) vector gene delivery systems have demonstrated great promise in clinical trials but continue to face durability and dose-related challenges. Unlike rAAV gene therapy, integrating gene addition approaches can provide curative expression in mitotically active cells and pediatric populations. We explored a novel in vivo delivery approach based on an engineered transposase, Sleeping Beauty (SB100X), delivered as an mRNA within a lipid nanoparticle (LNP), in combination with an rAAV-delivered transposable transgene. This combinatorial approach achieved correction of ornithine transcarbamylase deficiency in the neonatal Spfash mouse model following a single delivery to dividing hepatocytes in the newborn liver. Correction remained stable into adulthood, while a conventional rAAV approach resulted in a return to the disease state. In non-human primates, integration by transposition, mediated by this technology, improved gene expression 10-fold over conventional rAAV-mediated gene transfer while requiring 5-fold less vector. Additionally, integration site analysis confirmed a random profile while specifically targeting TA dinucleotides across the genome. Together, these findings demonstrate that transposable elements can improve rAAV-delivered therapies by lowering the vector dose requirement and associated toxicity while expanding target cell types.
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Interleukin-23 (IL-23) and IL-17 are cytokines currently being targeted in clinical trials. Although inhibition of both of these cytokines is effective for treating psoriasis, IL-12 and IL-23 p40 inhibition attenuates Crohn's disease, whereas IL-17A or IL-17 receptor A (IL-17RA) inhibition exacerbates Crohn's disease. This dichotomy between IL-23 and IL-17 was effectively modeled in the multidrug resistance-1a-ablated (Abcb1a(-/-)) mouse model of colitis. IL-23 inhibition attenuated disease by decreasing colonic inflammation while enhancing regulatory T (Treg) cell accumulation. Exacerbation of colitis by IL-17A or IL-17RA inhibition was associated with severe weakening of the intestinal epithelial barrier, culminating in increased colonic inflammation and accelerated mortality. These data show that IL-17A acts on intestinal epithelium to promote barrier function and provide insight into mechanisms underlying exacerbation of Crohn's disease when IL-17A or IL-17RA is inhibited.
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Colite/imunologia , Interleucina-17/fisiologia , Interleucina-23/fisiologia , Receptores de Interleucina-17/fisiologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Animais , Colite/tratamento farmacológico , Colite/etiologia , Colite/microbiologia , Modelos Animais de Doenças , Progressão da Doença , Epitélio/fisiopatologia , Feminino , Fatores de Transcrição Forkhead/análise , Regulação da Expressão Gênica/imunologia , Infecções por Helicobacter/complicações , Infecções por Helicobacter/imunologia , Imunização Passiva , Imunoglobulina G/uso terapêutico , Subunidade p40 da Interleucina-12/antagonistas & inibidores , Interleucina-17/imunologia , Interleucina-23/imunologia , Subunidade p19 da Interleucina-23/antagonistas & inibidores , Subunidade p19 da Interleucina-23/imunologia , Mucosa Intestinal/fisiopatologia , Camundongos , Camundongos Knockout , Permeabilidade , Receptores de Interleucina-17/antagonistas & inibidores , Receptores de Interleucina-17/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , TranscriptomaRESUMO
The discovery of brown adipose tissue (BAT) as a key regulator of energy expenditure has sparked interest in identifying novel soluble factors capable of activating inducible BAT (iBAT) to combat obesity. Using a high content cell-based screen, we identified fibroblast growth factor 16 (FGF16) as a potent inducer of several physical and transcriptional characteristics analogous to those of both "classical" BAT and iBAT. Overexpression of Fgf16 in vivo recapitulated several of our in vitro findings, specifically the significant induction of the Ucp1 gene and UCP1 protein expression in inguinal white adipose tissue (iWAT), a common site for emergent active iBAT. Despite significant UCP1 up-regulation in iWAT and dramatic weight loss, the metabolic improvements observed due to Fgf16 overexpression in vivo were not the result of increased energy expenditure, as measured by indirect calorimetric assessment. Instead, a pattern of reduced food and water intake, combined with feces replete with lipid and bile acid, indicated a phenotype more akin to that of starvation and intestinal malabsorption. Gene expression analysis of the liver and ileum indicated alterations in several steps of bile acid metabolism, including hepatic synthesis and reabsorption. Histological analysis of intestinal tissue revealed profound abnormalities in support of this conclusion. The in vivo data, together with FGF receptor binding analysis, indicate that the in vivo outcome observed is the likely result of both direct and indirect mechanisms and probably involves multiple receptors. These results highlight the complexity of FGF signaling in the regulation of various metabolic processes.
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Tecido Adiposo Branco/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Termogênese , Proteases Específicas de Ubiquitina/biossíntese , Tecido Adiposo Branco/patologia , Animais , Linhagem Celular , Gorduras na Dieta/efeitos adversos , Gorduras na Dieta/farmacologia , Fatores de Crescimento de Fibroblastos/genética , Humanos , Camundongos , Obesidade/induzido quimicamente , Obesidade/genética , Obesidade/metabolismo , Proteases Específicas de Ubiquitina/genéticaRESUMO
Asthma is a heterogeneous disease characterized by airway inflammation and hyperreactivity. IL-17 receptor A (IL-17RA) is a shared receptor subunit required for activity of IL-17 family cytokines, including IL-17A and IL-25. IL-17A and IL-25 induce different proinflammatory responses, and concentrations are elevated in subjects with asthma. However, the individual contributions of IL-17A and IL-25 to disease pathogenesis are unclear. We explored proinflammatory activities of the IL-17 pathway in models of pulmonary inflammation and assessed its effects on contractility of human bronchial airway smooth muscle. In two mouse models, IL-17RA, IL-17RB, or IL-25 blockade reduced airway inflammation and airway hyperreactivity. Individually, IL-17A and IL-25 enhanced contractility of human bronchial smooth muscle induced by methacholine or carbachol. IL-17A had more pronounced effects on methacholine-induced contractility in bronchial rings from donors with asthma compared with donors without asthma. Blocking the IL-17 pathway via IL-17RA may be a useful therapy for some patients with asthma by reducing pulmonary inflammation and airway hyperreactivity.
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Asma/metabolismo , Receptores de Interleucina-17/fisiologia , Animais , Asma/imunologia , Brônquios/imunologia , Brônquios/patologia , Células Cultivadas , Expressão Gênica , Humanos , Interleucina-17/fisiologia , Interleucinas/fisiologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Muscular , Miócitos de Músculo Liso/imunologia , Miócitos de Músculo Liso/metabolismo , Transdução de SinaisRESUMO
Naive T cell activation involves at least two signals from an APC, one through the TCR via interaction with peptide-MHC complexes and a second through ligation of CD28 with B7 ligands. Following activation, T cells upregulate a host of other membrane-bound costimulatory molecules that can either promote or inhibit further T cell maturation and proliferation. In some cases, it is necessary to attenuate T cell activation to prevent deleterious inflammation, and inhibitory members of the B7/butyrophilin family of ligands have evolved to balance the strong stimuli the activating B7 ligands confer. Human genetic association and in vitro studies have implicated one such ligand, BTNL2, in controlling inflammation at mucosal surfaces. In this study, we show that recombinant mouse BTNL2 modifies B7/CD28 signaling to promote expression of Foxp3, a transcription factor necessary for regulatory T cell (Treg) development and function. BTNL2 blocks Akt-mediated inactivation of Foxo1, a transcription factor necessary for Foxp3 expression. Immunophenotyping and gene profiling reveal that BTNL2-induced Treg share many properties with natural Treg, and in vivo they suppress enteritis induced by mouse effector T cells. These findings describe a mechanism by which environmental Ag-specific Tregs may be induced by APC expressing specific modulators of costimulatory signals.
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Antígenos B7/genética , Diferenciação Celular/efeitos dos fármacos , Fatores de Transcrição Forkhead/genética , Glicoproteínas de Membrana/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Antígenos B7/imunologia , Butirofilinas , Antígenos CD28/genética , Antígenos CD28/imunologia , Feminino , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/imunologia , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Imunofenotipagem , Ativação Linfocitária , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologiaRESUMO
Iron is a trace element important for the proper folding and function of various proteins. Physiological regulation of iron stores is of critical importance for RBC production and antimicrobial defense. Hepcidin is a key regulator of iron levels within the body. Under conditions of iron deficiency, hepcidin expression is reduced to promote increased iron uptake from the diet and release from cells, whereas during conditions of iron excess, induction of hepcidin restricts iron uptake and movement within the body. The cytokine IL-6 is well established as an important inducer of hepcidin. The presence of this cytokine during inflammatory states can induce hepcidin production, iron deficiency, and anemia. In this study, we show that IL-22 also influences hepcidin production in vivo. Injection of mice with exogenous mouse IgG1 Fc fused to the N terminus of mouse IL-22 (Fc-IL-22), an IL-22R agonist with prolonged and enhanced functional potency, induced hepcidin production, with a subsequent decrease in circulating serum iron and hemoglobin levels and a concomitant increase in iron accumulation within the spleen. This response was independent of IL-6 and was attenuated in the absence of the IL-22R-associated signaling kinase, Tyk2. Ab-mediated blockade of hepcidin partially reversed the effects on iron biology caused by IL-22R stimulation. Taken together, these data suggest that exogenous IL-22 regulates hepcidin production to physiologically influence iron usage.
Assuntos
Hepcidinas/fisiologia , Interleucinas/fisiologia , Ferro/metabolismo , Sequência de Aminoácidos , Anemia Ferropriva/sangue , Anemia Ferropriva/induzido quimicamente , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Células Cultivadas , Feminino , Hepatócitos/metabolismo , Hepcidinas/antagonistas & inibidores , Hepcidinas/biossíntese , Hepcidinas/genética , Hepcidinas/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Interleucina-6/fisiologia , Interleucinas/genética , Interleucinas/farmacologia , Interleucinas/toxicidade , Ferro/sangue , Deficiências de Ferro , Síndrome de Job/metabolismo , Fígado/metabolismo , Fígado/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Dados de Sequência Molecular , Fosforilação , Processamento de Proteína Pós-Traducional , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de IgG/deficiência , Receptores de Interleucina/agonistas , Receptores de Interleucina/fisiologia , Proteínas Recombinantes de Fusão/farmacologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Baço/metabolismo , Baço/patologia , TYK2 Quinase/deficiência , TYK2 Quinase/metabolismo , Interleucina 22RESUMO
This continuing education course was designed to provide an overview of the immunologic mechanisms involved in immunogenicity and hypersensitivity reactions following administration of biologics in nonclinical toxicity studies, the methods used to determine whether such reactions are occurring, and the associated clinical and anatomic pathology findings. Hypersensitivity reactions have classically been divided into type I, II, III, and IV reactions; type I and III reactions are those most often observed following administration of biologics. A variety of methods can be used to detect these reactions. Antemortem methods include hematology; detection of antidrug antibodies, circulating immune complexes and complement fragments, and immunoglobulin E in serum; tests for serum complement activity; and evaluation of complement receptor 1 on erythrocytes. Postmortem methods include routine light microscopy and electron microscopy, which can demonstrate typical findings associated with hypersensitivity reactions, and immunohistochemistry, which can detect the presence of immune complexes in tissues, including the detection of the test article. A final determination of whether findings are related to a hypersensitivity reaction in individual animals or across the entire study should rely on the overall weight of evidence, as findings indicative of these reactions are not necessarily consistent across all affected animals.
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Produtos Biológicos/administração & dosagem , Produtos Biológicos/efeitos adversos , Hipersensibilidade a Drogas , Animais , Anticorpos/sangue , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/complicações , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/imunologia , Humanos , Imunoglobulina E/sangueRESUMO
BACKGROUND: Asthma has been considered an immunologic disease mediated by T(H)2 cells and adaptive immunity. However, clinical and experimental observations suggest that additional pathways might regulate asthma, particularly in its nonallergic forms, such as asthma associated with air pollution, stress, obesity, and infection. OBJECTIVES: Our goal was to understand T(H)2 cell-independent conditions that might lead to airway hyperreactivity (AHR), a cardinal feature of asthma. METHODS: We examined a murine model of experimental asthma in which AHR was induced with glycolipid antigens, which activate natural killer T (NKT) cells. RESULTS: In this model AHR developed rapidly when mice were treated with NKT cell-activating glycolipid antigens, even in the absence of conventional CD4(+) T cells. The activated NKT cells directly induced alveolar macrophages to produce IL-33, which in turn activated NKT cells, as well as natural helper cells, a newly described non-T, non-B, innate lymphoid cell type, to increase production of IL-13. Surprisingly, this glycolipid-induced AHR pathway required not only IL-13 but also IL-33 and its receptor, ST2, because it was blocked by an anti-ST2 mAb and was greatly reduced in ST2(-/-) mice. When adoptively transferred into IL-13(-/-) mice, both wild-type natural helper cells and NKT cells were sufficient for the development of glycolipid-induced AHR. CONCLUSION: Because plant pollens, house dust, and some bacteria contain glycolipids that can directly activate NKT cells, these studies suggest that AHR and asthma can fully develop or be greatly enhanced through innate immune mechanisms involving IL-33, natural helper cells, and NKT cells.
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Imunidade Adaptativa , Asma/imunologia , Imunidade Inata , Interleucinas/metabolismo , Linfócitos/imunologia , Transferência Adotiva , Animais , Asma/induzido quimicamente , Asma/metabolismo , Células Dendríticas/imunologia , Modelos Animais de Doenças , Feminino , Galactosilceramidas/administração & dosagem , Glicolipídeos/imunologia , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-13/biossíntese , Interleucina-33 , Interleucinas/biossíntese , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células T Matadoras Naturais/imunologia , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Sphingomonas/imunologia , Células Th2/imunologiaRESUMO
Although chimeric antigen receptor (CAR) T cells have emerged as highly effective treatments for patients with hematologic malignancies, similar efficacy has not been achieved in the context of solid tumors. There are several reasons for this disparity including a) fewer solid tumor target antigens, b) heterogenous target expression amongst tumor cells, c) poor trafficking of CAR T cells to the solid tumor and d) an immunosuppressive tumor microenvironment (TME). Oncolytic viruses have the potential to change this paradigm by a) directly lysing tumor cells and releasing tumor neoantigens, b) stimulating the local host innate immune response to release cytokines and recruit additional innate and adaptive immune cells, c) carrying virus-encoded transgenes to "re-program" the TME to a pro-inflammatory environment and d) promoting an adaptive immune response to the neoantigens in this newly permissive TME. Here we show that the Tumor-Specific Immuno-Gene (T-SIGn) virus NG-347 which encodes IFNα, MIP1α and CD80 synergizes with anti-EGFR CAR T cells as well as anti-HER-2 CAR T cells to clear A549 human tumor xenografts and their pulmonary metastases at doses which are subtherapeutic when each is used as a sole treatment. We show that NG-347 changes the TME to a pro-inflammatory environment resulting in the recruitment and activation of both CAR T cells and mouse innate immune cells. We also show that the transgenes encoded by the virus are critical as synergy is lost in their absence.
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Neoplasias Pulmonares , Receptores de Antígenos Quiméricos , Animais , Antígenos de Neoplasias/genética , Xenoenxertos , Humanos , Imunoterapia Adotiva/métodos , Neoplasias Pulmonares/terapia , Camundongos , Receptores de Antígenos Quiméricos/genética , Linfócitos T , Microambiente TumoralRESUMO
Dickkopf-1 (DKK1), a secreted modulator of Wnt signaling, is overexpressed in many cancers, is often associated with worse clinical outcomes, and has been shown to have immunosuppressive effects. DKN-01 is an IgG4 clinical stage antibody that potently and specifically neutralizes human and murine DKK1 and has recently completed a promising study in combination with pembrolizumab in patients with gastric/gastroesophageal junction cancer. The purpose of this study is to characterize a murine version of DKN-01 (mDKN-01) and to better understand its mechanism of action. We examined the efficacy of mDKN-01 in both melanoma and metastatic breast cancer models. Immune depletion experiments revealed a requirement for natural killer (NK) but not B and T cells for tumor growth inhibition. mDKN-01 treatment promotes the induction of the NK-activating cytokines IL15 and IL33 as well as an enhanced recruitment of CD45+ cells. Other treatment-related changes include a reduction of Gr-1+CD11b+ myeloid-derived suppressor cells (MDSC) in the tumor and spleen and the upregulation of PD-L1 on MDSCs. In addition, mDKN-01 has a marked effect at reducing pulmonary metastases in the mouse 4T1 breast cancer model. Finally, the mDKN-01/anti-PD-1 combination was more effective at inhibiting melanoma growth than mDKN-01 alone. Taken together, our data demonstrate that mDKN-01 has efficacy by blocking the immunosuppressive effects of DKK1 in the tumor microenvironment (TME) and provides insight into the clinical activity observed with DKN-01-based treatment. IMPLICATIONS: mDKN-01 reverses a DKK1-mediated innate immune suppression in the TME and has additive efficacy with a PD-1 inhibitor.
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Anticorpos Monoclonais/uso terapêutico , Imunidade Inata/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Neoplasias/tratamento farmacológico , Animais , Anticorpos Monoclonais/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Camundongos , Neoplasias/patologia , Microambiente TumoralRESUMO
Dickkopf-1 (DKK1) is a secreted modulator of Wnt signaling that is frequently overexpressed in tumors and associated with poor clinical outcomes. DKN-01 is a humanized monoclonal therapeutic antibody that binds DKK1 with high affinity and has demonstrated clinical activity in gastric/gastroesophageal junction (G/GEJ) patients with elevated tumoral expression of DKK1. Here we report on the validation of a DKK1 RNAscope chromogenic in situ hybridization assay to assess DKK1 expression in G/GEJ tumor tissue. To reduce pathologist time, potential pathologist variability from manual scoring and support pathologist decision making, a digital image analysis algorithm that identifies tumor cells and quantifies the DKK1 signal was developed. Following CLIA guidelines the DKK1 RNAscope chromogenic in situ hybridization assay and digital image analysis algorithm were successfully validated for sensitivity, specificity, accuracy, and precision. The DKK1 RNAscope assay in conjunction with the digital image analysis solution is acceptable for prospective screening of G/GEJ adenocarcinoma patients. The work described here will further advance the companion diagnostic development of our DKK1 RNAscope assay and could generally be used as a guide for the validation of RNAscope assays with digital image quantification.
Assuntos
Adenocarcinoma/diagnóstico , Biomarcadores Tumorais/análise , Neoplasias Esofágicas/diagnóstico , Processamento de Imagem Assistida por Computador/métodos , Peptídeos e Proteínas de Sinalização Intercelular/análise , Neoplasias Gástricas/diagnóstico , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Ensaios Clínicos Fase II como Assunto , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Junção Esofagogástrica/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização In Situ/métodos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Pessoa de Meia-Idade , Estudos Multicêntricos como Assunto , Estudos Prospectivos , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Estudos Retrospectivos , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genéticaRESUMO
Therapeutic combinations targeting innate and adaptive immunity and predictive biomarkers of response in esophagogastric cancer (EGC) are needed. We assessed safety and clinical utility of DKN-01 (a novel DKK1-neutralizing IgG4 antibody) combined with pembrolizumab and retrospectively determined DKK1 tumoral expression as a biomarker. Patients with advanced EGC received intravenous DKN-01 (150 or 300 mg) on days 1 and 15 with pembrolizumab 200 mg on day 1 in 21-day cycles. Clinical response was assessed by RECIST v1.1. Association of tumoral DKK1 mRNA expression (H-score: high ≥ upper-tertile, low < upper-tertile) with response was assessed with PD-L1 levels as a covariate. Sixty-three patients received DKN-01 150 mg (n = 2) or 300 mg (n = 61) plus pembrolizumab. Common adverse events were fatigue, anemia, blood alkaline phosphatase elevation, aspartate aminotransferase elevation, and hyponatremia. Among evaluable anti-PD-1/PD-L1-naïve patients receiving DKN-01 300 mg and pembrolizumab, objective response rate (ORR) was 11.4% (5/44) and 18.5% (5/27) in patients with gastroesophageal junction or gastric cancer (GEJ/GC). Among response-evaluable anti-PD-1/PD-L1-naïve patients with GEJ/GC and known tumoral DKK1 expression, ORR was 50% in DKK1-high and 0% in DKK1-low patients, median PFS was 22.1 vs. 5.9 weeks (HR, 0.24; 95% CI, 0.08-0.67), respectively, and median OS was 31.6 weeks vs. 17.4 weeks (HR, 0.41; 95% CI, 0.16-1.07), respectively. Association of DKK1 expression with PFS was independent of PD-L1 expression (adjusted HR, 0.21; 95% CI, 0.06-0.69). DKN-01 combined with pembrolizumab was well tolerated with no new safety signals. Antitumor activity was enriched in anti-PD-1/PD-L1-naïve patients with GEJ/GC whose tumors expressed high DKK1.
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Anticorpos Monoclonais Humanizados/uso terapêutico , Biomarcadores Tumorais/metabolismo , Neoplasias Esofágicas/tratamento farmacológico , Receptor de Morte Celular Programada 1/uso terapêutico , Neoplasias Gástricas/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Humanizados/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
IL-25 (IL-17E) is a unique IL-17 family ligand that promotes Th2-skewed inflammatory responses. Intranasal administration of IL-25 into naive mice induces pulmonary inflammation similar to that seen in patients with allergic asthma, including increases in bronchoalveolar lavage fluid eosinophils, bronchoalveolar lavage fluid IL-5 and IL-13 concentrations, goblet cell hyperplasia, and increased airway hyperresponsiveness. IL-25 has been reported to bind and signal through IL-17RB (IL-17BR, IL-17Rh1). It has been demonstrated recently that IL-17A signals through a heteromeric receptor composed of IL-17RA and IL-17RC. We sought to determine whether other IL-17 family ligands also utilize heteromeric receptor complexes. The required receptor subunits for IL-25 biological activities were investigated in vitro and in vivo using a combination of knockout (KO) mice and antagonistic Abs. Unlike wild-type mice, cultured splenocytes from either IL-17RB KO or IL-17RA KO mice did not produce IL-5 or IL-13 in response to IL-25 stimulation, and both IL-17RB KO and IL-17RA KO mice did not respond to intranasal administration of IL-25. Furthermore, treatment with antagonistic mAbs to either IL-17RB or IL-17RA completely blocked IL-25-induced pulmonary inflammation and airway hyperresponsiveness in naive BALB/c mice, similar to the effects of an antagonistic Ab to IL-25. Finally, a blocking Ab to human IL-17RA prevented IL-25 activity in a primary human cell-based assay. These data demonstrate for the first time that IL-25-mediated activities require both IL-17RB and IL-17RA and provide another example of an IL-17 family ligand that utilizes a heteromeric receptor complex.
Assuntos
Interleucina-17/fisiologia , Interleucinas/fisiologia , Receptores de Interleucina-17/fisiologia , Receptores de Interleucina/fisiologia , Animais , Células Cultivadas , Humanos , Interleucina-17/metabolismo , Interleucinas/deficiência , Interleucinas/genética , Interleucinas/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Ratos Endogâmicos Lew , Receptores de Interleucina/deficiência , Receptores de Interleucina/genética , Receptores de Interleucina-17/deficiência , Receptores de Interleucina-17/genéticaRESUMO
PURPOSE: Angiogenesis plays a critical role in breast cancer development and progression. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that regulates endothelial cell proliferation and survival. We investigated the effects of motesanib, a novel, oral inhibitor of VEGF receptors 1, 2, and 3; platelet-derived growth factor receptor; and Kit receptor, on the growth of xenografts representing various human breast cancer subtypes. EXPERIMENTAL DESIGN: Athymic nude mice were implanted with MCF-7 (luminal) or MDA-MB-231 (mesenchymal) tumor fragments or Cal-51 (mixed/progenitor) tumor cells. Once tumors were established, animals were randomized to receive increasing doses of motesanib alone or motesanib plus cytotoxic chemotherapy (docetaxel, doxorubicin, or tamoxifen). RESULTS: Across all three xenograft models, motesanib treatment resulted in significant dose-dependent reductions in tumor growth, compared with vehicle-treated controls, and in marked reductions in viable tumor fraction and blood vessel density. No significant effect on body weight was observed with compound treatment compared with control-treated animals. Motesanib did not affect the proliferation of tumor cells in vitro. There was a significantly greater reduction in xenograft tumor growth when motesanib was combined with docetaxel (MDA-MB-231 tumors) or with the estrogen receptor modulator tamoxifen (MCF-7 tumors), compared with either treatment alone, but not when combined with doxorubicin (Cal-51 tumors). CONCLUSIONS: Treatment with motesanib alone or in combination with chemotherapy inhibits tumor growth in vivo in various models of human breast cancer. These data suggest that motesanib may have broad utility in the treatment of human breast cancer.
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Inibidores da Angiogênese/uso terapêutico , Indóis/uso terapêutico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Niacinamida/análogos & derivados , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Peso Corporal/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Humanos , Neoplasias Mamárias Experimentais/irrigação sanguínea , Camundongos , Camundongos Nus , Niacinamida/uso terapêutico , Oligonucleotídeos , Distribuição Aleatória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Development of next-generation oncolytic viruses requires the design of vectors that are potently oncolytic, immunogenic in human tumors, and well tolerated in patients. Starting with a joint-region deleted herpes simplex virus 1 (HSV-1) to create large transgene capability, we retained a single copy of the ICP34.5 gene, introduced mutations in UL37 to inhibit retrograde axonal transport, and inserted cell-type-specific microRNA (miRNA) target cassettes in HSV-1 genes essential for replication or neurovirulence. Ten miRNA candidates highly expressed in normal tissues and with low or absent expression in malignancies were selected from a comprehensive profile of 800 miRNAs with an emphasis on protection of the nervous system. Among the genes essential for viral replication identified using a small interfering RNA (siRNA) screen, we selected ICP4, ICP27, and UL8 for miRNA attenuation where a single miRNA is sufficient to potently attenuate viral replication. Additionally, a neuron-specific miRNA target cassette was introduced to control ICP34.5 expression. This vector is resistant to type I interferon compared to ICP34.5-deleted oncolytic HSVs, and in cancer cell lines, the oncolytic activity of the modified vector is equivalent to its parental virus. In vivo, this vector potently inhibits tumor growth while being well tolerated, even at high intravenous doses, compared to parental wild-type HSV-1.
RESUMO
We assessed the tolerability and antitumor activity of solitomab, a bispecific T-cell engager (BiTE®) antibody construct targeting epithelial cell adhesion molecule (EpCAM). Patients with relapsed/refractory solid tumors not amenable to standard therapy received solitomab as continuous IV infusion in a phase 1 dose-escalation study with six different dosing schedules. The primary endpoint was frequency and severity of adverse events (AEs). Secondary endpoints included pharmacokinetics, pharmacodynamics, immunogenicity, and antitumor activity. Sixty-five patients received solitomab at doses between 1 and 96 µg/day for ≥28 days. Fifteen patients had dose-limiting toxicities (DLTs): eight had transient abnormal liver parameters shortly after infusion start or dose escalation (grade 3, n = 4; grade 4, n = 4), and one had supraventricular tachycardia (grade 3); all events resolved with solitomab discontinuation. Six patients had a DLT of diarrhea: four events resolved (grade 3, n = 3; grade 4, n = 1), one (grade 3) was ongoing at the time of treatment-unrelated death, and one (grade 3) progressed to grade 5 after solitomab discontinuation. The maximum tolerated dose was 24 µg/day. Overall, 95% of patients had grade ≥3 treatment-related AEs, primarily diarrhea, elevated liver parameters, and elevated lipase. Solitomab half-life was 4.5 hours; serum levels plateaued within 24 hours. One unconfirmed partial response was observed. In this study of a BiTE® antibody construct targeting solid tumors, treatment of relapsed/refractory EpCAM-positive solid tumors with solitomab was associated with DLTs, including severe diarrhea and increased liver enzymes, which precluded dose escalation to potentially therapeutic levels.
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Designing drugs to treat diseases associated with articular joints, particularly those targeting chondrocytes, is challenging due to unique local environmental constraints including the avascular nature of cartilage, the absence of a closed joint compartment, and a highly cross-linked extracellular matrix. In an effort to address these challenges, we developed a novel strategy to prolong residence time of intra-articularly administered protein therapeutics. Avimer domains are naturally found in membrane polypeptides and mediate diverse protein-protein interactions. Screening of a phage Avimer domain library led to identification of several low affinity type II collagen-binding Avimers. Following several rounds of mutagenesis and reselection, these initial hits were transformed to high affinity, selective type II collagen-binding Avimers. One such Avimer (M26) persisted in rat knees for at least 1 month following intra-articular administration. Fusion of this Avimer to a candidate therapeutic payload, IL-1Ra, yielded a protein construct which simultaneously bound to type II collagen and to IL-1 receptor. In vitro, IL-1Ra_M26 bound selectively to cartilage explants and remained associated even after extensive washing. Binding appeared to occur preferentially to pericellular regions surrounding chondrocytes. An acute intra-articular IL-1-induced IL-6 challenge rat model was employed to assess in vivo pharmacodynamics. Whereas both IL-1Ra_M26 and native IL-1Ra inhibited IL-6 output when co-administered with the IL-1 challenge, only IL-1Ra_M26 inhibited when administered 1 week prior to IL-1 challenge. Collagen-binding Avimers thus represent a promising strategy for enhancing cartilage residence time of protein therapeutics. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1238-1247, 2018.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Artropatias/tratamento farmacológico , Proteínas/administração & dosagem , Animais , Colágeno Tipo II/metabolismo , Feminino , Humanos , Injeções Intra-Articulares , Masculino , Domínios Proteicos , Engenharia de Proteínas , Ratos Endogâmicos Lew , Ratos Sprague-DawleyRESUMO
Glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) has been identified in multiple genome-wide association studies (GWAS) as a contributor to obesity, and GIPR knockout mice are protected against diet-induced obesity (DIO). On the basis of this genetic evidence, we developed anti-GIPR antagonistic antibodies as a potential therapeutic strategy for the treatment of obesity and observed that a mouse anti-murine GIPR antibody (muGIPR-Ab) protected against body weight gain, improved multiple metabolic parameters, and was associated with reduced food intake and resting respiratory exchange ratio (RER) in DIO mice. We replicated these results in obese nonhuman primates (NHPs) using an anti-human GIPR antibody (hGIPR-Ab) and found that weight loss was more pronounced than in mice. In addition, we observed enhanced weight loss in DIO mice and NHPs when anti-GIPR antibodies were codosed with glucagon-like peptide-1 receptor (GLP-1R) agonists. Mechanistic and crystallographic studies demonstrated that hGIPR-Ab displaced GIP and bound to GIPR using the same conserved hydrophobic residues as GIP. Further, using a conditional knockout mouse model, we excluded the role of GIPR in pancreatic ß-cells in the regulation of body weight and response to GIPR antagonism. In conclusion, these data provide preclinical validation of a therapeutic approach to treat obesity with anti-GIPR antibodies.