RESUMO
The objective of the present study was to identify biological signatures of severe coronavirus disease 2019 (COVID-19) predictive of admission in the intensive care unit (ICU). Over 170 immunological markers were investigated in a 'discovery' cohort (n = 98 patients) of the Lausanne University Hospital (LUH-1). Here we report that 13 out of 49 cytokines were significantly associated with ICU admission in the three cohorts (P < 0.05 to P < 0.001), while cellular immunological markers lacked power in discriminating between ICU and non-ICU patients. The cytokine results were confirmed in two 'validation' cohorts, i.e. the French COVID-19 Study (FCS; n = 62) and a second LUH-2 cohort (n = 47). The combination of hepatocyte growth factor (HGF) and C-X-C motif chemokine ligand 13 (CXCL13) was the best predictor of ICU admission (positive and negative predictive values ranging from 81.8% to 93.1% and 85.2% to 94.4% in the 3 cohorts) and occurrence of death during patient follow-up (8.8 fold higher likelihood of death when both cytokines were increased). Of note, HGF is a pleiotropic cytokine with anti-inflammatory properties playing a fundamental role in lung tissue repair, and CXCL13, a pro-inflammatory chemokine associated with pulmonary fibrosis and regulating the maturation of B cell response. Up-regulation of HGF reflects the most powerful counter-regulatory mechanism of the host immune response to antagonize the pro-inflammatory cytokines including CXCL13 and to prevent lung fibrosis in COVID-19 patients.
Assuntos
COVID-19/imunologia , COVID-19/mortalidade , Quimiocina CXCL13/imunologia , Citocinas/imunologia , Fator de Crescimento de Hepatócito/imunologia , Biomarcadores/sangue , Linfócitos T CD4-Positivos , COVID-19/sangue , COVID-19/diagnóstico , Quimiocina CXCL13/genética , Citocinas/sangue , Fator de Crescimento de Hepatócito/genética , Hospitalização , Humanos , Unidades de Terapia Intensiva , Fibrose Pulmonar , SARS-CoV-2/isolamento & purificação , Índice de Gravidade de DoençaRESUMO
BACKGROUND: The receptor encoded by the MET oncogene and its ligand Hepatocyte Growth Factor (HGF) are at the core of the invasive-metastatic behavior. In a number of instances genetic alterations result in ligand-independent onset of malignancy (MET addiction). More frequently, ligand stimulation of wild-type MET contributes to progression toward metastasis (MET expedience). Thus, while MET inhibitors alone are effective in the first case, combination therapy with ligand inhibitors is required in the second condition. METHODS: In this paper, we generated hybrid molecules gathering HGF and MET inhibitory properties. This has been achieved by 'head-to-tail' or 'tail-to-head' fusion of a single chain Fab derived from the DN30 MET antibody with a recombinant 'ad-hoc' engineered MET extracellular domain (decoyMET), encompassing the HGF binding site but lacking the DN30 epitope. RESULTS: The hybrid molecules correctly bind MET and HGF, inhibit HGF-induced MET downstream signaling, and quench HGF-driven biological responses, such as growth, motility and invasion, in cancer cells of different origin. Two metastatic models were generated in mice knocked-in by the human HGF gene: (i) orthotopic transplantation of pancreatic cancer cells; (ii) subcutaneous injection of primary cells derived from a cancer of unknown primary. Treatment with hybrid molecules strongly affects time of onset, number, and size of metastatic lesions. CONCLUSION: These results provide a strategy to treat metastatic dissemination driven by the HGF/MET axis.
Assuntos
Imunoconjugados/farmacologia , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Células A549 , Animais , Sítios de Ligação de Anticorpos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Fator de Crescimento de Hepatócito/imunologia , Humanos , Imunoconjugados/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Camundongos , Camundongos SCID , Metástase Neoplásica , Neoplasias/imunologia , Proteínas Proto-Oncogênicas c-met/imunologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Intercellular communication through the secretion of soluble factors plays a vital role in a wide range of biological processes (e.g., homeostasis, immune response), yet identification and quantification of many of these factors can be challenging due to their degradation or sequestration in cell culture media prior to analysis. Here, we present a customizable bead-based system capable of simultaneously binding to live cells (through antibody-mediated cell tethering) and capturing cell-secreted molecules. Our functionalized beads capture secreted molecules (e.g., hepatocyte growth factor secreted by fibroblasts) that are diminished when sampled via traditional supernatant analysis techniques (p < 0.05), effectively rescuing a reduced signal in the presence of neutralizing components in the cell culture media. Our system enables capture and analysis of molecules integral to chemical communication that would otherwise be markedly decreased prior to analysis.
Assuntos
Antígenos de Superfície/metabolismo , Fator de Crescimento de Hepatócito/análise , Microesferas , Anticorpos/imunologia , Antígenos de Superfície/análise , Antígenos de Superfície/imunologia , Linhagem Celular , Meios de Cultura/química , Fibroblastos/citologia , Fibroblastos/metabolismo , Fluoresceínas/química , Fator de Crescimento de Hepatócito/imunologia , Fator de Crescimento de Hepatócito/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Imunoensaio/métodos , Antígenos Thy-1/análise , Antígenos Thy-1/imunologia , Antígenos Thy-1/metabolismoRESUMO
The objective of this manuscript is to provide the reader with two examples on how to present an immunogenicity risk assessment for a PEGylated therapeutic as part of Investigational New Drug (IND) application or during other stages of the drug development process. In order to provide context to the bioanalytical strategies used to support the PEGylated therapeutics presented here, a brief summary of information available for marketed PEGylated biologics is provided. Two case studies are presented, a PEGylated enzyme and a PEGylated growth factor. For the former, the risk assessment covers how to deal with a narrow therapeutic window and suggestions to utilize a PD marker as surrogate for neutralizing antibody assessments in Phase I. The latter has recommendations on additional analytes that should be monitored to mitigate risk of immunogenicity to endogenous counterparts.
Assuntos
Anticorpos Neutralizantes/imunologia , Produtos Biológicos/imunologia , Fator de Crescimento de Hepatócito/imunologia , Fenilalanina Amônia-Liase/imunologia , Polietilenoglicóis , Succinimidas/imunologia , Animais , Produtos Biológicos/química , Produtos Biológicos/toxicidade , Composição de Medicamentos , Fator de Crescimento de Hepatócito/química , Fator de Crescimento de Hepatócito/toxicidade , Humanos , Fenilalanina Amônia-Liase/química , Fenilalanina Amônia-Liase/toxicidade , Polietilenoglicóis/química , Polietilenoglicóis/toxicidade , Medição de Risco , Succinimidas/química , Succinimidas/toxicidadeRESUMO
Although human adipose tissue-derived stromal vascular fraction (SVF) has been considered a promising source of stem cells, its characteristics relevant to treatment of a damaged liver have not been fully elucidated. In the present study, we sought to characterize the property of human SVF and determine the therapeutic utility of SVF in the liver cirrhosis model. We performed microarray, quantitative (q)-PCR experiments, and in vivo therapeutic assays using a liver cirrhotic mouse model. q-PCR results revealed that hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF)-A, Interleukin (IL)-10 and microRNA (miR)-146 were more highly upregulated in SVF than in human adipose-derived mesenchymal stem cells (ASCs). The SVF culture medium (CM) inhibited the activation of hepatic stellate cells in vitro. Injection of SVF significantly suppressed TAA-induced liver fibrosis and repaired liver function by inhibition of infiltrating inflammatory cells and induction of capillary/hepatocyte regeneration in vivo. Injection of IL-10 siRNA treated SVF cells decreased anti-inflammation and anti-fibrotic effects in TAA-induced mice liver. Our data indicate that SVF show a high anti-inflammatory property for treating fibrotic liver diseases through IL-10 secretion. Therefore, SVF might be a novel therapeutic alternative for the treatment of liver cirrhosis in clinical settings.
Assuntos
Inflamação/terapia , Cirrose Hepática/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Animais , Linhagem Celular , Células Cultivadas , Modelos Animais de Doenças , Fator de Crescimento de Hepatócito/análise , Fator de Crescimento de Hepatócito/imunologia , Humanos , Inflamação/imunologia , Inflamação/fisiopatologia , Interleucina-10/análise , Interleucina-10/imunologia , Fígado/imunologia , Fígado/fisiopatologia , Cirrose Hepática/imunologia , Cirrose Hepática/fisiopatologia , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos Endogâmicos NOD , Camundongos SCID , Fator A de Crescimento do Endotélio Vascular/análise , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
: YYB-101 is a humanized rabbit anti-human hepatocyte growth factor (HGF)-neutralizing antibody currently in clinical trial. To test the effect of HGF neutralization with antibody on anti-cancer T cell immunity, we generated surrogate antibodies that are reactive to the mouse homologue of the epitope targeted by YYB-101. First, we immunized a chicken with human HGF and monitored changes in the B cell repertoire by next-generation sequencing (NGS). We then extracted the VH gene repertoire from the NGS data, clustered it into components by sequence homology, and classified the components by the change in the number of unique VH sequences and the frequencies of the VH sequences within each component following immunization. Those changes should accompany the preferential proliferation and somatic hypermutation or gene conversion of B cells encoding HGF-reactive antibodies. One component showed significant increases in the number and frequencies of unique VH sequences and harbored genes encoding antibodies that were reactive to human HGF and competitive with YYB-101 for HGF binding. Some of the antibodies also reacted to mouse HGF. The selected VH sequences shared 98.3% identity and 98.9% amino acid similarity. It is therefore likely that the antibodies encoded by them all react to the epitope targeted by YYB-101.
Assuntos
Anticorpos/genética , Anticorpos/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito B/imunologia , Fator de Crescimento de Hepatócito/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Sequência de Aminoácidos , Animais , Anticorpos/química , Linfócitos B/imunologia , Linfócitos B/metabolismo , Galinhas , Mapeamento de Epitopos/métodos , Biblioteca Gênica , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , CamundongosRESUMO
BACKGROUND: Cancer is a complex disease with profound genomic alterations and extensive heterogeneity. Recent studies on large-scale genomics have shed light on the impact of core oncogenic pathways, which are frequently dysregulated in a wide spectrum of cancer types. Aberrant activation of the hepatocyte growth factor (HGF) signaling axis has been associated with promoting various oncogenic programs during tumor initiation, progression, and treatment resistance. As a result, HGF-targeted therapy has emerged as an attractive therapeutic approach. However, recent clinical trials involving HGF-targeted therapies have demonstrated rather disappointing results. Thus, an alternative, in-depth assessment of new patient stratification is necessary to shift the current clinical course. METHODS: To address such challenges, we have evaluated the therapeutic efficacy of YYB-101, an HGF-neutralizing antibody, in a series of primary glioblastoma stem cells (GSCs) both in vitro and in vivo. Furthermore, we performed genome and transcriptome analysis to determine genetic and molecular traits that exhibit therapeutic susceptibility to HGF-mediated therapy. RESULTS: We have identified several differentially expressed genes, including MET, KDR, and SOX3, which are associated with tumor invasiveness, malignancy, and unfavorable prognosis in glioblastoma patients. We also demonstrated the HGF-MET signaling axis as a key molecular determinant in GSC invasion, and we discovered that a significant association in HGF expression existed between mesenchymal phenotype and immune cell recruitment. CONCLUSIONS: Upregulation of MET and mesenchymal cellular state are essential in generating HGF-mediated therapeutic responses. Our results provide an important framework for evaluating HGF-targeted therapy in future clinical settings.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genômica/métodos , Glioblastoma/tratamento farmacológico , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Transcriptoma , Animais , Apoptose , Movimento Celular , Proliferação de Células , Feminino , Glioblastoma/genética , Glioblastoma/patologia , Fator de Crescimento de Hepatócito/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fenótipo , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Immunosuppression following lung transplantation is a key aspect to the graft's survival. However, the well-known complications that are caused by immunosuppressive regimens present an opportunity to study ways to minimize the usage of these drugs. Recently, a promising discovery has been made pertaining to the immunomodulatory effects of adipose tissue-derived mesenchymal stem cells (ADMSCs) through their secretion of hepatocyte growth factor. In the hopes of mitigating the adverse effects of standard immunosuppressive regimens, our study aims to investigate the effects of ADMSCs on the immune response utilizing a rat lung transplantation model. METHODS: Each rat's own ADMSCs were intravenously administered immediately after orthotopic left lung transplantation. The experimental subjects were divided into four groups: 1) control group (group C) was administered no treatment following transplantation; 2) ADMSC group (group A), administered a single intravenous injection of ADMSCs following transplantation; 3) tacrolimus group (group T), administered tacrolimus (0.5 mg/kg) every 24 h following transplantation; and 4) ADMSC and tacrolimus group (AT group) administered a single intravenous injection of ADMSCs in combination with tacrolimus every 24 h following transplantation. RESULTS: The histologically proven rejection grade in group AT was significantly lower than that in group T. The serum levels of hepatocyte growth factor and the expression of cMet in group AT accompanied by low CD40 expression were also significantly higher than those of the lung grafts of group T. CONCLUSIONS: These results suggest that co-administration of ADMSCs with tacrolimus is a beneficial therapeutic approach in lung transplantation.
Assuntos
Tecido Adiposo/citologia , Rejeição de Enxerto/prevenção & controle , Imunoterapia/métodos , Transplante de Pulmão/efeitos adversos , Transplante de Células-Tronco Mesenquimais/métodos , Administração Intravenosa , Animais , Terapia Combinada/métodos , Modelos Animais de Doenças , Rejeição de Enxerto/sangue , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Fator de Crescimento de Hepatócito/sangue , Fator de Crescimento de Hepatócito/imunologia , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Imunossupressores/administração & dosagem , Imunossupressores/efeitos adversos , Pulmão/patologia , Pulmão/cirurgia , Masculino , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismo , Ratos , Ratos Endogâmicos Lew , Tacrolimo/administração & dosagem , Tacrolimo/efeitos adversos , Transplante Autólogo/métodos , Resultado do TratamentoRESUMO
Immunotherapies have revolutionized medical oncology following the remarkable and, in some cases, unprecedented outcomes observed in certain groups of patients with cancer. Combination with other therapeutic modalities, including anti-angiogenic agents, is one of the many strategies currently under investigation to improve the response rates and duration of immunotherapies. Such a strategy might seem counterintuitive given that anti-angiogenic agents can increase tumour hypoxia and reduce the number of blood vessels within tumours. Herein, we review the additional effects mediated by drugs targeting VEGF-dependent signalling and other pathways, such as those mediated by angiopoietin 2 or HGF, which might increase the efficacy of immunotherapies. In addition, we discuss the seldom considered possibility that immunotherapies, and immune-checkpoint inhibitors in particular, might increase the efficacy of anti-angiogenic or other types of antivascular therapies and/or promote changes in the tumour vasculature. In short, we propose that interactions between both therapeutic modalities could be considered a 'two-way street'.
Assuntos
Imunoterapia/tendências , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Inibidores da Angiogênese/uso terapêutico , Angiopoietina-2/antagonistas & inibidores , Angiopoietina-2/imunologia , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Fator de Crescimento de Hepatócito/imunologia , Humanos , Fatores Imunológicos , Neoplasias/imunologia , Neovascularização Patológica/imunologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
MP0250 is a multi-domain drug candidate currently being tested in clinical trials for the treatment of cancer. It comprises one anti-vascular endothelial growth factor-A (VEGF-A), one anti-hepatocyte growth factor (HGF), and two anti-human serum albumin (HSA) DARPin® domains within a single polypeptide chain. While there is first clinical validation of a single-domain DARPin® drug candidate, little is known about DARPin® drug candidates comprising multiple domains. Here, we show that MP0250 can be expressed at 15 g/L in soluble form in E. coli high cell-density fermentation, it is stable in soluble/frozen formulation for 2 years as assessed by reverse phase HPLC, it has picomolar potency in inhibiting VEGF-A and HGF in ELISA and cellular assays, and its domains are simultaneously active as shown by surface plasmon resonance. The inclusion of HSA-binding DARPin® domains leads to a favorable pharmacokinetic profile in mouse and cynomolgus monkey, with terminal half-lives of â¼ 30 hours in mouse and â¼ 5 days in cynomolgus monkey. MP0250 is thus a highly potent drug candidate that could be particularly useful in oncology. Beyond MP0250, the properties of MP0250 indicate that multi-domain DARPin® proteins can be valuable next-generation drug candidates.
Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Antineoplásicos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Administração Intravenosa , Animais , Repetição de Anquirina/genética , Repetição de Anquirina/imunologia , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Desenho de Fármacos , Feminino , Meia-Vida , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/imunologia , Humanos , Infusões Intravenosas , Macaca fascicularis , Masculino , Camundongos Endogâmicos BALB C , Ligação Proteica/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacocinética , Albumina Sérica Humana/genética , Albumina Sérica Humana/imunologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
Craniofacial mesenchymal stem cells (MSCs), isolated from an abundant and accessible source of craniofacial tissues, possess self-renewal and multilineage differentiation potential. It has been reported that craniofacial MSCs show elevated proliferation and regeneration capacities compared to bone marrow mesenchymal stem cells (BMMSCs). Furthermore, the immunomodulatory property has generated an emerging multidisciplinary research field that translates MSC-based therapies to the clinic for the treatment of inflammatory and autoimmune diseases. Due to tremendous unmet clinical needs, it was extensively investigated how craniofacial MSCs impose their therapeutic effects, especially by interacting with immune cells. Mechanically, MSCs take advantage of a variety of pathways to regulate immune cells, including paracrine signaling such as transforming growth factor (TGF)-ß and hepatocyte growth factor (HGF) pathways, and cell-cell contact Fas/FasL signaling-induced apoptosis. In return, immune cells attenuate MSC function by secreting inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1ß. This perspective review critically discusses the interaction of craniofacial MSCs with the immune milieu, as well as the underlying molecular mechanism contributing to the future improved therapeutic effects of craniofacial MSCs.
Assuntos
Doenças Autoimunes/terapia , Diferenciação Celular/imunologia , Proliferação de Células , Ossos Faciais/imunologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Ossos Faciais/patologia , Fator de Crescimento de Hepatócito/imunologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Inflamação/terapia , Interleucina-1beta/imunologia , Células-Tronco Mesenquimais/patologia , Comunicação Parácrina/imunologia , Fator de Crescimento Transformador beta/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
MST1 deficiency causes T and B cell lymphopenia, resulting in combined immunodeficiency. However, MST1-deficient patients also exhibit autoimmune-like symptoms such as hypergammaglobulinemia and autoantibody production. Recent studies have shown that the autoimmune responses observed in MST1-deficient patients were most likely attributable to defective regulatory T (Treg) cells instead of intrinsic signals in MST1-lacking B cells. Nevertheless, it is not determined how MST1 deficiency in T cells breaks B cell tolerance and causes systemic autoimmune-like phenotypes. In this study, we confirmed that Mst1-/- mice developed hypergammaglobulinemia associated with increased levels of IgG, IgA, and IgE. We also showed that uncontrolled B cell responses were resulted from the IL-4-rich environment created by CD4+ T cells. Defective MST1-FOXO1 signaling down-regulated Treg cells, resulting in the collapse of immune tolerance where the populations of Th2 and T follicular helper cells expanded. In conclusion, we suggest that MST1 acts as a molecular brake to maintain immune tolerance by regulating T cell-mediated B cell activation.
Assuntos
Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Fator de Crescimento de Hepatócito/imunologia , Hipergamaglobulinemia/imunologia , Interleucina-4/imunologia , Proteínas Proto-Oncogênicas/imunologia , Animais , Fator de Crescimento de Hepatócito/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas/deficiênciaRESUMO
BACKGROUND: Mesenchymal stromal cells (MSCs) have emerged as a more beneficial alternative to conventional therapy and may offer a potential cure for unmet medical needs. MSCs are known to possess strong immunomodulatory and anti-inflammatory properties. Moreover, they promote angiogenesis and tissue regeneration through the secretion of trophic factors. For these reasons, the past decade witnessed a sharp increase in the number of clinical trials conducted with stem cells for various vascular diseases requiring angiogenesis. In this study, we evaluated the in vitro angiogenic potency of Stempeucel®, which is an allogeneic pooled human bone marrow-derived mesenchymal stromal cell (phBMMSC) product. We previously established the safety of Stempeucel® in our pre-clinical studies, and clinical trials conducted for critical limb ischaemia and acute myocardial infarction. METHODS: Because the proposed mechanism of action of phBMMSCs is mainly through the secretion of pro-angiogenic cytokines, we developed a surrogate potency assay by screening various batches of large-scale expanded phBMMSCs for the expression of angiogenic factors and cytokines through gene expression and growth factor analyses, followed by in vitro functional assays. RESULTS: The well characterized angiogenic vascular endothelial growth factor (VEGF) was selected and quantified in twenty six manufactured batches of phBMMSCs to establish consistency following the United States Food and Drug Administration recommendations. According to recommendations 21 CFR 211.165(e) and 211.194(a)(2), we also established and documented the specificity and reproducibility of the test methods employed through validation. Moreover, we also attempted to elucidate the mechanism of action of the cell population to ensure appropriate biological activity. The functional role of VEGF has been established through in vitro angiogenic assays and a dose-dependent correlation was observed with in vitro functional results. CONCLUSIONS: The data generated from this study suggest the selection of VEGF as a single surrogate marker to test the angiogenic potency of phBMMSCs. Our study reports the quantification of VEGF in twenty six batches of large-scale manufactured phBMMSCs, and a concentration-dependent correlation of secreted VEGF to endothelial cell functions of migration, proliferation and tube formation, in the conditioned medium obtained from nine phBMMSC batches. To our cognizance, this is the first study in which a single angiogenic factor (VEGF) has been qualified as a surrogate potency marker through all three in vitro functional assays to determine the angiogenic potency of the phBMMSC population.
Assuntos
Indutores da Angiogênese/imunologia , Bioensaio/normas , Células da Medula Óssea/imunologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Fator A de Crescimento do Endotélio Vascular/imunologia , Indutores da Angiogênese/química , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quimiocina CXCL12/genética , Quimiocina CXCL12/imunologia , Meios de Cultivo Condicionados/química , Expressão Gênica , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/imunologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/imunologia , Humanos , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Cultura Primária de Células , Ribonuclease Pancreático/genética , Ribonuclease Pancreático/imunologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/imunologia , Transplante Homólogo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Dysregulated receptor tyrosine kinase signaling in human cancer cells leads to tumor progression, invasion and metastasis. The receptor tyrosine kinase cMET is frequently overexpressed in cancer tissue, and activation of cMET signaling is related to drug resistance and the processes of carcinogenesis, invasion and metastasis. For that reason, cMET and its ligand, hepatocyte growth factor (HGF), are considered prime targets for the development of anticancer drugs. At least eight anti-cMET and four anti-HGF antibodies have been tested or are being tested in clinical trials. However, to date none of these HGF/cMET inhibitors have shown significant efficacy in clinical trials. Furthermore, no receptor tyrosine kinase inhibitors primarily targeting cMET have been approved. Given that neutralization of HGF or cMET does not cause significant adverse effects, inhibition of the HGF/cMET signaling pathway appears to be safe. In this review, we summarized the completed and ongoing clinical trials testing antibody- or protein-based anticancer drugs targeting cMET and HGF.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Animais , Anticorpos Neutralizantes/efeitos adversos , Anticorpos Neutralizantes/farmacologia , Anticorpos Neutralizantes/uso terapêutico , Antineoplásicos Imunológicos/efeitos adversos , Antineoplásicos Imunológicos/uso terapêutico , Ensaios Clínicos como Assunto , Fator de Crescimento de Hepatócito/imunologia , Humanos , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-met/imunologia , Proteínas Recombinantes de Fusão/efeitos adversos , Proteínas Recombinantes de Fusão/uso terapêuticoRESUMO
Hepatocyte growth factor (HGF) and its receptor, cMET, play critical roles in cell proliferation, angiogenesis and invasion in a wide variety of cancers. We therefore examined the anti-tumor activity of the humanized monoclonal anti-HGF antibody, YYB-101, in nude mice bearing human glioblastoma xenografts as a single agent or in combination with temozolomide. HGF neutralization, The extracellular signal-related kinases 1 and 2 (ERK1/2) phosphorylation, and HGF-induced scattering were assessed in HGF-expressing cell lines treated with YYB-101. To support clinical development, we also evaluated the preclinical pharmacokinetics and toxicokinetics in cynomolgus monkeys, and human and cynomolgus monkey tissue was stained with YYB-101 to test tissue cross-reactivity. We found that YYB-101 inhibited cMET activation in vitro and suppressed tumor growth in the orthotopic mouse model of human glioblastoma. Combination treatment with YYB-101 and temozolomide decreased tumor growth and increased overall survival compared with the effects of either agent alone. Five cancer-related genes (TMEM119, FST, RSPO3, ROS1 and NBL1) were overexpressed in YYB-101-treated mice that showed tumor regrowth. In the tissue cross-reactivity assay, critical cross-reactivity was not observed. The terminal elimination half-life was 21.7 days. Taken together, the in vitro and in vivo data demonstrated the anti-tumor efficacy of YYB-101, which appeared to be mediated by blocking the HGF/cMET interaction. The preclinical pharmacokinetics, toxicokinetics and tissue cross-reactivity data support the clinical development of YYB-101 for advanced cancer.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Antineoplásicos Imunológicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Neutralizantes/efeitos adversos , Anticorpos Neutralizantes/imunologia , Antineoplásicos Imunológicos/efeitos adversos , Antineoplásicos Imunológicos/uso terapêutico , Cães , Feminino , Células Hep G2 , Fator de Crescimento de Hepatócito/imunologia , Humanos , Macaca fascicularis , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/imunologiaRESUMO
Hepatocyte growth factor (HGF) is a pleiotropic cytokine composed of an α-chain and a ß-chain, and these chains contain four kringle domains and a serine protease-like structure, respectively. The receptor for HGF was identified as the c-met proto-oncogene product of transmembrane receptor tyrosine kinase. HGF-induced signaling through the receptor Met provokes dynamic biological responses that support morphogenesis, regeneration, and the survival of various cells and tissues, which includes hepatocytes, renal tubular cells, and neurons. Characterization of tissue-specific Met knockout mice has further indicated that the HGF-Met system modulates immune cell functions and also plays an inhibitory role in the progression of chronic inflammation and fibrosis. However, the biological actions that are driven by the HGF-Met pathway all play a role in the acquisition of the malignant characteristics in tumor cells, such as invasion, metastasis, and drug resistance in the tumor microenvironment. Even though oncogenic Met signaling remains the major research focus, the HGF-Met axis has also been implicated in infectious diseases. Many pathogens try to utilize host HGF-Met system to establish comfortable environment for infection. Their strategies are not only simply change the expression level of HGF or Met, but also actively hijack HGF-Met system and deregulating Met signaling using their pathogenic factors. Consequently, the monitoring of HGF and Met expression, along with real-time detection of Met activation, can be a beneficial biomarker of these infectious diseases. Preclinical studies designed to address the therapeutic significance of HGF have been performed on injury/disease models, including acute tissue injury, chronic fibrosis, and cardiovascular and neurodegenerative diseases. Likewise, manipulating the HGF-Met system with complete control will lead to a tailor made treatment for those infectious diseases.
Assuntos
Fator de Crescimento de Hepatócito/imunologia , Fator de Crescimento de Hepatócito/fisiologia , Infecções/imunologia , Infecções/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Bactérias/imunologia , Bactérias/metabolismo , Bactérias/patogenicidade , Fator de Crescimento de Hepatócito/metabolismo , Fator de Crescimento de Hepatócito/uso terapêutico , Hepatócitos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-met/deficiência , Proteínas Proto-Oncogênicas c-met/genética , Transdução de Sinais , Vírus/imunologia , Vírus/metabolismo , Vírus/patogenicidadeRESUMO
Human UC-MSCs are regarded as an attractive alternative to BM-MSCs for clinical applications due to their easy preparation, higher proliferation and lower immunogenicity. However, the mechanisms underlying immune suppression by UC-MSCs are still unclear. We studied the mechanism of inhibition by UC-MSCs during the differentiation of monocytes into DCs and focused on the specific source and the role of the involved cytokines. We found that UC-MSCs suppressed monocyte differentiation into DCs and instructed monocytes towards other cell types, with clear decreases in the expression of co-stimulatory molecules, in the secretion of inflammatory factors and in allostimulatory capacity. IL6, HGF and IL10 might be involved in this process because they were detected at higher levels in a coculture system. UC-MSCs produce IL-6 and HGF, and neutralization of IL-6 and HGF reversed the suppressive effect of UC-MSCs. IL10 was not produced by UC-MSCs but was exclusively produced by monocytes after exposure to UC-MSCs, IL-6 or HGF. In summary, we found that the UC-MSC-mediated inhibitory effect was dependent on IL6 and HGF secreted by UC-MSCs and that this effect induced monocyte-derived cells to produce IL10, which might indirectly strengthen the suppressive effect of UC-MSCs.
Assuntos
Comunicação Celular , Sangue Fetal/metabolismo , Fator de Crescimento de Hepatócito/imunologia , Interleucina-10/imunologia , Interleucina-6/imunologia , Células-Tronco Mesenquimais/metabolismo , Monócitos/metabolismo , Diferenciação Celular , Técnicas de Cocultura , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Sangue Fetal/citologia , Regulação da Expressão Gênica , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Células-Tronco Mesenquimais/citologia , Monócitos/citologia , Fenótipo , Cultura Primária de Células , Transdução de SinaisRESUMO
Neutrophils need to penetrate the perivascular basement membrane for successful extravasation into inflamed tissue, but this process is incompletely understood. Recent findings have associated mammalian sterile 20-like kinase 1 (MST1) loss of function with a human primary immunodeficiency disorder, suggesting that MST1 may be involved in immune cell migration. Here, we have shown that MST1 is a critical regulator of neutrophil extravasation during inflammation. Mst1-deficient (Mst1-/-) neutrophils were unable to migrate into inflamed murine cremaster muscle venules, instead persisting between the endothelium and the basement membrane. Mst1-/- neutrophils also failed to extravasate from gastric submucosal vessels in a murine model of Helicobacter pylori infection. Mechanistically, we observed defective translocation of VLA-3, VLA-6, and neutrophil elastase from intracellular vesicles to the surface of Mst1-/- neutrophils, indicating that MST1 is required for this crucial step in neutrophil transmigration. Furthermore, we found that MST1 associates with the Rab27 effector protein synaptotagmin-like protein 1 (JFC1, encoded by Sytl1 in mice), but not Munc13-4, thereby regulating the trafficking of Rab27-positive vesicles to the cellular membrane. Together, these findings highlight a role for MST1 in vesicle trafficking and extravasation in neutrophils, providing an additional mechanistic explanation for the severe immune defect observed in patients with MST1 deficiency.
Assuntos
Fator de Crescimento de Hepatócito/imunologia , Neutrófilos/imunologia , Proteínas Proto-Oncogênicas/imunologia , Vesículas Secretórias/imunologia , Migração Transendotelial e Transepitelial/imunologia , Músculos Abdominais/irrigação sanguínea , Músculos Abdominais/imunologia , Animais , Membrana Basal/imunologia , Transporte Biológico Ativo/genética , Transporte Biológico Ativo/imunologia , Mucosa Gástrica/química , Mucosa Gástrica/imunologia , Fator de Crescimento de Hepatócito/genética , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Integrina alfa3beta1/genética , Integrina alfa3beta1/imunologia , Integrina alfa6beta1/genética , Integrina alfa6beta1/imunologia , Elastase de Leucócito/genética , Elastase de Leucócito/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Vesículas Secretórias/genética , Migração Transendotelial e Transepitelial/genética , Vênulas/imunologia , Proteínas de Transporte VesicularRESUMO
HGF-Met signaling contributes to various biological events by controlling cell migration. Since the abnormal activation of Met receptor causes cancer progression, inhibitors such as neutralizing antibodies are regarded as promising therapeutics. HGF is secreted as a single-chain (sc) precursor and is processed by extracellular proteases to generate disulfide-bonded two-chain (tc) HGF. Although this proteolytic processing of HGF is necessary for its biological activity, exactly how the proteolysis leads to the conversion of HGF to the active form is still unclar due to the lack of structural information. In order to gain insights about this point, we generated 6 antibodies against HGF. All antibodies recognized different epitopes on the native HGF protein and showed distinct effects when tested in a cell-based HGF-Met signaling assay. They included one antibody (t1E4) that strongly blocks Met activation by tcHGF, as well as one antibody (t8E4) exclusively recognizing the active tcHGF but not inactive scHGF. Thus, a panel of anti-HGF antibodies suitable for probing the structural mechanism of HGF activation were obtained.
Assuntos
Anticorpos Monoclonais Murinos/química , Epitopos/química , Fator de Crescimento de Hepatócito/química , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/farmacologia , Cães , Epitopos/imunologia , Fator de Crescimento de Hepatócito/imunologia , Humanos , Células Madin Darby de Rim Canino , Camundongos , Conformação Proteica , Proteínas Proto-Oncogênicas c-met/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologiaRESUMO
Non-alcoholic steatohepatitis (NASH), a metabolic disorder consisting of steatosis and inflammation, is considered the hepatic equivalent of metabolic syndrome and can result in irreversible liver damage. Macrophage-stimulating protein (MSP) is a hepatokine that potentially has a beneficial role in hepatic lipid and glucose metabolism via the activation of AMP-activated protein kinase (AMPK). In the current study, we investigated the regulatory role of MSP in the development of inflammation and lipid metabolism in various NASH models, both in vitro and ex vivo. We observed that MSP treatment activated the AMPK signaling pathway and inhibited lipopolysaccharide (LPS)- and palmitic acid (PA)-induced gene expression of pro-inflammatory cytokines in primary mouse hepatocytes. In addition, MSP treatment resulted in a significant reduction in PA-induced lipid accumulation and inhibited the gene expression of key lipogenic enzymes in HepG2 cells. Upon short hairpin RNA-induced knockdown of RON (the membrane-bound receptor for MSP), the anti-inflammatory and anti-lipogenic effects of MSP were markedly ablated. Finally, to mimic NASH ex vivo, we challenged bone marrow-derived macrophages with oxidized low-density lipoprotein (oxLDL) in combination with LPS. OxLDL+LPS exposure led to a marked inhibition of AMPK activity and a robust increase in inflammation. MSP treatment significantly reversed these effects by restoring AMPK activity and by suppressing pro-inflammatory cytokine gene expression and secretion under this condition. Taken together, these data suggest that MSP is an effective inhibitor of inflammation and lipid accumulation in the stressed liver, thereby indicating that MSP has a key regulatory role in NASH.