RESUMO
Trastuzumab, a targeted anti-human epidermal-growth-factor receptor-2 (HER2) monoclonal antibody, represents a mainstay in the treatment of HER2-positive (HER2+) breast cancer. Although trastuzumab treatment is highly efficacious for early-stage HER2+ breast cancer, the majority of advanced-stage HER2+ breast cancer patients who initially respond to trastuzumab acquire resistance to treatment and relapse, despite persistence of HER2 gene amplification/overexpression. Here, we sought to leverage HER2 overexpression to engage antibody-dependent cellular phagocytosis (ADCP) through a combination of trastuzumab and anti-CD47 macrophage checkpoint immunotherapy. We have previously shown that blockade of CD47, a surface protein expressed by many malignancies (including HER2+ breast cancer), is an effective anticancer therapy. CD47 functions as a "don't eat me" signal through its interaction with signal regulatory protein-α (SIRPα) on macrophages to inhibit phagocytosis. Hu5F9-G4 (magrolimab), a humanized monoclonal antibody against CD47, blocks CD47's "don't eat me" signal, thereby facilitating macrophage-mediated phagocytosis. Preclinical studies have shown that combining Hu5F9-G4 with tumor-targeting antibodies, such as rituximab, further enhances Hu5F9-G4's anticancer effects via ADCP. Clinical trials have additionally demonstrated that Hu5F9-G4, in combination with rituximab, produced objective responses in patients whose diffuse large B cell lymphomas had developed resistance to rituximab and chemotherapy. These studies led us to hypothesize that combining Hu5F9-G4 with trastuzumab would produce an anticancer effect in antibody-dependent cellular cytotoxicity (ADCC)-tolerant HER2+ breast cancer. This combination significantly suppressed the growth of ADCC-tolerant HER2+ breast cancers via Fc-dependent ADCP. Our study demonstrates that combining trastuzumab and Hu5F9-G4 represents a potential new treatment option for HER2+ breast cancer patients, even for patients whose tumors have progressed after trastuzumab.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Antineoplásicos Imunológicos/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Antígeno CD47/imunologia , Trastuzumab/administração & dosagem , Animais , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Antígeno CD47/antagonistas & inibidores , Antígeno CD47/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Quimioterapia Combinada , Feminino , Humanos , Imunoterapia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptor ErbB-2/genética , Receptor ErbB-2/imunologiaRESUMO
Pancreatic neuroendocrine tumors (PanNETs) are a type of pancreatic cancer with limited therapeutic options. Consequently, most patients with advanced disease die from tumor progression. Current evidence indicates that a subset of cancer cells is responsible for tumor development, metastasis, and recurrence, and targeting these tumor-initiating cells is necessary to eradicate tumors. However, tumor-initiating cells and the biological processes that promote pathogenesis remain largely uncharacterized in PanNETs. Here we profile primary and metastatic tumors from an index patient and demonstrate that MET proto-oncogene activation is important for tumor growth in PanNET xenograft models. We identify a highly tumorigenic cell population within several independent surgically acquired PanNETs characterized by increased cell-surface protein CD90 expression and aldehyde dehydrogenase A1 (ALDHA1) activity, and provide in vitro and in vivo evidence for their stem-like properties. We performed proteomic profiling of 332 antigens in two cell lines and four primary tumors, and showed that CD47, a cell-surface protein that acts as a "don't eat me" signal co-opted by cancers to evade innate immune surveillance, is ubiquitously expressed. Moreover, CD47 coexpresses with MET and is enriched in CD90(hi)cells. Furthermore, blocking CD47 signaling promotes engulfment of tumor cells by macrophages in vitro and inhibits xenograft tumor growth, prevents metastases, and prolongs survival in vivo.
Assuntos
Tumores Neuroendócrinos , Neoplasias Pancreáticas , Evasão Tumoral , Família Aldeído Desidrogenase 1 , Animais , Antígeno CD47/imunologia , Feminino , Humanos , Isoenzimas/imunologia , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Metástase Neoplásica , Proteínas de Neoplasias/imunologia , Tumores Neuroendócrinos/imunologia , Tumores Neuroendócrinos/patologia , Tumores Neuroendócrinos/terapia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Proto-Oncogene Mas , Retinal Desidrogenase/imunologia , Antígenos Thy-1/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mobilization of the T-cell response against cancer has the potential to achieve long-lasting cures. However, it is not known how to harness antigen-presenting cells optimally to achieve an effective antitumor T-cell response. In this study, we show that anti-CD47 antibody-mediated phagocytosis of cancer by macrophages can initiate an antitumor T-cell immune response. Using the ovalbumin model antigen system, anti-CD47 antibody-mediated phagocytosis of cancer cells by macrophages resulted in increased priming of OT-I T cells [cluster of differentiation 8-positive (CD8(+))] but decreased priming of OT-II T cells (CD4(+)). The CD4(+) T-cell response was characterized by a reduction in forkhead box P3-positive (Foxp3(+)) regulatory T cells. Macrophages following anti-CD47-mediated phagocytosis primed CD8(+) T cells to exhibit cytotoxic function in vivo. This response protected animals from tumor challenge. We conclude that anti-CD47 antibody treatment not only enables macrophage phagocytosis of cancer but also can initiate an antitumor cytotoxic T-cell immune response.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígeno CD47/imunologia , Neoplasias do Colo/imunologia , Neoplasias do Colo/terapia , Macrófagos/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Bloqueadores/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Citotoxicidade Imunológica/genética , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fagocitose/imunologia , Regulação para Cima/genética , Regulação para Cima/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Gastrointestinal stromal tumor (GIST) is the most common sarcoma of the gastrointestinal tract and arises from the interstitial cells of Cajal. It is characterized by expression of the receptor tyrosine kinase CD117 (KIT). In 70-80% of GIST cases, oncogenic mutations in KIT are present, leading to constitutive activation of the receptor, which drives the proliferation of these tumors. Treatment of GIST with imatinib, a small-molecule tyrosine kinase inhibitor, inhibits KIT-mediated signaling and initially results in disease control in 70-85% of patients with KIT-positive GIST. However, the vast majority of patients eventually develop resistance to imatinib treatment, leading to disease progression and posing a significant challenge in the clinical management of these tumors. Here, we show that an anti-KIT monoclonal antibody (mAb), SR1, is able to slow the growth of three human GIST cell lines in vitro. Importantly, these reductions in cell growth were equivalent between imatinib-resistant and imatinib-sensitive GIST cell lines. Treatment of GIST cell lines with SR1 reduces cell-surface KIT expression, suggesting that mAb-induced KIT down-regulation may be a mechanism by which SR1 inhibits GIST growth. Furthermore, we also show that SR1 treatment enhances phagocytosis of GIST cells by macrophages, indicating that treatment with SR1 may enhance immune cell-mediated tumor clearance. Finally, using two xenotransplantation models of imatinib-sensitive and imatinib-resistant GIST, we demonstrate that SR1 is able to strongly inhibit tumor growth in vivo. These results suggest that treatment with mAbs targeting KIT may represent an alternative, or complementary, approach for treating GIST.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/patologia , Piperazinas/uso terapêutico , Proteínas Proto-Oncogênicas c-kit/imunologia , Pirimidinas/uso terapêutico , Animais , Anticorpos Monoclonais/farmacologia , Benzamidas , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Mesilato de Imatinib , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fagocitose/efeitos dos fármacos , Piperazinas/farmacologia , Pirimidinas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Envelhecimento/fisiologia , Proteínas Reguladoras de Apoptose/fisiologia , Poluição Ambiental , Imunidade Inata/imunologia , Proteínas Adaptadoras de Sinalização NOD/química , Receptores Toll-Like/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Humanos , Inflamação/imunologia , Inflamação/microbiologia , Proteínas NLR , Receptores Toll-Like/genéticaRESUMO
Antibodies against CD47, which block tumor cell CD47 interactions with macrophage signal regulatory protein-α, have been shown to decrease tumor size in hematological and epithelial tumor models by interfering with the protection from phagocytosis by macrophages that intact CD47 bestows upon tumor cells. Leiomyosarcoma (LMS) is a tumor of smooth muscle that can express varying levels of colony-stimulating factor-1 (CSF1), the expression of which correlates with the numbers of tumor-associated macrophages (TAMs) that are found in these tumors. We have previously shown that the presence of TAMs in LMS is associated with poor clinical outcome and the overall effect of TAMs in LMS therefore appears to be protumorigenic. However, the use of inhibitory antibodies against CD47 offers an opportunity to turn TAMs against LMS cells by allowing the phagocytic behavior of resident macrophages to predominate. Here we show that interference with CD47 increases phagocytosis of two human LMS cell lines, LMS04 and LMS05, in vitro. In addition, treatment of mice bearing subcutaneous LMS04 and LMS05 tumors with a novel, humanized anti-CD47 antibody resulted in significant reductions in tumor size. Mice bearing LMS04 tumors develop large numbers of lymph node and lung metastases. In a unique model for neoadjuvant treatment, mice were treated with anti-CD47 antibody starting 1 wk before resection of established primary tumors and subsequently showed a striking decrease in the size and number of metastases. These data suggest that treatment with anti-CD47 antibodies not only reduces primary tumor size but can also be used to inhibit the development of, or to eliminate, metastatic disease.
Assuntos
Anticorpos/uso terapêutico , Antígeno CD47/imunologia , Modelos Animais de Doenças , Imunoterapia , Leiomiossarcoma/terapia , Animais , Anticorpos/imunologia , Linhagem Celular Tumoral , Leiomiossarcoma/patologia , Camundongos , Metástase Neoplásica , Fagocitose/imunologiaRESUMO
Current clinical judgment in bladder cancer (BC) relies primarily on pathological stage and grade. We investigated whether a molecular classification of tumor cell differentiation, based on a developmental biology approach, can provide additional prognostic information. Exploiting large preexisting gene-expression databases, we developed a biologically supervised computational model to predict markers that correspond with BC differentiation. To provide mechanistic insight, we assessed relative tumorigenicity and differentiation potential via xenotransplantation. We then correlated the prognostic utility of the identified markers to outcomes within gene expression and formalin-fixed paraffin-embedded (FFPE) tissue datasets. Our data indicate that BC can be subclassified into three subtypes, on the basis of their differentiation states: basal, intermediate, and differentiated, where only the most primitive tumor cell subpopulation within each subtype is capable of generating xenograft tumors and recapitulating downstream populations. We found that keratin 14 (KRT14) marks the most primitive differentiation state that precedes KRT5 and KRT20 expression. Furthermore, KRT14 expression is consistently associated with worse prognosis in both univariate and multivariate analyses. We identify here three distinct BC subtypes on the basis of their differentiation states, each harboring a unique tumor-initiating population.
Assuntos
Diferenciação Celular , Neoplasias da Bexiga Urinária/classificação , Neoplasias da Bexiga Urinária/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Diferenciação Celular/genética , Membrana Celular/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Queratinas/genética , Queratinas/metabolismo , Reprodutibilidade dos Testes , Fatores de Risco , Análise de Sobrevida , Neoplasias da Bexiga Urinária/genética , Urotélio/metabolismo , Urotélio/patologiaRESUMO
CD47, a "don't eat me" signal for phagocytic cells, is expressed on the surface of all human solid tumor cells. Analysis of patient tumor and matched adjacent normal (nontumor) tissue revealed that CD47 is overexpressed on cancer cells. CD47 mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer. CD47 is a ligand for SIRPα, a protein expressed on macrophages and dendritic cells. In vitro, blockade of CD47 signaling using targeted monoclonal antibodies enabled macrophage phagocytosis of tumor cells that were otherwise protected. Administration of anti-CD47 antibodies inhibited tumor growth in orthotopic immunodeficient mouse xenotransplantation models established with patient tumor cells and increased the survival of the mice over time. Anti-CD47 antibody therapy initiated on larger tumors inhibited tumor growth and prevented or treated metastasis, but initiation of the therapy on smaller tumors was potentially curative. The safety and efficacy of targeting CD47 was further tested and validated in immune competent hosts using an orthotopic mouse breast cancer model. These results suggest all human solid tumor cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination. These data, taken together with similar findings with other human neoplasms, show that CD47 is a commonly expressed molecule on all cancers, its function to block phagocytosis is known, and blockade of its function leads to tumor cell phagocytosis and elimination. CD47 is therefore a validated target for cancer therapies.
Assuntos
Antígenos de Diferenciação/metabolismo , Antígeno CD47/imunologia , Neoplasias/imunologia , RNA Mensageiro/genética , Receptores Imunológicos/metabolismo , Anticorpos/imunologia , Antígeno CD47/genética , Divisão Celular/imunologia , Citometria de Fluxo , Humanos , Neoplasias/patologia , Neoplasias/terapia , Fagocitose/imunologia , Prognóstico , Análise de SobrevidaRESUMO
The nucleotide-binding domain leucine-rich repeat-containing proteins, NLRs, are intracellular sensors of pathogen-associated molecular patterns and damage-associated molecular patterns. A subgroup of NLRs can form inflammasome complexes, which facilitate the maturation of procaspase 1 to caspase 1, leading to IL-1ß and IL-18 cleavage and secretion. NLRC5 is predominantly expressed in hematopoietic cells and has not been studied for inflammasome function. RNA interference-mediated knockdown of NLRC5 nearly eliminated caspase 1, IL-1ß, and IL-18 processing in response to bacterial infection, pathogen-associated molecular patterns, and damage-associated molecular patterns. This was confirmed in primary human monocytic cells. NLRC5, together with procaspase 1, pro-IL-1ß, and the inflammasome adaptor ASC, reconstituted inflammasome activity that showed cooperativity with NLRP3. The range of pathogens that activate NLRC5 inflammasome overlaps with those that activate NLRP3. Furthermore, NLRC5 biochemically associates with NLRP3 in a nucleotide-binding domain-dependent but leucine-rich repeat-inhibitory fashion. These results invoke a model in which NLRC5 interacts with NLRP3 to cooperatively activate the inflammasome.
Assuntos
Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Animais , Proteínas de Transporte/fisiologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Escherichia coli/imunologia , Escherichia coli/patogenicidade , Regulação da Expressão Gênica/imunologia , Humanos , Imunidade Inata , Inflamassomos/biossíntese , Inflamassomos/genética , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucina/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Estrutura Terciária de Proteína/genética , Shigella flexneri/imunologia , Shigella flexneri/patogenicidade , Staphylococcus aureus/imunologia , Staphylococcus aureus/patogenicidadeRESUMO
BACKGROUND: CD73 is widely expressed on immune cells playing a critical role in immunomodulatory functions including cell adhesion and migration, as a costimulatory molecule for T cells and in production of adenosine. The function of CD73 expressed on B cells has not been fully characterized. Mupadolimab is an anti-human CD73 antibody that activates B cells. We evaluated the characteristics of this antibody and its effects on immune cells in vitro and in vivo. METHODS: Mupadolimab binding to CD73, inhibition of CD73 enzymatic activity, and effects on lymphocyte activation were evaluated in vitro by measuring changes in immunophenotype by flow cytometry. Cryogenic-transmission electron microscopy was used to determine epitope binding. Effects on human B cells in vivo were evaluated in immunodeficient NSG-SGM3 mice immunized with SARS-CoV-2 and influenza viral antigens. Safety and immune effects were evaluated in the completed dose escalation portion of a phase 1 trial conducted in patients with cancer. RESULTS: Mupadolimab binds to a unique epitope on CD73POS B cells resulting in their activation and differentiation through B cell receptor signaling pathways. Mupadolimab induces expression of CD69, CD83, CD86 and MHC class II on B cells along with morphological transformation into plasmablasts and expression of CD27, CD38 and CD138. These effects are independent of adenosine. Mupadolimab binds to the N-terminal of CD73 in the closed position and competitively inhibits substrate binding. Mupadolimab enhanced antigen specific antibody response to SARS-CoV-2 spike protein and influenza hemagglutinin in humanized mouse models. Mupadolimab was evaluated as a monotherapy in a phase 1 trial (NCT03454451) in 34 patients with advanced cancer and demonstrated binding to CD73POS circulating cells and transient reduction in the number of B cells, with return of CD73NEG B cells with memory phenotype. No dose-limiting toxicities or changes in serum immunoglobulins were seen. CONCLUSIONS: Mupadolimab activates B cells and stimulates the production of antigen specific antibodies. The effects in patients with cancer suggest that activated, CD69POS B cells redistribute to lymphoid tissues. Minor tumor regression was observed in several patients. These results support further investigation of mupadolimab as an immunotherapy for cancer and its potential use as a vaccine adjuvant. TRIAL REGISTRATION NUMBER: NCT03454451.
Assuntos
Anticorpos Monoclonais , Linfócitos B , Imunidade Humoral , Neoplasias , Animais , Humanos , Camundongos , Adenosina , Anticorpos Monoclonais/imunologia , Antígenos Virais , Linfócitos B/imunologia , Epitopos , Neoplasias/imunologia , Neoplasias/terapiaRESUMO
Neisseria gonorrhoeae is a common sexually transmitted pathogen that significantly impacts female fertility, neonatal health, and transmission of HIV worldwide. N. gonorrhoeae usually causes localized inflammation of the urethra and cervix by inducing production of IL-1beta and other inflammatory cytokines. Several NLR (nucleotide-binding domain, leucine-rich repeat) proteins are implicated in the formation of pro-IL-1beta-processing complexes called inflammasomes in response to pathogens. We demonstrate that NLRP3 (cryopyrin, NALP3) is the primary NLR required for IL-1beta/IL-18 secretion in response to N. gonorrhoeae in monocytes. We also show that N. gonorrhoeae infection promotes NLRP3-dependent monocytic cell death via pyronecrosis, a recently described pathway with morphological features of necrosis, including release of the strong inflammatory mediator HMBG1. Additionally, N. gonorrhoeae activates the cysteine protease cathepsin B as measured by the breakdown of a cathepsin B substrate. Inhibition of cathepsin B shows that this protease is an apical controlling step in the downstream activities of NLRP3 including IL-1beta production, pyronecrosis, and HMGB1 release. Nonpathogenic Neisseria strains (Neisseria cinerea and Neisseria flavescens) do not activate NLRP3 as robustly as N. gonorrhoeae. Conditioned medium from N. gonorrhoeae contains factors capable of initiating the NLRP3-mediated signaling events. Isolated N. gonorrhoeae lipooligosaccharide, a known virulence factor from this bacterium that is elaborated from the bacterium in the form of outer membrane blebs, activates both NLRP3-induced IL-1beta secretion and pyronecrosis. Our findings indicate that activation of NLRP3-mediated inflammatory response pathways is an important venue associated with host response and pathogenesis of N. gonorrhoeae.
Assuntos
Proteínas de Transporte/imunologia , Catepsina B/imunologia , Proteínas do Citoesqueleto/imunologia , Inflamação/imunologia , Neisseria gonorrhoeae/imunologia , Transdução de Sinais/imunologia , Animais , Proteínas Reguladoras de Apoptose , Western Blotting , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Transporte/metabolismo , Catepsina B/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Ativação Enzimática/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Proteína HMGB1/imunologia , Proteína HMGB1/metabolismo , Humanos , Inflamação/metabolismo , Interleucina-18/imunologia , Interleucina-18/metabolismo , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Masculino , Camundongos , Camundongos Knockout , Monócitos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Necrose/imunologia , RNA Interferente PequenoRESUMO
Bacterial infection elicits a range of beneficial as well as detrimental host inflammatory responses. Key among these responses are macrophage/monocyte necrosis, release of the proinflammatory factor high-mobility group box 1 protein (HMGB1), and induction of the cytokine IL-1. Although the control of IL-1beta has been well studied, processes that control macrophage cell death and HMGB1 release in animals are poorly understood. This study uses Klebsiella pneumonia as a model organism because it elicits all three responses in vivo. The regulation of these responses is studied in the context of the inflammasome components NLRP3 and ASC, which are important for caspase-1 activation and IL-1beta release. Using a pulmonary infection model that reflects human infection, we show that K. pneumonia-induced mouse macrophage necrosis, HMGB1, and IL-1beta release are dependent on NLRP3 and ASC. K. pneumoniae infection of mice lacking Nlrp3 results in decreased lung inflammation and reduced survival relative to control, indicating the overall protective role of this gene. Macrophage/monocyte necrosis and HMGB1 release are controlled independently of caspase-1, suggesting that the former two responses are separable from inflammasome-associated functions. These results provide critical in vivo validation that the physiologic role of NLRP3 and ASC is not limited to inflammasome formation.
Assuntos
Proteínas de Transporte/fisiologia , Caspase 1/metabolismo , Proteínas do Citoesqueleto/fisiologia , Proteína HMGB1/metabolismo , Pneumonia/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Proteínas Adaptadoras de Sinalização CARD , Modelos Animais de Doenças , Inflamação/metabolismo , Inflamação/microbiologia , Interleucina-1beta/metabolismo , Klebsiella , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Necrose , Pneumonia/microbiologia , Pneumonia/patologiaRESUMO
Periodontal disease is a chronic inflammatory disorder that leads to the destruction of tooth-supporting tissue and affects 10-20 million people in the U.S. alone. The oral pathogen Porphyromonas gingivalis causes inflammatory host response leading to periodontal and other secondary inflammatory diseases. To identify molecular components that control host response to P. gingivalis in humans, roles for the NLR (NBD-LRR) protein, NLRP3 (cryopyrin, NALP3), and its adaptor apoptotic speck protein containing a C-terminal caspase recruitment domain (ASC) were studied. P. gingivalis strain A7436 induces cell death in THP1 monocytic cells and in human primary peripheral blood macrophages. This process is ASC and NLRP3 dependent and can be replicated by P. gingivalis LPS and Escherichia coli. P. gingivalis-induced cell death is caspase and IL-1 independent and exhibits morphological features consistent with necrosis including loss of membrane integrity and release of cellular content. Intriguingly, P. gingivalis-induced cell death is accompanied by the formation of ASC aggregation specks, a process not previously described during microbial infection. ASC specks are observed in P. gingivalis-infected primary human mononuclear cells and are dependent on NLRP3. This work shows that P. gingivalis causes ASC- and NLRP3-dependent necrosis, accompanied by ASC speck formation.
Assuntos
Infecções por Bacteroidaceae/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Macrófagos/microbiologia , Monócitos/microbiologia , Necrose/metabolismo , Infecções por Bacteroidaceae/imunologia , Infecções por Bacteroidaceae/patologia , Western Blotting , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Transporte/imunologia , Proteínas do Citoesqueleto/imunologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Microscopia Eletrônica de Transmissão , Monócitos/imunologia , Monócitos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Necrose/imunologia , Necrose/microbiologia , Porphyromonas gingivalis , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The immunosuppressive tumor microenvironment (TME) represents a challenge that all immunotherapies must overcome to enable a robust and durable anti-tumor response. One of the dominant mechanisms of immunosuppression in the TME is hypoxia and the generation of extracellular adenosine [1]. Pioneering work from Drs Ohta and Sitkovsky demonstrating that adenosine signaling through the adenosine 2A receptor (A2AR) inhibits T cells has led to the development of several agents designed to inhibit the production or downstream signaling of adenosine [2â¢â¢,3â¢â¢]. This review will focus on the safety, efficacy, and biomarkers associated with A2AR antagonists in clinical development.
Assuntos
Antagonistas do Receptor A2 de Adenosina/uso terapêutico , Neoplasias/tratamento farmacológico , Animais , Humanos , Neoplasias/metabolismo , Receptor A2A de Adenosina/metabolismoRESUMO
Neutrophils are the most abundant effector cells of the innate immune system and represent the first line of defense against infection. However, in many common pathologies, including autoimmune diseases, excessive recruitment and activation of neutrophils can drive a chronic inflammatory response leading to unwanted tissue destruction. Several strategies have been investigated to tackle pathologic neutrophil biology, and thus provide a novel therapy for chronic inflammatory diseases. The chemokine receptor CXCR2 plays a crucial role in regulating neutrophil homeostasis and is a promising pharmaceutical target. In this study, we report the discovery and validation of a humanized anti-human CXCR2 monoclonal antibody. To enable in vivo studies, we developed a surrogate anti-mouse CXCR2 antibody, as well as a human knock-in CXCR2 mouse. When administered in models of atopic dermatitis (AD) and rheumatoid arthritis (RA), the antibodies rapidly clear inflammation. Our findings support further developments of anti-CXCR2 mAb approaches not only for RA and AD, but also for other neutrophil-mediated inflammatory conditions where neutrophils are pathogenic and medical needs are unmet.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Artrite Experimental/tratamento farmacológico , Dermatite Atópica/tratamento farmacológico , Receptores de Interleucina-8B/antagonistas & inibidores , Animais , Artrite Experimental/genética , Artrite Experimental/metabolismo , Dermatite Atópica/genética , Dermatite Atópica/metabolismo , Feminino , Camundongos , Camundongos Transgênicos , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismoRESUMO
Adenosine mediates immunosuppression within the tumor microenvironment through triggering adenosine 2A receptors (A2AR) on immune cells. To determine whether this pathway could be targeted as an immunotherapy, we performed a phase I clinical trial with a small-molecule A2AR antagonist. We find that this molecule can safely block adenosine signaling in vivo. In a cohort of 68 patients with renal cell cancer (RCC), we also observe clinical responses alone and in combination with an anti-PD-L1 antibody, including subjects who had progressed on PD-1/PD-L1 inhibitors. Durable clinical benefit is associated with increased recruitment of CD8+ T cells into the tumor. Treatment can also broaden the circulating T-cell repertoire. Clinical responses are associated with an adenosine-regulated gene-expression signature in pretreatment tumor biopsies. A2AR signaling, therefore, represents a targetable immune checkpoint distinct from PD-1/PD-L1 that restricts antitumor immunity. SIGNIFICANCE: This first-in-human study of an A2AR antagonist for cancer treatment establishes the safety and feasibility of targeting this pathway by demonstrating antitumor activity with single-agent and anti-PD-L1 combination therapy in patients with refractory RCC. Responding patients possess an adenosine-regulated gene-expression signature in pretreatment tumor biopsies.See related commentary by Sitkovsky, p. 16.This article is highlighted in the In This Issue feature, p. 1.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células Renais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Renais/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Receptor A2A de Adenosina/química , Terapia de Salvação , Adulto , Idoso , Anticorpos Monoclonais Humanizados/administração & dosagem , Carcinoma de Células Renais/patologia , Feminino , Seguimentos , Furanos/administração & dosagem , Humanos , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Prognóstico , Piridinas/administração & dosagem , Pirimidinas/administração & dosagem , Receptor A2A de Adenosina/metabolismo , Taxa de SobrevidaRESUMO
Osteoarthritis (OA) is the leading cause of joint failure, yet the underlying mechanisms remain elusive, and no approved therapies that slow progression exist. Dysregulated integrin function was previously implicated in OA pathogenesis. However, the roles of integrin αVß3 and the integrin-associated receptor CD47 in OA remain largely unknown. Here, transcriptomic and proteomic analyses of human and murine osteoarthritic tissues revealed dysregulated expression of αVß3, CD47, and their ligands. Using genetically deficient mice and pharmacologic inhibitors, we showed that αVß3, CD47, and the downstream signaling molecules Fyn and FAK are crucial to OA pathogenesis. MicroPET/CT imaging of a mouse model showed elevated ligand-binding capacities of integrin αVß3 and CD47 in osteoarthritic joints. Further, our in vitro studies demonstrated that chondrocyte breakdown products, derived from articular cartilage of individuals with OA, induced αVß3/CD47-dependent expression of inflammatory and degradative mediators, and revealed the downstream signaling network. Our findings identify a central role for dysregulated αVß3 and CD47 signaling in OA pathogenesis and suggest that activation of αVß3 and CD47 signaling in many articular cell types contributes to inflammation and joint destruction in OA. Thus, the data presented here provide a rationale for targeting αVß3, CD47, and their signaling pathways as a disease-modifying therapy.
Assuntos
Antígeno CD47/metabolismo , Cartilagem Articular/patologia , Integrina alfaVbeta3/metabolismo , Osteoartrite/imunologia , Transdução de Sinais/imunologia , Animais , Antígeno CD47/genética , Antígeno CD47/imunologia , Cartilagem Articular/citologia , Cartilagem Articular/diagnóstico por imagem , Cartilagem Articular/imunologia , Células Cultivadas , Condrócitos , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Progressão da Doença , Perfilação da Expressão Gênica , Humanos , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/imunologia , Masculino , Camundongos , Osteoartrite/diagnóstico por imagem , Osteoartrite/patologia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Cultura Primária de Células , Proteômica , Membrana Sinovial/citologia , Membrana Sinovial/imunologia , Membrana Sinovial/patologia , Sinoviócitos , Microtomografia por Raio-XRESUMO
Adenosine signaling through A2A receptors (A2AR) expressed on immune cells suppresses antitumor immunity. CPI-444 is a potent, selective, oral A2AR antagonist. Blockade of A2AR with CPI-444 restored T-cell signaling, IL2, and IFNγ production that were suppressed by adenosine analogues in vitro CPI-444 treatment led to dose-dependent inhibition of tumor growth in multiple syngeneic mouse tumor models. Concentrations of extracellular adenosine in the tumor microenvironment, measured using microdialysis, were approximately 100-150 nmol/L and were higher than corresponding subcutaneous tissue. Combining CPI-444 with anti-PD-L1 or anti-CTLA-4 treatment eliminated tumors in up to 90% of treated mice, including restoration of immune responses in models that incompletely responded to anti-PD-L1 or anti-CTLA-4 monotherapy. Tumor growth was fully inhibited when mice with cleared tumors were later rechallenged, indicating that CPI-444 induced systemic antitumor immune memory. CD8+ T-cell depletion abrogated the efficacy of CPI-444 with and without anti-PD-L1 treatment, demonstrating a role for CD8+ T cells in mediating primary and secondary immune responses. The antitumor efficacy of CPI-444 with and without anti-PD-L1 was associated with increased T-cell activation, a compensatory increase in CD73 expression, and induction of a Th1 gene expression signature consistent with immune activation. These results suggest a broad role for adenosine-mediated immunosuppression in tumors and justify the further evaluation of CPI-444 as a therapeutic agent in patients with solid tumors. Cancer Immunol Res; 6(10); 1136-49. ©2018 AACR.
Assuntos
Antagonistas do Receptor A2 de Adenosina/uso terapêutico , Antineoplásicos/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Antígeno CTLA-4/antagonistas & inibidores , Neoplasias do Colo/tratamento farmacológico , Furanos/uso terapêutico , Piridinas/uso terapêutico , Pirimidinas/uso terapêutico , Antagonistas do Receptor A2 de Adenosina/farmacologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Avaliação Pré-Clínica de Medicamentos , Quimioterapia Combinada , Furanos/farmacologia , Humanos , Interferon gama/metabolismo , Interleucina-2/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Piridinas/farmacologia , Pirimidinas/farmacologia , Linfócitos T/efeitos dos fármacosRESUMO
CD47 is a cell surface molecule that inhibits phagocytosis of cells that express it by binding to its receptor, SIRPα, on macrophages and other immune cells. CD47 is expressed at different levels by neoplastic and normal cells. Here, to reveal mechanisms by which different neoplastic cells generate this dominant 'don't eat me' signal, we analyse the CD47 regulatory genomic landscape. We identify two distinct super-enhancers (SEs) associated with CD47 in certain cancer cell types. We show that a set of active constituent enhancers, located within the two CD47 SEs, regulate CD47 expression in different cancer cell types and that disruption of CD47 SEs reduces CD47 gene expression. Finally we report that the TNF-NFKB1 signalling pathway directly regulates CD47 by interacting with a constituent enhancer located within a CD47-associated SE specific to breast cancer. These results suggest that cancers can evolve SE to drive CD47 overexpression to escape immune surveillance.
Assuntos
Neoplasias da Mama/metabolismo , Antígeno CD47/fisiologia , Elementos Facilitadores Genéticos , Mediadores da Inflamação/metabolismo , Inflamação/metabolismo , Transdução de Sinais , Regulação para Cima , Animais , Neoplasias da Mama/patologia , Antígeno CD47/genética , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Subunidade p50 de NF-kappa B/metabolismo , Fagocitose , Ligação Proteica , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Morbidity and mortality associated with pediatric malignant primary brain tumors remain high in the absence of effective therapies. Macrophage-mediated phagocytosis of tumor cells via blockade of the anti-phagocytic CD47-SIRPα interaction using anti-CD47 antibodies has shown promise in preclinical xenografts of various human malignancies. We demonstrate the effect of a humanized anti-CD47 antibody, Hu5F9-G4, on five aggressive and etiologically distinct pediatric brain tumors: group 3 medulloblastoma (primary and metastatic), atypical teratoid rhabdoid tumor, primitive neuroectodermal tumor, pediatric glioblastoma, and diffuse intrinsic pontine glioma. Hu5F9-G4 demonstrated therapeutic efficacy in vitro and in vivo in patient-derived orthotopic xenograft models. Intraventricular administration of Hu5F9-G4 further enhanced its activity against disseminated medulloblastoma leptomeningeal disease. Notably, Hu5F9-G4 showed minimal activity against normal human neural cells in vitro and in vivo, a phenomenon reiterated in an immunocompetent allograft glioma model. Thus, Hu5F9-G4 is a potentially safe and effective therapeutic agent for managing multiple pediatric central nervous system malignancies.