RESUMO
The development of therapeutic cancer vaccines remains an active area, although previous approaches have yielded disappointing results. We have built on lessons from previous cancer vaccine approaches and immune checkpoint inhibitor research to develop VBIR, a vaccine-based immunotherapy regimen. Assessment of various technologies led to selection of a heterologous vaccine using chimpanzee adenovirus (AdC68) for priming followed by boosts with electroporation of DNA plasmid to deliver T cell antigens to the immune system. We found that priming with AdC68 rapidly activates and expands antigen-specific T cells and does not encounter pre-existing immunity as occurs with the use of a human adenovirus vaccine. The AdC68 vector does, however, induce new anti-virus immune responses, limiting its use for boosting. To circumvent this, boosting with DNA encoding the same antigens can be done repetitively to augment and maintain vaccine responses. Using mouse and monkey models, we found that the activation of both CD4 and CD8 T cells was amplified by combination with anti-CTLA-4 and anti-PD-1 antibodies. These antibodies were administered subcutaneously to target their distribution to vaccination sites and to reduce systemic exposure which may improve their safety. VBIR can break tolerance and activate T cells recognizing tumor-associated self-antigens. This activation lasts more than a year after completing treatment in monkeys, and inhibits tumor growth to a greater degree than is observed using the individual components in mouse cancer models. These results have encouraged the testing of this combination regimen in cancer patients with the aim of increasing responses beyond current therapies.
Assuntos
Vacinas Anticâncer , Neoplasias , Vacinas de DNA , Humanos , Animais , Camundongos , Linfócitos T CD8-Positivos , Antígenos de Neoplasias , Vacinação/métodos , Modelos Animais de Doenças , AutoantígenosRESUMO
The aspartic protease cathepsin D, a poor prognostic indicator of breast cancer, is abundantly secreted as procathepsin D by human breast cancer cells and self-activates at low pH in vitro, giving rise to catalytically active cathepsin D. Due to a lower extracellular pH in tumor microenvironments compared to normal tissues, cathepsin D may cleave pathophysiological substrates contributing to cancer progression. Here, we show by yeast 2-hybrid and degradomics analyses that cystatin C, the most potent natural secreted inhibitor of cysteine cathepsins, both binds to and is a substrate of extracellular procathepsin D. The amount of cystatin C in the extracellular environment is reduced in the secretome of mouse embryonic fibroblasts stably transfected with human cathepsin D. Cathepsin D extensively cleaved cystatin C in vitro at low pH. Cathepsin D secreted by breast cancer cells also processed cystatin C at the pericellular pH of tumors and so enhancing extracellular proteolytic activity of cysteine cathepsins. Thus, tumor derived cathepsin D assists breast cancer progression by reducing cystatin C activity, which, in turn, enhances cysteine cathepsin proteolytic activity, revealing a new link between protease classes in the protease web.
Assuntos
Catepsina D/metabolismo , Cistatina C/metabolismo , Fibroblastos/metabolismo , Microambiente Tumoral , Sequência de Aminoácidos , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Catepsina D/genética , Células Cultivadas , Cistatina C/genética , Embrião de Mamíferos/citologia , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Espaço Extracelular/metabolismo , Feminino , Fibroblastos/citologia , Humanos , Concentração de Íons de Hidrogênio , Immunoblotting , Células MCF-7 , Camundongos , Camundongos Knockout , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica , Proteólise , Interferência de RNA , Técnicas do Sistema de Duplo-HíbridoRESUMO
Interactions between cancer cells and fibroblasts are crucial in cancer progression. We have previously shown that the aspartic protease cathepsin D (cath-D), a marker of poor prognosis in breast cancer that is overexpressed and highly secreted by breast cancer cells, triggers mouse embryonic fibroblast outgrowth via a paracrine loop. Here, we show the requirement of secreted cath-D for human mammary fibroblast outgrowth using a three-dimensional co-culture assay with breast cancer cells that do or do not secrete pro-cath-D. Interestingly, proteolytically-inactive pro-cath-D remains mitogenic, indicating a mechanism involving protein-protein interaction. We identify the low-density lipoprotein (LDL) receptor-related protein-1, LRP1, as a novel binding partner for pro-cath-D in fibroblasts. Pro-cath-D binds to residues 349-394 of the ß chain of LRP1, and is the first ligand of the extracellular domain of LRP1ß to be identified. We show that pro-cath-D interacts with LRP1ß in cellulo. Interaction occurs at the cell surface, and overexpressed LRP1ß directs pro-cath-D to the lipid rafts. Our results reveal that the ability of secreted pro-cath-D to promote human mammary fibroblast outgrowth depends on LRP1 expression, suggesting that pro-cath-D-LRP1ß interaction plays a functional role in the outgrowth of fibroblasts. Overall, our findings strongly suggest that pro-cath-D secreted by epithelial cancer cells promotes fibroblast outgrowth in a paracrine LRP1-dependent manner in the breast tumor microenvironment.
Assuntos
Antígenos CD/metabolismo , Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Catepsina D/metabolismo , Precursores Enzimáticos/metabolismo , Fibroblastos/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Animais , Antígenos CD/genética , Neoplasias da Mama/patologia , Carcinoma/patologia , Processos de Crescimento Celular , Linhagem Celular Transformada , Técnicas de Cocultura , Feminino , Fibroblastos/patologia , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Microdomínios da Membrana/genética , Camundongos , Comunicação Parácrina , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , RNA Interferente Pequeno/genéticaRESUMO
Six allosteric HIV-1 entry inhibitor modulators of the chemokine (C-C motif) receptor 5 (CCR5) receptor are compared for their potency as inhibitors of HIV-1 entry [infection of human osteosarcoma (HOS) cells and peripheral blood mononuclear cells (PBMC)] and antagonists of chemokine (C-C motif) ligand 3-like 1 [CCL3L1]-mediated internalization of CCR5. This latter activity has been identified as a beneficial action of CCL3L1 in prolonging survival after HIV-1 infection ( Science 307: 1434-1440, 2005 ). The allosteric nature of these modulators was further confirmed with the finding of a 58-fold (HOS cells) and 282-fold (PBMC) difference in relative potency for blockade of CCL3L1-mediated internalization versus HIV-1 entry. For the CCR5 modulators, statistically significant differences in this ratio were found for maraviroc, vicriviroc, aplaviroc, Sch-C, TAK652, and TAK779. For instance, although TAK652 is 13-fold more potent as an HIV-1 inhibitor (over blockade of CCL3L1-mediated CCR5 internalization), this ratio of potency is reversed for Sch-C (22-fold more potent for CCR5-mediated internalization over HIV-1 entry). Quantitative analyses of the insurmountable antagonism of CCR5 internalization by these ligands suggest that all of them reduce the efficacy of CCL3L1 for CCR5 internalization. The relatively small magnitude of dextral displacement accompanying the depression of maximal responses for aplaviroc, maraviroc and vicriviroc suggests that these modulators have minimal effects on CCL3L1 affinity, although possible receptor reserve effects obscure complete interpretation of this effect. These data are discussed in terms of the possible benefits of sparing natural CCR5 chemokine function in HIV-1 entry inhibition treatment for AIDS involving allosteric inhibitors.
Assuntos
Inibidores da Fusão de HIV/farmacologia , Inibidores da Fusão de HIV/uso terapêutico , HIV-1/efeitos dos fármacos , HIV-1/patogenicidade , Receptores CCR5/metabolismo , Internalização do Vírus/efeitos dos fármacos , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Inibidores da Fusão de HIV/química , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologiaRESUMO
Bone morphogenetic protein 7 (BMP-7) regulates cellular metabolism in embryonic and adult tissues. Signal transduction occurs through the activation of intracellular Smad proteins. In this paper, using a yeast two-hybrid screen, Smad1 was found to interact with the cytoplasmic domain of CD44, a receptor for the extracellular matrix macromolecule hyaluronan. Coimmunoprecipitation experiments confirmed the interaction of Smad1 with full-length CD44-interactions that did not occur when CD44 receptors truncated within the cytoplasmic domain were tested. Chondrocytes overexpressing a truncated CD44 on a background of endogenous full-length CD44 no longer exhibited Smad1 nuclear translocation upon BMP-7 stimulation. Further, pretreatment of chondrocytes with Streptomyces hyaluronidase to disrupt extracellular hyaluronan-cell interactions inhibited BMP-7-mediated Smad1 phosphorylation, nuclear translocation of Smad1 or Smad4, and SBE4-luciferase reporter activation. These results support a functional link between the BMP signaling cascade and CD44. Thus, changes in hyaluronan-cell interactions may serve as a means to modulate cellular responsiveness to BMP.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Cartilagem/metabolismo , Condrócitos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Matriz Extracelular/metabolismo , Receptores de Hialuronatos/metabolismo , Transdução de Sinais/genética , Transativadores/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/genética , Animais , Proteína Morfogenética Óssea 7 , Proteínas Morfogenéticas Ósseas/genética , Células COS , Cartilagem/citologia , Cartilagem/crescimento & desenvolvimento , Bovinos , Condrócitos/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Genes Reporter/genética , Humanos , Receptores de Hialuronatos/genética , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/farmacologia , Mutagênese Sítio-Dirigida , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad , Proteína Smad1 , Proteína Smad4 , Transativadores/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Fator de Crescimento Transformador beta/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Recent advances in several areas are rekindling interest and enabling progress in the development of therapeutic cancer vaccines. These advances have been made in target selection, vaccine technology, and methods for reversing the immunosuppressive mechanisms exploited by cancers. Studies testing different tumor antigens have revealed target properties that yield high tumor versus normal cell specificity and adequate immunogenicity to affect clinical efficacy. A few tumor-associated antigens, normal host proteins that are abnormally expressed in cancer cells, have been demonstrated to serve as good targets for immunotherapies, although many do not possess the needed specificity or immunogenicity. Neoantigens, which arise from mutated proteins in cancer cells, are truly cancer-specific and can be highly immunogenic, though the vast majority are unique to each patient's cancer and thus require development of personalized therapies. Lessons from previous cancer vaccine expeditions are teaching us the type and magnitude of immune responses needed, as well as vaccine technologies that can achieve these responses. For example, we are learning which vaccine approaches elicit the potent, balanced, and durable CD4 plus CD8 T cell expansion necessary for clinical efficacy. Exploration of interactions between the immune system and cancer has elucidated the adaptations that enable cancer cells to suppress and evade immune attack. This has led to breakthroughs in the development of new drugs, and, subsequently, to opportunities to combine these with cancer vaccines and dramatically increase patient responses. Here we review this recent progress, highlighting key steps that are bringing the promise of therapeutic cancer vaccines within reach.
RESUMO
The adapter protein Crk contains an SH2 domain and two SH3 domains. Through binding of particular ligands to the SH2 domain and the N-terminal SH3 domain, Crk has been implicated in a number of signaling processes, including regulation of cell growth, cell motility, and apoptosis. We report here that the C-terminal SH3 domain, never shown to bind any specific signaling molecules, contains a binding site for the nuclear export factor Crm1. We find that a mutant Crk protein, deficient in Crm1 binding, promotes apoptosis. Moreover, this nuclear export sequence mutant [NES(-) Crk] interacts strongly, through its SH2 domain, with the nuclear tyrosine kinase, Wee1. Collectively, these data suggest that a nuclear population of Crk bound to Wee1 promotes apoptotic death of mammalian cells.
Assuntos
Apoptose/fisiologia , Proteínas de Ciclo Celular , Carioferinas/metabolismo , Proteínas Nucleares , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Receptores Citoplasmáticos e Nucleares , Transporte Ativo do Núcleo Celular , Compartimento Celular , Núcleo Celular/fisiologia , Humanos , Masculino , Mutação , Ligação Proteica , Proteínas Quinases/genética , Sinais Direcionadores de Proteínas , Proteínas Proto-Oncogênicas c-crk , Transdução de Sinais , Domínios de Homologia de src , Proteína Exportina 1RESUMO
Purpose: Locoregional recurrence is a frequent treatment outcome for patients with advanced head and neck squamous cell carcinoma (HNSCC). Emerging evidence suggests that tumor recurrence is mediated by a small subpopulation of uniquely tumorigenic cells, that is, cancer stem cells (CSC), that are resistant to conventional chemotherapy, endowed with self-renewal and multipotency.Experimental Design: Here, we evaluated the efficacy of MEDI0641, a novel antibody-drug conjugate targeted to 5T4 and carrying a DNA-damaging "payload" (pyrrolobenzodiazepine) in preclinical models of HNSCC.Results: Analysis of a tissue microarray containing 77 HNSCC with follow-up of up to 12 years revealed that patients with 5T4high tumors displayed lower overall survival than those with 5T4low tumors (P = 0.038). 5T4 is more highly expressed in head and neck CSC (ALDHhighCD44high) than in control cells (non-CSC). Treatment with MEDI0641 caused a significant reduction in the CSC fraction in HNSCC cells (UM-SCC-11B, UM-SCC-22B) in vitro Notably, a single intravenous dose of 1 mg/kg MEDI0641 caused long-lasting tumor regression in three patient-derived xenograft (PDX) models of HNSCC. MEDI0641 ablated CSC in the PDX-SCC-M0 model, reduced it by five-fold in the PDX-SCC-M1, and two-fold in the PDX-SCC-M11 model. Importantly, mice (n = 12) treated with neoadjuvant, single administration of MEDI0641 prior to surgical tumor removal showed no recurrence for more than 200 days, whereas the control group had 7 recurrences (in 12 mice; P = 0.0047).Conclusions: Collectively, these findings demonstrate that an anti-5T4 antibody-drug conjugate reduces the fraction of CSCs and prevents local recurrence and suggest a novel therapeutic approach for patients with HNSCC. Clin Cancer Res; 23(10); 2516-27. ©2016 AACR.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Imunoconjugados/administração & dosagem , Glicoproteínas de Membrana/imunologia , Animais , Benzodiazepinas/administração & dosagem , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Autorrenovação Celular/genética , Autorrenovação Celular/imunologia , Dano ao DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/imunologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Imunoconjugados/imunologia , Camundongos , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/patologia , Pirróis/administração & dosagem , Carcinoma de Células Escamosas de Cabeça e Pescoço , Análise Serial de Tecidos , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Antibody-drug conjugates (ADC) are used to selectively deliver cytotoxic agents to tumors and have the potential for increased clinical benefit to cancer patients. 5T4 is an oncofetal antigen overexpressed on the cell surface in many carcinomas on both bulk tumor cells as well as cancer stem cells (CSC), has very limited normal tissue expression, and can internalize when bound by an antibody. An anti-5T4 antibody was identified and optimized for efficient binding and internalization in a target-specific manner, and engineered cysteines were incorporated into the molecule for site-specific conjugation. ADCs targeting 5T4 were constructed by site-specifically conjugating the antibody with payloads that possess different mechanisms of action, either a DNA cross-linking pyrrolobenzodiazepine (PBD) dimer or a microtubule-destabilizing tubulysin, so that each ADC had a drug:antibody ratio of 2. The resulting ADCs demonstrated significant target-dependent activity in vitro and in vivo; however, the ADC conjugated with a PBD payload (5T4-PBD) elicited more durable antitumor responses in vivo than the tubulysin conjugate in xenograft models. Likewise, the 5T4-PBD more potently inhibited the growth of 5T4-positive CSCs in vivo, which likely contributed to its superior antitumor activity. Given that the 5T4-PBD possessed both potent antitumor activity as well as anti-CSC activity, and thus could potentially target bulk tumor cells and CSCs in target-positive indications, it was further evaluated in non-GLP rat toxicology studies that demonstrated excellent in vivo stability with an acceptable safety profile. Taken together, these preclinical data support further development of 5T4-PBD, also known as MEDI0641, against 5T4+ cancer indications. Mol Cancer Ther; 16(8); 1576-87. ©2017 AACR.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Benzodiazepinas/uso terapêutico , Imunoconjugados/uso terapêutico , Pirróis/uso terapêutico , Animais , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Benzodiazepinas/efeitos adversos , Benzodiazepinas/farmacologia , Linhagem Celular Tumoral , Humanos , Imunoconjugados/efeitos adversos , Imunoconjugados/farmacologia , Masculino , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Pirróis/efeitos adversos , Pirróis/farmacologia , Ratos Sprague-Dawley , Moduladores de Tubulina/efeitos adversos , Moduladores de Tubulina/farmacologia , Moduladores de Tubulina/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
HER3/ERBB3 is a kinase-deficient member of the EGFR family receptor tyrosine kinases (RTK) that is broadly expressed and activated in human cancers. HER3 is a compelling cancer target due to its important role in activation of the oncogenic PI3K/AKT pathway. It has also been demonstrated to confer tumor resistance to a variety of cancer therapies, especially targeted drugs against EGFR and HER2. HER3 can be activated by its ligand (heregulin/HRG), which induces HER3 heterodimerization with EGFR, HER2, or other RTKs. Alternatively, HER3 can be activated in a ligand-independent manner through heterodimerization with HER2 in HER2-amplified cells. We developed a fully human mAb against HER3 (KTN3379) that efficiently suppressed HER3 activity in both ligand-dependent and independent settings. Correspondingly, KTN3379 inhibited tumor growth in divergent tumor models driven by either ligand-dependent or independent mechanisms in vitro and in vivo Most intriguingly, while investigating the mechanistic underpinnings of tumor response to KTN3379, we discovered an interesting dichotomy in that PTEN loss, a frequently occurring oncogenic lesion in a broad range of cancer types, substantially blunted the tumor response in HER2-amplified cancer, but not in the ligand-driven cancer. To our knowledge, this represents the first study ascertaining the impact of PTEN loss on the antitumor efficacy of a HER3 mAb. KTN3379 is currently undergoing a phase Ib clinical trial in patients with advanced solid tumors. Our current study may help us optimize patient selection schemes for KTN3379 to maximize its clinical benefits. Mol Cancer Ther; 15(4); 689-701. ©2016 AACR.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Neoplasias/metabolismo , Receptor ErbB-3/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Expressão Gênica , Humanos , Ligantes , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Receptor ErbB-3/química , Receptor ErbB-3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Head and neck squamous cell carcinomas (HNSCC) exhibit a small population of uniquely tumorigenic cancer stem cells (CSC) endowed with self-renewal and multipotency. We have recently shown that IL-6 enhances the survival and tumorigenic potential of head and neck cancer stem cells (i.e. ALDH(high)CD44(high) cells). Here, we characterized the effect of therapeutic inhibition of IL-6 with a novel humanized anti-IL-6 antibody (MEDI5117) using three low-passage patient-derived xenograft (PDX) models of HNSCC. We observed that single agent MEDI5117 inhibited the growth of PDX-SCC-M1 tumors (P < .05). This PDX model was generated from a previously untreated HNSCC. In contrast, MEDI5117 was not effective at reducing overall tumor volume for PDX models representing resistant disease (PDX-SCC-M0, PDX-SCC-M11). Low dose MEDI5117 (3 mg/kg) consistently decreased the fraction of cancer stem cells in PDX models of HNSCC when compared to IgG-treated controls, as follows: PDX-SCC-M0 (P < .001), PDX-SCC-M1 (P < .001), PDX-SCC-M11 (P = .04). Interestingly, high dose MEDI5117 (30 mg/kg) decreased the CSC fraction in the PDX-SCC-M11 model (P = .002), but not in PDX-SCC-M0 and PDX-SCC-M1. MEDI5117 mediated a dose-dependent decrease in the number of orospheres generated by ALDH(high)CD44(high) cells cultured in ultra-low attachment plates (P < .05), supporting an inhibitory effect on head and neck cancer stem cells. Notably, single agent MEDI5117 reduced the overall recurrence rate of PDX-SCC-M0, a PDX generated from the local recurrence of human HNSCC. Collectively, these data demonstrate that therapeutic inhibition of IL-6 with low-dose MEDI5117 decreases the fraction of cancer stem cells, and that adjuvant MEDI5117 inhibits recurrence in preclinical models of HNSCC.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Interleucina-6/antagonistas & inibidores , Interleucina-6/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Animais , Biomarcadores , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Modelos Animais de Doenças , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/mortalidade , Humanos , Camundongos , Recidiva , Carcinoma de Células Escamosas de Cabeça e Pescoço , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Dendritic cell (DC)-based vaccine strategies aimed at targeting cancer stem-like cells (CSC) may be most efficacious if deployed in the adjuvant setting. In this study, we offer preclinical evidence that this is the case for a CSC-DC vaccine as tested in murine models of SCC7 squamous cell cancer and D5 melanoma. Vaccination of mice with an ALDH(high) SCC7 CSC-DC vaccine after surgical excision of established SCC7 tumors reduced local tumor relapse and prolonged host survival. This effect was augmented significantly by simultaneous administration of anti-PD-L1, an immune checkpoint inhibitor. In the minimal disease setting of D5 melanoma, treatment of mice with ALDH(high) CSC-DC vaccination inhibited primary tumor growth, reduced spontaneous lung metastases, and increased host survival. In this setting, CCR10 and its ligands were downregulated on ALDH(high) D5 CSCs and in lung tissues, respectively, after vaccination with ALDH(high) D5 CSC-DC. RNAi-mediated attenuation of CCR10 blocked tumor cell migration in vitro and metastasis in vivo T cells harvested from mice vaccinated with ALDH(high) D5 CSC-DC selectively killed ALDH(high) D5 CSCs, with additional evidence of humoral immunologic engagement and a reduction in ALDH(high) cells in residual tumors. Overall, our results offered a preclinical proof of concept for the use of ALDH(high) CSC-DC vaccines in the adjuvant setting to more effectively limit local tumor recurrence and spontaneous pulmonary metastasis, as compared with traditional DC vaccines, with increased host survival further accentuated by simultaneous PD-L1 blockade. Cancer Res; 76(16); 4661-72. ©2016 AACR.
Assuntos
Vacinas Anticâncer/farmacologia , Células Dendríticas/imunologia , Melanoma Experimental , Neoplasias de Células Escamosas , Células-Tronco Neoplásicas/imunologia , Adjuvantes Imunológicos/farmacologia , Aldeído Desidrogenase/metabolismo , Animais , Vacinas Anticâncer/imunologia , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BLRESUMO
Antibody-drug conjugates (ADCs) are among the most promising empowered biologics for cancer treatment. ADCs are commonly prepared by chemical conjugation of small molecule cytotoxic anti-cancer drugs to antibodies through either lysine side chains or cysteine thiols generated by the reduction of interchain disulfide bonds. Both methods yield heterogeneous conjugates with complex biophysical properties and suboptimal serum stability, efficacy, and pharmacokinetics. To limit the complexity of cysteine-based ADCs, we have engineered and characterized in vitro and in vivo antibody cysteine variants that allow precise control of both site of conjugation and drug load per antibody molecule. We demonstrate that the chemically-defined cysteine-engineered antibody-tubulysin conjugates have improved ex vivo and in vivo stability, efficacy, and pharmacokinetics when compared to conventional cysteine-based ADCs with similar drug-to-antibody ratios. In addition, to limit the non-target FcγRs mediated uptake of the ADCs by cells of the innate immune system, which may result in off-target toxicities, the ADCs have been engineered to lack Fc-receptor binding. The strategies described herein are broadly applicable to any full-length IgG or Fc-based ADC and have been incorporated into an ADC that is in phase I clinical development.
Assuntos
Anticorpos Monoclonais/química , Imunoconjugados/química , Animais , Anticorpos Monoclonais/farmacocinética , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Sobrevivência Celular , Cromatografia Líquida de Alta Pressão , Cisteína/química , Desenho de Fármacos , Feminino , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/farmacocinética , Camundongos Nus , Terapia de Alvo Molecular , Estabilidade Proteica , Receptores Fc/químicaRESUMO
Elevated levels of the proinflammatory cytokine IL6 are associated with poor survival outcomes in many cancers. Antibodies targeting IL6 and its receptor have been developed for chronic inflammatory disease, but they have not yet been shown to clearly benefit cancer patients, possibly due to antibody potency or the settings in which they have been tested. In this study, we describe the development of a novel high-affinity anti-IL6 antibody, MEDI5117, which features an extended half-life and potent inhibitory effects on IL6 biologic activity. MEDI5117 inhibited IL6-mediated activation of STAT3, suppressing the growth of several tumor types driven by IL6 autocrine signaling. In the same models, MEDI5117 displayed superior preclinical activity relative to a previously developed anti-IL6 antibody. Consistent with roles for IL6 in promoting tumor angiogenesis, we found that MEDI5117 inhibited the growth of endothelial cells, which can produce IL6 and support tumorigenesis. Notably, in tumor xenograft assays in mice, we documented the ability of MEDI5117 to enhance the antitumor activities of chemotherapy or gefitinib in combination treatment regimens. MEDI5117 also displayed robust activity on its own against trastuzumab-resistant HER2(+) tumor cells by targeting the CD44(+)CD24(-) cancer stem cell population. Collectively, our findings extend the evidence of important pleiotropic roles of IL6 in tumorigenesis and drug resistance, and offer a preclinical proof of concept for the use of IL6 antibodies in combination regimens to heighten therapeutic responses and overcome drug resistance.
Assuntos
Interleucina-6/metabolismo , Neoplasias/genética , Trastuzumab/uso terapêutico , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Transdução de Sinais , Trastuzumab/administração & dosagemRESUMO
MEDI9447 is a human monoclonal antibody that is specific for the ectoenzyme CD73 and currently undergoing Phase I clinical trials. Here we show that MEDI9447 is a potent inhibitor of CD73 ectonucleotidase activity, with wide ranging immune regulatory consequences. MEDI9447 results in relief from adenosine monophosphate (AMP)-mediated lymphocyte suppression in vitro and inhibition of mouse syngeneic tumor growth in vivo. In contrast with other cancer immunotherapy agents such as checkpoint inhibitors or T-cell agonists, MEDI9447 drives changes in both myeloid and lymphoid infiltrating leukocyte populations within the tumor microenvironment of mouse models. Changes include significant alterations in a number of tumor micro-environmental subpopulations including increases in CD8(+) effector cells and activated macrophages. Furthermore, these changes correlate directly with responder and non-responder subpopulations within animal studies using syngeneic tumors. Combination data showing additive activity between MEDI9447 and anti-PD-1 antibodies using human cells in vitro and mouse tumor models further demonstrate the potential value of relieving adenosine-mediated immunosuppression. Based on these data, a Phase I study to test the safety, tolerability, and clinical activity of MEDI9447 in cancer patients was initiated (NCT02503774).
RESUMO
Polo-like kinase 3 (Plk3, alternatively termed Prk) is involved in the regulation of DNA damage checkpoint as well as in M-phase function. Plk3 physically interacts with p53 and phosphorylates this tumor suppressor protein on serine-20, suggesting that the role of Plk3 in cell cycle progression is mediated, at least in part, through direct regulation of p53. Here we show that Plk3 is rapidly activated by reactive oxygen species in normal diploid fibroblast cells (WI-38), correlating with a subsequent increase in p53 protein level. Plk3 physically interacts with Chk2 and the interaction is enhanced upon DNA damage. In addition, Chk2 immunoprecipitated from cell lysates of Daudi (which expressed little Plk3) is capable of stimulating the kinase activity of purified recombinant Plk3 in vitro, and this stimulation is more pronounced when Plk3 is supplemented with Chk2 immunoprecipitated from Daudi after DNA damage. Furthermore, ectopic expression Chk2 activates cellular Plk3. Together, our studies suggest Chk2 may mediate direct activation of Plk3 in response to genotoxic stresses.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/genética , Transformação Celular Neoplásica/genética , Dano ao DNA/genética , Neoplasias/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Transformação Celular Neoplásica/metabolismo , Quinase do Ponto de Checagem 2 , Dano ao DNA/efeitos dos fármacos , Humanos , Neoplasias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Espécies Reativas de Oxigênio/farmacologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
The origins of the complex process of intratumoral heterogeneity have been highly debated and different cellular mechanisms have been hypothesized to account for the diversity within a tumor. The clonal evolution and cancer stem cell (CSC) models have been proposed as drivers of this heterogeneity. However, the concept of cancer stem cell plasticity and bidirectional conversion between stem and non-stem cells has added additional complexity to these highly studied paradigms and may help explain the tumor heterogeneity observed in solid tumors. The process of cancer stem cell plasticity in which cancer cells harbor the dynamic ability of shifting from a non-CSC state to a CSC state and vice versa may be modulated by specific microenvironmental signals and cellular interactions arising in the tumor niche. In addition to promoting CSC plasticity, these interactions may contribute to the cellular transformation of tumor cells and affect response to chemotherapeutic and radiation treatments by providing CSCs protection from these agents. Herein, we review the literature in support of this dynamic CSC state, discuss the effectors of plasticity, and examine their role in the development and treatment of cancer.
RESUMO
Excess production of the proinflammatory IL6 has both local and systemic tumor-promoting activity in many cancers, including ovarian cancer. However, treatment of advanced ovarian cancer patients with a neutralizing IL6 antibody yielded little efficacy in a previous phase II clinical trial. Here, we report results that may explain this outcome, based on the finding that neutralizing antibodies to IL6 and STAT3 inhibition are sufficient to upregulate the EGFR pathway in high-grade serous and other ovarian cancer cells. Cell treatment with the EGFR inhibitor gefitinib abolished upregulation of the EGFR pathway. Combining neutralizing IL6 antibodies and gefitinib inhibited malignant cell growth in 2D and 3D culture. We found that ErbB-1 was localized predominantly in the nucleus of ovarian cancer cells examined, contrasting with plasma membrane localization in lung cancer cells. Treatment with anti-IL6, gefitinib, or their combination all led to partial restoration of ErbB-1 on the plasma membrane. In vivo experiments confirmed the effects of IL6 inhibition on the EGFR pathway and the enhanced activity of a combination of anti-IL6 antibodies and gefitinib on malignant cell growth. Taken together, our results offer a preclinical rationale to combine anti-IL6 and gefitinib to treat patients with advanced stage ovarian cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Receptores ErbB/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Animais , Anticorpos Neutralizantes/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Núcleo Celular , Sinergismo Farmacológico , Receptores ErbB/genética , Feminino , Gefitinibe , Humanos , Interleucina-6/antagonistas & inibidores , Interleucina-6/imunologia , Interleucina-6/fisiologia , Sistema de Sinalização das MAP Quinases , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Ovarianas/metabolismo , Transporte Proteico , Quinazolinas/administração & dosagem , Fator de Transcrição STAT3/metabolismo , Carga Tumoral , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The cytokine IL6 has a number of tumor-promoting activities in human and experimental cancers, but its potential as an angiogenic agent has not been fully investigated. Here, we show that IL6 can directly induce vessel sprouting in the ex vivo aortic ring model, as well as endothelial cell proliferation and migration, with similar potency to VEGF. However, IL6-stimulated aortic ring vessel sprouts had defective pericyte coverage compared with VEGF-stimulated vessels. The mechanism of IL6 action on pericytes involved stimulation of the Notch ligand Jagged1 as well as angiopoietin2 (Ang2). When peritoneal xenografts of ovarian cancer were treated with an anti-IL6 antibody, pericyte coverage of vessels was restored. In addition, in human ovarian cancer biopsies, there was an association between levels of IL6 mRNA, Jagged1, and Ang2. Our findings have implications for the use of cancer therapies that target VEGF or IL6 and for understanding abnormal angiogenesis in cancers, chronic inflammatory disease, and stroke.
Assuntos
Interleucina-6/metabolismo , Interleucina-6/farmacologia , Neovascularização Patológica/metabolismo , Animais , Aorta/efeitos dos fármacos , Aorta/patologia , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Tumoral , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Interleucina-6/genética , Proteína Jagged-1 , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular/métodos , Neovascularização Patológica/induzido quimicamente , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Técnicas de Cultura de Órgãos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Pericitos/efeitos dos fármacos , Pericitos/patologia , Ratos Wistar , Proteínas Serrate-Jagged , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Proteínas de Transporte Vesicular/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
ADAM17 is the primary sheddase for HER pathway ligands. We report the discovery of a potent and specific ADAM17 inhibitory antibody, MEDI3622, which induces tumor regression or stasis in many EGFR-dependent tumor models. The inhibitory activity of MEDI3622 correlated with EGFR activity both in a series of tumor models across several indications as well in as a focused set of head and neck patient-derived xenograft models. The antitumor activity of MEDI3622 was superior to that of EGFR/HER pathway inhibitors in the OE21 esophageal model and the COLO205 colorectal model suggesting additional activity outside of the EGFR pathway. Combination of MEDI3622 and cetuximab in the OE21 model was additive and eradicated tumors. Proteomics analysis revealed novel ADAM17 substrates that function outside of the HER pathways and may contribute toward the antitumor activity of the monoclonal antibody.