RESUMEN
Preclinical and clinical studies demonstrate that T cell-dependent bispecific antibodies (TDBs) induce systemic changes in addition to tumor killing, leading to adverse events. Here, we report an in-depth characterization of acute responses to TDBs in tumor-bearing mice. Contrary to modest changes in tumors, rapid and substantial lymphocyte accumulation and endothelial cell (EC) activation occur around large blood vessels in normal organs including the liver. We hypothesize that organ-specific ECs may account for the differential responses in normal tissues and tumors, and we identify a list of genes selectively upregulated by TDB in large liver vessels. Using one of the genes as an example, we demonstrate that CD9 facilitates ICAM-1 to support T cell-EC interaction in response to soluble factors released from a TDB-mediated cytotoxic reaction. Our results suggest that multiple factors may cooperatively promote T cell infiltration into normal organs as a secondary response to TDB-mediated tumor killing. These data shed light on how different vascular beds respond to cancer immunotherapy and may help improve their safety and efficacy.
Asunto(s)
Anticuerpos Biespecíficos , Neoplasias , Ratones , Animales , Linfocitos T , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Neoplasias/tratamiento farmacológico , Comunicación Celular , Células EndotelialesRESUMEN
BACKGROUND: Cancer immunotherapy approaches that elicit immune cell responses, including T and NK cells, have revolutionized the field of oncology. However, immunosuppressive mechanisms restrain immune cell activation within solid tumors so additional strategies to augment activity are required. METHODS: We identified the co-stimulatory receptor NKG2D as a target based on its expression on a large proportion of CD8+ tumor infiltrating lymphocytes (TILs) from breast cancer patient samples. Human and murine surrogate NKG2D co-stimulatory receptor-bispecifics (CRB) that bind NKG2D on NK and CD8+ T cells as well as HER2 on breast cancer cells (HER2-CRB) were developed as a proof of concept for targeting this signaling axis in vitro and in vivo. RESULTS: HER2-CRB enhanced NK cell activation and cytokine production when co-cultured with HER2 expressing breast cancer cell lines. HER2-CRB when combined with a T cell-dependent-bispecific (TDB) antibody that synthetically activates T cells by crosslinking CD3 to HER2 (HER2-TDB), enhanced T cell cytotoxicity, cytokine production and in vivo antitumor activity. A mouse surrogate HER2-CRB (mHER2-CRB) improved in vivo efficacy of HER2-TDB and augmented NK as well as T cell activation, cytokine production and effector CD8+ T cell differentiation. CONCLUSION: We demonstrate that targeting NKG2D with bispecific antibodies (BsAbs) is an effective approach to augment NK and CD8+ T cell antitumor immune responses. Given the large number of ongoing clinical trials leveraging NK and T cells for cancer immunotherapy, NKG2D-bispecifics have broad combinatorial potential.
Asunto(s)
Neoplasias de la Mama , Linfocitos T CD8-positivos , Células Asesinas Naturales , Subfamilia K de Receptores Similares a Lectina de Células NK , Humanos , Animales , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Ratones , Linfocitos T CD8-positivos/inmunología , Células Asesinas Naturales/inmunología , Femenino , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/terapia , Receptor ErbB-2/inmunología , Línea Celular Tumoral , Inmunoterapia/métodos , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismoRESUMEN
Multiple myeloma (MM) arises from malignant immunoglobulin (Ig)-secreting plasma cells and remains an incurable, often lethal disease despite therapeutic advances. The unfolded-protein response sensor IRE1α supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may co-opt the IRE1α-XBP1s pathway; however, the validity of IRE1α as a potential MM therapeutic target is controversial. Genetic disruption of IRE1α or XBP1s, or pharmacologic IRE1α kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice and augmented efficacy of two established frontline antimyeloma agents, bortezomib and lenalidomide. Mechanistically, IRE1α perturbation inhibited expression of key components of the endoplasmic reticulum-associated degradation machinery, as well as secretion of Ig light chains and of cytokines and chemokines known to promote MM growth. Selective IRE1α kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138- cells derived from bone marrows of newly diagnosed or posttreatment-relapsed MM patients, in both US- and European Union-based cohorts. Effective IRE1α inhibition preserved glucose-induced insulin secretion by pancreatic microislets and viability of primary hepatocytes in vitro, as well as normal tissue homeostasis in mice. These results establish a strong rationale for developing kinase-directed inhibitors of IRE1α for MM therapy.
Asunto(s)
Endorribonucleasas/genética , Mieloma Múltiple/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/genética , Anciano , Animales , Bortezomib/farmacología , Estrés del Retículo Endoplásmico/genética , Endorribonucleasas/antagonistas & inhibidores , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Lenalidomida/farmacología , Masculino , Ratones , Persona de Mediana Edad , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Respuesta de Proteína Desplegada/genética , Proteína 1 de Unión a la X-Box/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Acute myeloid leukemia (AML) is a major unmet medical need. Most patients have poor long-term survival, and treatment has not significantly changed in 40 years. Recently, bispecific antibodies that redirect the cytotoxic activity of effector T cells by binding to CD3, the signaling component of the T-cell receptor, and a tumor target have shown clinical activity. Notably, blinatumomab is approved to treat relapsed/refractory acute lymphoid leukemia. Here we describe the design, discovery, pharmacologic activity, pharmacokinetics, and safety of a CD3 T cell-dependent bispecific (TDB) full-length human IgG1 therapeutic antibody targeting CLL-1 that could potentially be used in humans to treat AML. CLL-1 is prevalent in AML and, unlike other targets such as CD33 and CD123, is not expressed on hematopoietic stem cells providing potential hematopoietic recovery. We selected a high-affinity monkey cross-reactive anti-CLL-1 arm and tested several anti-CD3 arms that varied in affinity, and determined that the high-affinity CD3 arms were up to 100-fold more potent in vitro. However, in mouse models, the efficacy differences were less pronounced, probably because of prolonged exposure to TDB found with lower-affinity CD3 TDBs. In monkeys, assessment of safety and target cell depletion by the high- and low-affinity TDBs revealed that only the low-affinity CD3/CLL1 TDB was well tolerated and able to deplete target cells. Our data suggest that an appropriately engineered CLL-1 TDB could be effective in the treatment of AML.
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Antineoplásicos/uso terapéutico , Lectinas Tipo C/inmunología , Leucemia Mieloide Aguda/tratamiento farmacológico , Lectina 3 Similar a Ig de Unión al Ácido Siálico/inmunología , Animales , Anticuerpos Biespecíficos/efectos adversos , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacocinética , Antineoplásicos/efectos adversos , Antineoplásicos/inmunología , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Humanos , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/patología , Macaca fascicularis , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Pancreatic adenocarcinoma (PDAC) is a major unmet medical need and a deeper understanding of molecular drivers is needed to advance therapeutic options for patients. We report here that p21-activated kinase 1 (PAK1) is a central node in PDAC cells downstream of multiple growth factor signalling pathways, including hepatocyte growth factor (HGF) and MET receptor tyrosine kinase. PAK1 inhibition blocks signalling to cytoskeletal effectors and tumour cell motility driven by HGF/MET. MET antagonists, such as onartuzumab and crizotinib, are currently in clinical development. Given that even highly effective therapies have resistance mechanisms, we show that combination with PAK1 inhibition overcomes potential resistance mechanisms mediated either by activation of parallel growth factor pathways or by direct amplification of PAK1. Inhibition of PAK1 attenuated in vivo tumour growth and metastasis in a model of pancreatic adenocarcinoma. In human tissues, PAK1 is highly expressed in a proportion of PDACs (33% IHC score 2 or 3; n = 304) and its expression is significantly associated with MET positivity (p < 0.0001) and linked to a widespread metastatic pattern in patients (p = 0.067). Taken together, our results provide evidence for a functional role of MET/PAK1 signalling in pancreatic adenocarcinoma and support further characterization of therapeutic inhibitors in this indication.
Asunto(s)
Adenocarcinoma/metabolismo , Movimiento Celular , Resistencia a Antineoplásicos/fisiología , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Quinasas p21 Activadas/metabolismo , Adenocarcinoma/patología , Animales , Anticuerpos Monoclonales/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Azetidinas/farmacología , Movimiento Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Inmunohistoquímica , Ratones , Neoplasias Pancreáticas/patología , Piperidinas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Apo2L/TRAIL stimulates cancer cell death through the proapoptotic receptors DR4 and DR5, but the determinants of tumor susceptibility to this ligand are not fully defined. mRNA expression of the peptidyl O-glycosyltransferase GALNT14 correlated with Apo2L/TRAIL sensitivity in pancreatic carcinoma, non-small-cell lung carcinoma and melanoma cell lines, and up to 30% of samples from various human malignancies showed GALNT14 overexpression. RNA interference of GALNT14 reduced cellular Apo2L/TRAIL sensitivity, whereas overexpression increased responsiveness. Biochemical analysis of DR5 identified several ectodomain O-(N-acetyl galactosamine-galactose-sialic acid) structures. Sequence comparison predicted conserved extracellular DR4 and DR5 O-glycosylation sites; progressive mutation of the DR5 sites attenuated apoptotic signaling. O-glycosylation promoted ligand-stimulated clustering of DR4 and DR5, which mediated recruitment and activation of the apoptosis-initiating protease caspase-8. These results uncover a new link between death-receptor O-glycosylation and apoptotic signaling, providing potential predictive biomarkers for Apo2L/TRAIL-based cancer therapy.
Asunto(s)
Receptores de Muerte Celular/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Secuencia de Aminoácidos , Animales , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas , Línea Celular Tumoral , Supervivencia Celular , Predisposición Genética a la Enfermedad , Glicosilación , Humanos , Neoplasias Pulmonares , Melanoma , Ratones , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Pancreáticas , ARN Mensajero/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Trasplante HeterólogoRESUMEN
Insufficient quantity of functional T cells is a likely factor limiting clinical activity of T cell bispecific antibodies, especially in solid tumor indications. We hypothesized that XmAb24306 (efbalropendekin alfa), a lymphoproliferative interleukin (IL)-15/IL-15 receptor α (IL-15Rα) Fc-fusion protein, may potentiate the activity of T cell dependent (TDB) antibodies. Activation of human peripheral T cells by cevostamab, an anti-FcRH5/CD3 TDB, or anti-HER2/CD3 TDB resulted in upregulation of IL-2/15Rß (CD122) receptor subunit in nearly all CD8+ and majority of CD4+ T cells, suggesting that TDB treatment may sensitize T cells to the IL-15. XmAb24306 enhanced T cell bispecific antibody induced CD8+ and CD4+ T cell proliferation and expansion. In vitro combination of XmAb24306 with cevostamab or anti-HER2/CD3 TDB resulted in significant enhancement of tumor cell killing, which was reversed when T cell numbers were normalized, suggesting that T cell expansion is the main mechanism for the observed benefit. Pre-treatment of immune competent mice with a mouse-reactive surrogate of XmAb24306 (mIL-15-Fc) resulted in significant increase of T cells in blood, spleen and in tumors and converted transient anti-HER2/CD3 TDB responses to complete durable responses. In summary, our results support the hypothesis where the number of tumor infiltrating T cells is rate limiting for the activity of solid tumor targeting TDBs. Upregulation of CD122 by TDB treatment and the observed synergy with XmAb24306 and T cell bispecific antibodies supports clinical evaluation of this novel immunotherapy combination.
RESUMEN
Upper hinge is vulnerable to radical attacks that result in breakage of the heavy-light chain linkage and cleavage of the hinge of an IgG1. To further explore mechanisms responsible for the radical induced hinge degradation, nine mutants were designed to determine the roles that the upper hinge Asp and His play in the radical reactions. The observation that none of these substitutions could inhibit the breakage of the heavy-light chain linkage suggests that the breakage may result from electron transfer from Cys(231) directly to the heavy-light chain linkage upon radical attacks, and implies a pathway separate from His(229)-mediated hinge cleavage. On the other hand, the substitution of His(229) with Tyr showed promising advantages over the native antibody and other substitutions in improving the stability and function of the IgG1. This substitution inhibited the hinge cleavage by 98% and suggests that the redox active nature of Tyr did not enable it to replicate the ability of His to facilitate radical induced degradation. We propose that the lower redox potential of Tyr, a residue that may be the ultimate sink for oxidizing equivalents in proteins, is responsible for the inhibition. More importantly, the substitution increased the antibody's binding to FcγRIII receptors by 2-3-fold, and improved ADCC activity by 2-fold, while maintaining a similar pharmacokinetic profile with respect to the wild type. Implications of these observations for antibody engineering and development are discussed.
Asunto(s)
Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Células CHO , Cricetinae , Cricetulus , Estudios de Factibilidad , Humanos , Radical Hidroxilo/farmacología , Inmunoglobulina G/genética , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/química , Datos de Secuencia Molecular , Mutación , Estabilidad Proteica , Proteolisis/efectos de los fármacosRESUMEN
New therapeutics and combination regimens have led to marked clinical improvements for the treatment of a subset of colorectal cancer. Immune checkpoint inhibitors have shown clinical efficacy in patients with mismatch-repair-deficient or microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC). However, patients with microsatellite-stable (MSS) or low levels of microsatellite instable (MSI-L) colorectal cancer have not benefited from these immune modulators, and the survival outcome remains poor for the majority of patients diagnosed with mCRC. In this article, we describe the discovery of a novel T-cell-dependent bispecific antibody (TDB) targeting tumor-associated antigen LY6G6D, LY6G6D-TDB, for the treatment of colorectal cancer. RNAseq analysis showed that LY6G6D was differentially expressed in colorectal cancer with high prevalence in MSS and MSI-L subsets, whereas LY6G6D expression in normal tissues was limited. IHC confirmed the elevated expression of LY6G6D in primary and metastatic colorectal tumors, whereas minimal or no expression was observed in most normal tissue samples. The optimized LY6G6D-TDB, which targets a membrane-proximal epitope of LY6G6D and binds to CD3 with high affinity, exhibits potent antitumor activity both in vitro and in vivo. In vitro functional assays show that LY6G6D-TDB-mediated T-cell activation and cytotoxicity are conditional and target dependent. In mouse xenograft tumor models, LY6G6D-TDB demonstrates antitumor efficacy as a single agent against established colorectal tumors, and enhanced efficacy can be achieved when LY6G6D-TDB is combined with PD-1 blockade. Our studies provide evidence for the therapeutic potential of LY6G6D-TDB as an effective treatment option for patients with colorectal cancer.
Asunto(s)
Anticuerpos Biespecíficos , Neoplasias Colorrectales , Inmunoglobulinas , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inmunoglobulinas/inmunología , Ratones , Inestabilidad de Microsatélites , Linfocitos T/inmunologíaRESUMEN
The importance of Bax for induction of tumor apoptosis through death receptors remains unclear. Here we show that Bax can be essential for death receptor--mediated apoptosis in cancer cells. Bax-deficient human colon carcinoma cells were resistant to death-receptor ligands, whereas Bax-expressing sister clones were sensitive. Bax was dispensable for apical death-receptor signaling events including caspase-8 activation, but crucial for mitochondrial changes and downstream caspase activation. Treatment of colon tumor cells deficient in DNA mismatch repair with the death-receptor ligand apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selected in vitro or in vivo for refractory subclones with Bax frameshift mutations including deletions at a novel site. Chemotherapeutic agents upregulated expression of the Apo2L/TRAIL receptor DR5 and the Bax homolog Bak in Baxminus sign/minus sign cells, and restored Apo2L/TRAIL sensitivity in vitro and in vivo. Thus, Bax mutation in mismatch repair--deficient tumors can cause resistance to death receptor--targeted therapy, but pre-exposure to chemotherapy rescues tumor sensitivity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Apoptosis/fisiología , Glicoproteínas de Membrana/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Antineoplásicos Fitogénicos/farmacología , Proteínas Reguladoras de la Apoptosis , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Camptotecina/farmacología , Proteínas Portadoras/metabolismo , Caspasa 8 , Caspasa 9 , Caspasas/metabolismo , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Activación Enzimática , Etopósido/farmacología , Proteína de Dominio de Muerte Asociada a Fas , Femenino , Citometría de Flujo , Humanos , Glicoproteínas de Membrana/uso terapéutico , Ratones , Ratones Desnudos , Mitocondrias/metabolismo , Mutación , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/genética , Distribución Aleatoria , Ligando Inductor de Apoptosis Relacionado con TNF , Trasplante Heterólogo , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/uso terapéutico , Proteína X Asociada a bcl-2RESUMEN
Ovarian cancer is a diverse class of tumors with very few effective treatment options and suboptimal response rates in early clinical studies using immunotherapies. Here we describe LY6/PLAUR domain containing 1 (LYPD1) as a novel target for therapeutic antibodies for the treatment of ovarian cancer. LYPD1 is broadly expressed in both primary and metastatic ovarian cancer with â¼70% prevalence in the serous cancer subset. Bispecific antibodies targeting CD3 on T cells and a tumor antigen on cancer cells have demonstrated significant clinical activity in hematologic cancers. We have developed an anti-LYPD1/CD3 T-cell-dependent bispecific antibody (TDB) to redirect T-cell responses to LYPD1 expressing ovarian cancer. Here we characterize the nonclinical pharmacology of anti-LYPD1/CD3 TDB and show induction of a robust polyclonal T-cell activation and target dependent killing of LYPD1 expressing ovarian cancer cells resulting in efficient in vivo antitumor responses in PBMC reconstituted immune-deficient mice and human CD3 transgenic mouse models. Anti-LYPD1/CD3 TDB is generally well tolerated at high-dose levels in mice, a pharmacologically relevant species, and showed no evidence of toxicity or damage to LYPD1 expressing tissues.
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Complejo CD3/inmunología , Neoplasias Ováricas/tratamiento farmacológico , Secuencia de Aminoácidos , Animales , Anticuerpos Biespecíficos/farmacología , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Transgénicos , Neoplasias Ováricas/patologíaRESUMEN
The recent success of multiple immunomodulating drugs in oncology highlights the potential of relieving immunosuppression by directly engaging the immune system in the tumor bed to target cancer cells. Durable responses to immune checkpoint inhibitors experienced by some patients may be indicative of the formation of a T cell memory response. This has prompted the search for preclinical evidence of therapy-induced long-term immunity as part of the evaluation of novel therapeutics. A common preclinical method used to document long-term immunity is the use of tumor rechallenge experiments in which tumor growth is assessed in mice that have previously rejected tumors in response to therapy. Failure of rechallenge engraftment, typically alongside successful engraftment of the same tumor in naive animals as a control, is often presented as evidence of therapy-induced tumor immunity. Here, we present evidence that formation of tumor immunity often develops independent of therapy. We observed elevated rates of rechallenge rejection following surgical resection of primary tumors for four of five commonly used models and that such postexcision immunity could be adoptively transferred to treatment-naïve mice. We also show that tumor-specific cytolytic T cells are induced on primary tumor challenge independent of therapeutic intervention. Taken together these data call into question the utility of tumor rechallenge studies and the use of naïve animals as controls to demonstrate therapy-induced formation of long-term tumor immunity.
Asunto(s)
Inmunización/métodos , Neoplasias/terapia , Animales , Femenino , Humanos , Masculino , Ratones , Neoplasias/patologíaRESUMEN
Inhibiting the programmed death-1 (PD-1) pathway is one of the most effective approaches to cancer immunotherapy, but its mechanistic basis remains incompletely understood. Binding of PD-1 to its ligand PD-L1 suppresses T-cell function in part by inhibiting CD28 signaling. Tumor cells and infiltrating myeloid cells can express PD-L1, with myeloid cells being of particular interest as they also express B7-1, a ligand for CD28 and PD-L1. Here we demonstrate that dendritic cells (DCs) represent a critical source of PD-L1, despite being vastly outnumbered by PD-L1+ macrophages. Deletion of PD-L1 in DCs, but not macrophages, greatly restricted tumor growth and led to enhanced antitumor CD8+ T-cell responses. Our data identify a unique role for DCs in the PD-L1-PD-1 regulatory axis and have implications for understanding the therapeutic mechanism of checkpoint blockade, which has long been assumed to reflect the reversal of T-cell exhaustion induced by PD-L1+ tumor cells.
Asunto(s)
Antígeno B7-H1 , Neoplasias , Antígeno B7-H1/genética , Antígenos CD28/metabolismo , Células Dendríticas , Humanos , Ligandos , Neoplasias/genética , Receptor de Muerte Celular Programada 1/genéticaRESUMEN
The deubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with a high risk for mesothelioma and melanocytic tumors. Here, we show that pancreatic intraepithelial neoplasia driven by oncogenic mutant KrasG12D progressed to pancreatic adenocarcinoma in the absence of BAP1. The Hippo pathway was deregulated in BAP1-deficient pancreatic tumors, with the tumor suppressor LATS exhibiting enhanced ubiquitin-dependent proteasomal degradation. Therefore, BAP1 may limit tumor progression by stabilizing LATS and thereby promoting activity of the Hippo tumor suppressor pathway. SIGNIFICANCE: BAP1 is mutated in a broad spectrum of tumors. Pancreatic Bap1 deficiency causes acinar atrophy but combines with oncogenic Ras to produce pancreatic tumors. BAP1-deficient tumors exhibit deregulation of the Hippo pathway.See related commentary by Brekken, p. 1624.
Asunto(s)
Adenocarcinoma , Neoplasias Pancreáticas , Vía de Señalización Hippo , Humanos , Proteínas Serina-Treonina Quinasas , Transducción de Señal , Proteínas Supresoras de Tumor , Ubiquitina TiolesterasaRESUMEN
Cancer cells exploit the unfolded protein response (UPR) to mitigate endoplasmic reticulum (ER) stress caused by cellular oncogene activation and a hostile tumor microenvironment (TME). The key UPR sensor IRE1α resides in the ER and deploys a cytoplasmic kinase-endoribonuclease module to activate the transcription factor XBP1s, which facilitates ER-mediated protein folding. Studies of triple-negative breast cancer (TNBC)-a highly aggressive malignancy with a dismal posttreatment prognosis-implicate XBP1s in promoting tumor vascularization and progression. However, it remains unknown whether IRE1α adapts the ER in TNBC cells and modulates their TME, and whether IRE1α inhibition can enhance antiangiogenic therapy-previously found to be ineffective in patients with TNBC. To gauge IRE1α function, we defined an XBP1s-dependent gene signature, which revealed significant IRE1α pathway activation in multiple solid cancers, including TNBC. IRE1α knockout in TNBC cells markedly reversed substantial ultrastructural expansion of their ER upon growth in vivo. IRE1α disruption also led to significant remodeling of the cellular TME, increasing pericyte numbers while decreasing cancer-associated fibroblasts and myeloid-derived suppressor cells. Pharmacologic IRE1α kinase inhibition strongly attenuated growth of cell line-based and patient-derived TNBC xenografts in mice and synergized with anti-VEGFA treatment to cause tumor stasis or regression. Thus, TNBC cells critically rely on IRE1α to adapt their ER to in vivo stress and to adjust the TME to facilitate malignant growth. TNBC reliance on IRE1α is an important vulnerability that can be uniquely exploited in combination with antiangiogenic therapy as a promising new biologic approach to combat this lethal disease. SIGNIFICANCE: Pharmacologic IRE1α kinase inhibition reverses ultrastructural distension of the ER, normalizes the tumor vasculature, and remodels the cellular TME, attenuating TNBC growth in mice.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antineoplásicos Inmunológicos/farmacología , Estrés del Retículo Endoplásmico/fisiología , Endorribonucleasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Neoplasias de la Mama Triple Negativas/terapia , Animales , Antineoplásicos Inmunológicos/inmunología , Línea Celular Tumoral , Estrés del Retículo Endoplásmico/efectos de los fármacos , Endorribonucleasas/genética , Femenino , Técnicas de Inactivación de Genes , Humanos , Ratones , Ratones SCID , Neovascularización Patológica/terapia , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Microambiente Tumoral , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/inmunología , Proteína 1 de Unión a la X-Box/antagonistas & inhibidores , Proteína 1 de Unión a la X-Box/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Systemic cytokine release and on-target/off-tumor toxicity to normal tissues are the main adverse effects limiting the clinical utility of T cell-redirecting therapies. This study was designed to determine how binding affinity for CD3 and tumor target HER2 impact the efficacy and nonclinical safety of anti-HER2/CD3 T cell-dependent antibodies (TDBs). Affinity was found to be a major determinant for the overall tolerability. Higher affinity for CD3 associated with rapidly elevated peripheral cytokine concentrations, weight loss in mice, and poor tolerability in cynomolgus monkeys. A TDB with lower CD3 affinity was better tolerated in cynomolgus monkeys compared with a higher CD3-affinity TDB. In contrast to tolerability, T cell binding affinity had only limited impact on in vitro and in vivo antitumor activity. High affinity for HER2 was critical for the tumor-killing activity of anti-HER2/CD3 TDBs, but higher HER2 affinity also associated with a more severe toxicity profile, including cytokine release and damage to HER2-expressing tissues. The tolerability of the anti-HER2/CD3 was improved by implementing a dose-fractionation strategy. Fine-tuning the affinities for both the tumor target and CD3 is likely a valuable strategy for achieving maximal therapeutic index of CD3 bispecific antibodies.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Afinidad de Anticuerpos , Antineoplásicos Inmunológicos/inmunología , Receptor ErbB-2/inmunología , Animales , Anticuerpos Biespecíficos/química , Antineoplásicos Inmunológicos/química , Complejo CD3/química , Células CHO , Cricetulus , Evaluación Preclínica de Medicamentos , Humanos , Macaca fascicularis , Receptor ErbB-2/químicaRESUMEN
PURPOSE: Apomab is a fully human monoclonal antibody that induces programmed cell death through the proapoptotic receptor DR5 in various cancer cells but not in normal cells. Several lung cancer cell lines express DR5 and exhibit apoptosis in response to apomab in vitro. EXPERIMENTAL DESIGN: We investigated the efficacy of apomab and its interaction with chemotherapy in xenograft models based on human NCI-H460 non-small-cell lung carcinoma cells. In an established model of s.c. tumor xenografts, apomab or Taxol plus carboplatin chemotherapy delayed tumor progression, whereas combined treatment caused tumor regression and a substantially longer growth delay. To test apomab activity in a setting that may more closely mimic lung cancer pathology in patients, we developed a lung orthotopic model. RESULTS: In this model, microcomputed tomography imaging showed that apomab, chemotherapy, or combination treatment significantly inhibited tumor growth compared with vehicle, whereas the combination caused greater inhibition in tumor growth relative to chemotherapy or apomab. Similarly, histologic analysis revealed that apomab, chemotherapy, or the combination significantly reduced tumor size compared with vehicle, whereas the combination induced significantly greater reduction in tumor size than did chemotherapy or apomab. Furthermore, combined treatment improved 105-day survival relative to vehicle (P = 0.0023) as well as to apomab (P = 0.0445) or chemotherapy (P = 0.0415). CONCLUSION: These results show a positive interaction of apomab with chemotherapy, evidenced by significant inhibition of tumor growth as well as improved survival, thus supporting further investigation of this therapeutic approach in lung cancer patients.
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Anticuerpos Monoclonales/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Animales , Apoptosis/efectos de los fármacos , Carboplatino/administración & dosificación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Immunoblotting , Etiquetado Corte-Fin in Situ , Ratones , Ratones Desnudos , Paclitaxel/administración & dosificación , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Erlotinib (Tarceva), is an orally available, reversible inhibitor of epidermal growth factor receptor (EGFR; HER1) that exhibits inhibitory activity on purified HER2 kinase at much higher concentrations. Despite the minimal activity on purified protein in vitro, in vivo studies show that erlotinib inhibits the growth of HER2-driven systems effectively. Several hypotheses have been put forward to explain this discrepancy. In particular, it has been suggested that erlotinib might indirectly suppress the activity of HER2 by blocking the ability of EGFR to transactivate it when the two receptors are part of a heterodimer complex. However, an alternative possibility that has not been adequately addressed is whether the direct inhibitory action of erlotinib on the HER2 kinase might account for the observed biological responses. To distinguish between a direct effect of erlotinib on HER2 kinase in intact cells or an indirect effect of erlotinib on HER2 activity that is mediated through EGFR, we generated cell lines that express either EGFR-H2 chimeric receptor or HER2 and HER3 receptors in an EGFR-negative background. We show that dose-dependent inhibition of HER2 was achieved at the receptor level, on downstream signaling molecules, and more importantly was also translated into inhibition of cell growth. Our findings imply that the inhibitory effect of erlotinib in HER2-expressing cells may in part be mediated through direct interaction with HER2 rather than indirectly through a process that requires the presence of EGFR.
Asunto(s)
Receptores ErbB/biosíntesis , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Cetuximab , ADN Complementario/genética , Interacciones Farmacológicas , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Clorhidrato de Erlotinib , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/enzimología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Neurregulina-1/farmacología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2/biosíntesis , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-3/biosíntesis , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismoRESUMEN
The insulin-like growth factor (IGF) system consists of two ligands (IGF-I and IGF-II), which both signal through IGF-I receptor (IGF-IR) to stimulate proliferation and inhibit apoptosis, with activity contributing to malignant growth of many types of human cancers. We have developed a humanized, affinity-matured anti-human IGF-IR monoclonal antibody (h10H5), which binds with high affinity and specificity to the extracellular domain. h10H5 inhibits IGF-IR-mediated signaling by blocking IGF-I and IGF-II binding and by inducing cell surface receptor down-regulation via internalization and degradation, with the extracellular and intracellular domains of IGF-IR being differentially affected by the proteasomal and lysosomal inhibitors. In vitro, h10H5 exhibits antiproliferative effects on cancer cell lines. In vivo, h10H5 shows single-agent antitumor efficacy in human SK-N-AS neuroblastoma and SW527 breast cancer xenograft models and even greater efficacy in combination with the chemotherapeutic agent docetaxel or an anti-vascular endothelial growth factor antibody. Antitumor activity of h10H5 is associated with decreased AKT activation and glucose uptake and a 316-gene transcription profile with significant changes involving DNA metabolic and cell cycle machineries. These data support the clinical testing of h10H5 as a biotherapeutic for IGF-IR-dependent human tumors and furthermore illustrate a new method of monitoring its activity noninvasively in vivo via 2-fluoro-2-deoxy-d-glucose-positron emission tomography imaging.
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Anticuerpos Monoclonales/farmacología , Glucosa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor IGF Tipo 1/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Biomarcadores/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Sinergismo Farmacológico , Endocitosis/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/patología , Inhibidores de Proteasoma , Subunidades de Proteína/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
T cell-retargeting therapies have transformed the therapeutic landscape of oncology. Regardless of the modality, T cell activating therapies are commonly accompanied by systemic cytokine release, which can progress to deadly cytokine release syndrome (CRS). Because of incomplete mechanistic understanding of the relationship between T cell activation and systemic cytokine release, optimal toxicity management that retains full therapeutic potential remains unclear. Here, we report the cell type-specific cellular mechanisms that link CD3 bispecific antibody-mediated killing to toxic cytokine release. The immunologic cascade is initiated by T cell triggering, whereas monocytes and macrophages are the primary source of systemic toxic cytokine release. We demonstrate that T cell-generated tumor necrosis factor-α (TNF-α) is the primary mechanism mediating monocyte activation and systemic cytokine release after CD3 bispecific treatment. Prevention of TNF-α release is sufficient to impair systemic release of monocyte cytokines without affecting antitumor efficacy. Systemic cytokine release is only observed upon initial exposure to CD3 bispecific antibody not subsequent doses, indicating a biological distinction between doses. Despite impaired cytokine release after second exposure, T cell cytotoxicity remained unaffected, demonstrating that cytolytic activity of T cells can be achieved in the absence of cytokine release. The mechanistic uncoupling of toxic cytokines and T cell cytolytic activity in the context of CD3 bispecifics provides a biological rationale to clinically explore preventative treatment approaches to mitigate toxicity.