RESUMEN
Cancer immunotherapies are increasingly combined with targeted therapies to improve therapeutic outcomes. We show that combination of agonistic anti-CD40 with antiangiogenic antibodies targeting 2 proangiogenic factors, vascular endothelial growth factor A (VEGFA) and angiopoietin 2 (Ang2/ANGPT2), induces pleiotropic immune mechanisms that facilitate tumor rejection in several tumor models. On the one hand, VEGFA/Ang2 blockade induced regression of the tumor microvasculature while decreasing the proportion of nonperfused vessels and reducing leakiness of the remaining vessels. On the other hand, both anti-VEGFA/Ang2 and anti-CD40 independently promoted proinflammatory macrophage skewing and increased dendritic cell activation in the tumor microenvironment, which were further amplified upon combination of the 2 treatments. Finally, combined therapy provoked brisk infiltration and intratumoral redistribution of cytotoxic CD8+ T cells in the tumors, which was mainly driven by Ang2 blockade. Overall, these nonredundant synergistic mechanisms endowed T cells with improved effector functions that were conducive to more efficient tumor control, underscoring the therapeutic potential of antiangiogenic immunotherapy in cancer.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Antígenos CD40/agonistas , Neoplasias/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Microambiente Tumoral/efectos de los fármacos , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Angiopoyetina 2/antagonistas & inhibidores , Angiopoyetina 2/metabolismo , Animales , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Antígenos CD40/inmunología , Línea Celular Tumoral/trasplante , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Femenino , Humanos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Neoplasias/irrigación sanguínea , Neoplasias/inmunología , Neoplasias/patología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Microambiente Tumoral/inmunología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVES: To investigate whether the glycoengineered type II anti-CD20 monoclonal antibody obinutuzumab (GA101) combined with the selective MDM2 antagonist idasanutlin (RG7388) offers superior efficacy to monotherapy in treating B-lymphoid malignancies in preclinical models. METHODS: The combined effect of obinutuzumab or rituximab plus idasanutlin on direct cell death/apoptosis induction and antibody-dependent cellular cytotoxicity (ADCC) was evaluated using p53 wild-type Z-138 and DoHH-2 lymphoma cells. Furthermore, whole blood B-cell depletion was analysed, and tumour growth inhibition was evaluated in subcutaneous xenograft models. RESULTS: Idasanutlin induced concentration-dependent death of Z-138 and DoHH-2 cells. At concentrations >10-100 nm, idasanutlin enhanced obinutuzumab-induced death of DoHH-2 and Z-138 cells without negatively impacting obinutuzumab-mediated ADCC, natural killer cell activation or whole blood B-cell depletion. In the Z-138 xenograft model, a suboptimal dose of obinutuzumab with idasanutlin yielded substantial tumour growth inhibition and prolonged survival in a time-to-event analysis. In the DoHH-2 model, idasanutlin plus obinutuzumab showed superior tumour growth inhibition to idasanutlin plus rituximab. CONCLUSIONS: Obinutuzumab plus idasanutlin enhanced cell death of p53 wild-type tumour cells vs. rituximab plus idasanutlin without affecting obinutuzumab-mediated ADCC or B-cell depletion and showed robust antitumour efficacy in xenograft models, strongly supporting the investigation of this combination in clinical trials.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Pirrolidinas/farmacología , para-Aminobenzoatos/farmacología , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Depleción Linfocítica , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Ratones , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoAsunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos/farmacología , Linfocitos B/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Rituximab/uso terapéutico , Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/normas , Humanos , Depleción Linfocítica , Ingeniería de ProteínasRESUMEN
Advancing novel immunotherapy strategies requires refined tools in preclinical research to thoroughly assess drug targets, biodistribution, safety, and efficacy. Light sheet fluorescence microscopy (LSFM) offers unprecedented fast volumetric ex vivo imaging of large tissue samples in high resolution. Yet, to date laborious and unstandardized tissue processing procedures have limited throughput and broader applications in immunological research. Therefore, we developed a simple and harmonized protocol for processing, clearing and imaging of all mouse organs and even entire mouse bodies. Applying this Rapid Optical Clearing Kit for Enhanced Tissue Scanning (ROCKETS) in combination with LSFM allowed us to comprehensively study the in vivo biodistribution of an antibody targeting Epithelial Cell Adhesion Molecule (EpCAM) in 3D. Quantitative high-resolution scans of whole organs did not only reveal known EpCAM expression patterns but, importantly, uncovered several new EpCAM-binding sites. We identified gustatory papillae of the tongue, choroid plexi in the brain and duodenal papillae as previously unanticipated locations of high EpCAM expression. Subsequently, we confirmed high EpCAM expression also in human tongue and duodenal specimens. Choroid plexi and duodenal papillae may be considered as particularly sensitive sites due to their importance for liquor production or as critical junctions draining bile and digestive pancreatic enzymes into the small bowel, respectively. These newly gained insights appear highly relevant for clinical translation of EpCAM-addressing immunotherapies. Thus, ROCKETS in combination with LSFM may help to set new standards for preclinical evaluation of immunotherapeutic strategies. In conclusion, we propose ROCKETS as an ideal platform for a broader application of LSFM in immunological research optimally suited for quantitative co-localization studies of immunotherapeutic drugs and defined cell populations in the microanatomical context of organs or even whole mice.
Asunto(s)
Descubrimiento de Drogas , Proteínas Tirosina Quinasas Receptoras , Animales , Humanos , Ratones , Molécula de Adhesión Celular Epitelial , Distribución Tisular , Microscopía Fluorescente/métodos , FosforilaciónRESUMEN
PURPOSE: Disease progression in BRAF V600E/K positive melanomas to approved BRAF/MEK inhibitor therapies is associated with the development of resistance mediated by RAF dimer inducing mechanisms. Moreover, progressing disease after BRAFi/MEKi frequently involves brain metastasis. Here we present the development of a novel BRAF inhibitor (Compound Ia) designed to address the limitations of available BRAFi/MEKi. EXPERIMENTAL DESIGN: The novel, brain penetrant, paradox breaker BRAFi is comprehensively characterized in vitro, ex vivo, and in several preclinical in vivo models of melanoma mimicking peripheral disease, brain metastatic disease, and acquired resistance to first-generation BRAFi. RESULTS: Compound Ia manifested elevated potency and selectivity, which triggered cytotoxic activity restricted to BRAF-mutated models and did not induce RAF paradoxical activation. In comparison to approved BRAFi at clinical relevant doses, this novel agent showed a substantially improved activity in a number of diverse BRAF V600E models. In addition, as a single agent, it outperformed a currently approved BRAFi/MEKi combination in a model of acquired resistance to clinically available BRAFi. Compound Ia presents high central nervous system (CNS) penetration and triggered evident superiority over approved BRAFi in a macro-metastatic and in a disseminated micro-metastatic brain model. Potent inhibition of MAPK by Compound Ia was also demonstrated in patient-derived tumor samples. CONCLUSIONS: The novel BRAFi demonstrates preclinically the potential to outperform available targeted therapies for the treatment of BRAF-mutant tumors, thus supporting its clinical investigation.
Asunto(s)
Melanoma , Proteínas Proto-Oncogénicas B-raf , Encéfalo/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Terapia Molecular Dirigida , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Addressing resistance to third-generation EGFR TKIs such as osimertinib via the EGFRC797S mutation remains a highly unmet need in EGFR-driven non-small-cell lung cancer (NSCLC). Herein, we present the discovery of the allosteric EGFR inhibitor 57, a novel fourth-generation inhibitor to overcome EGFRC797S-mediated resistance in patients harboring the activating EGFRL858R mutation. 57 exhibits an improved potency compared to previous allosteric EGFR inhibitors. To our knowledge, 57 is the first allosteric EGFR inhibitor that demonstrates robust tumor regression in a mutant EGFRL858R/C797S tumor model. Additionally, 57 is active in an H1975 EGFRL858R/T790M NSCLC xenograft model and shows superior efficacy in combination with osimertinib compared to the single agents. Our data highlight the potential of 57 as a single agent against EGFRL858R/C797S and EGFRL858R/T790M/C797S and as combination therapy for EGFRL858R- and EGFRL858R/T790M-driven NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Acrilamidas , Compuestos de Anilina/farmacología , Compuestos de Anilina/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/patología , Resistencia a Antineoplásicos , Receptores ErbB/genética , Humanos , Indoles , Neoplasias Pulmonares/patología , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , PirimidinasRESUMEN
Simulating a viral infection in tumor cells is an attractive concept to eliminate tumor cells. We previously reported the molecular design and the in vitro potency of recombinant monoclonal antibodies fused to a virus-derived peptide MHC class I complex that bypass the peptide processing and MHC loading pathway and directly displays a viral peptide in an MHC class I complex on the tumor cell surface. Here, we show that a vaccination-induced single peptide-specific CD8 T cell response was sufficient to eliminate B16 melanoma tumor cells in vivo in a fully immunocompetent, syngeneic mouse tumor model when mice were treated with mouse pMHCI-IgGs fusion proteins targeting the mouse fibroblast activation protein. Tumor growth of small, established B16 lung metastases could be controlled. The pMHCI-IgG had similar potency as an analogous pan-CD3 T-cell bispecific antibody. In contrast to growth control of small tumors, none of the compounds controlled larger solid tumors of MC38 cancer cells, despite penetration of pMHCI-IgGs into the tumor tissue and clear attraction and activation of antigen-specific CD8 T cells inside the tumor. pMHCI-IgG can have a similar potency as classical pan-T-cell recruiting molecules. The results also highlight the need to better understand immune suppression in advanced solid tumors.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunoglobulina G/inmunología , Melanoma Experimental/inmunología , Animales , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
RG7787 is a re-engineered mesothelin-targeted immunotoxin with reduced immunogenicity composed of a humanized anti-mesothelin Fab fragment and a B-cell epitope silenced 24 kD fragment of Pseudomonas exotoxin A. High prevalence of mesothelin-positive cases and a large unmet medical need make ovarian cancer a promising indication for the clinical development of RG7787. However, ovarian cancer patients also frequently have elevated serum levels of the cancer antigen 125 (CA-125). In principle this could pose a problem, since the binding sites for CA-125 and RG7787 on mesothelin were reported to overlap. However, we show here that RG7787 can readily displace even excess amounts of CA-125 in different cellular assays. Moreover when tested in-vitro on a panel of 12 ovarian cancer cell lines, RG7787 had high cytotoxic activity on COV644, Caov-4, and SNU-119 cells and fully inhibited growth of EFO-21, KURAMOCHI, OVSAHO, and Caov-3 cells with potency values ranging from 1 to 86 pM. Finally, we evaluated the in-vivo efficacy of RG7787 in OvCa6668, a patient-derived ovarian cancer model with high levels of CA-125 expression. RG7787 had moderate monotherapy efficacy but in combination with standard chemotherapies (cisplatin, paclitaxel) achieved pronounced tumor regressions. In summary our data support clinical testing of RG7787 in ovarian cancer.
Asunto(s)
Antineoplásicos/uso terapéutico , Proliferación Celular/efectos de los fármacos , Inmunoconjugados/uso terapéutico , Inmunotoxinas/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológico , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , Inmunoconjugados/farmacología , Inmunotoxinas/farmacologíaRESUMEN
BACKGROUND: Although the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) system has become a prime target for antiangiogenic treatment, its biological role in glioblastoma beyond angiogenesis has remained controversial. METHODS: Using neutralizing antibodies to VEGF or placental growth factor (PlGF) or the tyrosine kinase inhibitor, cediranib, or lentiviral gene silencing, we delineated autocrine signaling in glioma cell lines. The in vivo effects of VEGFR1 and VEGFR2 depletion were evaluated in orthotopic glioma xenograft models. RESULTS: VEGFR1 and VEGFR2 modulated glioma cell clonogenicity, viability, and invasiveness in vitro in an autocrine, cell-line-specific manner. VEGFR1 silencing promoted mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling, whereas VEGFR2 silencing resulted in cell-type dependent activation of the protein kinase B (PKB)/AKT and MAPK/ERK pathways. These responses may represent specific escape mechanisms from VEGFR inhibition. The survival of orthotopic glioma-bearing mice was prolonged upon VEGFR1 silencing in the LNT-229, LN-308, and U87MG models and upon VEGFR2 silencing in LN-308 and U87MG. Disruption of VEGFR1 and VEGFR2 signaling was associated with decreased tumor size, increased tumor necrosis, or loss of matrix metalloproteinase 9 (MMP9) immunoreactivity. Neutralizing VEGF and PlGF by specific antibodies was superior to either antibody treatment alone in the VEGFR1-dependent LNT-229 model. CONCLUSIONS: Differential dependence on autocrine signaling through VEGFR1 and VEGFR2 suggests a need for biomarker-stratified VEGF(R)-based therapeutic approaches to glioblastoma.
Asunto(s)
Comunicación Autocrina , Proliferación Celular , Glioblastoma/patología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Apoptosis , Quinasas MAP Reguladas por Señal Extracelular , Femenino , Glioblastoma/metabolismo , Humanos , Técnicas In Vitro , Ratones , Ratones Desnudos , Neovascularización Patológica , Transducción de Señal , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mesothelin overexpression in lung adenocarcinomas correlates with the presence of activating KRAS mutations and poor prognosis. Hence SS1P, a mesothelin-targeted immunotoxin, could offer valuable treatment options for these patients, but its use in solid tumor therapy is hampered by high immunogenicity and non-specific toxicity. To overcome both obstacles we developed RG7787, a de-immunized cytotoxic fusion protein comprising a humanized SS1 Fab fragment and a truncated, B-cell epitope silenced, 24 kD fragment of Pseudomonas exotoxin A (PE24). Reactivity of RG7787 with sera from immunotoxin-treated patients was >1000 fold reduced. In vitro RG7787 inhibited cell viability of lung cancer cell lines with picomolar potency. The pharmacokinetic properties of RG7787 in rodents were comparable to SS1P, yet it was tolerated up to 10 fold better without causing severe vascular leak syndrome or hepatotoxicity. A pharmacokinetic/pharmacodynamic model developed based on NCI-H596 xenograft studies showed that for RG7787 and SS1P, their in vitro and in vivo potencies closely correlate. At optimal doses of 2-3 mg/kg RG7787 is more efficacious than SS1P. Even large, well established tumors (600 mm(3)) underwent remission during three treatment cycles with RG7787. Also in two patient-derived lung cancer xenograft models, Lu7336 and Lu7187, RG7787 showed anti-tumor efficacy. In monotherapy two treatment cycles were moderately efficacious in the Lu7336 model but showed good anti-tumor activity in the KRAS mutant Lu7187 model (26% and 80% tumor growth inhibition, respectively). Combination of RG7787 with standard chemotherapies further enhanced efficacy in both models achieving near complete eradication of Lu7187 tumors.
Asunto(s)
ADP Ribosa Transferasas/uso terapéutico , Toxinas Bacterianas/uso terapéutico , Exotoxinas/uso terapéutico , Proteínas Ligadas a GPI/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Ingeniería de Proteínas , Pseudomonas/metabolismo , Proteínas Recombinantes de Fusión/uso terapéutico , Factores de Virulencia/uso terapéutico , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Hígado/efectos de los fármacos , Hígado/patología , Neoplasias Pulmonares/patología , Mesotelina , Ratones SCID , Modelos Biológicos , Ratas , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Exotoxina A de Pseudomonas aeruginosaRESUMEN
Dysregulated cellular apoptosis and resistance to cell death are hallmarks of neoplastic initiation and disease progression. Therefore, the development of agents that overcome apoptosis dysregulation in tumor cells is an attractive therapeutic approach. Activation of the extrinsic apoptotic pathway is strongly dependent on death receptor (DR) hyperclustering on the cell surface. However, strategies to activate DR5 or DR4 through agonistic antibodies have had only limited clinical success. To pursue an alternative approach for tumor-targeted induction of apoptosis, we engineered a bispecific antibody (BsAb), which simultaneously targets fibroblast-activation protein (FAP) on cancer-associated fibroblasts in tumor stroma and DR5 on tumor cells. We hypothesized that bivalent binding to both FAP and DR5 leads to avidity-driven hyperclustering of DR5 and subsequently strong induction of apoptosis in tumor cells but not in normal cells. Here, we show that RG7386, an optimized FAP-DR5 BsAb, triggers potent tumor cell apoptosis in vitro and in vivo in preclinical tumor models with FAP-positive stroma. RG7386 antitumor efficacy was strictly FAP dependent, was independent of FcR cross-linking, and was superior to conventional DR5 antibodies. In combination with irinotecan or doxorubicin, FAP-DR5 treatment resulted in substantial tumor regression in patient-derived xenograft models. FAP-DR5 also demonstrated single-agent activity against FAP-expressing malignant cells, due to cross-binding of FAP and DR5 across tumor cells. Taken together, these data demonstrate that RG7386, a novel and potent antitumor agent in both mono- and combination therapies, overcomes limitations of previous DR5 antibodies and represents a promising approach to conquer tumor-associated resistance to apoptosis. Mol Cancer Ther; 15(5); 946-57. ©2016 AACR.
Asunto(s)
Anticuerpos Biespecíficos/metabolismo , Anticuerpos Biespecíficos/farmacología , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Gelatinasas/metabolismo , Proteínas de la Membrana/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Afinidad de Anticuerpos/inmunología , Antineoplásicos/inmunología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Endopeptidasas , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Gelatinasas/inmunología , Humanos , Proteínas de la Membrana/inmunología , Ratones , Unión Proteica/inmunología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Serina Endopeptidasas/inmunología , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Angiogenesis, the process by which new vessels form from existing ones, plays an important role in many developmental processes and pathological conditions. We study angiogenesis in the context of a highly controllable experimental environment: the cornea micropocket assay. Using a multidisciplinary approach that combines experiments, image processing and analysis, and mathematical modelling, we aim to provide mechanistic insight into the action of two angiogenic factors, vascular endothelial growth factor A (VEGF-A) and basic fibroblast growth factor (bFGF). We use image analysis techniques to extract quantitative data, which are both spatially and temporally resolved, from experimental images, and we develop a mathematical model, in which the corneal vasculature evolves in response to both VEGF-A and bFGF. The experimental data are used for model parametrization, while the mathematical model is used to assess the utility of the cornea micropocket assay and to characterize proposed synergies between VEGF-A and bFGF.
Asunto(s)
Neovascularización de la Córnea , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Modelos Cardiovasculares , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Neovascularización de la Córnea/metabolismo , Neovascularización de la Córnea/patología , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos BALB CRESUMEN
Obinutuzumab (GA101) is a novel glycoengineered type II CD20 antibody in development for non-Hodgkin lymphoma. We compared the anti-tumor activity of obinutuzumab and rituximab in preclinical studies using subcutaneous Z138 and WSU-DLCL2 xenograft mouse models. Obinutuzumab and rituximab were assessed alone and in combination with bendamustine, fludarabine, chlorambucil, doxorubicin and cyclophosphamide/vincristine. Owing to strong single-agent efficacy in these models, suboptimal doses of obinutuzumab were applied to demonstrate a combination effect. Obinutuzumab plus bendamustine achieved superior tumor growth inhibition versus rituximab plus bendamustine and showed a statistically significant effect versus the respective single treatments. Combinations of obinutuzumab with fludarabine, chlorambucil or cyclophosphamide/vincristine demonstrated significantly superior activity to rituximab-based treatment. Obinutuzumab monotherapy was at least as effective as rituximab plus chemotherapy in vivo, and obinutuzumab plus chemotherapy was superior to the respective monotherapies. These data support further clinical investigation of obinutuzumab plus chemotherapy.
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Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/farmacología , Linfoma/metabolismo , Animales , Anticuerpos Monoclonales Humanizados/administración & dosificación , Antígenos CD20/metabolismo , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Ciclofosfamida/administración & dosificación , Ciclofosfamida/farmacología , Modelos Animales de Enfermedad , Femenino , Humanos , Linfoma/tratamiento farmacológico , Linfoma/mortalidad , Linfoma/patología , Ratones , Carga Tumoral , Vincristina/administración & dosificación , Vincristina/farmacología , Ensayos Antitumor por Modelo de XenoinjertoAsunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Linfoma no Hodgkin/tratamiento farmacológico , Linfoma no Hodgkin/patología , Pirrolidinas/farmacología , Sulfonamidas/farmacología , para-Aminobenzoatos/farmacología , Animales , Modelos Animales de Enfermedad , Quimioterapia Combinada , Humanos , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
There is increasing experimental evidence for an important role of Angiopoietin-2 (Ang-2) in tumor angiogenesis and progression. In addition, Ang-2 is up-regulated in many cancer types and correlated with poor prognosis. To investigate the functional role of Ang-2 inhibition in tumor development and progression, we generated novel fully human antibodies that neutralize specifically the binding of Ang-2 to its receptor Tie2. The selected antibodies LC06 and LC08 recognize both rodent and human Ang-2 with high affinity, but LC06 shows a higher selectivity for Ang-2 over Ang-1 compared to LC08 which can be considered an Ang-2/Ang-1 cross-reactive antibody. Our data demonstrate that Ang-2 blockade results in potent tumor growth inhibition and pronounced tumor necrosis in subcutaneous and orthotopic tumor models. These effects are attended with a reduction of intratumoral microvessel density and tumor vessels characterized by fewer branches and increased pericyte coverage. Furthermore, anti-Ang-2 treatment strongly inhibits the dissemination of tumor cells to the lungs. Interestingly, in contrast to the Ang-2/Ang-1 cross-reactive antibody LC08 that leads to a regression of physiological vessels in the mouse trachea, the inhibition with the selective anti-Ang-2 antibody LC06 appears to be largely restricted to tumor vasculature without obvious effects on normal vasculature. Taken together, these data provide strong evidence for the selective Ang-2 antibody LC06 as promising new therapeutic agent for the treatment of various cancers.
Asunto(s)
Angiopoyetina 1/antagonistas & inhibidores , Angiopoyetina 2/antagonistas & inhibidores , Angiopoyetina 2/inmunología , Anticuerpos Neutralizantes/farmacología , Antineoplásicos/farmacología , Neoplasias del Colon/irrigación sanguínea , Neoplasias del Colon/tratamiento farmacológico , Angiopoyetina 1/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Especificidad de Anticuerpos , Antineoplásicos/inmunología , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Córnea/efectos de los fármacos , Córnea/inmunología , Femenino , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Microvasos/efectos de los fármacos , Microvasos/inmunología , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/inmunología , Fosforilación , Distribución Aleatoria , Receptor TIE-2/antagonistas & inhibidores , Receptor TIE-2/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
UNLABELLED: Placental growth factor (PlGF) is a member of the proangiogenic vascular endothelial growth factor family, which is upregulated in many tumors. RO5323441, a humanized monoclonal antibody against PlGF, showed antitumor activity in human tumor xenografts. We therefore aimed to radiolabel RO5323441 and preclinically validate this tracer to study drug tumor uptake and organ distribution by PET imaging. (89)Zr-RO5323441 was tested for stability and immunoreactivity in vitro. METHODS: The tumor uptake and organ distribution for 10, 50, and 500 µg of (89)Zr-RO5323441 was assessed in mice bearing human PlGF-expressing hepatocellular cancer (Huh7) xenografts or human renal cell carcinoma (ACHN) xenografts without detectable human PlGF expression. The effect of pretreatment with RO5323441 (20 mg/kg) on (89)Zr-RO5323441 tumor uptake was analyzed in Huh7 xenografts. (111)In-IgG served as a control for nonspecific tumor uptake and organ distribution. Cy5-RO5323441 was injected to study the intratumor distribution of RO5323441 with fluorescence microscopy. RESULTS: (89)Zr-RO5323441 showed a time- and dose-dependent tumor accumulation. Uptake in Huh7 xenografts at 10 µg of (89)Zr-RO5323441 was 8.2% ± 1.7% injected dose (ID)/cm(3) at 144 h after injection, and in ACHN xenografts it was 5.5 ± 0.3 %ID/cm(3) (P = 0.03). RO5323441 pretreatment of Huh7 xenograft-bearing mice reduced (89)Zr-RO5323441 tumor uptake to the level of nonspecific (111)In-IgG uptake. Cy5-RO5323441 was present in the tumors mainly in the microenvironment. CONCLUSION: The findings show that RO5323441 tumor uptake is PlGF-specific and time- and dose-dependent.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacocinética , Proteínas Gestacionales/inmunología , Proteínas Gestacionales/metabolismo , Radioisótopos , Microambiente Tumoral , Circonio , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Transporte Biológico/efectos de los fármacos , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Transformación Celular Neoplásica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Marcaje Isotópico , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Macrófagos/metabolismo , Masculino , Ratones , Factor de Crecimiento Placentario , Microambiente Tumoral/efectos de los fármacosRESUMEN
PURPOSE: VEGF-A blockade has been clinically validated as a treatment for human cancers. Angiopoietin-2 (Ang-2) expression has been shown to function as a key regulator of tumor angiogenesis and metastasis. EXPERIMENTAL DESIGN: We have applied the recently developed CrossMab technology for the generation of a bispecific antibody recognizing VEGF-A with one arm based on bevacizumab (Avastin), and the other arm recognizing Ang-2 based on LC06, an Ang-2 selective human IgG1 antibody. The potency of Ang-2-VEGF CrossMab was evaluated alone and in combination with chemotherapy using orthotopic and subcutaneous xenotransplantations, along with metastasis analysis by quantitative real-time Alu-PCR and ex vivo evaluation of vessels, hypoxia, proliferation, and apoptosis. The mechanism of action was further elucidated using Western blotting and ELISA assays. RESULTS: Ang-2-VEGF-A CrossMab showed potent tumor growth inhibition in a panel of orthotopic and subcutaneous syngeneic mouse tumors and patient or cell line-derived human tumor xenografts, especially at later stages of tumor development. Ang-2-VEGF-A CrossMab treatment led to a strong inhibition of angiogenesis and an enhanced vessel maturation phenotype. Neoadjuvant combination with chemotherapy resulted in complete tumor regression in primary tumor-bearing Ang-2-VEGF-A CrossMab-treated mice. In contrast to Ang-1 inhibition, anti-Ang-2-VEGF-A treatment did not aggravate the adverse effect of anti-VEGF treatment on physiologic vessels. Moreover, treatment with Ang-2-VEGF-A CrossMab resulted in inhibition of hematogenous spread of tumor cells to other organs and reduced micrometastatic growth in the adjuvant setting. CONCLUSION: These data establish Ang-2-VEGF-A CrossMab as a promising antitumor, antiangiogenic, and antimetastatic agent for the treatment of cancer.
Asunto(s)
Angiopoyetina 2/inmunología , Anticuerpos Biespecíficos/administración & dosificación , Neoplasias/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular/inmunología , Angiopoyetina 2/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales Humanizados/administración & dosificación , Bevacizumab , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/inmunología , Ratones , Metástasis de la Neoplasia , Neoplasias/inmunología , Neovascularización Patológica/inmunología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
We report the first preclinical in vitro and in vivo comparison of GA101 (obinutuzumab), a novel glycoengineered type II CD20 monoclonal antibody, with rituximab and ofatumumab, the two currently approved type I CD20 antibodies. The three antibodies were compared in assays measuring direct cell death (AnnexinV/PI staining and time-lapse microscopy), complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and internalization. The models used for the comparison of their activity in vivo were SU-DHL4 and RL xenografts. GA101 was found to be superior to rituximab and ofatumumab in the induction of direct cell death (independent of mechanical manipulation required for cell aggregate disruption formed by antibody treatment), whereas it was 10 to 1,000 times less potent in mediating CDC. GA101 showed superior activity to rituximab and ofatumumab in ADCC and whole-blood B-cell depletion assays, and was comparable with these two in ADCP. GA101 also showed slower internalization rate upon binding to CD20 than rituximab and ofatumumab. In vivo, GA101 induced a strong antitumor effect, including complete tumor remission in the SU-DHL4 model and overall superior efficacy compared with both rituximab and ofatumumab. When rituximab-pretreated animals were used, second-line treatment with GA101 was still able to control tumor progression, whereas tumors escaped rituximab treatment. Taken together, the preclinical data show that the glyoengineered type II CD20 antibody GA101 is differentiated from the two approved type I CD20 antibodies rituximab and ofatumumab by its overall preclinical activity, further supporting its clinical investigation.
Asunto(s)
Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales de Origen Murino/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Neoplasias/tratamiento farmacológico , Animales , Anticuerpos Monoclonales Humanizados/efectos adversos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Citotoxicidad Inmunológica/efectos de los fármacos , Humanos , Ratones , Neoplasias/inmunología , Neoplasias/patología , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Rituximab , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The cell surface glycoprotein CD44 plays an important role in the development and progression of various tumor types. RG7356 is a humanized antibody targeting the constant region of CD44 that shows antitumor efficacy in mice implanted with CD44-expressing tumors such as MDA-MB-231 breast cancer cells. CD44 receptor seems to function as the main receptor for hyaluronic acid and osteopontin, serving as coreceptor for growth factor pathways like cMet, EGFR, HER-2, and VEGFR and by cytoskeletal modulation via ERM and Rho kinase signaling. To assess the direct impact of RG7356 binding to the CD44 receptor, a global mass spectrometry-based phosphoproteomics approach was applied to freshly isolated MDA-MB-231 tumor xenografts. Results from a global phosphoproteomics screen were further corroborated by Western blot and ELISA analyses of tumor lysates from CD44-expressing tumors. Short-term treatment of tumor-bearing mice with RG7356 resulted in modifications of the MAPK pathway in the responsive model, although no effects on downstream phosphorylation were observed in a nonresponsive xenograft model. Taken together, our approach augments the value of other high throughput techniques to identify biomarkers for clinical development of targeted agents.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Receptores de Hialuranos/metabolismo , Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales Humanizados/administración & dosificación , Antineoplásicos/administración & dosificación , Biología Computacional/métodos , Femenino , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones SCID , Neoplasias/tratamiento farmacológico , Proteómica , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
GA101 is a novel glycoengineered Type II CD20 monoclonal antibody. When compared with rituximab, it mediates less complement-dependent cytotoxicity (CDC). As expected for a Type II antibody, GA101 appears not to act through CDC and is more potent than the Type I antibody rituximab in inducing cell death via nonclassical induction of apoptosis cytotoxicity, with more direct cytotoxicity and more antibody-dependent cell-mediated cytotoxicity. We evaluated the antitumor activity of GA101 against the human-transformed follicular lymphoma RL model in vivo in severe combined immunodeficient mice (SCID) mice. GA101 induced stronger inhibition of tumor growth than rituximab. Combination of GA101 with cyclophosphamide in vivo confirmed the superiority of GA101 over rituximab. Neutralizing the complement system with cobra venom factor partially impaired the antitumor activity of rituximab, but had no impact on the efficacy of GA101. In vitro GA101 more potently induced cell death of RL cells than rituximab. The expression of a limited number of genes was found to be induced by both antibodies after exposure in vitro. Among these, early growth response 1 and activation transcription factor 3 were confirmed to be increased at the protein level, suggesting a possible role of these proteins in the apoptotic signalling of anti-CD20 antibodies. These data imply that GA101 is superior to rituximab not only as a single agent, but also in combination with chemotherapy. These data suggest the presence of novel signalization pathways activated after exposure to anti-CD20 antibodies.