RESUMO
Advanced ovarian cancer is frequently treated with combination chemotherapy, but high recurrence rates show the need for therapies that can produce durable responses and extend overall survival. Bispecific antibodies that interact with tumor antigens on cancer cells and activating receptors on immune cells offer an innovative immunotherapy approach. Here, we describe a human bispecific antibody (REGN4018) that binds both Mucin 16 (MUC16), a glycoprotein that is highly expressed on ovarian cancer cells, and CD3, thus bridging MUC16-expressing cells with CD3+ T cells. REGN4018 induced T cell activation and killing of MUC16-expressing tumor cells in vitro. Binding and cytotoxicity of REGN4018 in vitro were minimally affected by high concentrations of CA-125, the shed form of MUC16, which is present in patients. In preclinical studies with human ovarian cancer cells and human T cells in immunodeficient mice, REGN4018 potently inhibited growth of intraperitoneal ovarian tumors. Moreover, in a genetically engineered immunocompetent mouse expressing human CD3 and human MUC16 [humanized target (HuT) mice], REGN4018 inhibited growth of murine tumors expressing human MUC16, and combination with an anti-PD-1 antibody enhanced this efficacy. Immuno-PET imaging demonstrated localization of REGN4018 in MUC16-expressing tumors and in T cell-rich organs such as the spleen and lymph nodes. Toxicology studies in cynomolgus monkeys showed minimal and transient increases in serum cytokines and C-reactive protein after REGN4018 administration, with no overt toxicity. Collectively, these data demonstrate potent antitumor activity and good tolerability of REGN4018, supporting clinical evaluation of REGN4018 in patients with MUC16-expressing advanced ovarian cancer.
Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/uso terapêutico , Antígeno Ca-125/imunologia , Antígeno Ca-125/metabolismo , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/imunologia , Linfócitos T/metabolismo , Animais , Antígenos CD13/imunologia , Antígenos CD13/metabolismo , Feminino , Citometria de Fluxo , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Células Jurkat , Macaca fascicularis , Camundongos , Neoplasias Ovarianas/metabolismo , Linfócitos T/imunologiaRESUMO
The Notch ligand delta-like 4 (Dll4) has been identified as a promising target in tumor angiogenesis in preclinical studies, and Dll4 inhibitors have recently entered clinical trials for solid tumors, including ovarian cancers. In this study, we report the development of REGN421 (enoticumab), a fully human IgG1 monoclonal antibody that binds human Dll4 with sub-nanomolar affinity and inhibits Notch signaling. Administering REGN421 to immunodeficient mice engineered to express human Dll4 inhibited the growth of several human tumor xenografts in association with the formation of nonfunctional tumor blood vessels. In ovarian tumor xenograft models, Dll4 was expressed specifically by the tumor endothelium, and Dll4 blockade by human-specific or mouse-specific Dll4 antibodies exerted potent antitumor activity, which relied entirely on targeting Dll4 expressed by tumor stromal cells but not by the tumor cells themselves. However, Dll4 blockade reduced Notch signaling in both blood vessels and tumor cells surrounding the blood vessels, suggesting that endothelial-expressed Dll4 might induce Notch signaling in adjacent ovarian tumor cells. The antitumor effects of targeting Dll4 were augmented significantly by simultaneous inhibition of VEGF signaling, whereas this combined blockade reversed normal organ vascular changes induced by Dll4 blockade alone. Overall, our findings deepen the rationale for antibody-based strategies to target Dll4 in ovarian cancers, especially in combination with VEGF blockade.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Terapia de Alvo Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Neovascularização Patológica/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Proteínas Adaptadoras de Transdução de Sinal , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Antineoplásicos/farmacologia , Proteínas de Ligação ao Cálcio , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos SCID , Proteínas de Neoplasias/fisiologia , Neoplasias Ovarianas/irrigação sanguínea , Neoplasias Ovarianas/metabolismo , Receptores Notch/fisiologia , Transdução de Sinais/efeitos dos fármacos , Especificidade da Espécie , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
EGFR blocking antibodies are approved for the treatment of colorectal cancer and head and neck squamous cell carcinoma (HNSCC). Although ERBB3 signaling has been proposed to limit the effectiveness of EGFR inhibitors, the underlying molecular mechanisms are not fully understood. To gain insight into these mechanisms, we generated potent blocking antibodies against ERBB3 (REGN1400) and EGFR (REGN955). We show that EGFR and ERBB3 are coactivated in multiple HNSCC cell lines and that combined blockade of EGFR and ERBB3 inhibits growth of these cell lines more effectively than blockade of either receptor alone. Blockade of EGFR with REGN955 strongly inhibited activation of ERK in HNSCC cell lines, whereas blockade of ERBB3 with REGN1400 strongly inhibited activation of Akt; only the combination of the 2 antibodies blocked both of these essential downstream pathways. We used a HER2 blocking antibody to show that ERBB3 phosphorylation in HNSCC and colorectal cancer cells is strictly dependent on association with HER2, but not EGFR, and that neuregulin 1 activates ERBB3/HER2 signaling to reverse the effect of EGFR blockade on colorectal cancer cell growth. Finally, although REGN1400 and REGN955 as single agents slowed the growth of HNSCC and colorectal cancer xenografts, the combination of REGN1400 plus REGN955 caused significant tumor regression. Our results indicate that activation of the Akt survival pathway by ERBB3/HER2 limits the effectiveness of EGFR inhibition, suggesting that REGN1400, which is currently in a phase I clinical trial, could provide benefit when combined with EGFR blocking antibodies.
Assuntos
Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Neoplasias de Cabeça e Pescoço/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Animais , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Camundongos , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-3/antagonistas & inibidores , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Murine cytomegalovirus (MCMV) m137 null mutants, Deltam137A and Deltam137B, were generated by inserting a gpt cassette into a deleted region of the open reading frame. A polyclonal antiserum produced to an Escherichia coli expressed gst-m137 fusion protein was used to show that a 38 kDa polypeptide corresponding to the predicted m137 gene product was present in NIH 3T3 fibroblasts infected with wild-type MCMV but was not detected in Deltam137 infected cells. The protein did not fractionate with infected cell membranes and was not detectable in purified wild-type virions. Plaque size, plaque morphology, and viral yield did not differ significantly between Deltam137 and wild-type MCMV infected 3T3 fibroblasts. The results showed that deletion of the 38 kDa protein did not negatively effect viral growth in 3T3 fibroblasts indicating that the m137 gene product is not essential for replication in these cells. In vivo analysis revealed that two independently isolated m137 mutants showed a significant delay in time until death but ultimately killed 100% of the mice in a SCID mouse model of virulence.