RESUMO
The ectodomain of matrix protein 2 (M2e) of influenza A virus is an attractive target for a universal influenza A vaccine: the M2e sequence is highly conserved across influenza virus subtypes, and induced humoral anti-M2e immunity protects against a lethal influenza virus challenge in animal models. Clinical phase I studies with M2e vaccine candidates have been completed. However, the in vivo mechanism of immune protection induced by M2e-carrier vaccination is unclear. Using passive immunization experiments in wild-type, FcRγ(-/-), FcγRI(-/-), FcγRIII(-/-), and (FcγRI, FcγRIII)(-/-) mice, we report in this study that Fc receptors are essential for anti-M2e IgG-mediated immune protection. M2e-specific IgG1 isotype Abs are shown to require functional FcγRIII for in vivo immune protection but other anti-M2e IgG isotypes can rescue FcγRIII(-/-) mice from a lethal challenge. Using a conditional cell depletion protocol, we also demonstrate that alveolar macrophages (AM) play a crucial role in humoral M2e-specific immune protection. Additionally, we show that adoptive transfer of wild-type AM into (FcγRI, FcγRIII)(-/-) mice restores protection by passively transferred anti-M2e IgG. We conclude that AM and Fc receptor-dependent elimination of influenza A virus-infected cells are essential for protection by anti-M2e IgG.
Assuntos
Imunoglobulina G/metabolismo , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Macrófagos Alveolares/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Domínios e Motivos de Interação entre Proteínas/imunologia , Receptores Fc/fisiologia , Proteínas da Matriz Viral/imunologia , Animais , Morte Celular/genética , Morte Celular/imunologia , Citotoxicidade Imunológica , Modelos Animais de Doenças , Feminino , Imunização Passiva , Imunoglobulina G/toxicidade , Vírus da Influenza A/genética , Vacinas contra Influenza/genética , Vacinas contra Influenza/uso terapêutico , Depleção Linfocítica/métodos , Macrófagos Alveolares/patologia , Macrófagos Alveolares/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/patologia , Domínios e Motivos de Interação entre Proteínas/genética , Receptores Fc/deficiência , Receptores Fc/uso terapêutico , Receptores de IgG/deficiência , Receptores de IgG/metabolismo , Receptores de IgG/fisiologia , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/uso terapêuticoRESUMO
Hepatocyte growth factor (HGF) and its receptor MET represent validated targets for cancer therapy. However, HGF/MET inhibitors being explored as cancer therapeutics exhibit cytostatic activity rather than cytotoxic activity, which would be more desired. In this study, we engineered an antagonistic anti-MET antibody that, in addition to blocking HGF/MET signaling, also kills MET-overexpressing cancer cells by antibody-dependent cellular cytotoxicity (ADCC). As a control reagent, we engineered the same antibody in an ADCC-inactive form that is similarly capable of blocking HGF/MET activity, but in the absence of any effector function. In comparing these two antibodies in multiple mouse models of cancer, including HGF-dependent and -independent tumor xenografts, we determined that the ADCC-enhanced antibody was more efficacious than the ADCC-inactive antibody. In orthotopic mammary carcinoma models, ADCC enhancement was crucial to deplete circulating tumor cells and to suppress metastases. Prompted by these results, we optimized the ADCC-enhanced molecule for clinical development, generating an antibody (ARGX-111) with improved pharmacologic properties. ARGX-111 competed with HGF for MET binding, inhibiting ligand-dependent MET activity, downregulated cell surface expression of MET, curbing HGF-independent MET activity, and engaged natural killer cells to kill MET-expressing cancer cells, displaying MET-specific cytotoxic activity. ADCC assays confirmed the cytotoxic effects of ARGX-111 in multiple human cancer cell lines and patient-derived primary tumor specimens, including MET-expressing cancer stem-like cells. Together, our results show how ADCC provides a therapeutic advantage over conventional HGF/MET signaling blockade and generates proof-of-concept for ARGX-111 clinical testing in MET-positive oncologic malignancies.
Assuntos
Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Fator de Crescimento de Hepatócito/metabolismo , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Ligação Competitiva , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Humanos , Camundongos Nus , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-met/imunologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Activation of MET by HGF plays a key role in tumor progression. Using a recently developed llama platform that generates human-like immunoglobulins, we selected 68 different antibodies that compete with HGF for binding to MET. HGF-competing antibodies recognized 4 distinct hotspots localized in different MET domains. We identified 1 hotspot that coincides with the known HGF ß chain binding site on blades 2-3 of the SEMA domain ß-propeller. We determined that a second and a third hotspot lie within blade 5 of the SEMA domain and IPT domains 2-3, both of which are thought to bind to HGF α chain. Characterization of the fourth hotspot revealed a region across the PSI-IPT 1 domains not previously associated with HGF binding. Individual or combined targeting of these hotspots effectively interrupted HGF/MET signaling in multiple cell-based biochemical and biological assays. Selected antibodies directed against SEMA blades 2-3 and the PSI-IPT 1 region inhibited brain invasion and prolonged survival in a glioblastoma multiforme model, prevented metastatic disease following neoadjuvant therapy in a triple-negative mammary carcinoma model, and suppressed cancer cell dissemination to the liver in a KRAS-mutant metastatic colorectal cancer model. These results identify multiple regions of MET responsible for HGF-mediated tumor progression, unraveling the complexity of HGF-MET interaction, and provide selective molecular tools for targeting MET activity in cancer.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Anticorpos Monoclonais , Afinidade de Anticorpos , Sítios de Ligação , Ligação Competitiva , Neoplasias Encefálicas/patologia , Camelídeos Americanos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Progressão da Doença , Glioblastoma/patologia , Fator de Crescimento de Hepatócito/química , Fator de Crescimento de Hepatócito/imunologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-met/químicaRESUMO
At present few vaccine candidates exists against potentially pandemic influenza virus infections. We provide compelling evidence that a targeted fusion protein based on the CTA1-DD adjuvant and containing tandem repeats of the matrix protein 2 (M2e) ectodomain epitope, CTA1-3M2e-DD, confers strong protective immunity against a potentially lethal challenge infection with influenza virus in mice. The formulation was highly effective for mucosal immunizations and promoted high M2e-specific serum IgG and mucosal IgA antibody titers and an hitherto unknown anti-M2e CD4 T cell immunity. This novel CTA1-3M2e-DD fusion protein combines adjuvant and a conserved influenza A antigen in a promising candidate for a universal anti-influenza vaccine.