RESUMO
Binding of hepatocyte growth factor (HGF) to the receptor tyrosine kinase MET is implicated in the malignant process of multiple cancers, making disruption of this interaction a promising therapeutic strategy. However, targeting MET with bivalent antibodies can mimic HGF agonism via receptor dimerization. To address this limitation, we have developed onartuzumab, an Escherichia coli-derived, humanized, and affinity-matured monovalent monoclonal antibody against MET, generated using the knob-into-hole technology that enables the antibody to engage the receptor in a one-to-one fashion. Onartuzumab potently inhibits HGF binding and receptor phosphorylation and signaling and has antibody-like pharmacokinetics and antitumor activity. Biochemical data and a crystal structure of a ternary complex of onartuzumab antigen-binding fragment bound to a MET extracellular domain fragment, consisting of the MET Sema domain fused to the adjacent Plexins, Semaphorins, Integrins domain (MET Sema-PSI), and the HGF ß-chain demonstrate that onartuzumab acts specifically by blocking HGF α-chain (but not ß-chain) binding to MET. These data suggest a likely binding site of the HGF α-chain on MET, which when dimerized leads to MET signaling. Onartuzumab, therefore, represents the founding member of a class of therapeutic monovalent antibodies that overcomes limitations of antibody bivalency for targets impacted by antibody crosslinking.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais/farmacologia , Fragmentos Fab das Imunoglobulinas/farmacologia , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/genética , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Desenho de Fármacos , Fator de Crescimento de Hepatócito/química , Fator de Crescimento de Hepatócito/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Nus , Camundongos SCID , Camundongos Transgênicos , Modelos Moleculares , Dados de Sequência Molecular , Neoplasias/patologia , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-met/química , Proteínas Proto-Oncogênicas c-met/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Hepatocyte growth factor (HGF) binds to its target receptor tyrosine kinase, Met, as a single-chain form (pro-HGF) or as a cleaved two-chain disulfide-linked α/ß-heterodimer. However, only two-chain HGF stimulates Met signaling. Proteolytic cleavage of the Arg(494)-Val(495) peptide bond in the zymogen-like pro-HGF results in allosteric activation of the serine protease-like ß-chain (HGF ß), which binds Met to initiate signaling. We use insights from the canonical trypsin-like serine protease activation mechanism to show that isolated peptides corresponding to the first 7-10 residues of the cleaved N terminus of the ß-chain stimulate Met phosphorylation by pro-HGF to levels that are â¼25% of those stimulated by two-chain HGF. Biolayer interferometry data demonstrate that peptide VVNGIPTR (peptide V8) allosterically enhances pro-HGF ß binding to Met, resulting in a K(D)(app) of 1.6 µm, only 8-fold weaker than the Met/HGF ß-chain affinity. Most notably, in vitro cell stimulation with peptide V8 in the presence of pro-HGF leads to Akt phosphorylation, enhances cell survival, and facilitates cell migration between 75 and 100% of that found with two-chain HGF, thus revealing a novel approach for activation of Met signaling that bypasses proteolytic processing of pro-HGF. Peptide V8 is unable to enhance Met binding or signaling with HGF proteins having a mutated activation pocket (D672N). Furthermore, Gly substitution of the N-terminal Val residue in peptide V8 results in loss of all activity. Overall, these findings identify the activation pocket of the serine protease-like ß-chain as a "hot spot" for allosteric regulation of pro-HGF and have broad implications for developing selective allosteric activators of serine proteases and pseudoproteases.
Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Oligopeptídeos/metabolismo , Precursores de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais/fisiologia , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Substituição de Aminoácidos , Animais , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Cricetinae , Cricetulus , Fator de Crescimento de Hepatócito/genética , Humanos , Mutação de Sentido Incorreto , Oligopeptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Precursores de Proteínas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Receptores de Fatores de Crescimento/genética , Serina Proteases/genética , Serina Proteases/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Antibody-dependent cellular cytotoxicity (ADCC) is a key mechanism by which therapeutic antibodies mediate their antitumor effects. The absence of fucose on the heavy chain of the antibody increases the affinity between the antibody and FcγRIIIa, which results in increased in vitro and in vivo ADCC compared with the fucosylated form. However, the cellular and molecular mechanisms responsible for increased ADCC are unknown. Through a series of biochemical and cellular studies, we find that human natural killer (NK) cells stimulated with afucosylated antibody exhibit enhanced activation of proximal FcγRIIIa signaling and downstream pathways, as well as enhanced cytoskeletal rearrangement and degranulation, relative to stimulation with fucosylated antibody. Furthermore, analysis of the interaction between human NK cells and targets using a high-throughput microscope-based antibody-dependent cytotoxicity assay shows that afucosylated antibodies increase the number of NK cells capable of killing multiple targets and the rate with which targets are killed. We conclude that the increase in affinity between afucosylated antibodies and FcγRIIIa enhances activation of signaling molecules, promoting cytoskeletal rearrangement and degranulation, which, in turn, potentiates the cytotoxic characteristics of NK cells to increase efficiency of ADCC.
Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Fucose/imunologia , Células Matadoras Naturais/imunologia , Receptores de IgG/imunologia , Actinas/metabolismo , Anticorpos Monoclonais Humanizados/química , Afinidade de Anticorpos/imunologia , Degranulação Celular/imunologia , Células Cultivadas , Citoesqueleto/metabolismo , Humanos , Transdução de Sinais/imunologia , Trastuzumab/química , Trastuzumab/imunologiaRESUMO
Onartuzumab, a humanized, monovalent monoclonal anti-MET antibody, antagonizes MET signaling by inhibiting binding of its ligand, hepatocyte growth factor (HGF). We investigated the effects of onartuzumab on cell-associated and circulating (shed) MET (sMET) and circulating HGF in vitro and nonclinically to determine their utility as pharmacodynamic biomarkers for onartuzumab. Effects of onartuzumab on cell-associated MET were assessed by flow cytometry and immunofluorescence. sMET and HGF were measured in cell supernatants and in serum or plasma from multiple species (mouse, cynomolgus monkey, and human) using plate-based immunoassays. Unlike bivalent anti-MET antibodies, onartuzumab stably associates with MET on the surface of cells without inducing MET internalization or shedding. Onartuzumab delayed the clearance of human xenograft tumor-produced sMET from the circulation of mice, and endogenous sMET in cynomolgus monkeys. In mice harboring MET-expressing xenograft tumors, in the absence of onartuzumab, levels of human sMET correlated with tumor size, and may be predictive of MET-expressing tumor burden. Because binding of sMET to onartuzumab in circulation resulted in increasing sMET serum concentrations due to reduced clearance, this likely renders sMET unsuitable as a pharmacodynamic biomarker for onartuzumab. There was no observed effect of onartuzumab on circulating HGF levels in xenograft tumor-bearing mice or endogenous HGF in cynomolgus monkeys. Although sMET and HGF may serve as predictive biomarkers for MET therapeutics, these data do not support their use as pharmacodynamic biomarkers for onartuzumab.