RESUMO
Activation of the Met receptor tyrosine kinase, either by its ligand, hepatocyte growth factor (HGF), or via ligand-independent mechanisms, such as MET amplification or receptor overexpression, has been implicated in driving tumor proliferation, metastasis, and resistance to therapy. Clinical development of Met-targeted antibodies has been challenging, however, as bivalent antibodies exhibit agonistic properties, whereas monovalent antibodies lack potency and the capacity to down-regulate Met. Through computational modeling, we found that the potency of a monovalent antibody targeting Met could be dramatically improved by introducing a second binding site that recognizes an unrelated, highly expressed antigen on the tumor cell surface. Guided by this prediction, we engineered MM-131, a bispecific antibody that is monovalent for both Met and epithelial cell adhesion molecule (EpCAM). MM-131 is a purely antagonistic antibody that blocks ligand-dependent and ligand-independent Met signaling by inhibiting HGF binding to Met and inducing receptor down-regulation. Together, these mechanisms lead to inhibition of proliferation in Met-driven cancer cells, inhibition of HGF-mediated cancer cell migration, and inhibition of tumor growth in HGF-dependent and -independent mouse xenograft models. Consistent with its design, MM-131 is more potent in EpCAM-high cells than in EpCAM-low cells, and its potency decreases when EpCAM levels are reduced by RNAi. Evaluation of Met, EpCAM, and HGF levels in human tumor samples reveals that EpCAM is expressed at high levels in a wide range of Met-positive tumor types, suggesting a broad opportunity for clinical development of MM-131.
Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Molécula de Adesão da Célula Epitelial/antagonistas & inibidores , Fator de Crescimento de Hepatócito/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Molécula de Adesão da Célula Epitelial/metabolismo , Humanos , Camundongos , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The full complement of molecular pathways contributing to the pathogenesis of Parkinson disease (PD) remains unknown. Here we address this issue by taking a broad approach, beginning by using functional MRI to identify brainstem regions differentially affected and resistant to the disease. Relying on these imaging findings, we then profiled gene expression levels from postmortem brainstem regions, identifying a disease-related decrease in the expression of the catabolic polyamine enzyme spermidine/spermine N1-acetyltransferase 1 (SAT1). Next, a range of studies were completed to support the pathogenicity of this finding. First, to test for a causal link between polyamines and α-synuclein toxicity, we investigated a yeast model expressing α-synuclein. Polyamines were found to enhance the toxicity of α-synuclein, and an unbiased genome-wide screen for modifiers of α-synuclein toxicity identified Tpo4, a member of a family of proteins responsible for polyamine transport. Second, to test for a causal link between SAT1 activity and PD histopathology, we investigated a mouse model expressing α-synuclein. DENSPM (N1, N11-diethylnorspermine), a polyamine analog that increases SAT1 activity, was found to reduce PD histopathology, whereas Berenil (diminazene aceturate), a pharmacological agent that reduces SAT1 activity, worsened the histopathology. Third, to test for a genetic link, we sequenced the SAT1 gene and a rare but unique disease-associated variant was identified. Taken together, the findings from human patients, yeast, and a mouse model implicate the polyamine pathway in PD pathogenesis.
Assuntos
Acetiltransferases/metabolismo , Tronco Encefálico/metabolismo , Doença de Parkinson/metabolismo , Poliaminas/metabolismo , alfa-Sinucleína/metabolismo , Acetiltransferases/genética , Animais , Tronco Encefálico/patologia , Diminazena/análogos & derivados , Diminazena/farmacologia , Variação Genética , Humanos , Imageamento por Ressonância Magnética , Camundongos , Camundongos Transgênicos , Doença de Parkinson/genética , Doença de Parkinson/patologia , Pemolina/análogos & derivados , Pemolina/farmacologiaRESUMO
Antibody-directed nanotherapeutics (ADNs) represent a promising delivery platform for selective delivery of an encapsulated drug payload to the site of disease that improves the therapeutic index. Although both single-chain Fv (scFv) and Fab antibody fragments have been used for targeting, no platform approach applicable to any target has emerged. scFv can suffer from intrinsic instability, and the Fabs are challenging to use due to native disulfide over-reduction and resulting impurities at the end of the conjugation process. This occurs because of the close proximity of the disulfide bond connecting the heavy and light chain to the free cysteine at the C-terminus, which is commonly used as the conjugation site. Here we show that by engineering an alternative heavy chain-light chain disulfide within the Fab, we can maintain efficient conjugation while eliminating the process impurities and retaining stability. We have demonstrated the utility of this technology for efficient ADN delivery and internalization for a series of targets, including EphA2, EGFR, and ErbB2. We expect that this technology will be broadly applicable for targeting of nanoparticle encapsulated payloads, including DNA, mRNA, and small molecules.
Assuntos
Nanopartículas , Anticorpos de Cadeia Única , Dissulfetos/química , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Nanopartículas/químicaRESUMO
Antibody-mediated tumour targeting and nanoparticle-mediated encapsulation can reduce the toxicity of antitumour drugs and improve their efficacy. Here, we describe the performance of a nanotherapeutic encapsulating a hydrolytically sensitive docetaxel prodrug and conjugated to an antibody specific for EphA2-a receptor overexpressed in many tumours. Administration of the nanotherapeutic in mice led to slow and sustained release of the prodrug, reduced exposure of active docetaxel in the circulation (compared with administration of the free drug) and maintenance of optimal exposure of the drug in tumour tissue. We also show that administration of the nanotherapeutic in rats and dogs resulted in minimal haematological toxicity, as well as the absence of neutropenia and improved overall tolerability in multiple rodent models. Targeting of the nanotherapeutic to EphA2 improved tumour penetration and resulted in markedly enhanced antitumour activity (compared with administration of free docetaxel and non-targeted nanotherapeutic controls) in multiple tumour-xenografted mice. This nanomedicine could become a potent and safe therapeutic alternative for cancer patients undergoing chemotherapy.
Assuntos
Antineoplásicos/uso terapêutico , Nanopartículas/uso terapêutico , Receptor EphA2/metabolismo , Animais , Antineoplásicos/farmacologia , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Hidrocarbonetos Aromáticos com Pontes/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Docetaxel/sangue , Docetaxel/química , Docetaxel/farmacocinética , Docetaxel/uso terapêutico , Humanos , Lipossomos , Camundongos Endogâmicos NOD , Camundongos SCID , Taxoides/farmacologia , Taxoides/uso terapêutico , Distribuição Tecidual/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Our objective is to produce a protein biosensor (or molecular switch) that is specifically activated in solution by a monoclonal antibody. Many effector-dependent enzymes have evolved in nature, but the introduction of a novel regulatory mechanism into a normally unregulated enzyme poses a difficult design problem. We used site-saturation mutagenesis and screening to generate effector-activated variants of the reporter enzyme beta-glucuronidase (GUS). The specific activity of the purified epitope-tagged GUS variant was increased by up to approximately 500-fold by the addition of an equimolar concentration of a monoclonal antibody. This molecular switch is modular in design, so it can easily be re-engineered for the detection of other peptide-specific antibodies. Such antibody-activated reporters could someday enable point-of-care serological assays for the rapid detection of infectious diseases.
Assuntos
Anticorpos/metabolismo , Glucuronidase/química , Glucuronidase/metabolismo , Estrutura Quaternária de Proteína , Anticorpos/química , Ativação Enzimática , Glucuronidase/genética , Humanos , Modelos Moleculares , Mutagênese , Engenharia de ProteínasRESUMO
The discovery of antibodies that bind to targets with high affinity is now a routine exercise. However, it is still challenging to screen for candidates that, in addition to having excellent biological properties, also have optimal biophysical characteristics. Here, we describe a simple HPLC-based screening method to assess for developability factors earlier in the discovery process.
Assuntos
Anticorpos/isolamento & purificação , Adsorção , Anticorpos/química , Anticorpos Monoclonais , Especificidade de Anticorpos , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Estabilidade ProteicaRESUMO
Antibody-targeted nanoparticles have great promise as anti-cancer drugs; however, substantial developmental challenges of antibody modules prevent many candidates from reaching the clinic. Here, we describe a robust strategy for developing an EphA2-targeting antibody fragment for immunoliposomal drug delivery. A highly bioactive single-chain variable fragment (scFv) was engineered to overcome developmental liabilities, including low thermostability and weak binding to affinity purification resins. Improved thermostability was achieved by modifying the framework of the scFv, and complementarity-determining region (CDR)-H2 was modified to increase binding to protein A resins. The results of our engineering campaigns demonstrate that it is possible, using focused design strategies, to rapidly improve the stability and manufacturing characteristics of an antibody fragment for use as a component of a novel therapeutic construct.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Efrina-A2/imunologia , Imunoconjugados/imunologia , Nanopartículas , Anticorpos de Cadeia Única/imunologia , Animais , Humanos , Região Variável de Imunoglobulina/imunologia , Engenharia de Proteínas/métodos , Estabilidade Proteica , Receptor EphA2 , Anticorpos de Cadeia Única/biossínteseRESUMO
Protein engineers can alter the properties of enzymes by directing their evolution in vitro. Many methods to generate molecular diversity and to identify improved clones have been developed, but experimental evolution remains as much an art as a science. We previously used DNA shuffling (sexual recombination) and a histochemical screen to direct the evolution of Escherichia coli beta-glucuronidase (GUS) variants with improved beta-galactosidase (BGAL) activity. Here, we employ the same model evolutionary system to test the efficiencies of several other techniques: recursive random mutagenesis (asexual), combinatorial cassette mutagenesis (high-frequency recombination) and a versatile high-throughput microplate screen. GUS variants with altered specificity evolved in each trial, but different combinations of mutagenesis and screening techniques effected the fixation of different beneficial mutations. The new microplate screen identified a broader set of mutations than the previously employed X-gal colony screen. Recursive random mutagenesis produced essentially asexual populations, within which beneficial mutations drove each other into extinction (clonal interference); DNA shuffling and combinatorial cassette mutagenesis led instead to the accumulation of beneficial mutations within a single allele. These results explain why recombinational approaches generally increase the efficiency of laboratory evolution.
Assuntos
Evolução Molecular Direcionada , Glucuronidase/genética , Engenharia de Proteínas , Escherichia coli/enzimologia , Escherichia coli/genética , Frequência do Gene , Genótipo , Modelos Moleculares , Fenótipo , beta-Galactosidase/metabolismoRESUMO
Monoclonal antibodies and antibody-like molecules represent a fast-growing class of bio-therapeutics that has rapidly transformed patient care in a variety of disease indications. The discovery of antibodies that bind to particular targets with high affinity is now a routine exercise and a variety of in vitro and in vivo techniques are available for this purpose. However, it is still challenging to identify antibodies that, in addition to having the desired biological effect, also express well, remain soluble at different pH levels, remain stable at high concentrations, can withstand high shear stress, and have minimal non-specific interactions. Many promising antibody programs have ultimately failed in development due to the problems associated with one of these factors. Here, we present a simple high-performance liquid chromatography (HPLC)-based screening method to assess these developability factors earlier in discovery process. This method is robust and requires only microgram quantities of proteins. Briefly, we show that for antibodies injected on a commercially available pre-packed Zenix HPLC column, the retention times are inversely related to their colloidal stability with antibodies prone to precipitation or aggregation retained longer on the column with broader peaks. By simply varying the salt content of running buffer, we were also able to estimate the nature of interactions between the antibodies and the column. We believe this approach should generally be applicable to assessment of the developability of other classes of bio-therapeutic molecules, and that the addition of this simple tool early in the discovery process will lead to selection of molecules with improved developability characteristics.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Humanos , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Antibody-targeted nanoparticles have the potential to significantly increase the therapeutic index of cytotoxic anti-cancer therapies by directing them to tumor cells. Using antibodies or their fragments requires careful engineering because multiple parameters, including affinity, internalization rate and stability, all need to be optimized. Here, we present a case study of the iterative engineering of a single chain variable fragment (scFv) for use as a targeting arm of a liposomal cytotoxic nanoparticle. We describe the effect of the orientation of variable domains, the length and composition of the interdomain protein linker that connects VH and VL, and stabilizing mutations in both the framework and complementarity-determining regions (CDRs) on the molecular properties of the scFv. We show that variable domain orientation can alter cross-reactivity to murine antigen while maintaining affinity to the human antigen. We demonstrate that tyrosine residues in the CDRs make diverse contributions to the binding affinity and biophysical properties, and that replacement of non-essential tyrosines can improve the stability and bioactivity of the scFv. Our studies demonstrate that a comprehensive engineering strategy may be required to identify a scFv with optimal characteristics for nanoparticle targeting.
Assuntos
Citotoxinas , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/química , Neoplasias/tratamento farmacológico , Anticorpos de Cadeia Única , Substituição de Aminoácidos , Animais , Anticorpos Antineoplásicos/química , Anticorpos Antineoplásicos/genética , Anticorpos Antineoplásicos/imunologia , Anticorpos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Citotoxinas/química , Citotoxinas/farmacologia , Humanos , Camundongos , Neoplasias/imunologia , Proteínas Recombinantes , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacologiaRESUMO
Parkinson's disease (PD), dementia with Lewy bodies and multiple system atrophy, collectively referred to as synucleinopathies, are associated with a diverse group of genetic and environmental susceptibilities. The best studied of these is PD. alpha-Synuclein (alpha-syn) has a key role in the pathogenesis of both familial and sporadic PD, but evidence linking it to other predisposition factors is limited. Here we report a strong genetic interaction between alpha-syn and the yeast ortholog of the PD-linked gene ATP13A2 (also known as PARK9). Dopaminergic neuron loss caused by alpha-syn overexpression in animal and neuronal PD models is rescued by coexpression of PARK9. Further, knockdown of the ATP13A2 ortholog in Caenorhabditis elegans enhances alpha-syn misfolding. These data provide a direct functional connection between alpha-syn and another PD susceptibility locus. Manganese exposure is an environmental risk factor linked to PD and PD-like syndromes. We discovered that yeast PARK9 helps to protect cells from manganese toxicity, revealing a connection between PD genetics (alpha-syn and PARK9) and an environmental risk factor (PARK9 and manganese). Finally, we show that additional genes from our yeast screen, with diverse functions, are potent modifiers of alpha-syn-induced neuron loss in animals, establishing a diverse, highly conserved interaction network for alpha-syn.
Assuntos
Resistência a Medicamentos/genética , Epistasia Genética/fisiologia , Manganês/toxicidade , ATPases Translocadoras de Prótons/genética , Proteínas de Saccharomyces cerevisiae/genética , alfa-Sinucleína/fisiologia , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Células Cultivadas , Sequência Conservada , Epistasia Genética/efeitos dos fármacos , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/fisiologia , Mutação/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Doença de Parkinson/etiologia , Doença de Parkinson/genética , ATPases Translocadoras de Prótons/metabolismo , ATPases Translocadoras de Prótons/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Distribuição Tecidual , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , alfa-Sinucleína/toxicidadeRESUMO
Cells respond to stimuli by changes in various processes, including signaling pathways and gene expression. Efforts to identify components of these responses increasingly depend on mRNA profiling and genetic library screens. By comparing the results of these two assays across various stimuli, we found that genetic screens tend to identify response regulators, whereas mRNA profiling frequently detects metabolic responses. We developed an integrative approach that bridges the gap between these data using known molecular interactions, thus highlighting major response pathways. We used this approach to reveal cellular pathways responding to the toxicity of alpha-synuclein, a protein implicated in several neurodegenerative disorders including Parkinson's disease. For this we screened an established yeast model to identify genes that when overexpressed alter alpha-synuclein toxicity. Bridging these data and data from mRNA profiling provided functional explanations for many of these genes and identified previously unknown relations between alpha-synuclein toxicity and basic cellular pathways.
Assuntos
Redes Reguladoras de Genes/fisiologia , alfa-Sinucleína/toxicidade , Algoritmos , Ergosterol/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Redes Reguladoras de Genes/genética , Resposta ao Choque Térmico/genética , Ácido Mevalônico/metabolismo , Modelos Biológicos , Compostos Nitrosos/toxicidade , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Transdução de Sinais/genética , Estresse Fisiológico/genética , Transcrição Gênica/fisiologia , Transfecção , alfa-Sinucleína/genéticaRESUMO
The dominant paradigm of protein engineering is structure-based site-directed mutagenesis. This rational approach is generally more effective for the engineering of local properties, such as substrate specificity, than global ones such as allostery. Previous workers have modified normally unregulated reporter enzymes, including beta-galactosidase, alkaline phosphatase, and beta-lactamase, so that the engineered versions are activated (up to 4-fold) by monoclonal antibodies. A reporter that could easily be "reprogrammed" for the facile detection of novel effectors (binding or modifying activities) would be useful in high throughput screens for directed evolution or drug discovery. Here we describe a straightforward and general solution to this potentially difficult design problem. The transcription factor p53 is normally regulated by a variety of post-translational modifications. The insertion of peptides into intrinsically unstructured domains of p53 generated variants that were activated up to 100-fold by novel effectors (proteases or antibodies). An engineered p53 was incorporated into an existing high throughput screen for the detection of human immunodeficiency virus protease, an arbitrarily chosen novel effector. These results suggest that the molecular recognition properties of intrinsically unstructured proteins are relatively easy to engineer and that the absence of crystal structures should not deter the rational engineering of this class of proteins.
Assuntos
Engenharia de Proteínas/métodos , Proteína Supressora de Tumor p53/fisiologia , Fosfatase Alcalina/metabolismo , Anticorpos Monoclonais/química , Bacillus anthracis/metabolismo , Membrana Celular/metabolismo , Cristalografia por Raios X , Escherichia coli/metabolismo , Genes Reporter , Variação Genética , Vetores Genéticos , HIV/metabolismo , Protease de HIV/metabolismo , Humanos , Modelos Genéticos , Mutagênese Sítio-Dirigida , Mutação , Peptídeos/química , Processamento Pós-Transcricional do RNA , Especificidade por Substrato , Fatores de Tempo , Proteína Supressora de Tumor p53/metabolismo , beta-Galactosidase/metabolismo , beta-Lactamases/metabolismoRESUMO
Protein engineers have widely adopted directed evolution as a design algorithm, but practitioners have not come to a consensus about the best method to evolve protein molecular recognition. We previously used DNA shuffling to direct the evolution of Escherichia coli beta-glucuronidase (GUS) variants with increased beta-galactosidase activity. Epistatic (synergistic) mutations in amino acids 557, 566, and 568, which are part of an active site loop, were identified in that experiment (Matsumura, I., and Ellington, A. D. (2001) J. Mol. Biol. 305, 331-339). Here we show that site saturation mutagenesis of these residues, overexpression of the resulting library in E. coli, and high throughput screening led to the rapid evolution of clones exhibiting increased activity in reactions with p-nitrophenyl-beta-d-xylopyranoside (pNP-xyl). The xylosidase activities of the 14 fittest clones were 30-fold higher on average than that of the wild-type GUS. The 14 corresponding plasmids were pooled, amplified by long PCR, self-ligated with T4 DNA ligase, and transformed into E. coli. Thirteen clones exhibiting an average of 80-fold improvement in xylosidase activity were isolated in a second round of screening. One of the evolved proteins exhibited a approximately 200-fold improvement over the wild type in reactivity (k(cat)/K(m)) with pNP-xyl, with a 290,000-fold inversion of specificity. Sequence analysis of the 13 round 2 isolates suggested that all were products of intermolecular recombination events that occurred during whole plasmid PCR. Further rounds of evolution using DNA shuffling and staggered extension process (StEP) resulted in modest improvement. These results underscore the importance of epistatic interactions and demonstrate that they can be optimized through variations of the facile whole plasmid PCR technique.