RESUMO
Biological therapeutic agents are highly targeted and potent but limited in their ability to reach intracellular targets. These limitations often necessitate high therapeutic doses and can be associated with less-than-optimal therapeutic activity. One promising solution for therapeutic agent delivery is use of cell-penetrating peptides. Canonical cell-penetrating peptides, however, are limited by low efficiencies of cellular uptake and endosomal escape, minimal proteolytic stability, and toxicity. To overcome these limitations, we designed a family of proprietary cyclic cell-penetrating peptides that form the core of our endosomal escape vehicle technology capable of delivering therapeutic agent-conjugated cargo intracellularly. We demonstrated the therapeutic potential of this endosomal escape vehicle platform in preclinical models of muscular dystrophy with distinct disease etiology. An endosomal escape vehicle-conjugated, splice-modulating oligonucleotide restored dystrophin protein expression in striated muscles in the mdx mouse, a model for Duchenne muscular dystrophy. Furthermore, another endosomal escape vehicle-conjugated, sterically blocking oligonucleotide led to knockdown of aberrant transcript expression levels in facioscapulohumeral muscular dystrophy patient-derived skeletal muscle cells. These findings suggest a significant therapeutic potential of our endosomal escape vehicle conjugated oligonucleotides for targeted upregulation and downregulation of gene expression in neuromuscular diseases, with possible broader application of this platform for delivery of intracellular biological agents.
RESUMO
With a completely reengineered and humanized glycosylation pathway, glycoengineered Pichia pastoris has emerged as a promising production host for the manufacture of therapeutic glycoproteins. However, the extensive genetic modifications have also negatively affected the overall fitness levels of the glycoengineered host cells. To make glycoengineered Pichia strains more compatible with a scalable industrial fermentation process, we sought to identify genetic solutions to broadly improve cell robustness during fermentation. In this study, we report that mutations within the Pichia pastoris ATT1 (PpATT1) gene (a homolog of the Saccharomyces cerevisiae GAL4 [ScGAL4] transcriptional activator) dramatically increased the cellular fitness levels of glycoengineered Pichia strains. We demonstrate that deletion of the PpATT1 gene enabled glycoengineered Pichia strains to improve their thermal tolerance levels, reduce their cell lysis defects, and greatly improve fermentation robustness. The extension of the duration of fermentation enabled the PpATT1-modified glycoengineered Pichia strains to increase their product yields significantly without any sacrifice in product quality. Because the ATT1 gene could be deleted from any Pichia strains, including empty hosts and protein-expressing production strains alike, we suggest that the findings described in this study are broadly applicable to any Pichia strains used for the production of therapeutic proteins, including monoclonal antibodies, Fc fusions, peptides, hormones, and growth factors.
Assuntos
Deleção de Genes , Regulação Fúngica da Expressão Gênica , Engenharia Metabólica , Pichia/genética , Pichia/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/genética , Glicosilação , Viabilidade Microbiana , Pichia/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência , Fatores de Transcrição/genética , Transcrição Gênica , VirulênciaRESUMO
Yeast is capable of performing posttranslational modifications, such as N- or O-glycosylation. It has been demonstrated that N-glycans play critical biological roles in therapeutic glycoproteins by modulating pharmacokinetics and pharmacodynamics. However, N-glycan sites on recombinant glycoproteins produced in yeast can be underglycosylated, and hence, not completely occupied. Genomic homology analysis indicates that the Pichia pastoris oligosaccharyltransferase (OST) complex consists of multiple subunits, including OST1, OST2, OST3, OST4, OST5, OST6, STT3, SWP1, and WBP1. Monoclonal antibodies produced in P. pastoris show that N-glycan site occupancy ranges from 75-85 % and is affected mainly by the OST function, and in part, by process conditions. In this study, we demonstrate that N-glycan site occupancy of antibodies can be improved to greater than 99 %, comparable to that of antibodies produced in mammalian cells (CHO), by overexpressing Leishmania major STT3D (LmSTT3D) under the control of an inducible alcohol oxidase 1 (AOX1) promoter. N-glycan site occupancy of non-antibody glycoproteins such as recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) was also significantly improved, suggesting that LmSTT3D has broad substrate specificity. These results suggest that the glycosylation status of recombinant proteins can be improved by heterologous STT3 expression, which will allow for the customization of therapeutic protein profiles.
Assuntos
Glicoproteínas/metabolismo , Glicosilação , Pichia/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Anticorpos Monoclonais/metabolismo , Células CHO , Cricetinae , Expressão Gênica , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Humanos , Leishmania major/enzimologia , Leishmania major/genética , Engenharia Metabólica , Proteínas Recombinantes/metabolismoRESUMO
Pichia pastoris is a methylotropic yeast that has gained great importance as an organism for protein expression in recent years. Here, we report the expression of recombinant human erythropoietin (rhEPO) in glycoengineered P. pastoris. We show that glycosylation fidelity is maintained in fermentation volumes spanning six orders of magnitude and that the protein can be purified to high homogeneity. In order to increase the half-life of rhEPO, the purified protein was coupled to polyethylene glycol (PEG) and then compared to the currently marketed erythropoiesis stimulating agent, Aranesp(®) (darbepoetin). In in vitro cell proliferation assays the PEGylated protein was slightly, and the non-PEGylated protein was significantly more active than comparator. Pharmacodynamics as well as pharmacokinetic activity of PEGylated rhEPO in animals was comparable to that of Aranesp(®). Taken together, our results show that glycoengineered P. pastoris is a suitable production host for rhEPO, yielding an active biologic that is comparable to those produced in current mammalian host systems.
Assuntos
Eritropoetina/biossíntese , Pichia/metabolismo , Engenharia de Proteínas/métodos , Animais , Proliferação de Células/efeitos dos fármacos , Darbepoetina alfa , Eritropoetina/análogos & derivados , Eritropoetina/sangue , Eritropoetina/genética , Eritropoetina/farmacocinética , Eritropoetina/farmacologia , Feminino , Glicosilação , Humanos , Masculino , Camundongos , Pichia/genética , Polietilenoglicóis , Polissacarídeos/química , Ratos Sprague-Dawley , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
The N-glycosylation pathway in Pichia pastoris has been humanized by the deletion of genes responsible for fungal-type glycosylation (high mannose) as well as the introduction of heterologous genes capable of forming human-like N-glycosylation. This results in a yeast host that is capable of expressing therapeutic glycoproteins. A thorough investigation was performed to examine whether glycoproteins expressed in glycoengineered P. pastoris strains may contain residual fungal-type high-mannose structures. In a pool of N-linked glycans enzymatically released by protein N-glycosidase from a reporter glycoprotein expressed in a developmental glycoengineered P. pastoris strain, an oligosaccharide with a mass consistent with a Hexose(9)GlcNAc(2) oligosaccharide was identified. When this structure was analyzed by a normal-phase high-performance liquid chromatography (HPLC), its retention time was identical to a Man(9)GlcNAc(2) standard. However, this Hexose(9)GlcNAc(2) oligosaccharide was found to be resistant to α-1,2-mannosidase as well as endomannosidase, which preferentially catabolizes endoplasmic reticulum oligosaccharides containing terminal α-linked glucose. To further characterize this oligosaccharide, we purified the Hexose(9)GlcNAc(2) oligosaccharide by HPLC and analyzed the structure by high-field one-dimensional (1D) and two-dimensional (2D) (1)H NMR (nuclear magnetic resonance) spectroscopy followed by structural elucidation by homonuclear and heteronuclear 1D and 2D (1)H and (13)C NMR spectroscopy. The results of these experiments lead to the identification of an oligosaccharide α-Man-(1 â 2)-ß-Man-(1 â 2)-ß-Man-(1 â 2)-α-Man-(1 â 2) moiety as part of a tri-antennary structure. The difference in enzymatic reactivity can be attributed to multiple ß-linkages on the α-1,3 arm of the Man(9)GlcNAc(2) oligosaccharide.
Assuntos
Manosidases/metabolismo , Proteínas de Membrana/metabolismo , Oligossacarídeos/biossíntese , Oligossacarídeos/química , Pichia/metabolismo , Humanos , Manosidases/genética , Proteínas de Membrana/genética , Pichia/genética , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
A robust and scalable purification process was developed to quickly generate antibody of high purity and sufficient quantity from glycoengineered Pichia pastoris fermentation. Protein A affinity chromatography was used to capture the antibody from fermentation supernatant. A pH gradient elution was applied to the Protein A column to prevent antibody precipitation at low pH. Antibody from Protein A chromatography contained some product related impurities, which were the misassembling of cleaved heavy chain, heavy chain and light chain. It also had some process related impurities, including Protein A residues, endotoxin, host cell DNA and proteins. Cation exchange chromatography with optimal NaCl gradient at pH 4.5-6.0 efficiently removed these product and process related impurities. The antibody from glycoengineered P. pastoris was comparable to its commercial counterpart in heterotetramer folding, physical stability and binding affinity.
Assuntos
Anticorpos Monoclonais/biossíntese , Pichia/metabolismo , Proteínas Recombinantes/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Organismos Geneticamente Modificados , Pichia/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
The methylotrophic yeast Pichia pastoris has recently been engineered to express therapeutic glycoproteins with uniform human N-glycans at high titers. In contrast to the current art where producing therapeutic proteins in mammalian cell lines yields a final product with heterogeneous N-glycans, proteins expressed in glycoengineered P. pastoris can be designed to carry a specific, preselected glycoform. However, significant variability exists in fermentation performance between genotypically similar clones with respect to cell fitness, secreted protein titer, and glycan homogeneity. Here, we describe a novel, multidimensional screening process that combines high and medium throughput tools to identify cell lines producing monoclonal antibodies (mAbs). These cell lines must satisfy multiple selection criteria (high titer, uniform N-glycans and cell robustness) and be compatible with our large-scale production platform process. Using this selection process, we were able to isolate a mAb-expressing strain yielding a titer (after protein A purification) in excess of 1 g/l in 0.5-l bioreactors.
Assuntos
Anticorpos Monoclonais/biossíntese , Engenharia Genética , Glicoproteínas/biossíntese , Pichia/isolamento & purificação , Proteínas Recombinantes/biossíntese , Anticorpos Monoclonais/genética , Reatores Biológicos , Técnicas de Cultura de Células , Linhagem Celular , DNA Fúngico/genética , Fermentação , Expressão Gênica , Glicoproteínas/genética , Glicosilação , Humanos , Técnicas Microbiológicas , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Seleção Genética , Transformação GenéticaRESUMO
Yeast is a widely used recombinant protein expression system. We expanded its utility by engineering the yeast Pichia pastoris to secrete human glycoproteins with fully complex terminally sialylated N-glycans. After the knockout of four genes to eliminate yeast-specific glycosylation, we introduced 14 heterologous genes, allowing us to replicate the sequential steps of human glycosylation. The reported cell lines produce complex glycoproteins with greater than 90% terminal sialylation. Finally, to demonstrate the utility of these yeast strains, functional recombinant erythropoietin was produced.
Assuntos
Eritropoetina/metabolismo , Pichia/genética , Engenharia de Proteínas , Sialoglicoproteínas/biossíntese , Animais , Linhagem Celular , Clonagem Molecular , Ácido N-Acetilneuramínico do Monofosfato de Citidina/metabolismo , Eritropoetina/química , Eritropoetina/genética , Vetores Genéticos , Glicosilação , Humanos , Pichia/metabolismo , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Ácidos Siálicos/metabolismo , Sialoglicoproteínas/química , Sialoglicoproteínas/genética , Transformação GenéticaRESUMO
Protein-based drugs constitute about a quarter of new approvals with a majority being glycoproteins. Increasing use of glycoproteins, such as monoclonal antibodies, at high therapeutic doses is challenging current production capacity. Mammalian cell culture, which is currently the production system of choice for glycoproteins, has several disadvantages including high cost of goods, long cycle times and, importantly, limited control over glycosylation. In view of this, several expression systems are currently being explored as alternatives to mammalian cell culture, these include yeast, plant and insect expression systems. Each of these has different merits for the production of therapeutic glycoproteins and can lead to enhanced therapeutic efficiency.
Assuntos
Biotecnologia/métodos , Glicoproteínas/biossíntese , Proteínas Recombinantes/biossíntese , Animais , Expressão Gênica/genética , Glicoproteínas/genética , Glicoproteínas/uso terapêutico , Glicosilação , Humanos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Polissacarídeos/metabolismo , Proteínas Recombinantes/uso terapêuticoRESUMO
As the fastest growing class of therapeutic proteins, monoclonal antibodies (mAbs) represent a major potential drug class. Human antibodies are glycosylated in their native state and all clinically approved mAbs are produced by mammalian cell lines, which secrete mAbs with glycosylation structures that are similar, but not identical, to their human counterparts. Glycosylation of mAbs influences their interaction with immune effector cells that kill antibody-targeted cells. Here we demonstrate that human antibodies with specific human N-glycan structures can be produced in glycoengineered lines of the yeast Pichia pastoris and that antibody-mediated effector functions can be optimized by generating specific glycoforms. Glycoengineered P. pastoris provides a general platform for producing recombinant antibodies with human N-glycosylation.
Assuntos
Anticorpos Monoclonais/biossíntese , Melhoramento Genético/métodos , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Pichia/genética , Pichia/metabolismo , Engenharia de Proteínas/métodos , Anticorpos Monoclonais/genética , Glicosilação , Humanos , Proteínas Recombinantes/biossínteseRESUMO
Angiogenesis, the formation of new blood vessels from preexisting microvasculature, is a highly regulated process. Angiogenesis is controlled by both positive and negative factors thus providing several targets for drug discovery. The inhibition of angiogenesis represents a new approach to cancer therapy and several agents and approaches are in different stages of clinical development. These inhibitors were recently shown to constitute a new modality for cancer treatment. In this article, we will review angiogenesis inhibitors-related patent literature for the years 2000-2005. This review will cover specifically the discovery and development disclosures of endogenous inhibitors. The scope of this review is to give the reader a well-structured patent literature review of these agents targeting different steps of the angiogenic process. Finally, we have summarized the key attributes of the emerging endogenous angiogenesis inhibitors that make them potent antitumor agents.