RESUMO
Patients with α-dystroglycanopathies, a subgroup of rare congenital muscular dystrophies, present with a spectrum of clinical manifestations that includes muscular dystrophy as well as CNS and ocular abnormalities. Although patients with α-dystroglycanopathies are genetically heterogeneous, they share a common defect of aberrant post-translational glycosylation modification of the dystroglycan alpha-subunit, which renders it defective in binding to several extracellular ligands such as laminin-211 in skeletal muscles, agrin in neuromuscular junctions, neurexin in the CNS, and pikachurin in the eye, leading to various symptoms. The genetic heterogeneity associated with the development of α-dystroglycanopathies poses significant challenges to developing a generalized treatment to address the spectrum of genetic defects. Here, we propose the development of a bispecific antibody (biAb) that functions as a surrogate molecular linker to reconnect laminin-211 and the dystroglycan beta-subunit to ameliorate sarcolemmal fragility, a primary pathology in patients with α-dystroglycan-related muscular dystrophies. We show that the treatment of LARGEmyd-3J mice, an α-dystroglycanopathy model, with the biAb improved muscle function and protected muscles from exercise-induced damage. These results demonstrate the viability of a biAb that binds to laminin-211 and dystroglycan simultaneously as a potential treatment for patients with α-dystroglycanopathy.
Assuntos
Anticorpos Biespecíficos/farmacologia , Distroglicanas/metabolismo , Laminina/metabolismo , Síndrome de Walker-Warburg/metabolismo , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/metabolismo , Modelos Animais de Doenças , Distroglicanas/imunologia , Expressão Gênica , Humanos , Imuno-Histoquímica , Injeções Intramusculares , Laminina/genética , Laminina/imunologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Ligação Proteica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas/genética , Sarcolema/efeitos dos fármacos , Sarcolema/metabolismo , Síndrome de Walker-Warburg/tratamento farmacológico , Síndrome de Walker-Warburg/etiologiaRESUMO
We report a modular approach to synthesize maleimido group containing hydrophilic dolastatin 10 (Dol10) derivatives as drug-linkers for the syntheses of antibody-drug conjugates (ADCs). Discrete polyethylene glycol (PEG) moieties of different chain lengths were introduced as part of the linker to impart hydrophilicity to these drug linkers. The synthesis process involved construction of PEG maleimido derivatives of the tetrapeptide intermediate (N-methylvaline-valine-dolaisoleucine-dolaproine), which were subsequently coupled with dolaphenine to generate the desired drug linkers. The synthetic method reported in this manuscript circumvents the use of highly cytotoxic Dol10 in its native form. By using trastuzumab (Herceptin®) as the antibody we have synthesized Dol10 containing ADCs. The presence of a discrete PEG chain in the drug linkers resulted in ADCs free from aggregation. The effect of PEG chain length on the biological activities of these Dol10 containing ADCs was investigated by in vitro cytotoxicity assays. ADCs containing PEG6 and PEG8 spacers exhibited the highest level of in vitro anti-proliferative activity against HER2-positive (SK-BR-3) human tumor cells. ADCs derived from Herceptin® and PEG8-Dol10, at a dose of 10 mg kg-1, effectively delayed the tumor growth and prolonged the survival time in mice bearing human ovarian SKOV-3 xenografts.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Depsipeptídeos/farmacologia , Imunoconjugados/efeitos dos fármacos , Animais , Anticorpos Monoclonais/química , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Depsipeptídeos/química , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Camundongos SCID , Conformação Molecular , Células Tumorais CultivadasRESUMO
Antibody-drug conjugates (ADCs) have been proven clinically to be more effective anti-cancer agents than native antibodies. However, the classical conjugation chemistries to prepare ADCs by targeting primary amines or hinge disulfides have a number of shortcomings including heterogeneous product profiles and linkage instability. We have developed a novel site-specific conjugation method by targeting the native glycosylation site on antibodies as an approach to address these limitations. The native glycans on Asn-297 of antibodies were enzymatically remodeled in vitro using galactosyl and sialyltransferases to introduce terminal sialic acids. Periodate oxidation of these sialic acids yielded aldehyde groups which were subsequently used to conjugate aminooxy functionalized cytotoxic agents via oxime ligation. The process has been successfully demonstrated with three antibodies including trastuzumab and two cytotoxic agents. Hydrophobic interaction chromatography and LC-MS analyses revealed the incorporation of ~1.6 cytotoxic agents per antibody molecule, approximating the number of sialic acid residues. These glyco-conjugated ADCs exhibited target-dependent antiproliferative activity toward antigen-positive tumor cells and significantly greater antitumor efficacy than naked antibody in a Her2-positive tumor xenograft model. These findings suggest that enzymatic remodeling combined with oxime ligation of the native glycans of antibodies offers an attractive approach to generate ADCs with well-defined product profiles. The site-specific conjugation approach presented here provides a viable alternative to other methods, which involve a need to either re-engineer the antibody sequence or develop a highly controlled chemical process to ensure reproducible drug loading.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos/química , Antineoplásicos/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Animais , Anticorpos Monoclonais Humanizados/química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Glicosilação , Humanos , Camundongos , Camundongos SCID , Estrutura Molecular , Neoplasias Experimentais/patologia , Polissacarídeos/química , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Sialiltransferases/química , Sialiltransferases/metabolismo , Relação Estrutura-Atividade , TrastuzumabRESUMO
A series of novel multivalent drug linkers (MDLs) containing cytotoxic agents were synthesized and conjugated to antibodies to yield highly potent antibody-drug conjugates (ADCs) with drug/antibody ratios (DARs) higher than those typically reported in the literature (10 vs. ≈4). These MDLs contain two copies of a cytotoxic agent attached to biocompatible scaffolds composed of a branched peptide core and discrete polyethylene glycol (PEG) chains to enhance solubility and decrease aggregation. These drug linkers produced well-defined ADCs, whose DARs could be accurately determined by LC-MS. Using this approach, ADCs with significantly lower aggregation and higher DAR than those of conventional drug linker design were obtained with highly hydrophobic cytotoxic agents such as monomethyldolastatinâ 10 (MMAD). The inâ vitro potencies of the MDL-derived conjugates matched that of ADCs of similar DAR with conventional linkers, and the potency increased proportionally with drug loading. This approach may provide a means to prepare highly potent ADCs from a broader range of drugs, including those with lower cytotoxicity or poor solubility, which otherwise limits their use for antibody-drug conjugates. This may also provide a means to further improve the potency achievable with cytotoxins currently used in ADCs.
Assuntos
Antineoplásicos Imunológicos/química , Imunoconjugados/química , Polietilenoglicóis/química , Trastuzumab/química , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Desenho de Fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunoconjugados/farmacologia , Neoplasias/tratamento farmacológico , Polietilenoglicóis/farmacologia , Agregados Proteicos , Solubilidade , Trastuzumab/farmacologiaRESUMO
Recombinant proteins are important tools for studying biological processes. Generating a recombinant protein requires the use of an expression system. Selection of an appropriate expression system is dependent on the characteristics and intended application of the recombinant protein and is essential to produce sufficient quantities of the protein. Over the last 30 years, there have been considerable advances in the technologies for expressing recombinant proteins. In this chapter the unique characteristics of four commonly used expression systems, Escherichia coli, Pichia pastoris, baculovirus/insect cell, and mammalian cells are described. The E. coli system is a rapid method for expressing proteins but lacks many of the posttranslational modifications found in eukaryotes. The capacity of E. coli for protein folding and forming disulfide bonds is not sufficient for many recombinant proteins although there are a number of tools developed to overcome these limitations. In contrast to E. coli, the eukaryotic P. pastoris, baculovirus/insect cell, and mammalian systems promote good protein folding and many posttranslational modifications. How the characteristics and the downstream application of a recombinant protein can influence the choice of an expression system is then reviewed.
Assuntos
Expressão Gênica , Técnicas de Transferência de Genes , Proteínas Recombinantes/genética , Animais , Baculoviridae/genética , Baculoviridae/crescimento & desenvolvimento , Baculoviridae/metabolismo , Comportamento de Escolha/fisiologia , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Pichia/genética , Pichia/crescimento & desenvolvimento , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Projetos de PesquisaRESUMO
The extracellular domain of the p55 TNF receptor (TNFrED) is an important therapeutic protein for targeting tumor necrosis factor-alpha (TNF-alpha). The expression level of the TNFrED is low for bioproduction, which is presumably associated with the complication of pairing 24 cysteine residues to form correct disulfide bonds. Here we report the application of the yeast display method to study expression of TNFrED, a multimeric receptor. Randomly mutated libraries of TNFrED were screened, and two mutants were identified that express several-fold higher protein levels compared with the wild type while still retaining normal binding affinity for TNF-alpha. The substituted residues responsible for the higher protein expression in both mutants were identified as proline, and both proline residues are adjacent to cysteine residues involved in disulfide bonds. Analysis of the mutant residues revealed that the improved level of expression is due to conformational restriction of the substituted residues to that of the folded state seen in the crystal structures of TNFrED thereby forcing the neighboring cysteine residues into the correct orientation for proper disulfide bond formation.