RESUMO
BACKGROUND: MUC18 is a glycoprotein highly expressed on the surface of melanoma and other cancers which promotes tumor progression and metastasis. However, its mechanism of action and suitability as a therapeutic target are unknown. METHODS: A monoclonal antibody (mAb) (JM1-24-3) was generated from metastatic melanoma tumor live cell immunization, and high-throughput screening identified MUC18 as the target. RESULTS: Analysis of molecular interactions between MUC18 and JM1-24-3 revealed that the downstream signaling events depended on binding of the mAb to a conformational epitope on the extracellular domain of MUC18. JM1-24-3 inhibited melanoma cell proliferation, migration and invasion in vitro and reduced tumor growth and metastasis in vivo. CONCLUSION: These results confirm that MUC18 is mechanistically important in melanoma growth and metastasis, suggest that the MUC18 epitope identified is a promising therapeutic target, and that the JM1-24-3 mAb may serve as the basis for a potential therapeutic agent.
Assuntos
Anticorpos Monoclonais/farmacologia , Melanoma/terapia , Animais , Anticorpos Monoclonais/imunologia , Antígeno CD146/imunologia , Linhagem Celular Tumoral , Humanos , Masculino , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos A , Camundongos Nus , Distribuição Aleatória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
HER2 overexpression is frequently associated with tumor metastasis and poor prognosis of breast cancer. More evidence indicates that HER3 is involved in HER2-resistant therapies. Combination treatments with two or more different monoclonal antibodies are a promising strategy to overcome resistance to HER2 therapies. We presented a novel fully human HER2-targeted monoclonal antibody, GB235, screened from a phage-display library against the HER2 antigen. GB235 in combination with Trastuzumab overcomes resistance in HER2-positive tumors and results in more sustained inhibition of tumor growth over time. The competition binding assay showed that the epitopes of GB235 do not overlap with those of Pertuzumab and Trastuzumab on HER2. Further HER2 mutagenesis results revealed that the binding epitopes of GB235 were located in the domain III of HER2. The mechanism of action of GB235 in blocking HER2-driven tumors is different from the mechanisms of Trastuzumab or Pertuzumab. GB235 does not affect the heterodimerization of HER2 and HER3, whereas the GB235 combined treatment with Trastuzumab significantly inhibited heregulin-induced HER3 phosphorylation and downstream signaling. Moreover, GB235 in combination with Trastuzumab reversed the resistance to heregulin-induced proliferation in HER2-overexpressing cancer cell lines. GB235 combined with Trastuzumab treatment in xenograft models resulted in improved antitumor activity. Complete tumor suppression was observed in the HER2-positive NCI-N87 xenograft model treated with the combination treatment with GB235 and Trastuzumab. In a Trastuzumab-resistant patient-derived tumor xenograft model GA0060, GB235 plus Trastuzumab reversed the resistance to Trastuzumab monotherapy. Because GB235 showed a different working mechanism with Pertuzumab and Trastuzumab, these agents can be considered complementary therapy against HER2 overexpression tumors.
Assuntos
Anticorpos Monoclonais/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Receptor ErbB-2/antagonistas & inibidores , Animais , Anticorpos Monoclonais/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Neoplasias/patologia , Neuregulina-1/metabolismo , Fosforilação/efeitos dos fármacos , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trastuzumab/farmacologia , Trastuzumab/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Aggregate removal is one of the most important aspects in monoclonal antibody (mAb) purification. Cation-exchange chromatography (CEX), a widely used polishing step in mAb purification, is able to clear both process-related impurities and product-related impurities. In this study, with the implementation of quality by design (QbD), a process development approach for robust removal of aggregates using CEX is described. First, resin screening studies were performed and a suitable CEX resin was chosen because of its relatively better selectivity and higher dynamic binding capacity. Second, a pH-conductivity hybrid gradient elution method for the CEX was established, and the risk assessment for the process was carried out. Third, a process characterization study was used to evaluate the impact of the potentially important process parameters on the process performance with respect to aggregate removal. Accordingly, a process design space was established. Aggregate level in load is the critical parameter. Its operating range is set at 0-3% and the acceptable range is set at 0-5%. Equilibration buffer is the key parameter. Its operating range is set at 40 ± 5 mM acetate, pH 5.0 ± 0.1, and acceptable range is set at 40 ± 10 mM acetate, pH 5.0 ± 0.2. Elution buffer, load mass, and gradient elution volume are non-key parameters; their operating ranges and acceptable ranges are equally set at 250 ± 10 mM acetate, pH 6.0 ± 0.2, 45 ± 10 g/L resin, and 10 ± 20% CV respectively. Finally, the process was scaled up 80 times and the impurities removal profiles were revealed. Three scaled-up runs showed that the size-exclusion chromatography (SEC) purity of the CEX pool was 99.8% or above and the step yield was above 92%, thereby proving that the process is both consistent and robust.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Cromatografia por Troca Iônica/métodos , Animais , Células CHO , Resinas de Troca de Cátion/química , Cátions/química , Cricetinae , Concentração de Íons de HidrogênioRESUMO
Process advancements driven through innovations have been key factors that enabled successful commercialization of several human therapeutic antibodies in recent years. The production costs of these molecules are higher in comparison to traditional medicines. In order to lower the development and later manufacturing costs, recent advances in antibody production technologies target higher throughput processes with increased clinical and commercial economics. In this review, essential considerations and trends for commercial process development and optimization are described, followed by the challenges to obtain a high titer cell culture process and its subsequent impact on the purification process. One of these recent technical advances is the development and implementation of a disposable Q membrane adsorber as an alternative to a Q-packed-bed column in a flow-through mode. The scientific concept and principles underlining Q membrane technology and its application are also reviewed.
Assuntos
Anticorpos Monoclonais/biossíntese , Engenharia de Proteínas/instrumentação , Engenharia de Proteínas/métodos , Anticorpos Monoclonais/isolamento & purificação , Reatores Biológicos , Técnicas de Cultura de Células , Cromatografia , Cromatografia de AfinidadeRESUMO
Once highly selective protein A affinity is chosen for robust mAb downstream processing, the major role of polishing steps is to remove product related impurities, trace amounts of host cell proteins, DNA/RNA, and potential viral contaminants. Disposable systems can act as powerful options either to replace or in addition to polishing column chromatography to ensure product purity and excellent viral clearance power for patients' safety. In this presentation, the implementation of three disposable systems such as depth filtration, membrane chromatography, and nanometer filtration technology in a commercial process are introduced. The data set of viral clearance with these systems is presented. Application advantages and disadvantages including cost analysis are further discussed.
Assuntos
Anticorpos Monoclonais/biossíntese , Biotecnologia , Equipamentos Descartáveis/estatística & dados numéricos , Contaminação de Medicamentos/prevenção & controle , Vírus/isolamento & purificação , Animais , Cromatografia/instrumentação , Cromatografia/métodos , Custos e Análise de Custo , Equipamentos Descartáveis/economia , Filtração/instrumentação , Filtração/métodos , Humanos , Concentração de Íons de Hidrogênio , Pressão , TemperaturaRESUMO
The commercial production of recombinant human monoclonal antibody therapeutics demands robust processes. In this article we describe the development of a pH-conductivity hybrid gradient for a cation-exchange chromatography step to obtain high binding capacity and consistent purification resolution in scale process. Operational parameters and their ranges were characterized with DOE statistical method. Aggregate, DNA and leached protein A removal were examined during development. The advantages and disadvantages of hybrid gradient elution compared to sodium chloride gradient elution were explored. As this step was designed as a good fit for the compatibility of the feed and operating pH/conductivity conditions for next step, the effects of elution by either changing sodium chloride concentration or changing pH of elution buffers on overall separation efficiency were compared. The operation condition was further confirmed in six 2000 L scale runs. The thorough evaluation demonstrated process reliability of hybrid gradient cation-exchange chromatography with high step purity and yield.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Cromatografia por Troca Iônica/métodos , Animais , Soluções Tampão , Células CHO , Cátions , Fracionamento Químico , Cromatografia em Gel , Cricetinae , Cricetulus , DNA/isolamento & purificação , Humanos , Concentração de Íons de Hidrogênio , Polímeros/química , Resinas Sintéticas/química , Acetato de Sódio/química , Cloreto de Sódio/química , Proteína Estafilocócica A/isolamento & purificação , TemperaturaRESUMO
Process-scale antibody production requires polishing steps with extremely high product throughput and robust operation. In this communication, the Sartobind Q membrane adsorber for process-scale antibody production is evaluated as an alternative to Q column chromatography. Although the capacity seen with large-scale membrane adsorbers is competitive with column chromatography, the same throughput is not achieved with the current scale-down models. The operational issues currently found in membrane scale-down models, including backpressure, which significantly compromises the membrane's capacity, were examined. A new scale-down model was designed to mimic the liquid flow path found in the large-scale capsule, and a new process capacity equivalent at both small and large scale was successfully achieved. Results of a 4-model virus study with a redesigned Sartobind Q absorber scale-down model at the new process capacity are presented.
Assuntos
Anticorpos/isolamento & purificação , Membranas Artificiais , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Camundongos , Modelos Biológicos , Reprodutibilidade dos Testes , Fenômenos Fisiológicos ViraisRESUMO
The large-scale production of recombinant human monoclonal antibodies demands economical purification processes with high throughputs. In this article we briefly describe a common antibody process and evaluate the Q membrane adsorber for process-scale antibody production as an alternative to a Q-packed-bed column in a flow-through mode. The scientific concepts underlining Q membrane technology and its application are reviewed. The disadvantages and advantages of using Q membrane chromatography as a purification unit in large-scale production are discussed, including problems initially seen with the Q membrane scale-down model but solved with the invention of a new scale-down model. The new Q-membrane unit operation has a process capacity greater than 3,000 g/m(2) or 10.7 kg/L with a LRV over 5 for four model viruses. In this Review, a cost analysis illustrates that Q membrane chromatography is a viable alternative to Q column chromatography as a polishing step in a flow-through mode for process-scale antibody production.
Assuntos
Anticorpos/isolamento & purificação , Cromatografia/métodos , Animais , Cromatografia/economia , Humanos , Modelos Biológicos , Temperatura , Fatores de Tempo , ViscosidadeRESUMO
Interferon-alpha (IFN-alpha) is indicated for the treatment of certain viral infections including hepatitis B and C, and cancers such as melanoma. The short circulating half-life of unmodified IFN-alpha makes frequent dosing (daily or three times weekly) over an extended period (6-12 months or more) necessary. To improve the pharmacokinetics of IFN-alpha and decrease dosing frequency, IFN-alpha was fused to human serum albumin producing a new protein, Albuferon. In vitro comparisons of Albuferon and IFN-alpha showed similar antiviral and antiproliferative activities, although Albuferon was less potent on a molar basis than IFN-alpha. Pharmacokinetic and pharmacodynamic properties of the fusion protein were enhanced in monkeys. After a single intravenous injection (30 microg/kg,) clearance was 0.9 ml/h/kg, and the terminal half-life was 68 h. After 30 microg/kg subcutaneous injection, apparent clearance (clearance divided by bioavailability) was 1.4 ml/h/kg, the terminal half-life was 93 h, and bioavailability was 64%. The rate of clearance of Albuferon was approximately 140-fold slower, and the half-life 18-fold longer, than for IFN-alpha given by the subcutaneous route in other monkey studies. Sera from Albuferon-treated monkeys demonstrated dose-related antiviral activity for > or =8 days based on an in vitro bioassay, whereas antiviral activity from IFN-alpha-treated animals was only slightly elevated relative to vehicle on day 0. Significant increases in 2',5'-oligoadenylate synthetase mRNA relative to IFN-alpha- or vehicle-treated animals were maintained for > or =10 days after subcutaneous dosing. The improved pharmacokinetics of Albuferon are accompanied by an improved pharmacodynamic response suggesting that Albuferon may offer the benefits of less frequent dosing and a potentially improved efficacy profile compared with IFN-alpha.
Assuntos
Antivirais/farmacologia , Antivirais/farmacocinética , Interferon-alfa/farmacologia , Interferon-alfa/farmacocinética , 2',5'-Oligoadenilato Sintetase/antagonistas & inibidores , Animais , Antivirais/administração & dosagem , Área Sob a Curva , Disponibilidade Biológica , Linfoma de Burkitt/patologia , Células COS , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Feminino , Humanos , Injeções Intravenosas , Injeções Subcutâneas , Interferon-alfa/administração & dosagem , Interferon-alfa/imunologia , Macaca fascicularis , Masculino , RNA Mensageiro/biossíntese , Albumina Sérica , Albumina Sérica HumanaRESUMO
DR3 is a death domain-containing receptor that is upregulated during T cell activation and whose overexpression induces apoptosis and NF-kappaB activation in cell lines. Here we show that an endothelial cell-derived TNF-like factor, TL1A, is a ligand for DR3 and decoy receptor TR6/DcR3 and that its expression is inducible by TNF and IL-1alpha. TL1A induces NF-kappaB activation and apoptosis in DR3-expressing cell lines, while TR6-Fc protein antagonizes these signaling events. Interestingly, in T cells, TL1A acts as a costimulator that increases IL-2 responsiveness and secretion of proinflammatory cytokines both in vitro and in vivo. Our data suggest that interaction of TL1A with DR3 promotes T cell expansion during an immune response, whereas TR6 has an opposing effect.