RESUMO
Inadequate T cell activation has severely limited the success of T cell engager (TCE) therapy, especially in solid tumors. Enhancing T cell activity while maintaining the tumor specificity of TCEs is the key to improving their clinical efficacy. However, currently, there needs to be more effective strategies in clinical practice. Here, we design novel superantigen-fused TCEs that display robust tumor antigen-mediated T cell activation effects. These innovative drugs are not only armed with the powerful T cell activation ability of superantigens but also retain the dependence of TCEs on tumor antigens, realizing the ingenious combination of the advantages of two existing drugs. Superantigen-fused TCEs have been preliminarily proven to have good (>30-fold more potent) and specific (>25-fold more potent) antitumor activity in vitro and in vivo. Surprisingly, they can also induce the activation of T cell chemotaxis signals, which may promote T cell infiltration and further provide an additional guarantee for improving TCE efficacy in solid tumors. Overall, this proof-of-concept provides a potential strategy for improving the clinical efficacy of TCEs.
Assuntos
Neoplasias , Linfócitos T , Humanos , Superantígenos/uso terapêutico , Antígenos de Neoplasias , Morte CelularRESUMO
Targeting single tumor antigens makes it difficult to provide sufficient tumor selectivity for T cell engagers (TCEs), leading to undesirable toxicity and even treatment failure, which is particularly serious in solid tumors. Here, we designed novel trispecific TCEs (TriTCEs) to improve the tumor selectivity of TCEs by logic-gated dual tumor-targeting. TriTCE can effectively redirect and activate T cells to kill tumor cells (â¼18 pM EC50) by inducing the aggregation of dual tumor antigens, which was â¼70- or 750- fold more effective than the single tumor-targeted isotype controls, respectively. Further in vivo experiments indicated that TriTCE has the ability to accumulate in tumor tissue and can induce circulating T cells to infiltrate into tumor sites. Hence, TriTCE showed a stronger tumor growth inhibition ability and significantly prolonged the survival time of the mice. Finally, we revealed that this concept of logic-gated dual tumor-targeted TriTCE can be applied to target different tumor antigens. Cumulatively, we reported novel dual tumor-targeted TriTCEs that can mediate a robust T cell response by simultaneous recognition of dual tumor antigens at the same cell surface. TriTCEs allow better selective T cell activity on tumor cells, resulting in safer TCE treatment.
Assuntos
Neoplasias , Linfócitos T , Camundongos , Animais , Neoplasias/metabolismo , Antígenos de NeoplasiasRESUMO
Metabolic syndrome (MetS) is a complex pathological state consisting of metabolic risk factors such as hypertension, insulin resistance, and obesity. The interconnectivity of cellular pathways within various biological systems suggests that each individual component of MetS may share common pathological sources. Additionally, MetS is closely associated with vasculopathy, including a reduction in microvessel density (MVD) (rarefaction) and elevated risk for various cardiovascular diseases. Microvascular impairments may contribute to perfusion-demand mismatch, where local metabolic needs are insufficiently met due to the lack of nutrient and oxygen supply, thus creating pathological positive-feedback loops and furthering the progression of disease. Sexual dimorphism is evident in these underlying cellular mechanisms, which places males and females at different levels of risk for cardiovascular disease and acute ischemic events. Estrogen exhibits protective effects on the endothelium of pre-menopausal women, while androgens may be antagonistic to cardiovascular health. This review examines MetS and its influences on MVD, as well as sex differences relating to the components of MetS and cardiovascular risk profiles. Finally, translational relevance and interventions are discussed in the context of these sex-based differences.
Assuntos
Doenças Cardiovasculares/etiologia , Síndrome Metabólica/patologia , Densidade Microvascular , Rarefação Microvascular , Microvasos/patologia , Animais , Fatores de Risco Cardiometabólico , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Doenças Cardiovasculares/fisiopatologia , Feminino , Humanos , Masculino , Síndrome Metabólica/complicações , Síndrome Metabólica/metabolismo , Síndrome Metabólica/fisiopatologia , Microvasos/metabolismo , Prognóstico , Medição de Risco , Caracteres Sexuais , Fatores SexuaisRESUMO
The novel anti-PD-L1/TGFBR2-ECD fusion protein (BR102) comprises an anti-PD-L1 antibody (HS636) which is fused at the C terminus of the heavy chain to a TGF-ß1 receptor â ¡ ectodomain (TGFBR2-ECD), and which can sequester the PD-1/PD-L1 pathway and TGF-ß bioactivity in the immunosuppressive tumor microenvironment. In the expression of TGFBR2-ECD wild-type fused protein (BR102-WT), a 50 kDa clipped species was confirmed to be induced by proteolytic cleavage at a "QKS" site located in the N-terminus of the ectodomain, which resulted in the formation of IgG-like clipping. The matrix metalloproteinase-9 was determined to be associated with BR102-WT digestion. In addition, it was observed that the N-glycosylation modifications of the fusion protein were tightly involved in regulating proteolytic activity and the levels of cleavage could be significantly suppressed by MMP-inhibitors. To avoid proteolytic degradation, eliminating protease-sensitive amino acid motifs and introducing potential glycosylation were performed. Three sensitive motifs were mutated, and the levels of clipping were strongly restrained. The mutant candidates exhibited similar binding affinities to hPD-L1 and hTGF-ß1 as well as highly purified BR102-WT2. Furthermore, the mutants displayed more significant proteolytic resistance than that of BR102-WT2 in the lysate incubation reaction and the plasma stability test. Moreover, the bifunctional candidate Mu3 showed an additive antitumor effect in MC38/hPD-L1 bearing models as compared to that of with anti-PD-L1 antibody alone. In conclusion, in this study, the protease-sensitive features of BR102-WT were well characterized and efficient optimization was performed. The candidate BR102-Mutants exhibited advanced druggability in drug stability and displayed desirable antitumor activity.
Assuntos
Anticorpos Antineoplásicos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Neoplasias do Colo/terapia , Processamento de Proteína Pós-Traducional , Receptor do Fator de Crescimento Transformador beta Tipo II/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Animais , Anticorpos Antineoplásicos/genética , Anticorpos Antineoplásicos/imunologia , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Células CHO , Neoplasias do Colo/genética , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Cricetulus , Feminino , Glicosilação , Humanos , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Domínios Proteicos , Proteólise , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/imunologia , Microambiente Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Despite the significant therapeutic advances in T-cell immunotherapy, many malignancies remain unresponsive, which might be because of the negative regulation of T cells by the tumor microenvironment (TME). T cells discriminate tumor cells and normal cells through T-cell receptors (TCRs); therefore, we generated a novel type of TCR-drug conjugates (TDCs) by referring antibody-drug conjugations (ADCs) to overcome the effects of the TME on T cells while preserving the specificity of TCR for tumor recognition. We selected HLA-A2/NY-ESO-1157-165 (peptide NY-ESO-1157-165 in complex with human leukocyte antigen serotype HLA-A*02:01) as the antigen and the antigen-specific TCR (1G4113) as the carrier. By sortase A-mediated ligation, we obtained three NY-TCR-vcMMAEs and further studied their properties, antitumor activity, and toxicity in vitro and in vivo. We found that all the NY-TCR-vcMMAEs had high endocytosis efficiency and specifically killed HLA-A2/NY-ESO-1157-165 positive tumor cells. In xenograft models, one of the TDCs, NY-TCR-2M, was effectively and specifically distributed into tumor tissues and inhibited tumor growth without inducing obvious toxicity. Our results demonstrated that TCRs can be carriers of toxic payloads and that the TDCs thus formed can specifically inhibit tumor growth, neglecting the immune microenvironment.
Assuntos
Antígenos de Neoplasias/imunologia , Antígenos de Superfície/imunologia , Proliferação de Células/efeitos dos fármacos , Imunoconjugados/farmacologia , Espaço Intracelular/efeitos dos fármacos , Proteínas de Membrana/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Humanos , Imunoconjugados/imunologia , Imunoconjugados/metabolismo , Imunoterapia , Espaço Intracelular/metabolismo , CamundongosRESUMO
BACKGROUND: Human leukocyte antigen (HLA) alleles are implicated in drug-induced hypersensitivity, including by nevirapine and abacavir. The purpose of this meta-analysis was to evaluate the relationship between HLA polymorphisms and hypersensitivity to antiretroviral therapy in human immunodeficiency virus (HIV)-infected patients. METHODS: We conducted a systematic search of PubMed, Embase, Web of Science, and the Cochrane Library for studies that evaluated the associations of HLA polymorphisms with antiretroviral therapy-induced hypersensitivity published in April 2019. The summary odds ratios (ORs) with 95% confidence intervals (CIs) were considered as estimates of the effect. RESULTS: The meta-analysis included 17 studies that assessed a total of 4273 patients. First, carriers of HLA-A *24 were associated with an increased risk of hypersensitivity among patients with HIV who received antiretroviral therapy (OR: 12.12; P = 0.018). Second, five SNPs of HLA-B genotypes, including *18 (OR: 1.63; P = 0.028), *35 (OR: 2.31; P = 0.002), *39 (OR: 11.85; P = 0.040), *51 (OR: 1.66; P = 0.028), and *81 (OR: 8.11; P = 0.021), were associated with an increased risk of hypersensitivity. Conversely, carriers of HLA-B *15 were associated with a reduced risk of hypersensitivity (OR: 0.43; P < 0.001). Third, HLA-C *04 was associated with an increased risk of hypersensitivity (OR: 3.09; P < 0.001), whereas a lower risk for hypersensitivity was observed in patients who were carriers of HLA-C *02 (OR: 0.22; P = 0.030), *03 (OR: 0.53; P = 0.049), and *07 (OR: 0.61; P = 0.044). Finally, carriers of HLA-DRB1 *05 (OR: 0.18; P = 0.006) and *15 (OR: 0.23; P = 0.013) were associated with a reduced risk of hypersensitivity among patients receiving antiretroviral therapy. CONCLUSIONS: The findings of this meta-analysis indicated patients carrying HLA-A *24, HLA-B *18, *35, *39, *51, *81, HLA-C *04 were associated with a higher risk of hypersensitivity. Conversely, subjects carrying HLA-B *15, HLA-C *02, *03, *07, HLA-DRB1 *05, *15 were associated with a reduced risk of hypersensitivity.
Assuntos
Fármacos Anti-HIV/efeitos adversos , Hipersensibilidade a Drogas/genética , Antígenos HLA/genética , Polimorfismo de Nucleotídeo Único , Fármacos Anti-HIV/uso terapêutico , Didesoxinucleosídeos/efeitos adversos , Didesoxinucleosídeos/uso terapêutico , Feminino , Infecções por HIV/tratamento farmacológico , Humanos , Masculino , Nevirapina/efeitos adversos , Nevirapina/uso terapêutico , Razão de ChancesRESUMO
Wilms tumor 1 (WT1) oncoprotein is an intracellular oncogenic transcription factor which is barely expressed in normal adult tissues but over expressed in a variety of leukemias and solid cancers. WT1-derived HLA-A*02:01 T cell epitope, RMFPNAPYL (RMF), is a validated target for T cell-based immunotherapy. We generated two T cell receptor mimic antibody-drug conjugates (TCRm-ADCs), ESK-MMAE, and Q2L-MMAE, against WT1 RMF/HLA-A*02:01 complex with distinct affinities, which mediate specific antitumor activity. Although ESK-MMAE showed higher tumor growth inhibition ratio in vivo, the efficacy of them was not so promising, which might be due to low expression of peptide/HLA targets. Therefore, we explored a bispecific TCRm-ADC that exerted more potent tumor cytotoxicity compared with TCRm-ADCs. Hence, our findings validate the feasibility of the presenting intracellular peptides as the targets of ADCs, which broadens the antigen selection range of antibody-based drugs and provides new strategies for precision medicine in tumor therapy.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Imunoconjugados/uso terapêutico , Neoplasias Renais/tratamento farmacológico , Proteínas WT1/imunologia , Tumor de Wilms/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Antígenos HLA-A/imunologia , Humanos , Neoplasias Renais/imunologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Receptores de Antígenos de Linfócitos T/imunologia , Tumor de Wilms/imunologiaRESUMO
Numerous studies have illustrated the relationship between SLCO1B1 T521C polymorphism and statin-induced myopathy risk; however, this association is not consistent. Three electronic databases (PubMed, EMBASE, and the Cochrane Library) were searched from inception to October 2017 to identify potential studies. The summary odds ratios (ORs) with 95% confidence intervals (CIs) were calculated from different genetic models by using a random-effects model. Fourteen studies comprising 3265 myopathy patients and 7743 controls were included. The summary ORs suggested that 521CC (OR: 2.31; 95% CI: 1.15-4.63; P = 0.019), 521TC (OR: 1.34; 95% CI: 1.02-1.76; P = 0.034), and 521CC + TC (OR: 1.82; 95% CI: 1.32-2.51; P < 0.001) were associated with a greater risk of statin-induced myopathy than 521TT. The higher incidence of statin-induced myopathy was found to be significantly correlated with the C allele compared with the T allele (OR: 1.89; 95% CI: 1.36-2.62; P < 0.001). In addition, we observed that 521CC + TC was associated with an increased risk of myopathy in individuals who received simvastatin (OR: 2.35; 95% CI: 1.08-5.12; P = 0.032) or rosuvastatin (OR: 1.69; 95% CI: 1.07-2.67; P = 0.024) when compared with 521TT. The 521C allele was associated with a greater risk of cerivastatin-induced myopathy than the T allele (OR: 1.95; 95% CI: 1.47-2.57; P < 0.001). The findings of this study indicated that SLCO1B1 T521C was associated with a significantly higher risk of statin-induced myopathy, especially for simvastatin, rosuvastatin, and cerivastatin. Future studies should be conducted in subjects receiving specific types of drugs, and any potential adverse events need to be explored.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Transportador 1 de Ânion Orgânico Específico do Fígado/genética , Doenças Musculares/induzido quimicamente , Doenças Musculares/genética , Variantes Farmacogenômicos , Polimorfismo Genético , Idoso , Feminino , Frequência do Gene , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Musculares/diagnóstico , Farmacogenética , Piridinas/efeitos adversos , Medição de Risco , Fatores de Risco , Rosuvastatina Cálcica/efeitos adversos , Sinvastatina/efeitos adversosRESUMO
As the population ages, neurodegenerative diseases such as Parkinson's disease (PD) and Alzheimer's disease (AD) impose a heavy burden on society and families. The pathogeneses of PD and AD are complex. There are no radical cures for the diseases, and existing therapeutic agents for PD and AD have diverse side effects. Tea contains many bioactive components such as polyphenols, theanine, caffeine, and theaflavins. Some investigations of epidemiology have demonstrated that drinking tea can decrease the risk of PD and AD. Tea polyphenols can lower the morbidity of PD and AD by reducing oxidative stress and regulating signaling pathways and metal chelation. Theanine can inhibit the glutamate receptors and regulate the extracellular concentration of glutamine, presenting neuroprotective effects. Additionally, the neuroprotective mechanisms of caffeine and theaflavins may contribute to the ability to antagonize the adenosine receptor A2AR and the antioxidant properties, respectively. Thus, tea bioactive components might be useful for neuronal degeneration treatment in the future. In the present paper, the neuro protection and the mechanisms of tea and its bioactive components are reviewed. Moreover, the potential challenges and future work are also discussed.
Assuntos
Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Compostos Fitoquímicos/química , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Chá/química , Animais , Antioxidantes/química , Antioxidantes/farmacologia , Biflavonoides/química , Biflavonoides/farmacologia , Cafeína/química , Cafeína/farmacologia , Catequina/química , Catequina/farmacologia , Glutamatos/química , Glutamatos/farmacologia , Interações Ervas-Drogas , Humanos , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/epidemiologia , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Compostos Fitoquímicos/uso terapêutico , Extratos Vegetais/uso terapêutico , Polifenóis/química , Polifenóis/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Malignant peritoneal mesothelioma (MPM) is an aggressive rare malignancy associated with asbestos exposure. A better understanding of the molecular pathogenesis of MPM will help develop a targeted therapy strategy. Oncogene targeted depth sequencing was performed on a tumor sample and paired peripheral blood DNA from a patient with malignant mesothelioma of the peritoneum. Four somatic base-substitutions in NOTCH2, NSD1, PDE4DIP, and ATP10B and 1 insert frameshift mutation in BAP1 were validated by the Sanger method at the transcriptional level. A 13-amino acids neo-peptide of the truncated Bap1 protein, which was produced as a result of this novel frameshift mutation, was predicted to be presented by this patient's HLA-B protein. The polyclonal antibody of the synthesized 13-mer neo-peptide was produced in rabbits. Western blotting results showed a good antibody-neoantigen specificity, and Immunohistochemistry (IHC) staining with the antibody of the neo-peptide clearly differentiated neoplastic cells from normal cells. A search of the Catalogue of Somatic Mutations in Cancer (COSMIC) database also revealed that 53.2% of mutations in BAP1 were frameshift indels with neo-peptide formation. An identified tumor-specific neo-antigen could be the potential molecular biomarker for personalized diagnosis to precisely subtype rare malignancies such as MPM.
Assuntos
Antígenos de Neoplasias/genética , Biomarcadores Tumorais/genética , Mutação da Fase de Leitura/genética , Neoplasias Pulmonares/genética , Mesotelioma/genética , Neoplasias Peritoneais/genética , Medicina de Precisão , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Idoso , Sequência de Aminoácidos , Apresentação de Antígeno/imunologia , Western Blotting , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Complexo Principal de Histocompatibilidade , Mesotelioma Maligno , Modelos Biológicos , Peso Molecular , Reprodutibilidade dos Testes , Transdução de Sinais , Proteínas Supressoras de Tumor/química , Ubiquitina Tiolesterase/químicaRESUMO
OBJECTIVE: To prepare rabbit monoclonal antibody (RabMab) against guanosine 3', 5'-cyclic monophosphate (cGMP) and to develop a competitive ELISA for the detection of cGMP. METHODS: New Zealand white rabbits were immunized with synthesized cGMP-keyhole limpet hemoeyanin (cGMP-KLH) to prepared a RabMAb with monoclonal antibody technique of Epitomics. A competitive ELISA kit was produced with cGMP RabMAb. The specificity, the precision and the recoveries of the method were determined. RESULTS: The RabMAb with high sensitivity towards cGMP were prepared with an antibody timer of 3.1 ng/mL and 50% inhibitive concentration (IC50) of 12.57 ng/mL. The cGMP RabMAb had 33% cross-reactivity to inosine 3', 5'-cyclic monophosphate (cIMP) and little or no cross-reactivity to other compounds. A competitive ELISA was developed for detection of cGMP. The range of detection was 0~120 ng/mL with a minimal limit of 1.95 ng/mL. The recovery of assay was 89%~103%. The inter-assay and intra-assay coefficient variations were below 11.68% and 13.85%, respectively. CONCLUSION: The RabMab against cGMP with high affinity and high specificity has been generated successfully, and a competitive ELISA for detection of cGMP has been developed with the prepared cGMP RabMAb.
Assuntos
Anticorpos Monoclonais/biossíntese , GMP Cíclico/imunologia , Ensaio de Imunoadsorção Enzimática , Animais , Especificidade de Anticorpos , Reações Cruzadas , CoelhosRESUMO
Utilization of antibodies to deliver highly potent cytotoxic agents to corresponding antigen-overexpressed tumor cells is a clinically validated therapeutic strategy. Ofatumumab (OFA, trade name Arzerra) is a fully human CD20-specific antibody that is active against CD20-positive B-cell lymphoma/chronic lymphocytic leukemia cells. In order to further enhance the anticancer effect of OFA, anti-CD20 OFA has been conjugated with highly cytotoxic monomethyl auristatin E (MMAE) through a cathepsin-B-cleavable valine-citrulline (vc) dipeptide linkage to form OFA-vcMMAE and the anti-tumor activity of OFA-vcMMAE against CD20-positive B lymphoma cells are then evaluated in vitro and in vivo. As a result, conjugation of OFA with MMAE has kept the initial effector functional activities of OFA such as binding affinity, complement-dependent cytotoxicity (CDC) as well as antibody-dependent cell-mediated cytotoxicity (ADCC). In addition, the conjugation of MMAE significantly improved the cytotoxic activity of OFA against CD20-positive cells (i.e., Raji, Daudi and WIL2-S cells) but not against CD20-negative K562 cells. On the other hand, OFA-vcMMAE was modulated from the CD20-positive cell surface and then entered the lysosomes by receptor-mediated endocytosis, underwent proteolytic degradation and released active drug MMAE to induce apoptotic cell death through a caspase-3-like protease-dependent pathway. Surprisingly, OFA-vcMMAE completely inhibited the growth of CD20-positive Daudi and Ramos lymphoma xenografts in vivo, and exhibited greater anti-tumor activity than unconjugated OFA, suggesting that the anti-tumor activity of anti-CD20 antibody can be enhanced by conjugation with MMAE. In the near future, this new approach might be used as a clinical treatment of CD20-positive B lymphoid malignancies.
Assuntos
Aminobenzoatos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Antígenos CD20/metabolismo , Linfoma de Células B/tratamento farmacológico , Terapia de Alvo Molecular , Oligopeptídeos/uso terapêutico , Aminobenzoatos/química , Aminobenzoatos/farmacologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/química , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Endocitose/efeitos dos fármacos , Humanos , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos SCID , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Trastuzumab , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
AIM: To develop a population pharmacokinetic model for the immunosuppressant ciclosporin in Chinese children with aplastic anemia and to identify covariates influencing ciclosporin pharmacokinetics. METHODS: A total of 102 children with either acquired or congenital aplastic anemia aged 8.8±3.6 years (range 0.9-17.6 years) were included. Therapeutic drug monitoring (TDM) data for ciclosporin were collected. The population pharmacokinetic model of ciclosporin was described using the nonlinear mixed-effects modeling (NONMEM) VI software. The final model was validated using bootstrap and normalized prediction distribution errors. RESULTS: A one-compartment model with first-order absorption and elimination was developed. The estimated CL/F was 15.1, which was lower than those of children receiving stem cell or kidney transplant reported in the West (16.9-29.3). The weight normalized CL/F was 0.45 (range: 0.27-0.70) Lh(-1)·kg(-1). The covariate analysis identified body weight, serum creatinine and concomitant administration of the anabolic steroid stanozolol as individual factors influencing the CL/F of ciclosporin. CONCLUSION: Our model could be used to optimize the ciclosporin dosing regimen in Chinese children with aplastic anemia.
Assuntos
Anemia Aplástica/fisiopatologia , Peso Corporal/fisiologia , Ciclosporina/farmacocinética , Imunossupressores/farmacocinética , Testes de Função Renal , Estanozolol/farmacocinética , Adolescente , Anemia Aplástica/tratamento farmacológico , Anemia Aplástica/etnologia , Povo Asiático/etnologia , Peso Corporal/efeitos dos fármacos , Criança , Pré-Escolar , Ciclosporina/administração & dosagem , Feminino , Humanos , Imunossupressores/administração & dosagem , Lactente , Testes de Função Renal/métodos , Masculino , Taxa de Depuração Metabólica/efeitos dos fármacos , Taxa de Depuração Metabólica/fisiologia , Estudos Prospectivos , Estanozolol/administração & dosagemRESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered to be a promising anticancer agent because its active form TRAIL trimer is able to induce apoptosis in different tumor cell lines while sparing normal cells. However, TRAIL trimer possesses a short half-life and low stability, which turns out to be a major obstacle for the development of clinical trials. In our present study, we constructed a recombined TRAIL trimer by genetic fusion of non-collagenous domain (NC1) of human collagen XVIII or its trimerization domain (TD) to C-terminus of TRAIL via a flexible linker, and then refolded the fusion proteins using a two-step refolding approach, namely a combination of dilution and gel filtration chromatography. As a result, both recombinant proteins, TRAIL-NC1 and TRAIL-TD, were expressed in Escherichia coli as inclusion bodies, and they exhibited difficultly to refold efficiently by conventional methods. Thereby, we applied a modified two-step refolding approach to refold fusion proteins. More than 55 % of TRAIL-NC1 and 90 % of TRAIL-TD protein activity was recovered during the two-step refolding approach, and their stability was also increased significantly. Also, size exclusion chromatography showed refolded TRAIL-NC1 was a trimer while TRAIL-TD, hexamer. However, both of them exerted good apoptosis activity on NCI-H460 cells.
Assuntos
Colágeno Tipo XVIII/metabolismo , Dobramento de Proteína , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Antineoplásicos/isolamento & purificação , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Cromatografia em Gel , Colágeno Tipo XVIII/genética , Colágeno Tipo XVIII/isolamento & purificação , Colágeno Tipo XVIII/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Escherichia coli/genética , Expressão Gênica , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
At present, multiple myeloma (MM) is still an essentially incurable hematologic malignancy. Although BCMA-targeted therapies have achieved remarkable results, BCMA levels were found to be downregulated in patients with MM who relapsed after these treatments. Therefore, the search for other antigens specific to MM has become a priority. Independently of BCMA expression, G-protein-coupled receptor family C group 5 member D (GPRC5D) is mainly expressed in the plasma cells of MM patients, while it is expressed in a limited number of normal tissues. Combining MM-specific antigen GPRC5D and T-cell-mediated therapies would be a promising therapeutic strategy for MM. Recently, we constructed a new anti-GPRC5D × anti-CD3 T-cell-engaging bispecific antibody (TCB), BR109, which was capable of binding to human GPRC5D and human CD3ε. Moreover, BR109 was proven to have relatively good stability and antitumor activity. BR109 could specifically trigger T-cell-mediated cytotoxicity against many GPRC5D-positive MM cells in vitro. Meanwhile, antitumor activity was demonstrated in MM cell line xenograft mouse models with human immune cell reconstitution. These preclinical studies have formed a solid foundation for the evaluation of MM treatment efficacy in clinical trials.
RESUMO
Interleukin-1 receptor antagonist (IL1ra) is known to treat a number of diseases such as rheumatoid arthritis and type 2 diabetes. However, the biological half-life of IL1ra is very short due to its rapid renal clearance. Our present study aimed to increase the biological half-life of IL1ra through fusion with human serum albumin (HSA), and then augmented expression of the IL1ra and HSA fusion protein (IH) in Pichia pastoris strain by increasing IH gene copy number or was co-expressed with chaperone. By comparing clones containing varying copy numbers of IH fusion gene, it was observed that higher levels of secretory IH fusion protein was produced in strain with higher IH gene copy number. In addition, IH protein yield was further improved after being co-expressed with protein disulfide isomerase (PDI). Conversely, it was significantly decreased (i.e., secretory IH in the culture medium) by co-expression of immunoglobulin binding protein. We have also discussed whether the multi-copy strain and co-expressed of PDI could enhance the levels of other secretory albumin fusion protein (e.g., HSA and human growth hormone fusion protein). Interestingly, the level of this fusion protein was apparently also increased by these approaches. In conclusion, our results have demonstrated that increasing copy number and co-expression of PDI may raise yield of albumin fusion protein in P. pastoris, which might probably contribute to the industry for the development of proteinous drugs.
Assuntos
Dosagem de Genes , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Chaperonas Moleculares/metabolismo , Pichia/genética , Pichia/metabolismo , Albumina Sérica/metabolismo , Biotecnologia/métodos , DNA Fúngico/química , DNA Fúngico/genética , Proteína Antagonista do Receptor de Interleucina 1/genética , Engenharia Metabólica , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Albumina Sérica/genética , Albumina Sérica HumanaRESUMO
CYP2D6 is an important drug-metabolizing enzyme. The polymorphism of CYP2D6 leads to metabolism difference and the different reactions of drugs in the individuals and different races are normal phenomenon in clinical medication. CYP2D6*10 is an important subtype in Asian people and 51.3% Chinese are classified with this subtype. To obtain recombinant active CYP2D6*1/CYP2D6*10 in baculovirus system by optimizing coexpression with CYPOR, and detect their activity to catalyze dextromethorphan, three recombinants pFastBac-CYP2D6*1, pFastBac-CYP2D6*10 and pFastBac-CYPOR were constructed and transformed into DH10Bac cell to obtain the recombinant Bacmid-CYPOR, Bacmid-CYP2D6*1 and Bacmid-CYP2D6*10. And then the recombinant CYP2D6*1 and CYP2D6*10 virus were obtained by transfecting Sf9. Then homogenate protein activity was determined with dextromethorphan as substrate. The multiple of infection (MOI) and its ratio of recombinant CYP2D6 virus to CYPOR virus were adjusted by detecting the activity of the homogenate protein. The Km and Vmax are 26.67 +/- 2.71 micromol x L(-1) (n=3) and 666.7 +/- 56.78 pmol x nmol(-1) (CYP2D6) x min(-1) (n=3) for CYP2D6*1 to catalyze dextromethaphan. The Km and Vmax are 111.36 +/- 10.89 micromol x L(-1) (n=3) and 222.2 +/- 20.12 pmol x nmol(-1) (CYP2D6) x min(-1) (n=3) for CYP2D6*10 to catalyze dextromethorphan. There is significant difference between CYP2D6*1 and CYP2D6*10 for Vmax and Km (P < 0.01). The clearance ratio of CYP2D6*1 is 25.0 and the clearance ratio of CYP2D6*10 is 2.0. The expressed CYP2D6*1 and CYP2D6*10 are useful tools to screen the metabolism profile of many xenobiotics and endobiotics in vitro, which are benefit to understand individual metabolism difference.
Assuntos
Citocromo P-450 CYP2D6/metabolismo , Dextrometorfano/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Animais , Baculoviridae/enzimologia , Baculoviridae/genética , Catálise , Células Cultivadas , Cromatografia Líquida de Alta Pressão/métodos , Citocromo P-450 CYP2D6/genética , Isoenzimas/metabolismo , NADPH-Ferri-Hemoproteína Redutase/genética , Plasmídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Spodoptera/citologia , Spodoptera/virologia , TransfecçãoRESUMO
New Chemical Entities (NCEs) development is a systematic long-term project that involves multiple disciplines. The translation research will help to build an advanced R&D system from the basic laboratory research, preclinical studies and clinical evaluation to clinical application of drug, for the purpose of shortening the R&D cycle and accelerate the launch of new drugs. In new drug R&D and its clinical application, drug disposition (absorption, distribution, metabolism, excretion, ADME) properties are important criteria for assessing drug-likeness of candidates. ADME evaluation of NCEs plays an important role in the translation research throughout innovative drug R&D process. Therefore, ADME evaluation at the early stage of drug design and development will be helpful to improve the success rate and reduce costs, and further access to safe, effective drugs.
Assuntos
Preparações Farmacêuticas/metabolismo , Farmacocinética , Pesquisa Translacional Biomédica , Absorção , Transporte Biológico , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Preparações Farmacêuticas/química , Distribuição TecidualRESUMO
OBJECTIVE: To enhance the expression level of staphylococcal enterotoxin O (SEO) by optimization of rare codons. METHODS: The gene of mature SEO (His-tag included) was cloned to pET28a, and 15 rare codons on the gene were optimized by PCR technology. These recombinant plasmids then were transformed into E.coli BL21(DE3), respectively. After IPTG induced, the expression levels of those mutants were analyzed by SDS-PAGE. The proteins were purified and their bioactivities were determined. RESULT: After the optimization of rare codons, the expression levels were increased from 7.49% to 19.8% in total cell proteins. The optimized SEO had bioactivity to stimulate the proliferation of murine lymphocytes, which was equivalent to that of non-optimized SEO in vitro. CONCLUSION: Optimization of rare codons can enhance the expression of SEO effectively.
Assuntos
Códon/genética , Enterotoxinas/biossíntese , Escherichia coli/metabolismo , Animais , Clonagem Molecular , Enterotoxinas/genética , Escherichia coli/genética , Camundongos , Mutação , Plasmídeos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Transformação BacterianaRESUMO
The specific recognition of T cell receptors (TCR) and peptides presented by human leukocyte antigens (pHLAs) is the core step for T cell triggering to execute anti-tumor activity. However, TCR assembly and soluble expression are challenging, which precludes the broad use of TCR in tumor therapy. Herein, we used heterodimeric Fc to assist in the correct assembly of TCRs to achieve the stable and soluble expression of several TCRs in mammalian cells, and the soluble TCRs enable us to yield novel bispecific T cell engagers (TCR/aCD3) through pairing them with an anti-CD3 antibody. The NY-ESO-1/LAGE-1 targeted TCR/aCD3 (NY-TCR/aCD3) that we generated can redirect naïve T cells to specific lysis antigen-positive tumor cells, but the potency of the NY-TCR/aCD3 was disappointing. Furthermore, we found that the activation of T cells by NY-TCR/aCD3 was mild and unabiding, and the activity of NY-TCR/aCD3 could be significantly improved when we replaced naïve T cells with pre-activated T cells. Therefore, we employed the robust T cell activation ability of staphylococcal enterotoxin C2 (SEC2) to optimize the activity of NY-TCR/aCD3. Moreover, we found that the secretions of SEC2-activated T cells can promote HLA-I expression and thus increase target levels, which may further contribute to improving the activity of NY-TCR/aCD3. Our study described novel strategies for soluble TCR expression, and the optimization of the generation and potency of TCR/aCD3 provided a representative for us to fully exploit TCRs for the precision targeting of cancers.