RESUMO
Cancer immunotherapies, such as checkpoint blockade of programmed cell death protein-1 (PD-1), represents a breakthrough in cancer treatment, resulting in unprecedented results in terms of overall and progression-free survival. Discovery and development of novel anti PD-1 inhibitors remains a field of intense investigation, where novel monoclonal antibodies (mAbs) and novel antibody formats (e.g., novel isotype, bispecific mAb and low-molecular-weight compounds) are major source of future therapeutic candidates. HLX10, a fully humanized IgG4 monoclonal antibody against PD-1 receptor, increased functional activities of human T-cells and showed in vitro, and anti-tumor activity in several tumor models. The combined inhibition of PD-1/PDL-1 and angiogenesis pathways using anti-VEGF antibody may enhance a sustained suppression of cancer-related angiogenesis and tumor elimination. To elucidate HLX10's mode of action, we solved the structure of HLX10 in complex with PD-1 receptor. Detailed epitope analysis showed that HLX10 has a unique mode of recognition compared to the clinically approved PD1 antibodies Pembrolizumab and Nivolumab. Notably, HLX10's epitope was closer to Pembrolizumab's epitope than Nivolumab's epitope. However, HLX10 and Pembrolizumab showed an opposite heavy chain (HC) and light chain (LC) usage, which recognizes several overlapping amino acid residues on PD-1. We compared HLX10 to Nivolumab and Pembrolizumab and it showed similar or better bioactivity in vitro and in vivo, providing a rationale for clinical evaluation in cancer immunotherapy.
Assuntos
Anticorpos Monoclonais/química , Imunoterapia , Neoplasias/imunologia , Neoplasias/terapia , Receptor de Morte Celular Programada 1/química , Receptor de Morte Celular Programada 1/imunologia , Inibidores da Angiogênese/uso terapêutico , Animais , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/uso terapêutico , Bevacizumab/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Epitopos/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Interferon gama/metabolismo , Interleucina-2/metabolismo , Ligantes , Macaca fascicularis , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Moleculares , Neoplasias/tratamento farmacológico , Nivolumabe/química , Nivolumabe/uso terapêutico , Ligação Proteica , Ratos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Mutations in the GRN (granulin precursor) are a frequent cause of frontotemporal dementia (FTD) and other atypical parkinsonian disorders. However, the frequency of GRN mutations in Asian patients with atypical parkinsonian disorders is still uncertain. METHODS: We screened GRN mutations by sequencing cDNA from 98 patients with FTD or atypical parkinsonian disorders. The functional properties of the identified mutation were evaluated by overexpression in human embryonic kidney (HEK)-293â¯cells. RESULTS: We identified a new missense (GRN p.T487I) mutation in a female patient with undefined atypical parkinsonism. The overexpression experiment further demonstrated that p.T487I mutation reduced the progranulin protein level and stability in HEK-293â¯cells. CONCLUSION: GRN p.T487I mutation, which decreases the stability of progranulin protein, could be a new causative mutation in patients with atypical parkinsonian disorders.
Assuntos
Demência Frontotemporal/genética , Transtornos Parkinsonianos/genética , Progranulinas/genética , Idoso , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , TaiwanRESUMO
BACKGROUND/PURPOSE: Leucine-rich repeat kinase 2 (LRRK2) is a large protein encoding multiple functional domains. Mutations within different LRRK2 domains have been considered to be involved in the development of Parkinson disease by different mechanisms. Our previous study found three LRRK2 mutations-p.R767H, p.S885N, and p.R1441H-in Taiwanese patients with Parkinson disease. METHODS: We evaluated the functional properties of LRRK2 p.R767H, p.S885N, and p.R1441H mutations by overexpressing them in human embryonic kidney 293 and neuroblastoma SK-N-SH cells. The common p.G2019S mutation in the kinase domain was included for comparison. RESULTS: In 293 cells, overexpressed p.R1441H-but not p.R767H, p.S885N, or p.G2019-increased GTP binding affinity to prolong the active state. Overexpressed p.R1441H and p.G2019S generated inclusions in 293 cells. In SK-N-SH cells, the α-synuclein was coexpressed with wild type as well as mutated p.R767H, p.S885N, p.R1441H, and p.G2019 LRRK2 proteins. Part of the perinuclear inclusions formed by p.R1441H and p.G2019S were colocalized with α-synuclein. Additionally, p.S885N and p.R1441H mutations caused reduced interaction between LRRK2 and ARHGEF7, a putative guanine nucleotide exchange factor for LRRK2, whereas this interaction was well preserved in p.R767H and p.G2019S mutations. CONCLUSION: Our study suggests that p.R1441H protein facilitates the formation of intracellular inclusions, compromises GTP hydrolysis by increasing its affinity for GTP, and reduces its interaction with ARHGEF7.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Doença de Parkinson/genética , Doença de Parkinson/fisiopatologia , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Células HEK293 , Humanos , Mutação de Sentido Incorreto , TaiwanRESUMO
Genetic variants of leucine-rich repeat kinase 2 (LRRK2) were reported to alter the risk for Parkinson's disease (PD). However, the genetic spectrum of LRRK2 variants has not been clearly disclosed yet in Taiwanese population. Herein, we sequenced LRRK2 coding region in 70 Taiwanese early onset PD patients (age at onset ≤ 50), and found six amino acid-changing single nucleotide polymorphisms (SNPs, N551K, R1398H, R1628P, S1647T, G2385R and M2397T), one reported (R1441H) and 2 novel missense (R767H and S885N) mutations. We examined the frequency of identified LRRK2 variants by genotyping 573 Taiwanese patients with PD and 503 age-matched control subjects. The results showed that PD patients demonstrated a higher frequency of G2385R A allele (4.6%) than control subjects (2.1%; odds ratio = 2.27, 95% confidence interval: 1.38-3.88, P = 0.0017). Fewer PD patients (27.7%) carried the 1647T-2397T haplotype as compared with the control subjects (33.0%; odds ratio = 0.80, 95% confidence interval: 0.65-0.97, P = 0.0215). However, the frequency of 1647T-2385R-2397T haplotype (4.3%) in PD patients was still higher than in control subjects (1.9%, odds ratio: 2.15, 95% confidence interval: 1.27-3.78, P = 0.0058). While no additional subject was found to carry R767H and R1441H, one more patient was observed to carry the S885N variant. Our results indicate a robust risk association regarding G2385R and a new possible protective haplotype (1647T-2397T). Gene-environmental interaction and a larger cohort study are warranted to validate our findings. Additionally, two new missense mutations (R767H and S885N) regarding LRRK2 in PD patients were identified. Functional studies are needed to elucidate the effects of these LRRK2 variants on protein function.
Assuntos
Alelos , Frequência do Gene , Mutação de Sentido Incorreto , Doença de Parkinson/genética , Polimorfismo de Nucleotídeo Único , Proteínas Serina-Treonina Quinases/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Povo Asiático , Feminino , Interação Gene-Ambiente , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/epidemiologia , Taiwan/epidemiologiaRESUMO
Spinocerebellar ataxia type 8 (SCA8) involves the expansion of CTG/CAG repeats from the overlapping ataxin 8 opposite strand (ATXN8OS) and ataxin 8 (ATXN8) genes located on chromosome 13q21. Although being transcribed, spliced and polyadenylated in the CTG orientation, ATXN8OS does not itself appear to be protein coding, as only small open reading frames (ORFs) were noted. In the present study we investigated the translation of a novel 102 amino acids containing-ORF in the ATXN8OS RNA. Expression of chimeric construct with an in-frame ORF-EGFP gene demonstrated that ATXN8OS RNA is translatable. Using antiserum raised against ORF, ATXN8OS ORF expression was detected in various human cells including lymphoblastoid, embryonic kidney 293, neuroblastoma IMR-32, SK-N-SH, SH-SY5Y cells and human muscle tissue. The biological role of the ATXN8OS ORF and its connection to SCA8 remains to be determined.
Assuntos
RNA Longo não Codificante/genética , RNA/genética , RNA/metabolismo , Linhagem Celular , Expressão Gênica , Ordem dos Genes , Humanos , Fases de Leitura Aberta , RNA Longo não Codificante/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequências Repetitivas de Ácido NucleicoRESUMO
A single nucleotide polymorphism GRN rs5848 (3'UTR+78 C>T) was reported to alter the risk for frontotemporal lobar degeneration. Herein, we investigated the effect of GRN rs5848 on the risk of Parkinson's disease (PD) by genotyping 573 Taiwanese patients with PD and 490 age-matched control subjects. Compared to subjects with CC genotype, those with TT genotype had a 1.58-fold increased risk of PD (95% CI: 1.77â¼2.34, Pâ=â0.021). PD patients demonstrate a higher frequency of T allele (37.2%) than controls (32.2%; odds ratio [OR]â=â1.24, 95% CI: 1.04â¼1.49, Pâ=â0.017). This susceptibility was particularly observed in female subjects, in which TT genotype had a 2.16-fold increased risk of PD as compared with controls(95% CI: 1.24â¼3.78, Pâ=â0.006). The frequency of T allele (39.3%) in female PD patients was higher than in female control subjects (31.1%; ORâ=â1.43, CI: 1.11â¼1.87, Pâ=â0.007). No association was observed between GRN rs5848 and susceptibility in male subjects. These findings show that the GRN rs5848 TT genotype and T allele are risk factors for female Taiwanese patients with PD.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/genética , Doença de Parkinson/genética , Polimorfismo Genético/genética , Alelos , Povo Asiático , Estudos de Casos e Controles , Feminino , Frequência do Gene/genética , Predisposição Genética para Doença/genética , Genótipo , Humanos , Masculino , Progranulinas , TaiwanRESUMO
Mutations in ATP13A2 have been reported to associate with Parkinson's disease (PD). This study investigates the contribution of genetic variants in ATP13A2 to Taiwanese PD. ATP13A2 cDNA fragments from 65 early onset PD (onset <50 years) were sequenced. The identified variants were validated in a cohort of PD (n = 493) and ethnically matched controls (n = 585). A novel heterozygous G1014S, located at the conserved seventh transmembrane domain of ATP13A2 protein, was identified in an early onset PD patient, which was absent in 585 normal controls. Additionally, a reported heterozygous A746T was found in two PD patients and four controls. The clinical features and 99mTc-TRODAT-1 single photon emission computed tomography (SPECT) image of the patients carrying G1014S and A746T were similar to that of idiopathic PD. One normal control with A746T showed an asymmetric reduction of 99mT TRODAT-1 uptake in the right striatum. Under oxidative stress or apoptotic stimulus, lymphoblastoid cells carrying either A764T or G1014S showed increased caspase 3 activity compared with the controls. The rates of decay for G1014S and A746T proteins were more or less reduced in cycloheximide chase experiment. In silico modeling of G1014S exhibited a more stable feature than wild-type, and G1014S is mislocalized mainly in the intralysosomal space, which is coherent with the prediction of prohibiting N-myristoylation and membrane association. We therefore hypothesize that rare variants of ATP13A2 may contribute to PD susceptibility in Taiwan. The role played by ATP13A2 variants in PD remains to be clarified.
Assuntos
Doença de Parkinson/genética , Doença de Parkinson/metabolismo , ATPases Translocadoras de Prótons/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Caspase 3/biossíntese , Caspase 3/metabolismo , Linhagem Celular Tumoral , Feminino , Variação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Estrutura Terciária de Proteína , ATPases Translocadoras de Prótons/química , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Análise de Sequência de DNA , TaiwanRESUMO
BACKGROUND: Impaired ubiquitin-proteasome system function may contribute to the pathogenesis of Parkinson's disease (PD). METHODS: We conducted a case-control study in a cohort of 517 PD cases and 518 ethnically matched controls to investigate the association of ubiquitin specific proteases USP24 rs487230 C>T, USP40 rs1048603 C>T, and ubiquitin thiolesterase UCHL1 rs5030732 A>C polymorphisms with the risk of PD. RESULTS: No significant difference in the genotype or allele distribution was found between PD and controls. After stratification by age, the genotype and allele frequencies of USP24 rs487230 are significantly different between PD and controls >or=60 years of age (P=0.035 and 0.013, respectively). Multivariable logistic regression with adjusting for onset age and sex showed that, in a dominant model, USP24 T-carrying genotype was associated with risk reduction in developing PD in individuals >or=60 years of age (OR=0.61; 95% CI=0.41-0.90, P=0.010). This is also true for T allele (OR=0.64; 95% CI=0.44-0.91, P=0.023). When examining the interaction between genes on PD risk without age stratification, the protective effect of USP24 CT/TT genotype on PD risks was strengthened by the USP40 T-carrying genotype (OR=0.42; 95% CI=0.22-0.81, P=0.009) and UCHL1 C-carrying genotype (OR=0.67; 95% CI=0.47-0.97, P=0.032). CONCLUSIONS: Our results suggest that USP24 alone plays a role in PD susceptibility among Taiwanese people >or=60 years of age, or acting synergistically with USP40 and UCHL1 in the total subjects.
Assuntos
Povo Asiático/genética , Predisposição Genética para Doença , Doença de Parkinson/genética , Polimorfismo Genético , Ubiquitina Tiolesterase/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Estudos de Casos e Controles , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Taiwan , Adulto JovemRESUMO
BACKGROUND: Expansion of the CAG repeat of the TATA-box binding protein (TBP) gene has been identified as the causative mutations in spinocerebellar ataxia 17 (SCA17). TBP is ubiquitously expressed in both central nervous system and peripheral tissues. The underlying molecular changes of SCA17 are rarely explored. METHODS: To study the molecular mechanisms underlying SCA17, we generated stably induced isogenic 293 cells expressing normal TBP-Q(36) and expanded TBP-Q(61) and analyzed the expressed proteins using two-dimensional difference in gel electrophoresis (2D-DIGE), followed by mass spectrometry and immunoblotting. RESULTS: Upon induction with doxycycline, the expanded TBP-Q(61) formed aggregates with significant increase in the cell population at subG1 phase and cleaved caspase-3. Proteomics study identified a total of 16 proteins with expression changes greater than 1.5 fold. Among the 16 proteins, PARK7, GLRX3, HNRNPA1, GINS1, ENO1, HNRPK and NPM1 are increased, and SERPINA5, HSPA5, VCL, KHSRP, HSPA8, HNRPH1, IMMT, VCP and HNRNPL are decreased in cells expressing TBP-Q(61) compared with those expressing TBP-Q(36). The altered expression of HSPA5, HSPA8 and PARK7 were further validated by 2D and Western immunoblot analyses. CONCLUSIONS: The results illustrate the utility of proteomics to identify alterations of proteins which underlie pathogenesis of SCA17, and may serve as potential therapeutic targets.