RESUMO
Programmed cell death-ligand 1 positive (PD-L1+) exosomes play a crucial role in the realm of cancer diagnosis and treatment. Nevertheless, due to the intricate nature of biological specimens, coupled with the heterogeneity, low refractive index (RI), and scant surface coverage density of exosomes, traditional surface plasmon resonance (SPR) sensors still do not meet clinical detection requirements. This study utilizes the exceptional electrical and optical attributes of single-walled carbon nanotubes (SWCNTs) as the substrate for SPR sensing, thereby markedly enhancing sensitivity. Furthermore, sp2 hybridized SWCNTs have the ability to load specific recognition elements. Additionally, through the coordination interaction of Ti with phosphate groups and the ferromagnetism of Fe3O4, efficient exosomes isolation and enrichment in complex samples are achievable with the aid of an external magnetic field. Owing to the high-quality and high-RI of Fe3O4@TiO2, the response signal experiences amplification, thus further improving the performance of the SPR biosensor. The linear range of the SPR biosensor constructed by this method is 1.0 × 103 to 1.0 × 107 particles/mL, with a limit of detection (LOD) of 31.9 particles/mL. In the analysis of clinical serum samples, cancer patients can be differentiated from healthy individuals with an Area Under Curve (AUC) of 0.9835. This study not only establishes a novel platform for exosomes direct detection but also offers new perspectives for the sensitive detection of other biomarkers.
Assuntos
Antígeno B7-H1 , Exossomos , Nanotubos de Carbono , Ressonância de Plasmônio de Superfície , Titânio , Humanos , Antígeno B7-H1/sangue , Antígeno B7-H1/análise , Antígeno B7-H1/isolamento & purificação , Técnicas Biossensoriais/métodos , Exossomos/química , Limite de Detecção , Nanotubos de Carbono/química , Neoplasias/sangue , Ressonância de Plasmônio de Superfície/métodos , Titânio/químicaRESUMO
Using the pathosystem Phaseolus vulgaris-tobacco necrosis virus (TNV), we demonstrated that PD-L1 and PD-L4, type-1 ribosome inactivating proteins (RIPs) from leaves of Phytolacca dioica L., possess a strong antiviral activity. This activity was exerted both when the RIPs and the virus were inoculated together in the same leaf and when they were inoculated or applied separately in the adaxial and abaxial leaf surfaces. This suggests that virus inhibition would mainly occur inside plant cells at the onset of infection. Histochemical studies showed that both PD-L1 and PD-L4 were not able to induce oxidative burst and cell death in treated leaves, which were instead elicited by inoculation of the virus alone. Furthermore, when RIPs and TNV were inoculated together, no sign of H2O2 deposits and cell death were detectable, indicating that the virus could have been inactivated in a very early stage of infection, before the elicitation of a hypersensitivity reaction. In conclusion, the strong antiviral activity is likely exerted inside host cells as soon the virus disassembles to start translation of the viral genome. This activity is likely directed towards both viral and ribosomal RNA, explaining the almost complete abolition of infection when virus and RIP enter together into the cells.
Assuntos
Antígeno B7-H1/farmacologia , Phaseolus/virologia , Phytolacca/química , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Tombusviridae/efeitos dos fármacos , Antivirais/farmacologia , Antígeno B7-H1/isolamento & purificação , Interações entre Hospedeiro e Microrganismos , Folhas de Planta/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificaçãoRESUMO
Aptamers are small-sized RNA or ssDNA ligands with a unique structure, which have high specificity and affinity to their cognate targets. Thus, in addition to the extensive values in various bio-medical fields, aptamers can also be alternatively used as affinity ligands in the bioprocess, such as for protein purification. In the present study, a hexahistidine specific aptamer named AptHis-C, was developed through the SELEX methodology, which has high affinity to hexahistidine, and its dissociation constant was as low as 20.8 nM. The structural prediction revealed that AptHis-C contains two connected stem-loop conformations. AptHis-C can only specifically recognize recombinant proteins with the hexahistidine-tag in simple or complex situations, and not to those with other tags. When immobilized on magnetic beads, AptHis-C can be used as a tool for hexahistidine-tagged recombinant protein purification. Its effectiveness is as good as traditional Ni-based beads. Besides, due to the intrinsic characteristics of nucleic acids, such as high thermal/chemical stability, immobilized aptamer-magnetic beads can be reused many times without an obvious decrease of purification effectiveness. This aptamer may represent a novel method for the detection and purification of hexahistidine-tagged recombinant proteins.
Assuntos
Aptâmeros de Nucleotídeos/química , DNA de Cadeia Simples/química , Histidina/química , Oligopeptídeos/química , Proteínas Recombinantes/isolamento & purificação , Antígeno B7-H1/genética , Antígeno B7-H1/isolamento & purificação , Cromatografia de Afinidade , Escherichia coli/química , Escherichia coli/genética , Fator 15 de Diferenciação de Crescimento/genética , Fator 15 de Diferenciação de Crescimento/isolamento & purificação , Imãs/química , Microesferas , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/isolamento & purificação , Proteínas Recombinantes/genética , Técnica de Seleção de Aptâmeros , Propriedades de SuperfícieRESUMO
Advances in in vitro display and protein engineering yield therapeutics with affinities in the picomolar range. The Gyrolab® microfluidics platform uses the kinetic exclusion assay principle to measure subnanomolar solution affinities. This work describes application of the Gyrolab solution affinity module and the new multi-curve analysis feature to determine affinity of the PD-L1 Adnectin™ positron emission tomography radioligand, which was measured as 20 pM for human PD-L1. We also report key parameters that affect assay signal-to-background ratio and data quality, such as detection reagent concentration. Gyrolab offers the necessary throughput for rapid assay development with low sample consumption, as demonstrated in this study, which also provides helpful tips for assay optimization for solution affinity measurement.
Assuntos
Antígeno B7-H1/isolamento & purificação , Microfluídica/métodos , Tomografia por Emissão de Pósitrons/métodos , Antígeno B7-H1/química , Antígeno B7-H1/genética , Humanos , Ligantes , Ligação Proteica/genéticaRESUMO
BACKGROUND: The purpose of this study is to test if functional multiparametric imaging with 18F-FDG-PET/MRI correlates spatially with immunohistochemical biomarker status within a lesion of head and neck squamous cell carcinoma (HNSCC), and also whether a biopsy with the highest FDG uptake was more likely to have the highest PD-L1 expression or the highest percentage of vital tumour cells (VTC) compared with a random biopsy. METHODS: Thirty-one patients with HNSCC were scanned on an integrated PET/MRI scanner with FDG prior to surgery in this prospective study. Imaging was quantified with SUV, ADC and Ktrans. A 3D-morphometric MRI scan of the specimen was used to co-register the patient and the specimen scans. All specimens were sectioned in consecutive slices, and slices from six different locations were selected randomly from each tumour. Core biopsies were performed to construct TMA blocks for IHC staining with the ten predefined biomarkers. The spatial correlation was assessed with a partial correlation analysis. RESULTS: Twenty-eight patients with a total of 33 lesions were eligible for further analysis. There were significant correlations between the three imaging biomarkers and some of the IHC biomarkers. Moreover, a biopsy taken from the most FDG-avid part of the tumour did not have a statistically significantly higher probability of higher PD-L1 expression or VTC, compared with a random biopsy. CONCLUSION: We found statistically significant correlations between functional imaging parameters and key molecular cancer markers.
Assuntos
Biomarcadores Tumorais/genética , Imageamento por Ressonância Magnética Multiparamétrica/métodos , Carcinoma de Células Escamosas de Cabeça e Pescoço/diagnóstico por imagem , Idoso , Antígeno B7-H1/genética , Antígeno B7-H1/isolamento & purificação , Biópsia , Feminino , Fluordesoxiglucose F18/uso terapêutico , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons , Estudos Prospectivos , Carcinoma de Células Escamosas de Cabeça e Pescoço/diagnóstico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologiaRESUMO
Cryptococcal meningitis remains one of the leading causes of death among HIV-infected adults in the fourth decade of HIV era in sub-Saharan Africa, contributing to 10%-20% of global HIV-related deaths. Despite widespread use and early induction of ART among HIV-infected adults, incidence of cryptococcosis remains significant in those with advanced HIV disease. Cryptococcus species that causes fatal infection follows systemic spread from initial environmental acquired infection in lungs to antigenaemia and fungaemia in circulation prior to establishment of often fatal disease, cryptococcal meningitis in the CNS. Cryptococcus person-to-person transmission is uncommon, and deaths related to blood infection without CNS involvement are rare. Keen to the persistent high mortality associated with HIV-cryptococcal meningitis, seizures are common among a third of the patients, altered mental status is frequent, anaemia is prevalent with ensuing brain hypoxia and at autopsy, brain fibrosis and infarction are evident. In addition, fungal burden is 3-to-4-fold higher in those with seizures. And high immune activation together with exacerbated inflammation and elevated PD-1/PD-L immune checkpoint expression is immunomodulated phenotypes elevated in CSF relative to blood. Lastly, though multiple Cryptococcus species cause disease in this setting, observations are mostly generalised to cryptococcal infection/meningitis or regional dominant species (C neoformans or gattii complex) that may limit our understanding of interspecies differences in infection, progression, treatment or recovery outcome. Together, these factors and underlying mechanisms are hypotheses generating for research to find targets to prevent infection or adequate therapy to prevent persistent high mortality with current optimal therapy.
Assuntos
Infecções por HIV/complicações , Meningite Criptocócica , Infecções Oportunistas Relacionadas com a AIDS/epidemiologia , Infecções Oportunistas Relacionadas com a AIDS/imunologia , Infecções Oportunistas Relacionadas com a AIDS/patologia , Infecções Oportunistas Relacionadas com a AIDS/terapia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Antígeno B7-H1/sangue , Antígeno B7-H1/isolamento & purificação , Encéfalo/imunologia , Encéfalo/parasitologia , Líquido Cefalorraquidiano/imunologia , Coinfecção , Criptococose/etiologia , Cryptococcus/isolamento & purificação , Cryptococcus/patogenicidade , Cryptococcus gattii/isolamento & purificação , Cryptococcus gattii/patogenicidade , Cryptococcus neoformans/isolamento & purificação , Cryptococcus neoformans/patogenicidade , Humanos , Imunidade , Incidência , Inflamação , Meningite Criptocócica/epidemiologia , Meningite Criptocócica/imunologia , Meningite Criptocócica/patologia , Meningite Criptocócica/terapia , Mortalidade , Prevalência , Receptor de Morte Celular Programada 1/sangue , Receptor de Morte Celular Programada 1/isolamento & purificação , Linfócitos T/imunologia , Linfócitos T/metabolismo , Resultado do TratamentoRESUMO
Circling exosomal PD-L1 can be expected as a predictor for the clinical responds of anti-PD-1/PD-L1 therapy. Here, we present a simple method integrating capture and analysis of exosomal PD-L1 directly from serum. Firstly, Fe3O4@TiO2 nanoparticles were used to enrich exosomes through the binding of TiO2 shell and hydrophilic phosphate head of the exosome phospholipids. Model exosomes can be enriched and separated from solution within 5â¯min with a capture efficiency of 96.5%. Secondly, anti-PD-L1 antibody modified Au@Ag@MBA SERS tags were added to label the exosomal PD-L1 for quantification. The whole process can be finished within 40â¯min with a detection limit of 1 PD-L1+exosome/µL. Furthermore, this method was used for personalized exosomal PD-L1quantification by using a 4⯵L clinical serum sample individually. Based on the personalized SERS signal analysis, NSCLC patients can be distinguished from the healthy controls easily. More important, the advantage of clearly individual quantification may help the doctor to discover the relationship of exosomal PD-L1 and the immnuotherapy responds in individual level.
Assuntos
Antígeno B7-H1/análise , Antígeno B7-H1/sangue , Exossomos/química , Óxido Ferroso-Férrico/química , Análise Espectral Raman/métodos , Titânio/química , Células A549 , Anticorpos Imobilizados/química , Antígeno B7-H1/isolamento & purificação , Técnicas Biossensoriais/métodos , Linhagem Celular , Humanos , Imunoensaio/métodosRESUMO
BACKGROUND: In melanoma, there is no companion diagnostic test to predict response to programmed cell death 1 (PD-1) axis immune checkpoint inhibitor (ICI) therapy. In the adjuvant setting, only one in five patients may benefit from ICI, so a biomarker is needed to select those that may or may not benefit. Here, we test a new 4-gene multiplex immunotherapy panel with research use only (RUO) prototype mRNA expression profile on the GeneXpert closed system using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) for association with clinical benefit after treatment with ICI therapy in metastatic melanoma patients. METHODS: Pretreatment formalin-fixed paraffin-embedded (FFPE) tissue sections from melanoma patients treated with anti-PD-1 therapy (pembrolizumab, nivolumab, or ipilimumab plus nivolumab) between 2011 and 17 were selected from the Yale Pathology archives. FFPE sections were macrodissected to enrich for tumor for quantitative assessment of CD274 (PD-L1), PDCD1LG2 (PD-L2), CD8A, and IRF1 by RT-qPCR multiplex mRNA panel. Multiplex panel transcript levels were correlated with clinical benefit (complete response [CR], partial response [PR], stable disease [SD]); disease outcomes (progression-free survival [PFS] and overall survival [OS]); and protein levels assessed by quantitative immunofluorescence (QIF). RESULTS: Transcript levels were significantly higher in responders (CR/PR/SD) than in nonresponders (PD) for CD8A (p = 0.0001) and IRF1 (p = 0.0019). PFS was strongly associated with high CD274 (p = 0.0046), PDCD1LG2 (p = 0.0039), CD8A (p = 0.0002), and IRF1 (p = 0.0030) mRNA expression. Similar associations were observed for OS with high CD274 (p = 0.0004), CD8A (p = 0.0030), and IRF1 (p = 0.0096) mRNA expression. Multivariate analyses revealed significant PFS and OS associations with immunotherapy panel markers independent of baseline variables. Exploratory analyses revealed a novel significant association of high combined CD274 & PDCD1LG2 (L1/L2) transcript expression with PFS (p < 0.0001) and OS (p = 0.0011), which remained significant at a multivariate level for both PFS (HR = 0.31) and OS (HR = 0.39). CONCLUSIONS: Individual immunotherapy panel markers CD274, PDCD1LG2, CD8A, IRF1 and a combined L1/L2 mRNA levels show promising associations with melanoma immunotherapy outcome. The turnaround time of the test (2 h) and easy standardization of the platform makes this an attractive approach for further study in the search for predictive biomarkers for ICI.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores Tumorais/isolamento & purificação , Melanoma/tratamento farmacológico , Monitorização Imunológica/métodos , Neoplasias Cutâneas/tratamento farmacológico , Idoso , Antineoplásicos Imunológicos/farmacologia , Antígeno B7-H1/genética , Antígeno B7-H1/isolamento & purificação , Biomarcadores Tumorais/genética , Antígenos CD8/genética , Antígenos CD8/metabolismo , Feminino , Seguimentos , Perfilação da Expressão Gênica/métodos , Humanos , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/metabolismo , Masculino , Melanoma/imunologia , Melanoma/mortalidade , Melanoma/secundário , Pessoa de Meia-Idade , Prognóstico , Proteína 2 Ligante de Morte Celular Programada 1/genética , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Intervalo Livre de Progressão , RNA Mensageiro/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologiaRESUMO
INTRODUCTION: Companion diagnostic tests (CDXs) are considered mandatory for decision-making for treatment with targeted therapies in thoracic oncology. The emergence of immunotherapy has also given rise to the development of CDXs. Some CDXs, in particular PD-L1 immunohistochemistry tests, have been questioned and re-examined for use with new combination therapies that are being evaluated in clinical trials. Current questions include: Can we establish therapeutic indications in thoracis oncology without CDXs? Would the addition of new tests benefit patient outcome? Areas covered: This review covers the use of CDXs for decision-making in the treatment of lung cancer but also covers the limits of certain tests. It discusses the major challenges for present and future development of CDXs in daily practice. Expert opinion: CDXs can predict the efficacy of drugs if crucial steps in development and validation are fully controlled. Future development of CDXs must consider the detection of biomarkers of resistance and toxicity that are complementary to CDXs predicting therapeutic drug efficacy. Certain CDXs that have already been developed may be of interest for new indications in the field of thoracic oncology.
Assuntos
Biomarcadores Tumorais/genética , Testes Diagnósticos de Rotina , Neoplasias Pulmonares/diagnóstico , Neoplasias Torácicas/diagnóstico , Antígeno B7-H1/isolamento & purificação , Antígeno B7-H1/uso terapêutico , Tomada de Decisão Clínica , Humanos , Imuno-Histoquímica/tendências , Imunoterapia/tendências , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Neoplasias Torácicas/imunologia , Neoplasias Torácicas/patologia , Neoplasias Torácicas/terapiaRESUMO
OBJECTIVE: Interest in immune therapies has exploded since the 2014 approval of first-generation programmed cell death 1 blocking antibodies for use in advanced melanoma. Clinical trials have focused primarily on histological material as the gold standard for evaluating programmed death ligand 1 (PD-L1) by immunoperoxidase (IPOX) studies. Studies validating the use of cytological specimens in the assessment of PD-L1 by IPOX staining are needed to optimise tissue utilisation in complementary diagnostic testing. METHODS: Twenty-three melanoma surgical biopsies (SBx) with an IPOX stain for PD-L1 clone 28-8, and a corresponding cytological specimen from the same patient, adequate for PD-L1 evaluation, were selected. Cell-transfer cell blocks (CBs) and conventional CBs were used to perform PD-L1 testing. Tumour proportion scores (TPS) were generated and the results were correlated with the corresponding SBx. RESULTS: Overall agreement (OA) using a ≥1% TPS cut-off for SBx compared to CB was 88.9%, positive percent agreement (PPA) was 87.5%, and negative percent agreement (NPA) was 100%, OA using a ≥5% TPS cut-off was 55.6%, PPA was 42.9%, and NPA was 100%. SBx compared to cell-transfer CB using a ≥1% TPS cut-off had an OA of 65.2%, a PPA of 55.6%, and a NPA of 100%, while a ≥5% TPS cut-off generated an OA of 52.2%, a PPA of 35.7%, and a NPA of 77.8%. CONCLUSION: Our results demonstrate that cytological material, particularly conventional CB, is a viable alternative for evaluating PD-L1 in melanoma cases and suggest that a lower threshold (≥1%) may be beneficial when evaluating cytological material.
Assuntos
Antígeno B7-H1/genética , Biomarcadores Tumorais/genética , Citodiagnóstico , Melanoma/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígeno B7-H1/isolamento & purificação , Biópsia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Imunoterapia/tendências , Masculino , Melanoma/genética , Melanoma/patologia , Pessoa de Meia-IdadeRESUMO
INTRODUCTION: PD-L1 detection with immunohistochemistry (IHC) is the only predictive biomarker available to date for PD-L1/PD1 immunotherapy in thoracic oncology. While many studies have been published on this biomarker, they raise a number of questions concerning mainly, (i) the type of antibody for use and its condition of utilization, (ii) the threshold to be used, (iii) the message and information to communicate to the thoracic oncologist and, (iv) the adoption of this methodology as part of the daily practices of a pathology laboratory. Areas covered: This review provides an update on the use of the different PD-L1 antibodies for IHC in the context of metastatic non-small cell lung cancer (NSCLC) and discusses their use as companion or complementary diagnostic tests. The limits of PD-L1 IHC as a predictive test, the precautions to be adopted as well as some perspectives will then be considered. Expert commentary: IHC for PD-L1 can be considered as a theranostic test, which implies providing an extremely reliable result that avoids any false positive and negative results. PD-L1 IHC requires considerable expertise and specific training of pathologists. PD-L1 IHC can be a companion or complementary diagnostic test depending on the clone employed, the molecular therapy prescribed and the indication of use.
Assuntos
Antígeno B7-H1/isolamento & purificação , Biomarcadores Tumorais/isolamento & purificação , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Imuno-Histoquímica , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Humanos , Imunoterapia/métodos , Metástase Neoplásica , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologiaRESUMO
Programmed cell death 1 ligand 1 (PD-L1) is a trans-membrane protein highly expressed on the membrane of cancer cell, which binds inhibitory receptor of PD-1 on the T cells and attenuates anti-tumor immune response.The strategy of blocking PD1 and PD-L1 interaction has been widely used for anti-cancer drug development. The DNA encoding extracellular domain of PD-L1 was cloned and expressed with the pET30(+) and Escherichia coli BL21(DE3) system. Cloning of PD-L1 extracellular domain was confirmed by PCR and enzymatic digestion. Sequence analysis of cloned targeted genes showed 100% homology of original sequence. The recombinant protein was expressed using 1mM/mL IPTG and purified by affinity chromatography on a column of Ni-NTA and confirmed by SDS-PAGE and western blot analysis. Results showed that our constructed pET30(+)/PDL1-ECD system efficiently produces desired recombinant protein with molecular weight of 38.1kDa. The prokaryotic expression system provides an easy method to express PD-L1 extracellular domain that further facilitate the role of PD-1/PD-L1 binding inhibition and helps in valuable drug and antibodies production.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígeno B7-H1/genética , Escherichia coli/genética , Imunoterapia/métodos , Neoplasias/terapia , Domínios Proteicos/genética , Antígeno B7-H1/isolamento & purificação , Western Blotting , Clonagem Molecular , Expressão Gênica , Vetores Genéticos , Humanos , Neoplasias/imunologia , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Análise de SequênciaRESUMO
AIM: Nivolumab, a fully human immunoglobulin G4 programmed death-1 (PD-1) immune checkpoint inhibitor antibody, has activity in melanoma, non-small-cell lung cancer (NSCLC), renal cell carcinoma (RCC), and Hodgkin lymphoma. Nivolumab is approved in the USA and EU for advanced melanoma, NSCLC, and RCC, and relapsed Hodgkin lymphoma in the USA. Programmed death-ligand 1 (PD-L1), a PD-1 ligand, is expressed on mononuclear leukocytes, myeloid cells, and tumor cells. PD-L1 is being investigated as a potential biomarker to predict the association of tumor PD-L1 expression with nivolumab efficacy. METHODS: Bristol-Myers Squibb and Dako previously reported on an automated PD-L1 immunohistochemical (IHC) assay that detects cell surface PD-L1 in formalin-fixed, paraffin-embedded, human tumor tissue specimens using Dako's Autostainer Link 48. The primary antibody for this assay is a rabbit monoclonal antihuman PD-L1 antibody, clone 28-8. Another rabbit monoclonal antihuman PD-L1 antibody, clone E1L3N, was compared with 28-8 for specificity and sensitivity using an identical detection method followed by vendor-recommended detection methods. RESULTS: Using PD-L1 null clones of L2987 and ES-2 tumor cell lines, both antibodies were specific for detection of PD-L1 on the plasma membrane, although E1L3N also stained cytoplasm in ES-2 knockout cells. Using the identical method, E1L3N was slightly more sensitive than 28-8 based on staining intensities. Using manufacturer-recommended detection methods and predefined scoring criteria for plasma membrane staining of tumor and immune cells, 28-8 demonstrated significantly improved detection compared with E1L3N. CONCLUSIONS: Epitope retrieval and highly sensitive detection reagents are key determinants in IHC detection of PD-L1.
Assuntos
Antígeno B7-H1/isolamento & purificação , Imuno-Histoquímica , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Especificidade de Anticorpos , Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Epitopos/química , Marcadores Genéticos , Humanos , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Nivolumabe , Sensibilidade e EspecificidadeRESUMO
Programmed death ligand 1 (PD-L1), is an important regulator of T-cell activation and has emerged as an important target for cancer immunotherapy. Single chain variable fragments (scFvs) have several desirable characteristics and are an attractive alternative to monoclonal antibodies for experimental or therapeutic purposes. Three chickens were immunized against murine PD-L1, and mRNA isolated from their spleens was used to generate an immunized immunoglobulin variable region library. Using splice-overlap extension PCR, variable region cDNAs were combined to generate full-length scFvs. M13 phage display of the resulting scFv library identified a functional scFv against PD-L1 (αPD-L1 scFv). The scFv was expressed as soluble protein in the periplasm and culture supernatant of recombinant Escherichia coli and purified with a 6×-His tag using immobile metal affinity chromatography. The dissociation constant of αPD-L1 scFv was determined to be 7.11×10(-10)M, and the scFv demonstrated inhibitory biological activity comparable to an antagonistic monoclonal antibody, providing an alternative agent for blocking PD-1/PD-L1 signaling.
Assuntos
Anticorpos Monoclonais/imunologia , Antígeno B7-H1/imunologia , Antígeno B7-H1/isolamento & purificação , Anticorpos de Cadeia Única/imunologia , Animais , Anticorpos Monoclonais/genética , Antígeno B7-H1/genética , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/imunologia , Humanos , Camundongos , Biblioteca de Peptídeos , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/isolamento & purificação , SolubilidadeRESUMO
AIMS AND BACKGROUND: The programmed death-1-ligand 1 (PD-L1) has been recently suggested to play a pivotal role in the immune evasion of tumors from host immune system. In the study, we tried to reveal the clinical significance of PD-L1 in patients with non-small cell lung cancer (NSCLC), which is one of the most aggressive and intractable malignant tumors. METHODS AND STUDY DESIGN: PD-L1 expression in 120 NSCLC tissue specimens and 10 benign control samples embedded with wax were retrospectively detected by immunohistochemistry. RESULTS: No PD-L1 was detected in the 10 benign controls, whereas 57.5% of NSCLC tissue specimens showed PD-L1 expression. There was no relationship between PD-L1 expression and patient age, gender or histopathological type. However, PD-L1 expression was significantly correlated to the degree of tumor cell differentiation, stage of tumor node metastasis (TNM) and patient survival. Poor tumor cell differentiation and advanced TNM stage were related to higher PD-L1 expression. PD-L1-negative NSCLC patients had longer overall 5-year survival than PD-L1-positive patients ( P <0.0001). PD-L1 status was a significant independent prognostic factor of NSCLC (χ2 = 18.153, RR = 2.946, P <0.001). CONCLUSIONS: Up-regulated PD-L1 expression in NSCLC is related to the degree of tumor cell differentiation and TNM stage. PD-L1 status may be a new predictor of prognosis for patients with NSCLC.