Construction and expression of chimeric antigen receptors targeting epidermal growth factor receptor (EGFR) and programmed cell death ligand-1(PD-L1) / 中华微生物学和免疫学杂志

Objective:To construct an expression system of lentivirus vector encoding epidermal growth factor receptor-specific chimeric antigen receptor (EGFR-CAR) and programmed cell death ligand-1 (PD-L1) antibody.Methods:Human PD-L1-Fc protein was used to immunize BALB/c mice. Cell-fusion and subcloning were performed to screen stable hybridoma strains with high secretion of PD-L1-specific antibodies, which were identified by both ELISA and Western blot. The activity of the antibodies in blocking the binding of programmed cell death-1 (PD-1) to PD-L1 was determined by fluorescence-activated cell sorting (FACS). Antibody affinity was analyzed by Fortebio Octet96. A single-chain variable fragment (scFv) was further constructed after antibody full-length sequencing and humanization using CDR grafting method. Meanwhile, the genes encoding the light and heavy chain variable regions (VL and VH) were cloned from a hybridoma secreting antibody against human EGFR by 5′ RACE technology to construct scFv gene. The expression of scFv was confirmed using pcDNA3.1 vector. EGFR-CAR containing CD137 intracellular function domain and PD-L1-scFv was ligated using 2A gene. The synthetic single molecule was cloned into pLVX-EF1a-IRES-ZsGreen1 lentivirus expression vector, and then transfected into 293T cells using Lenti-X Packaging Single Shots (VSV-G) to prepare infectious virus. Expression of CAR on cell surface and the soluble form of PD-L1-scFv in the supernatant of transfected 293V cells were detected by FACS and ELISA.Results:A PD-L1 antibody named 11E3 with high ligand-receptor blocking performance was obtained. The humanized antibody showed a stable affinity (2.67×10 -10 mol/L) after directly grafting the mouse CDRs (CDR1, CDR2 and CDR3) to human frameworks. EGFR-scFv was effectively expressed in a form of Fc-fusion. Secretory CAR (CTZ0431-1) and membrane CAR (CTZ0431-2) expression plasmids were constructed using lentivirus vector containing EGFR-CAR and PD-L1-scFv. The infection efficiency in 293V cells was around 10%. EGFR-scFv on the cell membranes and PD-L1-scfv in the culture supernatants were detected after 293V cells were infected with CTZ0431-1. EGFR-scFv and PD-L1-scfv were expressed on the cell membranes of 293V cells infected with CTZ0431-2. The expression rate of CAR in LV-CART46407-1-transfected activated T cells was 39.3%. Conclusions:The lentivirus vectors co-expressing EGFR-CAR with moderate binding affinity and PD-L1-scFv with high binding affinity were successful constructed, which provided an essential tool for investing EGFR- and PD-L1 double targeted CAR-T cell therapy against solid tumor.

Construction of lung tumor cells using RFP and Fluc dual-labeled system / 国际生物医学工程杂志

Objective To establish a human lung cancer cell line that can stably express firefly luciferase (Fluc) and red fluorescent protein (RFP) gene so as to lay the foundation for the further establishment of a live-imaging lung cancer xenograft model in nude mice and therapeutic research.Methods The lentiviral vector pHBLV-FlucRFP containing luciferase and red fluorescent protein was constructed and then transfected into 293T cells for virus packaging.The complete virus was used to infect human lung cancer cell lines A549,H1975 and human B-cell lymphoma cell line K562.The stable cell lines were obtained by puromycin selection.Fluorescence microscopy and quantitative PCR were used to confirm the RFP and Fluc expression.Results The lentiviral vector pHBLV-FlucRFP was successfully constructed.Cancer cell line A549,H1975 and K562 stably expressing Fluc and RFP was obtained.The real-time quantitative PCR results showed that the relative expression of Fluc gene in the three stable infected cells was much higher than that in the corresponding wild-type cells,and the differences were statistically significant(all P＜0.05).Conclusion The human lung cancer cell line A549,H1975 and human B-cell lymphoma cell line K562 with dual expression of RFP and Fluc were obtained,which provided a new model of fluorescent cells for in vivo imaging of immunodeficient mouse models such as nude mice.

Regulatory T cells are increased in tuberculosis patients and closely correlate with its immunopathology / 中华微生物学和免疫学杂志

Objective To determine whether regulatory T cells(Tr)are increased in patients with tuberculosis and whether they are associated with its immunopathology.Meantime,to investigate the possibility of tuberculosis(TB)as a model for studying Tr functions.Methods The lymphocyte subsets were isolated from peripheral blood mononuclear cells by sorting with flow cytometry.Total cellular RNA was extracted and RT-PCR was performed to detect the Foxp3 mRNA in purified CD3+CIM+T cells,CD3+CD8+T cells and non-CD3+CD4+CD8+T cells.Using FACS analysis.we further investigated the distribution of Foxp3+ population in CD4+ CD25+T cells.Finally,we compared the percentage of CD4+CD25highFoxp3+T cells present in 51 active patients with tuberculosis and 40 uninfected healthy control subjects by FACS.The detection of Tr infiltration of Foxp3+ cells were performed with immunohistochemistry(IHC)method on tuberculosis pathological sections.Results Foxp3 was specific expressed in CD3+CD4+T cells,either in tuberculosis patients or healthy control subjects.Foxp3+ T cells took about 85%fraction of CD4+ CD25highpopulation.We used CD4+CD25high Foxp3+as a detective markers for Tr in the FACS analysis.The results showed that patients with active TB had a 4.4 fold higher percentage within the CD4+T cells in peripheral blood compared to healthy control group(modian,1.01%vs 0.23%,P<0.01).Much higher frequency of Tr were found along with T cells infiltration at the tuberculosis pathological tissues.A few individuals that we can followed indicated the expanded Tr was declined after curative treatment with operation.Conclusion Tr cells are increased in tuberculosis patients and closely correlate with its immunopathology.Tuberculosis should be a valuable model for Tr functional study.

Location of extracellular cysteine-rich domains of 4-1BB binding to murine 4-1BB ligand and analysis of its possible structure / 中华微生物学和免疫学杂志

ObJective To locate the cysteine-rich domains(CRD) of murine 4-1BB binding to its natural ligand. Methods A serial soluble extracellular CRDs of routine 4-1BB and 4-1BBL fusion proteins was constructed and prepared. The binding of purified 4-1BB-Igs to 4-1BBL and 4-1BB monoclonal antibody were tested using ELISA assay and Western blot analysis. Blocking experiment with 4-1BBL and 4-1BB mon-oclonal antibody was performed by ELISA assay. Results All truncated overlapped proteins containing ex-tracellular CRD Ⅱ of murine 4-1BB were able to bind to 4-1BBL by ELISA assay, excepting the CRD Ⅰ do-main alone. A 4-1BB monoclonal antibody proved to block the interaction of 4-1BB and 4-1BBI, was also able to bind to CRD Ⅱ. Conclusion Murine 4-1BBL whose specificity was mapped to CRD Ⅱ of 4-1BB ex-tracellular region with a possible conformational structure.

Detection of metabolites of tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in lung cancer smokers' urine / 中国肺癌杂志

<p><b>BACKGROUND</b>It was reported that tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was a powerful pulmonary carcinogen, predominantly inducing adenocarcinoma of the lung in mouse. The aim of this study is to assay metabolites of NNK, which are 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its O-glucuronide (NNAL-Gluc), and their ratio (NNAL-Gluc/NNAL) in smokers and non-smokers' urine, and to explore the carcinogenicity of NNK among different people.</p><p><b>METHODS</b>Using high pressure liquid chromatograph (HPLC) and gas chromatograph-mass tadom (GC-MS/MS), NNAL-Gluc and NNAL in 24h urine were detected in 8 healthy smokers, 10 lung cancer smokers and 4 healthy non-smokers.</p><p><b>RESULTS</b>Both of the two metabolites were not found in non-smokers' urine. The ratios of urine NNAL-Gluc/NNAL were greatly different among different smokers. The mean ratio of NNAL-Gluc/NNAL in healthy smokers was 4.95, and 0.5 in lung cancer smokers.</p><p><b>CONCLUSIONS</b>The results provide the first evidence for metabolite detection of tobacco-specific nitrosamine in Chinese smokers' urine . The result suggests that detoxification ability of healthy smokers is higher than that of lung cancer smokers. It may provide a detective way to screen high risk people for lung cancer in smokers.</p>

Study on Quality Standards for Pugan Tablets / 中成药

Objective: To establish the quality standards for Pugan Tablets. Methods: Two main components, acetaminophen and chlorpheniramine maleate in Pugan Tablets were identified by physico chemical methods and TLC, and determined by ist derivative spectrography directly and TLC scanning, respectively. Results: The average recoveries were 98.23% (RSD was 1.38%, n was 9) for acetamnophen and 98.83% (RSD was 1.78%, n was 5) for chlorpheniramine maleate. Conclusion: These methods are simple and feasible with good results, and can be used for the quality control of the tablet.