Preparation of CD73/PD-L1 bispecific antibody and evaluation of its antitumor function in vitro / 中国生物制品学杂志

@#Objective To prepare bispecific antibody targeting cluster of differentiation 73(CD73) and programmed cell death-ligand 1(PD-L1),and evaluate its binding ability and killing ability in vitro.Methods Using genetic engineering method,PD-L1 single-chain fragment variable(scFv) was inserted into the hinge region of CD73 monoclonal antibody to construct anti-CD73/PD-L1 bispecific antibody(BS-21),which was screened by CHO GS expression system to obtain highly expressed cell line.After purified by Protein A and molecular sieve,the purity of antibody was detected by size exclusion chromatography-high performance liquid chromatography(SEC-HPLC),the binding ability of antibody in vitro was detected by flow cytometry,and the killing ability in vitro was detected by using peripheral blood mononuclear cell(PBMC) to kill Calu 1 lung cancer cells in vitro.Results High-yield cell lines were obtained by pressure screening.A bispecific antibody BS-21 with a purity of 99.6% was obtained by purification,which bound to CD73 and PD-L1 molecules simultaneously.Compared with anti CD73 and anti PD-L1 groups,BS-21 group significantly increased the killing rate of immune cells to Calu 1 tumor cells(F=30.36,each P<0.001).Conclusion Bispecific antibody BS-21 reduced the immunosuppressive effect of CD73 and PD-Ll on immune cells simultaneously,and showed good anti-tumor function.

Characterization and protective efficacy in an animal model of a novel truncated rotavirus VP8 subunit parenteral vaccine candidate.

The cell-attachment protein VP8* of rotavirus is a potential candidate parenteral vaccine. However, the yield of full-length VP8 protein (VP8*, residues 1-231) expressed in Escherichia coli was low, and a truncated VP8 protein (ΔVP8*, residues 65-231) cannot elicit efficient protective immunity in a mouse model. In this study, tow novel truncated VP8 proteins, VP8-1 (residues 26-231) and VP8-2 (residues 51-231), were expressed in E. coli and evaluated for immunogenicity and protective efficacy, compared with VP8* and ΔVP8*. As well as ΔVP8*, the protein VP8-1 and VP8-2 were successfully expressed in high yield and purified in homogeneous dimeric forms, while the protein VP8* was expressed with lower yield and prone to aggregation and degradation in solution. Although the immunogenicity of the protein VP8*, VP8-1, VP8-2 and ΔVP8* was comparable, immunization of VP8* and VP8-1 elicited significantly higher neutralizing antibody titers than that of VP8-2 and ΔVP8* in mice. Furthermore, when assessed using a mouse maternal antibody model, the efficacy of VP8-1 to protect against rotavirus-induced diarrhea in pups was comparable to that of VP8*, both were dramatically higher than that of VP8-2 and ΔVP8*. Taken together, the novel truncated protein VP8-1, with increased yield, improved homogeneity and high protective efficacy, is a viable candidate for further development of a parenterally administrated prophylactic vaccine against rotavirus infection.

Improved characteristics and protective efficacy in an animal model of E. coli-derived recombinant double-layered rotavirus virus-like particles.

Live rotavirus vaccines that are effective in middle- and high-income countries have been found to be less immunogenic and effective in infants in resource-limited settings. The virus-like particle (VLP) approach is promising for rotavirus vaccine development, but challenges remain for VLP production at large scale. In this study, rotavirus capsid VP2 and VP6 proteins were expressed in Escherichia coli and were assembled with high efficiency into homogeneous single-layered VP6-VLPs or double-layered VP2/6-VLPs (dl2/6-VLPs) through a post-purification assembly process. The dl2/6-VLPs were observed to have better thermal stability and antigenicity. Although the immunogenicity of VP6 trimers, VP6-VLPs and dl2/6-VLPs was comparable, the efficacy of the dl2/6-VLPs to protect against rotavirus-induced diarrhea in pups was significantly higher than that of the trimeric VP6 or the VP6-VLPs when assessed using a mouse maternal antibody model. Taken together, the recombinant dl2/6-VLP antigen, which is highly analogous to rotavirus virion-derived double-layered particles, is a viable candidate for vaccine development and has the potential to be a parenterally administered safe and efficacious rotavirus vaccine.

Serum miR-483-5p as a potential biomarker to detect hepatocellular carcinoma.

BACKGROUND AND GOALS: There are no highly sensitive and specific minimally invasive biomarkers for hepatocellular carcinoma (HCC) to date. The objective of this study was to identify serum microRNAs (miRNAs) as potential HCC biomarkers. METHODS: Using miRCURY LNA™ microRNA arrays, the levels of circulating miRNAs in the serum of patients with HCC were compared and controls were matched. Then 253 subjects (112 HCC, 85 chronic hepatitis B [CHB], and 56 healthy controls) were recruited and 12 serum miRNAs were compared by quantitative real-time polymerase chain reaction (qRT-PCR). It was followed by the comparison of serum miRNA concentrations before and after the surgical resection in HCC group. RESULTS: Median levels of miR-483-5p and miR-500a were higher in HCC patients than in patients with CHB and in healthy controls (p < 0.0001), but there were no differences between CHB patients and healthy controls (p > 0.05) and miR-483-5p levels were significantly reduced in serum samples obtained 30 days after surgical resection (p < 0.0001). The area under receiver operating characteristic curves of miR-483-5p and miR-500a was 74% (cutoff [Ct] value = 2.824, sensitivity = 74%, and specificity = 66%) and 66% (Ct value = 1.830, sensitivity = 74%, and specificity = 51%) for the prediction of HCC, respectively. In detecting HCC, combining α-fetoprotein (AFP) and serum miR-483-5p (sensitivity = 81% and specificity = 83%) was better than AFP alone (sensitivity = 78%, specificity = 70%). CONCLUSION: Our observations suggest that serum miR-483-5p and miR-500a might serve as novel, noninvasive biomarkers for HCC. Serum miR-483-5p might complement AFP in detecting HCC.