[Preparation and characterization of three novel monoclonal antibodies against human PD-L1].

AIM: To prepare a functional mouse anti-human PD-L1 monoclonal antibody and to characterize its biological activities. METHODS: A stable human PD-L1 transfected cell line L929/PD-L1 was used as an antigen to immunize BALB/c mice. By means of the cell fusion technique, multiple cell subcloning, repeated screening with L929/ PD-L1 as target cells and the L929/mock cells used as the negative control, the hybridomas specifically secreting mouse anti-PD-L1 monoclonal antibodies were generated. Then its biological characterization was investigated by rapid murine Ig-subclass typing, Western blotting, indirect immune of luorescene assay, mutual competitive inhibition test. By means of MTT incorporation assay, detected the infection of mAb to T cell proliferation. Three mouse anti-human PD-L1 monoclonal antibodies were generated, named as 11G8, 2G5 and 5C3. RESULTS: The results of characterization study showed that the monoclonal antibodies could recognize the PD-L1 on the activated T cells. The mAbs could promote T cells proliferation. CONCLUSION: It is evident that the functional monoclonal antibodies for human PD-L1 have been generated, and it would provide the initial material for further study on the role of PD-1/PD-L1 signaling pathways.

[Expression of human PD-1Deltaex3 and primary study of its biological activity].

AIM: To express human PD-1Deltaex3(DeltaPD-1) gene in eukaryotic expressing vector and identify the biological activity of the recombinant protein. METHODS: The target gene encoding full length human PD-1(PD-1) was cloned by RT-PCR, then two fragments of PD-1Deltaex3 gene were amplified and assembled by TP-PCR, PD-1Deltaex3 gene was obtained. Then the two genes PD-1 and DeltaPD-1 were inserted into the eukaryotic expressing vector pIRES2-EGFP respectively to construct the recombinant vectors pIRES2-EGFP/PD-1 and pIRES2-EGFP/DeltaPD-1. The recombinants were transfected into 293T cells with Lipofect2000 Reagent. The membrane PD-1 protein on the transfected cell surface was detected by flow cytometry. The expression of soluble PD-1 was also analysised by Western blot. The combination of DeltaPD-1 protein to the ligands of PD-1, PD-L1 and PD-L2, were determined by indirect immunofluorescence assay. RESULTS: The results of enzyme digestion of recombinant vectors and DNA sequencing showed the two genes PD-1 and PD-1Deltaex3 were inserted correctly into plasmid pIRES2-EGFP, the two recombinant vectors were constructed successfully. Flow cytometry and Western blot revealed that 293T cells transfected with vector pIRES2-EGFP/PD-1 could express PD-1 protein on the cell surface but no soluble PD-1 in the supernatant of transfected cells, on the contrary, 293T cells transfected with vector pIRES2-EGFP/DeltaPD-1 could express soluble PD-1 in the culture supernatant but no membrane PD-1. Indirect immunofluorescence assay indicated the DeltaPD-1 protein could bind to the two ligands of PD-1 on the cells surface. CONCLUSION: The recombinant eukaryotic expressing vector containing PD-1Deltaex3 was constructed successfully, and the PD-1Deltaex3 gene could encode a soluble form of PD-1 protein. DeltaPD-1 protein remained the biological activity as PD-1. This study provides the initial material for further study of PD-1Deltaex3 in PD-1/PD-L signaling pathway.