Preparation Of Shark Anti-PD-1 Single Domain Antibody And Preliminary Study On Anti-tumor Activity

Background:The striped bamboo shark is a small carnivorous shark found off the coast of eastern Fujian,China.It has the ability to produce Immunoglobulin New Antigen Receptor（IgNAR）.IgNAR is a heavy chain antibody first discovered in nurse sharks.Its V domain,VNAR,is the smallest antigen binding region known at present,and it has attracted much attention due to its small molecular weight,high binding ability,strong tissue penetration and strong stability.PD-1/PD-L1 is an important signaling pathway in the process of tumor immune escape,and blocking the PD-1/PD-L1 signaling pathway through antibodies can reactivate T lymphocytes and play the function of tumor specific clearance,which has a high commercial value.At present,PD-1/PD-L1 antibody blocking therapy is a research hot spots in clinical tumor therapy.However,the relevant studies on shark derived anti-PD-1 single domain antibody have not been reported.Objective:The VNAR amino acid sequence of striped bamboo shark anti-PD-1protein was obtained by immunizing sharks using transcriptome data.In vitro antigen-specific binding activity analysis and in vivo functional experiments proved that anti-PD-1 VNAR had immunosuppressive activity of binding and silencing target antigens,which provided a new strategy for the clinical development of single domain antibody drugs to block the PD-1/PD-L1 signaling pathway in the treatment of tumors.Methods:The recombinant PD-1 protein was prepared by constructing E.coli Rosetta（DE3）-pETduet-his-SUMO-mPD-1 expression strain.The purified PD-1protein was emulsified with CFA for preparing an antigen emulsion,which was immunized subcutaneously with the dose of 1μg per fish（about 600g）.After 60 days,shark blood was collected for transcriptome sequencing,and the transcriptome data of unimmunized shark blood was used as the control group.After transcriptome data analysis,the candidate sequence of VNAR was screened,and the synthetic gene was linked to the expression vector pET22b to construct the anti-PD-1 VNAR expression strain E.coli Rosetta（DE3）-pET22b-VNAR.The purified recombinant VNAR protein was assayed by enzyme-linked adsorption（ELISA）to verify its biological activity in vitro.A tumor transplantation mouse model was established and we set up 4 groups including:positive control group（rabbit anti-mPD-1 administration group）,full-dose experimental group（VNAR full-dose administration group）,half-dose experimental group（VNAR half-dose administration group）,negative control group（unrelated IgG administration group）.The targeted binding ability and tumor inhibition effects of recombinant VNAR in tumor transplantation mouse model were demonstrated by q PCR,flow cytometry and in vivo imaging.Results:Anti-PD-1 VNAR was obtained by immunizing striped bamboo shark with recombinant PD-1 protein.In vitro binding activity assayed,the recombinant VNAR and PD-1 showed significant biological activity by Elisa.In vivo experiments binding activity assayed,the recombinant VNAR could increase the proportion of CD₈⁺T cells in tumor-infiltrating lymphocytes,regulate the expression of inflammatory factors promoting anti-tumor micro-environment（IFN-γ,IL-2 and IL-10）formation in tumor micro-environment,and reduce tumor growth rate and proliferation.This study provides a new thoughts of drug development for the clinical treatment of tumor escape by antibody blocking.