The Research Of CD40ScFv In Regulation Of PD-1 Expression In Tumor Environment

Objective: To investigate the CD40 single-chain antibody(CD40ScFv)on up-regulating the expression of PD-1 and anti-tumor effect in tumor micro-environment.Finally,it provided experimental basis and theoretical support for tumor treatment with CD40 ScFv combined with anti-PD-1antibody.Methods: We used PCR amplification to get gene CD40 scFv from pCANTAB5E-CD40 ScFv phagemid.Then directed cloning was used to construct pET28a-CD40 ScFv plasmids.The recombinant plasmids were transformed into Escherichia coli Rosetta.The CD40 ScFv proteins were purified by Protein affinity chromatography.Then the purified proteins were measured by BCA protein content,and were identified by SDS-PAGE and Western blot.Balb/C mice models of right auxiliary 4T1 tumor were constructed.According to different drugs treatment,the mice were divided into 3 groups: CD40 ScFv group,CD40 mAb group and NS group.Tumor volume of mice were observed.The mice were sacrificed and the tumor tissues were separated after treatment.Then the concentration of INF-? in tumor tissue was measured by ELISA.PD-1 expression of tumor-infiltrating lymphocytes in tumor microenvironment was stained by Immunohistochemistry.Results:pET28a-CD40 ScFv recombinant plasmid was Constructed successfully.The molecular size of CD40 ScFv protein is about 27 KDa and the volume was about 1.12mg/ml.The concentration of INF-?in the supernatant of tumor tissue in group CD40 ScFv was(423.25±50.23)pg/mL,significantly higher than that of group NS(P<0.05),but there was no significant difference between group CD40 mAb agonist and CD40 ScFv group(P>0.05).PD-1 expression of tumor-infiltrating lymphocytes in group CD40 ScFv was 8.18%±2.01%,8.84%±2.04% in group agonist CD40 mAb and 3.15%±1.66% in group NS.Group CD40 ScFv was significantly higher than the group NS(P<0.05),but there was no significant difference between group CD40 mAb and CD40 ScFv group(P>0.05).Conclusion:pET28a-CD40 ScFv recombinant plasmid was successfully constructed and CD40 ScFv protein could be stably expressed by IPTG.CD40 ScFv can stimulate T lymphocytes activation in vivo to inhibit tumor growth.It also can up-regulate the PD-1 expression of infiltrating T lymphocytes in tumor micro-environment.