Selection and expression of CD40 single chain variable fragment by phage display and evaluation of tumor specific immune activation

Abstract

Immune escape is the final phase of the cancer immunoediting process. Researchers have found that cancer induces immune escape by inhibiting the expression of CD40L. The purpose of the present study was to select a high affinity CD40 single chain variable fragment (ScFv) and to evaluate its effect on tumor-specific immune activation. One Wistar rat was immunized with mouse CD40 antigen. CD40 ScFv with high affinity was constructed by overlap extension-polymerase chain reaction (SOE-PCR) and screened by three rounds of phage display. CD40 ScFv protein was expressed by the pET28a (+)-Rosetta prokaryotic expression system and purified using a nickel-nitrilotriacetic acid (Ni-NTA) column. CD40 ScFv significantly upregulated CD80, CD86, and MHC-II in vitro expression in dendritic cells (DCs) and upregulated the expression of IL-12 (p70) based on ELISA results. Cell counting kit-8 (CCK-8) results indicated that T lymphocytes were stimulated by DCs in an Ag + CD40 ScFv group, which also inhibited the proliferation of immortalized T6-17 cells. In an in vivo assay, 1 × 10⁶ T6-17 cells were subcutaneously injected into BALB/c mice in the hind flank. Tumor volume curves showed that CD40 ScFv exhibited a remarkable inhibition of tumor proliferation after 15 days of treatment. Hematoxylin-eosin (H&E) staining of tumor tissues indicated that CD40 ScFv enhanced lymphocyte infiltration, which remarkably inhibited the proliferation of T6-17 cells. Furthermore, immunohistochemistry (IHC) staining revealed that caspase-3 was abundantly expressed in the T6-17 cytoplasm after CD40 ScFv treatment. In conclusion, this study revealed that high affinity CD40 ScFv could be screened by phage display and had a significant stimulating effect on DCs and inhibited the proliferation of T6-17 cells in vivo and in vitro.

Keywords: CD40; Phage display; ScFv; Tumor immunity.