Purification of monoclonal anti-B7-H4 antibody and the blockade of B7-H4-mediated tumor immune evasion by the antibody

The purified 3C8 was obtained by two step column purification, including Protein G affinity purification and DEAE anion exchange purification. The purity of purified 3C8 was about 93% when analyzed by reverse column. SDS-PAGE showed that the purity of 3C8 was increased greatly by two step purification. By flowcytometry we found that 3C8 specifically binded with B7-H4/293T cells and did not bind with Mock/293T cells, moreover 3C8 did not bind with other B7 family members transgene cells. In confocal experiment 3C8 could specifically stained B7-H4/293T cells. In Western blot only B7-H4/293T cells showed positive band while Mock/293T cells showed negative result. The result of immunohistochemistry showed that B7-H4 was highly expressed in prostate cancer and renal cell carcinoma, while para-cancer tissues did not express B7-H4. The T cell proliferation experiment showed that B7-H4-Ig could bind to activate T cells and inhibit T cell proliferation, while 3C8 could block the binding of B7-H4-Ig and reverse the T cell proliferation inhibition effect of B7-H4-Ig by CFSE and CCK8 assay. The cytokine IFN-γ and IL-2 secreted by activating T cells was decreased by B7-H4-Ig and 3C8 could reverse the effect of B7-H4-Ig.