Fabrication and anti-cancer activity evaluation of a DNP-conjugated PD-L1 nanobody

• 1、Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122

Abstract：Objective：To sudy the site-specific hapten DNP conjugation of an PD-L1 nanobody (KN035) and evaluate the anticaner activities of the conjugates by recruiting endogenous antibodies. Methods: DNP haptens containing linkers of differentPEG repeatswere site-specifically conjugated to the C-terminus of KN035 using a sortase-mediated ligation reaction and the resultant conjugates were characterized by SDS-PAGE and Western blot; The affinity and antibody-recruiting capability of these conjugates were determined by flow cytometryand ELISA assay;The in vitro anticaner activity of the conjugates were evaluated by ADCC and CDC assay.Results: Three conjugates containing 1, 3, 6 PEGlinker (KNPn, n=1, 3, 6) were successfully prepared; All the KNP conjugateswere able to specifically target to tumor cells with high PD-L1 expression and KNP3 showed the best capability in recruiting anti-DNP antibodies; In the presence of anti-DNP antibodies, all the KNP conjugates were capable of killing tumor cells by mediating ADCC and CDC antivities, and KNP3 showed the highest tumor killing ratio of ca. 30%. Conclusion: Site-specific DNP conjugation of PD-L1 nanobody KN035 can indirectly reconstruct its Fc-related immune functions by recruiting anti-DNP antibodies and the conjugates displayed target-specific antitumor activity against PD-L1 expressing tumor cells.

Keywords： Immuno-therapy；PD-L1；Nanobody；DNP