NAase-Modified Whole-Cell Tumor Vaccines Elicit Host Anti-Tumor Immunity In Ovarian Cancer

Ovarian cancer（OC）is an immunogenic tumor,but with a low response rate to immune checkpoint inhibitors（ICIs）.Currently,ICIs are the primary immunotherapeutic strategies that block inhibitory signals of immune cells.However,tumor cells evade immune attacks not only through inhibitory immunity but also by concealing tumor-specific antigens,which may be the most significant barrier to the success of current immunological therapies.Thus,it is imperative to develop innovative immunological therapies to fix this problem.The aberrant sialoglycans on OC cells causing tumor antigen masking and immunosuppression,play an important role in the progression,metastasis,and relapse of OC.Sialoglycans,as the ligands of sialic acid-binding immunoglobulin-type lectins（Siglecs）,involve in all dendritic cells（DCs）functions,including antigen uptake,DC migration,and capacity to prime T cell responses.The terminal sialic acids of sialoglycans usually attach to galactose and N-acetylgalactosamine（Gal/GalNAc）by α-2,3/6-linkage,and Gal/GalNAc orchestrating immune responses distinct from sialic acids.The interaction between Gal/GalNAc and macrophage galactose-type lectin（MGL）triggers dual functions of MGL as a recognition molecule and as an endocytic receptor,and primes cellular immune responses to antigens specifically endocytosed by MGL.The therapeutic mechanism of tumor vaccine is to educate the immune system with the ability to recognize and eliminate tumor cells expressing specific tumor antigens,which makes tumor cells carrying all tumor antigens a suitable source of tumor vaccines.Based on these,desialylation is expected to abolish the masking effect of sialoglycans,reduce the sialic acid-mediated immunosuppression,and improve the recognition and presentation of newly exposed antigens by MGL on DCs,thus triggering specific immune responses against OC cells.Thus,desialylated whole-cell tumor vaccines are promising personal immunotherapy to improve the outcomes of OC patients.In this dissertation,membrane glycans on OC cells were modified with neuraminidase（sialidase,NAase）from Clostridium perfringens（α2-3NA）and Arthrobacter ureafaciens（α2-6NA）to cleaveα-2,3 andα-2,6 linked sialic acid residues（α-2,3Sia and α-2,6Sia）respectively,to generate NAase-modified whole-cell tumor vaccines.α-2,3Sia,α-2,6Sia,and Gal/GalNAc epitopes were determined by flow cytometry（FCM）to explore the optimal condition for generating NAase-modified OC cell vaccines.DCs were pulsed with these OC cell vaccines,and the activated matured DCs stimulated lymphocytes.The phenotypes of DCs and lymphocytes were detected by FCM;IFNγexpression and cytotoxicity were determined by RT-qPCR,and LDH assay,respectively;TRBV genes that code the TCR Vβchain were identified using Sanger sequencing,and the sequencing results were analyzed using IMGT/V-QUEST.Allogeneic immunocompetent mice were immunized with NAase-modified mouse OC cell vaccines,and the tumor formation time of parental live OC cells,tumor volume,and overall survival were observed.The IFNγand IL2 plasma levels of mice were detected using ELISA,the tumor-infiltrating lymphocytes（TILs）were measured by immunohistochemistry,and the phenotypes of DCs and lymphocytes in tumor-draining lymph nodes were detected by FCM.The results showed thatα2-3NA andα2-6NA treatment of OC cell vaccines reduced theα-2,3Sia andα-2,6Sia,respectively,and both increased the exposure of Gal/GalNAc residues,leading to the distinct glycan features.α2-3NA treated OC cell vaccines significantly reduced the Siglec-9 and MGL on DCs.Siglec-9^dim/MGL^bright DCs diversified TCR Vβrepertoires of T cells and enhanced the cytotoxicity of T cells against parental OC cells.All mouse OC cell vaccines elevated the IFNγand IL2 plasma levels.And theα2-3NA treated mouse OC cell vaccines kept a low level of Siglec E and increased the MGL expression level of DCs in tumor-draining lymph nodes;increased CD8⁺TILs and decreased CD8⁺CD39⁺TILs;significantly delayed the tumor formation time of parental tumor cells,slowed tumor growth and prolonged overall survival of tumor-bearing mice.In summary,whole-cell tumor vaccines were generated by modifying OC cells with selective NAases in this dissertation.These vaccines remove inhibitory sialic acids from OC cell membrane glycans and expose immune-activating antigens with Gal/GalNAc epitopes,thereby enhancing DC recognition and presentation of tumor antigens and eliciting the specific anti-tumor immune responses.Moreover,in vivo animal studies confirmed that TILs induced by α2-3NA-modified whole-cell OC vaccines were able to overcome the immunosuppressive niche to eliminate parental tumor cells.Therefore,α2-3NA-modified desialylated whole-cell tumor vaccines reshape anti-tumor immunity and are potential anti-OC immunotherapy strategies.