The Antitumor Effect Of Oncolytic Adenovirus Expressing MUC16-BiTE Combined With MUC16CAR-T Cells In Ovarian Cancer

BackgroundOvarian cancer(OC)is the deadliest tumor affecting the female reproductive system.Traditional treatments,including surgery,radiotherapy,and chemotherapy,have not significantly improved patient survival rates.Therefore,there is an urgent need for more effective treatments for ovarian cancer.Immunotherapy has shown great promise as a therapeutic approach for various cancers,including ovarian cancer.Previous studies have shown that ovarian cancer is an immunogenic tumor,and the presence of tumor-infiltrating lymphocytes,particularly CD8+T cells,can greatly improve patient survival rates.However,immune evasion mechanisms,such as those mediated by suppressor cells like T regulatory cells,are associated with poorer survival.Therefore,T cell-based immunotherapy may be beneficial for ovarian cancer patients.Oncolytic adenoviruses(OAd)have emerged as a promising treatment for solid tumors due to their direct oncolytic effects that selectively kill tumor cells and their indirect activation of anti-tumor immune responses.However,the high heterogeneity of malignant tumors and the body’s antiviral immune response limit the efficacy of OAd monotherapy.Therefore,a new strategy is needed to reduce the antiviral immune response and activate the antitumor immune response simultaneously.Bispecific T-Cell Engager(BiTE)is a bispecific antibody consisting of two single-chain variable fragments(ScFv)that simultaneously target tumor associated antigens(TAA)on the surface of cancer cells and CD3 on T cells,bridging them to activate T cells without the restriction of the major histocompatibility complex(MHC),which effectively kills tumor cells.BiTEs can also activate and proliferate exhausted tumor-specific T cells.However,the short half-life of BiTE necessitates long-term intravenous infusion,which can cause systemic toxicity and off-target effects.In solid tumors,the complex tumor microenvironment can also limit the penetration of BiTE,reducing its therapeutic effect.Therefore,to increase the efficacy of BiTE and reduce adverse side effects caused by continuous systemic administration,an effective optimization method is to use oncolytic adenovirus as a carrier to express BiTE molecules for sustained expression at the tumor site,stimulating T cells without causing systemic toxicity.Chimeric antigen receptor T-cell(CAR-T)therapy is a novel form of tumor immunotherapy.However,the immunosuppressive microenvironment of tumors and insufficient effective localization and persistence of CAR-T cells in solid tumors limit its efficacy as a monotherapy.Therefore,combining CAR-T therapy with other treatments can enhance its anti-tumor effect.MUC16 is commonly overexpressed in ovarian cancer and comprises a large cleavage and release domain(CA-125)with multiple repeats and a retention domain(MUC-CD).While CA125 is a serum marker for ovarian cancer,MUC-CD is retained on the cell surface and expressed at low levels in normal tissues,making it an attractive target for therapy.In this study,we initiated by loading BiTE against MUC16(MUC16-BiTE)into an oncolytic adenovirus,thereby successfully creating OAd-MUC16-BiTE.We then investigated its effectiveness in eliminating ovarian cancer and delved into its underlying mechanism.Additionally,we designed MUC16CAR-T cells targeting the same antigen and explored the combined application of oncolytic adenovirus and CAR-T cells targeting MUC16 for the treatment of ovarian cancer,both in vitro and in vivo.The specific research areas covered in this study are as follows:1.Successful construction of an oncolytic adenovirus expressing MUC16-BiTE(OAdMUC16-BiTE).2.The antitumor effect and mechanism of OAd-MUC16-BiTE in ovarian cancer.3.The antitumor effect of OAd-MUC16-BiTE combined with MUC16CAR-T cells in ovarian cancer.Part 1:Successful Construction of an Oncolytic Adenovirus Expressing MUC16-BiTE(OAd-MUC16-BiTE)Objectives:1.To construct an oncolytic adenovirus expressing MUC16-BiTE(OAd-MUC16-BiTE),and to determine the successful expression of MUC16-BiTE molecules by OAd-MUC16-BiTE.2.To clarify the effect of the insertion of MUC16-BiTE on the replication ability and oncolytic properties.3.To investigate the effect of MUC16-BiTE expressed and secreted by OAd-MUC16BiTE on T cells.Methods:1.The ScFv sequences targeting CD3ε and MUC16 were linked to produce the bispecific antibody MUC16-BiTE that targets MUC16.The MUC16-BiTE sequence was cloned into the hTERT promoter-driven parental OAd to obtain the MUC16-BiTE-containing OAd(OAdMUC16-BiTE).The expression of MUC16-BiTE was evaluated using Western blot.2.The replication ability and oncolytic properties of OAd-MUC16-BiTE were evaluated using q-PCR and CCK8 methods,respectively.3.The MUC 16-BiTE-mediated activation,proliferation,cytokine release,and cytotoxicity of T cells were evaluated using flow cytometry(FC)and luciferase assays.Results:1.The OAd-MUC16-BiTE was successfully created,and Western blot confirmed the expression of the 6×His tag in HEY cells infected with OAd-MUC16-BiTE.2.Q-PCR results indicated that both OAd-MUC16-BiTE and OAd exhibited a comparable number of viral genomes at the measured time points.CCK8 analysis revealed OAd-MUC16BiTE and OAd had similar lytic activity on OC cells.3.In the presence of MUC 16-positive OC cells,the secretion of MUC16-BiTE from OAdMUC16-BiTE yielded significant results:luciferase assay indicated a notable enhancement in T cell-mediated killing activity;FC revealed T cells in an activated state,characterized by increased rounds of proliferation,higher surface expression of CD 107a(a degranulation marker),and substantial release of cytokines.Conclusions:1.OAd-MUC16-BiTE is successfully constructed and is able to secrete MUC16-BiTE after infection of OC cells.2.The insertion of MUC16-BiTE will not affect the oncolytic properties and replication ability.3.The secretion of MUC16-BiTE by OAd-MUC16-BiTE facilitates the interaction between CD3+T cells and MUC16+tumor cells,leading to T cell activation,proliferation,secretion of cytokines,and specific killing of MUC 16-positive OC cells.Part 2:The Antitumor Effect and Mechanism of OAd-MUC16-BiTE in Ovarian CancerObjectives:To elucidate the antitumor effect and mechanism of OAd-MUC16-BiTE on ovarian cancer.Methods:1.The luciferase assay was used to evaluate the effect of OAd-MUC16-BiTE on T cellmediated cytotoxicity.2.In 5 cases of OC patient ex vivo tumor cultures:first,the expression of MUC16 was analyzed by IHC;second,the expression of T cell surface exhaustion markers was analyzed by FC.The CCK8 method was used to assess the toxicity of OAd-MUC16-BiTE to OC patient ex vivo tumor cultures,while the CBA multifactor method was used to evaluate its impact on the secretion of IL-10 and TGF-β.3.OAd-MUC16-BiTE’s antitumor effects were examined in ovarian cancer CDX and PDX models.4.The tumor-infiltrating T cells were studied by FC and IHC.The anti-tumor mechanism of OAd-MUC16-BiTE was explored using protein iTRAQ quantification and Luminex liquid phase suspension chip.Results:1.In the presence of T cells,OAd-MUC16-BiTE enhanced T cell-mediated anti-tumor effects.In addition,more significant cell death was observed when OAd-MUC16-BiTE-infected HEK293A cells were co-cultured with MUC 16-positive OC cells.2.IHC revealed high levels of MUC16 expression in all patients.FC analysis showed that T cells within the tumors exhibited high expression of PD-1,TIM-3,LAG-3,and TIGIT.Cytotoxicity experiments demonstrated that OAd-MUC16-BiTE exhibited comparable or even enhanced cytotoxicity compared to the parental virus.Additionally,OAds led to a reduction in the secretion of cytokines IL-10 and TGF-β within the cultures.3.In vivo,OAd-MUC16-BiTE demonstrated stronger antitumor activity when T cells were present,both in CDX and PDX models.4.FC analysis revealed that the tumor-infiltrating T cells primarily consisted of CD8+lymphocytes,while the presence of CD4+lymphocytes was limited.IHC findings demonstrated an increased presence of CD8+cells in tumor tissues treated with OAd-MUC16-BiTE,observed in both the PDX model and CDX model.Quantitative analysis using proteomic iTRAQ detected up-regulated expression of proteins associated with T cell migration to tumor tissues and the presentation of MHC class I antigens.Luminex analysis indicated an elevation in chemokines and a decrease in VEGF and Basic.FGF in tumor tissues treated with OAdMUC16-BiTE and infused with T cells.Additionally,an increase in pro-inflammatory factors such as IL-2,TNF-α,IL-5,IL-6,IL-17A,IL-9 was observed,while anti-inflammatory factors including IL-1.Ra,IL-10,IL-13 showed reduced levels.Conclusions:1.In vitro,OAd-MUC16-BiTE enhances T cell-mediated anti-tumor effects and induces bystander killing effects.2.In OC patient ex vivo tumor cultures,OAd-MUC16-BiTE overcomes the immunesuppressive tumor microenvironment to achieve cytotoxicity.3.In vivo,OAd-MUC16-BiTE demonstrates stronger antitumor activity when T cells are present.4.OAd-MUC16-BiTE increases chemokines,promotes blood vessel normalization,induces more T cells to infiltrate the tumor area,increases pro-inflammatory factors,reduces antiinflammatory factors,and reverses the tumor immunosuppressive microenvironment to enhance the antitumor response.Part 3:The Antitumor Effect of OAd-MUC16-BiTE Combined with MUC16CAR-T Cells in Ovarian CancerObjectives:1.Construction of MUC16CAR-T cells targeting MUC16.2.To investigate the anti-tumor effect of MUC16CAR-T cells on ovarian cancer.3.To evaluate the combined anti-tumor effect of OAd-MUC16-BiTE and MUC16CAR-T cells on ovarian cancer.Methods:1.MUC16CAR-T and CD19CAR-T cells were constructed by infecting lentiviral particles.The transduction efficiency and biological characteristics of MUC16CAR-T and CD19-CART cells were evaluated by FC.2.In vitro,the killing effect of MUC16CAR-T cells on OC cells was evaluated by luciferase assay,and the secretion of cytokines and the degranulation index CD 107a were analyzed by FC.In vivo,the anti-tumor effect of MUC16CAR-T cells was evaluated using in vivo bioluminescence imaging.3.In vitro,the killing effect of MUC16CAR-T cells on OC OV90 cells infected with OAdMUC16-BiTE was evaluated by luciferase assay,and cytokine secretion and degranulation were evaluated by FC.In vivo,the combined anti-tumor effect of OAd-MUC16-BiTE and MUC16CAR-T cells was evaluated using an NCG mouse ovarian cancer PDX model.Results:1.MUC16 CAR-T cells were constructed,with CD 19 CAR-T cells used as controls.FC revealed a high transduction efficiency of 73.7%72 hours after virus infection.Moreover,the CAR-T cells exhibited significantly low expression levels of PD-1,LAG-3,and TIGIT.2.MUC16CAR-T cells showed strong specific cytotoxicity、cytokine secretion and CD107a expression against MUC16+OC cells in vitro.In vivo,MUC16CAR-T cells induced better tumor regression compared with CD 19CAR-T.3.In vitro,the combination of OAd-MUC16-BiTE and MUC16CAR-T cells showed a stronger killing effect,and induced stronger cytokine secretion and CD 107a expression of MUC16CAR-T;in vivo,OAd-MUC16-BiTE and MUC16CAR-T cells combination showed a stronger antitumor effect.Conclusions:1.MUC16CAR-T cells have been successfully constructed.2.Both in vitro and in vivo,MUC16CAR-T cells exhibit specific killing capabilities against MUC 1 6-positive ovarian cancer.3.The combined application of OAd-MUC16-BiTE and MUC16CAR-T cells demonstrates a more potent killing effect on ovarian cancer,both in vitro and in vivo.Through the aforementioned three parts of the research,the final conclusions are as follows:1.The oncolytic adenovirus expressing MUC16-BiTE(OAd-MUC16-BiTE)has been successfully constructed,overcoming the limitations of bispecific antibodies and oncolytic adenoviruses individually.By combining two distinct cytotoxic mechanisms,it demonstrates a synergistic anti-tumor effect in the treatment of ovarian cancer.2.OAd-MUC16-BiTE exhibits an enhanced anti-tumor effect primarily by augmenting T cell-mediated cytotoxicity,promoting T cell infiltration into the tumor,and improving the immunosuppressive microenvironment.3.MUC16CAR-T cells specifically target MUC 16-positive ovarian cancer cells,resulting in a notable killing effect.When combined with OAd-MUC16-BiTE,a synergistic and cumulative effect is observed,offering a novel strategy for the combined immunotherapy of ovarian cancer and solid tumors.These findings lay a solid foundation for future clinical applications.Highlights1.MUC16-BiTE,a bispecific antibody targeting MUC16,was successfully constructed and linked to an oncolytic adenovirus to create OAd-MUC16-BiTE.2.This is the first time such a combination has been studied for the synergistic killing effects of bispecific antibodies and oncolytic adenoviruses on ovarian cancer in cancer PDX models.Furthermore,the study systematically analyzed changes in gene expression profiles,immune cell infiltration,and immune suppression by proteome sequencing.3.The research conducted in vitro and in vivo systematic investigation on the synergistic anti-tumor effect of OAd-MUC16-BiTE and MUC16CAR-T,thereby breaking the limitations of single immunotherapy and proposing a new strategy for the immunotherapy of ovarian cancer and solid tumors.Limitations1.This study has limitations due to the use of immunodeficient mouse models,which cannot fully replicate human innate antiviral responses and immune memory.2.The combination of oncolytic adenovirus and bispecific antibody,as well as CAR-T cell therapy,are still in the preclinical research stage and require further resolution of issues before clinical translation.These include addressing the safety of these therapies for human treatment,selecting appropriate patients,determining the best injection methods,and managing adverse reactions.3.When three immunotherapies are administered simultaneously,it may lead to heightened toxic side effects.Nevertheless,this approach allows for a reduction in the individual dosage of each immunotherapy,which holds the potential for enhanced safety and efficacy compared to a single high-dose immunotherapy.Extensive further studies are required to substantiate this perspective.