Study On Anti-tumor Activity And Mechanism Of DNA Vaccine Based On FAP? And Survivin

Cancer has become a major public health problem that threatens human life.Although the overall global cancer mortality rate has declined slightly with the advancement of cancer diagnosis and treatment technology,the number of deaths due to cancer has been increasing every year due to the increasing incidence.It remains high and shows an upward trend.The emergence of tumor immunotherapy represented by immune checkpoint blockers(ICBs)(such as PD-1/PD-L1 antibodies)has brought new hope for cancer treatment.However,ICBs are generally only effective for"hot"tumors with a large amount of anti-tumor T cell infiltration,but not for"cold"tumors without T cell infiltration.Studies have found that patients with large numbers of anti-tumor T-cell infiltration in tumors("hot"tumors)usually respond well to immunotherapy,while patients without T-cell infiltration in tumors("cold"tumors)usually do not Response;and the infiltration of T cells in tumors was positively correlated with the prognosis of cancer patients.This suggests that the presence of anti-tumor T cells is crucial for cancer treatment.Among many immunotherapies,cancer vaccines have attracted more and more attention because of their powerful tumor antigen-specific CD8~+T cell inducing ability.However,the presence of the immunosuppressive tumor microenvironment(TME)prevents the intratumoral infiltration and antitumor effects of vaccine-induced effector CD8~+T cells.Therefore,destroying the TME to promote the infiltration of CD8~+T cells into tumors is necessary to boost the effectiveness of immunotherapy such as cancer vaccines and ICBs.In addition to cancer cells,tumor also contains immune cells,pericytes,epithelial cells,adipocytes,cancer-related fibroblasts and cell matrix(ECM)components.These non-cancer cell components interact to form the tumor microenvironment,and occupy the majority of tumor quality.As an important part of TME,CAFs can promote tumor development by secreting chemokines(such as CXCL12 and CCL2),synthesizing ECM components(such as collagen I)and PD-1/PD-L1 signaling pathway.However,CAFs are a heterogeneous group of cells.Not all CAFs have a cancer-promoting effect.Some types of CAFs can inhibit cancer cell metastasis.It is gratifying that many studies have confirmed the cancer promoting effect of CAFs with fibroblast activating protein?(FAP?)on the surface,and the removal of CAFs in TME by gene editing and other means can effectively inhibit the growth of tumor.As a tumor matrix antigen,FAP?is expressed in more than 90%of solid tumors and is closely related to the progress of cancer.In previous studies,we constructed a DNA vaccine(CpVR-FAP)containing full-length human FAP?,and verified the anti-tumor effect of the vaccine in a 4T1 breast cancer model with abundant FAP?expression.Targeting FAP?~+CAFs with CpVR-FAP can effectively reduce the immunosuppressive factors in TME and promote the intratumoral infiltration of CD8~+T cells.Unfortunately,this vaccine only has a certain effect in delaying tumor growth in the model that simulate postoperative adjuvant therapy,while in the model of tumor formation,the therapeutic effect almost disappeared.There are three possible reasons for the above phenomena.1.The immunogenicity of the vaccine is not strong,and the induced FAP?-specific T cells are insufficient.2.The target of anti-tumor T cells induced by vaccine is only FAP?~+CAFs,but lack of direct killing effect on tumor cells.3.There are many immunosuppressive factors(such as MDSCs,Tregs,IL-10,etc.)in the established tumor model,but the vaccine does not have the function of eliminating immunosuppression.Therefore,in order to obtain more ideal therapeutic effects,the FAP?vaccine needs to be further optimized and combined with other therapeutic methods that can target tumor cells(such as other tumor vaccines).It is more important to find suitable combination drugs that can eliminate immunosuppressive factors to release the antitumor activity of the vaccine.First,we optimized the FAP?DNA vaccine.The new vaccine has significantly improved both the induction of FAP?-specific cellular immunity and the anti-4T1tumor effect,and it still has a tumor suppressive effect on the model of tumors already formed.The ability of the new vaccine to induce FAP?-specific cellular immunity and inhibit the growth of 4T1 tumors has been significantly improved,and it still has a tumor suppressing effect on the model in which tumors have formed.In addition,the new vaccine can effectively reduce the expression of CXCL12 and CCL2 in tumors,thereby reducing the infiltration of myeloid-derived suppressor cells(MDSCs)in the TME.Next,we constructed a DNA vaccine that can target both FAP?~+CAFs and tumor cells.Survivin,as an ideal tumor-associated antigen,has been used to construct various forms of cancer vaccines including DNA vaccines.The FAP?/survivin co-targeting vaccine(OsFS)has a stronger TME regulation and tumor suppressive effect,and compared with the FAP?-targeting vaccine(OsF)and survivin-targeting vaccine(OS),it further prolongs the survival time of 4T1 tumor-bearing mice.After combined with Doxorubicin(Dox),the antitumor effect of OsFS was further released.In the subcutaneous 4T1 tumor model,compared with the Dox and OsFS groups,the tumor inhibition rate of the combined group increased by 55.3%and 55.4%,respectively,and the survival time increased by 26.4%and 22.2%,respectively.In the orthotopic 4T1 tumor model,compared with the Dox group and the OsFS group,the tumor inhibition rate of the combined group increased by 61.5%and 100%,and the survival time increased by 33.1%and 23.6%,respectively.Subsequently,we verified the therapeutic effect of OsFS vaccine in a mouse pancreatic cancer tumor model(Panc02),which is also rich in FAP?~+CAFs.However,unlike the 4T1 tumor model,Panc02 tumor did not induce upregulation of MDSCs in tumor-bearing mice,but the Tregs content and the expression of antigen-specific IL-10 in the tumor-bearing mice's spleen were high.This indicates that different tumor types differ in their ability to induce immunosuppression.The elimination of immunosuppressive factors by gemcitabine(Gem)significantly improved the antitumor effect of the vaccine.In the subcutaneous Panc02 model,compared with the Gem group and the OsFS group,the tumor inhibition rate of the combined group increased by 75.5%and 72.8%,respectively,and the survival time increased by52.3%and 35.9%,respectively.In the orthotopic Panc02 model,compared with the Gem group and the OsFS group,the survival time of the combined group increased by55.7%and 37.9%,respectively.Finally,in order to further improve the therapeutic effect,we conducted preliminary explorations of the impact of Anti-PD-1 on the combination therapies(Dox/OsFS and Gem/OsFS).In the orthotopic Panc02 model,the addition of Anti-PD-1 significantly improved the therapeutic effect of combination therapy.Compared with the Anti-PD-1 group and the Gem/OsFS group,the survival time of the Gem/OsFS/Anti-PD-1 group increased by 159.2%and 32.2%,respectively.More importantly,90%of the tumor-bearing mice in the Gem/OsFS/Anti-PD-1 group survived 90 days after Panc02 injection.In summary,the optimal form of FAP?DNA vaccine was determined firstly,and a new therapeutic DNA vaccine,OSFs,which can target both FAP?~+CAFs and tumor cells simultaneously,with FAP?and survivin as antigens,was constructed for the first time.By targeting FAP?~+CAFs,OsFS can not only remodel the TME by eliminating FAP?~+CAFs,reducing immunosuppressive factors,but also enhance the killing effect of survivin-specific T cells induced by OsFS on tumor cells.By combining chemotherapy drugs to regulate the immune system of tumor bearing mice and reduce the immunosuppressive factors,the therapeutic effect of the vaccine on 4T1 breast cancer and panc02 pancreatic cancer was significantly improved.And the addition of Anti-PD-1 further prolonged the survival time of Panc02 tumor-bearing mice.The research content in this article provides new ideas for the clinical treatment of breast and pancreatic cancer.