Targeted Delivery Of Whole Tumor Antigens To Dendritic Cells Via Activating Fc Receptor Endocytic Pathway To Prepare Novel Tumor Vaccines

As we all know,colorectal cancer(CRC)is one of the common malignant tumors of the digestive tract.In the treatment of human cancers,traditional therapies such as surgery,chemotherapy,and radiotherapy have entered a bottleneck stage.Therefore,biological treatment methods such as immunotherapy have attracted more and more attention.Tumor vaccine is one of the research hotspots in recent years,and it has the advantages of high safety and good patient tolerance.With the in-depth understanding of the tumor microenvironment and tumor escape mechanism,mobilizing the body's immune system to resist tumors has gradually become a new research direction.In this study,a broad-spectrum CT26.WT mouse colorectal cancer cell antigen was cross-linked with specific phagocytic receptor ligands using chemical biological methods,and the obtained immune complexes were used to prepare tumor vaccines,and the effect on DCs was preliminarily studied,and by observing the tumor growth of vaccinated tumor-bearing mice to evaluate the therapeutic effect of DCs vaccine.First,genetic engineering is used to construct and express the fusion protein of Fc?R ligand and SA,that is,the gene sequences of mlg G2 a Fc and SA are recombined,cloned into pFUSE expression vector,and SA-mlg G2 a Fc expressed by eukaryotic cells is collected and purified.Then,the azide-modified unnatural sugars were incorporated into the glycosylation modification sites of mouse CRC tumor cells through the cellular sugar metabolism pathway,and the azide modification sites of the broad-spectrum tumor antigens were covalently made using a bioorthogonal reaction conjugated alkyne binding biotin,to obtain a biotin-TAg.Finally,in order to obtain as many cross-linked complexes of SA-mlg G2 a Fc and Biotin-TAg as possible,cross-link the two at a mass ratio of 1:96 to obtain the SA-mlg G2 a Fc-Biotin-TAg immune complex.Detect the specific antigens IGF-1R,CCR6,and E-cadherin contained in Biotin-TAg.The SA-mlg G2 a Fc-Biotin-TAg immune complex was loaded as an antigen on mouse bone marrow-derived DCs cultured in vitro to day 6,and TNF-? was used to further stimulate the maturation of DCs.Flow cytometry was used to detect the expression of DCs surface molecules before and after maturation and the targeting effect of SA-mlg G2 a Fc-Biotin-TAg on DCs.When mouse bone marrow mononuclear cells were induced to differentiate and loaded with antigen and cultured to the 7th day,there were short protrusions on the cell surface,with typical DCs morphological characteristics.At the same time,CD11 c positive cells accounted for about 70%,and the expression of MHC I,MHC II and the costimulatory molecules CD80 and CD86 were significantly increased,in line with the characteristics of m DCs.In addition,the expression of surface molecules in the SA-mlg G2 a Fc-Biotin-TAg group was significantly higher than that in the control group,indicating that mlg G2 a Fc combined with Fc?R to achieve SA-mlg G2 a Fc-Biotin-TAg specific targeting im DCs,stimulate DCs maturation and enhance their activity.Finally,a mouse CRC tumor model was established,DCs sensitized by immune complexes were collected in vitro,and tumor-bearing BALB/c mice were immunized three times next to the tumor.At the same time,a combined anti-PD-1 antibody treatment group was set up to observe Changes in tumor volume and lymphocyte subsets of mice in each group.In all the monotherapy groups,the tumors of the mice were gradually growing.In all the combination treatment groups,the tumor growth of mice was slower than that of the single-agent group.In the PBS combined with anti-PD-1 antibody group,the tumor in only one mouse completely resolved,and the tumors in the remaining four mice continued to grow in a similar manner to the mice in the single-drug group.In the TAg-DCs combined with anti-PD-1 antibody group,the tumor of one mouse completely regressed,and the tumor growth of the other four mice was slightly delayed,but the trend of tumor expansion and growth did not change significantly,and all tumors showed a central pit shape ulcer.However,in the SA-mlg G2 a Fc-Biotin-TAg-DCs combined with anti-PD-1 antibody group,the tumors of three mice completely resolved after treatment,and the tumor growth of the other two mice was significantly slower,and the envelope on the tumor surface remained intact without ulcers occur.In summary,this study achieved the cross-linking of specific activating phagocytic receptor ligands with a broad spectrum of tumor antigens through the bioorthogonal reaction,the specific binding reaction of SA and biotin,and the incorporation reaction of unnatural sugar metabolism.The obtained tumor vaccine can specifically target and activate DCs in vitro,thereby increasing the expression of DCs surface molecules.Isolate and culture DCs from mouse bone marrow in vitro,separate lymphocytes from spleen and peripheral blood of tumor-bearing mice,and immunize tumor-bearing mice with DCs sensitized by immune complexes.At the same time,the combination of PD-1 antibody is of great significance to slow down the growth of mouse tumors.This research has laid a good foundation for the development of personalized tumor vaccines in clinical application.