Anti-tumor Immune Responses And Mechanisms Of Allo-dribble Vaccines

In recent years,immune checkpoint blockade antibody in the clinical treatment of cancer has been an unprecedented success and entered the first-line drug choices that only contain traditional chemotherapy drugs before.However,for most patients with low responses to immune checkpoint blockade antibodies because of low mutation and less immune infiltration of the tumor,we need to find new therapeutic strategies to prime T cells target to the hidden epitopes that are ignored by the host immune system.Thus,therapeutic cancer vaccine is considered to be capable to induce T cells immune responses against neo-antigens.Although the duration of tumor vaccines study is not short,the therapeutic cancer vaccine alone is not very efficient in preclinical research or clinical trials,one possible reason is the T cell immune responses induced by therapeutic cancer vaccine is limited.In the preliminary study,we found that DRibble tumor vaccine had the potential to target hidden tumor epitopes and enhance T cell immunity.In order to break through the limitations of therapeutic tumor vaccine inducing T cell immune response,we hope to find a combination way for DRibble vaccine in order to improve its ability to induce T cell immune responses.In this study,we hypothesized that DRibble induced T cell immune responses can be enhanced by co-administration of co-stimulatory antibodies such as anti-OX40(CD134).At the same time,we also found in the MCA-induced and independent generated homologous tumors of mouse model,DRibble vaccine could prime immune responses not only to the tumor that generated DRibble,but also for other MCA-induced and independently generated tumors.These results suggest that DRibble vaccines contain the tumor antigen epitopes that shared by these MCA-induced but independently derived tumors,could cross-protect the DRibble vaccinated mice from the challenge of these MCA-induced tumors.In this study,we extended our research objects from syngeneic but independently derived sarcomas to allogenic tumors of different genetic background,examined the T cell immune responses and anti-tumor effects induced by allogenic DRibble vaccines.Purpose:To study the mechanism of the anti-tumor immune response induced by Allo-DRibble tumor vaccine,and to explore the mechanism of Allo-DRibble vaccine-induced immune response by ubiquitinated proteins,the essential component in DRibble vaccine.Methods:1.The OT-I T cells was stimulated by subcutaneous immunization and intranodal injection of DRibble vaccine,measure the proliferation by CFSE division to compare two different administrations;DRibble vaccine combined with OX40 co-stimulatory antibody stimulated the OT-I T cells,then examined the expression of CD 127 and KLRG1;naive BALB\c mice were immunized by syngeneic 4T1-DRibble vaccine and allogenic C57MG-DRibble and MMC-DRibble vaccine via intranodal injection,splenocyte were re-stimulated by 4T1 tumor and 4T1 DRibble 7 days later;BALB/c mice bearing 4T1 orthotopic breast cancer were immunotherapied by syngeneic 4T1-DRibble vaccine and allogenic C57MG-DRibble and MMC-DRibble vaccine combined with OX40 costimulatory antibody,observed tumor growth and mouse survival,then drawed the tumor growth curve and survival curve.2.Constructed the prokaryotic expression plasmid pUbiG 101-Vx3GFP,then transformed to LPS-deficient BL21(DE3)competent bacteria,expanded to 2L medium,induced expression of target protein Vx3GFP at low temperature;The crude extract was prepared by three-phase purification,then purified by Ni-Sepharose excel chromatography column;The tumor cells were treated with bortezomib for 16-24 hours,lysed by repeated freez/thaw cycles,then combined with Vx3GFP protein and Ni-Sepharose excel column particles,enriched tumor-derived ubiquitinated protein by affinity purification.3.The ubiquitinated short-lived proteins were isolated from the B78H1-GFP and B78H1-OVA-GFP tumor cells,or normal liver tissue,then stimulated antigen-specific OT-IT cells in vitro,measured the proliferation by CFSE division;ubiquitinated short-lived protein isolated from syngeneic 4T1 tumors and allogenic C57MG or CT26 tumors were intranodal injected in BALB/c mice,splenocyte were re-stimulated by 4T1 tumor 7 days later,the percentage of IFN-γexpressing tumor-specific T cells were determined by flow cytometry;the ubiquitinated short-lived protein isolated from B78H1-Db,B78H1-Kb and B78H1-DbKb containing different MHC-Ia molecules were intranodal injected in C57BL/6 mice,splenocyte were re-stimulated by B78H1 tumor without MHC-Ia molecules 7 days later,the percentage of IFN-γ expressing tumor-specific T cells were determined by flow cytometry.4.The ubiquitination short-lived proteins were isolated from the Mel-30 melanoma cells expressing the gp 100 melanoma antigen by Vx3GFP fusion protein,presented by in vitro induced moDCs,co-cultured with gp100 specific CD8+tumor infiltrating lymphocyte cell line(TIL-1520)generated from melanoma patient recognized HLA-A2 restricted gp100 epitope,the intracellular staining of IFN-γ+ was measured 18 hours later;the ubiquitination short-lived proteins isolated from UbiLT3 or UbiLT3pp65 tumor lysates were co-cultured with moDCs,then these activate moDCs pulsed with ubiquitinated proteins were co-cultured with pp65 MHC-I tetramer-positive CD8+ T cells,the intracellular staining of IFN-γ+ was measured 18 hours later;the ubiquitination short-lived proteins were conjugated with alumina adjuvant HS then loaded on dendritic cells,stimulated the pp65 specific T cells;prepared PFO permeate products and freeze-thaw lysate with same total protein content,compared their ability to stimulate human T cells by cross-presentation;the ubiquitination short-lived proteins isolated from allogenic lung cancer cell line UbiLT3 then loaded on dendritic cells,stimulated tumor-reactive tumor infiltrating lymphocytes from non-small cell lung cancer patients,the secreted GM-CSF and IFN-γ were measured by ELISA.Results:1.Directly intranodal injected DRibble vaccine without loading by dendritic cells could induce an approximately 20-fold increase in OT-I antigen specific T cell division than subcutaneous injection;after combined with OX40 co-stimulatory antibody,the percentage of OT-I T cells in spleen was nearly 5-fold higher than the DRibble alone group,which only expressed CD 127 on the surface on the day when the T cell proliferation reached peak level,whereas the OX40 combination group also expressed KLRG1,which changed the T cell composition;both syngeneic and allogenic DRibble vaccines could induce 4T1 tumor-specific CD8+effector T cells,more than 70%of CD8+ T cells secreting IFN-γ also produce granzyme A or B,indicating that these cells are typical effect/memory T cells;syngeneic 4T1 DRibble vaccine and OX40 co-stimulatory molecular antibody combination showed significant synergistic effect,not only tumor growth was significantly inhibited,and 60%of mice were completely cured,the median survival time was significantly higher than the control groups;the allogenic DRibble vaccine could also stimulate and produce 4T1 tumor-specific immune responses.2.By reconstructing the Vx3(A7)prokaryotic expression plasmid,Vx3(A7)was optimized to GFP fusion protein(Vx3GFP)and transformed into LPS-deficient BL21(DE3)bacteria.After optimization,0.1mM IPTG and low temperature induction at 16℃ was chose as the final expression condition;the three phase purification method could remove most of the non-specific protein,then the crude extract of Vx3GFP protein were purified by affinity chromatography;optimize the working conditions of combination ubiquitinated proteins with Vx3GFP by protein amount titration,the results of Western blotting showed that 10μg of Vx3GFP could effectively isolate ubiquitinated protein from lmg cell lysate.3.The naive OT-IT cells were labeled with CFSE and then stimulated by mutu-1940 DCs pulsed with ubiquitinated short-lived protein,OVA-specific T cell proliferation was only detected in ubiquitinated proteins derived from B78H1-OVA,whereas ubiquitinated proteins from B78H1-GFP or normal liver tissue did not activate OT-I T cells;CD8+ T cells induced by both syngeneic and allogenic ubiquitinated short-lived proteins could recognize 4T1 tumor cells and produce IFN-γ,they could also recognize syngeneic CT26 colorectal cancer cells and produce IFN-γ;B78H1 tumor cells expressed H-2Db,H-2Kb or H-2DbKb could stimulate the T cells induced by ubiquitinated proteins from B78H1,whereas original B78H1 tumor cells could not.Ubiquitinated proteins purified from Mel-30 tumor cell lysate could activate moDCs then be presented by them,which could stimulate TIL-1520 cells to produce IFN-y,the ubiquitination protein purified from UbiLT3 lung cancer cells could not stimulate TIL-1520 cells;ubiquitinated proteins purified from UbiLT3pp65 tumor cell lysate could activate both CD8+ and CD4+ T cells to produce IFN-γ,which stimulates the level of activated T cells to be similar to or greater than the recombinant CMV protein as a positive control,whereas the ubiquitin derived from the UbiLT3 lysate Protein can not activate pp65 MHC-I tetramer-positive T cells at all.Compared with the input and flowthrough in the purification of UbiLT3pp65 ubiquitinated protein,only enriched ubiquitinated proteins can effectively stimulate CD8+ and CD4+ T cells and produce an immune response;HS-alumina adjuvant combined with ubiquitinated protein can significantly increase the percentage of IFN-γ-producing CD8+ T cells,and IFN-γ-producing CD4+ T cells are not subject to the addition of HS;The percentages of IFN-γ+ CD8+ T cells produced by PFO permeate products were significantly higher than those produced by repeated freeze/thaw lysates,whereas IFN-γ CD4+ T cells produced by these two were similar;when ubiquitinated protein isolated from UbiLT3 cell lysates loading onto autologous DC or Mutz-3 DLC,could stimulate and activate the TIL-101 cells,the GM-CSF and IFN-γproduction could be significantly detected.Conclusions:1.The activation of OT-I T cells stimulated by lymph node injection of DRibble antigen was better than that of subcutaneous injection;syngeneic or allogenic DRibble vaccines combined with the OX40 co-stimulatory antibody all have the ability to induce 4T1 tumor-specific T cells and tumor regress on 4T1 tumor bearing mice.2.The constructed Vx3GFP fusion protein expression plasmid could be successfully expressed and purified,and a large number of Vx3GFP protein can be prepared;of prokaryotic expressed Vx3GFP proteins had biological activity and could specifically bind to the ubiquitinated protein in tumor cell lysates.3.DC-pulsed ubiquitinated short-lived proteins could stimulate antigen-specific CD8+ T cell with efficient activation and proliferation;tumor-derived ubiquitinated short-lived proteins could effectively cross-prime the tumor-specific T cells that could recognize cancer cells,which was not restricted by MHC-I molecules.4.Ubiquitinated proteins isolated from tumor cells could activate tumor antigen specific T cells;alumina adjuvants could facilitate cross-presentation ubiquitinated protein to CD8+ T cells;Ubiquitinated proteins derived from allogenic tumor cell lines could provide efficient and enriched antigens,which could cross-recognized TILs isolated from tumor tissue from patients with non-small cell lung cancer.