CpG ODN Formulated MF59as Adjuvants For Tumor Antigens To Induce Immunity Against Melanoma And Lung Cancer In Mice

Ideally, a potent cancer vaccine should be able to induce a strong T helper (Th)1cell mediated immune response and a vigorous generation of specific cytotoxic Tlymphocytes (CTLs). However, tumor antigens in cancer vaccines are usually poorlyimmunogenic and therefore required an adjuvant to enhance their ability to elicitstrong cellular immune response. In general, an adjuvant serves to enhance thespecific immune responses to antigens, composing of a vehicle and immunestimula-头nts usually. The vehicles, such as mineral salts, emulsions, liposomes, function tocontrol the release and delivery of antigens. The most common immunestimulatorsare pathogen-associated molecular patterns (PAMPs), which capable of activatingantigen presenting cells, especially dendritic cells (DC) to process and presentantigens, produce cytokines and chemokines, and up-regulate co-stimulatorymolecules more efficiently.MF59is an oil in water emulsion adjuvant approved for influenza vaccines ofhuman use in Europe, which is prepared with4.3%w/w of squalene as an oil phase.In addition to the depot effect, MF59acts through enhancing antigen uptake byactivated DCs and facilitating Th2-biased immune responses to the antigens. Due toits Th2biasing property, MF59is seldom tested in cancer vaccines. It was found thatsome toll like receptor (TLR) ligands could confer MF59with Th1immuneresponse-promoting properties.Synthetic oligodeoxynucleotides containing CpG motifs (CpG ODN), a TLR9agonist, have been explored as adjuvants for tumor vaccines and a number of themare being evaluated in clinical trials. Based on functional characteristics, CpG ODNsare divided into A, B and C types. A-type CpG ODN induces plasmacytoid dendriticcells (pDC) to secret large amount of interferon-α (IFN-α). B-type CpG ODN stimulates the proliferation and antibody production of B cells. C-type CpG ODNshares both of the properties of A-type and B-type CpG ODN. In the clinical trials,CpG ODNs, especially B-type CpG ODNs, have been evaluated for their efficaciesas adjuvants to cancer vaccines.To explore the possibility of using PAMP mimics to confer Th1biasingcapability to MF59as an efficient adjuvant to tumor antigens, in this study, MF59formulated with CpG ODN, polyinosinic-polycytidylic acid (ploy I:C) or Thymosinalpha1(Tα1) were tested for their Th1adjuvant activities to assist HSP65-MUC1, arecombinant fusion protein between mycobacterial heat shock protein (HSP65) andhuman MUC1VNTR peptides (MUC-1), and then a C-type CpG YW002as theoptimal PAMP mimic was selected to be formulated with MF59to evaluate whethercould assist HSP65-MUC1and Lewis tumor celll lysate (TCL) to induce anti-tumorimmunity against MUC1~+B16melanoma and Lewis lung cancer in mice,respectively.1. Preparation of recombinant HSP65-MUC1and Lewis TCL andestablishment of MUC1~+B16melanoma cellsTo assess whether MF59plus PAMP mimics could be used as a potent adjuvantfor cancer vaccines, we selected the recombinant fusion protein HSP65-MUC1andLewis TCL as antigens for tumor vaccines against MUC1~+B16melanoma cells andLewis lung cancer cells, respectively. HSP65-MUC1was prepared with purity over96%, and was recognized by anti-HSP65and anti-MUC1VNTR mAbs, respectively.MUC1~+B16melanoma cells were obtained by transfecting a fusion gene with theencoding genes for green fluorescence protein (GFP) and MUC1VNTR peptide intomurine B16melanoma cells. The transfected cells were identified by GFPfluorescence. The green transfected cells represent the cells expressing MUC1VNTRpeptide. Lewis TCL was prepared by freeze-thaw method. The Lewis lung caner cellswere destroyed after thefreezing and thawing and stained into blue by trypan blueunder microscope. The selected and identified tumor antigens and tumor cells wereused in tumor inhibition and cytotoxicity assays. 2. Effect of MF59formulated with immunostimulants on assistingHSP65-MUC1to induce Th1-biased immune response in miceTo investigate whether MF59formulated with various PAMP mimics couldinduce the Th1polarized immune response to HSP65-MUC1. Vaccines were preparedby mixing MF59with10μg/dose HSP65-MUC1and/or10μg/dose immunostimulants(CpG ODN, ploy I:C or Tα1) at1:1(v/v) ratio. C57BL/6mice were immunizedsubcutaneously (s.c.) with PBS, HSP65-MUC1, MF59/HSP65-MUC1, MF59-2216/HSP65-MUC1, MF59-2006/HSP65-MUC1, MF59-YW002/HSP65-MUC1, MF59-YW002/HSP65-MUC1or MF59-Tα1/HSP65-MUC1, respectively, for four times at a7-day interval. On day1after the last immunization, the mouse splenocytes wereisolated for analyzing mRNA expression of interferon-gamma (IFN-γ). The resultsindicated that MF59-YW002/HSP65-MUC1induced a significant higher level ofIFN-γ mRNA expression than that of HSP65-MUC1, MF59/HSP65-MUC1, MF59-2216/HSP65-MUC1and MF59-Tα1/HSP65-MUC1.Next, we tested whether MF59formulated with various PAMP mimics couldassist HSP65-MUC1to induce specific a higher level of IgG2c antibody. On day7post the last immunization, the mouse sera were collected and HSP65-MUC1specificIgG2c or IgG1antibodies were detected by ELISA. It was found that MF59-YW002/HSP65-MUC1induced much higher levels of IgG2c and slightly elevatedlevels of IgG1, resulting in a significant increase in the IgG2c/IgG1ratio, compared toother groups. Noticeably, compared to HSP65-MUC1, MF59/HSP65-MUC1significantly reduced the ratio of specific IgG2c/IgG1, which indicated that MF59itself assisted HSP65-MUC1to induce a Th2-baised immune response. However,CpG YW002could shift MF59to facilitate a Th1-biased immune response, andMF59-YW002may be the optimal combination could assist HSP65-MUC1tofacilitate a Th1-biased immune response.3. Immunization of mice with adjuvant containing MF59-YW002andHSP65-MUC1is prophylactic against anti-MUC1~+melanomaUpon the above testing, CpG YW002was selected to be formulated with MF59and the formulated MF59was assessed for its adjuvant capacity on assistingHSP65-MUC1to induce anti-MUC1~+B16melanoma in mice, both prophylactically and therapeutically. Mice were immunized s.c. with HSP65-MUC1, YW002/HSP65-MUC1, MF59/HSP65-MUC1or MF59-YW002/HSP65-MUC1on day-24,-17,-10and-3, and challenged with MUC1~+B16melanoma cells (1.2×105/per mouse) onday3after the last immunization. The results showed that MF59-YW002couldsignificantly boost the immunogenicity of HSP65-MUC1to induce anti-MUC1~+melanoma immunity. Using either MF59or YW002alone as an adjuvant forHSP65-MUC1failed to inhibit tumor growth. Surprisingly, when MF59was used asthe sole adjuvant with HSP65-MUC1it promoted significant growth of MUC1~+B16melanoma. Importantly, mice vaccinated with MF59-YW002/HSP65-MUC1had asignificantly prolonged survival rate, and on day75post-tumor challenge,50%ofvaccinated mice were alive. In contrast, only11.1%,20%and20%of the micesurvived in the PBS, HSP65-MUC1alone or YW002/HSP65-MUC1groups,respectively. In the group vaccinated with MF59/HSP65-MUC1, all mice died at anearly stage, consistent with the fast tumor growth. Tumor incidence was the highest(100%) in this group, compared with the PBS, HSP65-MUC1alone orYW002/HSP65-MUC1groups, where tumor incidence was88.9%,60%or70%,respectively. In comparison, vaccination with MF59-YW002/HSP65-MUC1significantly decreased tumor incidence to30%. Taken together, MF59-YW002maybe a promising adjuvant to promote immunogenicity of HSP65-MUC1toprophylactically inhibit MUC1~+tumors.Next, we tested whether vaccination with MF59-YW002/HSP65-MUC1couldinduce a therapeutic effect against anti-MUC1~+melanoma. Mice were inoculatedwith MUC1~+B16melanoma cells on day0, followed by immunization withHSP65-MUC1, MF59-YW002or MF59-YW002/HSP65-MUC1on days1,8,15and22, respectively. Immunization with MF59-YW002failed to enhanceimmunogenicity of HSP65-MUC1to induce tumor inhibition in the therapeuticmodel. The survival rate of mice immunized with MF59-YW002/HSP65-MUC1wasonly22.2%compared with38%in mice immunized with HSP65-MUC1alone,indicating that MF59-YW002may be not a suitable adjuvant for therapeuticapplication of HSP65-MUC1to induce anti-tumor immunity. 4. MF59-YW002enhances immunogenicity of HSP65-MUC1to induceimmunological memory to inhibit MUC1~+tumorsTo investigate whether MF59formulated with CpG YW002could promoteHSP65-MUC1to generate immune memory to MUC1~+melanoma, five miceimmunized with MF59-YW002/HSP65-MUC1and challenged with MUC1~+B16melanoma cells were re-challenged with MUC1~+B16melanoma cells on day75after the first challenge. Na ve mice inoculated with MUC1~+B16melanoma cellswere used as controls. Four na ve mice succumbed to melanoma on day39, whereasonly one mouse died in the re-challenged group. The average survival wassignificantly longer in re-challenged mice than na ve mice. This data suggested thatMF59-YW002could assist HSP65-MUC1to generate MUC1~+B16melanoma-specific immunologic memory in mice.5. MF59-YW002enhances the immunogenicity of HSP65-MUC1to induce acellular immune response in miceTo explore how MF59-YW002could enhance the anti-tumor effect ofHSP65-MUC1in mice, we also measured the sera of mice immunized withHSP65-MUC1, MF59/HSP65-MUC1, MF59/HSP65-MUC1or MF59-YW002/HSP65-MUC1in prophylactical setting, for HSP65-MUC1-specific IgG1and IgG2cantibodies. The results were same as the observations in immunostimulantsscreening, the ratio of IgG2c/IgG1was higher in mice immunized with MF59-YW002/HSP65-MUC1than that of other groups. Subsequently, spleens of miceimmunized with HSP65-MUC1, MF59/HSP65-MUC1, MF59/HSP65-MUC1orMF59-YW002/HSP65-MUC1were isolated on day3after the fourth immunization.Splenocytes were stained with FITC-labeled anti-CD4(NK1.1) mAb and PE-labeledanti-CD69mAb to analyze CD4~+T and NK cell activation. The ratio of activatedCD4~+T cells in splenocytes from mice immunized with MF59-YW002/HSP65-MUC1was higher than that of other groups, while the activated NK cells ofsplenocytes have no obvious difference in each group. The results suggest thatMF59-YW002enhances the immunogenicity of HSP65-MUC1to induce theactivation of CD4~+T cell and a Th1-baised immune response. To investigate whether MF59-YW002enhanced the immunogenicity of HSP65-MUC1to induce tumor-specific immune responses, splenocytes from immunizedmice were cultured with HSP65-MUC1for12h and then stained with FITC-labeledanti-CD8mAb and PE-labeled anti-CD69mAb to analyze CD8~+T cell activation. Theratio of CD69~+/CD8~+T cells in splenocytes from mice immunized with MF59-YW002/HSP65-MUC1was significantly higher compared with mice injected withPBS, HSP65-MUC1and MF59/HSP65-MUC1. Furthermore, splenocytes fromimmunized mice were cultured with HSP65-MUC1for5days and then used aseffector cells in a CTL killing assay. The result shown that splenocytes from miceimmunized with MF59-YW002/HSP65-MUC1lysed MUC1~+B16melanoma cellstested at different effector:target (E:T) ratios, the highest cytotoxicity (44.2%) wasobserved at an E: T ratio of45:1. Splenocytes from mice immunized with HSP65-MUC1, YW002/HSP65-MUC1or MF59/HSP65-MUC1could also lyse target cellscompared to the PBS control group, but there is an obviously lower cytotoxicity thanthe MF59-YW002/HSP65-MUC1group. This data indicates MF59-YW002as anadjuvant with HSP65-MUC1could contribute to the production of tumor-specificimmune responses, may be a promising adjuvant for the development of tumorvaccines.6. Effect of MF59-YW002on assisting Lewis TCL to induce anti-Lewis lungcancer immune responses in miceBased on the inducing of effective anti-tumor immunity on MUC1~+B16melanoma model, we tried to explore whether MF59-YW002could enhance theanti-Lewis lung cancer effect of TCL in mice. Fristly, it was necessary to establish theappropriate Lewis lung cancer model. We chose several different tumor challengesites (s.c. at back, axillary, groin; intraperitoneal and intrathoracic) for evaluating theaverage survival of tumor-bearing mice and observing the tumor rupture phenomenon.At last, the axillary s.c. and intrathoracic were chose for the best challenge sites, andthe suitable cell numbers for tumor inoculation were confirmed (6.0×10~5/per mousefor axillary s.c. model and3.75×10~4/per mouse for intrathoracic model).Next, we investigated the anti-tumor effect of the MF59-YW002/TCL. In the experiment,we immunized mice on day-24,-17,-10,-3,and inoculated Lewislung cancer cell through axillary s.c. and intrathoracic sites on day0, respectively.The results showed that immunization of MF59-YW002couldn't assist TCL toinhibit the growth of Lewis lung cancer in mice, compared with the TCL alone. Bythe results of mice survival, compared with other groups, we didn't found the thesurvival of the tumor-bearing mice was prolonged obviously in MF59-YW002/TCLgroup, both in axillary s.c. and intrathoracic model. The result indicated thatMF59-YW002may be not a suitable adjuvant for application of TCL to induceanti-Lewis lung cancer immunity.Collectively, MF59formulated with CpG YW002could facilitate HSP65-MUC1to induce a Th1-baised immune response and specific anti-MUC1~+B16melanomaimmunity in prophylacticly. The formulation could be applied to prepare tumorspecific antigen-based vaccines for cancer patients, particularly for postoperativecancer patients by using the prophylactic strategy to prevent tumor recurrence andeliminate the small amount of residual tumor cells.