腺病毒初免蛋白加强免疫策略联合DDA/MPL佐剂增强survivin/MUC1特异性抗肿瘤作用研究
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摘要
由于发病率和死亡率的逐年增加，肿瘤已经成为危害人类健康的头号杀手。生物治疗由于具有副作用小，作用范围广等特点已经成为继手术，化疗，放疗之后的第四大肿瘤治疗方法。肿瘤疫苗作为生物治疗的重要组成部分，目前已有很多肿瘤疫苗进入临床实验并且已经有个别疫苗被FDA批准用于临床治疗。
肿瘤疫苗主要是通过诱导机体产生肿瘤抗原特异性的免疫反应来提高机体免疫力，从而杀伤肿瘤细胞。理想的疫苗靶点应该具备以下几个特点：①特异性较强，即只在肿瘤细胞中表达，在正常细胞中不表达或低表达，若在正常组织中有表达，则在肿瘤组织中的表达要有显著的质和量的改变；②具有一定的广谱性，即在多数肿瘤中均有表达；③最重要的是能在肿瘤病人体内能检测到该抗原的特异性抗体和CTL，这表明肿瘤患者已将其视为异物，并对其产生了免疫应答，如用这样的抗原作为靶点设计疫苗将更容易突破机体的免疫耐受。肿瘤抗原Survivin和MUC1同时具备这三个条件，是非常理想的肿瘤疫苗靶抗原，因此，本论文拟采用MUC1和Survivin为靶点研制肿瘤疫苗。
Survivin是IAP(inhibitor of apoptosis protein)家族成员之一，具有抗凋亡的功能，广泛表达于肿瘤组织中而在正常的终末分化组织中不表达。研究表明，survivin的表达水平与病人的耐药，预后不良和生存期缩短之间有一定的关系，这使得survivin成为肿瘤疫苗的理想靶抗原。目前已报道的靶向survivin的疫苗有多种形式，但是未见survivin蛋白疫苗的报道。这可能是由于单独使用的蛋白疫苗诱导产生的细胞免疫反应较弱。本论文通过对其蛋白疫苗的佐剂和免疫策略的优化来达到增强蛋白疫苗诱导产生细胞免疫的效果。
常用的蛋白佐剂有很多种，包括CpG寡核苷酸，弗氏佐剂，Al(OH)3，MPL(monophosphoryl lipid A)等。DDA/MPL (MPL formulated with cationic dimethyldioctadecylammonium)佐剂由于能够显著增强疫苗的抗原提呈能力和疫苗的保护力而被用于结核和衣原体疫苗的研究中。目前DDA/MPL用做增强肿瘤疫苗的佐剂还未见报道。异源的prime-boost策略可以通过应用不同的抗原提呈系统增强蛋白疫苗的细胞免疫水平。本论文选用了DDA/MPL和Al(OH)_3作为蛋白疫苗的佐剂并联合应用重组腺病毒疫苗和蛋白疫苗的异源免疫策略形式来增强survivin抗原特异性的细胞免疫反应和抗肿瘤效应（出于疫苗安全性方面的考虑，本论文使用缺失了survivin蛋白抗凋亡功能的S8蛋白形式）。
研究结果显示，单独使用survivin的蛋白疫苗并不能诱导小鼠产生有效的细胞免疫，但DDA/MPL佐剂能显著提高survivin蛋白的抗原特异性细胞免疫反应。同时，与其它免疫策略相比，重组腺病毒初免，蛋白联合佐剂加强的免疫策略能够最大程度地抑制小鼠黑色素瘤的生长。对抗肿瘤效应机制的研究表明，这种抗肿瘤效应主要是由Th1型免疫反应介导的。
MUC1是粘蛋白家族成员之一，是一个跨膜的糖蛋白，其膜外的VNTR区是它主要的免疫原性区域，目前已在很多类型的肿瘤患者体内发现其蛋白的表达，是肿瘤治疗的理想靶抗原。目前许多针对MUC1VNTR区的疫苗正在进行临床实验。本论文应用重组腺病毒初免蛋白联合佐剂加强的免疫策略，评价了MUC1蛋白疫苗的免疫原性及其对黑色素瘤荷瘤小鼠的肿瘤抑制效应。结果显示，采用重组腺病毒初免，MUC1蛋白联合DDA/MPL佐剂加强的免疫策略能够产生有效的抗原特异性免疫反应，且能有效抑制肿瘤生长并延长荷瘤小鼠的生存时间，生命延长率约为33%。由于肿瘤细胞的异质性，应用单一抗原位点的疫苗不一定能够引起足够的肿瘤抑制效应。本论文评价了survivin与MUC1联合应用后对黑色素瘤荷瘤小鼠的肿瘤抑制效应和生命延长情况，结果表明，两种肿瘤相关抗原联用与二者单独使用时相比，尽管不能减缓小鼠肿瘤生长的速度，但是可以延长荷瘤小鼠的生存时间，与AD-S8/S8pro组相比生命延长率为8.82%；与AD-9M/9M pro组相比生命延长率为2.78%。
由于肿瘤细胞的异质性和肿瘤微环境的复杂性，很多单独使用的肿瘤疫苗临床实验宣告失败。研究表明，生物疗法与其他的肿瘤治疗方法的组合，比如化药治疗，可能会更有效。本论文中，我们采用了化疗药物奥沙利铂与疫苗联合应用的方式来治疗肿瘤，并且观察疫苗与奥沙利铂联合使用后荷瘤小鼠的生命质量是否有改善。结果显示，与PBS组相比，奥沙利铂组，疫苗组和联用组的抑瘤率分别为29.64%，42.59%和54.68%；生命延长率分别为22.22%，33.33%，48.15%。表明奥沙利铂可以增强survivin与MUC1联合疫苗对黑色素瘤荷瘤小鼠的抗肿瘤作用，且二者联用组小鼠体重较奥沙利铂组有一定程度的增大，表明疫苗可以减轻奥沙利铂对小鼠的副作用。
为了初步探索一下以survivin和MUC1为靶点的蛋白疫苗的肿瘤适应症，我们对乳腺癌，肠癌，卵巢癌，食管癌，肺癌，胃癌六种血清样本中survivin和MUC1的抗体进行了检测，结果发现不同类型肿瘤的血清样本survivin和MUC1抗体表达水平不同。肠癌，胃癌，肺癌，卵巢癌，食管癌，乳腺癌患者血清样本的MUC1抗体阳性率分别为18.5%,12%，2%,0,0,10.1%；survivin抗体的阳性率分别为31.1%,3%,0,0,2.9%,15.15%。可以看出，在肠癌和乳腺癌患者体内均产生了针对MUC1和survivin的特异性抗体，而且肠癌样本中的含量明显高于乳腺癌样本。因此，我们评价了survivin及MUC1联合使用疫苗对肠癌和乳腺癌荷瘤模型小鼠的抗肿瘤效应。
结果表明，AD-MS/S8+9M pro能够明显抑制结肠癌模型小鼠肿瘤生长(P<0.05)且能明显延长荷瘤小鼠的生存期（P<0.01）。对于乳腺癌模型小鼠，AD-MS/S8+9M pro能够抑制肿瘤的生长速度（P<0.01），但是并不能延长小鼠的生存期（P>0.05）。这一结果与肠癌患者血清抗体抗性阳性率要高于乳腺癌患者血清阳性率的结果是相呼应的。对于结肠癌模型小鼠，疫苗与化疗药物奥沙利铂有协同作用，能够协同抑制结肠癌荷瘤小鼠的肿瘤生长速度并延长荷瘤小鼠的生存期。但是对于乳腺癌模型小鼠，二者并没有协同作用。
总的来说，DDA/MPL能够增加蛋白疫苗诱导的抗原特异性细胞免疫反应，重组腺病毒初免蛋白联合佐剂加强免疫的策略具有最强的肿瘤抑制效率，这种肿瘤抑制效应是由Th1型免疫反应介导的。Survivin和MUC1两种抗原联合免疫的效果要优于单独免疫的效果。两种抗原联合疫苗对于黑色素瘤模型小鼠，乳腺癌模型小鼠，结肠癌模型小鼠的肿瘤生长都具有明显抑制作用，并能显著延长黑色素瘤模型小鼠和结肠癌模型小鼠的生命。化药与免疫治疗的联用对于黑色素瘤和结肠癌模型小鼠有显著的协同作用，但是不能增强乳腺癌模型小鼠的抗肿瘤效应。。
Because of the increasment of incidence and mortality year by year, cancer hasbecome one of the most serious diseases to human health. Due to less side-effect andwider range of treatment, biological therapy has now become the fourth mostfrequently used therapy for tumor treatment, except for surgery, chemotherapy andradiation. Many tumor vaccines have been under the clinical trials or permitted to beused for the clinical treatment so far.
The ideal vaccine targets should have the following characteristics:①strongspecificity. The targets are only expressed in tumor cells, but they are not or lowexpressed in normal cells. If they are expressed in normal tissues, the expressionlevels must have a significant change in quality and quantity in tumor tissue.②Thetumor targets must have a certain broad spectrum, which means that they areexpressed in the majority of tumors.③Antigen-specific antibody and CTL can bedetected in cancer patients. It is suggested that cancer patients has been regarded it asa foreign body and produced immune response against it. Such tumor antigens used asvaccine targets will be easier to break immune tolerance. Survivin and MUC1whichare broadly used have all the three conditions above. Therefore, this thesis intends toapply MUC1and Survivin as the targets of cancer vaccine.
Survivin, as a member of IAP families, could both inhibit cell apoptosis andcontrol cell division. Survivin is widely expressed in tumor tissues, but not expressedin terminal devision of normal tissues. It is reported that the expression levels ofsurvivin are associated with drug resistance, poor prognosis and shortened survival,and which make survivin an ideal target antigen of tumor vaccine. The current reportshave revealed that survivin-targeted vaccine has various forms, whereas no report on survivin protein vaccine can be found. This may be due to its poor ability to inducecellular immunity. A suitable immunoadjuvant and optimized immunization strategycan greatly enhance the cellular immune response to this protein vaccine.
In order to get a vaccine of higher immunogenicity and better antitumor effection,our research makes survivin as the target of tumor vaccine, uses the protein form ofS8which has lost the function of anti-apoptosis and optimizes the choice of adjuvantof S8proten and the vaccine immunization strategies.
Immunoadjuvants such as CpG ODN, incomplete Freund's adjuvant, aluminiumhydroxide [Al(OH)3] and monophosphoryl lipid A (MPL) have been used to enhanceresponses to a variety of vaccines. DDA/MPL has been shown to enhance antigenuptake and presentation and induce a significant level of protective immunity inChlamydia and tuberculosis vaccine research. However, whether DDA/MPL canenhance efficacy of tumor vaccines has not been reported. A heterologousprime-boost strategy can enhance the cellular immunity of a protein vaccine byapplying different antigen-presenting systems. DDA/MPL, Al(OH)3and anadenovirus prime-protein boost strategy were applied to enhance the survivin-specificimmunity and anti-tumor immunity of a truncated survivin protein vaccine.(Due tosafety concerns of full-length survivin, an anti-apoptosis protein, this vaccine wasbased on a truncated form of survivin (S8) with a deletion of seven amino acids at itsN-terminus, which was shown to be non-functional in our previous study.)
It was showed that survivin protein used alone couldn’t induce effective cellularimmunity, and DDA/MPL adjuvant induced stronger antigen-specific cellularresponses than Al(OH)3. Furthermore, comparing to other immunization regimens,recombinant adenovirus prime adjuvanted protein boosted immunization showed thestrongest anti-tumor effect in the murine melanoma model. The anti-tumor effect wasmediated by Th1-typed immune response.
MUC1is a transmembrane glycoprotein normally expressed on the apical surfaceof ductal epithelia. A variable number (20–125) of tandem repeats (VNTR) of a20 amino acid sequence which was contained in its extracellular domain is the dominantimmunogenic domain. MUC1(mucin1) is a classic target for cancer immunotherapythat is overexpressed in many adenocarcinomas. Many active immunotherapiestargeting MUC1are in clinical trials. We investigated the immunity response andanti-tumor effect in a murine melanoma model after administration of an AD-9Madenoviral vector prime-recombinant9M protein and DDA/MPL boost. Our resultsdemonstrated that the vaccination induced a significant inhibition of the growth ofB16melanoma in mice and prolonged the survival of B16melanoma–bearing mice.The survival rate was about33%. Due to the heterogeneity of tumor cells, theapplication of a single antigen site vaccine may not be able to induce enough tumorsuppressive effect. Our work evaluated the anti-tumor effect and survival extendanceof the survivin and MUC1combination vaccine in a murine melanoma-bearingmodel. The results showed that the combination could not inhibit the tumor growth,however, it could extend the survival time of tumor-bearing mice. The survival ratewas8.82%compared with AD-S8/S8pro group, and2.78%compared withAD-9M/9M pro group.
Due to the heterogeneity of tumor cells and complexity of tumormicroenvironments, many clinical trials of tumor vaccines alone have failed. It isreported that the administration of other therapies such as chemotherapy along with avaccine may enhance its efficacy. The tumor inhibitory effect of our cancer vaccinewas also tested in combination with a widely used anti-tumor chemotherapy drugoxaliplatin. Compared to PBS group, the tumor inhibition rate of oxaliplatin, vaccine,combination of oxaliplatin and vaccine are29.64%，42.59%and54.68%respectively;the survival rate of tumor-bearing mice are22.22%，33.33%and48.15%respectively.It is suggested that oxaliplatin may help to improve the outcome of survivin-basedimmunotherapy. Moreover, the vaccine also alleviated side effects of thechemotherapy drug oxaliplatin, such as emaciation and poor activity.
To investigate the most suitable cancer types of vaccine targeting survivin andMUC1, antibody levels of serum from breast cancer, colorectal cancer, overian cancer, esophageal cancer, lung cancer and gastric cancer are evaluated. Our results showedthat the antibody levels are different from cancer types. The sero-positive rate ofMUC1of colorectal cancer, gastric cancer, lung cancer overian cancer, esophagealcancer and breast cancer was18.5%,12%，2%,0,0,10.1%respectively; thesero-positive rate of surviving was31.1%,3%,0,0,2.9%,15.15%respectively. Theinduced anti-tumor effect of vaccine targeting survivin and MUC1was evaluated inmurine colorectal cancer and breast cancer models whose sero-positive rate wererelatively high.
The results showed that the AD-MS/S8+9M Pro could significantly inhibittumor growth in murine colon cancer model (P<0.05) and can significantly prolongthe survival time of colon carcinoma-bearing mice (P<0.01). However, AD-MS/S8+9M Pro could significantly inhibit tumor growth in murine breast cancer model(P<0.05) but can’t significantly prolong the survival time of colon carcinoma-bearingmice (P>0.05). The data was in correspondence with the serological test results. Thecombination of vaccine and oxaliplatin had synergistic effects in inhibition andsurvival time prolonging in murine colon cancer model. However, it didn’t havesynergistic effects in murine breast cancer model.
Overall, DDA/MPL possibly increased the protein vaccine induced antigen-specific cellular immuneresponse,the strategy adenovirus early immuned withprotein combined with adjuvant to enhance immunization has the strongest tumorinhibition efficiency, this tumor inhibitory effect is mediatedby Th1type immuneresponses.Survivin and MUC1combined immunization is better than the aloneimmune effects. Combined immunity inhibits tumor growth in the model ofmelanoma, mammary gland carcinoma and colon carcinoma. However, it cansignificantly extend the life of the tumor-bearing mice in nurine melanoma and coloncancer model.
肿瘤疫苗主要是通过诱导机体产生肿瘤抗原特异性的免疫反应来提高机体免疫力，从而杀伤肿瘤细胞。理想的疫苗靶点应该具备以下几个特点：①特异性较强，即只在肿瘤细胞中表达，在正常细胞中不表达或低表达，若在正常组织中有表达，则在肿瘤组织中的表达要有显著的质和量的改变；②具有一定的广谱性，即在多数肿瘤中均有表达；③最重要的是能在肿瘤病人体内能检测到该抗原的特异性抗体和CTL，这表明肿瘤患者已将其视为异物，并对其产生了免疫应答，如用这样的抗原作为靶点设计疫苗将更容易突破机体的免疫耐受。肿瘤抗原Survivin和MUC1同时具备这三个条件，是非常理想的肿瘤疫苗靶抗原，因此，本论文拟采用MUC1和Survivin为靶点研制肿瘤疫苗。
Survivin是IAP(inhibitor of apoptosis protein)家族成员之一，具有抗凋亡的功能，广泛表达于肿瘤组织中而在正常的终末分化组织中不表达。研究表明，survivin的表达水平与病人的耐药，预后不良和生存期缩短之间有一定的关系，这使得survivin成为肿瘤疫苗的理想靶抗原。目前已报道的靶向survivin的疫苗有多种形式，但是未见survivin蛋白疫苗的报道。这可能是由于单独使用的蛋白疫苗诱导产生的细胞免疫反应较弱。本论文通过对其蛋白疫苗的佐剂和免疫策略的优化来达到增强蛋白疫苗诱导产生细胞免疫的效果。
常用的蛋白佐剂有很多种，包括CpG寡核苷酸，弗氏佐剂，Al(OH)3，MPL(monophosphoryl lipid A)等。DDA/MPL (MPL formulated with cationic dimethyldioctadecylammonium)佐剂由于能够显著增强疫苗的抗原提呈能力和疫苗的保护力而被用于结核和衣原体疫苗的研究中。目前DDA/MPL用做增强肿瘤疫苗的佐剂还未见报道。异源的prime-boost策略可以通过应用不同的抗原提呈系统增强蛋白疫苗的细胞免疫水平。本论文选用了DDA/MPL和Al(OH)_3作为蛋白疫苗的佐剂并联合应用重组腺病毒疫苗和蛋白疫苗的异源免疫策略形式来增强survivin抗原特异性的细胞免疫反应和抗肿瘤效应（出于疫苗安全性方面的考虑，本论文使用缺失了survivin蛋白抗凋亡功能的S8蛋白形式）。
研究结果显示，单独使用survivin的蛋白疫苗并不能诱导小鼠产生有效的细胞免疫，但DDA/MPL佐剂能显著提高survivin蛋白的抗原特异性细胞免疫反应。同时，与其它免疫策略相比，重组腺病毒初免，蛋白联合佐剂加强的免疫策略能够最大程度地抑制小鼠黑色素瘤的生长。对抗肿瘤效应机制的研究表明，这种抗肿瘤效应主要是由Th1型免疫反应介导的。
MUC1是粘蛋白家族成员之一，是一个跨膜的糖蛋白，其膜外的VNTR区是它主要的免疫原性区域，目前已在很多类型的肿瘤患者体内发现其蛋白的表达，是肿瘤治疗的理想靶抗原。目前许多针对MUC1VNTR区的疫苗正在进行临床实验。本论文应用重组腺病毒初免蛋白联合佐剂加强的免疫策略，评价了MUC1蛋白疫苗的免疫原性及其对黑色素瘤荷瘤小鼠的肿瘤抑制效应。结果显示，采用重组腺病毒初免，MUC1蛋白联合DDA/MPL佐剂加强的免疫策略能够产生有效的抗原特异性免疫反应，且能有效抑制肿瘤生长并延长荷瘤小鼠的生存时间，生命延长率约为33%。由于肿瘤细胞的异质性，应用单一抗原位点的疫苗不一定能够引起足够的肿瘤抑制效应。本论文评价了survivin与MUC1联合应用后对黑色素瘤荷瘤小鼠的肿瘤抑制效应和生命延长情况，结果表明，两种肿瘤相关抗原联用与二者单独使用时相比，尽管不能减缓小鼠肿瘤生长的速度，但是可以延长荷瘤小鼠的生存时间，与AD-S8/S8pro组相比生命延长率为8.82%；与AD-9M/9M pro组相比生命延长率为2.78%。
由于肿瘤细胞的异质性和肿瘤微环境的复杂性，很多单独使用的肿瘤疫苗临床实验宣告失败。研究表明，生物疗法与其他的肿瘤治疗方法的组合，比如化药治疗，可能会更有效。本论文中，我们采用了化疗药物奥沙利铂与疫苗联合应用的方式来治疗肿瘤，并且观察疫苗与奥沙利铂联合使用后荷瘤小鼠的生命质量是否有改善。结果显示，与PBS组相比，奥沙利铂组，疫苗组和联用组的抑瘤率分别为29.64%，42.59%和54.68%；生命延长率分别为22.22%，33.33%，48.15%。表明奥沙利铂可以增强survivin与MUC1联合疫苗对黑色素瘤荷瘤小鼠的抗肿瘤作用，且二者联用组小鼠体重较奥沙利铂组有一定程度的增大，表明疫苗可以减轻奥沙利铂对小鼠的副作用。
为了初步探索一下以survivin和MUC1为靶点的蛋白疫苗的肿瘤适应症，我们对乳腺癌，肠癌，卵巢癌，食管癌，肺癌，胃癌六种血清样本中survivin和MUC1的抗体进行了检测，结果发现不同类型肿瘤的血清样本survivin和MUC1抗体表达水平不同。肠癌，胃癌，肺癌，卵巢癌，食管癌，乳腺癌患者血清样本的MUC1抗体阳性率分别为18.5%,12%，2%,0,0,10.1%；survivin抗体的阳性率分别为31.1%,3%,0,0,2.9%,15.15%。可以看出，在肠癌和乳腺癌患者体内均产生了针对MUC1和survivin的特异性抗体，而且肠癌样本中的含量明显高于乳腺癌样本。因此，我们评价了survivin及MUC1联合使用疫苗对肠癌和乳腺癌荷瘤模型小鼠的抗肿瘤效应。
结果表明，AD-MS/S8+9M pro能够明显抑制结肠癌模型小鼠肿瘤生长(P<0.05)且能明显延长荷瘤小鼠的生存期（P<0.01）。对于乳腺癌模型小鼠，AD-MS/S8+9M pro能够抑制肿瘤的生长速度（P<0.01），但是并不能延长小鼠的生存期（P>0.05）。这一结果与肠癌患者血清抗体抗性阳性率要高于乳腺癌患者血清阳性率的结果是相呼应的。对于结肠癌模型小鼠，疫苗与化疗药物奥沙利铂有协同作用，能够协同抑制结肠癌荷瘤小鼠的肿瘤生长速度并延长荷瘤小鼠的生存期。但是对于乳腺癌模型小鼠，二者并没有协同作用。
总的来说，DDA/MPL能够增加蛋白疫苗诱导的抗原特异性细胞免疫反应，重组腺病毒初免蛋白联合佐剂加强免疫的策略具有最强的肿瘤抑制效率，这种肿瘤抑制效应是由Th1型免疫反应介导的。Survivin和MUC1两种抗原联合免疫的效果要优于单独免疫的效果。两种抗原联合疫苗对于黑色素瘤模型小鼠，乳腺癌模型小鼠，结肠癌模型小鼠的肿瘤生长都具有明显抑制作用，并能显著延长黑色素瘤模型小鼠和结肠癌模型小鼠的生命。化药与免疫治疗的联用对于黑色素瘤和结肠癌模型小鼠有显著的协同作用，但是不能增强乳腺癌模型小鼠的抗肿瘤效应。。
Because of the increasment of incidence and mortality year by year, cancer hasbecome one of the most serious diseases to human health. Due to less side-effect andwider range of treatment, biological therapy has now become the fourth mostfrequently used therapy for tumor treatment, except for surgery, chemotherapy andradiation. Many tumor vaccines have been under the clinical trials or permitted to beused for the clinical treatment so far.
The ideal vaccine targets should have the following characteristics:①strongspecificity. The targets are only expressed in tumor cells, but they are not or lowexpressed in normal cells. If they are expressed in normal tissues, the expressionlevels must have a significant change in quality and quantity in tumor tissue.②Thetumor targets must have a certain broad spectrum, which means that they areexpressed in the majority of tumors.③Antigen-specific antibody and CTL can bedetected in cancer patients. It is suggested that cancer patients has been regarded it asa foreign body and produced immune response against it. Such tumor antigens used asvaccine targets will be easier to break immune tolerance. Survivin and MUC1whichare broadly used have all the three conditions above. Therefore, this thesis intends toapply MUC1and Survivin as the targets of cancer vaccine.
Survivin, as a member of IAP families, could both inhibit cell apoptosis andcontrol cell division. Survivin is widely expressed in tumor tissues, but not expressedin terminal devision of normal tissues. It is reported that the expression levels ofsurvivin are associated with drug resistance, poor prognosis and shortened survival,and which make survivin an ideal target antigen of tumor vaccine. The current reportshave revealed that survivin-targeted vaccine has various forms, whereas no report on survivin protein vaccine can be found. This may be due to its poor ability to inducecellular immunity. A suitable immunoadjuvant and optimized immunization strategycan greatly enhance the cellular immune response to this protein vaccine.
In order to get a vaccine of higher immunogenicity and better antitumor effection,our research makes survivin as the target of tumor vaccine, uses the protein form ofS8which has lost the function of anti-apoptosis and optimizes the choice of adjuvantof S8proten and the vaccine immunization strategies.
Immunoadjuvants such as CpG ODN, incomplete Freund's adjuvant, aluminiumhydroxide [Al(OH)3] and monophosphoryl lipid A (MPL) have been used to enhanceresponses to a variety of vaccines. DDA/MPL has been shown to enhance antigenuptake and presentation and induce a significant level of protective immunity inChlamydia and tuberculosis vaccine research. However, whether DDA/MPL canenhance efficacy of tumor vaccines has not been reported. A heterologousprime-boost strategy can enhance the cellular immunity of a protein vaccine byapplying different antigen-presenting systems. DDA/MPL, Al(OH)3and anadenovirus prime-protein boost strategy were applied to enhance the survivin-specificimmunity and anti-tumor immunity of a truncated survivin protein vaccine.(Due tosafety concerns of full-length survivin, an anti-apoptosis protein, this vaccine wasbased on a truncated form of survivin (S8) with a deletion of seven amino acids at itsN-terminus, which was shown to be non-functional in our previous study.)
It was showed that survivin protein used alone couldn’t induce effective cellularimmunity, and DDA/MPL adjuvant induced stronger antigen-specific cellularresponses than Al(OH)3. Furthermore, comparing to other immunization regimens,recombinant adenovirus prime adjuvanted protein boosted immunization showed thestrongest anti-tumor effect in the murine melanoma model. The anti-tumor effect wasmediated by Th1-typed immune response.
MUC1is a transmembrane glycoprotein normally expressed on the apical surfaceof ductal epithelia. A variable number (20–125) of tandem repeats (VNTR) of a20 amino acid sequence which was contained in its extracellular domain is the dominantimmunogenic domain. MUC1(mucin1) is a classic target for cancer immunotherapythat is overexpressed in many adenocarcinomas. Many active immunotherapiestargeting MUC1are in clinical trials. We investigated the immunity response andanti-tumor effect in a murine melanoma model after administration of an AD-9Madenoviral vector prime-recombinant9M protein and DDA/MPL boost. Our resultsdemonstrated that the vaccination induced a significant inhibition of the growth ofB16melanoma in mice and prolonged the survival of B16melanoma–bearing mice.The survival rate was about33%. Due to the heterogeneity of tumor cells, theapplication of a single antigen site vaccine may not be able to induce enough tumorsuppressive effect. Our work evaluated the anti-tumor effect and survival extendanceof the survivin and MUC1combination vaccine in a murine melanoma-bearingmodel. The results showed that the combination could not inhibit the tumor growth,however, it could extend the survival time of tumor-bearing mice. The survival ratewas8.82%compared with AD-S8/S8pro group, and2.78%compared withAD-9M/9M pro group.
Due to the heterogeneity of tumor cells and complexity of tumormicroenvironments, many clinical trials of tumor vaccines alone have failed. It isreported that the administration of other therapies such as chemotherapy along with avaccine may enhance its efficacy. The tumor inhibitory effect of our cancer vaccinewas also tested in combination with a widely used anti-tumor chemotherapy drugoxaliplatin. Compared to PBS group, the tumor inhibition rate of oxaliplatin, vaccine,combination of oxaliplatin and vaccine are29.64%，42.59%and54.68%respectively;the survival rate of tumor-bearing mice are22.22%，33.33%and48.15%respectively.It is suggested that oxaliplatin may help to improve the outcome of survivin-basedimmunotherapy. Moreover, the vaccine also alleviated side effects of thechemotherapy drug oxaliplatin, such as emaciation and poor activity.
To investigate the most suitable cancer types of vaccine targeting survivin andMUC1, antibody levels of serum from breast cancer, colorectal cancer, overian cancer, esophageal cancer, lung cancer and gastric cancer are evaluated. Our results showedthat the antibody levels are different from cancer types. The sero-positive rate ofMUC1of colorectal cancer, gastric cancer, lung cancer overian cancer, esophagealcancer and breast cancer was18.5%,12%，2%,0,0,10.1%respectively; thesero-positive rate of surviving was31.1%,3%,0,0,2.9%,15.15%respectively. Theinduced anti-tumor effect of vaccine targeting survivin and MUC1was evaluated inmurine colorectal cancer and breast cancer models whose sero-positive rate wererelatively high.
The results showed that the AD-MS/S8+9M Pro could significantly inhibittumor growth in murine colon cancer model (P<0.05) and can significantly prolongthe survival time of colon carcinoma-bearing mice (P<0.01). However, AD-MS/S8+9M Pro could significantly inhibit tumor growth in murine breast cancer model(P<0.05) but can’t significantly prolong the survival time of colon carcinoma-bearingmice (P>0.05). The data was in correspondence with the serological test results. Thecombination of vaccine and oxaliplatin had synergistic effects in inhibition andsurvival time prolonging in murine colon cancer model. However, it didn’t havesynergistic effects in murine breast cancer model.
Overall, DDA/MPL possibly increased the protein vaccine induced antigen-specific cellular immuneresponse,the strategy adenovirus early immuned withprotein combined with adjuvant to enhance immunization has the strongest tumorinhibition efficiency, this tumor inhibitory effect is mediatedby Th1type immuneresponses.Survivin and MUC1combined immunization is better than the aloneimmune effects. Combined immunity inhibits tumor growth in the model ofmelanoma, mammary gland carcinoma and colon carcinoma. However, it cansignificantly extend the life of the tumor-bearing mice in nurine melanoma and coloncancer model.
引文
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