【作者】 张艳；

【导师】 李荣秀； 刘建华；

【作者基本信息】 上海交通大学 ， 生物学， 2020， 博士

【摘要】 基因突变的积累驱动肿瘤的发生发展。由突变基因编码的变异蛋白(肽)一旦激发出特异的免疫反应,生成变异蛋白(肽)特异的免疫细胞,机体的免疫系统可以特异识别和杀伤肿瘤细胞,控制癌症发展,其中部分患者的治疗可维持长期效果。变异蛋白(肽)是肿瘤组织细胞特有,正常组织细胞中不存在,因此靶向肿瘤变异肽的免疫治疗不会损伤正常组织。肿瘤特异的变异蛋白(肽)是免疫治疗的理想靶标。但是,在癌症生理条件下,肿瘤变异蛋白(肽)自动引发免疫反应的几率低于0.5-1%。目前最先进的新抗原(neoantigen)策略是用计算机人工智能计算变异肽与主要组织相容性复合体-I(major histocompatibility complex-I,MHC-I)的亲和力数据,优选亲和力IC50<500 nmole/L的变异肽作为最有可能引发免疫反应的免疫靶标,称为新抗原(neoantigen)。用优选的新抗原变异肽制备疫苗制剂免疫动物或人体,实际能够引发免疫反应的比例也仅仅为30%,引发CD4+T细胞免疫反应的优选变异肽比例高达60%,引发CD8+T细胞免疫反应的优选变异肽比例仅为16%。鉴于CD8+T细胞是主要的免疫杀伤细胞(CTL),提高免疫治疗效果的优先途径应该是提高肿瘤变异肽,尤其是不属于新抗原的无(弱)免疫反应性变异肽,免疫激发CD8+T细胞免疫反应的强度和效率,是免疫治疗领域急需解决的问题。本论文以小鼠黑色素瘤B16F10为模型,参考文献研究数据,选取实测有强免疫反应的肿瘤变异肽和无(弱)免疫反应的肿瘤变异肽为研究靶标,选富含Th表位的外源载体蛋白DTT(白喉毒素T结构域)或CTB(霍乱毒素B亚单位)作为免疫增强辅助元件,与串联的无(弱)免疫反应肿瘤变异肽(neg neo Ag)融合,构建、制备重组蛋白抗原DTT-neg neo Ag和CTB-neg neo Ag。与免疫佐剂配制成疫苗后免疫小鼠,发现两种蛋白都激发了针对肿瘤变异肽的免疫反应,并在肿瘤预防模型和治疗模型上均检测到非常显著的肿瘤抑制效果。尤其是DTT-neg neo Ag疫苗在肿瘤预防模型上的免疫抑制肿瘤效率达到84%,在低剂量肿瘤细胞接瘤的免疫治疗模型上,完全抑制了肿瘤生长,再次给予高剂量细胞接瘤测试中,仍然观察到33%的小鼠维持无瘤生存状态。以上数据表明,本文设计的用DTT免疫活性蛋白实现了激发原本无(弱)免疫反应的肿瘤变异肽产生免疫应答和治疗肿瘤的效果。比较两种疫苗引发的抗肿瘤效果,证明DTT形式的抗原设计对肿瘤变异肽的免疫增强作用优于CTB。进一步通过用变异肽体外刺激DTT-neg neo Ag免疫的小鼠脾脏细胞,发现促进了CD8+IFN-γ+T增加;DTT-neg neo Ag疫苗干预(治疗)促进了肿瘤内CD8+T细胞和NK细胞的浸润,两种细胞都有杀伤肿瘤细胞的能力。本论文还测试了DTT融合串联肿瘤变异肽的设计对强免疫反应肿瘤变异肽(pos neo Ag)的免疫增强效果。首先设计构建和制备了重组蛋白抗原DTT-pos neo Ag。与免疫佐剂配制疫苗免疫小鼠,同样激发出了针对肿瘤变异肽的免疫应答,增强了CD8+T细胞反应,具有显著的抗肿瘤作用。联合多靶标(DTT-neg neo Ag和DTT-pos neo Ag)的免疫治疗结果显示,多个变异肽靶标联合免疫治疗,比强免疫反应肿瘤变异肽(pos neo Ag)单独免疫治疗的肿瘤抑制效果更优。总之,本论文利用疫苗成份蛋白(DTT或CTB)的强免疫反应性,设计了与个性化肿瘤变异肽联接的融合蛋白抗原,加入免疫佐剂制备研究性疫苗制剂,免疫小鼠激发了对无(弱)免疫反应肿瘤变异肽相当强烈的特异的免疫反应,在肿瘤细胞接廇的免疫治疗模型上表现出突出的治疗效果。本研究建立的疫苗设计技术把无(弱)免疫反应肿瘤变异肽转变为有效治疗的靶标,为肿瘤患者,尤其是低基因突变负荷患者,提供了免疫治疗的机会。研究结果为进一步开发个体化治疗性癌症疫苗新药奠定了基础。更多还原

【Abstract】 Tumorigenesis and cancer development were driven by the accumulation of gene mutations.These corresponding mutant proteins(peptides)are unique to tumor cells.So that the immune response elicited with mutant proteins(peptides)can specifically recognize and kill tumor cells without the concern of autoimmunity risk compared with self proteins.Therefore,tumor-specific mutant proteins(peptides)are ideal targets for immunotherapy.However,only less than 0.5-1%of mutant proteins(peptides)have been identified having triggered immune response of therapy value in cancerous physiology.The current neoantigen strategy is to caculate the affinity of mutant peptide with MHC-I complex,and prioritize the antigenicity of the mutant peptides using sensitive computation algorithms.The top list of mutant peptides most likely to elicite immune reaction are classified as neoantigens.Personalized cancer vaccines with neoantigens have showed great success in clinical trial of late stage melanoma immunotherapy.However,vaccination data showed that only 30%of the neoantigens are confirmed having elicited immune response,and 60%of neoantigens induced CD4+T cell immune responses,only 18%of neoantigens induced CD8+T cell immune responses,which are the CTL killers.So the mutant peptides of no(low)T-cell reactivity are reservoir of therapeutic targets.It is hoped that,if they are made CD8+T cell response,the therapy efficacy of cancer would be improved further.In present study,we designed mutant peptides targeting antigen by fusion multiple copy of no(low)T-cell reactivity mutant peptides(neg neo Ag)in tandem to the C terminal of immunoactive vaccine proteins of the translocation domain of diphtheria toxin(DTT)or Cholera Toxin B subunit(CTB).The immunoactive vaccine proteins contained confirmed universal Th epitopes.It is hoped that the designed antigen vaccine immunization would activate CD4+T help cells first,and in turn promote immune system mount reaction to these non-immunogenic mutant peptides and expand Cytotoxic T-lymphocytes.After formulation with adjuvants and immunization of mice,indeed,it is observed that both DTT-neg neo Ag and CTB-neg neo Ag elicite a strong immune response to non-immunogenic mutant peptides.And the immunization significantly deterred tumor growth in both the preventive tumor model and in therapeutic tumor model.It is further observed that DTT is the better than CTB in eliciting immune reponse to mutant peptides and in therapeutic efficacy.Furthermore,Anti-tumor mechanism was analized.The stimulation the spleen immune cells with mutant peptides promoted CD8+IFN-γ+T number.The tumor infiltrated CD8+T cells and NK cells were enhanced by imminization.The study design and assessed the immune reactivity and therapeutic efficacy of strong T-cell reactivity mutant peptides(pos neo Ag).Data showed that the antigen design worked well with strong T-cell reactivity mutant peptides by generating strong CD8+T immune response similar to no(low)T-cell reactivity mutant peptides(neg neo Ag).The combination of vaccination of both no(low)and strong CD8+T immune response mutant peptides,showed improved efficacy than either alone.In summary,the study achieved success of promotion the immune responses to no(low)and strong CD8+T reactive mutant peptides with vaccine componient proteins(DTT and CTB).The therapeutic efficacy in grafted tumor mouse model showed great promise for cancer patients with low mutation burden.The data accumulated in this study laid foundation to develop more efficacous personilzed cancer vaccines.更多还原