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水滑石纳米佐剂在肿瘤免疫治疗中的应用研究
张凌宵
Thesis Advisor刘瑞田
2020-07-01
Degree Grantor中国科学院大学
Degree Name博士
Degree Discipline生物化工
Keyword水滑石,肿瘤治疗性疫苗,纳米佐剂,肿瘤免疫耐受,疫苗免疫途径
Abstract

肿瘤治疗性疫苗可诱导机体特异性免疫杀伤肿瘤细胞,抑制恶性肿瘤的生长、转移及复发,具有良好的临床应用前景。然而,由于可高效诱导T细胞免疫反应的疫苗佐剂不足,同时由于肿瘤免疫耐受的存在和疫苗在体内递送效率低下等原因,造成肿瘤治疗性疫苗治疗效果不理想,严重限制了疫苗的临床应用。针对这些问题,本文使用了一种新型铝佐剂(镁铝水滑石,LDH),并联合应用免疫调节分子,设计得到了三类能够抵抗肿瘤抑制性免疫环境和肿瘤抗原多发性突变的多功能肿瘤治疗疫苗,并提出了新的疫苗皮下递送或注射途径优化策略,显著提高了疫苗在体内诱发免疫应答的效率,提高了疫苗的免疫治疗效果。具体开展的研究工作如下:1. 研究制备的能够共递送IDO抑制剂(siIDO)和抗原Trp2的LDH纳米疫苗(TLIs)有效缓解树突状细胞(DC)的免疫抑制状态,促进抗原特异性免疫应答。在肿瘤患者体内,肿瘤细胞释放的促炎性因子导致DC胞内吲哚胺2,3-双加氧酶(IDO)过度表达,进而影响色氨酸代谢,并造成DC由免疫激活态转变为免疫抑制态。TLIs具备快速的内涵体逃逸能力,能够迅速将siIDO与Trp2释放到细胞质。一方面,siIDO通过干扰IDO mRNA的表达实现IDO的下调,促进DC的激活;另一方面,Trp2与主要组织相容性复合体(MHC)I分子结合,并被呈递至细胞表面,从而有效促进抗原提呈、激活特异性T细胞免疫应答并对肿瘤组织进行杀伤。2. 研究制备的同时载有肿瘤经典抗原表位Trp2、突变表位M27和M30及CpG的LDH三价疫苗(s-BmALC),可引发广谱的抗肿瘤免疫应答,显著提升疫苗对肿瘤的杀伤效率。s-BmALC在被DC摄取后,能够在内涵体的酸化过程中释放CpG与抗原表位,并快速逃逸至细胞质中。其中,释放的CpG与内涵体TLR-9结合,促进DC细胞的激活;释放的抗原在细胞质中被进一步加工后与MHC I类分子结合,并被分别呈递至细胞表面,激活广谱的抗原特异性T细胞免疫应答。相对于传统的单价疫苗,三价疫苗能够更广谱地诱导T细胞免疫应答,更好地抑制肿瘤的生长,降低由于肿瘤细胞表面抗原持续突变与丢失所造成的抗原特异性T细胞免疫攻击脱靶的问题。3. 研究制备的联合光热疗法和化学疗法的多功能LDH纳米佐剂诱发强烈的原位抗肿瘤免疫应答。通过将FDA批准的吲哚菁绿(ICG)、盐酸表阿霉素/DNA复合前药(Dox/DNA)和CpG装载于LDH中得到多功能LDH纳米佐剂(IDCB-LDH)。IDCB-LDH能够高效地富集于肿瘤内,在808 nm激光的诱导下,ICG能够快速加热肿瘤至~55 °C,对肿瘤进行热杀伤。在温度升高的环境中,Dox/DNA中的DNA(Tm=40.5 °C)快速解旋并释放Dox,对肿瘤细胞进一步杀伤,释放大量肿瘤抗原。随后,抗原被LDH/CpG在原位结合并呈递至DC,引发强烈的抗原特异性T细胞应答,有效清除原位残余肿瘤组织,并预防肿瘤的复发与转移。 4. 研究阐明了LDH疫苗的分散性与其诱发抗肿瘤免疫应答效率之间的关系。基于“白蛋白包被技术”,可控地制备得到在生理环境下处于单颗粒分散状态的LDH疫苗(s-BTLC)和聚集状态的LDH疫苗(a-BTLC)。研究表明,s-BTLC可主动浸润淋巴结(LNs),短时间内促进疫苗被更多的抗原提呈细胞(APC)摄取,从而显著提升疫苗诱发细胞免疫应答的效率。相对地,聚集的a-BTLC则主要停留在注射位点,浸润LNs效率低下,从而无法有效诱发细胞免疫应答。5. 脾脏靶向性LDH疫苗可快速诱发强烈的抗肿瘤免疫应答。恶性肿瘤通常生长迅速且致死率高,对疫苗的时效性要求较高,而当前大多数肿瘤治疗性疫苗对于中晚期的实体瘤治疗效果较差。通过静脉注射(IV)优化的LDH疫苗能够迅速富集于脾脏,并诱发强烈的T细胞免疫应答。在这基础上,通过联合皮下注射(SC)的方式,设计了一种新型的疫苗注射策略IV+SC。即在肿瘤早期通过IV途径注射疫苗以快速引发免疫应答,随后通过SC途径注射疫苗来持续刺激免疫系统,实现“快速强效免疫反应”与“长期免疫激发”的结合,从而更高效地抑制肿瘤生长。更有意义的是,将疫苗以IV和SC途径同时注射,能够引发更强烈的T细胞免疫应答,显著抑制晚期肿瘤的生长。 ;Cancer therapeutic vaccine can promote body forms specific anti-tumor immunity to kill tumor cells, inhibit the growth, metastasis and recurrence of malignant tumors, showing ideal clinical application prospects. However, little vaccine adjuvants can efficiently induce T-cell immune responses, as well as the tumor immune tolerance and poor vaccine in vivo delivering process, resulting in limited therapeutic efficacy and clinical application of cancer therapeutic vaccines. To address these issues, a new type of aluminum adjuvant (magnesium-aluminum laydered double hydroxide; LDH) combined with immunoregulatory molecules was used in this study. Basedly, three kinds of multifunctional cancer therapeutic vaccines which aim to overcome tumor immune tolerance and loss or mutation of cancer epitopes, as well as optimized vaccine subcutaneous delivery and novel administration routes were developed, significantly enhancing the immune induction efficiency and therapeutic efficacy of cancer therapeutic vaccine. The specific research work is as follows:1. The LDH nanovaccines (TLIs) co-delivered with IDO inhibitor (siIDO) and the melanoma antigen Trp2 were prepared to alleviate the immunosuppressive status of dendritic cells (DC) and promote antigen-specific immune responses. In tumor patients,pro-inflammatory cytokines released by tumor cells leads to over-expression of indoleamine 2,3-dioxygenase (IDO) in DC cells, which in turn affects tryptophan metabolism and changes the DC function from immune activation to immunesuppressive. TLIs have rapid DC endosomal escape ability, and can quickly release siIDO and Trp2 to the cytoplasm. On the one hand, siIDO down-regulates the level of IDO and promotes DC activation by interfering with the expression of IDO mRNA. On the other hand, Trp2 binds with the major histocompatibility complex (MHC) I molecule and is presented to the cell surface, thereby effectively promoting antigen specific T cell immune response to eliminate tumor.2. The LDH trivalent vaccine (s-BmALC) which consists of tumor classical antigen Trp2, mutant epitopes M27 and M30, and CpG, can elicit a broad-spectrum antitumor immune response and significantly improve the therapeutic efficacy of cacner therapeutic vaccine. Once the s-BmALC was internalized by DC, the CpG and epitopes were released during the acidification of the endosome, and then can quickly promote these cargoes escape into the cytoplasm. Especially, the released CpG binds to the endosomal or lysosomal TLR-9 receptor to promote the activation of DC. The released antigens are further processed in the cytoplasm, then bind to MHC class I molecules and are presented by DC to induce a broad spectrum of antigen-specific T cell immune responses. Compared with traditional monovalent vaccines, trivalent vaccines can induce a broader spectrum of T-cell immune responses, more efficienctly inhibit tumor growth and avoid the off-targets of antigen-specific T-cell immune immune response.3. The multifunctional LDH nanoadjuvant combined with photothermotherapy and chemotherapy was prepared to induce a strong in situ anti-tumor immune response. FDA-approved indocyanine green (ICG), doxorubicin/ DNA complex prodrug (Dox/DNA), and CpG were loaded in LDH to obtain a multifunctional LDH nanoadjuvant (IDCB-LDH). IDCB-LDH can be efficiently enriched in tumors, and ICG can rapidly heat tumors to ~ 55 ° C under the induction of a 808 nm laser to thermally kill the tumors. Meanwhile,the DNA in Dox/DNA (Tm = 40.5 ° C) rapidly unwinds and releases Dox as the temperature of tumor increased, further killing tumor cells and releasing a large amount of tumor antigens. Subsequently, LDH/CpG binds these antigens in situ and presents them to DC, triggering strong antigen-specific T cell responses, effectively removing the residual tumor tissue, and preventing tumor recurrence and metastasis.4. The study clarified the relationship between the in vivo dispersity of LDH vaccine and its anti-tumor immune response inducing efficiency. Benefited from the albumin-coating technology, LDH vaccines at monodispersed statue (s-BTLC) and aggregated statue (a-BTLC) under physiological conditions were prepared. The results show that s-BTLC can actively infiltrate into lymph nodes (LNs) and promote the uptake of vaccines by more antigen-presenting cells (APC) in a short time, thereby significantly enhancing the efficiency of cellular immune response inducing efficiency. In contrast, a-BTLC aggregated mainly remained at the injection site and the infiltration of LNs was inefficient, resulting poorer anti-tumor immune response.5. The spleen-targeted LDH vaccine delivery can rapidly induce a strong anti-tumor immune response. Malignant tumors usually grow rapidly and have high lethality, which requires high timeliness of vaccines. However, most of the current cancer therapeutic vaccines are ineffective in the treatment of advanced tumors. Intravenous (IV) injected LDH vaccine can rapidly enrich the spleen and induce a strong T cell immune response. Based on this, a new vaccine administration strategy IV + SC was designed by combined with subcutaneous injection (SC). That is, in the early stage of the tumor, the vaccine is injected through the IV route to rapidly trigger an immune response, and then the vaccine is injected through the SC route to continuously stimulate the immune system to achieve a combination of “rapid and strong immune response” and “long-term immune stimulation”, thereby suppressing the tumor more efficiently. Interestingly, the simultaneous injection of the vaccine by IV and SC routes can trigger a stronger T cell immune response and significantly inhibit the growth of advanced tumors. 

Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/49661
Collection中国科学院过程工程研究所
Recommended Citation
GB/T 7714
张凌宵. 水滑石纳米佐剂在肿瘤免疫治疗中的应用研究[D]. 中国科学院大学,2020.
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