提高HPV融合蛋白疫苗疗效策略研究
发布时间：2018-09-01 05:38
【摘要】： 人乳头状瘤病毒(Human papillomavirus, HPV)与全世界5.3%的癌症相关,包括子宫颈癌,部分生殖器肿瘤和头颈肿瘤等。据估计有5千万妇女携带HPV,每年大约有50万妇女患癌症。因此,需要持续的发展治疗HPV相关疾病的策略。目前虽然已有多种肿瘤疫苗进入临床验证,但许多疗效不尽如人意,其原因一方面与肿瘤微环境持续地阻碍临床免疫有关,也与肿瘤细胞中人类白细胞抗原(Human leukocyte antigen, HLA)Ⅰ类分子和抗原提呈分子(Antigen processing machinery,APM)表达低下或缺失,造成逃逸宿主免疫监视密切相关。本研究探讨了通过改善肿瘤微环境来提高HPV治疗性疫苗的疗效,并研究了HPV治疗性疫苗在E7阳性、HLA-Ⅰ类分子表达下调的人舌癌移植瘤中的应用。 肿瘤微环境包括增殖的肿瘤细胞,肿瘤间质,血管,浸润的炎性细胞和各种相关的组织细胞。近年来研究确定髓样抑制细胞(Myeloid derived suppressor cells,MDSCs)为肿瘤诱导的免疫抑制的重要因素之一。肿癌组织中的各种因子阻止了此类细胞的分化和成熟,导致荷瘤小鼠及患者体内积聚了大量的MDSCs,阻碍了抗肿瘤免疫反应。治疗浓度的全反式维甲酸(All-trans retinoic acid, ATRA)能够明显降低荷瘤小鼠和肿瘤患者体内的MDSCs的数量,在体内和体外诱导MDSCs分化成树突状细胞和巨噬细胞。本实验室构建表达的融合蛋白疫苗mE6△/mE7/TBhsp70△在C57BL/6小鼠TC-1移植瘤模型中能够产生抗肿瘤免疫,但是这种抗肿瘤作用不足以抑制较大的TC-1肿瘤的生长。研究发现TC-1肿瘤中聚集了大量的MDSCs, ATRA能够诱导骨髓细胞的正常分化,从而提高了乳头瘤病毒治疗性疫苗的抗肿瘤免疫反应。研究表明,ATRA联合疫苗不仅减少了的MDSCs的数量,还能通过抑制MDSCs上CD80的表达来抑制其功能。此外,产生了大量CD11c+CD80+, CD11c+CD86+, CD11c+MHCII+成熟树突状细胞,增加了E7特异性分泌干扰素-γ(Interferon-γ,IFN-γ)的T细胞的数量及其杀伤活性。 另一个肿瘤免疫逃逸的重要机制是募集和活化的调节性T细胞(Regulatory T cell, Tregs)。这群细胞在正常情况下数量很少,但在恶性肿瘤的病人或动物体内大量扩增。研究显示在TC-1小鼠模型的脾脏和肿瘤中,聚集着大量的抑制性调节T细胞和不成熟的MDSCs。研究提出通过免疫调节剂量的环磷酰胺(Cyclophosphamide,CTX)和ATRA抑制调节T细胞和MDSCs而形成对治疗有利的肿瘤微环境,来增强HPV蛋白疫苗的疗效。正常小鼠给予CTX和ATRA共4周的观察期内,未发现体重改变,脱毛,毛色改变,活动减少或增多等毒性反应特征,说明药物剂量和用药频率是安全的。研究发现先注射CTX,然后免疫并持续14天给予ATRA能够使较大的TC-1肿瘤(200mm3)完全消退,C57BL/6小鼠长期生存并获得记忆性免疫应答。通过优化CTX, HPV融合蛋白疫苗和ATRA的序贯联合的给药时间和给药策略,研究发现在最优时间窗内治疗可以根治TC-1肿瘤,显著的降低了脾脏中的Tregs和MDSCs,对于错过最优治疗窗的荷瘤小鼠通过增强序贯联合治疗强度可以在一定程度上提高免疫应答,伴随着Tregs和MDSCs数量的降低。研究发现不同治疗方式中肿瘤微环境中的多种抑制性因子如IL-10,IL-6,VEGF,激活的STAT3均下调,与治疗效果密切相关。ATRA加CTX也能提高肽疫苗和过继治疗的效果。本研究提示免疫调节剂量的化疗药物和疫苗序贯联合可以通过改善肿瘤微环境来抑制肿瘤生长,为HPV相关恶性肿瘤的免疫治疗提供新的思路。 HPV致癌亚型(HPV16和HPV18)是部分头颈鳞状细胞癌病因之一,尤其是口咽肿瘤。其抗原加工和提呈的改变是头颈鳞状细胞肿瘤免疫逃逸重要机制。研究发现E7相关头颈部鳞状细胞中人白细胞抗原HLA-Ⅰ类分子和抗原加工提呈成分如β2-微球蛋白,低分子量蛋白,甲巯蛋白,抗原处理转运体的表达显著下降,而IFN-γ可以提高这些分子的表达。本研究发现该重组蛋白疫苗NCRT/E7/hsp可以在Hu-PBL-SCID小鼠E7阳性的舌鳞状细胞癌移植瘤中产生人IFN-γ,而不是小鼠IFN-γ。研究表明,NCRT/E7/hsp疫苗在体外能有效地提高头颈部鳞状细胞癌E7特异性的细胞毒性T淋巴细胞的产生,体内通过上调HLA-Ⅰ类分子和抗血管生成明显抑制肿癌生长。 综上,肿瘤是多因素诱导、多基因参与、多阶段发生与演进的极其复杂的病理过程,是一种全身性疾病。传统的肿瘤治疗方法虽已经取得了较大的进展,但对中晚期及复发转移患者的预后没有显著的改善。本研究为HPV相关的恶性肿瘤的免疫治疗以及提高生存率等提供了实验数据和初步理论依据。
[Abstract]:Human papillomavirus (HPV) is associated with 5.3% of the world's cancers, including cervical cancer, some genital and head and neck cancers. It is estimated that 50 million women carry HPV, and about 500,000 women suffer from cancer each year. Therefore, strategies for the treatment of HPV-related diseases need to be developed on a sustained basis. Although there are already many cancers Vaccines have been tested clinically, but many therapeutic effects are unsatisfactory. On the one hand, the reasons are related to the persistent obstruction of clinical immunity by tumor microenvironment, and the low or absent expression of human leukocyte antigen (HLA) class I molecules and antigen processing machinery (APM) in tumor cells, resulting in escape. Host immune surveillance is closely related. In this study, we investigated how to improve the efficacy of HPV therapeutic vaccines by improving tumor microenvironment. We also studied the application of HPV therapeutic vaccines in human tongue cancer xenografts with E7 positive and HLA-I down-regulated expression.
Tumor microenvironment includes proliferating tumor cells, tumor stroma, blood vessels, infiltrating inflammatory cells and various related tissue cells. Recent studies have identified Myeloid derived suppressor cells (MDSCs) as one of the important factors for tumor-induced immunosuppression. Factors in tumor tissues prevent these cells. All-trans retinoic acid (ATRA) can significantly reduce the number of MDSCs in tumor-bearing mice and tumor patients, and induce MDSCs to differentiate into dendritic cells and giant cells in vivo and in vitro. Phagocytes. The fusion protein vaccine mE6 (?) / mE7 (?) / TBhsp70 (?) constructed in our laboratory can produce anti-tumor immunity in C57BL / 6 mouse TC-1 transplanted tumor model, but this anti-tumor effect is not enough to inhibit the growth of larger TC-1 tumor. Studies have found that a large number of MDSCs are aggregated in TC-1 tumor, and ATRA can induce positive bone marrow cells. Studies have shown that ATRA combined vaccine not only reduces the number of MDSCs, but also inhibits their function by inhibiting the expression of CD80 on MDSCs. In addition, a large number of CD11c + CD80 +, CD11c + CD86 +, CD11c + MHCII + mature dendritic cells have been produced, which increase E7 specificity. The number and killing activity of T cells secreting interferon gamma (Interferon- gamma, IFN- gamma).
Another important mechanism of tumor immune escape is the recruitment and activation of regulatory T cells (Tregs). These cells are normally small in number, but proliferate in large numbers in patients or animals with malignant tumors. It was suggested that the therapeutic effect of HPV protein vaccine could be enhanced by the inhibition of T cells and MDSCs by immunomodulator Cyclophosphamide (CTX) and ATA. Normal mice received CTX and ATA for 4 weeks showed no changes in body weight, hair loss and hair color. Toxic reactions, such as decreased or increased activity, indicate that the dosage and frequency of drug use are safe. Studies have shown that CTX injection followed by ATRA Administration for 14 days can completely eliminate the large TC-1 tumor (200mm3). C57BL/6 mice survive for a long time and acquire memory immune response. By optimizing CTX, HPV fusion protein vaccine and ATR A sequential combination of administration time and strategy, the study found that the optimal time window treatment can eradicate TC-1 tumors, significantly reducing the spleen of Tregs and MDSCs, missing the optimal treatment window for tumor-bearing mice by enhancing the intensity of sequential combination therapy can improve the immune response to a certain extent, accompanied by Tregs and MDSCs number. It was found that many inhibitory factors such as IL-10, IL-6, VEGF and STAT3 activated in tumor microenvironment were down-regulated in different treatments, which were closely related to the therapeutic effect. ATRA plus CTX could also improve the effect of peptide vaccine and adoptive therapy. This study suggests that the sequential combination of immunomodulator dosage of chemotherapeutic drugs and vaccines can be modified. Good tumor microenvironment can inhibit tumor growth and provide new ideas for immunotherapy of HPV related malignancies.
HPV carcinogenic subtypes (HPV16 and HPV18) are one of the etiologies of some head and neck squamous cell carcinomas, especially oropharyngeal tumors. The changes of their antigen processing and presentation are important mechanisms of immune escape in head and neck squamous cell tumors. It was found that the recombinant protein NCRT/E7/hsp could produce human IFN-gamma in Hu-PBL-SCID mouse E7-positive tongue squamous cell carcinoma xenografts, not mouse IFN-gamma. SP vaccine can effectively enhance the production of E7-specific cytotoxic T lymphocytes in head and neck squamous cell carcinoma in vitro, and inhibit tumor growth by up-regulating HLA-I molecules and anti-angiogenesis in vivo.
In summary, tumor is an extremely complex pathological process involving multiple factors, multiple genes, and multiple stages of occurrence and evolution, and is a systemic disease. Although great progress has been made in traditional tumor therapy, there is no significant improvement in the prognosis of patients with advanced and recurrent HPV-related malignancies. It provides experimental data and preliminary theoretical basis for disease treatment and improving survival rate.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392
本文编号：2216268
[Abstract]:Human papillomavirus (HPV) is associated with 5.3% of the world's cancers, including cervical cancer, some genital and head and neck cancers. It is estimated that 50 million women carry HPV, and about 500,000 women suffer from cancer each year. Therefore, strategies for the treatment of HPV-related diseases need to be developed on a sustained basis. Although there are already many cancers Vaccines have been tested clinically, but many therapeutic effects are unsatisfactory. On the one hand, the reasons are related to the persistent obstruction of clinical immunity by tumor microenvironment, and the low or absent expression of human leukocyte antigen (HLA) class I molecules and antigen processing machinery (APM) in tumor cells, resulting in escape. Host immune surveillance is closely related. In this study, we investigated how to improve the efficacy of HPV therapeutic vaccines by improving tumor microenvironment. We also studied the application of HPV therapeutic vaccines in human tongue cancer xenografts with E7 positive and HLA-I down-regulated expression.
Tumor microenvironment includes proliferating tumor cells, tumor stroma, blood vessels, infiltrating inflammatory cells and various related tissue cells. Recent studies have identified Myeloid derived suppressor cells (MDSCs) as one of the important factors for tumor-induced immunosuppression. Factors in tumor tissues prevent these cells. All-trans retinoic acid (ATRA) can significantly reduce the number of MDSCs in tumor-bearing mice and tumor patients, and induce MDSCs to differentiate into dendritic cells and giant cells in vivo and in vitro. Phagocytes. The fusion protein vaccine mE6 (?) / mE7 (?) / TBhsp70 (?) constructed in our laboratory can produce anti-tumor immunity in C57BL / 6 mouse TC-1 transplanted tumor model, but this anti-tumor effect is not enough to inhibit the growth of larger TC-1 tumor. Studies have found that a large number of MDSCs are aggregated in TC-1 tumor, and ATRA can induce positive bone marrow cells. Studies have shown that ATRA combined vaccine not only reduces the number of MDSCs, but also inhibits their function by inhibiting the expression of CD80 on MDSCs. In addition, a large number of CD11c + CD80 +, CD11c + CD86 +, CD11c + MHCII + mature dendritic cells have been produced, which increase E7 specificity. The number and killing activity of T cells secreting interferon gamma (Interferon- gamma, IFN- gamma).
Another important mechanism of tumor immune escape is the recruitment and activation of regulatory T cells (Tregs). These cells are normally small in number, but proliferate in large numbers in patients or animals with malignant tumors. It was suggested that the therapeutic effect of HPV protein vaccine could be enhanced by the inhibition of T cells and MDSCs by immunomodulator Cyclophosphamide (CTX) and ATA. Normal mice received CTX and ATA for 4 weeks showed no changes in body weight, hair loss and hair color. Toxic reactions, such as decreased or increased activity, indicate that the dosage and frequency of drug use are safe. Studies have shown that CTX injection followed by ATRA Administration for 14 days can completely eliminate the large TC-1 tumor (200mm3). C57BL/6 mice survive for a long time and acquire memory immune response. By optimizing CTX, HPV fusion protein vaccine and ATR A sequential combination of administration time and strategy, the study found that the optimal time window treatment can eradicate TC-1 tumors, significantly reducing the spleen of Tregs and MDSCs, missing the optimal treatment window for tumor-bearing mice by enhancing the intensity of sequential combination therapy can improve the immune response to a certain extent, accompanied by Tregs and MDSCs number. It was found that many inhibitory factors such as IL-10, IL-6, VEGF and STAT3 activated in tumor microenvironment were down-regulated in different treatments, which were closely related to the therapeutic effect. ATRA plus CTX could also improve the effect of peptide vaccine and adoptive therapy. This study suggests that the sequential combination of immunomodulator dosage of chemotherapeutic drugs and vaccines can be modified. Good tumor microenvironment can inhibit tumor growth and provide new ideas for immunotherapy of HPV related malignancies.
HPV carcinogenic subtypes (HPV16 and HPV18) are one of the etiologies of some head and neck squamous cell carcinomas, especially oropharyngeal tumors. The changes of their antigen processing and presentation are important mechanisms of immune escape in head and neck squamous cell tumors. It was found that the recombinant protein NCRT/E7/hsp could produce human IFN-gamma in Hu-PBL-SCID mouse E7-positive tongue squamous cell carcinoma xenografts, not mouse IFN-gamma. SP vaccine can effectively enhance the production of E7-specific cytotoxic T lymphocytes in head and neck squamous cell carcinoma in vitro, and inhibit tumor growth by up-regulating HLA-I molecules and anti-angiogenesis in vivo.
In summary, tumor is an extremely complex pathological process involving multiple factors, multiple genes, and multiple stages of occurrence and evolution, and is a systemic disease. Although great progress has been made in traditional tumor therapy, there is no significant improvement in the prognosis of patients with advanced and recurrent HPV-related malignancies. It provides experimental data and preliminary theoretical basis for disease treatment and improving survival rate.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392
【参考文献】
相关期刊论文 前1条
1 王安训,许鸿生,连克乾,钟小龙;口腔鳞癌发生与人类乳头状瘤病毒感染关系的Meta分析[J];癌症;2004年09期
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