提高HPV融合蛋白疫苗療效策略研究
發(fā)布時(shí)間：2018-09-01 05:38
【摘要】： 人乳頭狀瘤病毒(Human papillomavirus, HPV)與全世界5.3%的癌癥相關(guān),包括子宮頸癌,部分生殖器腫瘤和頭頸腫瘤等。據(jù)估計(jì)有5千萬(wàn)婦女?dāng)y帶HPV,每年大約有50萬(wàn)婦女患癌癥。因此,需要持續(xù)的發(fā)展治療HPV相關(guān)疾病的策略。目前雖然已有多種腫瘤疫苗進(jìn)入臨床驗(yàn)證,但許多療效不盡如人意,其原因一方面與腫瘤微環(huán)境持續(xù)地阻礙臨床免疫有關(guān),也與腫瘤細(xì)胞中人類白細(xì)胞抗原(Human leukocyte antigen, HLA)Ⅰ類分子和抗原提呈分子(Antigen processing machinery,APM)表達(dá)低下或缺失,造成逃逸宿主免疫監(jiān)視密切相關(guān)。本研究探討了通過(guò)改善腫瘤微環(huán)境來(lái)提高HPV治療性疫苗的療效,并研究了HPV治療性疫苗在E7陽(yáng)性、HLA-Ⅰ類分子表達(dá)下調(diào)的人舌癌移植瘤中的應(yīng)用。 腫瘤微環(huán)境包括增殖的腫瘤細(xì)胞,腫瘤間質(zhì),血管,浸潤(rùn)的炎性細(xì)胞和各種相關(guān)的組織細(xì)胞。近年來(lái)研究確定髓樣抑制細(xì)胞(Myeloid derived suppressor cells,MDSCs)為腫瘤誘導(dǎo)的免疫抑制的重要因素之一。腫癌組織中的各種因子阻止了此類細(xì)胞的分化和成熟,導(dǎo)致荷瘤小鼠及患者體內(nèi)積聚了大量的MDSCs,阻礙了抗腫瘤免疫反應(yīng)。治療濃度的全反式維甲酸(All-trans retinoic acid, ATRA)能夠明顯降低荷瘤小鼠和腫瘤患者體內(nèi)的MDSCs的數(shù)量,在體內(nèi)和體外誘導(dǎo)MDSCs分化成樹(shù)突狀細(xì)胞和巨噬細(xì)胞。本實(shí)驗(yàn)室構(gòu)建表達(dá)的融合蛋白疫苗mE6△/mE7/TBhsp70△在C57BL/6小鼠TC-1移植瘤模型中能夠產(chǎn)生抗腫瘤免疫,但是這種抗腫瘤作用不足以抑制較大的TC-1腫瘤的生長(zhǎng)。研究發(fā)現(xiàn)TC-1腫瘤中聚集了大量的MDSCs, ATRA能夠誘導(dǎo)骨髓細(xì)胞的正常分化,從而提高了乳頭瘤病毒治療性疫苗的抗腫瘤免疫反應(yīng)。研究表明,ATRA聯(lián)合疫苗不僅減少了的MDSCs的數(shù)量,還能通過(guò)抑制MDSCs上CD80的表達(dá)來(lái)抑制其功能。此外,產(chǎn)生了大量CD11c+CD80+, CD11c+CD86+, CD11c+MHCII+成熟樹(shù)突狀細(xì)胞,增加了E7特異性分泌干擾素-γ(Interferon-γ,IFN-γ)的T細(xì)胞的數(shù)量及其殺傷活性。 另一個(gè)腫瘤免疫逃逸的重要機(jī)制是募集和活化的調(diào)節(jié)性T細(xì)胞(Regulatory T cell, Tregs)。這群細(xì)胞在正常情況下數(shù)量很少,但在惡性腫瘤的病人或動(dòng)物體內(nèi)大量擴(kuò)增。研究顯示在TC-1小鼠模型的脾臟和腫瘤中,聚集著大量的抑制性調(diào)節(jié)T細(xì)胞和不成熟的MDSCs。研究提出通過(guò)免疫調(diào)節(jié)劑量的環(huán)磷酰胺(Cyclophosphamide,CTX)和ATRA抑制調(diào)節(jié)T細(xì)胞和MDSCs而形成對(duì)治療有利的腫瘤微環(huán)境,來(lái)增強(qiáng)HPV蛋白疫苗的療效。正常小鼠給予CTX和ATRA共4周的觀察期內(nèi),未發(fā)現(xiàn)體重改變,脫毛,毛色改變,活動(dòng)減少或增多等毒性反應(yīng)特征,說(shuō)明藥物劑量和用藥頻率是安全的。研究發(fā)現(xiàn)先注射CTX,然后免疫并持續(xù)14天給予ATRA能夠使較大的TC-1腫瘤(200mm3)完全消退,C57BL/6小鼠長(zhǎng)期生存并獲得記憶性免疫應(yīng)答。通過(guò)優(yōu)化CTX, HPV融合蛋白疫苗和ATRA的序貫聯(lián)合的給藥時(shí)間和給藥策略,研究發(fā)現(xiàn)在最優(yōu)時(shí)間窗內(nèi)治療可以根治TC-1腫瘤,顯著的降低了脾臟中的Tregs和MDSCs,對(duì)于錯(cuò)過(guò)最優(yōu)治療窗的荷瘤小鼠通過(guò)增強(qiáng)序貫聯(lián)合治療強(qiáng)度可以在一定程度上提高免疫應(yīng)答,伴隨著Tregs和MDSCs數(shù)量的降低。研究發(fā)現(xiàn)不同治療方式中腫瘤微環(huán)境中的多種抑制性因子如IL-10,IL-6,VEGF,激活的STAT3均下調(diào),與治療效果密切相關(guān)。ATRA加CTX也能提高肽疫苗和過(guò)繼治療的效果。本研究提示免疫調(diào)節(jié)劑量的化療藥物和疫苗序貫聯(lián)合可以通過(guò)改善腫瘤微環(huán)境來(lái)抑制腫瘤生長(zhǎng),為HPV相關(guān)惡性腫瘤的免疫治療提供新的思路。 HPV致癌亞型(HPV16和HPV18)是部分頭頸鱗狀細(xì)胞癌病因之一,尤其是口咽腫瘤。其抗原加工和提呈的改變是頭頸鱗狀細(xì)胞腫瘤免疫逃逸重要機(jī)制。研究發(fā)現(xiàn)E7相關(guān)頭頸部鱗狀細(xì)胞中人白細(xì)胞抗原HLA-Ⅰ類分子和抗原加工提呈成分如β2-微球蛋白,低分子量蛋白,甲巰蛋白,抗原處理轉(zhuǎn)運(yùn)體的表達(dá)顯著下降,而IFN-γ可以提高這些分子的表達(dá)。本研究發(fā)現(xiàn)該重組蛋白疫苗NCRT/E7/hsp可以在Hu-PBL-SCID小鼠E7陽(yáng)性的舌鱗狀細(xì)胞癌移植瘤中產(chǎn)生人IFN-γ,而不是小鼠IFN-γ。研究表明,NCRT/E7/hsp疫苗在體外能有效地提高頭頸部鱗狀細(xì)胞癌E7特異性的細(xì)胞毒性T淋巴細(xì)胞的產(chǎn)生,體內(nèi)通過(guò)上調(diào)HLA-Ⅰ類分子和抗血管生成明顯抑制腫癌生長(zhǎng)。 綜上,腫瘤是多因素誘導(dǎo)、多基因參與、多階段發(fā)生與演進(jìn)的極其復(fù)雜的病理過(guò)程,是一種全身性疾病。傳統(tǒng)的腫瘤治療方法雖已經(jīng)取得了較大的進(jìn)展,但對(duì)中晚期及復(fù)發(fā)轉(zhuǎn)移患者的預(yù)后沒(méi)有顯著的改善。本研究為HPV相關(guān)的惡性腫瘤的免疫治療以及提高生存率等提供了實(shí)驗(yàn)數(shù)據(jù)和初步理論依據(jù)。
[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.
【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
本文編號(hào)：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.
【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 王安訓(xùn),許鴻生,連克乾,鐘小龍;口腔鱗癌發(fā)生與人類乳頭狀瘤病毒感染關(guān)系的Meta分析[J];癌癥;2004年09期
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