【作者】 于永生；

【导师】 王皓；

【作者基本信息】 第二军医大学 ， 肿瘤学， 2011， 硕士

【摘要】 DNA疫苗在肿瘤免疫治疗中具有巨大的应用前景,临床研究表明接受DNA疫苗免疫的病人能够产生抗原特异性抗肿瘤免疫应答,然而,目前的DNA疫苗免疫极少能诱导持久高效的抗肿瘤免疫反应,这表明DNA疫苗仍需不断优化。DNA疫苗起作用的一个很关键的环节是其表达的蛋白抗原需要被抗原提呈细胞（antigen presenting cells,APC）摄取,然后加工处理并提呈给抗原特异性T淋巴细胞,诱导T细胞活化及增殖。树突状细胞（dendritic cells,DC）是目前体内已知的功能最强的抗原提呈细胞,其能够刺激初始（na（?）ve）T细胞增殖进而启动机体免疫应答,诱导抗原特异性细胞免疫反应。皮下或肌肉注射质粒DNA疫苗后,大部分质粒被注射部位的皮肤及肌肉细胞摄取,其编码的免疫抗原在这些细胞表达或释放。皮肤和肌肉细胞因缺乏MHC II和共刺激分子表达不能有效递呈抗原而不能致敏抗原特异性T细胞,因此DC的抗原摄取效率直接与诱导的免疫反应相关。在DNA疫苗免疫中,DC获取免疫抗原主要有两种途径,一是局部少量DC直接摄取DNA质粒并表达免疫抗原,这一途径相对次要,毕竟局部能够直接摄取质粒DNA的DC比例极低。其二是局部DC摄取皮肤或肌肉细胞释放的免疫抗原或吞噬表达免疫抗原的死亡皮肤肌肉细胞,这是主要途径。因此,我们设想:通过DC靶向性分子（DC表面分子特异抗体或配体）把免疫抗原直接递送至DC将能够有效提高DC摄取抗原效率,进而有效增强DNA疫苗诱导的免疫应答。为验证上述假设,本研究中我们制备了DC靶向性及对照的非靶向DNA疫苗scFv^NLDC-145-HER2/neu和HER2/neu,其中scFv^NLDC-145编码DEC-205分子特异单链抗体片段,DEC-205是DC表面特异表达的吞噬受体,能够有效介导抗原内吞从而进入抗原加工递送通路;HER2/neu为原癌基因,编码HER2/neu生长因子受体,在乳腺癌等多种肿瘤中过表达,其直接促进肿瘤生长及发展。实验表明:DC靶向性DNA疫苗能够把编码的免疫抗原有效的递送至DC进而被加工递呈;在HER2/neu阳性的移植乳腺癌动物模型中,与非靶向HER2/neu DNA疫苗相比,DC靶向疫苗诱导了显著增强的HER2/neu特异细胞和体内免疫应答,能够有效预防HER2/neu阳性小鼠肿瘤生长,并产生抗原特异性免疫记忆;更重要的是,DC靶向疫苗联合低剂量环磷酰胺介导的调节性T细胞短暂清除,能够消除已生成的HER2阳性移植乳腺肿瘤,延缓BALB-neuT转基因小鼠中自发性乳腺肿瘤生长。第一部分:DNA疫苗的构建及表达一、DNA疫苗的构建:单链抗体片段NLDC-145(scFv^NLDC-145)通过参考其他学者前期发表的文章全基因合成。HER2/neu的胞外区片段分别从SK-BR-3或TUBO乳腺癌细胞系扩增出,测序正确后两胞外区分别与scFv^NLDC-145经overlapping PCR方法构建出scFv^NLDC-145-HER2和scFv^NLDC-145-neu,两PCR产物装入pGEM-Teasy载体扩增。我们用HindIII和XbaI双酶切上述载体分别获得scFv^NLDC-145-HER2和scFv^NLDC-145-neu片段,然后装入经相同酶切的pcDNA3.1（+）中成功构建出含上述两片段的表达载体。二、DNA疫苗的融合蛋白表达:抽提上述构建的表达载体质粒,瞬时转染293T细胞,72h后收获上清,用WB方法检测质粒疫苗的蛋白表达,两靶向疫苗表达的融合蛋白比预期的稍大,推测为融合蛋白在真核表达系统中存在糖基化的原因。第二部分:DNA疫苗的体内靶向性研究一、构建pcDNA3.1-scFv^NLDC-145-EGFP表达载体:从质粒载体pEGFP-N1中克隆出EGFP基因,然后用EGFP基因替代上述表达载体scFv^NLDC-145-HER2中HER2片段,构建成scFv^NLDC-145-EGFP表达载体。二、scFv^NLDC-145体内靶向性检测:用电击免疫的方法在BALB/c小鼠右后大腿肌肉注射scFv^NLDC-145-EGFP质粒,以PE标记的CD11c阳性细胞设门,用流式细胞术检测EGFP绿色荧光,结果显示scFv^NLDC-145具有良好的DC靶向性。第三部分:BALB/c小鼠体内抗肿瘤作用研究一、预防性抗肿瘤作用: BALB/c小鼠随机分为5组,对应每组分别用电击免疫的方法在小鼠右后大腿肌肉注射scFv^NLDC-145-HER2,scFv^NLDC-145-neu,HER2,neu和空载体pcDNA3.1,每只50μg/50μL。两周之后再用相同的方法免疫第二次。BALB/c小鼠经过两次免疫一周后,在相反侧背部皮下注射D2F2/E2细胞（D2F2/E2是稳定表达HER2的人的乳腺癌细胞株）,后观察并记录肿瘤生长情况。结果显示,靶向的scFv^NLDC-145-HER2组可有效预防肿瘤的生长,此组在120天时100%小鼠都成活,而其他组在80天时已全部死亡。有意思的是scFv^NLDC-145-neu组比非靶向的HER2,neu和pcDNA3.1组稍微有效的延缓了肿瘤生长。二、治疗性抗肿瘤作用:首先验证DNA疫苗单独应用的抗肿瘤作用,BALB/c小鼠背部皮下接种D2F2/E2细胞,当肿瘤直径约40mm³时,上述方法两次免疫小鼠,观察肿瘤生长情况。结果显示,scFv^NLDC-145-HER2组比其他组虽然有明显的抑瘤效果,但到120天时该组也才只有20%小鼠生存,所以scFv^NLDC-145-HER2单独应用作用有限。随后我们验证DNA疫苗联合应用环磷酰胺抗肿瘤作用,BALB/c小鼠接种D2F2/E2细胞,当肿瘤直径约3-4mm时,腹腔注射低剂量的环磷酰胺（100mg/kg）,四天后小鼠按照上述方法免疫两次,后观察肿瘤生长情况。结果显示,scFv^NLDC-145-HER2联合环磷酰胺组在120时能显著抑制80%小鼠肿瘤生长并能使肿瘤体积减小至消除,其他各联合应用组也比对应的单独应用质粒免疫有好的抑瘤效果。第四部分:DNA疫苗的抗肿瘤机制研究一、诱导HER2特异性T细胞反应:上述方法两次免疫BALB/c小鼠,一周后,取脾单细胞分别用HER2或TRP2蛋白体外刺激3天,通过液相闪烁计数仪检测后可知scFv^NLDC-145-HER2组经HER2蛋白刺激的比TRP2蛋白刺激的有显著的T细胞增殖。同样都是经HER2蛋白刺激的各组比较,结果显示scFv^NLDC-145-HER2组也比其他组有显著的T细胞增殖,而scFv^NLDC-145-neu组也比其他各非靶向对照组的T细胞增殖稍显著。ELISA检测上述经HER2蛋白刺激的各组的培养脾细胞的血清,scFv^NLDC-145-HER2组可检测IFN-γ, TNF-α的高表达,同样scFv^NLDC-145-neu组IFN-γ, TNF-α的表达也略高于其他各非靶向对照组。流式检测经HER2肽段刺激的scFv^NLDC-145-HER2组有CD4+ IFN-γ+,CD4+ TNF-α+,CD8+ IFN-γ+和CD8+ TNF-α+ T细胞高表达。二、诱导HER2特异性抗体反应:收集上述各组的血清,ELISA可检测到scFv^NLDC-145-HER2组比其他各组有高滴度的HER2特异性抗体,且主要为IgG2a亚型。另上清与D2F2/E2细胞孵育,后与FITC标记的二抗结合孵育后流式检测scFv^NLDC-145-HER2组D2F2/E2特异性抗体明显高于其他组,而scFv^NLDC-145-neu也略高于各非靶向组。第五部分:scFv^NLDC-145-neu在BALB-neuT小鼠中抗肿瘤作用研究一、预防性抗肿瘤作用:建立稳定表达neu抗原的小鼠乳腺癌细胞株TUBO可移植肿瘤模型;在BALB-neuT小鼠经过两次免疫一周后,在相反侧背部皮下注射TUBO细胞,后观察肿瘤生长情况。结果显示,靶向的scFv^NLDC-145-neu组可预防肿瘤的生长,此组在120天时约90%小鼠都成活,而其他组在不到60天时已全部死亡。二、抗小鼠自发性乳腺癌作用:BALB-neuT小鼠是表达neu抗原的可自发性生长乳腺癌的小鼠,在小鼠8周和10周时用电击免疫的方法肌肉注射scFv^NLDC-145-neu+CTX , scFv^NLDC-145-neu , neu , pcDNA3.1和PBS ,其中scFv^NLDC-145-neu+CTX组在第一次免疫的4天前注射低剂量的环磷酰胺（100mg/kg）,再肌肉注射scFv^NLDC-145-neu,后观察各组小鼠自发性肿瘤个数。结果显示,scFv^NLDC-145-neu联合环磷酰胺组可有效延缓自发性肿瘤生长,比其他组延缓约4周生长肿瘤。到第38周时,环磷酰胺联合应用组小鼠还都活着,而非靶向对照组在26周时就已死亡或肿瘤体积超过1500mm³,另scFv^NLDC-145-neu单独应用组效果也好于其他各非靶向组,该组绝大部分小鼠在30周时还生存着。结论:综上所述我们构建的DC靶向性的DNA疫苗在小鼠体内可以把肿瘤特异性抗原靶向到DC表面,从而引起抗原特异性的细胞和体液免疫,更重要的若联合低剂量的环磷酰胺短暂清除调节性T细胞后,其作为治疗性疫苗能在小鼠体内起到较好的抗肿瘤作用,从而为肿瘤治疗提供一个潜在可行的治疗策略。更多还原

【Abstract】 Although DNA vaccine holds a great potential for cancer immunotherapy,effective long-lasting antitumoral immunity sufficient for inducing objective responses in cancer patients remains to be achieved. Clinical trials for DNA vaccines have shown that immune responses can be generated in humans, but they also highlight the need for increased potency if this vaccine technology is to be effective. A critical initial step in DNA vaccination is that encoded proteins need to be taken up,processed,and presented by dendritic cells（DC）,which are most potent antigen-presenting cells that govern many aspects of the immune response. Dendritic cells are effective in activating host immune response through initiation of na（?）ve T cell activation,and they play an important role in induction of host anti-tumor specific immunity. Following intradermal or intramuscular injection of a plasmid DNA vaccine in mice,the encoded gene is expressed in transfected keratinocytes and myocytes at the site of injection as well as a small number of DC. Keratinocytes and myocytes are poorly effective at presenting antigen and priming naive immune cells due to lack of expression of MHC class II and costimulatory molecules,and they do not have ready access to T cells in lymphoid tissues, as is the case for DCs. There are two ways for DCs to obtain the immune antigen expressed by DNA vaccine, one of which is DCs absorb the immune antigen directly, but only a small percentage of the DCs injected in current trials actually migrate from the injection site into the draining lymph node to present the antigen to T cells. Also, the local DCs are able to absorb the immune antigen released by skin and muscle cells, or to devour the dead skin and muscle cells which express the immune antigen. The latter is thought as the main way.To increase the efficacies of DNA vaccine,we prepared dendritic cel（lDC）-targeting DNA vaccines by fusing tumor-associated antigen HER2/neu ectodomain to single chain antibody fragmen（tscFv）from NLDC-145(scFv^NLDC-145), a monoclonal antibody binding the mouse DC-restricted surface molecule DEC-205,and explored its antitumoral efficacy and underlying mechanisms in mouse breast cancer models. Compared with untargeted HER2/neu DNA vaccines,scFv^NLDC-145-HER2/neu vaccination markedly enhanced the HER2/neu-specific cellular and hormonal immune responses,resulting in much more efficient protection against tumor challenge and induction of long-lasting memory immune response in HER2/neu-expressing transplantable breast cancer models. More importantly, in combination with temporary depletion of regulatory T cells by low-dose cyclophosphamide, vaccination with scFv^NLDC-145-HER2/neu induced the regression of established tumor in HER2-positive transplantable breast cancer model and significantly retarded the development of spontaneous mammary carcinomas in transgenic BALB-neuT mice.Part One: Construction and expression of DNA vaccines1. Construction of DNA vaccines. The genes encoding the variable regions of the heavy （VH） and light （VL） chains of scFv^NLDC-145 were synthesized according to the published sequences. The sequence encoding for the extracellular domain of human HER2 or its rat homologue neu was amplified from cDNA of SK-BR-3 and TUBO cell lines.After that, overlapping PCR was performed to generate scFv^NLDC-145-HER2 and scFv^NLDC-145-neu. The PCR products of scFv^NLDC-145-HER2 and scFv^NLDC-145-neu were cloned into pGEM-Teasy vector to generate pGEM-scFv^NLDC-145-HER2 and pGEM-scFv^NLDC-145-neu, and sequence verified. scFv^NLDC-145-HER2 and scFv^NLDC-145-neu gene removed from pGEM-scFv^NLDC-145-HER2 and pGEM-scFv^NLDC-145-neu were doublely digested by HindIII and XbaI endonucleases and cloned into pcDNA3.1（+） expression vector previously digested with the same enzyme to creat pcDNA3.1- scFv^NLDC-145-HER2 and pcDNA3.1- scFv^NLDC-145-neu, and sequence verified.2. Expression of DNA vaccines. The pcDNA3.1-scFv^NLDC-145-HER2 and pcDNA3.1- scFv^NLDC-145-neu plasmids were transfected into 293T cells by the use of Lipofactamine 2000 according to the manufacturer’s protocol. After 72 hours, the supernatants were collected and assayed for fusion expression by Western blotting, and their molecular weights were a little bit larger than predicted, indicating certain extent of glycosylation.Part two: In vivo targeting assay1. Construction of pcDNA3.1-scFv^NLDC-145-EGFP. We generated pcDNA3.1-scFvNL DC-145-EGFP by replacing the HER2 fragment with EGFP sequence cloned from pEGFP-N1 plasmid.2. scFv^NLDC-145 targets antigen to DC in vivo. The 50μg pcDNA3.1-scFv^NLDC-145-E GFP and pcDNA3.1-EGFP plasmids in 50μL PBS were injected into the upper leg muscle of the left hind limb of the mice followed by in vivo electroporation as described previously. The GFP fluorescence in the CD11c-positive DC was analyzed by flow cytometry . Part three:Antitumor Activity of DNA vaccines in BALB/c mice1. Protection of mice from challenge with HER2-expressing tumor cells. For prophylactic vaccination, female BALB/c mice were vaccinated on days -21 and -7 by intramuscular injections of 50μg pcDNA3.1-scFv^NLDC-145-HER2/neu, pcDNA3.1-HER2 /neu plasmid DNA in 50μL PBS as described above. As control, 50μL pcDNA3.1 were injected. On day 0, animals were inoculated subcutaneously （s.c.） with 2×10⁵ D2F2/E2 tumor cells in the opposite flank. Tumor growth was monitored with a caliper by measuring two perpendicular tumor diameters every week. The date show that scFv^NLDC-145-HER2 vaccination protected mice from D2F2/E2 tumor challenge, resulting in 100% survival in all mice during the observation period （120 days after vaccination）.2. Therapeutic efficacy of scFv^NLDC-145-HER2 vaccine. We next evaluated the therapeutic effect of scFv^NLDC-145-HER2 vaccination on established tumors in D2F2/E2 breast tumor model. BALB/c mice were subcutaneously inoculated with D2F2/E2 tumor cells. On day 7, animals with tumors sizing 40 mm³ were randomized into groups treated with scFv^NLDC-145-HER2 or respective controls. Treatment was repeated once 2 week later. The date show that scFv^NLDC-145-HER2 vaccination substantially slowed tumor development and protected up to 20% of the mice from tumor growth at the end of experiment （120 days after tumor inoculation）. We therefore tested DC-targeted vaccines in combination with Treg depletion by low-dose CTX. As we expected, this combination significantly improved the therapeutic effects of scFv^NLDC-145-HER2 vaccine; at the end of experiment, 80% mice rejected the established tumor. Untargeted DC vaccines failed to exert therapeutic effects although this vaccine in combination with CTX mildly delayed tumor growth.Part four: Evaluation of T-cell and antibody responses1. Induction of HER2-specific T cells. To analyze the nature of the immune responses induced by scFv^NLDC-145-HER2, splenocytes were isolated from the vaccinated mice and cultured in the presence of recombinant HER2 or TRP2 protein for 3 days in vitro. Splenocytes obtained from scFv^NLDC-145-HER2-treated mice showed vigorous proliferation upon restimulation with recombinant HER2 protein, but not TRP2 protein. A slightly increased proliferation was also detected in the splenocytes from scFv^NLDC-145-neu-vaccinated mice. In contrast, no evident T-cell proliferation could be observed when mice were vaccinated with untargeted HER2 or neu. The supernatants of stimulated T cells were tested for the presence of cytokines by ELISA. Splenocytes obtained from scFv^NLDC-145-HER2-vaccinated mice produced substantial amounts of TNF-αand IFN-γ,similarly, a mildly higher level of IFN-γand TNF-αcytokine was also detected in the supernatant from scFv^NLDC-145-neu-vaccinated mice. Next, we checked for the induction of HER2-specific CD4+ and CD8⁺ T cells. The results show that a much higher percentage of CD4⁺ and CD8+ T cells producing IFN-γand TNF-αwas detected upon in vitro restimulation with HER2-derived synthetic p63-71 peptide TYLPTNASL in splenocytes from scFv^NLDC-145-HER2-vaccinated mice. 2. Induction of HER2-specific antibody. Vaccination with scFv^NLDC-145-HER2induced a high titer of HER2-specific antibody specifically binding to recombinant HER2 protein in ELISA experiments. Detailed analysis of antibody isotype demonstrated that antibody induced by scFv^NLDC-145-HER2 vaccine was mainly IgG2a, which is consistent with the cytokine profile of splenocytes.Part five: Antitumoral activity of the scFv^NLDC-145-neu DNA vaccine in immunotolerant BALB-neuT mice1. Preventive efficacy of scFv^NLDC-145-neu vaccine in BALB-neuT mice. We first evaluated the preventive efficacy of scFv^NLDC-145-neu vaccine using transplantable neu-expressing TUBO tumor model in BALB-neuT mice. BALB-neuT mice received twice scFv^NLDC-145-neu or control vaccination at 14 days interval. One week after last vaccination, the animals were challenged with TUBO tumor cells. The date show that the animals receiving scFv^NLDC-145-neu vaccination were significantly protected against a subsequent challenge with TUBO cells. Sixty days after tumor challenge, 80% mice in this group remained tumor free and the other groups all died.2. The effect of scFv^NLDC-145-neu vaccination in the prevention of spontaneous mammary tumors in BALB-neuT mice. The scFv^NLDC-145-neu was given to the mice at week 8 from birth when diffuse atypical hyperplasia is already evident in the mammary glands but before in situ carcinoma is evident and repeated at week 10. Mice in one group also received CTX injection 4 days before the first vaccination. As we expected, scFv^NLDC-145-neu/CTX vaccination resulted in a significant prolongation of tumor-free survival. This corresponded with a marked delay （⁴ weeks） in the appearance of macroscopically detectable tumors in the mammary glands of these mice. By week 38, all of the mice that were vaccinated with scFv^NLDC-145-neu/CTX remained alive. In contrast, by week 26, all of the mice in the control groups had large tumors and required euthanasia. We again observed a mildly protective effect in scFv^NLDC-145-neu vaccination group, in which the survival of most mice delayed to week 30.ConclusionThe results demonstrate that DC-targeted DNA vaccines for in vivo direct delivery of tumor antigens to DC can induce potent antigen-specific cellular and humoral immune responses, when in combination with systemic depletion of regulatory T cells, mount an impressively therapeutic antitumoral activity,providing a rationale for further development of this approach for cancer treatment.更多还原