超抗原SEA的GPI锚定修饰和在CHO-dhfr~-细胞膜上的表达

将GPI锚定修饰的免疫分子直接转移到肿瘤细胞膜上,是研究治疗性肿瘤疫苗的一种有潜力的新策略。通过将从衰变加速因子(DAF)来源的GPI修饰性信号序列与SEA嵌合,获得了GPI修饰的SEA分子,构建的真核表达载体pCIGPI-SEA,用脂质体方法转染到CHOdhfr-细胞中,并用氨甲喋呤(MTX)进行筛选,细胞免疫荧光分析证实,SEA能够在细胞膜上表达。上述GPI锚定修饰的SEA,可用于进一步研究治疗性肿瘤疫苗。     

用免疫刺激分子开发治疗性疫苗

将免疫刺激分子加至肿瘤细胞中据称这是一种潜在的诱导抗肿瘤免疫的有用方案。作使用共刺激分子B7—1(CD80)的GPI锚定形式的转移而对分离的肿瘤细胞膜进行修饰,从而开发一种无细胞肿瘤疫苗以期用于临床。分离的肿瘤细胞膜是用已建立的肿瘤细胞系制备出来,作对进行修饰所需的最优条件及临床应用进行了测定。发现于生理温度37℃下GPI—B7—1能够最大程度地转移至人肿瘤膜上并且是以剂量依赖方式进行,GPI—B7—1向分离的膜的转移导致稳定的表达及显示共刺激功能,这些经修饰的膜能够贮存以便进行反复免疫。分离的肿瘤膜可直接从外科手术除的人肿瘤组织制备,通过GPI—B7—1修饰并共刺激T细胞。最终,从肿瘤组织分离获得的膜表达Ⅱ类MHC化,这与同一病人体外建立的细胞系有所不同。这种得自病人肿瘤组织的所分离的膜上表达免疫刺激分子的新型方案将使自体性治疗性癌症疫苗的制备更加有利于那些取其肿瘤细胞系的病人,而这些肿瘤细胞还尚未建立起来。     

PSCA与前列腺癌研究进展

前列腺干细胞抗原(prostate stem cell antigen,PSCA)是一种前列腺癌相关肿瘤抗原,也是一种GPI(gly-cosyl phosphatidylinositol)锚定蛋白,通过其C端的GPI锚定结构锚定到细胞膜表面.PSCA在正常前列腺组织中的表达较低,提高的PSCA表达伴随着增加的肿瘤分期、分级以及雄激素非依赖性和转移癌的形成,且不随癌症进展而降低,是前列腺癌诊断和治疗的理想靶抗原.动物实验显示,PSCA抗体和疫苗可能在前列腺癌免疫靶向治疗中具有重要价值.     

GPI锚固型Met-RANTES真核表达载体的构建和表达

目的：在仓鼠卵巢细胞（CHO细胞）中高效表达糖基磷脂酰肌醇（GPI） 锚定修饰的Met-RANTES融合蛋白,以研制新型免疫抑制分子GPI锚固型 Met-RANTES。方法：构建真核表达载体PEF/GPI-Met-RANTES,利用电转化法转染CHO-DHFRˉ细胞,氨甲喋呤（MTX）筛选抗性克隆。用流式细胞仪、细胞免疫荧光和免疫金标记电镜检测细胞膜上 GPI 锚固型Met-RANTES融合蛋白的表达情况。结果：构建了阅读框完整的 GPI 锚固型Met-RANTES 嵌合分子,经测序是正确的,并在CHO-DHFRˉ细胞膜上稳定表达。结论：在CHO 细胞表面高效表达GPI修饰的Met-RANTES融合蛋白,GPI 锚固型Met-RANTES分子有可能作为新型的免疫抑制剂用于器官移植中,抑制移植排斥反应。     

肾癌特异性抗原G250/MN/CA Ⅸ的克隆及真核表达

目的:克隆人肾细胞癌特异性抗原G250基因,使其能够在真核细胞中表达并锚定修饰于细胞膜上。方法:提取人肾透明细胞癌细胞的总RNA,用RT-PCR扩增出G250基因,插入含有人Igκ链前导信号肽、IgG-Fc和GPI锚定信号肽基因序列的真核表达载体pCI-neo-GPI中;将构建的重组质粒pCI-neo-GPI-G250转染RenCa细胞,利用RT-PCR、Western印迹和免疫荧光检测G250在细胞中的表达。结果:构建的重组质粒pCI-neo-GPI-G250经测序正确;用各种方法对筛选得到的稳定转染细胞进行检测,均可以发现G250的表达,表达部位为细胞膜。结论:克隆出人肾细胞癌特异性抗原G250基因,且G250在RenCa细胞膜上可以正常表达,为进一步研究肾癌肿瘤疫苗的免疫原性提供了必要的前期准备。     

糖基化磷脂酰肌醇锚定型EGFP真核表达质粒的构建及表达

构建与增强型绿色荧光蛋白基因相连的糖基化磷脂酰肌醇(glycosyl phosphatidylinositol,GPI)序列的真核表达质粒,并检测其在A549细胞中的表达.分离人外周血淋巴细胞,提取总RNA,以RT-PCR法扩增CD24基因的243 bp GPI锚定序列,双酶切后定向克隆入pEGFP-C1质粒中,构建并鉴定pEGFP-C1-GPI质粒.经脂质体介导转染A549细胞后,在荧光显微镜下观察目的蛋白在真核细胞内的表达情况.经酶切和测序鉴定证实,所克隆的CD24 GPI序列正确,荧光显微镜观察pEGFP-C1-GPI质粒转染A549细胞可见围绕细胞膜的强绿色荧光,而对照pEGFP-C1质粒转染A549细胞仅见胞内均匀荧光.成功构建与EGFP相连的GPI真核表达质粒,且能在A549细胞膜上锚定表达EGFP-GPI融合蛋白,为构建锚定表达型肿瘤疫苗奠定基础.     

经NGR肽段遗传修饰的携带三种报告基因的腺病毒载体的构建及其实验研究

NGR(Asn-Gly-Arg)是通过噬菌体展示技术筛选出来的能够和肿瘤新生血管特异结合的三肽模体,可以将多种药物分子和病毒载体靶向运输到肿瘤或者进行血管再生的组织中。为此构建了腺病毒衣壳蛋白knob的HI环(HI-loop)经NGR肽段修饰的并同时表达三种报告基因的腺病毒载体Ad5/E1-mCherry/E3-luciferase-2A-eGFP/knob-NGR。体内、外实验研究表明,该病毒载体可成功表达三种报告基因;经NGR肽段修饰的腺病毒载体对人乳腺癌细胞系MDA-MB-231的感染效率高于未经修饰的对照腺病毒Ad5CMVeGFP。该载体的成功构建为进一步研究经NGR肽段修饰的腺病毒在肿瘤动物模型体内的靶向性及经NGR肽段修饰的并携带治疗基因的实验治疗研究奠定了基础。     

多靶点复合抗原抗肿瘤基因疫苗的构建及真核表达

目的：构建含有人存活蛋白（survivin）-2B主要T细胞表位区域、人和猴绒毛膜促性腺激素β链的核心片段CTP37区域融合基因的真核表达质粒,并在人胚肾293T细胞中进行表达。方法：通过基因合成和搭桥PCR技术构建含有Survivin2B主要T细胞表位区域、人和猴CTP37区域基因的融合基因2PAG,将其插入含有人IgK链前导信号肽（sig）、人IgG-Fc和糖基磷脂酰肌醇（GPI）锚定信号肽融合基因序列的细胞膜锚定修饰真核表达载体pCI—Fc—GPI中,继而又将酶切后的sig2PAG-FC-GPI融合基因导入含有细小病毒内部核糖体结合位点（IRES）基因且可以共表达人GM-CSF和B7．1融合基因的真核表达载体pVAX1-IRES-GM／B7中;将构建的重组质粒pVAX1-sig-2PAG-FC-GPI-GM／B7（简称pVAX1-2PFcGB）转染293T细胞,利用流式细胞仪和免疫荧光检测其表达情况。结果：2PAG融合基因经测序正确,PCR和酶切鉴定证明已成功连入真核表达载体pVAX-IRES-GM／B7中;流式细胞仪和免疫荧光的检测结果显示,重组质粒pVAX1-2PFcGB在293T细胞中得到很好的表达。结论：成功构建了重组质粒pVAX1-2PFcGB,且在293T细胞中可以有效表达,为对该基因疫苗的后续功能研究奠定了基础。     

表观遗传学修饰在血液肿瘤中的研究进展

血液肿瘤作为一类常见恶性肿瘤疾病主要包括各类白血病、多发性骨髓瘤以及恶性淋巴瘤.随着现代社会高速发展,人群发病率呈逐年升高趋势,且发病年龄逐渐低龄化,发病原因与环境因素以及遗传因素密不可分.近年来研究发现,表观遗传学修饰在血液肿瘤发生发展的过程中扮演了重要角色,一些相关修饰基因作为血液肿瘤的治疗靶点在临床应用上取得了重要进展.针对近年来表观遗传学修饰在血液肿瘤中的研究新进展,本文将系统综述DNA甲基化、组蛋白修饰、非编码RNA及RNA修饰等在血液肿瘤发病机制方面的研究进展.     

mRNA疫苗：战胜新型冠状病毒感染的重要突破

2023年诺贝尔生理学或医学奖授予医学家卡塔琳·卡里科（Katalin Karikó）和德鲁·韦斯曼（Drew Weissman）,以表彰他们在核苷碱基修饰方面的发现,这些修饰的发现对于开发针对严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的有效mRNA疫苗至关重要。疫苗接种是预防感染性疾病最经济最有效的措施。到目前为止,疫苗已经从灭活疫苗、重组蛋白疫苗进入到了第三代核酸疫苗。两位科学家的研究发现,掺入修饰碱基的体外转录mRNA可以逃避不良的免疫激活,解决了体外转录的mRNA过度引起炎症反应的问题;进一步的研究发现,含假尿苷的mRNA能更有效地进行翻译。同时 德鲁·韦斯曼对于递送系统的研究与发展也做出了重要贡献。新型冠状病毒感染（COVID-19）爆发后,以两位科学家的研究为基础,mRNA疫苗的研发技术体系被完善,在COVID-19疫情期间为人类抗击SARS-CoV-2起到非常重要的作用。本文介绍了疫苗发展的过程、mRNA疫苗中重要的核苷酸修饰和脂质纳米颗粒技术、针对SARS-CoV-2的mRNA疫苗以及技术发展的总结与展望。     

Development of a successful antitumor therapeutic model combining in vivo dendritic cell vaccination with tumor irradiation and intratumoral GM-CSF delivery

Vaccination of dendritic cells (DC) combined with GM-CSF secreting tumor cells has shown good therapeutic efficacy in several tumor models. Nevertheless, the engineering of GM-CSF secreting tumor cell line could represent a tedious step limiting its application for treatment in patients. We therefore developed in rats, an “all in vivo” strategy of combined vaccination using an in vivo local irradiation of the tumor as a source of tumor antigens for DC vaccines and an exogenous source of GM-CSF. We report here that supplying recombinant mGM-CSF by local injections or surgical implantation of osmotic pumps did not allow reproducing the therapeutic efficacy observed with in vitro prepared combined vaccines. To bypass this limitation possibly due to the short half-life of recombinant GM-CSF, we have generated adeno-associated virus coding for mGM-CSF and tested their efficacy to transduce tumor cells in vitro and in vivo. The in vivo vaccines combining local irradiation and AAV2/1-mGM-CSF vectors showed high therapeutic efficacy allowing to cure 60% of the rats with pre-implanted tumors, as previously observed with in vitro prepared vaccines. Same efficacy has been observed with a second generation of vaccines combining DC, local tumor irradiation, and the controlled supply of recombinant mGM-CSF in poloxamer 407, a biocompatible thermoreversible hydrogel. By generating a successful “all in vivo” vaccination protocol combining tumor radiotherapy with DC vaccines and a straightforward supply of GM-CSF, we have developed a therapeutic strategy easily translatable to clinic that could become accessible to a much bigger number of cancer patients.     

A prostate cancer vaccine comprising whole cells secreting IL-7, effective against subcutaneous challenge,requires local GM-CSF for intra-prostatic efficacy

A panel of cytokine-secreting RM-9 prostate cancer cells were tested as whole cell vaccines to determine their capacity to evoke an anti-prostate cancer immune response. In our model, vaccines secreting mGM-CSF or mIL-7 resulted in the highest increase in circulating T lymphocytes after vaccination, prolonged survival and, in a proportion of animals, tumor-free survival. Anti-tumor effects were more evident after a subcutaneous RM-9 challenge than after an intraprostatic challenge. However, when the RM-9/mGM-CSF cell line was used as intraprostatic tumor challenge, protection after RM-9/mIL-7 vaccination was restored.     

Tumor regression induced by intratumor therapy with a disabled infectious single cycle (DISC) herpes simplex virus (HSV) vector, DISC/HSV/murine granulocyte-macrophage colony-stimulating factor, correlates with antigen-specific adaptive immunity

Direct intratumor injection of a disabled infectious single cycle HSV-2 virus encoding the murine GM-CSF gene (DISC/mGM-CSF) into established murine colon carcinoma CT26 tumors induced a significant delay in tumor growth and complete tumor regression in up to 70% of animals. Pre-existing immunity to HSV did not reduce the therapeutic efficacy of DISC/mGM-CSF, and, when administered in combination with syngeneic dendritic cells, further decreased tumor growth and increased the incidence of complete tumor regression. Direct intratumor injection of DISC/mGM-CSF also inhibited the growth of CT26 tumor cells implanted on the contralateral flank or seeded into the lungs following i.v. injection of tumor cells (experimental lung metastasis). Proliferation of splenocytes in response to Con A was impaired in progressor and tumor-bearer, but not regressor, mice. A potent tumor-specific CTL response was generated from splenocytes of all mice with regressing, but not progressing tumors following in vitro peptide stimulation; this response was specific for the gp70 AH-1 peptide SPSYVYHQF and correlated with IFN-gamma, but not IL-4 cytokine production. Depletion of CD8(+) T cells from regressor splenocytes before in vitro stimulation with the relevant peptide abolished their cytolytic activity, while depletion of CD4(+) T cells only partially inhibited CTL generation. Tumor regression induced by DISC/mGM-CSF virus immunotherapy provides a unique model for evaluating the immune mechanism(s) involved in tumor rejection, upon which tumor immunotherapy regimes may be based.     

Recombinant murine GM-CSF from E. coli has biological activity and is neutralized by a specific antiserum.

We report the production and characterization of a mouse granulocyte-macrophage colony stimulating factor (mGM-CSF) made in Escherichia coli. The synthesis of mGM-CSF was directed by a plasmid containing a gene isolated from the EL-4 cell line. After induction of expression and accumulation of the protein in E. coli, mGM-CSF accounted for 10% of total cellular protein. This recombinant mGM-CSF was purified to 90% homogeneity by chaotrope extraction and gel filtration. Recombinant mGM-CSF, like the native molecule, stimulates the growth of granulocyte and macrophage colonies in serum-free cultures of mouse bone marrow cells. Antibodies raised against recombinant mGM-CSF not only reacted with the recombinant protein but also neutralized the biological activity of both native and recombinant mGM-CSF. These results indicate that the functional structure of the recombinant protein is similar to that of native mGM-CSF.     

PSPN/GFRalpha4 has a significantly weaker capacity than GDNF/GFRalpha1 to recruit RET to rafts, but promotes neuronal survival and neurite outgrowth

Previously, it was shown that the recruitment of RET into lipid rafts by glial cell line-derived neurotrophic factor (GDNF)/GFRalpha1 is crucial for efficient signal transduction. Here, we show that the mouse GFRalpha4 is a functional, N-glycosylated, glycosylphosphatidylinositol (GPI)-anchored protein, which mediates persephin (PSPN)-induced phosphorylation of RET, but has an almost undetectable capacity to recruit RET into the 0.1% Triton X-100 insoluble membrane fraction. In spite of this, PSPN/mGFRalpha4 promotes neurite outgrowth in PC6-3 cells and survival of cerebellar granule neurons. As we show that also human PSPN/GFRalpha4 is unable to recruit RET into lipid rafts, we propose that the mammalian GFRalpha4 in this respect differs from GFRalpha1.     

Saccharomyces cerevisiae CWH43 is involved in the remodeling of the lipid moiety of GPI anchors to ceramides

The glycosylphosphatidylinositol (GPI)-anchored proteins are subjected to lipid remodeling during their biosynthesis. In the yeast Saccharomyces cerevisiae, the mature GPI-anchored proteins contain mainly ceramide or diacylglycerol with a saturated long-fatty acid, whereas conventional phosphatidylinositol (PI) used for GPI biosynthesis contains an unsaturated fatty acid. Here, we report that S. cerevisiae Cwh43p, whose N-terminal region contains a sequence homologous to mammalian PGAP2, is involved in the remodeling of the lipid moiety of GPI anchors to ceramides. In cwh43 disruptant cells, the PI moiety of the GPI-anchored protein contains a saturated long fatty acid and lyso-PI but not inositolphosphorylceramides, which are the main lipid moieties of GPI-anchored proteins from wild-type cells. Moreover, the C-terminal region of Cwh43p (Cwh43-C), which is not present in PGAP2, is essential for the ability to remodel GPI lipids to ceramides. The N-terminal region of Cwh43p (Cwh43-N) is associated with Cwh43-C, and it enhanced the lipid remodeling to ceramides by Cwh43-C. Our results also indicate that mouse FRAG1 and C130090K23, which are homologous to Cwh43-N and -C, respectively, share these activities.     

Evolution of a tumorigenic property conferred by glycophosphatidyl-inositol membrane anchors of carcinoembryonic antigen gene family members during the primate radiation

GPI membrane anchors of cell surface glycoproteins have been shown to confer functional properties that are different from their transmembrane (TM)-anchored counterparts. For the human carcinoembryonic antigen (CEA) family, a subfamily of the immunoglobulin superfamily, conversion of the mode of membrane linkage from TM to GPI confers radical changes in function: from tumor suppression or neutrality toward inhibition of differentiation and anoikis and distortion of tissue architecture, thereby contributing to tumorigenesis. We show here that GPI anchorage in the CEA family evolved twice independently in primates, very likely from more primitive TM anchors, by different packages of mutations. Both mutational packages, one package found in many primates, including humans, and a second, novel package found only in the Cebidae radiation of New World monkeys, give rise to efficiently processed GPI-linked proteins. Both types of GPI anchors mediate inhibition of cell differentiation. The estimated rate of nonsynonymous mutations (Ka) in the anchor-determining domain for conversion from TM to GPI anchorage in the CEA family that were fixed during evolution in these primates is 7 times higher than the average Ka in primates, indicating positive selection. These results suggest therefore that the functional changes mediated by CEA GPI anchors, including the inhibition of differentiation and anoikis, could be adaptive and advantageous.     

Targeting tumor vasculature with novel <Emphasis Type="Italic">Listeria</Emphasis>-based vaccines directed against CD105

The FDA approval of bevacizumab (Avastin^®, Genentech/Roche), a monoclonal antibody raised against human VEGF-A, as second-line therapy for colon and lung carcinoma validated the approach of targeting human tumors with angiogenesis inhibitors. While the VEGF/VEGFR pathway is a viable target for anti-angiogenesis tumor therapy, additional targets involved in tumor neovascularization have been identified. One promising target present specifically on tumor vasculature is endoglin (CD105), a member of the TGF-β receptor complex expressed on vascular endothelium and believed to play a role in angiogenesis. Monoclonal antibody therapy and preventive vaccination against CD105 has met with some success in controlling tumor growth. This report describes the in vivo proof-of-concept studies for two novel therapeutic vaccines, Lm-LLO-CD105A and Lm-LLO-CD105B, directed against CD105 as a strategy to target neovascularization of established tumors. Listeria-based vaccines directed against CD105 lead to therapeutic responses against primary and metastatic tumors in the 4T1-Luc and NT-2 mouse models of breast cancer. In a mouse model for autochthonous Her-2/neu-driven breast cancer, Lm-LLO-CD105A vaccination prevented tumor incidence in 20% of mice by week 58 after birth while all control mice developed tumors by week 40. In comparison with previous Listeria-based vaccines targeting tumor vasculature, Lm-LLO-CD105A and Lm-LLO-CD105B demonstrated equivalent or superior efficacy against two transplantable mouse models of breast cancer. Support is provided for epitope spreading to endogenous tumor antigens and reduction in tumor vascularity after vaccination with Listeria-based CD105 vaccines. Reported here, these CD105 therapeutic vaccines are highly effective in stimulating anti-angiogenesis and anti-tumor immune responses leading to therapeutic efficacy against primary and metastatic breast cancer.     

Expression and release of stable and active forms of murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) targeted to different subcellular compartments

Cytokines have been used for several years as immunomodulators. However, one of the main drawbacks of systemically applied cytokines is their high toxicity. In addition, cytokines work in a paracrine form and frequently after cell-to-cell interaction. Therefore, a very restricted release of cytokines-in time and space-could be desired for most of their therapeutic applications. The murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) is one of the most promising cytokine candidates for cancer immunotherapy and as an adjuvant of DNA vaccines. With the aim of improving the administration and release of cytokines in a very restricted area, we have designed vectors expressing the mGM-CSF cDNA with different localization signals. Using this strategy we have shown that cytokines can be expressed and targeted to different subcellular compartments (i.e. the cytoplasm, the endoplasmic reticulum and the nucleus), stored inside the cells and released after cell lysis as stable active proteins. Moreover, a plasma membrane targeted form of mGM-CSF displayed substantial amount of biological activity. These vectors could have potential applications in immunotherapy for tumours and DNA vaccination protocols.