双重免疫分子膜表面修饰疫苗的制备及抗肿瘤作用的研究
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摘要
1971年Burnet提出肿瘤免疫监视概念，认为机体免疫系统能够识别并通过细胞免疫机制破坏肿瘤细胞。如果机体的免疫监视功能低下或缺陷，就有可能形成肿瘤。肿瘤细胞逃避机体免疫攻击的可能机制包括：1)MHC分子的下调或丢失；2)抗原处理通路的改变导致无法将肿瘤特异性抗原递呈到T细胞；3)激活宿主免疫系统必需的共刺激分子或粘附分子的缺失；4)肿瘤局部的细胞因子缺乏或肿瘤细胞分泌免疫抑制因子；5)肿瘤抗原丢失或抗原调变等。多种因素的共存导致机体处于一种局部免疫抑制状态，使机体的免疫效应细胞不能产生有效的抗肿瘤免疫应答。针对肿瘤免疫逃避的机制，用转基因手段，将目的基因导入靶细胞中，提高机体抗肿瘤免疫反应，这种治疗手段称为肿瘤免疫基因治疗。
抗肿瘤瘤苗是肿瘤免疫基因治疗的主要策略，在瘤苗制备过程中对自体或异体肿瘤细胞进行表面修饰以增强其免疫原性和免疫效能，目前瘤苗修饰主要通过基因转染手段实现。如Hodge等将B7.1基因导入肿瘤细胞中表达，提供共刺激因子，能增强瘤苗的抗肿瘤免疫反应；Shrayer等用SEA基因转染小鼠B16黑色素瘤细胞，使之表达SEA，应用该瘤细胞制成瘤苗，显示了较强的抗肿瘤作用。
虽然基因转染法在肿瘤免疫基因治疗上起着很重要作用，但是基因转染法亦有其局限性，如原代细胞不易转染，转染效率低、整合后基因的降解及其表达的不稳定性以及制备费时和临床应用困难。因此有必要探寻适合于原代细胞转染、转染效率高、制备简便、临床易推广应用的转染法。
通过基因工程手段将糖基化磷脂酰肌醇(GPI)或蛋白质的跨膜区(TM)与目的蛋白质嵌合表达，由此制备的目的蛋白质能够锚定到多种细胞膜上，这种不通过基因转染手段使目的蛋白在靶细胞膜上表达的方法叫蛋白转染法(protein transfer)。GPI锚定蛋白转染法和跨膜区锚定蛋白转染法是目前常用的蛋白转染法。
GPI锚定蛋白转染法是指将目的蛋白编码序列和GPI锚定信号肽编码序列重组，制备该目的蛋白的GPI重组衍生物，当它在适宜的温度下与靶细胞共同孵育时，可整合至细胞表面并表达活性。Mchugh等将hB7-1基因胞外区与CD16B的GPI锚定信号肽基因序列融合，表达出hB7.1-GPI蛋白，该蛋白能嵌入细胞膜，其瘤苗能激活T细胞，并刺激其增殖，在体内和体外显示出较强的抗肿瘤作用。
浙汀人学博卜学位论义 易小勇
Brunschwig等将mB7工基因胞外区与DAFpGPIf定信号肽基因序歹融合，表
达mB7－GPI，该蛋白能嵌入细胞膜，实验发现它能提供T细她增殖的共刺激信
号。
1982年Boeke等首次提出将分泌型蛋白基因序列与跨膜型蛋白的跨膜区基
因序列拼接成融合基因，可将分泌型蛋白表达成跨膜型蛋白。1995年Chen等通
过基因工程方法，将细菌的分泌型碱性磷酸酶转换为跨膜型的碱性磷酸酶。此方
法称为跨膜区锚定蛋白转染法。WahLsten等将癌基因CerbB－2的跨膜区编码序
列与TSSTI编码序列融合，在细菌中表达，得到融合蛋白TSSTI－TM，它能锚
定在儿种瘤细胞表而，其痈茁有较强的抗亲本肿痫作用。马文学等将癌基因
Cerb－B－2的跨吸区编码序列与超抗原SEA编码序列融合，在细菌中表达，得到
触合蛋肉SBA－＊M，它能伽定在痫别胞茨而，其痢冰在体内外均显示出较强的抗
肿概作用。
蛋白转染法有许多优点：（1）不依赖于细胞自身增殖潜能及可转染性，故可
用于多利。原代细胞的转染，与细胞的类型和组织器官的来源无关：（2）在同一细
胞中复合基因的共转染及协同表达还存在一定的困难，而蛋白转染可允许多种蛋
向同时或顺序地锚定在细胞表而，适合于多下免疫分了吸表而修饰痢m的制趴
（3）在体外，可皿过基因工程方法大量制备用于蛋白转染的免疫分子，这些免
疫分子与病人自体肿瘤细胞共同孵育后，可较简便地制备经免疫分子表而修饰的
自体肿瘤细胞的瘤苗，无需在自体肿瘤细胞中作个别化的基因转染，因此有较大
的临床应用潜力。
JI。I’俯的免疫抑制Z时］Z涉及到多种兔疫活性冈了的下调，研究表明多摧因共转
染痫苗在打破肿瘤微环境的免疫抑制、激发机体的免疫应方而优于单基因转染瘤
芮，们多从闪人转染撇作复卉、＿巳筛巡多个从闪均高没达的州性克隆困难。为此，
本课题设想通过蛋白转染法将多种免疫分子同时锚定到肿瘤细胞膜，制成瘤苗，
发挥抡疫协同作用，以期其疗效能优于单种免疫分子锚定的瘤茁。经文献检索，
h6iJ还没有这方而的研究报道。我们选用 SEA－TM和 ruB7．卫－GPI二种免疫分子，
作为日的蛋白，共同修饰抗肿瘤瘤苗。
本研究口的为：通过蛋内转染法，将 SEA－TM和 mB7．l－GPI锚定于小鼠T
细胞淋巴瘤细胞（EL4人制备双重免疫分子绷胞膜表面修饰瘤苗，以此为模型，
观察双贡免疫分子表而修饰瘤苗制各的可行性和瘤苗对亲本肿瘤的治疗作用和
免疫保护作用。
本课题研究内容包括：（）构建 mB7lGPI的表达载体，制策其融合蛋白，
仲之邯定广Z EL－4 9抉纠【）jbN而，制成二B7．l－GPI）JQ表mi修饰瘤苗：（2）将 SEA－TM
锚定在 EL－4瘤圳月b表而，制成 SEA－TM膜表面修?
The mechanisms for tumor cells to escape immune surveillance of the body include: weak tumor specific or associated antigens, defective antigen presentation process, down-regulated surface MHC molecules and lack or costimulatory molecules. Introduction of immunostimulatory molecules such as MHC I, MHCII and B7.1. to the tumor cells, and use of these surface-modified tumor cells as vaccine, can induce antilumor immunity as demonstrated by rejection of parental tumor in vivo. Typically, these approaches involving transfecu'on of gene is time consuming, gene expression is instability and primary tumor cells often do not grow well in vitro. Therefore, the practical application of this strategy to human tumors may be limited.
Alternatively, surface molecules can be introduced onto tumor cells through the following approach: the DNA sequence of target protein is fused with a glycosyl-phosphalidylinositol(GPI) signal sequence, the purified, recombinant GPI-linkcd fusion proteins are incubated with tumor cells so the target protein can be anchored onto extracellular membranes of the tumor cells. In vitro studies applying this approach demonstrated that GPI-anchored MHC I molecules effectively promoted T cell-mediated cytotoxicity, and GPI-anchored B7.1 co-stimulated lymphocyte proliferation.
However, as a prokaryotic protein such as bacteria superanligen is not well suited to a GPI-signal-sequence-fusion strategy for cell membrane anchoring because GPI linkage requires eukaryotic processing. Wahlsten developed a strategy for passively attaching the superanligen toxic shock syndrome toxin-1 (TSST1) onto tumor cells, this strategy was fusing TSST1 coding region to the transmembranc region (TM) sequence of the proto-oncogene c-erb-B2. TSST1-TM was expressed in bacteria. Purified TSSTI-TM can be anchored onto tumor cells, Ihe tumor vaccine derived from these tumor cells can stimulate proliferation of lymphocytes in vitro and induce a systemic anlitumor immunity. Ma ct al also reported that mice immunized with tumor cell vaccine, which was anchored by fusion protein slaphylococcal cnleroloxin A- transmembrane (SEA-TM), developed specific antilumor immunity.
The mechanisms of tumor escape from the immune surveillance indicate thai the tumor development is associate with lack or down-regulation of various immunological factors and the multiple-surface modified tumor cell vaccine may induce immune response more effectively.
Many studies have showed the tumor cell vaccine of multiple-gene transfection, can induce much stronger antitumor immunity than that of single-gene transfection. However, multiple-gene transfection is time consuming and less practicable.
We are intended to develop a new approach for passively attaching mB7.1-GPI and SEA-TM onto tumor cells by protein transfer, with this approach the tumor cell vaccine membrane-anchored with the two different immunologic molecule is prepared. Using this model, we will explore the feasibility for preparation of immune molecule dual-anchored lumor cell vaccine and observe the effectiveness of this novel approach in treatment and immnopretection of parental murine tumor growth in the experimental animals.
The objectives of the study includes: (1) to construct mB7.1-GPI fusion gene, to express and purify mB7.1-GPI fusion protein and incorporate it on lumor cells and to prepare mB7.1-GPI-anchoring tumor vaccine; (2) to prepare the vaccine of lumor cells membrane- anchored with SEA-TM; (3) to prepare SEA-TM and mB7.1-GPI dual-anchored tumor cell vaccine; (4) to observe the antitumor immunity induced by the prepared single or dual-anchored lumor cell vaccine in tumor-bearing mice.
The sludy is reported as follows:
Part 1: Cloning and expression of mB7.1-GPl fusion gene
A DNA fragment encoding the firsl 247 amino acids of mB7.1 and a DNA fragment encoding the signal for GPI-anchor allachmenl of hPLAP-1 were amplified by PCR. The two amplified gene sequence were annealed to form a chimeric GPI-anchored mB7.1 molecule by gene SOEing. The resulting chimera was clone
抗肿瘤瘤苗是肿瘤免疫基因治疗的主要策略，在瘤苗制备过程中对自体或异体肿瘤细胞进行表面修饰以增强其免疫原性和免疫效能，目前瘤苗修饰主要通过基因转染手段实现。如Hodge等将B7.1基因导入肿瘤细胞中表达，提供共刺激因子，能增强瘤苗的抗肿瘤免疫反应；Shrayer等用SEA基因转染小鼠B16黑色素瘤细胞，使之表达SEA，应用该瘤细胞制成瘤苗，显示了较强的抗肿瘤作用。
虽然基因转染法在肿瘤免疫基因治疗上起着很重要作用，但是基因转染法亦有其局限性，如原代细胞不易转染，转染效率低、整合后基因的降解及其表达的不稳定性以及制备费时和临床应用困难。因此有必要探寻适合于原代细胞转染、转染效率高、制备简便、临床易推广应用的转染法。
通过基因工程手段将糖基化磷脂酰肌醇(GPI)或蛋白质的跨膜区(TM)与目的蛋白质嵌合表达，由此制备的目的蛋白质能够锚定到多种细胞膜上，这种不通过基因转染手段使目的蛋白在靶细胞膜上表达的方法叫蛋白转染法(protein transfer)。GPI锚定蛋白转染法和跨膜区锚定蛋白转染法是目前常用的蛋白转染法。
GPI锚定蛋白转染法是指将目的蛋白编码序列和GPI锚定信号肽编码序列重组，制备该目的蛋白的GPI重组衍生物，当它在适宜的温度下与靶细胞共同孵育时，可整合至细胞表面并表达活性。Mchugh等将hB7-1基因胞外区与CD16B的GPI锚定信号肽基因序列融合，表达出hB7.1-GPI蛋白，该蛋白能嵌入细胞膜，其瘤苗能激活T细胞，并刺激其增殖，在体内和体外显示出较强的抗肿瘤作用。
浙汀人学博卜学位论义 易小勇
Brunschwig等将mB7工基因胞外区与DAFpGPIf定信号肽基因序歹融合，表
达mB7－GPI，该蛋白能嵌入细胞膜，实验发现它能提供T细她增殖的共刺激信
号。
1982年Boeke等首次提出将分泌型蛋白基因序列与跨膜型蛋白的跨膜区基
因序列拼接成融合基因，可将分泌型蛋白表达成跨膜型蛋白。1995年Chen等通
过基因工程方法，将细菌的分泌型碱性磷酸酶转换为跨膜型的碱性磷酸酶。此方
法称为跨膜区锚定蛋白转染法。WahLsten等将癌基因CerbB－2的跨膜区编码序
列与TSSTI编码序列融合，在细菌中表达，得到融合蛋白TSSTI－TM，它能锚
定在儿种瘤细胞表而，其痈茁有较强的抗亲本肿痫作用。马文学等将癌基因
Cerb－B－2的跨吸区编码序列与超抗原SEA编码序列融合，在细菌中表达，得到
触合蛋肉SBA－＊M，它能伽定在痫别胞茨而，其痢冰在体内外均显示出较强的抗
肿概作用。
蛋白转染法有许多优点：（1）不依赖于细胞自身增殖潜能及可转染性，故可
用于多利。原代细胞的转染，与细胞的类型和组织器官的来源无关：（2）在同一细
胞中复合基因的共转染及协同表达还存在一定的困难，而蛋白转染可允许多种蛋
向同时或顺序地锚定在细胞表而，适合于多下免疫分了吸表而修饰痢m的制趴
（3）在体外，可皿过基因工程方法大量制备用于蛋白转染的免疫分子，这些免
疫分子与病人自体肿瘤细胞共同孵育后，可较简便地制备经免疫分子表而修饰的
自体肿瘤细胞的瘤苗，无需在自体肿瘤细胞中作个别化的基因转染，因此有较大
的临床应用潜力。
JI。I’俯的免疫抑制Z时］Z涉及到多种兔疫活性冈了的下调，研究表明多摧因共转
染痫苗在打破肿瘤微环境的免疫抑制、激发机体的免疫应方而优于单基因转染瘤
芮，们多从闪人转染撇作复卉、＿巳筛巡多个从闪均高没达的州性克隆困难。为此，
本课题设想通过蛋白转染法将多种免疫分子同时锚定到肿瘤细胞膜，制成瘤苗，
发挥抡疫协同作用，以期其疗效能优于单种免疫分子锚定的瘤茁。经文献检索，
h6iJ还没有这方而的研究报道。我们选用 SEA－TM和 ruB7．卫－GPI二种免疫分子，
作为日的蛋白，共同修饰抗肿瘤瘤苗。
本研究口的为：通过蛋内转染法，将 SEA－TM和 mB7．l－GPI锚定于小鼠T
细胞淋巴瘤细胞（EL4人制备双重免疫分子绷胞膜表面修饰瘤苗，以此为模型，
观察双贡免疫分子表而修饰瘤苗制各的可行性和瘤苗对亲本肿瘤的治疗作用和
免疫保护作用。
本课题研究内容包括：（）构建 mB7lGPI的表达载体，制策其融合蛋白，
仲之邯定广Z EL－4 9抉纠【）jbN而，制成二B7．l－GPI）JQ表mi修饰瘤苗：（2）将 SEA－TM
锚定在 EL－4瘤圳月b表而，制成 SEA－TM膜表面修?
The mechanisms for tumor cells to escape immune surveillance of the body include: weak tumor specific or associated antigens, defective antigen presentation process, down-regulated surface MHC molecules and lack or costimulatory molecules. Introduction of immunostimulatory molecules such as MHC I, MHCII and B7.1. to the tumor cells, and use of these surface-modified tumor cells as vaccine, can induce antilumor immunity as demonstrated by rejection of parental tumor in vivo. Typically, these approaches involving transfecu'on of gene is time consuming, gene expression is instability and primary tumor cells often do not grow well in vitro. Therefore, the practical application of this strategy to human tumors may be limited.
Alternatively, surface molecules can be introduced onto tumor cells through the following approach: the DNA sequence of target protein is fused with a glycosyl-phosphalidylinositol(GPI) signal sequence, the purified, recombinant GPI-linkcd fusion proteins are incubated with tumor cells so the target protein can be anchored onto extracellular membranes of the tumor cells. In vitro studies applying this approach demonstrated that GPI-anchored MHC I molecules effectively promoted T cell-mediated cytotoxicity, and GPI-anchored B7.1 co-stimulated lymphocyte proliferation.
However, as a prokaryotic protein such as bacteria superanligen is not well suited to a GPI-signal-sequence-fusion strategy for cell membrane anchoring because GPI linkage requires eukaryotic processing. Wahlsten developed a strategy for passively attaching the superanligen toxic shock syndrome toxin-1 (TSST1) onto tumor cells, this strategy was fusing TSST1 coding region to the transmembranc region (TM) sequence of the proto-oncogene c-erb-B2. TSST1-TM was expressed in bacteria. Purified TSSTI-TM can be anchored onto tumor cells, Ihe tumor vaccine derived from these tumor cells can stimulate proliferation of lymphocytes in vitro and induce a systemic anlitumor immunity. Ma ct al also reported that mice immunized with tumor cell vaccine, which was anchored by fusion protein slaphylococcal cnleroloxin A- transmembrane (SEA-TM), developed specific antilumor immunity.
The mechanisms of tumor escape from the immune surveillance indicate thai the tumor development is associate with lack or down-regulation of various immunological factors and the multiple-surface modified tumor cell vaccine may induce immune response more effectively.
Many studies have showed the tumor cell vaccine of multiple-gene transfection, can induce much stronger antitumor immunity than that of single-gene transfection. However, multiple-gene transfection is time consuming and less practicable.
We are intended to develop a new approach for passively attaching mB7.1-GPI and SEA-TM onto tumor cells by protein transfer, with this approach the tumor cell vaccine membrane-anchored with the two different immunologic molecule is prepared. Using this model, we will explore the feasibility for preparation of immune molecule dual-anchored lumor cell vaccine and observe the effectiveness of this novel approach in treatment and immnopretection of parental murine tumor growth in the experimental animals.
The objectives of the study includes: (1) to construct mB7.1-GPI fusion gene, to express and purify mB7.1-GPI fusion protein and incorporate it on lumor cells and to prepare mB7.1-GPI-anchoring tumor vaccine; (2) to prepare the vaccine of lumor cells membrane- anchored with SEA-TM; (3) to prepare SEA-TM and mB7.1-GPI dual-anchored tumor cell vaccine; (4) to observe the antitumor immunity induced by the prepared single or dual-anchored lumor cell vaccine in tumor-bearing mice.
The sludy is reported as follows:
Part 1: Cloning and expression of mB7.1-GPl fusion gene
A DNA fragment encoding the firsl 247 amino acids of mB7.1 and a DNA fragment encoding the signal for GPI-anchor allachmenl of hPLAP-1 were amplified by PCR. The two amplified gene sequence were annealed to form a chimeric GPI-anchored mB7.1 molecule by gene SOEing. The resulting chimera was clone
引文
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13. Tykocinski ML,Kaplan DR,Medof ME.Antigen-presenting cell engineering. AJP January, 1996,148(1): 1-16.
14. Medof ME,Nagarajan S,Tykocinski ML. Cell-surface engineering with GPI-anchored proteins. FASEB-J., 1996,10(5): 574-86.
15. Medof ME,Kinoshita T, Nussenzweig V. Inhibition of complement activation on the surface of cells after incorporation of decay-accelerating factor (DAF) into their membranes. J-Exp-Med.,1984,160(5): 1558-78.
16. Zhang F, Schmidt WG,Hou Y, et al. Spontaneous incorporation of the glycosyl-phosphatidylinositol-linked protein Thy-1 into cell membranes. Proc-Natl-Acad-Sci-U-S-A., 1992,89(12): 5231-5.
17. Henthorn PS.Knoll BJ, Raducha M,et al. Products of two common alleles at the locus for human placental alkaline phosphatase differ by seven amino acids. Proc-Nall-Acad-Sci-U-S-A., 1986,83(15): 5597-601.
18. Mcdof ME, Kinoshita T,Nussenzweig V. Inhibition of complement activation on the surface of cells after incorporation of decay-accelerating factor (DAF) into their membranes. J-Exp-Med.,1984,160(5): 1558-78.
19. Mann KJ, Sevlever D. 1,10-Phenanthroline inhibits glycosylphosphatidylinositol anchoring by preventing phosphoethanolamine addition to glycosylphosphatidylinositol anchor precursors. Biochemistry, 2001,40(5): 1205-13.
20. Davitz MA.Hereld D,Shak S,et al.A glycan-phosphatidylinositol-specific phospholipase D in human serum. Science, 1987,238(4823): 81-4.
21. Bangs JD,Andrews NW, Hart GW, et al.Posttranslalional modification and intracellular transport of a trypanosomc variant surface glycoprotein. J-Cell-Biol., 1986,103(1): 255-63.
22. Tykocinski ML,Shu HK,Ayers DJ,et al.Glycolipid reanchoring of T-lymphocyte surface antigen CD8 using the 3' end sequence of decay-accelerating factor's mRNA. Proc-Natl-Acad-Sci-U-S-A., 1988,85(10): 3555-9.
23. Oda K,Cheng J,Saku T, et al. Conversion of secretory proteins into membrane proteins by fusing with a glycosylphosphatidylinositol anchor signal of alkaline phosphatase. Biochem-J., 1994,301(Pt 2): 577-83.
24. McHugh RS,Ahmed SN,Wang YC,et al. Construction, purification, and functional incorporation on tumor cells of glycolipid-anchored human B7-1 (CD80). Proc-Natl-Acad-Sci-U-S-A.,1995,92(17): 8059-63.
25. Brunschwig EB,Levine E,Trefzer U,et al. Glycosylphosphatidylinositol-modified murine B7-1 and B7-2 retain costimulator function. J-Immunol.,1995,155(12): 5498-505.
26. Boeke JD.Model P.A prokaryotic membrane anchor sequence:carboxyl terminus of bacteriophage fl gene Ⅲ protein retains it in the membrane.
Proc-Natl-Acad-Sci-U-S-A, 1982,79:5200-5204.
27. Chen H,Kendall DA. Artificial transmembrane segments. J-Biol-Chem, 1995,270:14115-14122.
28. Wahlsten JL, Mills CD, and Ramakrishnan S. Antitumor response clicited by a superantigen-transmembrane sequence fusion protein anchored onto tumor cells. J Immunol, 1998; 161(12): 6761-6767
29．马文学，余海，王青青等。跨膜型超抗原SEA融合蛋白的表达及其对小鼠黑色素瘤的治疗作用研究。中华微生物与免疫学杂志，2003，in press.
30. Pulaski BA, Terman DS, Khan S,et al.Cooperativity of staphylococcal aureus enterotoxin B superantigen,major hitocompatibility complex class Ⅱ,and CD80 for immunotherapy of advanced spontaneous metastases in a clinically relevant postoperative mouse breast cancer model.Cancer Res.2000,60:2710-2715.
31. Freeman GJ,Freedman AS,Segil JM,et al. B7,a new member of the Ig super-family with unique expression on activated and neoplastic Bcell.J Immunol,1989,143:2714
32. Scherer MT, Ignatowicz L, Winslow G, et al. Superantigens: bacterial and viral proteins that manipulate the immune system. Annu Rev Cell Biol, 1993; 9:101-128.
33. Parra E,Wingren AG, Hedlund G,et al.The role of B7-1 and LFA-3 in costimulation of CD8+ T cells. J-Immunol. 1997,158(2): 637-42
34. Nestle FO, ThomPson C, Shimizu Y; etal.Costimulation of superantigen-activated T lymphocytes by autologous dendritic cells is dependent on B7. Cell-Immunol. 1994, 156(1): 220-9
35. Goldbach-Mansky R, King PD, Taylor AP;etal. A co-stimulatory role for CD28 in the activation of CD4+ T lymphocytes by staphylococcal enlerotoxin B. Int-Immunol. 1992, 4(12): 1351-60
36. Chen L,McGowan P,.Tumor immunogenicity determines the effect of B7 Costimulation on T cell-mediated tumor immunity. J Exp Med. 1995,179:523-532.
37. Millan JL. Molecular Cloning and Sequence Analysis of Human Placental Alkaline Phosphatase. J.Biol,Chem.1986,261(7):3112-3115.
38. Freeman GJ,GrayGS,GimmiCD,et al.Structure,Expression,and T Cell Costimulatory Activity of the Murine Homologue of the Human B Lymphocyte Activation Antigen B7. J Exp Med, 1991, 174:625-631
39. Horton RM, Hunt HD.Ho SN,et al.Engineering hybrid gene without the use of restriction cnzymes:gene splicing by overlap extension. Gene, 1989,77:61-68.
40. Lehto Marty T.Sharom FJ.PI-specific phospholipase C cleavage of a reconstituted
GPI-anchored protein: modulation by the lipid bilayer. Biochemistry, 2002,41(4): 1398-408
41. Freeman GJ,GrayGS,GimmiCD,et al. Structure,Expression,and T Cell Costimulatory Activity of the Murine Homologue of the Human B Lymphocyte Activation Antigen B7. J Exp Med, 1991, 174:625-631
42. Kozak M.The scanning model for translation:An update.J cell Biol,108:229
43. Ferguson MA,Williams AF.Cell-surface anchoring of proteins via glycosyl-phosphatidylinositol structures. Annu-Rev-Biochem.,1988; 57285-320.
44. Goldstein DJ,Blasco L, Harris H. Placental alkaline phosphatase in nonmalignant human cervix. Proc-Natl-Acad-Sci-U-S-A.,1980,77(7): 4226-8.
45. Goldstein DJ,Rogers C,Harris H.A search for trace expression of placental-like alkaline phosphatase in non-malignant human tissues: demonstration of its occurrence in lung, cervix, testis and thymus. Clin-Chim-Acta., 1982,125(1): 63-75.
46. Abu-Hasan NS, Sutcliffe RG. Placental alkaline phosphatase integrates via its carboxy-terminus into the microvillous membrane: its allotypes differ in conformation. Placenta.,1985,6(5): 391-404.
47. Lange PH,Millan JL, Stigbrand T, et al. Placental alkaline phosphatase as a tumor marker for seminoma. Cancer-Res.,1982,42(8): 3244-7.
48. McDicken IW, McLaughlin PJ,Tromans PM,et al.Detection of placental-type alkaline phosphatase in ovarian cancer. Br-J-Cancer., 1985,52(1): 59-64.
49. Knoll BJ,Rothblum KN,Longley M. Nucleotide sequence of the human placental alkaline phosphatase gene. Evolution of the 5' flanking region by deletion/substitution. J-Biol-Chem., 1988,263(24): 12020-7.
50. Micanovic R,Bailey CA,Brink L,et al.Aspartic acid-484 of nascent placental alkaline phosphatase condenses with a phosphatidylinositol glycan to become the carboxyl terminus of the mature enzyme. Proc-Natl-Acad-Sci-U-S-A., 1988,85(5): 1398-402.
51. Micanovic R,Kodukula K,Gerber LD,et al.Selectivity at the cleavage/attachment site of phosphatidylinositol-glycan anchored membrane proteins is enzymatically determined. Proc-Natl-Acad-Sci-U-S-A., 1990,87(20): 7939-43.
52. Tiede A,Bastisch I,Schubert J,et al. Biosynthesis of glycosylphosphatidylinositols in mammals and unicellular microbes. Biol-Chem., 1999,380(5): 503-23.
53. Davitz MA,Hereld D,Shak S,et al.A glycan-phosphatidylinositol-specific phospholipase D in human serum. Science,1987,238(4823): 81-4.
54. Medof ME, Kinoshita T, Nussenzweig V. Inhibition of complement activation on the surface of cells after incorporation of decay-accelerating factor (DAF) into their membranes.
J-Exp-Med.,1984,160(5): 1558-78.
55. Zhang F, Schmidt WG,Hou Y, et al. Spontaneous incorporation of the glycosyl-phosphatidylinositol-linked protein Thy-1 into cell membranes. Proc-Natl-Acad-Sci-U-S-A.,1992,89(12): 5231-5.
56. Brodsky RA,Jane SM,Vanin EF, et al. Purified GPI-anchored CD4DAF as a receptor for HIV-mediated gene transfer. Hum-Gene-Ther.,1994,5(10): 1231-9.
57. Huang JH, Getty RR,Chisari FV, et al.Protein transfer of preformed MHC-peptide complexes sensitizes target cells to T cell cytolysis. Immunity, 1994,1(7): 607-13
58. Gimmi CD, Freeman GJ, Gribbenh JG,et al. B-cell surface antigen B7 provides a costimulatory signal that induces T cells to proliferate and secrete interleukin 2. Proc-Natl-Acad-Sci-U-S-A. 1991, 88(15): 6575-9.
59. Huang JH,Greenspan NS,Tykocinski ML. Alloantigenic recognition of artificial glycosyl phosphatidylinositol-anchored HLA-A2.1. Mol-Immunol. 1994,31(13): 1017-28.
60. Weber MC,Groger RK,Tykocinski ML. A glycosylphosphatidylinositol-anchored cytokine can function as an artificial cellular adhesin. Exp-Cell-Res. 1994,210(1): 107-12.
61. Dohlsten M, Abrahmsen L, Bjork P, et al. Monoclonal antibody-supcrantigcn fusion protein: tumor specific agents for T-cell-based tumor therapy. Proc Natl Acad Sci USA, 1994; 91(19): 8945-8949
62. Sakurai N,Kudo T, Suzuki M,et al. SEA-scFv as a bifunctional antibody: construction of a bacterial expression system and its functional analysis.
63. Robey E,Allison JP.T cell activation:intergration of signals from the antigen receptor and constimulatory molecules.Immunol Today, 1995,13:306-310.
64. Robey E,Allison JP.T cell activation:intergration of signals from the antigen receptor and constimulatory molecules.Immunol Today, 1995,13:306-310.
65. Kiessling R.Tumor-induced immune dysfunction.Cancer Immunol Immunother, 1999,48:353-362
66. Yang G, Hellstrom KE,Mizuno M T, et al.In vitro priming of tumor-reactive cytolytic T lymphocytes by combining IL-10 with B7-CD28 costimulation. J-Immunol. 1995, 155(8): 3897-903
67. Geldhof A B, Raes G, Bakkus M,et al. Expression of B7-1 by highly metastatic mouse T lymphomas induces optimal natural killer cell-mediated cytotoxicity. Cancer-Res. 1995, 55(13): 2730-3
68. Townsend SE, Su FW, Atherton JM, Allison JP. Specificity and longevity of antitumor immune responses induced by B7-transfected tumors. Cancer-Res. 1994, 54(24): 6477-83
69. GuoY, Wu M,Chen H,et al. Effective tumor vaccine generated by fusion of hepatoma cells with activated B cells. Science. 1994,263(5146): 518-20.
70. Kalland T, Hedlund G,Dohlsten M,et al. Staphylococcal enterotoxin-dependent cell-mediated cytotoxicity. Curr-Top-Microbiol-Immunol. 1991.174:81-92
71. Gaken JA, Hollingsworth SJ, Hirst WJ,et al. Irradiated NC adenocarcinoma cells transduced with both B7.1 and interleukin-2 induce CD4+-mediated rejection of established tumors. Hum-Gene-Ther. 1997,8(4): 477-88.
72. Dow-SW, Elmslie-RE,Willson-AP, et al.In vivo transfection with superantigen plus cytokine genes induces tumor regressionand prolongs survival in dogs with malignant melanoma. J-Clin-Invest, 1998,101(11): 2406-14
73. Labuda T, Wendt J, Hedlund G,et al.ICAM-1 costimulation induces IL-2 but inhibits IL-10 production in superantigen-activated human CD4+ T cells. Immunology. 1998, 94(4): 496-502
74. Muraille E,Devos S, Thielemans K,et al. Costimulation regulates the.kinetics of interleukin-2 response to bacterial superantigens. Immunology. 1996,89(2): 245-9.
75. Muraille E,Devos S, Thielemans K,et al. Costimulation regulates the kinetics of interleukin-2 response to bacterial superantigens. Immunology. 1996,89(2): 245-9.
76. Muraille E,Devos S, Thielemans K,et al. Costimulalion regulates the kinetics of interleukin-2 response to bacterial superantigens. Immunology. 1996,89(2): 245-9.
77. Tatsumi T, Takehara T,Katayama K,et al. Expression of costimulatory molecules B7-1 (CD80) and B7-2 (CD86) on human hepatocellular carcinoma. Hepatology. 1997,25(5): 1108-14.
78. Tatsumi T, Takehara T,Katayama K,et al. Expression of costimulatory molecules B7-1 (CD80) and B7-2 (CD86) on human hepatocellular carcinoma. Hepatology. 1997,25(5): 1108-14.
79. Tatsumi T, Takehara T,Katayama K,et al. Expression of costimulatory molecules B7-1 (CD80) and B7-2 (CD86) on human hepatocellular carcinoma. Hepatology. 1997,25(5): 1108-14.
80. Renno T, Hahne M, Tschopp J,et al. Peripheral T cells undergoing superantigen-induced apoptosis in vivo express B220 and upregulate Fas and Fas ligand. J-Exp-Med. 1996,183(2): 431-7.
81. Boise LH, Minn AJ, Noel PJ,et al.CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-XL. Boise LH, Minn AJ, Noel PJ,et al. Immunity. 1995, 3(1): 87-98
82. Parra E,Wingren AG, Hedlund G,et al.The role of B7-1 and LFA-3 in costimulation of CD8+ T cells. J-Immunol. 1997,158(2): 637-42
2. Seliger B, Harders C, Lohmann S, et al. Down-regulation of the MHC class I antigen-processing machinery after oncogenic transformation of murine fibroblasts. Eur-J-Immunol, 1998; 28(1): 122-133
3. Restifo NP, Esquivel F, Kawakami Y, et al. Identification of human cancers deficient in antigen processing. J-Exp-Med, 1993; 177(2): 265-272
4. Seliger B, Maeurer MJ, Ferrone S. TAP off--tumors on. Immunol-Today, 1997; 18(6): 292-299
5. Chen L, Ashe S, Brady WA. Costimulation of antitumor immunity by the B7 counter receptor for the T lymphocyte molecules CD28 and CTLA-4. Cell, 1992; 71(7): 1093-1102
6. Townsend SE, Allison JP. Tumor rejection after direct costimulation of CD8+ T cells by B7-transfected melanoma cells. Science, 1993; 259(5093): 368-370
7. Becker JC, Brocker EB. Lymphocyte-melanoma interaction: role of surface molecules. Recent-Results-Cancer-Res, 1995, 139:205-214
8. De Giovanni G,Lahaye T, Herin M,et al.Antigenic heterogeneity of a human melanoma tumor detected by autologous CTL clones. Eur J Immunol,1988,18:671.
9. Maeurer MJ, Gollin SM, Martin D, et al. Tumor escape from immune recognition: lethal recurrent melanoma in a patient associated with downregulation of the peptide transporter protein TAP-1 and loss of expression of the immunodominant MART-1/Melan-A antigen. J-Clin-Invest, 1996; 98(7): 1633-1641
10. Ostrand-Rosenberg S,Clemcnts VK,Thakur A,et al.Transfection of major hislocompatibility complex class Ⅰ and class Ⅱ gcne causes lumour rejection. J Immunogenet,r1989,16:34
11. Hodgc JW, Abrams S,schlom J, et al. Induction of antitumor immunity by recombinant vaccinia viruses expressing B7-1 and B7-2 costimulatory molecules. Cancer Res,1994,54:5552-5555.
12. Shrayer DP, Kouttab N, Hearing VJ, el al. Immunization of mice with melanoma cells transfected to secrete the superantigen, staphylococcal enterotoxin A. Cancer-Immunol-Immunother. 1998 Mar; 46(1): 7-13
13. Tykocinski ML,Kaplan DR,Medof ME.Antigen-presenting cell engineering. AJP January, 1996,148(1): 1-16.
14. Medof ME,Nagarajan S,Tykocinski ML. Cell-surface engineering with GPI-anchored proteins. FASEB-J., 1996,10(5): 574-86.
15. Medof ME,Kinoshita T, Nussenzweig V. Inhibition of complement activation on the surface of cells after incorporation of decay-accelerating factor (DAF) into their membranes. J-Exp-Med.,1984,160(5): 1558-78.
16. Zhang F, Schmidt WG,Hou Y, et al. Spontaneous incorporation of the glycosyl-phosphatidylinositol-linked protein Thy-1 into cell membranes. Proc-Natl-Acad-Sci-U-S-A., 1992,89(12): 5231-5.
17. Henthorn PS.Knoll BJ, Raducha M,et al. Products of two common alleles at the locus for human placental alkaline phosphatase differ by seven amino acids. Proc-Nall-Acad-Sci-U-S-A., 1986,83(15): 5597-601.
18. Mcdof ME, Kinoshita T,Nussenzweig V. Inhibition of complement activation on the surface of cells after incorporation of decay-accelerating factor (DAF) into their membranes. J-Exp-Med.,1984,160(5): 1558-78.
19. Mann KJ, Sevlever D. 1,10-Phenanthroline inhibits glycosylphosphatidylinositol anchoring by preventing phosphoethanolamine addition to glycosylphosphatidylinositol anchor precursors. Biochemistry, 2001,40(5): 1205-13.
20. Davitz MA.Hereld D,Shak S,et al.A glycan-phosphatidylinositol-specific phospholipase D in human serum. Science, 1987,238(4823): 81-4.
21. Bangs JD,Andrews NW, Hart GW, et al.Posttranslalional modification and intracellular transport of a trypanosomc variant surface glycoprotein. J-Cell-Biol., 1986,103(1): 255-63.
22. Tykocinski ML,Shu HK,Ayers DJ,et al.Glycolipid reanchoring of T-lymphocyte surface antigen CD8 using the 3' end sequence of decay-accelerating factor's mRNA. Proc-Natl-Acad-Sci-U-S-A., 1988,85(10): 3555-9.
23. Oda K,Cheng J,Saku T, et al. Conversion of secretory proteins into membrane proteins by fusing with a glycosylphosphatidylinositol anchor signal of alkaline phosphatase. Biochem-J., 1994,301(Pt 2): 577-83.
24. McHugh RS,Ahmed SN,Wang YC,et al. Construction, purification, and functional incorporation on tumor cells of glycolipid-anchored human B7-1 (CD80). Proc-Natl-Acad-Sci-U-S-A.,1995,92(17): 8059-63.
25. Brunschwig EB,Levine E,Trefzer U,et al. Glycosylphosphatidylinositol-modified murine B7-1 and B7-2 retain costimulator function. J-Immunol.,1995,155(12): 5498-505.
26. Boeke JD.Model P.A prokaryotic membrane anchor sequence:carboxyl terminus of bacteriophage fl gene Ⅲ protein retains it in the membrane.
Proc-Natl-Acad-Sci-U-S-A, 1982,79:5200-5204.
27. Chen H,Kendall DA. Artificial transmembrane segments. J-Biol-Chem, 1995,270:14115-14122.
28. Wahlsten JL, Mills CD, and Ramakrishnan S. Antitumor response clicited by a superantigen-transmembrane sequence fusion protein anchored onto tumor cells. J Immunol, 1998; 161(12): 6761-6767
29．马文学，余海，王青青等。跨膜型超抗原SEA融合蛋白的表达及其对小鼠黑色素瘤的治疗作用研究。中华微生物与免疫学杂志，2003，in press.
30. Pulaski BA, Terman DS, Khan S,et al.Cooperativity of staphylococcal aureus enterotoxin B superantigen,major hitocompatibility complex class Ⅱ,and CD80 for immunotherapy of advanced spontaneous metastases in a clinically relevant postoperative mouse breast cancer model.Cancer Res.2000,60:2710-2715.
31. Freeman GJ,Freedman AS,Segil JM,et al. B7,a new member of the Ig super-family with unique expression on activated and neoplastic Bcell.J Immunol,1989,143:2714
32. Scherer MT, Ignatowicz L, Winslow G, et al. Superantigens: bacterial and viral proteins that manipulate the immune system. Annu Rev Cell Biol, 1993; 9:101-128.
33. Parra E,Wingren AG, Hedlund G,et al.The role of B7-1 and LFA-3 in costimulation of CD8+ T cells. J-Immunol. 1997,158(2): 637-42
34. Nestle FO, ThomPson C, Shimizu Y; etal.Costimulation of superantigen-activated T lymphocytes by autologous dendritic cells is dependent on B7. Cell-Immunol. 1994, 156(1): 220-9
35. Goldbach-Mansky R, King PD, Taylor AP;etal. A co-stimulatory role for CD28 in the activation of CD4+ T lymphocytes by staphylococcal enlerotoxin B. Int-Immunol. 1992, 4(12): 1351-60
36. Chen L,McGowan P,.Tumor immunogenicity determines the effect of B7 Costimulation on T cell-mediated tumor immunity. J Exp Med. 1995,179:523-532.
37. Millan JL. Molecular Cloning and Sequence Analysis of Human Placental Alkaline Phosphatase. J.Biol,Chem.1986,261(7):3112-3115.
38. Freeman GJ,GrayGS,GimmiCD,et al.Structure,Expression,and T Cell Costimulatory Activity of the Murine Homologue of the Human B Lymphocyte Activation Antigen B7. J Exp Med, 1991, 174:625-631
39. Horton RM, Hunt HD.Ho SN,et al.Engineering hybrid gene without the use of restriction cnzymes:gene splicing by overlap extension. Gene, 1989,77:61-68.
40. Lehto Marty T.Sharom FJ.PI-specific phospholipase C cleavage of a reconstituted
GPI-anchored protein: modulation by the lipid bilayer. Biochemistry, 2002,41(4): 1398-408
41. Freeman GJ,GrayGS,GimmiCD,et al. Structure,Expression,and T Cell Costimulatory Activity of the Murine Homologue of the Human B Lymphocyte Activation Antigen B7. J Exp Med, 1991, 174:625-631
42. Kozak M.The scanning model for translation:An update.J cell Biol,108:229
43. Ferguson MA,Williams AF.Cell-surface anchoring of proteins via glycosyl-phosphatidylinositol structures. Annu-Rev-Biochem.,1988; 57285-320.
44. Goldstein DJ,Blasco L, Harris H. Placental alkaline phosphatase in nonmalignant human cervix. Proc-Natl-Acad-Sci-U-S-A.,1980,77(7): 4226-8.
45. Goldstein DJ,Rogers C,Harris H.A search for trace expression of placental-like alkaline phosphatase in non-malignant human tissues: demonstration of its occurrence in lung, cervix, testis and thymus. Clin-Chim-Acta., 1982,125(1): 63-75.
46. Abu-Hasan NS, Sutcliffe RG. Placental alkaline phosphatase integrates via its carboxy-terminus into the microvillous membrane: its allotypes differ in conformation. Placenta.,1985,6(5): 391-404.
47. Lange PH,Millan JL, Stigbrand T, et al. Placental alkaline phosphatase as a tumor marker for seminoma. Cancer-Res.,1982,42(8): 3244-7.
48. McDicken IW, McLaughlin PJ,Tromans PM,et al.Detection of placental-type alkaline phosphatase in ovarian cancer. Br-J-Cancer., 1985,52(1): 59-64.
49. Knoll BJ,Rothblum KN,Longley M. Nucleotide sequence of the human placental alkaline phosphatase gene. Evolution of the 5' flanking region by deletion/substitution. J-Biol-Chem., 1988,263(24): 12020-7.
50. Micanovic R,Bailey CA,Brink L,et al.Aspartic acid-484 of nascent placental alkaline phosphatase condenses with a phosphatidylinositol glycan to become the carboxyl terminus of the mature enzyme. Proc-Natl-Acad-Sci-U-S-A., 1988,85(5): 1398-402.
51. Micanovic R,Kodukula K,Gerber LD,et al.Selectivity at the cleavage/attachment site of phosphatidylinositol-glycan anchored membrane proteins is enzymatically determined. Proc-Natl-Acad-Sci-U-S-A., 1990,87(20): 7939-43.
52. Tiede A,Bastisch I,Schubert J,et al. Biosynthesis of glycosylphosphatidylinositols in mammals and unicellular microbes. Biol-Chem., 1999,380(5): 503-23.
53. Davitz MA,Hereld D,Shak S,et al.A glycan-phosphatidylinositol-specific phospholipase D in human serum. Science,1987,238(4823): 81-4.
54. Medof ME, Kinoshita T, Nussenzweig V. Inhibition of complement activation on the surface of cells after incorporation of decay-accelerating factor (DAF) into their membranes.
J-Exp-Med.,1984,160(5): 1558-78.
55. Zhang F, Schmidt WG,Hou Y, et al. Spontaneous incorporation of the glycosyl-phosphatidylinositol-linked protein Thy-1 into cell membranes. Proc-Natl-Acad-Sci-U-S-A.,1992,89(12): 5231-5.
56. Brodsky RA,Jane SM,Vanin EF, et al. Purified GPI-anchored CD4DAF as a receptor for HIV-mediated gene transfer. Hum-Gene-Ther.,1994,5(10): 1231-9.
57. Huang JH, Getty RR,Chisari FV, et al.Protein transfer of preformed MHC-peptide complexes sensitizes target cells to T cell cytolysis. Immunity, 1994,1(7): 607-13
58. Gimmi CD, Freeman GJ, Gribbenh JG,et al. B-cell surface antigen B7 provides a costimulatory signal that induces T cells to proliferate and secrete interleukin 2. Proc-Natl-Acad-Sci-U-S-A. 1991, 88(15): 6575-9.
59. Huang JH,Greenspan NS,Tykocinski ML. Alloantigenic recognition of artificial glycosyl phosphatidylinositol-anchored HLA-A2.1. Mol-Immunol. 1994,31(13): 1017-28.
60. Weber MC,Groger RK,Tykocinski ML. A glycosylphosphatidylinositol-anchored cytokine can function as an artificial cellular adhesin. Exp-Cell-Res. 1994,210(1): 107-12.
61. Dohlsten M, Abrahmsen L, Bjork P, et al. Monoclonal antibody-supcrantigcn fusion protein: tumor specific agents for T-cell-based tumor therapy. Proc Natl Acad Sci USA, 1994; 91(19): 8945-8949
62. Sakurai N,Kudo T, Suzuki M,et al. SEA-scFv as a bifunctional antibody: construction of a bacterial expression system and its functional analysis.
63. Robey E,Allison JP.T cell activation:intergration of signals from the antigen receptor and constimulatory molecules.Immunol Today, 1995,13:306-310.
64. Robey E,Allison JP.T cell activation:intergration of signals from the antigen receptor and constimulatory molecules.Immunol Today, 1995,13:306-310.
65. Kiessling R.Tumor-induced immune dysfunction.Cancer Immunol Immunother, 1999,48:353-362
66. Yang G, Hellstrom KE,Mizuno M T, et al.In vitro priming of tumor-reactive cytolytic T lymphocytes by combining IL-10 with B7-CD28 costimulation. J-Immunol. 1995, 155(8): 3897-903
67. Geldhof A B, Raes G, Bakkus M,et al. Expression of B7-1 by highly metastatic mouse T lymphomas induces optimal natural killer cell-mediated cytotoxicity. Cancer-Res. 1995, 55(13): 2730-3
68. Townsend SE, Su FW, Atherton JM, Allison JP. Specificity and longevity of antitumor immune responses induced by B7-transfected tumors. Cancer-Res. 1994, 54(24): 6477-83
69. GuoY, Wu M,Chen H,et al. Effective tumor vaccine generated by fusion of hepatoma cells with activated B cells. Science. 1994,263(5146): 518-20.
70. Kalland T, Hedlund G,Dohlsten M,et al. Staphylococcal enterotoxin-dependent cell-mediated cytotoxicity. Curr-Top-Microbiol-Immunol. 1991.174:81-92
71. Gaken JA, Hollingsworth SJ, Hirst WJ,et al. Irradiated NC adenocarcinoma cells transduced with both B7.1 and interleukin-2 induce CD4+-mediated rejection of established tumors. Hum-Gene-Ther. 1997,8(4): 477-88.
72. Dow-SW, Elmslie-RE,Willson-AP, et al.In vivo transfection with superantigen plus cytokine genes induces tumor regressionand prolongs survival in dogs with malignant melanoma. J-Clin-Invest, 1998,101(11): 2406-14
73. Labuda T, Wendt J, Hedlund G,et al.ICAM-1 costimulation induces IL-2 but inhibits IL-10 production in superantigen-activated human CD4+ T cells. Immunology. 1998, 94(4): 496-502
74. Muraille E,Devos S, Thielemans K,et al. Costimulation regulates the.kinetics of interleukin-2 response to bacterial superantigens. Immunology. 1996,89(2): 245-9.
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