CN1646153B - 抗血管生成的主动免疫疗法 - Google Patents
抗血管生成的主动免疫疗法 Download PDFInfo
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- CN1646153B CN1646153B CN03808567.4A CN03808567A CN1646153B CN 1646153 B CN1646153 B CN 1646153B CN 03808567 A CN03808567 A CN 03808567A CN 1646153 B CN1646153 B CN 1646153B
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- Emergency Medicine (AREA)
Abstract
本发明涉及血管通透因子(VPF)家族分子、受体与共同受体的寡核苷酸与肽序列及其变体在治疗与脉管系统增加有关的病理组织的主动免疫疗法中的应用。上述方法可以单独或者与其它方法联合用于治疗:肿瘤及其转移、急性与慢性炎症过程、传染病、自身免疫病、糖尿病性视网膜病变或者新生儿视网膜病变、器官移植排斥、黄斑变性、新生血管性青光眼、血管瘤和血管纤维瘤。
Description
发明背景
本发明涉及生物技术和制药工业领域,特别涉及使用血管发生相关分子作为靶标的主动免疫疗法。
从原有的血管形成新的血管的过程叫做血管发生。促(pro-)血管生成和抗血管生成因子之间的平衡对这一过程进行着广泛的调控。其过程与促血管生成因子(pro-angiogenic)的诱导作用和异常形式的新血管形成相关的疾病包括:(a)肿瘤(包括原发肿瘤及其转移灶),(b)急性与慢性炎症,例如哮喘、呼吸窘迫、子宫内膜异位症、动脉粥样硬化以及组织水肿,(c)感染引起的疾病,例如肝炎和Kaposi肉瘤(Kaposisarcoma)(d)自身免疫病例如糖尿病、牛皮癣、类风湿性关节炎、甲状腺炎和(e)其他疾病和状态,例如糖尿病性视网膜病变或者新生儿视网膜病变、器官移植排斥、黄斑变性、新生血管性青光眼、血管瘤和血管纤维瘤(Carmelliet P.y Jain RK.Nature 407:249,2000;Kuwano M等人,Intern Med40:565,2001)。许多这些疾病的潜在的治疗方法可能基于通过其中和作用抑制刺激血管的异常形成的促血管生成因子的活性或者其受体的活性,或者基于清除促血管生成因子的来源。
血管内皮生长因子是能够直接并特异性地诱导新血管形成的分子家族(LeungScience 246:1306,1989;Klagsburn M,Annual RevPhysiol 33:217,1991)。这个家族包括血管通透因子,也被称为血管内皮生长因子VPF/VEGF(现在命名为VEGF-A)、胎盘生长因子PIGF、血小板衍生生长因子PDGF-A和PDGF-B、以及其它四种与VEGF-A在结构和功能上相关的新分子VEGF-B/VRF、VEGF-C/VRP、VEGD-D/FIGF和VEGF-E(Olofsson B等人,PNAS USA 13:2576,1996;Joukov V等人,EMBO J 15:290,1996;Yamada Y等人,Genomics 42:483,1997;OgawaS等人,J Biol Chem 273:31273,1998)。
VEGF-A是由两个23-kDa的亚单位组成的同源二聚体糖蛋白质(Ferrara N等人,Biochem Biophys Res Comun 165:198,1989),有5种由同一RNA经过不同的拼接形成的单体同种型(isoform)。其中有两种固定在细胞膜上(VEGF 189和VEGF 206),其余三种是可溶性的(VEGF 121、VEGF145和VEGF 165)。VEGF 165是除了心脏和肺以外哺乳动物组织中含量最丰富的同种型,心脏和肺中含量最多的是VEGF189(Neufeld G等人,Canc Met Rev 15:153,1995),而胎盘中则以VEGF121的表达量占优(Shibuya MA等人,Adv Canc Res 67:281,1995)。
VEGF-A是家族中研究最多,特征最为人所知的,在多种疾病研究中都描述了它的改变。它的过度表达与多种疾病都相关,例如不同来源、不同位置的肿瘤以及肿瘤转移(Grunstein J等人,Caneer Res 59:1592,1999)、溃疡性结肠炎和克隆氏症(Crohn’sdisease)等慢性炎症(Kanazawa S等人,Am J Gastroentnterol 96:822,2001)、牛皮癣(Detmar M等人,J Exp Med 180:1441,1994)、呼吸窘迫(Thichett DR等人,Am J RespirCare Med 164:1601,2001)、动脉粥样硬化(CellettiFL等人,Nat Med 7:425,2001;Couffinhal T等人,Am J Pathol 150:1653,1997)、子宫内膜异位症(McLaren J.HumanReprod Update 6:45,200)、哮喘(Hoshino M等人,J Allergy Clin Immunol 107:295,2001)、类风湿性关节炎和骨关节炎(Pufe T等人,J Rheumatol 28:1482,2001)、甲状腺炎(Nagura S等人,Hum Pathol 32:10,2001)、糖尿病性视网膜病变和新生儿视网膜病变(Murata T等人,Lab Invest 74:819,1996;Reynolds JD.Paediatr Drugs 3:263,2001)、黄斑变性和青光眼(Wells JA等人,Br J Ophthalmol 80:363,1996;Tripathi RC等人,Ophthalmology105:232,1998)、组织水肿(Kaner RJ等人,Am J Respir Cell MolBiol.22:640 2000;Ferrara N,Endocrinol Rev 13:18,1992)、肥胖(Tonello C等人,FEBSlett 442:167,1999)、血管瘤(Wizigmann S y Plate KH HistolHistopathol 11:1049,1996),在炎性关节病人的滑液中存在(BottomleyMJ等人,Clin Exp Immunol 119:182,2000),并且与移植排斥相关(Vasir B等人,Transplantation 71:924,2001)。特别是在肿瘤中,表达 VEGF-A的3种基本同种型:121、165和189的细胞也是在体内生长比较快的细胞,然而到了末期,大多数肿瘤限制165同种型的表达,或者说,在缺乏165同种型的情况下,限制121和189表达而远不能促进细胞生长,从而证明几种同种型的联合作用能够增强肿瘤血管网络(Grunstein J.MolCell Biol 20:7282,2000)。
PIGF报道于1991年,它在同源二聚体状态下不能够诱导内皮细胞增殖(MaglioneD等人,Proc Natl Acad Sci USA88:9267,1991;DiSalvo J等人,J Biol Bhem 270:7717,1995)。通过VEGFR-1进行的信号转导使PIGF得以上调,血管内皮细胞放大了其对于VEGF在向与某些疾病相关的血管生成的表型转变中的反应(Carmeliet P等人,Nat Med 7:575,2001)。PIGF的表达与人脑膜瘤和神经胶质细胞瘤的血管形成过程有关(Numara M等人,JNeurooncol 40:123,1998)。这个分子与VEGF165形成异源二聚体,具有促进血管生成的活性,并描述了在一些在肿瘤细胞系的条件性培养液中它们的过度表达(Cao Y等人,J BioChem 271:3154,1996),这种过度表达通常还与类风湿性关节炎和原发的炎性关节病有关(Bottomley MJ等人,Clin Exp Immunol 119:182,2000)。
VEGF家族其它成员的过度表达的研究较少,也与一些病变相关。VEGF-B与乳腺、卵巢和肾脏肿瘤以及黑色素瘤和纤维组织瘤相关(Sowter HM等人,Lab.Invest.77:607,1997;Salven P Am.J.Pathol.153:103,1998;Gunningham SP等人,Cancer Res 61:3206,2001)。报道了在不同起源的肿瘤细胞中VEGF-B 167同种型的体外差异表达(Li X等人,Growth Factors 19:49,2001)。VEGF-C和VEGF-D参与了淋巴管形成的调节(Joukov V.等人,EMBO J.15:290,1996),VEGF-C的过度表达与下列疾病有关:组织水肿、乳腺、肺、头颈部、食道、胃等部位肿瘤、淋巴瘤、前列腺癌、转移性淋巴结(Kajita T等人,Br J Cancer85:93,2001;Kitadi Y等人,IntJ Cancer 93:662,2001;HashimotoI等人,BrJ Cancer 85:93,2001;KinoshitaJ等人,Breast Cancer Res Treat 66:159,2001;Ueda M,等人,GynecolOncol 82:162,2001;Salven P Am.J.Pathol.153:103,1998;O-Charoenrat P等人,Cancer92:556,2001)。关于 VEGF-D,它在肿瘤细胞中的过度表达与体内肿瘤的淋巴脉管系统增加和淋巴结内转移的提高有关(Stacker SA等人,Nat Med 7:186,2001;Marconcini L等人,Proc Natl Acad Sci U S A 96:9671,1999)。
VEGF家族分子诱导的内皮细胞功能变化是通过它们与酪氨酸激酶3型的细胞受体的结合所介导的,这类受体目前包括:VEGFR1(Flt1)、VEGFR2(KDR/Flk1)和VEGFR3(Flt4)(Kaipainen A J.Exp.Med.178:2077,1993)。N端的结构域2已经确认是负责与配体结合,它能促进胞浆结构域的磷酸化和信号转导(Davis-Smyth T等人,EMBO15:4919,1996)。
VEGFR1配体包括VEGF-A、PIGF和VEGF-B,其亲和力依次递减(Shibuya M Int JBiochem Cell Biol 33:409,2001)。在内皮细胞中,这个受体捕获循环中的VEGF(Gille H等人,EMBO J.19:4064,2000)。表达于造血细胞的VEGFR1和VEGF-A的结合显著影响B淋巴细胞、T淋巴细胞和树突细胞的前体细胞中转录因子NFκB的活化。上面这种相互作用与体内一种不利的免疫平衡的建立有关,其中树突细胞的成熟和T细胞的比例减少,在一些免疫抑制患者尤其是肿瘤患者中,观察到了这种现象(Dikov MM等人,Canc Res 61:2015,2001;Gabrilovich D等人,Blood 92:4150,1998)。VEGFR1的过度表达与牛皮癣、子宫内膜癌和肝细胞癌相关(Detmar M等人,J Exp Med 180:1141,1994:Ng IO Am JClin Patol 116:838,2001;Yokoyama Y等人,Gynecol Oncol 77:413,2000)。
VEGFR2受体(KDR/Flk1)介导VEGF-A的生物学功能,也能结合VEGF-C和VEGF-D。这个受体分别在活化的内皮细胞和肿瘤来源的细胞系上差异表达,在此建立VEGF的自分泌通路。除了涉及前面提到的那些与它的配体的过度表达相关的疾病以外,VEGFR2的过度表达与下列疾病的进展有关:子宫内膜癌(Giatromanolaki A等人,Cancer 92:2569,2001)、恶性间皮瘤(Strizzi L等人,J Pathol 193:468,2001)、星形细胞瘤(Carroll RS等人,Cancer 86:1335,1999)、原发乳腺癌(Kranz A等人,Int J Cancer 84:293,1999)、肠型胃癌(Takahashi Y等人,Clin Cancer Res 2:1679,1996)、多形性胶质母细胞瘤、退行性少突神经胶质细胞瘤(anaplastic oligodendroglioma)、坏死性室管膜瘤(necroticependimoma)(Chan AS等人,Am J Surg Pathol 22:816,1998)。KDR的过度表达与常染色体病VHL和成血管细胞瘤(Wizigmann-Voos S等人,Cancer Res 55:1358,1995)以及糖尿病性视网膜病变的进展有关(Ishibashi T.Jpn J Ophthalmol 44:323,2000),KDR与Flt-1共同过度表达还与迟发型超敏反应有关(Brown LF等人,J Immunol 154:2801,1995)。
VEGF-C和VEGF-D介导的淋巴血管生成(lymphangiogenesis)是它们与淋巴内皮细胞表面的FLT4受体或VEGFR3结合的结果。在有些情形中,甚至不出现配体的过度表达时,受体的过度表达同下列疾病过程中的不良预后相关联,这些疾病包括:糖尿病性视网膜病变(Smith G.BrJ Ophthalmol 1999 Apr;83(4):486-94)、慢性炎症与溃疡(Paavonen K等人,Am J Pathol 156:1499,2000)、淋巴结转移的建立与乳腺癌的发展(Gunningham SP.ClinCancer Res 6:4278,2000;Valtola R等人,Am JPathol 154:1381,1999),并且与鼻咽癌和口腔鳞癌有关(Saaristo A等人,Am J Pathol 157:7,2000;Moriyama M等人,Oral Oncol33:369,1997)。而且,VEGFR3的过度表达还是Kaposi肉瘤(Kaposi sarcoma)、Dabska型血管内皮瘤(type Dabska hemangioendothelioma)、皮肤淋巴管瘤(cutaneouslymphangiomatosis)一个灵敏的标志(Folpe AL等人,Mod Pathol 13:180,2000;Lymboussaki A等人,Am J Pathol 153:395,1998)。
最近确认了两个新的VEGF受体命名为NRP1和NRP2。它们属于neurophilin家族(NRP),并作为VEGF家族一些特定同种型VEGF-A145、VEGF-A165、VEGF-B167和PIGF蛋白质的共同受体(co-receptors),提高其促有丝分裂能力。NRP1的表达已经成为前列腺癌侵袭力的一个标志,还与黑色素瘤血管生成的增加以及乳腺癌的细胞凋亡逃逸有关(LatilA等人,Int J Cancer 89:167,2000;Lacal PM J Invest Dermatol 115:1000,2000;BachelderRE Cancer Res 61:5736,2001)。NRP1、KDR和 VEGF-A165的共同过度表达与糖尿病性视网膜病变中的纤维血管(fibrovascular)增生以及类风湿性关节炎有关(Ishida S.等人,InvestOphthalmol Vis Sci 41:1649,2000;Ikeda M.等人,J Pathol 191:426,2000)。NRP2在骨肉瘤中过度表达并促进血管生成和肿瘤的生长(HandaA等人,Int J Oncol 17:291,2000)。
多数以抑制血管生成为基础的治疗策略,尤其是肿瘤的治疗,是建立在阻断VEGF家族及其受体的基础上的,临床试验阶段使用:(1)阻断VEGF或者KDR受体的单克隆抗体、(2)金属蛋白质酶抑制剂,如Neovastat和Prinomastat、(3)VEGF抑制剂如Thalidomide、Suramin、Troponin I、IFN-α和Neovastat(4)VEGF受体阻断剂例如SU5416、FTK787和SU6668、(5)肿瘤内皮细胞凋亡诱导剂,如Endostatin和CA4-P、(6)降低VEGF或VEGF受体表达的核酶(ribozyme)(Angiozyme)。由于人类的VEGF及其受体KDR和Flt-1与小鼠的同源基因之间高度同源(分别为~90%,81%,89%),常规使用许多动物模型用以针对这个系统的抗血管生成化合物临床前药效评价(Hicklin DJ等人,DDT 6:517,2001)。
被动给药VEGF或VEGFR抗体在人体临床不同阶段的试验获得了成功(Hicklin DJ等人,DDT 6:517,2001)。抗VEGF人源化单克隆抗体A.4.6.1(Genentech,San Francisco,United States)已经进入治疗结肠、乳腺、肾脏和肺部肿瘤的三期临床试验(Dim,KJ等人,Nature 362:841,1993;Boersig C.R&D Directions Oct 7;44,2001)。尤其是KDR受体,已经开发了识别这个受体细胞外N端结构域的单克隆抗体(IMC-1C11,ImClone),这个单克隆抗体抑制人白血病细胞的增殖和转移并提高异种移植小鼠的存活。目前,正在研究它对结肠癌转移患者的作用(Dias S等人,J.Clin Invest 106:511,2000)。在上述试验中,已经证明这些抗体的使用中没有伴随副作用。
除了前面所述,还有一种尚没有应用于阻断血管生成的疗法,特异性的主动免疫疗法(SAI)。在肿瘤的SAI中,作为抗原的多肽、蛋白质或者DNA与合适的佐剂混合使用。SAI对体液免疫(B淋巴细胞活化)和细 胞免疫(T辅助细胞、细胞毒淋巴细胞和自然杀伤细胞活化)两种免疫反应进行刺激,并且与MHC I和MHC II类情况下,树突细胞作为专门的抗原呈递细胞的功能有关(Bystryn JC,Medscape Hematology-Oncology4:1,2001;Parker,KC等人,J.Immunol 152:163,1994;Nestle FO等人,Nature Medicine 7:761,2001;TimmermanJM,Annual Review Medicine50:507,1999)。
SAI是试验和临床研究中一个迅速增长的领域,它的应用前景很有吸引力,尤其是在肿瘤学中,已经报道了60多种基于SAI方法的临床试验正在进行中,已经超过了目前基于单克隆抗体的临床试验。特别是对于癌症而言,作为SAI免疫原的抗原选择,是建立在抗原的生理学特性(relevance)和它们在肿瘤表型漂变(phenotype drift)中不易被取代的特点(Bodey B等人,Anticancer Research 29:2665,2000),以及它们与肿瘤组织生长和演变高度特异的相关性的基础上的。
利用SAI治疗癌症的策略还优先考虑到表达于不同类型的肿瘤中的抗原确定,其可提高同一疫苗制剂的指征数目。例如癌胚抗原(CEA)、HER2-neu、人端粒酶以及神经节苷脂(Greener M.,Mol Med Today 6:257200;Rice J等人,J Immunol 167:1558,2001;CarrA等人,Melanoma Res11:219,2001;Murray JL等人,Semin Oncol 27:71,2000)。
在人类肿瘤中,VEGF过度表达于肿瘤区室中(Ferrara,N.Curr.Top.Microbiol.Immunol.237:1,1999),已经证实在肿瘤相关的脉管系统中有高水平的VEGF及其受体(Brekken RA.J Control Release 74:173,2001)。基质细胞也能在转化的细胞(transformed cells)的刺激下产生VEGF,结果是当肿瘤细胞被清除之后,患者仍保持原有的VEGF水平。在前列腺癌、宫颈癌和乳腺癌中,VEGF及其受体对于预后和病程发展(staging)有实用价值(George DJ等人,Clin Cancer Res 7:1932,2001;Dobbs SP等人,BrJ Cancer 76:1410,1997;Callagy G等人,Appl Immunohistochem MolMorphol 8:104,2000)。另一方面,VEGF也是可溶性因子,其与其它细胞因子例如IL-10、TNF-α、TGF-β(OhmJE和Carbone DP,ImmunolRes 23:263,2001)参与癌症患者特有的免疫抑制(Staveley K等人,Proc Natl Acad Sci USA 95:1178,1998;Lee KH等人,J Immunol 161:4183,1998)。该免疫抑制效果似乎与其结合F1t1受体相关(Gabrilovich D等人Blood 92:4150,1998)。
本发明描述了在试验肿瘤中使用VEGF家族分子及其受体的SAI方法。获得的抗肿瘤效果至少基于以下四种机制,不排除其可能的组合:(a)通过细胞毒淋巴细胞直接破坏产生VEGF的肿瘤和基质细胞、(b)通过抗体捕获或者中和循环中的VEGF从而破坏肿瘤相关血管的内皮细胞、(c)通过细胞毒淋巴细胞或者补体结合抗体直接破坏表达VEGF受体的内皮细胞、(d)通过捕获或者中和循环中的VEGF激活局部免疫反应,从而清除其免疫抑制效应。
在理想情况下,这些疗法可以清除或者避免肿瘤转移的出现,单独或者和其他抗肿瘤药剂结合用作一线或者二线疗法(first or second linetherapy)来减少或清除原发肿瘤。
针对VEGF家族分子及其受体的主动免疫,单独使用或者与其它疗法结合对于下列疾病也很有效:急性和慢性的炎症过程(哮喘、呼吸窘迫、子宫内膜异位症、动脉粥样硬化、组织水肿)、传染病(肝炎,Kaposi肉瘤)、自身免疫病(糖尿病、牛皮癣、类风湿性关节炎、甲状腺炎、滑膜炎)、糖尿病性视网膜病变与新生儿视网膜病变、器官移植排斥、黄斑变性、新生血管性青光眼、血管瘤和血管纤维瘤等等。
本发明的详细说明
根据本发明,体内给予编码VEGF家族蛋白质及其受体、共同受体或者片段的寡核苷酸序列或者其多肽变体,诱导具有抗血管生成和抗肿瘤作用的体液免疫和细胞免疫应答。
本发明的目的多肽性质的免疫原及其片段,可以从其天然来源中分离,或可以利用合成或DNA重组技术获得。这些多肽也可以与那些具有已知的佐剂活性的蛋白质如p64K(R.Silva等人,US 5286484和EP0474313)融合制备,或者单独制备以后再与它们共价结合。
在这些情况下其它可用的策略就是获得天然多肽、其突变的或者修饰 的变体以及它们的片段,作为突环(loop)的一部分暴露或不暴露在如OMP1的细菌蛋白,成为免疫激活制剂的一部分,在这种情况下特别指的是VSSP(R.Perez等人,US 5788985和6149921)。而且,还可以获得暴露在病毒粒子表面的多肽免疫原(HbsAg,细小病毒的VP2,等等),结合到特定的多肽,这些多肽以免疫应答诱导过程中特定的细胞和器官为靶标(CTLA4,Ig的Fc段,等等),也可以结合到能够提高生物分布的蛋白质如VP22。
本发明的目的蛋白质的最重要的天然来源优势表达于胎盘、激活的内皮细胞和肿瘤细胞。用这些细胞或组织的mRNA通过已知的方法来获得互补DNA(cDNA)。所提取的RNA用作多聚酶链式反应(PCR)的模板来扩增所选择的抗原所对应的cDNA。在每种情况下,所使用的引物都是根据将要插入cDNA的载体的特征设计的,或者根据以前报道的目的蛋白质的序列设计。或者,并且优选在PCR扩增用于本发明中最大的抗原的受体的情形中,其编码区是以两个或多个的重叠片段的形式扩增的。这些片段包含一个常用的连接位点,用来从片段开始组合成完整的DNA。
一种用来克隆目的抗原的替代方法是从市售的来源于人内皮或者同样来源的肿瘤的DNA库中选择。在某些情况下,可能希望突变本发明的目的抗原,目的是避免由接种疫苗尤其是在VEGF家族产生的血管生成诱导事件。这些突变优选在那些文献报道的受体结合位点进行。因此,所设计的引物要覆盖目的分子的两端,PCR产物用作模板来获得突变分子。这些突变变体缺乏生物学功能但是能够重现所选的抗原的免疫原性。
用前面提到的方法获得的cDNA分子插入合适的载体中,载体可以是病毒、质粒、细菌人工染色体或者类似的物质。载体带有在靶细胞中基因适当表达所需的元件,以及根据其性质使其在宿主细胞中复制的元件。本发明的DNA分子可能含有一个或者多个目的基因,这些基因由一个或者多个核酸组成(cDNA、gDNA、合成与半合成DNA等),其经过转录或者在适当的时候翻译就能在靶细胞中产生具有治疗或者疫苗价值的产物。
一般地,根据本发明的疫苗治疗产品的基因受到启动子的控制,该启 动子能在靶细胞或者有机体(哺乳动物)中发挥作用,还受到含有终止信号和目的基因产物mRNA中的多聚腺苷化的3’端区域的控制,使它能够表达。启动子可以是所述基因的天然的启动子或者异源的在靶细胞中有转录活性的启动子。启动子可以来源于真核生物或者病毒。在真核启动子中,可以使用任何启动子或者可以激活或者抑制基因转录的衍生的序列,可以是特异性的或者非特异的,可以是诱导性的或非诱导性的,可以是强或弱的。另外,启动子区域可以通过插入激活子(activator)或者诱导子(inductor)序列进行修饰,实现目的基因的组织特异性表达或优先表达。
另外,目的基因可以带有信号序列用于亚细胞定位,这样目的基因一旦合成,它在细胞内的定位或分泌可以在它所表达的细胞内得到改变。它还可以带有编码特定区域的基因,这个区域能结合到免疫组织特异的配体上,这样目的基因就被引导至产生应答的部位,从而获得治疗/疫苗效应。
此外,目的基因的前面可以带有编码mRNA复制元件(mRNAreplicationmachinery)的序列,使mRNA能够在靶细胞中复制,提高了所述基因的表达,从而提高了本发明的治疗/疫苗效果。这里提到的复制元件(replication machinery)可以来源于甲病毒属(alphavirus)(Schlesinger S.,Expert Opin Biol Ther.1:177,2001),特别是辛德毕斯病毒(Sinbis)、塞姆利基(Semliki)病毒等。在这种特殊情况下,目的基因在亚基因组启动子的转录控制下,使得本发明的分子一旦被内在化,它的mRNA就能够在靶细胞中复制。另外,DNA载体可能带有一些序列,这些序列能使本发明的目的分子在哺乳动物细胞中复制。这样就能够提高表达水平和/或治疗/疫苗效果(Collings A.,Vaccine 18:4601,1999)。
DNA载体可以用标准的质粒DNA纯化技术纯化。这些技术包括含有溴化乙啶的氯化铯密度梯度纯化法或者离子交换柱或其它用于分离DNA分子的交换物或方法(Ferreira GN等人,Trends Biotechnol.18:380,2000)。
本发明包括了质粒DNA载体的使用,尤其是用于人体基因治疗和DNA免疫的PAEC家族的紧凑型载体(compact vector)(Herrera等人,Biochem.Biophys.Res.Commu.279:548,2000)。这个家族包括载体 pAEC-K6(登录号AJ278712)、pAEC-M7(登录号AJ278713)、pAEC-Δ2(登录号AJ278714)、pAEC-SPE(登录号AJ278715)和pAEC-SPT(登录号AJ278716)。这些载体只含有在哺乳动物细胞包括人细胞中表达目的产物的最基本的元件,以及大肠杆菌中的复制单元。转录单元由人巨细胞病毒(CMV)的即早期启动子、用于目的产物插入的多克隆位点、以及来自猿猴病毒40(SV40)的转录终止和多聚腺苷化序列组成。复制单元中,该载体包括卡那霉素抗性基因(Tn903)、pUC 19复制起点(ColE1),目的是保证高拷贝数和选择带有目的质粒的细菌。
另外,本发明包括了质粒DNA载体的使用,优选用于人体DNA免疫的PMAE家族的紧凑型载体。其中包括与PAEC系列相同的细菌中的功能元件、人CMV的即早期启动子以及多克隆位点。此外,它们还带有合成的内含子以及合成的转录终止序列和来源于兔β-球蛋白的多聚腺苷化序列。据报道,利用与后者相似的序列可以获得所克隆基因的更高的表达水平(Norman JA等人,Vaccine 15:801,1997)。而且,这个系列的载体还含有连续的免疫刺激序列重复(CpG基序),这个序列能够激活人和小鼠的天然免疫系统,从而激活针对目的分子的体液和细胞免疫应答(Krieg SM,Vaccine 19:618,2001)。
用重组病毒(腺病毒、腺伴随病毒、牛痘病毒、禽痘病毒、金丝雀痘病毒等病毒)进行的免疫能在宿主体内产生较强的细胞毒性细胞反应。将目的基因引入具有整合序列以及每种病毒特异的启动子的重组病毒载体。本发明也包含了这种策略,并优选采用禽痘病毒和pFP67xgpt载体。pFP67xgpt载体用于在合成的强早/晚期启动子的作用下,位于禽痘病毒FP9的11.2kB的BamHI片段开放读码框架6和7之间克隆目的基因。这个质粒还含有牛痘病毒启动子p7.5K控制的Ecogpt,用来鉴定重组病毒。本发明包括的其它替代手段包括用VEGF家族的蛋白质及其受体和/或共同受体免疫。如前所描述获得的cDNA分子克隆到载体中在病毒、酵母、噬菌体、植物或者更高等细胞中表达,从而得到抗原的蛋白质变体,它们的序列都通过传统的自动测序法得到了验证。有几个表达载体已经报道并用于获得重组蛋白质。这些载体至少含有可操作性连接到所要表达的 DNA序列或片段上的调控表达的序列。用于控制表达的序列实例有:lac、trp、tac和trc系统、启动子区域、lambda噬菌体的主要操纵子、表面蛋白fd的调控子区域、酵母糖分解启动子(例如3-磷酸甘油酸酯激酶(3-phosphoglicerate kinase))、酵母酸性磷酸酶启动子(例如Pho5)、酵母α交配因子启动子、来源于多瘤病毒、腺病毒、反转录病毒、猿猴病毒的启动子(例如SV40的早/晚期启动子)、以及其它已知序列,这些序列能够调控基因在原核和真核细胞以及它们的病毒或其组合内的表达。
这些载体复制以及获得本发明的目的重组蛋白质所使用的宿主包括原核细胞与真核细胞。原核细胞包括大肠杆菌(E.Coli)(DHI、MRCI、HB101、W3110、SG-936、X1776、X2282、DH5α)、假单胞菌、枯草杆菌、链球菌等。真核细胞包括酵母和真菌、昆虫、动物细胞(例如COS-7和CHO)、人和植物细胞、组织培养物等。在适当的培养基中的所选择的系统中表达后,可以用已知的方法分离多肽或者肽。
佐剂的使用
即使用已经在一定的动物模型中显示有效的裸DNA或者蛋白质来免疫,本发明的治疗策在治疗肿瘤或者自身免疫病的患者的时候还是遇到了挑战。为了促进免疫应答,DNA或蛋白质疫苗可以与一些已经描述过的免疫增强剂共同使用,如:矿物盐(例如:氢氧化铝、磷酸铝、磷酸钙)、免疫刺激剂例如细胞因子(例如:IL-2、IL-12、GM-CSF、IFN-α、IFN-γ、IL-18)、分子(CD40、CD154、MHC I分子的稳定链、LFA3)、皂角苷(如QS21)、MDP衍生物、CpG寡聚物、LPS、MPL和聚磷腈、脂类颗粒如:乳剂(例如弗氏佐剂、SAF、MF59)、脂质体、病毒体、iscom、螯合剂、微粒佐剂如PLG微粒、poloxamer、病毒类(如HBcAg、HCcAg、HBsAg)、细菌类(如VSSP、OPC)、粘膜佐剂如热不稳定内毒素(LT)、霍乱毒素、以及突变毒素(例如LTK63和LTR72)、微粒和多聚脂质体。在DNA疫苗免疫中,目的抗原的表达可以在双顺反子载体上联合某些已经提到的免疫增强分子。
在实施例中详细列出的试验条件显示,DNA可以非共价的结合到某些提到的颗粒上,而这种混合物的使用降低了抗肿瘤应答的最适浓度,与报道的那些更高剂量的裸DNA相似。
对哺乳动物的用药
对于治疗性的应用,本发明的疫苗制剂以药物可接受的剂量通过以下途径给药哺乳动物,优选人:粘膜、皮下、肌肉内、腹腔、淋巴内、局部和吸入等。它们可以给药到组织间隙,包括:肌肉、皮肤、脑、肺、肝、骨髓、脾、胸腺、心脏、淋巴结、血液、骨、软骨、胰脏、肾脏、膀胱、胃、肠、睾丸、卵巢、子宫、直肠、眼、腺体以及结缔组织。对于转移寡核苷酸的载体,它们优选表达于分化的体细胞,尽管它们也可定位到未分化或分化程度较低的细胞例如皮肤成纤维细胞和血液多能细胞。
免疫原可以在药物学允许的不产生毒性或治疗效果的载体中给药。这些载体的例子包括:离子交换剂、氧化铝、aluminum esthearates、卵磷脂、血清蛋白质如白蛋白、缓冲液例如磷酸、甘氨酸、山梨酸、山梨酸钾、植物来源的饱和脂肪酸不完全甘油脂混合物、水、盐或者电解质例如鱼精蛋白硫酸盐、磷酸氢二钠、氯化钠、锌盐、胶体硅、三硅酸镁、聚乙烯吡咯烷酮(polivynil pirrolidone)、基于纤维素和聚乙二醇的物质。在本发明中,优选磷酸缓冲液作为疫苗制剂的载体使用。
在使用蛋白质和肽的时候,它们可以共价或者非共价地结合到已知有类似佐剂作用的的载体分子上。这些分子包括:KLH、p64K、OPC(Musacchio A等人,Vaccine 19;3692,2001)以及VSSP。本发明还有一种替代方法联合使用裸DNA、病毒载体和蛋白质免疫原。质粒DNA给药的优势在于能够在疫苗制剂中产生一种到多种目的分子。因此,本发明涉及的分子可以通过各种类型载体的组合在接种程序中给药(诱导再刺激的变体,使用DNA、蛋白质和病毒载体)。
DNA载体可以直接向患者给药,或者宿主细胞可以在体内或者体外用载体修饰。后一种策略可以联合位点特异重组插入或体细胞转基因免疫接种而指导载体至特定的细胞表达。而且,DNA载体的细菌宿主可以用 作它们体内转化的载体。
这样,带有本发明的基因的分子可以以裸DNA的形式或者联合不同的载体使用:化学/生物化学/生物学、天然/合成、或者重组的。这些分子可以偶联或者结合到阳离子肽、包装分子(如PEG、PEI)、核定位肽(NLP)等等。这些也可以与能够形成DNA沉淀的阳离子一起给药,作为脂质体制剂的一部分,所述分子膜融合前加到该制剂中,并在脂性的合成载体上,也可以由阳离子聚合物组成(例如DOGS或者DOTMA)。对于DNA载体的给药,也可以使用嵌合蛋白质,它们能包装DNA分子并且介导复合物的转运,以及其被特异细胞选择性内吞。带有本发明涉及的治疗/疫苗基因的DNA分子,可以通过物理转运方法例如颗粒轰击、电穿孔(离体、体内、体外)或者通过局部应用或者颗粒吸入等方法直接向体内给药而用于向细胞内的基因转运。活载体包括腺病毒颗粒或者本发明的分子所产生的相同宿主。
所使用的多肽和/或者寡核苷酸的剂量可以根据不同的参数建立,尤其是要依赖下列参数:作为抗原给药的基因或者蛋白质、给药的途径、预治疗的病理学、治疗时间,在使用寡核苷酸时的用于免疫的载体。与下面实施例描述的不同的剂量的改变或者给药途径的变化,不背离本发明的原则或者观点,可能达到最佳的免疫效果,获得更好的应答。
治疗应用
本发明比被动免疫疗法有很多优点,这些被动免疫方法已经进入高级临床阶段,它们使用相同分子作为靶标。相比给药单克隆抗体(如,Anti-VEGF)的被动免疫,使用蛋白质或寡核苷酸的免疫的优点在于能够诱导抗体的内源性生成并且诱导特异性的细胞毒性CD8+淋巴细胞的增殖与扩散。
本发明比直接阻断VEGF-VEGFR体系的治疗策略有很多优点,主要由于这些策略只能清除循环中的VEGF水平或阻断KDR。而这里所提出的策略不但具有上述作用,还能破坏VEGF的来源(也就是肿瘤细胞以及相关的基质)和/或表达它们的受体的细胞(肿瘤内皮细胞和一些肿瘤 细胞)。以前这个领域的工作只描述了体液应答作为所观察到的主要的效果。不想将本发明局限于一种特定的机制,实施例显示,除了特异性的体液应答之外,所述的疫苗制剂还能激发CD8+细胞应答,其与体液应签共同作用;在肿瘤环境中,两者的结合与获得抗肿瘤效应相关(在实施例9中观察到)。
细胞毒性细胞应答可能是通过对表1和表2中的一些肽的识别所介导的。其中某些肽段可能与针对所述择的VEGF家族及其受体和共同受体的靶标的细胞应答相关。这一信息是通过分别使用BIMAS和SYFPHEITI软件的NIH和Heidelberg研究所(http://bimas.dcrt.nih.gov/molbio/hla-bind和www.bmi-heidelberg.com/scripts/MHCServer.dll/home.htm)的公共数据库利用计算机分析得到的。标记的肽和其它由目的抗原衍生的序列可以用来对已经描述的病理状态进行主动免疫治疗,可以单独或联合治疗,含有或者不含有佐剂作用的分子。这些肽也可以以它们的寡核苷酸变体的形式用作疫苗。
用于抑制血管生成及相关的病理状态的方法,包括给予哺乳动物有效剂量的本发明所描述的DNA或者蛋白质分子,通过任何途径,并使用某些以前描述过的免疫增强剂或者佐剂。哺乳动物优选是人。
不可逆转并不受调控的血管生成的增加与许多疾病有关。这个包括VEGF家族及其受体和共同受体的体系在许多病理状态中都过度表达,正如以前所描述的。在这种情况下,本发明所提出的治疗策略有效治疗下列疾病:(a)癌症(原发肿瘤及其转移灶),(b)急性与慢性炎症过程如哮喘、呼吸窘迫、子宫内膜异位症、动脉粥样硬化、以及组织水肿,(c)传染性疾病如肝炎与Kaposi肉瘤,(d)自身免疫病如糖尿病、牛皮癣、类风湿性关节炎和甲状腺炎,(e)其它疾病与状态如糖尿病性视网膜病变或者新生儿视网膜病变、器官移植排斥、黄斑变性、新生血管性青光眼、血管瘤和血管纤维瘤。
尤其是在肿瘤情况下,用本发明所提出的免疫原免疫接种可有效治疗恶性肿瘤、肉瘤和血管化肿瘤。能够用所提出的策略治疗的一些肿瘤的实例包括:表皮样肿瘤、鳞状瘤如头颈部鳞状瘤、以及结直肠、前列腺、乳 腺、肺(包括小细胞和非小细胞)、胰腺、甲状腺、卵巢和肝肿瘤。这些方法在治疗其它类型的肿瘤中也有效,例如Kaposi肉瘤、中枢神经系统肿瘤(如成神经细胞瘤、毛细血管瘤、脑膜瘤和脑转移瘤)、黑色素瘤、肾脏和胃肠部癌、横纹肌肉瘤、胶质母细胞瘤、平滑肌肉瘤。
特别地,VEGF-A和/或其受体VEGFR-1和VEGFR-2作为免疫原用于治疗:不同来源和位置的肿瘤及其转移灶、血管瘤、子宫内膜异位症、组织水肿、急性与慢性炎症过程如溃疡性结肠炎和克隆氏症(Crohn’sdisease)、动脉粥样硬化、类风湿性关节炎、骨关节炎、炎性关节病、牛皮癣、呼吸窘迫、哮喘、甲状腺炎、糖尿病性视网膜病变或者新生儿视网膜病变、黄斑变性、青光眼、常染色体病VHL、肥胖、某些器官移植排斥。另一方面,针对PIGF的反应对于类风湿性关节炎有用,通常用于原发的炎性关节病的治疗。
对于VEGF-B而言,它作为免疫原对于乳腺、卵巢和肾脏肿瘤以及黑色素瘤和纤维肉瘤有效。VEGF-C和它的受体VEGFR-3对于治疗组织水肿、糖尿病性视网膜病变、慢性炎症、溃疡、乳腺肿瘤、肺部肿瘤、头颈部肿瘤、食道肿瘤、胃部肿瘤、淋巴瘤、前列腺肿瘤、转移性结节(metastatic nodule)、Kaposi肉瘤(Kaposi sarcoma)、Dabska型血管内皮瘤(Dabskatype hemangioendothelioma)、皮肤淋巴管瘤(cutaneouslymphangiomatosis)都有效。VEGF-D的免疫可以特异性的用于治疗淋巴结转移。
NRP1和NRP2共同受体用于哺乳动物免疫,能有效的治疗尤其是前列腺癌、黑色素瘤、骨肉瘤、乳腺癌转移、糖尿病性视网膜病变以及风湿性关节炎的纤维血管增生。
基于给予抗体的被动免疫治疗的研究显示,抗VEGF-A和KDR的抗体联合使用对于同源肿瘤(syngeneic tumors)模型更有效。因此,使用两种或多种本发明免疫原对抑制血管生成和肿瘤生长提供了特别有效的治疗。这些免疫原可以单独给药,也可以用双顺反子载体通过前面提过的途径成对给药。另外,本发明的疫苗组合物可以与药物或化学治疗剂一起或者先后使用,对于治疗的疾病有利。
下面描述的结果证明抗血管生成和抗肿瘤反应是通过体液和细胞应签的共同作用介导的。尤其是VEGF及其受体,它们参与了树突细胞成熟并且作用于B淋巴细胞和T淋巴细胞前体。实施例10表明,本发明所提出的治疗策略除了降低血清中的VEGF水平以外,也能促进B淋巴细胞和T淋巴细胞和树突细胞比例的正常化。这一效果能促进肿瘤抗原在MHC I的呈递,提高抗肿瘤免疫应答的质量和强度,使这种免疫应答不但指向免疫原,也能指向肿瘤环境中其它肿瘤相关、肿瘤特异和过度表达的抗原。
实施例
实施例1
抗原的克隆与瞬时表达
人VEGF及其同种型和功能突变体
用来自以前分离的CaSki细胞(ATCC CRL 1550)的mRNA的cDNA作为模板,通过多聚酶链式反应(PCR)克隆了VEGF的同种型,使用引物为SEQ ID1和SEQ ID2,操作按照制备商(Perkin-Elmer)的说明进行。从2%的琼脂糖凝胶上提取VEGF同种型121、165和189所对应的扩增产物带。用核酸内切酶BamHI和EcoRI消化电泳带后,纯化VEGF同种型的cDNA并独立克隆到PAECΔ2载体(CIGB所有的载体)中。所得到的质粒测序并确定克隆的同种型与EMBL(www.embl-heidelberg.de)所报道的氨基酸序列没有发生突变。与VEGF同种型对应的cDNA随后克隆到pMAE5Δ5的Kpnl/EcoRV中,这个载体因为有5’免疫刺激CpG位点的存在,而不同于pAECΔ2。
根据Seimeister G等人(Siemeister等人,J Biol Chem 273:11115,1998)所述,将前面克隆的VEGF121同种型直接突变得到与KDR受体结合能力缺陷的VEGF变体(VEGFKDR(-))的cDNA。
用下列引物通过PCR法得到该突变变体:
(A)5’端片段(315bp)的扩增:用引物SEQ ID3和SEQ ID4
(B)3’端片段(93bp)的扩增:用引物SEQ ID5和SEQ ID6
扩增的片段用文献中的方法纯化,并以等摩尔浓度作为融合PCR的模板,使用相应于序列SEQ ID7和SEQ ID8的引物。所得到的含有突变的cDNA用BamHI/EcoRI进行消化并纯化,再克隆到pAECΔ2载体中。用测序的方法验证所引入的突变,与VEGFKDR(-)对应的DNA亚克隆到pMAE5Δ5载体BamHI/EcoRI处,得到pMAE5Δ5VEGFKDR(-)。
用于转染和动物接种的质粒在不含内毒素的条件下纯化,如WhalenR.等人所述(Whalen RG和Davis HL,Clin Immunol Immunopathol 75:1,1995)。简言之,按照制造商说明使用QIAGEN Endo-free系统纯化DNA, 然后将该DNA进行第二次沉淀。最后,用不含内毒素的PBS(Sigma,USA)溶解DNA至终浓度4mg/mL。
1.2人VEGF受体(KDR/Flk1)
使用人VEGF(Sigma)和肝素(Sigma)处理的内皮细胞系HUVEC(Clonetic,USA)的mRNA,通过反转录PCR得到编码VEGF受体KDR细胞外结构域(KDR1-3)以及该受体跨膜和细胞内结构域(KDRTC)的cDNA。
对于细胞外结构域1到3,所使用的引物为序列SEQ ID9和SEQID10。用核酸内切酶BamHI和EcoRI消化所得到的扩增片段(943bp)后,将该编码KDR结构域1至3的cDNA纯化并克隆到pAECΔ2载体中。限制性酶分析阳性的克隆经过对相应的DNA的测序得到验证。KDR1-3对应的cDNA序列然后亚克隆到前述pMAE5Δ5载体BamHI/EcoRI处(pMAE5Δ5KDR1-3)。
设计一个两步法的策略克隆该受体的跨膜区和细胞内区域。为插入第一个片段,使用了相应于SEQ ID11和SEQ ID12的引物。经过Xbal/BgIII消化这个747bp的片段后,将该产物克隆到pMAE5载体中,载体事先也用同样的酶消化,得到了质粒pMAE5 KDR 747。再用BgIII/NotI消化该质粒以便于插入其余的1091bp的羧基端片段,这个1091bp的片段是用相应于序列SEQ ID13和SEQ ID14的引物扩增得到的。限制性酶分析阳性的克隆经过DNA测序验证,并命名为pMAE5 KDR C。
1.2.1在病毒载体中克隆KDP的跨膜区和细胞内区
为了在禽痘病毒上克隆VEGF受体(KDR)的跨膜区和细胞内区,使用对应于序列SEQID15和SEQ ID16的引物。用酶Stul/Smal消化该953bp片段后,将产物克隆到pFP67xgpt载体中,该载体事先也用同样的酶消化。在同一个载体上,用Smal/BamHI消化后,插入用对应于序列SEQ ID15和SEQ ID16的引物从原始cDNA扩增得到的919bp的片段。限制性酶分析阳性的克隆经过DNA测序验证,并命名为pFP67xgpt KDRC。
在补充有2%胎牛血清(FBS)的DMEM培养基中的鸡胚成纤维细胞(CEF)中培养禽痘病毒(FWPVs)。用Lipofectin(Gibco BRL,GrandIsland,USA)将pFP67xgpt KDR C转染到CEF中,CEF事先用减毒株FP9感染。24小时后,加入新鲜的培养基,细胞再培养3至4天。之后,细胞冻融3次。表达编码Ecogpt酶基因的重组病毒用含有霉酚酸(25μg/mL)、黄嘌呤(Xantine)(250μg/mL)和次黄嘌呤(hypoxantine)(15μg/mL)的选择性培养基(MXH)纯化。用PCR检查基因在重组病毒中的正确插入。重组病毒命名为FPKDRgpt,未重组病毒作为阴性对照FP。
实施例2
抗原的体内表达
为了确认所构建的载体在体内表达蛋白质的能力,将它们注射到C57BL/6小鼠(每组3只)的股四头肌中。
1.于pH 7.2的PBS中的pMAE5Δ5-VEGF121(每只小鼠10和50μg)
2.于pH 7.2的PBS中的pMAE5Δ5-VEGF165(每只小鼠10和50μg)
3.于pH 7.2的PBS中的pMAE5Δ5-VEGF189(每只小鼠10和50μg)
4.于pH 7.2的PBS中的pMAE5Δ5-VEGFKDR(-)(每只小鼠10和50μg)
5.于pH 7.2的PBS中的pMAE5Δ5-KDR 1-3(每只小鼠10和50μg)
6.于pH 7.2的PBS中的pMAE5 KDR C(每只小鼠10和50μg)
7.于pH 7.2的PBS中的FPKDRgpt(2.5*107cfu)
8.pH 7.2的PBS(阴性对照)
注射后48小时处死动物,分离一块所注射的肌肉。部分肌肉组织在蛋白酶抑制剂和去离子去污剂的存在下匀浆。用识别全部人VEGF同种型的多克隆抗体(sc-152G)通过Dot-Blot和Western-Blot,根据所描述的步骤分析蛋白质提取液中VEGF的存在。从其余的肌肉组织中用TRI-Reagent(SIGMA)提取RNA。每种试验条件的RNA共20μg在含甲醛的1%的琼脂糖凝胶中进行电泳。将RNA转到尼龙膜上(HYBOND),然后用ATP32标记的VEGF 121同种型的cDNA进行杂交,所述cDNA 识别所有的VEGF同种型,也可以用类似标记的KDR的cDNA杂交。在这两种情况下,滤膜都与一个组成型基因(constitutive gene)磷酸甘油醛脱氢酶(gliceraldehyde 3-phosphate deshydrogenase)(GAPDH)的cDNA重新杂交。在所有分析的构建体中,对与人VEGF和所克隆的KDR受体片段对应的电泳带都进行了鉴定。
实施例3
用含有VEGF受体KDR基因片段的质粒进行接种的体内保护试验
用或不用下列变体接种各组C57BL/6小鼠(每组10只):
1.于pH 7.2的PBS中的pMAE5Δ5-KDR 1-3(每只小鼠1、10、50和100μg)
2.于pH 7.2的PBS中的pMAE5Δ5KDR C(每只小鼠1、10、50和100μg)
3.FPKDRgpt(2.5*107cfu)
4.pH 7.2的PBS(阴性对照)
5.FP(2.5*107cfu)(阴性对照组3)
在每种情况下,小鼠在左后足处通过肌内注射免疫50微升总体积。15天后,所有动物用初始免疫方案重复免疫。在最后一次免疫后30天进行肿瘤攻击,在每只动物的右腹部皮下注射104个B16-F10黑色素瘤细胞(ATCC,CRL-6475)。每周进行三次测量监视肿瘤的生长直到动物开始死亡。
在pMAE5Δ5-KDR 1-3质粒免疫的小鼠中在每只小鼠50和100μgDNA的剂量下观察到了肿瘤大小的减少,与阴性对照相比有明显的降低(见表3)。第33天的生存分析显示,用上述DNA在每只小鼠50和100μg的剂量下免疫的动物与未免疫的小鼠(pH 7.2的PBS组)相比,这个参数明显的提高(与阴性对照组相比)。对于pMAE5Δ5KDR C(见表3),肿瘤体积在四个使用的剂量都观察到了明显的下降,每只动物100到10μg的剂量有存活的提高。在使用FPKDRgpt构建体的条件下(见表3)病毒载体的使用,与阴性对照相比(用没有插入FPgpt的载体免疫的小鼠组), 降低了肿瘤的体积并提高了存活。
表3.用VEGF受体(KDR)基因片段免疫的小鼠的肿瘤体积与存活
组 | [DNAμg] | 肿瘤体积(mm3) (第24天) | 存活 (第43天) |
pMAE5Δ5-KDR 1-3 | 100 | 424.0±119.2(***) | (***) |
50 | 756.32±435.9(***) | (**) | |
10 | 1024.2±397.1(*) | (ns) | |
1 | 1334.2±620.7(ns) | (ns) | |
pMAE5Δ5KDR C | 100 | 404.23±200.0(***) | (***) |
50 | 633.2±365.2(***) | (***) | |
10 | 924.3±437.1(**) | (*) | |
1 | 1114.2±665.7(*) | (ns) | |
FPKDRgpt | 2.5*107cfu | 304.23±152.0(***) | (***) |
FP gpt | 2.5*107cfu | 1891.0±726.0(ns) | (ns) |
pH 7.2的PBS | - | 1785.0±826.0 | - |
注意:肿瘤体积用从每组动物所测得得平均值±标准差(SD)表示,用一元ANOVA和Bonferroni post-test进行统计学比较。在存活方面,在所显示的天数用log-rank检验比较每组与对照组获得统计学意义。用ns表示p≤0.5,不明显;*表示p≤0.05;**表示p≤0.01;***表示p≤0.001。
实施例4
用含有VEGF同种型或突变株的质粒进行接种的体内保护试验
用或不用下列各变体接种各组C57BL/6小鼠(每组10只):
1.于pH 7.2的PBS中的pAECΔ2-VEGF121(每只小鼠1、10、50和100μg)
2.于pH 7.2的PBS中的pMAE5Δ5-VEGF121(每只小鼠1、10、50和100μg)
3.于H 7.2的PBS中的AE5Δ5-VEGF165(每只小鼠1、10、50和100μg)
4.于H 7.2的PBS中的AE5Δ5-VEGF189(每只小鼠1、10、50和100μg)
5.于H 7.2的PBS中的AE5Δ5-VEGF KDR(-)(每只小鼠1、10、50和100μg)
6.pH 7.2的PBS(阴性对照)
在每一情况下,小鼠都是在左后足处通过肌内注射免疫50微升总体积。15天后,所有小鼠用初始免疫方案重复免疫。在最后一次免疫后30天进行肿瘤攻击,每只动物在右腹部皮下注射104个B16-F10黑色素瘤细胞(ATCC,CRL-6475)。每周进行三次测量监视肿瘤的生长直到动物开始死亡。
PAEC系列的裸DNA在用每只动物100μg剂量免疫的小鼠中观察到了与对照组相比肿瘤生长的减少(见表4)。在带有5CpG位点的pMAE5Δ5系列载体中的小鼠组与阴性对照相比变体中,不论何种VEGF同种型,在免疫10、50和100μg DNA剂量的小鼠组与阴性对照相比肿瘤大小明显下降。在使用突变变体pMAE5Δ5-VEGF KDR(-)的情形中,在与pMAE5Δ5-VEGF121类似的剂量下获得肿瘤大小明显的减少。
第43天的存活分析表明在每只动物免疫50和100μg剂量的变体pMAE5Δ5-VEGF121、pMAE5Δ5-VEGF165、pMAE5Δ5-VEGF189和pMAE5Δ5-VEGF KDR(-)的动物(与阴性对照相比)得到了明显的提高(见表4)。
表4.用含有VEGF基因不同同种型以及突变变体的裸DNA的不同变体免疫的小鼠的肿瘤体积与存活
分组 | [DNAμg] | 肿瘤体积(mm3)(第24天) | 存活(第43天) |
PAECΔ2-VEGF121 | 100 | 991.5±354(*) | ns |
50 | 1429.2±396(ns) | ns | |
10 | 1506.6±442(ns) | ns | |
1 | 1660.5±456(ns) | ns | |
pMAE5Δ5-VEGF121 | 100 | 645.0±215(***) | *** |
50 | 850.1±463(***) | *** | |
10 | 992.1±410(*) | ns | |
1 | 1560.3±598(ns) | ns | |
pMAE5Δ5-VEGF165 | 100 | 799.2±335(***) | *** |
50 | 916.6±390(**) | ** | |
10 | 1000.5±662(*) | ns | |
1 | 1845.3±450(ns) | ns | |
pMAE5Δ5-VEGF189 | 100 | 790.1±235(***) | *** |
50 | 996.5±255(***) | ** | |
10 | 1050.2±362(*) | ns | |
1 | 1670.2±408(ns) | ns | |
pMAE5Δ5-VEGF KDR(-) | 100 | 550.1±335(***) | *** |
50 | 894.7±408(**) | *** | |
10 | 991.8±362(*) | ns | |
1 | 1489.3±510(ns) | ns | |
pH 7.2的PBS | 0 | 1673.9±712 |
注意:肿瘤体积用从每组动物所测得得平均值±标准差(SD)表示,使用一元ANOVA和Bonferroni post-test进行统计学比较。在存活方面,在所显示的天数使用log-ranktest比较每组与对照组获得统计学意义。统计学意义用ns表示p≤0.5不明显;*表示p≤0.05;**表示p≤0.01;*** 表示p≤0.001。
实施例5
在胶原诱导的关节炎模型中用pMAE5Δ5-VEGF121和pMAE5Δ5-KDR 1-3进行免疫的体内保护试验
用或不用下列变体接种各组C57BL/6小鼠(每组20只):
1.于pH 7.2的PBS中的pMAE5Δ5-VEGF121(每只小鼠50μgDNA)
2.于pH 7.2的PBS中的pMAE5Δ5-KDR 1-3(每只小鼠50μgDNA)
3.pH 7.2的PBS(阴性对照)
每种情况,在第0天在左后足处通过肌内注射免疫总体积为50微升。15天后,所有动物用初始免疫方案重复免疫。
在第5天,开始用鸡II型胶原(Sigma)诱导自身免疫性关节炎,这个模型以前被Campbell等人(Campbell IK等人,Eur.J.Immunol.30:1568,2000)报道过。在第26天重复这个免疫。基于日常标准,对每只小鼠的四肢进行测量,测量是根据对四肢是否存在以下迹象的检查而得到的关节炎指数,它是从0到3打分:红斑迹象(1)、炎症(2)、关节僵硬(3),最大值为12。诱导后23天小鼠开始出现关节炎的临床症状,50天时发病率较高。表5列出在不同试验组动物中关节炎发病率的分析。在第40和55天在接种组(1和2)观察到了与阴性对照组相比关节炎发病率的明显下降。
表5.在所选择的日期关节炎的发病率(第40和55天)
分组 | 第40天发病率 | 第55天发病率 |
1 | 20/8(40%) | 20/9(45%) |
2 | 20/6(30%) | 20/12(60%) |
3 | 20/10(50%) | 20/14(70%) |
实施例6
接种的体内抗血管生成效应
用或不用下列变体接种各组C57BL/6小鼠(每组15只):
1.于pH 7.2的PBS中的pMAE5Δ5-VEGF121(每只小鼠50μg/DNA)
2.于pH 7.2的PBS中的pMAE5Δ5-KDR 1-3(每只小鼠50μg/DNA)
3.于pH 7.2的PBS中的pMAE DR C(每只小鼠50μg/DNA)
4.pH 7.2的PBS(阴性对照)
在每种情况中,C57BL/6小鼠都是在左后足处通过肌内注射免疫总体积50微升。15天后,所有小鼠用初始免疫方案重复免疫。在最后一次免疫后30天用Coughlin MC等人(Coughlin MC等人,J.Clin.Invest.101:1441,1998)所描述的magrigel评价动物体内的血管生成。前面免疫过的动物分成5组并在腹中线处皮下注射500微升magrigel(BectonDickinson and Co.,Franklin Lakes,New Jersey,USA),其中含有:
1.50ng/mLVEGF,5U/mL肝素
2.105个B16-F10黑色素瘤细胞
3.PBS
六天后动物处死,提取matrigel塞。根据制造说明(Drabkin’s reagentkit;SigmaDiagnostics Co.,St.Louis,Missouri,USA)分析塞中的血红蛋白含量。编码VEGF及其受体KDR的质粒接种后能明显(p<0.001)抑制VEGF诱导的血管形成,也能抑制更复杂的体系:肿瘤细胞所诱导的血管形成。
实施例7
通过pMAE5Δ5-VEGF121与不同佐剂的非共价结合获得免疫原
以前报道过的各种不同的免疫刺激剂同pMAE5Δ5-VEGF121构建体按照下面描述的方法混合使用。
根据Musacchio等人(Musacchio A等人Vaccine,67:751,1997)报道的方法纯化脑膜炎奈瑟球菌(Neisseria meningitidis)外膜的Opc蛋白质。将50μg/mL的pMAE5Δ5-VEGF121加到10μg/mL的Opc中,在酸性pH下温和振荡。将所得到的混合物在不含内毒素的pH7.2的PBS(Sigma)中过夜透析。通过1%的琼脂糖凝胶检测DNA检查Opc蛋白质-质粒DNA的结合(Opc-pMAE5Δ5-VEGF121)水平。超过50%的质粒DNA与Opc 蛋白质结合。
使用来自脑膜炎奈瑟球菌外膜的蛋白质合体(OMPC)的非常小粒子(VSSP)(由分子免疫学中心(R.Perez等人United States PatentApplication 5788985,6149921)提供),与目的质粒DNA组合。VSSP(1mg)与5mg pMAE5Δ5-VEGF121孵育过夜并温和振荡。所得到的材料在不含内毒素的pH 7.2的PBS(Sigma)中过夜透析。用1%的琼脂糖凝胶检测DNA来检查VSSP-质粒DNA的结合(VSSP-pMAE5Δ5-VEGF121)水平。超过50%的质粒DNA结合VSSP粒子。
用一种以前报道的方法(Lorenzo LF等人,Biochem Biophys ResCommun 281:962,2001)制备乙型肝炎与丙型肝炎的核心颗粒化抗原(HCcAg与HBcAg)。每mg抗原与5mg质粒混合过夜孵育。用1%的琼脂糖凝胶检测DNA来检查HCcAg或HBcAg-质粒DNA的结合(分别为HCcAg-pMAE5Δ5-VEGF121与HBcAg-pMAE5Δ5-VEGF121)水平。在每种情形中,超过50%的DNA结合抗原粒子。
实施例8
用pMAE5Δ5-VEGF121构建体与免疫应答佐剂的体内保护试验
用或不用下列变体接种各组C57BL/6小鼠(每组10只):
1.于pH7.2的PBS中的pMAE5Δ5-VEGF121(每只小鼠1、10、和50μg DNA)
2.Opc-pMAE5Δ5-VEGF121(每只小鼠1、10、和50μg DNA)
3.VSSP-pMAE5Δ5-VEGF121(每只小鼠1、10、和50μg DNA)
4.HBcAg-pMAE5Δ5-VEGF121(每只小鼠1、10、和50μg DNA)
5.HCcAg-pMAE5Δ5-VEGF 121(每只小鼠1、10、和50μg DNA)
6.pH 7.2的PBS(第1组的阴性对照)
7.Opc(第2组的阴性对照)
8.VSSP(第3组的阴性对照)
9.HBcAg(第4组的阴性对照)
10.HCcAg(第5组的阴性对照)
免疫步骤、肿瘤攻击与肿瘤体积的测量与前面的实施例中所描述的类似。与各自阴性对照组相比各个疫苗变体在每只小鼠10μg或10μg以上DNA剂量下降低肿瘤生长(见表6)。用结合或不结合作为免疫增强剂载体的Opc、VSSP、HBcAg、HCcAg的VEGF基因免疫动物观察到了与各自对照组相比明显提高的存活。所有用了载体的变体与各自对照相比都显示了明显提高的存活,从每只小鼠10μg剂量开始,而pMAE5Δ5-VEGF121的裸DNA变体在每只小鼠50μg剂量下与对照组相比产生了明显的不同(见表6)。
表6.用不同免疫刺激剂免疫的小鼠的肿瘤体积与存活
分组 | [DNA μg] | 肿瘤体积(mm3) (第24天) | 存活 (第43天) |
pMAE5Δ5-VEGF | 50 | 1050.9±689(**) | ns |
10 | 1229.0±596(*) | ns | |
1 | 1895.3±596(ns) | ns | |
Opc-pMAE5Δ5-VEGF | 50 | 960.6±456(**) | ** |
10 | 1100.5±615(**) | * | |
1 | 1654.8±663(ns) | ns | |
VSSP-pMAE5Δ5-VEGF | 50 | 884.6±410(***) | ** |
10 | 1002.3±598(**) | * | |
1 | 1532.7±745(ns) | ns | |
HBcAg-pMAE5Δ5-VEGF | 50 | 950.1±570(**) | ** |
10 | 1230.5±662(*) | * | |
1 | 1867.2±652(ns) | ns | |
HCcAg-pMAE5Δ5-VEGF | 50 | 950.1±570(**) | ** |
10 | 1230.5±662(*) | * | |
1 | 1867.2±652(ns) | ns | |
Opc(5μg/小鼠/剂量) | 5μg | 2059.0±687(ns) | ns |
VSSP | 2156.0±759(ns) | ns | |
HBcAg(5μg/小鼠/剂量) | 1998.2±798(ns) | ns | |
HCcAg | 1897.0±812(ns) | ns | |
pH 7.2的PBS | 2073.0±816(ns) | ns |
注意:肿瘤体积用从每组动物所测得得平均值±标准差(SD)表示,用一元ANOVA和Bonferroni post-test进行统计学比较。在存活方面, 在所显示的天数用log-rank test比较每组与对照组获得报告的统计学意义。统计学意义用ns表示p≤0.5,不明显;*表示p≤0.05;**表示p≤0.01; ***表示p≤0.001。
实施例9
使用蛋白质形式的VEGF的体内保护试验
用或不用下列变体接种每组10只C57BL/6小鼠:
加完全和不完全弗氏佐剂的VEGF165(20μg/小鼠)。
完全或不完全弗氏佐剂(阴性对照)
VEGF165抗原来源是商品化的(Sigma),纯度达到97%以上。用弗氏完全佐剂(Sigma)通过腹腔内途径免疫小鼠,在第15天和第30天用弗氏不完全佐剂通过同样途径重复免疫。肿瘤攻击与肿瘤体积的测量与前面的实施例中所述类似。
观察到了VEGF免疫组与未免疫的对照组相比肿瘤体积明显减小,存活提高。效果与以前用VEGF DNA的试验结果相似。
实施例10
严重联合免疫缺陷(SCID)C57BL/6小鼠体内免疫保护转变试验
根据实施例5描述的方法用或不用每只小鼠50μg的pMAE5Δ5-VEGF121免疫C57BL/6小鼠。初次免疫后45天处死小鼠。根据制造商的说明用磁珠(Dynabeads,USA)分离所述小鼠的CD8+、CD4+和B淋巴细胞。
每组10只六周龄的C57BL/6SCID小鼠用下列前面提取的淋巴细胞的组合重建。
第1组:来自pMAE5Δ5-VEGF121免疫的小鼠的CD8+T淋巴细胞和CD4+的T淋巴细胞。B淋巴细胞不重建。
第2组:来自免疫小鼠的B淋巴细胞和CD4+T淋巴细胞,来自未免疫小鼠的CD8+的T淋巴细胞。
第3组:来自免疫小鼠的CD8+T淋巴细胞和CD4+的T淋巴细胞,以及B淋巴细胞,作为试验的阳性对照。
第4组:来自未免疫小鼠的CD8+T淋巴细胞和CD4+T淋巴细胞以及B淋巴细胞,作为试验的阴性对照。
重建的SCID小鼠皮下用104个B16-F10黑色素瘤细胞攻击。每周进行三次测量监视肿瘤的生长直到小鼠开始死亡。用实验室的ELISA方法分析抗VEGF抗体水平。用0.5μg/mL的VEGF165(Sigma)溶液将96孔板孵育过夜。用PBS-BSA 1%(BDH,UK)溶液封闭孔,然后用系列稀释的动物血清孵育。用PBS-吐温0.05%洗涤后加入商品化的的抗小鼠IgG多克隆抗体(Sigma,A0168)。通过商品化的底物邻苯二胺(OPD,Sigma)扩大信号。
表7反映了肿瘤攻击的小鼠肿瘤体积(第24天)和存活率(第40天)的结果。从重建后的第15天开始,第1到3组的动物与第4组用来自未免疫小鼠淋巴细胞重建的小鼠相比肿瘤大小减少。因此在被免疫的小鼠中激活免疫系统,从而降低肿瘤大小的作用,是与体液应答和细胞应答有关的,后一种是细胞毒性(CTL),因为在第1组中缺乏抗VEGF抗体。然而,在试验条件下只有第3组(免疫小鼠的B和T淋巴细胞)的存活提高(见表7)。在部分重建缺乏B或CTL型T应答的重建动物中(分别为第1组或第2组),其存活与阳性对照相比没有差别。这些结果表明体液应答与细胞应答的组合(第4组)能产生有效应答的协同作用,延长受肿瘤攻击的小鼠的存活。
表7.用来自pMAE5Δ5-VEGF121免疫的小鼠的淋巴细胞重建的SCID小鼠的肿瘤体积与存活
注意:供体小鼠用或不用每只小鼠50μg pMAE5Δ5-VEGF DNA免疫。肿瘤体积用从每组动物所测得得平均值±标准差(SD)表示,用一元ANOVA和Bonferroni post-test进行统计学比较。在存活方面,在所显示的天数用log-rank test比较每组与对照组获得统计学意义。统计学意义用ns表示p≤0.5,不明显;*表示p≤0.05;**表示p≤0.01;***表示p≤0.001。
实施例11
通过免疫反应去除循环中的VEGF显示免疫恢复
用或不用下列变体肌肉内注射每组15只C57BL/6雌性小鼠:
1.于pH7.2的PBS中的pMAE5Δ5-VEGF121(每只小鼠50μg)
2.pH 7.2的PBS
每种情况中,通过于左后足肌肉内注射总体积50微升免疫小鼠。15天后,所有动物用初次免疫方案重复免疫。最后一次免疫三十天后每组随机挑选5只处死,用宏观的和组织学的评价分析免疫与对照动物的免疫状态以及接种对器官和组织的毒性。
每组其余的动物在右腹部接受皮下注射104个B16-F10黑色素瘤细胞。在注射肿瘤细胞后的第15和30天,每组处死5只小鼠,并按照前面描述的方法进行评价。
在所有被评价的动物中宏观水平上都没有出现毒性现象,组织学分析表明在最后一次免疫后30天检测的任何器官没有发现损伤。免疫学评价包括:(1)小鼠血清中VEGF水平评价;(2)B淋巴细胞和T淋巴细胞的细胞构成,以及脾脏、腕腋(brachial axillary)和腹股沟淋巴结中树突细胞的成熟度。
未处理的动物血清中的鼠VEGF水平分析(用于鼠VEGF的R&D试剂盒)表明,随着暴露于肿瘤时间的增长,血清中VEGF水平升高,同肿瘤体积随时间的增长一致。在针对人VEGF免疫的一组,在肿瘤攻击后持续的30天内检测到了鼠VEGF水平的明显下降(p<0.001,方差分析,Bonferroni post-test)。
用Gabrilovich等人(Gabrilovich D等人,Blood 92:4150,1998)报道的方法研究了淋巴结和脾脏中细胞群的比例,从而分析了每个时间点处死的动物的免疫系统状况。在这些研究中,使用了商品化的识别CD3、CD19、CD11c和CD86(B7-2)分子的单克隆抗体(Pharmingen),这些单克隆抗体用异硫氰酸荧光素(FITC)和藻红蛋白(PE)标记,通过使用流式细胞仪(FACS)能够显现各个细胞群。所获得的结果显示在表8中。
表8.根据表面标记的FACS分析细胞群体结果总结
注意:在每种情况中,数值表示阳性细胞在总体计数细胞中所占的百分比。
免疫后30天动物淋巴细胞群和树突细胞成熟度的分析显示接种VEGF DNA不能诱导动物的免疫状态任何变化。然而肿瘤移植后30天,未接种动物出现了T淋巴细胞/B淋巴细胞比例(CD3/CD9)的下降,无论在淋巴结还是在脾脏,与肿瘤攻击前相比都有下降。而且,尤其是在脾脏中,淋巴样细胞数有了明显下降。在这些动物的淋巴结和脾脏中也都观察到了成熟树突细胞数的下降。在接种VEGF DNA的小鼠组中观察到了各个参数的明显恢复,这个现象可能与这组动物中观察到的血清VEGF水平的下降关。
Claims (4)
1.疫苗组合物,其含有与KDR受体结合能力缺陷但是能够重现VEGF多肽的免疫原性的人VEGF121突变变体或其编码寡核苷酸,并且任选地进一步含有药物可接受的佐剂,所述佐剂选自:乙型肝炎核心抗原的重组颗粒,丙型肝炎核心抗原的重组颗粒,OPC蛋白质,KLH蛋白质,脑膜炎奈瑟氏球菌(Neisseria meningitidis)p64k蛋白质或脑膜炎奈瑟氏球菌外膜衍生VSSP,其中所述突变由SEQ ID4和SEQ ID5定义。
2.疫苗组合物,其含有单独或通过使用双顺反子载体而成对的形式或单个蛋白或混合蛋白形式的与KDR受体结合能力缺陷但是能够重现VEGF多肽的免疫原性的人VEGF121突变变体或其编码寡核苷酸,和人VEGF受体多肽或其编码寡核苷酸,并且任选地进一步含有药物可接受的佐剂,所述佐剂选自:乙型肝炎核心抗原的重组颗粒,丙型肝炎核心抗原的重组颗粒,OPC蛋白质,KLH蛋白质,脑膜炎奈瑟氏球菌p64k蛋白质或脑膜炎奈瑟氏球菌外膜衍生VSSP,其中所述突变由SEQ ID4和SEQ ID5定义。
3.根据权利要求1的组合物的用途,用于制备用于治疗发生在人肿瘤、恶性瘤和其转移、良性瘤、慢性炎症过程、自身免疫过程和眼部改变中的与血管生成增加相关的病症的药物。
4.根据权利要求2的组合物的用途,用于制备用于治疗发生在人肿瘤、恶性瘤和其转移、良性瘤、慢性炎症过程、自身免疫过程和眼部改变中的与血管生成增加相关的病症的药物。
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EP3096769A1 (en) * | 2014-01-21 | 2016-11-30 | University of Helsinki | Therapeutic use of vegfr-3 ligands |
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US6149921A (en) * | 1993-12-29 | 2000-11-21 | Centro De Inmunologia Molecular | Vaccine compositions for eliciting an immune response against N-acetylated gangliosides and their use for cancer treatment |
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EP1032583A4 (en) * | 1998-03-06 | 2005-02-02 | Imclone Systems Inc | Active immunization against angiotens |
JP2002514573A (ja) * | 1998-05-08 | 2002-05-21 | スローン − ケッタリング インスティチュート フォー キャンサー リサーチ | 能動的なワクチン接種のための組成物および方法 |
AU776473C (en) | 1999-03-11 | 2005-04-14 | Entremed, Inc | Compositions and methods for treating cancer and hyperproliferative disorders |
JP2003528824A (ja) * | 2000-02-04 | 2003-09-30 | スプラテック ファーマ インコーポレイティド | 血管内皮成長因子受容体のためのリガンド |
US7094410B2 (en) * | 2002-03-02 | 2006-08-22 | The Scripps Research Institute | DNA vaccine against proliferating endothelial cells and methods of use thereof |
CU23178A1 (es) * | 2002-04-15 | 2006-09-22 | Ct Ingenieria Genetica Biotech | INMUNOTERAPIA ACTIVA ANTIANGIOGéNICA |
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RU2329824C2 (ru) | 2008-07-27 |
PT1502599E (pt) | 2011-08-03 |
AU2003218603A1 (en) | 2003-10-27 |
SI1502599T1 (sl) | 2011-10-28 |
US20080254050A1 (en) | 2008-10-16 |
AU2003218603B2 (en) | 2009-05-28 |
US8883724B2 (en) | 2014-11-11 |
KR100704127B1 (ko) | 2007-04-05 |
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JP2006501145A (ja) | 2006-01-12 |
WO2003086450A1 (es) | 2003-10-23 |
EP1502599A1 (en) | 2005-02-02 |
RU2004133338A (ru) | 2005-06-10 |
CU23178A1 (es) | 2006-09-22 |
EP1502599B1 (en) | 2011-06-08 |
US20100047265A1 (en) | 2010-02-25 |
CN1646153A (zh) | 2005-07-27 |
US20050175624A1 (en) | 2005-08-11 |
US7556809B2 (en) | 2009-07-07 |
CA2480079A1 (en) | 2003-10-23 |
JP4741800B2 (ja) | 2011-08-10 |
ZA200408460B (en) | 2005-08-30 |
ATE511851T1 (de) | 2011-06-15 |
ES2365329T3 (es) | 2011-09-29 |
KR20040111526A (ko) | 2004-12-31 |
AR039285A1 (es) | 2005-02-16 |
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