CN100384873C - Anth1嵌合拮抗剂 - Google Patents
Anth1嵌合拮抗剂 Download PDFInfo
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- CN100384873C CN100384873C CNB038141264A CN03814126A CN100384873C CN 100384873 C CN100384873 C CN 100384873C CN B038141264 A CNB038141264 A CN B038141264A CN 03814126 A CN03814126 A CN 03814126A CN 100384873 C CN100384873 C CN 100384873C
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Abstract
本发明涉及重组嵌合蛋白，它包括与IFN伽马(IFNγ)受体α链细胞外区域N-末端融合的人白细胞介素2(IL-2)N-末端60个氨基酸的片段。所述蛋白质在体外表现T细胞生长刺激活性，抑制IL-2在T细胞中的生长刺激活性，抑制IFNγ对HLA-DR的诱导并抑制IFNγ的抗增殖活性。本发明适宜在治疗各种病理，例如，自身免疫疾病、移植排斥、慢性炎症、脓毒症、缺血和再灌注综合征以及动脉粥样硬化的药物中使用。
Description
本发明涉及生物科学、生物技术和药物科学，特别涉及可以抑制白细胞介素-2(IL-2)和伽马干扰素(IFNγ)生物活性的药物，这两种细胞因子在对机体若干功能的调控中起作用，而且发现在病理状态下它们的含量提高。因而由此避免危及病人生命的免疫系统的失活。
辅助型T淋巴细胞(CD4+)和细胞毒性T淋巴细胞(CD8+)的细胞因子生产形成了一种被确定为Th1和Th2的细胞因子生产类型。Th1型的特征是产生IL-2、肿瘤坏死因子α(TNFα)和IFNγ，而Th2型则应答产生IL-4、IL-5、IL-6及其它。这一类型的应答在机体防御、以及作为不同免疫病理反应的促进物中起重要作用。
有许多由于炎症性和失控的免疫反应而导致炎症性和自身免疫疾病的出现、发展并永久的情况。已有经证实的、关于IL-2和IFNγ在某些此种疾病中的病理作用的例子。
多发性硬化是一种退行性脱髓鞘(degenerative demyelenating)自身免疫疾病。IFNγ对这种自身免疫疾病的作用是非常明显的。由此，业已在一项使用IFNγ的临床试验中证明，这种治疗导致该疾病恶化(Panitch HS.等，Exacerbations of multiple sclerosis in patientstreated with interferon gamma.Lancet 1，893-5，1987.)。还证明，在患此病的病人体内存在高水平的对应于IL-2和IFNγ的信使RNA以及蛋白质(Lin J.等，IL-2，IFN-gamma，and TNF-alpha mRNA expressionin peripheral blood mononuclear Rcells in patients with multiplesclerosis。Chung Kuo I Hsueh Ko Hsueh Yuan Hsueh Page19，24-8，1997)。病人细胞产生这两种细胞因子暗示着它们可作为多发性硬化复发的标记(Philippe J.等，In vitro TNF-alpha，IL-2 and IFN-gammaproduction as markers of relapses in multiple sclerosis。ClinNeurol Neurosurg 98，286-90，1996)。还观察到，IL-2和IFNγ参与对非特异性淋巴细胞的活化，该活化导致中枢神经系统的脱髓鞘作用(Martino G.等，Proinflammatory cytokines regulateantigen-independent T-cell activation by two separatecalcium-signaling pathways in multiple sclerosis patients。AnnNeurol 43，340-49，1998)。一项在患有对βIFN疗法没有应答的这种疾病的病人体内使用已知为Daclizumab的抗-IL-2抗体的临床试样正在进行中。
红斑狼疮是另一种系统性自身免疫疾病，其中高水平IL-2和IFNγ的存在与这一疾病的恶化有关(Viallard JF.等，Th1(IL-2，interferon-gamma(IFN-gamma))and Th2(IL-10，IL-4)cytokine productionby peripheral blood mononuclear cells(PBMC)from patients withsystemic lupus erythematosus(SLE)。Clin Exp Immunol 115，189-95，1999)。另一方面，在狼疮动物模型中由于不存在IFNγ受体而使得自身抗体的产生减少(Haas C.等，IFN-gamma receptor deletion preventsautoantibody production and glomerulonephritis in lupus-prone(NZB x NZW)Fl mice。J Immunol 160，3713-18，1998)，IFNγ可溶性受体的存在会抑制该疾病的出现(Ozmen L.等，Experimental therapyof systemic lupus erythematosus：the treatment of NZB/W mice withmouse soluble interferon(receptor inhibits the onset ofglomerunephritis。Eur J Immunol.25，6-12，1995)。最近用带有嵌合蛋白的鼠狼疮模型对此进行了检验，该嵌合蛋白包含与免疫球蛋白Fc片段融合的IFNγ细胞外区域(Lawson BR.等，Treatment of murine lupuswith cDNA encoding IFN-gammaR/FC. J Clin Invest. 106，207-15，2000)。然而，红斑狼疮病人体内已证实的的Fc受体功能紊乱能够限制此类分子在红斑狼疮中的功效(Frank MM.等，Defectivereticuloendothelial system Fc-receptor function in systemic lupuserythematosus。N Engl J Med 300，518-23，1979，Dijstelbloem HM.等，Fcgamma receptor polymorphisms in systemic lupuserythematosus：association with disease and in vivo clearance ofimmune complexes。Arthritis Rheum 43，2793-800，2000)。
重症肌无力被认为是一种由抗-乙酰胆碱(acetyl coline)受体自身抗体介导、且依赖于T细胞的器官特异性自身免疫疾病，其特征在于肌无力和疲乏。最近证实，IFNγ促使产生针对乙酰胆碱受体的自身抗体；而不存在IFNγ受体则降低动物模型对该疾病的易感性(Zhang GX.等，Mice with IFN-gamma receptor deficiency are less susceptible toexperimental autoimmune myasthenia gravis。J Immunol 162，3775-81，1999)。IL-2和其它细胞因子与IFNγ共同促进该疾病的发展(Zhang GX.等，Cytokines and the pathogenesis of myasthenia gravis。MuscleNerve。20，543-51，1997).
在1型糖尿病(胰岛素依赖的)或糖尿病中，胰腺的β细胞被自身免疫机制破坏。有体外证据表明，IFNγ对于胰腺β细胞可能是具有毒性的(Sternesjo J.等。Effects of prolonged exposure in vitro tointerferon gamma and tumor necrosis factor-alpha on nitric oxideand insulin production of rat pancreatic islets。Autoimmunity 20，185-90，1995，Dunger A.等，Tumor necrosis factor-alpha andinterferon-gamma inhibit insulin secretion and cause DNA damagein unweaned-rat islets。Extent of nitric oxide involvement.Diabetes 45，183-9，1996，Baldeon ME.等，interferon-gammaindependently activates the MHC class I antigen processing pa thwayand diminishes glucose responsiveness in pancreatic beta-celllines。Diabetes 46，770-8，1997)。然而，其它研究证明，IFNγ导致胰腺细胞产生胰岛素的作用是间接的(Sarventick N.等，Loss ofpancreatic islet tolerance induced by beta-cell expression ofinterferon-gamma.Nature，346，844-7，1990)。最为可能的是，它的作用是通过巨噬细胞而被激活以产生IL-1、TNFα和氧化氮，IL-1、TNFα和氧化氮直接作用于β细胞、并刺激MHC I在β细胞中表达、进而促进它们对细胞毒性淋巴细胞的破坏(Thomas HE.等，IFN-gamma action onpancreatic beta cells causes class I MHC upregulation but notdiabetes。J Clin Invest，102，1249-57，1998，Thomas HE等，Betacell destruction in the development of autoimmune diabetes in thenon-obese diabetic(NOD)mouse。Diabetes Metab Res Rev 16，251-61，2000)。还已被表明的是，尽管不存在IFNγ不能预防糖尿病的发生，但是可以使其延迟(Hultgren B.等，Genetic absence of gamma-interferondelays but does not prevent diabetes in NOD mice。Diabetes 45，812-7，1996)。几份报告显示了可以如何将对IFNγ生物活性的失活应用于预防糖尿病(Debray-Scahs M.等，Prevention of diabetes in NODmice treated with antibody to murine IFN gamma.J Autoimmun 4，237-48，1991，Moosmayer D.等，Abivalent immunoadhesin of the humaninterferon-gamma receptor is an effective inhibitor of IFN-gammaactivity。J Interferon Cytokine Res 15，1111-5，1995，Prud hommeGJ.等，Prevention of autoimmune diabetes by intramuscular genetherapy with a nonviral vector encoding an interferon -gammareceptor/IgGl fusion protein。Gene Ther 6，771-7，1999)。业已看到，IL-2作为T淋巴细胞的激活剂能在对产胰岛素细胞的破坏中促进这一反应。
最近启动了一项用抗IL-2的抗体Daclizumab治疗在近期被诊断患有1型糖尿病的儿童/10～21岁青少年所患1型糖尿病的临床试验(RileyHospital for Children.Project：Prevention of Diabetes ProgressionTrial(PDPT)。www.rileyhospital.org.)。这项研究被设计为在近期被确诊的儿童体内阻止β细胞进一步被破坏。
移植排斥是一个复杂的过程，在此过程中细胞介导的免疫和循环抗体起着重要作用。标准的抗排斥治疗使用例如环孢菌素、雷帕霉素、azatioprin、类固醇及其它药物的组合。然而，即使是用该疗法，超过50％接受肾移植的人在十年内也慢慢排斥他们的移植物。针对宿主的移植物疾病是接受骨髓移植的病人的主要死亡原因。在这些抵制移植物并危及接受移植的病人生命的反应中，已经证实IL-2和IFNγ都促进这些反应的发展(Hu HZ.等，Kinetics of interferon-gamma secretion and itsregulatory factors in the early phase of acute graft-versus-hostdisease。Immunology 98，379-85，1999，Nakamura H.等，Serum levelsof soluble IL-2 receptor，IL-12，IL-18，and IFN-gamma in patientswith acute graft-versus-host disease after allogeneic bone marrowtransplantation.J Allergy Clin Immunol.106，S45-50。2000).
类风湿性关节炎(RA)一种病因学(ethiology)未知的系统性疾病，其特征在于炎症、sinovial增生和对被感染关节的破坏。IL-2通常被看作是一种使Th1型疾病例如自身免疫关节炎状态恶化的早期炎症细胞因子。近期的研究表明，在动物模型由胶原诱导的关节炎急性期中IL-2信使RNA升高(Thornton S.等，Heterogeneous effects of IL-2 oncollagen-induced arthritis。J Immunol 165，1557-63，2000)。另一方面，在动物模型中发现疾病的恶化与IFNγ的升高有关(Tellander AC.等，Potent adjuvant effect by anti-CD40 in collagen-inducedarthritis。Enhanced disease is accompanied by increased productionof collagen type-II reactive IgG2a and IFN-gamma.J Autoimmun 14，295-302，2000)。在RA患者的sinovial组织中IL-2和IFNγ都显著升高(Canete JD等，Differential Th1/Th2 cytokine patterns in chronicarthritis：interferon gamma is highly expressed in synovium ofrheumatoid arthritis compared with seronegativespondyloarthropathies。Ann Rheum Dis 59，263-8，2000)。
肠胃炎性疾病包括两种肠胃疾病：节段性回肠炎和溃疡性结肠炎。这些疾病的特征在于肠胃慢性炎症。节段性回肠炎是一种在肠壁内线周围蔓延并穿入其最深层的炎性疾病。在消化系统的任何部位(食道、胃、小肠、大肠或肛门)都可以发现这种炎症。Protein Desig Labs公司已经宣布开始对中度或重度节段性回肠炎患者使用抗IFNγ抗体(SMARTAnti-Gamma Interferon Antibody)的I/II期临床试验(Fremont，CA.Protein Design Labs Announces Phase I/II Trial of SMART″Anti-GammaInterferon Antibody in Crohn′s Disease。Protein Design Labs，Inc.(Nasdaq)，January 10，2001)。溃疡性结肠炎限制在大肠(结肠或直肠)的粘膜或粘膜下层。最近在美国胃肠病学协会年会(Annual Congressof the American Association of Gastroenterology)中显示了，血流中IFNγ水平的降低是如何成为小鼠模型结肠炎免除标记的(Yaron I。Annual meeting of the American Gastroenterology Association.May20-23，2001。Georgia World Congress Center。Atlanta，Georgia.)。
感染性休克是严重感染性微生物经血流散播的结果。较为常见的是这是由在医院感染的革兰氏阴性杆菌引起的，而且在无免疫应答的病人和患有慢性疾病的病人中更为普遍。有1/3的病人是由革兰氏阳性细菌和白色念珠菌引起的。无论在由革兰氏阴性或阳性细菌引起的感染性休克中，IFNγ和IL-2都促使它们所参与的炎症反应致死。IFNγ在感染性休克动物模型中是一种致死介质(Heremans H.等，Interferon gamma，a mediatorof lethal lipopolysaccharide-induced Shwartzman-like shockreactions in mice。J Exp Med 171，1853-69，1990，Wysocka M.等，Interleukin-12 is required for interferon-gamma production andlethality in lipopolysaccharide-induced shock in mice。Eur JImmunol 25，672-6，1995，Kuschnaroff LM等，Increased mortalityand impaired clonal deletion after staphylococcal enterotoxin Binjection in old mice：relation to cytokines and nitric oxideproduction.Scand J Immunol 469-78，1997)。正如其它疾病动物模型的情况，动物模型中不存在IFNγ受体使得它们能够抗内毒素休克(Car BD等，Interferon gamma receptor deficient mice are resistant toendotoxic shock。Exp Med 179，1437-44，1994)。类似地，几份报告表明IL-2参与感染性休克的发展和致死(Micusan VV，等，Production ofhuman and murine interleukin-2 by toxic shock syndrome toxin-1。Immunology 58，203-8，1986，Arad G，等，Superantigen antagonistprotects against lethal shock and defines a new domain for T-cellactivation.Nat Med.6，378-9，2000，Stevens DL.等，Streptococcaltoxic shock syndrome associated with necrotizing fasciitis。AnnuRev Med 51，271-88，2000)。在体外与IL-2分离(segregate)次级细胞因子例如IL-1、TNFα和IFNγ生长的单核细胞涉及感染性休克的病理生理学。
普通牛皮癣是一种复杂的多基因皮肤病，它潜在地由受损T细胞产生的炎症细胞因子原介导。这些细胞因子的不适当慢性表达导致细胞的免疫激活和组织损伤。它的特征是过量产生皮肤细胞和形成血管——它们可能导致作为该疾病一部分的发红和形成小片。已有证据表明IFNγ和IL-2对牛皮癣的病理作用。普通牛皮癣的大部分表皮细胞都产生IL-2、IFNγ和TNFα，它们被定义为细胞毒性细胞。在牛皮癣病人中检测到了高水平的IFNγ和IL-2，而不是IL-4。这可能涉及促成这些细胞持续或慢性免疫激活的T细胞群体的失衡(Schaak JF 等，T cells involved inpsoriasis vulgaris belong to the Th1 subset.J Invest Dermatol 102，145-9，1994，Austin LM，等，The majority of epidermal T cells inPsoriasis vulgaris lesions can produce type 1 cytokines，interferon-gamma，interleukin-2，and tumor necrosis factor-alpha，defining Tc1(cytotoxic T lymphocyte)and TH1 effectorpopulations：a type 1 differentiation bias is also measured incirculating blood T cells in psoriatic patients。J Invest Dermatol113，752-9，1999)。用Daclizumab显示了一个令人振奋的治疗牛皮癣的结果(Krueger JM 等，Successful in vivo blockade of CD25(hihg-affinity interleukin 2 receptor)on T cells byadministration of humanized anti-Tac antibody to patients withpsoriasis。J Am.Acad.Dermatol.43，448-58，2000)。最近用该药物已开始了一项新的试验(Fremont，CA.Protein Design Labs PresentsThree Humanized Antibodies in Clinical Development for psoriasisat International psoriasis Symposium。June 22，2000。Protein DesignLabs，Inc.(PDL) Nasdaq)。
还有其它一些研究得较少的疾病，对这些疾病也可以使用抗IL-2和IFNγ的拮抗剂；这种情况有动脉粥样硬化和缺血/再灌注。动脉粥样硬化和移植后动脉粥样硬化的特征是动脉内膜扩张和存在IFNγ，动脉内膜扩张是单核细胞浸润、血管平滑肌细胞增殖和细胞外基质聚积的结果(RossR.Atherosclerosis--an inflamatory disease。N.Engl.J.Med.340，115-26，1999，Hansson GK等，Immune mechanismsin atherosclerosis。Arteriosclerosis 9，567-78，1989，y Libby P等，Functions of vascularwall cells related to development of transplantation-associatedcoronary arteriosclerosis。Transplant.Proc.21，3677-84，1989)。已经证明，外源IFNγ增加动物模型中的动脉粥样硬化(Whitman SC 等，Exogenous interferon -gamma enhances atherosclerosis inapolypoprotein E-/-mice。Am J Pathol 157，1819-24，2000)。另一方面，已经证明，中和血清中的IFNγ并缺失其基因会降低内膜扩张程度(Gupta S 等，IFNγ potentiates atherosclerosis in ApoE knock-outmice。J.Clin.Invest.99，2752-61，1997，Nagano H等，Interferonγdeficiency prevents coronary arteriosclerosis but not myocardialrejection in transplanted mouse hearts。J.Clin.Invest.100，550-57，1997，
-Sokolowski，A.等，Reduced transplantarteriosclerosis in murine cardiac allografts placed in interferonγknockout recipients。Am.J.Pathol.152，359-65，1998)。最近证明，无白细胞存在时IFNγ会促进动脉粥样硬化作用(Tellides G 等，Interferonγelicits arteriosclerosis in the absence of leukocytes。Nature 403，207-11，2000)。缺血和再灌注的特征是临床上常见的某一区域中的血流被中断，伴随着随后的氧和营养供应减少、和血流对缺血组织的再灌注和完全或部分恢复。这可以在血容量减少和感染性休克、心肌梗死、栓塞、腔隙综合征、冷冻、器官移植等过程中观察到。无论如何，组织低氧症会改变细胞代谢，由此衍生出恒定的更为熟知的复杂生化或分子改变。由再灌注引起的损伤导致细胞死亡和血液组织形成的内皮功能紊乱。已经报道IFNγ和IL-2是在经受缺血和再灌注的器官上造成损伤的介质(Serrick C 等，The early release of interleukin-2，tumornecrosis factor-alpha and interferon -gamma after ischemiareperfusion injury in the lung allograft.Transplantation 58，1158-62，1994，Marck ARC 等，Ischemia/Reperfusion Induced IFN-gamma Up-Regulation：Involvement of IL-12 and IL-18。The Journalof Immunology 162，5506-10，1999)。
几位作者已经描述了针对IFNγ的拮抗剂。欧洲专利EP 0 393 502 A1已经描述了人源人IFNγ通过重组可溶性受体的抗病毒活性。鼠IFNγ重组可溶性受体会抑制小鼠肾小球肾炎的发生，这也已得到描述(Ozmen L.等，Experimental therapy of systemic lupus erythematosus：thetreatment of NZB/W mice with mouse soluble interferon-gammareceptor inhibits the onset of glomerulonephritis。EuRJ Immunol.25，6-12，1995)。已经构建了三种鼠IFNγ。它们是由小鼠中IFNγ受体的细胞外区域和免疫球蛋白分子恒定区形成的嵌合蛋白质组成。这些构建体可中和小鼠IFNγ的抗病毒活性而且在血液中的平均寿命延长(Cornelia K.等，Construction，purification，and characterizationof new interferon gamma(IFNγ)inhibitor protein。J.Biol。Chemestry。267，9354-60，1992和欧洲专利EP 0 614 981 A1)。红斑狼疮这种自身免疫疾病中已证实的Fc受体功能障碍能够限制与可溶性IFNγ受体融合、将用于红斑狼疮的免疫球蛋白片段或其Fc区域的潜在用途(Frank MM 等，Defective reticuloendothelial system Fc-receptorfunction in systemic lupus erythema tosus。N Engl J Med.300，518-23，1979)。这种类型的抑制剂是单功能的，而且它因此而具有较小的作用范围。
在小鼠中，使用中和抗IFNγ抗体会降低针对宿主的移植疾病的表达(Mowat A.等，Antibodies to IFN gamma prevent immunologicallymediated intestinal damage in murine graft-versus-host reaction.Immunology 68，18-23，1989)。在一项同种异体移植研究中，只有当移植物与II类MHC抗原不相容时抗-IFNγ抗体才抑制排斥。这可能暗示着，IFNγ通过对II类MHC抗原的诱导来促进对同种异体移植的排斥(Rosenberg A.等，Specific prolongation of MHC class II disparateskin allografts by in vivo administration of anti-IFN gammamonoclonal antibody。J.Immunol.144，4648-50，1990)。细菌中表达的具有针对人IFNγ的可变区(scFv)的单链抗体也已被获得并已证实对中和鼠IFNγ的生物活性有效(Froyen G.等，Bacterial expressionof a single-strand antibody fragment(scFv)that neutralizesthe biological activity of human interferon γ。Mol Immunol.30，805-12，1993)。在人类治疗中使用抗体面临着针对这些异源分子的免疫原性区域产生应答的问题，而且对于嵌合或人源化抗体，将面临丧失亲和力和特异性(Merluzzi S 等，Humanized antibodies as potential drugsfor therapeutic use。Adv Clin Path，4，77-85，2000)以及细胞毒性表达(Clark M 等，Antibodies to IFN gamma prevent immunologicallymediated intestinal damage in murine graft-versus-host reaction.Immunology 68，18-23，2000)的问题。
其它类似的解决方案也已得到描述。已经提出了细胞因子及其可溶性受体的混合物，但其目的是增强细胞因子的效力。正如美国专利WO94/21282示范的，这种情况下，细胞因子及其受体是分别制备、并在之后混合于同一制剂中的。
环孢菌素A、FK506和雷帕霉素是潜在的、用于预防移植排斥的免疫系统(特别是T细胞)的抑制剂。前两种，抑制由针对T细胞的抗原受体引发的、从而导致早期激活基因转录的信号转导。这包括IL-2编码基因的转录，IL-2对于细胞周期从静息的G0态转换为G1相是必需的。雷帕霉素对T细胞早期合成细胞因子没有作用，但是它抑制这些细胞对从细胞周期的G1到S相的转换所必需的IL-2的应答(Waldmann T.A.等，TheIL-2/IL-2 receptor system：a target for rational immuneintervention.Immunology Today 14，264-70，1993)。雷帕霉素通过借助IL-2受体β链(RβIL-2)来干扰信号从而能够允许对T细胞的特异性激活并避免它们的克隆性扩增(Woerly G.等，Effect of rapamycinon the expression of the IL-2 receptor(CD25)。Clin Exp Immunol103，322-7，1996)。环孢菌素A显著提高肾同种异体移植物的存活，同时它也减少自身免疫疾病。由于这些免疫抑制剂的毒性效应包括肠胃并发症、牙龈肥大以及特别是剂量依赖性的中毒性肾损害和高血压，因此它们的使用已受到限制。(Hortelano S.等，Potentiation by nitric oxideof cyclosporin A and FK506-induced apoptosis in renal proximaltubule cells。J Am Soc Nephrol 11，2315-23，2000，Tsimaratos M.等，Kidney function in cyclosporine-treated paediatric pulmonarytransplant recipients。Transplantation 69，2055-9，2000)。
在动物模型中已经使用阻断IL-2与其受体互作的单克隆抗体来抑制移植物-对-宿主的疾病和同种异体排斥。这些抗体也用于啮齿类动物抑制自身免疫失调。临床试验中，针对IL-2受体α链的(RαIL-2)抗体改善了对类固醇治疗有抗性的宿主的移植疾病状况。然而，这些努力受到这些抗体抗原性的限制。正如欧洲专利EP 0 621 338 A2中所述，在对鼠细胞系CTLL-2的测试中已经发现，具有可变区的单链抗体可抑制IL-2与IL-2受体γ亚单位的结合并干扰IL-2的生物活性。可改善该类型治疗方法的人源化抗体的出现也仍然具有前述缺陷。在这一类型的治疗方法中，还测试了与毒素缀合或经放射活性标记的单克隆抗体(Waldmann T.A.Genetically engineered monoclonal antibodies armed withradionuclides.Year Immunol.7，205-12，1993)。在美国专利WO92/20701和欧洲专利EP 0 369 316 A2中已经描述了将与抗IL-2或IL-2抗体融合的毒素用于消除表达IL-2受体的细胞和促进病理阶段发展的用途。免疫毒素的非特异性毒性(FrankelA.E.等，Clinical trials oftarget toxins。Cancer Biology 6，307-17，1995)以及它们的免疫原性(Chen S-Y.等，Design of genetic immunotoxin to eliminate toxinimmunogenicity。Gene Therapy 2，116-23，1995)使得这些药物很难被推荐使用。抗IL-2抗体正被开发用于治疗RA(Simon LS.等，New andfuture drug therapies for rheumatoid arthritis。Rheumatology(Oxford)39 Suppl 1：36-42，2000)。正利用靶向RαIL-2抗体来避免肾移植排斥(Olyaei AJ等，Use of basiliximab and daclizumab inkidney transplantation.Prog Transplant 11，33-7，2001)。上面已经描述了在延长的治疗方法中使用抗体的限制。
在几种动物模型中，对炎症细胞因子原例如TNFα(Beutler B.等，Passiye immunization against cachectin/tumor necrosis factorprotects mice from lethal effect of endotoxin.Science 229，869-71，1989)或IL-1(Natanson C.等，Selected treatment strategies forseptic shock based on proposed mechanism of pathogenesis。Ann.Intern.Med.120，771-83，1994)的中和会降低由脓毒症导致的死亡率。然而，在临床试验中用IL-1的拮抗剂(IL-1受体拮抗剂)(Fischer C.J.等，Recombinant human interleukin-1 receptor antagonist in thetreatment of patients with sepsis syndrome：results fromrandomized，double-blind，placebo-contrrolled trial。JAMA 271，1836-43，1994)和TNFα拮抗剂(重组嵌合蛋白质：可溶性受体TNF/Fc)(Fischer C.J.等，Treatment of septic shock with tumor necrosisfactor receptor.Fc fusion protein。N. Engl J.Med.334，1697-1702，1996)不仅没有改善反而提高了由于免疫系统未被激活而导致的死亡率。FDA已经批准了两种已经证明对RA治疗有有益效果的TNFα拮抗剂(Lipsky PE.等，Infliximab and Methotrexate in the Treatment ofRheumatoid Arthritis。N Engl J Med，343，1594-1602，2000)。其中一种是针对TNFα受体亚基的抗体(Infliximab)，若对TNFα的中和高于一定水平则会象已经在患有RA的小鼠模型中证实的那样导致免疫系统不能被激活(Colagiovanni DB.等，TNF-alpha blockade by a dimericTNF type I receptor molecule selectively inhibits adaptive immuneresponses。Immunopharmacol Immunotoxicol 22，627-51，2000)。已经在用Infliximab治疗的病人中观察到比未用Infliximab的对照组(安慰剂组)更高的感染率(Schaible TF。Long term safety of infliximab。Can J Gastroenterol 14 Suppl C：29C-32C，2000)，以及出现自体抗体和狼疮发展(Markham A.等，Jnfliximab：a review of its use in themanagement of rheumatoid arthritis。Drugs 59，1341-59，2000)。Etanercept是一种与TNFα的可溶性受体以及IgG1的Fc部分结合的嵌合抗体(Moreland LW等，Etanercept therapy in rheumatoid arthritis。Arandomized，controlled trial。Ann Intern Med 130，478-86，1999)。暂停治疗会使病人疾病复发；因此，这种药物并不能治愈该疾病。这意味着需要对病人进行长期治疗。尽管在短期研究中没有出现不良效果，但是长期的治疗可能导致出现对抗该分子的抗体(Russell E.等，Patientsreceiving etanercept may develop antibodies that interfere withmonoclonal antibody laboratory assays。Arthritis Rheum 43，944-47，2000)。已经报道了用Etanercept治疗的几个病人发展出了致命的再生障碍性贫血、以及全血细胞减少症、和脱髓鞘综合征(Klippel JH，Biologic Therapy for Rheumatoid Arthritis。N.Engl J.Med.，343，1640-1，2000)。
到目前为止，临床上还未曾使用过针对IFNγ的拮抗剂。对于许多细胞因子，已经开发出了细胞因子拮抗剂用于预防、减弱或消除由于短期改变或长期存在而危害机体的炎症和自身免疫反应。它们中许多都是无效的。在有的病例中，拮抗剂被限制到一种细胞因子而且它的作用范围和潜力都受到限制，因为由于它们的毒性而不能提高它们的剂量。在那些能够中和细胞因子的病例中，这种效应被极端发挥并导致机体重要功能(免疫系统)未能被激活。
IL-2与其RαIL-2相互作用。该亚单位能将IL-2内化，已经证明是进入T和B淋巴细胞，并可能被重新循环到表面，而该受体的其它链在它们被内化后被降解(Hemar A.等，Endocytosis of interleukin 2receptors in human T lymphocytes：distinct intracellularlocalization and fate of the receptor alpha，beta，and gamma chains。J Cell Biol 129，55-64，1995)。IFNγ被内化并降解，而其受体得以再循环利用(Celada A.等，Internalization and degradation ofreceptor-bound interferon -gamma by murine macrophages。Demonstration of receptor recycling。J Immunol 139，147-53，1987)。IL-2被其IL-2Rα内化的特征、以及IFNγ被其受体内化的特征表明可以循环利用，至少是，IFNγ受体的细胞外区域以形成部分嵌合拮抗剂的潜力。这可以延长该分子在血流中的存在，并因此延长期效果。
两个分子的联合或缀合常常由于新分子形成而产生的构象改变或空间干涉导致相关分子生物活性的显著降低或丧失(Knusli，C 等，Polyethylene glycol(PEG)modification of granulocyte-macrophagecolony stimulating factor(GM-CSF)enhances neutrophil primingactivity but not colony stimulating activity。Br J Haematol 82，654-63，1992)。可是使用某些的替代物，例如在美国专利Americanpatent US # 5,073,627中描述了一种粒细胞巨噬细胞集落刺激因子(GM-CSF)与IL-3融合形成的联合肽。这种类型的肽的问题是，它们可能是刚性的而不灵活。这样的结果是，蛋白质不能活动以采取能够允许其产生正确生物活性的精确构象。因此仍然需要设计和寻找能够结合两个分子并同时允许它们各自采取正确构象的肽，以形成一个具有生物活性的分子。考虑到后者要更安全，则需要更有效和更加特异的免疫抑制剂和抗炎症药物。因此重要的是构思出一个功能更加多样的分子，该分子能抑制靶药物的所有病理活性和功能，同时，它可以允许机体产生重要功能，使它能够被长期使用且不会危及病人的生命。还未曾设计出一种可以干扰两种细胞因子同时产生的作用、并能避免免疫麻痹(immuno-paralization)的拮抗剂。
发明详述
本发明的实质是一种称作AnTH1的重组嵌合蛋白质，它由人IL-2N-末端区域60个氨基酸的片段通过一个4氨基酸的肽与含有228个氨基酸的IFNγ受体α亚单位N-末端细胞外区域融合形成。该蛋白质具有T细胞生长刺激活性，抑制由IL-2诱导的T细胞生长刺激活性，抑制IFNγ对HLA-II的诱导并抑制IFNγ的抗增殖活性。通过分别从Jurkat和Raji细胞反转录RNA多聚A并用特异引物进行PCR扩增得到(参见实施例1，SEQUENCE #1和SEQUENCE #2、SEQUENCE #3、SEQUENCE #4)，与人IL-2N-末端区域60个氨基酸的片段及结合肽对应的DNA序列(SEQUENCE#5)和结合肽及RαIFNγ细胞外区域228氨基酸的编码DNA的序列(SEQUENCE #7)。根据该目的设计对应于结合肽的DNA序列(SEQUENCE #6)并将其添加在用于扩增RαIFNγ可溶性受体区域的引物上。
本发明还描述了用于在大肠杆菌中生产重组蛋白AnTH1的载体，称作pHu(AnTH1)，它保藏在比利时微生物协调保藏中心(BelgianCoordinated Collections of Microoganism，BCCM)，保藏编号为LMBP4535，保藏日期为2002年5月6日。该载体是用重组DNA技术制备的(Sambrook 等，Molecular Cloning-A laboratory Manual，2nd ed。Cold Spring Harbor，New York，1989)。后一个载体经过了修饰并最终包含选择标记、色氨酸启动子、和编码人IL-2的60个氨基酸、结合肽和RαIFNγ细胞外区域228个氨基酸的序列(SEQUENCE #8)。所获的最终载体构建物称作pHu(AnTH1)，(参见实施例2，附图1).
本发明还描述了用pHu载体(AnTH1)转化的大肠杆菌菌株。用PHu载体(AnTH1)转化大肠杆菌W3110P3菌株。使含有pHu载体(AnTH1)的W3110P3菌株生长并诱导其表达重组嵌合蛋白质AnTH1。菌株高水平表达该蛋白质(参见实施例4)。该蛋白质也可以在其它宿主(用适当的表达载体，在昆虫细胞、哺乳动物细胞和植物细胞)中表达。本发明还描述了通过一种获得生物活性蛋白质纯度为80-90％的制剂的方法来生产纯度适宜以评估其生物活性的重组嵌合蛋白质。对诱导后从菌株培养物获得的细胞沉淀进行处理以提取重组蛋白。用溶解性试剂(变性)对沉淀进行连续洗涤，该试剂允许以最小量的大肠杆菌蛋白质污染提取目的蛋白。经这些程序达到适宜纯度(80-90％)后，重新折叠含有AnTH1蛋白质的制剂。该过程包括慢慢去除变性剂。该过程在实施例5中进行阐述。
本发明描述了重组蛋白质AnTH1的生物活性。该蛋白质具有T细胞生长刺激活性(实施例7)。它能在小鼠细胞系CTLL-2中干扰由人IL-2引起的对T细胞增殖的刺激(实施例8)。它能在细胞系Hep-2中抑制人IFNγ的抗增殖活性(实施例9)，还能在COLO 205细胞系中抑制IFNγ对HLA-II表达的刺激(实施例10)。人IL-2，与IFNγ相反，还能作用于鼠细胞。
该重组蛋白质可用于制备对疾病有效的药物组合物，在这些疾病中IL-2和IFNγ都起病理作用。这些疾病可能是自身免疫型的，例如多发性硬化、系统性红斑狼疮、重症肌无力、胰岛素依赖型糖尿病、活性慢性肝炎和爆发性肝炎；可能是自身免疫/炎症类型的，例如类风湿性关节炎和牛皮癣，以及在器官排斥中的疾病、移植物-对-宿主的疾病和炎症性疾病例如感染性休克，还有动脉粥样硬化。
用本发明创造了一种预防、干扰、和/或消除两种在不同疾病中具有病理功能的细胞因子的作用的新方法。由此，更特异地增加了目的性。对能抑制和干扰处理相同病理状况的两种细胞因子信号系统的嵌合蛋白质的使用将允许扩大它们的干扰方式及其功效的范围。基于其具有特定的T细胞生长刺激活性，这将能避免不期望的对免疫系统的失活，使得能够在病人和需要它的实体中使用更长时间。因此，该药物将更安全并更有效。本发明旨在创造一种异型二价拮抗剂，它可以干扰人IL-2和IFNγ的功能，扩大该分子的治疗可能性并避免不良反应。
微生物保藏：
根据微生物保存布达佩斯保藏条约，已将pHu(AnTH1)质粒保藏于比利时微生物协调保藏中心(Belgian Coordinated Collections ofMicrooganism，BCCM)，保藏编号为LMBP 4535，保藏日期为2002年5月6日。
附图说明
附图1表达重组嵌合蛋白质AnTH1的遗传构建质粒pHu(AnTH1)。
附图2嵌合蛋白质在AnTH1上的表达。还原条件下在12.5％的聚丙烯酰胺凝胶中进行电泳。a：分子量标准；b、c、d：诱导0、8和18小时的阴性对照菌株；e、f、g：诱导0、8和18小时的含有质粒pHu(AnTH1)的菌株。
附图3半纯化过程不同阶段中重组嵌合蛋白质AnTH1的存在状况。A：还原条件下在12.5％聚丙烯酰胺凝胶中的电泳。泳道a：分子量标准，b：发酵，c：8M脲提取，d：混合凝胶过滤层析中的级分，e：透析后的级分混合物。B：“Western印迹”用以1∶1000稀释于1％PBS/脱脂牛奶中的抗-AnTH1抗血清，来自抗大肠杆菌菌株W3110P3抗体。B：在泳道a、b、c、d中使用相同的样品并与图A电泳中的顺序相同(未使用提供分子量标准)。
附图4.用“western印迹”对嵌合蛋白质AnTH1的鉴定。用抗-IL-2(A)和抗-AnTH1(B)抗血清进行鉴定。稀释比例均为1∶1000。使用的是透析后重折叠的AnTH1制剂(泳道a)，在泳道b中使用的是IL-2。
附图5.用“斑点印迹”对I125标记的人IFNγ与嵌合蛋白质AnTH1的结合检测。硝化纤维素膜中使用了约1ul来自凝胶过滤层析的稀释级分。将条带与I125(35μCi/μg)标记的人IFNγ在不含(泳道a)或含(泳道b)过量(超过100倍)人IFNγ的情况下温育。
附图6.重组嵌合蛋白质AnTH1对T细胞的生长刺激活性。柱1：2.8ng/ml的IL-2，柱2：1.5μg/ml的AnTH1，柱3：培养基。
附图7.重组嵌合蛋白质AnTH1对人IL-2对小鼠细胞系CTLL-2生长刺激活性的抑制(结果以IL-2的国际单位表示)。
附图8.重组嵌合蛋白质AnTH1对人IFNγ抗增殖活性的抑制结果。柱1：4IU/ml的IFNγ(4ng/ml)，柱2：4IU/ml的IFNγ+50μg/mlAnTH1，柱3：50μg/ml AnTH1。
附图9.重组嵌合蛋白质AnTH1对IFNγ诱导的HLA II的抑制。柱1：基本水平(IFNγ＝0IU/ml)，柱2：IFNγ 500IU/ml(0.5μg/ml)，柱3：IFNγ 500IU/ml+1.5μg/ml AnTH1。
附图10.重组嵌合蛋白质AnTH1的氨基酸序列。指示了由质谱法定义的肽。
实施例：
实施例1
编码人IL-2前60个氨基酸和人IFNγ可溶性受体α亚单位细胞外区域(RαIFNγs)的互补DNA链的分离
为分离人互补DNA，在补加了10％胎牛血清的RPMI 1640培养基中生长Raji(Burkitt lymphoma，ATCC：CCL-86)和Jurkat(人急性T细胞白血病，ATCC：TIB-152)细胞，于5L培养瓶中、5％CO2气氛条件下5℃缓慢振荡。用Chomczyski法从Jurkat和Raji细胞提取总RNA(ChomczyskiP.等，Single-step method of RNA isolation by acid guanidiumthiocyanate-phenol-chloroform extraction.Anal.Biochem.162，156-9，1987)。之后，用商品化的信使RNA分离系统(Isolation SystemLife Technologies Messagemakerr caT.#10551-018)提取RNA-poly A。经反转录并PCR扩增分别来自Jurkat和Raji细胞的poly A RNA分离出编码人IL-2前60个氨基酸和人RαIFNγs细胞外氨基末端部分288个氨基酸的互补DNA。用随机六聚物处理了约1-2μg的poly A RNA。
用于扩增人IL-2前60个氨基酸DNA互补链的寡核苷酸(引物)序列描述于SEQUENCE #1(引物1)和SEQUENCE #2(反向引物1)。用于扩增人RαIFNγs氨基末端部分288个氨基酸DNA互补链的寡核苷酸(引物)序列描述于SEQUENCE #3(引物2)和SEQUENCE #4(反向引物2)。
用限制酶Nco I(引物1序列中下划线所示，SEQUENCE #1)消化编码人IL-2前60个氨基酸和结合肽的扩增DNA(SEQUENCE #5)。之后用酚氯仿抽提以去除酶和缓冲液。沉淀并重悬于适宜的缓冲液中，-70℃保存。参见结合肽序列(SEQUENCE #6)。
用Klenow聚合酶补齐扩增的编码结合肽和RαIFNγ 231氨基酸的DNA(SEQUENCE #7)粘末端，并如前所述进行纯化。之后用限制性酶BamHI(反向引物2序列中下划线所示)消化，并如前所述进行纯化。参见结合肽序列(SEQUENCE #6)。
将扩增带(SEQUENCES #5和#7)以计算的比例混合于适当的缓冲液中以获得新条带扩增，该新条带包含经结合肽(SEQUENCE #6)结合于人RαIFNγ细胞外区域231个氨基酸对应序列的人IL-2前60个氨基酸对应DNA序列。PCR扩增新条带。
如前述纯化扩增的DNA(SEQUENCE #8)。用限制性酶BamH I消化纯化的DNA，并按所述对其进行处理以去除酶和缓冲液。之后，再用NcoI酶消化DNA并根据所述进行纯化。
实施例2
表达重组嵌合蛋白质AnTH1的遗传构建体
表达载体包含强色氨酸启动子。载体经BamH I酶消化。之后经酚氯仿抽提以去除酶和缓冲液，并沉淀和重悬于适当的缓冲液。之后，用NcoI酶消化。最后如前所述凝胶分离载体。因此，载体包含色氨酸启动子、一个自由粘性位点Nco I、一个自由粘性位点BamH I、T4终止子和氨苄青霉素抗性基因。
用T4连接酶将对应于SEQUENCE #8的DNA与载体连接。用该遗传构建体转化大肠杆菌细胞。经Nco I和EcoR I酶限制性分析，鉴定了含有与色氨酸启动子同向的人IL-2前端氨基酸、以及结合肽和人RαIFNγs231个氨基酸的互补DNA片段的转化子。所得质粒称作pHu(AnTH1)(参见附图1)。
实施例3
测序
对最终的遗传构建体进行测序。基于Sanger程序(Sanger F 等，DNA sequencing with chain-terminating inhibitors。ProC. Natl.Acad.Sci.USA 74，5463-67，1977)在该方案中进行测序。已证明，该构建体具有受控制编码IL-2的基因的部分(从氨基末端基团起始的60个氨基酸)，以及之后是，与编码RαIFNγs氨基末端228个氨基酸的区域结合的编码联合肽的核苷酸序列(SEQUENCE #6)。参见SEQUENCE #8。
实施例4。
重组嵌合蛋白质AnTH1在大肠杆菌中的表达
宿主菌株是大肠杆菌W3110 P3(原养型F-)，质粒是pHu(AnTh1)。为进行表达，将质粒接种于含有氨苄青霉素(50μg/mL)和L-色氨酸(100μg/mL)的5mL LB培养基中，并在37℃下振荡培养6小时。将培养物加入50mL LB培养基中，并于37℃置于100r.p.m的摇床上6小时。将培养物以0.3的初始光密度(620nm)加至500mL M9培养基(33mM Na2HPO4、2mM KH2PO4、8.5mM NaCl、18mM NH4CL、0.1mM CaCl2、1mM MgSO4)，培养基中添加了0.2％水解酪蛋白、0.4％葡萄糖、50μg/mL氨苄青霉素。之后在上述相同条件下培养8小时，最后，离心收集细胞沉淀(参见附图2)。
实施例5
重组嵌合蛋白质AnTH1的抽提、纯化和复性
用polytron以每mL TE缓冲液(10mM Tris HCl，1mM EDTA pH 7.2)中含0.1g湿生物质的浓度匀浆细胞。用溶菌酶对该悬浮液进行酶消化处理。用不同摩尔浓度的脲对前一步骤中获得的沉淀进行细胞洗涤，脲在50mM Tris pH 7.2、1mM EDTA中，浓度为1M～8M。用polytron匀浆。最初匀浆1分钟，放置3分钟，之后再匀浆一分钟。整个过程在4℃下进行。约150mL用脲溶解的蛋白质以3mL/分钟的流速上K9/60柱(Pharmacia，Sweden)，柱中含有预先用3倍体积的50mM Tris HCL pH9、4M脲平衡的Sephadex G-100树脂。用相同的缓冲液进行洗脱。将含有蛋白质的级分混合并用0.1 M Tris HCl pH 9透析。之后，再用磷酸盐缓冲液(PBS)pH 7.4进行透析。(参见附图3和4)
实施例6
IFNγ与重组嵌合蛋白质AnTH1的结合。
硝化纤维条带上用了约一微升(约1μg总蛋白质)折叠后的重组嵌合蛋白质AnTH1。将条带与10％脱脂牛奶在室温下(RT)一起温育2小时。用Tris缓冲液盐水(TBS)洗膜两次5分钟。洗涤后，在存在或不存在过量未标记的IFNγ的条件下将条带与碘放射性标记的IFNγ(125I-IFNγ)35μci/μg在室温下温育1小时。之后，用TBS洗涤硝化纤维条带两次5分钟，之后用TBS+0.03％Tween 20洗涤5分钟。最后，将X光胶片对条带曝光，并保持在-70℃ 72小时，之后显影。(参见附图5)。
实施例7
重组嵌合蛋白质AnTH1对T细胞的生长刺激活性。
用依赖于IL-2的鼠T淋巴细胞细胞系检测重组嵌合蛋白质AnTH1的生物活性。细胞在含有1mM丙酮酸盐、2mM L-谷胺酰胺、40mM HEPES、100U/mL of青霉素、50μg/mL链霉素、50μM 2-巯基乙醇和10％胎牛血清、并补加了比活性为1.2×107IU/mg的8IU/mL人重组白细胞介素-2的RPMI-1640培养基上生长。使用之前将洗涤3次，重悬于不含IL-2的完全培养基中并于37℃在湿润CO2气氛中培养1小时。之后洗涤细胞，以4×105细胞/mL的密度重悬，并分置于(100μl每孔)含有添加了100μl以1∶2连续稀释的rhIL-2或样品(重组嵌合蛋白质AnTH1)的完全培养基的96孔板中。该试验中所用的国际标准是IL-2010397。37℃孵育36小时后，向各孔中加入20μl 5mg/mL MTT(C18H16N5SBr)，并在相同条件下孵化板4小时。最后，加入50μl/pozo 10％ SDS、0.1N HCl、50％异丙醇，37℃下摇板一小时，用读板器读取570nm处的吸光度。(参见附图6)
实施例8
用重组嵌合蛋白质AnTH1对IL-2的T细胞生长刺激活性的抑制
用依赖于IL-2的鼠淋巴细胞系测试IL-2的生物活性。以与前述试验相似的形式生长细胞。细胞在使用前，先洗涤3次，重悬于不含IL-2的完全培养基中并于37℃在湿润CO2气氛中培养1小时。之后洗涤细胞，以4×105细胞/mL的密度重悬，并分置于(100μl每孔)含有添加了100μl以1∶2连续稀释的rhIL-2或样品(rhIL-2+重组嵌合蛋白质AnTH1或仅为重组嵌合蛋白质AnTH1)的完全培养基的96孔板中。该试验中所用的国际标准是IL-2010397。37℃孵育36小时后，向各孔中加入20μl5mg/mL MTT(C18H16N5SBr)，并在相同条件下温育板4小时。最后，加入50μl/孔10％ SDS、0.1N HCl、50％异丙醇的溶液。37℃下摇板一小时，用读板器读取570nm处的吸光度。基于对rhIL-2的标准稀释曲线和系列稀释样品的数据的分析以rhIL-2单位表示结果。(参见附图7)
实施例9
重组嵌合蛋白质AnTH1对IFNγ抗增殖活性的抑制
在96孔板中以2.5×103细胞/孔培养Hep-2以使细胞生长，板孔中含有补加了10％胎牛血清的MEM CANE培养基(含有非必需氨基酸的必需基本培养基)。细胞在37℃、5％ CO2的培养箱中培养24小时。之后更换培养基，并加入以系列方式稀释的样品以及它们各自的对照。孵育72小时后，用0.5％的结晶紫对细胞染色2分钟，在读板器中读板。(参见附图8)
实施例10
重组嵌合蛋白质AnTH1对由IFNγ诱导的HLA II的抑制。
这是一个描述于Seeling G.等，Development of receptor peptideantagonist to human γ-interferon and characterization of itsligand-bound conformation using transferred nuclear overhausereffect spectroscopy.J.Biol.Chem.270，9241-53，1995的对细胞的ELISA试验。使用了Colo 205细胞系。它们生长于96孔培养平板内的0.1mL补加有10％胎牛血清的RPMI 1640中，密度为2.5×105细胞/孔。细胞于37℃5％CO2的培养箱中培养12小时。之后，在存在重组嵌合蛋白质和IFNγ下加入0.1mL在培养基中生长的细胞，随后在37℃下孵育1小时。培养后，去除培养基并用培养基洗涤板孔三次。之后，向板孔中加入等分量的1.2mL培养基，37℃下将板孵育48小时以允许诱导HLA-DR抗原。用PBS洗涤板孔，并用纯乙醇固定细胞2分钟。重复洗涤，并于室温下用稀释于PBS 0.5％牛血清白蛋白的鼠单克隆抗体抗HLA-DR孵育板孔1小时。板孔用PBS洗涤，并在相同条件下与抗-鼠IgG过氧化物酶缀合物孵育。重复洗涤3次，并通过加入100μL/孔0.15％H2O2+5mg/mL邻苯二胺显色。用50μL/孔2M H2SO4完成对反应的检测，在读板器中测定492nm处的吸光度。(附图9)
实施例11
质谱分析
用SDS-PAGE和Zn-immidazol反向染色分析等分量(0.5μg)的纯化蛋白质(Castellanos-Serra L，y cols。Detection of biomoleculesin electrophoresis gels with salts of imidazole and zinc II：adecade of research。Electrophoresis。2001，22，864-7)。将条带切出，并用柠檬酸溶液(1％)温育5分钟直到完全脱色，在水中再温育10分钟以除去过量螯合剂。另外将透明带切成约1mm3的小方块，在不含TFA的90％乙氰水溶液中脱水，并在speed-vac中完全干燥。将胶块在含有12.5ng Promega(MA，USA)的测序级改良胰蛋白酶20-30μL 50mMNH4HCO3溶液中重新水合。在37℃混温器(termomixer)(Eppendorf，USA)中温育过夜进行胶内消化。再加入20μL 50mM NH4HCO3溶液，并再温育45分钟，并用Millipore(USA)的ZipTips C18提取胰蛋白酶消化的肽，该ZipTips C18已根据制造商推荐被预先活化并平衡。进行二十次载样循环以提取胰蛋白酶肽。用甲酸将消化物酸化，于室温下温育45分钟，以及再进行二十次载样循环。Ziptips用5％甲酸溶液充分洗涤，并将蛋白水解肽稀释于2μL含有1％甲酸的60％乙氰。
用Micromass(Manchester，UK)的装有Z-喷射纳流电喷射离子源(Z-spray nanoflow electrospray ion source)杂交四极直角(hydridquadrupole orthogonal)加速串连质谱仪QTOF获得低能MS/MS光谱。用80℃的源和50L/h的干燥气流操作质谱仪。将肽溶解至约5pmole/μL浓度。将两微升胰蛋白酶解的肽加载至硼硅酸盐纳流头上(nanoflowtip)，并分别对纳流头和entrance cone加以900V和35V电压。使用了由Gonz ález，L.y cols.Differentiating alpha-and beta-asparticacids by electrospray ionization and low-energy tandem massspectrometry.Rapid.Commun.Mass Spectrom.2000，14，2092-210所述的方法来获得MS/MS光谱。在4-5Th窗口中用第一个四极来选择前体离子。在六极碰撞室(hexapole collision cell)中使用～3×10-2Pa碰撞气体(氩)的压力以产生离子碎片。用适宜的碰撞能将前体离子密度降低其初始密度一半多。用Micromass的MassLynx系统(v3.5)完成数据获得和处理。
对从AnTh1蛋白质衍生的胰蛋白酶肽的ESI-MS分析
| # | 肽序列 <sup>a)</sup> | m/zexp. | m/z teor. | Z<sup>b)</sup> | Abs.ErroR<sup>c)</sup> |
| 1 | 11T-K<sup>33 d)</sup> | 908.81 | 908.83 | 3 | 0.02 |
| 2 | 40M-K<sup>44</sup> | 639.34 | 639.35 | 1 | 0.01 |
| 3 | 45F-K<sup>50</sup> | 685.33 | 685.33 | 1 | 0.00 |
| 4 | 56H-R<sup>66 f)</sup> | 442.87 | 442.88 | 3 | 0.01 |
| 5 | 67A-K<sup>114</sup> | 1781.52 | 1781.54 | 3 | 0.02 |
| 6 | 101Q-K<sup>114 d)，e)</sup> | 807.93 | 807.95 | 2 | 0.02 |
| 7 | 120N-R<sup>151 f)</sup> | 921.14 | 921.17 | 4 | 0.03 |
| 8 | 166S-R<sup>173</sup> | 506.23 | 506.23 | 2 | 0.00 |
| 9 | 177I-K<sup>181</sup> | 511.31 | 511.28 | 1 | 0.03 |
| 10 | 190Q-R<sup>220 g)</sup> | 1225.20 | 1225.23 | 3 | 0.03 |
| 11 | 221V-R<sup>227 d)</sup> | 456.74 | 456.75 | 2 | 0.01 |
| 12 | 228M-K<sup>236 d)</sup> | 535.24 | 535.25 | 2 | 0.01 |
a.肽序列的标号基于附图10所示的AnTh1蛋白质
b.单个肽的带电状态
c.指出所检测肽的理论和试验分子质量间的绝对质量差异
d.ESI-MS/MS测序的肽
e.由胰蛋白酶非特异性切割产生的肽
f.含有自由半胱氨酸的肽
所提出溶液的优点
本发明将在两个影响免疫调控和炎症机制信号系统中进行干涉的能力结合到了一个分子。嵌合蛋白质的设计包括，配体(IL-260)经一个4氨基酸肽与细胞外受体(RαIFNγ)的融合。这种结合使得重组嵌合蛋白质能与在其表面含有RαIL-2的细胞结合。该亚单位主要存在于未激活的T细胞和已激活T细胞的高亲和力IL-2受体(RαβγIL-2)中(Smith，K.A.The interleukin-2 receptor.Annu.Rev.Cell.Biol.5，397-403，1989和Strom，T.B.等，Interleukin-2 receptor-directed therapies：antibody-or cytokine-based targeting molecules。Annu.Rev.Med.44，343-50，1993)。
如果在静息细胞中AnTH1蛋白与RαIL-2结合，它就能将蛋白质内化，并在被再循环出去的条件下将RαIFNγ留在细胞质中，以及干扰由细胞激活产生的IFNγ。已经报道了IFNγ从细胞质中与产生IFNγ生物活性的膜受体细胞内区域的相互作用(Szente B.E.等，IdentificationofIFN(receptor binding sites for JAK2 and enhancement of bindingbyIFNγand its C-terminal peptideIFNγ(95-133)。J.Immunol.155，5617-22，1995)。可能会需要减弱由机体产生的IL-2的作用的疾病中，在加入AnTH1蛋白质时，其将通过IL-260部分与已激活细胞中的高亲和力复合物α亚单位结合(RαβγIL-2)，它将干扰由免疫系统的细胞分泌的完整IL-2(天然)的结合并干扰其生物活性。另一方面，已由被激活T细胞分泌的IFNγ可被嵌合蛋白质的RαI FNγ部分封闭，避免它与膜受体的附着。通过这种方式，自身免疫、和/或炎症反应可以在两种不同的时刻得到控制，即，激活过程中以及反应增殖过程中。
本发明提供了可以干扰IL-2和IFNγ生物活性的异型二价嵌合蛋白质。考虑到该重组嵌合蛋白质AnTH1还具有T细胞生长刺激活性，可以预计到免疫系统的非极度的失活，它将激发产生已被临床验证的拮抗剂抗细胞因子。
序列表
<110>遗传工程与生物技术中心
<120>嵌合拮抗剂ANTH1
<130>拮抗剂
<140>
<141>
<160>8
<170>Patent in See. 2.1
<210>1
<211>30
<212>ADN
<213>人(Homo sapiens)
<220>
<221>引物_结合
<222>(1)..(30)
<223>用于扩增人IL-2前60个氨基酸互补DNA链的引物1。
<400>1
ccatggcacc tactttcaag ttctacaaag 30
<210>2
<211>32
<212>DNA
<213>人
<220>
<221>引物_结合
<222>(1)..(32)
<223>用于扩增人IL-2前60个氨基酸互补DNA链的反向引物1。
<400>2
catcatatgg gtctagacac tgaagatgtt tc 32
<210>3
<211>29
<212>DNA
<213>人
<220>
<221>引物_结合
<222>(1)..(29)
<223>用于扩增人RαIFNγ氨基末端部分231个氨基酸互补DNA链的引物2。
<400>3
cccatatgat gagcagggct gagatgggc 29
<210>4
<211>31
<212>DNA
<213>人
<220>
<221>引物_结合
<222>(1)..(31)
<223>用于扩增人RαIFNγ氨基末端部分231个氨基酸互补DNA链的反向引物2。
<400>4
ggatccttat tttatactgc tattgaaaat g 31
<210>5
<211>193
<212>DNA
<213>人
<220>
<221>cDNA(基因片段)
<222>(1)..(193)
<223>与所扩增的编码人IL-2前60个氨基酸和结合肽的DNA对应的序列。
<400>5
atggcaccta cttcaagttc tacaaagaaa acacagctac aactggagca tttactgctg 60
gatttacaga tgattttgaa tggaattaat aattacaaga atcccaaact caccaggatg 120
ctcacattta agttttacat gcccaagaag gccacagaac tgaaacatct tcagtgtcta 180
gacccatatg atg 193
<210>6
<211>11
<212>DNA
<213>人
<220>
<221>引物_结合
<222>(1)..(11)
<223>与结合肽DNA对应的序列。
<400>6
cccatatgat g 11
<210>7
<211>703
<212>DNA
<213>人
<220>
<221>cDNA(基因片段)
<222>(1)..(703)
<223>与所扩增的编码结合肽和RαIFNγ区231个氨基酸的DNA对应的序列。
<400>7
cccatatgat gagcagggct gagatgggca ccgcggatct ggggccgtcc tcagtgccta 60
caccaactaa tgttacaatt gaatcctata acatgaaccc tatcgtatat tgggagtacc 120
agatcatgcc acaggtccct gtttttaccg tagaggtaaa gaactatggt gttaagaatt 180
cagaatggat tgatgcctgc atcaatattt ctcatcatta ttgtaatatt tctgatcatg 240
ttggtgatcc atcaaattct ctttgggtca gagttaaagc cagggttgga caaaaagaat 300
ctgcctatgc aaagtcagaa gaatttgctg tatgccgaga tggaaaaatt ggaccaccta 360
aactggatat cagaaaggag gagaagcaat catgattgac atatttcacc cttcagtttt 420
tgtaaatgga gacgagcagg aagtcgatta tgatcccgaa actacctgtt acattagggt 480
gtacaatgtg tatgtgagaa tgaacggaag tgagatccag tataaaatac tcacgcagaa 540
ggaagatgat tgtgacgaga ttcagtgcca gttagcgatt ccagtatcct cactgaattc 600
tcagtactgt gtttcagcag aaggagtctt acatgtgtgg ggtgttacaa ctgaaaagtc 660
aaaagaagtt tgtattacca ttttcaatag cagtataaaa taa 703
<210>8
<211>885
<212>DNA
<213>人
<220>
<221>cDNA(基因片段)
<222>(1)..(885)
<223>人IL-2氨基末端前60个氨基酸的序列，之后是编码结合肽的核苷酸序列，接着是编码RαIFNγ氨基末端231个氨基酸的区域。
<400>8
atggcaccta cttcaagttc tacaaagaaa acacagctac aactggagca tttactgctg 60
gatttacaga tgattttgaa tggaattaat aattacaaga atcccaaact caccaggatg 120
ctcacattta agttttacat gcccaagaag gccacagaac tgaaacatct tcagtgtcta 180
gacccatatg atgagcaggg ctgagatggg caccgcggat ctggggccgt cctcagtgcc 240
tacaccaact aatgttacaa ttgaatccta taacatgaac cctatcgtat attgggagta 300
ccagatcatg ccacaggtcc ctgtttttac cgtagaggta aagaactatg gtgttaagaa 360
ttcagaatgg attgatgcct gcatcaatat ttctcatcat tattgtaata tttctgatca 420
tgttggtgat ccatcaaatt ctctttgggt cagagttaaa gccagggttg gacaaaaaga 480
atctgcctat gcaaagtcag aagaatttgc tgtatgccga gatggaaaaa ttggaccacc 540
taaactggat atcagaaagg aggagaagca atcatgattg acatatttca cccttcagtt 600
tttgtaaatg gagacgagca ggaagtcgat tatgatcccg aaactacctg ttacattagg 660
gtgtacaatg tgtatgtgag aatgaacgga agtgagatcc agtataaaat actcacgcag 720
aaggaagatg attgtgacga gattcagtgc cagttagcga ttccagtatc ctcactgaat 780
tctcagtact gtgtttcagc agaaggagtc ttacatgtgt ggggtgttac aactgaaaag 840
tcaaaagaag tttgtattac cattttcaat agcagtataa aataa 885
Claims (6)
1.一种称作ANTH1的重组嵌合蛋白质，由通过具有多于4个氨基酸的肽共价结合于细胞因子受体细胞外区域的细胞因子或细胞因子片段组成，其特征在于包含由序列表中序列#5所确定的、编码人白细胞介素2N-末端区域60个氨基酸的细胞因子片段，以及特征在于由序列表中序列#7所确定的、编码人γ干扰素受体α链细胞外N-末端部分的细胞因子受体细胞外区域。
2.根据权利要求1的重组嵌合蛋白质，其特征在于形成该重组嵌合蛋白质的结合肽由序列表中序列#6所确定的序列编码。
3.根据权利要求1或2的重组嵌合蛋白质，其特征在于它由序列表中序列#8所确定的核苷酸序列编码且为该序列的表达产物。
4.包含根据权利要求1、2、3之任一项的重组嵌合蛋白质的药物组合物，其中该药物组合物用于治疗由白细胞介素-2、γ干扰素的作用或这两种细胞因子的结合作用介导的疾病。
5.包含根据权利要求1、2、3之任一项的重组嵌合蛋白质的药物组合物，其中该药物组合物用于治疗自身免疫疾病、炎症疾病、移植物排斥或由微生物引起的感染。
6.包含根据权利要求1、2和3之任一项的重组嵌合蛋白质的药物组合物，其中该药物组合物用于治疗多发性硬化、系统性红斑狼疮、重症肌无力、糖尿病、类风湿性关节炎、肠道炎症疾病、脓毒症、牛皮癣、动脉粥样硬化或由缺血再灌注引起的疾病。
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CU20020095A CU23229A1 (es) | 2002-05-10 | 2002-05-10 | ANTAGONISTA QUIMéRICO ANTH1 |
| CU2002/0095 | 2002-05-10 |
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| US5073627A (en) * | 1989-08-22 | 1991-12-17 | Immunex Corporation | Fusion proteins comprising GM-CSF and IL-3 |
| EP0614981A1 (en) * | 1993-03-05 | 1994-09-14 | F. Hoffmann-La Roche Ag | Chimeric human interferon-gamma-receptor/immunoglobulin polypeptides |
| WO1994021282A1 (en) * | 1993-03-19 | 1994-09-29 | Immunex Corporation | Pharmaceutical compositions comprising a ligand complexed with a soluble receptor |
| EP0621338A2 (en) * | 1993-04-21 | 1994-10-26 | Ajinomoto Co., Inc. | Immunosuppressant able to block the interleukin - 2 response |
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| RU2004136170A (ru) | 2005-05-27 |
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| KR20040108786A (ko) | 2004-12-24 |
| JP4275065B2 (ja) | 2009-06-10 |
| ZA200409073B (en) | 2005-05-09 |
| BR0304827A (pt) | 2004-08-17 |
| US7429384B2 (en) | 2008-09-30 |
| EP1550672B1 (en) | 2009-08-12 |
| DE60328807D1 (de) | 2009-09-24 |
| AU2003223832B2 (en) | 2009-02-19 |
| RU2322455C2 (ru) | 2008-04-20 |
| WO2003095488A1 (es) | 2003-11-20 |
| CU23229A1 (es) | 2007-09-26 |
| CN1662555A (zh) | 2005-08-31 |
| CA2485439A1 (en) | 2003-11-20 |
| MXPA04011143A (es) | 2005-01-25 |
| JP2006506958A (ja) | 2006-03-02 |
| AU2003223832A1 (en) | 2003-11-11 |
| ATE439379T1 (de) | 2009-08-15 |
| UA81908C2 (uk) | 2008-02-25 |
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