CN101720230A - 用于治疗或预防类风湿性关节炎的含有抗-4-1bb抗体的药物组合物 - Google Patents
用于治疗或预防类风湿性关节炎的含有抗-4-1bb抗体的药物组合物 Download PDFInfo
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Abstract
本发明涉及一种药物组合物,所述药物组合物含有有效量的抗4-1BB抗体作为活性成分,以及药学上可接受的载体。所述抗4-1BB抗体通过CD11c+CD8+T细胞的增殖和诱导CD4+T细胞抑制以预防和治疗类风湿性关节炎。本发明的组合物在通常免疫应答中无毒性并且可以通过诱导抗原特异性的免疫抑制显著地减轻进行性,炎性的或者自体-免疫性关节炎症状。因此在预防或治疗关节炎疾病上很有用,并且没有副反应。
Description
技术领域
本发明涉及药物组合物,所述药物组合物是含有一种用于治疗和预防类风湿性关节炎的药物组合物。
背景技术
类风湿性关节炎(RA),是一种慢性,退化性系统炎性疾病,其特征是关节增生及炎性细胞的产生,关节内纤维蛋白的沉积,和在其晚期,软骨和骨质破损。尽管对RA的病原学仍旧存在争论,但似乎已经建立了该疾病发病的事件顺序以及末期效应机理。CD4+辅助性T细胞控制最初的炎性损伤。巨噬滑膜内衬细胞以及并指滑膜成纤维使大量的II类主要组织相容性复合体(MHC)增殖及表达。活性的辅助T细胞似乎驱使B细胞渗入到滑膜中以产生免疫球蛋白。大多数这些局部合成的抗体的专一性未知,但是一些是与其他IgG分子在关节处结合形成免疫复合体的IgG类风湿因子。最终,中性粒细胞和巨噬细胞的大量补充和侵入,一连串降解酶的合成以及肿瘤坏死因子α(TNF-α),IL-1和IL-6的产生将侵蚀类风湿关节的软骨和其他组分(Moreland,L.W.et al.Treatment of rheumatoid arthritis with a recombinant human tumor necrosis factorreceptor(p75)-Fc fusion protein.N.Engl.J.Med.337,pp141-147,1997)。
有可能这些不同的疾病路线汇集成一条普通路径进行破坏。
在RA上应用最广泛的动物模型是II型胶原诱导性关节炎(CIA),它是DBA/1小鼠关节的Th1细胞依赖性慢性炎症。这一模型由于其可重复性以及定义明确而被接受,并且已经证明在开发RA新疗法方面很有用,例如TNF-α中和治疗(Williams,R.O.,Mason,L.J.,Feldmann,M.&Maini,R.N.Synergybetween anti-CD4 and anti-TNF in the amelioration of establishedcollagen-induced arthritis.Proc.Natl.Acad.Sci.USA 92,pp2762-2766,1994)。适应性免疫的两种细胞,T细胞和B细胞,在CIA的病原发生学上起着核心的作用,但是它们在引动免疫激活以及关节损伤方面的相对重要性还不清楚。B细胞的主要作用是产生至关节炎抗-CII抗体,现已清楚的显示该抗体与CII作用可以同软骨结合并诱导关节炎。T细胞在CIA中的作用更复杂并且可以分成两种主要途径,这两种途径在关节炎的发展过程中是协同作用的。首先,T细胞在B细胞产生至关节炎抗-CII抗体中提供帮助。第二,T细胞自身通过产生细胞因子及活化其他细胞而在关节炎症方面起作用。
目前大多数的治疗是针对于矫正可能引起滑膜细胞增殖及软骨侵蚀的免疫畸变。本发明的关节炎治疗包括用于控制疼痛和发炎的一线药物,这类药物列为非类固醇消炎药(NSAIDs),例如,阿司匹林,布洛芬,萘普生,甲氨蝶呤等。二线治疗药包括皮质激素,慢作用抗风湿药(SAARDs)和疾病改善药物(DMs),例如,青霉胺,环磷酰胺,金盐类,叠氮胸苷,左旋咪唑等。然而,至今大多数有效的甾类激素都被报道有各种不同的副作用,例如色素沉着过度,闭经,痤疮,肌张力缺乏等。近来,已尝试新的抗关节炎治疗法,该方法利用遗传重组技术生产在炎症机理上起重要作用的TNF受体。然而,至今仍需要用以治疗和减轻各种综合症,例如,炎症,水肿,血管生成异常以及骨和软骨侵蚀的先进治疗药物。
4-1BB,一种TNF受体,作用于主要存在于T淋巴细胞中的协同刺激受体并且在T细胞收到抗原-特异性信号时被诱导。而且,有报道4-1BB通过其他淋巴样和骨髓样细胞谱系,例如NK(自然杀伤)细胞,CD4+CD25+常规T细胞,单核细胞及其他乳头状细胞(DCs)。4-1BB协同刺激T细胞以实现效应功能,例如消除肿瘤的产生,扩大初级CD8+T细胞应答,和增强抗原特异性CD8+T细胞的记忆池。另外,4-1BB介导的信号主要通过抑制CD4+导致炎性和T-细胞-依赖性抗体应答的这一T细胞功能,改善自身免疫疾病,例如系统性红斑狼疮(SLE)和实验性自体免疫性脑炎(EAE)。
对于吲哚胺2,3-加双氧酶(IDO)-介导的免疫调节的重要性已在Munn etal的开创性工作中重新发现(Munn,D.H.,et al.Prevention of allogeneic fetalrejection by tryptophan catabolism.Science 281,pp1191-1193,1998)。
因此,本发明的发明人力图研究4-1BB的生理作用并确认4-1BB-介导的RA抑制是由于以一种抗原-依赖性方式诱导CD11c+CD8+T细胞而产生的。另外,我们发现这一组新的诱导的T细胞产生一种丰富的IFN-Y,IFN-Y反过来诱导DCs中的IDO以及巨噬细胞,而抗原-特异性CD4+T细胞被IDO-依赖的机制所抑制。因此,通过确认抗4-1BB抗体在治疗或预防关节炎疾病上是有用的完成了本发明。
发明内容
技术问题
如前所说,本发明的一个目的是提供一种药物组合物,所述药物组合物含有有效量的抗4-1BB抗体作为活性成分,以及药学上可接受的载体。所述抗4-1BB抗体通过CD11c+CD8+T细胞增殖和诱导CD4+T细胞抑制以预防和治疗类风湿性关节炎。
技术方案
所述用于治疗关节炎疾病的药物组合物可以含有基于组合物总重量的大约0.01~80w/w%,优选0.1~50w/w%的上述本发明的抗4-1BB抗体。
本发明公开的抗4-1BB抗体可以按照本领域技术人员公知的方法制备。例如,杂交瘤细胞生产4-1BB(3H3)和4-1BBL(TKS-1)抗体可以按照本领域技术人员公知的方法制备,例如,文献中公开的制备方法(Shuford,W.W.et al.4-1BB constimulatory signals preferentially induce CD8+T cell proliferation andlead to amplification in vivo of cytotoxic T cell responses.J.Exp.Med.186,pp47-55,1997;Futagawa,T.et al.Expression and function of 4-1BBligand on murine dendritic cells.Int.Immunol.14,pp275-286,2002)。
在本发明的一个优选实施方案中,为了理解抗4-1BB在胶原诱导性CIA过程中的作用,对CIA诱导的小鼠给予激动性抗4-1BB抗体(3H3)并且观察了关节炎症的平均临床评分指数。在结果中,通过激动性抗4-1BB给药强烈抑制了疾病的发展。CIA的所有特点,例如过度关节增生,血管翳形成,软骨破坏和骨侵蚀以及各种类风湿性关节炎因子的表达,例如,MCP-1,MCP-2,酸性细胞趋化素,MIP-1a,RANTES等趋化性细胞因子和细胞因子IL-6,IL-5,TNF-α或IL-1β,均未发现并且它诱导抗原特异性抑制反应,该反应完全抑制了在抗体与抗-CII作用再生的IgG和IgG2b。
在本发明的一个优选实施方案中,就在类风湿性关节炎的发展中,抗4-1BB降低了疾病指数并抑制了抗-CII抗体的再生,这使得可以通过4-1BB交联来抑制CIA。
由于抗4-1BB抗体治疗的活性抑制机制抑制了CD4+T细胞的诱导并导致淋巴结细胞内的CD11c+CD8+T细胞的增加。所诱导的CD11c+CD8+T细胞是新的CD8+T淋巴细胞,表达不同于其他白细胞表面标记,例如CD11c-CD8+,CD11c+CD8+,CD8-CD11c+,DCs细胞的CD3+,TCR Vβ+,Thy1.1+,CD11c和II型抗原1-Aq。当CD11c+CD8+T细胞被分离并且被继承性转移后,它们抑制CIA的发展。因此由抗4-1BB-处理所诱导的CD11c+CD8+T细胞的增加可以抑制关节性关节炎症。那时,CD11c+CD8+T细胞产生在CD11b+巨噬细胞和CD11c+乳头细胞中诱导IDO(吲哚胺2,3-加双氧酶)表达的IFN-Y,而1-甲基色氨酸处理使抗4-1BB的效应复原。CD11c+CD8+T细胞增殖引起CIA的抑制和表达的IFN-Y的IDO-依赖反应抑制了抗原-特异性CD4+T细胞。
根据本发明的另一方面,还提供了抗4-1BB抗体在制备治疗和预防哺乳动物类风湿性关节炎的治疗药剂上的应用,所述哺乳动物包括需要的人。
根据本发明的另一方面,还提供了治疗或预防哺乳动物关节炎疾病的方法,所述方法包括向所述哺乳动物给予有效量的抗4-1BB抗体,以及其药学上可接受的载体。
本发明的组合物按照使用方法可以另外包括常规的载体,佐剂或稀释剂。按照用法和给药方法,优选所述载体作为适宜的物质,但不限于此。适宜的稀释剂在Remington的药物科学一文中列出(Mack Publishing co,Easton PA)。
下文中,下述制剂方法和赋形剂仅仅是示例且并不限制本发明。
根据本发明的组合物可以作为药物组合物,所述组合物含有药学上可接受的载体,佐剂或稀释剂,例如乳糖,葡萄糖,蔗糖,山黎醇,甘露醇,木糖醇,赤藓醇,麦芽糖醇,淀粉,洋槐胶,藻酸盐,明胶,磷酸钙,硅酸钙,纤维素,甲基纤维素,聚乙烯基吡咯烷酮,水,羟甲基苯甲酸盐,羟丙基苯甲酸盐,滑石,硬脂酸镁和矿物油。所述制剂可以另外含有填充物,抗凝结剂,润滑剂,湿润剂,香味剂,乳化剂,防腐剂等等。本发明的组合物可以通过本领域技术人员公知的制备方法制成以提供活性成分在给药于患者后速效,长效或缓释的制剂。
例如,本发明的组合物可以溶于油,丙二醇或其他通常用于生产注射剂的溶剂。载体适宜的例子包括生理盐水,聚乙二醇,乙醇,植物油,异丙基豆蔻酸盐等,但并不限于此。对于局部给药,本发明的复合物可以制成软膏和霜的形式。
含有本发明组合物的药物制剂可以以任何形式制备,例如口服剂型(粉末,片剂,胶囊,软胶囊,液体药物,酏剂,香粉,颗粒),或者局部制剂(霜,软膏,乳液,胶,油膏,塞片,涂胶,喷雾,溶液,气雾剂等),或者注射制剂(溶液,悬浮液,乳化液)。
本发明的组合物的药学剂型可以以其药学上可接受的盐的形式使用,也可以独自使用或者以适宜的组合,以及和其他药学上活性复合物结合的形式使用。
本发明组合物的理想剂量由于患者的情况和体重,病情,药物形式,给药途径和时间的不同而变化,并且可以由本领域技术人员选择。然而,为了获得理想的效果,通常建议以按重量/天计的0.01~10g/kg本发明的抗体复合物给药,优选1~5g/kg。所述剂量可以每日一次或者一日多次给药。就组合物而言,本发明的组合物应该以按重量计基于组合物总重量的0.01~80%,优选0.5~50%的量存在。
本发明的药物组合物可以通过不同途径给药于被试动物,例如哺乳动物(大鼠,小鼠,家畜或人类)。可以考虑所有给药方式,例如,可以口服给药,直肠给药或者通过静脉,肌肉,皮下,皮内,鞘内,硬脑膜或脑室内注射。
有益效果
根据本发明的另一方面,还提供抗4-1BB抗体在制备治疗和预防哺乳动物类风湿性关节炎的治疗药剂上的应用,所述哺乳动物包括需要的人。
根据本发明的另一方面,还提供了一种治疗或预防哺乳动物关节炎疾病的方法,所述方法包括给予所述哺乳动物有效量的抗4-1BB抗体,以及其药学上可接受的载体。
附图说明
图1:显示经免疫原攻击后每个测试组分别的临床分数(a)和爪厚度(b),测试组即对照IgG,抗-4-1BBL和抗-4-1BB;
图2:显示对CIA诱导的小鼠注射对照IgG(a),抗-4-1BBL(b)和抗-4-1BB(c)后,踝关节的组织病理;
图3:显示RPA法(RNase保护试验)对每个测试组踝关节细胞因子表达的测定,测试组即对照IgG,抗-4-1BBL和抗-4-1BB;
图4:显示抗-CII抗体的产生以及通过ELISA法测定的分别用抗-CII IgG1(a)和抗-CII IgG2b(b)处理的各组的血清水平,测试组即对照IgG,抗-4-1BBL和抗-4-1BB;
图5:显示每个测试组分别的CIA(胶原蛋白型II诱导的关节炎)临床分数,测试组即对照IgG,抗-4-1BBL和抗-4-1BB;
图6:显示通过ELISA测定的每个测试组分别的抗-CII IgG1(a)和抗-CIIIgG2b(b)水平,测试组即对照IgG,抗-4-1BBL和抗-4-1BB;
图7:显示通过[3H]标记测定的每个测试组分别的CII-特异的CD4+T细胞,测试组即对照IgG,抗-4-1BBL和抗-4-1BB;
图8:显示FACS(荧光活性细胞分类仪)观察的用CII+抗-4-1BB(a),CII+对照IgG(b),基因剔除小鼠中的CII+抗-4-1BB(c),CII-抗-4-1BB的F(ab’)2片断(d)以及CFA+抗-4-1BB(e)处理的CIA诱导的小鼠的照片;
图9:显示FACS观察的CD11c-CD8+T细胞(a),CD11c+CD8+T细胞(b)和CD8a-CD11c+DCs细胞(c)的细胞表面标记表达;
图10:显示诱导CD11c+CD8+T细胞的TCR Vβ表达谱;
图11:显示每个测试组分别在HSV-1处理后注射抗-4-1BB抗体后的CD11c+CD8+T细胞诱导,测试组即对照IgG,抗-4-1BBL和抗-4-1BB;
图12:显示在对DBA/1小鼠注射CII抗原和抗-4-1BB抗体后,每个测试组分别在每个时间点CD11c+CD8+T细胞(a)和CD11c-CD8+T细胞(b)的增加,测试组即对照IgG,抗-4-1BB;
图13:显示通过激光共聚焦显微观察观察到的诱导的CD11c+CD8+T细胞的类型(绿色:抗-CD3,红色:CD11c)((a)枝状大细胞;(b)中型枝状细胞;(c)没有枝状结构的小细胞;(d)无枝状结构);
图14:显示在用CII+对照IgG(a)和CII+抗-4-1BB(b)处理后用碘化丙啶染色的淋巴结切片;
图15:显示对测试组分别由抗-4-1BB处理所诱导的CD11c+CD8+T细胞的IFN-Y产生,该处理包括观察细胞内染色(a)和通过ELISA法测定的上清培养物(b),测试组即对照IgG和抗-4-1BB;
图16:显示由与CD11c+CD8+T细胞和CD11c-CD8+T细胞转化相关的[3H]标记测定的每个测试组分别的CII-特异的CD4+T细胞抑制反应,测试组即无转化,CD11c-CD8+T/对照IgG,CD11c-CD8+T/抗-IgG和CD11c+CD8+T/抗-IgG;
图17:显示将CD11c+CD8+T细胞和CD11c-CD8+T细胞转化到新的DBA/1小鼠后,每个测试组的临床分数,测试组即无转化,CD11c-CD8+T/对照IgG,CD11c-CD8+T/抗-IgG和CD11c+CD8+T/抗-IgG;
图18:通过RT-PCR和电泳,展示在抗-4-1BB处理和IDO,iNOS以及GAPDH诱导之间的相关性(泳道1:CD11b+T细胞用对照IgG处理,泳道2:CD11c-CD8+T细胞用抗-4-1BB处理,泳道3:CD11c+CD 8+T细胞用抗-4-1BB处理,泳道4:CD11b+T细胞用对照IgG处理,泳道5:CD11b+T细胞用抗-4-1BB处理,泳道6:CD11chigh T细胞用对照IgG处理,泳道7:CD11chigh T细胞用抗-4-1BB处理;
图19:通过RT-PCR和电泳显示IFN-γ在诱导IDO和iNOS的作用(泳道1:CD11b+T细胞用对照IgG处理,泳道2:CD11b+T细胞用对照IgG和抗-IFN-γ处理,泳道3:CD11b+T细胞用抗-4-1BB处理,泳道4:CD11b+T细胞用抗-IFN-γ和抗-4-1BB处理,泳道5:IFN-γ处理作为阳性对照);
图20:显示通过BrdU掺入法测定的每个测试组在抗IFN-γ处理和CII-特异性CD4+T细胞增殖之间的相关性;测试组即对照IgG,对照IgG,/抗IFN-γ,抗-4-1BB,抗-4-1BB/抗IFN-γ;
图21:显示对CIA诱导的小鼠进行对照IgG,抗-4-1BB,抗-4-1BB和抗IFN-γ处理后的临床分数;
图22:显示通过4-1BB处理后,1-甲基-D,L-色氨酸(1-MT)CIA的CIA抑制效果,包括(a)每个测试组分别的剂量依赖型1-MT的平均临床分数,测试组即对照IgG,抗-4-1BB/1-MT,抗-4-1BB/1-MT,抗-4-1BB/安慰剂,和(b)每个测试组抗-4-1BB-处理-依赖的的平均临床分数,测试组即对照IgG/1-MT,抗-4-1BB/1-MT,抗-4-1BB/安慰剂。
最佳实施方式
很明显,本领域技术人员可以对组合物进行各种修饰和改变,可以在不背离本发明的精神和范围的前提下使用和制备本发明。
通过下面的图和实例对本发明进行了更具体的解释,然而,应该理解为本发明并不以任何方式限制于这些实例。
发明实施方式
通过下面的图和实例对本发明进行了更具体的解释,然而,应该理解为本发明并不以任何方式限制于这些实例。
【实施例1】抗体的制备
产生4-1BB(3H3)和4-1BB(TKL-1)抗体的杂交瘤细胞分别由Drs.RobertMittler(Emory university,Atlanta,Georgia)和Hideo Yagita以及KoOkumura(Juntendo university,Tokyo,Japan)惠赠。通过蛋白G-柱(sigma,St.Louis,Missouri)从艾氏腹水癌细胞中纯化抗体。LAL(Cambrex,Walkersville,Maryland)测定内毒素水平低于0.05单位,在抗-CD3mAb-刺激的T细胞或4-1BBL转染的P815细胞中检测mAbs的结合活性。在用胃蛋白酶消化抗体后使用Sephacryl s-200HR柱(sigma)纯化3H3的F(ab’)2片段,购自Sigma的纯化的鼠IgG作为对照抗体。随后的mAbs购自BDPharMingen(San Diego,California)用于流式细胞仪分析:FITC-,PE-.PerCP-,和生物素-抗-CD8(53-6.7);PE-抗-CD4(GK1.5);PE-和生物素-抗-CD11c(HL3);FITC-和生物素-抗-CD11b(M1/70);PE-抗-1L12(C15.6);生物素-抗-H-2Kg(KH14);生物素-抗-1-Aq(KH116);生物素-抗-CD80(16-10A1);生物素-抗-CD86(GL1);生物素-抗-CD40(3/23);生物素-抗-CD45RA(14.8);纯化的抗-CD16/CD32(2.4G2);PE-,FITC-和Cy-链霉亲和素;和含有FITC-标记的mAb的抗Vβ2,3,4,5.1/5.2,6,7,8.1/8.2,8.3,9,10b,11,12,13,14和17TCRs的鼠VβTCR筛板。FITC-抗-DEX205(NLDG-145)购自Serotec(Kidlington,Oxford,United Kingdom)。DX5购自eBioscience(SanDiego,California)。
【实施例2】CIA诱导和抗4-1BB处理
为了理解4-1BB在CIA过程中的作用,检测了是激动性抗4-1BB抗体(3H3)还是阻断抗4-1BB配体可以改变CIA过程。
通过对6-7周的DBA/1雄性小鼠尾巴底部皮内注射100D CFA乳化的牛胶原II(CII)(Chondrex,Redmond,Washington)引起CIA。通过M.tuberculosisH37RA(2.0mg/ml,Chondrex)补充抗原。每日对小鼠关节的炎症进行检查并按下述方法计分:0,正常;1,局限于踝关节的红斑和轻度肿胀;2,延伸到踝关节外的红斑和轻度肿胀;3,延伸到踝关节外的红斑和中度肿胀;4,延伸到踝关节外的红斑和重度肿胀;每个爪子最大的关节炎评分是4,而每只小鼠最大的疾病评分是16。
使用对照IgG处理的小鼠在后免疫(PI)大约28天患上重度关节炎。在PI第42天得到关节炎严重程度的峰值(严重程度=13.4±2.7;发病率=9/10;爪子厚度=3.5±0.26)。使用抗-4-1BBL处理阻断4-1BB和4-1BBL相互作用的小鼠也患上关节炎,尽管比IgG处理对照组的程度轻(严重程度=9.6±3.2,P<0.05;发病率=8/10;爪子厚度=2.7±0.25,P<0.05)。激动性抗4-1BB处理强烈抑制了疾病的发展(严重程度=1.6±1.6,P<0.0001;发病率=2/10;爪子厚度=1.7±0.2,P<0.001)(见图1)。
【实施例3】组织学和免疫染色
将免疫原性后爪固定于10%福尔马林中,脱钙并装入石蜡中。制备关节切片(5-7D)并用常规方法进行苏木精和曙红染色。光学显微镜检测切片。对于双荧光染色,将纯化的CD11c+CD8+T细胞于L-赖氨酸包被的载片上37℃培养1小时。随后用1D未标记的2.4G2抗-FcγR Ab(BD PharMingen)阻断。用FITC-抗-CD3加上PE-抗-CD8或者FITC-抗-CD3加上PE-抗-CD11c或者FITC-抗-CD8加上PE-抗-CD11c对细胞着色。在最终清洗后,载片被装载到GVA使用支架上(Zymed,San Francisco,California)并使用激光扫描共聚焦显微镜观察(FV500,Olympus,Tokyo,Japan)。对于DLN的免疫染色,用PBS清洗DLN切片(8D),用FITC-抗-CD3加上PE-抗-CD11c或者FITC-抗-CD8加上PE-抗-CD11c染色。载片按上述方法处理。为了使双色样品通道间的交扰最小化,使用一次只能引发一种染料的顺次扫描技术。
组织学检测显示同型-或者抗4-1BBL-处理的小鼠大量渗入白细胞并且产生关节增生,血管翳形成,软骨破坏和骨侵蚀等所有CIA的特征。相反,抗4-1BB-处理的小鼠表现正常并且无疾病,反映为低的严重程度分数(见图2)。
【实施例4】细胞因子表达
为了检测含有IL-6,IL-5,TNF-α和IL-1β等周知的类风湿性关节炎特征的高水平细胞因子是否可以表达,通过从小鼠关节组织中分离RNA,利用RPA对细胞因子表达进行了分析。
RNA酶保护试验
使用TRIzolR(InVitrogen,Carlsbad,California)从每一组免疫40天后的小鼠踝组织中分离总RNA,按照操作指南(Riboquant,BD PharMingen)利用RNA酶保护试验定量测定细胞因子和趋化性细胞因子mRNA水平。简言之,15D总RNA与[32P]UTP-标记的RNA探针(mCK-1和mCK-5;BD PharMingen)杂交,56℃过夜。杂交后,通过RNA酶处理消化未杂交的单链RNA。保护的RNA通过酚/氯仿提取和乙醇沉淀进行纯化。将样品进行6%聚丙烯酰胺/7M尿素胶电泳。将胶干燥并进行放射自显影分析。
RT-PCR
通过使用SuperscriptII(LifeTechnologies,Gaithersburg,Maryland)和oligo(dT)引物从1D总RNA中合成单链cDNA,cDNA随后用10单位的RNase-H处理(LifeTechnologies)。在含有0.5D cDNA和每种引物0.5μM的20D反应混合物中进行PCR反应。使用下述引物:
<IL-1β>
序列号1:5’-CTGAAAGCTCTCCACCTC-3’(正义引物)
序列号2:5’-GGTGCTGATGTACCAGTTC-3’(反义引物)
<TNF-α>
序列号3:5’CCACCACGTCTTTG-3’(正义引物)
序列号4:5’-ATGGGCTCATACCCAGGG-3’(反义引物)
<GAPDH>
序列号5:5’-GAACGGGAAGCTTGTCATCAA-3’(正义引物)
序列号6:5’-CTAAGCAGTTGGTGGTGCAG-3’(反义引物)
关节组织细胞因子产物图谱也反映疾病严重程度:对照IgG或者抗-4-1BBL-处理的小鼠检测到高水平趋化性细胞因子,包括MCP-1,MCP-2,酸性细胞趋化素,MIP-1a,RANTES,而抗-4-1BB-处理的小鼠的上述趋化性细胞因子的量低至检测不到的水平。对照IgG-处理的小鼠显示高水平的IL-6,IL-5,TNF-α和IL-1mRNA。有趣的是,抗-4-1BBL-处理的小鼠产生高水平的IL-5,TNF-α和IL-1mRNA,但是IL-6mRNA的水平却非常低。相反,抗-4-1BB-处理的小鼠上述细胞因子的量低至检测不到的水平。高水平的表达IL-1β,TNF-α和IL-6是类风湿性关节炎众所周知的特征。(见图3)
【实施例5】胶原特异性抗体的测定
接下来测定了抗-4-1BB处理是否可以抑制抗-CII抗体产生。
通过酶联免疫测定法(ELISA)测定了抗-牛CII IgG1,IgG2a,IgG2b,IgG2,IgM和IgE的血清浓度。简言之,使用牛CII(10D/ml,Chondrex)包被微量滴定板,封闭并用一系列稀释的测试乳清培养。通过和HRP-标记的鼠-抗鼠IgG1,IgG2a,IgG2b,IgG3,IgM或IgE(BD PharMingen)以及底物,四甲基联苯胺培养而检测到边际IgG。使用ELISA读板器(Wallac,Turku,Finland)在450nm处测定光密度。
在PI第19,28,35和42天测定抗-CII抗体的血清水平。抗-4-1BB处理完全抑制了抗-CII IgG1和IgG2b(p<0.001)的产生,而抗-4-1BBL处理某种程度上减少了抗-CII抗体的产生(p<0.05)。其他同型的CII的IgG,IgA和IgE则几乎检测不到(数据没有显示)。这些结果再次反映了疾病的严重程度。
对于CIA模式研究的结果提示用抗-4-1BB引发4-1BB可诱导与阻断4-1BB/4-1BBL相互作用不同的活性抑制机制。(见图4)
【实施例6】抗4-1BB(3H3)改善已确诊的CIA
在DBA/1小鼠中诱导CIA,在PI第28天将小鼠分成三组以至于每组的平均关节炎评分相等,在PI第28,30,32,34和36天对它们进行对照IgG,抗-4-1BB或抗-4-1BBL处理。(见图5)
只有用抗-4-1BB处理组的关节炎消退;这一效果在PI第52天更为显著(p<0.05)。也检测了CII-特异抗体的水平。抗-4-1BB处理迅速并且几乎完全清除了血清抗-CII抗体(p<0.001)。这些数据再次说明交联4-1BB诱导抗CIA的抑制机制。(见图6)
【实施例7】抗-4-1BB抑制CII-特异CD4+T细胞增殖
测试抗-4-1BB处理所诱导的活性抑制是否针对于CD4+T细胞。从PI第14天CII-免疫的小鼠的引流淋巴结(DLN)分离CD4+T细胞并且在体外测定CD4+T细胞对CII的记忆应答。利用磁珠(MiltenylBiotech)纯化DLN(腋和腹股沟淋巴结)的CD4+T细胞,其来源于CII免疫后第12天的对照IgG,抗4-1BB或抗-4-1BBL mAb-处理的小鼠。纯化的CD4+T细胞(1×105)在变性CII(0.5D/ml)存在或不存在的情况下和丝裂霉素c(sigma)处理(50D/ml,37℃,20min)的同基因型脾APCs(2×105)共培养。在5%CO2中37℃培养72小时后,培养物在最后的12小时中用[3H]胸腺嘧啶脉冲(1.0μCi/well)(Amersham Pharmacia,Piscataway,New Jersey)。使用闪烁计数器(Wallac)计算掺入的放射性。按照操作指南的建议,通过ELISA法使用Endogen的细胞因子-特异抗体对评估上述培养物中的细胞因子产物。
经抗-4-1BB-处理的小鼠的CD4+T细胞没有显示记忆应答,而作为对CII的应答,对照IgG-处理的小鼠的CD4+T细胞大量增殖。在CIA模式中源于抗-4-1BBL-处理的小鼠的CD4+T细胞显示比对照IgG-处理组更低的记忆应答(p<0.05)。这些数据表明CIA模式中抗-4-1BB-介导的抑制是针对于CD4+T细胞(见图7)。
【实施例8】抗-4-1BB处理诱导DLN中CD11c+CD8+T细胞群体的膨胀
8-1流式细胞术
为研究在CIA模式中抗-4-1BB-介导的CD11c+CD8+T细胞抑制机制,对PI第12天,源于DLN和脾脏的白细胞亚群进行了分析。和对照IgG-处理组相比,期待在抗-4-1BB处理组找到淋巴细胞的特殊群体的增加或者减少。抗-4-1BBL处理组对于结果提供了进一步的对照和确认。
从DLN中获得红细胞游离单核细胞悬浮液,并将白细胞(100D中有1×106细胞)在首先用1D未标记的抗-FcY R Ab(BD PharMingen)封闭步骤后和指示的荧光标记抗体在4℃进行流式细胞仪分析,随后进行FaCSCalibur(BDBioscicnecs)分析。对于细胞内的细胞因子染色,将细胞用CII(50D/ml)或者CII加PMA(50ng/ml;Sigma)以及钙离子导入剂(500ng/ml;Sigma)刺激18小时。在最后的6小时加入GolgiPlug(BD PharMingen)。细胞首先用表面标记着色,固定,透化,并按照操作指南的说明用FITC-标记的抗-IFN-γ,抗-TGF-β,抗-IL-4,抗-IL-10或IL-12mAb的Cytofix/Cytoperm试剂盒(BD PharMingen)培养。
按照操作指南说明进行BrdU掺入法测定(BrdU流式试剂盒,BDPharMingen)。简言之,在CII(50D/ml)存在下培养DLN细胞(2×105)。细胞经PE-标记的抗-CD4染色,固定,透化,DNaseI处理并进一步用FITC-标记的抗-BrdU(BD PharMingen)培养。样品立即通过FaCSCalibur(BDBioscicnecs)分析。
利用FACS检测了白细胞表面标记的多种组合并发现CD11c+CD8+细胞在抗-4-1BB处理的小鼠的DLN和脾脏中都显著的膨胀。这一群体的CD11c表达水平比CD11c+DCs的要低。(尽管这一群体的表型是CD11clowCD8+,为了简单则认为这些细胞是CD11c+CD8+细胞群体。)在对照IgG-处理组没有发现CD11c+CD8+细胞的膨胀。
接下来试图确定引起CD11c+CD8+细胞膨胀的条件。如上文所述,当DBA/1小鼠用CII和对照IgG免疫后,CD11c+CD8+细胞并没有膨胀。当4-1BB KO小鼠用CII和对照IgG免疫后,CD11c+CD8+细胞也没有膨胀。当DBA/1小鼠用CII免疫和注射抗4-1BB的(Fab’)2片段后,CD11c+CD8+群体没有膨胀,膨胀需要通过与完整的抗-4-1BB mAb交联而产生的4-1BB信号。最后,当DBA/1小鼠用抗-4-1BB和完整的不含CII的Freund佐剂免疫后,也没有膨胀。CD11c+CD8+细胞的膨胀仅发生在当CII和4-1BB信号同时存在时。因此,CD11c+CD8+细胞的膨胀是一个4-1BB-依赖以及抗原驱动的效应。(见图8)
8-2 CD11c+CD8+细胞的表型
为了确定CD11c+CD8+细胞的表型,对于CD11c-CD8+T细胞,CD11c+CD8+T细胞以及CD8a-CD11c+DCs的表面标记的表达进行了比较。CD11c+CD8+细胞表达T细胞标记,例如,CD3+,TCR Vβ+和Thy1.2+。这些细胞由于他们除了表达CD11c和II型抗原1-Aq还表达33D1乳头细胞标记而与常规的CD11c-CD8+T细胞不同。他们由于不表达DC标记,如CD205,B220和CD40以及不吸收荧光葡糖颗粒而与CD11c+CD8+DCs也不同。
结论是这一新型细胞群体是CD8+T淋巴细胞的亚型。
对于CD11c+CD8+T细胞的TCR Vβ表达图谱也进行了确定。用CII免疫小鼠以诱导CIA,随后用对照抗4-1BB处理小鼠。从DLN细胞中分离CD11c-CD8+T细胞,CD11c+CD8+T细胞以及CD4+T细胞。TCR单克隆抗体在板上染色并通过FaCSCalibur(BD Bioscicnecs,San Jose,California)分析。
在结果中,30%以上的细胞表现Vβ8.1/8.2并且每个Vβ8.3,Vβ5.1/5.2和Vβ7为28%,22%和17%。CD4+T细胞的Vβ表达谱和CD11c+CD8+T细胞中的类似(见图10)。
8-3 HSV-1感染后的CD11c+CD8+T细胞产物。
确定了即使在细胞已经收到由外部抗原引起的免疫原性后给予其抗-4-1BB抗体,是否诱导CD11c+CD8+T细胞产物。
通过腹腔注射盐酸氯胺酮(1mg/kg,维生素;Phoenix Scientific Inc.,St.Joseph,Missouri)和甲苯噻嗪(0.5mg/kg,Ben Venue Laboratories,Bedford,Ohio)麻醉小鼠,并在每只后爪垫注射含4×105PFU HSV-1的20D的PBS。在第0和2天对HSV-1-感染的小鼠腹腔注射纯化的抗-4-1BB(3H3,200D)或鼠IgG。在PI第5天从DLN制备单核细胞悬浮液。细胞首先用2.4G2培养然后用PE-标记的抗-鼠CD11c和Cy-标记的抗-CD8mAbs染色。染色后的细胞利用FaCSCalibur(BD Bioscicnecs,San Jose,California)分析。将一部分已经用对照IgG或抗-4-1BB处理的HSV-1-感染的小鼠细胞置于96孔板中并在1D/ml gB肽存在或不存在的情况下培养3天。细胞用10μM BrdU标记1小时并用PE-抗-CD4或PE-抗-CD8mAb染色,随后用FITC-抗-BrdU细胞内染色。
CD11c+CD8+T细胞也由其他抗原诱导,包括HSV-1感染,当抗原和抗-4-1BB一起出现时,抗-4-1BB再次在很大程度上减少CD4+T细胞对HSV-1的记忆应答,尽管和IgG-处理组相比CD8+T细胞应答增强了。
8-4膨胀的动力学和CD11c+CD8+T细胞的特性
对于注射了CII的DBA/1小鼠的CD11c+CD8+T细胞膨胀的时间过程也进行了调查。
CD11c+CD8+T细胞膨胀的时间过程由注射了CII和抗-4-1BB的DBA/1小鼠决定。膨胀从PI第5天开始并在第12天达到峰值;在第18天细胞再次小到无法检测。当在第24天再次给予抗-4-1BB时,膨胀在4天的滞后调整后重新开始。常规CD11c+CD8+T细胞的百分含量也有所增加(见图12)。
通过使用抗-CD3和抗-CD8或者抗-CD3和抗-CD11c或者抗-CD8和抗-CD11c染色,共聚焦显微观察到CD11c+CD8+T细胞。所有这些标记-CD3,CD8和CD11c-均在细胞表面检测到并且可以消失。CD11c的膨胀水平比CD3和CD8要低(图3b1)。碘化丙啶染色以及FACS分析表明CD11c+CD8+T细胞的DNA含量为2N(图3b2)。在淋巴结切片上发现大量CD11c+CD8+T细胞;在其峰值,这些细胞占总DLN细胞的将近22%(图13和14)。
通过细胞内染色和ELISA,使用经过带有CIICD11c+CD8+T细胞刺激后的培养悬浮液测定了CD11c+CD8+T细胞产生的细胞因子。IFN-v是CD11c+CD8+T细胞产物中最丰富的。
8-5通过CD11c+CD8+T细胞继承性转移治疗CIA
对CD11c+CD8+T细胞继承性转移是否可以抑制CII-特异性CD4+T细胞进行了测定。由CII-免疫的和抗-4-1BB-处理的小鼠的DLN制备CD11c+CD8+T细胞和CD11c-CD8+T细胞,并且通过对CII-免疫的和对照IgG-处理的小鼠纯化得到CD11c-CD8+T细胞。这些细胞被继承性转移到也在同一天接受了CII免疫的新的DBA/1小鼠中。7天后,从每一组小鼠中制备DLN中的CD4+T细胞并且在CII(50D/ml)存在或者不存在的情况下用y-照射抗原提呈细胞(APCs)培养72小时。将脾脏切成小段并在II型胶原酶(1mg/ml;Sigma)和DNaseI(15D/ml;Roche)存在下37℃孵育40min。引流淋巴结在1mg/ml胶原酶和5mM EDTA存在下37℃孵育5min。制备单核细胞悬浮液并通过MACS分离柱(Miltenyi Biotec)分离CD11c+CD8+T细胞。由于CD11c+CD8+T细胞对于CD4,F4/80,CD40和B220细胞为阴性,而常规CD11c+DCs并不是(数据没有显示),通过免疫磁性检测CD4,F4/80,CD40和B220群体而完成CD11c+CD8+T细胞的纯化,所述CD4,F4/80,CD40和B220群体在含有这些分子抗体的混合物中孵育。在一些实验中,CD49b特异的DX5在混合物中孵育。阴性选择细胞(DC-)进一步和抗-鼠CD11c(N418)-微珠孵育并分成CD11c+CD8+和CD11c-细胞。上述步骤得到的阴性细胞部分(CD11c-)与抗-鼠CD8(Ly-2)微珠孵育并且通过MACS柱完成CD11c-CD8+细胞的分离。选择的细胞群体的纯度为87-90%。对于继承性转移,5×106纯化的细胞通过静脉注射转移到DBA/1小鼠中。
在结果中,来源于小鼠的接受了CD11c+CD8+T细胞的CD4+T细胞并没有表明出CII的增殖记忆应答,而来源于小鼠的接受了CD11c-CD8+T细胞的CD4+T细胞表现出正常的CII的增殖记忆应答(见图16)。
然后对CD11c+CD8+T细胞继承性转移是否可以抑制CIA的发展进行了调查。制备来源于抗-4-1BB-处理的小鼠的CD11c+CD8+T细胞或来源于抗-4-1BB-处理或者对照IgG-处理的小鼠的CD11c-CD8+T细胞并且在PI第0,10,25和35天继承性转移到CII-免疫的DBA/1小鼠中。CD11c+CD8+T细胞改善了CIA的发展(P<0.001)(见图17)。
【实施例9】抗-IFN-γ抵消了抗-4-1BB-介导的IDO和iNOS的诱导并且抑制CD4+T细胞
由于CD11c+CD8+T细胞产生IFN-γ,对于IFN-γ诱导效应分子例如IDO和诱导一氧化氮合成酶(iNOS)是否在DLN细胞产生进行了调查。IDO(吲哚胺2,3-加双氧酶)表达细胞控制了在妊娠期由母系T细胞发生的免疫反应。
具体地,将CII+对照IgG和CII+抗-4-1BB用于小鼠,14天后,从用对照IgG处理的小鼠中分离CD8+T细胞,CD11b+单核细胞和CD11c+DCs。从用CII和抗-4-1BB处理的小鼠中分离CD11c-CD8+T细胞,CD11c+CD8+T细胞,CD11b+单核细胞和CD11c+DCs。从纯化的细胞中提取RNA并且使用IDO,iNOS和GAPDH-特异性引物进行RT-PCR。使用引物如下:
<IDO>
序列号5:5’-CACTGTACCAGTGCAGTAG-3’(正义引物)
序列号6:5’-ACCATTCACACACTCGTTAT-3’(反义引物)
<iNOS>
序列号7:5’-AAGTCAAATCCTACCAAAGTGA-3’(正义引物)
序列号8:5’-CCATAATATGGTTGATGAACT-3’(反义引物)
<GAPDH>
序列号9:5’-GAACGGGAAGCTTGTCATCAA-3’(正义引物)
序列号10:5’-CTAAGCAGTTGGTGGTGCAG-3’(反义引物)
将处理的小鼠的脾脏切成小片并在II型胶原酶(1mg/ml;Sigma)和DNaseI(15D/ml;Roche Biomedical,Mannhein,Germany)存在下37℃孵育40min。清洗后,使用抗CD-11b免疫磁珠纯化巨噬细胞并随后在MACS柱(MiltenyiBiotec,Auburn,California)上分离。对于分离的DCs,将细胞在抗CD-90微珠上孵育并在MACS上再次选择。阴性选择细胞群体(CD11c+CD8-)进一步用抗CD-11微珠孵育,从而分离出CD11c+DCs。分离的巨噬细胞和DCs的纯度为87-90%。
在结果中,对照IgG处理的小鼠并不产生任何IDO和iNOS mRNA并且不同于IDO,iNOS在CD11c+CD8+T细胞中表达。IgG处理的对照小鼠的CD11c+DC低水平地表达IDO和iNOS。然而,IDO和iNOS mRNA在CD11b+单核细胞和CD11c+DC中被强烈的诱导,而他们都是用抗-4-1BB处理的(见图18)。
【实施例10】抗-IFN-γ逆转了抗-4-1BB-介导的IDO和iNOS的诱导并且抑制CD4+T细胞
对于IDO和iNOS是否由IFN-γ介导进行了检测。用抗-IFN-γ结合IgG或者抗-4-1BB处理CII-免疫的或者未免疫的小鼠。从每组小鼠的淋巴结中制备巨噬细胞和DCs并检测其IDO和iNOS mRNA的表达。
具体地,用CII免疫DBA/1诱导CIA,然后用对照IgG或者抗-4-1BB结合抗-IFN-γ处理小鼠。在PI第14天收获DLN细胞中的CD11b+单核细胞并且使用从对照IgG处理的CD11b+细胞中分离的每个RNA对IDO,iNOS和GAPD进行RT-PCR。对照IgG和抗-IFN-γ,抗-4-1BB,抗-4-1BB和抗-IFN-γ以及用抗-IFN-γ处理的264个原始细胞作为阳性对照。
结果表明抗-IFN-γAb中和了抗-4-1BB-介导的IDO和iNOS,这表明抗-IFN-γAb抑制CD11b+单核细胞中的IDO和iNOS的表达。
为了进一步明确IFN-γ在抑制抗-4-1BB-处理的小鼠中CD4+T细胞对CII的应答,用CII免疫DBA/1诱导CIA,然后用对照IgG或者抗-4-1BB结合抗-IFN-γ处理小鼠。在PI第14天收获总DLN细胞并在体外用CII刺激。
用BrdU渗入法测定CII-特异的CD4+T细胞。来源于抗-4-1BB-处理的小鼠的CD4+T细胞在对CII应答时并没有增殖。当抗-IFN-γ和抗-4-1BB一起给药时,这种抑制被逆转。
这些数据表明由抗-4-1BB发挥的抗-关节炎效应主要由IFN-γ的诱导调节。IFN-γ看起来由CD11c+CD8+T细胞产生,所述CD11c+CD8+T细胞由抗-4-1BB和抗原引起膨胀。
为了确定抗-IFN-γ是否逆转抗-4-1BB-介导CIA的抑制,将三组DBA/1小鼠用CII免疫并用对照IgG,抗-4-1BB或者抗-4-1BB加抗-IFN-γ处理。结果表明抗-IFN-γ中和了抗-4-1BB-介导的CIA的改善(见图21)。
【实施例11】1-甲基色氨酸(1-MT)完全逆转4-1BB-介导的CIA的抑制
由于施加抗-4-1BB可以诱导IDO,而IDO可以是IFN-γ的分子效应器,因此调查了抗-4-1BB-介导的CD4+T细胞和CIA的抑制中是否包括IDO。
每只小鼠在PI第0,2,4,6和8天腹腔注射200D纯化的抗-4-1BB(3H3,鼠IgG1),抗-4-1BBL(TKS-1,鼠IgG1),或者对照鼠IgG。通过测量抗-4-1BB的血清IFN-Y水平检测体内抗体的剂量应答并且检测了对抗-4-1BBL记忆应答的抑制。当以每只小鼠150-200D的剂量给予抗体时观察到最大效应。为了治疗确诊的CIA,在PI第28,30,32,34和36天注射。为了阻断IFN-γ,对小鼠在PI第0,4,8和12天每隔4天腹腔注射500D纯化的R4-6A2。再次用鼠IgG作为对照。
为了抑制IDO的活性,在免疫前1天将混有1-MT(10mg/天释放率)或者安慰剂(InnovativeResearch of America,Sarasota,Florida)的缓释聚合颗粒由指背皮肤注入。在一些实验中,各含有210mg 1-MT的两种颗粒分别注入每只小鼠,并在21-天的周期内提供20mg/天的剂量。由于相对于小鼠的小体积,这些颗粒的尺寸大,又进行了另外的实验,即对每只小鼠注入两种较小的颗粒(10mg/天释放率),每个含有120mg的1-MT,并在12-天的周期内提供20mg/天的剂量。在第13天,再次注入2粒颗粒,持续24天的总计量周期。
在结果中,1-MT 20mg/天的剂量完全逆转了抗-4-1BB对CIA的抑制效果。由于用5mg/天处理小鼠时的逆转是不完全的,故而该逆转是剂量-依赖型的。1-MT对3H3效应的逆转被延迟;在1-MT处理的小鼠中,直到PI第48天,才可见该疾病的发生,而对照小鼠在PI第30天就有该疾病的迹象。(见图22)。
在这些实验中,1-MT对抗-4-1BB抑制效应的逆转导致这些小鼠表现出与同时处理的对照小鼠一样的CIA。
【实施例12】毒性试验
为了检测抗-4-1BB抗体的细胞毒性,按下述方法进行实验:
方法
使用抗-4-1BB抗体对平均体重为108.3-126的SPF Sprague-Dawley鼠(Bopgenomics)的急性毒性进行测定。每组由5只小鼠组成,口服给予8000mg/kg的抗-4-1BB抗体并观察14天。这些试验按照韩国食品药品管理局发布的药品安全性评估检测指南(通告号1999-61)和韩国食品药品管理局发布的药物非临床研究优良实验室规范法规(通告号2000-63)以及优良实验室规范的OECD原理实行。
结果
在使用8000mg/kg抗-4-1BB抗体的任何组或者每种性别中均没有发现死亡率,临床体征,体重改变以及总数的治疗相关效应。这些结果表明本发明制备的复合物是有效并安全的。
因此,将对制备方法和各种赋形剂进行描述,但是本发明并不限于此。典型的制备实例描述如下:
注射剂制备
抗-4-1BB抗体100mg
偏重亚硫酸氢钠3.0mg
苯甲酸甲脂0.8mg
羟苯甲酸丙脂0.1mg
注射用蒸馏水适宜量
通过溶解活性物质,控制pH在大约7.5然后将所有组分填入2安瓿中,通过常规注射制剂制备方法消毒以制备注射制剂。
粉剂制备
抗-4-1BB抗体500mg
玉米淀粉100mg
乳糖100mg
滑石10mg
通过混合上述组分并装入密封包中制备粉末制剂。
片剂制备
抗-4-1BB抗体200mg
玉米淀粉100mg
乳糖100mg
硬脂酸镁适宜量
通过混合上述组分并压片制备片剂。
胶囊制备
抗-4-1BB抗体100mg
乳糖50mg
玉米淀粉50mg
滑石2mg
硬脂酸镁适宜量
通过混合上述组分并用常规的胶制备方法装入胶质胶囊中制备胶囊。
液体制备
抗-4-1BB抗体100mg
蔗糖20g
多糖20g
柠檬香料20g
通过溶解活性组分,然后将所有组分填入1000D安瓿中,通过常规液体制剂制备方法消毒以制备液体制剂。
显而易见,本发明如此的描述,可以以多种方式变化。这些变化不被认为背离本发明的精神和范围,并且这些对于本领域技术人员显见的修饰包括在本发明权利要求的范围内。
Claims (4)
1.一种药物组合物,包含有效量的抗4-1BB抗体作为活性成分,以及一种药学上可接受的载体,所述抗4-1BB抗体通过CD11c+CD8+T细胞增殖和诱导CD4+T细胞抑制以预防和治疗类风湿性关节炎疾病。
2.根据权利要求1的药物组合物,其中所述组合物含有基于组合物总重量的大约0.01~50w/w%的抗体。
3.根据权利要求1的药物组合物,其中所述类风湿性关节炎疾病是胶原诱导性关节炎。
4.抗-4-1BB抗体在制备治疗和预防哺乳动物类风湿性关节炎的治疗药物中的应用,所述哺乳动物包括需要的人。
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PCT/KR2005/001680 WO2005120568A1 (en) | 2004-06-09 | 2005-06-04 | Pharmaceutical composition comprising the anti-4-1bb antibody for treating or preventing rheumatoid arthritis |
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KR101894597B1 (ko) | 2009-03-11 | 2018-10-04 | 오거렉스 라이프 사이언시스 코포레이션 | 관절염 상태를 특성화하는 조성물 및 방법 |
EP2768851B1 (en) | 2011-10-21 | 2017-05-31 | Augurex Life Sciences Corporation | Antigens derived from citrullinated 14-3-3 and uses thereof in the diagnosis of rheumatoid arthritis |
MX2017014699A (es) * | 2015-05-21 | 2018-04-11 | Alligator Bioscience Ab | Polipeptidos novedosos. |
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WO2019104716A1 (en) * | 2017-12-01 | 2019-06-06 | Adagene Inc. | Methods for using cd137 ligand as biomarker for treatment with anti-cd137 antibody |
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WO2019148445A1 (en) | 2018-02-02 | 2019-08-08 | Adagene Inc. | Precision/context-dependent activatable antibodies, and methods of making and using the same |
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CA2172165C (en) * | 1993-09-16 | 2003-12-02 | Byoung S. Kwon | Human receptor h4-1bb |
US6210669B1 (en) * | 1996-10-11 | 2001-04-03 | Bristol-Myers Squibb Co. | Methods and compositions for immunomodulation |
US20030096976A1 (en) * | 1998-11-17 | 2003-05-22 | Hong Hyo Jeong | Humanized antibodies LB-00503 and LB-00506 specific for human 4-1BB and pharmaceutical compositions comprising said humanized antibodies |
DE60232895D1 (de) * | 2001-10-09 | 2009-08-20 | Mayo Foundation | Verwendung von agonistischen 4-1bb antikörpern zur erhöhung der immunantwort |
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KR20080107050A (ko) * | 2007-06-05 | 2008-12-10 | 울산대학교 산학협력단 | 항-cd137 단일클론 항체를 포함하는 만성이식편대숙주 질환의 예방 또는 치료용 약학적 조성물 |
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