CN1246125A - 含有特异于胶原ⅱ的t细胞表位的肽 - Google Patents
含有特异于胶原ⅱ的t细胞表位的肽 Download PDFInfo
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- CN1246125A CN1246125A CN98802141A CN98802141A CN1246125A CN 1246125 A CN1246125 A CN 1246125A CN 98802141 A CN98802141 A CN 98802141A CN 98802141 A CN98802141 A CN 98802141A CN 1246125 A CN1246125 A CN 1246125A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
本发明提供含有氨基酸序列通式(Ⅰ):A1-Xaa-Gly-A4-A5-Gly-A7-Xaa-Gly的分离的肽,其中A1代表具有芳香或脂肪族侧链的氨基酸,A4代表天冬酰胺或精氨酸残基或具有芳香或脂肪族侧链的氨基酸残基,A5代表任何现存的氨基酸,A7代表具有带负电的残基的氨基酸,Xaa代表氨基酸残基,Gly代表甘氨酸残基;和这样的肽在医学治疗中的用途,特定地在自身免疫病如类风湿关节炎的治疗中的用途。
Description
本发明提供了起源于胶原CII的新肽,他们的制备方法和他们在医学治疗,特别是类风湿关节炎和相关的自身免疫病的治疗中的用途。
胶原分子是结缔组织的一些主要的结构蛋白。他们包括形成具有独特的X-Y-Gly重复氨基酸序列的延伸的三股螺旋结构的三个多肽链。软骨的细胞外基质独特地主要含有II型胶原,但也含有IX和XI型胶原。最近从小鼠已经克隆了CII,并且它的1419个氨基酸的全部序列已经确定(Metsarantadm等人,1991,生物化学杂志,266:16862-9)。
认为II型胶原“躲避”了免疫系统的细胞,因为它只有在无血管组织如软骨和眼睛的玻璃体中发现。所以,免疫系统不完全是耐受它自身的II型胶原的,作为隔离的蛋白质,CII具有当在Freund完全佐药中注射时,诱导组织特异的自身免疫病的能力。它诱导的疾病命名为胶原诱导关节炎(CIA)。CIA通常是注射外源CII诱导的,它引起能够识别对应的自我蛋白质的T细胞和抗体的产生。
CIA在小鼠中的敏感性局限于I-Aq和I-Ar型MHC。这些分子的结合基序相似于人DR4和DR1的那些。所以,相信CII是类风湿关节炎(RA)中的自身抗原。
对于RA目前没有原因治疗(causal therapy)。现存的治疗方法例如施用皮质类固醇是非特异的,正如他们一般抑制免疫应答。目前的研究提出在RA的发病机理中自身反应T细胞的重要作用,这导致了一个概念,鼻/口腔给药免疫原性肽导致这些致病原T细胞的耐受性可能具有治疗潜力的。
Miyahara,H.等人(免疫学1995,86:110-115)叙述了在小鼠中的关节炎的抑制中II型胶原的片断的用途。但是,利用的片断(CII607-621)不含有人的RA的HLA-DR的任何分子的结合基序,并且所以在人RA治疗中用作耐受原将没有效果。
WO96/20950的目的是叙述能够结合人HLA DRB1MHC蛋白质的II型胶原肽,这表明将可用于RA治疗中。但是,这些存在于胶原CII蛋白质的273-404区的肽表明在推断的RA的耐受原的有关测试中只有微弱的活性。本发明的肽的治疗效力明显更大。
本发明提供了已经发现在诱导起源于T细胞表位的胶原CII的免疫耐受性中特别有效的肽。这些肽可以用于自身免疫症状如类风湿关节炎的治疗中。
所以,根据本发明提供了分离的肽,具有或包含通式(I):
A1-Xaa-Gly-A4-A5-Gly-A7-Xaa-Gly (I)的氨基酸序列
其中:
A1代表具有芳香或脂肪族侧链的氨基酸残基,
A4代表天冬酰胺或精氨酸残基,或具有芳香或脂肪族侧链的氨基酸残基,
A5代表任何天然存在的氨基酸,
A7代表具有带负电的残基的氨基酸,
Xaa代表任何氨基酸残基,和
Gly代表甘氨酸残基。
本发明的肽优选地是9,10,11,12,13,14或15个氨基酸肽;和更优选地是9个氨基酸的肽。
在本发明的肽中,下面独立地优选用:
-A1是F,I,L,A或P并且最优选地是F
-A4是F,I,L,A,R,N或P并且最优选地是F
-A5是K或R
-A7是E,D,Q,P或N并且最优选地是Q。
根据本发明,优选的肽包括那些包括或具有下面序列的一个的这些:ESG SPG ENG,PPG ADG QPG,ARG NDG QPG,QPG AKG DQG,APG AKG EAG,PTG VTG PKG,AQG SRG EPG,RVG PPG ANG,PAG ASG NPG,ANG NPG PAG,TDG IPG AKG,DPG LQG PAG,SAG APG IAG,APG EKG EPG,IAG APG FPG,PQGLAG QRG,FPG PRG PPG,PKG ANG DPG,APG ASG DRG,LPG ARG LTG,DAGPQG KVG,ALG APG APG,PAG ANG EKG,KQG DRG EAG,or ARG APG EPG(分别是SEQ ID No1至25)更优选是 RVG PPG ANG(SEQ ID No8)或ANG NPG PAG(SEQ ID No10)。
根据本发明,优选的例子也包括那些含有或具有下面的氨基酸序列的一个:VKG ESG SPG ENG SPG;FAG PPG ADG QPG AKG;AAG ARG NDG QPG PAG;ADG QPG AKG DQG EAG;APG AKG EAG PTG ARG;PQG PTG VTG PKG ARG;PEG AQG SRG EPG NPG;AAG RVG PPG ANG NPG;PAG ASG NPG TDG IPG;PPGANG NPG PAG PPG;NPG TDG IPG AKG SAG;RAG DPG LQG PAG APG;AKGSAG APG IAG APG;PAG APG EKG EPG DDG;APG IAG APG FPG PRG;PPG PQGLAG QRG IVG;APG FPG PRG PPG PQG;LAG PKG ANG DPG RPG;KQG APG ASGDRG PPG;EPG LPG ARG LTG RPG;RPG DAG PQG KVG PSG;ETG ALG APG APGPPG;PPG PAG ANG EKG EVG;PTG KQG DRG EAG AQG;or STG ARG APG EPGETG(分别是SEQ ID No26至52)更优选 AAG RVG PPG ANG NPG(SEQ ID No33),最优选 PPG ANG NPG PAG PPG(SEQ ID No35)。
根据本发明,优选的肽含有不中断地存在于胶原II的701-721区的9到15个氨基酸序列。
本发明同时涉及含有特异于胶原II的T细胞表位的肽,该肽至少含有9个氨基酸,这9个氨基酸具有相似于和连续地选自胶原II的110-239,338-379和587-895部分的序列的序列,其中功能等同氨基酸非强制性地替代每个氨基酸,其中肽是如上确定的通式I。
根据本发明,进一步提供了药物组合物,包括结合可药用载体或稀释剂的本发明的肽。该组合物优选地用于提供抗自我免疫症状,如类风湿关节炎和复发性多发性软骨炎的耐受性。
本发明进一步提供利用本发明的肽或本发明的组合物制造用于治疗自身免疫病的药剂。
根据本发明,同时提供了治疗患有自身免疫病如类风湿关节炎和复发性多发性软骨炎的人或动物的方法,该方法包括给予人或动物治疗有效量的肽或组合物。
利用熟练技术人员已知的方法可以制备本发明的肽。例如通过利用标准的固相的顺序偶联技术,利用了自动肽合成仪(参见例如,Jones,J.肽的化学合成,132-156,第一版,牛津大学出版社,1991和R.Epton,固相肽合成的革新和前景,SPCC(UK)公司,1990)。制备从可以获得的C末端氨基酸移植到甲基二苯甲基胺树脂或氯甲基化树脂或适当的固体支持物开始。在已经保护了侧链后,其它氨基酸一步一步移植。在这一偶联方法中,利用F-moc或t-Boc方法保护氨基酸的α-氨基基团。氨基酸的侧链保护基团是本领域已知的。整个保护肽是从氯化甲基化树脂通过溶铵基释放以便获得保护的酰胺,或从甲基二苯甲基胺树脂或二苯甲基胺树脂通过酸溶解释放。
利用溶液方法,通过逐步法或片断缩合(参见例如,Jones J.肽的化学合成,115-131,第一版,牛津大学出版社,1991)也可以制备本发明的肽。将适当的α-氨基保护氨基酸与适当的α-羧基保护氨基酸(这样的保护可能不是根据选择的偶联方法需要的)利用二亚胺,对称或非对称酸酐,或其它偶联试剂或本领域已知的技术偶联。这些技术可以是化学的或酶的。除去的α-氨基酸和/或α-羧基保护基团,并且偶联下一个适当的保护氨基酸或氨基酸嵌段以便扩展增长的肽。在每个合成中可以利用保护基团和化学和/或酶技术和组装的方案的各种联合。
确定了本发明的肽含有T细胞表位。用另一句话说,他们能够活化T细胞。存在几个已知技术确定结合长度。优选地,本发明的肽可以活化在IFN-γ释放测试中至少具有2的结合长度和/或具有刺激指标至少3的T细胞。利用本领域已知的方法或利用本文的实施例中叙述的方法可以进行IFN-γ释放测试。同样地,利用已知的增殖测试,例如在“在小鼠中的II型胶原反应T细胞的分析”Andersson和Holmdahl,当前免疫学杂志,20:1061-1066,1990中叙述的确定刺激指标,优选地,利用本文实施例中利用的方法进行测试。
本发明的肽没有修饰地用于治疗中,可替代地,肽可以修饰,例如他们可以偶联,例如共价结合递送系统,例如结合到粘膜结合结构,这些结构中包括辅助肠吸收的霍乱β毒素。
本发明的肽可以用于治疗,预防或诊断自身免疫病,并且术语“治疗”如本文所用也应该包括预防和诊断。
本发明的肽可以是分离的肽,其中本发明的肽不包括生物体中存在的肽。本发明的肽可以是从天然或重组产生的肽或蛋白质分离的或者可以如本文所用化学地合成。
化合物可以每天剂量10微克到10毫克,每天单个剂量或分开2到4次剂量给药。所以,单位剂量含有本发明的组合物2.5微克到10毫克。化合物可以溶液,悬浮液,HFA气雾剂和干燥粉末配方例如,Turbuhaler配方形式鼻内给药,或系统地例如通过口服,以片剂,丸剂,胶囊,糖浆,粉末或颗粒形式给药,或通过肠胃外以无菌肠胃外溶液,或悬浮液形式给药,或通过直肠以栓剂形式给药。
本发明的化合物可以以其自身给药,或作为含有本发明的化合物结合药物可接受稀释剂,佐药或载体的药物组合物给药。特别优选的是没有含有能够引起不利的,例如变应性反应的物质的组合物。
通过鼻吸入可以给药干燥粉末配方和本发明的化合物的压力化HFA气雾剂。为了吸入,化合物如愿地精细分裂。精细分裂的化合物优选地具有质量中等直径为小于10微米,并且可以悬浮于具有分散抗性的推进剂混合物,如C8-C20脂肪酸或其盐(例如,油酸),胆汁盐,磷脂,烷基多糖,全氟化或多聚乙氧基化表面活性剂,或其它药物可接受分散剂。
通过干燥粉末吸入剂也可以给药本发明的化合物。吸入剂可以是单个或多个剂量吸入剂,并且可以是呼吸驱动的干燥粉末吸入剂。
一种可能性是将精细分裂的化合物与载体物质,例如,单,二聚或多聚糖,糖醇或另一个多元醇混合。适当的载体是糖,例如乳糖,葡萄糖,棉子糖,松三糖,lactitol,maltitol,海藻糖,蔗糖,甘露醇;和淀粉。可替代地,精细分裂的化合物可以通过另一个物质包衣。粉末混合物也可以分散进入硬的明胶胶囊,每个含有活性化合物的需要的剂量。
含有本发明的化合物的药物组合物可以是常规的片剂,丸剂,胶囊,糖浆,粉末或颗粒用于口服给药;无菌肠胃外溶液或悬浮液用于肠胃外给药或栓剂用于直肠给药。
对于口服给药,活性化合物可以与佐药或载体例如,乳糖,多糖,山梨醇,甘露醇,淀粉如土豆淀粉,玉米淀粉或支链淀粉,纤维素衍生物,结合剂如明胶或聚乙烯吡咯烷酮,和润滑剂如硬脂酸镁,硬脂酸钙,聚乙二醇,蜡,石蜡,和诸如此类混合,然后,压缩成片剂。如果需要包衣片剂,如上所述制备的核心可以利用浓缩的蔗糖溶液包衣,蔗糖溶液可以含有例如阿拉伯树胶,明胶,滑石粉,二氧化钛,和诸如此类。可替代地,可以利用溶解于容易挥发的有机溶剂的适当多聚物包衣片剂。
对于制备软明胶胶囊,可以利用例如植物油或聚乙二醇混合化合物。硬的明胶胶囊可能含有利用上面提到的片剂赋形剂例如,乳糖,多糖,山梨醇,甘露醇,淀粉,纤维素衍生物或明胶的化合物的胶囊。同样,可以将药物的液体或半固体配方填充进入硬的明胶胶囊。
用于口服应用的液体制剂可以是糖浆或悬浮液形式,例如含有化合物的溶液的形式,平衡剂是糖和乙醇,水,甘油,丙二醇的混合物。非强制性地,这样的液体制剂可以含有色剂,调味剂,糖精和作为增稠剂的羧甲基纤维素或本领域已知的其它赋形剂。
现在通过下面的实施例解释本发明,这些实施例不应该解释为本发明的限制。
实施例1
利用SMPS350自动合成仪(Zinsser,Frankfurt/Main,德国),利用已知的Fmoc化学合成每个含有15个氨基酸的表1列出的CII肽链(参见Atherton和Sheppard,固相肽合成-一个实验途径,IRL出版社,牛津)。然后,在N末端乙酰化和在C末端酰胺化每个肽。通过HPLC和证实预期的分子量的肽的样品的质谱学评估肽的质量。
表1
肽 | 氨基酸残基 | 肽 | 氨基酸残基 |
110-124 | VKG ESG SPG ENG SPG | 635-649 | FAG PPG ADG QPG AKG |
140-154 | AAG ARG NDG QPG PAG | 641-655 | ADG QPG AKG DQG EAG |
161-175 | GPG FPG APG AKG EAG | 665-679 | PSG APG PQG PTG VTG |
170-184 | APG AKG EAG PTG ARG | 677-691 | PQG PTG VTG PKG ARG |
185-199 | PEG AQG SRG EPG NPG | 701-715 | AAG RVG PPG ANG NPG |
203-217 | PAG ASG NPG TDG IPG | 707-721 | PPG ANG NPG PAG PPG |
209-223 | NPG TDG IPG AKG SAG | 740-754 | RAG DPG LQG PAG APG |
218-232 | AKG SAG APG IAG APG | 749-763 | PAG APG EKG EPG DDG |
224-238 | APG IAG APG FPG PRG | 770-784 | PPG PQG LAG QRG IVG |
230-244 | APG FPG PRG PPG PQG | 779-793 | QPG IVG LPG QPG ERG |
338-352 | LAG PKG ANG DPG RPG | 806-820 | KQG APG ASG DRG PPG |
353-367 | EPG LPG ARG LTG RPG | 821-835 | PVG PPG LTG PAG EPG |
365-379 | RPG DAG PQG KVG PSG | 857-871 | ETG ALG APG APG PPG |
593-607 | PPG PAG ANG EKG EVG | 881-895 | PTG KQG DRG EAG AQG |
614-628 | STG ARG APG EPG ETG |
发现黑体表示的氨基酸部分是核心序列,即比较结构关系最近的肽的结合强度结合最强的氨基酸部分。
实施例2
利用已知的技术通过胃蛋白酶消化从剑突提取小鼠CII,然后进一步通过盐沉淀纯化。利用已知技术再次从Swarm软骨肉瘤纯化小鼠CII。在0.1摩尔/升的乙酸中溶解胶原。通过在56℃温育30分钟变性在再刺激致敏的淋巴结细胞中利用的胶原。
实施例3
为了测试在引流的淋巴结细胞中的增殖应答和细胞因子的释放,利用在CFA(含有H37Ra,Difco,底特律,MI)中乳化的50微克小鼠CII或合成肽在每个后脚垫免疫7-10星期大的雄性(B10.QX DBA/1)F1小鼠。当它们是7-10星期大时,通过利用如实施例2制备的100微克小鼠CII乳化CFA在尾巴的基部免疫小鼠,诱导关节炎。
在5星期后,利用50微克在1∶1重量比例利用IFA(DIFCO,底特律,MI)乳化CII加强免疫的小鼠。然后除去来自这些小鼠的严重关节炎关节引流的淋巴结细胞,并且合并以便在下面的方法中的增殖测试中实施例1中制备的小鼠CII肽的小组中测试反应性。
利用IFN-γ和IL-4小试剂盒(Endogen,剑桥,MA),测试根据实施例1制备的CII肽的小组体外再刺激的从50微升小鼠CII致敏淋巴结细胞的原代培养物的上清液释放的细胞因子。
表2显示了结果。
表2
肽 | SI | IFN-γ释放(ng/ml) | 肽 | SI | IFN-γ释放(ng/ml) |
110-124 | 2.2 | 2.2 | 635-649 | 2.7 | 0.4 |
140-154 | 2.1 | 1.5 | 641-655 | 2.0 | 0.9 |
161-175 | 3.1 | 4.2 | 665-679 | 3.5 | 1.5 |
170-184 | 1.5 | 677-691 | 2.2 | 2.9 | |
185-199 | 4.1 | 1.9 | 701-715 | 18.0 | 2.5 |
203-217 | 3.6 | 6.5 | 707-721 | 19.4 | 5.8 |
209-223 | 5.2 | 740-754 | 2.8 | 2.4 | |
218-232 | 3.9 | 2.3 | 749-763 | 4.8 | 1.0 |
224-238 | 5.0 | 4.4 | 770-784 | 2.7 | 1.4 |
230-244 | 3.9 | 5.8 | 779-793 | 3.9 | |
338-352 | 4.4 | 2.0 | 806-820 | 2.3 | |
353-367 | 3.1 | 1.5 | 821-835 | 1.8 | |
365-379 | 2.8 | 857-871 | 2.4 | 0.3 | |
593-607 | 2.9 | 0.7 | 881-895 | 2.6 | 0.6 |
614-628 | 2.7 | 2.3 |
实施例4
如制造商推荐,除了PBS/BSA缓冲液替代含有2%FCS,5毫摩尔/升EDTA,50微摩尔/升2-ME和10毫摩尔/升的PBS,利用与单克隆小鼠抗小鼠L3T4(CD4)抗体或小鼠抗小鼠B220(CD45R)(两者来自Miltenyi BiotecGmbH,Bergisch Gladbach,德国)偶联的超顺磁微珠,通过磁性活化细胞分拣器(MACS)耗尽来自利用小鼠CII致敏的小鼠的淋巴结细胞的T细胞或B细胞。对于染色的T细胞利用FITC共轭抗小鼠CD3-ε和对于B细胞利用FITC共轭抗小鼠κ轻链(Pharmigen,圣迭戈,CA),在FACScan流动细胞计(BectonDickinson)上分析这些组分。在分离后利用没有血清的DMEM培养基洗涤细胞3次,然后,利用实施例3所述的方法放入增殖测试。以1∶2的重量比例将富集的CD4+T细胞与作为抗原存在细胞的来自同源的小鼠的脾的脾细胞混合。以来自0.84%NH4Cl在pH7.4处理的脾的单个细胞悬浮液的形式利用AP细胞以便溶解红血细胞。
表3
细胞类型 | CPM×10-3 |
未分级 | 13.6 |
CD4+富集 | 21.4 |
CD4+耗尽 | 0.3 |
CD4+耗尽 | 12.5 |
CD4+富集 | 0.4 |
序列表SEQUENCE ID No.1:ESG SPG ENGSEQUENCE ID No.2:PPG ADG QPGSEQUENCE ID No.3:ARG NDG QPGSEQUENCE ID No.4:QPG AKG DQGSEQUENCE ID No.5:APG AKG EAGSEQUENCE ID No.6:PTG VTG PKGSEQUENCE ID No.7:AQG SRG EPGSEQUENCE ID No.8:RVG PPG ANGSEQUENCE ID No.9:PAG ASG NPGSEQUENCE ID No.10:ANG NPG PAGSEQUENCE ID No.11:TDG IPG AKGSEQUENCE ID No.12:DPG LQG PAGSEQUENCE ID No.13:SAG APG IAGSEQUENCE ID No.14:APG EKG EPGSEQUENCE ID No.15:IAG APG FPGSEQUENCE ID No.16:PQG LAG QRGSEQUENCE ID No.17:FPG PRG PPGSEQUENCE ID No.18:PKG ANG DPGSEQUENCE ID No.19:APG ASG DRGSEQUENCE ID No.20:LPG ARG LTGSEQUENCE ID No.21:DAG PQG KVGSEQUENCE ID No.22:ALG APG APGSEQUENCE ID No.23:PAG ANG EKGSEQUENCE ID No.24:KQG DRG EAGSEQUENCE ID No.25:ARG APG EPGSEQUENCE ID No.26:VKG ESG SPG ENG SPGSEQUENCE ID No.27:FAG PPG ADG QPG AKGSEQUENCE ID No.28:AAG ARG NDG QPG PAGSEQUENCE ID No.29:ADG QPG AKG DQG EAGSEQUENCE ID No.30:APG AKG EAG PTG ARGSEQUENCE ID No.31:PQG PTG VTG PKG ARGSEQUENCE ID No.32:PEG AQG SRG EPG NPGSEQUENCE ID No.33:AAG RVG PPG ANG NPGSEQUENCE ID No.34:PAG ASG NPG TDG IPGSEQUENCE ID No.35:PPG ANG NPG PAG PPGSEQUENCE ID No.36:NPG TDG IPG AKG SAGSEQUENCE ID No.37:RAG DPG LQG PAG APGSEQUENCE ID No.38:AKG SAG APG LAG APGSEQUENCE ID No.39:PAG APG EKG EPG DDGSEQUENCE ID No.40:APG IAG APG FPG PRGSEQUENCE ID No.41:PPG PQG LAG QRG IVGSEQUENCE ID No.42:APG FPG PRG PPG PQGSEQUENCE ID No.43:LAG PKG ANG DPG RPGSEQUENCE ID No.44:KQG APG ASG DRG PPGSEQUENCE ID No.45:EPG LPG ARG LTG RPGSEQUENCE ID No.46:RPG DAG PQG KVG PSGSEQUENCE ID No.47:EXG ALG APG APG PPGSEQUENCE ID No.48:PPG PAG ANG EKG EVGSEQUENCE ID No.49:PTG KQG DRG EAG AQGSEQUENCE ID No.50:STG ARG APG EPG ETGSEQUENCE ID No.51:AAG RVG PPG ANG NPGSEQUENCE ID No.52:PPG ANG NPG PAG PPG
Claims (19)
1.具有通式(I)的氨基酸序列的分离的肽:
A1-Xaa-Gly-A4-A5-Gly-A7-Xaa-Gly(I)
其中;
A1代表具有芳香或脂肪侧链的氨基酸残基,
A4代表天冬酰胺或精氨酸残基或具有芳香或脂肪侧链的氨基酸残基,
A5代表任何天然存在的氨基酸,
A7代表具有带负电的残基的氨基酸,
Xaa代表任何氨基酸残基,和
Gly代表甘氨酸残基。
2.含有权利要求1中要求保护的任何肽的氨基酸序列的分离的肽。
3.根据权利要求2中的肽,含有10和15个氨基酸。
4.根据权利要求1中所述的肽,其中A5代表赖氨酸或精氨酸残基并且A7代表谷氨酸残基。
5.具有下面的序列的任何一个的分离的肽:
ESG SPG ENG,
PPG ADG QPG,
ARG NDG QPG,
QPG AKG DQG,
APG AKG EAG,PTG VTG PKG,AQG SRG EPG,RVG PPG ANG,PAG ASG NPG,ANG NPG PAG,TDG IPG AKG,DPG LQG PAG,SAG APG IAG,APG EKG EPG,IAG APG FPG,PQG LAG QRG,FPG PRG PPG,PKG ANG DPG,APG ASG DRG,LPG ARG LTG,DAG PQG KVG,ALG APG APG,PAG ANG EKG,KQG DRG EAG,或ARG APG EPG(分别是SEQ ID No1至25)
6.含有权利要求3中的任何一个肽的氨基酸序列的分离的肽。
7.具有下面的序列的任何一个的分离的肽。
VKG ESG SPG ENG SPG;
FAG PPG ADG QPG AKG;
AAG ARG NDG QPG PAG;
ADG QPG AKG DQG EAG;
APG AKG EAG PTG ARG;
PQG PTG VTG PKG ARG;
PEG AQG SRG EPG NPG;
AAG RVG PPG ANG NPG;
PAG ASG NPG TDG IPG;
PPG ANG NPG PAG PPG;
NPG TDG IPG AKG SAG;
RAG DPG LQG PAG APG;
AKG SAG APG IAG APG;
PAG APG EKG EPG DDG;
APG IAG APG FPG PRG;
PPG PQG LAG QRG IVG;
APG FPG PRG PPG PQG;
LAG PKG ANG DPG RPG;
KQG APG ASG DRG PPG;
EPG LPG ARG LTG RPG;
RPG DAG PQG KVG PSG;
ETG ALG APG APG PPG;
PPG PAG ANG EKG EVG;
PTG KQG DRG EAG AQG;或
STG ARG APG EPG ETG(分别是SEQ ID No26至52)
8.权利要求7中要求保护的任何一个肽的10到14个氨基酸片断的分离的肽。
9.根据前面的权利要求的任何一个所述的肽,其结合到在IFN-γ释放测试中至少具有2的结合长度的T细胞上。
10.根据前面权利要求的任何一个的肽,其结合到具有刺激指标至少3的T细胞上。
11.根据前面的权利要求所述的任何一个的肽,其中肽的序列存在于位置110-239,338-379或587-895之间的胶原II的序列中。
12.根据权利要求11所述的肽,其中肽序列存在于胶原II的701-721区。
13.根据前面的权利要求的任何一个所述的分离的肽,用于医疗。
14.根据权利要求13所述的肽,其中医学治疗包括耐受性的诱导。
15.根据前面的权利要求的任何一个所述的分离的肽在制造可用于自身免疫病的治疗中的药剂的用途。
16.根据权利要求10所述的用途,其中自身免疫病是类风湿关节炎。
17.含有根据权利要求1到12的任何一个所述的肽的药物组合物,其结合有药物可接受载体或稀释剂。
18.治疗患有或有倾向患有自身免疫病的人或动物的方法,该方法包括给人或动物施用治疗有效量的根据权利要求1到12的任何一个的肽或根据权利要求17的组合物。
19.包括特异于胶原II的T-细胞表位的肽,该肽至少包括相似于和连续性地选自胶原序列的110-239,338-379或587-895部分的序列的9个氨基酸,其中每个氨基酸非强制性地利用功能等价的氨基酸替代,并且其中肽的通式为
A1-Xaa-Gly-A4-A5-Gly-A7-Xaa-Gly (I)
其中;
A1代表具有芳香或脂肪侧链的氨基酸残基,
A4代表天冬酰胺或精氨酸残基或具有芳香或脂肪侧链的氨基酸残基,
A5代表任何天然存在的氨基酸,
A7代表具有带负电的残基的氨基酸,
Xaa代表任何氨基酸残基,和
Gly代表甘氨酸残基。
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CN1169829C (zh) * | 2002-06-27 | 2004-10-06 | 北京大学人民医院 | 非t细胞结合肽及其用途 |
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IS5138A (is) | 1999-07-28 |
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