CN101229370A - 自体t-细胞疫苗材料和方法 - Google Patents
自体t-细胞疫苗材料和方法 Download PDFInfo
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- CN101229370A CN101229370A CNA2007101469717A CN200710146971A CN101229370A CN 101229370 A CN101229370 A CN 101229370A CN A2007101469717 A CNA2007101469717 A CN A2007101469717A CN 200710146971 A CN200710146971 A CN 200710146971A CN 101229370 A CN101229370 A CN 101229370A
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Abstract
本发明涉及自体T-细胞疫苗材料和方法。具体地,本发明涉及包含T细胞的组合物,其中所述T细胞对髓磷脂碱性蛋白表位有反应,并且所述髓磷脂碱性蛋白表位由髓磷脂碱性蛋白的氨基酸83-99和氨基酸151-170组成。
Description
本申请是申请日为2002年9月12日的中国专利申请02817672.3“自体T-细胞疫苗材料和方法”的分案申请。
技术领域
本发明涉及生产自体T细胞疫苗的方法,通过那些方法生产的T细胞疫苗以及使用那些疫苗治疗T细胞相关疾病的方法。
背景技术
越来越多的证据表明,自体免疫T细胞可应答涉及多发性硬化(MS)致病机理的髓磷脂抗原,包括髓磷脂碱性蛋白(MBP)(Stinissen等,Crit.Rev.Immunol.1997,17:33-75)。人们发现MBP-反应性T细胞经过体内活化,以较高的前体出现率出现在MS患者的血液和脑脊液中(Zhang等,J.Exp.Med.1994,179:973-984;Chou等,J.Neuroimmunol.1992,38:105-114;Allegretta等,Science,1990,247:718-721)。这些MBP-反应性T细胞可产生促炎Th1细胞因子(IL-2,TNF-α和γ-干扰素),并认为它们可促进中枢神经系统中的髓磷脂-破坏性炎症(Sharief等,N.Eng l.J.Med.1991,325:467-472;Selmaj等,J.Clin.Inves t.1991,87:949-954)。已经显示MBP-反应性T细胞可诱导实验性自体免疫性脑脊髓炎(EAE),一种MS的动物模型(Ben-Nun等,Eur.J.Immunol.1981,11:195-204)。EAE还可通过反复接种MBP-反应性T细胞而预防或治疗,其中的MBP-反应性T细胞已经通过化学处理或辐射而灭活,这一治疗过程被称作T细胞接种(Ben-Nun等,Nature,1981,292:60-61)。已经证实,T细胞接种可诱导由抗-独特型T细胞和抗-ergotypic T细胞构成的调节型免疫应答,有助于对EAE和其它实验性自体免疫疾病模型的治疗作用(Lider等,Science,1988,239:820-822;Lohse等,Science,1989,244:820-822)。
近来,T细胞接种已经发展用于MS患者的临床试验,它是以减少MBP-反应性T细胞可改善疾病临床进程的假设为基础的。在先导临床试验中,我们证实了接种辐射过的自体MBP-反应性T细胞克隆可引发CD8+细胞溶解性T细胞应答,而该应答可特异性识别并溶解用于接种的MBP-反应性T细胞(Zhang等,Science.1993,261:1451-1454,Medear等,Lancet 1995,346:807-808)。辐射过的MBP-反应性T细胞克隆的三次皮下接种可减少MS患者中的循环MBP-反应性T细胞。由T细胞接种导致的MBP-反应性T细胞的减少似乎与临床改善相关,这是由复发率、扩大残疾等级评分(EDSS),和缓解-复发患者中MRI病变活动度的降低证实的(Medaer等,1995)。虽然由于所研究的MS患者数有限而不能从先导试验得出结论,但是极好的安全性分布和潜在的临床益处可鼓励进一步的临床研究。进行初步临床试验是为了研究循环MBP-反应性T细胞的减少在临床上是否对MS患者有益。
发明内容
本发明涉及生产自体T细胞疫苗的方法,通过那些方法生产的T细胞疫苗以及使用那些疫苗治疗T细胞相关疾病的方法。本发明一方面涉及自体T细胞疫苗的生产和那些疫苗用于治疗多发性硬化的用途。本发明另一方面涉及使用T细胞疫苗治疗类风湿性关节炎。
另一方面,本发明包括一种自体T细胞疫苗。
本发明的优选实施方案包括利用称作直接扩展法(ditectexpansion method,DEM)的方法制备的自体T细胞疫苗,该方法可提供一种更快速,更简便,而且更经济的制备T细胞疫苗的方法。当鉴定T细胞对髓磷脂蛋白或其片段有反应,并且具有5或更高刺激指数(S.I.)时,直接扩展法是生产疫苗的优选方法。直接扩展法包括从将要进行治疗的MS患者获得外周血单核细胞(PBMC)或从患者的脑脊液获得单核细胞(CSFMC)。然后在有多发性硬化相关抗原,如髓磷脂碱性蛋白(MBP)或MBP的一个或多个免疫原性片段存在的条件下,温育从患者获得的PBMC或CSFMC。可用于本发明的其它多发性硬化相关抗原包括髓磷脂蛋白脂质脂蛋白,髓磷脂少突神经胶质细胞糖蛋白和glatiramer,及其片段。在更优选的实施方案中,MBP的免疫原性片段是免疫显性片段。最优选的MBP片段包括相应于MBP的氨基酸83-99的片段,和相应于MBP的氨基酸151-170的片段。在本发明其它实施方案中,还可在没有多发性硬化相关抗原和/或其片段存在的条件下温育细胞。用MBP或其片段温育后,在有抗原呈递细胞(APC)存在的条件下,用MBP和/或其片段再次温育PBMC或CSFMC。用于本发明的优选抗原呈递细胞包括从患者获得的辐射过的PBMC。然后使用促分裂原,优选植物凝集素和IL-2使处理过的细胞经过交替的刺激循环。用于本发明过程中的其它促分裂原分子包括但不限制于伴刀豆球蛋白A和美洲商陆促分裂原。用于本发明中的其它促分裂原分子包括T细胞表面受体的抗体,如CD3的单克隆抗体。交替的刺激循环可重复一次或多次。
本发明还涉及使用自体T细胞疫苗治疗MS的方法。该方法包括给予需要的患者有效剂量的自体T细胞疫苗。优选剂量约含40×106-80×106个细胞。所述疫苗可经由任何一种给药途径给药,包括但不限制于静脉内,肌内,腹膜内,真皮内和皮下。皮下注射是优选的疫苗给药途径。本发明范围内的有效剂量是导致患者循环中髓磷脂反应性T细胞数或前体出现率下降所需的剂量。有效的其它指标包括利用众所周知的标准测量的疾病临床原因的改变,包括EDSS降低,防止EDSS增加,或延迟EDSS进展。有效性的其它指标包括临床恶化率的降低,通过由MRI或其它诊断方法学检测的脑病变大小的稳定或减少。
同样,本发明还包括使用按照这里所述制备的T细胞疫苗治疗类风湿性关节炎的方法。
本发明的其它实施方案提供一种自体T细胞疫苗和通过“克隆法”生产疫苗的方法。当鉴定T细胞对髓磷脂碱性蛋白或其片段有反应,并具有低于5的刺激指数时,优选克隆法。
所述克隆法包括鉴定对MBP或髓磷脂蛋白脂质脂蛋白,髓磷脂少突神经胶质细胞糖蛋白,glatiramer和/或上述任何一种的片段有反应的T细胞系。S.I.低于5的T细胞系是通过有限稀释克隆的。该方法包括在不更换培养基的条件下,用MBP或其片段(优选与氨基酸83-99和151-170相应的片段)温育PBMC或CSFMC7小时而获得可与MBP和/或其片段反应的T细胞。将所有孔的大约50%等分成两组孔(抗原孔和对照孔)。将两组孔中的细胞在含有5%v/v人AB+血清的培养基中用APC(辐射的新鲜或融解的PBMC)温育,而抗原孔则按照上面所述接受MBP或其片段。刺激指数(S.I.)是按照这里所述,使用[3H]胸苷掺入增殖测定法测定的。然后利用有限稀释克隆含有抗原且S.I.小于5的孔,其中将对T细胞系有反应的细胞混合,稀释,并以约0.3-20个细胞/孔的密度接种在孔中的培养基中,培养基含有10%人AB+血清,和白介素,优选白介素2和凝集素,优选植物凝集素(PHA),并含有APC。每3-4天,使用含IL-2的培养基更换培养基。大约14天后,按照上面所述再次测试细胞的S.I.然后使用MBP(或其片段)和PHA,利用交替刺激循环使细胞扩展。
本发明还涉及一种用于治疗其它T细胞相关疾病,如类风湿性关节炎的自体T细胞疫苗。这种T细胞疫苗的制备和使用与上述用于治疗MS的自体T细胞疫苗的制备和使用相似。然而,T细胞的最初来源是类风湿关节炎患者的滑膜液。与MS疫苗制品不同的是,来源于滑膜液的T细胞是由PHA刺激的;CD3的单克隆抗体或其它促分裂原没有经过MS相关抗原的刺激。
附图说明
图1说明接种前后,循环MBP-反应性T细胞的估计前体出现率的改变。前体出现率是在进行接种方案前和完成接种方案后2-3个月,在所有患者中评估的。
具体实施方式
虽然MBP-反应性T细胞经过体内活化及克隆扩展,并在给定个体中表达限制性T细胞受体V基因,但是MBP-反应性T细胞的T细胞受体是高度多样性的,并因不同的MS患者而不同(Vandevyver等,Eur.J.Immunol.1995,25:958-968;Wucherpfennig等,J.Immunol.1994,152:5581-5592;Hong等,J.Immunol.1999,163:3530-3538)。因此,目前有效减少MS患者中MBP-反应性T细胞的策略需要治疗个体化。本发明可提供这种个体化的治疗,并考虑到了个体患者内T细胞的多样性,以便提供效果更持久的疫苗。
与先前的研究(Zhang等,J.Immunol.1993,164:4011-4017;Medaer等,1995)相一致,这里的数据证实了接种自体MBP-反应性T细胞可为患者提供始终如一、且强效的免疫方法,以减少循环MBP-反应性T细胞。虽然还不完全了解由T细胞接种诱导的免疫调节机理,但仍可清楚看出T细胞接种可作用于多种调节网,从而诱导CD8+抗-独特型T细胞应答(Zhang等,1993;Zhang等,1995)和Th2免疫偏离(Zhang等,2000)。特别是由T细胞接种诱导的这些抗-独特型T细胞可在T细胞可变区的识别和T细胞受体可变区的识别中溶解免疫T细胞,其代表使MBP-反应性T细胞减少的显性免疫调节(Zhang等,2000)。可以理解,由T细胞接种诱导的这些调节反应有助于T细胞接种在MS中的有益作用。
虽有间接证据表明髓磷脂-反应性T细胞与MS疾病过程的潜在关联(Zhang等,1994;Chou等,1992;Allegretta等,1990),但很难确定或否认髓磷脂-反应性T细胞在MS发病机理中的作用。就这点而论,T细胞接种可为评估减少髓磷脂-反应性T细胞是否对MS临床进程产生有益的影响提供独特的机会。
这里所述的实施例描述了利用克隆选择法制备的自体T细胞疫苗用于治疗MS的用途以及利用直接扩展法制备的自体T细胞疫苗。这里提供的数据可表明T细胞接种与临床指标改善的有益关联。首先,该结果表明,与MS自然史相比,减少MBP-反应性T细胞与缓解-复发和SP-MS队列中进展时间的延迟相一致,并且通过所需扩展法制备了自体T细胞疫苗。然而,应当注意的是,在最后一次注射12个月后,在某些患者中观察到加速进展的趋势。这种明显加速进展的意义还未可知,但它可能与由针对MBP-反应性T细胞的T细胞接种最初诱导的免疫性逐渐下降有关。事实上,在大约10-12%的免疫患者中,MBP-反应性T细胞都在那一时间左右再现,从而支持了这种可能性。在某些病例中,再现的MBP-反应性T细胞来源于不同的克隆群,接种之前不能检测到,这些还可在先前的研究中观察到(Zhang等,1995)。结果表明,MBP-反应性T细胞经过可能与进行性疾病过程有关的克隆转变或表位扩散(Touhy等,J.Exp.Med.1999,189:1033)。该观察结果表明,额外的加强注射可能是相同或新出现的T细胞克隆维持足够免疫性所必需的。还表明它可用于提供多克隆来源的T细胞疫苗,如通过这里所述的直接扩展法提供,从而避免与克隆转变或表位扩散有关的问题,这是因为可由这种疫苗识别的表位的专有阵列较之由克隆群识别的阵列更大。
使用本发明T细胞疫苗治疗的患者每年的MRI检查显示,第一年的MRI病变活动度稍稍降低,第二年仅增加了3.3%。MRI结果表明,使用T细胞接种治疗的患者明显更稳定。MRI结果与进展时间最初延迟,然后在第2年显著加速相一致,从而加强了T细胞接种的最初作用在第2年消失的可能性。
本发明的方法还可导致其它临床指标,包括接种患者每年的复发率和EDSS的有利改变,表明T细胞接种对MS临床进程的有益作用。研究结果在很大程度上与在先导临床试验中报道的结果相一致(Medaer等,1995)。然而,与其它临床指标相反,在两个研究组中,T细胞接种对通过EDSS测量的临床残疾的影响都非常小。它可反映EDSS在相对较短的时间(24个月)内,缺少对测量改变的灵敏性。这种可能性甚至在自体免疫成分被T细胞接种除去或抑制后仍然存在,炎性病变仍然要用较长的时间来消除,某些现有的组织病变将是永久性的。鉴于这些结果,本发明提供用于治疗MS的自体T细胞疫苗以及使用疫苗治疗MS的方法。
应当指出的是,将这里报道的临床结果与患者自体的治疗前状态以及在先前MS试验中证明的MS自然史的评估进行比较,但没有安慰剂对照。该研究也受到与研究的开放式-标记临床设计有关的潜在安慰剂作用的限制。因此,虽然研究提供了有利于T细胞接种在MS中作用的重要临床指标,但是T细胞接种的治疗效果还是在双盲和安慰剂对照的临床试验中可得到最好评估。
本发明还提供用于制备自体T细胞疫苗的新方法,其比早期的T细胞疫苗更容易制备,并可提供细胞的异源群(非-克隆),它们共同发挥作用,在患者中提供增强的免疫应答,避免与表位扩散或克隆转换有关的潜在问题,而且该方法可被设计更好地除去与疾病有关的T细胞的较大多样性。
实施例1
测定MS患者血液中MBP-反应性T细胞的出现率
MBP-反应性T细胞在MS患者血液中的出现率是使用Zhang等,1994;Zhang等,1993;Medaer等,1995(每篇参考文献都引入此处作为参考)描述的方法测定的。在每个病例中,用于细胞加工和细胞培养的物质是严格自体的。外周血单核细胞(PBMC)是通过标准Ficoll梯度分离从肝素化的静脉血制备的。在有浓度为20μg/ml、分别与两个免疫显性区(氨基酸残基83-99和151-170,Tejada-Simon等,Eur.J.Immunol.2001,Mar;31(3)907-917)对应的人髓磷脂碱性蛋白的两个合成肽存在的条件下,以200,000个细胞/孔(共96个孔)将PBMC铺板在补充了10%热灭活自体血清和50IU/ml重组白介素-2(IL-2)的RPMI 1640(Hyclone,Logan,Utah)中。温育是在37℃时进行的。7天后,用自体脉冲辐射过的PBMC(冷冻或新鲜)再次刺激所有培养物。PBMC的脉冲是通过37℃时,温育100μg/ml浓度的PBMC各肽3小时,然后在使用前用60CO源以4,000拉德辐射而进行的。再温育1周后,在下述增殖测定法中检测应答MBP肽的各培养物的特异性增殖。
简言之,将每个孔分成4份等分试样(每份等分试样大约104个细胞),并在有和没有(对照)上述MBP肽存在的条件下,与105个自体脉冲的辐射过的PBMC一式两份进行培养。将培养物温育3天,并在培养的最后16个小时内,以1μCi/孔用[3H]-胸苷(Amersham,ArlingtonHeights,IL)脉冲。然后使用自动细胞采集器采集细胞,在β平板计数器中测量掺入的[3H]-胸苷。当掺入到细胞中的[3H]-胸苷的每分钟计数大于1,500,并超过对照组的每分钟计数至少3倍时,确定细胞对MBP肽是反应性的。然后通过用显示反应性的孔数除以接种在起始培养物中的总PBMC数(19.2×106个细胞)而确定MBP-反应性T细胞的出现率(见,例如,Zhang等,1994;Zhang等,1993;Medaer等,1995)。使用相同的计算方法比较研究过程中MBP-反应性T细胞出现率的改变。
如图1所示,在这些MS患者中检测的循环MBP-反应性T细胞的出现率约为14×10-5,与由Zhang等,(1994),和Ota等,Nature,346:183-187(1990)(还可见实施例5)报道的约10×10-5的出现率相似。
实施例2
用于T细胞接种的髓磷脂-反应性T细胞的产生
PBMC的制备和最初刺激
在收集的2小时内加工新鲜的血液样品。可选择性地,单核细胞可从MS患者的脑脊液(CSFMC)获得。外周血单核细胞(PBMC)是利用标准Ficoll梯度分离法从全血分离的。具体而言,使用Hanks平衡盐溶液(HBSS)(1∶1血液/HBSS)稀释肝素化的血液,然后缓慢覆盖在离心管中的Ficoll-hypaque溶液上,18℃-25℃、1800rpm不间断离心20分钟。通过加入过量的HBSS并于18℃-25℃、1700rpm离心10分钟而清洗PBMS。纯化的PBMC是通过离心,随后重悬浮在AIM V培养基(Gibco,Grand Island,N.Y.)中,而在RPMI 1640培养基中清洗3次。计算细胞数,然后以200,000个细胞/孔的浓度将细胞铺板在96-孔U形底培养平板上。所有平板都用患者号和患者姓名的首字母标记的。分别以20μg/ml,将实施例1中讨论的髓磷脂肽加入到培养物中。将平板放在CO2温育箱中,每天目测检查。在不更换培养基的条件下,培养细胞7天,从而选择性生长肽-特异性T细胞。
MBP肽-特异性T细胞系的鉴定和选择
除去所有孔中大约50%的细胞,等分在两个孔(抗原和对照孔)中。以8,000拉德(使用60Co源)照射新鲜或融解的PBMC,作为抗原呈递细胞(APC)的来源,以100,000个细胞/孔使用。将细胞在含有5%人AB+血清的RPMI 1640中培养。分别以20μg/ml将实施例1中所述的髓磷脂肽加入到抗原孔中。将没有髓磷脂肽的培养基加入到成对的对照孔中。可选择性地,还可使用其它多发性硬化相关抗原,即,髓磷脂抗原和/或其片段,包括由Markovic-Plese等,J.Immunol.(1995),982-992(蛋白脂质蛋白表位);Genain等,J.Clin.Invest.(1995),2966-2974;Kerlero de Rosbo等,J.Clin.Invest.(1993)92:2602-2608;Trotter等,J.Neuroimmunol.(1998)84:172-178和Trotter等,J.Neuroimmunol.(1997)75:95(髓磷脂蛋白脂质蛋白);Linder等,Brain,(1999)122:2089(髓磷脂少突神经胶质细胞糖蛋白);和Johnson等,Neurol.(1995)45:1264(glatiramer[共聚物1])描述的那些。本发明还考虑了前述抗原和/或其片段组合的用途。
然后使用自动细胞采集器采集细胞,并在β-平板计数器中测量掺入的[3H]胸苷。各T细胞系/孔对相应髓磷脂肽的反应性是利用[3H]胸苷掺入增殖测定法测定的。具体而言,将每个孔的细胞分成4份等分试样(约104个细胞/等分试样),并在有和没有髓磷脂肽存在的条件下,一式两份,与作为APC来源的105个辐射过的自体PBMC一起培养。将培养物温育3天,在培养的最后16个小时内,以1μCi/孔用[3H]-胸苷(Amersham,Arlington Heights,IL)脉冲。当抗原孔每分钟计数(cpm)与对照孔cpm的商大于或等于3;且抗原孔的总cpm大于1,500时,可将T细胞系确定为髓磷脂肽-特异性的。髓磷脂-反应性T细胞的出现率是按照泊松统计学测定的。使用辐射过的PBMC再次刺激剩余50%已经鉴定过的髓磷脂-反应性T细胞,进行扩展。
所选T细胞系/克隆的扩展和建立
在鉴定T细胞系是髓磷脂肽反应性的,并且随后再次刺激一次后,可使用下列之一的方法:直接扩展法和T克隆法使它进一步繁殖,从而产生用于接种的足量细胞。繁殖法的选择取决于T细胞系对髓磷脂肽的特异性和反应性。这些性质是利用刺激指数(SI)测量的,该指数是根据上述[3H]胸苷掺入增殖测定法的结果计算的。SI是抗原孔每分钟计数(cpm)/对照孔cpm的商。当SI等于或高于5时,使用直接扩展法。当SI低于5时,使用克隆法。
直接扩展法
简言之,经鉴定S.I.等于或高于5的髓磷脂反应性T细胞是通过直接扩展法(DEM),在有辐射过的自体PBMC存在的条件下,使用相应的髓磷脂肽和PHA进行交替刺激循环而扩展的。每个刺激循环进行7-10天。更具体而言,在有辐射过的PBMC(APC)(100,000个细胞/孔)存在的条件下,以20,000-40,000个细胞/孔对上述鉴定的髓磷脂反应性T细胞铺板。对于抗原刺激循环而言,加入20μg/ml的相应髓磷脂肽,对于每次的PHA刺激循环而言,加入1μg/ml的PHA。在刺激循环的第2天,加入100IU/ml的重组人IL-2。每3-4天,用含有10%人AB+血清和100IU/ml rIL-2的RPMI 1640培养基更新培养物。髓磷脂反应性T细胞系在交替刺激循环中繁殖,直到总细胞数约达到2千万。
利用DEM制备的T细胞系的反应性
T细胞系 | 抗原 | 扩展次数 | CPM Ag/对照组 | S.I. | 细胞数(106) |
3E5 | MBP83-99 | 0 | 2,399/410 | 5.8 | 0.2 |
MBP8 3-99 | 1 | 6,991/2,021 | 3.4 | 3.4 | |
PHA | 2 | 5,804/1,266 | 4.5 | 23.5 | |
2C9 | MBP83-99 | 0 | 4,421/312 | 14 | 0.16 |
MBP83-99 | 1 | 8,220/1,882 | 4.3 | 4.2 | |
PHA | 2 | 10,221/3,142 | 3.2 | 21.4 |
在克隆法中,使用有限稀释测定法克隆T细胞系。将每种髓磷脂肽反应性T细胞系的细胞合并,以大约0.3-20个细胞/孔接种在含有10%人AB+血清和100IU/mL rIL-2的RPMI 1640培养基中。加入1μg/ml的PHA,然后以100,000个细胞/孔加入辐射过的自体APC。每3-4天更换含有rIL-2的RMPI 1640培养基。培养14天后,测定生长-阳性孔,从而确定它们对上述相应髓磷脂肽的特异反应性。这些肽-特异性T细胞系的进一步扩展是通过下列直接扩展法进行的,其中使用相应的髓磷脂肽和PHA进行交替刺激循环。
实施例3
T细胞接种可减少MBP-反应性T细胞
征募54名RR-MS(n=28)和SP-MS(n=26)患者进行此项开放标记研究。患者的基线临床特征在表1中给出。每位患者接受3个疗程的辐射过的自体MBP-反应性T细胞克隆(通过克隆法制备)的皮下注射,每个疗程间隔2个月。在24个月内,监测患者中MBP-反应性T细胞的前体出现率,复发率,EDSS和MRI病变活动度。以自我配对的方式,将结果与预接种值进行比较。此外,还包括β-干扰素-1a临床试验(Jacobs等,1996)中的RR-MS的安慰剂和近来β-干扰素-1b研究(European Study Group,Lancet,352:1491-1497(1998))中的SP-MS的临床数据,从而提供MS的自然史数据用于比较。在研究中描述的安慰剂对照组被试者的基线特性与这里研究的患者群体的那些相似,区别在于平均EDSS较低。
如图1所示和实施例1所述,与先前研究中报道的相比(在外周血单核细胞中约为10×10-5)(Zhang等,1994;Ota等,1990),在这些MS患者中检测的基线循环MBP-反应性T细胞的前体出现率较高(14×10-5)。在RR-MS和SP-MS队列之间没有发现MBP-反应性T细胞前体出现率的显著性差异。在完成3个疗程接种后的2-3个月,T细胞出现率在92%的患者中检测不到,或在剩余的患者中显著降低(14×10-5与1.9×10-5,p<0.0001)。结果证实了给MS患者进行T细胞接种可减少MBP-反应性T细胞。
实施例4
使用自体MBP-反应性T细胞给MS患者接种
在本试验中,有54名MS患者加入。纳入标准是临床上明确患有MS至少两年,RR-MS的基线扩大残疾等级评分(EDSS)为1.5-6.5,继发的进展性MS(SP-MS)患者为4.0-8.0,以及对于缓解一复发MS(RR-MS)队列,在进行研究之前的前两年中有至少1次恶化。大约有25%的患者先前不能应答或耐受使用β-干扰素或glatiramer进行的治疗,其余患者在进入并完成研究前至少1个月,没有用这些药剂治疗。患者在参与研究前至少3个月,没有服用任何免疫抑制药物,包括类固醇。如果出现恶化,那么在研究过程中允许使用类固醇。没有禁止疲劳,强直和膀胱病的对症治疗。在解释了实验过程之后,获得患者的知情同意。该方法由Institutional Human Subject Committeeat Baylor College of Medicine批准。
接种方案与先前临床研究中所用的相似(Zhahg等,1993,Medaer等,1995)。简言之,在有作为佐细胞源的辐射过的PBMC存在的条件下,使用植物凝集素(PHA)(1μg/ml)将通过上述克隆法制备的MBP-反应性T细胞预活化。然后,将细胞在补充了10%热灭活自体血清和50个单位rIL-2的RPMI 1640培养基中培养5-6天。随后,用无菌盐水清洗活化的MBP-反应性T细胞3次,除去残留的PHA和细胞碎屑。照射(8,000拉德,60Co源)后,将细胞重悬浮在2ml盐水中,在两臂上进行皮下注射(1ml/臂)。用于接种的T细胞数为每次注射40×106-80×106个细胞,这是以相对皮肤表面积为基础,根据对实验动物有效的T细胞剂量外推而选择(Ben-Nun等,1981)的。每位患者以两个月的间隔接受3次皮下注射。
然后,观察患者残疾确实进展的开始,EDSS,复发率和MRI病变活动度。将结果与患者自体的治疗前过程以及RR-MS和SP-MS患者中,最近两次临床试验注射了安慰剂的手臂进行比较,其中注射了安慰剂的手臂可作为MS自然史的评估(Jacobs等,1996),European StudyGroup,1998)。进展时间是通过EDSS增加至少1.0的情况(Poser等,1983)持续至少2个月而确定的。研究过程中出现的恶化是通过新的神经症状的出现或现有神经症状恶化至少持续48小时,同时伴有神经检查的客观改变(EDSS恶化至少0.5分)而确定的。指导患者报告有规律观察之间的事件,如果症状暗示恶化,则由神经病学家进行检查。安全性评估包括副作用,生命体征和有规律的体检。在T细胞接种前后,所研究患者临床指标的差异是使用Wilcoxon’s秩和检验分析的。
实施例5
接种后MS临床进程的改变
进行实验以确定通过T细胞接种减少循环MBP-反应性T细胞是否能够改变MS的临床进程。患者接受按照上面所述制备的自体T细胞疫苗。注射后,除了在某些患者注射位置见到的轻度和瞬时红斑外,没有与T细胞接种有关的副作用,且所有患者都是在门诊治疗的。如表2所示,RR-MS患者的平均EDSS在接种24个月的时间中稍稍降低(入组时为3.21出组时为3.1)。通过比较可知,在相同的观察期内,RR-MS自然史(n=56)的平均EDSS增加了0.61,正如在使用β-IFN-1a进行的试验中所报道的(Jacobs等,1996)。此外,EDSS未改变或提高的患者比例明显比自然MS史的要高(75%与50%)。在治疗过的RR-MS组中,只有1名患者(3.5%)在24个月内进展至EDSS超过2.0,而在MS自然史患者中为18%(表2)。
在SP-MS队列中,24个月内的平均EDSS稍稍增加(+0.12),而在SP-MS自然史中的记录为+0.6(European Study Group,Lancet1998;352:1491-1497)。此外,与MS自然史患者相比(RR-MS在12个月内,SP-MS在9个月内增加了20%),使用Kaplan-Meier方法评估的确定进展时间明显延迟(两个治疗组都在18个月内增加了20%)(Jacobs等,Ann.Neurol,1996;39:285-294,European StudyGroup,1998)。然而,两个研究组的进展似乎都在18个月(最后一次接种后12个月)后加速。
实施例6
临床恶化率的改变
如表3所示,T细胞接种后,RR-MS患者的年复发率下降,比基线复发率降低了40%。试验第1年和第2年间的复发率没有显著差异。有比较可知,在RR-MS自然史中观察到的年复发率降低了25%(Jacobs等,1996)。此外,不发作或较少发作患者的比例明显比MS自然史要高(表3)。虽然SP-MS队列的复发率降低了50%,但在进入研究前的2年内,只有少数继发的进展性患者复发(6/26)。
实施例7
利用磁共振成像检查脑病变活动度
磁共振成像(MRI)是根据钆-增强的T2加权像进行的。以半定量方式记录信号强度较高的区域(Scheltens等,Brain 1992;115:735-748;Truyen等,J.Neurol.Sci.1990;96:173-182)。这种记录方法可产生与信号高强度增加病灶的大小和数量有关的评分。信号高强度是在下列区中记录的:(i)室周,在额及枕区中,并与侧脑室平行;(ii)叶白质,分别在额,颞、顶和枕区;(iii)基底神经节,尾状核,豆状核,苍白球和丘脑;以及(iv)幕下区,小脑,中脑,脑桥和髓质。病变如下记录:病变的直径小于0.5cm时记录为‘1’,0.5cm-1.0cm记录为‘2’,1.0cm-1.5cm记录为‘3’,1.5cm-2.0cm记录为‘4’,大于2.0cm记录为‘5’。汇合病变是如下测量的:当少于25%的上述目标区被认为信号强度异常时,记录为‘5’,当超过50%的目标可见区受到影响时,分别将25%和50%记录为‘10’和‘15’。然后将这些值加到‘各个’病变评分中。
三次钆-增强的T2加权MRI检查是在入组(基线),12个月和出组(24个月)时进行的,从而监测作为疾病进展指数的脑病变活动度的改变。由于在不同医疗中心进行的某些扫描的技术不相容性,只能分析34名患者的MRI扫描。所有MRI扫描是由外来的、没有参与临床试验的神经放射学家评估的。使用先前在我们的先导临床试验中所用的半定量评分法和其它相关研究来评估病变活动度(Medaer等,1995;Scheltens等,1992;Truyen等,1990)。这种评分法可产生与T2加权像的信号高强度增加病灶的大小和数量有关的评分。如表4所示,结果显示,有70%受检患者的MRI病变评分没有改变或者提高,这是由病变评分至少降低1分确定的,而剩下30%患者的病变评分在研究过程中增加。作为一组平均MRI病变评分的改变在第1年降低了1.2%,第2年比基线MRI增加了3.3%。然而改变并不显著(p>0.4)。结果反映出可归因于T细胞接种的稳定或某些改善,这是因为正如在先前临床试验中证实的那样,在没有进行治疗的RR-MS患者中,通常每年大约有10%的MRI病变进一步发展(European Study Group,1998,IFNB Multiple Sclerosis Study Group,Neurol.1993:43:655-661)。综合考虑,结果表明T细胞接种所致MBP-反应性T细胞的减少与所检查MS患者临床改善的有利关联。
本发明已经通过非限制性实施例和优选实施方案描述,它们不是对本发明范围的限制,本发明的范围在权利要求书中提出。
表1
患者治疗前的临床特性
患者组 | #病例数 | 平均年龄 | 男/女 | 持续时间(年) | 入组时的EDSS | 复发率 |
研究组RR-MSSP-MS | 2826 | 45±9.749±8.1 | 13/1510/16 | 7.4±7.315.5±9.3 | 3.2±2.16.1±0.9 | 1.25 |
MS的自然史RR-MSaSP-MSb | 143358 | 36.9±0.0540.9±7.2 | 40/103128/230 | 6.4±0.513.4±7.5 | 2.3±0.075.2±1.1 | 1.2 |
aβ-IFN-1a试验[7]的安慰剂对照组 bβ-IFN-1b试验[5]的安慰剂对照组
表2
2年中EDSS的持续改变量
患者组 | 改变 | EDSS | #病例数 | 百分比 |
研究组RR-MS(n=28)SP-MS(n=26)自然史RR-MSb(n=56)SP-MSc(n=187) | 没有改变更好更差平均EDSS改变没有改变更好更差平均EDSS改变没有改变更好更差平均EDSS改变平均EDSS改变 | 0.0≥0.5>1.00.51.01.5>2.0-0.110.0≥0.5>1.00.51.01.5>2.0+0.120.0≥0.5>1.00.51.01.5>2.0+0.61+0.60 | 15624001124151121495114210 | 53.521.47.114.2003.546.115.33.819.23.83.87.62516.18.919.67.13.617.9 |
a个体内EDSS从基线到第2年的改变bβ-IFN-1a试验[7]的安慰剂对照组
cβ-IFN-1b试验[5]的安慰剂对照组
表3
临床恶化频率
患者组 | 每年的复发率 | #复发数 | #患者数 | 百分比 |
研究组RR-MS(n=28)自然史RR-MS(n=87) | 1.25(研究前)0.75(24个月)1.2(研究前)0.9(24个月) | 0123≥40123≥4 | 1145532326101216 | 39.214.217.817.810.72630111417 |
aβ-IFN-1a试验[7]的安慰剂对照组
表4
利用半定量分析平均MRI病变评分和从基线MRI改变的百分比
患者 | 基线 | 12个月(改变%) | 24个月(改变%) |
共34 | 14.94 | 14.76(-1.2%) | 15.44(+3.3%) |
19/34(55%)10/34(29%)5/34(14%) | 没有改变在24个月内MRI病变评分值至少增加1分在24个月内MRI病变评分值至少减少1分 |
Claims (8)
1.包含T细胞的组合物,其中所述T细胞对髓磷脂碱性蛋白表位有反应,并且所述髓磷脂碱性蛋白表位由髓磷脂碱性蛋白的氨基酸83-99和氨基酸151-170组成。
2.权利要求1的组合物,其中所述T细胞对基本由髓磷脂碱性蛋白的氨基酸83-99和氨基酸151-170组成的表位有反应。
3.权利要求1的组合物,其中所述组合物进一步包含对蛋白脂质脂蛋白、髓磷脂少突神经胶质细胞糖蛋白或其组合有反应的T细胞。
4.权利要求1的组合物,其中所述T细胞是从受试者的外周血获得的。
5.权利要求1的组合物,其中所述T细胞是从受试者的脑脊液获得的。
6.权利要求1的组合物,其中所述组合物包含40×106-80×106个T细胞。
7.有效剂量的权利要求1的组合物在制备用于治疗人的多发性硬化的药物中的用途。
8.权利要求7的用途,其中所述有效剂量的组合物是足够减少人的循环中的髓磷脂反应性T细胞的数目的剂量。
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