CN102186880A - 炎症的治疗 - Google Patents
炎症的治疗 Download PDFInfo
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- CN102186880A CN102186880A CN2009801414529A CN200980141452A CN102186880A CN 102186880 A CN102186880 A CN 102186880A CN 2009801414529 A CN2009801414529 A CN 2009801414529A CN 200980141452 A CN200980141452 A CN 200980141452A CN 102186880 A CN102186880 A CN 102186880A
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Abstract
一种装载了固相载体的分离柱，所述固相载体含有直接或间接固定于所述载体上的一种或多种趋化因子，尤其是生物素化的趋化因子和携有链霉亲和素的载体。本发明还揭示了用所述分离柱和载体从炎症患者尤其炎性肠病(IBD)患者外周血中清除细胞尤其是白细胞的用途和方法。
Description
发明领域
本发明涉及治疗炎症的产品和方法以及治疗手段，所述炎症例如炎性肠病，尤其是溃疡性结肠炎(UC)和克罗恩病(Crohn’s disease，CD)，更尤其是暴发型溃疡性结肠炎和克罗恩病。
发明背景
暴发型溃疡性结肠炎即溃疡性结肠炎的恶化，表现为白血球数升高和剧烈腹痛。目前用高剂量类固醇来治疗暴发型溃疡性结肠炎。抗-TNFα的治疗也已进入三期研究。这两类药都是炎症抑制剂。它们的有效率约为50％，但有严重的副作用。甚至，曾经治愈的暴发型溃疡性结肠炎仍有复发的可能。
在对药物治疗无反应的暴发型溃疡性结肠炎的患者中，及时的手术干预刻不容缓。溃疡性结肠炎通常见于大肠(结肠)。终极手段之一是切除结肠和迴肠造口。至少6个月的恢复期后，期间有时还包括对直肠残端炎症的药物治疗，大多数患者还需接受迴肠直肠吻合术或骨盆囊腔(pelvic pouch)修复术来恢复肠道的连续性。两种手术都会造成每天6次左右的腹泻，并导致水和矿物质失衡。克罗恩病也会有暴发性发作(暴发型克罗恩结肠炎)，也属重症，需要立即进行药物和/或手术干预。
虽说炎症可能位于克罗恩病患者胃肠道的任何部位，但通常多发于小肠的终末端和大肠的起始端(迴肠区)。虽然例如类固醇和硫唑嘌呤(aza-thioprine)等消炎药能够缓解症状，但药物治疗无法治愈该病。50％的患者需要再狭窄切除术后造瘘手术，其中半数复发而需要再手术。因此，亟需一种能够特异性消除患者IBD中的炎症从而避免复发的方法。
WO2008/038785描述了一种细胞吸附柱，能够从血液中分离去除细胞和细胞因子，所述细胞尤指白细胞和癌细胞。
发明内容
炎性肠病表现为受患肠道内的炎症和白细胞浸润。
趋化因子是参与炎症中细胞聚集和活化的一类细胞因子。趋化因子造成免疫系统内各种细胞亚群的趋化性和活化。趋化因子的活性是通过紧密结合其位于白细胞表面的受体来介导的。本发明是基于实现了以下发现：可利用趋化因子与表达其受体的细胞之间的相互作用来治疗炎症，尤其是表现为趋化因子受体表达细胞向炎症部位聚集增强的慢性炎症。因此，本发明能够减少炎性因子高表达诱导所致的炎性白细胞向炎症部位聚集。这是通过用促炎趋化因子来捕捉患者的促炎白细胞而实现的。更具体的说，白细胞，尤其是(活化)T淋巴细胞，(活化)单核细胞，(活化)中性粒细胞，(活化)嗜酸粒细胞中的一种或多种，引发并维持IBD中的炎症，因此，将它们从循环系统中清除或许能够减轻甚至消除这样的炎症。通过对活跃IBD患者肠道活检样本的流式细胞分析，本发明发明人发现(活化)T淋巴细胞，单核细胞，中性粒细胞和嗜酸粒细胞不仅在炎症部位富集，而且存在于循环外周血中。
因此，本发明内容之一是提供一种固相载体(solid support)，具有直接或间接固定于所述载体的一种或多种趋化因子，从而能够从患者外周血中清除表达所述一种或多种趋化因子的相应受体的细胞，所述细胞尤指本文所述的(活化)白细胞(例如单核细胞或淋巴细胞)。所述趋化因子是促炎趋化因子，即创伤或感染应激中在细胞或组织中高水平表达的那些趋化因子(它们还诱导促炎白细胞的聚集)。
本发明中，“趋化因子”包括生物素化或其它方式标记的趋化因子。“趋化因子”还包括趋化因子的修饰和截短型，条件是所述修饰或截短型保留结合其相应受体的能力(因此就本发明而言仍然有功能的)。修饰可以是以改进蛋白质合成为目的的，例如提高产品的一致性和产率。修饰可以包括趋化因子内一个或多个氨基酸位置上的氨基酸插入、取代、缺失或其它修饰。修饰可以是用非天然氨基酸例如正亮氨酸(NLeu)和衍生氨基酸例如焦谷氨酸(pyroGlu)取代野生型氨基酸。这样的修饰能够尽可能减少储存期间和本发明分离柱使用期间副产物的形成。修饰可以改善标记过程为目的，例如引入聚乙二醇(PEG)间隔基有利于生物素化过程。趋化因子的生物素化和/或趋化因子与荧光染料或其它标记基团偶联的进行方式应基本上不影响受体的结合能力。优选定点生物素化或其它定点标记，因为非选择性标记会削弱趋化因子的受体结合活性。优选在蛋白质的C-末端或靠近C-末端进行生物素化或其它方式标记，因为本发明发明人发现，蛋白质对这一区域的修饰具有良好的耐受性(即，对受体结合能力影响最小)。截短可以是N末端和/或C末端的氨基酸缺失，根据需要而定。通常，趋化因子的截短型保留了其正确折叠必需的残基，例如，保留趋化因子原折叠结构所需的残基，这与截短型必须保留结合相应受体(表达于白细胞表面)的能力这一要求是一致的。具体实施方式中，与野生型氨基酸序列相比，截短型缺少1至100个氨基酸，例如少1、2、3、4、5的氨基酸等。当然，截短型可以含有进一步的修饰，如后文所述。具体实施方式中，与全长野生型趋化因子相比，所述修饰形式或截短型具有40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或更高的整体氨基酸序列同一性(缺失计作氨基酸序列差异)。具体实施方式中，在共有序列区段(即未被缺失的氨基酸)，分子间的氨基酸序列同一性可达80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％。本文详细描述了一个本发明趋化因子的例子，它是含修饰的截短型，特别适用于本发明。该截短TECK对应于全长成熟蛋白的残基1至74(因此缺少氨基酸75至127和23个氨基酸的N-末端信号肽)，因此保留了趋化因子的折叠结构。此外还含有一处正亮氨酸取代甲硫氨酸，用以避免肽链组装过程中残基氧化。以焦谷氨酸取代N末端的谷氨酸残基能够在合成过程中获得均一的产品。通过一个PEG间隔基在第72位赖氨酸残基的ε-官能团进行生物素化。线性分子的氨基酸序列(即不包括第72位氨基酸上的PEG间隔基和生物素分子)包含SEQ ID NO：1所示的氨基酸序列，或基本上由SEQ ID NO：1所示的氨基酸序列构成，或由SEQ ID NO：1所示的氨基酸序列构成。本发明的趋化因子可用任何一种合适的方法来合成。较好的是化学合成，因为这便于对趋化因子进行修饰和标记等。然而，根据需要，也可以采用基于重组DNA的方法，并结合适当的标记和修饰技术。因此，本发明还提供编码截短CCL25蛋白的核酸分子，所述截短CCL25蛋白包含CCL25(天然形式)的1-74位氨基酸(即缺少信号肽的形式，所述信号肽包含头23个氨基酸)，基本上由所述1-74位氨基酸构成，或由所述1-74位氨基酸构成。因此，该蛋白缺少天然CCL25氨基酸序列的75至127位氨基酸。本发明还涉及包含所述核酸分子的载体，以及包含所述载体的宿主细胞。所述载体还可以包含与所述核酸分子操作性连接的合适的启动子，从而促进相应mRNA分子的转录。所述宿主细胞能够通过转录后翻译编码截短CCL25蛋白编码的核酸分子来表达该蛋白，所述的截短CCL25蛋白包含CCL25的氨基酸1至74(因此缺失氨基酸75至127和23个氨基酸的N-末端信号肽)。
趋化因子受体在许多迁移性细胞上表达，例如淋巴细胞、粒细胞和抗原呈递细胞，也在许多非迁移性细胞上表达，例如上皮细胞和成纤维细胞。如前所述，本发明的趋化因子以一定的方式固定在固相载体上，从而能够从患者的外周血中清除表达一种或多种趋化因子的相应受体的细胞。表1汇集了本发明优选趋化因子的相应受体。有时，一个趋化因子可结合一个以上受体。趋化因子的这一特性使之能够捕捉多种参与炎症的受体表达细胞从而提供更有效的治疗。具体实施方式中，所述固相载体带有多种趋化因子，旨在扩大捕捉促炎细胞的范围。
IBD患者的炎症迁延不愈是因为血液循环中的抗原呈递细胞(APCs)和T细胞不断进入肠粘膜。这一连续的细胞积累是由趋化因子及其受体调控的。因此，趋化因子分子参与炎症过程中多种免疫细胞亚群的聚集和活化。发炎肠粘膜内产生的一种本地(local)趋化因子是TECK(胸腺表达趋化因子，又名CCL25)。循环血中的单核细胞和淋巴细胞通过TECK受体，趋化因子受体9(CCR9)，识别趋化因子。细胞通过CCR9受体结合TECK，并开始向肠道炎症部位迁移(1)。到达发炎粘膜后，单核细胞转化为APCs。循环表达CCR9的T细胞也向肠道炎症部位迁移，并在那里活化。在小鼠模型中，通过基因删除或抗体抑制消除CCR9或其配体TECK能减弱T细胞向肠道迁移(2)。CCR9-TECK相互作用似乎在单核细胞和T细胞向整个肠道-包括小肠以及结肠-的迁移中起着主要作用。最近发现，健康人和IBD患者的大肠内都有CCR9和TECK。CD患者或UC患者结肠组织的免疫组化染色检查发现有表达CCR9的T细胞存在。并且在结肠中观察到有受体激动剂TECK存在(3)。IBD患者中，遵循的原理是在循环系统中的表达CCR9的单核细胞和T细胞到达肠道炎症部位之前将它们清除，从而减轻炎症。采用带有固定于固相载体上的生物素化TECK(bTECK)的分离柱，由TECK从循环系统中捕捉表达CCR9的单核细胞和T细胞，应该能够抑制所述细胞造成的促炎作用，从而使粘膜得以愈合。这一原理适用于参与白细胞向肠道炎症区聚集的多种趋化因子。例如，肠道粘膜分泌IL-8，其通过与的IL-8受体的相互作用来吸引中性粒细胞。CCR6调节Th17向肠道的迁移以及发炎组织内效应者T细胞的平衡/分布(14)。因此，固定于固相载体的CCL20也能够在本发明中用来治疗炎症，例如IBD。
本发明挑选趋化因子的根据是它们在炎症中的表达上调。而且，特定类型细胞上的趋化因子受体显示出与所述炎性疾病的发生相关，表现为这些受体表达细胞向炎症部位聚集。因此，本发明的趋化因子包括以下一种或多种：MIP-1a，MIP-1b，MCP-1，MCP-2，MCP-3，MCP-4，TARC，MDC，MIP-3，MIP-3a，MIP3b，MIP-4，I-309，HCC-1，HCC-2，SLC，IL-8，GROa，GROb，GROg，RANTES，NAP-2，ENA78，GCP-2，IP-10，MIG，I-TAC，SDF，CX3C趋化因子(fractalkine)，淋巴细胞趋化因子(lymphotactin)，嗜酸粒细胞趋化蛋白(eotaxin)，嗜酸粒细胞趋化蛋白-2，I-309，BLC，CCL25。优选的本发明趋化因子有：MIP-1a，MIP-1b，MIP-3a，MIP-3b，MIP-4，SLC，MCP-1，MCP-2，MCP-3，MCP-4，TARC，MDC，IL-8，IP-10，MIG，I-TAC，CX3C趋化因子，CCL-25，RANTES。最优选的是优先结合活化T淋巴细胞的趋化因子，尤其是MIP-1a，MCP，IP-10，MIG，ITAC，CCL25。“优先结合”指：趋化因子结合活化T淋巴细胞的倾向性大于结合非活化T淋巴细胞和/或其它血细胞。具体实施方式之一中的趋化因子是CCL25。CCL25优先结合表达CCR9的细胞，尤其是(活化)淋巴细胞(CD4和CD8淋巴细胞)和单核细胞(例如CD14-阳性单核细胞)。
表1提供本发明所用部分趋化因子的详细信息，包括认证的基因记号(根据HUGO基因命名委员会)、名称和序列信息。还列出了各的一个或多个相应受体。
本发明的趋化因子可用已知方法生物素化，例如WO 00/50088 A2中所述的方法，该文献通过引用纳入本文。如前所述，优选对本发明的趋化因子进行定点标记，但是，各种标记技术，只要不显著影响趋化因子的受体结合能力，都可采用。多种定点生物素化的趋化因子和天然趋化因子可通过商业途径获得，向Almac，Craigavon，UK获取。部分具体实施方式中，一种或多种趋化因子通过间隔基生物素化。间隔基可用来避免生物素基团对趋化因子活性的影响，尤其是趋化因子与相应受体的结合。本发明可采用有利于保留趋化因子受体结合特性的各种合适的间隔基。部分具体实施方式中，间隔基是聚乙二醇(PEG)间隔基。本发明发现，PEG间隔基能够让生物素与趋化因子(可以通过与链霉亲和素的相互作用固定于固相载体)结合而不损伤受体结合能力。
用于固定本发明趋化因子的固相载体是已知的。可用的载体材料不能另血细胞尤其是淋巴细胞活化而导致它们凝集或粘附在载体上。优选经处理而具有抗凝聚特性的载体，尤其是肝素化的载体。或者，可以在上样到载体上之前先对患者的血液进行抗凝剂如肝素处理。可用的载体材料包括高分子糖类，尤其是分子量100kDa或以上的糖类，例如琼脂糖，颗粒型的，可选交联型的，还有纤维素。优选的载体材料还有聚合物，例如羧化聚苯乙烯和玻璃。本发明的载体优选颗粒或纤维形式。载体颗粒可以是规则形状的，例如球形或珠状，或者是不规则形状的。它们可以是多孔性的，也可以是非多孔性的。载体优选的平均粒径是50μm至2mm。将趋化因子固定到固相载体上的方法是已知的。趋化因子可直接或间接固定到载体上。直接固定可采用合适的接头(linker)来进行。间接固定趋化因子的一种优选方法是利用生物素与亲和素之间的相互作用。因此，趋化因子的生物素化和采用固定于固相载体上的链霉亲和素是将趋化因子固定于固相载体的可靠方法。具体地说，该方法可包括：提供生物素化的趋化因子，提供表面固定有链霉亲和素的固相载体，将该载体与生物素化趋化因子的水性溶液接触，用水性溶剂洗涤载体。此外，还可利用抗体-抗原相互作用进行趋化因子在载体上的间接固定。此类实施方式中，可用已知对某特定肽序列或小分子半抗原具有亲和力的抗体或其片段或衍生物对载体进行衍生处理。将所述肽序列或抗原加到趋化因子之上或之内能促进其固定到涂覆了相应抗体或其片段或衍生物的固相载体上。因此，可以修饰趋化因子使之带上肽序列或半抗原，所述肽序列或半抗原可以包含在线性分子内，或作为侧链或标记。本发明可采用各种合适的抗体-抗原对。所述抗体片段或衍生物可以是保留了对相应抗原特异性结合亲和力的各种片段或衍生物，例如Fab，scFV，VH结构域，纳米抗体(Nanobodies)，重链抗体和人源化非人抗体等等。还可利用其它高亲和力相互作用来固定趋化因子，条件是，趋化因子能够用相互作用中的一方进行衍生化，而固相载体则可用另一方衍生化，且不造成结合活性(趋化因子与其相应受体的结合)的损失。
或者，可通过业已成熟的生物偶联技术将趋化因子直接固定到固相载体上，例如蛋白质通过其一级序列内的氨基官能团直接固定到溴化氰活化的固相载体上。或者，可利用蛋白质内的巯基官能团将蛋白质直接固定在卤代烃衍生的载体上或含有游离硫醇官能团的载体上。另一些实施方式中，可利用固有的化学连接反应，将含有α-巯酯官能团的蛋白质直接固定在含有1，2-氨基硫醇基团(例如N-末端半胱氨酸)的载体上。或者，可利用腙/肟成键连接反应，将酮修饰和醛修饰的蛋白质固定在肼基、酰肼和氨氧基衍生的固相载体上，反之亦然。或者，可用“点击化学”(Click chemistry)将蛋白质固定到固相载体上，其中，蛋白质和载体以合适的相互反应的官能团(叠氮和炔)衍生。其它一些实施方式中，可用施陶丁格(Staudinger)连接反应将适当衍生的蛋白质固定到适当衍生的固相载体上。
本发明揭示了一种装载了上述固相载体的分离柱，所述固相载体上固定有一种或多种趋化因子。所述分离柱装载了含有一种或多种趋化因子的固相载体，所述一种或多种趋化因子直接或间接固定在所述载体上，从而能够从患者的外周血中去除表达所述一种或多种趋化因子受体的细胞，尤其是本文所述的(活化)白细胞，例如单核细胞或淋巴细胞。优选实施方式之一中，本发明的分离柱装载了这样的载体，所述载体上固定有链霉亲和素，一种或多种生物素化趋化因子结合在这些固定在载体上的链霉亲和素上。优选高分子糖作为载体，可选交联型糖，例如琼脂糖。“装载”表示所述分离柱以这样一种方式携带或含有所述固相载体，即(外周)血能够通过分离柱并与固相载体接触。因此，固相载体构成分离柱中血液流经的基质，在某些实施方式中是连续流经。
本发明分离柱因此被用来承载能够从血样中清除表达相应趋化因子受体的细胞的载体。更具体的说，这样的分离柱可用来从(IBD)患者的外周血中清除一种或多种(活化)白细胞，尤其是一种或多种以下细胞：(活化)T淋巴细胞、(活化)单核细胞、(活化)中性粒细胞、(活化)嗜酸粒细胞。根据具体患者的细胞分布，可挑选特定的趋化因子来制备载体，从而特异性地清除T淋巴细胞、单核细胞、中性粒细胞和嗜酸粒细胞，或其它参与肠道炎症的活化细胞。
因此，本发明还提供一种从患者的外周血中清除表达相应趋化因子受体的细(例如白细胞)的方法，所述细胞例如一种或多种以下细胞：(活化)T淋巴细胞、(活化)单核细胞、(活化)中性粒细胞和(活化)嗜酸粒细胞，所述患者尤指炎症患者，尤其是炎性肠病(IBD)，所述方法包括：将采集到的外周血与固定在固相载体的一种或多种趋化因子接触足够长的时间，使得所述细胞附着到载体上；将清除了所述细胞的血液与载体分离。该方法可以是体外(ex vivo)或离体(in vitro)方法。然而，部分实施方式中，所述方法还包括在接触步骤前从患者采集外周血的步骤。另一种实施方式中，本发明方法还包括在分离步骤之后将清除了所述细胞的血液回输给患者。这样就构成了完整的分离性输血疗法。因此，分离性输血包括：采集患者的外周血；将采得的外周血与固定在固相载体的一种或多种趋化因子接触足够的时间，使得表达相应趋化因子受体的细胞附着于载体，所述细胞例如一种或多种白细胞，尤其是(活化)T淋巴细胞、(活化)核细胞、(活化)中性粒细胞或(活化)嗜酸粒细胞；将清除了所述趋化因子受体表达细胞的血液与载体分离，所述细胞例如一种或多种白细胞，尤其是(活化)T淋巴细胞、(活化)核细胞、(活化)中性粒细胞或(活化)嗜酸粒细胞；和将清除了所述细胞的血液回输给患者。
可以连续地从患者采血。同样，采用本文中所述的合适的回路也可以连续地将清除了所述细胞的血液回输给患者。因此，可将载体装在一个血液流经的分离柱中。例如，这可以用合适的泵来实现。血液流经分离柱，固定在固相载体上的趋化因子就能够捕捉表达相应受体的白细胞，于是，就能够将它们从血液中清除，从而避免它们加剧炎症。
因此，概括地说，可将本发明的分离柱或载体用于治疗炎症例如IBD，或用于治疗以外周血中存在相应趋化因子受体表达细胞(尤其白细胞)为特征的疾病，所述细胞例如一种或多种以下细胞：(活化)T淋巴细胞、(活化)单核细胞、(活化)中性粒细胞、(活化)嗜酸粒细胞。本发明还提供用于治疗的趋化因子，尤其是用于治疗例如IBD等炎症的趋化因子，所述趋化因子固定在固相载体上。同样，本发明涉及趋化因子在制造治疗IBD等炎症的药物中的用途，所述趋化因子固定在固相载体上。
本发明载体和分离柱的所有实施方式均比照适用于所有这些方面，不在赘述。
本发明还揭示了一种制造结合了生物素化趋化因子的磁性链霉亲和素-包被微珠的方法。该方法包括：提供磁性链霉亲和素-包被的微珠，微珠悬浮在水性溶剂中，提供生物素化趋化因子的水性溶液，将上述悬浮液和溶液混合并孵育，任选通过磁性方式分离出由此形成的结合了生物素化趋化因子的磁性链霉亲和素-包被的微珠，和用水性溶剂洗涤所述结合了生物素化趋化因子的磁性链霉亲和素-包被的微珠。
本发明结合了生物素化趋化因子的磁性链霉亲和素-包被的微珠可用于将具有(表达)相应趋化因子受体的血细胞与没有所述受体的血细胞分离。较好的是，用磁性分离机对外周血进行这样的分离。根据本发明的优选内容之一，将分离后的外周血回输给同一患者。
本发明的方法和医药用途可根据特定的患者个体或群体进行调整。通过从循环系统中清除向肠粘膜细胞活化迁移的细胞，实现了对IBD炎症过程中一个重要因子的控制。本发明的方法对于治疗或逆转溃疡性结肠炎或克罗恩病，尤其是暴发型(溃疡性)结肠炎或暴发型克罗恩病特别有效。
本发明的方法和医药用途还可以用来通过清除表达相应趋化因子受体的细胞尤其是白细胞来治疗克罗恩病，所述细胞例如(活化)T细胞、(活化)单核细胞、(活化)中性粒细胞、(活化)嗜酸粒细胞，以及向肠壁深处抗原活化迁移的其他类型细胞。
本发明更广义的实施是用本发明分离柱或载体治疗以外周血中存在表达相应趋化因子受体的细胞尤其是白细胞为特征的疾病，所述细胞例如(活化)T淋巴细胞、(活化)核细胞、(活化)中性粒细胞和(活化)嗜酸粒细胞中的一种或多种。
以下，将通过非限定性实施方式和实施例来进一步阐述本发明。
附图说明
图1a、1b & 1c-生物素化的MIP-1α分别被CD4+、CD8+T细胞和CD14+单核细胞所结合，所述细胞来自健康供者的外周血；
图1d、1e & 1f-生物素化的MCP-1分别被CD4+、CD8+T细胞和CD14+单核细胞所结合，所述细胞来自健康供者的外周血；
图2a、2b & 2c-生物素化的CCL25分别被CD4+、CD8+T细胞和CD14+单核细胞所结合，所述细胞来自健康供者的外周血；
图2d、2e & 2f-生物素化的CCL25CD4+、CD8+T细胞和CD14+单核细胞所结合，所述细胞来自CD患者的外周血；
图3a、3b & 3c-IL-8分别被CD4+、CD8+T细胞和CD16+单核细胞所结合，所述细胞来自健康供者的外周血；
图4-塑料壳体和顶盖，显示分布板(2)和安全过滤单元(3和4)。
图5-整个分离输血系统。
图6-配有空气检测器和光学检测器(4)的泵。
图7-清除供血者的表达CCR9的细胞群。整体细胞群过柱后未受影响。
图8-清除IBD患者的表达CCR9的细胞群。整体细胞群过柱后未受影响。
图9-过柱前和过柱后某供血者血细胞上的活化标记。
图10-经过小型工具之前和之后，细胞上的IFN-γ分泌。
图11-过柱前和过柱后，PHA抗原刺激细胞的结果。
图12-与高浓度bTECK孵育后的细胞死亡情况。
图13-纯化的折叠生物素-TECK(Nleu)的HPLC。
图14-纯化的折叠生物素-TECK(Nleu)的电喷离子化串联质谱(ES/MS)数据。
优选实施方式
材料和方法
外周血白细胞的分离。健康供血者或IBD患者外周血肝素化，然后用4％多聚甲醛固定4分钟，0.83％氯化铵溶液溶血15分钟，然后用FACS缓冲液洗涤两次，得到白细胞悬浮液。
趋化因子。白细胞与生物素化并以Alexa647 Fluor标记的以下趋化因子4℃暗培养30分钟：CCL25(浓度分别为0.1ng/μL、0.5ng/μL和5ng/μL)，MIP-1α或MCP-1(浓度分别为10ng/μL和50ng/μL)。然后用FACS-缓冲液洗涤细胞，并进行流式细胞分析。实施例所用化因子均来自Almac Sciences Scotland Ltd，苏格兰爱丁堡。
流式细胞分析。流式细胞分析在两台激光FACS Calibur流式细胞仪(BD Immunocytometry systems，美国加利福尼亚圣胡塞)。每份样品计数并分析1万个细胞。用Becton Dickinson的Cell Quest Pro软件进行数据分析。
实施例1-单核细胞与MIP-1α的结合。在用生物素化MIP-1α进行的实验中发现，30分钟的孵育后，健康供血者外周血的单核细胞中约90％与细胞因子结合(图1c)，CD4+和CD8+淋巴细胞则没有结合(图1a和1b)。
实施例2-单核细胞与MCP-1的结合。在用生物素化MCP-1进行的实验中发现，健康供血者外周血的单核细胞中约90％与细胞因子结合(图1f)，CD4+和CD8+淋巴细胞则没有结合(图1d和1e)。
实施例3-血细胞对CCL25的亲和性。在用生物素化CCL25进行的实验中发现，健康供血者外周血的T细胞(CD4+淋巴细胞，CD8+淋巴细胞)和单核细胞(CD14+单核细胞)都不结合该生物素化的趋化因子(图2a，2b和2c)。与此相对，克罗恩病患者约80％的CD8+淋巴细胞和约90％的CD4+淋巴细胞和单核细胞与CCL25结合(图2d，2e和2f)。
实施例4-血细胞对生物素化IL-8的亲和性。图3显示了健康供血者的CD4+淋巴细胞(图3a)、CD8+淋巴细胞(图3b)和CD16+中性粒细胞(图3c)与生物素化IL-8(CXCL8)的结合。孵育30分钟后，CD16+中性粒细胞全部与IL-8结合。对此相对，未见CD4+淋巴细胞和CD8+淋巴细胞结合。
实施例5-用于血细胞分离的趋化因子分离柱的制备。链霉亲和素交联的75μm至300μm琼脂糖(ProZyme，San Leandro，美国加利福尼亚)珠粒悬浮在含有25mM磷酸钠(pH 7.0)和150mM NaCl的水性溶液中(200ml，～50％，v/v)，22℃，向其中加入相同缓冲液配制的75μg生物素化MIP-1α(Almac Sciences)溶液，手动轻柔搅拌3分钟。接着静置20分钟，然后过滤去除载体，用中性的磷酸钠/氯化钠水溶液洗涤三次后装入玻璃柱(内径25mm，长12cm)。
实施例6-用实施例5制备的趋化因子分离柱从健康供血者的外周血中分离单核细胞。一名健康男性供血者的外周血肝素化后进行关于CD4+淋巴细胞、CD8+淋巴细胞和CD14单核细胞的流式细胞分析。用所述分离柱过滤100ml血液，流速约每分钟8ml，用FACS缓冲液洗涤。再次分析过滤后血液中的上述细胞。结果发现，约95％的单核细胞被分离柱截留，90％以上的CD4+和CD8+淋巴细胞回收。
实施例7-制备结合了生物素化MIP-1α的链霉亲和素偶联的磁性珠粒。偶联了链霉亲和素的磁性珠粒(MagCellect Streptavidin Ferrofluid，1ml；R&D Systems，美国明尼苏达明尼阿波利斯)水性悬浮液与30μg MIP-1α(Almac Sciences)在50ml含25mM磷酸钠(pH 7.0)和150mM NaCl的溶剂中混合并低速搅拌1小时。所得颗粒用上述溶剂洗涤3次，每次20ml，以悬浮液形式4℃保存。
实施例8-用实施例7的链霉亲和素磁性珠粒从健康供血者的外周血中分离CD14+单核细胞。100ml实施例7中所述的肝素化的健康人血与结合了生物素化MIP-1α的链霉亲和素偶联磁性珠粒混合，低速搅拌40分钟。用磁性分离仪从血液中分离出珠粒颗粒，对血液进行CD14+单核细胞和CD4+和CD8+淋巴细胞检测。基本上未见CD14+单核细胞，但CD4+和CD8+淋巴细胞的量基本未变。
实施例9-定制的分离性输血
分离柱设计和性能
介绍
血液分离术是一种成熟的用于去除某些血液成分的处理方法，例如去除某些抗体、低密度脂蛋白(LDL)和血细胞。白细胞分离术即用来去除白细胞的分离术。将患者连通体外血液循环系统；血液从一条手臂的静脉抽出，流经柱设备，然后从另一条手臂回输给患者。白细胞分离术的副作用包括一些轻症，例如头疼、头晕、低血压、心悸和潮红，可见于0.1-5％受治者。
分离柱
所述分离柱将用于IBD的白细胞分离术治疗。它采用含bTECK树脂，利用CCR9-TECK相互作用，特异性清除表达CCR9的肠道归巢白细胞。该柱由三部分组成：塑料壳体，链霉亲和素(SA)琼脂糖TM大珠粒(SepharoseTM BigBeads)基质和结合于该基质的bTECK。治疗过程采用与标准分离术相同的技术进行。
塑料壳体(图4)
塑料壳体用来保持血流连续地流经基质，它由透明壳身和红色顶盖构成。顶盖具有分布板(2)，位于流入进口处(1)，用于将血液均匀地分布到整个基质表面。分布板是第一道安全屏障，可阻止大颗粒流经分离柱而进入患者体内。安全过滤单元(3和4)安装在塑料壳体的进口处(1)和出口处(5)。安全过滤单元含有三层滤材，从而构成牢固的屏障，并阻止所有大于血细胞的颗粒流经分离柱。塑料壳体的设计如图4所示。柱体两端的安全过滤单元(3和4)设计能够最大程度降低颗粒漏过从而进入患者体内的风险，即便装置倒置，血液反向流动时也是如此。链霉亲和素琼脂糖TM大珠粒(SepharoseTM BigBeads)
该装置的第二项元素是称为“链霉亲和素琼脂糖TM大珠粒”(SepharoseTM GE Healthcare，瑞典)的亲和性基质。琼脂糖TM是一种交联珠状琼脂糖，是从海藻中提取的多糖。琼脂糖TM和琼脂糖是生物医药亲和技术中的常用装柱基质。选择它是因为其适宜的分布能力以及能够为亲和性结合提供较大的可用面积。
bTECK
与所述基质偶联的是所述装置的第三项元素，bTECK。bTECK肽是人趋化因子TECK的合成和改造(engineered)版，经截短并且被生物素化，但它保留了与TECK受体CCR9结合的能力。通过对改造的TECK进行生物素化使之能够与琼脂糖TM基质上的链霉亲和素分子结合。生物素-链霉亲和素结合被认为是最强的生物学相互作用之一，其Kd约为4x10-14M。柱内链霉亲和素∶生物素结合位点的计算所得比率为10∶1。因此，基质与bTECK之间将立即偶联，由此最大限度降低了bTECK从基质上解离的风险。
分离系统
进行白细胞分离术需以下器件：分离柱，管路系统和4008 ADS泵(FM护理公司(Fresenius Medical Care))。
回路
系统如图5所示。患者(1)通过无菌Venflon针头左右臂静脉进针与体外回路连通。还接入了盐水袋(3)，用ACD泵(2)泵送盐水溶液。血液从患者的一条手臂抽出，在血液泵(4)作用下流经无菌管路系统，流经分离柱(6)，然后回输给患者。管路系统与分离柱通过标准透析Luer锁(LuerLock)接头连接。柱上的接头以不同颜色标记以便正确接装；红色管是由柱顶进入的流入流，蓝色管是返回患者的流出流。有一个空气检测器(8)。用入口压力(5)和Pven传感器(7)来监测回路内的压力。4008 ADS泵
FM护理公司(Fresenius Medical Care)出品的分离泵监测患者血液的流入和流出，体外循环中的压力，并能用气泡捕捉器和空气检测器来隔除空气。气泡捕捉器内置有血栓捕捉过滤器。该泵还有一个光学检测器，用来区分管路系统中浅的组分(例如盐水溶液或空气)和暗的组分(例如血液)。
图6是一例泵的图解，其中显示有空气检测器和光学过滤器。如果泵系统检测到气泡和光学波动，或者，如果体外压力值超出了预设范围，泵将立即停止并发出视觉或声音警报。
图6中的图例：
1.监测仪
2.废液袋支架
3.模块(从左至右-血液泵，ACD泵，空气检测器)
4.模块扩展槽
5.吸收块(Absorber)支架
6.滴流检测器
7.IV杆
患者的准备
每次治疗前给患者使用抗凝剂。用含有5000IE肝素的无菌盐水灌洗体外循环系统，然后在每次治疗开始时前向回路中推注4000IE肝素。白细胞分离的时间和流速
分离系统的运行流速为30-60mL/min。循环血液达1800mL后结束治疗。
保存条件
分离柱装置应于1℃至25℃保存，避免结冰，也避免温度更高。超过3个月的稳定性数据显示功能未因时间和温度而改变(室温和冷藏)。分离柱在用前应冷藏保存。应避免剧烈震荡或冲击等机械损伤。非如上建议条件下保存的分离柱不可用。
运输条件
所述分离柱装置应冷藏条件下运输，避免结冰和高温。应避免剧烈震荡或冲击等机械损伤。
实施例10-非临床研究
介绍
早在上世纪70年代就发现，供体羊肠系膜淋巴结中回收的淋巴细胞中移植入受体动物后在肠道中积累(4，5)。这些最初的动物研究提示，循环中的淋巴细胞具有特异性归巢能力，靶向体内不同的区室(compartments)。此后的鼠类模型研究显示数条信号转导路径导致了不同T细胞子集的器官特异性。L-选择素(L-selectin)(又称CD62L)被发现是导致淋巴细胞向肠系膜淋巴结迁移的一种细胞表面蛋白(6)。肠道血管的内皮层中，MadCAM1和TECK参与结合粘膜的淋巴细胞和单核细胞的粘附和迁移。这些研究让人注意到这些免疫系统的相应受体，α4β7和CCR9(2)。在这方面，克罗恩病的小鼠模型之一，TNFDeltaARE，提示：α4β7路径与TECKCCR9-依赖性转导无关，并且似乎是肠道归巢的主要机制(7，8)。然而，其它小鼠模型确认，TECK-CCR9相互作用对于靶向发炎粘膜的肠道归巢来说同样重要。TECK-/-和CCR9-/-鼠模型以及抗体介导的TECK-CCR9结合的抑制表现出粘膜炎症减弱(9-12)。因此，不同归巢机制的影响似乎随动物模型的不同而不同。数项鼠模型研究显示，表达CCR9的T细胞优先向小肠归巢。然而，溃疡性结肠炎小鼠模型MDR1a-/-所见的结肠内粘膜炎症表现出对于表达CCR9的淋巴细胞的依赖性。在给予CCR9阻抑蛋白CCX282-B之后，结肠的炎性损伤明显缓解，这表明TECK-CCR9相互作用在结肠粘膜也起着重要作用(3)。鉴于TECK-CCR9归巢机制的治疗意义，开展了数项人体研究，如同小鼠中所见，表达CCR9的T细胞聚集于人的小肠(2，3，13)。CD患者中，与健康对照相比，肠系膜淋巴结中表达CCR9的淋巴细胞显著升高(13)。其它研究描述了CD或UC患者发炎结肠粘膜内的TECK和表达CCR9的T细胞的存在。健康对照也被发现结肠粘膜中具有表达CCR9的免疫细胞存在，由此可知该受体在肠道相关免疫系统的正常运作中起着重要作用(3)。就炎性肠病而言，现有的动物模型与人肠道炎症之间的对应性并非最佳。因此，研究重点已转移到采用对采自IBD患者的血样进行离体(in-vitro)实验，从而获取非临床概念测试证据。此外，白细胞分离柱中所用的bTECK蛋白是人CCR9表面蛋白特异性的，因此不太适合动物体内(in-vivo)功效研究。
离体(In-vitro)清除靶细胞群
为了检测清除表达CCR9的细胞的能力，对偶联了bTECK的基质进行体外测试。血液采自供血者和IBD患者，让其流经含有bTECK偶联基质的分离柱装置。过柱前和过柱后分别提取血样，用流式细胞分析(FACS)检测CCR9表达细胞的清除结果。
结果显示，经基质灌注后，靶细胞群即CD14-阳性的CCR9表达细胞显著减少；同时，CD14-阳性细胞总数保持不变。对健康供血者和IBD患者血液进行的清除试验证实了相近的效果。上述结果可见于图7和图8。
结论：体外试验结果表明分离柱能够特异性地减少50-75％CCR9表达细胞。不表达CCR9的细胞不受影响。
实施例11-毒理学测评和安全性测试
接触
患者通过两种不同的方式与分离柱装置发生接触。首先是局部接触，即血液及其中的细胞与装置内包括bTECK在内的化学物质接触，其次是系统性地与装置所释放的和经血液回输进入患者的包括bTECK在内的化学物质接触。两种情况下都难以测评总体接触情况，但是，对于基质稳定性的研究将能揭示与琼脂糖、链霉亲和素和bTECK的系统性接触，见后文。然而，因为塑料和滤材符合FDA/ISO 10993标准以及USP第VI级生物学评价要求，即使在辐射消毒后，可以确定，由装置的这些部件造成的有毒化合物接触时可以忽略不计的。并且，没有数据表明分离柱不同部件之间存在相互作用。
基质的稳定性
研究了基质的稳定性，检测了在柱上实际检测期间是否有材料漏逸。将充填了基质的分离柱接入泵系统，用2L PBS(磷酸盐缓冲液)灌洗，以30-100ml/min的流速洗去装柱步骤中残留的颗粒。采集过柱前和过柱后的样品，通过显微镜检和ELISA检测产物漏逸。
结果，2L PBS洗柱后未见基质材料漏逸。
ELISA检测结合稳定性。我们通过在测试孔用链霉亲和素抗体4℃孵育1小时来检测解吸的bTECK。我们通过在测试孔用生物素化过氧化物酶室温孵育1小时来检测解吸的链霉亲和素。结果显示，没有琼脂糖颗粒、链霉亲和素或bTECK从基质中漏出。
生物学(毒理学)数据
期望的生物学结果是特异性地清除靶向胃肠道的活化白细胞(肠道归巢细胞)，这是由于bTECK通过强烈的受体-配体亲和性吸引并结合细胞上的特异性受体CCR9。不表达该受体的细胞通过分离柱返回患者。分离柱按需使用过程中的接触可能导致诸多不良生物(毒性)效应，按照此类医疗器械的ISO 10993-1对此进行了测评。基于血液均匀分布于整个柱表面时的假定局部接触，血液尤其是其中的细胞可能受到不利影响。趋化因子bTECK或装置内的其它化学物质可能产生局部影响，例如细胞毒性和造血障碍。最重要的是检查各种免疫细胞的活化情况。患者还会与bTECK或灌注期间分离柱装置或塑料管路释放的各种化学物质发生系统性接触，这些可能产生生物学(毒理学)效应。这些化学物质可能引起各种系统性反应，其中，必须按照ISO 10993-1对细胞毒性、致敏性、刺激性和皮内刺激，系统毒性(急性)，亚急性和亚慢性毒性和造血障碍进行测评。为了确定bTECK的生物学效应，合成了一个9kDa的截短型并将其生物素化，进行了一系列另外的研究。体外特异性细胞清除和FACS(荧光活化细胞分选)分析
对于用基质和bTECK对IBD患者血液进行的细胞清除试验，用一小型工具来模拟实际大小分离柱上的运行。用顶部装有尼龙滤膜组的塑料管进行模拟。将血液温和地与基质混合，流过滤膜，进入采集管。对未过滤血液和过滤血液进行裂解，然后用抗体染色，接着进行FACS分析。
采集供血者的血样，用含bTECK偶联基质的标准柱进行细胞清除试验。采集过柱前和过柱后的样品，裂解，然后用抗体染色，接着进行FACS分析。
小型工具和标准分离柱装置都能成功地特异性清除表达bTECK受体的细胞。还可显示，这种清除术特异性针对表达CCR9的细胞群。例如，图8和7中，CCR9阳性是细胞CD14和淋巴细胞(CD4和CD8)显著减少，不到1/5通过分离柱，但过柱后CD14和淋巴细胞群总数不变。
活化，增殖和细胞死亡
目的在于研究通过分离柱装置的细胞的活化和功能性特征。
活化标记
裂解后的细胞室温下用10％HUS(人抗血清)孵育15分钟，以避免与表面具有Fc-受体的细胞发生非特异性结合。对细胞进行针对活化标记的染色：CD69(淋巴细胞)，CD66b(粒细胞)和HLA-DR(单核细胞)。采集细胞，上样到FACSAria流式细胞分选仪上，用FACSDiva软件进行分析。过柱前后，带活化标记的被检细胞数相同(见图9)。
细胞因子释放
用炎性细胞因子释放试验，γ干扰素(IFNγ)分泌测试试剂盒，检查与基质接触前和接触后的细胞活化情况。该分泌试验将显示受激后产生IFNγ的细胞数量和类型。Ficoll分离获取4名供血者的外周单核血细胞(PBMC)。将细胞重悬于细胞培养基(RPMI，含1％Pest+1％L-Glut+5％HUS)中，浓度为1x106细胞/毫升。基质经PBS洗涤后与0.1μg/ml bTECK混合。让半量的细胞悬浮液流过装载了基质的小型工具。将未过滤的和滤过的细胞按500 000/孔的密度接种到48孔板上，37℃孵育16小时。在细胞中加入PMA(50ng/ml)+离子霉素(1μg/ml)作为阳性对照。16小时后，分析细胞的IFNγ表面结合量和IFNγ分泌量。用于FACS分析的其他单抗(Mab)是CD3、CD14和DAPI单抗。IFNγ分泌未见明显改变(见图10)。
利用[3H]掺入的增殖试验
从一供血者的肝素化全血中Ficoll分离和制备PBMC。细胞计数按照2x106细胞/毫升的浓度用培养基(RPMI+10％BGS+1％pest和1％L-glut)稀释。让半量的细胞悬液流过装载了偶联有200nM(0,2μg/ml)bTECK的基质(SA浓度4mg/ml)小型工具。按照规程，将2x106细胞/毫升的细胞悬液(100 000个细胞)按照50μl/孔接种到96-孔微滴板上，每三孔为一组平行试验。50μl/孔的细胞培养基作为阴性对照，50μl/孔的PHA(植物凝血素)抗原(5μg/ml)为阳性对照。细胞于37℃孵育至第2天、第3天和第4天。收获细胞前，向孔中加入25μl胸腺嘧啶[3H]并于37℃孵育18小时。18小时后，收获细胞，用闪烁计数仪计数掺入的[3H]。过柱前后，作为增殖指标的[3H]掺入没有变化(见图11)。
用膜联素(Annexin)V进行的细胞凋亡试验
用Ficoll分离法自3名供血者分离PBMC。细胞用PBS洗涤两次，悬浮在培养基(RPMI，含的1％L-glut、1％pest和10％BGS)，浓度为1x106/ml。细胞用5μg/ml或10μg/ml bTECK进行刺激。细胞在24孔微滴板上孵育16小时。用地塞米松(Dexametason)(1μM)作为阳性对照。细胞用PBS洗涤两次，按照BD膜联素V试剂盒说明书用膜联素V染色。在FACS分析前向细胞中加入100μl DAPI。用FACS Aria流式细胞分选仪和FACS Diva软件分析样品。膜联素V阳性且DAPI阴性的细胞为凋亡细胞。与5或10μg/ml bTECK接触后，膜联素V阳性细胞数没有显著增加(见图12)。
概述
在过柱前和过柱后，或者与偶联有bTECK的链霉亲和素琼脂糖基质直接接触前和接触后对细胞进行检查。结果显示，与基质和bTECK接触过的细胞未受或几乎未受影响。过柱前后，活化标记的细胞CD69(淋巴细胞)、CD66b(粒细胞)和HLA-DR(单核细胞)的数量没有改变。PBMC的增殖能力未受影响，释放细胞因子的细胞数很低。高剂量的bTECK，比原临床研究分离柱装置所用剂量高5-10倍，对细胞死亡的影响极小。
毒理学研究
离体(in vitro)细胞毒性试验
在哺乳动物细胞(L929小鼠成纤维细胞)培养物中检测分离柱基质的体外细胞毒性。该试验按照ISO 10993-5稀释试验指南(Elution Test guideline)进行。测试物为有涂层的琼脂糖珠粒用20％乙醇配制的浆状物。所述琼脂糖珠粒经洗涤后用等体积的无菌等渗盐水(0.9％NaCl)重悬，从而在试验前去除乙醇，如同分离柱基质在实际使用前需经洗涤一样。制备分离柱的抽样样品：经洗涤的测试物在完全细胞培养基(HAM F12培养基，含10％胎牛血清和50μg/ml艮它霉素)中于37℃孵育24小时，期间温和搅拌。抽样比为0.2ml测试物/ml培养基(约0.2g/ml)。用于测试的抽样样本不稀释，或者用新鲜细胞培养基1+3稀释。本测试包括阴性对照(聚丙烯样本，6cm2/ml)，阳性对照(锡-稳定化的聚氯乙烯样本，0.3cm2/ml)和作为未处理对照的以完全细胞培养基处理的培养物。在各时间点取3份细胞培养物进行平行处理，共48小时。对照处理表现正常表明测试系统功能和灵敏度正常。非稀释样本和稀释样本都表现为无毒(细胞毒性级数为0)。
溶血试验
检测分离柱基质的体外溶血活性(红细胞裂解)。按照ISO 10993-4指南的要求进行溶血试验。根据材料科学研究会(Material Science Institute，MSI，美国田纳西)的建议(1979)设计实验，测试物直接与无菌盐水中的兔血混合物接触。测试物是有涂层的琼脂糖珠粒用20％乙醇配制的浆状物。所述琼脂糖珠粒经洗涤后用等体积的无菌等渗盐水(0.9％NaCl)重悬，从而在试验前去除乙醇，如同分离柱基质在实际使用前需经洗涤一样。将测试物加入无菌等渗盐水中，比例为0.2ml测试物/ml盐水(约0.2g/ml)。37℃孵育39分钟后，加入兔血(20μl血/ml盐水)，继续孵育60分钟。实验包括阴性对照(等渗盐水)和阳性对照(蒸馏水)。所有处理方式进行一式三份。孵育期末，将混合物500xg离心5分钟。检测上清液的545nm吸光度。计算溶血百分比。本实验条件下，以分离柱基质处理的血样中，溶血量为-0.3％。因此认为，分离柱基质符合MSI溶血测试要求(溶血＜5％)。
以人血进行的凝血试验
体外检测分离柱基质影响人血样本凝血速度的能力。测试物是有涂层的琼脂糖珠粒用20％乙醇配制的浆状物。所述琼脂糖珠粒经洗涤后用等体积的无菌等渗盐水(0.9％NaCl)重悬，从而在试验前去除乙醇，如同分离柱基质在实际使用前需经洗涤一样。将洗涤后的测试物样本(0.2ml)加入测试管。制备了阴性对照(无处理)和阳性对照(富勒土(Fuller’s Earth))测试管。向各管中加入新鲜人血(1ml)。比例为每毫升(ml)血液约0.2ml测试物(约0.2g/ml)。将试管放入约37℃的水浴，规则性振荡。记录血样完全凝结所需的时间。用4人的血样各自对测试物和每个对照进行处理。对照处理的结果反映测试系统的有效性和灵敏度。
以基质处理的血样的平均凝血时间略有缩短，为阴性对照均值的91％。然而，这一缩短不算显著，因为四名供血者之间存在很大的试验偏差。由此认为，本试验中，分离柱基质不影响人血的凝血时间。
概述
基于进行的试验和对分离柱的评价可以认为，分离柱毒性极低，未见特异性毒性或靶器官。
实施例12-TECK-PEG-生物素合成概述
靶分子：
TECK(Met→Nleu取代)，对Lys72的ε-氨基侧链官能基进行PEG-生物素化(TFA盐)
修饰：
截短型人TECK对应于成熟蛋白的残基1-74，包含了趋化因子折叠的相应序列。成熟蛋白全长127个氨基酸(在150个氨基酸长的非成熟蛋白中，信号肽长23个氨基酸)。序列中的单个甲硫氨酸被改为正亮氨酸，避免链组装期间该残基的氧化，天然序列衍生物的合成中曾发现有这一氧化反应。由蛋白质N-末端的Gln在生理条件下形成焦谷氨酸(pyroGlu)。这样，序列的Gln1被取代为焦谷氨酸，避免产生N-末端Gln和N-末端pyroGlu混合物。这能够提高合成得率，并确保分离柱制造和使用过程中的趋化因子均一性。在树脂上，对第72位上的天然赖氨酸进行生物素化修饰。在ε-氨基官能基与生物素之间引入PEG间隔基。
线性氨基酸序列(SEQ ID NO：1)如下所示，在氨基酸72(K)上接装PEG间隔基和生物素分子之前：
H-PyrGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKRHRK VCGNPKSREVQRANleKLLDARNKVF-OH
在固相载体上组装经改造的TECK序列，采用固相肽合成Fmoc方案：
H-PyrGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKRHRK VCGNPKSREVQRANleKLLDARNK(Dde)VF-树脂
引入FmocLys(Dde)-OH作为残基72，以利该位置上的定点标记。
Met→Nle取代
N-末端Gln的焦谷氨酸取代
去除Dde保护：
去除Dde保护基：全部树脂(2.5g)用2％的肼DMF(100ml)溶液处理1小时以上，得到2.0g树脂。
标记步骤：
1.Fmoc-8-氨基-3，6-二辛酸(Fmoc-8-amino-3，6-dioctanoic acid)偶联
树脂(1.5g)在DMF(2ml)中溶胀，然后加入Fmoc-8-氨基-3，6-二辛酸溶液(0.38g，1mmol)，DIC溶液(2ml，0.2M，以DMF配制)和HOCt溶液(2ml，0.2M，以DMF配制)。对混合物超声处理2小时，然后以DMF洗涤。
2.封端(Cap)
用0.5M无水乙酸/DMF溶液(20ml)对树脂进行封端处理5分钟，然后用DMF洗涤。
3.Fmoc去保护
Fmoc去保护：用20％的哌啶DMF溶液(2x50ml)处理，每次15分钟。然后用DMF洗涤树脂。
4.生物素-OSu偶联
将生物素-NHS酯(341mg，1mmol)与DIPEA(348ul)在DMF(10ml)中形成的溶液加入树脂，对混合物超声处理3小时。用DMF和DCM彻底清洗树脂，然后真空干燥。得干树脂1.5g。
切割：
干燥的肽树脂(1.5g)和混合物用含有由TIS、硫代苯甲醚、水、EDT和苯酚构成的组合清除剂的TFA(30ml)切割，混合物于室温下搅拌6小时。将溶液滤入冷醚中，用TFA洗涤树脂。肽经离心、醚洗涤、离心和冷冻干燥后得1.0g粗肽。
折叠：
将粗肽(100mg)溶于6M GnHCl(233ml)，然后加入50mM含0.5mM GSSG和5mM GSH的TRIS pH8(467ml)迅速稀释到2M GnHCl浓度。混合物室温下搅拌2.5天，然后进行HPLC(Jupiter C18，250x4.6mm型柱，10-60％B，30分钟以上。HPLC验证所需产物和误折叠副产物的形成。
纯化：
反相HPLC纯化折叠蛋白，用Jupiter C18，250x21mm柱，9ml/min，10-60％B，50分钟以上。得到11.1mg纯的折叠Nle-TECK-生物素。
图13显示经纯化折叠生物素-TECK(Nleu)的HPLC。蛋白质为第21.6分钟洗出的单峰。
图14显示经纯化折叠生物素-TECK(Nleu)的电喷离子化串联质谱(ES/MS)数据。预计分子量为8959.4Da。
功能试验数据
用Aequorin试验检测TECK-生物素-Nle的抗hCCR9(Euroscreen)活性，测得EC50值为63.6nM。参考值：天然TECK的EC50为67.87nM。
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本发明不受所述具体实施方式的限制。事实上，根据以上描述和附图，本文所述之外的各种本发明改换模式对于本领域技术人员来说是显而易见的。这些改换都属于权利要求所述范围之内。并且，文中所述的所有实施方式都广泛适合于彼此联合。
本文所引用的诸多参考文献都通过引用纳入本文。
Claims (37)
1.一种装载了固相载体的分离柱，所述固相载体含有直接或间接固定于所述载体上的一种或多种趋化因子，从而能够从患者的外周血中去除表达所述一种或多种趋化因子的相应受体的细胞。
2.如权利要求1所述的分离柱，其中，所述一种或多种趋化因子是生物素化的，所述载体上固定有链霉亲和素，并且，所述一种或多种生物素化的趋化因子结合于载体上的链霉亲和素。
3.如权利要求1或2所述的分离柱，其中，所述一种或多种趋化因子通过间隔基生物素化。
4.如权利要求3所述的分离柱，其中，所述间隔基是聚乙二醇间隔基。
5.如前述权利要求中任一项所述的分离柱，其中，所述载体含有分子量大于100kDa的糖，或由分子量大于100kDa的糖构成，所述糖可选交联糖。
6.如权利要求5所述的分离柱，其中，所述糖是交联琼脂糖。
7.如前述权利要求中任一项所述的分离柱，其中，所述一种或多种趋化因子选自：CCL25，MIP-1a，MIP-1b，MCP-1，MCP-2，MCP-3，MCP-4，TARC，MDC，MIP-3，MIP-3a，MIP3b，MIP-4，I-309，HCC-1，HCC-2，SLC，IL-8，GROa，GROb，GROg，RANTES，NAP-2，ENA78，GCP-2，IP-10，MIG，I-TAC，SDF，CX3C趋化因子，淋巴细胞趋化因子，嗜酸粒细胞趋化蛋白，嗜酸粒细胞趋化蛋白-2，I-309，BLC。
8.如权利要求7所述的分离柱，其中，所述一种或多种趋化因子是CCL25。
9.如前述权利要求中任一项所述的分离柱，其中，所述载体的形式为球形、珠粒或不规则形的颗粒，平均粒径为50μm至2mm。
10.如前述权利要求中任一项所述的分离柱，其中，所述载体经试剂处理而具有抗凝血特性，例如肝素化的载体。
11.如前述权利要求中任一项所述的分离柱的固相载体。
12.一种从患者的外周血中去除表达一种或多种趋化因子相应受体的细胞方法，包括：
a.将采集的外周血与固定在固相载体上的一种或多种趋化因子接触足够长的时间以使所述细胞附着在所述载体上；和
b.将去除了所述细胞的血液与所述载体分离。
13.如权利要求12所述的方法，还包括在步骤a之前采集患者的外周血。
14.如权利要求12或13所述的方法，还包括在步骤b之后将去除了所述细胞的血液回输给所述患者。
15.如权利要求12至14中任一项所述的方法，其中，所述细胞是白细胞。
16.如权利要求15所述的方法，其中，所述白细胞选自：T淋巴细胞，单核细胞，中性粒细胞或嗜酸粒细胞。
17.如权利要求12至16中任一项所述的方法，该方法被用于治疗炎症。
18.如权利要求17所述的方法，其中，所述炎症炎性肠病(IBD)。
19.如权利要求18所述的方法，其中，所述IBD是溃疡性结肠炎或克罗恩病。
20.权利要求12至19中任一项所述的方法，其中，所述一种或多种趋化因子是生物素化的，所述载体上固定有链霉亲和素，并且，所述一种或多种生物素化的趋化因子结合于载体上的链霉亲和素。
21.如权利要求20所述的方法，其中，所述一种或多种趋化因子通过间隔基生物素化。
22.如权利要求21所述的方法，其中，所述间隔基是聚乙二醇间隔基。
23.如权利要求12至22中任一项所述的方法，其中，所述一种或多种趋化因子选自：CCL25，MIP-1a，MIP-1b，MCP-1，MCP-2，MCP-3，MCP-4，TARC，MDC，MIP-3，MIP-3a，MIP3b，MIP-4，I-309，HCC-1，HCC-2，SLC，IL-8，GROa，GROb，GROg，RANTES，NAP-2，ENA78，GCP-2，IP-10，MIG，I-TAC，SDF，CX3C趋化因子，淋巴细胞趋化因子，嗜酸粒细胞趋化蛋白，嗜酸粒细胞趋化蛋白-2，I-309，BLC。
24.如权利要求23所述的方法，其中，所述一种或多种趋化因子是CCL25。
25.如权利要求12至24中任一项所述的方法，其中，连续地自患者采集外周血，并且清除了所述细胞的血液连续地回输给所述患者。
26.如权利要求12至25中任一项所述的方法，其中，所述载体设置在一柱体内，血液流动通过所述柱体。
27.如权利要求12至26中任一项所述的方法，其中，所述载体的形式为球形、珠粒或不规则形的颗粒，平均粒径为50μm至2mm。
28.如权利要求12至27中任一项所述的方法，其中，所述载体经试剂处理而具有抗凝血特性，例如肝素化的载体，并且/或着，外周血是经抗凝剂处理过的。
29.权利要求1至10中任一项所述的分离柱或权利要求11所述的载体用于治疗炎症的用途。
30.如权利要求29所述的用途，其中，所述炎症是IBD。
31.权利要求1至10中任一项所述的分离柱或权利要求11所述的载体用于治疗以表达一种或多种趋化因子的相应受体的细胞含量升高为特征的疾病的用途。
32.如权利要求31所述的用途，其中，所述细胞数是患者外周血中的白细胞，尤其是一种或多种以下细胞：淋巴细胞，单核细胞，中性粒细胞或嗜酸粒细胞。
33.用于治疗炎症的趋化因子，其中，所述趋化因子固定在固相载体上。
34.固定在固相载体上的趋化因子在制造治疗炎症的药物中的用途。
35.如权利要求33所述的趋化因子或如权利要求34所述的用途，其中，所述炎症是IBD。
36.截短型CCL25趋化因子，含有如SEQ ID NO：1所示的氨基酸序列，其第72位(赖氨酸)生物素化，目的是让该趋化因子能够固定到固相载体上。
37.如权利要求36所述的趋化因子，还包含位于生物素与所述赖氨酸残基之间的PEG间隔基。
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| AU2009290694A1 (en) | 2010-03-18 |
| US20110224645A1 (en) | 2011-09-15 |
| EP2331568A2 (en) | 2011-06-15 |
| WO2010029317A3 (en) | 2011-03-31 |
| WO2010029317A8 (en) | 2011-05-05 |
| CA2736632C (en) | 2018-07-31 |
| KR20110057222A (ko) | 2011-05-31 |
| EA201100452A1 (ru) | 2011-12-30 |
| JP5611209B2 (ja) | 2014-10-22 |
| CA2736632A1 (en) | 2010-03-18 |
| ZA201101823B (en) | 2014-08-27 |
| AU2009290694B2 (en) | 2014-09-11 |
| EP2331568B1 (en) | 2014-09-03 |
| JP2012501708A (ja) | 2012-01-26 |
| IL211587A0 (en) | 2011-05-31 |
| WO2010029317A2 (en) | 2010-03-18 |
| KR101703785B1 (ko) | 2017-02-07 |
| EA023912B1 (ru) | 2016-07-29 |
| US8877177B2 (en) | 2014-11-04 |
| CN106823033A (zh) | 2017-06-13 |
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