CN1186352C - HAb18G/CD147肽类拮抗剂及其制备方法和用途 - Google Patents
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Abstract
HAb18G(/CD147)肽类拮抗剂及其制备方法和用途涉及生物技术及生物医药领域。本发明是能抗间质降解和抗炎的一组多肽类物质。本发明首次以肝癌转移相关因子HAb18G为靶向分子，设计、筛选并合成它的肽类拮抗剂；阻断效应细胞(成纤维细胞、滑膜细胞)诱导分泌基质金属蛋白酶(MMPs)的功能；减少肿瘤组织间质的降解、关节软骨侵蚀、新生血管(血管翳)的形成；达到治疗肝癌等肿瘤的复发、转移，以及类风湿性关节炎和骨关节炎的目的。经体内外实验证明，这类肽类拮抗剂对肝癌细胞转移的抑制率为4.35%～65.22%，HAb18G(/CD147)分子在滑膜细胞上有明确定位。因此，该类肽类拮抗剂极具新药开发的前景。
Description
技术领域
本发明涉及生物技术及生物医药领域，特别是HAb18G/CD147肽类拮抗剂及其制备方法和用途。
背景技术
目前全世界每年新增1000万癌症患者，而且癌症的发病率呈急剧上升趋势。据世界卫生组织(WHO)估计，每年全世界死于癌症患者约600万。我国1995年主要疾病死亡率及死亡原因构成中，恶性肿瘤排列第2位，占死亡总人数的21.85％。其中发病率较高的肿瘤有胃癌(占癌总死亡率21.76％)、肝癌(17.83％)、肺癌(15.19％)、食管癌(15.02％)等。长期以来，由于肿瘤高复发、高转移的恶性特点，一直是治疗疗效亟待解决的问题。
目前国内外抗癌治疗关注的热点之一是抗肿瘤的浸润、转移。肿瘤的转移规律极其复杂，涉及诸多因素，如：(1)蛋白水解酶；(2)血管生成因子；(3)粘附因子，如整合素，选择素，钙粘合素等；(4)相关运动因子；(5)瘤细胞信号转导。在肿瘤的侵袭、转移过程中，癌细胞诱导效应细胞分泌蛋白水解酶，降解细胞外基质，其中基质金属蛋白酶(MMPs)最为重要，目前已发现了20余种，它们几乎能降解细胞外基质的所有成分。MMPs的作用可概括为(1)降解间质，破坏胶原限制肿瘤生长的机械屏障作用；(2)破坏基底膜完整性，利于癌细胞穿过血管壁进入血流；(3)通过对细胞外基质的改建，促进肿瘤新生血管或转移灶的形成。由于MMPs在肿瘤侵袭转移中的重要作用，因而肿瘤组织中MMPs的来源一直是人们研究的重点问题。近年来多个实验室研究发现，CD147分子与肿瘤细胞膜有关，可刺激肿瘤间质成纤维细胞分泌MMPs，且最近的一篇文献报道，CD147分子不仅能刺激MMPs的产生，而且还与MMPs在肿瘤细胞表面形成复合物，这种复合物的形成相当于将MMPs浓缩在肿瘤细胞周围，有利于肿瘤细胞周围基质的降解，使肿瘤细胞易于扩散和转移。
目前，抗间质降解多采用酶抑制剂，如天然酶抑制剂—组织型金属蛋白酶抑制剂(TIMPs)，能够与MMPs结合并抑制酶活性，但TIMPs在组织中含量极微且难于提取，人工重组TIMPs是大分子蛋白质，存在降解和口服吸收不良等问题。人工合成的拟肽类金属蛋白酶抑制剂，如Batimastat(BB-94)，只能抑制早期肿瘤诱导产生的MMPs的活性，几乎不能抑制晚期肿瘤诱导产生的大量MMPs的活性，导致III期临床实验的停止。
类风湿性关节炎(rheumatoid arthritis，RA)在我国患病人数达300余万。发病年龄轻，致残率高。其临床特点为累及到周身关节的增殖性和侵蚀性滑膜炎，呈进行性病变终生反复不断。被认为是类似“局限性恶性肿瘤”的增生性和破坏性病变，自发性缓解极为罕见。晚期由于关节软骨及骨的损伤而导致关节强直、畸形和功能障碍，病变亦可侵犯浆膜、心肺、动脉、神经及眼等结缔组织。
RA组织病理学特征是滑膜衬里过度增生和衬里下层大量炎性单个核细胞浸润。成纤维细胞样滑膜细胞和滑膜组织巨噬细胞在关节损伤中起最重要的作用。这两种细胞均能产生大量MMPs。此外，骨关节炎与老龄化密切相关，随着人类寿命的延长发病率增高，软骨细胞、滑膜细胞和成纤维细胞产生MMPs，亦在骨关节炎发病中起重要作用。这些酶以协同作用方式能够消化滑膜、关节软骨和软骨下骨的所有主要的细胞外基质组分。RA滑膜中EMMPRIN(CD147)的表达上调，可诱导局部产生MMPs 1、2、3等蛋白水解酶，且可导致MMPs和TIMPs的不平衡最终引起RA关节的破坏。
此病至今尚无特效治疗。90年代以来，国内外学者采用仿效结核病和恶性肿瘤的联合疗法，是此类疾病治疗学上的一大突破，使部分患者病情得到较长期缓解，生活质量得到提高，但并未治愈疾病。抑制软骨和骨侵蚀仍是此病治疗亟待解决的问题。但迄今有关应用酶抑制剂抗间质降解的临床实验结果与上述抗肿瘤转移治疗一样令人失望。
发明内容
本发明提供一种具有抗肿瘤复发、转移和抗类风湿性关节炎、骨关节炎的作用的HAb18G/CD147肽类拮抗剂及其制备方法和用途，从高位阻断效应细胞分泌MMPs，则对肿瘤复发转移的治疗可能更为有效。筛选有效的EMMPRIN(CD147)肽类拮抗剂，在高位阻断效应细胞分泌MMPs，也是类风温性关节炎和骨关节炎治疗的新途径和发展趋势。
本发明是以下述方式实现的：HAb18G/CD147肽类拮抗剂，针对靶抗原HAb18G/CD147的肽类拮抗剂具有如下其中一种氨基酸序列，
(1)Met Thr His Asp Pro Val Ile Ser Leu Pro Thr Thr
(2)Lys Tyr Pro His Gln His Leu His Met His Asp Ser
(3)Tyr Pro Phe His His Lys His Trp His Arg Pro Asn
(4)Ala Asn Ile Val Pro Ile His Ala Asn His Phe Gln
(5)Tyr Lys Leu Pro Gly His His His His Tyr Arg Pro
所述的HAb18G/CD147肽类拮抗剂的制备方法如下所述：
用人新鲜肝癌组织免疫小鼠而得到鼠源性肝癌高特异性单克隆抗体HAb18，将该单抗交联在凝胶颗粒Sepharose-4B上，制成亲和层析柱，将大量培养的肝癌细胞裂解，裂解液经上述的亲和层析柱结合、洗脱、收集过程，得到纯的肝癌抗原HAb18G。
肝癌抗原HAb18G经SDS-PAGE及Western blot检测，为单一条带，分子量约60kd。
用此单抗从人肝癌组织cDNA文库中筛选得到其相应抗原分子HAb18G，其核苷酸序列与白细胞分化抗原CD147分子核苷酸序列高度同源。
用纯的肝癌抗原HAb18G筛选噬菌体随机12肽库，筛选到5条HAb18G结合肽段序列，通过固相合成法合成这5条多肽。
HAb18G/CD147肽类拮抗剂作为制备抗肿瘤复发、转移和抗类风湿关节炎、骨关节炎治疗剂的应用。
本发明人在长期从事肝癌免疫导向药物研究的基础上，获得肝癌高亲和力单抗HAb18的相应膜抗原HAb18G的cDNA全长序列，发现其与CD147的序列高度同源，通过基底膜侵袭实验和明胶酶谱实验分析，体外实验表明HAb18G(/CD147)与其效应细胞(癌周成纤维细胞)结合，诱导产生MMPs，使肿瘤组织中MMPs含量及活性显著提高，间质成分和血管基底膜的胶原蛋白过度降解，癌细胞穿过基底膜及结缔组织屏障不断侵袭扩散，证实HAb18G分子在肝癌中发挥的作用同CD147分子在其它肿瘤上发挥的作用相同，可大大促进肝癌细胞的侵袭和转移。基于以上研究基础，以及HAb18G在正常肝细胞中不表达，在肝癌上为高表达的特点，故可通过阻断HAb18G(/CD147)分子的作用来抑制肝癌转移。本发明首次以肝癌转移相关因子HAb18G为靶向分子，设计、筛选并合成它的肽类拮抗剂；阻断效应细胞(成纤维细胞、滑膜细胞)诱导分泌基质金属蛋白酶(MMPs)的功能；减少肿瘤组织间质的降解、关节软骨侵蚀、新生血管(血管翳)的形成；达到治疗肝癌等肿瘤的复发、转移，以及类风湿性关节炎和骨关节炎的目的。明胶酶谱实验结果与细胞侵袭力抑制实验结果相符。体内功能实验用合成多肽对荷人肝细胞癌裸鼠进行治疗，发现其中3号和5号多肽对肝癌成瘤性有明显抑制作用。经体内外实验证明，这类肽类拮抗剂对肝癌细胞转移的抑制率为4.35％～65.22％，因此，该类肽类拮抗剂极具新药开发的前景。
鉴于MMPs通过直接降解软骨、骨组织和间接促进血管形成而参与RA关节破坏。因此抑制MMPs是治疗RA的基本途径之一。我们经免疫组化法证明HAb18G(/CD147)分子在滑膜细胞上有明确定位。所以，该拮抗剂可能在类风湿关节炎及骨关节炎等疾病的治疗中具有潜在的应用价值。
附图说明
附图为HAb18G核苷酸序列和CD147核苷酸序列。
具体实施方式
1.制备HAb18G纯抗原：
(1)将用人新鲜肝癌组织免疫小鼠而得到鼠源性肝癌高特异性单克隆抗体HAb18交联在Sepharose-4B凝胶介质上，制成亲和层析柱；
(2)将大量培养的肝癌细胞(HHCC)裂解，经离心、透析后，上亲和层析柱，结合、洗脱、收集，得到HAb18G纯抗原。
2.肝癌抗原HAb18G经SDS-PAGE及Western blot检测，为单一条带，分子量约60kd。
3.用单克隆抗体HAb18筛选肝癌细胞cDNA表达文库，得到HAb18G的cDNA序列，其序列与Genbank中CD147分子的序列高度同源，参见附图。
4.用HAb1gG纯抗原筛选噬菌体随机12肽库，获得5条HAb18G结合肽段，它们的序列如下：
(1)Met Thr His Asp Pro Val Ile Ser Leu Pro Thr Thr
(2)Lys Tyr Pro His Gln His Leu His Met His Asp Ser
(3)Tyr Pro Phe His His Lys His Trp His Arg Pro Asn
(4)Ala Asn Ile Val Pro Ile His Ala Asn His Phe Gln
(5)Tyr Lys Leu Pro Gly His His His His Tyr Arg Pro
5.将这5条肽进行体外化学合成、纯化，经FPLC鉴定纯度达99％以上。
6.体外功能实验：采用细胞侵袭力抑制实验，即将待验证的肽类拮抗剂加入到底层铺有Matrigel胶的肝癌细胞和人成纤维细胞的共培养物中，经过20h左右的培养后，检测穿过Matrigel胶的肝癌细胞较对照是否有减少，并计算肽类拮抗剂对肝癌细胞转移的抑制率。结果如下：
组别 穿过细胞数 抑制率(％)
对照组 23
1号肽 14 39.13
2号肽 13 43.48
3号肽 8 65.22
4号肽 22 4.35
5号肽 10 56.52
这些肽类拮抗剂中4条肽具有明显抑制肝癌侵袭、转移的功能，最高抑制率可达65.22％。我们还采用了明胶酶谱方法结果与细胞侵袭力抑制实验一致。
7.体内功能实验：将肝癌细胞HHCC接种到裸鼠双侧胁部，接种后立即经尾静脉注入HAb18G(/CD147)肽类拮抗剂(2.5mg/kg)，同时设立对照。实验证明，其中1号和3号肽类拮抗剂对肝癌成瘤性有明显抑制作用。当对照组在第11天每只裸鼠都成瘤时，其中3号肽类拮抗剂试验组裸鼠均未成瘤。此外，治疗组平均生存期比对照组延长一倍以上。
8.免疫组化法证明HAb18G(/CD147)在滑膜中表达：将HAb18G(/CD147)单抗与类风湿关节炎和骨关节炎滑膜组织经免疫组化染色，发现滑膜细胞有HAb18G(/CD147)分子的表达。
9.新型制剂的研究：对HAb18G(/CD147)肽类拮抗剂进行改造根据三维定量构效关系分析的结果，将拮抗剂序列中的L-型氨基酸置换为D-型氨基酸，或用纳米脂质体包裹制备制剂，以延长其半衰期，增加其在体内的生物利用度及其活性。
Claims (3)
1、HAb18G/CD147肽类拮抗剂，其特征在于：针对靶抗原HAb18G/CD147的肽类拮抗剂具有如下其中一种氨基酸序列，
(1)Met Thr His Asp Pro Val Ile Ser Leu Pro Thr Thr
(2)Lys Tyr Pro His Gln His Leu His Met His Asp Ser
(3)Tyr Pro Phe His His Lys His Trp His Arg Pro Asn
(4)Ala Asn Ile Val Pro Ile His Ala Asn His Phe Gln
(5)Tyr Lys Leu Pro Gly His His His His Tyr Arg Pro.
2、制备如权利要求1所述的HAb18G/CD147肽类拮抗剂的方法，其特征在于：
用人新鲜肝癌组织免疫小鼠而得到鼠源性肝癌高特异性单克隆抗体HAb18，将该单抗交联在凝胶颗粒Sepharose-4B上，制成亲和层析柱，将大量培养的肝癌细胞裂解，裂解液经上述的亲和层析柱结合、洗脱、收集过程，得到纯的肝癌抗原HAb18G；
肝癌抗原HAb18G经SDS-PAGE及Western blot检测，为单一条带，分子量约60kd；用肝癌单抗HAb18从人肝癌组织cDNA文库中筛选得到其相应抗原分子HAb18G，其核苷酸序列与白细胞分化抗原CD147分子核苷酸序列高度同源；
用纯的肝癌抗原HAb18G筛选噬菌体随机12肽库，筛选到5条HAb18G结合肽序列，用固相合成法合成这5条短肽。
3、权利要求1所述的HAb18G/CD147肽类拮抗剂作为制备抗肿瘤复发、转移和抗类风湿关节炎、骨关节炎治疗剂的应用。
Priority Applications (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01131735 CN1186352C (zh) | 2001-09-28 | 2001-09-28 | HAb18G/CD147肽类拮抗剂及其制备方法和用途 |
| JP2002592349A JP2005504516A (ja) | 2001-05-25 | 2002-05-27 | ＨＡｂ１８Ｇ／ＣＤ１４７における拮抗剤及びその応用 |
| EP02734987A EP1403284B1 (en) | 2001-05-25 | 2002-05-27 | HAb18G/CD147, ITS ANTAGONIST AND APPLICATION |
| DE60230269T DE60230269D1 (de) | 2001-05-25 | 2002-05-27 | HAb18G/CD147, DESSEN ANTAGONIST UND ANWENDUNG |
| US10/478,647 US20050026841A1 (en) | 2001-05-25 | 2002-05-27 | Hab18g/cd147 its antagonist and application |
| AT02734987T ATE417064T1 (de) | 2001-05-25 | 2002-05-27 | Hab18g/cd147, dessen antagonist und anwendung |
| PCT/CN2002/000356 WO2002094875A1 (fr) | 2001-05-25 | 2002-05-27 | Hab18g/cd147, son agoniste et son utilisation |
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| CN 01131735 CN1186352C (zh) | 2001-09-28 | 2001-09-28 | HAb18G/CD147肽类拮抗剂及其制备方法和用途 |
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