CN107827984B - 嵌合抗ROR1抗体Fab分子及其制备方法和应用 - Google Patents
嵌合抗ROR1抗体Fab分子及其制备方法和应用 Download PDFInfo
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Abstract
嵌合抗ROR1抗体Fab分子及其制备方法和应用，包括通过链间二硫键连接的重组轻链L和重组重链Fd，所述重组轻链L的氨基酸序列如SEQ ID NO.1所示，重组重链Fd的氨基酸序列如SEQ ID NO.2所示。本发明大大降低了抗ROR1抗体的鼠源性成分，仍保存人鼠交叉反应的特点，有利于进一步开展体内试验。保留了鼠源性抗体的模拟抗原特性，可潜在用于高表达ROR1的肿瘤的靶向诊疗。原核表达载体易于构建，可大量表达，生产快速且便于纯化。有利于制备嵌合全分子抗体或抗体的人源化改造，进一步降低抗ROR1抗体中的鼠源成分，以开展高表达ROR1的肿瘤的靶向诊疗研究。
Description
技术领域：
本发明属于基因工程技术和免疫靶向诊疗领域，具体涉及一种嵌合抗ROR1抗体Fab分子及其制备方法和应用。
背景技术：
自1986年美国FDA首次批准单克隆抗体药物—莫罗单抗(Muromomab-CD3)应用于临床治疗以来，已有数十种靶向制剂用于肿瘤治疗，均展现出良好的肿瘤特异性和低毒性，成为肿瘤生物治疗的一大亮点。靶向治疗具有较好的选择性，通过抗体对靶分子的识别，可特异性结合肿瘤细胞膜受体，阻断信号传递过程，进而抑制肿瘤细胞的生长和扩散。近年来，抗体介导的肿瘤靶向治疗技术已在大肠癌、乳腺癌、肺癌等多种肿瘤的临床治疗中取得了令人瞩目的成就。然而在卵巢癌领域，大多数单抗介导的靶向治疗还处于临床前试验阶段，尽管针对肿瘤血管生成的单克隆抗体(Bevacizumab等)已用于卵巢癌的治疗，但由于组织特异性差，会在不同程度上与正常组织结合，而其所针对的靶分子在卵巢癌组织中的平均表达量和表达率较低，故临床治疗应用前景不明。因此，积极寻找卵巢癌组织中合适的靶分子已成为妇科肿瘤防治的当务之急。
多项研究表明ROR1在慢性淋巴细胞白血病、乳腺癌、神经母细胞瘤、肾癌等多种肿瘤细胞中过表达，本课题组的前期研究中分析了ROR1与卵巢癌之间的关系，结果发现：①组织芯片技术检测结果显示，卵巢癌组织中ROR1表达的阳性率高达55％，而正常卵巢组织中仅为6％；②定量PCR结果表明不同组织类型的卵巢癌组织中ROR1的表达水平显著高于正常卵巢组织；③ROR1主要存在于卵巢癌细胞胞膜，少部分见于胞核；④ROR1的表达水平与卵巢癌的FIGO分期及预后密切相关，可作为卵巢癌预后的一个独立预测。以上结果提示，ROR1可能成为卵巢癌免疫治疗新的特异性靶点。
抗体药物是以细胞工程技术和基因工程技术为主体的抗体工程技术制备的药物，其在感染、心血管疾病、自身免疫性疾病、肿瘤治疗中有巨大的潜力与应用前景。由于鼠源单克隆抗体的临床应用的产生人抗鼠抗体反应(HAMA)和过敏反应，减低了药物的疗效，因而运用基因工程技术将鼠源抗体进行改造，降低HAMA反应同时提高治疗效果，成为该领域的研究热点。
发明内容
解决的技术问题：本发明提供一种嵌合抗ROR1抗体Fab分子及其制备方法和应用，该蛋白保持了鼠源性抗ROR1的抗原特异性，在用于高表达ROR1肿瘤的靶向诊疗药物中具有潜在的应用前景。
技术方案：嵌合抗ROR1抗体Fab分子，包括通过链间二硫键连接的重组轻链L和重组重链Fd，所述重组轻链L的氨基酸序列如SEQ ID NO.1所示，重组重链Fd的氨基酸序列如SEQ ID NO.2所示。
编码重组轻链L的核酸序列如SEQ ID NO.3所示，编码重组重链Fd的核酸序列如SEQ IDNO.4所示。
嵌合抗ROR1抗体Fab分子的制备方法，将鼠源性单克隆抗体的重链可变区VH、轻链可变区VL通过重叠延伸PCR技术分别和人IgG1抗体的重链恒定区CH1和轻链恒定区CL片段连接，获得重组轻链L和重组重链Fd；经过限制性核酸内切酶酶切，L和Fd与表达载体pETDUET-1连接，构建得到嵌合Fab片段的原核表达载体pETDuet-ROR1-cFab；将表达载体pETDuet-ROR1-cFab转入大肠杆菌BL21后加入IPTG诱导表达嵌合的Fab抗体片段，该嵌合Fab抗体的重组重链Fd和重组轻链L通过链间二硫键连接组成抗ROR1抗体人鼠嵌合Fab分子。
所述嵌合抗ROR1抗体Fab分子在制备诊疗高表达ROR1的肿瘤药物中的应用。
一种诊疗高表达ROR1的肿瘤药物，有效成分为上述嵌合抗ROR1抗体Fab分子。
本发明是利用基因重组技术对一株鼠源性的抗ROR1抗体进行改造，即将鼠源性单克隆抗体的重链可变区(VH)、轻链可变区(VL)通过重叠延伸PCR技术分别和人IgG1抗体的重链恒定区(CH1)和轻链恒定区(CL)片段连接，获得重组轻链(L)和重链(Fd)。经过限制性核酸内切酶酶切，L和Fd与表达载体pETDUET-1连接，构建嵌合Fab片段的原核表达载体pETDuet-ROR1-cFab。将表达载体pETDuet-ROR1-cFab转入大肠杆菌BL21后加入IPTG诱导表达嵌合的Fab抗体片段，该嵌合Fab抗体的重链(Fd)和轻链(L)通过链间二硫键连接组成抗ROR1抗体人鼠嵌合Fab分子，由于重链(Fd)分子量和轻链(L)分子量相近，经蛋白纯化系统得到抗ROR1抗体Fab段在WB图上的条带大小约为27KDa。该蛋白保持了鼠源性抗ROR1的抗原特异性，在用于高表达ROR1肿瘤的靶向诊疗药物中具有潜在的应用前景。
鉴于我室建立的抗ROR1抗体为鼠源性的IgG型单克隆抗体，我们通过基因重组技术，将抗ROR1抗体鼠源性抗体的重链、轻链可变区和人IgG1抗体的CH1和CL片段进行重组，扩增了嵌合的Fab分子，并构建了嵌合Fab抗体片段的原核表达质粒并进行可溶性表达，建立了表达蛋白的纯化方法，获得了高纯度的可溶性蛋白，在高表达ROR1的肿瘤的靶向诊疗中具有潜在的应用前景。
有益效果：由于鼠源单克隆抗体的临床应用的产生人抗鼠抗体反应(HAMA)和过敏反应，减低了药物的疗效，因而运用基因工程技术将鼠源抗体进行改造，降低HAMA反应同时提高治疗效果，成为该领域的研究热点。抗体Fab片段由重链Fd段和完整的轻链组成，是完整抗体分子的三分之一，属于小分子抗体，具穿透力强，半衰期短的优势，尤其适用于人体疾病的靶向诊疗。申请人制备的抗ROR1抗体人鼠嵌合Fab分子，通过试验表明保留了鼠源性抗ROR1抗体的模拟抗原性质和特异性，可用于高表达ROR1的肿瘤的靶向诊疗。我们使用基因重组技术对该鼠源单克隆抗体进行改造制备嵌合Fab抗体片段，制备的抗体片段有以下优点：(1)大大降低了抗ROR1抗体的鼠源性成分，仍保存人鼠交叉反应的特点，有利于进一步开展体内试验。(2)保留了鼠源性抗体的模拟抗原特性，可潜在用于高表达ROR1的肿瘤的靶向诊疗。(3)原核表达载体易于构建，可大量表达，生产快速且便于纯化。(4)有利于制备嵌合全分子抗体或抗体的人源化改造，进一步降低抗ROR1抗体中的鼠源成分，以开展高表达ROR1的肿瘤的靶向诊疗研究。
附图说明
图1：鼠源性单克隆抗ROR1抗体轻链可变区(VL)、人IgG1抗体的轻链恒定区(CL)及重组轻链(L)PCR扩增产物电泳。M：核酸标准分子量(Takara DL2000)；1：VL基因片段；2：CL基因片段；3：L基因片段。
图2：鼠源性单克隆抗ROR1抗体重链可变区(VH)、人IgG1抗体的重链恒定区(CH1)及重组重链(Fd)PCR扩增产物电泳。M：核酸标准分子量(Takara DL2000)；1：VH基因片段；2：CH1基因片段；3：Fd基因片段。
图3：构建重组原核表达载体pETDuet-ROR1-cFab电泳结果。M：核酸标准分子量(TakaraDL10000)；1：空载体pETDUET-1质粒；2：连接重组轻链(L)的pETDUET-1质粒；3：连接重组轻链(L)的pETDUET-1质粒酶切得重组轻链(L)；4：连接重组轻链(L)和重组重链(Fd)的pETDUET-1质粒(pETDuet-ROR1-cFab)；5：pETDuet-ROR1-cFab质粒酶切得重组重链(Fd)。
图4：以原核表达载体pETDuet-ROR1-cFab为模板，扩增轻链(L)和重链(Fd)。M：核酸标准分子量(Takara DL2000)；1：L基因片段；2：Fd基因片段；
图5：嵌合Fab蛋白表达的SDS-PAGE电泳结果。M为蛋白质标准(Fermentas，#26616)，1：不含pETDuet-ROR1-cFab的大肠杆菌超声沉淀；2：不含pETDuet-ROR1-cFab的大肠杆菌超声上清；3：含pETDuet-ROR1-cFab的大肠杆菌超声沉淀；4：含pETDuet-ROR1-cFab的大肠杆菌超声上清；5：经IPTG过夜诱导的含pETDuet-ROR1-cFab的大肠杆菌超声沉淀；6:经IPTG过夜诱导的含pETDuet-ROR1-cFab的大肠杆菌超声上清。
图6：嵌合Fab蛋白表达的western-blot结果，以标记HRP的羊抗人Fab抗体为一抗。M为蛋白质标准(Fermentas，#26616)，1：不含pETDuet-ROR1-cFab的大肠杆菌超声沉淀；2：不含pETDuet-ROR1-cFab的大肠杆菌超声上清；3：含pETDuet-ROR1-cFab的大肠杆菌超声沉淀；4：含pETDuet-ROR1-cFab的大肠杆菌超声上清；5：经IPTG过夜诱导的含pETDuet-ROR1-cFab的大肠杆菌超声沉淀；6:经IPTG过夜诱导的含pETDuet-ROR1-cFab的大肠杆菌超声上清。
图7：嵌合Fab蛋白纯化的SDS-PAGE电泳结果。M为蛋白质标准(Fermentas，#26616)；1：纯化的Fab蛋白；2：经IPTG过夜诱导的含pETDuet-ROR1-cFab的大肠杆菌超声沉淀；3：经IPTG过夜诱导的含pETDuet-ROR1-cFab的大肠杆菌超声上清；4：纯化流穿。
图8：嵌合Fab蛋白纯化的western-blot结果，以标记HRP的羊抗人Fab抗体为一抗。M为蛋白质标准(Fermentas，#26616)；1：纯化的Fab蛋白；2：经IPTG过夜诱导的含pETDuet-ROR1-cFab的大肠杆菌超声沉淀；3：经IPTG过夜诱导的含pETDuet-ROR1-cFab的大肠杆菌超声上清；4：纯化流穿。
图9：以重组人ROR1蛋白为抗原，抗ROR1抗体嵌合Fab分子为一抗的ELISA结果图。
图10：以A2780和Iose386细胞总蛋白为天然抗原，抗ROR1抗体嵌合Fab分子为一抗的western-blot结果。1为A2780细胞总蛋白，2为Iose386细胞总蛋白。
图11：以抗ROR1抗体嵌合Fab分子为一抗FACS结果。
图12：以抗ROR1抗体嵌合Fab分子为一抗的免疫荧光结果。
具体实施方式
实施例1、抗ROR1抗体Fab分子的制备方法：
1)抗体可变区基因片段的扩增及验证：
用重组人ROR1蛋白(购于北京义翘神州科技有限公司)免疫3只8周雌性BALB/c小鼠(购于上海斯莱克实验动物有限责任公司)，每隔2周免疫一次，共3次。第三次免疫后，取小鼠血清测效价，取效价较高的小鼠冲刺免疫，3天后取该小鼠脾脏与骨髓瘤细胞进行细胞融合。经过三次亚克隆，筛选出高表达抗ROR1抗体的鼠源性杂交瘤细胞。将该鼠源性抗ROR1抗体杂交瘤细胞培养至对数生长期，用RNA提取试剂盒提取细胞总RNA，反转录获得单链cDNA。设计4条VL上游引物和1条VL下游引物，6条VH下游引物和2条VH下游引物进行抗体可变区基因片段的扩增：
将轻链可变区(VL)上游引物和下游引物终浓度调至100pmol/μL，然后以1:1体积比例混匀用来扩增轻链可变区(VL)；重链可变区(VH)上游引物和下游引物终浓度调至100pmol/μL，然后以1:1体积比例混匀用来扩增重链可变区(VH)。扩增条件为94℃5分钟，94℃30秒，56℃30秒，72℃50秒，30个循环；最后72℃延伸10分钟，16℃，停止。电泳回收、纯化扩增的基因片段，连至pMD-18T载体，转化大肠杆菌DH5α，测序后获得轻链、重链可变区基因序列。
2)嵌合Fab片段的扩增：
(1)抗ROR1抗体重链可变区(VH)和轻链可变区(VL)的扩增：
将测序正确的单克隆菌落，提取其质粒作为模板，分别扩增重链和轻链可变区。轻链可变区(VL)的上游引物为VLF：5′-CCATGGGCGAGCTCGTGATGACCCAG-3’，下游引物为VLR：5′-CAGCCTTGGGCTGACCTTTTATTTCCAACTTTGTC-3′，在VLF的5′端引入了NcoI的酶切识别位点即下划线部分序列，VLR的5′端引入了与人IgG1的CL上游引物5′端互补的16个碱基即斜体部分序列，以利于鼠源性抗ROR1抗体的VL与人IgG1的CL片段的重叠PCR扩增；重链可变区(VH)的上游引物为VHF：5′-CATATGGAGGTGCAGCTGGTGCAGTCTG-3′，下游引物为VHR：5′-GAACCCTGGTCACCGTCTCCGCCTCCACCAAGGGCCCA-3′，在VHF的5′端引入了NdeI的酶切识别位点即下划线部分序列，VHR的5′端引入了和人IgG1的CH1上游引物互补的20个碱基序列，即斜体部分，以利于抗ROR1抗体的VH与人IgG1的CH1片段的重叠PCR扩增。扩增条件均为94℃5分钟，94℃30秒，56℃30秒，72℃50秒，30个循环；最后72℃延伸10分钟，16℃，停止。琼脂糖凝胶电泳，分别扩增出约400bp、400bp的条带(见图1，2)，胶回收纯化扩增条带，溶于去离子水内，-20℃冻存备用。
(2)人IgG1抗体重链恒定区(CH1)和轻链恒定区(CL)段的扩增：
以本实验室保存的pFUSE-hIgG1载体为模板，分别扩增人IgG1抗体的CL和CH1片段；CL段的上游引物为CLF：5′-GACAAAGTTGGAAATAAAAGGTCAGCCCAAGGCTG-3′，下游引物为CLR:5′-AAGCTTTTATGAACATTCTGTAGGGGCCACT-3′，在CLR的5′端引入了HindIII的酶切识别位点即下划线部分序列；CH1的上游引物为CH1F:5′-GAACCCTGGTCACCGTCTCCGCCTCCACCAAGGGCCCA-3′，下游引物为CH1R:5′-GGTACCTTAAGAAGCGTAGTCCGGAAC-3′，在CH1R的5′端引入了kpnI的酶切识别位点即下划线部分序列。扩增条件均为94℃5分钟，94℃30秒，56℃30秒，72℃50秒，30个循环；最后72℃延伸10分钟，16℃，停止。琼脂糖凝胶电泳，分别扩增出约350bp、400bp的条带(见图1，2)，胶回收纯化扩增条带，溶于去离子水内，-20℃冻存备用。
(3)L链、Fd段基因片段的扩增：
以鼠源性抗ROR1抗体的VL及人IgG1抗体的CL的PCR扩增产物为模板，用上游引物LF:5′-CCATGGGCGAGCTCGTGATGACCCAG-3′和下游引物LR:5′-AAGCTTTTATGAACATTCTGTAGGGGCCACT-3′进行重叠延伸PCR扩增嵌合L链，扩增条件均为94℃5分钟，94℃30秒，56℃30秒，72℃50秒，30个循环；最后72℃延伸10分钟，16℃，停止。琼脂糖凝胶电泳，扩增出约750bp的条带(见图1)，胶回收扩增条带，连至pMD-18T载体，转化大肠杆菌DH5α，测序，序列正确的菌保存，并提质粒，用限制性内切酶NcoI/HindIII双酶切(37℃酶切2h)，琼脂糖凝胶电泳，胶回收，溶于去离子水内，-20℃冻存备用。
以鼠源性抗ROR1抗体的VH及人IgG1抗体的CH1的PCR扩增产物为模板，用上游引物FdF:5′-CATATGCAGGTGCAGCTGGTGCAGTCTG-3′和下游引物FdR:5′-GGTACCTTAAGAAGCGTAGTCCGGAAC-3′进行重叠延伸PCR扩增嵌合Fd片段(见图2)，扩增条件均为94℃5分钟，94℃30秒，56℃30秒，72℃50秒，30个循环；最后72℃延伸10分钟，16℃，停止。琼脂糖凝胶电泳，扩增出约800bp的条带(见图2)，胶回收扩增条带，连至pMD-18T载体，转化大肠杆菌DH5α，测序，序列正确的菌保存，提质粒，用限制性内切酶NdeI/kpnI双酶切(37℃酶切2h)，琼脂糖凝胶电泳，胶回收，溶于去离子水内，-20℃冻存备用。
3)嵌合Fab原核表达载体的构建和鉴定：
提取质粒pETDuet-1，用限制性内切酶NcoI/HindIII双酶切(37℃酶切2h)，电泳后胶回收酶切的质粒大片段，溶于去离子水中。取pETDuet-1、L链的酶切产物按1:7摩尔比混匀，在同一离心管内用T4连接酶16℃过夜连接。将连接产物转化感受态大肠杆菌DH5α，涂布含氨苄青霉素(100μg/mL)LB平板，倒置37℃12-16h。次日随机挑取转化菌及空质粒转化对照菌，37℃摇菌5小时后，用L链引物对菌液进行PCR扩增鉴定，菌液PCR验证扩增出大小正确条带的菌液送生物公司进行测序，序列正确的菌保存，并提质粒(pETDuet-1-L)。
用限制性内切酶NdeI/kpnI双酶切连有L链的质粒(pETDuet-1-L)(37℃酶切2h)，电泳后胶回收酶切的质粒大片段，溶于去离子水中。取pETDuet-1-L、Fd段按1:7摩尔比混匀，在同一离心管内用T4连接酶16℃过夜连接。将连接产物转化感受态大肠杆菌DH5α，涂布含氨苄青霉素(100μg/mL)LB平板，倒置37℃12-16h。次日随机挑取转化菌及空质粒转化对照菌，37℃摇菌5小时后，用Fd段引物对菌液进行PCR扩增鉴定，菌液PCR验证扩增出大小正确条带的菌液送生物公司进行测序，序列正确的菌保存，提质粒(pETDuet-1-L-H)即pETDuet-ROR1-cFab(见图3)。以载体pETDuet-ROR1-cFab为模板，分别用L链引物和Fd段引物进行PCR扩增鉴定，证明重组原核表达载体pETDuet-ROR1-cFab构建成功(见图4)。
将含有正确重组质粒的菌液按1:100转入2mL LB溶液中，氨苄青霉素工作浓度为100μg/mL，37℃摇菌过夜；次日按1：100转接入2mL LB中(氨苄青霉素终浓度为100μg/mL)，37℃培养OD600＝1.0左右，加入异丙基-Β-D-硫代半乳糖苷(IPTG)至终浓度为1mmol/L，16℃振荡培养12h。离心收集菌体，菌体超声后收集超声上清及超声沉淀，进行SDS-PAGE及western-blot检测，结果嵌合Fab片段在菌体超声上清及超声沉淀中均有表达，其中可溶性蛋白主要是在菌体超声上清中，Fab片段分子量约为27KD，而对照菌中无目的蛋白条带，未加IPTG诱导的转化菌低表达(见图5，6)。
4)蛋白表达的纯化
细菌大量诱导表达后菌液10000g离心15分钟，弃培养上清，沉淀中加入原菌液1/10体积的PBS磷酸盐平衡缓冲液，将细菌重悬；而后对菌液进行超声，超5秒，停5秒，共超180次，而后4℃12000rpm离心30分钟，弃沉淀，超声上清用0.22μm滤膜过滤，然后用1mLproteinL柱子进行纯化。先用5倍柱体积的水以0.5mL/min的速度洗柱子，而后用至少10倍柱体积的平衡缓冲液平衡柱子至基线，然后以0.2mL/min的速度上样，用0.1mol pH3.0的柠檬酸缓冲液洗脱，收集的蛋白洗脱液加入约十分之一体积的1mol pH8.0的Tris-base调节蛋白pH至7.0左右。将收集的蛋白样品用10kD的超滤管超滤3次，用PBS定容，分装，-80℃保存。进行10％SDS-PAGE电泳及western-blot检测，观察蛋白纯化情况(见图7，8)。
实施例2嵌合Fab抗体片段的活性鉴定：
I酶联免疫法
用包被液(0.1M碳酸盐缓冲液，pH9.6)稀释重组人ROR1蛋白(购自北京义翘神州生物技术有限公司，产品编号13968-H08H)至0.5μg/mL包被ELISA 96孔板，每孔加入100μL，4℃过夜；PBST(PBS含0.5％Tween20)5％脱脂牛奶-洗涤缓冲液封闭，37℃孵育2h；PBST洗涤3次后，每个孔中加入100μL抗ROR1抗体Fab分子(200μg/mL起始浓度，11个浓度梯度稀释)4℃过夜；以1:5000稀释的羊抗人Fab二抗100μL/孔加入到孔内，37℃孵育1h；过氧化物酶底物显色液100μL/孔，室温下10分钟后用2mol硫酸中止反应，酶标仪读取OD450值。结果如图9示，嵌合Fab抗体片段能与重组人ROR1蛋白起抗原抗体反应。
II western-blot
高表达ROR1的人卵巢癌细胞系(A2780)、低或无表达人正常卵巢癌细胞(Iose386)细胞总蛋白为天然抗原。细胞分别培养至1×107，4℃条件下去除培养基用PBS清洗，用500μLRIPA裂解液充分裂解细胞，10000g离心5分钟取上清及细胞总蛋白，测定各细胞总蛋白的浓度。将各细胞总蛋白浓度制成相等浓度(1mg/mL)后分装冻存-20℃备用。将A2780，Iose386细胞总蛋白进行10％SDS-PAGE电泳后电转到硝酸纤维膜上，此膜经TBST(TBS含0.5％Tween20)5％脱脂牛奶-洗涤缓冲液封闭，37℃封闭2h后，与抗ROR1抗体Fab分子(30μg/mL)4℃孵育过夜，TBST洗涤间隔15分钟3次，羊抗人Fab二抗37℃孵育1h，western-blot结果如图10所示，A2780总蛋白泳道在100KD到1300KD之间出现明显条带，Iose386细胞总蛋白泳道相应位置条带不明显；内参western-blot方法同上。
III流式细胞术
采用流式细胞仪(FSCS)分析抗ROR1抗体Fab分子与ROR1天然抗原的结合活性。具体方法为：分别收集1×106个A2780和Iose386细胞，PBS洗3次，1000rpm，5min。每种细胞平均分成3管，编号为1号，2号，3号。每管加5％脱脂牛奶37℃封闭1h，PBS洗3次，1000rpm，5min。1号用300μL PBS重悬；2号用300μL PBS重悬；3号用含抗ROR1抗体Fab分子(30μg/mL)PBS重悬。37℃孵育1h，PBS洗3次，1号用300μL PBS重悬；2号用含FITC标记的羊抗人Fab二抗的PBS重悬；3号用含FITC标记的羊抗人Fab二抗的PBS重悬，避光室温孵育1h，PBS洗3次后，每管均用400μL PBS重悬，在FACS上进行免疫荧光分析，结果如图11：抗ROR1抗体Fab分子与A2780结合率分别约为97.4％；同样浓度的抗ROR抗体Fab分子与Iose386细胞结合率分别为1.6％。
Ⅳ免疫荧光
采用荧光显微镜直观地分析抗ROR1抗体Fab分子与ROR1天然抗原的结合活性。具体方法为：将人卵巢癌细胞A2780和人正常卵巢癌细胞Iose386计数，调整细胞密度为1×105/mL左右，滴加细胞悬液到6孔细胞培养板内，置于37℃、5％CO2、饱和湿度的细胞培养箱培养过夜；吸去上清液，PBS洗涤三次；加入4％多聚甲醛1mL，室温固定15min；吸去固定液，再用PBS洗三次；每孔加入新鲜配制5％脱脂牛奶1mL，37℃条件下封闭1h；吸掉牛奶，每孔加入30ug/mL的抗ROR1抗体人鼠嵌合Fab分子600μL，37℃孵育1h；PBS洗涤三次，加羊抗人FITC标记荧光二抗(1:100)，37℃避光敷育40min，用DAPI复染细胞核，室温避光3min，PBS洗涤三次，荧光显微镜拍照。结果如图12：能直观的观察到抗ROR1抗体Fab分子与A2780很好的结合；不能直观的观察到同样浓度的抗ROR抗体Fab分子与Iose386细胞结合。
以上结果显示，抗ROR1抗体Fab分子能与ROR1抗原结合，说明该嵌合Fab片段保留了鼠源性抗ROR1抗体的抗原性质和特异性。
序列表
<110> 张慧林
朱进
<120> 嵌合抗ROR1抗体Fab分子及其制备方法和应用
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Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser
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<213> 人工序列(rengongxulie)
<400> 2
Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Lys Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Met
65 70 75 80
Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Val Ser Pro Ala Tyr Tyr Gly Asn Tyr Val Gly Phe Ala Tyr
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ser Thr Lys Gly Pro
115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
210 215 220
<210> 3
<211> 642
<212> DNA
<213> 人工序列(rengongxulie)
<400> 3
gagctcgtga tgacccagtc tccaaccacg atggctgcat ctcccggaga gaagatcact 60
ttcacctgca gtgccagctc aagtataagt tccacttact tgcattggtt tcagcagaag 120
ccaggattct cccctaaact cttgatttat aggacatcca atctggcttc tggagtccca 180
gctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaattgg cacgatggag 240
gctgaagatg ttgccactta ctactgccag cagggtagta gtttaccatt cacgttcggc 300
tcggggacaa agttggaaat aaaaggtcag cccaaggctg ccccctcggt cactctgttc 360
ccgccctcct ctgaggagct tcaagccaac aaggccacac tggtgtgtct cataagtgac 420
ttctacccgg gagccgtgac agtggcctgg aaggcagatg gcagccccgt caaggcggga 480
gtggagacca ccacaccctc caaacaaagc aacaacaagt acgcggccag cagctatctg 540
agcctgacgc ctgagcagtg gaagtcccac agaagctaca gctgccaggt cacgcatgaa 600
gggagcaccg tggagaagac agtggcccct acagaatgtt ca 642
<210> 4
<211> 660
<212> DNA
<213> 人工序列(rengongxulie)
<400> 4
gaggtgcagc tggtgcagtc tggtggagga ttggtgcagc ctaaagggtc attgaaactc 60
tcatgtgcag cctctggatt caccttcaat acctacgcca tgaactgggt ccgccaggct 120
ccaggaaagg gtttggaatg ggttgctcgc ataagaagta aaagtaataa ttatgcaaca 180
tattatgccg attcagtgaa agacaggttc accatctcga gagatgattc acaaagcatg 240
ctctatctgc aaatgaacaa cttgaaaact gaggacacag ccatgtatta ctgtgtgagc 300
ccggcctact atggtaacta cgtggggttt gcttactggg gccaaggaac cctggtcacc 360
gtctccgcct ccaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc 420
tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg 480
gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 540
tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 600
cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagaaggtt 660
<210> 5
<211> 44
<212> DNA
<213> 人工序列(VHF1)
<400> 5
gctgcccaac cagccatggc ccaggtgcag ctggtgcagt ctgg 44
<210> 6
<211> 44
<212> DNA
<213> 人工序列(VHF2)
<400> 6
gctgcccaac cagccatggc ccagatcacc ttgaaggagt ctgg 44
<210> 7
<211> 44
<212> DNA
<213> 人工序列(VHF3)
<400> 7
gctgcccaac cagccatggc cgaggtgcag ctggtgsagt ctgg 44
<210> 8
<211> 43
<212> DNA
<213> 人工序列(VHF4)
<400> 8
gctgcccaac cagccatggc cgaggtgcag ctgktggagt ctg 43
<210> 9
<211> 44
<212> DNA
<213> 人工序列(VHF5)
<400> 9
gctgcccaac cagccatggc ccaggtgcag ctgcaggagt cggg 44
<210> 10
<211> 44
<212> DNA
<213> 人工序列(VHF6)
<400> 10
gctgcccaac cagccatggc ccaggtgcag ctacagcagt gggg 44
<210> 11
<211> 45
<212> DNA
<213> 人工序列(VHR1)
<400> 11
cgatgggccc ttggtggagg ctgaggagac ggtgaccagg gttcc 45
<210> 12
<211> 45
<212> DNA
<213> 人工序列(VHR2)
<400> 12
cgatgggccc ttggtggagg cwgrggagac ggtgaccagg gtbcc 45
<210> 13
<211> 37
<212> DNA
<213> 人工序列(VLF1)
<400> 13
gggcccaggc ggccgagctc cagatgaccc agtctcc 37
<210> 14
<211> 37
<212> DNA
<213> 人工序列(VLF2)
<400> 14
gggcccaggc ggccgagctc gtgatgacyc agtctcc 37
<210> 15
<211> 37
<212> DNA
<213> 人工序列(VLF3)
<400> 15
gggcccaggc ggccgagctc gtgwtgacrc agtctcc 37
<210> 16
<211> 37
<212> DNA
<213> 人工序列(VLF4)
<400> 16
gggcccaggc ggccgagctc acactcacgc agtctcc 37
<210> 17
<211> 24
<212> DNA
<213> 人工序列(VLR1)
<400> 17
gaagacagat ggtgcagcca cagt 24
<210> 18
<211> 26
<212> DNA
<213> 人工序列(VLF)
<400> 18
ccatgggcga gctcgtgatg acccag 26
<210> 19
<211> 35
<212> DNA
<213> 人工序列(VLR)
<400> 19
cagccttggg ctgacctttt atttccaact ttgtc 35
<210> 20
<211> 28
<212> DNA
<213> 人工序列(VHF)
<400> 20
catatggagg tgcagctggt gcagtctg 28
<210> 21
<211> 38
<212> DNA
<213> 人工序列(VHR)
<400> 21
gaaccctggt caccgtctcc gcctccacca agggccca 38
<210> 22
<211> 35
<212> DNA
<213> 人工序列(CLF)
<400> 22
gacaaagttg gaaataaaag gtcagcccaa ggctg 35
<210> 23
<211> 31
<212> DNA
<213> 人工序列(CLR)
<400> 23
aagcttttat gaacattctg taggggccac t 31
<210> 24
<211> 38
<212> DNA
<213> 人工序列(CH1F)
<400> 24
gaaccctggt caccgtctcc gcctccacca agggccca 38
<210> 25
<211> 27
<212> DNA
<213> 人工序列(CH1R)
<400> 25
ggtaccttaa gaagcgtagt ccggaac 27
<210> 26
<211> 26
<212> DNA
<213> 人工序列(LF)
<400> 26
ccatgggcga gctcgtgatg acccag 26
<210> 27
<211> 31
<212> DNA
<213> 人工序列(LR)
<400> 27
aagcttttat gaacattctg taggggccac t 31
<210> 28
<211> 28
<212> DNA
<213> 人工序列(FdF)
<400> 28
catatgcagg tgcagctggt gcagtctg 28
<210> 29
<211> 27
<212> DNA
<213> 人工序列(FdR)
<400> 29
ggtaccttaa gaagcgtagt ccggaac 27
Claims (1)
1.嵌合抗ROR1抗体Fab分子在制备诊疗卵巢癌药物中的应用，所述嵌合抗ROR1抗体Fab分子包括通过链间二硫键连接的重组轻链L和重组重链Fd，所述重组轻链L的氨基酸序列如SEQ ID NO.1所示，重组重链Fd的氨基酸序列如SEQ ID NO.2所示。
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| TW202342539A (zh) * | 2022-03-09 | 2023-11-01 | 大陸商三優生物醫藥（上海）有限公司 | 一種靶向ｒｏｒ１的結合分子及其應用 |
| CN118063612B (zh) * | 2024-04-18 | 2024-07-26 | 上海宏成药业有限公司 | 抗ror1抗体或其抗原结合片段及其用途 |
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| CN104817642B (zh) * | 2015-05-04 | 2018-03-27 | 北京百普赛斯生物科技有限公司 | 抗人ror1单克隆抗体及其制备方法与应用 |
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