CN114920830A - Vκ4-1-IgLC多肽及其用途 - Google Patents
Vκ4-1-IgLC多肽及其用途 Download PDFInfo
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- CN114920830A CN114920830A CN202210492107.7A CN202210492107A CN114920830A CN 114920830 A CN114920830 A CN 114920830A CN 202210492107 A CN202210492107 A CN 202210492107A CN 114920830 A CN114920830 A CN 114920830A
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Abstract
本发明公开了带有独特的Vκ4‑1的免疫球蛋白κ型轻链(Vκ4‑1‑IgLC)多肽及其用途，该Vκ4‑1‑IgLC多肽氨基酸序列如SEQ ID NO.1所示，该多肽可作为靶标或标志物用于制备诊断和/或治疗肿瘤或炎症性疾病药物，也可以用于制备Integrin‑FAK信号通路抑制剂。利用本发明多肽作为抗原制备的抗体能够有效抑制肿瘤的发展，为肿瘤的临床诊断和治疗提供新思路。
Description
技术领域
本发明涉及免疫及癌症的诊断和治疗技术领域，具体涉及一种带有独特的Vκ4-1的免疫球蛋白κ型轻链(Vκ4-1-IgLC)多肽及其用途。
背景技术
经典免疫理论认为，免疫球蛋白(Immunoglobulin,Ig)的基本结构由四条肽链组成，即由两条完全相同的重链和两条完全相同的轻链通过二硫键连接，形成完整的Ig分子。除了经典四肽链结构的Ig分子外，正常人外周血和多种体液中还普遍存在一部分不与Ig重链结合的轻链，被称为游离轻链(free light chain，FLC)，它们以单体形式(分子量22～27kDa)、作为共价或非共价结合的二聚体(44～55kDa)形式、或以聚合物的形式存在，如尿液中的本周氏蛋白(Bence-Jones protein，BJP)。
传统理解上，FLC被认为是B细胞产生的一种过量副产物，在生理条件下没有任何功能。然而，越来越多的证据表明FLC与炎症性疾病(如自身免疫性疾病、糖尿病和中枢神经系统炎症)的进展和严重程度显著相关。特别是两种难治性疾病：轻链淀粉样变性(Amyloidprotein immunoglobulin light chain，AL)和轻链沉积病(Light Chain DepositionDisease，LCDD)，已被确定是由异常折叠的不溶性FLC所介导。AL和LCDD都是由不可逆的游离κ(kappa)或λ(lamda)链沉积在细胞外空间导致的一种难治性疾病，其特点是游离轻链发生错误折叠并沉积在组织和器官中，常发生在心、肾、肝和肺，导致组织结构损伤、器官功能障碍和进行性进展。AL相关FLC在组织中显示出独特的丝状结构，LCDD相关FLC在组织中显示出非晶态沉积。AL和LCDD均表现出共同的单克隆特征，但不具有多样性。此外，AL或LCDD相关FLC的κ链通常表现出相同的Vκ4-1/Jκ3重排。到目前为止，AL和LCDD相关的FLC被认为与B细胞畸形有关，然而，越来越多的病例显示没有B细胞或浆细胞异常增殖，表明该FLC可能有其他来源。
免疫球蛋白轻链存在к和λ两种类型，在B淋巴细胞编码Ig过程中，к链的使用频率往往较高。并且к链也同重链一样，在Ig分子中存在多样性，这是因为к链基因存在40个V片段，这些片段可随机与5个J片断重组形成完整的к链可变区编码序列，故不同的B淋巴细胞表达的к链呈现不同的Vк与VJ组合，同样，由部分Vк片段与部分VJ片断编码的高变区(CDR3区，抗体的特异性主要由该区决定的)也不相同。然而，我们的研究结果发现(CN1940069A)，不同类型肿瘤细胞之间有着完全相同或极为相似的轻链可变区序列，即呈现出Vк4-1/J3组合特异性序列；并且Vк4-1/J3组合中的CDR3序列在肿瘤细胞来源的Ig轻链可变区中具有极高的保守性，它和B淋巴细胞来源的CDR3相比具有特异性，表现为肿瘤细胞与B淋巴细胞之间的不同。研究表明其CDR3序列能够作为靶标用于诊断或者治疗肿瘤。
然而在后续研究中，我们又有了新的发现，进而获得本发明。
发明内容
我们研究发现带有独特的Vκ4-1的免疫球蛋白κ型轻链在不同谱系的癌细胞中广泛表达，在非癌细胞中低表达。另外我们获得了游离轻链Vκ4-1的一段序列，其能够用于标记带Vκ4-1的游离Ig轻链。基于此，本发明技术方案详述如下：
第一方面，本发明提供了一种Vκ4-1-IgLC多肽，氨基酸序列如5'-TQSPDSLVVSLGERASINCKSQRVSLG-3'(SEQ ID NO:1)所示。
第二方面，本发明提供了上述多肽在制备诊断和/或治疗恶性肿瘤和/或炎症药物的用途。
可选或优选的，上述用途中，所述恶性肿瘤包括以下至少一种：神经胶质瘤、髓母细胞瘤、大细胞肺癌、食管癌、胃癌、结直肠癌、乳腺癌、肾细胞癌、前列腺癌、肝癌、胰腺癌、皮肤癌、口腔癌、精原细胞癌、骨肉瘤、平滑肌肉瘤、血管肉瘤、脂肪肉瘤、滑膜肉瘤、横纹肌肉瘤、B细胞淋巴瘤、T细胞淋巴瘤、白血病及骨髓瘤。
可选或优选的，上述用途中，所述炎症性疾病，包括以下至少一种：系统性红斑狼疮、类风湿性关节炎、多发性硬化症、炎性肠病、炎性肾病、系统性淀粉样变、阿尔兹海默症(早老性痴呆)、帕金森病。
第三方面，本发明提供了一种抗体药物，是以上述多肽作为抗原制备而成，所述抗体药物为识别性抗体或者是阻断性抗体。
可选或优选的，上述抗体药物为单克隆抗体，其重链可变区氨基酸序列如SEQ IDNO:3或SEQ ID NO:4所示。
可选或优选的，上述抗体药物的轻链可变区氨基酸序列如SEQ ID NO:5所示。
第四方面，本发明还提供了一种小分子药物，是以上述多肽的基因序列或者mRNA序列作为靶点制备。小分子药物包括但不限于gRNA、siRNA，能够特异性靶向上述多肽的基因序列或者mRNA序列进而阻断其表达。
第五方面，本发明还提供了上述多肽在制备Integrin-FAK信号通路抑制剂中的用途。
本发明具有如下有益效果：
本发明基于带有独特Vκ4-1的免疫球蛋白κ型轻链的序列分析，获得了一段功能性多肽，位于Vκ4-1的保守区域，能够特异性标记含有独特Vκ4-1的游离Ig轻链序列，基于该多肽制备的抗体或可与该靶点特异性结合的多肽或小分子能够特异性识别恶性肿瘤和/或炎症性疾病组织中带Vκ4-1的免疫球蛋白κ型轻链，并与其结合抑制其功能，进而可以显著抑制体内外癌细胞的生长或抑制炎症；经过进一步发分子研究发现，基于该多肽的抗体还能够抑制Integrin-FAK信号通路活化，作为Integrin-FAK信号通路抑制剂应用。
附图说明
图1为带有独特Vκ4-1的免疫球蛋白κ型轻链的全氨基酸序列及各部分功能标注。
图2为实施例2单克隆抗体在细胞裂解液中特异性识别Vκ4-1/Jκ3-FLC，但不识别Vκ1-5/Jκ3-LC，Lane1和Lane2分别为转染Vκ1-5/Jκ3-LC和Vκ4-1/Jκ3-FLC质粒的293T细胞裂解物，并与Myc标记融合，以抗Myc标记抗体作为对照。
图3为实施例2单克隆抗体6G5(Ighv2-3)与5D3(Ighv2-9)重链可变区序列比对。
图4为实施例3用5D3对四种癌症组织的Vκ4-1/Jκ-FLC免疫组化检测结果。
图5为实施例3用6G5对更多种类的癌症组织中Vκ4-1/Jκ-FLC免疫组化检测评分统计。
图6为实施例3用6G5和5D3对结肠直癌患者不同部位组织中Vκ4-1/Jκ-FLC免疫组化检测结果，左图为两个病例的免疫组化照片，右上为所有病例的6G5检测组织学评分，右下为所有病例的5D3检测组织学评分。
图7为实施例4不同癌细胞系用5D3进行免疫荧光染色结果图。
图8为实施例4结直肠癌细胞系用6G5进行western blot检测结果图。
图9为实施例5CHO表达系统得到Vκ4-1/Jκ-FLC重组蛋白鉴定，其中(A)转染第6天检测CHO细胞分泌上清中的Vκ4-1/Jκ-FLC重组蛋白，(B)从培养上清中纯化Vκ4-1/Jκ-FLC，(C)Western blot及PAGE检测纯化获得的Vκ4-1/Jκ-FLC重组蛋白，(D)利用superdex200分子筛柱对Vκ4-1/Jκ-FLC重组蛋白进行分析。
图10为实施例5r-Vκ4-1/Jκ-FLC促进肿瘤细胞增殖的实验结果，*，P<0.05；***,P<0.001。
图11为实施例5r-Vκ4-1/Jκ-FLC促进肿瘤细胞迁移和侵袭的实验结果，**,P<0.01；***,P<0.001；****,P<0.0001。标尺，200μm。
图12为实施例5中HT-29细胞和SW480细胞敲除Igκ后进行Transwell实验检测细胞迁移能力的结果，A为SW480细胞，B为HT-29细胞，**,P<0.01；***,P<0.001；****,P<0.0001。标尺，200μm。
图13为实施例5外源加入重组蛋白r-Vκ4-1/Jκ-FLC可部分挽救敲减Igκ导致的迁移抑制Transwell实验结果，A是SW480敲除Igκ后，在培养体系加入1μg/ml r-Vκ4-1/Jκ-FLC，Transwell检测细胞迁移能力结果；B是HT-29敲除Igκ后，在培养体系加入1μg/ml r-Vκ4-1/Jκ-FLC，Transwell检测细胞迁移能力结果；*，P<0.05；**,P<0.01；***,P<0.001；****,P<0.0001；标尺，200μm。
图14为实施例5用单抗5D3可以阻断Vκ4-1/Jκ-FLC抑制细胞迁移实验结果，A是SW480细胞，B是HT-29细胞，ns表示not significant，不明显；*,P<0.05；****,P<0.0001。标尺，200μm。
图15实施例5中外源加入5D3和外源加入5D3+r-Vκ4-1/Jκ-FLC对肿瘤细胞迁移能力检测的Transwel结果。A是SW480细胞，B是HT-29细胞，ns表示not significant，不明显；*,P<0.05；**,P<0.01；****,P<0.0001。标尺，200μm。
图16为实施例5中用SW480细胞构建的肿瘤模型注射r-Vκ4-1/Jκ-FLC后肿瘤照片，以及肿瘤体积和重量统计结果，ns表示not significant，不明显；*,P<0.05；**,P<0.01。
图17为实施例6中CDX模型用6G5治疗后的肿瘤照片，以及在体检测和实验结束后取出的肿瘤体积和重量统计结果，***,P<0.001，****,P<0.0001。
图18为实施例6中PDX模型用6G5治疗后的肿瘤照片，以及在体检测和实验结束后取出的肿瘤体积和重量统计结果，***,P<0.001，****,P<0.0001。
图19为实施例6中CDX模型用5D3治疗后的肿瘤照片，以及在体检测和实验结束后取出的肿瘤体积和重量统计结果，***,P<0.001，****,P<0.0001。
图20为实施例6中PDX模型用5D3治疗后的肿瘤照片，以及在体检测和实验结束后取出的肿瘤体积和重量统计结果，***,P<0.001，****,P<0.0001。
图21为实施例7肿瘤细胞加入单抗培养后Integrin-FAK信号通路主要分子的Western blot检测结果，A为SW480细胞，B为HT-29细胞。
图22为实施例7单抗6G5对两种肿瘤模型干预后Integrin-FAK信号通路主要分子的Western blot检测结果，A为CDX模型，B为PDX模型。
图23为实施例7单抗5D3对两种肿瘤模型干预后Integrin-FAK信号通路主要分子的Western blot检测结果，A为CDX模型，B为PDX模型。
图24为实施例8中单抗5D3对骨肉瘤在裸鼠体内生长的影响，上图为5D3组和mIgG对照组小鼠随治疗天数增加体内肿瘤体积的变化，下图为两组小鼠取出的肿瘤大小比较。
图25为实施例9中利用特异性识别28肽的抗体检测MS患者和健康人血清中游离的Vκ4-1/Jκ轻链的电泳结果。
图26为实施例10中使用6G5检测AD患者和正常人脑组织的染色结果，其中A展示了AD患者树枝状强染色，B为AD患者灶状及斑状染色，C为AD患者弥散沉淀染色，D为正常对照。
图27为实施例11中使用6G5检测心脏淀粉样变患者和正常人血清的SDS-PAGE电泳结果。
图28为实施例12中用6G5和小鼠IgG检测局灶性肾病患者肾小球基底膜中Vκ4-1模式游离Ig轻链多肽，A为6G5的荧光染色结果，B为小鼠IgG的荧光染色结果。
图29为实施例13中使用5D3检测不同炎性肠胃病的病理切片的染色结果，其中A是炎症性结肠息肉，B是溃疡性结肠炎，C是慢性胃炎，D是正常结肠组织。
具体实施方式
下面结合较佳的具体实施例对本发明的技术方案进行详细地解释说明以使本领域技术人员能够更好地理解并予以实施。
实施例1具有独特Vκ4-1重排模式的轻链发现
我们前期对乳腺癌、结直肠癌、肺鳞癌等肿瘤上皮细胞的研究结果显示，Igκ轻链的所有可变区均显示出带有Vκ4-1，这些模式已经公开(参见GenBank：AY505537-AY505541)，此外，我们发现Vκ4-1与疏水性FLC高度同源，在LCDD或AL中具有Vκ4-1。
随后，我们选取5例结直肠癌组织样本，包括癌组织、癌旁组织和癌远端正常组织。样本从北京大学人民医院获得并经过书面知情同意。该研究按照机构审查委员会批准的方案进行，并得到北京大学人民医院临床研究伦理委员会的批准(2015PHB212-01)。对获取的癌组织，通过流式细胞术对EpCAM+癌细胞进行分选，分选方法如下：
首先将组织切成小块(约1mm3)并用1×PBS清洗。通过在37℃下培养1小时，并在添加5mmol/L EDTA和5mmol/L DTT的1×PBS中摇动，将上皮细胞与组织分离。消化后的上皮细胞由gentleMACS分离器(Miltenyi Biotec)分离，并通过尼龙网过滤。然后用含有2％胎牛血清(FBS)(10099141，Gibco)的1×PBS洗涤细胞3次，在含有5％胎牛血清的1×PBS中4℃下封闭细胞30分钟，并在4℃下用抗人CD19(11-0199-41，eBioscience)和抗人EpCAM(12-9326-42，eBioscience)染色30分钟。然后由FACSAria II(BD Biosciences)对EpCAM+细胞进行荧光激活细胞分选(FACS)，获得EpCAM+癌细胞。
接下来，以EpCAM+癌细胞为研究对象，通过多重RT-PCR扩增和IR-Seq测序技术研究了Igκ轻链的可变区转录物。
IR-Seq的样品制备及测序：通过Trizol Reagent(15596018,Life Technology)提取分选的EpCAM+癌细胞总RNA。使用
公司商品试剂盒，用Igκ(iRepertoireInc.)引物组，在试剂盒说明书指定的反应条件下进行两轮PCR。在第一轮中，完成逆转录，并使用与V和C基因互补的嵌套基因特异性引物将标签和测序引物引入PCR产物。第二轮PCR使用公用(测序)引物进行指数扩增。检测洗脱的PCR产物的DNA浓度，并收集100ng DNA用于测序。后续的质量控制和测序由Novogene公司完成。
在5例患者中，发现了占绝对优势的带有独特Vκ4-1模式的轻链。
根据IR-Seq测序结果，获得带有独特Vκ4-1可变区的Igκ链全长序列如下：
DIVMTQSPDSLVVSLGERASINCKSSQRVSLGSNNKNYLAWYQHQPGQPPRLLIYWASTQESGVPDQFSGSGSGTDFTLTINSLQAEDVAVYYCQQYYDTPVTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：2)，是一种带有独特的Vκ4-1的免疫球蛋白κ型轻链，其各部分功能区标记如图1所示。
其中，第5-32位的28个氨基酸(SEQ ID NO:1，全部在Vκ4-1区域内)具有特异性靶点功能，能够标记带有独特Vκ4-1可变区的游离免疫球蛋白κ链。
实施例2靶向带有独特Vκ4-1模式的Ig轻链的抗体制备
为了了解这些非B细胞来源的带Vκ4-1模式Igκ轻链可变区的功能，我们以SEQ IDNO:1所示序列(人工合成)作为抗原，制备单克隆抗体。
单克隆抗体制备：
使用上述28个氨基酸的多肽抗原与人白蛋白偶联，免疫6-8周龄的BALB/c雌性小鼠(购自北京维通利华实验动物技术有限公司)，免疫结束后获取脾细胞，与骨髓瘤细胞融合，用上述28个氨基酸的多肽筛选阳性克隆杂交瘤细胞株。最终筛选到的杂交瘤细胞的阳性克隆分别命名为5D3和6G5。
10周龄雌性BALB/c经产小鼠，腹腔注射不完全弗氏佐剂500μL/只，一周后可腹腔注射杂交瘤细胞。5D3和6G5杂交瘤细胞分别培养于RPMI 1640+10％FBS+1％PS培养基，在37℃，5％CO2培养孵箱培养。检测培养上清中抗体表达情况。确认表达后，取培养至对数生长期的杂交瘤细胞，离心弃上清，用预热的无血清1640培养基重悬并洗涤细胞2次，按照每只小鼠腹腔内注射杂交瘤细胞2.0×106个/500μL分装细胞，分别进行腹腔注射。待小鼠腹水产生明显时(约7～10天后)，用9号注射针头在无菌条件下收取腹水，于4℃，1200rpm离心10min去除腹水中的杂交瘤细胞，腹水上清加入30％甘油充分混匀，之后分装贮存于-20℃备用。
取适量protein G柱料装填层析柱，并用10～20倍体积冷PBS(pH 7.4)洗涤并平衡。将单抗腹水10000rpm，4℃离心5min后，除去有形杂质，用PBS以3：1稀释后与Protein G柱料孵育，4℃旋转过夜。次日，收集穿柱液体并重复过柱10～20次，之后让穿过液自然流出，再用10～20倍柱体积的冷PBS(pH 7.4)洗去非特异结合的杂蛋白，然后用0.1mol/L甘氨酸-HCl缓冲液(pH 3.0)洗脱结合的抗体，并立即用1mol/L Tris-HCl(pH 11.0)中和至pH7.4。洗脱完毕后用10～20倍体积PBS(pH 7.4)洗涤纯化柱，将纯化柱保存于20％乙醇中。洗脱的抗体经PBS超滤，定量后加入30％～50％甘油，贮存于-20℃待用。全程操作在4℃冷室或冰上进行。通过SDS-PAGE检测抗体纯度。所得抗体对应其杂交瘤细胞株，分别命名为5D3和6G5。
单克隆抗体特异性评价：
用分别转染了Vκ1-5/Jκ3-LC和Vκ4-1/Jκ3-LC质粒(均带Myc标签)的两种293T细胞裂解物作为实验对象，将5D3和6G5分别与经SDS-PAGE分离的细胞裂解物孵育，并用抗Myc标签抗体作为对照，检测单抗对Vκ1-5模式和Vκ4-1模式的识别能力(LC：light chain，轻链)。
结果参见图2，电泳结果显示，6G5及5D3都特异识别Vκ4-1/Jκ3-LC，而不识别Vκ1-5/Jκ3-LC，表明其能够特异性识别Vκ4-1第5-32位氨基酸序列。图中anti-Myc即表示抗Myc标签抗体。
经测序，单克隆抗体5D3的重链可变区氨基酸序列如SEQ ID NO:4所示，编码基因如SEQ ID NO:7所示，轻链可变区氨基酸序列如SEQ ID NO:5所示，编码基因如SEQ ID NO:8所示。单克隆抗体6G5的重链可变区氨基酸序列如SEQ ID NO:3所示，编码基因如SEQ IDNO:6所示。两个单克隆抗体的重链可变区DNA序列及氨基酸序列比对如图3所示。
实施例3单克隆抗体对不同癌症样本带有独特的Vκ4-1的免疫球蛋白κ型轻链的检测
使用单克隆抗体5D3，对子宫内膜癌(Endometrial cancer)、结直肠癌(Coloncancer)、乳腺癌(Breast cancer)、食管癌(Esophageal cancer)组织样本中带独特Vκ4-1模式的轻链Igκ，标记为Vκ4-1/Jκ-Igκ，进行免疫组化检测，同时用正常组织作为阴性对照。
免疫组化评分标准：细胞质染色评分利用四个强度等级(0，无；1，弱；2，中；3，强)和阳性细胞的百分比(0％-100％)。最终的评分为强度等级和阳性细胞的百分比的乘积(范围：0-300)。最终评分>100被认为是强阳性染色，最终评分≤100被认为是弱阳性染色。
检测结果见图4，带有独特Vκ4-1的免疫球蛋白κ型轻链(免疫球蛋白κ链)在四种癌症组织中都高表达，在非恶性肿瘤细胞中低表达(normal组正常组织染色非常浅)。
另外，我们又选取了更多肿瘤组织，包括神经胶质瘤(glioma)、髓母细胞瘤(medulloblastoma)、大细胞肺癌(Large cell lung cancer)、食管癌(esophagealcancer)、胃癌(gastric cancer)、结直肠癌(Colon cancer)、乳腺癌(Breast cancer)、肾细胞癌(renal cell carcinoma)、前列腺癌(prostate cancer)、肝癌(liver cancer)、胰腺癌(pancreatic carcinoma)、皮肤癌(skin cancer)、精原细胞癌(seminoma)、骨肉瘤(osteosarcoma)、子宫平滑肌肉瘤(lelomyosarcoma)、横纹肌肉瘤(rhabdomyosarcoma)、脂肪肉瘤(liposarcoma)、血管肉瘤(angiosarcoma)、滑膜肉瘤(synovialsarcoma)、霍奇金淋巴瘤(hodgkin lymphoma)、B细胞淋巴瘤(B lymphoma)、T细胞淋巴瘤(T lymphoma)，以正常组织作为对照，使用单克隆抗体6G5进行Vκ4-1/Jκ-Igκ免疫组化检测。
检测结果统计见图5，单抗6G5识别的Vκ4-1/Jκ-Igκ高水平表达在多种恶性肿瘤细胞，低表达在非恶性肿瘤细胞(正常组织)。
为了进一步验证癌细胞中Vκ4-1/Jκ-Igκ是否过表达，我们比较了同一结直肠癌个体中癌细胞和正常上皮细胞之间Vκ4-1/Jκ-Igκ的表达频率。
收集10例结直肠癌患者的组织，每例患者取手术断端(癌远端组织，是正常组织)、癌旁和癌组织，分别用6G5及5D3进行免疫组化染色。结果参见图6，该图展示了两例病例，正常组织未见阳性染色，癌旁组织显示弱阳性，癌组织呈现较强的染色，且两个单抗显示一致结果。结果表明，结直肠癌组织和正常组织相比Vκ4-1/Jκ-Igκ表达频率显著升高，与癌旁组织相比也差异显著。表明Vκ4-1/Jκ-Igκ只在癌细胞中广泛表达。
实施例4Vκ4-1/Jκ-Igκ的性质研究
我们使用单抗5D3作为检测工具，以细胞系MDA-MB-231(乳腺癌细胞)、NCI-H520(肺鳞癌细胞)、HT-29(结直肠癌细胞)、U2OS(骨肉瘤细胞)(从美国菌种保藏中心ATCC获得，并由北京大学人类疾病基因组学中心维护)作为实验对象，通过免疫荧光进行Vκ4-1/Jκ-Igκ的细胞定位分析，并检测Vκ4-1/Jκ-Igκ在两种癌细胞系中是否表现出疏水性和游离性。
免疫荧光：细胞系用PBS快速清洗，并在室温下用4％多聚甲醛中固定15分钟。固定后的细胞用5％BSA封闭，并在室温下用0.02％Triton X-100作用30分钟在细胞膜打孔(使抗体能够进入细胞)。然后用5D3(10μg/mL)在室温下在封闭缓冲液中稀释3小时对细胞进行免疫染色，并用PBS洗涤三次，每次5分钟。将细胞与Alexa594抗鼠二抗和Alexa488抗兔二抗(分子探针)在室温下孵育1小时后清洗。使用徕卡STED成像仪(徕卡微系统)通过共焦显微镜捕获图像。
免疫荧光参见图7，结果显示Vκ4-1/Jκ-Igκ可被分泌并沉积到细胞外基质。
接下来，我们又以SW480和HT-29细胞系(均为结肠癌细胞系)作为研究对象，进行western blot检测。还原电泳的四个泳道样本分别为细胞培养上清(Culturesupernatant)、阴性对照(Medium control)、细胞裂解液(Cell lysate)、ECM提取物(ECMextract)。非还原电泳样本分别为血清(serum)、SW480细胞、HT-29细胞。非还原电泳使用的抗体分别为抗人IgG抗体(anti IgG Fc)、抗人游离Igκ抗体(anti kappa free lightchain)、6G5；还原电泳均使用6G5。
结果如图8所示，左侧为还原电泳结果，右侧为非还原电泳结果。带有Vκ4-1/Jκ序列的Igκ轻链主要以游离多聚体的形式存在于细胞质、培养上清及细胞外基质。还原(左)及非还原(右)电泳都显示带有Vκ4-1/Jκ序列的Igκ轻链呈现游离形式且主要以多聚体形式存在。在非还原电泳条件下用抗人IgG抗体(anti IgG Fc)检测证明带有Vκ4-1/Jκ序列的Igκ轻链没有与IgG重链形成4肽链结构，而抗人游离Igκ抗体(anti kappa free light chain)检测证明带有Vκ4-1/Jκ序列的Igκ轻链呈现游离形式(与6G5信号一致)。
以上结果表明，在还原或非还原状态下，Vκ4-1/Jκ-Igκ显示出一种自由特征，其具有多种形式，包括培养上清液中和ECM提取物中的单体、二聚体和聚合物等。这提示非B细胞源性Vκ4-1/Jκ可能是一种ECM蛋白。组织中带有独特Vκ4-1的轻链Igκ即Vκ4-1/Jκ(Vκ4-1与其他Jκ基因进行组合重排)，实际上是一种游离Ig轻链，后文标记为Vκ4-1/Jκ-FLC。
实施例5Vκ4-1/Jκ-FLC促进肿瘤侵袭和转移
为了探索Vκ4-1/Jκ-FLC对癌症发生进展的影响，我们利用中国仓鼠卵巢细胞真核表达平台(Chinese Hamster Ovary cells，CHO)，制备了具有生物学活性的重组蛋白r-Vκ4-1/Jκ-FLC。
将含有Vκ4-1的全长序列基因(SEQ ID NO:2，带有C区和V区)克隆到pGEX-4T-2载体，再通过相同的限制酶切位点进一步亚克隆到pcDNA3.1 myc-His(-)B载体中，重组蛋白r-Vκ4-1/Jκ-FLC在C末端含有His标签，在CHO细胞中表达，并通过CaptoTM L亲和层析(GEHealthcare，美国)纯化。
真核重组蛋白的纯化及鉴定(CHO表达系统)：以3000rpm，4℃，15min离心，留取培养上清。Capto L可以特异性结合κ型轻链的可变区，可以用来纯化Igκ轻链真核重组蛋白。取适量Capto L装填至层析柱内，10倍体积的冷平衡液(20mmol/L磷酸二氢钠，150mmol/LNaCl，pH 7.2)进行预平衡，将培养上清(input)加入柱内，封闭好在4℃旋转孵育过夜，之后使柱内液体流出(穿流液，flow through)，冷平衡液洗涤以去除未结合和非特异性结合的蛋白，然后用冷洗脱液(0.1mol/L柠檬酸钠，pH 2.0～3.5)进行洗脱，洗脱液用中和液(1mmol/LTris-HCl，pH 11.0)及时中和至pH 7.4，洗脱液(washing)通过超滤浓缩，BCA定量后分装并贮存于-80℃备用。纯化得到的蛋白进行SDS-PAGE和Western blot进行鉴定。
在成功建立该细胞的培养和表达体系之后，我们发现CHO细胞表达的r-Vκ4-1/Jκ-FLC可以分泌在细胞培养上清中(图9A)。我们将收集到的培养上清用CaptoL亲和层析柱(特异性结合Igκ型轻链可变区)进行亲和层析，再分别用SDS-PAGE、Western blot及Superdex200层析柱对纯化得到的重组蛋白进行验证，证实我们获得了纯度较高的重组蛋白r-Vκ4-1/Jκ-FLC(图9B-D)。
以SW480和HT-29细胞作为研究对象，加入r-Vκ4-1/Jκ-FLC进行干预培养。实验结果参见图10和图11，其中图10中A为用1μg/mL r-Vκ4-1/Jκ-FLC处理SW480细胞，进行克隆形成实验检测细胞增殖及自我更新能力。B为用1μg/mL r-Vκ4-1/Jκ-FLC处理HT-29细胞，进行克隆形成实验检测细胞增殖及自我更新能力。图11中A和B为用1μg/mL r-Vκ4-1/Jκ-FLC处理SW480细胞，分别用Transwell实验(A)和Matrigel-Transwell实验(B)检测r-Vκ4-1/Jκ-FLC对SW480细胞迁移和侵袭能力的影响。C和D为用1μg/mL r-Vκ4-1/Jκ-FLC处理HT-29细胞，分别用Transwell实验(C)和Matrigel-Transwell实验(D)检测r-Vκ4-1/Jκ-FLC对SW480细胞迁移和侵袭能力的影响。实验结果发现与阴性对照相比，r-Vκ4-1/Jκ-FLC的处理增加了SW480和HT-29细胞的存活/增殖和迁移能力。
接着，我们利用三条针对Igκ恒定区的gRNA敲除SW480和HT-29细胞中Igκ。
结果显示，敲除Igκ后两种细胞的迁移能力均下降，参见图12，A中SW480细胞敲除Igκ后，进行Transwell实验检测细胞迁移能力和B中HT-29细胞敲除Igκ后进行Transwell实验检测细胞迁移能力，与对照组空载体(vector)相比，经三条gRNA敲除Igκ后，细胞迁移能力明显下降。
为了进一步证明Vκ4-1/Jκ-FLC主要通过分泌形式而不是细胞内Igκ发挥其促肿瘤作用，我们首先在细胞中敲除Igκ，然后在培养体系中加入重组蛋白r-Vκ4-1/Jκ-FLC。Transwell实验结果显示外源加入重组蛋白r-Vκ4-1/Jκ-FLC可以部分逆转由于敲除Igκ导致的细胞迁移能力的抑制(图13)。以上结果表明Vκ4-1/Jκ-FLC主要以分泌形式发挥促肿瘤作用。
更进一步，为了证明Vκ4-1/Jκ-FLC主要通过分泌形式发挥促肿瘤作用，我们还在培养体系中加入5D3特异性抗体用来封闭分泌型Vκ4-1/Jκ-FLC(细胞自身分泌的Igκ)。结果显示，加入5D3封闭分泌的Vκ4-1/Jκ-FLC后能明显抑制细胞迁移。结果参见图14，A为SW480细胞培养上清加入50μg/mL 5D3，Transwell检测细胞迁移能力实验结果，B为HT-29细胞培养上清加入50μg/mL 5D3，Transwell检测细胞迁移能力实验结果。结果显示，相较于阴性对照PBS和mIgG，加入5D3后，两种细胞迁移能力都显著下降。
随后，我们在SW480和HT-29细胞培养上清分别加入50μg/mL 5D3，同时在培养体系分别加入10μg/mL r-Vκ4-1/Jκ-FLC，进行Transwell实验检测细胞迁移能力，实验结果参见图15，被5D3封闭后，再外源加入重组蛋白r-Vκ4-1/Jκ-FLC可逆转抗体阻断对于细胞迁移的抑制作用。
进一步，我们利用SW480细胞在裸鼠中建立皮下移植瘤模型，瘤周注射不同剂量的重组蛋白r-Vκ4-1/Jκ-FLC，检测r-Vκ4-1/Jκ-FLC的促肿瘤生长能力。结果显示，重组蛋白r-Vκ4-1/Jκ-FLC以剂量依赖的方式促进肿瘤生长，并且肿瘤体积及重量均高于对照组。参见图16，A为实验结束时三组肿瘤图片，及肿瘤体积的生长曲线；B为实验结束时三组肿瘤体积统计图及肿瘤重量统计图。
综上，我们利用体内、体外实验证实，肿瘤细胞分泌的Vκ4-1/Jκ-FLC在结直肠癌的增殖与转移过程中发挥重要作用。
实施例6单抗5D3和6G5能够抑制肿瘤生长
鉴于实施例5已经验证了单抗5D3可以在体外特异性阻断Vκ4-1/Jκ-FLC的促癌特性，我们接下来检测了单抗6G5和5D3对结肠癌移植瘤在SCID小鼠体内生长的影响。
动物模型分为两组，一组模型用SW480细胞注射SCID小鼠皮下构建(CDX)，另一组模型用结肠癌患者的肿瘤细胞注射SCID小鼠皮下构建(PDX)。成瘤后每隔4天静脉给药(5mg/kg)，以小鼠IgG(mIgG)为对照，动态观察肿瘤生长速度，同时也动态监测小鼠体重变化。在实验结束后，比较6G5与对照组肿瘤体积及肿瘤重量的差别、5D3与对照组肿瘤体积及肿瘤重量的差别。
两组实验结果如图17～18(6G5)和图19～20(5D3)所示。
结果表明，单抗6G5和5D3都具有抗肿瘤效果，相较于对照组，明显抑制肿瘤体积和重量的增加，对小鼠体重则没有明显影响，与对照组相当。
实施例7单抗5D3和6G5能够抑制Integrin-FAK信号通路
在SW480细胞、HT-29细胞培养上清加入对照抗体mIgG(50μg/ml)或者抗体5D3、6G5。24h收取细胞，Western blot检测Integrin-FAK信号通路中主要分子的磷酸化水平，以检测抗体对Integrin-FAK通路的活化的影响。结果参见图21，显示6G5和5D3均可使整合素信号通路下游主要分子FAK、Src、Erk、Akt的磷酸化水平下降，抑制Integrin-FAK信号通路活化。
体内实验过程如实施例6，在实验结束后，比较6G5和5D3与对照组Integrin-FAK信号通路活化的差别。结果参见图22和图23，与体外细胞水平检测结果基本一致。
实施例8单抗5D3能够抑制骨肉瘤在体内生长
我们接下来检测了单抗5D3对骨肉瘤在裸鼠体内生长的影响。
动物模型用人骨肉瘤细胞系143B注射裸鼠小鼠皮下构建(CDX)，成瘤后分为两组，每组6只。一组模型每隔4天静脉给药单抗5D3，剂量为5mg/kg，另一组以小鼠IgG(mIgG)为对照，给药方式和剂量同5D3。动态观察两组小鼠体内肿瘤生长速度，同时也动态监测小鼠体重变化。在实验结束后，比较5D3治疗组与对照组肿瘤体积及肿瘤重量的差别。
实验结果如图24(5D3)所示，与对照组相比，实验组小鼠体内肿瘤生长速度明显减慢。取出的肿瘤体积比较也可以看出，实验组整体上肿瘤体积明显小于mIgG对照组。
结果表明，单抗5D3都具有抗肿瘤效果，相较于对照组，明显抑制肿瘤体积和重量的增加。
实施例9Vκ4-1-IgLC多肽用于辅助诊断多发性硬化症
多发性硬化症(MS)是最常见的一种中枢神经脱髓鞘疾病。本病急性活动期中枢神经白质有多发性炎性脱髓鞘斑，好发于视神经、脊髓和脑干，为神经内科常见的难治性疾病。该病的血清及脑脊液中还有高水平Igκ游离Ig轻链，是鉴别MS的重要指标。然而，该Igκ游离Ig轻链一直被认为是B细胞产生的。我们使用能够识别Vκ4-1的28肽(全长的第5-32位氨基酸，SEQ ID NO:1所示)特异性抗体进行检测实验。
选择10例多发性硬化症(MS)患者血清，以8例正常人血清作对照，冻存的样品取1μL并用10μL PBS稀释，进行变性SDS-PAGE电泳，而后将蛋白电转到硝酸纤维素膜上，再用Vκ4-1/Jκ的28肽(SEQ ID NO:1)免疫获得的兔特异性抗体进行检测。
结果如图25所示，针对Vκ4-1的28肽特异性抗体特异性识别MS患者血清中游离的Vκ4-1/Jκ轻链4聚体，而该4聚体在正常人(健康对照组)没有检测到。结果表明出现在MS患者血清中的Igκ游离Ig轻链存在独特的Vκ4-1重排，该28肽可以作为MS的辅助诊断标志物。
实施例10Vκ4-1-IgLC多肽用于辅助诊断阿尔茨海默症
我们在前期工作中发现，Ig游离轻链广泛存在于非B细胞中，而且在肿瘤细胞、增殖旺盛的细胞及炎性细胞高水平表达。现已经证明其是一个细胞骨架相关蛋白，既可以存在于细胞质形成丝状结构，又可以因疏水特性沉积于细胞外基质。尽管此前没有证据提示其与阿尔茨海默症(Alzheimer disease，AD)的淀粉样变相关，但我们用6G5染检测10例AD患者的病理切片却有了新发现。
取10例AD患者的病理切片，用二甲苯常规脱蜡、用梯度酒精脱二甲苯后，置煮沸的枸橼酸钠缓冲液中5分钟，待自然冷却后，用1％过氧化氢抑制过氧化物酶(室温10分钟)，PBS洗后用10％马血清封闭切片(室温10分钟)，而后用6G5与切片孵育(4℃过夜)，PBS洗后用HRP标记的抗小鼠IgG染色后，显微镜观察，结果如图26所示。
结果发现，10例患者的脑皮层区都出现不同的树枝状强染色(图26A)，或出现灶状及斑状(老年斑)染色(图26B)，远离皮层的部位也有少量轻链沉积，但大多呈现弥散的沉积状态(图26C)，而在正常脑组织没有阳性反应(图26D)。
实施例11Vκ4-1-IgLC多肽用于辅助诊断系统性淀粉样变
系统性淀粉样变是以游离Ig轻链在多种组织沉积导致的不可逆性脏器衰竭为特征的难治性疾病。该致病性游离Ig轻链一直被认为是B细胞产生的，然而，我们前期工作(包括蛋白提取、氨基酸和核苷酸序列比对等)发现，非B细胞产生的带有Vκ4-1可变区的Ig游离轻链与导致系统性淀粉样变的游离Ig轻链高度同源。随后我们用Vκ4-1的28肽(5-32位)特异性单克隆抗体6G5进行了检测实验。
选择3例心脏淀粉样变患者血清，以2例正常人血清作对照，取1μL血清样品并用10μLPBS稀释，进行变性SDS-PAGE电泳，而后将蛋白电转到硝酸纤维素膜上，再用6G5进行检测，结果如图27所示。
结果发现，针对Vκ4-1的特异性抗体可特异性识别系统性淀粉样变患者血清中游离的Igκ轻链，而不识别正常人Igκ，表明该28氨基酸的靶点多肽作为系统性淀粉样变患者的辅助诊断标志物。
实施例12Vκ4-1-IgLC多肽用于辅助诊断炎性肾病
几乎90％的肾病都与致病性Ig有关，如IgA肾病是由IgA沉积在肾小球系膜区、膜型肾病是由IgG沉积在肾小球足细胞与基底膜之间、肾脏淀粉样变是游离轻链沉积于肾小球基底膜。但领域内一直认为这些Ig是来源于B细胞，而发明人发现这些致病Ig主要是局部非免疫细胞产生的。
取5例局灶性肾病的冰冻病理切片，用1％过氧化氢抑制过氧化物酶(室温10分钟)，PBS洗后用10％马血清封闭切片(室温10分钟)，而后用针对带Vκ4-1的多肽的单克隆抗体6G5与切片孵育(4度过夜)，并设置小鼠IgG作为对照。PBS洗后用FITC标记的抗小鼠IgG染色后，显微镜观察，结果如图28所示。结果发现，5例患者的肾小球都出现基底膜强荧光染色(A)，而无关小鼠IgG未显示阳性染色(B)，表示6G5可特异识别人局灶性肾病患者肾小球基底膜中沉积Vκ4-1/Jκ-FLC。
实施例13Vκ4-1-IgLC多肽用于辅助诊断炎性肠胃病
溃疡性结肠炎及克隆氏病是临床上常见的难治性慢性炎症，常常会导致炎-癌转化；慢性胃炎也常转变为胃癌。发明人发现上述两种疾病组织的腺上皮细胞中存在高水平游离Ig轻链(Vκ4-1/Jκ-Igκ)，并可促进炎症进程。
分别取炎症性结肠炎性息肉、溃疡性结肠炎及慢性胃炎的病理切片，用二甲苯常规脱蜡、用梯度酒精脱二甲苯后，置煮沸的枸橼酸钠缓冲液中5分钟，待自然冷却后，用1％过氧化氢抑制过氧化物酶(室温10分钟)，PBS洗后用10％马血清封闭切片(室温10分钟)，而后用5D3与切片孵育(4度过夜)，PBS洗后用HRP标记的抗小鼠IgG染色后，显微镜观察。参见图29，结果发现，炎症性结肠息肉(A)、溃疡性结肠炎(B)及慢性胃炎上皮细胞(C)均呈现阳性染色(棕色)，而正常结肠组织(D)为阴性染色(蓝色)。
本文中应用了具体个例对发明构思进行了详细阐述，以上实施例的说明只是用于帮助理解本发明的核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离该发明构思的前提下，所做的任何显而易见的修改、等同替换或其他改进，均应包含在本发明的保护范围之内。
序列表
<110> 北京大学
北京艾赛吉生物医药科技有限公司
<120> Vκ4-1-IgLC多肽及其用途
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Claims (9)
1.Vκ4-1-IgLC多肽，其特征在于，氨基酸序列如5'-TQSPDSLVVSLGERASINCKSQRVSLG-3'(SEQ ID NO:1)所示。
2.权利要求1所述多肽在制备诊断和/或治疗恶性肿瘤或炎症性疾病药物的用途。
3.根据权利要求2所述的用途，其特征在于，所述恶性肿瘤包括以下至少一种：神经胶质瘤、髓母细胞瘤、大细胞肺癌、食管癌、胃癌、结直肠癌、乳腺癌、肾细胞癌、前列腺癌、肝癌、胰腺癌、皮肤癌、口腔癌、精原细胞癌、骨肉瘤、平滑肌肉瘤、血管肉瘤、脂肪肉瘤、滑膜肉瘤、横纹肌肉瘤、B细胞淋巴瘤、T细胞淋巴瘤、白血病及骨髓瘤。
4.根据权利要求2所述的用途，其特征在于，所述炎症性疾病包括以下至少一种：系统性红斑狼疮、类风湿性关节炎、多发性硬化症、炎性肠病、炎性肾病、系统性淀粉样变、阿尔茨海默病、帕金森病。
5.一种抗体药物，其特征在于，以权利要求1所述的多肽作为抗原制备，所述抗体药物为识别性抗体或者是阻断性抗体。
6.根据权利要求5所述的抗体药物，其特征在于，为单克隆抗体，其重链可变区氨基酸序列如SEQ ID NO:3或SEQ ID NO:4所示。
7.根据权利要求6所述的抗体药物，其特征在于，轻链可变区氨基酸序列如SEQ ID NO:5所示。
8.一种小分子药物，其特征在于，以权利要求1所述的多肽对应的基因序列或者mRNA序列作为靶点制备。
9.权利要求1所述多肽在制备Integrin-FAK信号通路抑制剂中的用途。
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| 邱晓彦: "非B细胞来源的免疫球蛋白促进肿瘤的发生和发展", 《中国肿瘤生物治疗杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023217020A1 (zh) * | 2022-05-07 | 2023-11-16 | 北京艾赛吉生物医药科技有限公司 | Vκ4-1-IgLC多肽及其用途 |
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| CN114920830B (zh) | 2023-05-16 |
| WO2023217020A1 (zh) | 2023-11-16 |
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