CN114957476A - 一种半胱氨酸工程化的结合人5t4的全人源纳米抗体 - Google Patents
一种半胱氨酸工程化的结合人5t4的全人源纳米抗体 Download PDFInfo
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Abstract
本发明属于生物技术领域，具体涉及一种半胱氨酸工程化的结合人5T4的全人源纳米抗体及其抗体偶联物，以及构建该抗体所需的核酸、质粒、宿主细胞，相应的药用组合物及其应用。本发明基于人的5T4抗原是表达于胚胎滋养层细胞的糖蛋白，在多种实体瘤中高度表达，而正常组织中却很少存在，通过对可结合人的5T4靶点的全人源纳米抗体进行半胱氨酸工程化改造，使之产生游离的巯基，将细胞毒性剂、酶或检测标记物偶联至改造后的全人源纳米抗体上，将其应用于癌症的治疗或免疫分析和检测。本发明中采用位点特异偶联技术能保证将一定数目药物分子定点偶联至抗体的特定位点，很大程度上在生产上保证药物同质性和批量生产稳定性。
Description
技术领域
本发明属于生物技术领域，涉及一种半胱氨酸工程化的结合人5T4的全人源纳米抗体，具体涉及一种半胱氨酸工程化的结合人5T4的全人源纳米抗体及其抗体偶联物，以及构建该抗体所需的核酸、质粒、宿主细胞，相应的药用组合物及其应用。
背景技术
现有技术公开了基于抗体的免疫治疗在治疗多种癌症中是非常有效的，而开发成功的免疫治疗主要取决于发现肿瘤细胞中优先表达的细胞表面蛋白的抗体。研究显示，胚胎滋养层细胞和恶性肿瘤细胞具有一些共同的生理过程，包括局部组织的入侵和免疫监视的逃逸，该特性使得许多在滋养层细胞和肿瘤细胞共同表达的抗原被发展为肿瘤特异性免疫治疗的靶点，其中人的5T4抗原，是表达于胚胎滋养层细胞的糖蛋白，在多种实体瘤中高度表达，而正常组织中却很少存在，被认定是一种肿瘤相关抗原，是治疗肿瘤的潜在靶点。
近年来，有研究在羊驼血清中发现了一类仅含重链可变区的抗体，它是自然界存在的具有与抗原结合性能的最小抗体片段，称为纳米抗体(nanobody,VHH)或单域抗体(sdAb)(Protein Engineering,1994.7(9):p.1129-1135.)，这些纳米抗体的抗原亲和力和特异性很高，与全长IgG单抗相类似。更重要的是，这些抗体更易于改造并且能在原核系统中表达，因此制备成本很低廉(Front Immunol,2017.8:p.1802.)。然而，这些小分子抗体目前主要来源于骆驼或鲨鱼，在人体内容易产生免疫反应从而限制了其在临床上的应用。虽然动物源的纳米抗体可以通过基因工程技术进行人源化，但此过程中抗体容易失去热稳定性以及很难保持抗体的亲和力。有研究发现，人的某一重链可变区亚家族也具有较优异的稳定性、可溶性、低聚集性、优异的表达和纯化产率以及适用于噬菌体展示系统的特性(JMol Biol,2008.382(3):p.779-789.)。
在癌症的治疗中，抗体偶联物(ADC)是近年来发展起来的新型抗癌药物。传统的抗体偶联物的制备一般通过将抗体链间二硫键还原，将payloads偶联至半胱氨酸或赖氨酸上。赖氨酸偶联会导致每个抗体偶联0-8个药物分子，肽图实验显示偶联位点一般发生在重链和轻链的大约20个赖氨酸残基上(一个抗体上约有40个赖氨酸)，因此，此种偶联方式将会产生数百万计的不同形式的ADC药物分子。另外，ADC混合物在药物分子载量和偶联位点的不同也会使异质多样性数目翻番。不同位点偶联的多样异质ADC在体内PK动力学性质也不同，后期工业化生产难度将增加，批次间样品一致性将面临非常大的生产挑战。
位点特异偶联技术能保证将一定数目药物分子定点偶联至抗体的特定位点，很大程度上在生产上保证药物同质性和批量生产稳定性。另外DAR值也可以精确控制。位点特异性ADCs技术不仅使得在不同位点进行偶联对ADC药效影响的研究成为可能，而且可以广泛应用于其他分子如核素，免疫毒素，蛋白，前体酶等分子与抗体的偶联药物开发上，大大提高这些偶联药物的治疗效果和应用。目前位点特异性ADC技术一般通过工程化改造半胱氨酸位点，非天然氨基酸，硒代半胱氨酸和酶(谷氨酰胺，糖工程，FGE)偶联等技术将药物分子特异偶联至靶向抗体上。
基于现有技术的研究基础，本申请的研究团队以所述的重链可变区亚家族为骨架，引入源于健康成年人与新生儿的所有抗体亚家族的重链CDR区(CDR1,CDR2,CDR3)，构建得全人源纳米抗体噬菌体展示库；进一步，本申请利用所述的抗体库从中筛选提供针对人的5T4靶点的特异性全人源纳米抗体。
发明内容
本发明的目的是基于现有技术的研究基础，为解决现有技术存在的问题，通过对可结合人的5T4靶点的全人源纳米抗体进行半胱氨酸工程化改造，构建了一种半胱氨酸工程化的结合人5T4的全人源纳米抗体。本发明的光胱氨酸工程化全人源纳米抗体在人5T4全人源纳米抗体的第63、85和/或120位氨基酸被半胱氨酸所取代，所取代的半胱氨酸提供了能用于偶联的游离巯基，从而实现将细胞毒性剂、酶或检测标记物等偶联至改造后的全人源纳米抗体上，并将其应用于癌症的治疗或免疫分析和检测。
具体的，
本发明提供了一种结合人5T4抗原的半胱氨酸工程化全人源纳米抗体，其特征在于，所述的半胱氨酸工程化全人源纳米抗体的序列包含SEQ ID NO:1的63、85和/或120位氨基酸被半胱氨酸取代后的氨基酸序列。
优选的，本发明所述半胱氨酸工程化全人源纳米抗体包含至少一个游离的巯基。
优选的，本发明所述游离的巯基能与细胞毒性剂、化疗剂、放射性核素、核酸或可检测标记物进行化学偶联。
本发明所述的细胞毒性剂包括任何对细胞有害的试剂，包括但不限于细胞松弛素B、短杆菌肽D、溴乙啶、丝裂霉素、长春新碱、长春碱、秋水仙碱、道诺霉素、嘌呤霉素、环磷酰胺、利多卡因等，以及他们的类似物或同源物。
本发明所述的化疗剂，包括但不限于紫杉醇、阿霉素、卡铂、美法仑、长春花生物碱、甲氨喋呤、丝裂霉素C以及其他。
本发明所述的放射性核素，包括但不限于，锌、硫、锰、钯、磷、氟、钴、碘等；采用各种正电子发射断层扫描的正电子发射金属，以及非放射性顺磁性金属离子。
本发明所述的核酸可以选自DNA、RNA、短链干扰RNA(siRNA)、小分子RNA、发夹或核酸模拟物例如肽核酸。
本发明所述的可检测标记物包括但不限于各种酶，所述酶包括但不限于辣根过氧化物酶、碱性磷酸酶、β半乳糖苷酶、乙酰胆碱酯酶；辅基，所述辅基包括但不限于联袂亲和素/生物素、抗生素蛋白/生物素；荧光物质，所述荧光物质包括但不限于7-羟基香豆素、荧光素、异硫氰酸酯荧光素、若丹明、二氯三嗪胺荧光素、丹磺酰氯、藻红蛋白；发光物质，所述发光物质包括但不限于生物发光物质，所述生物发光物质包括但不限于荧光素酶、荧光素、发光蛋白。
本发明提供了编码所述结合人5T4抗原的半胱氨酸工程化全人源纳米抗体的核酸。本发明提供了包含所述核酸的质粒，任选地，可操作地连接调控序列，如启动子、增强子等。本发明提供了包含该质粒的宿主细胞，以及用于生产和任选地回收该结合人5T4抗原的半胱氨酸工程化全人源纳米抗体的方法。本发明所述宿主细胞可以是任何原核细胞或真核细胞，包括但不限于细菌细胞(例如大肠杆菌、枯草杆菌)、昆虫细胞(例如利用杆状病毒表达系统)、酵母或哺乳动物细胞(例如CHO或BHK细胞系)。其它合适的宿主细胞为本领域技术人员所知。
本发明提供了一种结合人5T4抗原的半胱氨酸工程化全人源纳米抗体的抗体偶联物，其特征在于，包含本发明所述的结合人5T4抗原的半胱氨酸工程化全人源纳米抗体和偶联物。
优选的，本发明所述偶联物为细胞毒性剂、化疗剂、放射性核素、核酸或可检测标记物。
本发明提供了一种药用组合物，由有效预防或治疗剂量的本发明的结合人5T4抗原的半胱氨酸工程化全人源纳米抗体或其核酸分子、质粒或结合人5T4抗原的半胱氨酸工程化全人源纳米抗体的抗体偶联物，和生理学上或药学上可接受的载体、赋形剂或稳定剂混合制备而成，所述组合物包括但不限于冻干剂型、水溶液剂型、脂质体或胶囊剂型等。本发明的结合人5T4抗原的半胱氨酸工程化全人源纳米抗体或其核酸分子、质粒或结合人5T4抗原的半胱氨酸工程化全人源纳米抗体的抗体偶联物的浓度可从约0.1％变化为100％(重量)。
本发明提供了一种制备结合人5T4抗原的半胱氨酸工程化全人源纳米抗体的抗体偶联物的方法，该方法包括如下步骤：
(1)表达和纯化结合人5T4抗原的半胱氨酸工程化的全人源纳米抗体；
(2)加入能与游离巯基进行化学偶联的偶联物，进行反应，制备抗体偶联物；
(3)分子筛过滤未反应的偶联物。
优选的，本发明所述方法中的偶联物包括但不限于细胞毒性剂、化疗剂、放射性核素、核酸或可检测标记物。
本发明提供了一种检测试剂盒，含有本发明所述的半胱氨酸工程化的全人源纳米抗体、其核酸分子或质粒、或其抗体偶联物。该检测试剂盒用于检测肿瘤细胞，所述肿瘤细胞包括各种良性肿瘤细胞、恶性肿瘤细胞(即癌细胞)、实体瘤细胞、血源性癌细胞。
本发明提供了一种诊断、预防或治疗疾病的方法，包括向受试者施用本发明所述的结合人5T4抗原的半胱氨酸工程化全人源纳米抗体、核酸分子、质粒、结合人5T4抗原的半胱氨酸工程化全人源纳米抗体的抗体偶联物以及药用组合物。
本发明所述疾病为癌症。本发明所述癌症包括但不限于淋巴瘤、胚细胞瘤、肉瘤(包括脂肉瘤)、神经内分泌肿瘤、间皮瘤、神经鞘瘤、脑膜瘤、腺瘤、黑素瘤以及非白血性白血病或淋巴恶性肿瘤。上述癌症更具体的实例包括鳞状细胞癌(如，鳞状上皮细胞癌)、肺癌、小细胞肺癌、非小细胞肺癌、肺腺癌以及肺鳞状细胞癌、腹膜癌、肝细胞癌、胃癌、胃肠癌、胰腺癌、恶性胶质瘤、宫颈癌、卵巢癌、肝癌、膀胱癌、肝细胞瘤、乳腺癌、结肠癌、直肠癌、结肠直肠癌、子宫内膜或子宫癌、唾液腺癌、肾癌、前列腺癌、外阴癌、甲状腺癌、肝癌、肛门癌、阴茎癌、睾丸癌、食道癌、胆管肿瘤，头癌、颈癌、骨髓基质瘤、破骨细胞瘤、多发性骨髓瘤、溶骨性癌(osteolytic bone cancers)、中枢神经系统肿瘤、脑肿瘤(神经胶质瘤、成神经细胞瘤、星细胞瘤、成神经管细胞瘤、室管膜细胞瘤和视网膜成神经细胞瘤)、鼻咽癌、基底细胞癌、胆管癌、卡波氏肉瘤、原发性肝癌或子宫内膜癌、以及血管系统肿瘤(血管肉瘤和hemagiopericytoma)。
本发明的结合人5T4抗原的半胱氨酸工程化全人源纳米抗体、核酸分子、质粒、结合人5T4抗原的半胱氨酸工程化全人源纳米抗体的抗体偶联物以及药用组合物可以通过各种不同的给药途径给予人或动物受试者，所述给药途径通常取决于待治疗的疾病本身的特征。一般而言，可利用医学上可接受的任何给药模式来实施本发明的方法，所述给药模式包括经口、直肠、局部、眼内、脑池内、脑室内、气管内、鼻内滴入、透皮、皮下、鞘内、肌内、腹腔、腹膜内、颅内输注或者静脉输注。
本发明通过对可结合人的5T4靶点的全人源纳米抗体进行半胱氨酸工程化改造，即在人5T4全人源纳米抗体的第63、85和/或120位氨基酸被半胱氨酸所取代，所取代的半胱氨酸提供了能用于偶联的游离巯基，从而实现将细胞毒性剂、酶或检测标记物等偶联至改造后的全人源纳米抗体上，并将其应用于癌症的治疗或免疫分析和检测。本发明的位点特异偶联技术能保证将一定数目药物分子定点偶联至抗体的特定位点，很大程度上在生产上保证药物同质性和批量生产稳定性。
为了能更彻底地理解发明，以下列出一些定义。上述定义意在包含语法等同成分。
本文中使用的“氨基酸”意指20种天然存在的氨基酸之一或任一非天然类似物，它们可位于具体规定的位置。“氨基酸”也包括诸如脯氨酸和羟脯氨酸的亚氨基酸残基。侧链可以是(R)或(S)构型。在优选的实施方案中，氨基酸以(S)或L-构型存在。如果使用非天然存在的侧链，可使用非氨基酸取代，例如以阻止或延迟体内降解。
本文中“游离的巯基”意指半胱氨酸中由一个硫原子和一个氢原子相连而成的官能团，其末端没有连接基团。
本文中“抗体”意指由基本上为公认的免疫球蛋白基因的全部或部分所编码的一种或多种多肽组成的蛋白质。所述公认的免疫球蛋白基因，例如在人中，包括kappa(κ)、lambda(λ)和重链基因座，其中包含了无数的可变区基因，以及分别编码IgM、IgD、IgG、IgE和IgA同种型的恒定区基因mu(μ)、delta(δ)、gamma(γ)、epsilon(ε)、alpha(α)。本文中的抗体意指包括全长抗体和抗体片段，以及来自任意生物体的天然抗体，工程抗体，或为试验、治疗目的或其它如下所进一步规定的目的而重组产生的抗体。术语“抗体”包括抗体片段，为本领域所公知，诸如Fab、Fab’、F(ab’)2、Fv，scFv或抗体的抗原结合的其它亚序列，或通过修饰完整抗体或使用重组DNA技术重新合成的那些抗体而产生的抗体片段。术语“抗体”包括单克隆以及多克隆抗体。抗体可以是拮抗剂、激动剂、中和性抗体、或抑制性抗体、或刺激性抗体。本发明的抗体可以是非人抗体，嵌合抗体，人源化抗体或完全人抗体。
本文中使用的“抗原”意指可以在动物体内刺激抗体产生或T细胞反应的化合物、组合物或物质，包括注射或吸收到动物体内的组合物，可以是蛋白质、糖类、脂质或其它病原体。
本文所使用的“核酸”意指由核苷酸单元(核糖核苷酸，脱氧核糖核苷酸，相关的天然存在的结构变体及其合成的非天然存在的类似物)通过磷酸二酯键组成的聚合物。因此，该术语包括核苷酸聚合物，其中核苷酸和它们之间的键包括非天然存在的合成类似物，例如但不限于硫代磷酸酯，氨基磷酸酯，甲基磷酸酯，手性甲基磷酸酯，2'-O-甲基核糖核苷酸，肽核酸(PNA)等。例如，可以使用自动DNA合成仪合成这些多核苷酸。术语“寡核苷酸”通常是指短多核苷酸，通常不大于约50个核苷酸。应当理解，当核苷酸序列由DNA序列(即A，T，G，C)表示时，这也包括其中“U”取代“T”的RNA序列(即A，U，G，C)。
本文使用常规符号来描述核苷酸序列：单链核苷酸序列的左手末端5'末端；双链核苷酸序列的左手方向称5'方向。向新生RNA转录物添加5'至3'核苷酸的方向称为转录方向。具有与mRNA相同序列的DNA链被称为编码链。
本文中所使用的“编码”意指多核苷酸中特定核苷酸序列的固有特性，例如基因，cDNA或mRNA，用作在具有确定的核苷酸序列的生物过程中合成其他聚合物和大分子的模板，或确定的氨基酸序列和由此产生的生物学特性。因此，如果由该基因产生的mRNA的转录和翻译在细胞或其他生物系统中产生蛋白质，则基因编码蛋白质。编码链(其核苷酸序列与mRNA序列相同并且通常在序列表中提供)和非编码链(用作转录模板，基因或cDNA)可以被称为编码蛋白质。或该基因或cDNA的其他产物。除非另有说明，否则“编码氨基酸序列的核苷酸序列”包括彼此简并形式且编码相同氨基酸序列的所有核苷酸序列。编码蛋白质和RNA的核苷酸序列可包括内含子。
本文中使用的“质粒”意指在天然质粒的基础上为适应实验室操作而进行人工构建的质粒。可将核酸分子导入宿主细胞，从而产生转化的宿主细胞。载体可包括允许其在宿主细胞中复制的核酸序列，例如复制起点，还可以包括一种或多种选择标记基因和本领域已知的其他遗传元件。
本文所使用的“宿主细胞”也称为受体细胞，是指在转化和转导(感染)中接受外源基因的宿主细胞。
本文中使用的“药学可接受载体”意指常规的药学上可接受的载体。Remington'sPharmaceutical Sciences，EWMartin，Mack Publishing Co.，Easton,Pa.，第15版(1975)，描述了适用于药物递送一种或多种治疗化合物或分子(例如一种或多种抗体)的组合物和制剂，以及另外的药剂。
本文中所使用的“诊断”疾病是指在给病人做检查之后判定病人的病症及其发展情况。“预防”疾病是指抑制疾病的完全发展。“治疗”是指在其开始发展后改善疾病或病理状况的体征或症状的治疗性干预。
本文中“施用”意指选择合适的途径将所述物质引入受试者。例如，如果所选择的途径是静脉内的，则通过将所述物质引入受试者的静脉来施用组合物。
本文中“有效预防/治疗剂量”意指足以在用该药剂治疗的受试者中达到所需效果的一定量的特定药剂。精确的剂量将依赖于治疗的目的，并可为本领域技术人员通过使用公知技术所确定。剂量范围可为0.01-100mg/kg体重或更大，例如0.1、1、10或50mg/kg体重，优选1-10mg/kg。如本领域所公知，对于抗体或Fc融合体降解、全身性或局部性递药和新蛋白酶合成速率，以及年龄、体重、大致健康状况、性别、饮食、给药时间、药物相互作用以及病症的严重程度而言，调整可以是必需的，并可由本领域那些技术人员通过常规的实验方法来确定。此类试剂包括本文所述的单体Fc结构域分子。在一个非限制性实例中，这可以是用于预防，治疗或改善HIV感染的HIV特异性单体Fc结构域(或HIV特异性CH3结构域分子)的量。理想地，治疗有效量的抗体是足以预防，治疗或改善感染或疾病的量，例如由受试者中的HIV感染引起而不会在受试者中引起显着的细胞毒性作用。用于预防，改善和/或治疗受试者的治疗有效量的药剂将取决于所治疗的受试者，痛苦的类型和严重程度，以及治疗组合物的施用方式。
本文中的“癌症”为实体瘤或血源性癌。本发明所述的实体瘤是肉瘤或癌，例如纤维肉瘤，粘液肉瘤，脂肪肉瘤，软骨肉瘤，成骨肉瘤，或另一种肉瘤，滑膜瘤，间皮瘤，尤文氏瘤，平滑肌肉瘤，横纹肌肉瘤，结肠癌，淋巴恶性肿瘤，胰腺癌，乳腺癌，肺癌，卵巢癌，前列腺癌，肝细胞癌，鳞状细胞癌，基底细胞癌，腺癌，汗腺癌，皮脂腺癌，乳头状癌，乳头状腺癌，髓样癌，支气管癌，肾细胞癌，肝细胞癌，胆管癌，绒毛膜癌，肾母细胞瘤，宫颈癌，睾丸肿瘤，膀胱癌或中枢神经系统肿瘤(如胶质瘤，星形细胞瘤，成神经管细胞瘤，颅咽管瘤，室管膜瘤，松果体，血管母细胞瘤，听神经瘤，少突神经胶质瘤，血管瘤，黑素瘤，神经母细胞瘤或视网膜母细胞瘤)。本发明所述的血源性癌症是白血病，如急性白血病(如急性淋巴细胞白血病，急性髓细胞白血病，急性髓性白血病和成髓细胞，早幼粒细胞，髓单核细胞，单核细胞和红白血病)；慢性白血病(如慢性粒细胞性(粒细胞)白血病，慢性粒细胞白血病和慢性淋巴细胞白血病)，真性红细胞增多症，淋巴瘤，霍奇金病，非霍奇金淋巴瘤(惰性和高级形式)，多发性骨髓瘤，瓦尔登斯特伦巨球蛋白血症，重链性疾病，骨髓增生异常综合征，多毛细胞白血病或骨髓增生异常。
除非另外说明，否则本文使用的所有技术和科学术语具有与本公开所属领域的普通技术人员通常理解的含义相同的含义。除非上下文另有明确说明，否则单数术语“一”，“一个”和“该”包括复数指示物。还应理解，对于核酸或多肽给出的所有碱基大小或氨基酸大小，以及所有分子量或分子量值是近似的，并且提供用于描述。尽管与本文描述的那些类似或等同的方法和材料可用于本公开的实践或测试，但下文描述了合适的方法和材料。术语“包含”表示“包括”。本文提及的所有出版物、专利申请、专利和其他参考文献均通过引用整体并入。如果发生冲突，将以本说明书(包括术语解释)为准。另外，材料，方法和实施例仅是说明性的而不是限制性的。
附图说明
图1.全人源纳米抗体n501和5T4抗原的复合物结构图，其中，发生突变的氨基酸残基S63、S85、S120用深黑色表示。
图2.利用ForteBio检测全人源纳米抗体n501以及半胱氨酸改造的n501与5T4抗原的结合亲和力。
图3.流式细胞仪检测全人源纳米抗体n501(S85C)与各肿瘤细胞系的结合。
图4.抗5T4纳米抗体与MMAE的抗体偶联物。
图5.抗体偶联物与5T4的结合。
图6.抗体偶联物对细胞的杀伤活性。
图7.抗体偶联物对胰腺癌类器官的杀伤。
图8.n501-MMAE对异种移植瘤小鼠模型的抗肿瘤活性。
具体实施方式
实施例中使用的标准的重组DNA技术和分子克隆技术是本领域所熟知的(Ausubel，F.M等人，Current Protocols in Molecular Biology,Greene PublishingAssoc.和Wiley-Interscience出版)，适用于微生物生长的材料和方法是本领域熟知的。主要化学、生物试剂购自KAPA Biosystems，New England Biolabs，TransGen Biotech，Thermo Fisher Scientific，OMEGA bio-tek等。
下面结合具体实施例对本发明进行详细说明。
实施例1人的5T4抗原的生产
编码人的5T4的60-344位氨基酸(GenBank:CAA09930.1)的基因由Genscript公司合成，并构建到带有His标签的昆虫表达载体pFastBac1(Invitrogen，产品号：10712024)中。参照Cellfectin Reagent转染试剂说明书，将重组质粒和转染试剂于300uL无血清培养基中室温孵育30min后，将两溶液混合于室温再孵育20min。转染昆虫细胞Sf9(ThermoFisher，产品号：B82501)后5h，将培养基换成含10％FBS的培养基继续培养。待细胞出现明显病变时，收集细胞及上清液，500g离心5min，收集上清，即为P1重组杆状病毒。将P1代病毒感染Sf9细胞，48h后，离心收集上清，即P2代重组杆状病毒。将P2代病毒感染Sf9细胞5-7天，收集上清。用平衡缓冲液(含有2.5M NaCl的PBS缓冲液)平衡Ni-NTA树脂(GE Healthcare，产品号：170531801)，将含有5T4蛋白的细胞上清液加入Ni-NTA树脂中，控制流速在1ml/min。用2倍柱体积的平衡缓冲液以及20mM咪唑(购自SigmaAldrich)依次洗脱杂蛋白，最终用250mM咪唑洗脱结合的抗体。将洗脱后的溶液转移至3kD MWCO的Amicon Ultra离心超滤管(购自Millipore)中，于4℃、6500g超滤20min，并用PBS溶液置换洗脱缓冲液，重复超滤3～4次，收集浓缩后的蛋白。蛋白浓度定量用分光光度计(购自BioTek)测定280nm处的吸光值，SDS-PAGE电泳检测纯度。
实施例2针对5T4的全人源纳米抗体的生产
针对5T4的全人源纳米抗体的可溶性表达产物的制备基本按文献进行(Cell HostMicrobe.2017.22(4):471-483.e5.)。具体为：将全人源纳米抗体n501(抗体序列为SEQ IDNO:1)的重组质粒(见中国专利：2016110409818)转入HB2151感受态细胞(来自于质粒载体菌株细胞基因保藏中心)，从过夜生长的氨苄平皿(胰蛋白胨16g，酵母提取物10g，NaCl5g，琼脂粉2g，加蒸馏水至1L，高压灭菌。加入100μg/ml Amp和2％Glucose，保存于4℃)中挑取单个菌落，接种SB细菌培养液(胰蛋白胨30g，酵母提取物20g，3-(N-吗啉)丙磺酸(MOPS)10g，加蒸馏水至1L，并调节pH至7.0，高压灭菌，保存于4℃)，在30度IPTG(23.8g IPTG溶于100mL去离子水中，0.22μm滤器过滤除菌后分装于无菌1.5mL Eppendorf管中，保存于-20℃，使用浓度为1mM)诱导条件下表达12-14小时。用平衡缓冲液(含有2.5M NaCl的PBS缓冲液)平衡Ni-NTA树脂(GE Healthcare，产品号：170531801)，将含有抗体的细菌裂解液的上清液加入Ni-NTA树脂中，控制流速在1ml/min。用2倍柱体积的平衡缓冲液以及20mM咪唑(购自SigmaAldrich)依次洗脱杂蛋白，最终用250mM咪唑洗脱结合的抗体。将洗脱后的溶液转移至3kD MWCO的Amicon Ultra离心超滤管(购自Millipore)中，于4℃、6500g超滤20min，并用PBS溶液置换洗脱缓冲液，重复超滤3～4次，收集浓缩后的抗体。蛋白浓度定量用分光光度计(购自BioTek)测定280nm处的吸光值，SDS-PAGE电泳检测纯度。
实施例3人的5T4抗原和全人源纳米抗体n501的复合物晶体解析
4℃条件下将纯化的全人源纳米抗体n501与5T4蛋白按照摩尔比1：1进行混合使之形成抗原-抗体复合物，用Superdex 200column(购自GE Healthcare)通过尺寸排阻色谱法(具体方法参考Cell Host Microbe.2017.22(4):471-483.e5.)将复合物进行纯化。纯化后的复合物超滤浓缩(具体方法参考Cell Host Microbe.2017.22(4):471-483.e5.)至5mg/ml，利用悬滴法(具体方法参考Cell Host Microbe.2017.22(4):471-483.e5.)将晶体置于96孔板中于20℃下生长直至晶体长至合适尺寸。利用X-ray衍射技术(具体方法参考CellHost Microbe.2017.22(4):471-483.e5.)对抗原抗体复合物的精细作用模式进行解析(见图1)。选取抗体上一些不参与5T4相互作用的氨基酸位点(S63，S85，以及S120)进行半胱氨酸改造。
实施例4全人源纳米抗体与5T4的结合动力学检测
使用BLI技术(具体方法参见Emerg Microbes Infect.2017.6(10):e89.)测定全人源纳米抗体n501以及半胱氨酸改造后的全人源纳米抗体n501(S63C)，n501(S85C)，n501(S120C)以及n501(S63/S85/S120C)与5T4的结合活性。将5T4固定于AHC传感器(购自PallFortebio)上，浓度梯度稀释的抗体溶液作为分析样品。步骤如下：将5T4蛋白用PBST溶液(含有0.02％Tween-20的PBS溶液)稀释至10μg/ml，然后固化到AHC biosensor上，与3倍梯度稀释的抗体溶液(浓度范围为100～1.24nM)在running buffer(PBST：含有0.02％Tween-20的PBS溶液)中进行结合，其中空白对照孔中不加入抗体。程序设定为：Association，300s；Dissociation，300s，温度设定为37℃。应用ForteBio Data analysis 10.1软件对数据进行分析。以对应的空白对照孔作为对照扣减背景，各浓度的曲线按照1:1结合模式进行拟合，得到动力学分析结果，报告中显示结合的平衡常数(KD)值以及结合速率常数(kon)和解离速率常数(koff)(见图2)。结果显示，n501抗体在85和120位进行半胱氨酸改造后，亲和力与改造前相当，但进行S63C改造后，亲和力下降。三个位点同时进行半胱氨酸改造后，亲和力与改造前相比有所下降。
实施例5流式细胞术检测抗体与细胞的结合活性
将荧光标记物DyLight 650(ThermoFisher，产品号：84535)与全人源纳米抗体n501(S85C)于室温下进行孵育1小时使n501(S85C)偶联上荧光标记物DyLight 650。将细胞培养至对数生长期，用胰酶(Gibco，产品号：25200072)消化细胞后，PBS(Gibco，产品号：10010049)洗3遍，加入3％BSA(上海翊圣生物科技有限公司，产品号：36106ES25)稀释的带有荧光的抗体n501(S85C)(100nM)，室温避光放置1.5h。PBS洗3遍，0.2ml PBS溶液重悬细胞，然后上流式细胞仪(BD Calibur)检测。结果显示，全人源纳米抗体n501(S85C)能跟表达5T4的细胞进行结合，与不表达5T4的FCHO细胞则不结合(见图3)。所用的人胰腺癌细胞PANC-1和BxPc-3均来自复旦IBS细胞资源中心(FDCC)，人乳腺癌细胞系MDA-MB-468，人卵巢癌细胞系PA-1，人肝癌细胞系Huh-7和仓鼠卵巢细胞FCHO均来自中国科学院细胞库。
实施例6制备抗5T4纳米抗体与MMAE抗体偶联物
抗5T4全人源纳米抗体半胱氨酸改造后的n501(S63C)，n501(S85C)，n501(S120C)以及n501(S63/S85/S120C)溶解于含有1mM TCEP(购自SIGMA)的PBS溶液中，维持其游离的巯基。加入2摩尔的vc-MMAE(来自上海美雅珂生物技术有限责任公司)并于室温下连续摇动20分钟。然后将过量的未结合的vcMMAE用2倍摩尔比的NAC(N-乙酰-L-半胱氨酸)(上海翊圣生物科技有限公司，产品号：50303ES05)封闭5分钟。取出反应液，采用脱盐柱(GEHealthcare)去除反应体系中残留的小分子并将缓冲液置换成PBS溶液。经质谱检测(方法见The AAPS,2014,16(3))，DAR值分别为：n501(S63C)-MMAE为1，n501(S85C)-MMAE为1，n501(S120C)-MMAE为1，n501(S63/S85/S120C)-MMAE为3(见图4)。
实施例7抗体偶联物与5T4的结合
将5T4蛋白包被在ELISA板(购自Corning)上，加入起始浓度为1000nM，3倍梯度浓度稀释的n501(S85C)-MMAE孵育，加入抗FLAG标签抗体(购自SIGMA)来检测抗体n501(S85C)-MMAE与抗原5T4的结合能力(具体方法见Cell Host Microbe.2020.27(6):891-898.e5.)。图5的ELISA结果显示，偶联了MMAE的n501(S85C)保持与5T4的结合能力。
实施例8抗体偶联物对细胞的杀伤活性
将细胞培养至对数生长期，用胰酶(购自Gibco)消化细胞后，将细胞密度调整为10E6细胞/毫升。将细胞以每孔5000个细胞接种于96孔培养板中，37℃，5％CO2培养过夜。弃去培养基，加入200ul的抗体稀释液(起始浓度为1000nM)，继续培养72h。弃去培养基，加入含有10％CCK8(购自碧云天生物有限公司)的培养基静置1-4h，检测450nm下的吸收值。设置空白对照孔和未处理孔，根据公式：细胞存活率(％)＝(OD处理组-OD空白对照孔)/(OD未处理孔-OD空白对照孔)*100％，计算细胞存活率。结果显示，连接单个MMAE分子的n501(S85C)-MMAE和n501(S120C)-MMAE的杀伤活性最好，n501(S63C)-MMAE的杀伤活性偏弱。连接了3个MMAE分子的n501(S63/S85/S120C)-MMAE杀伤活性最强(图6)。
实施例9抗体偶联物对胰腺癌类器官的杀伤
正常和肿瘤胰腺组织在含有9mL DMEM培养基(GIBCO，产品号：C1199500BT)、500U/ml胶原酶IV(购自Sigma-Aldich)、1.5mg胶原酶II(购自Solarbio)、20μm/ml透明质酸酶(购自Solarbio)、0.1mg/ml dispase II(购自Sigma-Aldrich)、10μM RHOK抑制剂Y-27632(购自Sigma-Aldrich)和1％胎牛血清的10ml溶液中在37℃下放置30分钟进行消化。细胞250g离心5min后，接种于Matrigel(购自Corning)48孔平底细胞培养板中，用300μl培养基于37℃、5％CO2培养箱中孵育10min。对于肿瘤源性类器官，其培养基为含30％Wnt3a(购自Sino-Biological)、500ng/ml R-spondin 1(购自Sino-Biological)、100ng/ml Noggin(购自Sino-Biological)、50ng/ml EGF(购自Sino-Biological)、100ng/ml FGF-10(购自Sino-Biological)、1×HEPES(购自GIBCO)、1×谷氨酰胺(购自GIBCO)、100μg/ml诺莫辛(购自InvivoGen)，1×庆大霉素/两性激素B(购自GIBCO)，1×B27(购自Invitrogen)，1mM n-乙酰半胱氨酸(购自Sigma-Aldrich)，10mM烟酰胺(购自Sigma-Aldrich)，0.5μM A-83-01(购自Tocris)，10μM Y-27632(购自Sigma-Aldrich)和10nM胃泌素(购自Sigma-Aldrich)的DMEM/F12培养基。对于正常胰腺类器官，其培养基需额外加入1μM前列腺素E2(购自Sigma-Aldrich)。培养基每三天更新一次。一旦类器官建立立即接种到96孔板中。当类器官生长到直径为100μm时，用培养基稀释的浓度为1000nM、100nM和10nM的n501(S85C)-MMAE进行处理。每三天更新一次含有1000nM、100nM和10nM的n501(S85C)-MMAE的新鲜培养基。在第0天、第3天、第6天和第9天拍照。实验结束时，通过CellTiter Glo(购自Promega)对类器官活性进行测定。结果显示，n501(S85C)-MMAE可以有效的杀伤胰腺癌类器官，但对正常组织没有明显的杀伤活性(图7)。
实施例10抗体偶联物的体内抗肿瘤活性
将人胰腺癌BxPC-3细胞培养至对数生长期，用胰酶(购自Gibco)消化细胞后，用无血清无抗生素的培养基(购自Gibco)清洗两次，并进行细胞计数。用无血清无抗生素的培养基和基质胶(购自Corning)将细胞密度调整为10E7或细胞/毫升。
实验动物为BALB/c无胸腺裸鼠(购自上海灵畅生物科技有限公司)，6-8周龄，体重20g左右。实验动物饲养：饲养于SPF级，每周更换一次饲料、饮用水、垫料和笼具，同时观察动物生理状态。
将10E6个细胞接种于裸鼠背部，接种体积均为100ul。当肿瘤长至200mm3左右，可进行分组和给药。总共分为5组，分别为PBS组，G12-MMAE对照组(G12为靶向乙肝病毒表面抗原的全人源单抗，参见文献：Shi B et al.,EBioMedicine 2019；49:247-57；Wang W etal.,Mab 2016；8:468-77，偶联MMAE后作为无关抗体的对照)，n501抗体组以及n501-MMAE抗体组。称量各组裸鼠体重，测量肿瘤体积。开始以尾静脉注射给药，每两天给药一次，连续给药5次。肿瘤大小计算公式：V＝长径*短径*短径/2。对照小鼠肿瘤体积大于2000mm3情况下，停止治疗，处死小鼠。结果显示，偶联了小分子药物的n501-MMAE抗体组具有很好的抗肿瘤活性(如图8所示)。
序列表
<110> 复旦大学
<120> 一种半胱氨酸工程化的结合人5T4的全人源纳米抗体
<130> 20210223
<160> 1
<170> SIPOSequenceListing 1.0
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<212> PRT
<213> 5T4
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Ala Arg Leu Arg Asp Gly Phe Asn Asn Gly Phe Asp Tyr Trp Gly Gln
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Gly Thr Leu Val Thr Val Ser Ser
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Claims (13)
1.一种结合人5T4抗原的半胱氨酸工程化全人源纳米抗体，其特征在于，所述的半胱氨酸工程化全人源纳米抗体的序列包含SEQ ID NO:1的63、85和/或120位氨基酸被半胱氨酸取代后的氨基酸序列。
2.根据权利要求1所述的半胱氨酸工程化全人源纳米抗体，其特征在于，所述半胱氨酸工程化全人源纳米抗体包含至少一个游离的巯基。
3.根据权利要求2所述的半胱氨酸工程化全人源纳米抗体，其特征在于，所述游离的巯基能与细胞毒性剂、化疗剂、放射性核素、核酸或可检测标记物进行化学偶联。
4.一种核酸分子，其特征在于编码权利要求1-3中任一项所述的半胱氨酸工程化全人源纳米抗体的核苷酸序列。
5.一种质粒，其特征在于含有权利要求4所述的核酸分子。
6.一种宿主细胞，其特征在于含有权利要求5所述的质粒。
7.一种结合人5T4抗原的半胱氨酸工程化全人源纳米抗体的抗体偶联物，其特征在于，包含如权利要求1-3中所述的任一结合人5T4抗原的半胱氨酸工程化全人源纳米抗体和偶联物。
8.根据权利要求7所述的结合人5T4抗原的半胱氨酸工程化全人源纳米抗体的抗体偶联物，其特征在于，所述偶联物为细胞毒性剂、化疗剂、放射性核素、核酸或可检测标记物。
9.一种药用组合物，其特征在于，含有有效预防或治疗剂量的如权利要求1-3中所述的任一半胱氨酸工程化全人源纳米抗体或如权利要求4中所述的核酸分子或如权利要求5中所述的质粒或如权利要求7-8中所述的任一结合人5T4抗原的半胱氨酸工程化全人源纳米抗体的抗体偶联物，和一种药学可接受载体。
10.一种制备如权利要求7-8中所述的任一结合人5T4抗原的半胱氨酸工程化全人源纳米抗体的抗体偶联物的方法，其特征在于，所述方法包括如下步骤：
(1)表达和纯化如权利要求1-3中所述的任一结合人5T4抗原的半胱氨酸工程化的全人源纳米抗体；
(2)加入能与游离巯基进行化学偶联的偶联物，进行反应，制备抗体偶联物；
(3)分子筛过滤未反应的偶联物。
11.根据权利要求10所述的制备结合人5T4抗原的半胱氨酸工程化全人源纳米抗体的抗体偶联物的方法，其特征在于，所述偶联物为细胞毒性剂、化疗剂、放射性核素、核酸或可检测标记物。
12.一种检测试剂盒，其特征在于，含有如权利要求1-3中所述的任一半胱氨酸工程化全人源纳米抗体，或权利要求4中所述的核酸分子，或权利要求5中所述的质粒，或权利要求7-8中所述的任一结合人5T4抗原的半胱氨酸工程化全人源纳米抗体的抗体偶联物。
13.根据权利要求12所述的检测试剂盒，其特征在于，所述的检测试剂盒用于检测肿瘤细胞。
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