CN115975039A - 重组融合抗体和抗体-药物偶联物及其用途 - Google Patents

重组融合抗体和抗体-药物偶联物及其用途 Download PDF

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CN115975039A
CN115975039A CN202111201616.1A CN202111201616A CN115975039A CN 115975039 A CN115975039 A CN 115975039A CN 202111201616 A CN202111201616 A CN 202111201616A CN 115975039 A CN115975039 A CN 115975039A
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蔡晓青
蒋先兴
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Sun Yat Sen University
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Priority to EP22880163.5A priority patent/EP4417627A1/en
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Abstract

本发明公开一种重组融合抗体和抗体‑药物偶联物及其用途，该重组融合抗体包括抗体、至少一溶酶体靶向肽以及至少一接头肽，所述溶酶体靶向肽与所述抗体通过所述接头肽融合形成所述重组融合抗体；其中所述溶酶体靶向肽为高尔基体定位的含γ‑适应蛋白耳的生长素响应因子结合蛋白(GGA)、甘露糖‑6‑磷酸受体(MPR)和溶酶体关联膜蛋白(LAMP)的溶酶体分选信号肽，或在其基础上取代、缺失和/或添加一个或多个氨基酸。相较于原抗体，本发明的重组融合抗体的细胞内化效率及在溶酶体的富集能力都有明显提高。本发明重组融合抗体‑药物偶联物在实验中表现出优良的抗肿瘤活性，可用于癌症治疗。

Description

重组融合抗体和抗体-药物偶联物及其用途
技术领域
本发明属于生物药物技术领域，涉及一种重组融合抗体及其药物偶联物，具体涉及一类新型的重组融合抗体、包含该重组融合抗体的抗体-药物偶联物及其用途。
背景技术
抗体偶联药物又称抗体-药物偶联物(antibody-drug conjugate,ADC)，是一种通过接头小分子将细胞毒性剂偶联在抗体结构上的大分子偶联物，该偶联物利用抗体的靶向作用将毒性小分子呈递到肿瘤细胞，从而降低传统化疗药物的非特异性所引起的毒副作用。ADC结合了“化学疗法”和“抗体靶向疗法”的优势，为肿瘤的“精准治疗”提供新的思路和研究途径。在过去二十年里，ADC发展迅速，逐渐成为全球抗肿瘤药物研发的热点之一。ADC具有高效、低毒、药物适应症丰富等特点，应用市场潜力巨大。
如何提高ADC的内吞及在溶酶体的富集效率是ADC设计的瓶颈之一。与小分子药物不同的是，ADC药效的发挥需要抗体与细胞表面抗原产生特异性结合，通过抗原介导的方式发生内吞并转运至溶酶体降解，最终释放出毒性小分子药物，从而杀死肿瘤细胞。ADC递呈至肿瘤细胞表面后，并非所有分子都能够实现有效内吞；而内吞进入细胞内的ADC也只有一部分最终通过溶酶体途径实现降解。大多数ADC是经由网格蛋白介导的内吞、小窝蛋白介导的内吞和巨胞饮这三种方式进入胞内。其中，只有网格蛋白介导的内吞最能有效地将抗体运输至溶酶体中。因此，ADC能否有效内吞并进入溶酶体对其发挥药效至关重要。
ADC的内吞和在溶酶体富集效率还受限于肿瘤细胞表面抗原的表达水平。当前临床阶段的大多数ADC平均抗体载药量为2–4个；一般来说，每个细胞表面需要至少有104个抗原，才能保证由ADC输送到肿瘤细胞的药物量达到致死水平。理想的靶抗原不仅在肿瘤细胞表面有足够的表达量(>105抗原/细胞)，而且还能够有效诱导ADC内吞。事实上，肿瘤细胞表面抗原的数量有限，而且总是处于往返于胞膜内外的动态中，导致一部分内吞进入胞内的ADC又被运输到胞膜外，因而，在胞内释放的毒性小分子数量非常有限；尤其是在抗原表达量本身就低的肿瘤细胞中，进入细胞内的小分子药物的量就更少，杀伤效果更不明显。如上所述，由ADC输送到肿瘤细胞内并最终发挥作用的毒性药物往往非常有限，这也被认为是大多数ADC临床失败的主要原因之一。
如何提高ADC在溶酶体的富集效率以实现毒性药物的有效释放，是制约ADC技术发展的瓶颈之一。
发明内容
为解决现有技术存在的问题，本发明的一个目的是提供一种重组融合抗体，该重组融合抗体能够提高细胞内化效率及在溶酶体的富集能力；本发明的另一个目的是提供一种包含该重组融合抗体的抗体-药物偶联物，该重组融合抗体-药物偶联物能够表现出优良的抗肿瘤活性，可用于癌症治疗；本发明的又一个目的是提供该重组融合抗体-药物偶联物在制备用于治疗癌症的药物中的用途。
为实现上述目的，本发明主要通过以下技术方案实现：
一方面，本发明提供一种重组融合抗体，该重组融合抗体包括抗体、至少一溶酶体靶向肽以及至少一接头肽，所述溶酶体靶向肽与所述抗体通过所述接头肽融合形成所述重组融合抗体；其中所述溶酶体靶向肽为高尔基体定位的含γ-适应蛋白耳的生长素响应因子结合蛋白(GGA)、甘露糖-6-磷酸受体(MPR)和溶酶体关联膜蛋白(LAMP)的溶酶体分选信号肽，或在其基础上取代、缺失和/或添加一个或多个氨基酸。
本发明的溶酶体靶向肽选自以下氨基酸序列中的至少一种：ASVSLLDDELMSL(SEQID NO：1)，RRRASVSLLDDELMSL(SEQ ID NO：2)，ASVSLLDDEL(SEQ ID NO：3)，ASSGLDDLDLLGK(SEQ ID NO：4)，VQNPSADRNLLDL(SEQ ID NO：5)，NALSWLDEELLCL(SEQ ID NO：6)，SDEDLLHI(SEQ ID NO：7)，SFHDDSDEDLLHI(SEQ ID NO：8)，EESEERDDHLLPM(SEQ ID NO：9)，SYKYSKVNKE(SEQ ID NO：10)，PAAYRGVGDD(SEQ ID NO：11)，RRRSDEDLLHI(SEQ ID NO：12)，RRLRKSDEDLLHI(SEQ ID NO：13)，RRRRKSDEDLLHI(SEQ ID NO：14)，RRRSFHDDSDEDLLHI(SEQID NO：15)，RRLRKSFHDDSDEDLLHI(SEQ ID NO：16)，RRRRKSFHDDSDEDLLHI(SEQ ID NO：17)，RKRSHAGYQTI(SEQ ID NO：18)，RRRRKRKRSHAGYQTI(SEQ ID NO：19)，KHHHAGYEQF(SEQ IDNO：20)和RRLRKHHHAGYEQF(SEQ ID NO：21)。
本发明的接头肽的氨基酸序列为(Leu-Pro-Glu-Thr)x-(Glyy1-Sery2)z，其中x＝0或1，y1＝3、4或5，y2＝0或1，z＝1、2或3。
优选地，本发明的接头肽选自以下氨基酸序列中的至少一种：Leu-Pro-Glu-Thr-Gly-Gly-Gly(SEQ ID NO:22)，Gly-Gly-Gly，Gly-Gly-Gly-Gly-Gly(SEQ ID NO:23)，Gly-Gly-Gly-Gly-Ser(SEQ ID NO:24)，Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(SEQ IDNO:25)，和Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(SEQ IDNO:26)。
本发明的抗体具有肿瘤抗原特异性，该抗体为能够结合肿瘤细胞表面的靶标蛋白的抗体，优选地，所述肿瘤细胞表面的靶标蛋白选自以下中的至少一种：EGFR、EGFRvIII、HER2、ErB3、CD19、CD20、CD30、CD33、CD37、CD46、CD48、CD74、CD79B、CD27 ligand、TROP2、Nectin-4、PSMA、BCMA、HGFR、FOLR1、CA125、ALCAM、SLC1A5、Siglec-2、Siglec-3、IL-2Ralpha、IL-3R alpha、TNF-alpha、B7-H3、GUCY2C、TPBG、ROR2、ENPP3、Coagulation FactorIII、Mesothelin、Transferrin R、CEACAM-5、IGF-IR、Syndecan-1、DLL3、EpCAM、LIV-1、ROR1、TIM-1、Ly6E和NCAM-1。
本发明的抗体为单特异性抗体、双特异性抗体或多特异性抗体。
本发明的抗体为嵌合抗体、人源化抗体或人源抗体。
本发明的抗体包含单克隆抗体或其抗原结合片段。
优选地，抗原结合片段为Fab、Fab′、F(ab′)2、Fv、dsFv、scFv、sc(Fv)2或VHH。
本发明的溶酶体靶向肽的融合位置包括所述单克隆抗体的重链的C末端、所述单克隆抗体的重链的N末端、所述单克隆抗体的轻链的C末端和所述单克隆抗体的轻链的N末端中的至少一种。
本发明的溶酶体靶向肽的融合位置包括所述抗原结合片段的C末端和所述抗原结合片段的N末端中的至少一种。
另一方面，本发明提供一种重组融合抗体-药物偶联物，其中所述重组融合抗体-药物偶联物的结构如下所示：
Drn1Ab；
其中，Dr为药物；Ab为上述重组融合抗体；n1为大于或等于1的整数。
本发明的药物为细胞毒性剂，选自以下任一种：微管蛋白抑制剂的药物或前药，DNA损伤剂的药物或者前药，RNA聚合酶II抑制剂，和细菌、真菌或动物来源的毒素。
优选地，微管蛋白抑制剂选自以下任一种：奥里斯他汀(auristatin)或奥里斯他汀衍生物及类似物、美登素(maytansinoid)或美登素衍生物及类似物、紫杉醇(paclitaxel)或紫杉醇衍生物及类似物、长春花生物碱(vinca-alkaloid)或长春花生物碱衍生物及类似物、念珠藻素(cryptophycin)或念珠藻素衍生物及类似物、陶素(tubulysin)或陶素衍生物及类似物。
优选地，DNA损伤剂选自以下任一种：多柔比星(doxorubicin)或多柔比星衍生物及类似物、卡奇霉素(calicheamicin)或卡奇霉素衍生物及类似物、喜树碱(camptothecin)或喜树碱衍生物及类似物、吡咯并苯并二氮杂卓(pyrrolobenzodiazepines，PBD)或PBD衍生物及类似物、倍癌霉素(duocarmycin)或倍癌霉素衍生物及类似物。
本发明的药物通过共价键偶联在所述重组融合抗体上的反应性基团。
优选地，重组融合抗体上的反应性基团为赖氨酸、半胱氨酸或生物正交基团。
优选地，半胱氨酸为所述重组融合抗体自身的半胱氨酸，或人为突变得到的半胱氨酸。
优选地，生物正交基团为叠氮基团、炔基、醛基、酮基和氟代磺酸酯基团中的一种。
本发明的重组融合抗体与所述药物经由可裂解或不可裂解的接头小分子偶联。
优选地，接头小分子选自以下任一种：多肽、寡糖、-(CH2)n-、-(CH2CH2O)n-、Val-Cit-PABC、Val-Ala-PABC、Val-Lys(Ac)-PABC、Phe-Lys-PABC、Phe-Lys(Ac)-PABC、D-Val-Leu-Lys、Gly-Gly-Arg、Ala-Ala-Asn-PABC、Ala-PABC、PABC或其组合；其中，n为1-10的整数，Cit为瓜氨酸，Ac为乙酰基，PABC为对氨基苄醇。
又一方面，本发明提供一种重组融合抗体-药物偶联物在制备用于治疗癌症的药物中的用途。
本发明的癌症为胃肿瘤、肠肿瘤、肝脏肿瘤、肺肿瘤、胰腺肿瘤、乳腺肿瘤、宫颈肿瘤、子宫内膜肿瘤、卵巢肿瘤、前列腺肿瘤、膀胱肿瘤、鼻咽喉或软组织肿瘤、血液或淋巴肿瘤以及皮肤肿瘤。
本发明的技术方案相比现有技术具有以下优点：
本发明的重组融合抗体通过在抗体的C末端和/或N末端融合具有溶酶体靶向作用的溶酶体靶向肽。该溶酶体靶向肽是人的溶酶体分选信号肽或以人的溶酶体分选信号肽为模版获得的突变体。相较于原抗体，本发明的重组融合抗体能增强重组融合抗体-药物偶联物的内吞和在溶酶体富集的效率，有利于药物在细胞内的有效释放，进而提高重组融合抗体-药物偶联物的抗肿瘤活性。本发明的重组融合抗体-药物偶联物具有很好的细胞内化效率及在溶酶体的富集能力，并在实验中表现出优良的抗肿瘤活性，可用于癌症治疗。
附图说明
图1为本发明的实施例1的溶酶体靶向肽融合于纳米抗体的三种结构示意图。
图2为本发明的实施例1的溶酶体靶向肽融合于单链抗体的六种结构示意图。
图3为本发明的实施例1的溶酶体靶向肽融合于全长抗体的十五种结构示意图。
图4为本发明实施例1的重组融合纳米抗体及实施例4的重组融合纳米抗体-药物偶联物的SDS-PAGE分析图。
图5为本发明实施例1的重组融合全长抗体及实施例4的重组融合全长抗体-药物偶联物的SDS-PAGE分析图。
图6A为本发明实施例2的重组融合纳米抗体VHH1-VHH6的内化情况的表征结果图。
图6B为本发明实施例2的重组融合纳米抗体VHH1-VHH6的内化情况的量化结果图。
图7A为本发明实施例2的重组融合纳米抗体VHH7-VHH22的内化情况的表征结果图。
图7B为本发明实施例2的重组融合纳米抗体VHH7-VHH22的内化情况的量化结果图。
图8A为本发明实施例2的重组融合纳米抗体VHH23-VHH26的内化情况的表征结果图。
图8B为本发明实施例2的重组融合纳米抗体VHH23-VHH26的内化情况的量化结果图。
图9A为本发明实施例2的重组融合全长抗体的内化情况的表征结果图。
图9B为本发明实施例2的重组融合全长抗体的内化情况的量化结果图。
图10A为本发明实施例3的融合VHH1-VHH6的共聚焦激光扫描显微镜图片。
图10B为本发明实施例3的融合VHH1-VHH6与溶酶体之间的皮尔斯相关系数测试结果。
图11A为本发明实施例3的融合VHH7-VHH22的共聚焦激光扫描显微镜图片。
图11B为本发明实施例3的融合VHH7-VHH22与溶酶体之间的皮尔斯相关系数测试结果。
图12A为本发明实施例3的融合VHH23-VHH26的共聚焦激光扫描显微镜图片。
图12B为本发明实施例3的融合VHH23-VHH26与溶酶体之间的皮尔斯相关系数测试结果。
图13A为本发明实施例3的重组融合全长抗体的共聚焦激光扫描显微镜图片。
图13B为本发明实施例3的重组融合全长抗体与溶酶体之间的皮尔斯相关系数测试结果。
图14A为本发明实施例5中SKBR3细胞对重组融合纳米抗体-药物偶联物融合VHH1-MMAF至融合VHH6-MMAF的活力结果图。
图14B为本发明实施例5中SKBR3细胞对重组融合纳米抗体-药物偶联物融合VHH7-MMAF至融合VHH22-MMAF的活力结果图。
图14C为本发明实施例5中SKBR3细胞对重组融合纳米抗体-药物偶联物融合VHH23-MMAF至融合VHH26-MMAF的活力结果图。
图15为本发明实施例5中阴性对照组MDA-MB-231细胞对融合VHH7-MMAF和融合VHH8-MMAF的活力结果图。
图16为本发明实施例5中SKBR3细胞对重组融合全长抗体-药物偶联物的活力结果图。
图17为本发明实施例5中SKOV3细胞对重组融合全长抗体-药物偶联物的活力结果图。
图18为本发明实施例5中SKOV3细胞在重组融合全长抗体-药物偶联物给药后的细胞成像图。
图19为本发明实施例6中重组融合抗体-药物偶联物对人乳腺癌株BT474的抗肿瘤活性评价。
图20为本发明实施例6中重组融合抗体-药物偶联物对人胃癌株NCI-N87的抗肿瘤活性评价。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
溶酶体靶向肽
本发明的溶酶体靶向肽为高尔基体定位的含γ-适应蛋白耳的生长素响应因子结合蛋白(Golgi-localized,γ-adaptin ear-containing,ARF-binding proteins,GGA)、甘露糖-6-磷酸受体(mannose 6-phosphate receptor,MPR)和溶酶体关联膜蛋白(lysosome-associated membrane proteins,LAMP)的溶酶体分选信号肽，或在其基础上取代、缺失和/或添加一个或多个氨基酸。本发明优选的溶酶体靶向肽的氨基酸序列如下表1所示，但本发明的溶酶体靶向肽包括但不限于表1示出的氨基酸序列。
Figure BDA0003305047290000081
接头肽
本发明的接头肽的氨基酸序列为(Leu-Pro-Glu-Thr)x-(Glyy1-Sery2)z，其中x＝0或1，y1＝3、4或5，y2＝0或1，z＝1、2或3。本发明优选的接头肽包括但不于限以下表2示出的氨基酸序列：
Figure BDA0003305047290000082
注：Linker2只有3个氨基酸，由于后附的说明书氨基酸《序列表》中要求每条多肽至少有4个氨基酸，因此接头肽Linker2未在《序列表》中示出，接头肽Linker2无对应的SEQID NO:。
实施例1重组融合抗体的制备
为了方便阐述本发明的重组融合抗体，本实施例选用抗-人表皮生长因子受体2(Human Epidermal Growth Factor Receptor 2,HER2)的抗原结合片段(纳米抗体，以下简称VHH)和单克隆抗体(全长抗体，以下简称mAb)为模板抗体，mAb具有重链(Heavy Chain,HC)mAb-HC和轻链(Light Chain,LC)mAb-LC。需注意的是，本发明的抗体包括但并不限于上述两种抗体。
请参照图1至图3，图1为本发明的溶酶体靶向肽融合于纳米抗体的三种结构示意图，图2为本发明的溶酶体靶向肽融合于单链抗体(简称scFv)的六种结构示意图，图3为本发明的溶酶体靶向肽融合于全长抗体的十五种结构示意图。本发明的溶酶体靶向肽与抗体的融合包括但不限于图1至图3所示的结构，本发明的溶酶体靶向肽可选择地融合在抗体的抗原结合片段的C末端、抗原结合片段的N末端、单克隆抗体的重链的C末端、单克隆抗体的重链的N末端、单克隆抗体的轻链的C末端和单克隆抗体的轻链的N末端中的至少一种。
1)抗体的位点特异性标记
为了方便药物与抗体连接，可以首先将抗体进行位点特异性标记，例如，将VHH的84位丝氨酸突变为半胱氨酸得到VHH-S84C，以及将mAb的重链mAb-HC的121位丙氨酸突变为半胱氨酸得到mAb-HC-A121C。VHH、VHH-S84C、mAb-HC、mAb-HC-A121C、mAb-LC的氨基酸序列如下表3所示：
Figure BDA0003305047290000091
Figure BDA0003305047290000101
2)重组融合抗体质粒的构建
本实施例采用基因重组的方式将溶酶体靶向肽通过接头肽融合到抗体的N末端或C末端。
a)溶酶体靶向肽融合VHH的重组融合抗体表达质粒的构建
VHH的C末端可引入c-Myc、组氨酸、GST、MBP、脂类标签等，使表达产物更易于检测和纯化，本实施例选用His6作为蛋白纯化标签。将His6标签融合到VHH-S84C的C末端且未作融合，得到VHH-S84C-His6，记作VHH0；选用Linker1-Linker4和Linker6作为接头肽，P1-P21作为溶酶体靶向肽，并将纯化标签His6、接头肽以及溶酶体靶向肽融合到位点特异性突变抗体的C末端，制备得到重组融合抗体：融合VHH1-VHH26，其氨基酸序列如表4所示，但本发明的重组融合抗体并不限于本实施例给出的氨基酸序列，本发明的接头肽Linker1-Linker6与溶酶体靶向肽P1-P21可任意组合，得到的重组融合抗体均在本发明的保护范围内。
本实施例的溶酶体靶向肽融合VHH的重组融合抗体质粒的构建具体包括以下步骤：
首先将His6标签融合到VHH-S84C的C末端，构建片段VHH-S84C-His6，之后将接头肽、溶酶体靶向肽分别融合到His6后端或前端，得到重组融合抗体的目的片段，将重组融合抗体的目的片段整合到载体pET-28a上，得到重组融合抗体质粒。
Figure BDA0003305047290000111
Figure BDA0003305047290000121
Figure BDA0003305047290000131
Figure BDA0003305047290000141
Figure BDA0003305047290000151
通过PCR分别扩增目的片段和载体，要求设计目的片段和载体引物时需同时包含NcoI和BstYI酶切位点。然后根据引物设计的酶切位点，分别对PCR产物和质粒载体进行酶切。之后通过连接酶将酶切后的PCR产物和质粒载体进行连接，并将连接后的产物转入DH5α感受态细胞中，涂板，放入培养箱过夜。次日挑取单个菌斑培养，最后测序验证是否构建成功。
b)溶酶体靶向肽融合mAb的重组融合抗体表达质粒的构建
溶酶体靶向肽融合于mAb的位置包括mAb重链的C末端、mAb重链的N末端、mAb轻链的C末端和mAb轻链的N末端中的至少一种。为了方便阐述，本实施例将未作融合的mAb记作mAb0，mAb0的轻链为mAb-LC，mAb0的重链为mAb-HC-A121C；将Linker1作为接头肽、P7作为溶酶体靶向肽，并将Linker1以及P7融合到位点特异性突变抗体轻链和/或重链的C末端，制备得到重组融合抗体：融合mAb1-mAb3，其氨基酸序列如表6所示，但本发明的重组融合抗体并不限于本实施例给出的氨基酸序列，本发明的接头肽Linker1-Linker6与溶酶体靶向肽P1-P21可任意组合，且溶酶体靶向肽也可以融合于重链和/或轻链的N末端，其得到的重组融合抗体均在本发明的保护范围内。
本实施例的溶酶体靶向肽融合mAb的重组融合抗体质粒的构建具体包括以下步骤：
首先将Linker1和P7融合到重链的C末端和轻链的C末端，构建片段mAb-LC-Linker1-P7和mAb-HC-A121C-Linker1-P7，其氨基酸序列如表5所示，并将mAb-LC-Linker1-P7和mAb-HC-A121C-Linker1-P7分别整合到载体pcDNA 3.4。
Figure BDA0003305047290000161
Figure BDA0003305047290000171
mAb轻链(mAb-LC)的表达质粒和重链(mAb-HC-A121C)的表达质粒用于表达全长抗体mAb0；溶酶体靶向肽融合的mAb轻链(mAb-LC-Linker1-P7)的表达质粒和重链(mAb-HC-A121C)的表达质粒用于表达融合mAb1；mAb轻链(mAb-LC)的表达质粒和融合的重链(mAb-HC-A121C-Linker1-P7)的表达质粒用于表达融合mAb2；融合的mAb轻链(mAb-LC-Linker1-P7)的表达质粒和融合的重链(mAb-HC-A121C-Linker1-P7)的表达质粒用于表达融合mAb3，融合mAb1-mAb3的氨基酸序列如表6所示。
Figure BDA0003305047290000172
Figure BDA0003305047290000181
Figure BDA0003305047290000191
通过PCR分别扩增目的片段和载体，要求设计目的片段和载体引物时需同时包含NcoI和BstYI酶切位点。然后根据引物设计的酶切位点，分别对PCR产物和质粒载体进行酶切。之后通过连接酶将酶切后的PCR产物和质粒载体进行连接，并将连接后的产物转入DH5α感受态细胞中，涂板，放入培养箱过夜。次日挑取单个菌斑培养，最后测序验证是否构建成功。
3)重组融合抗体的表达与纯化
本实施例中VHH0、融合VHH1-VHH26选用原核表达系统进行抗体表达；mAb0、融合mAb1-mAb3选用真核表达系统进行抗体表达。
a)VHH0、融合VHH1-VHH26在原核表达系统的表达与纯化
在构建上述重组融合抗体表达质粒后，将重组质粒转入E.coli BL21(DE3)宿主细胞中，表达上述融合蛋白，并用Ni-NTA beads进行纯化。简言之，先用平衡缓冲溶液(buffer)(400mM NaCl，50mM Tris-HCl pH 8.0，20mM咪唑)平衡Ni-NTAbeads，然后上样(菌体裂解液上清)，再用平衡buffer洗去非特异性结合的蛋白，最后用含200mM咪唑的洗脱buffer洗脱目的蛋白，蛋白浓缩换液后采用BCA法确定蛋白浓度。突变后蛋白与野生型蛋白相比产率没有明显变化。纯化后的蛋白用液相色谱-质谱联用仪(LC-MS)、基质辅助激光解吸电离飞行时间质谱(MALDI-TOF Mass)、SDS-PAGE(其结果如图4所示)、HPLC进行结构表征，结果显示VHH0、融合VHH1-VHH26已在原核表达系统中表达。
b)mAb0、融合mAb1-mAb3在真核表达系统的表达与纯化
将人胚胎肾细胞293(HEK293)接种于摇瓶中，待细胞密度达到2×106/mL时，可以进行转染，转染试剂为聚乙烯亚胺(PEI)。转染时，取两支离心管分别加入2mL磷酸盐缓冲盐(PBS)溶液，一支离心管加入100μL PEI，另一支离心管加入80μg质粒(重链：轻链＝2：3)，充分混匀后，将稀释液加入PEI稀释液中，充分混匀，静置20min，转入摇瓶，转染5天后收集上清。上清液经0.45μm滤膜过滤后，通过蛋白A(Protein A)亲和层析柱纯化。纯化后的蛋白通过LC-MS、SDS-PAGE(其结果如图5所示)、HPLC进行结构表征，结果显示mAb0、融合mAb1-mAb3已在真核表达系统中表达。
实施例2重组融合抗体内化情况的研究
为了方便观察重组融合抗体的内化情况，将重组融合抗体与荧光分子连接，该荧光分子包括但不限于为罗丹明(Rhodamine)类、CY3(Cyanine 3)类、德克萨斯红(TexasRed)类荧光分子中的一种。
本实施例中，荧光分子选用四甲基罗丹明(TAMRA)，TAMRA通过三聚乙二醇-马来酰亚胺(PEG3-maleimide)接头小分子与重组融合抗体上突变的半胱氨酸发生迈克尔加成反应。
1)细胞培养
采用BT474、SKBR3、SKOV3、MDA-MB-231细胞系(来源：美国模式菌种收集中心(ATCC))，将细胞培养在添加10％胎牛血清的达尔伯克改良伊格尔培养基(DMEM)(Gibco，USA)培养基中，外加青霉素(100U/mL)-链霉素(100g/mL)。所有的细胞均培养在37℃含有5％CO2的湿润环境下，细胞形态正常，生长状态良好。
2)采用流式细胞术检测重组融合抗体的内化情况
将荧光分子标记到抗体上，通过流式细胞术，检测经荧光分子标记的重组融合抗体在细胞内化过程中的表现，并通过荧光分子来检测细胞中重组融合抗体的含量。
本实施例使用高灵敏度的荧光分子TAMRA标记VHH0和融合VHH1-VHH26，具体操作步骤如下：首先用5mM三羧基乙基膦(TCEP；赛默飞世尔科技公司)在室温下分别还原VHH0和融合VHH1-VHH26的巯基约30min，然后用含有1mM乙二胺四乙酸(EDTA)的PBS换液去除TCEP，在抗体溶液中加入2eq的Maleimide-PEG3-TAMRA在4℃下反应2h，最后换液除去多余小分子，得到TAMRA标记的VHH0和融合VHH1-VHH26。
本实施例使用高灵敏度的荧光分子TAMRA标记mAb0和融合mAb1-mAb3，其具体操作步骤如下：二硫苏糖醇(DTT)在20℃的条件下反应16h，还原抗体上的巯基，然后用50mMTris-HCl，pH 7.5，150mM NaCl换液去除DTT，在蛋白溶液中加入100mM脱氢抗坏血酸(DHAA)(＞15eq)，室温反应3h，使天然的链间二硫键重新氧化，同时保持工程化半胱氨酸不配对；然后加入4eq的Maleimide-PEG3-TAMRA在4℃下连接2h，最后换液去除多余小分子，得到TAMRA标记的mAb0和融合mAb1-mAb3。
本实施例采用SKBR3细胞系，SKBR3细胞系是一种HER2高表达的人乳腺癌细胞系。将荧光分子TAMRA标记的VHH0、融合VHH1-VHH26、mAb0、融合mAb1-mAb3与SKBR3细胞共孵育6h，用无血清培养基将几种修饰后的抗体稀释至终浓度为2μM，随后使用酸洗去细胞膜上结合的抗体，通过流式细胞仪观察荧光强度。
a)融合VHH1-VHH26的内化情况
图6A、图7A和图8A为本发明实施例2的重组融合纳米抗体VHH1-VHH26的内化情况的表征结果图，图中Control为细胞的背景吸收；图6B、图7B和图8B为本发明实施例2的重组融合纳米抗体VHH1-VHH26的内化情况的量化结果图，图6B、图7B和图8B中的数据表示为平均值±SEM(n＝3)，ns：没有显著性，*P<0.05；**P<0.01；***P<0.001；****P<0.0001。通过对流式细胞术分析和平均细胞荧光值的分析清楚地表明，融合了溶酶体靶向肽的融合VHH1-VHH26比未作融合的VHH0具有更强的内化能力，且具有显著性差异，该结果证明溶酶体靶向肽能够增强抗体的内化能力。此外，荧光分子标记的融合VHH7和荧光分子标记的融合VHH8的内化能力没有显著性差异。由此可以证明，His6标签的位置不会影响溶酶体靶向肽的内化能力。
再者，通过观察融合VHH1-VHH7以及VHH13-VHH26的内化情况可以看出，融合VHH1-VHH7以及VHH13-VHH26具有比未作融合的VHH0更强的内化能力，由此可以证明，本发明的溶酶体靶向肽能够增加抗体的内化能力。
最后，通过观察融合VHH7、融合VHH9-VHH12的内化情况可以看出，VHH7、融合VHH9-VHH12均表现出比未作融合的VHH0更强的内化能力，由此可以证明，本发明的接头肽均可用于重组融合抗体的构建。
b)融合mAb1-mAb3的内化情况
图9A为本发明实施例2的重组融合全长抗体的内化情况的表征结果图，图中Control为细胞的背景吸收；图9B为本发明实施例2的重组融合全长抗体的内化情况的量化结果图，图9B中的数据表示为平均值±SEM(n＝3)，ns：没有显著性，**P<0.01；***P<0.001；****P<0.0001。通过对流式细胞术分析和平均细胞荧光值的分析清楚地表明，融合了溶酶体靶向肽的融合mAb1-mAb3比未作融合的mAb0具有更为明显的内化水平。
此外，mAb的轻链和重链同时融合了溶酶体靶向肽P7的融合mAb3表现出最为明显的内化效率。仅在mAb的轻链或重链融合了P7所得到的融合mAb1和mAb2同样能够增强内化水平，但两者的内化水平没有显著性差异。由此可以证明，本发明的溶酶体靶向肽对抗体进行重组融合可以明显增强抗体的内化能力。
实施例3重组融合抗体与溶酶体共定位情况的研究
本发明采用共聚焦激光扫描显微镜(Confocal Laser Scanning Microscope，CLSM)观察重组融合抗体与溶酶体共定位情况，通常重组融合抗体-药物偶联物内吞途径中的运输和加工过程具体为：重组融合抗体-药物偶联物与其表面肿瘤抗原结合，重组融合抗体-药物偶联物被内化成内体，随后成熟并与溶酶体融合。在溶酶体中，酸性的环境和特异性蛋白酶(例如组织蛋白酶B)导致ADC的降解，从而释放药物。释放的药物穿过溶酶体膜到达细胞质并与其靶标结合，例如微管蛋白或DNA，最终诱导细胞死亡。因此，重组融合抗体-药物偶联物内化后是否进入溶酶体，并且能否在溶酶体累积，是决定其发挥药效的重要因素。
本实施例将实施例2所制备的荧光分子TAMRA标记的VHH0、融合VH H1-VHH26、mAb0、融合mAb1-mAb3与SKBR3细胞共孵育4小时后，使用Hoechst 33342染细胞核，溶酶体绿色荧光探针(LysoTracker Green)染SKB R3细胞中的溶酶体，通过共聚焦激光扫描显微镜观察VHH0、融合VHH1-VHH26、mAb0、融合mAb1-mAb3靶向溶酶体的情况。在任何可比较的图像中，图像缩放和拍照条件是相同的，在定量分析中，相同的阈值用于所有图像。
1)融合VHH1-VHH26与溶酶体共定位情况
图10A为本发明实施例3的融合VHH1-VHH6的共聚焦激光扫描显微镜图片，从左到右依次为溶酶体信号、重组融合抗体信号、以及溶酶体与重组融合抗体重合后的图像。图11A为本发明实施例3的融合VHH7-VHH22的共聚焦激光扫描显微镜图片，从左到右依次为细胞核信号、溶酶体信号、重组融合抗体信号、以及溶酶体与重组融合抗体重合后的图像。图12A为本发明实施例3的融合VHH23-VHH26的共聚焦激光扫描显微镜图片，从左到右依次为细胞核信号、溶酶体信号、重组融合抗体信号、以及溶酶体与重组融合抗体重合后的图像。图10B、图11B和图12B为重组融合纳米抗体VHH1-VHH26与溶酶体之间的皮尔斯相关系数(Pearson’s correlation coefficients)测试结果，平均值±SEM；ns：没有显著性；*P<0.05；**P<0.01；***P<0.001；****P<0.0001。
从图10A、图11A和图12A的结果可以看出，融合了溶酶体靶向肽的融合VHH1-VHH26比未作融合的VHH0具有更强的溶酶体共定位效果，说明抗体上融合溶酶体靶向肽会增加抗体与溶酶体的共定位能力。融合VHH7和融合VHH8的溶酶体共定位能力没有显著性差异，说明His6标签的位置不会影响溶酶体靶向肽的溶酶体靶向能力。从图10B、图11B和图12B的结果可以看出，重组融合抗体与溶酶体之间共定位能力的皮尔斯相关系数结果也与上述一致，再次证明溶酶体靶向肽修饰的抗体可以增强抗体靶向溶酶体的能力。此外，图11A、图11B、图12A和图12B的结果还表明上述重组融合抗体具有靶向HER2的抗原特异性，在HER2低表达的阴性对照MDA-MB-231细胞系中，重组融合抗体在细胞膜上不显示特异性结合。
2)融合mAb1-mAb3与溶酶体共定位情况
图13A为本发明实施例3的重组融合全长抗体的共聚焦激光扫描显微镜图片，从左到右依次为细胞核信号、溶酶体信号、重组融合抗体信号、以及溶酶体与重组融合抗体重合后的图像。图13B为本发明实施例3的重组融合全长抗体与溶酶体之间的皮尔斯相关系数测试结果，平均值±SEM；ns：没有显著性；**P<0.01；***P<0.001；****P<0.0001。
从图13A的结果可以看出，融合了溶酶体靶向肽的融合mAb1-mAb3比未作融合的mAb0具有更强的溶酶体共定位效果，说明在抗体中融合了溶酶体靶向肽可以显著增加抗体的溶酶体靶向能力。从图13B的结果可以看出，重组融合抗体与溶酶体之间共定位能力的皮尔斯相关系数结果也与上述一致，再次证明溶酶体靶向肽修饰的抗体可以增强抗体靶向溶酶体的能力。同样，结果还表明上述重组融合抗体具有靶向HER2的抗原特异性，在HER2低表达的阴性对照MDA-MB-231细胞系中，重组融合抗体在细胞膜上不显示特异性结合。
实施例4重组融合抗体-药物偶联物的制备
药物通过接头小分子与重组融合抗体偶联形成重组融合抗体-药物偶联物。药物通过共价键偶联在重组融合抗体上的反应性基团，该反应性基团为赖氨酸、半胱氨酸或生物正交基团；该半胱氨酸为重组融合抗体自身的半胱氨酸或人为突变得到的半胱氨酸；该生物正交基团为叠氮基团、炔基、醛基、酮基和氟代磺酸酯基团中的一种。药物与重组融合抗体经由可裂解或不可裂解的接头小分子偶联；该接头小分子选自以下任一种：多肽、寡糖、-(CH2)n-、-(CH2CH2O)n-、Val-Cit-PABC、Val-Ala-PABC、Val-Lys(Ac)-PABC、Phe-Lys-PABC、Phe-Lys(Ac)-PABC、D-Val-Leu-Lys、Gly-Gly-Arg、Ala-Ala-Asn-PABC、Ala-PABC、PABC或其组合；其中，n为1-10的整数，Cit为瓜氨酸，Ac为乙酰基，PABC为对氨基苄醇。
本发明的药物为细胞毒性剂，包括但不限于以下任一种：微管蛋白抑制剂的药物或前药，DNA损伤剂的药物或者前药，RNA聚合酶II抑制剂，或细菌、真菌或动物来源的毒素。微管蛋白抑制剂包括但不限于为奥里斯他汀(auristatin)或奥里斯他汀衍生物及类似物，例如单甲基澳瑞他汀F(MMAF)和单甲基澳瑞他汀E(MMAE)、美登素(maytansinoid)或美登素衍生物及类似物、紫杉醇(paclitaxel)或紫杉醇衍生物及类似物、长春花生物碱(vinca-alkaloid)或长春花生物碱衍生物及类似物、念珠藻素(cryptophycin)或念珠藻素衍生物及类似物、陶素(tubulysin)或陶素衍生物及类似物。DNA损伤剂包括但不限于为多柔比星(doxorubicin)或多柔比星衍生物及类似物、卡奇霉素(calicheamicin)或卡奇霉素衍生物及类似物、喜树碱(camptothecin)或喜树碱衍生物及类似物、吡咯并苯并二氮杂卓(pyrrolobenzodiazepines，PBD)或PBD衍生物及类似物、倍癌霉素(duocarmycin)或倍癌霉素衍生物及类似物中的一种。
本实施例中，药物选用MMAF，荧光分子选用TAMRA。其中，MMAF通过6-马来酰亚胺己酸基(6-maleimidocaproyl,mc)接头小分子与抗体上突变的半胱氨酸发生迈克尔加成反应，TAMRA通过三聚乙二醇-马来酰亚胺(PEG3-maleimide)接头小分子与抗体上突变的半胱氨酸发生迈克尔加成反应。
1)重组融合纳米抗体-药物偶联物的制备
将未作融合的纳米抗体VHH0和融合VHH1-VHH26分别与mc-MMAF进行偶联，得到VHH0-MMAF和重组融合纳米抗体-药物偶联物融合VHH1-MMAF至融合VHH26-MMAF，该制备过程采用以下方式进行：
首先用5mM TCEP(赛默飞世尔科技公司)在室温下分别还原VHH0和融合VHH1-VHH26的巯基约30min，然后用含有1mM EDTA的PBS换液去除TCEP，在蛋白溶液中加入2eq的mc-MMAF在4℃下连接2h，最后换液除去多余小分子，得到VHH0-MMAF和重组融合纳米抗体-药物偶联物融合VHH1-MMAF至融合VHH26-MMAF。所得到的重组融合纳米抗体-药物偶联物通过SDS-PAGE进行表征(其结果如图4所示)，并通过MALDI-TOF Mass确定分子量。
2)重组融合全长抗体-药物偶联物的制备
将未作融合的全长抗体mAb0和融合mAb1-mAb3分别与mc-MMAF进行偶联，得到mAb0-MMAF和重组融合全长抗体-药物偶联物融合mAb1-MMAF、融合mAb2-MMAF和融合mAb3-MMAF，该制备过程采用以下方式进行：
首先用100mM的DTT在20℃的条件下反应16h，还原抗体上的巯基，然后用50mMTris-HCl，pH 7.5，150mM NaCl换液去除DTT，在蛋白溶液中加入100mM DHAA(＞15eq)，室温反应3h，使天然的链间二硫键重新氧化，同时保持工程化半胱氨酸不配对；然后加入4eq的mc-MMAF在4℃下连接2h，最后换液去除多余小分子，得到mAb0-MMAF和重组融合全长抗体-药物偶联物融合mAb1-MMAF、融合mAb2-MMAF和融合mAb3-MMAF。所得到的重组融合全长抗体-药物偶联物通过SDS-PAGE进行表征(其结果如图5所示)，并通过LC-MS确定分子量。
实施例5
体外细胞毒性
本实施例选择HER2高表达的人乳腺癌SKBR3细胞和人卵巢癌SKOV3细胞评估重组融合抗体-药物偶联物抑制癌细胞增殖的能力。不同浓度的重组融合抗体-药物偶联物被加入种有SKBR3细胞或SKOV3细胞的96孔板中，处理48h后，使用CCK8试剂盒，计算细胞的存活率。
1)重组融合纳米抗体-药物偶联物的毒性分析
以细胞活力相对于MMAF、VHH0-MMAF或者所示不同重组融合纳米抗体-药物偶联物融合VHH1-MMAF至融合VHH26-MMAF的浓度，绘制浓度曲线，并计算半抑制浓度IC50(其结果如图14A至图14C、表7所示)。图14A、图14B和图14C分别为SKBR3细胞对重组融合纳米抗体-药物偶联物融合VHH1-MMAF至融合VHH6-MMAF、融合VHH7-MMAF至融合VHH22-MMAF以及融合VHH23-MMAF至融合VHH26-MMAF的活力结果图。表7显示MMAF、VHH0、VHH0-MMAF、融合VHH1-MMAF至融合VHH26-MMAF的IC50值，数据表示为平均值(n＝3)。
Figure BDA0003305047290000261
Figure BDA0003305047290000271
结果表明，首先MMAF小分子对细胞具有毒性，细胞存活率约为50％；VHH0对细胞几乎没有毒性，存活率约为100％；VHH0-MMAF对细胞有显著的杀伤能力，存活率约为50％，值得注意的是，重组融合纳米抗体-药物偶联物融合VHH1-MMAF至VHH26-MMAF比VHH0-MMAF的杀伤能力更强。结果与溶酶体共定位结果一致，融合了溶酶体靶向肽的重组融合纳米抗体-药物偶联物融合VHH1-MMAF至融合VHH26-MMAF比未融合溶酶体靶向肽的重组融合纳米抗体-药物偶联物VHH0-MMAF具有更强的肿瘤细胞杀伤活力。总结来说，融合了溶酶体靶向肽的重组融合纳米抗体-药物偶联物的细胞毒性与其内化和溶酶体靶向能力相关。
此外，本实施例还选择HER2阴性的人乳腺癌MDA-MB-231细胞作为阴性对照组，比较融合VHH7-MMAF、融合VHH8-MMAF、VHH0-MMAF、纳米抗体VHH0和毒性药物MMAF抑制癌细胞的增殖能力，其结果如图15所示，在HER2阴性的人乳腺癌MDA-MB-231细胞中，只有MMAF能诱导显著的细胞死亡；融合VHH7-MMAF、融合VHH8-MMAF、VHH0-MMAF和VHH0均不具有显著的细胞毒性。该结果表明，本发明的重组融合抗体-药物偶联物的细胞毒性是依赖于HER2的，因此具有肿瘤细胞靶向特异性。
2)重组融合全长抗体-药物偶联物的毒性分析
选择SKBR3细胞和SKOV3细胞分别评估重组融合全长抗体-药物偶联物的抑制癌细胞增殖的能力。以细胞活力相对于MMAF、mAb0-MMAF或者所示不同重组融合全长抗体-药物偶联物融合mAb1-MMAF至融合mAb3-MMAF的浓度绘制浓度曲线，并计算半抑制浓度IC50。
SKBR3
Figure BDA0003305047290000281
SKOV3
Figure BDA0003305047290000282
重组融合抗体-药物偶联物在SKBR3细胞的细胞毒性如图16和表8所示。图16为本发明实施例5中SKBR3细胞对重组融合全长抗体-药物偶联物的活力结果图。表8显示MMAF、mAb0、mAb0-MMAF融合mAb1-MMAF至融合mAb3-MMAF在SKBR3细胞的IC50值，数据表示为平均值(n＝3)。结果表明，融合了溶酶体靶向肽的重组融合全长抗体-药物偶联物融合mAb1-MMAF至融合mAb3-MMAF显示出优异的肿瘤杀伤活性，其活性达到皮摩尔范围，相较于未做融合的全长抗体-药物偶联物mAb0-MMAF有明显提高。
重组融合抗体-药物偶联物在SKOV3细胞的细胞毒性如图17和表9所示。图17为本发明实施例5中SKOV3细胞对重组融合全长抗体-药物偶联物的活力结果图。表9显示MMAF、mAb0、mAb0-MMAF、融合mAb1-MMAF至融合mAb3-MMAF在SKOV3细胞的IC50值，数据表示为平均值(n＝3)。结果再次证明，融合了溶酶体靶向肽的重组融合全长抗体-药物偶联物融合mAb1-MMAF至融合mAb3-MMAF相比未作融合的全长抗体-药物偶联物mAb0-MMAF具有更强的肿瘤细胞杀伤活力。
最后，通过细胞成像技术观察SKOV3细胞在不同给药组中的细胞状态。具体操作如下：将SKOV3细胞接种于96孔板，每孔细胞约为8000个，将细胞过夜贴壁培养；将细胞分为六组(空白对照组、mAb0组、mAb0-MMAF组、融合mAb1-MMAF组、融合mAb2-MMAF组和融合mAb3-MMAF组)，分别配制给药浓度为0.01nM的所需药物。除空白对照组外，对剩余五组进行给药；给药48小时后，吸弃原培养基，加入4％的多聚甲醛在室温下固定细胞15分钟，最后吸弃4％的多聚甲醛，加入100μL PBS。在细胞成像仪上观察细胞状态并拍照。不同给药组的SKOV3细胞成像结果如图18所示。
从图18的结果可以看出，未加任何对照药物的空白对照组，细胞生长状态良好；mAb0组的细胞状态变化不明显。相比空白对照组和mAb0组，mAb0-MMAF组、融合mAb1-MMAF组、融合mAb2-MMAF组和融合mAb3-MMAF组明显地影响细胞的健康状况。其中，重组融合全长抗体-药物偶联物组的细胞形态变得更不规则；说明相比mAb0-MMAF，融合mAb1-MMAF、融合mAb2-MMAF和融合mAb3-MMAF对SKOV3细胞的活力影响更为明显，细胞毒性更为显著。
综上所述，融合了溶酶体靶向肽的重组融合全长抗体-药物偶联物具有显著的肿瘤细胞杀伤活性，活性达到皮摩尔范围。通过溶酶体靶向肽对抗体进行重组融合能够有效提高其相应的全长抗体-药物偶联物的细胞毒性。重组融合抗体-药物偶联物的细胞毒性与其细胞内化和溶酶体靶向能力相关。
实施例6
体内抗肿瘤活性
本实施例分别评估重组融合抗体-药物偶联物对人乳腺癌BT474和人胃癌NCI-N87的抗肿瘤活性。
将6到8周龄的雌性裸小鼠分成以下3组：生理盐水组、mAb0-MMAF组和融合mAb3-MMAF组。将BT474或者NCI-N87细胞株接种到裸小鼠前右侧部位皮下，细胞接种量为5×106～7×106，待成瘤后，用游标卡尺测量移植瘤直径。肿瘤长至100-300mm3后，按照上述分组给药。给药方式为尾静脉注射，剂量为10mg/kg(100μL)。接种人乳腺癌BT474裸小鼠的给药周期为5天一次，共6次；接种人胃癌NCI-N87裸小鼠的给药周期为5天一次，共8次。定期测量裸鼠体重和肿瘤体积，长和宽分别用L和W表示，计算肿瘤体积：V＝[长度(mm)×宽度2(mm)2]/2。最后一次给药后24小时，称体重后脱颈椎处死小鼠。
1)重组融合抗体-药物偶联物对人乳腺癌BT474的抗肿瘤活性
图19为本发明实施例6中重组融合抗体-药物偶联物对人乳腺癌BT474的抗肿瘤活性评价，横坐标表示给药天数，纵坐标表示肿瘤体积。结果表明，相比生理盐水组，mAb0-MMAF组和融合mAb3-MMAF组均能抑制肿瘤的增殖；但是融合mAb3-MMAF组对肿瘤增值的抑制效果更为明显，由此可以证明，本发明的重组融合抗体-药物偶联物能够更好的抑制人乳腺癌肿瘤的增值。
此外，mAb0-MMAF和融合mAb3-MMAF给药均未导致小鼠体重减少，由此可以证明，本发明的重组融合抗体-药物偶联物具有良好的安全性。
2)重组融合抗体-药物偶联物对人胃癌NCI-N87的抗肿瘤活性
图20为本发明实施例6中重组融合抗体-药物偶联物对人胃癌NCI-N87的抗肿瘤活性评价，横坐标表示给药天数，纵坐标表示肿瘤体积。结果表明，相比生理盐水组，mAb0-MMAF组和融合mAb3-MMAF组均有抗肿瘤效果，但融合mAb3-MMAF组对肿瘤的增值抑制效果更为明显，由此可以证明，本发明的重组融合抗体-药物偶联物能够更好的抑制人胃癌肿瘤的增值。
另外，并未观察到小鼠因mAb0-MMAF或融合mAb3-MMAF给药而导致体重减少，再次证明，本发明的重组融合抗体-药物偶联物良好的安全性。
虽然本发明已以实施方式揭露如上，然而其并非用以限定本发明，任何熟习此技艺者，在不脱离本发明的精神和范围内，当可作各种的更动与润饰，因此本发明的保护范围当视后附的申请专利范围所界定者为准。
序列表
<110> 中山大学
<120> 重组融合抗体和抗体-药物偶联物及其用途
<130> GD1899-21P126183
<160> 66
<170> PatentIn version 3.5
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Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
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Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
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Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
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Gly Gly Gly Ala Ser Val Ser Leu Leu Asp Asp Glu Leu Met Ser Leu
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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
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Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
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Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
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Gln Val Thr Val Ser Ser His His His His His His Leu Pro Glu Thr
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Gly Gly Gly Arg Arg Arg Ala Ser Val Ser Leu Leu Asp Asp Glu Leu
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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn
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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
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Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
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Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser His His His His His His His Leu Pro Glu
115 120 125
Thr Gly Gly Gly Ala Ser Ser Gly Leu Asp Asp Leu Asp Leu Leu Gly
130 135 140
Lys
145
<210> 37
<211> 144
<212> PRT
<213> 人工序列
<400> 37
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Cys Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys
85 90 95
Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser His His His His His His Leu Pro Glu Thr
115 120 125
Gly Gly Gly Val Gln Asn Pro Ser Ala Asp Arg Asn Leu Leu Asp Leu
130 135 140
<210> 38
<211> 145
<212> PRT
<213> 人工序列
<400> 38
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Cys Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys
85 90 95
Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser His His His His His His His Leu Pro Glu
115 120 125
Thr Gly Gly Gly Asn Ala Leu Ser Trp Leu Asp Glu Glu Leu Leu Cys
130 135 140
Leu
145
<210> 39
<211> 139
<212> PRT
<213> 人工序列
<400> 39
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Cys Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys
85 90 95
Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser His His His His His His Leu Pro Glu Thr
115 120 125
Gly Gly Gly Ser Asp Glu Asp Leu Leu His Ile
130 135
<210> 40
<211> 139
<212> PRT
<213> 人工序列
<400> 40
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Cys Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys
85 90 95
Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser Leu Pro Glu Thr Gly Gly Gly Ser Asp Glu
115 120 125
Asp Leu Leu His Ile His His His His His His
130 135
<210> 41
<211> 135
<212> PRT
<213> 人工序列
<400> 41
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Cys Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys
85 90 95
Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser His His His His His His Gly Gly Gly Ser
115 120 125
Asp Glu Asp Leu Leu His Ile
130 135
<210> 42
<211> 137
<212> PRT
<213> 人工序列
<400> 42
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Cys Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys
85 90 95
Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser His His His His His His Gly Gly Gly Gly
115 120 125
Gly Ser Asp Glu Asp Leu Leu His Ile
130 135
<210> 43
<211> 137
<212> PRT
<213> 人工序列
<400> 43
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Cys Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys
85 90 95
Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser His His His His His His Gly Gly Gly Gly
115 120 125
Ser Ser Asp Glu Asp Leu Leu His Ile
130 135
<210> 44
<211> 147
<212> PRT
<213> 人工序列
<400> 44
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Cys Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys
85 90 95
Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser His His His His His His Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Glu Asp Leu
130 135 140
Leu His Ile
145
<210> 45
<211> 144
<212> PRT
<213> 人工序列
<400> 45
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Cys Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys
85 90 95
Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser His His His His His His Leu Pro Glu Thr
115 120 125
Gly Gly Gly Ser Phe His Asp Asp Ser Asp Glu Asp Leu Leu His Ile
130 135 140
<210> 46
<211> 144
<212> PRT
<213> 人工序列
<400> 46
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Cys Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys
85 90 95
Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser His His His His His His Leu Pro Glu Thr
115 120 125
Gly Gly Gly Glu Glu Ser Glu Glu Arg Asp Asp His Leu Leu Pro Met
130 135 140
<210> 47
<211> 141
<212> PRT
<213> 人工序列
<400> 47
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Cys Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys
85 90 95
Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser His His His His His His Leu Pro Glu Thr
115 120 125
Gly Gly Gly Ser Tyr Lys Tyr Ser Lys Val Asn Lys Glu
130 135 140
<210> 48
<211> 141
<212> PRT
<213> 人工序列
<400> 48
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Cys Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys
85 90 95
Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser His His His His His His Leu Pro Glu Thr
115 120 125
Gly Gly Gly Pro Ala Ala Tyr Arg Gly Val Gly Asp Asp
130 135 140
<210> 49
<211> 142
<212> PRT
<213> 人工序列
<400> 49
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Cys Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys
85 90 95
Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser His His His His His His Leu Pro Glu Thr
115 120 125
Gly Gly Gly Arg Arg Arg Ser Asp Glu Asp Leu Leu His Ile
130 135 140
<210> 50
<211> 143
<212> PRT
<213> 人工序列
<400> 50
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Cys Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys
85 90 95
Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser His His His His His His Leu Pro Glu Thr
115 120 125
Gly Gly Gly Arg Arg Leu Arg Ser Asp Glu Asp Leu Leu His Ile
130 135 140
<210> 51
<211> 144
<212> PRT
<213> 人工序列
<400> 51
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Cys Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys
85 90 95
Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser His His His His His His Leu Pro Glu Thr
115 120 125
Gly Gly Gly Arg Arg Arg Arg Lys Ser Asp Glu Asp Leu Leu His Ile
130 135 140
<210> 52
<211> 147
<212> PRT
<213> 人工序列
<400> 52
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Cys Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys
85 90 95
Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser His His His His His His Leu Pro Glu Thr
115 120 125
Gly Gly Gly Arg Arg Arg Ser Phe His Asp Asp Ser Asp Glu Asp Leu
130 135 140
Leu His Ile
145
<210> 53
<211> 149
<212> PRT
<213> 人工序列
<400> 53
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Cys Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys
85 90 95
Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser His His His His His His Leu Pro Glu Thr
115 120 125
Gly Gly Gly Arg Arg Leu Arg Lys Ser Phe His Asp Asp Ser Asp Glu
130 135 140
Asp Leu Leu His Ile
145
<210> 54
<211> 149
<212> PRT
<213> 人工序列
<400> 54
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Cys Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys
85 90 95
Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser His His His His His His Leu Pro Glu Thr
115 120 125
Gly Gly Gly Arg Arg Arg Arg Lys Ser Phe His Asp Asp Ser Asp Glu
130 135 140
Asp Leu Leu His Ile
145
<210> 55
<211> 143
<212> PRT
<213> 人工序列
<400> 55
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Cys Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys
85 90 95
Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser His His His His His His His Leu Pro Glu
115 120 125
Thr Gly Gly Gly Arg Lys Arg Ser His Ala Gly Tyr Gln Thr Ile
130 135 140
<210> 56
<211> 148
<212> PRT
<213> 人工序列
<400> 56
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Cys Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys
85 90 95
Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser His His His His His His His Leu Pro Glu
115 120 125
Thr Gly Gly Gly Arg Arg Arg Arg Lys Arg Lys Arg Ser His Ala Gly
130 135 140
Tyr Gln Thr Ile
145
<210> 57
<211> 142
<212> PRT
<213> 人工序列
<400> 57
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Cys Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys
85 90 95
Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser His His His His His His His Leu Pro Glu
115 120 125
Thr Gly Gly Gly Lys His His His Ala Gly Tyr Glu Gln Phe
130 135 140
<210> 58
<211> 146
<212> PRT
<213> 人工序列
<400> 58
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Cys Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys
85 90 95
Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser His His His His His His His Leu Pro Glu
115 120 125
Thr Gly Gly Gly Arg Arg Leu Arg Lys His His His Ala Gly Tyr Glu
130 135 140
Gln Phe
145
<210> 59
<211> 229
<212> PRT
<213> 人工序列
<400> 59
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Leu Pro Glu Thr Gly Gly Gly Ser Asp Glu
210 215 220
Asp Leu Leu His Ile
225
<210> 60
<211> 465
<212> PRT
<213> 人工序列
<400> 60
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Cys Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys Leu Pro Glu Thr Gly Gly Gly Ser Asp Glu Asp Leu Leu His
450 455 460
Ile
465
<210> 61
<211> 229
<212> PRT
<213> 人工序列
<400> 61
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Leu Pro Glu Thr Gly Gly Gly Ser Asp Glu
210 215 220
Asp Leu Leu His Ile
225
<210> 62
<211> 450
<212> PRT
<213> 人工序列
<400> 62
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Cys Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 63
<211> 214
<212> PRT
<213> 人工序列
<400> 63
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 64
<211> 465
<212> PRT
<213> 人工序列
<400> 64
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Cys Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys Leu Pro Glu Thr Gly Gly Gly Ser Asp Glu Asp Leu Leu His
450 455 460
Ile
465
<210> 65
<211> 229
<212> PRT
<213> 人工序列
<400> 65
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Leu Pro Glu Thr Gly Gly Gly Ser Asp Glu
210 215 220
Asp Leu Leu His Ile
225
<210> 66
<211> 465
<212> PRT
<213> 人工序列
<400> 66
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Cys Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys Leu Pro Glu Thr Gly Gly Gly Ser Asp Glu Asp Leu Leu His
450 455 460
Ile
465

Claims (23)

1.一种重组融合抗体，包括：抗体、至少一溶酶体靶向肽以及至少一接头肽，所述溶酶体靶向肽与所述抗体通过所述接头肽融合形成所述重组融合抗体；其中所述溶酶体靶向肽为高尔基体定位的含γ-适应蛋白耳的生长素响应因子结合蛋白(GGA)、甘露糖-6-磷酸受体(MPR)和溶酶体关联膜蛋白(LAMP)的溶酶体分选信号肽，或在其基础上取代、缺失和/或添加一个或多个氨基酸。
2.根据权利要求1所述的重组融合抗体，其中所述溶酶体靶向肽选自以下氨基酸序列中的至少一种：ASVSLLDDELMSL(SEQ ID NO：1)，RRRASVSLLDDELMSL(SEQ ID NO：2)，ASVSLLDDEL(SEQ ID NO：3)，ASSGLDDLDLLGK(SEQ ID NO：4)，VQNPSADRNLLDL(SEQ ID NO：5)，NALSWLDEELLCL(SEQ ID NO：6)，SDEDLLHI(SEQ ID NO：7)，SFHDDSDEDLLHI(SEQ ID NO：8)，EESEERDDHLLPM(SEQ ID NO：9)，SYKYSKVNKE(SEQ ID NO：10)，PAAYRGVGDD(SEQ ID NO：11)，RRRSDEDLLHI(SEQ ID NO：12)，RRLRKSDEDLLHI(SEQ ID NO：13)，RRRRKSDEDLLHI(SEQID NO：14)，RRRSFHDDSDEDLLHI(SEQ ID NO：15)，RRLRKSFHDDSDEDLLHI(SEQ ID NO：16)，RRRRKSFHDDSDEDLLHI(SEQ ID NO：17)，RKRSHAGYQTI(SEQ ID NO：18)，RRRRKRKRSHAGYQTI(SEQ ID NO：19)，KHHHAGYEQF(SEQ ID NO：20)和RRLRKHHHAGYEQF(SEQ ID NO：21)。
3.根据权利要求1所述的重组融合抗体，其中所述接头肽的氨基酸序列为(Leu-Pro-Glu-Thr)x-(Glyy1-Sery2)z，其中x＝0或1，y1＝3、4或5，y2＝0或1，z＝1、2或3。
4.根据权利要求3所述的重组融合抗体，其中所述接头肽选自以下氨基酸序列中的至少一种：Leu-Pro-Glu-Thr-Gly-Gly-Gly(SEQ ID NO:22)，Gly-Gly-Gly，Gly-Gly-Gly-Gly-Gly(SEQ ID NO:23)，Gly-Gly-Gly-Gly-Ser(SEQ ID NO:24)，Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(SEQ ID NO:25)，和Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(SEQ ID NO:26)。
5.根据权利要求1所述的重组融合抗体，其中所述抗体为能够结合肿瘤细胞表面的靶标蛋白的抗体，所述肿瘤细胞表面的靶标蛋白选自以下中的至少一种：EGFR、EGFRvIII、HER2、ErB3、CD19、CD20、CD30、CD33、CD37、CD46、CD48、CD74、CD79B、CD27 ligand、TROP2、Nectin-4、PSMA、BCMA、HGFR、FOLR1、CA125、ALCAM、SLC1A5、Siglec-2、Siglec-3、IL-2Ralpha、IL-3R alpha、TNF-alpha、B7-H3、GUCY2C、TPBG、ROR2、ENPP3、Coagulation FactorIII、Mesothelin、Transferrin R、CEACAM-5、IGF-IR、Syndecan-1、DLL3、EpCAM、LIV-1、ROR1、TIM-1、Ly6E和NCAM-1。
6.根据权利要求1所述的重组融合抗体，其中所述抗体为单特异性抗体、双特异性抗体或多特异性抗体。
7.根据权利要求1所述的重组融合抗体，其中所述抗体为嵌合抗体、人源化抗体或人源抗体。
8.根据权利要求1所述的重组融合抗体，其中所述抗体包含单克隆抗体或其抗原结合片段。
9.根据权利要求8所述的重组融合抗体，其中所述抗原结合片段为Fab、Fab′、F(ab′)2、Fv、dsFv、scFv、sc(Fv)2或VHH。
10.根据权利要求8所述的重组融合抗体，其中所述溶酶体靶向肽的融合位置包括所述单克隆抗体的重链的C末端、所述单克隆抗体的重链的N末端、所述单克隆抗体的轻链的C末端和所述单克隆抗体的轻链的N末端中的至少一种。
11.根据权利要求8所述的重组融合抗体，其中所述溶酶体靶向肽的融合位置包括所述抗原结合片段的C末端和所述抗原结合片段的N末端中的至少一种。
12.一种重组融合抗体-药物偶联物，其结构如下所示：
Drn1Ab；
其中，Dr为药物；
Ab为权利要求1-11任一项所述的重组融合抗体；
n1为大于或等于1的整数。
13.根据权利要求12所述的重组融合抗体-药物偶联物，其中所述药物为细胞毒性剂，选自以下任一种：
微管蛋白抑制剂的药物或前药，
DNA损伤剂的药物或者前药，
RNA聚合酶II抑制剂，和
细菌、真菌或动物来源的毒素。
14.根据权利要求13所述的重组融合抗体-药物偶联物，其中所述微管蛋白抑制剂选自以下任一种：奥里斯他汀(auristatin)或奥里斯他汀衍生物及类似物、美登素(maytansinoid)或美登素衍生物及类似物、紫杉醇(paclitaxel)或紫杉醇衍生物及类似物、长春花生物碱(vinca-alkaloid)或长春花生物碱衍生物及类似物、念珠藻素(cryptophycin)或念珠藻素衍生物及类似物、陶素(tubulysin)或陶素衍生物及类似物。
15.根据权利要求13所述的重组融合抗体-药物偶联物，其中所述DNA损伤剂选自以下任一种：多柔比星(doxorubicin)或多柔比星衍生物及类似物、卡奇霉素(calicheamicin)或卡奇霉素衍生物及类似物、喜树碱(camptothecin)或喜树碱衍生物及类似物、吡咯并苯并二氮杂卓(pyrrolobenzodiazepines，PBD)或PBD衍生物及类似物、倍癌霉素(duocarmycin)或倍癌霉素衍生物及类似物。
16.根据权利要求12所述的重组融合抗体-药物偶联物，其中所述药物通过共价键偶联在所述重组融合抗体上的反应性基团。
17.根据权利要求16所述的重组融合抗体-药物偶联物，其中所述重组融合抗体上的反应性基团为赖氨酸、半胱氨酸或生物正交基团。
18.根据权利要求17所述的重组融合抗体-药物偶联物，其中所述半胱氨酸为所述重组融合抗体自身的半胱氨酸，或人为突变得到的半胱氨酸。
19.根据权利要求17所述的重组融合抗体-药物偶联物，其中所述生物正交基团为叠氮基团、炔基、醛基、酮基和氟代磺酸酯基团中的一种。
20.根据权利要求12所述的重组融合抗体-药物偶联物，其中所述重组融合抗体与所述药物经由可裂解或不可裂解的接头小分子偶联。
21.根据权利要求20所述的重组融合抗体-药物偶联物，其中所述接头小分子选自以下任一种：多肽、寡糖、-(CH2)n-、-(CH2CH2O)n-、Val-Cit-PABC、Val-Ala-PABC、Val-Lys(Ac)-PABC、Phe-Lys-PABC、Phe-Lys(Ac)-PABC、D-Val-Leu-Lys、Gly-Gly-Arg、Ala-Ala-Asn-PABC、Ala-PABC、PABC或其组合；其中，n为1-10的整数，Cit为瓜氨酸，Ac为乙酰基，PABC为对氨基苄醇。
22.一种根据权利要求12-21任一项所述的重组融合抗体-药物偶联物在制备用于治疗癌症的药物中的用途。
23.根据权利要求22所述的用途，其中所述癌症为胃肿瘤、肠肿瘤、肝脏肿瘤、肺肿瘤、胰腺肿瘤、乳腺肿瘤、宫颈肿瘤、子宫内膜肿瘤、卵巢肿瘤、前列腺肿瘤、膀胱肿瘤、鼻咽喉或软组织肿瘤、血液或淋巴肿瘤以及皮肤肿瘤。
CN202111201616.1A 2021-10-15 2021-10-15 重组融合抗体和抗体-药物偶联物及其用途 Pending CN115975039A (zh)

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