CN115210262B - 人源化抗-cd22重组免疫毒素及其应用 - Google Patents

人源化抗-cd22重组免疫毒素及其应用 Download PDF

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CN115210262B
CN115210262B CN202180018578.8A CN202180018578A CN115210262B CN 115210262 B CN115210262 B CN 115210262B CN 202180018578 A CN202180018578 A CN 202180018578A CN 115210262 B CN115210262 B CN 115210262B
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郝振平
朱建伟
徐溧
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KUNMING SINOWAY NATURAL PHARMACEUTICALS CO Ltd
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Abstract

提供一种人源化抗‑CD22重组免疫毒素，具有降低的免疫原性，并保持了人源化改造前的CD22结合亲和力和相应的生物学活性，用于制备治疗CD22相关的B细胞恶性肿瘤的药物。

Description

人源化抗-CD22重组免疫毒素及其应用
本专利申请要求于2020年9月29日提交的申请号为CN2020110487230的中国发明专利申请的优先权权益，在此将其全部内容引入作为参考。
技术领域
本发明涉及生物医药领域，具体而言，本发明涉及与截短的假单胞菌外毒素A融合的人源化抗-CD22抗体片段的重组免疫毒素以及所述重组免疫毒素的应用。
背景技术
CD22(Siglec-2)是一种结合唾液酸的免疫球蛋白样凝集素(Siglec)受体，其与含唾液酸(Sia)的多糖特异性结合，从而促进细胞粘附和/或细胞信号传导[1]。CD22的表达局限于B细胞，并在建立B细胞抑制的基线水平中起关键作用，因此CD22是体液免疫中体内稳态的关键决定因素。CD22在多种B细胞恶性肿瘤的肿瘤细胞中表达，包括B淋巴细胞性白血病/淋巴瘤和成熟的B细胞白血病/淋巴瘤[2]。在毛细胞白血病(HCL)和前淋巴细胞性白血病中，CD22的表达尤其强烈。
毛细胞白血病(HCL)是一种具有典型锯齿状细胞质边界的成熟肿瘤性B细胞的慢性恶性肿瘤，由Bouroncle及其同事于1958年首次描述[3,4]。在美国，HCL患者占全部白血病患者的2％(美国每年有500-800例新病例[5])，其特征是全血细胞减少症和脾肿大。针对HCL，嘌呤类似物(克拉屈滨或喷司他丁)是初始治疗的标准疗法并与持续多年的持久缓解相关；然而，许多患者出现复发并需要另外的治疗[6]；随后的治疗通常采用改进的嘌呤类似物方案，但是治疗效果降低，患者的缓解期较短并最终成为难治性的[7]。此外，嘌呤类似物与神经毒性相关[8]并且具有很高的免疫抑制作用，这可能会增加机会感染的风险[4]。
针对毛细胞白血病，已证明免疫毒素是治疗有效的。免疫毒素是抗体缀合的治疗剂，其靶向癌细胞并利用有效的细胞毒性载荷(比如细菌毒素[9]、植物衍生毒素[10]和合成化学品[11])杀死癌细胞。上世纪90年代后期在美国国家癌症研究所开发了第一代抗-CD22免疫毒素，名称为BL22(RFB4(dsFv)-PE38或CAT-3888)。它采用与假单胞菌外毒素A(PE38)融合的鼠抗-CD22抗体，其中PE38为假单胞菌外毒素A的38kDa片段。2001年，CAT-3888进入了I期试验，并报道了其缓解白血病[12-16]。但之后，CAT-3888被moxetumomab pasudotox(HA22或CAT-8015)取代。HA22是一种在PE38和抗-CD22抗体片段中包含修饰的改良的免疫毒素。与CAT-3888相比，HA22对抗体片段中的三个氨基酸进行了改变，以增加对靶分子的结合亲和力。
结构组成上，HA22由与PE38融合的鼠抗-CD22单克隆抗体的Fv片段组成。从机理上讲，HA22的Fv部分结合CD22，而由于CD22作为一种细胞表面受体在多种恶性B细胞上表达，由此毒素部分PE38可以被直接递送至肿瘤细胞；内化后，PE38将催化延伸因子2(EF-2)中白喉酰胺残基的ADP核糖基化，使得凋亡蛋白髓样细胞白血病1(Mcl-1)的水平迅速下降，由此导致细胞凋亡[17,18]。2018年美国FDA批准了HA22用于治疗患有复发性或难治性毛细胞白血病的成人，这些成人之前接受过至少两次包括嘌呤核苷类似物治疗的系统性治疗。
早期的免疫毒素设计缺乏足够的治疗窗来保证进一步的临床开发，其中，固有的免疫原性和抗药物抗体的快速发展是关键因素[19]。而对HA22而言，所包含的靶向部分即该鼠抗-CD22单克隆抗体的Fv部分，由于为鼠源的，将在人体内具有一定的免疫原性[20]，并由此造成不期望的中和性抗药物抗体(ADA)的产生，并不利地影响药代动力学分布，包括影响药物在给定剂量下的活性，加速药物在体内的清除，同时限制重复给药的次数和有效性。
发明内容
为了解决上述技术问题，本发明的目的是提供一种基于假单胞菌外毒素A的新型重组免疫毒素，其中该假单胞菌外毒素A与人源化抗-CD22单克隆抗体片段相融合，相对于原鼠源抗-CD22单克隆抗体片段，人源化的抗-CD22单克隆抗体片段将具有降低的免疫原性且保留对靶标CD22的亲和力；同时，所形成的新型重组免疫毒素具有强的免疫毒素有效性，例如CD22表达肿瘤细胞的生长抑制与细胞凋亡效应。
本发明的另一个目的是提供所述新型重组免疫毒素的应用。
本发明的技术方案如下：
一方面，本发明提供一种人源化抗-CD22重组免疫毒素，所述人源化抗-CD22重组免疫毒素为包含以下两个多肽链的多肽分子：
(1)第一多肽链，其包含抗-CD22抗体的轻链可变区(VL)，所述轻链可变区(VL)包含SEQ ID NO.16或SEQ ID NO.18所示氨基酸序列或与其具有至少75％序列同一性的氨基酸序列同系物；
(2)第二多肽链，其包含抗-CD22抗体的重链可变区(VH)以及与所述重链可变区(VH)直接或间接连接的细胞毒素，其中所述重链可变区(VH)包含SEQ ID NO.3或SEQ IDNO.4所示氨基酸序列或与其具有至少75％同一性的氨基酸序列同系物。
在本文中，“氨基酸序列同系物”是指源自相应的氨基酸序列但相对于其具有一个或多个氨基酸替换、添加或缺失的氨基酸序列。
在第一多肽链中，优选地，所述氨基酸序列同系物与SEQ ID NO.16或SEQ IDNO.18所示氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性。进一步优选地，所述氨基酸序列同系物包含SEQ ID NO.31、SEQ ID NO.32和SEQ ID NO.33所示氨基酸序列，并且在对应SEQ ID NO.14所示氨基酸序列的第3、5、36、37、47、48、49、50、65、67、69、70和72位与SEQ ID NO.16或SEQ ID NO.18具有相同的氨基酸残基(氨基酸位置编号根据SEQ ID NO.14所示氨基酸序列)，且不是SEQ ID NO.14所示氨基酸序列。
在第二多肽链中，优选地，所述氨基酸序列同系物与SEQ ID NO.3或SEQ ID NO.4所示氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性。进一步优选地，所述氨基酸序列同系物包含SEQ ID NO.28、SEQ ID NO.29和SEQ ID NO.30所示氨基酸序列，并且在对应SEQ ID NO.2所示氨基酸序列的第3、48、49、50、69、71、73、75和80位与SEQ IDNO.3或SEQ ID NO.4具有相同的氨基酸残基(氨基酸位置编号根据SEQ ID NO.2所示氨基酸序列)，且不是SEQ ID NO.2所示氨基酸序列。
优选地，在本发明的人源化抗-CD22重组免疫毒素中，所述第一多肽链中的轻链可变区(VL)包含SEQ ID NO.16所示氨基酸序列或所述氨基酸序列同系物；所述第二多肽链中的重链可变区(VH)包含SEQ ID NO.3所示氨基酸序列或所述氨基酸序列同系物；
优选地，在本发明的人源化抗-CD22重组免疫毒素中，所述第一多肽链中的轻链可变区(VL)包含SEQ ID NO.18所示氨基酸序列或所述氨基酸序列同系物；所述第二多肽链中的重链可变区(VH)包含SEQ ID NO.3所示氨基酸序列或所述氨基酸序列同系物；
优选地，在本发明的人源化抗-CD22重组免疫毒素中，所述第一多肽链中的轻链可变区(VL)包含SEQ ID NO.16所示氨基酸序列或所述氨基酸序列同系物；所述第二多肽链中的重链可变区(VH)包含SEQ ID NO.4所示氨基酸序列或所述氨基酸序列同系物。
优选地，在本发明提供的人源化抗-CD22重组免疫毒素中，所述第一多肽链中的轻链可变区(VL)与所述第二多肽链中的重链可变区(VH)共价连接，例如经由一个二硫键相连接。
优选地，在本发明提供的人源化抗-CD22重组免疫毒素中，所述第二多肽链中的细胞毒素为假单胞菌外毒素A或者为保留细胞毒性的假单胞菌外毒素A的突变体或片段。
天然的假单胞菌外毒素A(PE)是由铜绿假单胞菌(Pseudomonas aeruginosa)分泌的细菌毒素，为单体蛋白，具有66kD的分子量。PE分子可以通过进行修饰或去除其一部分序列，从而减少或消除毒素的非特异性结合，同时保持细胞毒性。例如，所述假单胞菌外毒素A的突变体是保留了细胞毒性的经修饰的假单胞菌外毒素A，该修饰可以为保守修饰。例如所述突变体与未经修饰的天然PE具有至少75％、优选至少80％、进一步优选至少90％、91％、92％、93％、94％、95％的氨基酸序列同一性。此外，例如，所述假单胞菌外毒素A的片段是保留了细胞毒性的截短形式的假单胞菌外毒素A。优选地，本发明所述的假单胞菌外毒素A的突变体或片段例如PE40、PE38、PE35、PE24、mPE24，或者T19、T20、M11[21,22,23,24,25]。根据本发明的具体实施方式，所述片段为PE38，氨基酸序列示于SEQ ID NO.1。
优选地，在所述第二多肽链中，所述细胞毒素位于抗-CD22抗体的重链可变区(VH)的C端。例如，所述细胞毒素的N端与所述重链可变区(VH)的C端直接融合或经由接头连接。所述接头能够分别与重链可变区(VH)和细胞毒素形成共价键，同时不影响该人源化抗-CD22重组免疫毒素中各个功能构件发挥作用。合适的接头是本领域中已知的，例如直链或支链碳接头、杂环碳接头和肽接头。在本发明提供的人源化抗-CD22重组免疫毒素中，优选不采用接头，或采用肽接头即连接肽，例如包含一个或多个(GGGGS)氨基酸序列的柔性连接肽。
在本发明提供的人源化抗-CD22重组免疫毒素中，所述第一多肽链和第二多肽链可构成Fab形式，即所述第一多肽链还在所述轻链可变区(VL)的C端包含kappa型轻链恒定区，优选人kappa型轻链恒定区；或者，所述第二多肽链还在所述重链可变区(VH)的C端包含CH1重链恒定区，优选人CH1重链恒定区。
根据本发明的具体实施方式，在本发明提供的人源化抗-CD22重组免疫毒素中，所述第一多肽链包含SEQ ID NO.16所示氨基酸序列或其氨基酸序列同系物，所述第二多肽链包含SEQ ID NO.9所示氨基酸序列或其氨基酸序列同系物。
或者，所述第一多肽链包含SEQ ID NO.18所示氨基酸序列或其氨基酸序列同系物，所述第二多肽链包含SEQ ID NO.9所示氨基酸序列或其氨基酸序列同系物。
或者，所述第一多肽链包含SEQ ID NO.16所示氨基酸序列或其氨基酸序列同系物，所述第二多肽链包含SEQ ID NO.10所示氨基酸序列或其氨基酸序列同系物。
另一方面，本发明提供一种核酸分子，所述核酸分子包含编码本发明提供的人源化抗-CD22重组免疫毒素中的第一多肽链的核苷酸序列和/或编码本发明提供的人源化抗-CD22重组免疫毒素中的第二多肽链的核苷酸序列。本发明提供的核酸分子可以为单独的一条核苷酸序列，编码所述第一多肽链或第二多肽链；或者，本发明提供的核酸分子可以单独的一条核苷酸序列，但是同时编码所述第一多肽链或第二多肽链。或者，本发明提供的核酸分子可以为两条核苷酸序列的组合，分别编码所述第一多肽链或第二多肽链。
再一方面，本发明提供一种载体，其包含本发明提供的所述核酸分子。所述载体可以为真核表达载体、原核表达载体、人工染色体及噬菌体载体等。根据本发明的具体实施方式，所述载体为原核表达载体，例如为包含所述核酸分子的表达质粒。
根据本发明的具体实施方式，分别包含第一多肽链编码核苷酸和第二多肽链编码核苷酸的质粒图谱示于图3。
又一方面，本发明提供一种宿主细胞。所述宿主细胞被本发明的核酸分子和/或载体转化或转染，因此包含本发明的核酸分子和/或载体，用于保存或表达目的。所述宿主细胞可以是任何原核或真核细胞，例如细菌或昆虫、真菌、植物或动物细胞。根据本发明的具体实施方式，所述宿主细胞为原核细胞，例如大肠杆菌。
基于本发明的公开内容，本发明提供的人源化抗-CD22重组免疫毒素、核酸分子、载体和/或宿主细胞可以通过利用本领域已知的任何常规技术方法获得。例如，可以先由本发明提供的核酸分子或载体在宿主细胞中表达所述第一多肽链和第二多肽链，通过回收、体外重折叠，形成所述第一多肽链中的轻链可变区(VL)与所述第二多肽链中的重链可变区(VH)经由一个二硫键相连接的异二聚体。
本发明提供的人源化抗-CD22重组免疫毒素、核酸分子、载体和/或宿主细胞可以被包含在组合物(例如药物组合物)中，更特别地被包含在药物制剂中，从而根据实际需要用于各种目的。
因此，在又一方面，本发明还提供一种组合物，所述组合物包含本发明所述的人源化抗-CD22重组免疫毒素、核酸分子、载体和/或宿主细胞。优选地，所述组合物为药物组合物，其任选地还包含药学上可接受的辅料。
作为结合靶标B细胞表面标志物CD22、尤其是人CD22的抗-CD22重组免疫毒素，本发明还提供上述主题的相关应用。
具体而言，一方面，发明提供所述人源化抗-CD22重组免疫毒素、核酸分子、载体、宿主细胞或组合物在制备药物中的用途，所述药物用于治疗CD22相关的B细胞恶性肿瘤。
优选地，所述CD22相关的B细胞恶性肿瘤为以CD22高表达为特征的B细胞恶性肿瘤，例如CD22高表达的淋巴瘤和白血病。优选地，所述淋巴瘤为非何金杰氏淋巴瘤、小淋巴细胞淋巴瘤和套细胞淋巴瘤；所述白血病为慢性淋巴细胞性白血病、毛细胞白血病和急性淋巴细胞性白血病。
又一方面，本发明提供一种CD22相关的B细胞恶性肿瘤的治疗方法，所述方法包括给有此需要的受试者施用本发明提供的人源化抗-CD22重组免疫毒素、核酸分子、载体和/或宿主细胞。所述受试者为哺乳动物，包括人或非人的灵长类动物以及犬、猫、牛、猪、羊、马、鼠、兔等家养、畜牧或实验室哺乳动物。优选地，所述受试者为人。
本发明的人源化VH、VL也可形成单独的dsFv抗体，因此还一方面，本发明提供一种人源化抗-CD22的抗体或抗体片段，所述抗体或抗体片段包含重链可变区(VH)和轻链可变区(VL)，其中所述重链可变区(VH)包含SEQ ID NO.3或SEQ ID NO.4所示氨基酸序列或与其具有至少75％同一性的氨基酸序列同系物，所述轻链可变区(VL)包含示于SEQ ID NO.16或SEQ ID NO.18所示氨基酸序列或与其具有至少75％序列同一性的氨基酸序列同系物。
所述轻链可变区(VL)中，优选地，所述氨基酸序列同系物与SEQ ID NO.16或SEQID NO.18所示氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性。进一步优选地，所述氨基酸序列同系物包含SEQ ID NO.31、SEQ ID NO.32和SEQ ID NO.33所示氨基酸序列，并且在对应SEQ ID NO.14所示氨基酸序列的第3、5、36、37、47、48、49、50、65、67、69、70和72位与SEQ ID NO.16或SEQ ID NO.18具有相同的氨基酸残基(氨基酸位置编号根据SEQ ID NO.14所示氨基酸序列)，且不是SEQ ID NO.14所示氨基酸序列。
所述重链可变区(VH)中，优选地，所述氨基酸序列同系物与SEQ ID NO.3或SEQ IDNO.4所示氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性。进一步优选地，所述氨基酸序列同系物包含SEQ ID NO.28、SEQ ID NO.29和SEQ ID NO.30所示氨基酸序列，并且在对应SEQ ID NO.2所示氨基酸序列的第3、48、49、50、69、71、73、75和80位与SEQID NO.3或SEQ ID NO.4具有相同的氨基酸残基(氨基酸位置编号根据SEQ ID NO.2所示氨基酸序列)，且不是SEQ ID NO.2所示氨基酸序列。
优选地，在本发明的人源化抗-CD22的抗体或抗体片段中，所述轻链可变区(VL)包含SEQ ID NO.16所示氨基酸序列或所述氨基酸序列同系物；所述重链可变区(VH)包含SEQID NO.3所示氨基酸序列或所述氨基酸序列同系物；
优选地，在本发明的人源化抗-CD22的抗体或抗体片段中，所述轻链可变区(VL)包含SEQ ID NO.18所示氨基酸序列或所述氨基酸序列同系物；所述重链可变区(VH)包含SEQID NO.3所示氨基酸序列或所述氨基酸序列同系物；
优选地，在本发明的人源化抗-CD22的抗体或抗体片段中，所述轻链可变区(VL)包含SEQ ID NO.16所示氨基酸序列或所述氨基酸序列同系物；所述重链可变区(VH)包含SEQID NO.4所示氨基酸序列或所述氨基酸序列同系物。
优选地，本发明提供的人源化抗-CD22的抗体为单克隆抗体，其还包含人源的重链恒定区(CH)和轻链恒定区(CL)；优选地，所述单克隆抗体包含重链(HC)和轻链(LC)。
根据单克隆抗体的重链恒定区序列，该抗体可以为IgA、IgD、IgE、IgG和IgM类别，甚至可以进一步为不同的亚类(同种型)，例如IgG1、IgG2、IgG3、IgG4。根据单克隆抗体的轻链恒定区序列，该抗体可以为kappa型或lambda型。
优选地，本发明提供的抗体片段为单克隆抗体片段，优选为Fab、Fab’、F(ab’)2、scFv、dsFv或抗体保留CD22识别或结合能力的其他片段。根据上下文，本发明中的“抗体片段”可以与“抗体”互换使用。
更优选地，本发明提供的抗体片段为单克隆抗体的dsFv片段，即重链可变区和轻链可变区由二硫键连接的二硫键稳定的抗体片段。根据本发明的具体实施方式，所述dsFv片段是一种由一个二硫键连接的重链可变区和轻链可变区组成的异二聚体。
还一方面，本发明还提供所述人源化抗-CD22的抗体在制备重组免疫毒素中的用途。例如，可以将所述人源化抗-CD22的抗体的任一条链与本发明所述的细胞毒素相连接，制备重组免疫毒素。
本发明提供了一种新的人源化抗-CD22重组免疫毒素，其由与截短的假单胞菌外毒素A融合的人源化抗-CD22单克隆抗体片段组成，具体是通过一个二硫键连接的重链V区和κ轻链V区组成的异二聚体，其中重链V区与截短的假单胞菌外毒素A融合。
与人源化改造前的重组免疫毒素相比，本发明提供的人源化抗-CD22重组免疫毒素通过特定的人源化策略获得，从而与人的对应物更相似，由此潜在地降低免疫毒素的Fv部分在人体中的免疫原性。当在临床中使用时，这种降低的免疫原性能够阻止不期望的中和性抗药物抗体(ADA)的产生，并有助于改善药代动力学分布。
并且，本发明的人源化抗-CD22重组免疫毒素在具有最大人源化的同时，保持了针对靶标CD22的最佳亲和力和相应的生物学活性。与人源化改造前相比，本发明的人源化抗-CD22重组免疫毒素保持了与CD22受体之间的结合亲和力以及对效应细胞的细胞生长抑制和凋亡作用。此外，显示了与改造前相当的药代动力学和肿瘤抑制功效。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1为本发明提供的人源化抗-CD22重组免疫毒素的结构示意图。
图2为本发明进行的不同版本人源化序列示例的比对结果，其中，2A：VH；2B：VL。
图3为本发明的人源化抗-CD22重组免疫毒素的表达质粒图谱，其中，3A：第一多肽链表达质粒；3B：第二多肽链表达质粒。
图4示出了抗-CD22重组免疫毒素的PK图，其中，4A：HA22；4B：H1L2。
图5示出了抗-CD22重组免疫毒素的肿瘤抑制功效。
实施发明的最佳方式
本发明采用人源化策略来优化HA22的抗CD22部分的Fv区，使其与人的对应物更相似，并由此潜在地降低免疫毒素的Fv部分在人体中的免疫原性。当在临床中使用时，这种降低的免疫原性能够阻止不期望的中和性抗药物抗体(ADA)的产生，并有助于改善药代动力学分布。
由此本发明提供一种人源化抗-CD22重组免疫毒素，其包含与截短的假单胞菌外毒素A融合的人源化抗-CD22单克隆抗体片段，具体是由通过一个二硫键连接的κ轻链V区(第一多肽链)和重链V区组成的异二聚体，并且该重链V区与截短的假单胞菌外毒素A融合(第二多肽链)，其结构示意图见图1，该图中截短的假单胞菌外毒素A示例为PE38。
根据本发明，人源化抗-CD22重组免疫毒素与恶性B细胞表面的CD22受体结合，从而将毒素部分例如PE38直接递送至肿瘤细胞。内化后，该毒素部分将催化延伸因子2(EF-2)中白喉酰胺残基的ADP核糖基化，使得抗凋亡蛋白髓样细胞白血病1(Mcl-1)的水平迅速下降，由此导致细胞凋亡。
采用含有第一多肽链编码核苷酸的表达质粒和含有第二多肽链编码核苷酸的表达质粒，在例如增强型BL21衍生菌株(T7 Express)中，可以生产本发明的人源化抗-CD22重组免疫毒素。所得菌株对卡那霉素具有抗性，并且通过异丙基β-d-1-硫代吡喃半乳糖苷(IPTG)诱导该人源化抗-CD22重组免疫毒素的产生。产物表达为包涵体。回收两种包涵体，并在体外将其重折叠以形成异二聚体。随后，先通过疏水相互作用色谱，然后通过阴离子交换色谱纯化所述重折叠蛋白。最后将纯化的蛋白浓缩并渗滤到配制的缓冲液中储存。
本发明提供的人源化抗-CD22重组免疫毒素是HA22的人源化版本，因此预期其通过相同/相似的作用机制与表达CD22的细胞相互作用。本发明中进行了体外和体内实验，来评估本发明的免疫毒素的CD22结合亲和力、在表达CD22的细胞中的细胞毒性、药代动力学和肿瘤抑制效果。在这些研究中，所使用的HA22采用与本发明的重组免疫毒素相同的生产工艺生产。
本发明中，进行体外结合分析，确定了本发明的人源化抗-CD22重组免疫毒素的抗体片段与CD22受体之间的相互作用；基于细胞的分析检测了其生长抑制和凋亡细胞死亡效应。此外，进行了体内研究，确定了本发明的人源化抗-CD22重组免疫毒素的药代动力学和肿瘤抑制功效。
本发明中采用的序列如下：
SEQ ID NO.1：PE38 polypeptide sequence
KASGGP EGGSLAALTA HQACHLPLET FTRHRQPRGW EQLEQCGYPV QRLVALYLAARLSWNQVDQV IRNALASPGS GGDLGEAIRE QPEQARLALT LAAAESERFV RQGTGNDEAG AANGPADSGDALLERNYPTG AEFLGDGGDV SFSTRGTQNW TVERLLQAHR QLEERGYVFV GYHGTFLEAA QSIVFGGVRARSQDLDAIWR GFYIAGDPAL AYGYAQDQEP DARGRIRNGA LLRVYVPRSS LPGFYRTSLT LAAPEAAGEVERLIGHPLPL RLDAITGPEE EGGRLETILG WPLAERTVVI PSAIPTDPRN VGGDLDPSSI PDKEQAISALPDYASQPGKP PREDLK
SEQ ID NO.2：HA22，VH polypeptide sequence(HA22 VH)
MEVQLVESGG GLVKPGGSLK LSCAASGFAF SIYDMSWVRQ TPEKCLEWVA YISSGGGTTYYPDTVKGRFT ISRDNAKNTL YLQMSSLKSE DTAMYYCARH SGYGTHWGVL FAYWGQGTLV TVSA
SEQ ID NO.3：Version 1 VH polypeptide sequence(VH ver 1)
MEVQLVESGG GLVKPGGSLR LSCAASGFAF SIYDMSWVRQ APGKCLEWVA YISSGGGTTYYPDSVKGRFT ISRENAKNSL YLQMNSLKSE DTAMYYCARH SGYGTHWGVL FAYWGQGTMV TVSS
SEQ ID NO.4：Version 2 VH polypeptide sequence(VH ver 2)
MEVQLVESGG GLVKPGGSLR LSCAASGFAF SIYDMSWVRQ TPEKCLEWVA YISSGGGTTYYPDSVKGRFT ISRENAKNSL YLQMNSLKSE DTAMYYCARH SGYGTHWGVL FAYWGQGTMV TVSS
SEQ ID NO.5：Version 3 VH polypeptide sequence(VH ver 3)
MEVQLVESGG GLVKPGGSLR LSCAASGFAF SIYDMSWIRQ APGKCLEWVS YISSGGGTTYYPGSVKGRFT ISRENAKNSL YLQMNSLRAG DTAVYYCARH SGYGTHWGVL FAYWGQGTMV TVSS
SEQ ID NO.6：Version 4 VH polypeptide sequence(VH ver 4)
MEVQLVESGG GLVKPGGSLR LSCAASGFAF SIYDMSWIRQ APGKCLEWVA YISSGGGTTYYPGSVKGRFT ISRENAKNSL YLQMNSLRAG DTAVYYCARH SGYGTHWGVL FAYWGQGTMV TVSS
SEQ ID NO.7：Version 5 VH polypeptide sequence(VH ver 5)
MEVQLVESGG GLVKPGGSLR LSCAASGFAF SIYDMSWIRQ APGKCLEWVA YISSGGGTTYYPGSVKGRFT ISRENAKNSL YLQMNSLRSE DTAVYYCARH SGYGTHWGVL FAYWGQGTMV TVSS
SEQ ID NO.8：HA22 VH-PE38 polypeptide sequence
MEVQLVESGG GLVKPGGSLK LSCAASGFAF SIYDMSWVRQ TPEKCLEWVA YISSGGGTTYYPDTVKGRFT ISRDNAKNTL YLQMSSLKSE DTAMYYCARH SGYGTHWGVL FAYWGQGTLV TVSAKASGGPEGGSLAALTA HQACHLPLET FTRHRQPRGW EQLEQCGYPV QRLVALYLAA RLSWNQVDQV IRNALASPGSGGDLGEAIRE QPEQARLALT LAAAESERFV RQGTGNDEAG AANGPADSGD ALLERNYPTG AEFLGDGGDVSFSTRGTQNW TVERLLQAHR QLEERGYVFV GYHGTFLEAA QSIVFGGVRA RSQDLDAIWR GFYIAGDPALAYGYAQDQEP DARGRIRNGA LLRVYVPRSS LPGFYRTSLT LAAPEAAGEV ERLIGHPLPL RLDAITGPEEEGGRLETILG WPLAERTVVI PSAIPTDPRN VGGDLDPSSI PDKEQAISAL PDYASQPGKP PREDLK
SEQ ID NO.9：Version 1 VH-PE38 polypeptide sequence
MEVQLVESGG GLVKPGGSLR LSCAASGFAF SIYDMSWVRQ APGKCLEWVA YISSGGGTTYYPDSVKGRFT ISRENAKNSL YLQMNSLKSE DTAMYYCARH SGYGTHWGVL FAYWGQGTMV TVSSKASGGPEGGSLAALTA HQACHLPLET FTRHRQPRGW EQLEQCGYPV QRLVALYLAA RLSWNQVDQV IRNALASPGSGGDLGEAIRE QPEQARLALT LAAAESERFV RQGTGNDEAG AANGPADSGD ALLERNYPTG AEFLGDGGDVSFSTRGTQNW TVERLLQAHR QLEERGYVFV GYHGTFLEAA QSIVFGGVRA RSQDLDAIWR GFYIAGDPALAYGYAQDQEP DARGRIRNGA LLRVYVPRSS LPGFYRTSLT LAAPEAAGEV ERLIGHPLPL RLDAITGPEEEGGRLETILG WPLAERTVVI PSAIPTDPRN VGGDLDPSSI PDKEQAISAL PDYASQPGKP PREDLK
SEQ ID NO.10：Version 2 VH-PE38 polypeptide sequence
MEVQLVESGG GLVKPGGSLR LSCAASGFAF SIYDMSWVRQ TPEKCLEWVA YISSGGGTTYYPDSVKGRFT ISRENAKNSL YLQMNSLKSE DTAMYYCARH SGYGTHWGVL FAYWGQGTMV TVSSKASGGPEGGSLAALTA HQACHLPLET FTRHRQPRGW EQLEQCGYPV QRLVALYLAA RLSWNQVDQV IRNALASPGSGGDLGEAIRE QPEQARLALT LAAAESERFV RQGTGNDEAG AANGPADSGD ALLERNYPTG AEFLGDGGDVSFSTRGTQNW TVERLLQAHR QLEERGYVFV GYHGTFLEAA QSIVFGGVRA RSQDLDAIWR GFYIAGDPALAYGYAQDQEP DARGRIRNGA LLRVYVPRSS LPGFYRTSLT LAAPEAAGEV ERLIGHPLPL RLDAITGPEEEGGRLETILG WPLAERTVVI PSAIPTDPRN VGGDLDPSSI PDKEQAISAL PDYASQPGKP PREDLK
SEQ ID NO.11：Version 3 VH-PE38 polypeptide sequence
MEVQLVESGG GLVKPGGSLR LSCAASGFAF SIYDMSWIRQ APGKCLEWVS YISSGGGTTYYPGSVKGRFT ISRENAKNSL YLQMNSLRAG DTAVYYCARH SGYGTHWGVL FAYWGQGTMV TVSSKASGGPEGGSLAALTA HQACHLPLET FTRHRQPRGW EQLEQCGYPV QRLVALYLAA RLSWNQVDQV IRNALASPGSGGDLGEAIRE QPEQARLALT LAAAESERFV RQGTGNDEAG AANGPADSGD ALLERNYPTG AEFLGDGGDVSFSTRGTQNW TVERLLQAHR QLEERGYVFV GYHGTFLEAA QSIVFGGVRA RSQDLDAIWR GFYIAGDPALAYGYAQDQEP DARGRIRNGA LLRVYVPRSS LPGFYRTSLT LAAPEAAGEV ERLIGHPLPL RLDAITGPEEEGGRLETILG WPLAERTVVI PSAIPTDPRN VGGDLDPSSI PDKEQAISAL PDYASQPGKP PREDLK
SEQ ID NO.12：Version 4 VH-PE38 polypeptide sequence
MEVQLVESGG GLVKPGGSLR LSCAASGFAF SIYDMSWIRQ APGKCLEWVA YISSGGGTTYYPGSVKGRFT ISRENAKNSL YLQMNSLRAG DTAVYYCARH SGYGTHWGVL FAYWGQGTMV TVSSKASGGPEGGSLAALTA HQACHLPLET FTRHRQPRGW EQLEQCGYPV QRLVALYLAA RLSWNQVDQV IRNALASPGSGGDLGEAIRE QPEQARLALT LAAAESERFV RQGTGNDEAG AANGPADSGD ALLERNYPTG AEFLGDGGDVSFSTRGTQNW TVERLLQAHR QLEERGYVFV GYHGTFLEAA QSIVFGGVRA RSQDLDAIWR GFYIAGDPALAYGYAQDQEP DARGRIRNGA LLRVYVPRSS LPGFYRTSLT LAAPEAAGEV ERLIGHPLPL RLDAITGPEEEGGRLETILG WPLAERTVVI PSAIPTDPRN VGGDLDPSSI PDKEQAISAL PDYASQPGKP PREDLK
SEQ ID NO.13：Version 5 VH-PE38 polypeptide sequence
MEVQLVESGG GLVKPGGSLR LSCAASGFAF SIYDMSWIRQ APGKCLEWVA YISSGGGTTYYPGSVKGRFT ISRENAKNSL YLQMNSLRSE DTAVYYCARH SGYGTHWGVL FAYWGQGTMV TVSSKASGGPEGGSLAALTA HQACHLPLET FTRHRQPRGW EQLEQCGYPV QRLVALYLAA RLSWNQVDQV IRNALASPGSGGDLGEAIRE QPEQARLALT LAAAESERFV RQGTGNDEAG AANGPADSGD ALLERNYPTG AEFLGDGGDVSFSTRGTQNW TVERLLQAHR QLEERGYVFV GYHGTFLEAA QSIVFGGVRA RSQDLDAIWR GFYIAGDPALAYGYAQDQEP DARGRIRNGA LLRVYVPRSS LPGFYRTSLT LAAPEAAGEV ERLIGHPLPL RLDAITGPEEEGGRLETILG WPLAERTVVI PSAIPTDPRN VGGDLDPSSI PDKEQAISAL PDYASQPGKP PREDLK
SEQ ID NO.14：HA22，VL polypeptide sequence(HA22 VL)
MDIQMTQTTS SLSASLGDRV TISCRASQDI SNYLNWYQQK PDGTVKLLIY YTSILHSGVPSRFSGSGSGT DYSLTISNLE QEDFATYFCQ QGNTLPWTFG CGTKLEIK
SEQ ID NO.15：Version 1 VL polypeptide sequence(VL ver 1)
MDIQMTQSPS TLSASVGDRV TITCRASQDI SNYLNWYQQR PGQSVKLLIY YTSILHSGVPSRFSGSGSGT DYTLTISSLQ PEDFATYFCQ QGNTLPWTFG CGTKVEIK
SEQ ID NO.16：Version 2 VL polypeptide sequence(VL ver 2)
MDIQMTQSTS TLSASVGDRV TITCRASQDI SNYLNWYQQR PDGSVKLLIY YTSILHSGVPSRFSGSGSGT DYTLTISSLQ DEDFATYFCQ QGNTLPWTFG CGTKVEIK
SEQ ID NO.17：Version 3 VL polypeptide sequence(VL ver 3)
MDIQMTQSPS TLSASVGDRV TITCRASQDI SNYLNWFQQR PGQSPRRLIY YTSILHSGVPSRFSGSGSGT DYTLTISSLQ PEDFATYYCQ QGNTLPWTFG CGTKVEIK
SEQ ID NO.18：Version 4 VL polypeptide sequence(VL ver 4)
MDIQMTQSPS TLSASVGDRV TITCRASQDI SNYLNWFQQR PGQSPRLLIY YTSILHSGVPSRFSGSGSGT DYTLTISSLQ PEDFATYYCQ QGNTLPWTFG CGTKVEIK
SEQ ID NO.19：Vers ion 1 VH-PE38 DNA sequence
ATGGAAGTGCAGCTGGTGGAATCCGGCGGAGGCCTGGTCAAGCCCGGCGGCTCTCTGAGACTGTCCTGCGCTGCTTCTGGCTTCGCCTTCTCCATCTACGACATGTCCTGGGTCAGACAGGCCCCTGGCAAGTGCCTGGAATGGGTGGCCTACATCTCCTCTGGCGGAGGCACCACCTACTACCCTGACTCCGTGAAGGGCAGATTCACCATCTCTAGAGAGAACGCCAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAAGTCCGAGGACACCGCCATGTACTACTGCGCCAGACACTCCGGCTACGGCACACACTGGGGCGTGCTGTTCGCTTACTGGGGCCAGGGCACCATGGTGACCGTGTCCTCCAAAGCGTCAGGCGGTCCGGAAGGCGGTTCGCTGGCAGCTCTGACCGCACATCAGGCATGCCACCTGCCGCTGGAAACCTTTACCCGCCATCGTCAACCGCGCGGCTGGGAACAGCTGGAACAATGTGGTTATCCGGTGCAGCGCCTGGTTGCCCTGTACCTGGCCGCACGTCTGAGCTGGAACCAGGTTGATCAAGTCATTCGTAATGCGCTGGCATCACCGGGCTCGGGCGGTGACCTGGGTGAAGCAATCCGCGAACAGCCGGAACAAGCGCGCCTGGCCCTGACCCTGGCAGCTGCGGAATCCGAACGCTTTGTGCGTCAGGGCACCGGTAATGATGAAGCCGGTGCAGCAAATGGTCCGGCTGATTCAGGTGACGCGCTGCTGGAACGCAACTATCCGACGGGCGCCGAATTTCTGGGTGATGGCGGTGACGTGAGTTTCTCCACCCGCGGCACGCAGAATTGGACCGTTGAACGTCTGCTGCAGGCGCATCGCCAACTGGAAGAACGTGGTTATGTTTTTGTCGGCTATCATGGCACCTTTCTGGAAGCTGCGCAGTCGATTGTCTTTGGCGGTGTGCGTGCACGCAGCCAGGATCTGGATGCAATTTGGCGCGGCTTCTACATCGCAGGTGATCCGGCTCTGGCGTATGGCTACGCTCAGGATCAAGAACCGGATGCGCGTGGCCGCATCCGTAATGGTGCCCTGCTGCGTGTGTATGTTCCGCGTTCATCGCTGCCGGGTTTTTACCGTACCTCTCTGACCCTGGCGGCACCGGAAGCTGCCGGCGAAGTGGAACGCCTGATTGGTCACCCGCTGCCGCTGCGTCTGGATGCAATCACCGGTCCGGAAGAAGAAGGCGGCCGTCTGGAAACGATTCTGGGTTGGCCGCTGGCCGAACGTACCGTGGTTATTCCGAGCGCAATCCCGACGGACCCGCGCAATGTTGGCGGTGATCTGGACCCGAGCTCTATTCCGGATAAAGAACAGGCCATCTCCGCACTGCCGGACTATGCGTCACAACCGGGCAAACCGCCGCGTGAAGACCTGAAA
SEQ ID NO.20：Vers ion 2 VH-PE38 DNA sequence
ATGGAAGTGCAGCTGGTGGAATCCGGCGGAGGCCTGGTCAAGCCCGGCGGCTCTCTGAGACTGTCCTGCGCTGCTTCTGGCTTCGCCTTCTCCATCTACGACATGTCCTGGGTCAGACAGACCCCTGAAAAGTGCCTGGAATGGGTGGCCTACATCTCCTCTGGCGGAGGCACCACCTACTACCCTGACTCCGTGAAGGGCAGATTCACCATCTCTAGAGAGAACGCCAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAAGTCCGAGGACACCGCCATGTACTACTGCGCCAGACACTCCGGCTACGGCACACACTGGGGCGTGCTGTTCGCTTACTGGGGCCAGGGCACCATGGTGACCGTGTCCTCCAAAGCGTCAGGCGGTCCGGAAGGCGGTTCGCTGGCAGCTCTGACCGCACATCAGGCATGCCACCTGCCGCTGGAAACCTTTACCCGCCATCGTCAACCGCGCGGCTGGGAACAGCTGGAACAATGTGGTTATCCGGTGCAGCGCCTGGTTGCCCTGTACCTGGCCGCACGTCTGAGCTGGAACCAGGTTGATCAAGTCATTCGTAATGCGCTGGCATCACCGGGCTCGGGCGGTGACCTGGGTGAAGCAATCCGCGAACAGCCGGAACAAGCGCGCCTGGCCCTGACCCTGGCAGCTGCGGAATCCGAACGCTTTGTGCGTCAGGGCACCGGTAATGATGAAGCCGGTGCAGCAAATGGTCCGGCTGATTCAGGTGACGCGCTGCTGGAACGCAACTATCCGACGGGCGCCGAATTTCTGGGTGATGGCGGTGACGTGAGTTTCTCCACCCGCGGCACGCAGAATTGGACCGTTGAACGTCTGCTGCAGGCGCATCGCCAACTGGAAGAACGTGGTTATGTTTTTGTCGGCTATCATGGCACCTTTCTGGAAGCTGCGCAGTCGATTGTCTTTGGCGGTGTGCGTGCACGCAGCCAGGATCTGGATGCAATTTGGCGCGGCTTCTACATCGCAGGTGATCCGGCTCTGGCGTATGGCTACGCTCAGGATCAAGAACCGGATGCGCGTGGCCGCATCCGTAATGGTGCCCTGCTGCGTGTGTATGTTCCGCGTTCATCGCTGCCGGGTTTTTACCGTACCTCTCTGACCCTGGCGGCACCGGAAGCTGCCGGCGAAGTGGAACGCCTGATTGGTCACCCGCTGCCGCTGCGTCTGGATGCAATCACCGGTCCGGAAGAAGAAGGCGGCCGTCTGGAAACGATTCTGGGTTGGCCGCTGGCCGAACGTACCGTGGTTATTCCGAGCGCAATCCCGACGGACCCGCGCAATGTTGGCGGTGATCTGGACCCGAGCTCTATTCCGGATAAAGAACAGGCCATCTCCGCACTGCCGGACTATGCGTCACAACCGGGCAAACCGCCGCGTGAAGACCTGAAA
SEQ ID NO.21：Vers ion 3 VH-PE38 DNA sequence
ATGGAAGTGCAGCTGGTTGAAAGCGGTGGCGGTCTGGTGAAACCGGGCGGTAGCCTGCGTCTGAGCTGCGCGGCGAGCGGTTTCGCGTTTAGCATCTACGACATGAGCTGGATTCGTCAAGCGCCGGGCAAATGCCTGGAGTGGGTGAGCTATATCAGCAGCGGCGGTGGCACCACCTACTATCCGGGTAGCGTTAAGGGCCGTTTCACCATTAGCCGTGAAAACGCGAAAAACAGCCTGTACCTGCAGATGAACAGCCTGCGTGCGGGTGATACCGCTGTGTACTATTGCGCGCGTCACAGCGGTTACGGCACCCACTGGGGCGTTCTGTTTGCGTATTGGGGTCAAGGCACCATGGTGACCGTTAGCAGCAAAGCGTCAGGCGGTCCGGAAGGCGGTTCGCTGGCAGCTCTGACCGCACATCAGGCATGCCACCTGCCGCTGGAAACCTTTACCCGCCATCGTCAACCGCGCGGCTGGGAACAGCTGGAACAATGTGGTTATCCGGTGCAGCGCCTGGTTGCCCTGTACCTGGCCGCACGTCTGAGCTGGAACCAGGTTGATCAAGTCATTCGTAATGCGCTGGCATCACCGGGCTCGGGCGGTGACCTGGGTGAAGCAATCCGCGAACAGCCGGAACAAGCGCGCCTGGCCCTGACCCTGGCAGCTGCGGAATCCGAACGCTTTGTGCGTCAGGGCACCGGTAATGATGAAGCCGGTGCAGCAAATGGTCCGGCTGATTCAGGTGACGCGCTGCTGGAACGCAACTATCCGACGGGCGCCGAATTTCTGGGTGATGGCGGTGACGTGAGTTTCTCCACCCGCGGCACGCAGAATTGGACCGTTGAACGTCTGCTGCAGGCGCATCGCCAACTGGAAGAACGTGGTTATGTTTTTGTCGGCTATCATGGCACCTTTCTGGAAGCTGCGCAGTCGATTGTCTTTGGCGGTGTGCGTGCACGCAGCCAGGATCTGGATGCAATTTGGCGCGGCTTCTACATCGCAGGTGATCCGGCTCTGGCGTATGGCTACGCTCAGGATCAAGAACCGGATGCGCGTGGCCGCATCCGTAATGGTGCCCTGCTGCGTGTGTATGTTCCGCGTTCATCGCTGCCGGGTTTTTACCGTACCTCTCTGACCCTGGCGGCACCGGAAGCTGCCGGCGAAGTGGAACGCCTGATTGGTCACCCGCTGCCGCTGCGTCTGGATGCAATCACCGGTCCGGAAGAAGAAGGCGGCCGTCTGGAAACGATTCTGGGTTGGCCGCTGGCCGAACGTACCGTGGTTATTCCGAGCGCAATCCCGACGGACCCGCGCAATGTTGGCGGTGATCTGGACCCGAGCTCTATTCCGGATAAAGAACAGGCCATCTCCGCACTGCCGGACTATGCGTCACAACCGGGCAAACCGCCGCGTGAAGACCTGAAA
SEQ ID NO.22：Vers ion 4 VH-PE38 DNA sequence
ATGGAAGTGCAGCTGGTTGAAAGCGGTGGCGGTCTGGTGAAACCGGGCGGTAGCCTGCGTCTGAGCTGCGCGGCGAGCGGTTTCGCGTTTAGCATCTACGACATGAGCTGGATTCGTCAAGCGCCGGGCAAATGCCTGGAGTGGGTGGCGTATATCAGCAGCGGCGGTGGCACCACCTACTATCCGGGTAGCGTTAAGGGCCGTTTCACCATTAGCCGTGAAAACGCGAAAAACAGCCTGTACCTGCAGATGAACAGCCTGCGTGCGGGTGATACCGCTGTGTACTATTGCGCGCGTCACAGCGGTTACGGCACCCACTGGGGCGTTCTGTTTGCGTATTGGGGTCAAGGCACCATGGTGACCGTTAGCAGCAAAGCGTCAGGCGGTCCGGAAGGCGGTTCGCTGGCAGCTCTGACCGCACATCAGGCATGCCACCTGCCGCTGGAAACCTTTACCCGCCATCGTCAACCGCGCGGCTGGGAACAGCTGGAACAATGTGGTTATCCGGTGCAGCGCCTGGTTGCCCTGTACCTGGCCGCACGTCTGAGCTGGAACCAGGTTGATCAAGTCATTCGTAATGCGCTGGCATCACCGGGCTCGGGCGGTGACCTGGGTGAAGCAATCCGCGAACAGCCGGAACAAGCGCGCCTGGCCCTGACCCTGGCAGCTGCGGAATCCGAACGCTTTGTGCGTCAGGGCACCGGTAATGATGAAGCCGGTGCAGCAAATGGTCCGGCTGATTCAGGTGACGCGCTGCTGGAACGCAACTATCCGACGGGCGCCGAATTTCTGGGTGATGGCGGTGACGTGAGTTTCTCCACCCGCGGCACGCAGAATTGGACCGTTGAACGTCTGCTGCAGGCGCATCGCCAACTGGAAGAACGTGGTTATGTTTTTGTCGGCTATCATGGCACCTTTCTGGAAGCTGCGCAGTCGATTGTCTTTGGCGGTGTGCGTGCACGCAGCCAGGATCTGGATGCAATTTGGCGCGGCTTCTACATCGCAGGTGATCCGGCTCTGGCGTATGGCTACGCTCAGGATCAAGAACCGGATGCGCGTGGCCGCATCCGTAATGGTGCCCTGCTGCGTGTGTATGTTCCGCGTTCATCGCTGCCGGGTTTTTACCGTACCTCTCTGACCCTGGCGGCACCGGAAGCTGCCGGCGAAGTGGAACGCCTGATTGGTCACCCGCTGCCGCTGCGTCTGGATGCAATCACCGGTCCGGAAGAAGAAGGCGGCCGTCTGGAAACGATTCTGGGTTGGCCGCTGGCCGAACGTACCGTGGTTATTCCGAGCGCAATCCCGACGGACCCGCGCAATGTTGGCGGTGATCTGGACCCGAGCTCTATTCCGGATAAAGAACAGGCCATCTCCGCACTGCCGGACTATGCGTCACAACCGGGCAAACCGCCGCGTGAAGACCTGAAA
SEQ ID NO.23：Vers ion 5 VH-PE38 DNA sequence
ATGGAAGTGCAGCTGGTTGAAAGCGGTGGCGGTCTGGTGAAACCGGGCGGTAGCCTGCGTCTGAGCTGCGCGGCGAGCGGTTTCGCGTTTAGCATCTACGACATGAGCTGGATTCGTCAAGCGCCGGGCAAATGCCTGGAGTGGGTGGCGTATATCAGCAGCGGCGGTGGCACCACCTACTATCCGGGTAGCGTTAAGGGCCGTTTCACCATTAGCCGTGAAAACGCGAAAAACAGCCTGTACCTGCAGATGAACAGCCTGCGTTCTGAGGATACCGCTGTGTACTATTGCGCGCGTCACAGCGGTTACGGCACCCACTGGGGCGTTCTGTTTGCGTATTGGGGTCAAGGCACCATGGTGACCGTTAGCAGCAAAGCGTCAGGCGGTCCGGAAGGCGGTTCGCTGGCAGCTCTGACCGCACATCAGGCATGCCACCTGCCGCTGGAAACCTTTACCCGCCATCGTCAACCGCGCGGCTGGGAACAGCTGGAACAATGTGGTTATCCGGTGCAGCGCCTGGTTGCCCTGTACCTGGCCGCACGTCTGAGCTGGAACCAGGTTGATCAAGTCATTCGTAATGCGCTGGCATCACCGGGCTCGGGCGGTGACCTGGGTGAAGCAATCCGCGAACAGCCGGAACAAGCGCGCCTGGCCCTGACCCTGGCAGCTGCGGAATCCGAACGCTTTGTGCGTCAGGGCACCGGTAATGATGAAGCCGGTGCAGCAAATGGTCCGGCTGATTCAGGTGACGCGCTGCTGGAACGCAACTATCCGACGGGCGCCGAATTTCTGGGTGATGGCGGTGACGTGAGTTTCTCCACCCGCGGCACGCAGAATTGGACCGTTGAACGTCTGCTGCAGGCGCATCGCCAACTGGAAGAACGTGGTTATGTTTTTGTCGGCTATCATGGCACCTTTCTGGAAGCTGCGCAGTCGATTGTCTTTGGCGGTGTGCGTGCACGCAGCCAGGATCTGGATGCAATTTGGCGCGGCTTCTACATCGCAGGTGATCCGGCTCTGGCGTATGGCTACGCTCAGGATCAAGAACCGGATGCGCGTGGCCGCATCCGTAATGGTGCCCTGCTGCGTGTGTATGTTCCGCGTTCATCGCTGCCGGGTTTTTACCGTACCTCTCTGACCCTGGCGGCACCGGAAGCTGCCGGCGAAGTGGAACGCCTGATTGGTCACCCGCTGCCGCTGCGTCTGGATGCAATCACCGGTCCGGAAGAAGAAGGCGGCCGTCTGGAAACGATTCTGGGTTGGCCGCTGGCCGAACGTACCGTGGTTATTCCGAGCGCAATCCCGACGGACCCGCGCAATGTTGGCGGTGATCTGGACCCGAGCTCTATTCCGGATAAAGAACAGGCCATCTCCGCACTGCCGGACTATGCGTCACAACCGGGCAAACCGCCGCGTGAAGACCTGAAA
SEQ ID NO.24：Vers ion 1 VL DNA sequence
ATGGACATCCAGATGACCCAGTCTCCTTCTACACTGTCTGCTTCTGTGGGCGACAGAGTGACCATCACCTGCAGAGCCTCTCAGGACATCTCCAACTACCTGAACTGGTACCAGCAGAGACCTGGCCAGTCCGTGAAGCTGCTGATCTACTACACCTCCATCCTGCACTCCGGCGTGCCTTCCAGATTCTCCGGCTCTGGATCTGGCACCGACTACACCCTGACCATCTCCTCCCTGCAGCCTGAGGACTTCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCTTGGACCTTCGGCTGCGGCACCAAGGTGGAAATCAAG
SEQ ID NO.25：Vers ion 2 VL DNA sequence
ATGGACATCCAGATGACCCAGTCTACTTCTACACTGTCTGCTTCTGTGGGCGACAGAGTGACCATCACCTGCAGAGCCTCTCAGGACATCTCCAACTACCTGAACTGGTACCAGCAGAGACCTGATGGCTCCGTGAAGCTGCTGATCTACTACACCTCCATCCTGCACTCCGGCGTGCCTTCCAGATTCTCCGGCTCTGGATCTGGCACCGACTACACCCTGACCATCTCCTCCCTGCAGGATGAGGACTTCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCTTGGACCTTCGGCTGCGGCACCAAGGTGGAAATCAAG
SEQ ID NO.26：Vers ion 3 VL DNA sequence
ATGGACATCCAGATGACCCAGAGCCCGAGCACCCTGAGCGCGAGCGTGGGCGACCGTGTTACCATCACCTGCCGTGCGAGCCAGGATATTAGCAACTACCTGAACTGGTTTCAACAACGTCCGGGTCAAAGCCCGCGTCGTCTGATCTACTATACCAGCATTCTGCACAGCGGTGTGCCGAGCCGTTTTAGCGGTAGCGGCAGCGGTACCGACTATACCCTGACCATCAGCAGCCTGCAGCCGGAGGATTTCGCGACCTACTATTGCCAGCAGGGTAACACCCTGCCGTGGACCTTTGGCTGCGGTACCAAGGTTGAAATTAAA
SEQ ID NO.27：Version 3VL DNA sequence
ATGGACATCCAGATGACCCAGAGCCCGAGCACCCTGAGCGCGAGCGTGGGCGACCGTGTTACCATCACCTGCCGTGCGAGCCAGGATATTAGCAACTACCTGAACTGGTTTCAACAACGTCCGGGTCAAAGCCCGCGTctgCTGATCTACTATACCAGCATTCTGCACAGCGGTGTGCCGAGCCGTTTTAGCGGTAGCGGCAGCGGTACCGACTATACCCTGACCATCAGCAGCCTGCAGCCGGAGGATTTCGCGACCTACTATTGCCAGCAGGGTAACACCCTGCCGTGGACCTTTGGCTGCGGTACCAAGGTTGAAATTAAA
SEQ ID NO.28：HA22，H-CDR1
GFAFSIYD
SEQ ID NO.29：HA22，H-CDR2
ISSGGGTTY
SEQ ID NO.30：HA22，H-CDR3
CARHSGYGTHWGVLFAY
SEQ ID NO.31：HA22，L-CDR1
QDISNY
SEQ ID NO.32：HA22，L-CDR2
YTSILHSG
SEQ ID NO.33：HA22，L-CDR3
QQGNTLP
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的原料、试剂材料等，如无特殊说明，均为市售购买产品。
实施例1人源化设计
使用框架改组(shuffling)策略将HA22中重链可变区(VH，SEQ ID NO.2)和轻链可变区(VL，SEQ ID NO.14)的框架区中的氨基酸由原始的鼠的改变为人的。全部CDR(均是采用Kabat和Chothia编号系统以及在线工具(http://www.bioinf.org.uk/abs/)确定的)均保持不变。将HA22的VH和VL中每个框架和J区均与国际ImMunoGeneTics
Figure GDA0004126405920000171
(IMGT：http://www.imgt.org)中的人抗体种系(germline)序列进行比对，以找到最接近的人抗体匹配。不改变被看作为Vernier残基的鼠残基或对VH/VL相互作用很重要的鼠残基[26]，以保留对CD22的特异性和亲和力。
制备并评估一组人源化设计，以选择具有最大人源化且同时保持最佳亲和力和有效性的最终候选物。改良后的VH和VL形成与假单胞菌外毒素A(PE38，SEQ ID NO.1)融合的二硫键连接的抗CD22抗体的Fv部分(图1)。
比对结果显示，人种系序列IGHV3-15*01的框架1、IGHV3-11*04的框架2、IGHV3-13*01的框架3和IGHJ3*02的框架4的组合将提供与HA22中VH的框架最相似的人源化框架；类似地，IGKV1-5*01的框架1、IGKV2D-30*01的框架2、IGKV1D-43*01的框架3和IGKJ1*01的框架4的组合提供与HA22中VL的框架最相似的人源化框架。经过数轮迭代(iteration)后，将Vernier残基回复(revert)为原始的鼠残基，这表明它们对抗原结合至关重要。
图2中的序列比对结果给出了人源化过程的示例。其中，HA22VH和HA22VL表示用在HA22中的Fv片段。版本3(V3)为完全人源化的VH和VL版本。版本1、2、4和5(V1、V2、V4、V5)是设计的不同版本，这些设计中具有被回复为鼠残基的关键残基以恢复丢失的活性。以阴影表示的残基是Vernier残基。带下划线的残基在基于IMGT标准的CDR中。
实施例2人源化抗-CD22重组免疫毒素的生产
构建能够表达本发明的不同VL人源化版本(第一多肽链)的表达质粒；另外，将PE38编码核苷酸的5’端与本发明的不同VH人源化版本编码核苷酸的3’端连接，形成单一的核苷酸链，从而构建第二多肽链的表达质粒。结构示意图见图3(由Genescript根据设计合成构建)。
采用该第一多肽链的表达质粒和第二多肽链的表达质粒，在增强型BL21衍生菌株(T7 Express)中生产本发明的人源化抗-CD22重组免疫毒素。
1.发酵
在发酵培养基(每升含：30g大豆蛋白胨，30g酵母提取物，40g甘油，2g NH4Cl，2g(NH4)2SO4，0.973g MgSO4，3g葡萄糖，1g NaCl)中分别制备表达第一多肽链和第二多肽链的细胞种子培养物。将种子培养物在37℃、250RPM下温育12-16小时。使用New BrunswickBioFlo 3000发酵罐进行第一多肽链和第二多肽链的发酵。以2％的浓度将相应的第一多肽链和第二多肽链的种子接种至发酵罐。将pH设置为6.9，并在运行过程中通过添加氨(NH3·H2O)对pH进行控制。将溶氧(DO)设定为30％，并通过搅拌、通气和补充氧气进行DO控制。将容器压力设定为2psi。在运行期间每小时监测一次细胞密度(OD600)和葡萄糖浓度，以及pH、DO和温度。接种后4小时，OD600为8-9时，培养基中的葡萄糖被耗竭，将另外的葡萄糖补料至发酵罐中。葡萄糖进料持续一个小时，总体积为原始培养基体积的1.7％。接种后约5.5小时，另外的葡萄糖被耗尽。然后加入0.1％体积的1M IPTG开始诱导/表达阶段。在诱导阶段，使pH逐渐增加至7.0。诱导后三小时，通过在4℃下以8,000×g离心20分钟以去除培养基，收获细胞。在处理之前将细胞浆储存在-80℃。
2.回收和重折叠
通过使用高压均质器破坏细胞来分离第一多肽链和第二多肽链的包涵体。然后通过采用离心和去污剂的若干洗涤步骤纯化含有包涵体的裂解物沉淀，以移除细胞碎片和可溶性杂质。
将洗涤后的包涵体重悬于含有强变性剂(8M尿素)和还原剂(10mM DTT)的溶解缓冲液(50mM乙醇胺、8M尿素、0.5M精氨酸、2mM EDTA、10mM DTT，pH 9.3)中，以使全部半胱氨酸处于还原状态并打开在制备过程中形成的二硫键。于2-8℃下，通过能够促进溶解过程的搅拌实施溶解达>2小时。溶解后，将包涵体溶液以13,000×g离心30分钟，并将包含溶解的包涵体的上清液过滤以除去残留的聚集体和不溶性杂质。
基于A280读数确定总蛋白浓度，并通过SDS-PAGE凝胶的光密度分析法估算第一多肽链和第二多肽链的百分比。然后将第一多肽链和第二多肽链的以1:1的摩尔比混合，用溶解缓冲液稀释至10mg/mL，并将其以1:10的比例经24小时连续加入至重折叠缓冲液(50mM乙醇胺、0.5M精氨酸、2mM EDTA、0.9mM氧化型谷胱甘肽，pH 9.4)中，并在2-8℃下另外温育48-72小时。通过将pH调节至～7.40来终止重折叠反应。然后将重折叠的蛋白浓缩并通过切向流过滤将其制备用于色谱柱纯化。
3.苯基HP疏水相互作用色谱
苯基HP疏水相互作用色谱在17cm台式高度的色谱柱(GE Healthcare，Marlborough，MA USA)中进行。使用来自GE Healthcare(Marlborough，MA USA)的AKTAavant液相色谱系统进行全部运行，并且在95cm/hr下操作柱子。用20mM Tris-HCl，0.6MNa2SO4，pH 7.4平衡色谱柱。通过用1份20mM Tris-HCl，1.2M Na2SO4，pH 7.4来稀释1份(以重量计)蛋白溶液来制备加载液。上样后，用平衡缓冲液洗涤色谱柱，然后用从0.6M到0M的Na2SO4线性梯度经过16个柱体积洗脱。将产物峰收集在级分中。
4.POROS HQ阴离子交换色谱
POROS HQ阴离子交换色谱在填充至30cm床高的色谱柱(Thermo Scientific,Waltham,MA USA)中进行。使用来自GE Healthcare的AKTA avant液相色谱系统进行全部运行，并且在275cm/hr下操作柱子。用20mM Tris-HCl，pH 7.4平衡柱子，加载蛋白，然后用平衡缓冲液洗涤。用从0M到0.5M的NaCl线性梯度经25个柱体积洗脱。基于在产物峰前部和尾部的5mAU处的吸光度标准来收集产物峰。
5.CAPTO Q阴离子交换色谱
Capto Q阴离子交换色谱在流通模式下在填充至10cm床高的色谱柱(GEHealthcare，Marlborough，MA USA)中进行。使用来自GE Healthcare的AKTA avant液相色谱系统进行全部运行，并在250-350cm/hr下操作柱子。用20mM TrisHCl，250mM NaCl，pH7.4平衡色谱柱，加载蛋白，然后用平衡缓冲液洗涤。基于在产物流通峰前部和尾部的5mAU处的吸光度标准来收集产物峰。
实施例3人源化抗-CD22重组免疫毒素的非临床药理学
按照实施例2所述，基于本发明VH、VL不同人源化版本连同HA22中VH、VL的两两配对，制备各种重组免疫毒素，命名方式为“HmLn”，其中m、n分别是VH、VL人源化版本的编号。
3.1靶向作用
采用重组人Siglec-2/CD22 Fc嵌合蛋白(R&D systems，Cat#1968-SL)作为固定抗原用于捕获本发明的人源化抗-CD22重组免疫毒素或HA22，采用抗-假单胞菌外毒素A抗体(Sigma-Aldrich，Cat#P2318)作为第二抗体，其具有用于检测的酶缀合物，进行ELISA。
采用与生物素化的CD22缀合的链霉亲和素(SA)生物传感器(ForteBio，Cat#18-5019)进行Octet分析，进行本发明的重组免疫毒素或HA22的KD测量。
如表1所示，重组免疫毒素H1L2(包含VH ver 1+VL ver 2)和H2L2(包含VH ver 2+VL ver 2)显示出了与HA22相当的EC50和KD。
表1：CD22结合分析结果
Figure GDA0004126405920000201
Figure GDA0004126405920000211
不同版本的人源化重组免疫毒素与CD22结合亲和力如表2和表3所示.表2中，当分别对两批或三批以上产物进行单独分析时，EC50(ELISA)的范围以平均值±SD表示或给出。表3为各人源化重组免疫毒素EC50相对于HA22的结果。
表2：CD22结合亲和力(nM)
Figure GDA0004126405920000212
表3：CD22结合亲和力(EC50针对HA22标准化)
Figure GDA0004126405920000213
3.2基于细胞的效力分析
基于细胞的分析采用表达CD22的细胞模型来评估发明的人源化抗-CD22重组免疫毒素的生长抑制和细胞凋亡效应。这是使用细胞生长/抑制作为效力指标的终点分析。与结合分析相比，细胞模型包括了全部的CD22结合、内化和PE38催化的EF-2的ADP核糖基化，以驱动所观察的抑制作用，因此更好地模拟了体内条件。
将表达高水平CD22的Daudi细胞系(ATCC)与本发明的重组免疫毒素或HA22共培养一定时间，然后在分析结束时，用比色法对培养物中的活细胞进行定量。操作如下：
在RPMI-1640完全培养基中培养上述Daudi细胞，收集细胞并用HBSS洗一次。将细胞以2.5×105个/mL的密度重悬于RPMI-1640培养基中，以100μL/孔将细胞转移到96孔板中(相当于2.5×104个活细胞/孔)。将本发明的重组免疫毒素或HA22待测样品稀释到预定浓度，并制备系列稀释液。
取100μL系列稀释液加入每孔细胞中，然后将细胞在37℃、5％ CO2下孵育48-72小时。之后向每孔中加入10μL CCK-8试剂，在37℃下孵育4-8小时。为了针对背景活性进行校正，将细胞在10μg/mL环己酰亚胺存在下培养细胞直到100％死亡。
检测450nm吸光度来测量Formazan生成。在环己酰亚胺与培养基对照之间使数值标准化，50％细胞死亡时本发明的人源化重组免疫毒素或HA22的浓度为EC50。
不同版本的人源化重组免疫毒素与CD22的结合亲和力如表4和表5所示。表4中，当分别对两批或三批以上产物进行单独分析时，EC50的范围以平均值±SD表示或给出。表5为各人源化重组免疫毒素EC50相对于HA22的结果。如结果所示，在基于细胞的体内效力分析中，本发明的人源化重组免疫毒素、尤其是H1L2显示出的EC50与HA22相当。
表4：体外效力分析(pM)
Figure GDA0004126405920000221
表5：体外效力分析(EC50针对HA22标准化)
Figure GDA0004126405920000222
Figure GDA0004126405920000231
实施例4人源化抗-CD22重组免疫毒素的非临床药代动力学和药物代谢
4.1体内PK研究
使用C57BL/6小鼠模型(4-6周龄，雌性)。将45只小鼠随机分为3组：1)对照组(制剂缓冲液)；2)500μg/kg HA22；3)500μg/kg H1L2。对照组和治疗组均通过尾静脉注射给药，并在注射后的指定时间点(5min、15min、30min、45min、1h、2h、4h、8h、24h、48h、96h)通过眼眶后采样收集100μL全血。每个时间点分配三只重复的动物。从血样中回收50微升血浆进行分析。
如表6和表7所示，结果表明，在小鼠模型中H1L2与HA22显示出了相当的药代动力学特性：两种分子具有相似的半衰期(T1/2)、达到最大浓度的时间(Tmax)、最大浓度(Cmax)以及药时曲线下面积(AUC0-t)。PK图见图4。
表6：HA22 PK结果
T1/2(min) Tmax(min) Cmax(ng/mL) AUC0-t(ng/mL×min)
重复1 44.38 5 1.671×104 7.329×105
重复2 46.32 5 1.275×104 6.385×105
重复3 57.26 5 1.863×104 7.125×105
平均 49.32 5 1.603×104 6.947×105
表7：H1L2 PK结果
T1/2(min) Tmax(min) Cmax(ng/mL) AUC0-t(ng/mL×min)
重复1 41.69 5 1.906×104 7.416×105
重复2 41.76 5 1.581×104 7.458×105
重复3 42.48 5 1.910×104 7.474×105
平均 41.98 5 1.799×104 7.449×105
4.2体内功效
采用异种移植动物模型和CD22阳性Raji B淋巴细胞用于肿瘤构建。给药后，重组免疫毒素进入循环系统，与恶性B细胞表面的CD22结合，并通过凋亡途径抑制细胞生长。因此，本实施例将肿瘤生长的抑制指定为功效研究的终点。
使用无胸腺的NCr裸鼠模型(4-6周龄，雌性)。将25只小鼠随机分为5组，如表8所示。将CD-22阳性智人成淋巴细胞Raji细胞系(ATCC CCL-86)用于建立异种移植物。在进行任何治疗之前，以5×107个细胞/mL的终浓度皮下注射Raji细胞，并使其生长至100-200mm3。建立异种移植肿瘤后，通过尾静脉注射以指定的剂量水平给予单一剂量治疗。治疗后观察并测量异种移植小鼠的肿瘤大小达24天。
如图5所示，与对照组(第1组)相比，HA22高剂量组显示出84.9％的肿瘤生长抑制，低剂量组显示出95.8％的肿瘤生长抑制；H1L2高剂量组显示出87.7％的肿瘤生长抑制，低剂量组显示出34.2％的肿瘤生长抑制。经T检验，除了低剂量的人源化重组免疫毒素H1L2，所有治疗均显示出显著的肿瘤抑制功效(图5*P<0.05，**P<0.01，***P<0.005)。HA22和H1L2高剂量组之间没有统计学差异。
表8：功效研究中组别和给药信息
组 治疗 剂量水平(μg/kg) 动物数量
1 制剂缓冲液 0 5
2 HA22 100 5
3 HA22 300 5
4 H1L2 100 5
5 H1L2 300 5
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。
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序列表
<110> 昆明赛诺制药股份有限公司
<120> 人源化抗-CD22重组免疫毒素及其应用
<130> LC21210002P
<150> CN2020110487230
<151> 2020-09-29
<160> 33
<170> PatentIn version 3.3
<210> 1
<211> 352
<212> PRT
<213> artificial
<220>
<223> PE38 polypeptide sequence
<400> 1
Lys Ala Ser Gly Gly Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala
1 5 10 15
His Gln Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln
20 25 30
Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg
35 40 45
Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp
50 55 60
Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu
65 70 75 80
Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr
85 90 95
Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn
100 105 110
Asp Glu Ala Gly Ala Ala Asn Gly Pro Ala Asp Ser Gly Asp Ala Leu
115 120 125
Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly
130 135 140
Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg
145 150 155 160
Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val
165 170 175
Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly
180 185 190
Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe
195 200 205
Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln
210 215 220
Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val
225 230 235 240
Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr
245 250 255
Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His
260 265 270
Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly
275 280 285
Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val
290 295 300
Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp
305 310 315 320
Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu
325 330 335
Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Lys
340 345 350
<210> 2
<211> 124
<212> PRT
<213> artificial
<220>
<223> HA22，VH polypeptide sequence
<400> 2
Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly
1 5 10 15
Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ile
20 25 30
Tyr Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Cys Leu Glu Trp
35 40 45
Val Ala Tyr Ile Ser Ser Gly Gly Gly Thr Thr Tyr Tyr Pro Asp Thr
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu
65 70 75 80
Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr
85 90 95
Cys Ala Arg His Ser Gly Tyr Gly Thr His Trp Gly Val Leu Phe Ala
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
115 120
<210> 3
<211> 124
<212> PRT
<213> artificial
<220>
<223> Version 1 VH polypeptide sequence
<400> 3
Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ile
20 25 30
Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp
35 40 45
Val Ala Tyr Ile Ser Ser Gly Gly Gly Thr Thr Tyr Tyr Pro Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr
85 90 95
Cys Ala Arg His Ser Gly Tyr Gly Thr His Trp Gly Val Leu Phe Ala
100 105 110
Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 4
<211> 124
<212> PRT
<213> artificial
<220>
<223> Version 2 VH polypeptide sequence
<400> 4
Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ile
20 25 30
Tyr Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Cys Leu Glu Trp
35 40 45
Val Ala Tyr Ile Ser Ser Gly Gly Gly Thr Thr Tyr Tyr Pro Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr
85 90 95
Cys Ala Arg His Ser Gly Tyr Gly Thr His Trp Gly Val Leu Phe Ala
100 105 110
Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 5
<211> 124
<212> PRT
<213> artificial
<220>
<223> Version 3 VH polypeptide sequence
<400> 5
Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ile
20 25 30
Tyr Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp
35 40 45
Val Ser Tyr Ile Ser Ser Gly Gly Gly Thr Thr Tyr Tyr Pro Gly Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Arg His Ser Gly Tyr Gly Thr His Trp Gly Val Leu Phe Ala
100 105 110
Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 6
<211> 124
<212> PRT
<213> artificial
<220>
<223> Version 4 VH polypeptide sequence
<400> 6
Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ile
20 25 30
Tyr Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp
35 40 45
Val Ala Tyr Ile Ser Ser Gly Gly Gly Thr Thr Tyr Tyr Pro Gly Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Arg His Ser Gly Tyr Gly Thr His Trp Gly Val Leu Phe Ala
100 105 110
Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 7
<211> 124
<212> PRT
<213> artificial
<220>
<223> Version 5 VH polypeptide sequence
<400> 7
Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ile
20 25 30
Tyr Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp
35 40 45
Val Ala Tyr Ile Ser Ser Gly Gly Gly Thr Thr Tyr Tyr Pro Gly Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Arg His Ser Gly Tyr Gly Thr His Trp Gly Val Leu Phe Ala
100 105 110
Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 8
<211> 476
<212> PRT
<213> artificial
<220>
<223> HA22 VH-PE38 polypeptide sequence
<400> 8
Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly
1 5 10 15
Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ile
20 25 30
Tyr Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Cys Leu Glu Trp
35 40 45
Val Ala Tyr Ile Ser Ser Gly Gly Gly Thr Thr Tyr Tyr Pro Asp Thr
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu
65 70 75 80
Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr
85 90 95
Cys Ala Arg His Ser Gly Tyr Gly Thr His Trp Gly Val Leu Phe Ala
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Lys Ala Ser Gly
115 120 125
Gly Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys
130 135 140
His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp
145 150 155 160
Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu
165 170 175
Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg
180 185 190
Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile
195 200 205
Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala
210 215 220
Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly
225 230 235 240
Ala Ala Asn Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn
245 250 255
Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe
260 265 270
Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala
275 280 285
His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly
290 295 300
Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala
305 310 315 320
Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly
325 330 335
Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala
340 345 350
Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg
355 360 365
Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro
370 375 380
Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu
385 390 395 400
Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu
405 410 415
Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser
420 425 430
Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser
435 440 445
Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala
450 455 460
Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Lys
465 470 475
<210> 9
<211> 476
<212> PRT
<213> artificial
<220>
<223> Version 1 VH-PE38 polypeptide sequence
<400> 9
Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ile
20 25 30
Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp
35 40 45
Val Ala Tyr Ile Ser Ser Gly Gly Gly Thr Thr Tyr Tyr Pro Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr
85 90 95
Cys Ala Arg His Ser Gly Tyr Gly Thr His Trp Gly Val Leu Phe Ala
100 105 110
Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Lys Ala Ser Gly
115 120 125
Gly Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys
130 135 140
His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp
145 150 155 160
Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu
165 170 175
Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg
180 185 190
Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile
195 200 205
Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala
210 215 220
Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly
225 230 235 240
Ala Ala Asn Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn
245 250 255
Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe
260 265 270
Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala
275 280 285
His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly
290 295 300
Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala
305 310 315 320
Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly
325 330 335
Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala
340 345 350
Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg
355 360 365
Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro
370 375 380
Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu
385 390 395 400
Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu
405 410 415
Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser
420 425 430
Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser
435 440 445
Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala
450 455 460
Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Lys
465 470 475
<210> 10
<211> 476
<212> PRT
<213> artificial
<220>
<223> Version 2 VH-PE38 polypeptide sequence
<400> 10
Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ile
20 25 30
Tyr Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Cys Leu Glu Trp
35 40 45
Val Ala Tyr Ile Ser Ser Gly Gly Gly Thr Thr Tyr Tyr Pro Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr
85 90 95
Cys Ala Arg His Ser Gly Tyr Gly Thr His Trp Gly Val Leu Phe Ala
100 105 110
Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Lys Ala Ser Gly
115 120 125
Gly Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys
130 135 140
His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp
145 150 155 160
Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu
165 170 175
Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg
180 185 190
Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile
195 200 205
Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala
210 215 220
Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly
225 230 235 240
Ala Ala Asn Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn
245 250 255
Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe
260 265 270
Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala
275 280 285
His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly
290 295 300
Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala
305 310 315 320
Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly
325 330 335
Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala
340 345 350
Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg
355 360 365
Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro
370 375 380
Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu
385 390 395 400
Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu
405 410 415
Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser
420 425 430
Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser
435 440 445
Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala
450 455 460
Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Lys
465 470 475
<210> 11
<211> 476
<212> PRT
<213> artificial
<220>
<223> Version 3 VH-PE38 polypeptide sequence
<400> 11
Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ile
20 25 30
Tyr Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp
35 40 45
Val Ser Tyr Ile Ser Ser Gly Gly Gly Thr Thr Tyr Tyr Pro Gly Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Arg His Ser Gly Tyr Gly Thr His Trp Gly Val Leu Phe Ala
100 105 110
Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Lys Ala Ser Gly
115 120 125
Gly Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys
130 135 140
His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp
145 150 155 160
Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu
165 170 175
Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg
180 185 190
Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile
195 200 205
Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala
210 215 220
Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly
225 230 235 240
Ala Ala Asn Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn
245 250 255
Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe
260 265 270
Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala
275 280 285
His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly
290 295 300
Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala
305 310 315 320
Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly
325 330 335
Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala
340 345 350
Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg
355 360 365
Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro
370 375 380
Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu
385 390 395 400
Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu
405 410 415
Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser
420 425 430
Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser
435 440 445
Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala
450 455 460
Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Lys
465 470 475
<210> 12
<211> 476
<212> PRT
<213> artificial
<220>
<223> Version 4 VH-PE38 polypeptide sequence
<400> 12
Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ile
20 25 30
Tyr Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp
35 40 45
Val Ala Tyr Ile Ser Ser Gly Gly Gly Thr Thr Tyr Tyr Pro Gly Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Arg His Ser Gly Tyr Gly Thr His Trp Gly Val Leu Phe Ala
100 105 110
Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Lys Ala Ser Gly
115 120 125
Gly Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys
130 135 140
His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp
145 150 155 160
Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu
165 170 175
Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg
180 185 190
Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile
195 200 205
Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala
210 215 220
Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly
225 230 235 240
Ala Ala Asn Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn
245 250 255
Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe
260 265 270
Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala
275 280 285
His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly
290 295 300
Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala
305 310 315 320
Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly
325 330 335
Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala
340 345 350
Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg
355 360 365
Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro
370 375 380
Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu
385 390 395 400
Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu
405 410 415
Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser
420 425 430
Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser
435 440 445
Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala
450 455 460
Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Lys
465 470 475
<210> 13
<211> 476
<212> PRT
<213> artificial
<220>
<223> Version 5 VH-PE38 polypeptide sequence
<400> 13
Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ile
20 25 30
Tyr Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp
35 40 45
Val Ala Tyr Ile Ser Ser Gly Gly Gly Thr Thr Tyr Tyr Pro Gly Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Arg His Ser Gly Tyr Gly Thr His Trp Gly Val Leu Phe Ala
100 105 110
Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Lys Ala Ser Gly
115 120 125
Gly Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys
130 135 140
His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp
145 150 155 160
Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu
165 170 175
Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg
180 185 190
Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile
195 200 205
Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala
210 215 220
Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly
225 230 235 240
Ala Ala Asn Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn
245 250 255
Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe
260 265 270
Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala
275 280 285
His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly
290 295 300
Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala
305 310 315 320
Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly
325 330 335
Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala
340 345 350
Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg
355 360 365
Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro
370 375 380
Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu
385 390 395 400
Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu
405 410 415
Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser
420 425 430
Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser
435 440 445
Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala
450 455 460
Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Lys
465 470 475
<210> 14
<211> 108
<212> PRT
<213> artificial
<220>
<223> HA22，VL polypeptide sequence
<400> 14
Met Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu
1 5 10 15
Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn
20 25 30
Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu
35 40 45
Ile Tyr Tyr Thr Ser Ile Leu His Ser Gly Val Pro Ser Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu
65 70 75 80
Gln Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro
85 90 95
Trp Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 15
<211> 108
<212> PRT
<213> artificial
<220>
<223> Version 1 VL polypeptide sequence
<400> 15
Met Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val
1 5 10 15
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn
20 25 30
Tyr Leu Asn Trp Tyr Gln Gln Arg Pro Gly Gln Ser Val Lys Leu Leu
35 40 45
Ile Tyr Tyr Thr Ser Ile Leu His Ser Gly Val Pro Ser Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln
65 70 75 80
Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro
85 90 95
Trp Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys
100 105
<210> 16
<211> 108
<212> PRT
<213> artificial
<220>
<223> Version 2 VL polypeptide sequence
<400> 16
Met Asp Ile Gln Met Thr Gln Ser Thr Ser Thr Leu Ser Ala Ser Val
1 5 10 15
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn
20 25 30
Tyr Leu Asn Trp Tyr Gln Gln Arg Pro Asp Gly Ser Val Lys Leu Leu
35 40 45
Ile Tyr Tyr Thr Ser Ile Leu His Ser Gly Val Pro Ser Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln
65 70 75 80
Asp Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro
85 90 95
Trp Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys
100 105
<210> 17
<211> 108
<212> PRT
<213> artificial
<220>
<223> Version 3 VL polypeptide sequence
<400> 17
Met Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val
1 5 10 15
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn
20 25 30
Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser Pro Arg Arg Leu
35 40 45
Ile Tyr Tyr Thr Ser Ile Leu His Ser Gly Val Pro Ser Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln
65 70 75 80
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro
85 90 95
Trp Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys
100 105
<210> 18
<211> 108
<212> PRT
<213> artificial
<220>
<223> Version 4 VL polypeptide sequence
<400> 18
Met Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val
1 5 10 15
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn
20 25 30
Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser Pro Arg Leu Leu
35 40 45
Ile Tyr Tyr Thr Ser Ile Leu His Ser Gly Val Pro Ser Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln
65 70 75 80
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro
85 90 95
Trp Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys
100 105
<210> 19
<211> 1428
<212> DNA
<213> artificial
<220>
<223> Version 1 VH-PE38 DNA sequence
<400> 19
atggaagtgc agctggtgga atccggcgga ggcctggtca agcccggcgg ctctctgaga 60
ctgtcctgcg ctgcttctgg cttcgccttc tccatctacg acatgtcctg ggtcagacag 120
gcccctggca agtgcctgga atgggtggcc tacatctcct ctggcggagg caccacctac 180
taccctgact ccgtgaaggg cagattcacc atctctagag agaacgccaa gaacagcctg 240
tacctgcaga tgaactccct gaagtccgag gacaccgcca tgtactactg cgccagacac 300
tccggctacg gcacacactg gggcgtgctg ttcgcttact ggggccaggg caccatggtg 360
accgtgtcct ccaaagcgtc aggcggtccg gaaggcggtt cgctggcagc tctgaccgca 420
catcaggcat gccacctgcc gctggaaacc tttacccgcc atcgtcaacc gcgcggctgg 480
gaacagctgg aacaatgtgg ttatccggtg cagcgcctgg ttgccctgta cctggccgca 540
cgtctgagct ggaaccaggt tgatcaagtc attcgtaatg cgctggcatc accgggctcg 600
ggcggtgacc tgggtgaagc aatccgcgaa cagccggaac aagcgcgcct ggccctgacc 660
ctggcagctg cggaatccga acgctttgtg cgtcagggca ccggtaatga tgaagccggt 720
gcagcaaatg gtccggctga ttcaggtgac gcgctgctgg aacgcaacta tccgacgggc 780
gccgaatttc tgggtgatgg cggtgacgtg agtttctcca cccgcggcac gcagaattgg 840
accgttgaac gtctgctgca ggcgcatcgc caactggaag aacgtggtta tgtttttgtc 900
ggctatcatg gcacctttct ggaagctgcg cagtcgattg tctttggcgg tgtgcgtgca 960
cgcagccagg atctggatgc aatttggcgc ggcttctaca tcgcaggtga tccggctctg 1020
gcgtatggct acgctcagga tcaagaaccg gatgcgcgtg gccgcatccg taatggtgcc 1080
ctgctgcgtg tgtatgttcc gcgttcatcg ctgccgggtt tttaccgtac ctctctgacc 1140
ctggcggcac cggaagctgc cggcgaagtg gaacgcctga ttggtcaccc gctgccgctg 1200
cgtctggatg caatcaccgg tccggaagaa gaaggcggcc gtctggaaac gattctgggt 1260
tggccgctgg ccgaacgtac cgtggttatt ccgagcgcaa tcccgacgga cccgcgcaat 1320
gttggcggtg atctggaccc gagctctatt ccggataaag aacaggccat ctccgcactg 1380
ccggactatg cgtcacaacc gggcaaaccg ccgcgtgaag acctgaaa 1428
<210> 20
<211> 1428
<212> DNA
<213> artificial
<220>
<223> Version 2 VH-PE38 DNA sequence
<400> 20
atggaagtgc agctggtgga atccggcgga ggcctggtca agcccggcgg ctctctgaga 60
ctgtcctgcg ctgcttctgg cttcgccttc tccatctacg acatgtcctg ggtcagacag 120
acccctgaaa agtgcctgga atgggtggcc tacatctcct ctggcggagg caccacctac 180
taccctgact ccgtgaaggg cagattcacc atctctagag agaacgccaa gaacagcctg 240
tacctgcaga tgaactccct gaagtccgag gacaccgcca tgtactactg cgccagacac 300
tccggctacg gcacacactg gggcgtgctg ttcgcttact ggggccaggg caccatggtg 360
accgtgtcct ccaaagcgtc aggcggtccg gaaggcggtt cgctggcagc tctgaccgca 420
catcaggcat gccacctgcc gctggaaacc tttacccgcc atcgtcaacc gcgcggctgg 480
gaacagctgg aacaatgtgg ttatccggtg cagcgcctgg ttgccctgta cctggccgca 540
cgtctgagct ggaaccaggt tgatcaagtc attcgtaatg cgctggcatc accgggctcg 600
ggcggtgacc tgggtgaagc aatccgcgaa cagccggaac aagcgcgcct ggccctgacc 660
ctggcagctg cggaatccga acgctttgtg cgtcagggca ccggtaatga tgaagccggt 720
gcagcaaatg gtccggctga ttcaggtgac gcgctgctgg aacgcaacta tccgacgggc 780
gccgaatttc tgggtgatgg cggtgacgtg agtttctcca cccgcggcac gcagaattgg 840
accgttgaac gtctgctgca ggcgcatcgc caactggaag aacgtggtta tgtttttgtc 900
ggctatcatg gcacctttct ggaagctgcg cagtcgattg tctttggcgg tgtgcgtgca 960
cgcagccagg atctggatgc aatttggcgc ggcttctaca tcgcaggtga tccggctctg 1020
gcgtatggct acgctcagga tcaagaaccg gatgcgcgtg gccgcatccg taatggtgcc 1080
ctgctgcgtg tgtatgttcc gcgttcatcg ctgccgggtt tttaccgtac ctctctgacc 1140
ctggcggcac cggaagctgc cggcgaagtg gaacgcctga ttggtcaccc gctgccgctg 1200
cgtctggatg caatcaccgg tccggaagaa gaaggcggcc gtctggaaac gattctgggt 1260
tggccgctgg ccgaacgtac cgtggttatt ccgagcgcaa tcccgacgga cccgcgcaat 1320
gttggcggtg atctggaccc gagctctatt ccggataaag aacaggccat ctccgcactg 1380
ccggactatg cgtcacaacc gggcaaaccg ccgcgtgaag acctgaaa 1428
<210> 21
<211> 1428
<212> DNA
<213> artificial
<220>
<223> Version 3 VH-PE38 DNA sequence
<400> 21
atggaagtgc agctggttga aagcggtggc ggtctggtga aaccgggcgg tagcctgcgt 60
ctgagctgcg cggcgagcgg tttcgcgttt agcatctacg acatgagctg gattcgtcaa 120
gcgccgggca aatgcctgga gtgggtgagc tatatcagca gcggcggtgg caccacctac 180
tatccgggta gcgttaaggg ccgtttcacc attagccgtg aaaacgcgaa aaacagcctg 240
tacctgcaga tgaacagcct gcgtgcgggt gataccgctg tgtactattg cgcgcgtcac 300
agcggttacg gcacccactg gggcgttctg tttgcgtatt ggggtcaagg caccatggtg 360
accgttagca gcaaagcgtc aggcggtccg gaaggcggtt cgctggcagc tctgaccgca 420
catcaggcat gccacctgcc gctggaaacc tttacccgcc atcgtcaacc gcgcggctgg 480
gaacagctgg aacaatgtgg ttatccggtg cagcgcctgg ttgccctgta cctggccgca 540
cgtctgagct ggaaccaggt tgatcaagtc attcgtaatg cgctggcatc accgggctcg 600
ggcggtgacc tgggtgaagc aatccgcgaa cagccggaac aagcgcgcct ggccctgacc 660
ctggcagctg cggaatccga acgctttgtg cgtcagggca ccggtaatga tgaagccggt 720
gcagcaaatg gtccggctga ttcaggtgac gcgctgctgg aacgcaacta tccgacgggc 780
gccgaatttc tgggtgatgg cggtgacgtg agtttctcca cccgcggcac gcagaattgg 840
accgttgaac gtctgctgca ggcgcatcgc caactggaag aacgtggtta tgtttttgtc 900
ggctatcatg gcacctttct ggaagctgcg cagtcgattg tctttggcgg tgtgcgtgca 960
cgcagccagg atctggatgc aatttggcgc ggcttctaca tcgcaggtga tccggctctg 1020
gcgtatggct acgctcagga tcaagaaccg gatgcgcgtg gccgcatccg taatggtgcc 1080
ctgctgcgtg tgtatgttcc gcgttcatcg ctgccgggtt tttaccgtac ctctctgacc 1140
ctggcggcac cggaagctgc cggcgaagtg gaacgcctga ttggtcaccc gctgccgctg 1200
cgtctggatg caatcaccgg tccggaagaa gaaggcggcc gtctggaaac gattctgggt 1260
tggccgctgg ccgaacgtac cgtggttatt ccgagcgcaa tcccgacgga cccgcgcaat 1320
gttggcggtg atctggaccc gagctctatt ccggataaag aacaggccat ctccgcactg 1380
ccggactatg cgtcacaacc gggcaaaccg ccgcgtgaag acctgaaa 1428
<210> 22
<211> 1428
<212> DNA
<213> artificial
<220>
<223> Version 4 VH-PE38 DNA sequence
<400> 22
atggaagtgc agctggttga aagcggtggc ggtctggtga aaccgggcgg tagcctgcgt 60
ctgagctgcg cggcgagcgg tttcgcgttt agcatctacg acatgagctg gattcgtcaa 120
gcgccgggca aatgcctgga gtgggtggcg tatatcagca gcggcggtgg caccacctac 180
tatccgggta gcgttaaggg ccgtttcacc attagccgtg aaaacgcgaa aaacagcctg 240
tacctgcaga tgaacagcct gcgtgcgggt gataccgctg tgtactattg cgcgcgtcac 300
agcggttacg gcacccactg gggcgttctg tttgcgtatt ggggtcaagg caccatggtg 360
accgttagca gcaaagcgtc aggcggtccg gaaggcggtt cgctggcagc tctgaccgca 420
catcaggcat gccacctgcc gctggaaacc tttacccgcc atcgtcaacc gcgcggctgg 480
gaacagctgg aacaatgtgg ttatccggtg cagcgcctgg ttgccctgta cctggccgca 540
cgtctgagct ggaaccaggt tgatcaagtc attcgtaatg cgctggcatc accgggctcg 600
ggcggtgacc tgggtgaagc aatccgcgaa cagccggaac aagcgcgcct ggccctgacc 660
ctggcagctg cggaatccga acgctttgtg cgtcagggca ccggtaatga tgaagccggt 720
gcagcaaatg gtccggctga ttcaggtgac gcgctgctgg aacgcaacta tccgacgggc 780
gccgaatttc tgggtgatgg cggtgacgtg agtttctcca cccgcggcac gcagaattgg 840
accgttgaac gtctgctgca ggcgcatcgc caactggaag aacgtggtta tgtttttgtc 900
ggctatcatg gcacctttct ggaagctgcg cagtcgattg tctttggcgg tgtgcgtgca 960
cgcagccagg atctggatgc aatttggcgc ggcttctaca tcgcaggtga tccggctctg 1020
gcgtatggct acgctcagga tcaagaaccg gatgcgcgtg gccgcatccg taatggtgcc 1080
ctgctgcgtg tgtatgttcc gcgttcatcg ctgccgggtt tttaccgtac ctctctgacc 1140
ctggcggcac cggaagctgc cggcgaagtg gaacgcctga ttggtcaccc gctgccgctg 1200
cgtctggatg caatcaccgg tccggaagaa gaaggcggcc gtctggaaac gattctgggt 1260
tggccgctgg ccgaacgtac cgtggttatt ccgagcgcaa tcccgacgga cccgcgcaat 1320
gttggcggtg atctggaccc gagctctatt ccggataaag aacaggccat ctccgcactg 1380
ccggactatg cgtcacaacc gggcaaaccg ccgcgtgaag acctgaaa 1428
<210> 23
<211> 1428
<212> DNA
<213> artificial
<220>
<223> Version 5 VH-PE38 DNA sequence
<400> 23
atggaagtgc agctggttga aagcggtggc ggtctggtga aaccgggcgg tagcctgcgt 60
ctgagctgcg cggcgagcgg tttcgcgttt agcatctacg acatgagctg gattcgtcaa 120
gcgccgggca aatgcctgga gtgggtggcg tatatcagca gcggcggtgg caccacctac 180
tatccgggta gcgttaaggg ccgtttcacc attagccgtg aaaacgcgaa aaacagcctg 240
tacctgcaga tgaacagcct gcgttctgag gataccgctg tgtactattg cgcgcgtcac 300
agcggttacg gcacccactg gggcgttctg tttgcgtatt ggggtcaagg caccatggtg 360
accgttagca gcaaagcgtc aggcggtccg gaaggcggtt cgctggcagc tctgaccgca 420
catcaggcat gccacctgcc gctggaaacc tttacccgcc atcgtcaacc gcgcggctgg 480
gaacagctgg aacaatgtgg ttatccggtg cagcgcctgg ttgccctgta cctggccgca 540
cgtctgagct ggaaccaggt tgatcaagtc attcgtaatg cgctggcatc accgggctcg 600
ggcggtgacc tgggtgaagc aatccgcgaa cagccggaac aagcgcgcct ggccctgacc 660
ctggcagctg cggaatccga acgctttgtg cgtcagggca ccggtaatga tgaagccggt 720
gcagcaaatg gtccggctga ttcaggtgac gcgctgctgg aacgcaacta tccgacgggc 780
gccgaatttc tgggtgatgg cggtgacgtg agtttctcca cccgcggcac gcagaattgg 840
accgttgaac gtctgctgca ggcgcatcgc caactggaag aacgtggtta tgtttttgtc 900
ggctatcatg gcacctttct ggaagctgcg cagtcgattg tctttggcgg tgtgcgtgca 960
cgcagccagg atctggatgc aatttggcgc ggcttctaca tcgcaggtga tccggctctg 1020
gcgtatggct acgctcagga tcaagaaccg gatgcgcgtg gccgcatccg taatggtgcc 1080
ctgctgcgtg tgtatgttcc gcgttcatcg ctgccgggtt tttaccgtac ctctctgacc 1140
ctggcggcac cggaagctgc cggcgaagtg gaacgcctga ttggtcaccc gctgccgctg 1200
cgtctggatg caatcaccgg tccggaagaa gaaggcggcc gtctggaaac gattctgggt 1260
tggccgctgg ccgaacgtac cgtggttatt ccgagcgcaa tcccgacgga cccgcgcaat 1320
gttggcggtg atctggaccc gagctctatt ccggataaag aacaggccat ctccgcactg 1380
ccggactatg cgtcacaacc gggcaaaccg ccgcgtgaag acctgaaa 1428
<210> 24
<211> 324
<212> DNA
<213> artificial
<220>
<223> Version 1 VL DNA sequence
<400> 24
atggacatcc agatgaccca gtctccttct acactgtctg cttctgtggg cgacagagtg 60
accatcacct gcagagcctc tcaggacatc tccaactacc tgaactggta ccagcagaga 120
cctggccagt ccgtgaagct gctgatctac tacacctcca tcctgcactc cggcgtgcct 180
tccagattct ccggctctgg atctggcacc gactacaccc tgaccatctc ctccctgcag 240
cctgaggact tcgccaccta cttctgccag cagggcaaca ccctgccttg gaccttcggc 300
tgcggcacca aggtggaaat caag 324
<210> 25
<211> 324
<212> DNA
<213> artificial
<220>
<223> Version 2 VL DNA sequence
<400> 25
atggacatcc agatgaccca gtctacttct acactgtctg cttctgtggg cgacagagtg 60
accatcacct gcagagcctc tcaggacatc tccaactacc tgaactggta ccagcagaga 120
cctgatggct ccgtgaagct gctgatctac tacacctcca tcctgcactc cggcgtgcct 180
tccagattct ccggctctgg atctggcacc gactacaccc tgaccatctc ctccctgcag 240
gatgaggact tcgccaccta cttctgccag cagggcaaca ccctgccttg gaccttcggc 300
tgcggcacca aggtggaaat caag 324
<210> 26
<211> 324
<212> DNA
<213> artificial
<220>
<223> Version 3 VL DNA sequence
<400> 26
atggacatcc agatgaccca gagcccgagc accctgagcg cgagcgtggg cgaccgtgtt 60
accatcacct gccgtgcgag ccaggatatt agcaactacc tgaactggtt tcaacaacgt 120
ccgggtcaaa gcccgcgtcg tctgatctac tataccagca ttctgcacag cggtgtgccg 180
agccgtttta gcggtagcgg cagcggtacc gactataccc tgaccatcag cagcctgcag 240
ccggaggatt tcgcgaccta ctattgccag cagggtaaca ccctgccgtg gacctttggc 300
tgcggtacca aggttgaaat taaa 324
<210> 27
<211> 324
<212> DNA
<213> artificial
<220>
<223> Version 3 VL DNA sequence
<400> 27
atggacatcc agatgaccca gagcccgagc accctgagcg cgagcgtggg cgaccgtgtt 60
accatcacct gccgtgcgag ccaggatatt agcaactacc tgaactggtt tcaacaacgt 120
ccgggtcaaa gcccgcgtct gctgatctac tataccagca ttctgcacag cggtgtgccg 180
agccgtttta gcggtagcgg cagcggtacc gactataccc tgaccatcag cagcctgcag 240
ccggaggatt tcgcgaccta ctattgccag cagggtaaca ccctgccgtg gacctttggc 300
tgcggtacca aggttgaaat taaa 324
<210> 28
<211> 8
<212> PRT
<213> artificial
<220>
<223> HA22，H-CDR1
<400> 28
Gly Phe Ala Phe Ser Ile Tyr Asp
1 5
<210> 29
<211> 9
<212> PRT
<213> artificial
<220>
<223> HA22，H-CDR2
<400> 29
Ile Ser Ser Gly Gly Gly Thr Thr Tyr
1 5
<210> 30
<211> 17
<212> PRT
<213> artificial
<220>
<223> HA22，H-CDR3
<400> 30
Cys Ala Arg His Ser Gly Tyr Gly Thr His Trp Gly Val Leu Phe Ala
1 5 10 15
Tyr
<210> 31
<211> 6
<212> PRT
<213> artificial
<220>
<223> HA22，L-CDR1
<400> 31
Gln Asp Ile Ser Asn Tyr
1 5
<210> 32
<211> 8
<212> PRT
<213> artificial
<220>
<223> HA22，L-CDR2
<400> 32
Tyr Thr Ser Ile Leu His Ser Gly
1 5
<210> 33
<211> 7
<212> PRT
<213> artificial
<220>
<223> HA22，L-CDR3
<400> 33
Gln Gln Gly Asn Thr Leu Pro
1 5

Claims (9)

1.一种人源化抗-CD22重组免疫毒素，所述重组免疫毒素为包含以下两个多肽链的多肽分子：
(1) 第一多肽链，其包含抗-CD22抗体的轻链可变区(VL)；
(2) 第二多肽链，其包含抗-CD22抗体的重链可变区(VH)以及与所述重链可变区(VH)直接或间接连接的细胞毒素；其中：
所述第一多肽链包含SEQ ID NO. 16所示氨基酸序列，所述第二多肽链包含SEQ IDNO. 9所示氨基酸序列；或
所述第一多肽链包含SEQ ID NO. 16所示氨基酸序列，所述第二多肽链包含SEQ IDNO. 10所示氨基酸序列。
2.一种核酸分子，所述核酸分子包含编码权利要求1所述的人源化抗-CD22重组免疫毒素中的第一多肽链的核苷酸序列和第二多肽链的核苷酸序列。
3.一种载体，其包含权利要求2所述的核酸分子。
4.一种宿主细胞，所述宿主细胞被权利要求2所述的核酸分子或权利要求3所述的载体转化或转染。
5.一种药物组合物，所述药物组合物包含权利要求1所述的人源化抗-CD22重组免疫毒素。
6.根据权利要求5所述的药物组合物，其特征在于，所述药物组合物还包含药学上可接受的辅料。
7.权利要求1所述的人源化抗-CD22重组免疫毒素、权利要求2所述的核酸分子、权利要求3所述的载体、权利要求4所述的宿主细胞或权利要求5或6所述的药物组合物在制备药物中的用途，所述药物用于治疗以CD22高表达为特征的B细胞恶性肿瘤。
8.根据权利要求7所述的用途，其特征在于，所述以CD22高表达为特征的B细胞恶性肿瘤为CD22高表达的淋巴瘤或白血病。
9.根据权利要求8所述的用途，其特征在于，所述淋巴瘤为非何金杰氏淋巴瘤、小淋巴细胞淋巴瘤或套细胞淋巴瘤；所述白血病为慢性淋巴细胞性白血病、毛细胞白血病或急性淋巴细胞性白血病。
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