CN112236167A - 结合至ssea4的单株抗体及其用途 - Google Patents

结合至ssea4的单株抗体及其用途 Download PDF

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CN112236167A
CN112236167A CN201980020978.5A CN201980020978A CN112236167A CN 112236167 A CN112236167 A CN 112236167A CN 201980020978 A CN201980020978 A CN 201980020978A CN 112236167 A CN112236167 A CN 112236167A
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陈良博
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Abstract

一种经分离的单株抗体或抗原‑结合片段，其专一性结合至阶段特异性胚胎抗原4。该单株抗体或抗原‑结合片段包含：具有序列辨识编号：33或序列辨识编号：40的序列的重链CDR1、具有序列辨识编号：34或序列辨识编号：39的序列的重链CDR2、具有序列辨识编号：35或序列辨识编号：41的序列的重链CDR3、具有序列辨识编号：36或序列辨识编号：42的序列的轻链CDR1、具有序列辨识编号：37或序列辨识编号：43的序列的轻链CDR2，具有序列辨识编号：38或序列辨识编号：44的序列的轻链CDR3。本发明揭示一种抗‑肿瘤方法，由具有表现阶段特异性胚胎抗原4的肿瘤个体投予上面的单株抗体或抗原‑结合片段来施行。进一步提供的是编码上面序列的核酸以及含有该等核酸的重组型细胞。

Description

结合至SSEA4的单株抗体及其用途
技术领域
本发明是有关于结合至SSEA4的单株抗体及其用途。
背景技术
针对癌症的免疫疗法的目的是提高病患自身对抗肿瘤的免疫反应的强度。免疫疗法会刺激对抗癌细胞的免疫系统的特定成分的活性，或者可对抗由癌细胞所产生的抑制免疫反应的讯号。
例如，已研究出专一性结合至在癌细胞表面上的肿瘤关联性抗原(TAAs)(例如，蛋白质抗原以及糖类抗原)的抗体来作为癌症疫苗，其导致抗体依赖型细胞毒杀作用、抗体依赖型吞噬作用、补体依赖型细胞溶解，以及直接的细胞生长抑制作用和/或细胞毒性作用。
已研究出对抗许多糖类TAAs(例如，Globo H、阶段特异性胚胎抗原3，以及阶段特异性胚胎抗原4)的单株抗体为基础的抗癌疫苗。然而，为了促进更有效地杀死癌细胞并且对于癌症复发提供持久的抗性，需要对糖类TAA具有高亲和力的改良的单株抗体来作为抗癌疫苗。
发明内容
为了满足上述需求，本发明提供一种经分离的单株抗体或其抗原-结合片段，其专一性结合至阶段特异性胚胎抗原4(SSEA4)。该单株抗体或抗原-结合片段，包含：具有序列辨识编号：33或序列辨识编号：40的序列的重链CDR1(H-CDR1)、具有序列辨识编号：34或序列辨识编号：39的序列的重链CDR2(H-CDR2)、具有序列辨识编号：35或序列辨识编号：41的序列的重链CDR3(H-CDR3)、具有序列辨识编号：36或序列辨识编号：42的序列的轻链CDR1(L-CDR1)、具有序列辨识编号：37或序列辨识编号：43的序列的轻链CDR2(L-CDR2)，以及具有序列辨识编号：38或序列辨识编号：44的序列的轻链CDR3(L-CDR3)。
在本发明的范畴的经分离的单株抗体包括选自下列所构成的群组中的重链序列：序列辨识编号：2、序列辨识编号：6、序列辨识编号：10、序列辨识编号：12、序列辨识编号：14、序列辨识编号：22、序列辨识编号：24以及序列辨识编号：26的序列；以及选自下列所构成的群组中的轻链序列：序列辨识编号：4、序列辨识编号：8、序列辨识编号：16、序列辨识编号：18、序列辨识编号：20、序列辨识编号：28、序列辨识编号：30以及序列辨识编号：32的序列。
本发明也提供的是一种用来治疗肿瘤的方法，其中在该肿瘤中的细胞表现SSEA4。该方法是借由投予有效量的上述单株抗体或抗原-结合片段来施行。
另外揭示的是一种编码上述单株抗体或其抗原-结合片段核酸建构物，以及一种含有该核酸建构物的重组型细胞。该重组型细胞表现专一性结合至SSEA4的单株抗体或其抗原-结合片段。
本发明的许多具体例的细节已描述在下面的说明与附图中。透过说明且亦透过检附的申请专利范围，本发明的其他特征、目的以及优点将会是明显的。最后，此处所引用的文献以其整体并入作为参考资料。
附图说明
下面说明是针对本发明解释的附图，其中：
图1A是生殖腺人类IGHV4-59x01重链可变区(huIGHV4-59x01，序列辨识编号：60)与抗-SSEA4鼠单株抗体1(muMAb1 H，序列辨识编号：2的残基20-138)以及人类单株抗体1序列(huMAb1 H，序列辨识编号：10的残基20-138；huMAb1 H1，序列辨识编号：12的残基20-138；huMAb1 H2，序列辨识编号：14的残基20-138)所对应的氨基酸序列的氨基酸序列比对，在此图以及其他的所有图中，CDR区被框在方框中；
图1B是生殖腺人类IGKV3-11x01轻链可变区(huIGKV3-11x01，序列辨识编号：61)与抗-SSEA4鼠单株抗体1(muMAb1 L，序列辨识编号：4的残基23-128)以及人类单株抗体1序列(huMAb1 L，序列辨识编号：16的残基23-128；huMAb1 L1，序列辨识编号：18的残基23-128；huMAb1 L2，序列辨识编号：20的残基23-128)所对应的氨基酸序列的氨基酸序列比对；
图2A是生殖腺人类IGHV4-59x01重链可变区(huIGHV4-59x01，序列辨识编号：60)与抗-SSEA4鼠单株抗体2(muMAb2 H，序列辨识编号：6的残基20-139)以及人类单株抗体2序列(huMAb2 H，序列辨识编号：22的残基20-139；huMAb2 H1，序列辨识编号：24的残基20-139；huMAb2 H2，序列辨识编号：26的残基20-139)所对应的氨基酸序列的氨基酸序列比对；以及
图2B是生殖腺人类IGKV3-11x01轻链可变区与抗-SSEA4鼠单株抗体2(muMAb2 L，序列辨识编号：8的残基23-128)以及人类单株抗体2序列(huMAb2 L，序列辨识编号：28的残基23-128；huMAb2 L1，序列辨识编号：30的残基23-128；huMAb2 L2，序列辨识编号：32的残基23-128)所对应的氨基酸序列的氨基酸序列比对。
具体实施方式
较佳实施例的详细说明
如上所述，本发明提供一种具有所指定的CDR区的单株抗体或抗原-结合片段(例如，单链Fv)，其专一性结合至阶段特异性胚胎抗原4(SSEA4)。在一个示范性单株抗体或抗原-结合片段中，所述重链CDR1(H-CDR1)具有序列辨识编号：33的序列；所述重链CDR2(H-CDR2)具有序列辨识编号：34的序列；所述重链CDR3(H-CDR3)具有序列辨识编号：35的序列；所述轻链CDR1(L-CDR1)具有序列辨识编号：42的序列；所述轻链CDR2(L-CDR2)具有序列辨识编号：43的序列；以及所述轻链CDR3(L-CDR3)具有序列辨识编号：44的序列。
具有与在前面段落中相同的CDR区的单株抗体的实例具有下列重链序列与轻链序列的组合：(i)序列辨识编号：14的重链序列与序列辨识编号：28的轻链序列、(ii)序列辨识编号：14的重链序列与序列辨识编号：30的轻链序列、(iii)序列辨识编号：14的重链序列与序列辨识编号：32的轻链序列、(iv)序列辨识编号：2的重链序列与序列辨识编号：4的轻链序列，以及(v)序列辨识编号：6的重链序列与序列辨识编号：8的轻链序列。
一种编码该单株抗体或结合片段的核酸建构物包含编码上面所列出的H-CDR1、H-CDR2、H-CDR3、L-CDR1、L-CDR2以及L-CDR3区的序列。在一个实例中，该核酸建构物编码序列辨识编号：33、序列辨识编号：34、序列辨识编号：35、序列辨识编号：42、序列辨识编号：43以及序列辨识编号：44的氨基酸。
在另一个方面，该核酸建构物编码具有上述重链与轻链序列的单株抗体。这类核酸建构物的具体例分别包含下列序列组合中的任意一组：(i)序列辨识编号：13与序列辨识编号：27、(ii)序列辨识编号：13与序列辨识编号：29、(iii)序列辨识编号：13与序列辨识编号：31、(iv)序列辨识编号：1与序列辨识编号：3，以及(v)序列辨识编号：5与序列辨识编号：7的序列。
此外，提供一种重组型细胞，其含有任何上述核酸建构物，并且表现专一性结合至SSEA4的单株抗体或结合片段。特定的重组型细胞表现具有下列CDR序列的单株抗体或结合片段：序列辨识编号：33、序列辨识编号：34、序列辨识编号：35、序列辨识编号：42、序列辨识编号：43，以及序列辨识编号：44。这类重组型细胞的一个实例表现具有序列辨识编号：14的重链序列与序列辨识编号：28的轻链序列的单株抗体。
上述用于治疗肿瘤的方法可借由投予本申请案中所揭示的任何单株抗体来施行。在一个实例中，该单株抗体包含序列辨识编号：33、序列辨识编号：34、序列辨识编号：35、序列辨识编号：42、序列辨识编号：43，以及序列辨识编号：44的序列。该单株抗体可具有序列辨识编号：14的重链序列与序列辨识编号：28的轻链序列。
该肿瘤治疗方法可有效治疗乳癌、结肠癌、肠胃道癌、肾癌、肺癌、肝癌、卵巢癌、胰腺癌、直肠癌、胃癌、睾丸癌、胸腺癌、子宫颈癌、前列腺癌、膀胱癌、皮肤癌、鼻咽癌、食道癌、口腔癌、头颈部癌、骨癌、软骨癌、肌肉癌、淋巴结癌、骨髓癌，以及脑癌。
相信的是，不用进一步详细阐述，熟习此技术者可基于此处的揭示内容，将此揭示内容应用至最完整的程度。因此，无论如何下列具体例并非解释为仅是其余揭示内容的说明而非限制。此处所引用的所有刊物与专利文件以其整体并入此处作为参考资料。
实施例
实施例1：融合瘤生产
抗SSEA4的鼠单株抗体是使用标准操作程序来生产。简言之，对小鼠一起注射已经融合至牛血清蛋白的SSEA4(BSA-SSEA4)与佐剂每两周一次直到10周。在血液样本中的抗-SSEA4抗体效价是借由酵素结合免疫吸附分析法(ELISA)使用具有结合的BSA-SSEA4的ELISA培养盘在标准条件下进行分析。
由BSA-SSEA4 ELISA所得到的具有最高效价的小鼠血液样本也是借由ELISA并使用表现SSEA4的人类胰腺癌(HPAC)细胞来进行测试。
使用标准技术将具有最高抗体效价的脾细胞从两只小鼠中分离出来并且与骨髓瘤细胞进行融合形成融合瘤。生产高效价的抗-SSEA4抗体的融合瘤是使用标准的亚克隆操作程序与ELISA分析并使用BSA-SSEA4与HPAC细胞来进行鉴定。生产结合至HPAC以及不表现SSEA4的人类骨髓瘤细胞株A375这两者的抗体的融合瘤则不再进一步追踪。
结合至细胞表面的SSEA4的抗体是借由使HPAC细胞与A375细胞培育融合瘤上清液，继而培育荧光标记的二次抗体，并使用荧光-活化的细胞分选分析来进行确认。挑选出两个单株抗体[被称为鼠单株抗体1(muMAb1)与鼠单株抗体2(muMAb2)]来供进一步分析。这些单株抗体皆结合至BSA-SSEA4与HPAC细胞并且不结合至A375细胞。
同型分析显示muMAb1是IgG1κ抗体，而muMAb2是IgG3κ抗体。
融合瘤上清液中的单株抗体浓度以及对SSEA4的结合亲合力是下面两个部分中所述来进行测定。muMAb1的Kd值是0.22nM，而muMAb2的Kd值是0.08nM。
实施例2：借由ELISA来进行的抗体浓度的测定
培养基样本中的muMAb抗体浓度是借由ELISA并使用下面涂覆于ELISA培养盘上的一次抗体来进行测定。针对MAbs，将ELISA培养盘涂覆以呈1μg/ml、100μl/井的AffiniPureGoat Anti-Mouse IgG(H+L)(Jackson ImmunoResearch#115-005-062)过夜。在培育连续稀释的融合瘤上清液或得自于瞬时转染的培养基样本(参见下面)之后，将井彻底清洗并且借由添加以1:8,000稀释的Peroxidase AffiniPure Goat Anti-Mouse IgG(H+L)(JacksonImmunoResearch#115-035-062)与所检测的抗体进行结合。在彻底清洗后，添加ABTSPeroxidase Substrate(1Component)(KPL#50-66-06)并且测量各井在405nm下的吸光值。
针对人源化mAbs(参见下面)，将ELISA培养盘涂覆以1μg/ml、100μl/井的Fcγ片段专一性的AffiniPureF(ab')2Fragment Goat Anti-Human IgG(Jackson ImmunoResearch#109-006-098)过夜。结合的人源化抗体的检测是如同上面针对鼠抗体所述使用1:10,000稀释的Fcγ片段专一性的Peroxidase AffiniPure Goat Anti-Human IgG(JacksonImmunoResearch#109-035-008)来进行。
实施例3：借由ELISA来进行的抗体亲合力的测定
针对抗原结合亲和力的测定是使用下面试剂组来进行标准的ELISA操作程序。将ELISA培养盘涂覆以1:1,000稀释的BSA-SSEA4。将连续稀释的融合瘤上清液的样本或瞬时转染的样本添加至各井中，继而彻底清洗。使用下列二次抗体来侦测结合至培养盘上的抗原的muMAbs与huMAbs：针对muMAb1，1：2,500稀释的FcγSubclass 1专一性的AffiniPureGoat Anti-Mouse IgG(Jackson ImmunoResearch#115-005-205)，针对muMAb2，1：2,500稀释的FcγSubclass3专一性的AffiniPure Goat Anti-MouseIgG(JacksonImmunoResearch#115-005-209)，针对所有人源化抗体，1：2,500稀释的AffiniPure GoatAnti-Human IgG(H+L)(Jackson ImmunoResearch#109-005-088)。
培养盘-结合的二次抗体是使用1：6,000稀释的Peroxidase AffiniPure BovineAnti-Goat IgG(H+L)(Jackson ImmunoResearch#805-035-180)并如上所述来进行检测与显色。
实施例4：抗体基因的选殖
VH与VL抗体序列是选殖自融合瘤如下。总RNA是使用TRIZOL试剂(Invitrogen,cat#15596-026)并按照制造商的指示而分离自大约5-7x106个融合瘤细胞。第一股cDNA合成是使用Maxima Universal First Strand cDNA合成套组(Thermo Fisher cat.#K1661)以及6μg的总RNA与下列的基因专一性引子来进行：鼠IgG1 VH：TATGCAAGGCTTACAACCACA(序列辨识编号：45)、鼠IgG3 VH：GGGGGTACTGGGCTTGGGTAT(序列辨识编号：46)，以及鼠γVL：CTCATTCCTGTTGAAGCTCTTGAC(序列辨识编号：47)。
随着第一股cDNA合成，聚A序列(poly A sequence)借由末端转移酶附接至各个第一股cDNA的5’端。使所得到的5’端加尾的cDNA产物进行两轮的PCR扩增反应。第一轮PCR反应是使用Herculase II DNA聚合酶(Agilent Technologies cat#600679)以及下列引子来进行：5’GTGACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT(序列辨识编号：48)、针对鼠IgG1VH的3’引子：CTTCCGGAATTCCTCAATTTTCTTGTCCACCTTGGTGC(序列辨识编号：49)、针对鼠IgG3VH的3’引子：CTTCCGGAATTCCTCGATTCTCTTGATCAACTCAGTCT(序列辨识编号：50)，以及针对鼠γVL的3’引子：CTTCCGGAATTCCTCATTCCTGTTGAAGCTCTTGACAATGGG(序列辨识编号：51)。
第一轮PCR产物的一部分(1/20)是使用Phusion High Fidelity DNA聚合酶(Fisher Scientific cat#F530L)以及下列引子而在第二轮PCR中进一步扩增：5’GTGACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT(序列辨识编号：48)、针对鼠IgG1VH的3’引子：ATTAAGTCGACATAGACAGATGGGGGTGTCGTTTTGGC(序列辨识编号：52)、针对鼠IgG3VH的3’引子：ATTAAGTCGACAGGGACCAAGGGATAGACAGATGG(序列辨识编号：53)，以及针对鼠γVL的3’引子：CTACCTCGAGGGATACAGTTGGTGCAGCATC(序列辨识编号：54)。
所得到的PCR产物是使用一对限制酵素来进行消化：针对VH PCR片段为ClaI/SalI，而针对VLPCR片段为ClaI/XhoI，并且使用标准重组DNA操作程序而被插入至选殖载体中。为了鉴定全长的VH/VL选殖株，将含有插入物的选殖株以适当大小范围来进行定序。
实施例5：全长的鼠抗体表现载体的构筑
全长的鼠IgG重链与轻链表现质体是如下来进行构筑。
第一股cDNA合成是借由反转录酶(RT)并使用Maxima Universal First StrandcDNA合成套组(Thermo Fisher cat#K1661)与来自上面所鉴定的含有全长的VH与VL序列的融合瘤的6μg总RNA来进行。在个别的RT反应中采用寡dT引子(oligo dT primer)与随机六聚物引子(random hexamer primers)。将该寡dT与随机六聚物引子作为引子的RT产物合并，并且通过使用Phusion High Fidelity DNA聚合酶(Fisher Scientific cat#F530L)与下列引子对的一轮的PCR扩增反应，用来作为muMAb1与muMAb2的全长cDNA的来源，而该等引子对是使用上面所得到的序列信息来进行设计。
针对muMAb1与muMAb2γ轻链，5’引子CAGTCCGCGGCCACCATGGATTTTCAAGTGCAGATTTTC(序列辨识编号：55)，3’引子AGGAAGATCTAACACTCATTCCTGTTGAAGC(序列辨识编号：56)。针对muMAb1鼠IgG1重链，5’引子CAGTCCGCGGCCACCATGGCTGTCCTGGTGCTGTT(序列辨识编号：57)，3’引子CTGGACAGGGATCCAGAGTTCCA(序列辨识编号：58)。针对muMAb2鼠IgG3重链，5’引子CAGTCCGCGGCCACCATGGCTGTCCTGGTGCTGTT(序列辨识编号：57)，3’引子CATGAGATCTCATTTACCAGGGGAGCGAGA(序列辨识编号：59)。
将所扩增的muMAb1与muMAb2鼠γ轻链序列以及muMAb2鼠IgG3重链序列用SacII/BglII限制片段的形式直接选殖至哺乳动物表现质体中。该muMAb1鼠IgG1重链扩增产物呈现于基因编码序列的5’端，正好穿过对应于大约CH1区中央的内部BamHI位置。以SacII/BamHI限制片段的形式将它选殖至先前所构筑的全长鼠IgG1重链表现质体中。所有的选殖株是借由定序来进行验证。
对应于muMAb1与muMAb2的氨基酸序列的序列辨识编号显示在下面表1中。
表1.全长的重链与轻链单株抗体序列
mAb 重链 mAb 轻链
muMAb1 H 序列辨识编号：2 muMAb1 L 序列辨识编号：4
muMAb2 H 序列辨识编号：6 muMAb2 L 序列辨识编号：8
huMAb1 H 序列辨识编号：10 huMAb1 L 序列辨识编号：16
huMAb1 H1 序列辨识编号：12 huMAb1 L1 序列辨识编号：18
huMAb1 H2 序列辨识编号：14 huMAb1 L2 序列辨识编号：20
huMAb2 H 序列辨识编号：22 huMAb2 L 序列辨识编号：28
huMAb2 H1 序列辨识编号：24 huMAb2 L1 序列辨识编号：30
huMAb2 H2 序列辨识编号：26 huMAb2 L2 序列辨识编号：32
实施例6：人源化
为了使muMAb1与muMAb2人源化，与鼠抗体序列之间具有最高同源度的人类免疫球蛋白生殖腺序列是借由将鼠序列与人类免疫球蛋白基因数据库进行比对来鉴定。结果显示与muMAb1以及muMAb2的重链这两者最相近的人类生殖腺序列是huIGHV4-59x01，而与muMAb1以及muMAb2这两者最相近的轻链是huIGKV3-11x01。
为了使muMAb1与muMAb2人源化，使CDR区移植至人类序列分别产生huMAb1与huMAb2。对应在huMAb1与huMAb2重链与轻链的氨基酸序列的序列辨识编号显示在上面表1中。
在huMAb1与huMAb2序列中的突变是在计算机模拟(in silico)下来设计成最大程度的人源化并且提供CDR环的结构性支持。在重链与轻链序列中的氨基酸改变与其位置显示在下面表2-5以及图1A、1B、2A与2B中。
表2.在huMAb1重链序列中的突变
Figure BDA0002693516720000091
Figure BDA0002693516720000101
表3.在huMAb1轻链序列中的突变
残基 L1 L2
Ile 2 Asn Asn
Leu 47 Trp
Ser 65 Arg
Thr 69 Asn
Phe 71 Tyr Tyr
表4.在huMAb2重链序列中的突变
残基 H1 H2
Ile 37 Val
Ala 62 Ser
Val 67 Leu
Val 71 Lys Lys
Thr 73 Asn
Asn 76 Ser
Phe 78 Val Val
Arg 97 Lys Lys
表5.在huMAb2轻链序列中的突变
殘基 L1 L2
Ile 2 Asn Asn
Leu 47 Trp
Thr 69 Asn Asn
Phe 71 Tyr Tyr
对应于由突变的全长重链与轻链基因所编码的氨基酸序列的序列辨识编号显示在上面表1中。
上述抗体的所有CDR序列的序列辨识编号显示在下面表6中。
表6.对应于CDR区的序列辨识编号
CDR muMAb1/hMAb1 muMAb2/hMAb2
HCDR1 序列辨识编号：33 序列辨识编号：40
HCDR2 序列辨识编号：34 序列辨识编号：34
HCDR3 序列辨识编号：35 序列辨识编号：41
HCDR2 H1<sup>a</sup> 序列辨识编号：39 序列辨识编号：39
LCDR1 序列辨识编号：36 序列辨识编号：42
LCDR2 序列辨识编号：37 序列辨识编号：43
LCDR3 序列辨识编号：38 序列辨识编号：44
实施例7：人源化抗体表现载体的构筑
全长人源化重链与轻链表现质体的构筑是使用标准的重组DNA技术来进行。将片段适当地选殖至含有下列的建构物中：(i)全长的人类IgG1重链，其带有导入的缄默突变以产生NheI限制位置在CH1区的起始处，以及(ii)全长的人类κ轻链，其带有导入的缄默突变以产生BsiWI限制位址于CL恒定区的起始处。
使用标准技术来合成具有以上面所讨论的突变变异型为基础的序列的DNA片段并且选殖至适当的表现载体中。
实施例8：通过瞬时转染于293T细胞中来生产重组型抗体
将人类293T胚胎肾脏上皮细胞株维持于补充有10％的热失活的胎牛血清、2mML-麸酰胺酸以及青霉素/链霉素(GIBCO cat#15140)的DMEM(HyClonecat#SH30243.02)中。通过聚(乙烯亚胺)(PEI)媒介的转染将表现建构物导入细胞中。简言之，DNA混合物是借由将重链与轻链表现质体以1:1的比例合并来制备。接而以1.5:1的PEI对DNA的比例来形成PEI/DNA错合物，并且在培育历时20分钟之后，添加至所培育的细胞中。在转染两天之后，将培养基移除并且置换新鲜的培养基。在转染之后的第五天收取上清液来进行分析。
重组型抗体的大规模生产是借由将重链与轻链表现质体转染至如上所述的人类293T细胞中来进行。在三天之后，收取经转染的细胞并接种至摇瓶中补充有6mML-麸酰胺酸的无血清培养基(HyClone CDM4HEK293 cat#SH3085802)。监控细胞生长与抗体生产并且在细胞可活性降到50％以下时收取培养上清液来供抗体纯化。
不同组合的重链与轻链的表现是借由共转染如上所述的重链与轻链的表现质体来进行。这些重组型抗体的亲和力显示于下面表7中。
表7.重组型抗体的结合亲合力表
抗-SSEA4 Ab 重链 轻链 K<sub>d</sub>(nM) 指数<sup>a</sup>
muMAb1 muMAb1 H muMAb1 L 0.17 0.9
muMAb2 muMAb2 H muMAb2 L 0.19 1.1
huMAb1 huMAb1 H huMAb1 L N.D.<sup>b</sup> -<sup>c</sup>
huMAb1a huMAb1 H2 huMAb1 L1 4.3 23.9
huMAb1b huMAb1 H2 huMAb1 L2 4.7 26.1
huMAb2 huMAb2 H huMAb2 L N.D.<sup>b</sup> -<sup>c</sup>
huMAb2a huMAb2 H2 huMAb2 L2 5.7 31.7
huMAb3 huMAb1 H2 huMAb2 L 0.09 0.5
huMAb4 huMAb1 H2 huMAb2 L1 0.10 0.6
huMAb5 huMAb1 H2 huMAb2 L2 0.14 0.8
a指数是借由使用muMAb1以及muMAb2的平均Kd(0.18)来将Kd进行标准化而得到。
b未侦测到结合
c未测定
两个重组型人源化抗体(亦即huMAb3与huMAb4)的结合亲合力也是借由使用标准操作程序的表面电浆共振分析来进行测定。huMAb3的Kd值是介于50nM与90nM之间。huMAb4的Kd值是大约0.4μM。
无论借由ELISA或借由表面电浆共振，结果都显示huMAb3对于SSEA4具有高亲和力。
其他具体例
在本说明书中所揭示的所有特征可以任何的组合来结合。在此说明书中所揭示的各个特征可置换为提供相同、等效或相似目的的替代特征。除非另外明确指出，所揭示的各个特征仅为同类系列的等效或相似特征的一个实例。
通过上面说明，熟习此技术者可轻易地确定本发明的必要特征，并且可在没有背离其精神与范畴下作出本发明的各种不同的变化与修饰而使其适合于各种不同的使用与情况。因此，其他具体例也落在本申请专利范围的范畴内。
序列表
<110> 美国糖基生医股份有限公司
<120> 结合至SSEA4的单株抗体及其用途
<130> 218498-0001PCT
<160> 61
<170> PatentIn version 3.5
<210> 1
<211> 1389
<212> DNA
<213> Mus musculus
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<223> heavy chain
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gtc ctg tcc cag gtg cag ctg aag gag tca gga cct ggc ctg gtg gcg 96
Val Leu Ser Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala
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Glu Trp Leu Gly Val Ile Trp Gly Gly Gly Ser Thr Asn Tyr Asn Ser
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Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro
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Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val
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ccc tcc agc acc tgg ccc agc gag acc gtc acc tgc aac gtt gcc cac 672
Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His
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Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe
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Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro
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aag gtc acg tgt gtt gtg gta gac atc agc aag gat gat ccc gag gtc 864
Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val
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cag ttc agc tgg ttt gta gat gat gtg gag gtg cac aca gct cag acg 912
Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr
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caa ccc cgg gag gag cag ttc aac agc act ttc cgc tca gtc agt gaa 960
Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu
305 310 315 320
ctt ccc atc atg cac cag gac tgg ctc aat ggc aag gag ttc aaa tgc 1008
Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys
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agg gtc aac agt gca gct ttc cct gcc ccc atc gag aaa acc atc tcc 1056
Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser
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Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro
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Val Leu Ser Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala
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Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu
35 40 45
Thr Ser Tyr Gly Val Asp Trp Val Arg Gln Pro Pro Gly Lys Gly Leu
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Glu Trp Leu Gly Val Ile Trp Gly Gly Gly Ser Thr Asn Tyr Asn Ser
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Ala Leu Met Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln
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Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr
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Tyr Cys Ala Lys His Glu Val Leu Arg Gly Tyr Ala Leu Asp Tyr Trp
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Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro
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Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met
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Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr
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Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro
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Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val
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Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His
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Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys
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Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe
245 250 255
Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro
260 265 270
Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val
275 280 285
Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr
290 295 300
Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu
305 310 315 320
Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys
325 330 335
Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser
340 345 350
Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro
355 360 365
Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile
370 375 380
Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly
385 390 395 400
Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp
405 410 415
Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp
420 425 430
Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His
435 440 445
Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys
450 455 460
<210> 3
<211> 708
<212> DNA
<213> Mus musculus
<220>
<221> CDS
<222> (1)..(708)
<223> light chain
<400> 3
atg gat ttt caa gtg cag att ttc agc ttc ctg cta atc agt gcc tca 48
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
gtc ata atg tcc aga gga gaa aat gtt ctc acc cag tct cca gca atc 96
Val Ile Met Ser Arg Gly Glu Asn Val Leu Thr Gln Ser Pro Ala Ile
20 25 30
atg tct gca tct cca ggg gaa aag gtc acc atg acc tgc agt gcc agc 144
Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser
35 40 45
tca agt gta agt tac atg cac tgg tac cag cag aag tca aac acc tcc 192
Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Asn Thr Ser
50 55 60
ccc aaa ctc tgg att tat gac aca tcc aaa ctg gct tct gga gtc cca 240
Pro Lys Leu Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro
65 70 75 80
ggt cgc ttc agt ggc agg ggg tct gga aac tct tat tct ctc acg atc 288
Gly Arg Phe Ser Gly Arg Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile
85 90 95
agc agc atg gag gct gaa gat gtt gcc act tat tac tgt ttt cag ggg 336
Ser Ser Met Glu Ala Glu Asp Val Ala Thr Tyr Tyr Cys Phe Gln Gly
100 105 110
agt ggg tac cca ctc acg ttc ggc tcg ggg aca aag ttg gaa ata aaa 384
Ser Gly Tyr Pro Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
cgg gct gat gct gca cca act gta tcc atc ttc cca cca tcc agt gag 432
Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu
130 135 140
cag tta aca tct gga ggt gcc tca gtc gtg tgc ttc ttg aac aac ttc 480
Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
145 150 155 160
tac ccc aaa gac atc aat gtc aag tgg aag att gat ggc agt gaa cga 528
Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg
165 170 175
caa aat ggc gtc ctg aac agt tgg act gat cag gac agc aaa gac agc 576
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
180 185 190
acc tac agc atg agc agc acc ctc acg ttg acc aag gac gag tat gaa 624
Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
195 200 205
cga cat aac agc tat acc tgt gag gcc act cac aag aca tca act tca 672
Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser
210 215 220
ccc att gtc aag agc ttc aac agg aat gag tgt tag 708
Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
225 230 235
<210> 4
<211> 235
<212> PRT
<213> Mus musculus
<400> 4
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Glu Asn Val Leu Thr Gln Ser Pro Ala Ile
20 25 30
Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Asn Thr Ser
50 55 60
Pro Lys Leu Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro
65 70 75 80
Gly Arg Phe Ser Gly Arg Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile
85 90 95
Ser Ser Met Glu Ala Glu Asp Val Ala Thr Tyr Tyr Cys Phe Gln Gly
100 105 110
Ser Gly Tyr Pro Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu
130 135 140
Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
145 150 155 160
Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg
165 170 175
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
180 185 190
Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
195 200 205
Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser
210 215 220
Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
225 230 235
<210> 5
<211> 1410
<212> DNA
<213> Mus musculus
<220>
<221> CDS
<222> (1)..(1410)
<223> heavy chain
<400> 5
atg gct gtc ctg gtg ctg ttc ctc tgc ctg gtt gca ttt cca agc tgt 48
Met Ala Val Leu Val Leu Phe Leu Cys Leu Val Ala Phe Pro Ser Cys
1 5 10 15
gtc ctg acc cag gtg cag ctg aag gag tca gga cct ggc ctg gtg gcg 96
Val Leu Thr Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala
20 25 30
ccc tca cag agc ctg tcc atc act tgc act gtc tct ggg ttt tca tta 144
Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu
35 40 45
gcc agc tat ggt gta gac tgg gtt cgc cag cct cca gga aag ggt ctg 192
Ala Ser Tyr Gly Val Asp Trp Val Arg Gln Pro Pro Gly Lys Gly Leu
50 55 60
gag tgg ctg gga gta ata tgg ggt ggt gga agt aca aat tat aat tca 240
Glu Trp Leu Gly Val Ile Trp Gly Gly Gly Ser Thr Asn Tyr Asn Ser
65 70 75 80
gct ctc atg tcc aga ctt acc atc aac aaa gac aac tcc aag agc caa 288
Ala Leu Met Ser Arg Leu Thr Ile Asn Lys Asp Asn Ser Lys Ser Gln
85 90 95
ctt ttc tta aaa atg aac agt ctg caa act gat gac aca gcc atg tat 336
Leu Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr
100 105 110
tac tgt gcc aaa cat gga gat agt aat tcc ggt tat gct atg gac tac 384
Tyr Cys Ala Lys His Gly Asp Ser Asn Ser Gly Tyr Ala Met Asp Tyr
115 120 125
tgg ggt caa gga atc tca gtc acc gtc tcc tca gct aca aca aca gcc 432
Trp Gly Gln Gly Ile Ser Val Thr Val Ser Ser Ala Thr Thr Thr Ala
130 135 140
cca tct gtc tat ccc ttg gtc cct ggc tgc ggt gac aca tct gga tcc 480
Pro Ser Val Tyr Pro Leu Val Pro Gly Cys Gly Asp Thr Ser Gly Ser
145 150 155 160
tcg gtg aca ctg gga tgc ctt gtc aaa ggc tac ttc cct gag ccg gta 528
Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val
165 170 175
act gta aaa tgg aac tat gga gcc ctg tcc agc ggt gtg cgc aca gtc 576
Thr Val Lys Trp Asn Tyr Gly Ala Leu Ser Ser Gly Val Arg Thr Val
180 185 190
tca tct gta ctg cag tct ggg ttc tat tcc ctc agc agc ttg gtg act 624
Ser Ser Val Leu Gln Ser Gly Phe Tyr Ser Leu Ser Ser Leu Val Thr
195 200 205
gta ccc tcc agc acc tgg ccc agc cag act gtc atc tgc aac gta gcc 672
Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val Ile Cys Asn Val Ala
210 215 220
cac cca gcc agc aag act gag ttg atc aag aga atc gag cct aga ata 720
His Pro Ala Ser Lys Thr Glu Leu Ile Lys Arg Ile Glu Pro Arg Ile
225 230 235 240
ccc aag ccc agt acc ccc cca ggt tct tca tgc cca cct ggt aac atc 768
Pro Lys Pro Ser Thr Pro Pro Gly Ser Ser Cys Pro Pro Gly Asn Ile
245 250 255
ttg ggt gga cca tcc gtc ttc atc ttc ccc cca aag ccc aag gat gca 816
Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Ala
260 265 270
ctc atg atc tcc cta acc ccc aag gtt acg tgt gtg gtg gtg gat gtg 864
Leu Met Ile Ser Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Val
275 280 285
agc gag gat gac cca gat gtc cat gtc agc tgg ttt gtg gac aac aaa 912
Ser Glu Asp Asp Pro Asp Val His Val Ser Trp Phe Val Asp Asn Lys
290 295 300
gaa gta cac aca gcc tgg acg cag ccc cgt gaa gct cag tac aac agt 960
Glu Val His Thr Ala Trp Thr Gln Pro Arg Glu Ala Gln Tyr Asn Ser
305 310 315 320
acc ttc cga gtg gtc agt gcc ctc ccc atc cag cac cag gac tgg atg 1008
Thr Phe Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met
325 330 335
agg ggc aag gag ttc aaa tgc aag gtc aac aac aaa gcc ctc cca gcc 1056
Arg Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Ala Leu Pro Ala
340 345 350
ccc atc gag aga acc atc tca aaa ccc aaa gga aga gcc cag aca cct 1104
Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Arg Ala Gln Thr Pro
355 360 365
caa gta tac acc ata ccc cca cct cgt gaa caa atg tcc aag aag aag 1152
Gln Val Tyr Thr Ile Pro Pro Pro Arg Glu Gln Met Ser Lys Lys Lys
370 375 380
gtt agt ctg acc tgc ctg gtc acc aac ttc ttc tct gaa gcc atc agt 1200
Val Ser Leu Thr Cys Leu Val Thr Asn Phe Phe Ser Glu Ala Ile Ser
385 390 395 400
gtg gag tgg gaa agg aac gga gaa ctg gag cag gat tac aag aac act 1248
Val Glu Trp Glu Arg Asn Gly Glu Leu Glu Gln Asp Tyr Lys Asn Thr
405 410 415
cca ccc atc ctg gac tcg gat ggg acc tac ttc ctc tac agc aag ctc 1296
Pro Pro Ile Leu Asp Ser Asp Gly Thr Tyr Phe Leu Tyr Ser Lys Leu
420 425 430
act gtg gat aca gac agt tgg ttg caa gga gaa att ttt acc tgc tcc 1344
Thr Val Asp Thr Asp Ser Trp Leu Gln Gly Glu Ile Phe Thr Cys Ser
435 440 445
gtg gtg cat gag gct ctc cat aac cac cac aca cag aag aac ctg tct 1392
Val Val His Glu Ala Leu His Asn His His Thr Gln Lys Asn Leu Ser
450 455 460
cgc tcc cct ggt aaa tga 1410
Arg Ser Pro Gly Lys
465
<210> 6
<211> 469
<212> PRT
<213> Mus musculus
<400> 6
Met Ala Val Leu Val Leu Phe Leu Cys Leu Val Ala Phe Pro Ser Cys
1 5 10 15
Val Leu Thr Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala
20 25 30
Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu
35 40 45
Ala Ser Tyr Gly Val Asp Trp Val Arg Gln Pro Pro Gly Lys Gly Leu
50 55 60
Glu Trp Leu Gly Val Ile Trp Gly Gly Gly Ser Thr Asn Tyr Asn Ser
65 70 75 80
Ala Leu Met Ser Arg Leu Thr Ile Asn Lys Asp Asn Ser Lys Ser Gln
85 90 95
Leu Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr
100 105 110
Tyr Cys Ala Lys His Gly Asp Ser Asn Ser Gly Tyr Ala Met Asp Tyr
115 120 125
Trp Gly Gln Gly Ile Ser Val Thr Val Ser Ser Ala Thr Thr Thr Ala
130 135 140
Pro Ser Val Tyr Pro Leu Val Pro Gly Cys Gly Asp Thr Ser Gly Ser
145 150 155 160
Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val
165 170 175
Thr Val Lys Trp Asn Tyr Gly Ala Leu Ser Ser Gly Val Arg Thr Val
180 185 190
Ser Ser Val Leu Gln Ser Gly Phe Tyr Ser Leu Ser Ser Leu Val Thr
195 200 205
Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val Ile Cys Asn Val Ala
210 215 220
His Pro Ala Ser Lys Thr Glu Leu Ile Lys Arg Ile Glu Pro Arg Ile
225 230 235 240
Pro Lys Pro Ser Thr Pro Pro Gly Ser Ser Cys Pro Pro Gly Asn Ile
245 250 255
Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Ala
260 265 270
Leu Met Ile Ser Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Val
275 280 285
Ser Glu Asp Asp Pro Asp Val His Val Ser Trp Phe Val Asp Asn Lys
290 295 300
Glu Val His Thr Ala Trp Thr Gln Pro Arg Glu Ala Gln Tyr Asn Ser
305 310 315 320
Thr Phe Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met
325 330 335
Arg Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Ala Leu Pro Ala
340 345 350
Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Arg Ala Gln Thr Pro
355 360 365
Gln Val Tyr Thr Ile Pro Pro Pro Arg Glu Gln Met Ser Lys Lys Lys
370 375 380
Val Ser Leu Thr Cys Leu Val Thr Asn Phe Phe Ser Glu Ala Ile Ser
385 390 395 400
Val Glu Trp Glu Arg Asn Gly Glu Leu Glu Gln Asp Tyr Lys Asn Thr
405 410 415
Pro Pro Ile Leu Asp Ser Asp Gly Thr Tyr Phe Leu Tyr Ser Lys Leu
420 425 430
Thr Val Asp Thr Asp Ser Trp Leu Gln Gly Glu Ile Phe Thr Cys Ser
435 440 445
Val Val His Glu Ala Leu His Asn His His Thr Gln Lys Asn Leu Ser
450 455 460
Arg Ser Pro Gly Lys
465
<210> 7
<211> 708
<212> DNA
<213> Mus musculus
<220>
<221> CDS
<222> (1)..(708)
<223> light chain
<400> 7
atg gat ttt caa gtg cag att ttc agc ttc ctg cta atc agt gcc tca 48
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
gtc ata atg tcc aga gga gaa aat gtt ctc acc cag tct cca gca atc 96
Val Ile Met Ser Arg Gly Glu Asn Val Leu Thr Gln Ser Pro Ala Ile
20 25 30
atg tct gca tct cca ggg gaa aag gtc acc atg acc tgc agt gcc agc 144
Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser
35 40 45
cca agt gta agt tac atg cac tgg tac cag cag aag tca agc acc tcc 192
Pro Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Ser Thr Ser
50 55 60
ccc aaa ctc tgg att tat gac aca tac aaa ctg gct tct gga gtc cca 240
Pro Lys Leu Trp Ile Tyr Asp Thr Tyr Lys Leu Ala Ser Gly Val Pro
65 70 75 80
ggt cgc ttc agt ggc agt ggg tct gga aac tct tac tct ctc acg atc 288
Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile
85 90 95
aga acc atg gag gct gaa gat gtt gcc act tat tac tgt ttt cag ggg 336
Arg Thr Met Glu Ala Glu Asp Val Ala Thr Tyr Tyr Cys Phe Gln Gly
100 105 110
agt ggg ttc cca ctc acg ttc ggc tcg ggg aca aag ttg gaa ata aaa 384
Ser Gly Phe Pro Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
cgg gct gat gct gca cca act gta tcc atc ttc cca cca tcc agt gag 432
Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu
130 135 140
cag tta aca tct gga ggt gcc tca gtc gtg tgc ttc ttg aac aac ttc 480
Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
145 150 155 160
tac ccc aaa gac atc aat gtc aag tgg aag att gat ggc agt gaa cga 528
Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg
165 170 175
caa aat ggc gtc ctg aac agt tgg act gat cag gac agc aaa gac agc 576
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
180 185 190
acc tac agc atg agc agc acc ctc acg ttg acc aag gac gag tat gaa 624
Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
195 200 205
cga cat aac agc tat acc tgt gag gcc act cac aag aca tca act tca 672
Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser
210 215 220
ccc att gtc aag agc ttc aac agg aat gag tgt tag 708
Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
225 230 235
<210> 8
<211> 235
<212> PRT
<213> Mus musculus
<400> 8
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Glu Asn Val Leu Thr Gln Ser Pro Ala Ile
20 25 30
Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser
35 40 45
Pro Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Ser Thr Ser
50 55 60
Pro Lys Leu Trp Ile Tyr Asp Thr Tyr Lys Leu Ala Ser Gly Val Pro
65 70 75 80
Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile
85 90 95
Arg Thr Met Glu Ala Glu Asp Val Ala Thr Tyr Tyr Cys Phe Gln Gly
100 105 110
Ser Gly Phe Pro Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu
130 135 140
Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
145 150 155 160
Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg
165 170 175
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
180 185 190
Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
195 200 205
Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser
210 215 220
Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
225 230 235
<210> 9
<211> 1407
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic polynucleotide
<220>
<221> CDS
<222> (1)..(1407)
<223> humanized heavy chain
<400> 9
atg gct gtc ctg gtg ctg ttc ctc tgc ctg gtt gca ttt cca agc tgt 48
Met Ala Val Leu Val Leu Phe Leu Cys Leu Val Ala Phe Pro Ser Cys
1 5 10 15
gtc ctg tcc cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag 96
Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
20 25 30
cct tcg gag acc ctg tcc ctc acc tgc act gtc tct ggg ttt tca tta 144
Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu
35 40 45
acc agc tat ggt gta gac tgg atc cgg cag ccc cca ggg aag gga ctg 192
Thr Ser Tyr Gly Val Asp Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu
50 55 60
gag tgg att ggg gta ata tgg ggt ggt gga agc aca aat tat aat tca 240
Glu Trp Ile Gly Val Ile Trp Gly Gly Gly Ser Thr Asn Tyr Asn Ser
65 70 75 80
gct ctc atg tcc cga gtc acc ata tca gta gac acg tcc aag aac cag 288
Ala Leu Met Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln
85 90 95
ttc tcc ctg aag ctg agc tct gtg acc gct gcg gac acg gcc gtg tat 336
Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
100 105 110
tac tgt gcg aga cat gag gta cta cgg ggg tat gct ctg gac tac tgg 384
Tyr Cys Ala Arg His Glu Val Leu Arg Gly Tyr Ala Leu Asp Tyr Trp
115 120 125
ggc caa ggt acc ctg gtc acc gtc tcg agt gct agc acc aag ggc cca 432
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
130 135 140
tcg gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc aca 480
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
145 150 155 160
gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg acg 528
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
165 170 175
gtg tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccg 576
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
180 185 190
gct gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc 624
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
195 200 205
gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat 672
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
210 215 220
cac aag ccc agc aac acc aag gtg gac aag aga gtt gag ccc aaa tct 720
His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser
225 230 235 240
tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc ctg 768
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
245 250 255
ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc 816
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
260 265 270
atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc 864
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
275 280 285
cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag 912
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
290 295 300
gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg 960
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
305 310 315 320
tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat 1008
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
325 330 335
ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc 1056
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
340 345 350
atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag 1104
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
355 360 365
gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc 1152
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
370 375 380
agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg 1200
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
385 390 395 400
gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct 1248
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
405 410 415
ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc 1296
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
420 425 430
gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg 1344
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
435 440 445
atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg 1392
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
450 455 460
tct ccg ggt aaa tga 1407
Ser Pro Gly Lys
465
<210> 10
<211> 468
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 10
Met Ala Val Leu Val Leu Phe Leu Cys Leu Val Ala Phe Pro Ser Cys
1 5 10 15
Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
20 25 30
Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu
35 40 45
Thr Ser Tyr Gly Val Asp Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu
50 55 60
Glu Trp Ile Gly Val Ile Trp Gly Gly Gly Ser Thr Asn Tyr Asn Ser
65 70 75 80
Ala Leu Met Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln
85 90 95
Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
100 105 110
Tyr Cys Ala Arg His Glu Val Leu Arg Gly Tyr Ala Leu Asp Tyr Trp
115 120 125
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
130 135 140
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
145 150 155 160
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
165 170 175
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
180 185 190
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
195 200 205
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
210 215 220
His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser
225 230 235 240
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
245 250 255
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
260 265 270
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
275 280 285
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
290 295 300
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
305 310 315 320
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
325 330 335
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
340 345 350
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
355 360 365
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
370 375 380
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
385 390 395 400
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
405 410 415
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
420 425 430
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
435 440 445
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
450 455 460
Ser Pro Gly Lys
465
<210> 11
<211> 1407
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic polynucleotide
<220>
<221> CDS
<222> (1)..(1407)
<223> humanized heavy chain
<400> 11
atg gct gtc ctg gtg ctg ttc ctc tgc ctg gtt gca ttt cca agc tgt 48
Met Ala Val Leu Val Leu Phe Leu Cys Leu Val Ala Phe Pro Ser Cys
1 5 10 15
gtc ctg tcc cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag 96
Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
20 25 30
cct tcg gag acc ctg tcc ctc acc tgc act gtc tct ggg ttt tca tta 144
Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu
35 40 45
acc agc tat ggt gta gac tgg atc cgg cag ccc cca ggg aag gga ctg 192
Thr Ser Tyr Gly Val Asp Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu
50 55 60
gag tgg att ggg gta ata tgg ggt ggt gga agc aca aat tat aat tca 240
Glu Trp Ile Gly Val Ile Trp Gly Gly Gly Ser Thr Asn Tyr Asn Ser
65 70 75 80
tcc ctc atg tcc cga gtc acc ata tca aaa gac acg tcc aag aac cag 288
Ser Leu Met Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln
85 90 95
gtt tcc ctg aag ctg agc tct gtg acc gct gcg gac acg gcc gtg tat 336
Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
100 105 110
tac tgt gcg aaa cat gag gta cta cgg ggg tat gct ctg gac tac tgg 384
Tyr Cys Ala Lys His Glu Val Leu Arg Gly Tyr Ala Leu Asp Tyr Trp
115 120 125
ggc caa ggt acc ctg gtc acc gtc tcg agt gct agc acc aag ggc cca 432
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
130 135 140
tcg gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc aca 480
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
145 150 155 160
gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg acg 528
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
165 170 175
gtg tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccg 576
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
180 185 190
gct gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc 624
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
195 200 205
gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat 672
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
210 215 220
cac aag ccc agc aac acc aag gtg gac aag aga gtt gag ccc aaa tct 720
His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser
225 230 235 240
tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc ctg 768
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
245 250 255
ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc 816
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
260 265 270
atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc 864
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
275 280 285
cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag 912
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
290 295 300
gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg 960
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
305 310 315 320
tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat 1008
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
325 330 335
ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc 1056
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
340 345 350
atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag 1104
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
355 360 365
gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc 1152
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
370 375 380
agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg 1200
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
385 390 395 400
gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct 1248
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
405 410 415
ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc 1296
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
420 425 430
gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg 1344
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
435 440 445
atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg 1392
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
450 455 460
tct ccg ggt aaa tga 1407
Ser Pro Gly Lys
465
<210> 12
<211> 468
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 12
Met Ala Val Leu Val Leu Phe Leu Cys Leu Val Ala Phe Pro Ser Cys
1 5 10 15
Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
20 25 30
Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu
35 40 45
Thr Ser Tyr Gly Val Asp Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu
50 55 60
Glu Trp Ile Gly Val Ile Trp Gly Gly Gly Ser Thr Asn Tyr Asn Ser
65 70 75 80
Ser Leu Met Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln
85 90 95
Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
100 105 110
Tyr Cys Ala Lys His Glu Val Leu Arg Gly Tyr Ala Leu Asp Tyr Trp
115 120 125
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
130 135 140
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
145 150 155 160
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
165 170 175
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
180 185 190
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
195 200 205
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
210 215 220
His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser
225 230 235 240
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
245 250 255
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
260 265 270
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
275 280 285
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
290 295 300
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
305 310 315 320
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
325 330 335
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
340 345 350
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
355 360 365
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
370 375 380
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
385 390 395 400
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
405 410 415
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
420 425 430
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
435 440 445
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
450 455 460
Ser Pro Gly Lys
465
<210> 13
<211> 1407
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic polynucleotide
<220>
<221> CDS
<222> (1)..(1407)
<223> humanized heavy chain
<400> 13
atg gct gtc ctg gtg ctg ttc ctc tgc ctg gtt gca ttt cca agc tgt 48
Met Ala Val Leu Val Leu Phe Leu Cys Leu Val Ala Phe Pro Ser Cys
1 5 10 15
gtc ctg tcc cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag 96
Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
20 25 30
cct tcg gag acc ctg tcc ctc acc tgc act gtc tct ggg ttt tca tta 144
Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu
35 40 45
acc agc tat ggt gta gac tgg gtt cgg cag ccc cca ggg aag gga ctg 192
Thr Ser Tyr Gly Val Asp Trp Val Arg Gln Pro Pro Gly Lys Gly Leu
50 55 60
gag tgg att ggg gta ata tgg ggt ggt gga agc aca aat tat aat tca 240
Glu Trp Ile Gly Val Ile Trp Gly Gly Gly Ser Thr Asn Tyr Asn Ser
65 70 75 80
gct ctc atg tcc cga ctg acc ata tca aaa gac aac tcc aag agc cag 288
Ala Leu Met Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln
85 90 95
gtt tcc ctg aag ctg agc tct gtg acc gct gcg gac acg gcc gtg tat 336
Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
100 105 110
tac tgt gcg aaa cat gag gta cta cgg ggg tat gct ctg gac tac tgg 384
Tyr Cys Ala Lys His Glu Val Leu Arg Gly Tyr Ala Leu Asp Tyr Trp
115 120 125
ggc caa ggt acc ctg gtc acc gtc tcg agt gct agc acc aag ggc cca 432
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
130 135 140
tcg gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc aca 480
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
145 150 155 160
gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg acg 528
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
165 170 175
gtg tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccg 576
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
180 185 190
gct gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc 624
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
195 200 205
gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat 672
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
210 215 220
cac aag ccc agc aac acc aag gtg gac aag aga gtt gag ccc aaa tct 720
His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser
225 230 235 240
tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc ctg 768
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
245 250 255
ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc 816
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
260 265 270
atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc 864
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
275 280 285
cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag 912
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
290 295 300
gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg 960
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
305 310 315 320
tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat 1008
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
325 330 335
ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc 1056
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
340 345 350
atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag 1104
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
355 360 365
gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc 1152
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
370 375 380
agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg 1200
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
385 390 395 400
gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct 1248
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
405 410 415
ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc 1296
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
420 425 430
gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg 1344
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
435 440 445
atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg 1392
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
450 455 460
tct ccg ggt aaa tga 1407
Ser Pro Gly Lys
465
<210> 14
<211> 468
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 14
Met Ala Val Leu Val Leu Phe Leu Cys Leu Val Ala Phe Pro Ser Cys
1 5 10 15
Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
20 25 30
Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu
35 40 45
Thr Ser Tyr Gly Val Asp Trp Val Arg Gln Pro Pro Gly Lys Gly Leu
50 55 60
Glu Trp Ile Gly Val Ile Trp Gly Gly Gly Ser Thr Asn Tyr Asn Ser
65 70 75 80
Ala Leu Met Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln
85 90 95
Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
100 105 110
Tyr Cys Ala Lys His Glu Val Leu Arg Gly Tyr Ala Leu Asp Tyr Trp
115 120 125
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
130 135 140
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
145 150 155 160
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
165 170 175
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
180 185 190
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
195 200 205
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
210 215 220
His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser
225 230 235 240
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
245 250 255
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
260 265 270
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
275 280 285
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
290 295 300
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
305 310 315 320
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
325 330 335
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
340 345 350
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
355 360 365
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
370 375 380
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
385 390 395 400
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
405 410 415
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
420 425 430
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
435 440 445
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
450 455 460
Ser Pro Gly Lys
465
<210> 15
<211> 708
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic polynucleotide
<220>
<221> CDS
<222> (1)..(708)
<223> humanized light chain
<400> 15
atg gat ttt caa gtg cag att ttc agc ttc ctg cta atc agt gcc tca 48
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
gtc ata atg tcc aga gga gaa att gtg ttg aca cag tct cca gcc acc 96
Val Ile Met Ser Arg Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
20 25 30
ctg tct ttg tct cca ggg gaa aga gcc acc ctc tcc tgc agt gcc agc 144
Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser
35 40 45
tca agt gta agt tac atg cac tgg tac caa cag aaa cct ggc cag gct 192
Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala
50 55 60
ccc agg ctc ctc atc tat gac aca tcc aaa ctg gct tct ggc atc cca 240
Pro Arg Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro
65 70 75 80
gcc agg ttc agt ggc agt ggg tct ggg aca gac ttc act ctc acc atc 288
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
85 90 95
agc agc cta gag cct gaa gat ttt gca gtt tat tac tgt ttt cag ggg 336
Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly
100 105 110
agt ggg tac cca ctc acg ttt ggc cag ggg acc aag gtg gaa atc aaa 384
Ser Gly Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
115 120 125
cgt acg gtg gct gca cca tct gtc ttc atc ttc ccg cca tct gat gag 432
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
130 135 140
cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg ctg aat aac ttc 480
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
145 150 155 160
tat ccc aga gag gcc aaa gta cag tgg aag gtg gat aac gcc ctc caa 528
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
165 170 175
tcg ggt aac tcc cag gag agt gtc aca gag cag gac agc aag gac agc 576
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
180 185 190
acc tac agc ctc agc agc acc ctg acg ctg agc aaa gca gac tac gag 624
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
195 200 205
aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg 672
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
210 215 220
ccc gtc aca aag agc ttc aac agg gga gag tgt tag 708
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235
<210> 16
<211> 235
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 16
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
20 25 30
Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala
50 55 60
Pro Arg Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
85 90 95
Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly
100 105 110
Ser Gly Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
115 120 125
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
130 135 140
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
145 150 155 160
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
165 170 175
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
180 185 190
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
195 200 205
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
210 215 220
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235
<210> 17
<211> 708
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic polynucleotide
<220>
<221> CDS
<222> (1)..(708)
<223> humanized light chain
<400> 17
atg gat ttt caa gtg cag att ttc agc ttc ctg cta atc agt gcc tca 48
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
gtc ata atg tcc aga gga gaa aat gtg ttg aca cag tct cca gcc acc 96
Val Ile Met Ser Arg Gly Glu Asn Val Leu Thr Gln Ser Pro Ala Thr
20 25 30
ctg tct ttg tct cca ggg gaa aga gcc acc ctc tcc tgc agt gcc agc 144
Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser
35 40 45
tca agt gta agt tac atg cac tgg tac caa cag aaa cct ggc cag gct 192
Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala
50 55 60
ccc agg ctc ctc atc tat gac aca tcc aaa ctg gct tct ggc atc cca 240
Pro Arg Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro
65 70 75 80
gcc agg ttc agt ggc agt ggg tct ggg aac gac tat act ctc acc atc 288
Ala Arg Phe Ser Gly Ser Gly Ser Gly Asn Asp Tyr Thr Leu Thr Ile
85 90 95
agc agc cta gag cct gaa gat ttt gca gtt tat tac tgt ttt cag ggg 336
Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly
100 105 110
agt ggg tac cca ctc acg ttt ggc cag ggg acc aag gtg gaa atc aaa 384
Ser Gly Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
115 120 125
cgt acg gtg gct gca cca tct gtc ttc atc ttc ccg cca tct gat gag 432
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
130 135 140
cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg ctg aat aac ttc 480
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
145 150 155 160
tat ccc aga gag gcc aaa gta cag tgg aag gtg gat aac gcc ctc caa 528
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
165 170 175
tcg ggt aac tcc cag gag agt gtc aca gag cag gac agc aag gac agc 576
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
180 185 190
acc tac agc ctc agc agc acc ctg acg ctg agc aaa gca gac tac gag 624
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
195 200 205
aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg 672
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
210 215 220
ccc gtc aca aag agc ttc aac agg gga gag tgt tag 708
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235
<210> 18
<211> 235
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 18
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Glu Asn Val Leu Thr Gln Ser Pro Ala Thr
20 25 30
Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala
50 55 60
Pro Arg Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Asn Asp Tyr Thr Leu Thr Ile
85 90 95
Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly
100 105 110
Ser Gly Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
115 120 125
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
130 135 140
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
145 150 155 160
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
165 170 175
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
180 185 190
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
195 200 205
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
210 215 220
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235
<210> 19
<211> 708
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic polynucleotide
<220>
<221> CDS
<222> (1)..(708)
<223> humanized light chain
<400> 19
atg gat ttt caa gtg cag att ttc agc ttc ctg cta atc agt gcc tca 48
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
gtc ata atg tcc aga gga gaa aat gtg ttg aca cag tct cca gcc acc 96
Val Ile Met Ser Arg Gly Glu Asn Val Leu Thr Gln Ser Pro Ala Thr
20 25 30
ctg tct ttg tct cca ggg gaa aga gcc acc ctc tcc tgc agt gcc agc 144
Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser
35 40 45
tca agt gta agt tac atg cac tgg tac caa cag aaa cct ggc cag gct 192
Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala
50 55 60
ccc agg ctc tgg atc tat gac aca tcc aaa ctg gct tct ggc atc cca 240
Pro Arg Leu Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro
65 70 75 80
gcc agg ttc agt ggc agg ggg tct ggg aac gac tat act ctc acc atc 288
Ala Arg Phe Ser Gly Arg Gly Ser Gly Asn Asp Tyr Thr Leu Thr Ile
85 90 95
agc agc cta gag cct gaa gat ttt gca gtt tat tac tgt ttt cag ggg 336
Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly
100 105 110
agt ggg tac cca ctc acg ttt ggc cag ggg acc aag gtg gaa atc aaa 384
Ser Gly Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
115 120 125
cgt acg gtg gct gca cca tct gtc ttc atc ttc ccg cca tct gat gag 432
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
130 135 140
cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg ctg aat aac ttc 480
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
145 150 155 160
tat ccc aga gag gcc aaa gta cag tgg aag gtg gat aac gcc ctc caa 528
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
165 170 175
tcg ggt aac tcc cag gag agt gtc aca gag cag gac agc aag gac agc 576
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
180 185 190
acc tac agc ctc agc agc acc ctg acg ctg agc aaa gca gac tac gag 624
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
195 200 205
aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg 672
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
210 215 220
ccc gtc aca aag agc ttc aac agg gga gag tgt tag 708
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235
<210> 20
<211> 235
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 20
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Glu Asn Val Leu Thr Gln Ser Pro Ala Thr
20 25 30
Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala
50 55 60
Pro Arg Leu Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro
65 70 75 80
Ala Arg Phe Ser Gly Arg Gly Ser Gly Asn Asp Tyr Thr Leu Thr Ile
85 90 95
Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly
100 105 110
Ser Gly Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
115 120 125
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
130 135 140
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
145 150 155 160
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
165 170 175
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
180 185 190
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
195 200 205
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
210 215 220
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235
<210> 21
<211> 1410
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic polynucleotide
<220>
<221> CDS
<222> (1)..(1410)
<223> humanized heavy chain
<400> 21
atg gct gtc ctg gtg ctg ttc ctc tgc ctg gtt gca ttt cca agc tgt 48
Met Ala Val Leu Val Leu Phe Leu Cys Leu Val Ala Phe Pro Ser Cys
1 5 10 15
gtc ctg acc cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag 96
Val Leu Thr Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
20 25 30
cct tcg gag acc ctg tcc ctc acc tgc act gtc tct ggg ttt tca tta 144
Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu
35 40 45
gcc agc tat ggt gta gac tgg atc cgg cag ccc cca ggg aag gga ctg 192
Ala Ser Tyr Gly Val Asp Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu
50 55 60
gag tgg att ggg gta ata tgg ggt ggt gga agt aca aat tat aat tca 240
Glu Trp Ile Gly Val Ile Trp Gly Gly Gly Ser Thr Asn Tyr Asn Ser
65 70 75 80
gct ctc atg tcc cga gtc acc ata tca gta gac acg tcc aag aac cag 288
Ala Leu Met Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln
85 90 95
ttc tcc ctg aag ctg agc tct gtg acc gct gcg gac acg gcc gtg tat 336
Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
100 105 110
tac tgt gcg aga cat gga gat agt aat tcc ggt tat gct atg gac tac 384
Tyr Cys Ala Arg His Gly Asp Ser Asn Ser Gly Tyr Ala Met Asp Tyr
115 120 125
tgg ggc caa ggt acc ctg gtc acc gtc tcg agt gct agc acc aag ggc 432
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
130 135 140
cca tcg gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc 480
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
145 150 155 160
aca gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg 528
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
165 170 175
acg gtg tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc 576
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
180 185 190
ccg gct gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg 624
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
195 200 205
acc gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg 672
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
210 215 220
aat cac aag ccc agc aac acc aag gtg gac aag aga gtt gag ccc aaa 720
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys
225 230 235 240
tct tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc 768
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
245 250 255
ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc 816
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
260 265 270
ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg 864
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
275 280 285
agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg 912
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
290 295 300
gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc 960
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
305 310 315 320
acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg 1008
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
325 330 335
aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc 1056
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
340 345 350
ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca 1104
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365
cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag 1152
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
370 375 380
gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc 1200
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
385 390 395 400
gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg 1248
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
405 410 415
cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc 1296
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
420 425 430
acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc 1344
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
435 440 445
gtg atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc 1392
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
450 455 460
ctg tct ccg ggt aaa tga 1410
Leu Ser Pro Gly Lys
465
<210> 22
<211> 469
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 22
Met Ala Val Leu Val Leu Phe Leu Cys Leu Val Ala Phe Pro Ser Cys
1 5 10 15
Val Leu Thr Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
20 25 30
Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu
35 40 45
Ala Ser Tyr Gly Val Asp Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu
50 55 60
Glu Trp Ile Gly Val Ile Trp Gly Gly Gly Ser Thr Asn Tyr Asn Ser
65 70 75 80
Ala Leu Met Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln
85 90 95
Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
100 105 110
Tyr Cys Ala Arg His Gly Asp Ser Asn Ser Gly Tyr Ala Met Asp Tyr
115 120 125
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
130 135 140
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
145 150 155 160
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
165 170 175
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
180 185 190
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
195 200 205
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
210 215 220
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys
225 230 235 240
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
245 250 255
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
260 265 270
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
275 280 285
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
290 295 300
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
305 310 315 320
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
325 330 335
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
340 345 350
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
370 375 380
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
385 390 395 400
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
405 410 415
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
420 425 430
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
435 440 445
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
450 455 460
Leu Ser Pro Gly Lys
465
<210> 23
<211> 1410
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic polynucleotide
<220>
<221> CDS
<222> (1)..(1410)
<223> humanized heavy chain
<400> 23
atg gct gtc ctg gtg ctg ttc ctc tgc ctg gtt gca ttt cca agc tgt 48
Met Ala Val Leu Val Leu Phe Leu Cys Leu Val Ala Phe Pro Ser Cys
1 5 10 15
gtc ctg acc cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag 96
Val Leu Thr Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
20 25 30
cct tcg gag acc ctg tcc ctc acc tgc act gtc tct ggg ttt tca tta 144
Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu
35 40 45
gcc agc tat ggt gta gac tgg atc cgg cag ccc cca ggg aag gga ctg 192
Ala Ser Tyr Gly Val Asp Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu
50 55 60
gag tgg att ggg gta ata tgg ggt ggt gga agt aca aat tat aat tca 240
Glu Trp Ile Gly Val Ile Trp Gly Gly Gly Ser Thr Asn Tyr Asn Ser
65 70 75 80
tcc ctc atg tcc cga gtc acc ata tca aaa gac acg tcc aag aac cag 288
Ser Leu Met Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln
85 90 95
ctt tcc ctg aag ctg agc tct gtg acc gct gcg gac acg gcc gtg tat 336
Leu Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
100 105 110
tac tgt gcg aaa cat gga gat agt aat tcc ggt tat gct atg gac tac 384
Tyr Cys Ala Lys His Gly Asp Ser Asn Ser Gly Tyr Ala Met Asp Tyr
115 120 125
tgg ggc caa ggt acc ctg gtc acc gtc tcg agt gct agc acc aag ggc 432
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
130 135 140
cca tcg gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc 480
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
145 150 155 160
aca gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg 528
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
165 170 175
acg gtg tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc 576
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
180 185 190
ccg gct gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg 624
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
195 200 205
acc gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg 672
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
210 215 220
aat cac aag ccc agc aac acc aag gtg gac aag aga gtt gag ccc aaa 720
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys
225 230 235 240
tct tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc 768
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
245 250 255
ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc 816
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
260 265 270
ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg 864
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
275 280 285
agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg 912
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
290 295 300
gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc 960
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
305 310 315 320
acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg 1008
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
325 330 335
aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc 1056
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
340 345 350
ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca 1104
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365
cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag 1152
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
370 375 380
gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc 1200
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
385 390 395 400
gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg 1248
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
405 410 415
cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc 1296
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
420 425 430
acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc 1344
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
435 440 445
gtg atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc 1392
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
450 455 460
ctg tct ccg ggt aaa tga 1410
Leu Ser Pro Gly Lys
465
<210> 24
<211> 469
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 24
Met Ala Val Leu Val Leu Phe Leu Cys Leu Val Ala Phe Pro Ser Cys
1 5 10 15
Val Leu Thr Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
20 25 30
Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu
35 40 45
Ala Ser Tyr Gly Val Asp Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu
50 55 60
Glu Trp Ile Gly Val Ile Trp Gly Gly Gly Ser Thr Asn Tyr Asn Ser
65 70 75 80
Ser Leu Met Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln
85 90 95
Leu Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
100 105 110
Tyr Cys Ala Lys His Gly Asp Ser Asn Ser Gly Tyr Ala Met Asp Tyr
115 120 125
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
130 135 140
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
145 150 155 160
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
165 170 175
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
180 185 190
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
195 200 205
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
210 215 220
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys
225 230 235 240
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
245 250 255
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
260 265 270
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
275 280 285
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
290 295 300
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
305 310 315 320
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
325 330 335
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
340 345 350
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
370 375 380
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
385 390 395 400
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
405 410 415
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
420 425 430
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
435 440 445
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
450 455 460
Leu Ser Pro Gly Lys
465
<210> 25
<211> 1410
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic polynucleotide
<220>
<221> CDS
<222> (1)..(1410)
<223> humanized heavy chain
<400> 25
atg gct gtc ctg gtg ctg ttc ctc tgc ctg gtt gca ttt cca agc tgt 48
Met Ala Val Leu Val Leu Phe Leu Cys Leu Val Ala Phe Pro Ser Cys
1 5 10 15
gtc ctg acc cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag 96
Val Leu Thr Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
20 25 30
cct tcg gag acc ctg tcc ctc acc tgc act gtc tct ggg ttt tca tta 144
Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu
35 40 45
gcc agc tat ggt gta gac tgg gtt cgg cag ccc cca ggg aag gga ctg 192
Ala Ser Tyr Gly Val Asp Trp Val Arg Gln Pro Pro Gly Lys Gly Leu
50 55 60
gag tgg att ggg gta ata tgg ggt ggt gga agt aca aat tat aat tca 240
Glu Trp Ile Gly Val Ile Trp Gly Gly Gly Ser Thr Asn Tyr Asn Ser
65 70 75 80
gct ctc atg tcc cga ctt acc ata tca aaa gac aac tcc aag agc cag 288
Ala Leu Met Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln
85 90 95
ctt tcc ctg aag ctg agc tct gtg acc gct gcg gac acg gcc gtg tat 336
Leu Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
100 105 110
tac tgt gcg aaa cat gga gat agt aat tcc ggt tat gct atg gac tac 384
Tyr Cys Ala Lys His Gly Asp Ser Asn Ser Gly Tyr Ala Met Asp Tyr
115 120 125
tgg ggc caa ggt acc ctg gtc acc gtc tcg agt gct agc acc aag ggc 432
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
130 135 140
cca tcg gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc 480
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
145 150 155 160
aca gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg 528
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
165 170 175
acg gtg tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc 576
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
180 185 190
ccg gct gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg 624
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
195 200 205
acc gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg 672
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
210 215 220
aat cac aag ccc agc aac acc aag gtg gac aag aga gtt gag ccc aaa 720
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys
225 230 235 240
tct tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc 768
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
245 250 255
ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc 816
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
260 265 270
ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg 864
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
275 280 285
agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg 912
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
290 295 300
gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc 960
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
305 310 315 320
acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg 1008
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
325 330 335
aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc 1056
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
340 345 350
ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca 1104
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365
cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag 1152
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
370 375 380
gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc 1200
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
385 390 395 400
gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg 1248
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
405 410 415
cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc 1296
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
420 425 430
acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc 1344
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
435 440 445
gtg atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc 1392
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
450 455 460
ctg tct ccg ggt aaa tga 1410
Leu Ser Pro Gly Lys
465
<210> 26
<211> 469
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 26
Met Ala Val Leu Val Leu Phe Leu Cys Leu Val Ala Phe Pro Ser Cys
1 5 10 15
Val Leu Thr Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
20 25 30
Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu
35 40 45
Ala Ser Tyr Gly Val Asp Trp Val Arg Gln Pro Pro Gly Lys Gly Leu
50 55 60
Glu Trp Ile Gly Val Ile Trp Gly Gly Gly Ser Thr Asn Tyr Asn Ser
65 70 75 80
Ala Leu Met Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln
85 90 95
Leu Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
100 105 110
Tyr Cys Ala Lys His Gly Asp Ser Asn Ser Gly Tyr Ala Met Asp Tyr
115 120 125
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
130 135 140
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
145 150 155 160
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
165 170 175
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
180 185 190
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
195 200 205
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
210 215 220
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys
225 230 235 240
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
245 250 255
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
260 265 270
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
275 280 285
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
290 295 300
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
305 310 315 320
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
325 330 335
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
340 345 350
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
370 375 380
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
385 390 395 400
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
405 410 415
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
420 425 430
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
435 440 445
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
450 455 460
Leu Ser Pro Gly Lys
465
<210> 27
<211> 708
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic polynucleotide
<220>
<221> CDS
<222> (1)..(708)
<223> humanized light chain
<400> 27
atg gat ttt caa gtg cag att ttc agc ttc ctg cta atc agt gcc tca 48
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
gtc ata atg tcc aga gga gaa att gtg ttg aca cag tct cca gcc acc 96
Val Ile Met Ser Arg Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
20 25 30
ctg tct ttg tct cca ggg gaa aga gcc acc ctc tcc tgc agt gcc agc 144
Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser
35 40 45
cca agt gta agt tac atg cac tgg tac caa cag aaa cct ggc cag gct 192
Pro Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala
50 55 60
ccc agg ctc ctc atc tat gac aca tac aaa ctg gct tct ggc atc cca 240
Pro Arg Leu Leu Ile Tyr Asp Thr Tyr Lys Leu Ala Ser Gly Ile Pro
65 70 75 80
gcc agg ttc agt ggc agt ggg tct ggg aca gac ttc act ctc acc atc 288
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
85 90 95
agc agc cta gag cct gaa gat ttt gca gtt tat tac tgt ttt cag ggg 336
Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly
100 105 110
agt ggg ttc cca ctc acg ttt ggc cag ggg acc aag gtg gaa atc aaa 384
Ser Gly Phe Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
115 120 125
cgt acg gtg gct gca cca tct gtc ttc atc ttc ccg cca tct gat gag 432
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
130 135 140
cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg ctg aat aac ttc 480
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
145 150 155 160
tat ccc aga gag gcc aaa gta cag tgg aag gtg gat aac gcc ctc caa 528
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
165 170 175
tcg ggt aac tcc cag gag agt gtc aca gag cag gac agc aag gac agc 576
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
180 185 190
acc tac agc ctc agc agc acc ctg acg ctg agc aaa gca gac tac gag 624
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
195 200 205
aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg 672
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
210 215 220
ccc gtc aca aag agc ttc aac agg gga gag tgt tag 708
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235
<210> 28
<211> 235
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 28
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
20 25 30
Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser
35 40 45
Pro Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala
50 55 60
Pro Arg Leu Leu Ile Tyr Asp Thr Tyr Lys Leu Ala Ser Gly Ile Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
85 90 95
Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly
100 105 110
Ser Gly Phe Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
115 120 125
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
130 135 140
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
145 150 155 160
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
165 170 175
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
180 185 190
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
195 200 205
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
210 215 220
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235
<210> 29
<211> 708
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic polynucleotide
<220>
<221> CDS
<222> (1)..(708)
<223> humanized light chain
<400> 29
atg gat ttt caa gtg cag att ttc agc ttc ctg cta atc agt gcc tca 48
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
gtc ata atg tcc aga gga gaa aat gtg ttg aca cag tct cca gcc acc 96
Val Ile Met Ser Arg Gly Glu Asn Val Leu Thr Gln Ser Pro Ala Thr
20 25 30
ctg tct ttg tct cca ggg gaa aga gcc acc ctc tcc tgc agt gcc agc 144
Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser
35 40 45
cca agt gta agt tac atg cac tgg tac caa cag aaa cct ggc cag gct 192
Pro Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala
50 55 60
ccc agg ctc ctc atc tat gac aca tac aaa ctg gct tct ggc atc cca 240
Pro Arg Leu Leu Ile Tyr Asp Thr Tyr Lys Leu Ala Ser Gly Ile Pro
65 70 75 80
gcc agg ttc agt ggc agt ggg tct ggg aac gac tac act ctc acc atc 288
Ala Arg Phe Ser Gly Ser Gly Ser Gly Asn Asp Tyr Thr Leu Thr Ile
85 90 95
agc agc cta gag cct gaa gat ttt gca gtt tat tac tgt ttt cag ggg 336
Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly
100 105 110
agt ggg ttc cca ctc acg ttt ggc cag ggg acc aag gtg gaa atc aaa 384
Ser Gly Phe Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
115 120 125
cgt acg gtg gct gca cca tct gtc ttc atc ttc ccg cca tct gat gag 432
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
130 135 140
cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg ctg aat aac ttc 480
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
145 150 155 160
tat ccc aga gag gcc aaa gta cag tgg aag gtg gat aac gcc ctc caa 528
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
165 170 175
tcg ggt aac tcc cag gag agt gtc aca gag cag gac agc aag gac agc 576
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
180 185 190
acc tac agc ctc agc agc acc ctg acg ctg agc aaa gca gac tac gag 624
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
195 200 205
aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg 672
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
210 215 220
ccc gtc aca aag agc ttc aac agg gga gag tgt tag 708
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235
<210> 30
<211> 235
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 30
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Glu Asn Val Leu Thr Gln Ser Pro Ala Thr
20 25 30
Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser
35 40 45
Pro Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala
50 55 60
Pro Arg Leu Leu Ile Tyr Asp Thr Tyr Lys Leu Ala Ser Gly Ile Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Asn Asp Tyr Thr Leu Thr Ile
85 90 95
Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly
100 105 110
Ser Gly Phe Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
115 120 125
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
130 135 140
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
145 150 155 160
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
165 170 175
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
180 185 190
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
195 200 205
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
210 215 220
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235
<210> 31
<211> 708
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic polynucleotide
<220>
<221> CDS
<222> (1)..(708)
<223> humanized light chain
<400> 31
atg gat ttt caa gtg cag att ttc agc ttc ctg cta atc agt gcc tca 48
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
gtc ata atg tcc aga gga gaa aat gtg ttg aca cag tct cca gcc acc 96
Val Ile Met Ser Arg Gly Glu Asn Val Leu Thr Gln Ser Pro Ala Thr
20 25 30
ctg tct ttg tct cca ggg gaa aga gcc acc ctc tcc tgc agt gcc agc 144
Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser
35 40 45
cca agt gta agt tac atg cac tgg tac caa cag aaa cct ggc cag gct 192
Pro Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala
50 55 60
ccc agg ctc tgg atc tat gac aca tac aaa ctg gct tct ggc atc cca 240
Pro Arg Leu Trp Ile Tyr Asp Thr Tyr Lys Leu Ala Ser Gly Ile Pro
65 70 75 80
gcc agg ttc agt ggc agt ggg tct ggg aac gac tac act ctc acc atc 288
Ala Arg Phe Ser Gly Ser Gly Ser Gly Asn Asp Tyr Thr Leu Thr Ile
85 90 95
agc agc cta gag cct gaa gat ttt gca gtt tat tac tgt ttt cag ggg 336
Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly
100 105 110
agt ggg ttc cca ctc acg ttt ggc cag ggg acc aag gtg gaa atc aaa 384
Ser Gly Phe Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
115 120 125
cgt acg gtg gct gca cca tct gtc ttc atc ttc ccg cca tct gat gag 432
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
130 135 140
cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg ctg aat aac ttc 480
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
145 150 155 160
tat ccc aga gag gcc aaa gta cag tgg aag gtg gat aac gcc ctc caa 528
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
165 170 175
tcg ggt aac tcc cag gag agt gtc aca gag cag gac agc aag gac agc 576
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
180 185 190
acc tac agc ctc agc agc acc ctg acg ctg agc aaa gca gac tac gag 624
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
195 200 205
aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg 672
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
210 215 220
ccc gtc aca aag agc ttc aac agg gga gag tgt tag 708
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235
<210> 32
<211> 235
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 32
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Glu Asn Val Leu Thr Gln Ser Pro Ala Thr
20 25 30
Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser
35 40 45
Pro Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala
50 55 60
Pro Arg Leu Trp Ile Tyr Asp Thr Tyr Lys Leu Ala Ser Gly Ile Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Asn Asp Tyr Thr Leu Thr Ile
85 90 95
Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly
100 105 110
Ser Gly Phe Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
115 120 125
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
130 135 140
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
145 150 155 160
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
165 170 175
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
180 185 190
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
195 200 205
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
210 215 220
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235
<210> 33
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic polypeptide
<220>
<221> MISC_FEATURE
<223> H-CDR1
<400> 33
Gly Phe Ser Leu Thr Ser Tyr Gly Val Asp
1 5 10
<210> 34
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic polypeptide
<220>
<221> MISC_FEATURE
<223> H-CDR2
<400> 34
Val Ile Trp Gly Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met Ser
1 5 10 15
<210> 35
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic polypeptide
<220>
<221> MISC_FEATURE
<223> H-CDR3
<400> 35
His Glu Val Leu Arg Gly Tyr Ala Leu Asp Tyr
1 5 10
<210> 36
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic polypeptide
<220>
<221> MISC_FEATURE
<223> L-CDR1
<400> 36
Ser Ala Ser Ser Ser Val Ser Tyr Met His
1 5 10
<210> 37
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic polypeptide
<220>
<221> MISC_FEATURE
<223> L-CDR2
<400> 37
Asp Thr Ser Lys Leu Ala Ser
1 5
<210> 38
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic polypeptide
<220>
<221> MISC_FEATURE
<223> L-CDR3
<400> 38
Phe Gln Gly Ser Gly Tyr Pro Leu Thr
1 5
<210> 39
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic polypeptide
<220>
<221> MISC_FEATURE
<223> H-CDR2
<400> 39
Val Ile Trp Gly Gly Gly Ser Thr Asn Tyr Asn Ser Ser Leu Met Ser
1 5 10 15
<210> 40
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic polypeptide
<220>
<221> MISC_FEATURE
<223> H-CDR1
<400> 40
Gly Phe Ser Leu Ala Ser Tyr Gly Val Asp
1 5 10
<210> 41
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic polypeptide
<220>
<221> MISC_FEATURE
<223> H-CDR3
<400> 41
His Gly Asp Ser Asn Ser Gly Tyr Ala Met Asp Tyr
1 5 10
<210> 42
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic polypeptide
<220>
<221> MISC_FEATURE
<223> L-CDR1
<400> 42
Ser Ala Ser Pro Ser Val Ser Tyr Met His
1 5 10
<210> 43
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic polypeptide
<220>
<221> MISC_FEATURE
<223> L-CDR2
<400> 43
Asp Thr Tyr Lys Leu Ala Ser
1 5
<210> 44
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic polypeptide
<220>
<221> MISC_FEATURE
<223> L-CDR3
<400> 44
Phe Gln Gly Ser Gly Phe Pro Leu Thr
1 5
<210> 45
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic primer
<400> 45
tatgcaaggc ttacaaccac a 21
<210> 46
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic primer
<400> 46
gggggtactg ggcttgggta t 21
<210> 47
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic primer
<400> 47
ctcattcctg ttgaagctct tgac 24
<210> 48
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic primer
<400> 48
gtgactcgag tcgacatcga tttttttttt ttttttt 37
<210> 49
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic primer
<400> 49
cttccggaat tcctcaattt tcttgtccac cttggtgc 38
<210> 50
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic primer
<400> 50
cttccggaat tcctcgattc tcttgatcaa ctcagtct 38
<210> 51
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic primer
<400> 51
cttccggaat tcctcattcc tgttgaagct cttgacaatg gg 42
<210> 52
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic primer
<400> 52
attaagtcga catagacaga tgggggtgtc gttttggc 38
<210> 53
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic primer
<400> 53
attaagtcga cagggaccaa gggatagaca gatgg 35
<210> 54
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic primer
<400> 54
ctacctcgag ggatacagtt ggtgcagcat c 31
<210> 55
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic primer
<400> 55
cagtccgcgg ccaccatgga ttttcaagtg cagattttc 39
<210> 56
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic primer
<400> 56
aggaagatct aacactcatt cctgttgaag c 31
<210> 57
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic primer
<400> 57
cagtccgcgg ccaccatggc tgtcctggtg ctgtt 35
<210> 58
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic primer
<400> 58
ctggacaggg atccagagtt cca 23
<210> 59
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic primer
<400> 59
catgagatct catttaccag gggagcgaga 30
<210> 60
<211> 108
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> huIGHV4-59x01
<400> 60
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr
20 25 30
Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
100 105
<210> 61
<211> 105
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> huIGKV3-11x01
<400> 61
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Phe
85 90 95
Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105

Claims (20)

1.一种经分离的单株抗体或其抗原-结合片段，其特征在于，包含：具有序列辨识编号：33或序列辨识编号：40的序列的重链H-CDR1、具有序列辨识编号：34或序列辨识编号：39的序列的重链H-CDR2、具有序列辨识编号：35或序列辨识编号：41的序列的重链H-CDR3、具有序列辨识编号：36或序列辨识编号：42的序列的轻链L-CDR1、具有序列辨识编号：37或序列辨识编号：43的序列的轻链L-CDR2以及具有序列辨识编号：38或序列辨识编号：44的序列的轻链L-CDR3；
所述单株抗体或抗原-结合片段专一性结合至阶段特异性胚胎抗原4。
2.根据权利要求1所述的经分离的单株抗体或抗原-结合片段，其特征在于，所述H-CDR1具有序列辨识编号：33的序列；所述H-CDR2具有序列辨识编号：34的序列；所述H-CDR3具有序列辨识编号：35的序列；所述L-CDR1具有序列辨识编号：42的序列；所述L-CDR2具有序列辨识编号：43的序列；所述L-CDR3具有序列辨识编号：44的序列。
3.一种经分离的单株抗体，其特征在于，包括选自下列所构成的群组中的重链序列：序列辨识编号：2、序列辨识编号：6、序列辨识编号：10、序列辨识编号：12、序列辨识编号：14、序列辨识编号：22、序列辨识编号：24以及序列辨识编号：26的序列；以及选自下列所构成的群组中的轻链序列：序列辨识编号：4、序列辨识编号：8、序列辨识编号：16、序列辨识编号：18、序列辨识编号：20、序列辨识编号：28、序列辨识编号：30以及序列辨识编号：32的序列，所述单株抗体专一性结合至阶段特异性胚胎抗原4。
4.根据权利要求3所述的经分离的单株抗体，其特征在于，所述重链序列为序列辨识编号：14的序列；所述轻链序列为序列辨识编号：28的序列。
5.根据权利要求3所述的经分离的单株抗体，其特征在于，所述重链序列为序列辨识编号：14的序列；所述轻链序列是序列辨识编号：30的序列。
6.根据权利要求3所述的经分离的单株抗体，其特征在于；所述重链序列为序列辨识编号：14的序列；所述轻链序列为序列辨识编号：32的序列。
7.根据权利要求3所述的经分离的单株抗体，其特征在于，所述重链序列为序列辨识编号：2的序列；所述轻链序列为序列辨识编号：4的序列。
8.根据权利要求3所述的经分离的单株抗体，其特征在于，所述重链序列为序列辨识编号：6的序列；所述该轻链序列为序列辨识编号：8的序列。
9.一种核酸建构物，其特征在于，用于编码权利要求1所述的单株抗体或其抗原-结合片段。
10.根据权利要求9所述的核酸建构物，其特征在于，所述H-CDR1具有序列辨识编号：33的序列；所述H-CDR2具有序列辨识编号：34的序列；所述H-CDR3具有序列辨识编号：35的序列；所述L-CDR1具有序列辨识编号：42的序列；所述L-CDR2具有序列辨识编号：43的序列；所述L-CDR3具有序列辨识编号：44的序列。
11.一种核酸建构物，其特征在于，用于编码权利要求3所述的单株抗体。
12.根据权利要11所述的核酸建构物，其特征在于，所述重链序列为序列辨识编号：14的序列；所述轻链序列为序列辨识编号：28的序列。
13.一种重组型细胞，其特征在于，包含权利要求9所述的核酸建构物，所述细胞表现专一性结合至阶段特异性胚胎抗原4的单株抗体。
14.根据权利要求13所述的重组型细胞，其特征在于，所述H-CDR1具有序列辨识编号：33的序列；所述H-CDR2具有序列辨识编号：34的序列；所述H-CDR3具有序列辨识编号：35的序列所述L-CDR1具有序列辨识编号：42的序列；所述L-CDR2具有序列辨识编号：43的序列；所述L-CDR3具有序列辨识编号：44的序列。
15.一种重组型细胞，其特征在于；包含权利要求11所述的核酸建构物；所述细胞表现专一性结合至阶段特异性胚胎抗原4的单株抗体。
16.根据权利要求15所述的核酸，其特征在于，所述重链序列为序列辨识编号：14的序列；所述轻链序列为序列辨识编号：28的序列。
17.一种用于治疗肿瘤的方法，其特征在于，所述方法包括识别出带有肿瘤的个体以及对所述个体投予有效量权利要求1所述的单株抗体；所述的肿瘤中的细胞表现阶段特异性胚胎抗原4。
18.根据权利要求17所述的方法，其特征在于，所述H-CDR1具有序列辨识编号：33的序列；所述H-CDR2具有序列辨识编号：34的序列；所述H-CDR3具有序列辨识编号：35的序列；所述L-CDR1具有序列辨识编号：42的序列；所述L-CDR2具有序列辨识编号：43的序列；所述L-CDR3具有序列辨识编号：44的序列。
19.一种用于治疗肿瘤的方法，其特征在于，所述方法包括识别出具有肿瘤的个体以及对所述个体投予有效量权利要求3所述的单株抗体，其中在该肿瘤中的细胞表现阶段特异性胚胎抗原4。
20.根据权利要求19所述的方法，其特征在于，所述重链序列为序列辨识编号：14的序列；所述轻链序列为序列辨识编号：28的序列。
CN201980020978.5A 2018-03-29 2019-03-25 结合至ssea4的单株抗体及其用途 Pending CN112236167A (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116773813A (zh) * 2023-06-20 2023-09-19 北京健平金星医疗器械有限公司 一种vegfr1检测试剂盒的制备及应用

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220289805A1 (en) * 2018-09-07 2022-09-15 Nantbio, Inc. Targeted IL-12 Treatments and Methods to Stimulate haNK and NK92mi Cells
CN113817051B (zh) * 2020-06-19 2023-08-29 武汉生物制品研究所有限责任公司 一种抗SARS-CoV-2的单克隆抗体1B6
JP2023553313A (ja) * 2020-11-30 2023-12-21 チョ ファーマ インコーポレイテッド 細胞を富化するための抗体
WO2024040195A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015142675A2 (en) * 2014-03-15 2015-09-24 Novartis Ag Treatment of cancer using chimeric antigen receptor
CN106459920A (zh) * 2014-01-16 2017-02-22 中央研究院 治疗及检测癌症的组合物及方法
US20170283488A1 (en) * 2016-03-29 2017-10-05 Obi Pharma, Inc. Antibodies, pharmaceutical compositions and methods
WO2018039274A1 (en) * 2016-08-22 2018-03-01 CHO Pharma Inc. Antibodies, binding fragments, and methods of use

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7534605B2 (en) 1999-06-08 2009-05-19 Yissum Research Development Company Of The Hebrew University Of Jerusalem CD44 polypeptides, polynucleotides encoding same, antibodies directed thereagainst and method of using same for diagnosing and treating inflammatory diseases
WO2007048849A1 (en) 2005-10-28 2007-05-03 Novo Nordisk A/S Fusion proteins that bind effector lymphocytes and target cells
WO2015015489A1 (en) 2013-07-30 2015-02-05 Biolinerx Ltd. Antibody for treating diabetes and autoimmune diseases
WO2015053381A1 (ja) 2013-10-10 2015-04-16 幸成 加藤 抗ポドプラニン抗体
US10150818B2 (en) 2014-01-16 2018-12-11 Academia Sinica Compositions and methods for treatment and detection of cancers
US20180030124A1 (en) 2016-07-29 2018-02-01 Lan Bo Chen Methods and compositions for enhancing anti-ssea4 immunotherapy

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106459920A (zh) * 2014-01-16 2017-02-22 中央研究院 治疗及检测癌症的组合物及方法
WO2015142675A2 (en) * 2014-03-15 2015-09-24 Novartis Ag Treatment of cancer using chimeric antigen receptor
US20170283488A1 (en) * 2016-03-29 2017-10-05 Obi Pharma, Inc. Antibodies, pharmaceutical compositions and methods
WO2018039274A1 (en) * 2016-08-22 2018-03-01 CHO Pharma Inc. Antibodies, binding fragments, and methods of use

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116773813A (zh) * 2023-06-20 2023-09-19 北京健平金星医疗器械有限公司 一种vegfr1检测试剂盒的制备及应用
CN116773813B (zh) * 2023-06-20 2024-01-30 北京健平金星生物医药有限公司 一种vegfr1检测试剂盒的制备及应用

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