Construction, affinity maturation, and biological characterization of an anti-tumor-associated glycoprotein-72 humanized antibody
Construction, affinity maturation, and biological characterization of an anti-tumor-associated glycoprotein-72 humanized antibody
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DOI：
10.1074/jbc.m511165200
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发表时间：
2006-03-17
影响因子：
4.8
通讯作者：
Hong, HJ
中科院分区：
文献类型：
--
作者：
Yoon, SO;Lee, TS;Hong, HJ
肿瘤相关糖蛋白（TAG）-72在大多数人腺癌中表达，但在大多数正常组织中很少表达，这使其成为诊断和治疗多种人类癌症的潜在靶点。在这里，我们描述了具有最小潜在免疫原性的抗TAG-72人源化抗体的构建、亲和力成熟和生物学表征。通过仅将互补决定区（CDR）内的特异性决定残基（SDR）移植到同源人免疫球蛋白种系区段上，同时保留支持CDR构象的两个小鼠重链框架残基，构建人源化抗体。所得的人源化抗体（AKA）显示出与原始鼠单克隆抗体CC 49相比仅低约2倍的亲和力和与通过CDR移植构建的人源化抗体HuCC 49相比低27倍的对患者血清的反应性。通过随机诱变重链CDR 3（HCDR 3）来提高AKA的亲和力。最高亲和力变体（3E 8）显示出比AKA高22倍的亲和力，并保留了原始表位特异性。HCDR 3残基的突变分析显示，Asn（97）被异亮氨酸或缬氨酸取代对于亲和力成熟是关键的。用I-125或I-131标记的3E 8在具有人结肠癌异种移植物的无胸腺小鼠中分别显示出有效的肿瘤靶向或治疗效果，这表明3E 8可能有利于诊断和治疗表达TAG-72的肿瘤。
The tumor-associated glycoprotein (TAG)-72 is expressed in the majority of human adenocarcinomas but is rarely expressed in most normal tissues, which makes it a potential target for the diagnosis and therapy of a variety of human cancers. Here we describe the construction, affinity maturation, and biological characterization of an anti-TAG-72 humanized antibody with minimum potential immunogenicity. The humanized antibody was constructed by grafting only the specificity-determining residues (SDRs) within the complementarity-determining regions (CDRs) onto homologous human immunoglobulin germ line segments while retaining two mouse heavy chain framework residues that support the conformation of the CDRs. The resulting humanized antibody ( AKA) showed only about 2-fold lower affinity compared with the original murine monoclonal antibody CC49 and 27-fold lower reactivity to patient serum compared with the humanized antibody HuCC49 that was constructed by CDR grafting. The affinity of AKA was improved by random mutagenesis of the heavy chain CDR3 (HCDR3). The highest affinity variant (3E8) showed 22-fold higher affinity compared with AKA and retained the original epitope specificity. Mutational analysis of the HCDR3 residues revealed that the replacement of Asn(97) by isoleucine or valine was critical for the affinity maturation. The 3E8 labeled with I-125 or I-131 showed efficient tumor targeting or therapeutic effects, respectively, in athymic mice with human colon carcinoma xenografts, suggesting that 3E8 may be beneficial for the diagnosis and therapy of tumors expressing TAG-72.