Sanders Jane, Evans Michele, Betterle Corrado, Sanders Paul, Bhardwaja Anshu, Young Stuart, Roberts Emma, Wilmot Jane, Richards Tonya, Kiddie Angela, Small Kasemsri, Platt Hayley, Summerhayes Sara, Harris Rebecca, Reeve Magnus, Coco Graziella, Zanchetta Renato, Chen Shu, Furmaniak Jadwiga, Smith Bernard Rees
FIRS Laboratories, RSR Ltd, Llanishen, Cardiff, United Kingdom.
Thyroid. 2008 Jul;18(7):735-46. doi: 10.1089/thy.2007.0327.
Human monoclonal autoantibodies (MAbs) are valuable tools to study autoimmune responses. To date only one human MAb to the thyrotropin (TSH) receptor (TSHR) with stimulating activity has been available. We now describe the detailed characterization of a blocking type human MAb to the TSHR.
A single heterohybridoma cell line was isolated from the peripheral blood lymphocytes of a patient with severe hypothyroidism (TSH 278 mU/L) using standard techniques. The line stably expresses a TSHR autoantibody (5C9; IgG1/kappa). Ability of 5C9 to bind and compete with 125I-TSH or TSHR antibodies binding to the TSHR was tested using tubes coated with solubilized TSHR. Furthermore, the blocking effects of 5C9 on stimulation of cyclic AMP production was assessed using Chinese hamster ovary (CHO) cells expressing the wild-type human TSHR or TSHRs with amino acid mutations.
5C9 IgG bound to the TSHR with high affinity (4 x 10(10) L/mol) and inhibited binding of TSH and a thyroid-stimulating human monoclonal autoantibody (M22) to the receptor. 5C9 IgG preparations inhibited the cyclic AMP-stimulating activities of TSH, M22, serum TSHR autoantibodies and thyroid-stimulating mouse monoclonal antibodies. Furthermore 5C9 reduced the constitutive activity of wild-type TSHR and TSHR with some activating mutations. The effect of different amino acid mutations in the TSHR on 5C9 biological activity was studied and TSHR Lys129Ala or Asp203Ala completely abolished the ability of 5C9 to block TSH-mediated stimulation of cyclic AMP production.
The availability of 5C9 provides new opportunities to investigate the binding and biological activity of TSHR blocking type autoantibodies including studies at the molecular level. Furthermore, monoclonal antibodies such as 5C9 may well provide the basis of new drugs to control TSHR activity including applications in thyroid cancer and Graves' ophthalmopathy.
人源单克隆自身抗体（MAb）是研究自身免疫反应的重要工具。迄今为止，仅有一株具有刺激活性的人源促甲状腺激素（TSH）受体（TSHR）单克隆抗体。我们现在描述一株针对TSHR的阻断型人源单克隆抗体的详细特性。
采用标准技术，从一名严重甲状腺功能减退患者（TSH 278 mU/L）的外周血淋巴细胞中分离出单一的异源杂交瘤细胞系。该细胞系稳定表达一种TSHR自身抗体（5C9；IgG1/kappa）。使用包被有可溶性TSHR的试管，检测5C9与125I-TSH或TSHR抗体结合TSHR的能力以及竞争结合能力。此外，使用表达野生型人TSHR或带有氨基酸突变的TSHR的中国仓鼠卵巢（CHO）细胞，评估5C9对环磷酸腺苷（cAMP）产生刺激的阻断作用。
5C9 IgG以高亲和力（4×10(10) L/mol）结合TSHR，并抑制TSH和一种促甲状腺的人源单克隆自身抗体（M22）与受体的结合。5C9 IgG制剂抑制TSH、M22、血清TSHR自身抗体和促甲状腺的小鼠单克隆抗体的cAMP刺激活性。此外，5C9降低野生型TSHR和带有一些激活突变的TSHR的组成性活性。研究了TSHR中不同氨基酸突变对5C9生物学活性的影响，TSHR Lys129Ala或Asp203Ala完全消除了5C9阻断TSH介导的cAMP产生刺激的能力。
5C9的获得为研究TSHR阻断型自身抗体的结合和生物学活性提供了新机会，包括在分子水平上的研究。此外，诸如5C9这样的单克隆抗体很可能为控制TSHR活性的新药提供基础，包括在甲状腺癌和格雷夫斯眼病中的应用。
