Development of soluble TCR and soluble MHC/peptide compelx with high affinity for their ligands

开发对其配体具有高亲和力的可溶性 TCR 和可溶性 MHC/肽复合物

• 批准号: 08557026
• 负责人: SASAZUKI Takehiko
• 金额: $ 7.68万
• 依托单位: Kyushu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08557026/
• 关键词: soluble TCR; soluble MHC; peptide complex; leucine zipper; off rate; affinity; ペプチド複合体; MHC; GPIアンカー; PI-PLC; 高親和性; Leucin Zipper

We tried in this project to develop soluble MHC class II/peptide complex and soluble TCR with so high affinity as to directly identify and/or isolate their ligands. To facilitate the heterodimer formation, we connected leucine zipper sequence to the extracellular domain of alpha and beta chain of TCR or MHC class II.In addition, the antigenic peptide was covalently bound to MHC class II molecules. In each case, free cysteine was introduced at the C-terminal of one of the leucine zipper sequence, which was used for the target for the specific biotinylation.Finally, we developed multivalent TCR or MHC class II/peptide complex by coupling the biotinylated molecule with streptavidin. Such multivatent TCR or MHC class II/peptide complex showed remarkably slow off-rate for their ligands in BIAcore analysis and detected the cells expressing the ligand by flow cytometry. Thus, this approach would have wide application for several immunological fields, including T cell development, autoimmunity, transplantation and tumor immunity.

本项目中，我们试图开发具有如此高亲和力的可溶性MHC II类/肽复合物和可溶性TCR，以直接鉴定和/或分离它们的配体。为了促进异二聚体的形成，我们将亮氨酸拉链序列连接到TCR或MHC II类分子的α和β链的胞外结构域。此外，抗原肽与MHC II类分子共价结合。在每种情况下，在亮氨酸拉链序列之一的C-末端引入游离半胱氨酸，该亮氨酸拉链序列用作特异性生物素化的靶点。最后，我们通过将生物素化的分子与链霉亲和素偶联来开发多价TCR或MHC II类/肽复合物。这种多功能TCR或MHC II类/肽复合物在BIAcore分析中显示出对其配体的显著缓慢的解离速率，并通过流式细胞术检测表达配体的细胞。因此，这种方法将在包括T细胞发育、自身免疫、移植和肿瘤免疫在内的几个免疫学领域具有广泛的应用。

项目成果

Gyotoku T.: "An endogenously processed self peptide and the coresponding exogenous peptide bound to the same MHC class II molecule could be distinct ligands for TCR with different kinetic stability." Eur.J.Immunol.28. 4050-4061 (1998)

Gyotoku T.：“内源加工的自身肽和与相同 MHC II 类分子结合的相应外源肽可能是具有不同动力学稳定性的 TCR 不同配体。”

Fukui Y.: "Positive and negative CD4+ thymocyte selection by a single MHC class II/peptide liqand affected by its expression level in the thymus." Immunity. 6. 401-410 (1997)

Fukui Y.：“单一 MHC II 类/肽配体对阳性和阴性 CD4 胸腺细胞的选择受其在胸腺中表达水平的影响。”

Date Y.: "DNA typing of the HLA-A gene:population studay and identification of four new alleles in Japanese." Tissue Antigens. 47. 93-101 (1996)

Date Y.：“HLA-A 基因的 DNA 分型：日语中四个新等位基因的群体研究和鉴定。”

Hori T.: "Japanese cedar pollinosis and HLA-DP5" Tissue Antigens. 47. 485-491 (1996)

Hori T.：“日本雪松花粉病和 HLA-DP5”组织抗原。

Gapin L.: "Quantitaive analysis of the T-cell repertoire by a single peptide/MHC complex." J.Exp.Med.187. 1871-1883 (1998)

Gapin L.：“通过单个肽/MHC 复合物对 T 细胞库进行定量分析。”