Using solubel peptide-MHC tetramer, the trial of staining and cloning the melanoma antigen-specific cytotoxic lymphocytes

利用可溶性肽-MHC四聚体染色和克隆黑色素瘤抗原特异性细胞毒性淋巴细胞的试验

• 批准号: 11670849
• 负责人: SAKURAI Toshiharu
• 金额: $ 2.43万
• 依托单位: Keio University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670849/
• 关键词: HLA tetramer; HLA-A2; HLA-A24; Tumor infiltration lymphocyte (TIL)

The purpose of this project is to construct the soluble class I MHC-peptide complex ("tetramers") and to visualize the melanoma antigen-specific cytotoxic lymphocytes (CTL) using the produced tetramers.1. We have produced HLA-A2/gp100_<(209-217)>, HLA-A2/MART-1_<(27-35)>, and HLA-A24/EBV-P tetramers. The β2-microglobulin (β2m) and soluble HLA-A2 and A24 heavy chains linked at its carboxyl terminus to a BirA substrate peptide were expressed separately in Escherichia coli. The expressed HLA-A2 or A24-BirA substrate peptide and β2m subunits were refolded together in vitro in the presence of synthetic antigenic peptides, corresponding to the HLA-A2-restricted melanoma-immunogenic epitopes of gp100_<(209-217)> (ITDQVPFSV) and MART-1_<(27-35)> (AAGIGILTV), and HLA-A24 restricted viral-immunogenic epitope of EBV-P (TYGPVFMCL). Folded materials were then subjected to enzymatic biotinylation with BirA enzyme. The HLA-A2 or A24/peptide complexes were purified first on a get filtration column and … More subsequently on a Mono Q ion exchange column. Tetrameric complexes of the biotinylated HLA-A2 or A24/peptide were finally produced by mixing the purified biotinylated heterodimers with Streptavidine-phycoerythrin (PE) conjugates at a molar ratio of 4 : 1.2. The specific binding of the HLA-A2/gp100_<(209-217)> and HLA-A2/MART-1_<(27-35)> tetramers were assessed by staining the relevant peptide-specific tumor-infiltrating T-lymphocytes (TILs), TIL1520 and TIL1235 respectively, and analyzing them on flow cytometry. The HLA-A2/gp100_<(209-217)> tetramer specifically stained more than 90% of TIL1520 specific for the gp100_<(209-217)> peptide, but, did not stain TIL1370 specific for irrelevant gp100_<(154-162)> (G154 : KTWGQYWQV) peptide. In addition, using HLA-A24/EBV-P tetramer, we have analyzed EBV-P specific CTL that was generated by in vitro stimulation with the peptide from peripheral blood mononuclear cells (PBMCs) of HLA-A24 healthy donor and the CTL were found to stain with the tetramer.These results indicate that the produced peptide-MHC tetramers can be used to specifically bind to antigen-specific CTL restricted by both HLA-A2 and HLA-A24, and possibly to enrich specific CTL among a heterogeneous population. Less

本项目的目的是构建可溶性I类mhc肽复合物（“四聚体”），并利用所产生的四聚体可视化黑色素瘤抗原特异性细胞毒性淋巴细胞（CTL）。我们已经生产了HLA-A2/gp100_<(209-217)>, HLA-A2/ mat -1_<(27-35)>和HLA-A24/EBV-P四聚体。β2微球蛋白（β2m）及其羧基末端与BirA底物肽连接的可溶性HLA-A2和A24重链在大肠杆菌中分别表达。在体外合成的抗原肽存在下，将表达的HLA-A2或A24-BirA底物肽和β2m亚基重新折叠在一起，对应于HLA-A2限制性黑色素瘤免疫原性表位gp100_<(290 -217)> (ITDQVPFSV)和mat -1_<(27-35)> (AAGIGILTV)和HLA-A24限制性EBV-P病毒免疫原性表位（TYGPVFMCL）。折叠后的材料用BirA酶进行生物素化处理。HLA-A2或A24/肽复合物首先在get过滤柱上纯化，然后在Mono Q离子交换柱上纯化。将纯化的生物素化异二聚体与链亲和素-植红蛋白（PE）偶联物以4∶1.2的摩尔比混合，最终得到生物素化HLA-A2或A24/肽的四聚体配合物。分别对相关肽特异性肿瘤浸润t淋巴细胞（TILs）、TIL1520和TIL1235进行染色，流式细胞术分析HLA-A2/gp100_<(209-217)>和HLA-A2/ mar -1_<(27-35)>四聚体的特异性结合情况。HLA-A2/gp100_<(209-217)>四聚体特异性染色超过90%的TIL1520特异性gp100_<(209-217)>肽，但对不相关的gp100_<(154-162)> (G154: KTWGQYWQV)肽没有特异性染色TIL1370。此外，利用HLA-A24/EBV-P四聚体，我们分析了HLA-A24健康供者外周血单个核细胞（PBMCs）在体外刺激下产生的EBV-P特异性CTL，发现CTL被四聚体染色。这些结果表明，产生的肽- mhc四聚体可用于特异性结合受HLA-A2和HLA-A24限制的抗原特异性CTL，并可能在异质群体中富集特异性CTL。少

项目成果

YUTAKA KAWAKAMI: "Recognition of shared melanoma antigens in association with major HLA-A alleles by tumor infiltrating T lymphocytes from 123 patients with melanoma"Journal of Immunotherapy. 23. 17-27 (2000)

YUTAKA KAWAKAMI：“来自 123 名黑色素瘤患者的肿瘤浸润 T 淋巴细胞对与主要 HLA-A 等位基因相关的共有黑色素瘤抗原的识别”《免疫治疗杂志》。

Y.Kawakami, X.Wang, T.Shofuda, H.Suminoto, J.P.Tupesis, E.Fitzgerald, and S.A.Rosenberg.: "Isolation of a new melanoma antigen, MART-2, containing a mutated epitope recognized by autologous tumor infiltrating T lymphocytes."J Immunology. 166. 2871-2877 (2

Y.Kawakami、X.Wang、T.Shofuda、H.Suminoto、J.P.Tupesis、E.Fitzgerald 和 S.A.Rosenberg.：“分离出一种新的黑色素瘤抗原 MART-2，其中含有可被自体肿瘤浸润 T 识别的突变表位

Kawakami, Y.: "Development of new immunotherapy using cancer antigens recognized by Tlymphocytes."New Strategy for Cancer Treatment, Proceedings of the 3rd Shizuoka Forum on Health and Longevity. 62-68 (2000)

Kawakami, Y.：“利用 T 淋巴细胞识别的癌症抗原开发新的免疫疗法。”癌症治疗新策略，第三届静冈健康与长寿论坛论文集。

Coulie PG: "Antitumor immunity at work in a melanoma"Advances in Cancer Research. 76. 213-242 (1999)

Coulie PG：“抗肿瘤免疫在黑色素瘤中发挥作用”癌症研究进展。

YUTAKA KAWAKAMI: "Identification of human melanoma antigens recognized by tumor infiltrating T lymphocytes and their use for immunotherapy"Gann Monograph on Cancer Research. 48. 179-189 (1999)

YUTAKA KAWAKAMI：“肿瘤浸润 T 淋巴细胞识别的人类黑色素瘤抗原的鉴定及其在免疫治疗中的应用”江恩癌症研究专着。