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”）并使用所产生的四聚体可视化黑素瘤抗原特异性细胞毒性淋巴细胞（CTL）。我们制备了HLA-A2/gp 100_<（209-217）>、HLA-A2/MART-1_<（27-35）>和HLA-A24/EBV-P四聚体。在大肠杆菌中分别表达β2-微球蛋白（β 2 m）和可溶性HLA-A2和A24重链，其羧基末端连接到BirA底物肽。表达的HLA-A2或A24-BirA底物肽和β 2 m亚基在合成抗原肽存在下在体外重折叠在一起，所述合成抗原肽对应于HLA-A2限制性黑素瘤免疫原性表位gp 100 <（209-217）>（ITDQVPFSV）和MART-1 <（27-35）>（AAGIGILTV）以及HLA-A24限制性EBV-P病毒免疫原性表位（TYGPVFMCL）。然后用BirA酶对折叠的材料进行酶促生物素化。首先在凝胶过滤柱上纯化HLA-A2或A24/肽复合物， ...更多信息 随后在Mono Q离子交换柱上。生物素化的HLA-A2或A24/肽的四聚体复合物最终通过将纯化的生物素化的异源二聚体与链霉亲和素-藻红蛋白（PE）缀合物以4：1.2的摩尔比混合来产生。通过对肿瘤浸润性T淋巴细胞（TIL）、TIL 1520和TIL 1235进行相关肽特异性染色并在流式细胞仪上分析，来评估HLA-A2/gp 100 <（209-217）>和HLA-A2/MART-1 <（27-35）>四聚体的特异性结合。HLA-A2/gp 100_<（209-217）>四聚体对gp 100_<（209-217）>肽特异性染色超过90%的TIL 1520，但对不相关的gp 100_<（154-162）>（G154：KTWGQYWQV）肽特异性不染色TIL 1370。此外，使用HLA-A24/EBV-P四聚体，我们分析了由来自HLA-A24健康供体的外周血单个核细胞（PBMC）的肽体外刺激产生的EBV-P特异性CTL，发现CTL被四聚体染色。这些结果表明所产生的肽-MHC四聚体可用于特异性结合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。