Identification and elimination of an immunodominant T-cell epitope in recombinant immunotoxins based on Pseudomonas exotoxin A
Identification and elimination of an immunodominant T-cell epitope in recombinant immunotoxins based on Pseudomonas exotoxin A
复制标题
DOI：
10.1073/pnas.1218138109
复制
发表时间：
2012-12-18
影响因子：
11.1
通讯作者：
Pastan, Ira
中科院分区：
文献类型：
--
作者：
Mazor, Ronit;Vassall, Aaron N.;Pastan, Ira
重组免疫毒素（RIT）是正在开发用于癌症治疗的嵌合蛋白。我们已经生产了含有PE 38的RIT，PE 38是细菌蛋白假单胞菌外毒素A的一部分。由于毒素是细菌性的，它通常会诱导中和抗体，这限制了治疗周期的数量和治疗的有效性。因为T细胞对于抗体对蛋白质的应答是必需的，所以我们采用了一种测定法来定位PE 38中的CD 4（+）T细胞表位。我们将外周血单核细胞与免疫毒素一起孵育以刺激T细胞扩增，然后暴露于PE 38的重叠肽片段和IL-2 ELISpot测定以测量反应。我们在50个个体中的50个个体中观察到的T细胞反应与具有正常免疫系统的患者的抗体形成频率相关。我们在46%（23/50）的供体中发现了一个单一的高度免疫显性表位。免疫显性表位是DRB 1限制性的，在具有不同HLA等位基因的受试者中观察到，表明滥交。我们鉴定了两种氨基酸，当缺失或突变为丙氨酸时，消除了免疫显性表位，并且我们使用这些信息来构建具有高度细胞毒性并且在许多供体中不刺激T细胞应答的突变RIT。
Recombinant immunotoxins (RITs) are chimeric proteins that are being developed for cancer treatment. We have produced RITs that contain PE38, a portion of the bacterial protein Pseudomonas exotoxin A. Because the toxin is bacterial, it often induces neutralizing antibodies, which limit the number of treatment cycles and the effectiveness of the therapy. Because T cells are essential for antibody responses to proteins, we adopted an assay to map the CD4(+) T-cell epitopes in PE38. We incubated peripheral blood mononuclear cells with an immunotoxin to stimulate T-cell expansion, followed by exposure to overlapping peptide fragments of PE38 and an IL-2 ELISpot assay to measure responses. Our observation of T-cell responses in 50 of 50 individuals correlates with the frequency of antibody formation in patients with normal immune systems. We found a single, highly immunodominant epitope in 46% (23/50) of the donors. The immunodominant epitope is DRB1-restricted and was observed in subjects with different HLA alleles, indicating promiscuity. We identified two amino acids that, when deleted or mutated to alanine, eliminated the immunodominant epitope, and we used this information to construct mutant RITs that are highly cytotoxic and do not stimulate T-cell responses in many donors.