Meng G, Jiang Y, Hubbard W, Thomas J M
University of Alabama at Birmingham, USA.
Transpl Immunol. 1998 Mar;6(1):53-9. doi: 10.1016/s0966-3274(98)80035-x.
Recent advances in the design of immunotoxins (IT) have yielded significant improvements. FN18-CRM9, a construct of anti-CD3 epsilon mAb FN18 and mutated diphtheria toxin CRM9 has exhibited high specificity, low systemic toxicity and unusual efficacy compared to previous iterations of immunotoxins. Others and we have examined this anti-CD3-IT for the purpose of inducing immunological tolerance through selective ablation of T cells in rhesus macaques and have obtained encouraging results. In order to characterize its mode of action, we have examined its effects on peripheral blood and lymph node T cell killing in vitro. We have studied the cytotoxic mechanism induced by this anti-CD3-IT as well as its effects on proliferation, phenotypic changes and cytokine production (IL2, IFN gamma and TNF alpha). The results indicate that anti-CD3-IT was highly specific for T cell killing at doses as low as 1 x 10(6) micrograms/ml and showed a maximal effect at 48 h after exposure. The toxicity was restricted to T cells, as B cells and other bystander cells were spared. This immunotoxin was shown to induce T cell apoptosis, as assessed by TUNEL assay, DNA content and cytotoxicity. Fas expression was upregulated on T cells within 24 h after in vitro exposure to anti-CD3-IT, suggesting an early T cell activation phase prior to T cell death. T cell killing was manifest as an early cell cycle arrest at the G1/S phase transition, which appeared to virtually eliminate the production of cytokines. These findings corroborate the temporal, specificity and quantitative patterns for anti-CD3 immunotoxin administration previously observed in vivo.
免疫毒素（IT）设计方面的最新进展已带来显著改善。FN18-CRM9是抗CD3ε单克隆抗体FN18与突变白喉毒素CRM9的构建体，与之前的免疫毒素迭代产品相比，它表现出高特异性、低全身毒性和非凡疗效。我们和其他人已研究了这种抗CD3免疫毒素，目的是通过选择性清除恒河猴体内的T细胞来诱导免疫耐受，并取得了令人鼓舞的结果。为了阐明其作用方式，我们在体外研究了它对外周血和淋巴结T细胞杀伤的影响。我们研究了这种抗CD3免疫毒素诱导的细胞毒性机制及其对增殖、表型变化和细胞因子产生（IL-2、IFN-γ和TNF-α）的影响。结果表明，抗CD3免疫毒素在低至1×10⁻⁶微克/毫升的剂量下对T细胞杀伤具有高度特异性，且在暴露后48小时显示出最大效应。毒性仅限于T细胞，因为B细胞和其他旁观者细胞未受影响。通过TUNEL检测、DNA含量和细胞毒性评估表明，这种免疫毒素可诱导T细胞凋亡。体外暴露于抗CD3免疫毒素后24小时内，T细胞上的Fas表达上调，这表明在T细胞死亡之前存在一个早期T细胞激活阶段。T细胞杀伤表现为在G1/S期转换时早期细胞周期停滞，这似乎几乎消除了细胞因子的产生。这些发现证实了先前在体内观察到的抗CD3免疫毒素给药的时间、特异性和定量模式。
