Izquierdo M, Balboa M A, Lamana M L, López-Botet M
Department of Immunology, Hospital de la Princesa, Madrid, Spain.
Int J Cancer. 1989 Apr 15;43(4):697-702. doi: 10.1002/ijc.2910430426.
Immunotoxins (ITs) were prepared by covalently coupling ricin to monoclonal antibodies (MAbs) directed against: (a) 2 different epitopes of the T-cell receptor (TcR) expressed by the Jurkat leukemia T-cell line (JTi2 and JTi4 MAb), (b) 2 epitopes of the CD3 complex (SpV-T3b and 11D8 MAb), (c) the CD2 and the CD8 cell-surface molecules. Conjugates were assayed for their cytotoxic activity by pre-incubating the Jurkat cell line with different concentrations (10-250 ng/ml) of each IT for 2 hr at 37 degrees C in the presence of 0.1 M lactose. After washing, cells were cultured for 24 hr and their protein synthesis and proliferative capacities were assessed. Dose-response experiments indicated that JTi2, JTi4 and anti-CD3 (11D8) ITs inhibited by greater than 90% the cell line proliferation at 50 ng/ml, a 5-fold lower concentration than that required to achieve a similar effect when anti-CD2 and anti-CD3 (SpVT3b) were used. After 4 hr of culture subsequent to treatment with JTi2 or JTi4 ITs (250 ng/ml), protein synthesis was inhibited (greater than 80%). By limiting dilution analysis (LDA) we estimated that the frequency of proliferating Jurkat cells (1/1.5) was reduced to 1/20, 1/460 and 1/300 after treatment with anti-CD3 (SpVT3b), JTi4 and JTi2 ITs, respectively. Phenotypic analysis of 13 clones derived from JTi2 IT-treated Jurkat cells showed that 50% were CD7+ CD3- JTi- variants. When bone-marrow mononuclear cells, previously mixed with low concentrations of Jurkat cells, were treated with anti-JTi ITs, the toxic efficiency estimated by LDA was maintained whereas the growth of CFU-GM remained unaltered.
免疫毒素（ITs）是通过将蓖麻毒素与针对以下靶点的单克隆抗体（MAbs）共价偶联制备而成：（a）由Jurkat白血病T细胞系表达的T细胞受体（TcR）的2个不同表位（JTi2和JTi4单克隆抗体），（b）CD3复合物的2个表位（SpV-T3b和11D8单克隆抗体），（c）CD2和CD8细胞表面分子。通过在0.1M乳糖存在下，于37℃将Jurkat细胞系与不同浓度（10 - 250ng/ml）的每种IT预孵育2小时，来检测缀合物的细胞毒性活性。洗涤后，将细胞培养24小时，并评估其蛋白质合成和增殖能力。剂量反应实验表明，JTi2、JTi4和抗CD3（11D8）ITs在50ng/ml时可抑制细胞系增殖超过90%，这一浓度比使用抗CD2和抗CD3（SpVT3b）时达到类似效果所需浓度低5倍。在用JTi2或JTi4 ITs（250ng/ml）处理后培养4小时，蛋白质合成受到抑制（超过80%）。通过有限稀释分析（LDA），我们估计在用抗CD3（SpVT3b）、JTi4和JTi2 ITs处理后，增殖的Jurkat细胞频率（1/1.5）分别降至1/20、1/460和1/300。对源自JTi2 IT处理的Jurkat细胞的13个克隆进行表型分析表明，50%是CD7 + CD3 - JTi - 变体。当预先与低浓度Jurkat细胞混合的骨髓单个核细胞用抗JTi ITs处理时，通过LDA估计的毒性效率得以维持，而CFU - GM的生长保持不变。
