EVALUATION OF RICIN A-CHAIN IMMUNOTOXINS DIRECTED AGAINST HUMAN T-CELLS
EVALUATION OF RICIN A-CHAIN IMMUNOTOXINS DIRECTED AGAINST HUMAN T-CELLS
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DOI：
10.1016/0008-8749(86)90321-7
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发表时间：
1986-10-01
影响因子：
4.3
通讯作者：
MARTIN, PJ
中科院分区：
文献类型：
--
作者：
PRESS, OW;VITETTA, ES;MARTIN, PJ
我们通过将蓖麻毒素A链与小鼠单克隆抗体共价偶联，合成了四种免疫毒素（ITs），这些单克隆抗体可识别人T细胞表面抗原。用针对CD5 （Tp67）抗原的10.2-A或针对CD3 （Tp19）抗原的64.1-A治疗人外周血淋巴细胞，可消除随后用植物血凝素（PHA）培养的细胞中的蛋白质合成。相比之下，另外两种靶向CD2 （Tp50）抗原的ITs （9.6-A和35.1-A）对pha刺激细胞中蛋白质合成的影响最小。通过荧光素偶联山羊抗小鼠免疫球蛋白（FITC-GAMIg）和荧光素偶联兔抗蓖麻毒素a链（FITC-RAR）抗体的免疫荧光显示每种IT与T细胞的结合。蓖麻毒素A链在每个IT中的活性通过其在无细胞网织红细胞裂解试验中抑制蛋白质合成的能力来证明。IT内化的超微结构免疫过氧化物酶分析显示，无效ITs和有效ITs的内吞率相同（15分钟后50%的细胞标记了内体）。然而，无效的IT 35.1-A比有效的ITs (10.2-A和64.1-A) （.gtoreq）更快地传递到溶酶体（15-30分钟）。30分钟)。这些数据支持这样的假设，即ITs的内化有几种不同的途径，蓖麻毒素A链到达核糖体并使其失活的能力可能取决于与特定IT结合的特定膜受体、其内化途径以及IT进入溶酶体的速度。
We have synthesized four immunotoxins (ITs) by covalently coupling the A chain of ricin to murine monoclonal antibodies that recognize surface antigens on human T cells. Treatment of human peripheral blood lymphocytes with either 10.2-A, directed against the CD5 (Tp67) antigen, or 64.1-A, directed against the CD3 (Tp19) antigen, abolished protein synthesis in cells subsequently cultured with phytohemagglutinin (PHA). In contrast, two other ITs (9.6-A and 35.1-A), both directed against the CD2 (Tp50) antigen, had minimal effects on protein synthesis in PHA-stimulated cells. The binding of each IT to T cells was shown by immunofluorescence with fluorescein-conjugated goat anti-mouse immunoglobulin (FITC-GAMIg) and fluorescein-conjugated rabbit anti-ricin A-chain (FITC-RAR) antibodies. Activity of the ricin A chain in each IT was demonstrated by its ability to inhibit protein synthesis in a cell-free reticulocyte lysate assay. Ultrastructural immunoperoxidase analysis of IT internalization showed that ineffective and effective ITs were endocytosed at the same rate (50% of cells had labeled endosomes after 15 min). However, ineffective IT 35.1-A was more rapidly delivered to lysosomes (15-30 min) than effective ITs (10.2-A and 64.1-A) (.gtoreq. 30 min). The data support the hypothesis that there are several distinct pathways for internalization of ITs and that the ability of ricin A chain to reach and inactivate ribosomes may depend upon the specific membrane receptor involved in binding a given IT, its route of internalization, and the rate of entry of the IT into lysosomes.