Press O W, Vitetta E S, Farr A G, Hansen J A, Martin P J
Cell Immunol. 1986 Oct 1;102(1):10-20. doi: 10.1016/0008-8749(86)90321-7.
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) (greater than or equal to 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.
我们通过将蓖麻毒素A链与识别人类T细胞表面抗原的鼠单克隆抗体共价偶联，合成了四种免疫毒素（ITs）。用针对CD5（Tp67）抗原的10.2-A或针对CD3（Tp19）抗原的64.1-A处理人外周血淋巴细胞，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，另外两种均针对CD2（Tp50）抗原的ITs（9.6-A和35.1-A）对PHA刺激的细胞中的蛋白质合成影响极小。通过用异硫氰酸荧光素偶联的山羊抗小鼠免疫球蛋白（FITC-GAMIg）和异硫氰酸荧光素偶联的兔抗蓖麻毒素A链（FITC-RAR）抗体进行免疫荧光，显示了每种IT与T细胞的结合。通过其在无细胞网织红细胞裂解物测定中抑制蛋白质合成的能力，证明了每种IT中蓖麻毒素A链的活性。对IT内化的超微结构免疫过氧化物酶分析表明，无效和有效的ITs以相同的速率被内吞（15分钟后50%的细胞有标记的内体）。然而，无效的IT 35.1-A比有效的ITs（10.2-A和64.1-A）更快地被递送至溶酶体（15 - 30分钟）（有效ITs为大于或等于30分钟）。数据支持这样的假设，即ITs有几种不同的内化途径，并且蓖麻毒素A链到达并使核糖体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进入溶酶体的速率。
