Duke-Cohan J S, Morimoto C, Schlossman S F
Division of Tumor Immunology, Dana-Farber Cancer Institute, Boston, MA 02115.
Blood. 1993 Oct 1;82(7):2224-34.
We have developed a bispecific antibody that recognizes the CD4 and CD26 antigens simultaneously and that was examined for its ability to target CD4+CD26+T cells. These latter cells constitute the activated component of the CD4+ CD29highCD45RO+ memory T-cell subset that provides help for B-cell Ig synthesis and help for responses against recall antigens. The purified bispecific antibody exhibited an estimated dissociation constant (kd) of 2.4 x 10(-9) mol/L, on comparison with 1.1 x 10(-9) mol/L for anti-CD26, and 1.6 x 10(-10) mol/L for anti-CD4. Surface plasmon resonance was used to show the bifunctional capacity of the antibody. On binding 125I-bispecific antibody to phytohemagglutinin (PHA)-activated T cells, 54.4% of the bound antibody was internalized. This was the result of bispecific binding, because monovalent fragments of anti-CD4 and anti-CD26 were not able to modulate antigen or induce internalization using both a fluorescent assay and an 125I-internalization assay. The ability of the bispecific antibody to be internalized was used to deliver a toxin, blocked ricin, specifically to cells that are CD4+CD26+. The inability of monovalent fragments to be internalized formed the basis for our hypothesis that monovalent binding by the bispecific immunotoxin would not result in internalization. Against resting E+ T cells, the bispecific immunotoxin developed a minimal effect. On preactivating the same cells, using phorbol myristate acetate (PMA)/ionomycin on concanavalin A (ConA) or especially PHA, levels of CD26 were upregulated and the immunotoxin effectively inhibited the ability to provide help for B-cell Ig synthesis while leaving intact the CD4-CD26+ and CD4+CD26- populations; an effect observed both functionally and by phenotype. The bispecific antibody proved to be most effective at inhibiting a heterologous mixed leukocyte reaction. We propose that this reagent may form the basis for the rational design of toxins designed to modulate activated T cells from, or directed against, tissue grafts.
我们研发了一种双特异性抗体，它能同时识别CD4和CD26抗原，并对其靶向CD4+CD26+T细胞的能力进行了检测。后一种细胞构成了CD4+CD29highCD45RO+记忆T细胞亚群的活化成分，该亚群为B细胞免疫球蛋白合成提供帮助，并对回忆抗原的反应提供帮助。纯化后的双特异性抗体的解离常数（kd）估计为2.4×10（-9）mol/L，相比之下，抗CD26抗体的解离常数为1.1×10（-9）mol/L，抗CD4抗体的解离常数为1.6×10（-10）mol/L。表面等离子体共振用于显示该抗体的双功能能力。将125I标记的双特异性抗体与植物血凝素（PHA）激活的T细胞结合后，54.4%的结合抗体被内化。这是双特异性结合的结果，因为抗CD4和抗CD26的单价片段在荧光检测和125I内化检测中均无法调节抗原或诱导内化。双特异性抗体被内化的能力被用于将一种毒素——封闭的蓖麻毒素——特异性地递送至CD4+CD26+细胞。单价片段无法被内化，这构成了我们的假设基础，即双特异性免疫毒素的单价结合不会导致内化。对于静止的E+T细胞，双特异性免疫毒素产生的影响极小。在用佛波酯肉豆蔻酸酯乙酸盐（PMA）/离子霉素或伴刀豆球蛋白A（ConA），尤其是PHA预激活相同细胞后，CD26水平上调，免疫毒素有效抑制了为B细胞免疫球蛋白合成提供帮助的能力，同时使CD4-CD26+和CD4+CD26-群体保持完整；这一效应在功能和表型上均有观察到。双特异性抗体在抑制异源混合淋巴细胞反应方面被证明是最有效的。我们认为，这种试剂可能为合理设计旨在调节来自组织移植物或针对组织移植物的活化T细胞的毒素奠定基础。
