Novel anti-CD30 recombinant immunotoxins containing disulfide-stabilized Fv fragments.
Novel anti-CD30 recombinant immunotoxins containing disulfide-stabilized Fv fragments.
复制标题
含有二硫键稳定的 Fv 片段的新型抗 CD30 重组免疫毒素。
作者：
S. Nagata;M. Onda;Y. Numata;K. Santora;R. Beers;R. Kreitman;I. Pastan
目的
为了开发一种针对霍奇金氏病和间变性大细胞淋巴瘤CD30表达的新型靶向试剂，我们利用新制备的抗CD30单抗和假单胞菌外毒素M(R)38000截短突变体的Fv制备了一组针对CD30的重组免疫毒素。
实验设计
制备了一组抗CD30的单抗，并对其反应性和表位进行了鉴定。用从杂交瘤中克隆的Fv基因制备重组免疫毒素。在不同的CD30阳性细胞系上检测它们的细胞毒活性。
结果
共制备了6株单抗。它们都能与重组的可溶性CD30和CD30-Fc融合蛋白反应，并与霍奇金淋巴瘤细胞系上表达的天然CD30结合。通过与细胞表面CD30结合的相互竞争实验，将6株单抗的表位分为两组。测序表明，除了两个单抗共享的一个可验证的光之外，所有的可变链都是独一无二的。我们制备了四种二硫键稳定的Fv重组免疫毒素，其中可验证的Heavy与截短的假单胞菌外毒素突变体在基因上融合，与可验证的Light形成二硫键。纯化的免疫毒素结合在生物传感器芯片上的重组可溶性CD30上，K(D)S为4-400 nm。荧光激活的细胞分类分析证实了它们的特异性结合。体外细胞毒性试验表明，免疫毒素对多种CD30阳性淋巴瘤细胞株以及CD30基因转导的A431细胞具有特异性杀伤作用。IC50为0.3~100 ng/ml。
结论
成功制备了4种抗CD30二硫键稳定的Fv免疫毒素。其中两种化合物对各种CD30阳性细胞株表现出良好的细胞毒活性。这些新产生的免疫毒素应该额外评估CD30阳性淋巴瘤的治疗。
PURPOSE
To develop a novel targeting reagent to CD30 expressed on Hodgkin'sdisease and anaplastic large cell lymphoma, we made a panel of recombinant immunotoxins specific for CD30 using Fvs of newly produced anti-CD30 monoclonal antibodies (MAbs) and a M(r) 38,000 truncated mutant of Pseudomonas exotoxin.
EXPERIMENTAL DESIGN
A group of MAbs against CD30 was produced and characterized for their reactivity and epitopes. Recombinant immunotoxins were made using the Fv genes cloned from the hybridomas. Their cytotoxic activities were examined on various CD30-positive cell lines.
RESULTS
Six MAbs were produced. All reacted with recombinant soluble CD30 and to a CD30-Fc fusion protein, and bound to native CD30 expressed on Hodgkin's lymphoma-derived cell lines. The epitopes of the six MAbs were classified into two groups by a mutual competition assay for the binding to CD30 on cells. Sequencing the cDNAs revealed that all of the variable chains are unique except one valiable light that is shared by two MAbs. We made four disulfide stabilized Fv-based recombinant immunotoxins, in which the valiable heavy, which is genetically fused with truncated mutant of Pseudomonas exotoxin, forms a disulfide bond with the valiable light. The purified immunotoxins bound to recombinant soluble CD30 immobilized on a biosensor chip with K(d)s of 4-400 nM. Fluorescence-activated cell sorter analysis confirmed their specific binding. In vitro cytotoxicity tests showed that the immunotoxins specifically kill a variety of CD30-positive lymphoma cell lines as well as CD30-transfected A431 cells. The IC(50) ranged from 0.3 to 100 ng/ml.
CONCLUSIONS
Four anti-CD30 disulfide stabilized Fv immunotoxins were successfully produced. Two of these showed good cytotoxic activity to various CD30-positive cell lines. These newly produced immunotoxins should be additionally evaluated for the treatment of CD30-positive lymphomas.
通过 Fv 框架区突变降低等电点，减少抗 Tac(Fv)-PE38 的非特异性动物毒性。
DOI：
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发表时间：
1999
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
--
作者：
Onda,M;Kreitman,RJ;Vasmatzis,G;Lee,B;Pastan,I
通讯作者：
Pastan,I
影响因子：
3.5
作者：
HO, SN;HUNT, HD;PEASE, LR
通讯作者：
PEASE, LR
DOI：
10.1073/pnas.140210597
发表时间：
2000-07-18
影响因子：
11.1
作者：
Tsutsumi, Y;Onda, M;Pastan, I
通讯作者：
Pastan, I