Molecular modification of a recombinant anti-CD3epsilon-directed immunotoxin by inducing terminal cysteine bridging enhances anti-GVHD efficacy and reduces organ toxicity in a lethal murine model.
Molecular modification of a recombinant anti-CD3epsilon-directed immunotoxin by inducing terminal cysteine bridging enhances anti-GVHD efficacy and reduces organ toxicity in a lethal murine model.
复制标题
DOI：
10.1182/blood.v96.3.1157
复制
发表时间：
2000-08
期刊：
影响因子：
20.3
通讯作者：
D. Vallera;D. Kuroki;A. Panoskaltsis‐Mortari;D. Buchsbaum;B. Rogers;B. Blazar
中科院分区：
文献类型：
--
作者：
D. Vallera;D. Kuroki;A. Panoskaltsis‐Mortari;D. Buchsbaum;B. Rogers;B. Blazar
免疫毒素（IT）疗法显示出在体内选择性消除引起GVHD的T细胞的潜力，但该领域一直受到毒性的阻碍。以前，我们表明，由单链蛋白组成的基因工程IT，包括与白喉毒素（DT（390））的一部分剪接的抗CD 3sFv，具有抗GVHD作用，但这类药物常见的明显器官毒性。对先前显示具有抗GVHD活性的重组DT（390）抗CD 3sFv蛋白进行修饰，通过在蛋白c-末端sFv部分下游基因插入半胱氨酸残基，减少其向肾脏的滤过。这种修饰产生了一个分子间二硫键，产生了一个二价的，而不是一个单价的IT，称为SS 2，选择性地抑制体外T细胞增殖。尽管单体和SS 2在体外活性方面相似，但SS 2在体内具有更好的上级治疗指数，耐受性至少高8倍，肾毒性降低。最重要的是，在致死性GVHD模型中，给予40微克SS 2 1天，保护100%的小鼠免于致死性GVHD 3个月，而单体的最大耐受剂量（MTD）仅保护33%。据我们所知，这是第一次以这种方式产生二硫键结合的IT，这种简单的分子修饰可以解决IT领域的几个问题，因为它（1）在每天一次治疗后显著增加了治愈小鼠GVHD的功效，（2）显著降低了器官毒性，（3）增加了耐受剂量，以及（4）创造了以前不存在的治疗窗口。
Immunotoxin (IT) therapy shows potential for selectively eliminating GVHD-causing T cells in vivo, but the field has been hampered by toxicity. Previously, we showed that a genetically engineered IT consisting of a single-chain protein, including the anti-CD3sFv spliced to a portion of diphtheria-toxin (DT(390)) has anti-GVHD effects, but pronounced organ toxicity common to this class of agent. A recombinant DT(390) anti-CD3sFv protein previously shown to have anti-GVHD activity was modified to reduce its filtration into kidney by genetically inserting a cysteine residue downstream of the sFv moiety at the c-terminus of the protein. This modification produced an intermolecular disulfide bridge, resulting in a bivalent, rather than a monovalent IT, termed SS2, that selectively inhibited T-cell proliferation in vitro. Although monomer and SS2 were similar in in vitro activity, SS2 had a superior therapeutic index in vivo with at least 8-fold more being tolerated with reduced kidney toxicity. Most importantly, in a lethal model of GVHD, 40 microg SS2 given for 1 day, protected 100% of the mice from lethal GVHD for 3 months, whereas the maximum tolerated dose (MTD) of monomer protected only 33%. To our knowledge, this is the first time disulfide bonded ITs have been created in this way and this simple molecular modification may address several problems in the IT field because it (1) markedly increased efficacy curing mice of GVHD after a single daily treatment, (2) markedly decreased organ toxicity, (3) increased the tolerated dosage, and (4) created a therapeutic window where none existed before.