人源化双抗体-RNase融合蛋白对CD22+肿瘤细胞的高效杀伤作用

Efficient killing of CD22+ tumor cells by a humanized diabody-RNase fusion protein.

作者信息

Krauss Jürgen, Arndt Michaela A E, Vu Bang K, Newton Dianne L, Seeber Siegfried, Rybak Susanna M

机构信息

Department of Medical Oncology and Cancer Research, University of Essen, D-45122 Essen, Germany.

出版信息

Biochem Biophys Res Commun. 2005 Jun 3;331(2):595-602. doi: 10.1016/j.bbrc.2005.03.215.

DOI:10.1016/j.bbrc.2005.03.215

PMID:15850802

Abstract

We report on the generation of a dimeric immunoenzyme capable of simultaneously delivering two ribonuclease (RNase) effector domains on one molecule to CD22(+) tumor cells. As targeting moiety a diabody derived from the previously humanized scFv SGIII with grafted specificity of the murine anti-CD22 mAb RFB4 was constructed. Further engineering the interface of this construct (V(L)36(Leu-->Tyr)) resulted in a highly robust bivalent molecule that retained the same high affinity as the murine mAb RFB4 (K(D)=0.2 nM). A dimeric immunoenzyme comprising this diabody and Rana pipiens liver ribonuclease I (rapLRI) was generated, expressed as soluble protein in bacteria, and purified to homogeneity. The dimeric fusion protein killed several CD22(+) tumor cell lines with high efficacy (IC(50)=3-20 nM) and exhibited 9- to 48-fold stronger cytotoxicity than a monovalent rapLRI-scFv counterpart. Our results demonstrate that engineering of dimeric antibody-ribonuclease fusion proteins can markedly enhance their biological efficacy.

摘要

我们报道了一种二聚体免疫酶的产生，该酶能够在一个分子上同时将两个核糖核酸酶（RNase）效应结构域递送至CD22(+)肿瘤细胞。作为靶向部分，构建了一种双抗体，其来源于先前人源化的scFv SGIII，并移植了鼠抗CD22单克隆抗体RFB4的特异性。对该构建体的界面进行进一步工程改造（V(L)36(Leu→Tyr)），得到了一种高度稳定的二价分子，其保留了与鼠单克隆抗体RFB4相同的高亲和力（K(D)=0.2 nM）。产生了一种包含该双抗体和牛蛙肝脏核糖核酸酶I（rapLRI）的二聚体免疫酶，在细菌中表达为可溶性蛋白，并纯化至同质。该二聚体融合蛋白高效杀死了几种CD22(+)肿瘤细胞系（IC(50)=3-20 nM），并且比单价rapLRI-scFv对应物表现出强9至48倍的细胞毒性。我们的结果表明，二聚体抗体-核糖核酸酶融合蛋白的工程改造可显著增强其生物学功效。