Effector molecules of the apoptosis-inducing signaling cascade as active components in cytotoxic antibody fusion proteins for cancer therapy

该项目的主要目标是产生高活性的免疫毒素样抗体融合蛋白，利用人类来源的细胞-死亡诱导效应蛋白来消除肿瘤细胞。利用细菌毒素作为细胞毒效应的重组分子的临床研究证明了免疫毒素在癌症治疗中的潜在用途。然而，这些研究也发现，细菌蛋白结构域的高免疫原性是一个关键限制因素。我们之前描述了原型，人源化抗体融合蛋白，接近当前免疫毒素的高度特异性和细胞杀伤活性。这些分子含有人类促凋亡丝氨酸蛋白酶颗粒酶B(GRB)作为细胞毒性效应因子，与与临床相关的肿瘤相关抗原ErbB2和EGF受体结合的细胞识别结构域融合。基于这些原型分子，我们将引入定向修饰，旨在消除效应域剩余的非特异性细胞结合活性，并将特异性细胞识别扩展到肿瘤细胞上的其他重要表面抗原，如CD20和EpCAM。此外，通过包括额外的功能结构域，可以改善摄取后内体的有效释放，以及细胞内到关键靶底物的路线。为了增强抗肿瘤活性，将结合单独的修饰，这种优化的分子将在体内的人类肿瘤异种移植模型中进行测试。我们期望在该项目中建立的原则不仅将导致肿瘤特异性GRB融合蛋白适合于进一步开发用于临床应用，而且还将为改进来自其他人类促凋亡分子的免疫毒素样试剂提供基础。

Major objective of this project is the generation of highly active, immunotoxin-like antibody fusion proteins, that utilize a cell-death inducing effector protein of human origin to eliminate tumor cells. The potential utility of immunotoxins for cancer therapy was demonstrated in clinical studies with recombinant molecules, that employ a bacterial toxin as a cytotoxic effector. However, these studies also identified the high immunogenicity of the bacterial protein domain as a critical limitation. We previously described prototypic, humanized antibody fusion proteins, that approach the high specificity and cell killing activity of current immunotoxins. Such molecules contain the human pro-apoptotic serine protease Granzyme B (GrB) as a cytotoxic effector, fused to cell recognition domains that bind to the clinically relevant tumor-associated antigens ErbB2 and EGF receptor. Based on such prototypic molecules, we will introduce well directed modifications intended to eliminate remaining nonspecific cell binding activity of the effector domain, and extend specific cell recognition to other important surface antigens on tumor cells such as CD20 and EpCAM. Furthermore, efficient release from endosomes after uptake, and intracellular routing to critical target substrates will be improved by including additional functional domains. To enhance antitumoral activity, individual modifications will be combined, and such optimized molecules will be tested in human tumor xenograft models in vivo. We expect that principles established during this project will not only result in tumor-specific GrB fusion proteins suitable for further development towards clinical applications, but will also provide a basis to improve immunotoxin-like reagents derived from other human pro-apoptotic molecules.