Computer-aided design of novel immunotoxins for the treatment of malignant diseases

计算机辅助设计治疗恶性疾病的新型免疫毒素

• 批准号: 243918739
• 负责人: Professor Dr. Stefan Barth
• 金额: --
• 依托单位: Bereich Computational Biomedicine (IAS-5/INM-9)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/243918739?language=en
• 关键词: Computer; aided; design; novel; immunotoxins

Conventional treatments for cancer including chemotherapy, radiation and surgery have been limited by several factors such as drug resistance, non-specificity deleterious to normal cells, relapse due to the often-remaining metastatic cancer cells and failure in non-resectable tumors. Immunotoxins (ITs) as a target therapy can combat these problems by specifically killing cancer cells. The current third-generation ITs are recombinant proteins composed of a cell-selective recombinant antibody fragment genetically fused to a mutated bacterial or plant toxin. The antibody directs the IT to bind its target antigen, which is selectively expressed on cancer cells. The IT is subsequently internalized in the cells, which enables the toxin to kill the target cells. Several such ITs are in clinical trials and denileukin difitox (trade name Ontak) has been approved by FDA for treatment of cutaneous T-cell lymphoma. However, third-generation ITs suffer from major drawbacks due to the plant or bacteria toxins used: (I) their potential immunogenicity: upon repeated application, the immune system of the patients can react with the generation of neutralizing antibodies leading to reduced therapeutic efficiency. (II) Their potential in inducing vascular leak syndrome, due to non-specific binding to endothelial cells. (III) Their off-target activities towards healthy cells. To circumvent these problems, a promising IT strategy is to develop a new generation of ITs containing human enzymes instead of plant or bacteria toxins as the cytotoxic moiety. Previously, we have developed the first human cytolytic fusion proteins (hCFP) with promising specific cytotoxicity for CD30-positive and CD64-positive malignancies in vitro. These hCFP contain the human pancreatic ribonuclease (RNase) angiogenin (HuAng), which eliminates the target cells by degrading their RNA. These HuAng-containing hCFP may lead to novel immunotherapeutic agents with an extensive therapeutic index, provided that two major issues are resolved: (I) The hCFPs must be able to evade the inhibition of intracellular human placental RNase inhibitor RNH1 which binds with high affinity to human RNases. (II) The low catalytic activity of HuAng must be improved to enable high efficiency of the hCFPs. In this project, we will address these two issues by modifying the molecular structure of HuAng to obtain variants with both high catalytic activity and weak binding to RNH1. Based on known experimental data, computational approaches will enable us to investigate the molecular mechanism of such catalysis and binding. This will lead to rational design of the desired HuAng variants. Novel hCFP will be generated using these variants. Their specific cytotoxicity towards target cancer cells will be examined in vitro using our well-established experimental procedure. By combining the advanced computational and experimental techniques, this project will lead to successful development of HuAng-derived anti-cancer agents.

肿瘤的常规治疗包括化疗、放射治疗和手术治疗，受到多种因素的限制，如耐药性、对正常细胞有害的非特异性、由于经常残留的转移癌细胞而导致的复发以及对不可切除肿瘤的失败。免疫毒素(ITS)作为一种靶向治疗方法，可以通过特异性地杀死癌细胞来对抗这些问题。目前的第三代ITS是由细胞选择性重组抗体片段与突变的细菌或植物毒素基因融合而成的重组蛋白。该抗体指导IT结合其目标抗原，该抗原选择性地表达在癌细胞上。随后，IT被内化到细胞中，这使得毒素能够杀死目标细胞。几种这样的ITS正在进行临床试验，Deileukin difitox(商标为Ontak)已被FDA批准用于治疗皮肤T细胞淋巴瘤。然而，第三代ITS的主要缺点是所使用的植物或细菌毒素：(I)其潜在的免疫原性：反复应用时，患者的免疫系统可能会与产生的中和抗体发生反应，从而降低治疗效率。(2)由于与内皮细胞的非特异性结合，它们有可能导致血管渗漏综合征。(3)它们对健康细胞的非目标活动。为了绕过这些问题，一个有前途的IT策略是开发新一代ITS，其中包含人类酶，而不是植物或细菌毒素作为细胞毒部分。在此之前，我们已经开发了第一个人细胞溶解融合蛋白(HCFP)，在体外对CD30阳性和CD64阳性的恶性肿瘤具有潜在的特异性细胞毒作用。这些HCFP含有人胰腺核糖核酸酶(RNase)血管生成素(Huang)，它通过降解靶细胞的RNA来消除它们。如果解决两个主要问题：(1)HCFPS必须能够逃避细胞内与人RNase高亲和力结合的人胎盘RNase抑制物RNH1的抑制；(2)这些含黄素的HCFP可能导致具有广泛治疗指数的新型免疫治疗药物。(Ii)必须改善黄原胶的低催化活性，才能使氟氯烃氟氯烃生产效率高。在本项目中，我们将通过改变黄素的分子结构来解决这两个问题，以获得既具有高催化活性又与RNH1结合较弱的变异体。基于已知的实验数据，计算方法将使我们能够研究这种催化和结合的分子机制。这将导致所需的黄变种的合理设计。使用这些变体将产生新的HCFP。它们对靶癌细胞的特异性细胞毒性将使用我们成熟的实验程序在体外进行检测。通过将先进的计算和实验技术相结合，该项目将导致成功开发黄衍生抗癌药物。