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

包括化疗、放疗和手术在内的传统癌症治疗方法受到一些因素的限制，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性癌细胞而导致的复发以及不可切除肿瘤的失败。免疫毒素（ITs）作为一种靶向疗法可以通过特异性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基因融合突变的细菌或植物毒素组成的重组蛋白。该抗体引导IT结合其目标抗原，该抗原选择性地在癌细胞上表达。它随后被内化在细胞中，这使得毒素能够杀死目标细胞。一些这样的ITs正在临床试验中，denileukin difitox（商品名Ontak）已被FDA批准用于治疗皮肤t细胞淋巴瘤。然而，由于使用的植物或细菌毒素，第三代ITs存在主要缺点：(1)其潜在的免疫原性：反复使用后，患者的免疫系统可能与产生中和抗体发生反应，导致治疗效率降低。（II）由于与内皮细胞的非特异性结合，可能诱发血管渗漏综合征。（三）它们对健康细胞的脱靶活性。为了避免这些问题，一种有前途的IT策略是开发新一代的ITs，其中含有人类酶而不是植物或细菌毒素作为细胞毒性部分。在此之前，我们已经在体外开发了首个对cd30阳性和cd64阳性恶性肿瘤具有特异性细胞毒性的人细胞溶解融合蛋白（hCFP）。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来消除靶细胞。这些含黄酮的hCFP可能会成为具有广泛治疗指数的新型免疫治疗剂，前提是解决两个主要问题：(1)hCFP必须能够逃避细胞内人类胎盘rna酶抑制剂RNH1的抑制，RNH1与人类rna酶具有高亲和力。(2)必须改进HuAng的低催化活性，使hCFPs的效率更高。在本项目中，我们将通过修改HuAng的分子结构来解决这两个问题，以获得具有高催化活性和与RNH1弱结合的变体。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。这将导致合理的设计所需的黄变体。新的hCFP将使用这些变体生成。它们对目标癌细胞的特异性细胞毒性将在体外使用我们完善的实验程序进行检查。通过结合先进的计算和实验技术，本项目将成功开发黄嘌呤衍生的抗癌药物。

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.