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

包括化疗、放疗和手术在内的癌症常规治疗受到几个因素的限制，例如耐药性、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导致的复发和不可切除肿瘤的失败。免疫毒素（IT）作为靶向治疗可以通过特异性杀死癌细胞来解决这些问题。目前的第三代IT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组成的重组蛋白。该抗体指导IT结合其靶抗原，该抗原选择性地在癌细胞上表达。IT随后在细胞中内化，这使得毒素能够杀死靶细胞。几种这样的IT正在进行临床试验，并且地尼白介素（商品名Ontak）已被FDA批准用于治疗皮肤T细胞淋巴瘤。然而，第三代IT由于所使用的植物或细菌毒素而具有主要缺点：（I）其潜在的免疫原性：在重复应用时，患者的免疫系统可以与中和抗体的产生反应，导致治疗效率降低。(II)由于与内皮细胞的非特异性结合，它们可能诱导血管渗漏综合征。(III)它们对健康细胞的脱靶活性。为了避免这些问题，一个有前途的IT策略是开发新一代的IT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，我们已经开发了第一个人溶细胞融合蛋白（hCFP），在体外对CD 30阳性和CD 64阳性恶性肿瘤具有有希望的特异性细胞毒性。这些hCFP含有人胰腺核糖核酸酶（RNase）血管生成素（HuAng），其通过降解靶细胞的RNA来消除靶细胞。这些含HuAng-hCFP可能导致具有广泛治疗指数的新型免疫抑制剂，前提是解决两个主要问题：（I）hCFP必须能够逃避细胞内人胎盘RNA酶抑制剂RNH 1的抑制，RNH 1以高亲和力结合人RNA酶。(II)必须改善HuAng的低催化活性以实现hCFPs的高效率。在本项目中，我们将通过修饰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RNH 1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华昂变种。使用这些变体将产生新的hCFP。将使用我们成熟的实验程序在体外检查它们对靶癌细胞的特异性细胞毒性。通过结合先进的计算和实验技术，该项目将导致成功开发黄衍生的抗癌药物。

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.