Molecular Requirements for Recombinant Cytotoxins Efficacy

在绝大多数高级别胶质瘤（HGG）中发现了白细胞介素13（IL 13）的限制性受体（R）。 患者IL 13的HGG相关受体在分子上被鉴定为IL 13 Ra 2，并且其属于 肿瘤抗原家族，称为癌症/睾丸肿瘤抗原（CTA）。CTA提供了高特异性， 分子靶向/识别癌症。此外，基于IL 13的细菌毒素、假单胞菌外毒素 含A（PE）的重组细胞毒素被证明是前胶质瘤中有效的抗胶质瘤药物。 这些细胞毒素的第一代正在进行III期临床试验。结构- IL 13的功能关系分析及其最近揭示的溶液结构证明，α-螺旋 D在IL 13与HGG相关受体结合中起关键作用。提出要 产生新的高度特异性和高度有效的基于IL 13的细胞毒素， 关于IL 13结构-功能关系的知识，（ii）关于神经胶质瘤细胞生物学的新信息，和（iii） 在临床环境中使用细胞毒素的既往经验。因此，这些新的细胞毒素将 由基因工程形式的IL 13和另一种细菌毒素白喉的衍生物组成 DT 390毒素设计这些细胞毒素背后的主要思想是产生新的细胞毒素， 使IL-13的结合区，D-螺旋，可自由地被HGG相关的IL-13受体利用 并消除细胞因子上与表达的正常生理受体相互作用的位点 包括中枢神经系统在内的许多重要器官中IL 13将被改造成具有DT毒素 在一些实施方案中，IL-13 Rx 2可以与远离其与IL 13 Rx 2的结合位点的部分结合，这不能用PE完全实现。此外，IL 13 已经鉴定了α-螺旋D突变体，IL 13 Ra 2的超级激动剂，并且它们将用于本发明。 优化细胞毒素的设计。细胞毒素的限制性结合位点的数量和 细胞内蛋白酶激活细菌毒素（弗林蛋白酶）在细胞毒素的肿瘤细胞杀伤中起主要作用。 因此，将检查它们的表达水平的调节，并将表达水平与表达水平相关。 新的IL 13-DT细胞毒素功效。新的基于DT 390-IL 13突变体的细胞毒素将在体外进行测试， 为了证明在生物学背景下分子设计中的变化如何 靶和毒素的活化部分的表达反映在它们的抗肿瘤功效中。预计 收集的新信息将在重组抗癌药物的进一步分子设计中发挥重要作用。 细胞毒素

A restricted receptor (R) for interleukin 13 (IL13) was found in a vast majority of high-grade gliomas (HGG) patients. The HGG-associated receptor for IL13 was identified molecularly to be the IL13Ra2, and it belongs to a family of tumor antigens, termed cancer/testis tumor antigens (CTA). CTA provide high specificity for molecular targeting/recognition of cancer. Furthermore, IL13-based bacterial toxin, Pseudomonas exotoxin A (PE)-containing recombinant cytotoxins were documented to be the potent anti-glioma agents in pre- clinical evaluation, and the first generation of these cytotoxins is in Phase III clinical trials. The structure- function relationship analysis of IL13 and its recently revealed solution structure documented that alpha-helix D appears to play pivotal role in the binding of IL13to its HGG-associated receptor. It is proposed to generate novel highly specific and highly efficacious IL13-based cytotoxins that will incorporate (i) new knowledge on IL13 structure-function relationship, (ii) new information on glioma cell biology, and (iii) previous experience with the use of cytotoxins in a clinical setting. Thus, these new cytotoxins will be composed of genetically engineered forms of IL13and a derivative of another bacterial toxin, Diphtheria toxin, DT390. The principal idea behind the design of these cytotoxins is to produce novel cytotoxins that allow the binding region of IL13, the D-helix, to be freely available to the HGG-associated receptor forIL13 and to eliminate the site on the cytokine that interacts with its normal physiological receptor that is expressed in many vital organs, including the central nervous system. IL13will be engineered to have the DT toxin moiety remote form its binding site to the IL13R<x2, which cannot be fully achieved with PE. Moreover, IL13 alpha-helix D mutants, super agonists of the IL13Ra2, have been identified and they will be used in the design of optimized cytotoxins. The number of the restricted binding sites for the cytotoxins and an intracellular protease activating bacterial toxins (furin) play major roles in the cytotoxins' tumor cell killing. Thus, the regulation of their expression levels will be examined and the expression levels correlated with the novel IL13-DT cytotoxins efficacy. The novel DT390-IL13 mutant-based cytotoxins will be tested in vitro and in vivo in order to demonstrate how the changes in the molecular design in the context of biological expression of the target and toxin's activating moiety are reflected in their anti-tumor efficacy. It is expected that the new information gathered will be invaluable in further molecular design of recombinant anti-cancer cytotoxins.