Molecular Requirements for Recombinant Cytotoxins Efficacy

描述（由申请人提供）：在绝大多数高级别胶质瘤（HGG）患者中发现了白细胞介素13（IL 13）的限制性受体（R）。IL 13的HGG相关受体在分子上被鉴定为IL 13 R β 2，它属于一个肿瘤抗原家族，称为癌症/睾丸肿瘤抗原（CTA）。CTA为癌症的分子靶向/识别提供高特异性。此外，基于IL 13的细菌毒素，含有假单胞菌外毒素A（PE）的重组细胞毒素在临床前评价中被证明是有效的抗胶质瘤剂，并且这些细胞毒素的第一代处于III期临床试验中。IL 13的结构-功能关系分析及其最近揭示的溶液结构证明，α-螺旋D似乎在IL 13与其HGG相关受体的结合中起关键作用。提出了产生新的高度特异性和高度有效的基于IL 13的细胞毒素，其将结合（i）关于IL 13结构-功能关系的新知识，（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先前经验。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毒素DT 390的衍生物组成。这些细胞毒素设计背后的主要思想是产生新的细胞毒素，其允许IL 13的结合区域（D-螺旋）自由地可用于IL 13的HGG相关受体，并消除细胞因子上与其在许多重要器官（包括中枢神经系统）中表达的正常生理受体相互作用的位点。IL 13将被工程化以使DT毒素部分远离其与IL 13 R β 2的结合位点，这不能用PE完全实现。此外，已经鉴定了IL 13 α-螺旋D突变体，即IL 13 R β 2的超级激动剂，它们将用于优化细胞毒素的设计。细胞毒素和细胞内蛋白酶激活细菌毒素（弗林蛋白酶）的限制性结合位点的数量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，并且表达水平与新的IL 13-DT细胞毒素功效相关。将在体外和体内测试基于新型DT 390-IL 13突变体的细胞毒素，以证明在靶标和毒素的活化部分的生物表达的背景下分子设计的变化如何反映在其抗肿瘤功效中。我们希望所收集的新信息将对重组抗癌细胞毒素的进一步分子设计具有宝贵的价值。

DESCRIPTION (provided by applicant): 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 IL13R(2, 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 IL13 to 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 IL13 and 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 for IL13 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. IL13 will be engineered to have the DT toxin moiety remote form its binding site to the IL13R(2, which cannot be fully achieved with PE. Moreover, IL13 alpha-helix D mutants, super agonists of the IL13R(2, 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.