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）患者中发现白细胞介素13 （IL13）的限制性受体(R)。从分子上鉴定出IL13的hgg相关受体为IL13R(2)，它属于肿瘤抗原家族，称为癌症/睾丸肿瘤抗原（CTA）。CTA为肿瘤的分子靶向/识别提供了高特异性。此外，基于il - 13的细菌毒素，含假单胞菌外毒素A （PE）的重组细胞毒素在临床前评估中被证明是有效的抗胶质瘤药物，第一代这些细胞毒素正在III期临床试验中。IL13的结构-功能关系分析及其最近发现的溶液结构表明，α -螺旋D似乎在IL13与其hgg相关受体的结合中起关键作用。我们建议产生新的高特异性和高效的基于IL13的细胞毒素，这些细胞毒素将结合(i)关于IL13结构-功能关系的新知识，（ii）关于胶质瘤细胞生物学的新信息，以及（iii）在临床环境中使用细胞毒素的经验。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DT390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，使白细胞介素13的结合区域，d -螺旋，对白细胞介素13的hkg相关受体自由可用，并消除细胞因子上与其正常生理受体相互作用的位点，这些受体在许多重要器官中表达，包括中枢神经系统。将对IL13进行工程设计，使DT毒素片段远离其结合位点到IL13R(2)上，这是PE无法完全实现的。此外，IL13 α -螺旋D突变体，IL13R(2)的超级激动剂，已经被确定，它们将用于优化细胞毒素的设计。细胞毒素的限制性结合位点数量和激活细菌毒素的胞内蛋白酶（furin）在细胞毒素杀伤肿瘤细胞的过程中起主要作用。因此，我们将研究其表达水平的调控，并研究其表达水平与新型IL13-DT细胞毒素功效的相关性。新型基于DT390-IL13突变体的细胞毒素将在体外和体内进行测试，以证明在靶点和毒素激活片段的生物学表达背景下，分子设计的变化如何反映在其抗肿瘤功效中。期望这些新信息对进一步设计重组抗癌细胞毒素具有重要意义。

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.