Molecular Requirements for Recombinant Cytotoxins Efficacy

描述(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(IL13)的限制性受体(R)。IL13的HGG相关受体被分子鉴定为IL13R(2)，属于肿瘤抗原家族，称为肿瘤/睾丸肿瘤抗原(CTA)。CTA为肿瘤的分子靶向/识别提供了高度的特异性。此外，基于IL13的细菌毒素，含假单胞菌外毒素A(PE)的重组细胞毒素被证明是临床前评估中有效的抗胶质瘤药物，这些细胞毒素的第一代正处于第三阶段临床试验。IL13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IL13与其HGG相关受体的结合中发挥关键作用。建议产生新的高度特异和高效的基于IL13的细胞毒素，包括(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)先前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IL13和另一种细菌毒素白喉毒素DT390的衍生物组成。设计这些细胞毒素的主要思想是产生新的细胞毒素，允许IL13的D-螺旋结合区自由地被HGG相关的IL13受体获得，并消除细胞因子上与其正常生理受体相互作用的部位，该受体在许多重要器官中表达，包括中枢神经系统。IL13将被设计成使DT毒素部分远离其与IL13R(2)的结合部位，这是PE无法完全实现的。此外，IL13α-螺旋D突变体，IL13R(2)的超级激动剂已经被鉴定出来，它们将用于优化细胞毒素的设计。细胞毒素的限制性结合部位的数量和一种激活细菌毒素的胞内蛋白酶(Furin)在细胞毒素对肿瘤细胞的杀伤中起主要作用。因此，它们的表达水平的调节将被检测，并且表达水平与新的IL13-DT细胞毒素的有效性相关。新的基于DT390-IL13突变体的细胞毒素将在体外和体内进行测试，以展示在靶标和毒素激活部分的生物表达背景下分子设计的变化如何反映在它们的抗肿瘤效果上。预计收集到的新信息将对重组抗癌细胞毒素的进一步分子设计具有重要价值。

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.