Treating Melanoma with a Molecularly Engineered, Long Circulating Immunotoxin

 产品说明：我们已经开发了新一代scFv重组免疫毒素（RIT），由于其效力和低免疫原性，有望用于治疗黑色素瘤。尽管其有希望的体内功效，但其从体循环中迅速清除，使得其在体内的浓度增加。 并没有达到其对靶点的高亲和力所暗示的治疗潜力， 强效细胞毒性。为了释放其全部治疗潜力，我们假设RIT的药代动力学和生物分布的改善是必要的。聚乙二醇化（PEG）的连接-是一种有效的方法，以改善蛋白质药物的药代动力学和肿瘤蓄积。不幸的是，目前的聚乙二醇化方法产率低，位点选择性有限，导致生物活性受损的异质缀合物。我们已经开发了一种新的方法-称为InStealth-通过原子转移自由基聚合（ATRP）直接从感兴趣的蛋白质的N-或C-末端“生长”具有高产率的位点特异性和化学计量（1：1）的蛋白质-聚合物缀合物来解决这些问题。在以前的研究与模型蛋白质，我们已经表明，聚（聚乙二醇甲基丙烯酸甲酯）（POEGMA）共轭物具有显着增强的药代动力学和更好的肿瘤积累比母体蛋白质。该提议的总体目标是通过原位ATRP从F6 V-IT融合体的N-或C-末端直接生长POEGMA缀合物，以产生具有长循环和改善的肿瘤积累的位点特异性、化学计量（1：1）的蛋白质-聚合物缀合物。我们的具体目标是：1）、F6 V-IT的产生和表征。将遗传合成表现出极低的B细胞和T细胞表位免疫原性的F6 V-IT重组免疫毒素，并将在不同的黑素瘤细胞系上评估其体外活性。2）：F6 V-IT POEGMA缀合物的合成和体外表征。ATRP将用于直接从具有系统变化的POEGMA分子量的F6 V-IT的N-或C-末端合成POEGMA缀合物。将在体外表征缀合物对相关黑素瘤细胞系的靶向和细胞毒性活性。3）：F6 V-IT POEGMA缀合物的体内评价。将在无胸腺小鼠的人黑色素瘤异种移植物中研究特定目标2中确定的先导候选药物的体内药代动力学、器官生物分布和疗效。该提案的结果将是一种用于黑色素瘤患者的新型治疗策略：InStealth（TM）结合用于具有极低免疫原性的新型免疫毒素的肿瘤靶向递送。

 DESCRIPTION: We have developed a new-generation scFv recombinant immunotoxin (RIT) that holds promise for treatment of melanoma due to its potency and low immunogenicity. Despite its promising in vivo efficacy, it is rapidly cleared from systemic circulation, so that it does not live up to its therapeutic potential suggested by its high affinity for its target and its potent cytotoxicity. In order to unleash its full therapeutic potential, we hypothesize that improvement in the pharmacokinetics and biodistribution of the RIT are necessary. PEGylation-the attachment of poly (ethylene glycol (PEG)- is a powerful method to improve the pharmacokinetics and tumor accumulation of protein drugs. Unfortunately, current methods of PEGylation have poor yield, limited site-selectivity and result in heterogeneous conjugates with compromised bioactivity. We have developed a new method -termed InStealth- that solves these problems by "growing" a site-specific and stoichiometric (1:1) protein-polymer conjugate with high yield directly from the N- or C-terminus of a protein of interest by atom transfer radical polymerization (ATRP). In previous studies with model proteins, we have shown that a poly(oligoethylene glycol methyl methacrylate) (POEGMA) conjugate has significantly enhanced pharmacokinetics and better tumor accumulation than the parent protein. The overall objective of this proposal is to directly grow a POEGMA conjugate from the N- or C- terminus of the F6V-IT fusion by in situ ATRP to create a site-specific, stoichiometric (1:1) protein-polymer conjugate with long circulation, and improved tumor accumulation. Our Specific Aims are: 1): Generation and characterization of F6V-IT. The F6V-IT recombinant immunotoxin that exhibits extremely low B cell and T cell epitope immunogenicity will be genetically synthesized and it's in vitro activity will be evaluated on different melanoma cell lines. 2): Synthesis and in vitro characterization of F6V-IT POEGMA conjugates. ATRP will be used to synthesize POEGMA conjugates directly from the N- or C-terminus of the F6V-IT with systematically varied POEGMA molecular weight. The targeting and cytotoxic activity of the conjugates on relevant melanoma cell lines will be characterized in vitro. 3): In vivo evaluation of F6V-IT POEGMA conjugates. The in vivo pharmacokinetics, organ bio distribution, and therapeutic efficacy of lead candidates identified in Specific Aim 2 will be investigated in human melanoma xenografts in athymic mice. The outcome of this proposal will be a novel therapeutic strategy for melanoma patients: InStealth(tm) conjugation for tumor targeted delivery of a new immunotoxin with extremely low immunogenicity.