TUMOR TARGETING BY SINGLE CHAIN FV MOLECULES

描述：（申请人摘要）本研究的长期目标 计划是通过改善人类癌症的放射免疫治疗（RIT）， 使用抗体预靶向策略。基本假设是 这项工作是，优化的单链抗体分子（scFv）为基础的蛋白质将 是预靶向放射免疫治疗的理想载体，因为选择性 基于scFv的分子的靶向优势最好在末端期间看到， 体内生物分布阶段。在目前的支助期间， 申请人在确定亲属方面取得了实质性进展 抗体大小、效价和结合亲和力对定量分析的贡献 和选择性肿瘤滞留。C6.5 scFv分离自 一个人噬菌体展示文库，针对 HER 2/neu是乳腺癌和其他肿瘤的重要靶点。 使用链改组和定点突变产生C6.5亲和变体。 诱变以产生一系列scFv，其对HER 2/neu的亲和力为 10（-6）-10（-11）M。使用这些分子，申请人已经确定， 在鼠模型中可检测的体内肿瘤靶向的阈值亲和力 为10（-8）M。将scFv的亲和力增加到超过10（-9）M并不 进一步改善定量的、选择性的肿瘤保留。累积靶向 选择性，通过测量肿瘤与正常器官曲线下面积确定 比率，不超过与较大的IgG分子，虽然选择性， scFv分子的肿瘤靶向在终末阶段基本上更好， 生物分布的阶段。增加化合价的效果比 对定量肿瘤靶向的亲和力，即使结果得到校正 抗体大小。二价scFv显示出显著改善的肿瘤靶向 当以双抗体形式放置时。然而，还需要作出更多的改进 有效的RIT。申请人将创建一个抗体预靶向 使用靶向HER 2/neu的scFv融合蛋白的放射免疫治疗策略 并且半抗原将放射性金属定位于肿瘤部位。这将是完成 通过淘选人scFv噬菌体文库以分离与所述抗体反应的scFv， 螯合物，CHX-A。“这些scFv将被亲和力成熟，然后融合到C6.5 双抗体以产生可以系统性捕获 施用螯合至CHX-A”的90钇，并将放射性核素浓缩至 肿瘤部位。肿瘤靶向和临床前放射免疫治疗研究将在 进行。这种预先瞄准战略的优点也将由 进行临床前放射免疫治疗研究， 缀合的融合蛋白或C6.5 IgG 1。这些研究将确定候选人 分子的临床开发，并将提供一个基础 临床上有效的实体瘤放射免疫治疗。

DESCRIPTION: (Applicant's Abstract) The long-term objective of this research program is to improve radioimmunotherapy (RIT) for human cancers through the use of antibody-pretargeting strategies. The fundamental hypothesis underlying this work is that optimized single-chain Fv molecule (scFv) based proteins will be ideal vehicles for pre-targeted radioimmunotherapy, since the selective targeting advantages of scFv-based molecules are best seen during the terminal phases of in vivo biodistribution. In the current period of support, the applicant has made substantial progress in identifying the relative contributions of antibody size, valence and binding affinity to quantitative and selective tumor retention in murine models. The C6.5 scFv was isolated from a human phage display library panned against the extracellular domain of HER2/neu, which is an important target in breast cancer and other neoplasms. C6.5 affinity variants were generated using chain-shuffling and site-directed mutagenesis to yield a series of scFv with affinities for HER2/neu ranging from 10(-6) - 10(-11) M. Using these molecules, the applicant has determined that the threshold affinity for detectable in vivo tumor targeting in a murine model is 10(-8) M. Increasing the affinity of scFv to more than 10(-9) M does not further improve quantitative, selective tumor retention. Cumulative targeting selectivity, determined by measuring tumor to normal organ area-under-the-curve ratios, does not exceed that seen with larger IgG molecules, although selective tumor targeting by scFv molecules is substantially better during the terminal phases of biodistribution. Increasing valence has more effect than does affinity on quantitative tumor targeting, even when the results are corrected for antibody size. Divalent scFv exhibit profoundly improved tumor targeting when placed in a diabody format. However, additional improvements are required for effective RIT. The applicant will create an antibody pre-targeted radioimmunotherapy strategy employing scFv fusion proteins targeting HER2/neu and haptens to localize radiometals to tumor sites. This will be accomplished by panning a human scFv phage library to isolate scFv reactive with the chelate, CHX-A." These scFv will be affinity matured and then fused to the C6.5 diabody to create a bispecific fusion protein that can capture systemically administered 9OYttrium chelated to CHX-A" and concentrate the radionuclide at tumor sites. Tumor targeting and preclinical radioimmunotherapy studies will be conducted. The advantages of this pretargeting strategy will be sought by also conducting preclinical radioimmunotherapy studies employing the directly conjugated fusion protein or C6.5 IgG1. These studies will identify candidate molecules for clinical development, and will provide a foundation for the clinically effective radioimmunotherapy of solid tumors.