TUMOR TARGETING BY SINGLE CHAIN FV MOLECULES

描述：(申请者摘要)本研究的长期目标 该计划旨在通过以下途径改善人类癌症的放射免疫治疗(RIT) 使用抗体预靶向策略。潜在的基本假设 这项工作是基于优化的单链抗体分子(ScFv)的蛋白质 是预靶向放射免疫治疗的理想载体，因为选择性 单链抗体分子的靶向优势在末端表现得最明显。 体内生物分布的阶段。在本支助期内， 申请人在确定亲属身份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 以及在小鼠模型中选择性肿瘤滞留。C6.5单链抗体是从 人源噬菌体展示文库的抗原性研究 HER2/neu，它是乳腺癌和其他肿瘤的重要靶点。 C6.5亲和变异体是使用链洗牌和位点定向产生的 诱变产生一系列与HER2/neu有亲和力的单链抗体 使用这些分子，申请人已经确定 小鼠体内肿瘤靶向检测的阈值亲和力 是10(-8)M，将单链抗体的亲和力提高到10(-9)M以上不会 进一步提高数量，选择性肿瘤滞留。累积目标 选择性，通过测量肿瘤与正常器官的曲线下面积来确定 比率，不超过较大的免疫球蛋白分子，尽管有选择性 在终末阶段，单链抗体分子靶向肿瘤的效果明显更好。 生物分布的阶段。增加化合价的影响比 在定量肿瘤靶向上的亲和力，即使结果被校正 以确定抗体大小。二价ScFv显著改善了肿瘤靶向性 当被放置在diabody格式中时。然而，还需要进一步改进 以获得有效的RIT。申请者将产生一种预先靶向的抗体 靶向HER2/neu单链抗体融合蛋白的放射免疫治疗策略 和半抗原将放射性金属定位于肿瘤部位。这将会实现的 通过平移人源ScFv噬菌体文库来分离与 这些单链抗体将被亲和成熟，然后与C6.5融合。 Diabody将创造一种能够系统捕获的双特异性融合蛋白 将90Y与CHX-A络合，并将放射性核素浓缩到 肿瘤部位。肿瘤靶向和临床前放射免疫治疗研究将 指挥。这种预先确定目标的策略的优势也将被寻求 进行临床前放射免疫治疗研究直接使用 结合融合蛋白或C6.5免疫球蛋白1。这些研究将确定候选人 用于临床开发的分子，并将为 实体瘤的临床有效放射免疫治疗。

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.