HIV ENVELOPE SPECIFIC DARPIN-BASED MICROBICIDE

该子项目是利用资源的许多研究子项目之一 由NIH/NCRR资助的中心拨款提供。次级项目的主要支助 子项目的主要研究者可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 表示子项目使用的中心基础设施的估计数量， NCRR赠款不直接向子项目或子项目工作人员提供资金。 背景技术背景：最近描述的设计锚蛋白重复蛋白（DARPin）技术可以以低生产成本生产针对各种生物靶标的高选择性配体。 方法/主要结果：为了研究DARPin的体内用途以用于未来应用于新的抗HIV策略，我们鉴定了识别恒河猴CD 4的有效的CD 4特异性DARPin，并追踪了静脉注射的CD 4特异性DARPin 57.2在恒河猴中的命运。人CD 4特异性DARPin 57.2结合猕猴CD 4（+）细胞，并在体外显示出对SIV感染的有效抑制活性。将DARPin 57.2或对照E3_5DARPin注射到恒河猴中，并评估无细胞和细胞结合的CD 4特异性DARPin的命运。早在注射后30分钟就在外周血中检测到DARPin结合的CD 4（+）细胞，6小时内减少，24小时内几乎检测不到。结合的DARPin的量取决于注射的DARPin的量。在30分钟内还在淋巴结中的CD 4（+）细胞上检测到CD 4特异性DARPin，其以与血液相似的动力学持续存在。使用血液进行的更广泛的分析显示，DARPin 57.2在体内和体外与所有CD 4（+）细胞类型（T细胞、单核细胞、树突细胞）结合，结合量与细胞表面上的CD 4的量成正比。在血浆中也检测到无细胞DARPin，但迅速从循环中清除。结论/意义：我们证明了CD 4特异性DARPin可以在体内快速和选择性地结合其靶细胞，从而为DARPin技术的可能临床应用提供了进一步的研究。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. BACKGROUND: The recently described Designed Ankyrin Repeat Protein (DARPin) technology can produce highly selective ligands to a variety of biological targets at a low production cost. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the in vivo use of DARPins for future application to novel anti-HIV strategies, we identified potent CD4-specific DARPins that recognize rhesus CD4 and followed the fate of intravenously injected CD4-specific DARPin 57.2 in rhesus macaques. The human CD4-specific DARPin 57.2 bound macaque CD4(+) cells and exhibited potent inhibitory activity against SIV infection in vitro. DARPin 57.2 or the control E3_5 DARPin was injected into rhesus macaques and the fate of cell-free and cell-bound CD4-specific DARPin was evaluated. DARPin-bound CD4(+) cells were detected in the peripheral blood as early as 30 minutes after the injection, decreasing within 6 hours and being almost undetectable within 24 hours. The amount of DARPin bound was dependent on the amount of DARPin injected. CD4-specific DARPin was also detected on CD4(+) cells in the lymph nodes within 30 minutes, which persisted with similar kinetics to blood. More extensive analysis using blood revealed that DARPin 57.2 bound to all CD4(+) cell types (T cells, monocytes, dendritic cells) in vivo and in vitro with the amount of binding directly proportional to the amount of CD4 on the cell surface. Cell-free DARPins were also detected in the plasma, but were rapidly cleared from circulation. CONCLUSIONS/SIGNIFICANCE: We demonstrated that the CD4-specific DARPin can rapidly and selectively bind its target cells in vivo, warranting further studies on possible clinical use of the DARPin technology.