HIV ENVELOPE SPECIFIC DARPIN-BASED MICROBICIDE

这个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。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 背景：最近报道的设计型Ankyrin Repeat Protein(DARPin)技术能够以较低的生产成本生产出对多种生物靶标具有高度选择性的配体。方法/主要发现：为了研究DARPins在体内的使用，以便将来应用于新的抗HIV策略，我们发现了识别恒河猴CD4的有效的CD4特异性DARPins，并跟踪了恒河猴静脉注射CD4特异性DARPin 57.2的命运。人CD4特异性DARPin 57.2能与猕猴CD4()细胞结合，并在体外表现出较强的抑制SIV感染的活性。将DARPIN 57.2或对照E3_5DARPin注射到恒河猴体内，观察无细胞DARPin和细胞结合DARPin的命运。外周血中早在注射后30分钟就检测到与Darpin结合的CD4()细胞，6小时内下降，24小时内几乎检测不到。DARPin的结合量取决于注射的DARPin的量。在30分钟内，在淋巴结中的CD4()细胞上也检测到了CD4特异性DARPin，其持续的动力学与血液相似。更广泛的血液分析显示，DARPin 57.2在体内和体外与所有类型的CD4()细胞(T细胞、单核细胞、树突状细胞)结合，结合量与细胞表面的CD4量成正比。在血浆中也检测到无细胞的DARPins，但很快就从循环中清除出来。结论/意义：我们证明了CD4特异性DARPin可以在体内快速和选择性地结合其靶细胞，这为DARPin技术可能的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.