Engineered Protein Nanocarriers for Intracellular Antibody Delivery

用于细胞内抗体递送的工程蛋白纳米载体 有一长串“无法下药”的靶点，就是这么小的细胞内与疾病相关的蛋白质-蛋白质相互作用 分子药物无法阻止。特别是，抗体可以被改造成与几乎任何蛋白质结合并抑制 蛋白质具有功能，但尚未被用于对抗这些靶点。这主要是由于重大的、未得到满足的 在细胞内传递足够数量的折叠的功能性抗体是一项挑战。这项提案的目标是 是利用蛋白质自组装构建细胞内抗体载体，并评估其治疗活性 递送的抗体。通过利用六聚体蛋白束(HEX)和通用抗体结合肽 (SPAB)，只含有蛋白质的纳米级复合体可以与抗体形成。我们的假设是 与可溶性相比，HEX抗体纳米载体将显著增加细胞内抗体的摄取 抗体，并将使治疗性抗体与胞浆靶标结合。我们的初步数据表明 六角SPAB蛋白纳米载体确实将功能性抗体输送到胞浆中，因此，可以广泛地 适用于未来的细胞内抗体疗法。设定了三个目标，以满足目标和测试 假设。(1)制备和鉴定HEX抗体的大小、稳定性和抗体载量 纳米载体。(2)测定抗癌模型的细胞摄取、胞浆定位和治疗功能 由HEX纳米载体传递的抗体。(3)通过以下方法确定HEX纳米载体的治疗潜力 添加抗HER2靶向抗体将载体定向到乳腺癌细胞，并对血液进行评估 HEX纳米载体体内循环时间和免疫原性的研究预计会出现两个主要结果。首先，一个 创新的蛋白质纳米载体将被创造出来，以促进细胞内功能性治疗药物的输送 抗体。它将为细胞内抗体疗法抑制蛋白质提供概念证明，包括 与靶向抗体的组合。其次，这项工作将测量必要的关键属性 翻译、血液循环时间和免疫原性。这将为细胞内抗体打开大门 可与目前成功治疗人类疾病的细胞外抗体相媲美的工程技术。

Engineered Protein Nanocarriers for Intracellular Antibody Delivery There is a long list of “undruggable” targets, disease related protein-protein interactions inside cells that small molecule drugs cannot block. Antibodies, in particular, can be engineered to bind almost any protein and inhibit protein function but have not yet been used against these targets. This is primarily due to the significant, unmet challenge in delivering sufficient amounts of folded, functional antibodies inside cells. The goal of this proposal is to build an intracellular antibody carrier using protein self-assembly and assess the therapeutic activity of delivered antibodies. By utilizing a hexameric protein bundle (HEX) and generic antibody-binding peptides (SPABs), nanoscale complexes containing only protein can be formed with antibodies. Our hypothesis is that the HEX antibody nanocarrier will significantly increase intracellular antibody uptake compared to soluble antibodies, and will enable therapeutic antibody binding to cytosolic targets. Our preliminary data suggests that Hex-SPAB protein nanocarriers do deliver functional antibody into the cytosol and, therefore, could be broadly applicable to future intracellular antibody therapeutics. Three aims have been set to meet the objective and test the hypothesis. (1) Fabricate and characterize the size, stability and antibody loading of HEX antibody nanocarriers. (2) Measure cellular uptake, cytosolic localization, and therapeutic function of model anti-cancer antibodies delivered by HEX nanocarriers. (3) Determine the therapeutic potential of HEX nanocarriers by adding anti-HER2 targeting antibodies to direct carriers to breast cancer cells, and evaluate the blood circulation time and immunogenicity of HEX nanocarriers in vivo. Two main outcomes are expected. First, an innovative protein nanocarrier will be created that facilitates cytosolic delivery of functional therapeutic antibodies. It will provide proof of concept for intracellular antibody therapeutics to inhibit proteins, including combinations with targeting antibodies. Second, this work will measure key properties necessary for translation, blood circulation time and immunogenicity. This will open the door for intracellular antibody engineering comparable to current extracellular antibodies that are successfully treating human disease.