Engineering antibodies for intracellular targeting

项目摘要 抗体已经成为非常成功的癌症治疗方法，部分原因是它们可以中和它们的抗体。 抗原的生物活性，但它们不能穿过质膜，限制了分泌或分泌的靶点。 膜相关抗原。细胞内递送蛋白质的一种通用方法是 将货物缀合至细胞穿透肽（CPP），其是短的聚阳离子肽，以诱导细胞免疫。 摄取。然而，CPP遭受两个主要限制：由于内体导致的差的细胞质递送。 吸收后的截留和缺乏任何内在的组织特异性靶向能力。内体 包埋可以通过使用内体溶解肽（ELP）来解决，其在pH- 依赖性方式，与CPP一起诱导内体逃逸。相对较少的CPP-ELP对具有 然而，已经进行了测试，并且对各个组件如何相互作用和相互合作知之甚少 其他. CPP可以通过用多聚阴离子序列掩蔽它们来获得组织特异性， 仅在感兴趣的组织中特异性表达的细胞外蛋白酶的存在下。但只有 迄今为止，已经设计了一种基于聚精氨酸CPP的可活化CPP（aCPP 荧光探针和纳米颗粒。我们以前已经开发了一种小的衔接蛋白（pG）， 可以与任何现成IgG的恒定区进行位点特异性光交联，同时保留 其可变区的结合亲和力。通过将CPP、ELP或其它肽序列引入pG 通过重组，不仅可以以高通量的方式测试额外的功能， 可以很容易地换掉。本提案的目标是利用该技术开发aCPP-ELP 可以将天然IgG递送到活细胞的细胞质中以抑制细胞内蛋白质。在目标1中，我将 创建含有不同CPP-ELP对的pG变体文库。第一肽（CPP或ELP）将是 第二个将通过使用分选酶A（一种细菌分选酶）与pG缀合， 转肽酶在目标2中，我将通过使用自组装splitGFP来测试文库的细胞质递送， 报告系统，其中较大的splitGFP一半在细胞质中表达，而较小的一半融合到 PG.为了证明递送的IgG-pG缀合物是功能性的，我将抑制多药耐药性- 相关蛋白1（MRP 1），与化疗耐药相关的外排泵，与QCRL 3， 单克隆IgG，一旦与其胞质结构域之一结合，就强烈抑制MRP 1活性。最后在 目的3，我将测试良好表征的聚精氨酸aCPP以及基于其他设计的新的聚精氨酸aCPP。 在文库中用于细胞递送的CPP依赖于基质金属蛋白酶-2/9（MMP-2/9），其是高度依赖于细胞的基质金属蛋白酶。 在肿瘤中表达。该方案的完成将为抑制细胞内 活细胞中的蛋白质，并将形成开发治疗性细胞内抗体的基础。最优 aCPP-ELP对也可用于递送用于其它应用的大蛋白质货物。

Project Summary Antibodies have been tremendously successful cancer therapeutics partly because they can neutralize their antigen’s biological activity, but their inability to cross the plasma membrane has limited targets to secreted or membrane-associated antigens. One general approach for delivering proteins intracellularly has been to conjugate cargos to cell-penetrating peptides (CPPs), which are short poly-cationic peptides, to induce cellular uptake. However, CPPs suffer from two major limitations: poor cytoplasmic delivery due to endosome entrapment following uptake and a lack of any intrinsic tissue-specific targeting capability. Endosome entrapment can be addressed by using endosomolytic peptides (ELPs), which disrupt membranes in a pH- dependent manner, to induce endosome escape in conjunction with CPPs. Relatively few CPP-ELP pairs have been tested, though, and little is known about how the individual components interact and cooperate with each other. CPPs can be granted tissue-specificity by masking them with polyanionic sequences that dissociate solely in the presence of extracellular proteases specifically expressed in the tissue of interest. However, only one activatable CPP (aCPP) based on the polyarginine CPP has been designed thus far and only for delivery of fluorescent probes and nanoparticles. We have previously developed a small adaptor protein (pG) that can be site-specifically photo-crosslinked to the constant region of any off-the-shelf IgG while preserving the binding affinity of its variable region. By introducing CPP, ELP, or other peptide sequences into pG recombinantly, not only can additional functionalities can be tested in a high-throughput manner, but the cargo can be easily swapped out. The goal of this proposal is to leverage this technology to develop an aCPP-ELP pair that can delivery native IgGs into the cytoplasm of living cells to inhibit intracellular proteins. In Aim 1, I will create a library of pG variants containing different CPP-ELP pairs. The first peptide (CPP or ELP) will be introduced into pG recombinantly while the second will be conjugated to pG by using sortase A, a bacterial transpeptidase. In Aim 2, I will test the library for cytoplasmic delivery by using a self-assembling splitGFP reporter system in which the larger splitGFP half is cytoplasmically expressed while the smaller half is fused to pG. To demonstrate that delivered IgG-pG conjugates are functional, I will inhibit multidrug resistance- associated protein 1 (MRP1), an efflux pump associated with chemotherapy resistance, with QCRL3, a monoclonal IgG that robustly inhibits MRP1 activity once bound to one of its cytoplasmic domains. Finally, in Aim 3, I will test the well-characterized polyarginine aCPP as well as novel ones designed based on other CPPs used in the library for cell delivery dependent on matrix metalloproteinase-2/9 (MMP-2/9), which is highly expressed in tumors. Completion of this proposal will provide a new approach for inhibiting intracellular proteins in living cells and would form the basis for developing therapeutic intracellular antibodies. Optimal aCPP-ELP pairs could also be utilized to deliver large protein cargos for other applications.