Engineering antibodies for intracellular targeting

用于细胞内靶向的工程抗体

• 批准号: 9396448
• 负责人: Hejia Henry Wang
• 金额: $ 4.9万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9396448
• 关键词: Adaptor Signaling Protein; Address; Adopted; Adoption; Affinity; Amino Acid Sequence; Antibodies; Antigens; Binding; Biological; Blood group antigen S; Cations; Cell membrane; Cells; Cytoplasm; Cytoplasmic Tail; Endosomes; Engineering; Extracellular Matrix; Fluorescent Probes; Gelatinase A; Goals; Grant; Immunoglobulin Constant Region; Immunoglobulin G; Immunoglobulin Variable Region; Individual; Length; Libraries; Light; MALDI-TOF Mass Spectrometry; Malignant Neoplasms; Masks; Membrane; Methods; Molecular Conformation; Multidrug Resistance-Associated Proteins; Peptide Hydrolases; Peptides; Peptidyltransferase; Protein Engineering; Proteins; Recombinants; Reporter; Resistance; Site; Specificity; System; Technology; Testing; Therapeutic; Therapeutic antibodies; Tissues; Variant; antibody engineering; base; benzoylphenylalanine; cancer cell; chemotherapy; crosslink; design; efflux pump; extracellular; improved; in vitro testing; interest; nanoparticle; novel; novel strategies; polyarginine; protein aminoacid sequence; sortase; therapeutic target; tumor; unnatural amino acids; uptake

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.

项目摘要 抗体在癌症治疗中取得了巨大的成功，部分原因是它们可以中和体内的 抗原的生物活性，但它们不能穿透质膜仅限于分泌或 膜相关抗原。一种在细胞内传递蛋白质的通用方法是 将货物与细胞穿透肽(CPPs)偶联，CPP是一种短的多阳离子多肽，可诱导细胞 领悟。然而，CPP受到两个主要限制：由于内体导致细胞质递送不良 摄取后的圈闭和缺乏任何固有的组织特异性靶向能力。内吞体体 可以通过使用内溶多肽(ELPs)来解决包埋问题，内溶肽在pH-1的条件下破坏膜。 依赖方式，与CPPS联合诱导内体逃逸。相对较少的CPP-ELP对具有 然而，经过测试，人们对各个组件如何交互和协作知之甚少 其他的。CPP可以通过用解离的多阴离子序列来掩蔽它们而被赋予组织特异性 仅在感兴趣组织中特异表达的胞外蛋白水解酶存在的情况下。然而，只有 到目前为止，已经设计了一种基于多精氨酸CPP的可激活CPP(ACPP)，并且仅用于递送 荧光探针和纳米颗粒。我们之前已经开发出一种小接头蛋白(PG)，它可以 与任何现成的免疫球蛋白的恒定区进行定点相交，同时保留 其可变区的结合亲和力。通过将CPP、ELP或其他肽序列引入PG 重新组合，不仅可以以高吞吐量的方式测试附加功能，而且货物 可以很容易地换出。这项提议的目标是利用这项技术来开发aCPP-ELP 可以将天然的免疫球蛋白运送到活细胞的细胞质中以抑制细胞内蛋白质的对。在《目标1》中，我将 创建包含不同CPP-ELP对的PG变异体文库。第一个肽(CPP或ELP)将是 重组导入PG，第二个通过细菌分类酶A与PG偶联 转肽酶。在目标2中，我将使用自组装的SplitGFP测试文库的细胞质递送 一种报告系统，其中较大的分裂GFP一半在细胞质中表达，而较小的一半融合到 加分。为了证明交付的免疫球蛋白-PG偶联物具有功能，我将抑制多药耐药- 相关蛋白1(MRP1)，一种与化疗耐药相关的外排泵，与QCRL3，a 一旦结合到其细胞质结构域之一，就能强有力地抑制MRP1活性的单克隆性抗体。最后，在 目的3.对表征良好的多聚精氨酸aCPP以及在此基础上设计的新型聚精氨酸aCPP进行测试。 文库中用于细胞递送的CPP依赖于基质金属蛋白酶-2/9(MMP2/9)，它高度依赖于 在肿瘤中表达。这一提议的完成将为抑制细胞内信号提供一种新的方法 在活细胞中的蛋白质，并将成为开发治疗性细胞内抗体的基础。最优 ACPP-ELP对也可用于运送用于其他应用的大型蛋白质货物。