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.

项目概要 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 抗原的生物活性，但它们无法穿过质膜，因此限制了分泌或 细胞内递送蛋白质的一种通用方法是 将货物与细胞穿透肽（CPP）（一种短聚阳离子肽）结合，诱导细胞 然而，CPP 存在两个主要限制：内体导致的细胞质递送不良。 摄取后的截留和缺乏任何内在的组织特异性靶向能力。 可以通过使用内体肽 (ELP) 来解决包埋问题，该肽会在 pH 值范围内破坏细胞膜。 依赖的方式，与 CPP 一起诱导内体逃逸，相对较少的 CPP-ELP 对具有。 尽管已经过测试，但对于各个组件如何交互和相互协作知之甚少 其他 CPP 可以通过用解离的聚阴离子序列掩蔽它们来获得组织特异性。 仅在目标组织中特异性表达的细胞外蛋白酶存在的情况下。 迄今为止，已设计出一种基于聚精氨酸 CPP 的可激活 CPP (aCPP)，且仅用于交付 我们之前开发了一种小型接头蛋白（pG），可以 可与任何现成 IgG 的恒定区进行位点特异性光交联，同时保留 通过将 CPP、ELP 或其他肽序列引入 pG 来增强其可变区的结合亲和力。 通过重组，不仅可以以高通量方式测试附加功能，而且可以测试货物 该提案的目标是利用该技术来开发 aCPP-ELP。 一对可以将天然 IgG 传递到活细胞的细胞质中以抑制细胞内蛋白质。 创建包含不同 CPP-ELP 对的 pG 变体库 第一个肽（CPP 或 ELP）将是。 重组引入 pG，而第二个将通过使用分选酶 A（一种细菌）与 pG 缀合 在目标 2 中，我将使用自组装 splitGFP 测试文库的细胞质传递。 报告系统，其中较大的 splitGFP 一半在细胞质中表达，而较小的一半则融合到 为了证明所递送的 IgG-pG 缀合物具有功能性，我将抑制多药耐药性 - 相关蛋白 1 (MRP1) 是一种与化疗耐药性相关的外排泵，而 QCRL3 是一种 一旦结合到其胞质结构域之一，单克隆 IgG 就会强烈抑制 MRP1 活性。 目标 3，我将测试经过充分表征的聚精氨酸 aCPP 以及基于其他材料设计的新型聚精氨酸 aCPP 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)，该酶高度依赖于基质金属蛋白酶-2/9 (MMP-2/9) 该提案的完成将为抑制细胞内表达提供新的方法。 活细胞中的蛋白质，并将构成开发最佳治疗性细胞内抗体的基础。 aCPP-ELP 对还可用于为其他应用输送大量蛋白质货物。