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; 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 抗体巨大成功的癌症治疗部分是因为它们可以中和 抗原的生物学活动，但它们无法越过质膜的生物学活动有限的靶标的分泌或 膜相关抗原。细胞内传递蛋白质的一种一般方法是 与细胞穿透肽（CPP）（CPP）的共轭兑（是短多阳离子肽），以诱导细胞 吸收。但是，CPP遭受了两个主要局限性：内体引起的细胞质递送不良 在摄取摄入后的诱捕和缺乏任何固有的组织特异性靶向能力。内体 可以通过使用内溶液辣椒（ELP）来解决夹带 依赖方式，诱导内体与CPP结合。相对较少的CPP-ELP对具有 不过，经过测试，对各个组件如何与每个组件进行互动和合作知之甚少 其他。可以通过用分离的聚苯二醇序列掩盖组织特异性的CPP 仅在特异性在感兴趣的组织中表达的细胞外蛋白酶。但是，只有 到目前为止，已经设计了一个基于多精氨酸CPP的可激活的CPP（ACPP），仅用于交付 荧光问题和纳米颗粒。我们以前已经开发了一种小型适配器蛋白（PG） 在保留任何现成的IgG的恒定区域时，将现场特定于现场链接到保留 其可变区域的结合亲和力。通过将CPP，ELP或其他肽序列引入PG 重组，不仅可以以高通量方式对其他功能进行测试，还可以货物测试 可以很容易地交换。该建议的目的是利用这项技术开发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的对。在AIM 1中，我会 创建一个包含不同CPP-ELP对的PG变体库。第一个胡椒（CPP或ELP）是 通过使用分子酶A（细菌 转肽酶。在AIM 2中，我将使用自组装splitgfp测试库进行细胞质传递 较大的splitgfp一半被细胞质表达的记者系统，而较小的一半被融合到 pg。为了证明递送的IgG-PG偶联物是功能性的，我将抑制多药电阻 - 相关蛋白1（MRP1），一种与化学疗法抗性相关的外排泵，QCRL3，A 一旦与其细胞质结构域之一结合的MRP1活性，可抑制MRP1活性的单克隆IgG。最后，在 AIM 3，我将测试特征良好的多精灵ACPP以及基于其他的新颖的ACPP 库中用于细胞输送的CPP取决于基质金属蛋白酶2/9（MMP-2/9），这是高度的 在肿瘤中表达。该提案的完成将为抑制细胞内提供新的方法 活细胞中的蛋白质，将成为发展治疗性细胞内抗体的基础。最佳的 ACPP-ELP对也可用于为其他应用提供大型蛋白质兑。