Evaluation and optimisation of new engineered human human apoferritins: protein nanocages for targeted drug delivery and intracellular cargo release

自从Paul埃利希在1907年创造了“魔术子弹”一词以来，将药物的治疗效果最大化同时将副作用最小化的靶向递送一直是一个重要目标。1975年单克隆抗体技术的出现提供了一种可以靶向特定细胞的蛋白质，并被誉为“神奇子弹”的例子。抗体本身可以用作治疗剂，如乳腺癌治疗赫赛汀所证明的，或者最近，作为药物载体和治疗剂（抗体药物偶联物）将选择性较低的药物靶向肿瘤细胞，如Kadcyla所证明的。虽然基于抗体的系统在临床上使用，但它们确实具有显著的缺点，包括每个抗体仅能够递送少量的药物，并且由于这需要哺乳动物细胞而生产昂贵。我们希望开发一种新的靶向给药系统的基础上，人类蛋白质，脱铁铁蛋白。这种蛋白质由24个亚基组成，在pH 2.0以上自组装形成直径为12 nm的空心球（纳米笼）。我们可以在一个纳米笼中捕获多达500个药物分子（相比之下，每个抗体附着在它们的外表面上3-8个）。脱铁蛋白通过一种称为TfR 1的膜受体被细胞自然吸收。它将铁离子包裹起来，并将其输送到细胞的细胞质中，使其生长。虽然一些癌症随着生长速度的加快而表达升高量的TfR 1，这使得它们能够被天然脱铁铁蛋白靶向，但许多癌症表达其他表面蛋白（生物标志物）。这些可以被抗体或其他蛋白质靶向，包括最近开发的一种更小的蛋白质，称为抗体。已经鉴定了选择性结合在一些癌症中以高得多的水平发现的表皮生长因子受体（EGFR、HER 2、HER 3）的亲和体。通过使用合成生物学将抗体的靶向能力与脱铁铁蛋白的药物包封和跨膜能力相结合，我们可以产生新的药物递送系统，其递送每个蛋白质的药物量高得多，包括如果不被包封保护则对在血液中代谢敏感的药物。在这个项目中，我们将建立一个抗体-脱铁铁蛋白融合蛋白库，这些蛋白可以以不同的比例混合在一起，以优化针对一系列常见癌细胞类型的靶向药物递送特性。在一项初步研究中，我们已经表明，脑癌药物替莫唑胺的脱铁蛋白包封使其有效对抗那些对药物作用产生耐药性的细胞。如果其他药物也出现这种情况，它提供了延长药物有效期的机会。作为研究的一部分，我们将研究两类化合物，它们在体内对癌细胞具有良好的活性，但由于它们不能被细胞有效吸收，因此不能用作治疗药物。使命奖将使我们能够全面评估抗体-脱铁铁蛋白系统，以确定它是否可以成为“下一代”靶向药物输送系统或“特洛伊木马”，继抗体之后，用于更广泛的不同药物类型。与免疫球蛋白G抗体不同，抗体-脱铁铁蛋白亚基可以在细菌或其他非哺乳动物细胞中产生。这意味着它们可以以低成本和更大规模生产，并且比抗体更具可持续性，如果它们在世界范围内容易获得，则需要这些抗体。

The targeted delivery of drugs which maximises their therapeutic efficacy whilst minimising the side effects has been a significant goal since Paul Ehrlich coined the term 'magic bullet' in 1907. The advent of monoclonal antibody technology in 1975 provided a protein that could target specific cells and was heralded as an example of a 'magic bullet'. Antibodies can be used as therapeutic agents on their own as demonstrated with the breast cancer treatment Herceptin or more recently, as drug carriers and therapeutics (Antibody Drug Conjugates) to target less selective drugs to tumour cells as demonstrated by Kadcyla. Whilst antibody-based systems are used clinically, they do have significant disadvantages which include only being able to deliver a small number of drugs per antibody and being expensive to produce because this requires mammalian cells. We wish to develop a new targeted drug delivery system based on the human protein, apoferritin. This protein is made up of 24 subunits and self-assembles above pH 2.0 to form a hollow sphere (nanocage) 12 nm in diameter. We can trap up to 500 drug molecules in a single nanocage (compared with 3-8 per antibody, attached to their external surface). Apoferritin is naturally taken up into cells using a membrane receptor called TfR1. It does this as encapsulates iron ions and delivers these to the cytoplasm of the cell for them to grow. Whilst some cancers express elevated amounts of TfR1 as they grow faster and this allows them to be targeted by natural apoferritin, many cancers express other surface proteins (biomarkers). These can be targeted by antibodies or other proteins including a recently developed much smaller protein called an affibody. Affibodies that selectively bind epidermal growth factor receptors (EGFR, HER2, HER3) that are found at much higher levels in some cancers have been identified. By combining the targeting ability of affibodies with the drug encapsulation and membrane crossing ability of apoferritin using synthetic biology, we can generate new drug delivery systems that delivery much higher amounts of drugs per protein including ones that are sensitive to being metabolised in the blood if not protected by encapsulation. In this project we will make a library of affibody-apoferritin fusion proteins that can be mixed together in different ratios to optimise the targeted drug delivery properties against a range of common cancer cell types. In a preliminary study, we have shown that apoferritin encapsulation of the brain cancer drug temozolomide makes it effective against cells that have developed resistance to the action of the drug if delivered on its own. If this is seen with other drugs, it offers the opportunity to extend the period a drug is effective. As part of the study we will examine two classes of compounds that have good activity against cancer cells in vivo but because they are not taken up by cells efficiently, they cannot be used as therapeutics. The Mission award will allow us to comprehensively evaluate the affibody-apoferritin system to determine if it can become the 'next generation' targeted drug delivery system or 'trojan horse' following on from antibodies, for a wider variety of different drug types. Unlike immunoglobulin G antibodies, the affibody-apoferritin subunits can be produced in bacteria or other non-mammalian cells. This means that they can be produced at low cost and on a larger scale and much more sustainably than antibodies as will be required if they are to be readily available worldwide.