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

自从保罗·埃利希在1907年创造了“神奇子弹”一词以来，靶向递送药物，使其治疗效果最大化，同时将副作用降到最低，一直是一个重要的目标。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“灵丹妙药”的一个例子。抗体可以单独用作治疗剂，如乳腺癌治疗赫赛汀，或最近作为药物载体和治疗剂（抗体药物偶联物）靶向选择性较低的药物到肿瘤细胞，如Kadcyla。虽然基于抗体的系统在临床上使用，但它们确实有明显的缺点，包括每个抗体只能提供少量药物，而且生产成本昂贵，因为这需要哺乳动物细胞。我们希望开发一种新的靶向药物递送系统基于人蛋白，载铁蛋白。这种蛋白质由24个亚基组成，在pH值高于2.0时自组装形成直径为12纳米的空心球体（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，只能捕获3-8个药物分子）。载铁蛋白通过一种叫做TfR1的膜受体被自然地吸收到细胞中。它将铁离子包裹起来，并将其运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高量的TfR1，这使得它们成为天然载铁蛋白的目标，但许多癌症表达其他表面蛋白（生物标志物）。它们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，称为附着体。选择性结合表皮生长因子受体（EGFR, HER2, HER3）的粘附体在某些癌症中含量更高。通过利用合成生物学将修饰体的靶向能力与载铁蛋白的药物包封和跨膜能力结合起来，我们可以产生新的药物输送系统，这种系统可以为每个蛋白质输送更多的药物，包括那些在没有包封保护的情况下对血液代谢敏感的药物。在这个项目中，我们将制作一个修饰蛋白-载铁蛋白融合蛋白的文库，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载铁蛋白包封使其对那些对药物本身产生耐药性的细胞有效。如果其他药物也出现这种情况，就有机会延长药物的有效时间。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有良好的活性，但由于它们不能被细胞有效地吸收，因此不能用作治疗药物。该项目将使我们能够全面评估贴片-载铁蛋白系统，以确定它是否可以成为“下一代”靶向药物递送系统或抗体之后的“特洛伊木马”，用于更广泛的不同药物类型。与免疫球蛋白G抗体不同的是，抗原-载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，如果它们要在世界范围内随时可用。

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.