<br>通过设计分子大小和半衰期来优化蛋白质-药物缀合物的抗肿瘤功效,Journal of Controlled Release

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通过设计分子大小和半衰期来优化蛋白质-药物缀合物的抗肿瘤功效
Journal of Controlled Release ( IF 10.5 ) Pub Date : 2020-08-06 , DOI: 10.1016/j.jconrel.2020.08.004
Fabian Brandl ₁ , Sarah Busslinger ₂ , Uwe Zangemeister-Wittke ₁ , Andreas Plückthun ₂

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尽管抗体药物偶联物已获得一些用于癌症治疗的批准，但其临床成功率并不令人满意，因为治疗窗非常小，并且受到偶联物和释放毒素的靶向和脱靶毒性的影响。因此，必须探索具有系统研究的分子参数的其他形式，以增加其治疗窗口。这里我们重点关注有效分子量。为了生成具有精确定义的载药量和可调药代动力学的缀合物，我们使用设计的锚蛋白重复蛋白 (DARPins)，与不同长度的非结构化多肽融合，以产生具有任何所需半衰期的蛋白质，以确定具有最佳功效的蛋白质。我们生成了一种 EpCAM 靶向 DARPin-MMAF 缀合物，与不同长度的 PAS 或 XTEN 融合，以及一系列匹配的非结合 DARPin 对照，以解释增强渗透性和保留 (EPR) 效应，涵盖了小鼠中的分钟至 20.6 小时。所有缀合物均以高纯度生产，并在人肿瘤细胞培养物中表现出高特异性和细胞毒性，IC 值在低 nM 范围内，与多肽类型和长度无关。由于其更容易纯化，PASylated缀合物在携带HT29肿瘤异种移植物的裸鼠中进行了测试。无论其大小如何，所有 PASylated 缀合物在重复全身给药 300 nmol/kg 后都具有良好的耐受性。我们发现具有中等大小和半衰期的缀合物表现出最强的抗肿瘤作用，并推断这种作用是血清半衰期和肿瘤内扩散的折衷，因为结合率和亲和力基本上相同，外渗只起很小的作用。

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Optimizing the anti-tumor efficacy of protein-drug conjugates by engineering the molecular size and half-life

Despite some approvals of antibody-drug conjugates for cancer therapy, their clinical success rate is unsatisfactory because of very small therapeutic windows, influenced by on-target and off-target toxicities of conjugate and liberated toxin. Additional formats with systematically investigated molecular parameters must therefore be explored to increase their therapeutic window. Here we focused on the effective molecular weight. To generate conjugates with exactly defined drug loads and tunable pharmacokinetics, we used Designed Ankyrin Repeat Proteins (DARPins), fused to unstructured polypeptides of different lengths, to produce proteins with any desired half-life, to identify those with the best efficacy. We generated an EpCAM-targeting DARPin-MMAF conjugate, fused to PAS or XTEN of different lengths, and a matched series of controls of a non-binding DARPin to account for the enhanced permeability and retention (EPR) effect, covering half-lives of minutes to 20.6 h in mice. All conjugates were produced at high purity, and demonstrated high specificity and cytotoxicity in human tumor cell cultures, with IC values in the low nM range, independent of the polypeptide type and length. Due to their more facile purification, the PASylated conjugates were tested in nude mice bearing HT29 tumor xenografts. Independent of their size, all PASylated conjugates were very well tolerated after repeated systemic administration of 300 nmol/kg. We found that the conjugates with intermediate size and half-life showed the strongest anti-tumor effects, and deduced that this effect is a compromise of serum half-life and diffusion within the tumor, as on-rates and affinities are essentially identical, with extravasation playing only a very minor role.

更新日期：2020-08-06

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