School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju 61005, Republic of Korea.
Mol Pharm. 2021 Jun 7;18(6):2397-2405. doi: 10.1021/acs.molpharmaceut.1c00231. Epub 2021 May 13.
Human serum albumin (HSA) has been used to extend the serum half-life of therapeutic proteins owing to its exceptionally long serum half-life via the neonatal Fc receptor (FcRn)-mediated recycling mechanism. In most cases, only one HSA molecule was conjugated to a therapeutic protein, leading to a limited extension of the serum half-life. In this study, we hypothesized that conjugation of multiple HSA molecules to a therapeutic protein significantly further extends the serum half-life via multivalent HSA-FcRn interactions. We chose urate oxidase (Uox), a tetrameric therapeutic protein used for the treatment of gout, as a model. In previous studies, only one HSA molecule was site-specifically conjugated to one Uox because of poor conjugation yield of the relatively slow bio-orthogonal chemistry, strain-promoted azide-alkyne cycloaddition (SPAAC). To increase the number of HSA molecules conjugated to one Uox, we employed the faster bio-orthogonal chemistry, inverse electron demand Diels-Alder reaction (IEDDA). We site-specifically introduced the phenylalanine analog with a fast-reacting tetrazine group (frTet) into position 174 of each subunit of Uox. We then achieved site-specific HSA conjugation to each subunit of Uox via IEDDA, generating Uox conjugated to four HSA molecules (Uox-HSA4), with a small portion of Uox conjugated to three HSA molecules (Uox-HSA3). We characterized Uox-HSA4 as well as Uox variants conjugated to one or two HSA molecules prepared via SPAAC (Uox-HSA1 or Uox-HSA2). The enzyme activity of all three Uox-HSA conjugates was comparable to that of unmodified Uox. We found out that an increase in HSA molecules conjugated to Uox (multiple albumin-conjugated therapeutic protein) enhanced FcRn binding and consequently prolonged the serum half-life in vivo. In particular, the conjugation of four HSA molecules to Uox led to a prominent extension of the serum half-life (over 21 h), which is about 16-fold longer than that of Uox-WT.
人血清白蛋白（HSA）由于其通过新生 Fc 受体（FcRn）介导的循环机制具有异常长的血清半衰期，已被用于延长治疗性蛋白的血清半衰期。在大多数情况下，只有一个 HSA 分子与治疗性蛋白结合，导致血清半衰期的延长有限。在这项研究中，我们假设通过多价 HSA-FcRn 相互作用将多个 HSA 分子与治疗性蛋白结合可显著进一步延长血清半衰期。我们选择尿酸氧化酶（Uox）作为模型，Uox 是一种四聚体治疗蛋白，用于治疗痛风。在之前的研究中，由于相对较慢的生物正交化学应变促进叠氮化物-炔烃环加成（SPAAC）的缀合产率低，只能将一个 HSA 分子定点连接到一个 Uox 上。为了增加一个 Uox 上结合的 HSA 分子数量，我们采用了更快的生物正交化学物逆电子需求 Diels-Alder 反应（IEDDA）。我们在 Uox 的每个亚基的 174 位特异性引入带有快速反应四嗪基团（frTet）的苯丙氨酸类似物。然后，我们通过 IEDDA 实现了 Uox 各亚基的定点 HSA 缀合，生成了与四个 HSA 分子缀合的 Uox（Uox-HSA4），其中一小部分 Uox 与三个 HSA 分子缀合（Uox-HSA3）。我们对 Uox-HSA4 以及通过 SPAAC 制备的与一个或两个 HSA 分子缀合的 Uox 变体（Uox-HSA1 或 Uox-HSA2）进行了表征。所有三种 Uox-HSA 缀合物的酶活性均与未修饰的 Uox 相当。我们发现，与 Uox 缀合的 HSA 分子数量的增加（多白蛋白缀合的治疗性蛋白）增强了 FcRn 的结合，从而延长了体内的血清半衰期。特别是，将四个 HSA 分子缀合到 Uox 上导致血清半衰期显著延长（超过 21 小时），大约是 Uox-WT 的 16 倍。
