Human serum albumin (HSA) has been investigated as a serum half-life extender of therapeutic proteins thanks to its unusually long serum half-life. However, in mice, the serum half-life of a HSA-conjugated protein was much shorter than that of HSA in humans, likely due to the species-dependent nature of albumin–FcRn interactions. Herein, we investigated species-dependent albumin–FcRn interactions using species-matched albumin (mouse serum albumin) and species-mismatched albumin (HSA) in non-transgenic mice. We site-specifically introduced a clickable non-natural amino acid to a target protein followed by conjugation to an albumin species via a hetero-bifunctional linker. Using in vitro binding assays, we showed that both HSA- and MSA-conjugated proteins bound mouse FcRns. Conjugation of HSA led to very limited extension of the serum half-life of sfGFP in mice (16.3 h), compared to that of HSA in transgenic mice harboring an allele of mouse FcRn knock-out and expressing humn FcRn (67 h) reported previously. These results suggest that the FcRn-mediated recycling of HSA is not effective in mice. However, conjugation of mouse serum albumin (MSA) resulted in a serum half-life of sfGFP (27.7 h) comparable to that of MSA in mice (28.8 h). Altogether, our study supported that albumin–FcRn interactions are species dependent in vivo.

中文翻译：

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通过位点特异性缀合至物种匹配或不匹配的白蛋白 的蛋白质的血清半衰期延长的比较研究†

人类血清白蛋白（HSA）由于其异常长的血清半衰期，已被研究作为治疗性蛋白的血清半衰期延长剂。然而，在小鼠中，HSA偶联蛋白的血清半衰期比人类中HSA的半衰期短得多，这可能是由于白蛋白-FcRn相互作用的物种依赖性所致。本文中，我们在非转基因小鼠中使用物种匹配的白蛋白（小鼠血清白蛋白）和物种不匹配的白蛋白（HSA）研究了物种依赖性白蛋白-FcRn相互作用。我们位点特异性地引入一个可点击的非天然氨基酸与靶蛋白，随后偶联至白蛋白物种通过一个异-双功能连接子。体外使用结合检测，我们显示HSA和MSA结合蛋白都结合了小鼠FcRns。与先前报道的携带小鼠FcRn敲除等位基因并表达腐殖质FcRn的转基因小鼠中的HSA相比，HSA的缀合导致小鼠sfGFP的血清半衰期延长非常有限（16.3 h）。 。这些结果表明，FcRn介导的HSA回收在小鼠中无效。但是，小鼠血清白蛋白（MSA）的缀合导致sfGFP的血清半衰期（27.7 h）与小鼠MSA的血清半衰期（28.8 h）相当。总之，我们的研究支持白蛋白-FcRn相互作用在体内具有物种依赖性。