New approaches for the site-selective chemical modification of therapeutic proteins under mild conditions

蛋白质治疗的位点特异性化学修饰，例如抗体、蛋白质激素和细胞因子，在癌症和疾病治疗、诊断和成像方面的应用非常重要。化学修饰可以赋予蛋白质疗法新的和定制的特性，例如更长的血液循环时间，靶向细胞杀伤（免疫偶联物），并报告其定位（例如通过荧光团或示踪剂）。在这个项目中，我们探索了由分裂内链催化的蛋白质反式剪接。分裂内链将其融合的肽或蛋白质序列与天然肽键连接，并在反应过程中同时去除自己，从而使该方法几乎无迹可寻。我们已经开发并继续改进两种方法来使用蛋白质反式剪接来制备化学修饰的蛋白质。合成片段通过与重组蛋白的合成肽剪接（半合成蛋白反式剪接）或通过在独特的半胱氨酸或非天然氨基酸残基上预标记的短标签剪接（化学标签剪接）转移到目标蛋白上。在第二个资助期，我们将生成新的功能，化学修饰的蛋白质，并分析它们的生化和生物医学特性。重点将放在抗体片段上，作为蛋白质治疗和细胞成像的潜在用途。我们还将继续开发基于分裂蛋白的新技术，在温和和自然条件下对蛋白质进行选择性化学修饰。这些蛋白质化学工具有望具有广泛的应用价值。

The site-specific chemical modification of protein therapeutics, for example antibodies, protein hormones, and cytokines, is of great importance for applications in cancer and disease treatment, diagnostics, and imaging. Chemical modifications can endow the protein therapeutics with new and tailored properties, for example longer circulation time in the blood-stream, targeted cell-killing (immunoconjugates), and reporting their localization (e.g. by fluorophores or tracers). In this project, we explore protein trans-splicing catalyzed by split inteins. Split inteins ligate their fused peptide or protein sequences with a native peptide bond and concomitantly remove themselves during the reaction, thus making the approach virtually traceless. We have developed and continue to improve two types of approaches to use protein trans-splicing for the preparation of chemically modified proteins. The synthetic moiety is transferred to the target protein either by splicing of a synthetic peptide with a recombinant protein (semi-synthetic protein trans-splicing) or by splicing of a short tag prelabeled at a unique cysteine or unnatural amino acid residue (chemical-tag splicing). In the second funding period we will generate new functional, chemically modified proteins and analyze them for biochemical and biomedical properties. The focus will be on antibody fragments potentially useful as protein therapeutics and for cellular imaging. We will also continue to develop new split-intein based technologies for selective chemical modification of proteins under mild and native conditions. These protein chemical tools are expected to be of high interest for a broad range of applications.