In this work, we show that a prodrug enzyme covalently photoconjugated to live cell receptors survives endosomal proteolysis and retains its catalytic activity over multiple days. Here, a fusion protein was designed with both an antiepidermal growth factor receptor (EGFR) affibody and the prodrug enzyme cytosine deaminase, which can convert prodrug 5-fluorocytosine to the anticancer drug 5-fluorouracil. A benzophenone group was added at a site-specific mutation within the affibody, and the fusion protein was selectively photoconjugated to EGFR receptors expressed on membranes of MDA-MB-468 breast cancer cells. The fusion protein was next labeled with two dyes for tracking uptake: AlexaFluor 488 and pH-sensitive pHAb. Flow cytometry showed that fusion proteins photo-cross-linked to EGFR first underwent receptor-mediated endocytosis within 12 h, followed by recycling back to the cell membrane within 24 h. These findings were also confirmed by confocal microscopy. The unique cross-linking of the affibody-enzyme fusion proteins was utilized for two anticancer treatments. First, the covalent linking of the protein to the EGFR led to inhibition of ERK signaling over a two-day period, whereas conventional antibody therapy only led to 6 h of inhibition. Second, when the affibody-CodA fusion proteins were photo-cross-linked to EGFR overexpressed on MDA-MB-468 breast cancer cells, prodrug conversion was found even 48 h postincubation without any apparent decrease in cell killing, while without photo-cross-linking no cell killing was observed 8 h postincubation. These studies show that affinity-mediated covalent conjugation of the affibody-enzymes to cell receptors allows for prolonged expression on membranes and retained enzymatic activity without genetic engineering.

中文翻译：

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与活细胞受体光交联的酶在内化和回收后仍保留活性和 EGFR 抑制作用。

在这项工作中，我们表明，与活细胞受体共价光缀合的前药酶能够在内体蛋白水解作用中存活下来，并在多天内保留其催化活性。在此，设计了一种具有抗表皮生长因子受体（EGFR）亲和体和前药酶胞嘧啶脱氨酶的融合蛋白，该融合蛋白可以将前药5-氟胞嘧啶转化为抗癌药物5-氟尿嘧啶。在亲和体内的位点特异性突变处添加二苯甲酮基团，并且融合蛋白选择性地光缀合至MDA-MB-468乳腺癌细胞膜上表达的EGFR受体。接下来用两种染料标记融合蛋白以跟踪摄取：AlexaFluor 488 和 pH 敏感的 pHAb。流式细胞术显示与 EGFR 光交联的融合蛋白在 12 小时内首先经历受体介导的内吞作用，然后在24小时内循环回到细胞膜。这些发现也得到了共焦显微镜的证实。亲和体-酶融合蛋白的独特交联被用于两种抗癌治疗。首先，该蛋白与 EGFR 的共价连接导致 ERK 信号传导在两天内受到抑制，而传统抗体疗法仅导致 6 小时的抑制。其次，当 affibody-CodA 融合蛋白与 MDA-MB-468 乳腺癌细胞上过表达的 EGFR 光交联时，即使在孵育 48 小时后也发现前药转化，细胞杀伤力没有任何明显减少，而没有光交叉连接时，发现前药转化。孵育8小时后未观察到细胞死亡。