A general strategy for generating intact, full-length IgG antibodies that penetrate into the cytosol of living cells
A general strategy for generating intact, full-length IgG antibodies that penetrate into the cytosol of living cells
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DOI：
10.4161/mabs.36389
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发表时间：
2014-12-01
期刊：
影响因子：
5.3
通讯作者：
Kim, Yong-Sung
中科院分区：
文献类型：
--
作者：
Choi, Dong-Ki;Bae, Jeomil;Kim, Yong-Sung
全长的免疫球蛋白抗体不能穿过活细胞的细胞膜；这限制了它们直接靶向胞浆蛋白的使用。在这里，我们描述了一种产生完整的、全长的抗体的一般策略，这里称为细胞转移性抗体，它内化到活细胞中并定位于胞浆中。我们首先产生了一个人源化的轻链可变区(VL)，它可以穿透活细胞的胞浆，并被设计成与人类重链可变区(VHS)的不同亚型相关联。当含有人源化VL的轻链与3个重链(包括临床批准的阿达利单抗(Humira(R))和贝伐单抗(Avestin(R))的2个重链)共表达时，所有3个纯化的抗体都内化到活细胞的细胞质中。细胞转运蛋白主要通过与细胞表面表达的硫酸肝素蛋白多糖相互作用，通过网状蛋白介导的内吞途径进入活细胞。细胞转运体从早期的内小体逃逸到胞浆中，而不被进一步运输到其他细胞室，如溶酶体、内质网、高尔基体和细胞核。此外，当靶细胞与活细胞孵育时，我们产生了一种与靶细胞浆蛋白共定位的细胞传递体，表明该细胞传递体可以直接靶向胞浆蛋白。内化的细胞转导抗体未表现出明显的细胞毒性，在被蛋白小体降解前在胞浆中停留6h以上。这些结果表明，细胞转导抗体可以有效地进入活细胞并到达胞浆空间，将被广泛用作研究、诊断和治疗试剂。
Full-length IgG antibodies cannot cross cell membranes of living cells; this limits their use for direct targeting of cytosolic proteins. Here, we describe a general strategy for the generation of intact, full-length IgG antibodies, herein called cytotransmabs, which internalize into living cells and localize in the cytosol. We first generated a humanized light chain variable domain (VL) that could penetrate into the cytosol of living cells and was engineered for association with various subtypes of human heavy chain variable domains (VHs). When light chains with humanized VL were co-expressed with 3 heavy chains (HCs), including 2 HCs of the clinically approved adalimumab (Humira (R)) and bevacizumab (Avastin (R)), all 3 purified IgG antibodies were internalized into the cytoplasm of living cells. Cytotransmabs primarily internalized into living cells by the clathrin-mediated endocytic pathway through interactions with heparin sulfate proteoglycan that was expressed on the cell surface. The cytotransmabs escaped into the cytosol from early endosomes without being further transported into other cellular compartments, like the lysosomes, endoplasmic reticulum, Golgi apparatus, and nucleus. Furthermore, we generated a cytotransmab that co-localized with the targeted cytosolic protein when it was incubated with living cells, demonstrating that the cytotransmab can directly target cytosolic proteins. Internalized cytotransmabs did not show any noticeable cytotoxicity and remained in the cytosol for more than 6 h before being degraded by proteosomes. These results suggest that cytotransmabs, which efficiently enter living cells and reach the cytosolic space, will find widespread uses as research, diagnostic, and therapeutic agents.