Targeted induction of apoptosis by chimeric granzyme B fusion proteins carrying antibody and growth factor domains for cell recognition
Targeted induction of apoptosis by chimeric granzyme B fusion proteins carrying antibody and growth factor domains for cell recognition
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DOI：
10.1038/sj.cdd.4401773
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发表时间：
2006-04-01
影响因子：
12.4
通讯作者：
Wels, WS
中科院分区：
文献类型：
--
作者：
Dälken, B;Giesübel, U;Wels, WS
细胞毒性淋巴细胞的丝氨酸蛋白酶颗粒酶B （GrB）通过直接激活caspase和切割中心caspase底物有效地诱导细胞凋亡。我们将人GrB作为嵌合融合蛋白的效应功能，这些嵌合融合蛋白也含有EGFR配体TGF α或erbb2特异性单链抗体片段（scFv），用于选择性靶向肿瘤细胞。酵母毕赤酵母中表达的GrB- tgf α (GrB- t)和GrB- scfv (FRP5) （GrB-5）分子具有双功能，可切割合成和天然GrB底物，并特异性结合表达EGFR或ErbB2靶受体的细胞。在细胞结合后，嵌合分子被内化到细胞内囊泡中，但可以通过内溶试剂氯喹释放到细胞质中。以皮摩尔至纳摩尔浓度的GrB-5和GrB-T治疗可选择性和快速杀死肿瘤细胞，并伴有明显的凋亡迹象，如染色质凝聚、膜泡、凋亡小体的形成以及内源性引发剂和效应剂半胱天蛋白酶的激活。
The serine protease granzyme B (GrB) of cytotoxic lymphocytes efficiently induces apoptosis by direct activation of caspases and cleavage of central caspase substrates. We employed human GrB as an effector function in chimeric fusion proteins that also contain the EGFR ligand TGF alpha or an ErbB2-specific single-chain antibody fragment (scFv) for selective targeting to tumor cells. GrB-TGF alpha (GrB-T) and GrB-scFv(FRP5) (GrB-5) molecules expressed in the yeast Pichia pastoris were bifunctional, cleaving synthetic and natural GrB substrates, and binding specifically to cells expressing EGFR or ErbB2 target receptors. Upon cell binding the chimeric molecules were internalized into intracellular vesicles, but could be released into the cytosol by the endosomolytic reagent chloroquine. Treatment with picomolar to nanomolar concentrations of GrB-5 and GrB-T resulted in selective and rapid tumor cell killing, accompanied by clear signs of apoptosis such as chromatin condensation, membrane blebbing, formation of apoptotic bodies and activation of endogenous initiator and effector caspases.