Stahnke Bettina, Thepen Theo, Stöcker Michael, Rosinke Reinhard, Jost Edgar, Fischer Rainer, Tur Mehmet K, Barth Stefan
Fraunhofer IME, Department of Pharmaceutical Product Development, Forckenbeckstr. 6, 52074 Aachen, Germany.
Mol Cancer Ther. 2008 Sep;7(9):2924-32. doi: 10.1158/1535-7163.MCT-08-0554.
Acute myeloid leukemia (AML) cells of subtypes M4 and M5 show enhanced expression of CD64 (FcgammaRI), the high-affinity receptor for IgG, which is normally expressed at high levels only on activated cells of the myeloid lineage. CD64 is therefore a prime target for the specific delivery of cytotoxic agents. A promising toxin candidate is granzyme B, a human serine protease originating from cytotoxic granules of CD8+ T lymphocytes and natural killer cells. After evaluating the sensitivity of the AML-related cell line U937 toward cytosolic granzyme B, we genetically fused granzyme B to H22, a humanized single-chain antibody fragment (scFv) specific for CD64, to obtain Gb-H22(scFv), a fusion protein lacking the immunogenic properties of nonhuman immunofusions. Gb-H22(scFv) was successfully expressed in human 293T cells, secreted, and purified from cell culture supernatants. The purified protein bound specifically to CD64+ U937 cells. Despite linkage to the binding domain, the proteolytic activity of functional Gb-H22(scFv) was identical to that of free granzyme B. Target cell-specific cytotoxicity was observed with a half-maximal inhibitory concentration (IC50) between 1.7 and 17 nmol/L. In addition, the induction of apoptosis in U937 cells was confirmed by Annexin A5 staining and the detection of activated caspase-3 in the cytosol. Finally, apoptosis was observed in primary CD64+ AML cells, whereas CD64(-) AML cells were unaffected. This is the first report of a completely human granzyme B-based immunotoxin directed against CD64, with activity against an AML-related cell line and primary AML cells.
M4和M5亚型的急性髓系白血病（AML）细胞显示出CD64（FcγRI）的表达增强，CD64是IgG的高亲和力受体，通常仅在髓系谱系的活化细胞上高水平表达。因此，CD64是细胞毒性药物特异性递送的主要靶点。一种有前景的毒素候选物是颗粒酶B，它是一种源自CD8 + T淋巴细胞和自然杀伤细胞的细胞毒性颗粒的人丝氨酸蛋白酶。在评估AML相关细胞系U937对胞质颗粒酶B的敏感性后，我们将颗粒酶B与H22基因融合，H22是一种对CD64具有特异性的人源化单链抗体片段（scFv），以获得Gb-H22（scFv），一种缺乏非人免疫融合物免疫原性的融合蛋白。Gb-H22（scFv）在人293T细胞中成功表达、分泌，并从细胞培养上清液中纯化。纯化的蛋白特异性结合CD64 + U937细胞。尽管与结合域相连，但功能性Gb-H22（scFv）的蛋白水解活性与游离颗粒酶B相同。观察到靶细胞特异性细胞毒性，半数最大抑制浓度（IC50）在1.7至17 nmol/L之间。此外，通过膜联蛋白A5染色和胞质中活化的半胱天冬酶-3的检测证实了U937细胞中凋亡的诱导。最后，在原发性CD64 + AML细胞中观察到凋亡，而CD64（ - ）AML细胞未受影响。这是第一份关于针对CD64的完全基于人颗粒酶B的免疫毒素的报告，该毒素对AML相关细胞系和原发性AML细胞具有活性。
