In:
Molecular Cancer Therapeutics, American Association for Cancer Research (AACR), Vol. 7, No. 9 ( 2008-09-01), p. 2924-2932
Abstract:
Acute myeloid leukemia (AML) cells of subtypes M4 and M5 show enhanced expression of CD64 (FcγRI), 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. [Mol Cancer Ther 2008;7(9):2924–32]
Type of Medium:
Online Resource
ISSN:
1535-7163
,
1538-8514
DOI:
10.1158/1535-7163.MCT-08-0554
Language:
English
Publisher:
American Association for Cancer Research (AACR)
Publication Date:
2008
detail.hit.zdb_id:
2062135-8
SSG:
12
