Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes.
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.
Stahnke
Bettina
B
Fraunhofer IME, Department of Pharmaceutical Product Development, Forckenbeckstr. 6, 52074 Aachen, Germany.
Thepen
Theo
T
Stöcker
Michael
M
Rosinke
Reinhard
R
Jost
Edgar
E
Fischer
Rainer
R
Tur
Mehmet K
MK
Barth
Stefan
S
D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D004354:Drug Screening Assays, Antitumor; D004789:Enzyme Activation / Q000187:drug effects; D053804:Granzymes / Q000494:pharmacology*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000494:pharmacology*; D000922:Immunotoxins / Q000494:pharmacology*; D007948:Leukemia, Monocytic, Acute / Q000201:enzymology / Q000276:immunology / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017452:Receptors, IgG / Q000037:antagonists & inhibitors*; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D020298:U937 Cells
D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; D053804:Granzymes; D053148:Caspase 3