Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes

Abstract

Acute myeloid leukemia (AML) cells of subtypes M4 and M5 show enhanced expression of CD64 (Fc;RI), the highaffinity 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 (IC 50 ) 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 À 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]

Targeting CD64 mediates elimination of M1 but not M2 macrophages in vitro and in cutaneous inflammation in mice and patient biopsies

“…Furthermore, 2 human H22(scFv)-based CFPs, H22(scFv)-Angiogenin 25 and Granzyme B-H22(scFv), 26 were tested and again selectively killed the human M1 macrophages. This shows that the cytotoxic specificity is independent of the effector protein (Fig.…”

EpCAM-Selective Elimination of Carcinoma Cells by a Novel MAP-Based Cytolytic Fusion Protein

“…To reduce immunogenic potential and offtarget effects, proapoptotic human enzymes have been exploited in the context of fully human CFPs. In 2008, an entirely human granzyme B-based CFP directed against CD64 was found to be toxic for an acute myeloid leukemia (AML)-related cell line and primary AML cells with IC 50 values ranging between 1.7 and 17 nmol/L (27). The human RNase angiogenin was shown to be specifically cytotoxic toward CD30-overexpressing Hodgkin lymphoma-derived cell lines, when fused to the CD30 ligand (26).…”

“…Several human enzymes, including the protease granzyme B, RNase angiogenin, or death-associated protein kinase 2, have been demonstrated to induce apoptosis in target cells, when delivered by antigen-specific ligands or antibody fragments (26)(27)(28)(29). Similarly, death ligands of the tumor necrosis factor (TNF) family have been genetically fused to anticancer scFvs to selectively induce apoptosis in various forms of cancer (30,31).…”

Human microtubule‐associated protein tau mediates targeted killing of CD30+ lymphoma cells in vitro and inhibits tumour growth in vivo

“…In addition to human granzyme B, human RNases, such as angiogenin, have been used to replace nonhuman toxins (Hetzel et al, 2008;Stahnke et al, 2008;Mathew & Verma, 2009;Schiffer et al, 2013). The specific cytotoxicity of human angiogenin towards CD30-overexpressing HL-derived cell lines has been demonstrated by fusion with the CD30 ligand (Huhn et al, 2001).…”

“…In 2008, an entirely human granzyme B-based CFP directed against CD64 was found to be toxic for an acute myeloid leukaemia (AML)-related cell line and primary AML cells with IC 50 values ranging between 1Á7 and 17 nmol/l (Stahnke et al, 2008). Recently, our group also reported on an improved version of granzyme B, which shows resistance against the endogenous inhibitor serpin B9 and thus overcomes one potential escape strategy of CD30 + lymphoma cells (Schiffer et al, 2013).…”