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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]

Currently, the treatment options for patients with neuroendocrine carcinomas (NECs) are limited. Stem cell factor is found to have a trophic role in neuroendocrine cancer cells through an autocrine growth loop, which leads to the activation of the c-kit receptor. NECs like neuroblastomas, small cell lung carcinomas and poorly differentiated human colon carcinoma cells (CRCs) over-express both the c-kit receptor and its ligand, stem cell factor (SCF), as compared to normal cells. The role of the known c-kit inhibitor imatinib has already been investigated in these cell types and has shown promising results. We developed SCF-based c-kit targeting protein fusion toxins against neuroendocrine carcinomas, such as neuroblastomas and colorectal carcinomas. In this study, SCF was cloned from the HepG2 cell line and mutated in order to optimize its expression. The mutated SCF was recombinantly fused with bacterial toxins (Diphtheria toxin (DT) and Pseudomonas exotoxin A (PE)) to form c-kit targeting fusion toxins. These proteins were expressed in E.coli and purified by affinity chromatography followed by size exclusion chromatography. Flow cytometric analysis was used to monitor receptor expression on c-kit positive neuroblastoma cell lines (IMR 32 and SHSY5Y), colon carcinoma cell lines (HT-29, HCT 116 and DLD-1), and the c-kit negative cell line, MCF-7. In vitro binding, internalization and toxicity studies were performed on the above cell lines in order to characterize the purified chimeric toxins. Annexin V binding assays and cell cycle analysis were performed additionally, to prove their efficacy. The ID50 values for the toxins were found to correlate with the receptor expression on these cell lines. The novel c-kit targeting protein fusion toxins reported in our study offer a promising strategy for therapeutics against neuroendocrine tumors and warrant further testing in such tumors with c-kit autocrine and paracrine loops. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B240. Citation Format: Swati Choudhary, Alessa Pardo, Reinhard Rosinke, Stefan Barth, Janendra K. Batra, Rama S. Verma. Novel protein fusion toxins targeting c-kit positive neuroendocrine tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B240.