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Comparative Study
, 95 (1), 71-8

Targeting C-type Lectin-Like molecule-1 for Antibody-Mediated Immunotherapy in Acute Myeloid Leukemia

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Comparative Study

Targeting C-type Lectin-Like molecule-1 for Antibody-Mediated Immunotherapy in Acute Myeloid Leukemia

Xiaoxian Zhao et al. Haematologica.

Abstract

Background: C-type lectin-like molecule-1 is a transmembrane receptor expressed on myeloid cells, acute myeloid leukemia blasts and leukemic stem cells. To validate the potential of this receptor as a therapeutic target in acute myeloid leukemia, we generated a series of monoclonal antibodies against the extracellular domain of C-type lectin-like molecule-1 and used them to extend the expression profile analysis of acute myeloid leukemia cells and to select cytotoxic monoclonal antibodies against acute myeloid leukemia cells in preclinical models.

Design and methods: C-type lectin-like molecule-1 expression was analyzed in acute myeloid leukemia cell lines, and in myeloid derived cells from patients with acute myeloid leukemia and healthy donors. Anti-C-type lectin-like molecule-1 antibody-mediated in vitro cytotoxic activity against acute myeloid leukemia blasts/cell lines and in vivo anti-cancer activity in a mouse xenograft model were assessed. Internalization of C-type lectin-like molecule-1 monoclonal antibodies upon receptor ligation was also investigated.

Results: C-type lectin-like molecule-1 was expressed in 86.5% (45/52) of cases of acute myeloid leukemia, in 54.5% (12/22) of acute myeloid leukemia CD34(+)/CD38(-) stem cells, but not in acute lymphoblastic leukemia blasts (n=5). Selected anti-C-type lectin-like molecule-1 monoclonal antibodies mediated dose-dependent complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity specifically against acute myeloid leukemia-derived cell lines. Exogenous expression of the transmembrane receptor in HEK293 cells rendered the cells susceptible to antibody-mediated killing by monoclonal antibodies to the receptor. Furthermore, these monoclonal antibodies demonstrated strong complement-dependent cytotoxicity against freshly isolated acute myeloid leukemia blasts (15/16 cases; 94%). The monoclonal antibodies were efficiently internalized upon binding to C-type lectin-like molecule-1 in HL-60 cells. Moreover, a lead chimeric C-type lectin-like molecule-1 monoclonal antibody reduced the tumor size in xenograft mice implanted with HL-60 cells. Conclusions Our results demonstrate that targeting C-type lectin-like molecule-1 with specific cytotoxic monoclonal antibodies is an attractive approach which could lead to novel therapies for acute myeloid leukemia.

Figures

Figure 1.
Figure 1.
Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) of CLL-1 monoclonal antibodies. (A) CDC assay using human embryonic kidney (HEK) 293 cells stably expressing CLL-1 compared to wild-type HEK293 cells and lymphoma CA46 cells. Expression of CLL-1 on 293 parental and transfectants is represented by the histograms in the top left corner. (B) Ex vivo CDC of chimeric monoclonal antibody 1075.7 against freshly isolated AML blasts. (C) ADCC activity of anti-CLL-1 chimeric monoclonal antibody 1075.7 against HL-60 cells.
Figure 2.
Figure 2.
Internalization of CLL-1 upon monoclonal antibody binding. (A) HEK293 cells stably expressing CLL-1 were stained with fluorescently labeled CLL-1 mouse monoclonal antibody 1075.7, incubated for the indicated times at 37°C, fixed and examined using fluorescence microscopy. (B) CLL-1 internalization in HL-60 cells. After staining with monoclonal antibody 1075.7, cells were incubated for the indicated times at 37°C after which they were (squares) or were not (diamonds) subjected to protease treatment prior to flow cytometry analysis. The staining observed in the protease-treated cells represents only intracellular staining.
Figure 3.
Figure 3.
In vivo anti-tumor activity of anti-CLL-1 chimeric monoclonal antibody 1075.7 in HL-60 cell-inoculated SCID mice. Animals were dosed twice weekly with the indicated amount of 1075.7 monoclonal antibody upon establishment of subcutaneous tumors. (A) Growth curves of control and experimental treatments as measured 3 times/week. (B) Summary of HL-60 xenograft tumors treated with chimeric monoclonal antibody 1075.7 at 0.5 and 5 mg/kg. TTE=time to end-point in days, TGD=tumor growth delay as percentage compared to growth in animals administered saline control treatment.

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