Novel Human Monoclonal Antibody Against CD47 Induces Cell Death in Lymphoid Malignant Cells In Vitro and In Vivo: Potential Novel Therapeutic Agent for B-CLL.

CD47 belongs to immunoglobulin superfamily, and is expressed as a 50 kDa cell surface antigen in wide variety of tissues. CD47 associates with integrins of β1, β2, and β3, and it serves as a receptor for thrombospondin (TSP) and also as a ligand for transmembrane signal regulatory protein SIRP-α. CD47 has a number of different functions such as platelet activation, cell motility, leukocyte adhesion and migration. Recently, it was reported that the ligation of CD47 induces cell death in T-cells and chronic lymphocytic leukemic B cells (B-CLL) in a caspase-independent manner. B-CLL is the most common hematological malignancy in Western countries. Although new chemotherapeutic agents including fludarabine and 2-chlorodeoxyadenosine have been introduced into the clinic, B-CLL is not curable, and therefore, there is a strong need of new effective drugs. In an attempt to establish a novel therapeutic agent for B-CLL, we generated a murine monoclonal antibody against an extracellular domain of human CD47 (designated MABL). Soluble MABL (10 μg/ml) induced apoptosis in CD47-positive CCRF-CEM and MOLT-4 cells but not in CD47-negative cells as judged by annexin V staining. Caspase-3 was not activated by MABL, confirming that the cell death mediated by CD47 was caspase independent. In addition, administration of the F(ab’)2 of MABL (200 μg/mouse twice a day on day 3, 4 and 5) significantly prolonged the survival of the SCID mice inoculated CCRF-CEM (>150 days in MABL-treated group vs. <50 days in vehicle control group), indicating that even in vivo MABL caused tumor cell death independently of ADCC and CDC. Because both MABL and F(ab’)2 of MABL caused hemoagglutination, we created a disulfide-stabilized dimmer of a single-chain antibody fragment (scFv/S-S) of MABL to get rid of such an adverse effect. scFv/S-S indeed did not cause hemoagglutination, whereas MABL and the F(ab’)2 of MABL did. Induction of apoptosis was augmented with MABL cross-linked with 50 mg/ml goat anti-mouse IgG (GAM). In primary B-CLL cells, the percentage of annexin V-positive cells was 36.3% in the MABL (10 mg/ml, for 24 h)-treated cells, but was increased to 68.1% in the MABL plus GAM-treated cells. In addition, the same degree of apoptosis was achieved by scFv/S-S of MABL alone. These results demonstrate that scFv/S-S of MABL induced the ligation of CD47 more efficiently than original MABL without causing hemoagglutination, and therefore, scFv/S-S can be used as a therapeutic agent. In order to gain insight into the mechanism underlying the cell death by the ligation of CD47, we carried out gene expression profiling analysis. Gene expression profiling with Affymetrix gene chips of MOLT-4 cells revealed 54 up-regulated genes and 313 down-regulated genes by the treatment of MABL plus GAM. Involvement of these genes in cell death mediated by the ligation of CD47 is speculated. In conclusion, newly established human monoclonal antibody MABL and scFv/S-S against CD47 induced cell death in lymphoid malignant cells including B-CLL cells in a caspase-independent manner. In addition, MABL antibody showed anti-tumor activity in vivo. Taken together, these new antibodies against CD47 may have a potential as a novel therapeutic agent for the treatment of incurable lymphoid malignancies including B-CLL.