CD47 as a potential target for molecular imaging

Objectives The goal of this study was to evaluate CD47, a protein up-regulated on cancer cells where it protects them against attack by the host immune system, as a PET imaging target for cancer diagnosis and prognosis.

Methods OV10 is a CD47 deficient clone of the human ovarian carcinoma parental cell line, OVM1. This clone was stably transfected with the most commonly expressed human CD47 splice variant 2. Anti-CD47 antibody B6H12 was conjugated to the Zr chelator, desferroxamine and the resulting complex labeled with Zr-89 at 37°C for 1 hour, followed by purification with a desalting column. CD47 sufficient and deficient cells were grown to about 50% confluency on 6-well tissue culture plates and incubated with 2 µCi (0.5 µg) of radiolabeled antibody for 35 minutes in humidified atmosphere supplemented with 5% CO2. Unbound antibody was removed and the cells were washed three times with phosphate buffered saline. Cells were then solubilized in PBS containing 0.1% SDS, the lysate was transferred to microfuge tubes and cell associated activity detected with a gamma-counter. Protein content was measured with the bicinchoninic acid assay and the data expressed as counts per milligram of protein. These cell lines were also used at 2E6 per animal to generate subcutaneous xenographt models in athymic NCR nu/nu mice.

Results The antibody-chelate complex was efficiently labeled with Zr-89 at 4 µCi/µg specific activity. The complex was taken up by the CD47 sufficient and not by deficient cells. The same cell lines developed tumors of about 1cm in diameter within 14 days from the time of implant. Importantly, CD47 sufficient and deficient cell lines presented tumors of similar size.

Conclusions These results indicate that the antibody against CD47 can be effectively labeled with Zr-89 and used as an imaging probe for tumors over expressing this marker. Animal studies are underway to determine the feasibility of in vivo imaging with this novel radiotracer