Induction of antibodies against prostate-specific membrane antigen (PSMA) by vaccination with a PSMA DNA vector

Prostate-specific membrane antigen (PSMA) is a 750 amino acid surface protein expressed primarily in prostate epithelium, and is upregulated 10-fold in prostate cancer. It is therefore an attractive target for immunotherapy. However, most reported antibodies to PSMA apparently recognize epitopes in the residue 43-570 region of the extracellular domain, and upon binding are rapidly removed from the cell surface by internalization. This would potentially limit their ability to mediate Fc-dependent cytoxicity. In this study, we constructed a DNA expression vector, pV/TM-PSMc, in which this region was deleted from full-length PSMA cDNA. Mice were vaccinated with pV/TM-PSMc DNA to determine whether humoral responses directed against PSMA-positive human prostate cancer cells could be induced by this C-terminal region.

Polymerase chain reaction (PCR)-based techniques were used to delete codons 50-570 from the coding region of human PSMA cDNA, thereby joining the C-terminal end (PSMc) to the N-terminal cytoplasmic/transmembrane domain (TM). This truncated product, TM-PSMc, was cloned into the vector pNGVL3 (pV). The resulting vector, pV/TM-PSMc, was confirmed by DNA sequencing, and by expression studies using reverse transcriptase (RT)-PCR for transcripts and immunohistochemical (IHC) staining with the PSMA monoclonal antibody (mAb) 7E11.C5. BALB/c mice were injected in the tibialis anterior muscle four times, at biweekly intervals, with 100 microg vector DNA per injection. One week after the last injection, blood was drawn for serum preparation. The serum was assayed for antibodies against PSMA by IHC staining of LNCaP, a PSMA-positive human prostate cancer line. Expression in vaccinated muscle cells was determined by RT-PCR assay for TM-PSMc transcripts.

NIH3T3 cells transfected with pV/TM-PSMc stained positively by IHC reaction with mAb 7E11.C5. 48h after one intramuscular (i.m.) injection of mice with 100 microg pV/TM-PSMc vector DNA, TM-PSMc transcripts were detectable in muscle RNA by RT-PCR analysis. Anti-serum from pV/TM-PSMc-DNA vaccinated mice, at a dilution of 1:20, intensely IHC-stained both live and fixed LNCaP cells.

These results demonstrate that anti-PSMA humoral responses were induced by i.m. injection of mice with pV/TM-PSMc DNA. Antibodies in the anti-serum were directed against extracellular epitopes of native PSMA expressed by human prostate cancer cells. Vaccination with DNA expression vectors such as pV/TM-PSMc may provide an immunotherapeutic approach for the treatment of prostate cancer.