Strong humoral response elicited by a DNA vaccine targeting gastrin-releasing peptide with optimized adjuvants inhibits murine prostate carcinoma growth in vivo

Previous studies demonstrated that the elevated expression and receptor binding of gastrin-releasing peptide (GRP) in various types of cancer suggest that GRP might be a putative target for immunotherapy in neoplastic diseases. DNA vaccine for hormone/growth factor immune deprivation represents a feasible and attractive approach for cancer treatment; nevertheless, there is still a need to increase the potency of the DNA vaccine. Here, based on six copies of the B cell epitope GRP18–27 in a linear alignment as an immunogen, we designed several anti-GRP DNA vaccines containing different combinations of immunoadjuvants, such as HSP65, tetanus toxoid830–844 (T), pan HLA-DR-binding epitope (PADRE) (P), and mycobacterial HSP70407–426 (M), on a backbone of pCR3.1 plasmid vector with eight 5′-GACGTT-3′ CpG motifs and the VEGF183 signal peptide (VS). The effects of these immunoadjuvants in enhancing GRP-specific humoral immune response were then evaluated by comparing the respective immunogenicity and antitumor effects. Immunization of mice with pCR3.1-VS-HSP65-TP-GRP6-M2 elicited much higher levels of specific anti-GRP antibodies and more effectively inhibited the growth of a GRP-dependent tumor RM-1 in vivo. Interestingly, plasmids encoding for 2HSP70407–426, but not the one with 1 or 3HSP70407–426 showed stronger immune stimulatory potential as well as impressive antitumor activity, suggesting that 2HSP70407–426 is an efficient molecular adjuvant for developing self-epitope vaccines. The highly immunogenic, potent anti-tumorigenic and antiangiogenesis activities of the anti-GRP DNA vaccine offered a novel immunotherapeutic approach in the treatment of GRP-dependent tumors and their complications.

Specific Antibodies Elicited by a Novel DNA Vaccine Targeting Gastrin-Releasing Peptide Inhibit Murine Melanoma Growth In Vivo

ABSTRACT The elevated expression and receptor binding of gastrin-releasing peptide (GRP) in various types of cancer, especially in malignant melanoma of the skin, suggest that GRP might be a putative target for immunotherapy in neoplastic diseases. We have therefore constructed a novel DNA vaccine coding for six tandem repeats of a fragment of GRP from amino acids 18 to 27 (GRP6) flanked by helper T-cell epitopes for increased immunogenicity, including HSP65, a tetanus toxoid fragment from amino acids 830 to 844 (T), pan-HLA-DR-binding epitope (PADRE) (P), and two repeats of a mycobacterial HSP70 fragment from amino acids 407 to 426 (M). The anti-GRP DNA vaccine (pCR3.1-VS-HSP65-TP-GRP6-M2) was constructed on a backbone of a pCR3.1 plasmid vector with eight 5′-GACGTT-3′ CpG motifs and the VEGF183 signal peptide (VS). Intramuscular (IM) injections of anti-GRP vaccine in mice stimulated the production of high titers of specific antibodies against GRP and suppressed the growth of subcutaneous tumors of B16-F10 melanoma cells. Parallel results were obtained in vitro, showing inhibition of B16-F10 cell proliferation by GRP antisera. IM injections of the DNA vaccine also significantly attenuated tumor-induced angiogenesis associated with intradermal tumors of B16-F10 cells. In addition, lung invasion of intravenously injected cells was highly diminished, suggesting potent antimetastatic activity of the DNA vaccine. These findings support the highly immunogenic and potent antitumorigenic activity of specific anti-GRP antibodies elicited by the anti-GRP DNA vaccine.