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.
Yong Lu, Didier J L Mekoo, Kedong Ouyang, Xiangbing Hu, Yanhua Liu, Ming Lin, Liang Jin, Rongyue Cao, Taiming Li, Yankai Zhang, Hao Fan and Jingjing Liu
Wu Guojun, Guo Wei, Ouyan Kedong, He Yi, Xie Yanfei, Chen Qingmei, Zhang Yankai, Wu Jie, Fan Hao, Li Taiming, Liu Jingjing and Cao Rongyue
Gastrin-releasing peptide (GRP), a bombesin-like peptide, is an autocrine growth factor that can stimulate the growth of various cancer cells. We developed a novel protein vaccine HSP65-(GRP-10)6 (HG6) that consists of six copies of a 10-amino acid residue epitope of GRP C-terminal fragment carried by mycobacterial 65 kDa HSP65 and then immunized mice via subcutaneous injection. Strong humoral and cell-mediated immune responses were induced. High titer of anti-GRP antibodies was detected in immunized mice sera by ELISA and verified by Western blot analysis. Activity of CD4+T lymphocytes, especially high levels of interferon (INF)-γ, were developed in mice immunized with HG6 when compared with HSP65 or PBS. We found that immunogene tumor therapy with a vaccine based on GRP was effective at both protective and therapeutic antitumor immunity in breast tumor models in mice. The purified GRP monoclonal antibody (McAb) was proved to be potential in inhibiting EMT-6 tumor cell proliferation in vitro. The attenuation induced by active immune responses on tumor-induced angiogenesis was observed with an intradermal tumor model in mice. Taken together, we demonstrate for the first time that immune responses that are elicited by a novel chimeric protein vaccine targeting GRP can suppress the proliferation of breast tumor cell EMT-6 in mice, and it may be of importance in the further exploration of the applications of other autocrine growth factor identified in human and other animal in cancer therapy.