| The use of plasmid DNA to elicit the immune system against disease provides a variety of practical benefits for large scale vaccine production that are not as easily manageable with other forms of vaccines including recombinant protein or whole tumor cells. DNA vectors are capable of encoding a number of needed immunological components and are easily engineered and produced for administration using bacterial expression systems. Their safety in terms of adverse reactions after injection has also been demonstrated in animal models and human clinical trials. More importantly, neutralizing immune responses to plasmid DNA is rarely observed, making repeated injections possible. DNA immunization studies in animal models involving cancer and infectious disease have demonstrated preventative and therapeutic success. Direct inoculation of plasmid DNA encoding tumor-associated antigens is known to induce both antigen-specific cellular as well as humoral immune responses. The generation of T cell-mediated cytotoxicity or antibody-mediated cytotoxicity against tumor cells can inhibit tumor growth and lead to tumor rejection.In contrast, the crossover application of DNA vaccines in humans has faced many obstacles and difficulties, leading to their less-than-desired efficacy in the clinical setting. One of the reasons is the inefficious activation of CD4~+T cells. Epitopes of endogenous proteins expressed in the cytoplasm of transfected cells are commonly presented by MHC class I molecules to cytotoxic CD8~+T cells. Hower, MHC II-directed antigen activation of CD4~+T cells is vital to the function of genetic vaccines. In general, antibody class switching, clonal expansion of antigen specific B cells, T cell expansion and memory cell formation, and many other functions of the immune response require the costimulatory signals and cytokines released by antigen-activated CD4~+ T cells. Based on these observations, we reasoned that a molecular approach that directly routes the antigen into the MHC class II pathway, such as the endocytic or lysosomal compartments, might facilitate endogenous presentation to CD4~+T cells. The lysosome-associated membrane protein (LAMP-1) is a type 1 transmembrane protein which is localized predominantly in the outer membrane of lysosomes. It has a trafficking pathway from the Golgi complex mediated by adaptor protein 3(AP-3) binding to the carboxyl-terminal YXX(?) (where the (?) symbol represents any hydropholic amino acid) recognition sequence of an 11-amino acid cytoplasmic tail.Human chorionic gonadotropin (hCG) and Survivin are two promising TAAs, both of them are overexpressed in most kinds of cancers including colon, prostate, bladder, breast and lung, while the normal tissues from these same organs did not express them. The extremely high expression of hCG and Survivin in cancer carries prognostic and predictive implications and, through molecular profiling, is consistently associated with advanced disease, high grade, abbreviated survival, resistance to therapy, and accelerated recurrences. Thus, immunization against hCG or Survivin may result in effective immune effectors capable of lysing tumor cells. CTP37-DT is a synthetic subunit vaccine composed of a 37-amino acid peptide from the COOH terminal end ofβ-hCG conjugated to the carrier, DT. More recently, it has been through both phase I and phase II clinical trials in patients with colorectal cancer. The vaccine was safe and was associated with longer survival of patients with advanced colorectal cancer. While on the other hand, it has been observated that T cells mount a vigorous cytolytic response against Survivin peptides in vitro and in vivo, and that HLA class I-restricted cytolytic T cells against Survivin peptides exist in patients with breast cancer, leukaemia and melanoma in vivo.To date, multiple Survivin epitopes presented by HLA-A*0201 or HLA-A*0301 have been identified and used as targets of specific CTL responses.Accumulating experimental and clinical studies have implicated uncontrolled angiogenesis as a major contributor in tumor growth and metastasis. Compelling evidence suggest that vascular endothelial growth factor (VEGF) and its receptors, especially receptor2 (VEGFR2), play a critical role in angiogenesis under both physiological and pathological conditions.VEGF and VEGFR2 are frequently up-regulated in cancer. Inhibition of angiogenes with antagonists to either VEGF or VEGFR2, by using antibodies, immuno-toxins, ribozyme, soluble receptors, and small molecule tyrosine kinase inhibitors, has led to signicant therapeutic efficacy both in preclinical studies in animal models and in clinical trials in patients with cancer. Taken together, these findings indicate that VEGFR2 represent excellent targets for therapeutic intervention of human cancer. VEGFR2 is characterized by seven immunoglobulin (Ig)-like domains within its extracellular region. Study suggests that the third Ig-like domain is critical for ligand binding, binding ability was abolished completely with removal of the third domain; the fourth domain is important for ligand association, deletion of the fourth domain will cause a drastic decrease in the association rate. We therefore designed DNA vaccines encoding 37 amino acid from the COOH terminal end ofβ-hCG,5 HLA-restricted CTL epitopes from Survivin molecule and the corresponding amino acid from the third and fourth extracellular domain of human VEGFR2 to inserted proximal to the transmembrane domain of the complete LAMP-1 cDNA, designately p-L/HSV, with the aim of targeting the tumor antigens to the endosomal and lysosomal compartments.Mice were vaccined with plasmid p-L/HSV and control constructs for three times. 3 weeks after the last immunization, mice were sacrificed for ELISPOT and Cytotoxicity assay. The IFN-γELISPOT assay revealed that strong, HSV-specific T cell responses were elicited in mice immunized with p-L/HSV when splenocytes were stimulated with peptide 11,18,22,24; while in p-L/HS and p-L/V group, antigen specific T cell responses was seen when splenocytes were stimulated with peptide 4,8,9 and 19,24 separately. In addition, the peptide24-specific T cell response was stronger in p-L/HSV group compared with p-L/V group. We used CTL assays with splenocytes from plasmid p-L/HSV vaccinated mice as effector cells to determine whether target cells (p815 cells pulsed with different peptide pool) can be killed more efficiently than mice vaccinated with various control groups. When p815 cells pulsed with peptide pool inclouding peptide1-4 was used as the target cells, splenocytes from mice vaccined with p-L/HSV DNA generated significantly higher percentages of specific lysis compared with those vaccinated with wild-type p-HSV DNA. Compared with p-HS and p-V group, target cell lysis rate was enhanced in group p-L/HS and p-L/V when p815 cell was pulsed with peptide pool inclouding peptide1-4 and peptide23-26 separately. The splenocytes from p-L/HSV generated a higher lysis rate compared with p-L/HS group when p815 cell was loaded with peptide 1-4 mixture. Mice blood samples were collected two weeks after every vaccination to detect the HSV specific antibody tilter. Levels of antibody specific for 1-4,10-14,15-18,23-26 peptide pool were higher in mice that received p-L/HSV DNA vaccination (P < 0.05) compared with those received p-HSV vaccination. The antibody tilter was increased with time, and to the maximum after the third immunization, confirming that the p-L/HSV vaccine elicited an anti-HSV specific antibody response. The specific antibody level was also increased in p-L/HS group compare with p-HS. Additionly, 1-4 peptide-specific antibody was higher in p-L/HSV group than in p-L/HS group.The data presented in this study clearly demonstrate that; by enhancing endogenous antigen trafficking to the cellular MHC-II compartment of antigen-presenting cells, LAMP can enhance cellular and humoral response through greater antigen-specific activation of CD4+T cells; combination of antiangiogenic therapy and anti tumor cell therapy is highly synergistic.
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