Heat shock protein gp96 is a highly conserved and monomorphic glycoprotein in the endoplasmic reticulum. It functions as molecular chaperone and can associate with a variety of antigenic peptides noncovalently in vivo and in vitro. Recent studies have indicated that gp96 molecules participate in major histocompatibility complex class I-restricted antigen presentation pathway. Immunization of mice with gp96 preparations isolated from cancer cells can elicit a cancer-specific protective T cell immune response that is recallable, which is a prerequisite for gp96 as a therapeutic vaccine against cancers. The immunogenicity of gp96 molecules has been attributed to the antigenic peptides associated with them. These phenomena provide a new pathway for cancer immunotherapy. The mechanism that the gp96-peptide complex induces specific immune response and the explorations for gp96-peptide complex as a therapeutic cancer vaccine are reviewed.
Animals ; Antigens, Neoplasm ; immunology ; therapeutic use ; Cancer Vaccines ; therapeutic use ; Humans ; Immunotherapy ; Membrane Glycoproteins ; immunology ; metabolism ; Molecular Chaperones ; immunology ; Neoplasms ; immunology ; therapy ; Peptides ; immunology ; metabolism

Adjuvants, Immunologic ; biosynthesis ; therapeutic use ; Animals ; Antigen Presentation ; immunology ; Antigens, Neoplasm ; metabolism ; Antigens, Tumor-Associated, Carbohydrate ; biosynthesis ; immunology ; therapeutic use ; Cancer Vaccines ; Humans ; Neoplasms ; immunology ; therapy ; Vaccines, Synthetic

OBJECTIVETo construct a replicative anti-tumor DNA vaccine PSCK-2PFcGB based on Semliki Forest Virus (SFV) replicon vector and observe its expression in vivo and in vitro.

METHODSThe plasmid pVAX1-2PFcGB was digested with Nhe I, and the digestion product was blunted prior to further digestion with BssH II to obtain the fragment 2PFcGB, a fusion gene containing the multitarget complex antigen 2PAG encoding both the most cytotoxic T lymphocyte epitopes of human survivin and chorionic gonadotropin β chain-CTP37 of human and monkey. The 2PFcGB fragment was inserted into the PSCK vector digested by Sma I. The products with the expected size were extracted and ligated, and the positive clones were screened by kanamycin and amplified. The recombinant PSCK-2PFcGB, following identification by colony PCR and restriction endonuclease Nde I, was transfected into 293T cells via lipofectamine 2000 and its expression was detected. The recombinant plasmid was also transfected into mouse quadriceps femoris muscle to observe its expression in vivo by immunohistochemistry.

RESULTSNde I digestion resulted in a fragment of the expected size. Transfection with the recombinant plasmid PSCK-2PFcGB resulted in successful expression of the antigen and adjuvant molecular protein in 293T cells, with the positivity rates of 5.70% and 19.75%, respectively. The fusion tumor antigen survivin and hCGβ-CTP37 were also detected in the muscular tissues of the mice.

CONCLUSIONA novel replicative anti-tumor DNA vaccine PSCK-2PFcGB has been successfully constructed and can be expressed in 293T cells and in the muscular tissues of immunized mice, which provide a basis for further studies of the antitumor activity and immunological mechanism of the DNA vaccine.

Animals ; Antibodies, Antinuclear ; immunology ; Cancer Vaccines ; biosynthesis ; immunology ; Gene Expression ; Genetic Vectors ; HEK293 Cells ; Humans ; Mice ; Muscle, Skeletal ; metabolism ; Plasmids ; Semliki forest virus ; genetics ; Vaccines, DNA ; biosynthesis ; immunology

OBJECTIVESTo construct a novel virus-like particulate peptide-nucleic acid vaccine (VPNV) of human telomerase reverse transcriptase (hTERT), and to study its anti-liver cancer immunity.

METHODSA cationic antigenic peptide was synthesized and purified, and then human granulocyte macrophage colony stimulating factor (hGM-CSF) and TERT gene were cloned into the eukaryotic expression vector pTCAE. The peptide was combined with the nucleic acid vaccine to make a VPNV, which was transfected into eukaryotic cell COS-7. The immunogenicity of hGM-CSF and hTERT were detected using ELISA and Western blot. The efficacy of VPNV for inducing antigen specific CTL response was determined using the lactate dehydrogenase release method.

RESULTSVPNV was verified capable to trigger specific CTL responses and has shown a specific cytolytic activity to liver cancer cell HepG2.

CONCLUSIONA VPNV which can stimulate antigen specific CTL response was successfully constructed. This paves the way for our further investigation of anti-liver cancer immunity in mice.

Animals ; Cancer Vaccines ; immunology ; Cloning, Molecular ; Eukaryotic Cells ; metabolism ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; immunology ; Liver Neoplasms ; immunology ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Peptide Nucleic Acids ; genetics ; immunology ; Telomerase ; genetics ; immunology ; Vaccines, DNA ; immunology

OBJECTIVETo prepare the anticolon carcinoma vaccine with rich chaperone peptide and to examine its anticancer immunological efficacy.

METHODSCT26 colon carcinoma cells were cultured in 1 mg/L Trichosanthin 1640 medium at different temperatures to induce the chaperone expression and promote the synthesis of antigen peptides. Groups of these cells treated under the different condition were lysed by the sonic disintegration, and the lysates were centrifuged. The rawpurified proteins were obtained from the supernatants by precipitating with saturated ammonium sulfate and removing the molecules below 50,000 and above 300,000 in molecular weight via dialysis. Furthermore, the proteins with the molecular weights in 70,000, 90,000, 95,000, 110,000 and 170,000 were collected through gel filtration and SDS-PAGE. The purified proteins were analysed by Western blotting, and inspected on the anticancer immunological effects including lymphocyte proliferation and the activities of NK and CTL.

RESULTSMajor of the chaperone peptides of anticancer effects in CT26 cells, including antigen peptides joining with HSP70, HSP90, gp96, HSP110 and HSP170, was satisfactorily extracted and condensed, and rich chaperone peptide composites were successfully obtained. The composites prepared under various condition could all enhance lymphocyte proliferation and the activities of CTL and NK(P<0.01).

CONCLUSIONSThe rich chaperone peptide composites are successfully prepared via dialysis, salt fractionation and gel filtration combined with SDS-PAGE. Both the heat stress and Trichosanthin can increase the composites, which treated by 42 centi-degree heat stress and Trichosanthin are found to possess the strongest anticancer efficacy.

Animals ; Cancer Vaccines ; immunology ; Cells, Cultured ; Heat Stress Disorders ; metabolism ; Heat-Shock Proteins ; metabolism ; Mice ; Mice, Inbred BALB C ; Molecular Chaperones ; immunology ; Trichosanthin ; pharmacology

To study the influence of vaccine of bcr-abl fusion gene fragment on inoculated SP2/0/bcr-abl tumor cells in mice, BALB/c mice were immunized with pVbcr-abl, pVbcr-abl/mIL7 plasmids, respectively, then SP2/0/bcr-abl cells expressing the fragment of bcr-abl fusion gene were inoculated subcutaneously into the groin of BALB/c mice in order to observe the effect of vaccine on growth of inoculated SP2/0/bcr-abl tumor cells. The results showed that there were distinct differences on the time of tumor growth, the time of tumor ulceration, tumor volume and survival time of mice bearing tumor between two immunized groups and two control groups (blank and vacant plasmid groups). The mice immunized with pVbcr-abl/mIL7 lived longer as compared to mice immunized with pVbcr-abl. The tissue of inoculated tumor was more compact, tumor organ was larger, tumor form was irregular in 2 control groups, while the tissue of inoculated tumor was looser, tumor volume was smaller, and with mass inflammatory infiltration in two immunized groups. Moreover, the metastatic tumor cells were found in the livers of control groups, but not observed in two immunized groups. It is concluded that the protection occurred in immunized mice which inhibited the growth of SP2/0/bcr-abl tumor cell in vivo.
Animals ; Cancer Vaccines ; immunology ; metabolism ; Cell Line, Tumor ; Female ; Fusion Proteins, bcr-abl ; biosynthesis ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Multiple Myeloma ; genetics ; immunology ; pathology ; Neoplasm Transplantation ; Random Allocation ; Vaccines, DNA ; immunology

The purpose of this study was to construct the IgHV and IL-2 coexpressed vector. The IgHV gene fragments were obtained from the peripheral blood of patients with lymphoma, and were cloned into eukaryotic expression vector. Meanwhile, the gene fragments of IgHV linked with gene of IL-2 were inserted into pcDNA3.0 to form a fusion gene of IgHV-IL-2. Then fusion genes were transfected into COS cells by Lipofectin and the expression of IL-2 was detected by ELISA. The results showed that the IgHV/pcDNA3.0 expression vector was successfully constructed. The 3' end of IgHV was linked to IL-2 gene, and IL-2 could be correctly expressed. In conclusion, the expression vector of IgHV-IL-2 can express IL-2 correctly in COS cells.
Cancer Vaccines ; immunology ; Eukaryotic Cells ; metabolism ; Genes, Immunoglobulin ; Genetic Vectors ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Interleukin-2 ; biosynthesis ; genetics ; Lymphoma ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Vaccines, DNA ; biosynthesis ; genetics ; immunology

OBJECTIVETo obtain streptavidin-tagged human interleukin-15 (SA/hIL15) fusion protein and evaluate its bioactivity.

METHODSpET24a-6His-SA-hIL-15 and pET32a-hIL-15-SA-6His plasmids were constructed and expressed in BL 21(DE3) host bacteria to generate the fusion protein. The recombinant fusion protein IL-15/SA was purified using Ni-NTA affinity chromatography and refolded, and the efficiency of surface modification of the fusion protein on biotinylated cells was examined by fluorescence-activated cell sorting. CCK-8 method was used to evaluate the effect of IL-15/SA fusion protein in inducing the proliferation of human peripheral-blood lymphocyte (PBL) cells stimulated by PHA.

RESULTSThe recombinant SA-hIL-15 and hIL15-SA fusion proteins were highly expressed in BL21(DE3) at about 20% of the total bacterial proteins. The purified hIL15-SA fusion protein exhibited a bifunctionality by promoting the proliferation of PBL cells activated by PHA and high-affinity binding to biotinylated cell surface mediated by SA, with a cell surface modification efficiency exceeding 95%. SA-hIL-15 showed a 4-fold higher hIL15 bioactivity than hIL15-SA.

CONCLUSIONSA/hIL-15 bifunctional fusion protein has been successfully obtained to facilitate the future development of hIL-15-surface-modified cancer cell vaccine.

Cancer Vaccines ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; Interleukin-15 ; biosynthesis ; genetics ; Lymphocyte Activation ; drug effects ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Streptavidin ; biosynthesis ; genetics

OBJECTIVETo obtain streptavidin-tagged human interleukin-21 (hIL21) fusion protein and evaluate its bioactivities.

METHODShIL21-SA-pET21 and pET24a-SA- hIL21 plasmids were constructed and expressed in BL21(DE3) host bacteria. The hIL21-SA and SA- hIL21 fusion protein were purified through Ni-NTA affinity chromatography and refolded by dialysis. Flow cytometry was used to detect hIL21-SA and SA- hIL21 fusion protein on the biotinylated MB49 tumor cells. MTT assay was used to evaluate the effect of the fusion protein on the proliferation of human peripheral blood lymphocytes (PBLs) stimulated by Anti-CD3.

RESULTSThe recombinant fusion proteins were highly expressed in BL21(DE3) at about 30% of the total bacterial proteins. The two fusion proteins exhibited bifunctional activities, i.e. both biotin-binding property and hIL21 activity and SA-mediated high-affinity binding to biotinylated cell surfaces (with anchoring modified rate of about 95.18% and 96.91%).

CONCLUSIONWe have successfully obtained bifunctional fusion protein hIL21-SA and SA- hIL21,which will provide a basis for further study of tumor biotherapy using the proteins.

Cancer Vaccines ; immunology ; Cell Line, Tumor ; Escherichia coli ; genetics ; metabolism ; Humans ; Interleukins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Streptavidin ; biosynthesis ; genetics

OBJECTIVETo develop a new vaccine expressing membrane-bound heat shock protein 70 (mbHSP70) and further study its antitumor therapeutic effect.

METHODThe pre- established vector expressing mbHSP70 was transfected into CT26 cells of colorectal cancer. After the CT26 cells were incubated with 900 microg/ml G418, the sub-clones resistant to G418 were harvested and the HSP70 positive clones were selected by limiting dilution. The clones were amplified and inactivated, thereby the vaccine expressing mbHSP70 was prepared. Lymphocyte proliferation stimulated by the vaccines, NK and CTL activity was observed. The antitumor efficacy of vaccine was observed in BALB/c mice model with colorectal cancer.

RESULTSThe laser confocal microscopy and flow cytometry showed that there existed much HSP70 on the vaccine membrane surface. The HSP70 gene-modified vaccine displayed augmented lymphocyte proliferation and higher NK and CTL activity in vitro,and marked tumor suppression and prolonged survival time of the vaccinated micein vivo, when compared with its counterpart. Furthermore, mbHSP70-expression vaccine elicited lymphocyte proliferation most intensively, generated the highest NK and CTL activity as well as the strongest antitumor effect, and prolonged survival time of the vaccinated mice.

CONCLUSIONA new vaccine expressing mbHSP70 has more potent antitumor immunity and better therapeutic efficacy than HSP70 gene-modified vaccine did.

Animals ; Cancer Vaccines ; therapeutic use ; Cell Membrane ; immunology ; metabolism ; HSP70 Heat-Shock Proteins ; immunology ; Immunotherapy ; Mice ; Mice, Inbred BALB C ; Neoplasms, Experimental ; therapy ; Transfection