Listeria monocytogenes (LM), a Gram-positive facultative intracellular bacterium, can be used as an effective exogenous antigen expression vector in tumor-target therapy. But for successful clinical application, it is necessary to construct attenuated LM stain that is safe yet retains the potency of LM based on the full virulent pathogen. In this study, attenuated LM and recombinants of LM expressing melanoma inhibitory activity (MIA) were constructed successfully. The median lethal dose (LD(50)) and invasion efficiency of attenuated LM strains were detected. The recombinants were utilized for immunotherapy of animal model of B16F10 melanoma. The level of MIA mRNA expression in tumor tissue was detected by using real-time polymerase chain reaction (PCR) with specific sequence, meanwhile the anti-tumor immune response was assayed by flow cytometric analysis and enzyme-linked immunosorbent spot (ELISPOT) assay. The results showed the toxicity and invasiveness of attenuated LM were decreased as compared with LM, and attenuated LM expressing MIA, especially the double-genes attenuated LM recombinant, could significantly induce anti-tumor immune response and inhibit tumor growth. This study implicates attenuated LM may be a safer and more effective vector for immunotherapy of melanoma.
Animals ; Cancer Vaccines ; genetics ; immunology ; Extracellular Matrix Proteins ; genetics ; immunology ; Listeria monocytogenes ; immunology ; Male ; Melanoma ; genetics ; immunology ; Mice ; Mice, Inbred C57BL ; Vaccines, Attenuated ; genetics ; immunology

OBJECTIVETo study the effection of suppression murine melanoma growth by Intratumor injection of recombinant attenuated salmonella carrying heat shock protein 70 and herpes simplex virus thymidine kinase genes.

METHODSPlasmids PCMV-mtHSP70-IRES-TK were electro-transferred into salmonella typhimurium SL7207 to construct recombinant salmonella typhimurium. In vivo, Recombinant bacteria were injected into the mouse melanoma and the antitumor effection was observed. The survival period was recorded and safety analysis for this vaccine in each group.

RESULTSIn vivo, the mtHSP70/HSV-tk recombinant bacteria can suppress tumor growth significantly and extend survival. After recombinant Salmonella, 10(9) CFU/mL, was administered as an intratumoral injection, No diarrhea were observed. During therapy, body weight did not change markedly.

CONCLUSIONResults of the animal experiment suggests intratumor injection of recombinant attenuated salmonella typhimurium containing mtHSP70 and HSV-tk genes, has targeting ability against B16 tumor cell and could significantly inhibit tumor growth .

Animals ; Bacterial Proteins ; genetics ; immunology ; Cancer Vaccines ; genetics ; immunology ; pharmacology ; Genetic Therapy ; methods ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Melanoma, Experimental ; microbiology ; pathology ; therapy ; Mice ; Mice, Inbred C57BL ; Mycobacterium tuberculosis ; genetics ; Salmonella typhimurium ; genetics ; immunology ; Simplexvirus ; enzymology ; genetics ; Skin Neoplasms ; therapy ; Thymidine Kinase ; genetics ; immunology ; Vaccines, Attenuated ; genetics ; immunology ; pharmacology ; Vaccines, DNA ; genetics ; immunology ; pharmacology

Heat shock protein gp96 isolated from tumor tissues holds great promise for tumor immunotherapy. However, at present only very limited amount of gp96 protein can be isolated from tumor tissues. Here, we reconstituted the yeast-expressed gp96 (recombinant gp96, rgp96) with B16.F10 melanoma antigens in vitro to prepare new gp96 tumor vaccine on large-scale, and analyzed its induction of specific anti-tumor immunoresponses by ELISPOT, IFN-gamma intracellular staining and cytotoxicity assays. Immunization with rgp96-tumor antigen complexes significantly inhibited B16 tumor growth compared with either rgp96 or tumor antigens alone and led to enhancement of tumor-specific T-cell activities, which was found similar to that of tumor tissue derived gp96. Our results therefore may provide bases for large-scale preparation of the new generation of gp96 tumor vaccines.
Animals ; Cancer Vaccines ; biosynthesis ; genetics ; immunology ; therapeutic use ; Female ; Heat-Shock Proteins ; biosynthesis ; genetics ; immunology ; therapeutic use ; Melanoma, Experimental ; therapy ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; therapeutic use ; Skin Neoplasms ; therapy ; Yeasts ; genetics ; metabolism

OBJECTIVE(1) To investigate the expression and gene diversity of the 7 major cancer/testis (CT) antigens, MAGE-1, MAGE-3, MAGE-4, MAGE-10, NY-ESO-1, SSX-2 and SCP-1, in hepatocellular carcinoma (HCC). (2) To analyze the correlations between the clinical characters and CT antigens' expression.

METHODSThe cancer and para-cancer tissues were collected from 30 HCC patients. The mRNAs of seven CT antigens were detected by reverse transcription-polymerase chain reaction (RT-PCR) with the specific primers. The PCR products were sequenced to analyze the CT genes.

RESULTSThe MAGE-1, MAGE-3, MAGE-4, MAGE-10, NY-ESO-1, SSX-2 and SCP-1 were expressed in 66.7%, 70.0%, 20.0%, 36.7%, 40.0%, 33.3% and 33.3% of the tumor tissues from HCC patients respectively, however, they were not expressed in the para-cancer tissues. Among the 30 patients investigated, 90.0% expressed one CT gene at least, 70.0% expressed two CT genes, and 53.3% expressed three CT genes of the seven CT genes. The coding genes of these CT antigens were highly conserved between in Chinese patients and patients abroad. There were discernible correlations between alpha-fetoprotein level and MAGE-10 or SCP-1 expression level, as well as between average age and MAGE-3 or SSX-2 expression levels (P<0.05).

CONCLUSIONSWith a highly conserved coding gene, seven CT antigens were expressed in 20.0% - 70.0% of Chinese HCC patients. CT antigens' expression had correlations with some clinical characters.

Antigens, Neoplasm ; biosynthesis ; genetics ; Carcinoma, Hepatocellular ; genetics ; immunology ; Female ; Gene Expression Regulation, Neoplastic ; genetics ; Humans ; Liver Neoplasms ; genetics ; immunology ; Male ; Melanoma-Specific Antigens ; Neoplasm Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVETo investigate the possibility of using melanoma antigen-1 (MAGE-1) peptide as a tumor vaccine to treat hepatocellular carcinoma (HCC).

METHODSThe expressions of MAGE-1 in 8 HCC cell lines and in liver cancer tissue from a patient were detected using RT-PCR. The type of human leucocyte antigen I(HLA I) of both 8 HCC cell lines and peripheral blood mononuclear cells of the patient was detected using a microcytotoxicity method to screen out target cell lines for the cytotoxicity assay. Peripheral blood mononuclear cells from the HCC patient pulsed with an MAGE-1 peptide (NYKCRFPEI) were used as antigen presenting cells. Autogenous peripheral blood mononuclear cells were stimulated with antigen presenting cells every 7 days for 4 times to elicit cytotoxic T lymphocytes. The phenotype of effector cells was analyzed using flow cytometry. The cytotoxicity of effector cells was detected with a lactate dehydrogenase releasing assay.

RESULTSThe expressions of both MAGE-1 and HLA-A24 were detected in BEL7405 cell line which were used as the positive target cell line in the cytotoxicity assay. The expression of MAGE-1 alone was detected in HLE, BEL7402, BEL7404, QGY7703 and SMMC7721 cell lines, and the expression of neither MAGE-1 nor HLA-A24 was shown in QGY 7701 and HpG2 cell lines. The last 7 cell lines could be used as negative target cell lines in the cytotoxicity assay. Peripheral blood mononuclear cells expanded 32 folds during 28-day culture. The ratio of CD3(+) T cells increased by 16% (from 54% to 70%), and the ratio of CD8(+) T cells increased by 20% (from 36% to 56%) during 28-day culture. When the ratio of effector cells to target cells was 10:1, effector cells exhibited 62.5% cytotoxicity against autogenous lymphoblasts pulsed with the peptide (NYKCRFPEI) of MAGE-1 antigen, 40.25% cytotoxicity against BEL7405 cells, compared with 17.88% cytolysis observed against autogenous lymphoblasts, 19.55% against HLE cells, and 1.6% against QGY7701 cells. When the ratio of effector cells to target cells was 3.3:1, the cytotoxicity of effector cells against the peptide pulsed autogenous lymphoblasts was 53.6%, which was much higher against autogenous lymphoblasts, HLE cells and QGY7701 cells at 15.6%, 13% and 1%, respectively.

CONCLUSIONThe results demonstrate that cytotoxic T lymphocytes with the ability to specifically lyse target cells expressing both MAGE-1 and HLA-A24 could be successfully induced by the MAGE-1 peptide NYKCRFPEI in vitro. This indicates that a good result might be anticipated if this peptide is used as a tumor vaccine to treat HLA-A24 HCC patients.

Adult ; Antigens, Neoplasm ; Cancer Vaccines ; immunology ; Carcinoma, Hepatocellular ; immunology ; HLA-A Antigens ; analysis ; HLA-A24 Antigen ; Humans ; Liver Neoplasms ; immunology ; Male ; Melanoma-Specific Antigens ; Neoplasm Proteins ; genetics ; immunology ; RNA, Messenger ; analysis ; T-Lymphocytes, Cytotoxic ; immunology ; Tumor Cells, Cultured

We examined the effect of class II transactivator (CIITA) down-modulation on allograft rejection. To inhibit the function of CIITA, we constructed a series of CIITA mutants and found one exhibiting the dominant-negative effect on the regulation of major histocompatibility complex (MHC) class II expression. To test whether the CIITA dominant-negative mutant reduces immunogenecity, CIITA-transfected melanoma cells were injected into allogeneic host and assessed for immune evading activity against host immune cells. We demonstrated that the CIITA dominant-negative mutant allowed tumor nodules to develop earlier in the lung than control by this tumor challenge study. Furthermore, skin grafts deficient for CIITA also survived longer than wild-type in allogeneic hosts. Both the tumor challenge and skin graft studies suggest the inhibition of CIITA molecules in donor tissue would be beneficial to the control of allo-response.
Transplantation, Homologous ; Transfection ; Trans-Activators/genetics/*immunology/metabolism ; Trans-Activation (Genetics)/genetics/immunology ; Skin Transplantation ; Nuclear Proteins/genetics/*immunology/metabolism ; Mutation ; Mice, Transgenic ; Mice, Knockout ; Mice, Inbred C57BL ; Mice, Inbred BALB C ; Mice ; Melanoma, Experimental/genetics/immunology/pathology ; Male ; Interferon Type II/pharmacology ; Humans ; Histocompatibility Antigens Class II/genetics/*immunology/metabolism ; Graft Survival/genetics/immunology ; Graft Rejection/genetics/*immunology ; Genes, MHC Class II/genetics/immunology ; Flow Cytometry ; DNA, Complementary/genetics ; Cell Proliferation/drug effects ; Cell Line, Tumor ; Animals

OBJECTIVETo observe the inhibitory effect of dendritic cells (DCs) activated cytotoxicity T lymphocyte (CTL) combined with MAGE-1 nonapeptide on transplanted human hepatocyte carcinoma (HCC) in nude mice.

METHODSA model of HCC transplanted tumor was established by injecting BEL-7402 cell line HCC cells subcutaneously on the back of nude mice. Successful transplantation rate was 73%. Specific CTLs (1 x 10(6)), which were activated by DCs combined with MAGE-1 nonapeptide, were injected into the site of transplanted tumor (group A, n = 5). Another group of 17 mice were treated with same amounts of different kinds of cells, and they were divided into groups B, C, D, E, and F. The growth of tumor was observed, and pathological examination was also done.

RESULTS(1) The activated lymphocytes induced by DCs combined with MAGE-1 nonapeptide could suppress the growth of tumor and reduce the tumor size. In group A, 5/5 mice survived for at least two weeks, while the tumors grew rapidly and the majority of the mice died within two weeks in other groups (groups B, C, D, E, F) (P < 0.01). (2) Extensive necrosis and apoptosis were found in transplanted tumors in group A.

CONCLUSIONSThe DCs combined with MAGE-1 nonapeptide could not only inhibit the growth of HCC, but also result in produce death and apoptosis of HCC, hence preventing tumor metastasis and recurrence. The mechanism underlying tumor immunization resulted from DCs might be enhanced in apoptosis of tumor cells. MAGE-1 nonapeptide combined with DCs might be a potential novel tumor vaccine for the treatment of HCC.

Animals ; Antigens, Neoplasm ; Apoptosis ; Cancer Vaccines ; immunology ; Dendritic Cells ; immunology ; Humans ; Liver Neoplasms, Experimental ; immunology ; pathology ; therapy ; Melanoma-Specific Antigens ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Proteins ; administration & dosage ; genetics ; Neoplasm Transplantation ; T-Lymphocytes, Cytotoxic ; immunology ; Transplantation, Heterologous

OBJECTIVETo investigate the enhanced effect of bone marrow-derived dendritic cells (DC) pulsed with SVYDFFVWL, a MHC class I peptide located in 180-188 amino acid residues of human melanoma-associated antigen tyrosinase- related protein 2 ( hTRP2) on the immunity against melanomas elicited by adenovirus encoding hTRP2 (Ad hTRP2).

METHODSThe mice were intradermally immunized with Ad hTRP2, and three weeks later with Ad hTRP2 or DC/SVYDFFVWL once more. Analysis of CTL killing activity and IFN-gamma-producing CD8 + T cells in the total CD8 + T cells of spleen were made using in vivo CTL and intracellular staining of IFN-gamma, respectively. Additionally, the survival of mice was checked after the subcutaneous inoculation with mouse melanoma B16. F10 cells.

RESULTSThe 6 h CTL killing and IFN-gamma producing CD8 +T cells in the total CD8 ' T cells of spleens were 68. 40%+/-5. 50% and 0. 67%+/-0.16% in Ad hTRP2 (priming)-Ad hTRP2 (boosting) group,28. 50%+/-6.40% and 0.22%+/-0.07% in DC/SVYDFFVWL (priming)-DC/ SVYDFFVWL (boosting) group,and 98. 90%+/-0.90% and 1.05%+/-0.21% in Ad hTRP2 (priming)-DC/ SVYDFFVWI, (boosting) group, respectively. In the tumor-bearing model, none of mice survived in DC/SVYDFFVWL (priming)-DC/SVYDFFVWL (boosting) group, and just only 40% of mice were tumor-free in Ad hTRP2 (priming) -Ad hTRP2 (boosting) group, whereas 100% of mice survived in Ad hTRP2 (priming)-DC/SVYDFFVWL (boosting) group.

CONCLUSIONBoosting with DC/ SVYDFFVWL can significantly enhance the immunity against melanomas elicited by priming with Ad hTRP2, indicating that first priming with Ad hTRP2 and then boosting with DC/SVYDFFVWL is a potentially effective regimen for overcoming the disadvantage that anti-tumor immune response can not be significantly increased by readministration of adenovirus.

Adenoviridae ; genetics ; Animals ; CD8-Positive T-Lymphocytes ; immunology ; Cancer Vaccines ; immunology ; Cell Line, Tumor ; Cells, Cultured ; Cytotoxicity Tests, Immunologic ; Dendritic Cells ; cytology ; immunology ; Female ; Genetic Vectors ; genetics ; Humans ; Immunization, Secondary ; methods ; Intramolecular Oxidoreductases ; immunology ; Melanoma, Experimental ; immunology ; pathology ; therapy ; Membrane Proteins ; immunology ; Mice ; Mice, Inbred C57BL ; Peptide Fragments ; immunology ; Survival Analysis ; T-Lymphocytes, Cytotoxic ; immunology

This study sought to construct a recombinant vector that expresses anti-GD2/anti-CD16 bispecific single-chain antibody(sc-BsAb), and to assess its biological activities. The anti-GD2 gene and the anti-CD16 gene (NM3E2) were obtained using PCR amplification technique, and then the fusion gene was constructed by overlapping PCR. The sc-BsAb gene was subcloned into the pET-22b(+) plasmid from the pMD18-T easy vector by digestion with NcoI, Hind III restriction endonucleases, whose sites exist in both the vectors. Then the combinant plasmids were transferred into E. coli BL21 (DE3). The expression product in the periplasmic was analyzed by both SDS-PAGE and Western blot technique, then was purified with Ni2+ -NTA superflow affinity chromatography. It was demonstrated that the linker in the sc-BsAb fusion protein is SerGly4Ser. and the molecular is 53 KD.
Antibodies, Bispecific ; biosynthesis ; genetics ; Antibodies, Neoplasm ; biosynthesis ; genetics ; Base Sequence ; Cell Line, Tumor ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gangliosides ; immunology ; HeLa Cells ; Humans ; Melanoma ; pathology ; Molecular Sequence Data ; Receptors, IgG ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics

OBJECTIVESTo investigate the correlation between MAGE-A1 mRNA expression and genic demethylation in hepatoma cell lines.

METHODSTotal RNA and genomic DNA were prepared from 10 human hepatoma cell lines. MAGE-1 mRNA expression was determined with RT-PCR and the level of genome-wide demethylation was evaluated by enzyme digestion and Southern blot assay. The genomic DNA was digested by HpaII, then the promoter of MAGE-A1 gene was amplified with primers CDS21, EDP4 and CDS20. EDP4 and the PCR products were further hybridized with a probe to detect the methylation in the promoter of the MAGE-A1 gene. HLA-A locus was typed using SSP kit.

RESULTSIn cell lines QGY-7703, SMMC-7721, HLE, BEL-7402, BEL-7404 and BEL-7405, MAGE-A1 mRNA expression was positive and cell differentiation was moderate or low. In cell lines of HepG2 2.2.15, HepG2, QGY-7701 and Huh7, MAGE-A1 mRNA expression was negative and cell differentiation was well or moderate. The level of genomic demethylation in MAGE-A1 mRNA positive cell lines was much higher than that in MAGE-A1 mRNA negative cell lines (t = 2.896, P = 0.02). The methylation analysis showed that methylation in the promoters of MAGE-A1 gene of HepG2 2.2.15, HepG2, QGY-7701 and Huh7 was high, and that methylation in those of SMMC-7721, HLE, BEL-7402, BEL-7404, and BEL-7405 was low.

CONCLUSIONThe results suggest that MAGE-A1 mRNA expression in the human hepatoma cell lines is associated with genic hypomethylation.

Antigens, Neoplasm ; Carcinoma, Hepatocellular ; genetics ; immunology ; metabolism ; DNA Methylation ; Humans ; Liver Neoplasms ; genetics ; immunology ; metabolism ; Melanoma-Specific Antigens ; Neoplasm Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Tumor Cells, Cultured