Transmembrane protein p185 (the product of Her2/c-erbB-2 gene) is a member of the epidermal growth factor receptor (EGFR) family. Its overexpression was found in about 30% of breast cancer. It is essential to obtain soluble extracellular domain (ECD) of p185, especially disulfide bond rich domains, for identifying the epitopes of anti-p185 antibodies and researching the interrelationship between the antigen and antibody. The disulfide bond rich domain I-II and domain IV of p185 ECD were amplified from plasmid pBabe/erbB-2 by PCR respectively. These two fragments were inserted into pGEX/4T-1 vector, transfected into E. coli Origami B (DE3) pLysS and expressed inductively by low concentration of IPTG and low temperature overnight. After the pressure lysis of cells, the supernatants were analyzed by SDS-PAGE and the result demonstrated that this GST-fusion protein was expressed solubly in the amount of 10-15 mg/L. By the ELISA, Western blot and other immunological assays, the fusion proteins and their GST cut-off derivates both showed binding activities with several anti-p185 antibodies respectively. These results indicated that it was a feasible and effectual method to express disulfide bond rich domain I-II and domain IV of p185 ECD and this method may also be used to express other disulfide bond rich proteins.
Antibodies, Monoclonal ; immunology ; Disulfides ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Receptor, ErbB-2 ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Solubility ; Transfection

This research was to construct the lentiviral expression vector for anti- p185(erbB2) mouse/human chimeric antibody and to determine the expression of the chimeric antibody gene in 293T cells transfected with this vector. The genes (vL and vH) coding light and heavy chain of variable regions of anti-p185(erbB2) mAb and the constant regions of human IgG1 (kappa and gamma1) were cloned with PCR method. The target genes were assembled by three-primers PCR method to obtain the chimeric light chain (L) and the chimeric heavy chain (H). Both chains inserted into the down stream and upper stream of IRES gene of the plasmid pVAX1/IRES respectively. We digested the plasmid pVAX1/ H-IRES-L with endoenzyme and subcloned H-IRES-L into the lentiviral vector pWPI. The enzyme digestion and sequence analysis showed that the lentiviral expression vector pWPI/H-IRES-L was constructed correctly. Then, it was transfected into 293T cells and after 48h, GFP protein expression in 293T cells were detected by fluorescent microscope and the chimeric antibody expression was detected by RT-PCR and direct ELISA. The results showed that after 293T cells were transfected with recombination plasmid, both light and heavy chains of the chimeric antibody genes could express together. The chimeric antibody expressed could bind to p185(erbB2) specifically. This research may lay a sound foundation for further study of anti-p185(erbB2) engineered antibody.
Animals ; Antibodies, Monoclonal ; biosynthesis ; genetics ; Cell Line ; Chimera ; Cloning, Molecular ; Humans ; Lentivirus ; genetics ; metabolism ; Mice ; Receptor, ErbB-2 ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Transfection

We studied the aggregation of a recombinant engineering antibody (chA21). Anti-ErbB2 antibody chA21 was produced by fusing single-chain Fv (scFv) with human IgG1 Fc fragment, and it was proved to be a drug candidate for cancer therapy. We characterized the aggregation of chA21 by high performance sized-exclusive chromatography (HPSEC), dynamic light scattering (DLS), SDS-PAGE, indirect ELISA assay, and compared the influence of temperature and additive on the level of aggregation and binding activity. Conformation changes of different levels of aggregation were also analyzed via circular dichroism (CD). Finally, we analyzed which part of chA21 was involved in aggregation by cleaving it into scFv and Fc fragments. The results showed that chA21 could form aggregates in the storage solution. The aggregates interacted through non-covalent bonds and remained binding activity. Temperature and additive could slightly affect the level of aggregation and binding activity, while the conformations of chA21 were stable. Aggregation propensity of scFv fragment was almost same as chA21, indicating that scFv may be the major part to form the aggregates. The research on aggregation may be helpful to develop a suitable formulation for chA21 clinical application as well as provide direction for future antibody design and reconstruction.
Antibodies ; chemistry ; metabolism ; Humans ; Immunoglobulin Fc Fragments ; chemistry ; metabolism ; Immunoglobulin Variable Region ; chemistry ; metabolism ; Protein Conformation ; Protein Engineering ; methods ; Receptor Aggregation ; immunology ; Receptor, ErbB-2 ; chemistry ; immunology ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics ; immunology

OBJECTIVETo investigate the inhibitory effect of DNA vaccine immunization on neu-overexpressed melanoma growth in prophylactic treatment and anti-lung-metastasis experiments in C57BL/6 mice.

METHODSpcDNA-neu transfected into B16F10 with transfection reagent Fugene 6, neu-overexpressed cell clone B16F10-neu was selected with limited dilution method. The growth curve was drawn to analyse its proliferating character in vitro. With Helios gene gun system, DNA vaccine pWRG-neu was immunized to 8-week-old C57BL/6 mice in the shaved abdominal skin for 3 times at two-weekly interval. After immunization, the life span was analyzed. Using MTT assay, the cytolysis activity of the DNA immunized mice spleen cells was compared.

RESULTSOne clone of neu-overexpressed B16F10-neu was selected and its proliferating character was the same as B16F10 and B16F10-pcDNA. In prophylactic, treatment and anti-lung-metastasis experiments, gene gun-mediated pWRG-neu immunization could exhibit antitumor effects. The growth and metastasis of neu-overexpressed melanoma was reduced dramatically. The spleen cells of the immuned mice showed cytotoxic T lymphocyte (CTL) activity.

CONCLUSIONGene gun-mediated gene transfer is effective and practicable. DNA vaccine pWRG-neu is potent in preventing subsequent tumor cells challenge, inhibiting the tumor growth and metastasis.

Animals ; Biolistics ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; Genes, erbB-2 ; Immunization ; Lung Neoplasms ; prevention & control ; secondary ; Melanoma, Experimental ; metabolism ; pathology ; therapy ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Plasmids ; Receptor, ErbB-2 ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; Vaccines, DNA

OBJECTIVETo investigate the specific anti-breast cancer immune response induced by dendritic cells (DC) loaded with trastuzumab and apoptotic Her-2+ breast cancer cells.

METHODSDCs were generated from healthy peripheral blood mononuclear cells (PBMCs) in the presence of recombinant cytokines GM-CSF, IL-4 and TNF-alpha. Mature DCs were harvested after 7 days' co-culture of PBMCs and trastuzumab-treated apoptotic SKBr3 cells. The morphologic characteristics and ultrastructure of the DC were observed under the inverted phase-contrast microscope and transmission electron microscope (TEM), respectively. Flow cytometry (FCM) was used to check the expression of several DC specific markers: CD14, CD1a, CD64, CD80, CD83, CD86, HLA-ABC and HLA-DR. DC-cytokine induced killer (DC-CIK) cells were prepared by co-culture of DCs and peripheral blood lymphocytes in the presence of anti-CD3 antibodies and human IL-2 at an appropriate concentration. The number of antigen-specific T cells was analyzed by human interferon gamma enzyme linked immunospot (ELISPOT) assay. MTT assay was employed to assess the lysis of breast cancer cell line induced by DC-CIK cells.

RESULTS5 minutes after the adding of DCs to SKBr3 cells pretreated with trastuzumab, the apoptotic SKBr3 cells were found to be circled by DCs. 48 hours later, many membrane-wrapped organelles of the apoptotic target cells in the cytoplasm of DCs were found by TEM. The majority of the organelles were degraded. Fewer organelles from the apoptotic cells were found in DCs without Herceptin. More than 60% in every group of DCs expressed a high-affinity receptor for IgG (FcgammaRI or CD64). CD14 expression on the mature DCs were comparatively lower, and HLA-DR and HLA-ABC expressions were higher in the trastuzumab group. The expression of CD1a, CD80, CD83 and CD86 in trastuzumab group were higher than those in immature DCs group (P < 0.05). ELISPOT assay suggests that the spot number of antigen-specific T cells were higher in trastuzumab group than that in the antigen unloaded DCs group (P < 0.05). The lysis of SKBr3 cells induced by the SKBr3 antigen loaded DC-CIK cells were 1.7 times higher than that of CIK.

CONCLUSIONThe lysis of SKBr3 cells induced by DC-CIK was increased after that DCs were combined with trastuzumab to capture antigen from SKBr3 cells. These findings support further investigation into the use of combination immunotherapy of the humanized monoclonal antibody, DC vaccines and immunological effector cells.

Antibodies, Monoclonal ; pharmacology ; Antibodies, Monoclonal, Humanized ; Apoptosis ; Cell Line, Tumor ; Coculture Techniques ; Cytokine-Induced Killer Cells ; immunology ; Cytokines ; metabolism ; Cytotoxicity, Immunologic ; immunology ; Dendritic Cells ; cytology ; immunology ; metabolism ; ultrastructure ; Humans ; Receptor, ErbB-2 ; metabolism ; Receptors, IgG ; metabolism ; Trastuzumab

To produce many epitopes of peptide mut, different tandem repeats containing HER-2 mimetic peptide mut were constructed and expressed. The oligonucleotide coding mut sequence was inserted into pET28a(+) to construct the prokaryotic recombinant plasmid pET28a-mutl. The mut coding sequence was repeatedly inserted into the vector in tandem to obtain a series of plasmids pET28a-mut2, pET28a-mut4, pET28a-mut8 and pET28a-mut16. The plasmids were transformed into E. coli. BL21 and induced by IPTG. The recombinant proteins were expressed in E. coli. The binding analysis verified that the interaction between the tandem peptides mut and Herceptin was increased. In conclusion, the tandem peptides consisted of many antigen epitopes, and laid the foundation for the further study of HER-2 peptide vaccine for biotherapy of cancer with HER-2 overexpression.
Breast Neoplasms ; metabolism ; Cancer Vaccines ; biosynthesis ; immunology ; Epitopes ; Escherichia coli ; genetics ; metabolism ; Female ; Gene Expression ; Humans ; Peptides ; genetics ; immunology ; metabolism ; Receptor, ErbB-2 ; biosynthesis ; chemistry ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology

BACKGROUND/AIMS: The aim of this study was to investigate the immunohistochemical overexpression of c-erbB-2 and c-met proteins according to the histopathological parameters such as grade of dysplasia, histological type, depth of invasion, lymph node metastasis, and TNM stage in gastric adenoma and gastric adenocarcinoma. METHODS: Immunohistochemical staining using monoclonal c-erbB-2 and c-met antibodies was performed on paraffin embedded specimens in 43 adenomas and 44 adenocarcinomas. RESULTS: The expression rate of c-erbB-2 was higher in adenomas (91%) than adenocarcinomas (30%). The expression rate of c-met was higher in adenocarcinomas (77%) than adenomas (49%). In adenoma, the expression rate of c-met was higher in high grade dysplasia (94%) than in low grade dysplasia (22%). In adenocarcinoma, c-met expression was significantly related with lymph node metastasis. CONCLUSIONS: c-erbB-2 would be involved in the development of relatively early stage gastric carcinogenesis. c-erbB-2 is related with histologic type and c-met with lymph node metastasis in gastric carcinomas. Although meaning for the experession of these proteins in gastric carcinomas would be different, these proteins may play as important oncogenes in gastric carcinogenesis.
Adenocarcinoma/*metabolism/pathology ; Adenoma/*metabolism/pathology ; Aged ; Antibodies, Monoclonal ; Disease Progression ; Female ; Humans ; Immunohistochemistry ; Lymphatic Metastasis ; Male ; Middle Aged ; Proto-Oncogene Proteins c-met/immunology/*metabolism ; Receptor, erbB-2/immunology/*metabolism ; Stomach Neoplasms/*metabolism/pathology ; Tumor Markers, Biological

OBJECTIVETo explore the cytotoxic responses of spleen T lymphocytes (CTL) in BALB/c mice induced by recombinant HSP110-HER2/neu ICD complex.

METHODSTumor-bearing mouse model was immunized by HSP110-HER2/neu ICD complex. The IFN-γ level secreted by activated spleen T lymphocytes was detected by enzyme linked immunospot assay (ELISPOT). The corresponding CTL activity was measured by granzyme release assay.

RESULTSThe BALB/c mouse model of human mammary tumor highly expressing HER2/neu was established. HSP110-HER2/neu ICD complex immunization led to a significantly higher level of INF-γ than that in HSP110-P(789-797) immunized and HER2/neu ICD immunized mice. HSP110-HER2/neu ICD complex immunized animals also show significant CTL activity. The results of immunohistochemical staining showed that the number of blue spots in the PBS group was 4.57 ± 1.33, HSP110 group 6.83 ± 2.08, HER2/neu ICD group 16.17 ± 2.86, HSP110-P(789-797) group 43.67 ± 4.78, and SP110-HER2/neu ICD group 76.51 ± 8.17. The number of IFN-γ-secreting spleen lymphocytes in the HSP110-HER2/neu ICD group was significantly higher than that in the HSP110-P(789-797) group, and that of HSP110-P(789-797) group was significantly higher than that of HER2/neu ICD group (P < 0.01). The target cell-killing rate of the PBS group was (8.15 ± 1.27)%, HSP110 group (9.51 ± 1.51)%, HER2/neu ICD group (14.03 ± 2.45)%, HSP110-P(789-797) group (25.99 ± 3.04)% and HSP110-HER2/neu ICD group (38.15 ± 3.95)% (all P < 0.01).

CONCLUSIONSHSP110-HER2/neu ICD complex can promote the proliferation and maturation of T lymphocytes into CTLs, and might be used as anti-tumor vaccine to induce potent cytotoxic T lymophocyte immunoresponse against specific tumor cells.

Animals ; Breast Neoplasms ; metabolism ; pathology ; Cancer Vaccines ; immunology ; Cell Line, Tumor ; Cell Proliferation ; Female ; HSP110 Heat-Shock Proteins ; immunology ; Humans ; Interferon-gamma ; metabolism ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Random Allocation ; Receptor, ErbB-2 ; immunology ; metabolism ; Recombinant Proteins ; immunology ; Spleen ; cytology ; immunology ; T-Lymphocytes ; cytology ; immunology ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; Vaccines, Synthetic ; immunology

Breast Neoplasms ; metabolism ; pathology ; Cytokines ; metabolism ; Female ; Fluorescent Antibody Technique ; Humans ; Microscopy, Fluorescence ; Paraffin Embedding ; Proliferating Cell Nuclear Antigen ; metabolism ; Quantum Dots ; Receptor, ErbB-2 ; metabolism ; Receptors, Estrogen ; metabolism ; Stomach Neoplasms ; immunology ; pathology

OBJECTIVETo study the expression of a glycoprotein of plant origin in normal, benign and malignant breast tissues.

METHODSExpression of a plant glycoprotein was examined in 5 samples of normal breast tissues, 20 fibro-adenoma and 136 breast cancer by SABC immunohistochemical staining and the results were analyzed by SPSS statistics software.

RESULTSNo positive staining was detected in normal breast tissues (0/5). Weak staining was observed in 4 of 20 (20.0%) breast fibro-adenoma. Positive staining was demonstrated in 116 out of 136 (85.3%) breast cancer specimens. The differences were statistically significant. The expression of plant-associated human cancer antigen was related to pathological grade (P < 0.05), tissue invasiveness (P < 0.01) and recurrence (P < 0.05), but not to patients' age, tumor size and c-erbB-2 expression.

CONCLUSIONThe plant glycoprotein studied may be a human cancer-associated antigen which might be a potential marker of breast cancer.

Adenocarcinoma, Mucinous ; immunology ; pathology ; Adult ; Aged ; Antigens, Neoplasm ; metabolism ; Biomarkers, Tumor ; metabolism ; Breast ; immunology ; Breast Neoplasms ; immunology ; pathology ; Carcinoma, Ductal, Breast ; immunology ; pathology ; Carcinoma, Intraductal, Noninfiltrating ; immunology ; pathology ; Female ; Fibroadenoma ; immunology ; pathology ; Humans ; Middle Aged ; Neoplasm Invasiveness ; Neoplasm Recurrence, Local ; metabolism ; Plants ; immunology ; Receptor, ErbB-2 ; metabolism