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

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

In carcinoma ex pleomorphic adenoma (CXPA), pleomorphic adenoma (PA) and diverse carcinoma components showing luminal (ductal) or non-luminal (myoepithelial) differentiation coexist. To elucidate the clinicopathological implications of cellular differentiation in CXPA and the potential role of p53, vascular endothelial growth factor (VEGF), c-erbB-2, c-kit, and glucose transporter 1 (Glut-1) in carcinogenesis, we analyzed 11 CXPAs with luminal differentiation (CXPAs-LD) and 6 CXPAs with non-luminal differentiation (CXPAs-NLD) and compared protein expressions in residual PAs and carcinomas by immunohistochemistry. Among the CXPAs-LD, 5 were invasive and 8 were histologically high-grade tumors. The 5-year survival rate was 72.7%. P53, c-erbB-2, VEGF, and Glut-1 were more immunoreactive in carcinoma components than in PAs (P = 0.008, 0.004, 0.002, and 0.024, respectively); c-erbB-2 overexpression was associated with high histological grade (P = 0.024). Carcinoma components frequently lacked c-kit expression (P = 0.009). CXPAs-NLD were all low-grade and invasive with a larger mean tumor size (5.2 cm) than CXPAs-LD (3.3 cm) (P = 0.040). The patients remained disease-free without significant immunohistochemical expression. The immunoprofiles and clinical course of CXPA differed according to cellular differentiation. Therefore, it is important to report the histological subtype and to assess potential biomarkers in diagnostic and therapeutic trials.
Adenoma, Pleomorphic/*immunology/metabolism/*pathology ; Adult ; Aged ; Carcinoma/*immunology/metabolism/*pathology ; Cell Differentiation ; Female ; Glucose Transport Proteins, Facilitative/metabolism ; Humans ; Male ; Middle Aged ; Proto-Oncogene Proteins c-kit/metabolism ; Receptor, erbB-2/metabolism ; Salivary Gland Neoplasms/*immunology/metabolism/*pathology ; Tumor Markers, Biological/*analysis ; Tumor Suppressor Protein p53/metabolism ; Vascular Endothelial Growth Factor A/metabolism

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

OBJECTIVETo evaluate the immunostimulatory capacity of human peripheral blood dendritic cells (DCs) with Her2/neu gene transfection mediated by adeno-associated virus.

METHODSThe HLA genotypes of the breast cancer cells SK-BR-3 and MCF7 were determined, and the mononuclear cells from healthy donors with matching HLA genotype were isolated by Ficoll-Hypaque density gradient separation. The isolated cells were divided into two groups with or without transfection with the recombinant virus harboring Her2/neu gene. The cells were cultured for 7 days in RPMI 1640 medium supplemented with 10% AB human serum, GM-CSF, interleukin-4 (IL-4) and tumor necrosis factor-alpha (TNF-alpha). The mature DCs were then harvested from the cell culture and their phenotypes were identified using flow cytometry. MTT assay was employed to examine the specific killing activity of the T cells induced by the DCs.

RESULTSThe DCs transfected with the recombinant adeno-associated virus expressed CD1a, CD86 and CD83 at the rate of 98.10%, 99.42%, and 84.59%, and those without the viral transfection expressed the markers at the rate 92.69%, 98.07%, and 82.72%, respectively, showing no obvious differences in the phenotypes of the two DCs. The transfected DCs, however, showed markedly higher expression rates of CD40 and CD80 than the non-transfected DCs (61.02% vs 36.19%, and 97.61% vs 55.5%, respectively). The DCs, irrespective of the transfection, showed comparable capacities in stimulating T cell proliferation. The transfected DCs exhibited the capacity of inducing the T cells to specifically kill the target tumor cells, with the highest killing rate of (39.7-/+7.2)%.

CONCLUSIONThe immunostimulatory capacity of human peripheral blood DCs are enhanced by Her2/neu gene transfection mediated by adeno-associated virus.

Breast Neoplasms ; pathology ; Cell Line, Tumor ; Cells, Cultured ; Dendritic Cells ; cytology ; immunology ; metabolism ; Dependovirus ; genetics ; metabolism ; Genes, erbB-2 ; genetics ; Genetic Vectors ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Leukocytes, Mononuclear ; cytology ; Receptor, ErbB-2 ; biosynthesis ; genetics ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Transfection

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

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 study the clinicopathologic characteristics of basal-like immunophenotype breast cancer (BLBC).

METHODS458 cases of female infiltrative breast cancer were studied using immunohistochemical staining with an antibody panel of ER, PR, HER2, Ki-67, CK5/6, CK14 and epidermal growth factor receptor (EGFR) and were classified basing on the immunophenotypes. The clinicopathologic characteristics were compared with other immunophenotypes of breast cancer. 228 of 458 cases of breast cancer were followed up.

RESULTS46 cases of BLBC were screened out among the 458 breast cancers. And histological features of BLBC were analysed including the larger diameter of cancer foci (average 3.3 cm), appearance of squeezing phenomenon of neighboring cell borders (58.7%, 27/46), geography-like distribution of necrosis (52.2%, 24/46), central zone fibrosis (30.4%, 14/46) and lymphoplasmacytic infiltration at the margin and stroma (63.0%, 29/46). There were nuclear pleomorphism with numerous mitoses. The cancer cells were closely arranged, forming irregular solid architectures. There was a high expression (> 25%) of Ki-67 (43.5%, 20/46). CK5/6, CK14 and EGFR were positive in 58.7% (27/46), 43.5% (20/46) and 65.2% (30/46) respectively. 3-year survival rate of BLBC was 66.9%, lower than the luminal A breast cancer and similar to HER2 over-expression breast cancer.

CONCLUSIONSThe proportion of BLBC in the group of breast cancers is 10%. BLBC has its distinct histological and cytological features. Currently, it is still necessary to depend on immunophenotyping in making a BLBC diagnosis. BLBC is the one of breast cancer subtypes with the poorest prognosis.

Adult ; Aged ; Aged, 80 and over ; Biomarkers, Tumor ; analysis ; immunology ; Breast Neoplasms ; diagnosis ; immunology ; metabolism ; pathology ; Female ; Humans ; Immunophenotyping ; methods ; Middle Aged ; Prognosis ; Receptor, Epidermal Growth Factor ; analysis ; immunology ; Receptor, ErbB-2 ; analysis ; immunology ; Receptors, Estrogen ; analysis ; immunology ; Receptors, Progesterone ; analysis ; immunology ; Survival Rate

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

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