OBJECTIVETo construct human phage single-chain antibody (scFv) library against breast cancer, and to identify anti-HER2 specific antibodies from the human phage display scFv library to offer a stronger affinity sequence targeting HER2 for fusion protein targeting HER2 and CXCR4.

METHODSTotal RNA was extracted from the adjacent lymphatic tissue harvested from breast cancer patients. The variable regions of the whole antibody were amplified by using RT-PCR and were cloned into the vector pCANTAB-5E through a linker. The products were electroporated into competent E.coli TG1 cells. Recombinant phages specific for breast cancer cells were enriched in SKBR-3 after four rounds. The antigen-positive clones were selected by ELISA and immunohistochemistry.

RESULTSThe fragment of VH and VL were about 375 and 330 bp and were linked in vitro to form scFv of 750 bp that was resistant to the breast cancer HER2 single strand. A fusion phage display library that contained total of 2.48×10(8) pfu /ml was established. ELISA and immunohistochemical results confirmed that the antibody has a strong affinity with HER2 antigen in breast cancer tissue. Compared to human IgG antibody, a scFv phage library against human breast cancer was successfully constructed with high capacity. The scFv was highly specific to HER2 antigen and the sequencing results indicated that VL and VH genes were highly homologous with the variable region of human antibody.

CONCLUSIONThis strategy may achieve new targeted antibody resistant to the breast cancer for clinical treatment and provide a carrier that uses HER2 as a target of the fusion protein for anti-tumor therapy.

Breast Neoplasms ; genetics ; immunology ; Female ; Humans ; Peptide Library ; Receptor, ErbB-2 ; immunology ; Single-Chain Antibodies ; immunology

This phase I clinical trial (NCT01935843) is to evaluate the safety, feasibility, and activity of chimeric antigen receptor-engineered T cell (CART) immunotherapy targeting human epidermal growth factor receptor 2 (HER2) in patients with advanced biliary tract cancers (BTCs) and pancreatic cancers (PCs). Eligible patients with HER2-positive (>50%) BTCs and PCs were enrolled in the trial. Well cultured CART-HER2 cells were infused following the conditioning treatment composed of nab-paclitaxel (100-200 mg/m) and cyclophosphamide (15-35 mg/kg). CAR transgene copy number in the peripheral blood was serially measured to monitor the expansion and persistence of CART-HER2 cells in vivo. Eleven enrolled patients received 1 to 2-cycle CART-HER2 cell infusion (median CAR T cell 2.1 × 10/kg). The conditioning treatment resulted in mild-to-moderate fatigue, nausea/vomiting, myalgia/arthralgia, and lymphopenia. Except one grade-3 acute febrile syndrome and one abnormal elevation of transaminase (>9 ULN), adverse events related to the infusion of CART-HER2 cells were mild-to-moderate. Post-infusion toxicities included one case of reversible severe upper gastrointestinal hemorrhage which occurred in a patient with gastric antrum invaded by metastasis 11 days after the CART-HER2 cell infusion, and 2 cases of grade 1-2 delayed fever, accompanied by the release of C-reactive protein and interleukin-6. All patients were evaluable for assessment of clinical response, among which 1 obtained a 4.5-months partial response and 5 achieved stable disease. The median progression free survival was 4.8 months (range, 1.5-8.3 months). Finally, data from this study demonstrated the safety and feasibility of CART-HER2 immunotherapy, and showed encouraging signals of clinical activity.
Aged ; Biliary Tract Neoplasms ; immunology ; therapy ; Female ; Humans ; Immunotherapy, Adoptive ; Male ; Middle Aged ; Pancreatic Neoplasms ; immunology ; therapy ; Receptor, ErbB-2 ; immunology ; Receptors, Chimeric Antigen ; immunology ; T-Lymphocytes ; immunology

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

Tumor surface antigen human epidermal growth factor receptor 2 (Her2) is a type I receptor tyrosine kinase, which belongs to human epidermal growth factor receptor family. Her2-overexpression is associated with tumorigenesis and metastasis. Due to significant clinical effects, Her2-targeted cancer therapy especially therapeutic antibody has become the hot spot in the field of cancer treatment. Anti-Her2 antibody drugs include monoclonal antibodies, antibody-drug conjugates, bispecific antibodies and emerging "two in one" antibody. Based on structure and function of Her2, this review focuses on recent advances in active mechanisms and clinical researches of these antibodies.
Antibodies, Bispecific ; therapeutic use ; Antibodies, Monoclonal ; therapeutic use ; Humans ; Immunoconjugates ; therapeutic use ; Neoplasms ; drug therapy ; Receptor, ErbB-2 ; immunology

OBJECTIVETo explore the immune response induced by HER2/neu oncogene in the breast cancer (BC) carcinogenesis process and the immunological mechanism of Ru'ai Shuhou Recipe (RSR) in the prevention and treatment of BC.

METHODSHER2/neu transgenic spontaneous breast tumor model mice were fed with RSR from 5 weeks old, the occurrence of breast tumor in them was observed, and the changes of T cell-mediated immune response and associated cytokines were detected during the carcinogenesis process, i. e., when mice aged between 15 and 25 weeks.

RESULTSRSR showed significant effects in postponing and reducing the carcinogenesis of primary breast tumor, up-regulating the amount of T cell in splenic lymphocyte in tumor-bearing mice, promoting the proliferation of T lymphocyte, and inducing the secretion of cytokines such as interleukin-2, interleukin-12 and interferon-y.

CONCLUSIONSA serial immune response reveals in the carcinogenesis process. The immunologic function of HER-2/neu transgenic mice is significantly different to that of the same strain non-transgenic mice. Effect of RSR in preventing and postponing breast cancer carcinogenesis is possibly realized through enhancing the anti-tumor immune response of transgenic mice themselves.

Animals ; Breast Neoplasms ; immunology ; prevention & control ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Female ; Genes, erbB-2 ; genetics ; Mice ; Mice, Transgenic ; Receptor, ErbB-2 ; genetics ; Spleen ; cytology ; immunology ; T-Lymphocytes ; cytology ; immunology

Monoclonal antibodies (mAbs) have become a major class of therapeutic agents providing effective alternatives to treating various human diseases. To date, 15 mAbs have been approved by regulatory agencies in the world for clinical use in oncology indications. The selectivity and specificity, the unique pharmacokinetics, and the ability to engage and activate the host immune system differentiate these biologics from traditional small molecule anticancer drugs. mAb-based regimens have brought clinical benefits, including improvements in overall survival, to patients with a variety of cancers. Many challenges still remain, however, to fully realize the potential of these new medicines. With our further understanding of cancer biology, mechanism of antibody action, and advancement of antibody engineering technologies, many novel antibody formats or antibody-derived molecules are emerging as promising new generation therapeutics. Carefully designed and engineered, they retain the advantage of specificity and selectivity of original antibodies, but in the meantime acquire additional special features such as improved pharmacokinetics, increased selectivity, and enhanced anticancer efficacy. Promising clinical results are being generated with these newly improved antibody-based therapeutics.
Antibodies, Monoclonal ; therapeutic use ; Antibodies, Monoclonal, Humanized ; therapeutic use ; Antigens, CD20 ; immunology ; Humans ; Immunoconjugates ; therapeutic use ; Neoplasms ; drug therapy ; immunology ; Protein Engineering ; RANK Ligand ; immunology ; Receptor, Epidermal Growth Factor ; immunology ; Receptor, ErbB-2 ; immunology ; Vascular Endothelial Growth Factor A ; immunology

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 develop and identify the monoclonal antibodies (mAb) against c-erbB-2 p185 intracellular domain for detection of c-erbB-2 protein overexpression in breast tumor cells.

METHODSBALB/C mice were immunized with a synthesized p185 peptide of intracellular domain. The biological characteristics and immunoactivities were identified by different techniques.

RESULTSThree hybridoma cell strains secreting mAbs to c-erbB-2 protein were established and one of the mAbs, No. 035-E61 was tested for c-erbB-2 protein immunostaining on 39 breast carcinoma sections, 30 breast fibroadenoma sections and 16 sections from various normal organs. The results showed that the positive detection frequency of protein expression was 26% (10/39) in breast cancer, none (0/30) in fibroadenoma and 3 sections from normal organs were positively stained. The consistency between the 035-E61 and the DAKO reagent approved by FDA was 74%.

CONCLUSIONSNo. 035-E61 mAb can specifically recognize the p185 intracellular domain and may be useful in guiding clinical Herceptin treatment.

Animals ; Antibodies, Monoclonal ; immunology ; Antibody Specificity ; Binding Sites ; immunology ; Breast Neoplasms ; immunology ; pathology ; Female ; Fibroadenoma ; immunology ; pathology ; Humans ; Immunoglobulin G ; immunology ; Immunohistochemistry ; Mice ; Mice, Inbred BALB C ; Receptor, ErbB-2 ; immunology ; Tumor Cells, Cultured

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

It has been well established that immune surveillance plays critical roles in preventing the occurrence and progression of tumor. More and more evidence in recent years showed the host anti-tumor immune responses also play important roles in the chemotherapy and radiotherapy of cancers. Our previous study found that tumor- targeting therapy of anti-HER2/neu mAb is mediated by CD8(+) T cell responses. However, we found here that enhancement of CD8(+) T cell responses by combination therapy with IL-15R/IL-15 fusion protein or anti-CD40, which are strong stimultors for T cell responses, failed to promote the tumor therapeutic effects of anti-HER2/neu mAb. Analysis of tumor microenviornment showed that tumor tissues were heavily infiltrated with the immunosuppressive macrophages and most tumor infiltrating T cells, especially CD8(+) T cells, expressed high level of inhibitory co-signaling receptor PD-1. These data suggest that tumor microenvironment is dominated by the immunosuppressive strategies, which thwart anti-tumor immune responses. Therefore, the successful tumor therapy should be the removal of inhibitory signals in the tumor microenvironment in combination with other therapeutic strategies.
Animals ; Antibodies, Monoclonal ; immunology ; therapeutic use ; Breast Neoplasms ; drug therapy ; immunology ; pathology ; Cell Line, Tumor ; Female ; Humans ; Immune Tolerance ; immunology ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Receptor, ErbB-2 ; immunology ; Tumor Microenvironment ; immunology