ABSTRACT
Human epidermal growth factor receptor 2 (HER2) is overexpressed in 15-20 % of breast cancer patients and is an appropriate target for immunotherapy in these patients. Monoclonal antibodies (mAbs) specific to HER2 are currently applied to treat breast cancer patients with HER2 overexpression. Active immunization with HER2 DNA or protein has been considered as a suitable alternative. The aim of this study is to evaluate anti-HER2 antibody response in serum of mice immunized with DNA constructs containing full extracellular domain (fECD) or subdomains of human HER2. Four extracellular subdomains and also fECD of HER2 were cloned into pCMV6-Neo vector. Different groups of Balb/C mice were immunized with HER2 DNA constructs and boosted with HER2 recombinant protein. The anti-HER2 antibody was subsequently determined by ELISA, flow cytometry, and immunohistochemistry. Anti-HER2 antibody was detected only in serum of mice immunized with fECD DNA. None of HER2 extracellular subdomains induced appreciable levels of anti-HER2 antibody. However, boosting with fECD or extracellular subdomain III (DIII) recombinant protein resulted in enhanced anti-HER2 fECD as well as anti-HER2 subdomain antibody responses. In this regard, almost all (99 %) of HER2-overexpressing BT474 cells could be detected by serum antibody from mice immunized with HER2 subdomain DNA and boosted with recombinant HER2 protein by flow cytometry. Similarly, serum of mice immunized with DIII DNA construct and boosted with recombinant DIII protein could also recognize these cells, but to a lesser extent (50 %). Our findings suggest that combination of HER2 DNA and protein immunization could effectively induce anti-HER2 antibody response in Balb/C mice.
Subject(s)
Antibody Formation , Cancer Vaccines/immunology , Receptor, ErbB-2/immunology , Vaccines, DNA/immunology , Animals , Antibodies, Monoclonal/immunology , Breast Neoplasms/immunology , Breast Neoplasms/metabolism , DNA Primers/genetics , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Genetic Vectors , Humans , Immunohistochemistry , Immunotherapy/methods , Mice , Mice, Inbred BALB C , Protein Domains , Recombinant Proteins/immunologyABSTRACT
BACKGROUND: Prostate Specific Antigen (PSA) is an important laboratory marker for diagnosis of prostatic cancer. Thus, development of diagnostic tools specific for PSA plays an important role in screening, monitoring and early diagnosis of prostate cancer. In this paper, the production and characterization of a panel of murine monoclonal antibodies (mAbs) against PSA have been presented. METHODS: Balb/c mice were immunized with PSA, which was purified from seminal plasma. Splenocytes of hyperimmunized mice were extracted and fused with Sp2/0 cells. By adding selective HAT medium, hybridoma cells were established and positive clones were selected by ELISA after four times of cloning. The isotypes of produced mAbs were determined by ELISA and then purified from ascitic fluids using Hi-Trap protein G column. The reactivities of the mAbs were examined with the purified PSA and seminal plasma by ELISA and western blot techniques. Furthermore, the reactivities of the mAbs were assessed in Prostate Cancer (PCa), Benign Prostatic Hyperplasia (BPH) and brain cancer tissues by Immunohistochemistry (IHC). RESULTS: Five anti-PSA mAbs (clones: 2G2-B2, 2F9-F4, 2D6-E8, IgG1/Ð) and clones (2C8-E9, 2G3-E2, IgG2a/Ð) were produced and characterized. All mAbs, except 2F9-F4 detected the expression of PSA in PCa and BPH tissues and none of them reacted with PSA in brain cancer tissue in IHC. Besides, all mAbs could detect a protein band around 33 kDa in human seminal plasma in western blot. CONCLUSION: These mAbs can specifically recognize PSA and may serve as a component of PSA diagnostic kit in various biological fluids.
