Antibody response to HER2 extracellular domain and subdomains in mouse following DNA immunization.

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.

Immunization with HER2 extracellular subdomain proteins induces cellular response and tumor growth inhibition in mice.

AIM: We investigated cellular and protective immune responses in mice vaccinated with recombinant HER2 extracellular subdomains. MATERIALS & METHODS: Balb/C mice were immunized with recombinant full HER2 extracellular domain and subdomain proteins. Humoral and cellular immune response and antitumor effect was evaluated using a syngeneic mice tumor model. RESULTS: All recombinant proteins induced secretion of IL-4 and particularly IFN-γ and IL-17 cytokines. Challenging of immunized mice with stable 4T1-HER2 transfected cells resulted in partial but significant tumor growth inhibition in all groups of mice particularly those immunized with fHER2-ECD together with CPG. CONCLUSION: Our results suggest that the recombinant HER2-ECD subdomains induce mainly Th1 and Th17 responses, which seem to contribute to tumor growth inhibition in syngeneic mice.

Antibody Response to Human Extracellular HER2 Subdomain Proteins in Mice.

BACKGROUND: In addition to passive immunotherapy using anti-HER2 monoclonal antibodies, active immunotherapy via HER2 targeting is an interesting approach to inducing specific anti-tumor immune responses. We have recently reported the immunogenicity of HER2 subdomains following DNA immunization and HER2 protein boosting. In the present study, we evaluated the immunogenicity of different HER2 extracellular subdomains for the induction of anti-HER2 antibody response in BALB/c mice. OBJECTIVE: To investigate and characterize antibody responses to human recombinant proteins of HER2 extracellular subdomains in immunized mice. METHODS: Four subdomains of HER2 extracellular domain were expressed in E.coli; subsequently, purified recombinant proteins were intraperitoneally injected in BALB/c mice with Freund's adjuvant. The anti-HER2 antibody response was detected by ELISA, immunoblotting and flow cytometry. RESULTS: All the four HER2 subdomains along with the full extracellular domain (fECD) were able to induce specific anti-HER2 antibodies. Although anti-HER2 subdomains antibodies could not react with eukaryotic recombinant fECD protein by ELISA, they were able to recognize this protein by immunoblotting under both reduced and non-reduced conditions. Furthermore, only the sera of mice immunized with fECD protein could recognize native HER2 on HER2 overexpressing tumor cells (>99%) by flow cytometry. Moreover, fECD immunized mice sera inhibited the proliferation of tumor cells by XTT assay. CONCLUSION: The prokaryotic recombinant proteins of HER2 extracellular subdomains are immunogenic, yet the induced specific antibodies do not react with the native HER2 protein due to the paucity of post-translation modifications and /or distortion of the native conformation of isolated HER2 extracellular subdomains which might be potentially effective for induction of cell mediated immune response against HER2.