title: In vitro synthesis of primary specific anti-breast cancer antibodies by normal human peripheral blood mononuclear cells.
abstract: In this study, we developed a unique in vitro model to mimic the endogenous tumor microenvironment to understand the effect of immunotherapy with activated T-cells (ATC) armed with anti-CD3 × anti-Her2 bispecific antibody (aATC) on antibody response by naive immune cells. This model contained a co-culture of naïve peripheral blood mononuclear cells (PBMC), breast cancer cells (SK-BR-3), ATC or aATC and CpG ODNs. Culture supernatants were tested at various time points for anti-SK-BR-3 antibodies by ELISA, Western blot and flow cytometry. PBMC cocultured with non-irradiated aATC or irradiated (*) aATC showed significant increases in anti-tumor antibody production at day 14 (P < 0.0001) in the presence of CpG-ODN compared to unstimulated PBMC cultures (n = 9). Antibody specificity was confirmed by ELISA, Western blot and flow cytometry. Co-cultures containing *aATC and CpG showed significantly enhanced levels of IgG(2) (P < 0.001) and cytokines that promote IgG(2) synthesis including IL-13 (P < 0.02), IFNγ (P < 0.01) and GM-CSF (P < 0.05) compared to unstimulated PBMC control (n = 3). We show that aATC targeting and lysis of tumor cells induces an anti-tumor antibody response in our in vitro model. This model provides a unique opportunity to evaluate the interactions of T-cells, B-cells, and antigen-presenting cells leading to specific anti-tumor antibody responses.
authors.affiliation: Departments of Oncology, Medicine, Immunology and Microbiology, Wayne State University School of Medicine, 731 Hudson Webber Cancer Research Center, 4100 John R., Detroit, MI 48201, USA. thakur@karmanos.org
authors.forename: Archana
authors.initials: A
authors.lastname: Thakur
authors.forename: Oxana
authors.initials: O
authors.lastname: Norkina
authors.forename: Lawrence G
authors.initials: LG
authors.lastname: Lum
mesh_terms: D018033:Antibodies, Bispecific / Q000096:biosynthesis* / Q000276:immunology; D000912:Antibodies, Neoplasm / Q000096:biosynthesis* / Q000276:immunology; D000920:Antibody-Dependent Cell Cytotoxicity / Q000276:immunology; D000951:Antigens, Neoplasm / Q000276:immunology; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000276:immunology*; D017252:CD3 Complex / Q000276:immunology; D045744:Cell Line, Tumor; D002469:Cell Separation; D018920:Coculture Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007167:Immunotherapy / Q000379:methods; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D009838:Oligodeoxyribonucleotides; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology
chemical_list: D018033:Antibodies, Bispecific; D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D017252:CD3 Complex; D009838:Oligodeoxyribonucleotides; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human