Abstract
In our ongoing phase I dose-escalation trial in women with metastatic breast cancer, multiple infusions of ATC armed with Her2Bi (aATC) induced elevated levels of IL-2, IFNγ TNFα, GM-CSF, and IL-12 as well as the development of cytotoxic activity directed at breast cancer cell lines lasting up to 4 months (Grabert et al. Clin Cancer Res. 2006). These results suggested that aATC infusions “vaccinated” the endogenous immune system of the patients. In this study, we explored mechanisms of the increased cytotoxicity after immunotherapy with armed ATC by testing for cell-mediated cytotoxicity and in vitro anti-tumor antibody synthesis. We co-cultured irradiated aATC with fresh PBMC in presence or absence of SKBR3 for 7 days, collected the PBMC, and tested for cytotoxicity in MTT and Cr51 release assays directed at SKBR3, Daudi and A-431 cell lines. PBMC mediated high levels of non-specific cytotoxicity against all tested cell lines and there were no phenotypic changes in the PBMC after 7 days of co-culture. When PBMC were co-cultured with irradiated aATC and SKBR3 for 21 days in presence of IL-2, the B cells in the PBMC produced significantly higher amounts of specific Abs directed SKBR3 (ELISA for antibodies binding to SKBR3) compared to PBMC co-cultured with SKBR3 alone. CpG ODN type C augmented in vitro anti-SKBR3 Ab synthesis. These studies show that Her2Bi-armed ATCco-cultured with PBMC enhanced nonspecific cytotoxicity and induced in vitro specific antibody synthesis directed at SKBR3 cells. Evidence that Her2Bi armed ATC can induce a vaccination response is supported by dendritic cell (DC) loading experiments in which aATC were transiently mixed with SKBR3 cells to generate tumor lysate. IL-4 and GM-CSF generated DC were exposed to the lysate for 24 h, washed, and co-cultured with fresh PBMC for 14 and 21 days. At the end of co-culture, cytotoxicity assays against SKBR3 increased markedly whereas cytotoxicity directed at Daudi targets was low. In addition, there were considerable levels of Abs directed to SKBR3 in the supernatants of PBMC co-cultured with DC loaded with SKBR3 lysate, but not with DC loaded with SKBR3-culture media or RPMI alone. These studies established that the lysate produced as a result of aATC cytotoxicity against SKBR3 is immunogenic for DC. In summary, when PBMC were co-cultured with DC exposed to SKBR3 lysate, there was induction of specific cytotoxicity and in vitro tumor specific Abs synthesis. Together with experiments involving primary co-cultures of irradiated aATC, PBMC, and SKBR3, our studies show that there are both non-specific and specific cellular and humoral responses generated as a result of co-culture with Her2Bi armed ATC. These studies provide evidence that aATC infusions can induce both specific and non-specific cellular, humoral, and cytokine responses from the endogenous immune systems of patients.
Please credit the grants R01CA92344, 5P30CA022453-25, 1819 from Michigan Economic Development Corporation and 6066-06 from Leukemia and Lymphoma Society
Immunoregulatory effects of alpha-interferon. Effects on in-vitro antibody synthesis by lymphocytes from patients with rheumatoid arthritis.
