Construction and characterisation of a stably transfected BHK cell line permanently secreting the canine interleukin 12 as a source for adoptive cancer immunotherapy in dogs
BACKGROUND AND AIM OF THE STUDY: The dog represents the most important tumor patient in the veterinary medicine. Furthermore, the growing knowledge of tumor biology and immunology in dogs increases the interest of this species as a promising model in studies of tumor immunotherapy in human. Concerning the tumor treatment, one of the latest therapeutical approaches is the tumor immunotherapy, especially the adoptive immunotherapy, which is based on in vitro lymphocyte activation by cytokines. Among the cytokines showing very potent anti-tumor activity is the heterodimeric interleukin-12 (IL-12). However, in contrast to mice and human, detailed studies in dogs are lacking. In order to provide reliable sources for future investigations and applications on the field of cancer immunotherapy in dogs, we (1) constructed a canine single-chain IL-12 cDNA, which (2) was stably transfected into the baby hamster kidney (BHK)-Tet-On cell line and (3) confirmed and investigated the biological activities of the canine single-chain IL-12.
METHODS: Both cDNA sequences coding for the canine IL-12 chains p35 and p40 were amplified by PCR. The cDNA fragments were ligated to encode a single chain IL-12 in Tet-On expression vector pTRE/luciferase. Following the transfection into a BHK-Tet-On cell line, an anti-luciferase antibody was used to screen for stably transfected cell clones. Additionally, with help of an anti-canine IL-12 polyclonal antibody the presence of the canine single-chain IL-12 protein in the cell clones was investigated by Western blot. Using canine interferon-gamma (IFN-gamma) ELISA, BrdU ELISA-based proliferation assay and cytotoxicity assay (Rose Bengal Assay), the bioactivity of the IL-12 containing supernatants was investigated.
RESULTS: The anti-luciferase antibody showed transgene expression in almost 100 % of the cells of the obtained cell lines. Accordingly, these cell clones provide a uniform source of IL-12 for further investigations. Moreover, the single chain canine IL-12 protein was demonstrated by Western blot in the supernatants and in the cell lysates of the established clones. Canine IL-2 blasts incubated with the supernatant of the transfected clone showed significantly increased IFN-gamma production; the latter response was completely blocked by anti-canine IL-12 neutralizing monoclonal antibody. Further on, it was shown that the presence of IL-2 is necessary for the single-chain IL-12 to induce IFN-gamma production by the canine lymphoblasts. However, in contrast to the canine IL-2 lymphoblasts, the single-chain IL-12 did not induce IFN-gamma production in freshly isolated canine peripheral blood mononuclear cells. In canine IL-2 lymphoblasts the single chain IL-12 containing culture supernatant induced an increased cytolytic activity against canine thyroid adenocarcinomas (CTAC) target cell line.
CONCLUSIONS: The established cell lines release canine single-chain IL-12 into the supernatant and when compared with commercially available canine IL-12 the recombinant protein produced by our cell lines displayed the full panel of bioactivities. Perspectively, the BHK cell clones carrying the canine IL-12 could be used for further investigation of IL-12 induced anti-tumor effects on canine lymphocytes, especially NK cells. Moreover, canine single-chain IL-12 cDNA can be tested in gene delivery studies, like intra-tumor application.
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