540. Re-Targeting of Adenoviral Vector-Mediated Interferon-γ Gene Transfer to Colon Cancer Cells That Express the Human Carcinoembryonic Antigen

Abstract

We hypothesized that re-targeting of an Ad5 vector encoding the interferon-|[gamma]| (IFN-|[gamma]|) cDNA to tumor cells that express human carcinoembryonic antigen (CEA) can be accomplished. In order to test the feasibility of this schema, we conducted a proof-of-principle experiment. In this experiment, we utilized a recombinant adapter protein (sCAR-MFE), consisting of the extracellular portion of the native Ad receptor, the coxsackievirus-adenovirus receptor (CAR), fused to a single chain antibody (scFv) specific for the human carcinoembryonic antigen (CEA). We tested the ability of this adapter protein to re-target an adenovirus expressing Ad5-IFN-|[gamma]| to cells expressing CEA. We chose as a model, a mouse colon cancer cell line derived from the MC38 cell line that expresses the human CEA constitutively and forms tumors in syngeneic immunocompetent C57BL6 mice (MC38-CEA2). The Ad5-IFN-|[gamma]| vector (1|[times]|107 viral particles) was first incubated in the absence or presence of 100 ng of the recombinant adapter protein sCAR-MFE for 15 min at room temperature. As a negative control, the Ad5-IFN-|[gamma]| vector was also incubated in the absence or presence of a non-specific recombinant adapter protein, CFm40L. The untargeted or re-targeted Ad5-IFN-g vector was used to infect either the MC38 cell line or the MC38-CEA2 cell line at a low multiplicity of infection (0.1 infectious units/cell). The cells were infected for 1 hour, and then the infected cells were cultured for 48 hours. At the end of the culture period, the levels of Ad-IFN-|[gamma]| in the culture media were determined by ELISA analysis. The levels of IFN-|[gamma]| in the culture media from MC38 cells infected with Ad5-IFN-g was low in the culture media, ranging from 454 to 572 pg/ml, regardless of the presence or absence of recombinant adapter proteins. In contrast, in the culture media from MC38-CEA2 cells infected with Ad5-IFN-g the level of IFN-g was low, at 1159 pg/ml. Similarly, in the culture media from MC38-CEA2 cells infected with the Ad5-IFN-|[gamma]| vector pre-incubated with the irrelevant adapter protein CFm40L, the level of IFN-|[gamma]| was 1648 pg/ml. In contrast, in the culture media from MC38-CEA2 cells infected with the Ad5-IFN-|[gamma]| vector pre-incubated with the re-targeting adapter protein sCAR-MFE, the level of IFN-|[gamma]| was nearly three orders of magnitude higher at 732,000 pg/ml. Importantly, the observation that re-targeting was inefficient on CEA-negative MC38 cells demonstrates the CEA-targeted mechanism of the targeting complex uptake by MC38-CEA2 cells. Therefore, we have shown that the re-targeting is highly specific. These data clearly show that our approach can be exploited to re-target an Ad vector, and support our hypothesis that re-targeting of an Ad5-IFN-|[gamma]| vector to CEA-expressing tumor cells can be accomplished. We anticipate that the selective transduction of tumor cells with Ad5-IFN-|[gamma]| in vivo will allow this cytokine to act locally while minimizing any systemic toxicity.