Ad-ISF35 Intratumoral Administration Induces a Bystander Effect and Immune-Mediated Tumor Rejection with a Safe Vector Biodistribution and Toxicology Profile In a NHL Mouse Model.

Abstract 1470

Ad-ISF35 is a replication-defective adenovirus (Ad) vector encoding a membrane-stable chimeric CD154 (ISF35) that can induce expression of costimulatory and death receptor molecules in chronic lymphocytic leukemia (CLL) cells in vitro and in vivo.

Ad-ISF35 has shown to be safe and to have potential activity in two clinical studies in CLL patients (Wierda et al, Leukemia, 2010 in press; Castro et al. Blood 2008 112: Abstract 2100). However, the activity, immunogenicity, safety and biodistribution of Ad-ISF35 intratumoral administration have not been evaluated in a solid tumor model.

To study these questions, we use BALB/c mice bearing subcutaneous A20 Non-Hodgkin Lymphoma (NHL) tumors as an in vivo cancer vaccine model. A20 tumor bearing mice were injected intratumoraly with single or repeat dose administration of Ad-ISF35 [3×10⁹ or 3×10¹⁰ viral particles (vp)], Ad-Empty or control vehicle on days 0, 7 and 14. Treated animals were sacrificed on days 2, 5, 19 and 39. Clinical observations, comprehensive necropsy, hematology, serum chemistry, histopathology, cytokine and protein expression as well as Ad-ISF35 vector biodistribution and ISF35 transgene expression were analyzed.

Ad-ISF35 injection in A20 tumor-bearing mice was well tolerated with mild toxicities that included mild and transient elevation in RBC, hemoglobin and hematocrit treated males. In both sexes we observed a transient increase in neutrophils on day 2 after Ad-ISF35 injection, and there were no abnormalities in serum chemistry suggestive of major organ dysfunction. Histopathology assessment of ten different organs did not show differences between the controls and Ad-ISF35 injected mice.

Ad-ISF35 biodistribution showed that the viral vector was primarily found in the injected tumors and underwent a rapid clearance with no evidence of accumulation or persistence in the injected tumor or peripheral organs. Half of the Ad-ISF35 positive samples showed evidence of ISF35 transgene expression measured by real time q-PCR.

Immunohistochemistry analysis of A20 tumors 48 hours after Ad-ISF35 intratumoral injection showed a massive infiltration of monocytes, neutrophils and T-cells as well as evidence of tumor cell apoptosis that correlated with tumor shrinkage followed by complete regression in 80% of Ad-ISF35 injected mice but not in the control animals. Following Ad-ISF35 injection, we observed CD154 upregulation in 0.25% of the tumor cells while there was a 100-fold increase in the number of cells expressing CD40 suggesting a potent bystander effect in non-transduced tumor cells. Analysis of the cytokine expression profile in tumor cells from mice treated with Ad-ISF35 intratumoral injection showed increased expression of IL-6, IFN-γ and MCP-1 as early as 48 hours after injection; this was not observed in the control mice. Mice that were cured from A20 tumors after intratumoral Ad-ISF35 injection developed antibodies and cytotoxic T cells that reacted specifically with A20 cells and prevented the development of A20 tumors after rechallenge.

In summary, we show here that Ad-ISF35 intratumoral injection in a solid tumor NHL mouse model can function as an in vivo cancer vaccine breaking tumor tolerance and inducing complete tumor regression and immunity to tumor rechallenge. Ad-ISF35 intratumoral administration was well tolerated and showed a favorable biodistribution viral vector profile with rapid clearance and no evidence of vector persistence. Ad-ISF35 intratumoral injection induced expression of immunomodulatory and chemotactic cytokines as well as humoral and cellular anti-tumoral responses. Moreover, the effect of intratumoral Ad-ISF35 administration appears to be associated with a potent bystander effect in non-transduced cells. These data provide the rationale for development of Ad-ISF35 intratumoral administration as an in vivo cancer vaccine strategy in patients with NHL and other solid tumors.