Intratumoral injection of Ad-ISF35 (Chimeric CD154) breaks tolerance and induces lymphoma tumor regression

Abstract

Ad-ISF35, an adenovirus vector encoding a membrane-bound engineered CD154 chimeric protein (ISF35), induces complete A20 lymphoma tumor regression in mice after intratumoral direct injection (IDI). Ad-ISF35 induced durable local and systemic antitumor responses associated with a rapid tumor infiltration of macrophages and neutrophils as well as increased levels of proinflammatory cytokines in the tumor microenvironment. Ad-ISF35 IDI transduced preferentially fibroblasts and macrophages present in the tumor microenvironment, and ISF35 protein expression was observed in only 0.25% of cells present in the tumor. Moreover, Ad-ISF35 IDI induced upregulation of CD40 in tumor and immune regulatory cells, including those that did not express ISF35, suggesting the presence of a strong bystander effect. These responses resulted in the generation of IFN-γ-secreting cytotoxic lymphocytes and the production of specific cytotoxic antibodies against lymphoma cells. Overall, cellular immune therapy based on ISF35 induced phenotypic changes in the tumor cells and tumor microenvironment that were associated with a break in tumor immune tolerance and a curative antitumor effect in this lymphoma mouse model. Our data highlight the potential activity that modulation of costimulatory signaling has in cancer therapy.

FIG. 1.

Ad-ISF35 intratumoral direct injection (IDI) results in tumor regression by inducing local and systemic immune responses. (A) A20 tumor-bearing mice received three weekly IDI of the vehicle, Ad5 (3×10¹⁰ vp), or Ad-ISF35 (3×10¹⁰ vp) as shown by the arrows. Tumor size was measured over time and statistical analysis was performed. (B) Survival curve of mice receiving the vehicle, Ad5, and Ad-ISF35. (C) Tumor growth of bilateral injection of A20 cells. The tumor present on the right flank was injected with Ad-ISF35 or the vehicle (control). The tumor on the left was not injected. (D) Percentage of tumor-free mice on the right flank (Ad-ISF35 injected) and left flank (noninjected). (E) Survival curve of mice rechallenged with A20 cells 3 months after Ad-ISF35 IDI-induced tumor regression. Age-matched control group previously untreated was also injected with the same batch of A20 cells. Error bars indicate the standard deviation.

FIG. 2.

Ad-ISF35 induces A20 tumor necrosis and early infiltration of neutrophils and macrophages. Immunochemistry analysis of tumors harvested 24 hr after IDI with Ad-ISF35, Ad5, vehicle, and Ad-ISF35 heat inactivated at 72°C for 1 hr. Staining with anti-CD31 (endothelial cells), anti-GR1 (macrophages and neutrophils), anti-CD11b (granulocytes, macrophages, and monocytes), anti-F4/80 (macrophages), and anti-CD3 (T cells) antibodies is shown. Color images available online at www.liebertpub.com/hum

FIG. 3.

Expression of chimeric CD154- ISF35 induces cellular activation in the tumor microenvironment. (A) Flow cytometry analysis of the percentage of myeloid cells and macrophages present in the tumor 48 hr after Ad-ISF35 IDI. (B) Ad-ISF35 DNA copy numbers per 10,000 cells was measured by qPCR in different tumor cell types 4 hr after Ad-ISF35 IDI. Asterisks indicate a p<0.01 compared with the other cell types. Bone marrow cells from mice treated with the vehicle were used as a negative control. (C) Tumors injected with the vehicle, Ad5, or Ad-ISF35 were harvested 12 and 48 hr after IDI. Cells were dissociated and flow cytometry was performed. Analysis of CD40 and CD154 was performed in all the tumor population. A more detailed analysis was performed on macrophages and myeloid cells expressing CD154 (D) and CD40 (E). Error bars indicate the standard deviation. Statistical analysis was performed using t-test with Welch's correction.

FIG. 4.

Ad-ISF35 IDI induces cytokine and chemokine release from the tumor microenvironment and spleen. We evaluated the level of expression of IL-2, IL-6, IL-10, IL12, GMCSF, IFNγ, and MCP-1 (pg/ml), among others, in samples collected from tumor, spleen, and serum from A20-bearing mice that received the vehicle, Ad5, or Ad-ISF35 IDI. The level of expression was quantified using the Luminex platform as described in the Materials and Methods section. Statistical analysis was performed using t-test with Welch's correction. *p<0.05; **p<0.01.

FIG. 5.

Ad-ISF35 IDI-mediated induction of late T-cell infiltration and humoral–cellular immune response. (A) Analysis of tumor-infiltrating lymphocytes and (B) their expression of CD25. (C–F) Serum and splenocytes from mice cured by Ad-ISF35 treatment were tested in vitro. (C) Sera from Ad-ISF35-treated mice, but not control animals, contained antibodies that recognize A20-derived tumor proteins detected by Western blot. A single-well electrophoresis was performed with A20 protein lysate. (D) Complement-dependent cytotoxicity (CDC) of A20 cells was determined using sera from A20-bearing mice treated with the vehicle, Ad5, or Ad-ISF35. As a control, serum was inactivated at 56°C for 1 hr. (E) A20 cell apoptosis induced by splenocytes from vehicle-, Ad5-, or Ad-ISF35-treated mice at different effector (E)-to-target (T) ratios was determined by flow cytometry. (F) Splenocytes from vehicle-, Ad5-, and Ad-ISF35-treated mice were cultured with A20 cells, and IFN-γ production was determined by ELISPOT. Statistical analysis was performed using t-test with Welch's correction.