IL-12 is an effective adjuvant to recombinant vaccinia virus-based tumor vaccines: enhancement by simultaneous B7-1 expression

Abstract

A number of cytokines and costimulatory molecules involved in immune activation have recently been identified including IL-12, a heterodimeric cytokine that supports the development of cell-mediated immunity, and B7-1, a costimulatory molecule involved in the activation of T lymphocytes. We explored the use of these immunomodulants as molecularly defined adjuvants in the function of recombinant anticancer vaccines using a murine model adenocarcinoma, CT26, transduced with a model Ag, beta-galactosidase (beta-gal). Although IL-12 given alone to mice bearing tumors established for 3 days did not have consistent antitumor activity, a profound therapeutic effect was observed when IL-12 administration was combined with a recombinant vaccinia virus (rVV) encoding beta-gal called VJS6. On the basis of the reported synergistic effects of IL-12 and the costimulatory molecule B7-1 (CD80) in vitro, we immunized mice with a double recombinant vaccinia encoding both the model tumor Ag and the costimulatory molecule B7-1, designated B7-1 beta-gal rVV. The adjuvant administration of IL-12 after immunization with this virus significantly enhanced survival in tumor-bearing animals. T cell subset depletions demonstrated that the in vivo activity of IL-12 was largely independent of CD4+ T lymphocytes, whereas the in vivo activity of a B7-1 rVV required both CD4+ and CD8+ T cells to elicit maximal therapeutic effect. To our knowledge, this is the first description of B7-1 and IL-12 cooperation in vivo and represents a novel strategy to enhance the efficacy of recombinant anticancer vaccines.

FIGURE 1. Effect of IL-12 adjuvant with rVV vaccination on mice bearing 3- and 6-day-old pulmonary metastases

Nonirradiated BALB/c mice (10 per group) were injected i.v. with 10⁵ tumor cells of CT26.WT (A) or CT26.CL25 (B to D) and then randomized. Three (A and B) or 6 (C and D) days later, mice were immunized with either 10⁶ PFU of rVV encoding β-gal (VJS6) or nothing. Adjuvant HBSS or IL-12 (0.5 mg i.p.) once a day for 5 days was started 12 h after immunization. Mice were then followed for survival. This figure represents data pooled from two distinct, but identically performed experiments in which all experimental groups were included in each experiment. A, IL-12 plus vaccination did not prolong the survival of mice bearing TAA-negative tumors established for 3 days. B, IL-12 plus vaccination prolonged the survival of mice bearing tumors established for 3 days, which expressed the model TAA. Treatment with VJS6 + IL-12 was superior to the next best treatment, VJS6 alone (p < 0.0024). C, IL-12 plus vaccination with a rVV expressing the relevant TAA prolonged the survival of mice bearing tumor established for 6 days which expressed the model TAA. V69NP refers to a rVV that expressed NP and did not express β-gal. Treatment with MHA β-gal rVV + IL-12 was superior to the next best treatment, MHAβ-gal rVV alone (p < 0.048). This experiment rendered similar results when VJS6 was used as the model Ag-expressing virus (not shown). D, IL-12 plus vaccination prolonged survival in mice bearing 6-day-old pulmonary metastases. Treatment with VJS6 + IL-12 was superior to all other groups (p < 0.0018). This experiment also yielded similar results when IL-12 was started on day 4 or 12 h after viral administration (not shown).

FIGURE 2. Adjuvant effect of IL-12 enhances antitumor responses against low levels of Ag

Nonirradiated BALB/c mice (10 per group) were injected i.v. with CT26.CL25 tumor cells and then randomized. Three days later, mice were immunized i.v. with varying doses (10⁴ to 10⁷ PFU) of rVV encoding β-gal (VJS6) or nothing. Adjuvant HBSS or IL-12 (0.5 mg i.p.) once a day for 5 days was started 12 h after immunization. Mice were randomized and killed 12 days after tumor injection. Lungs were harvested and pulmonary metastases were enumerated in a blinded procedure. *, p < 0.05 comparing 10⁵ PFU VJS6 + IL-12 vs 10⁵ PFU VJS6 alone; **, p < 0.05 comparing 10⁶ PFU VJS6 + IL-12 vs 10⁶ PFU VJS6 alone; ***, p < 0.05 comparing 10⁷ PFU VJS6 + IL-12 vs 10⁷ PFU VJS6 alone. This figure represents data pooled from two distinct, but identically performed experiments.

FIGURE 3. IL-12 plus vaccination with a virus expressing B7-1 and tumor Ag prolongs survival of mice bearing 3-day-old pulmonary metastases

Nonirradiated BALB/c mice (10 per group) were injected i.v. with CT26.CL25 tumor cells and then randomized. Three days later, mice were immunized with 10⁵ PFU of MHAβ-gal rVV, B7-1β-gal rVV, B7-1 NP rVV, or nothing. Adjuvant IL-12 (0.5 mg i.p.) once a day for 5 days was started 12 h after immunization. Mice were followed for survival. This figure represents data pooled from two distinct, but identically performed experiments, each of which included all experimental groups. A, Experimental groups receiving no treatment or immunized with rVV without adjuvant IL-12. B, Treatment groups receiving IL-12 alone or as an adjuvant to rVV immunization. Treatment with B7-1 β-gal rVV + IL-12 was superior to treatment with any other group (B7-1β-gal rVV + IL-12 vs B7-1 β-gal rVV alone, p < 0.0018).

FIGURE 4. Primary adoptive transfer of lymphocytes after in vivo priming with rVV

Nonirradiated BALB/c mice (10 per group) were injected i.v. with B7-1β-gal rVV, MHAβ-gal rVV, or HBSS. Twentyone days later a splenectomy was performed on all immunized mice. Splenocytes from designated groups of immunized mice were immediately adoptively transferred to similar mice (10 per group) injected i.v. 3 days earlier with either CT26.CL25 or CT26.WT tumor cells. Mice were randomized and then killed 12 days after tumor injection. Lungs were harvested and stained, and pulmonary metastases were enumerated in blinded procedure. All mice inoculated with a CT26.WT had large tumor burdens (data not shown). The adoptive transfer of lymphocytes from animals immunized with either HBSS or MHAβ-gal rVV did not mediate a statistically significant reduction in tumor burden. The adoptive transfer of lymphocytes from mice immunized with B7-1β-gal rVV (*) resulted in a statistically significant reduction in pulmonary metastases vs both the MHA β-gal rVV group and the HBSS group (p < 0.05 and p < 0.007, respectively). This figure represents data pooled from two distinct, but identically performed experiments. The x-axis designates the rVV and the dose (PFU) used for immunization.

FIGURE 5. Effect of in vivo T cell subset depletions on the therapeutic effectiveness of rVV vectors

Two days before tumor administration, nonirradiated BALB/c mice (five per group) were injected with anti-CD4 and anti-CD8 mAbs (GK1.5 (anti-CD4) and 2.43 (anti-CD8)). Mice were then inoculated i.v. with CT26.CL25 tumor cells. Three days after tumor inoculation, mice were immunized i.v. with B7-lβ-gal rVV, MHAβ-gal rVV, B7-1 NP rVV, or HBSS. Repeat treatment with anti-CD4 and anti-CD8 mAbs was done 3 days after immunization. Mice were randomized and then killed on day 12. Lungs were harvested and pulmonary metastases were enumerated in a blinded procedure. FACS analysis was pet formed 1 day before immunization and again at day 7 to verify depletion. Only data from B7-1β-gal rVV (designated virus containing B7-1) and MHAβ-gal rVV (designated virus not containing B7-1) vaccination groups are shown. In the absence of any depleting Ab, immunization with both MHAβ-gal rVV and B7-lβ-gal rVV was able to mediate a significant reduction in the number of pulmonary metastases (*, p < 0.02 and **, p < 0.005, respectively), although the therapeutic effect elicited after immunization with B7-1β-gal rVV was superior to that with MHAβ-gal rVV immunization (p < 0.03). No significant tumor rejection was found in any of the B7-1 NP rVV or HBSS treatment groups (data not shown). This figure represents data pooled from two distinct, but identically performed experiments.

FIGURE 6. Effect of IL-12 on mice treated with rVV and depleted of CD4⁺ and CD8⁺ T lymphocytes

Nonirradiated BALB/c mice(10 per group) were depleted of CD8⁺ T cells or CD4⁺ T cells 2 days before and 5 days after tumor administration. Mice were injected i.v. with CT26.CL25 tumor cells and then randomized. Three days later mice were immunized with 10⁶ PFU of a rVV encoding β-gal, VJS6, or nothing. Adjuvant HBSS or IL-12 (0.5 mg i.p.) once a day for 5 days was started 12 h after immunization. Mice were randomized and killed 12 days after tumor injection. Lungs were harvested and pulmonary metastases were enumerated in a blinded procedure. In the absence of any depleting Ab, treatment with VJS6 + IL-12 (*) was superior to treatment with VJS6 alone (p < 0.05). In the absence of CD4⁺ T cells, treatment with VJS6 + IL-12 (**) was superior to treatment with VJS6 alone (p < 0.05). This figure represents data pooled from two distinct but identically performed experiments.