IL-2 enhances the function of recombinant poxvirus-based vaccines in the treatment of established pulmonary metastases

Abstract

Neoplastic cells are generally poor immunogens. Transfection of the murine tumor CT-26 with beta-galactosidase (beta-gal), a protein from Escherichia coli, did not alter its growth rate in vivo, or its lethality, and did not elicit a measurable anti-beta-gal immune response. Immunization with beta-gal-expressing recombinant vaccinia viruses (rVV) elicited specific anti-beta-gal cytolytic T lymphocytes, but rVV-beta-gal was only marginally therapeutic when given to tumor-bearing mice. With the aim of expanding the immune response against beta-gal, used here as a model tumor Ag, we gave mice exogenous IL-2 starting 12 h after the poxvirus. The therapeutic effectiveness of the combination of poxvirus and IL-2 was far greater than either of these treatments alone. When the cDNA for IL-2 was inserted into the viral genome of the rVV construct to make a double recombinant (drVV), antitumor activity was further augmented. One mechanism of action may be the enhanced activation or expansion of cytotoxic T cells, because a marked increase in primary cytotoxic responses against vaccinia determinants was observed. Interestingly, other cytokines (mGM-CSF, mTNF-alpha, and mIFN-gamma) inserted into the rVV genome did not modify the efficacy of the rVV constructs. The increase in specific CTL responses against beta-gal by drVV expressing the tumor-associated Ags (TAA) and IL-2 was more pronounced in mice bearing the lacZ-transduced tumor than in those bearing the parental cell line, suggesting that the TAA presented by growing tumor cells can either pre-activate or otherwise amplify the immune response induced by the rVV. Unfortunately, in several long-term surviving mice, tumor recurred that no longer expressed beta-gal. These results indicate that treatment of disseminated tumors by using recombinant viruses expressing TAA can be enhanced by IL-2 provided exogenously, or encoded within the recombinant virus.

FIGURE 1

Active immunotherapy is enhanced when exogenous rIL-2 and rVV are given in concert. BALB/c mice (five per group) were challenged i.v. with 5 × 10⁵ CT26.WT or CT26.CL25 tumor cells. After 3 days they received a single i.v. injection of medium alone (HBSS) or medium containing 5 × 10⁶ PFU of different TK⁻ rVV either expressing (VJS6) or not expressing (V69) β-gal. Two different regimens of rIL-2 administration were started 12 h after rVV injection: high dose (HD, 100,000 Cetus U, twice a day, i.p. for 3 days) or low dose (LD, 15,000 Cetus U, twice a day, i.p. for 5 days). Mice were checked twice a day for survival.

FIGURE 2

Exogenous rIL-2 enhances the function of rFPV. BALB/c mice (five per group) were inoculated i.v. with 5 × 10⁵ CT26.WT or CT26.CL25 tumor cells. On day 3 after tumor injection, they received a single i.v. injection of the following viruses: no virus (HBSS alone), 10⁷ PFU of FPV.bg40k (rFPV), or FPV wild-type (FPVwt). rIL-2 (100,000 Cetus U, twice a day) was administered i.p. starting 12 h after FPV injection and continued for 3 days. Mice were checked twice a day for survival.

FIGURE 3

Exogenous rIL-2 plus rVV is therapeutic in the more advanced 6-day tumor model. BALB/c mice (five per group) were inoculated i.v. with 10⁵ CT26.CL25 tumor cells. Six days after tumor injection, they received the same treatments described in Figure 1 with the exception that only the highest dose of rIL-2 was administered. Mice were checked daily for survival. No prolongation of survival was obtained by the various treatments in mice bearing 6-day-old pulmonary metastases of CT26.WT tumor (data not shown).

FIGURE 4

A drVV expressing IL-2,. but not GM-CSF, IFN-γ, or TNF-α, significantly reduces the number of pulmonary metastases in a 3-day model. Five BALB/c mice per group were injected i.v. with 5 × 10⁵ tumor cells of either CT26.WT or CT26.CL25 cell lines. Three days later they received a single i.v. injection of HBSS alone (none) or containing 5 × 10⁶ PFU/mouse of different rVV, as indicated. On day 12 post tumor challenge, lungs were harvested and pulmonary nodules were enumerated in a blind fashion. An independent repeat of this experiment gave identical results.

FIGURE 5

The function of a drVV expressing IL-2 is further enhanced when additional exogenous IL-2 is provided. BALB/c mice (five per group) were challenged i.v. with 5 × 10⁵ CT26.WT or CT26.CL25 tumor cells. After 3 days they received a single i.v. injection of plain medium (HBSS) or medium containing 5 × 10⁶ PFU of rVV encoding β-gal alone (VJS6) or together with IL-2 (IL-2 rVV). Twelve hours after rVV, 100,000 rIL-2 U were inoculated, according to the regimen described in Figure 1. Mice were checked twice a day for survival.

FIGURE 6

Expression of IL-2 by drVV enhances the antivaccinia CTL response. Two BALB/c mice were immunized with 5 × 10⁶ PFU/mouse of different rVV. After 6 days the spleens were aseptically removed, mixed together, and tested directly in a 6-h ⁵¹Cr release assay against CT26.WT tumor cell line, either infected (CT26 vaccinia) or noninfected (CT26.WT) during the isotope labeling with more than 10:1 moi of crude 19 VV preparation. Spontaneous release of target cells never exceeded 20%. E:T cell ratio was 100:1 and then 1:3 dilutions. Lytic units 30% (L.U. 30%) indicate the number of effector cells required to obtain 30% lysis of 10,000 target cells. L.U. 30% were normalized for the total number of cells recovered for each spleen and expressed as total L.U./spleen.

FIGURE 7

The presence of tumor cells specifically enhance the CTL response elicited by IL-2 rVV in a dose-dependent manner. BALB/c mice were injected with HBSS alone or with varying doses of CT26.WT or CT26.C25 as specified. After 3 days, mice were immunized with 5 × 10⁶ PFU/mouse of either VJS6 or IL-2 rVV. O n day 9 after tumor challenge (day 6 after vaccination) the primary cytotoxic response was evaluated in a 6-h ⁵¹Cr release assay against CT26.WT, CT26.WT pulsed with the β-gal L^d-restricted peptide (CT26T + peptide), CT26.CL25, and the irrelevant target cells E22 as shown. The effects of escalating doses of tumor on the generation of the primary cytotoxic response is shown for the immunization with IL-2 rVV in nontumor-bearing mice (▲), or in mice bearing 5 × 10⁴ CT26.CL25 (△), 1 × 10⁵ CT26.CL25 (•), 5 × 10⁵ CT26.CL25 (○), or the highest dose, 5 × 10⁵ of the parental (non-β-gal-expressing) CT26.WT cell line (□). An additional control is the non-IL-2-expressing VJS6 virus injected in mice bearing 5 × 10⁵ CT26.CL25 (▪).