Therapeutic antitumor response after immunization with a recombinant adenovirus encoding a model tumor-associated antigen

Abstract

Recombinant adenovirus (rAd), deleted of critical genes that enable viral replication and replaced with genes encoding heterologous proteins, has been shown to be a safe and effective vector in gene therapy studies. To evaluate a potential role for rAd as an immunogen, we used two different replication-defective type 2 rAds encoding the model Ag, beta-galactosidase (beta-gal). To determine whether rAd elicited the kind of immune responses therapeutic in an anti-tumor setting, the beta-gal-expressing adenocarcinoma, CT26.CL25, was used. Splenocytes from BALB/c mice immunized with 1 x 10(7) infectious units (iu) of rAd demonstrated anti-beta-gal activity after in vitro culture with the relevant L(d) beta-gal peptide. Adoptive transfer of these same splenocytes produced dramatic regression of established pulmonary metastases. However, when tumor-bearing mice were treated with 1 x 10(7) iu of rAd, no reduction in established disease was observed even when rAd was given with exogenous IL-2. To increase the viral dose delivered to each animal, we used an E1-E4-deleted rAd that could be grown to much higher titers. Significant reduction occurred with 10-fold more rAd (1 x10(8) iu) was administered. Exogenous IL-2 administration with 1 x 10(8) iu of rAd resulted in augmentation of this anti-tumor effect. These findings demonstrate that when using a nonreplicating virus, the viral dose is directly related to the immune response generated. These data constitute the first reported use of rAd in the treatment of an established experimental cancer and may have implication for the treatment of human cancer.

FIGURE 1

Two types of rAd, rAd-CMV1 and rAd-CMV2, were used in these studies. The back-bone vector is derived from the type 2 adenovirus. Both vectors have a complete E1A and partial E1B deletion spanning 356 to 3,327 bp. The LacZ gene is inserted into the E1 region under the control of the CMV immediate early promoter. rAd-CMV1 has no polyadenylation, while rAd-CMV2 has SV40 polyadenylation. In addition to the deletions in the E1 region, rAd-CMV2 has deletions in the E4 region spanning 32,177 to 32,815 bp and 34,082 to 35,575 bp.

FIGURE 2

rAd can elicit a specific CTL response after a single immunization. BALB/c mice (three per group) were immunized i.v. with 1 × 10⁵ or 1 × 10⁷ iu of rAd-CMV1 and 1 × 10⁵ or 1 × 10⁷ iu of rAd-CMV2. Twenty-one days after immunization, splenocytes were harvested and cultured for 6 days in the presence of 1 μg/ml of β-gal peptide. A ⁵¹Cr release assay was then performed. This experiment was repeated independently, and similar results were obtained.

FIGURE 3

A–C, rAd administered i.v. (A), i.m. (B), and intranasally (C) can elicit specific CTL responses. BALB/c mice were immunized with 10⁷ iu of rAd-CMV1 or rAd-mOVγ i.v. (A) or i.m. (B), or with 10⁶ iu of rAd-CMV1 or rAd-mOVγ intranasally (C). Twenty-one days later, spleens were harvested and cultured in the presence of 1 γg/ml of β-gal peptide. After 6 days in culture, a ⁵¹Cr release assay was performed on a portion of the splenocytes, while another portion was adoptively transferred by the i.v. route to mice bearing 3-day pulmonary metastases (see Fig. 4).

FIGURE 4

After in vitro stimulation with the relevant peptide, splenocytes from mice immunized 21 days previously were adoptively transferred into tumor-bearing mice. Immediately after adoptive transfer, those groups randomized to receive rIL-2 were given 90,000 IU of rIL-2 i.p. twice a day for 3 days. Eight days after the adoptive transfer of effector cells, mice were killed, lungs were excised, and metastases were counted in a blinded fashion. The results of the counts for each mouse are depicted in A, B, and C. The specific in vivo tumor response can be seen with splenocytes from mice immunized i.v. (A), i.m. (B), and intranasally (C). Moreover, in those groups that received only 2 × 10⁶ effector cells that had been primed with rAd-CMV1 (all panels marked with *), the addition of exogenous rIL-2 gave antitumor responses similar to those seen in mice receiving the higher dose of effector cells. Nonparametric statistical analysis was performed using two-tailed Wilcoxon’s test. An independent repetition of these experiments yielded identical results.

FIGURE 5

Active treatment of established pulmonary metastases correlated with viral titer administered and was improved with the addition of exogenous rIL-2. Groups of five or six mice were inoculated i.v. with 1 × 10⁵ cells/mouse of CT26.CL25 or CT26.WT. Three days later, mice were immunized with rAd-CMV2 or rAd-Empty in doses of 1 × 10⁷ and 1 × 10⁸ iu/mouse. Additionally, some mice were randomized to receive 90,000 IU of exogenous rIL-2 twice a day for a total of 3 days. Fourteen days after tumor administration, mice were killed, and pulmonary metastases were counted blindly. The magnitude of the anti-tumor response, as represented by the mean number of pulmonary metastases, correlated in a dose-dependent fashion with the viral titer administered. Exogenous rIL-2 shifted the dose-response curve such that lower viral titers were needed to achieve equivalent results. Nonparametric statistical analysis was performed with a two-tailed Wilcoxon’s test. An independent repetition of this experiment confirmed these results.

FIGURE 6

BALB/c mice were immunized with HBSS, 1 × 10⁷ iu of rAd-CMV1 (β-gal-expressing rAd), or rAd-mOV_γ (control). Fourteen days later, mice were reimmunized with HBSS or rAd-CMV1. On day 28, splenocytes from all mice were restimulated in culture with a 1 μM concentration of the synthetic peptide TPHPARIGL for 7 days and then assayed for specific anti-β-gal lysis in a ⁵¹Cr release assay. The experiment was repeated with similar results.