Cytokine enhancement of DNA immunization leads to effective treatment of established pulmonary metastases

Abstract

DNA immunization can result in the induction of Ag-specific cellular and humoral immune responses and in protective immunity in several Ag systems. To evaluate the utility of DNA-based immunization as a potential cancer treatment strategy, we employed an experimental murine tumor, CT26, expressing the model tumor-associated Ag, beta-galactosidase (beta-gal), designated CT26.CL25. A plasmid expressing beta-gal (pCMV/beta-gal) administered by particle-mediated gene delivery to the epidermis using a hand-held, helium-driven "gene gun" induced beta-gal-specific Ab and lytic responses. Immunization with this construct prevented the growth of pulmonary metastatic tumor, and the adoptive transfer of splenocytes generated by pCMV/beta-gal in vivo immunization and cultured in vitro with the beta-gal876-884 immunodominant peptide reduced the number of established pulmonary nodules. DNA immunization alone had little or no impact on the growth of established lung metastases. To enhance the function of DNA immunization for active immunotherapy, a panel of cytokines was added as adjuvants following DNA administration. Significant reduction in the number of established metastases was observed when human rIL-2, mouse rIL-6, human rIL-7, or mouse rIL-12 were given after DNA inoculation; mouse rIL-12 as an adjuvant had the most profound effect. These findings suggest that the cytokines involved in the activation and expansion of lymphocyte populations may improve the therapeutic effects of DNA immunization. Given the ease with which plasmid DNA can be prepared to high purity for safe use in humans with infectious diseases and cancers, DNA immunization administered together with cytokine adjuvant may be an attractive alternative to recombinant viral vaccines.

FIGURE 1

Induction of humoral immunity elicited with gene gun vaccination of pCMV/β-gal. BALB/c mice (three per group) were immunized two times at 2-wk intervals into the epidermis; each immunization consisted of four shots of 0.25 mg of gold delivering a total of either 1.0 μg of control pCMV/NP, or 0.01 μg, 0.1 μg, or 1.0 μg of pCMV/β-gal. Fourteen days following the boost, sera were tested by ELISA for the presence of Abs against recombinant β-gal protein. The relative concentration of Abs reactive against β-gal were calculated from a standard curve of an anti-β-gal mAb and expressed as μg/ml .

FIGURE 2

Secondary in vitro T_CD8+ induced by immunization with gene gun vaccination of pCMV/β-gal. BALB/c mice were immunized one time in the epidermis. Each immunization consisted of four shots of 0.25 mg of gold delivering a total of 1.0 μg pCMV/NP control DNA, 0.01 μg of pCMV/β-gal, 0.1 μg of pCMV/β-gal, or 1.0 μg of pCMV/β-gal. Fourteen days later pooled splenocytes (two mice per group) were restimulated in vitro with 1 μg of β-gal_876–884 peptide for 6 days and then assayed for specific lytic activity in a ⁵¹Cr release assay against CT26.WT(β-gal⁻), CT26.CL25 (β-gal⁺), or CT26.WT pulsed with β-gal_876–884 peptide target cells. These experiments have been repeated two times with similar results.

FIGURE 3

Immunization with DNA vaccine prevents the growth of i.v. tumors. On day 0, BALB/c mice were immunized one time in the epidermis. Each immunization consisted of five shots of 0.25 mg of gold delivering 1.0, 0.10, or 0 .01 μg of pCMV/β-gal or 1.0 μg of control DNA alone. Fourteen days later, mice (5 to 10 per group) were challenged, i.v., with 2.0 × 10⁵ CT26.CL25 (β-gal⁺) or CT26.WT (β-gal⁻) tumor cells. On day 17, lungs were harvested and tumor nodules were enumerated in a blind fashion. Statistical analysis was carried out using the nonparametric Kruskal-Wallis test; therefore error bars are not shown. This graph represents a summary of all the data from three separate experiments. In the first experiment, the control DNA utilized was pCMV/hGH, while in the remaining experiments, pCMV/NP was used.

FIGURE 4

Adoptive immunotherapy of tumor-bearing mice with immune splenocytes induced by gene gun vaccination. On day 0, CT26.WT (β-gal⁻, ○) and CT26.CL25 β-gal⁺, ●) tumor cell lines were each injected i.v. into BALB/c mice to create lung metastases. On day 3, tumor-bearing mice were treated with effector splenocytes from donor mice. The donor cells were generated by prior gene gun immunization with 1 μg of either pCMV/β-gal or pCMV/NP followed 14 days later by in vitro incubation for 6 days with 1 μg/ml of either β-gal_876–884 or NP_147–155 peptide. On day 17, pulmonary metastases were enumerated in a coded, blind fashion. This experiment has been repeated one time with similar findings.

FIGURE 5

Active immunotherapy of established pulmonary metastases with the pCMV/β-gal vaccine plus systemic administration of rhIL-2, rmIL-6, or rhIL-7. BALB/c mice were injected i.v. with 5 × 10⁵ CT26.CL25 (βgal⁺) tumor cells. On day 2 following tumor challenge, treated mice were immunized with 10 μg of pCMV/β-gal. Each mouse received 10 shots of 0.5 mg of gold, each shot delivering 1 μg of DNA. On day 3, mice (5 to 10 mice per group) began regimens of i.p. cytokine injections as described in Materials and Methods. On day 12, lungs were harvested and the pulmonary metastases were enumerated in a coded, blind fashion. Nonparametric statistical analysis was done using the Kruskal-Wallis test. The group with asterisks above were found to be statistically significant compared with either cytokine alone (*, p₂ = 0.001; **, p₂ = 0.003; ***, p₂ = 0.0002) or to β-gal DNA alone (*, p₂ = 0.003; **, p₂ = 0.0003; ***, p₂ = 0.001). The graph represents a summary of two experiments.

FIGURE 6

Active immunotherapy of established pulmonary metastases with the pCMV/β-gal vaccine plus systemic administration of rmIL-12. BALB/c mice were injected i.v. with 5 × 10⁵ CT26.CL25 (βgal⁺) or CT26 (βgal⁻) tumor cells. On day 2 following tumor challenge, treated mice were immunized with 10 μg of pCMV/β-gal or 10 μg of pCMV/NP. Each mouse received 10 shots of 0.5 mg of gold, each shot delivering 1 μg of DNA. On day 3, mice began regimens of i.p. rmIL-12 injections as described in Materials and Methods. On day 12, lungs were harvested and the pulmonary metastases were enumerated in a coded, blind fashion. Nonparametric statistical analysis was done using the Kruskal-Wallis test. The groups with asterisks were found to be statistically significant compared with either cytokine alone (*, p₂= 0.004; **, p₂ = 0.002; ***, p₂ = 0.002) or β-gal DNA (*, p₂ = 0.036; **, p₂ = 0.036; ***, p₂ = 0.012) alone. The adjuvant effects of IL-12 have been observed three times, whereas the effect of β-gal has been observed only one time.