Effective transfer of interleukin-12 gene to solid tumors using a novel gene delivery system, poly [D,L-2,4-diaminobutyric acid]

Abstract

Delivery of the interleukin-12 (IL-12) gene to solid tumors is a promising anticancer therapy. Vectors are currently being developed to achieve safe and effective intratumoral delivery of the IL-12 gene. Poly [D,L-2,4-diaminobutyric acid] (PDBA) is a novel gene carrier that was recently described. The goal of this study was to use this gene delivery system for treatment of solid tumors. To determine the optimal conditions for transfection, established B16F10-melanomas in C57BL/6 mice were treated with intratumoral injection of the PDBA/plasmid luciferase (pLuc) complex. We determined that the optimal complex composition was 50 μg/ml pLuc and 150 μg/ml PDBA. High levels of IL-12 protein were expressed in tumors after a single injection of PDBA/murine IL-12 (pmIL-12) complex, whereas serum levels of IL-12 in treated mice were below the limits of detection. IL-12 gene therapy with the PDBA system significantly inhibited tumor growth in comparison with the controls (P<.001). Moreover, both natural killer and cytotoxic T lymphocyte activities from draining lymph nodes of PDBA/pmIL-12-treated mice were increased substantially in comparison with those of controls (P<.05). These results suggest that PDBA-mediated IL-12 gene therapy is a potential strategy for treatment of patients with solid tumors.

Main

Cytokine-mediated immunotherapy is considered a promising strategy for treatment of cancer. Although a number of different cytokines have been tested, IL-12 has proven to be effective in the induction of potent antitumor immunity. IL-12 was initially identified and isolated as a natural killer (NK) cell stimulatory factor.1 In addition to the stimulatory effect on NK cells, bioactive IL-12 can activate cytotoxic T lymphocytes (CTLs).2,3,4 Induction of cytokines, such as interferon-γ (IFN-γ), IFN-inducible protein-10 (IP-10), and monokine induced by IFN-γ (MIG), has also been implicated as a mechanism of antitumor activity of IL-12.5,6 These diverse effects make IL-12 an attractive candidate as a therapeutic agent for cancer treatment. Systemic administration of IL-12 significantly suppressed the growth of established mouse tumors and prolonged the survival of tumor-bearing mice.7,8 However, systemic administration of recombinant IL-12 revealed potential side effects in mice9 and humans.10 Thus, delivery of the IL-12 gene to specific sites may be a useful strategy, and local expression of the protein may be less toxic than systemic delivery of recombinant protein.

Current methods for transfer of genes for experimental purposes rely largely on viral vectors because of their high transfection efficiencies. Intratumoral delivery of adeno-11,12 or retroviruses13 containing the IL-12 gene caused regression of some established tumors in mice. However, such viral vectors can have some harmful antigenicity, and serious concerns have been raised regarding the use of viral vectors, especially in clinical trials. Nonviral carriers, such as cationic liposomes,14,15 avoid such problems. Furthermore, nonviral vectors may be good clinical options because they may be more efficient and less labor intensive than viral vectors, although the present gene transfection efficiencies still need to be improved. Recently, nonviral gene delivery systems with polyvinylpyrrolidone (PVP),16 poly[α-(4-aminobutyl)-L-glycolic acid] (PAGA),17,18 and water-soluble lipopolymers (WSLP)19 have been developed and reported. However, the best method for intratumoral delivery of the IL-12 gene remains to be determined.

Poly [D,L-2,4-diaminobutyric acid] (PDBA) was recently synthesized and described as a novel gene delivery carrier. It differs from polylysine in the length of the side chain, resulting in high-efficiency transfection of a reporter gene (Nakanishi et al. data not yet published). Therefore, the objectives of this study were to evaluate the effectiveness of PDBA-mediated IL-12 gene delivery for treatment of solid tumors.

Materials and methods

Tumor cell lines and animals

B16F10 mouse melanoma and YAC-1 mouse lymphoma cell lines (both from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine (all from Life Technologies, Grand Island, NY). Female C57BL/6Nsea mice (7 weeks old) were purchased from Seac Yoshitomi, Ltd. (Fukuoka, Japan) and housed in the Laboratory Animal Research Center of Oita Medical University (Oita, Japan). Mice were maintained on ad libitum rodent feed and water at 23oC, 50% humidity. All mice were acclimated in the research center for at least 1 week before tumor implantation. All studies were performed in accordance with an animal protocol approved by the Oita Medical University Institutional Animal Care and Use Committee.

Plasmids and preparation of PDBA/pDNA complex

Mouse IL-12 expression plasmid, designated pCAGGS-mIL-12, was a kind gift from Dr Jun-ichi Miyazaki of Osaka University Medical School (Japan). Plasmid encoding the Luc gene was provided by Hisamitsu Pharmaceutical Co. (Ibaraki, Japan). Plasmid DNAs (pDNAs) were amplified in the E. coli strain DH5α and isolated and purified with the QIAGEN Plasmid Maxi kit (Hilden, Germany). The concentration of pDNA was measured by UV absorption at 260 nm. PDBA (PDBA8000, provided by Hisamitsu Pharmaceutical Co.) and pDNAs were diluted in phosphate-buffered saline (PBS) to various concentrations, and the PDBA/pDNA complexes were prepared by gentle vortexing. PDBA/pDNA complexes were used for gene transfection within 60 minutes of preparation.

Tumor implantation and treatment

To generate tumors, mice were challenged intradermally (i.d.) in the right flank with 0.1 ml of single-cell suspension containing 2.0 × 105 B16F10 cells. Gene transfer to the tumors was started approximately 7 days later when tumors reached a volume of about 100–125 mm3. To evaluate the antitumor effect of PDBA-mediated mIL-12 gene transfer, mice were randomized into four groups: controls (received 250 μl PBS), pDNA group (received 250 μl pmIL-12 solution), PDBA/pLuc group (received 250 μl PDBA/pLuc complex), and PDBA/pmIL-12 group (received 250 μl PDBA/pmIL-12 complex). Injections were performed twice on days 7 and 8. Each group consisted of six animals. Tumor size was measured every 4 days for a period of 19 days after tumor inoculation, and tumor volume was calculated according to the following formula: V=A × B2/2 (mm3), where A is the largest diameter (mm), and B is the smallest diameter (mm) of the tumor.

Luciferase activity

Luciferase activity was measured as described previously.20 Briefly, the PDBA/pLuc complex was transferred to tumors on day 7 after inoculation when tumors reached a volume of 100–125 mm3. Mice were killed on day 9 after inoculation, and tumors were isolated for luciferase assay. Tumors were washed in PBS, placed in 400-μl T-PER Tissue Protein Extraction Reagent (Pierce Biotechnology, Rockford, IL), and then dissociated until it was completely dissolved. Samples were centrifuged for 3 minutes at 3000 × g, and supernatants were then collected. An aliquot of 10 μl of each sample was added to 40 μl of substrate (Promega, Madison, WI) and assayed for luciferase activity in a luminometer (LUMAT LB 9507; EG&G Berthold, Tokyo, Japan). The total protein content for each sample was determined by the DC Protein Assay Kit (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. The results were normalized to yield luciferase activity in luciferase units (LU)/μg protein.

Detection of mIL12p70 and IFN-γ by ELISA

Blood and treated tumor sites were collected from mice at various times after a single injection of the PDBA/pmIL-12 complex or pmIL-12. Samples of the tumor sites were handled as described above. Concentrations of bioactive murine IL-12p70 and IFN-γ were measured with ELISA kits (both purchased from Biosource, Camarillo, CA) according to the manufacturer's instructions.

Cytotoxicity assay

Lymphoid cells were obtained from the right groin lymph nodes or splenocytes harvested from mice that had received intratumoral injection of PDBA/pmIL-12 complex or pmIL-12 alone. To obtain CTLs from these isolated cells, 2 × 106 cells were stimulated with 2 × 105 Mitomycin C-treated B16F10 in the presence of rmIL-2 (Sigma, St Louis, MO; 25 IU/ml) for 5 days. Lymphoid cells were then harvested, washed three times in serum-free media, and applied as effectors at various effector:target (E:T) ratios. B16F10 cells or YAC-1 cells (natural killer-sensitive) were used as targets. To measure the levels of lactate dehydrogenase (LDH) released from target cells after 4 hours incubation in 96-well round-bottomed plates, 100 μl of culture media from each assay was transferred to flat-bottomed enzymic assay plates. Cytotoxic activity was examined with an LDH Cytotoxicity Detection Kit (Takara, Tokyo, Japan) according to the manufacturer's instructions. Briefly, 100 μl of LDH substrate was added to the enzymatic assay plates and incubated for 30 min at room temperature. The optical absorbances of red formazan at 490 and 620 nm were recorded. The percent-specific toxicity was then calculated with the following formula: % cytotoxicity=100 × [(experimental−effector spontaneous−target spontaneous) OD/(target maximum – target spontaneous) OD].

Statistical analysis

Statistical significance of differences between groups was determined by the nonparametric Mann–Whitney U test. Differences in tumor growth and cytotoxic activity were analyzed with the repeated measures analysis of variance (ANOVA) test. P<.05 was considered to be statistically significant.

Results

Optimal conditions of in vivo PDBA-mediated gene delivery to mouse melanoma

When pDNA was complexed with PDBA at a molecular weight of 1:3, it was condensed completely (data not shown). We also found that complexes generated from higher concentrations (≥200 μg/ml pDNA; ≥600μg/ml PDBA) became turbid with white scum and showed lower gene transfection efficiencies. Therefore, we used complexes generated with 50 μg/ml pDNA and 150 μg/ml PDBA as our standard condition. When B16F10 tumors were transfected with such complexes, a mean luciferase activity of 5.0±4.0 × 105 LU/mg protein was observed. This level was higher than that observed when 25 μg/ml pDNA/75 μg/ml PDBA complexes (1.6±0.6 × 105 LU/mg protein) or 75 μg/ml pDNA/225 μg/ml PDBA (3.4±2.0 × 105 LU/mg protein) were used. Thus, the optimal condition for PDBA-mediated gene transfer was 50 μg/ml pDNA and 150 μg/ml PDBA (Fig 1) and were used for the remaining experiments.

Figure 1
figure1

Optimal condition for PDBA-mediated intratumoral gene delivery. Luciferase gene was transferred to the established B16F10 tumors in the condition at a molecular weight of 1:3 and concentrations of 25–75 μg/ml pDNA and 75–150 μg/ml PDBA. Luciferase activity was the highest when 50 μg/150 μg of pLuc/PDBA complex was given. Data are the mean±SD of luciferase activity. *P<.01 versus control.

Expression of IL-12 and induction of IFN-γ

To confirm that injected PDBA/pmIL-12 complexes led to the expression of IL-12p70 protein in vivo, we examined serum and intratumoral levels of IL-12p70 by ELISA. IL-12 levels in sera and tumor lysates in two mice per group at 24 hours after single injection of PDBA/pmIL-12 complex or pmIL-12 are shown in Table 1. Because many of the biological effects of IL-12 are known to be mediated via induction of IFN-γ, we measured IFN-γ concentrations in the same samples. IL-12p70 protein was detected only in PDBA/pmIL-12-injected tumors (130.4±42 pg/mg protein). The mean level of IFN-γ in PDBA/pmIL-12-injected tumors was 72 times higher than that of pmIL-12-injected tumors. The kinetics of IFN-γ expression are depicted in Figure 2. The levels of IFN-γ peaked at 24 hours after injection, and thereafter, IFN-γ levels decreased gradually to undetectable levels.

Table 1 IL-12 and IFN-γ concentration 24 hours after transfection
Figure 2
figure2

Detection of IFN-γ protein in sera and tumors. On day 0, mice received 2 × 105 B16F10 cells i.d. in the right flank. On day 7, mice were injected intratumorally with PDBA/pmIL-12 complex, and IFN-γ protein levels were measured by ELISA on days 1, 4, 7, and 9 after injection of complexes. Data are presented as mean±SD.

Significant anticancer effect of PDBA-mediated IL-12 gene delivery

We then examined whether PDBA-mediated IL-12 gene delivery inhibits tumor growth in this mouse melanoma model. C57BL/6 mice were challenged i.d. with 2 × 105 B16F10 cells. On day 7, tumors had reached approximately 100–125 mm3 in size. Mice were given intratumoral injections of the PDBA/pmIL-12 complex, PDBA/pLuc complex, pmIL-12 alone, or PBS on days 7 and 8. The growth of B16F10 tumors in the PDBA/pmIL-12-treated group was significantly lower than that in the mice treated with other protocols (P<.001). There were no significant differences between the remaining three groups. These results indicate that naked pmIL-12 alone is not transfected efficiently into B16F10 tumors and that PDBA-mediated gene delivery is non-toxic to B16F10 tumors (Fig 3).

Figure 3
figure3

Antitumor effect of intratumoral injection of PDBA/pmIL-12 complex. On day 0, mice received 2 × 105 B16F10 cells i.d. in the right flank. On days 7 and 8, mice received intratumoral injections of the PDBA/pmIL-12 complex, PDBA/pLuc complex, pmIL-12 alone, or PBS. Each group consisted of six animals. Data are the mean±SE of tumor volume. *P<.001.

Augmentation of NK and CTL activities after PDBA-mediated IL-12 gene therapy

To examine the mechanisms that underly the antitumor effect of IL-12 gene therapy, effector cells from groin lymph node cells or splenic cells of treated animals were collected and assessed. Cytolytic activities of effector cells against both YAC-1 and B16F10 cells were observed. Specifically, the cytolytic activity of cells from the right groin lymph nodes against both YAC-1 and B16F10 cells was greater than that of control (Fig 4, P<.05), indicating that IL-12 induced activation of both CTL and NK activities.

Figure 4
figure4

Augmentation of the cytolytic activities of draining lymph node cells after PDBA-mediated IL-12 gene delivery. (a) Cytolytic activities against YAC-1 cells at various effector:target (E:T) ratios from mice treated with PDBA/pmIL-12 complex (filled circles) or pmIL-12 alone (open circles). (b) Cytolytic activities against B16F10 cells at various E:T ratios from mice injected with the PDBA/pmIL-12 complex (filled circles) or pmIL-12 alone (open circles). Data are presented as mean±SD. *P<.05. Similar results were obtained in three separate experiments.

Discussion

IL-12 is an effective agent for immune modulation and for the treatment of cancer. Here, we report that a novel strategy for delivery of the IL-12 gene, PDBA-mediated gene delivery, inhibits the growth of established tumors via local expression of active IL-12 protein.

It was recently reported that the transfer and expression of the IL-12 gene to established tumors is an effective nonviral treatment for cancer. PDBA is a recently developed gene delivery carrier. PDBA differs from polylysine in the length of the side chain, resulting in a high transfection efficiency. In the present study, we found that the optimal condition for PDBA-mediated gene transfer was 50 μg/ml pDNA and 150 μg/ml PDBA. When tumors were treated with a single injection of optimized PDBA/pmIL-12 complex, active IL-12 protein was detected only in injected tumors and not in blood. As described previously, systemic elevation of IL-12 appears to be more efficacious than is local elevation in the cure of established tumors.21 However, high levels of IL-12 in sera were toxic in a clinical trial.10 The present findings support those of previous studies, suggesting that successful tumor regression is dependent on local rather than systemic expression of IL-12.22,23 Therefore, IL-12 gene therapy with the PDBA-mediated gene delivery system may be an ideal approach to avoid systemic toxicity.

Recently, several nonviral gene delivery methods have developed. However, the best method for intratumoral delivery of the IL-12 gene remains to be determined. In general, it is said that the particle size of the complex affects gene transfection efficiency. Nomura et al24 demonstrated that the large size of the cationic liposome complex caused poor dissemination in tumor tissue, leading to poor gene expression. The particle size of PDBA/pDNA complex was about 200 nm. This is smaller than that of cationic liposome complexes (1135±235 nm),24 but larger than that of PAGA (100 nm)18 or WSLP (26–62 nm).19 In our preliminary experiments, the size can be decreased to 100 nm, when the complex was prepared in MQ water (not in PBS). However, the luciferase activity of the complex in MQ water was extremely (1/180) lower than that of the complex prepar in PBS (data not shown). Therefore, we assume that the determining factors of gene transfer efficiency are not first the particle size. Further studies, such as direct comparison of the in vivo transfection efficiency between PDBA-mediated IL-12 gene transfer and other alternatives, are necessary to determine the best method for intratumoral delivery of the IL-12 gene.

In the present study, PDBA-mediated IL-12 gene therapy significantly inhibited the growth of established tumors in a mouse model of melanoma. The antitumor mechanism of PDBA-mediated IL-12 gene delivery is consistent with the mode of IL-12 action proposed by other groups. Brunda et al8 reported that the potent antitumor activity of IL-12 is due to induction of IFN-γ and activation of effector cells such as CTLs and NK cells. Therefore, we evaluated the levels of IFN-γ protein and augmentation of NK and CTL activities as parameters that reflect systemic IL-12 activity. Elevation of IL-12 levels following direct injection of PDBA/pmIL-12 complex increased the expression of IFN-γ in tumors (Table 1). Cytotoxicity of effector cells from treated animals against both B16F10 and YAC-1 cells was increased, indicating that the IL-12 induced activation of CTL and NK activities.

In summary, the PDBA-mediated gene delivery system could significantly enhance the efficiency of in vivo transfer of pDNAs into cancer cells. Furthermore, transfer and expression of the IL-12 gene into established tumors with this system significantly inhibited tumor growth. Thus, PDBA-mediated IL-12 gene therapy may be a useful option for treatment of solid tumors.

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Acknowledgements

We thank Dr Jun-ichi Miyazaki (Department of Nutrition and Physiological Chemistry, Osaka University Medical School, Osaka, Japan) for the mIL-12 plasmid, Dr Yo-ichi Yamashita (Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan) for helpful advice regarding statistical analyses, and Michiyo Hisaka and Fusayo Kawamura for excellent technical support.

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Correspondence to Shigeru Goto.

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Iwashita, Y., Ogawa, T., Goto, S. et al. Effective transfer of interleukin-12 gene to solid tumors using a novel gene delivery system, poly [D,L-2,4-diaminobutyric acid]. Cancer Gene Ther 11, 103–108 (2004). https://doi.org/10.1038/sj.cgt.7700669

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Keywords

  • interleukin-12
  • poly[D,L-2,4-diaminobutyric acid]
  • intratumoral delivery
  • B16F10

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