Murine carcinoma induced by MK 16 cells expressing HPV 16 E6/E7 oncogenes was utilized to examine the therapeutic effect of dendritic cell-based tumour vaccines. Mice carrying 5-day MK 16 tumours were injected peritumorally with either dendritic cells (DC) or DC pulsed with MK 16 tumour lysate. Both the unpulsed and MK 16 lysate-pulsed DC vaccines inhibited growth of the MK 16 transplants, the pulsed DC being more efficient than the unpulsed vaccines. In vitro priming of the effector cell-mediated anti-MK 16 responses by DC pulsed with MK 16 tumour lysate and a synthetic HPV 16 E7(49-57) peptide RAHYNIVTF was compared. The priming activity of the lysate was substantially higher than that of the HPV 16 E7(49-57) peptide; the priming activity was similar to that of a standard moderately immunogenic chemically-induced sarcoma. Taken collectively, these results suggest that DC vaccines pulsed with HPV 16-associated tumour lysates represent a prospective modality for treatment of HPV 16-associated carcinomas.

Amino Acid Sequence↗

PubMed2001

Genetically engineered dendritic cell-based cancer vaccines (review).

Dendritic cells (DCs) are the most potent professional antigen-presenting cells with exquisite capacity to interact with T cells and initiate their responses; the antigen-presenting capabilities of DCs make them attractive vehicles for the delivery of therapeutic cancer vaccines. The working hypothesis for utilization of DC-based cancer vaccines is that lack of efficient tumour antigen presentation on mature DCs, which is frequently observed in tumour-bearing individuals, can be bypassed by direct loading of DCs with oncoproteins in vitro, thus ensuring the transfer of immunostimulatory peptides on the respective antigen-presenting molecules. To enhance loading of DCs with oncoproteins in vitro and to increase the efficacy of the vaccines, a variety of genetic manipulations have been proposed and shown to be efficient in experimental tumour models. DCs were transfected either with polynucleotides, DNA or RNA, coding for tumour-associated antigens (TAAs), or with DNA encoding immunostimulatory cytokines and co-stimulatory molecules. The delivery of genes coding for antigenic epitopes or other molecules with a recombinant retrovirus, adenovirus, or poxvirus into dendritic cells has also been used for transduction and therapy. As an alternative method for TAA delivery into DCs, fusion of DCs with tumour cells has been utilized and the hybrid cell-based vaccines have been found to be highly therapeutically active, even in cancer patients. The purpose of this review is to summarize the approaches used for making and utilization of the genetically engineered DC-based cancer vaccines, to evaluate the therapeutic results obtained with the vaccines, and to discuss prospects and limitations of the vaccination.

Animals↗

PubMed2000

Local cytokine therapy of cancer: interleukin-2, interferons and related cytokines.

Local therapy with interleukin-2 (IL-2) and other cytokines may be a very effective way to treat cancer. This was the theme of the First Symposium on Local Cytokine Therapy of Cancer: Interleukin-2, Interferons and Related Cytokines, in Hamburg, 29 April-1 May 1999. The abstracts are published in Anticancer Research 19: 1995-2016 (1999). Here we present a report.

Animals↗

PubMed2000

Irradiation of genetically modified plasmacytoma vaccines results in upregulation of CD80 molecule expression, IL-2 production and higher therapeutic efficacy of the vaccines.

It has been found previously that irradiated, IL-2 gene-modified plasmacytoma (X63-m-IL-2) vaccines are more efficient in the therapy of the parental (X63-Ag8.653) plasmacytoma than live plasmacytoma vaccines. In this communication, we have demonstrated that irradiation of murine IL-2-producing plasmacytoma vaccines resulted in upregulation of CD80 molecule expression and IL-2 production. The expression of MHC class I antigens was not altered. The upregulation of the CD80 membrane molecule expression in X63-m-IL-2 cells was higher after irradiation with 150 Gy than after irradiation with 50 Gy. Comparable upregulation of the CD80 molecule expression has also been demonstrated after irradiation of the parental murine X63-Ag8.653 plasmacytoma cells. The results indicate that upregulation of the CD80 molecule expression and enhanced IL-2 production in irradiated X63-m-IL-2 cells was responsible for the higher therapeutic effectiveness of the irradiated plasmacytoma vaccine.

Animals↗

PubMed2000

Peritumoral administration of antigen-unstimulated bone marrow-derived dendritic cells inhibits tumour growth.

Murine BM cells from B6 mice were grown in vitro in medium supplemented with GM-CSF and IL-4 to differentiate DC from DC precursors. After 10 days of culture, approximately 20% of the cell population exhibited the characteristic morphology of BMDC. In cytofluorometric analysis the morphological changes of cells were accompanied by upregulation of the expression of the MHC class II, CD11c, CD80, and CD86 molecules. The BMDC were pulsed with a lysate of syngeneic MK16 carcinoma cells and used for in vitro activation of SC. Co-cultivation of the carcinoma lysate-pulsed BMDC with SC induced a proliferative response of the syngeneic SC. Priming of the proliferative responses was more efficient when the BMDC were grown in the presence of GM-CSF and IL-4 for 10 days than for 7 days. The in vivo effect of mature, tumour lysate-unstimulated BMDC was examined in mice carrying syngeneic MK16 carcinoma transplants. It has been found that local pretreatment with BMDC inhibits growth of a subsequent challenge inoculum of the MK16 cells. Similarly, treatment of mice carrying small MK16 tumours and of those with MK16 surgical minimal residual disease performed with BMDC significantly inhibited tumour growth. It can be concluded from these results that local concentration of mature BMDC at the tumour site can control the development and growth of the transplanted tumour inoculum.

Animals↗

PubMed2000

Cross-contamination of cell lines in culture.

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Cell Culture Techniques↗

PubMed2000

Intratumoral IL-12 gene transfer improves the therapeutic efficacy of IL-12 but not IL-19.

We have compared the therapeutic activity of IL-12 and IL-18 in mice carrying IL-2 gene-transduced syngeneic sarcoma Mc12. The IL-2 gene-transduced sarcoma has previously been utilized as an irradiated, genetically modified tumour vaccine. Murine recombinant IL-12 was capable of suppressing growth of the IL-2 gene-modified sarcoma Mc12 in syngeneic mice more efficiently than growth of the parental Mc12 sarcoma. In contrast, murine recombinant IL-18 could neither inhibit growth of the parental Mc12 sarcoma, nor suppress growth of its IL-2 gene-modified transfectant. These results suggest that although both of these cytokines are functionally related and participate in the induction of IFN gamma production as well as in cell-mediated immune cytotoxicity, in the murine sarcoma system only IL-12 is therapeutically active and exerts its therapeutic effect in concert with the IL-2 gene. Thus, intratumoral IL-2 gene transfer improves the therapeutic efficacy of IL-12; administration of recombinant IL-12 should therefore be considered as adjuvant in IL-2 gene therapy with irradiated, genetically modified tumour vaccines.

Animals↗

PubMed1999

Interleukin 2 gene therapy of residual disease in mice carrying tumours induced by HPV 16.

Experiments were designed to examine the efficacy of IL-2 gene therapy in a surgical minimal residual tumour disease, using moderately immunogenic MK16/1/IIIABC murine cells transformed by activated ras and HPV 16 E6/E7 oncogenes (MK16 cells). Previously we demonstrated that surgical minimal residual tumour disease (SMRTD) could be effectively cured when murine Mc12 sarcoma had been resected and the operated mice were treated with irradiated Mc12 sarcoma cells engineered to secrete IL-2. In this study we performed IL-2 gene therapy of MK16 carcinoma with two types of irradiated MK16-unrelated tumour cell vaccines. One type of vaccine was derived from MHC class I-matched Mc12 sarcoma cells engineered to secrete IL-2 and the other from MHC class I-discordant IL-2 producing plasmacytoma X63-m-IL-2. The vaccines did not share any tumour rejection antigen with the MK16 cells and served exclusively as a local source of IL-2 production. Both vaccines were capable of inhibiting MK16 tumours when administered peritumorally up to 15 days after MK16 tumour challenge. The irradiated MHC class I-matched and IL-2-producing Mc12 sarcoma vaccine was then selected for therapy of MK16 SMRTD. Whereas the recurrence rate in the operated MK16 carcinoma bearers was 80%, so that only 20% of mice were cured by surgery, approximately 65% of the MK16 carcinoma bearers were permanently protected when the surgery was followed by local administration of the IL-2-producing Mc12 sarcoma vaccine.

Animals↗

PubMed1999

Granulocyte-macrophage colony-stimulating factor-producing tumour vaccines.

Murine sarcoma MC12 cells were transfected with the gene coding for murine granulocyte-macrophage colony-stimulating factor (GM-CSF). Tumorigenicity of a variety of cell clones with different expression of the inserted gene was assessed. All of the genetically manipulated MC12 cell clones examined were found to be less tumorigenic than the parental MC12 cell population. No correlation was observed between the production of GM-CSF by the clones and their tumorigenicity. It has been found that irradiation of the GM-CSF-producing cells with the dose of 150 Gy did not significantly inhibit the GM-CSF production during the period of 5 days after irradiation. These findings provided us with the rationale for using the irradiated GM-CSF-producing MC12 sarcoma vaccine for therapy. It has further been found than immunosensitivity of the genetically manipulated, GM-CSF-producing tumour targets to the IL-2-activated killer (LAK) cell-mediated cytolysis was significantly increased, as compared to the parental target cell population. Irradiated, GM-CSF-producing tumour vaccines were used for therapy of 3-day-old MC12 sarcoma transplants in syngeneic mice and for therapy of surgically induced minimal residual tumour disease. Neither small tumour transplants, nor tumour residua after surgery were significantly sensitive to the therapy with GM-CSF-producing tumour vaccines.

Animals↗

PubMed1999

Dendritic-cell-based cancer vaccines.

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Antigen Presentation↗

PubMed1999

IL-2 gene-modified tumour vaccines: monitoring of IL-2 levels in serum and peritoneal cavity of vaccinated mice.

IL-2 kinetics was assessed in mice vaccinated with irradiated syngeneic tumour vaccines carrying an inserted IL-2 gene and producing constitutively IL-2. For comparison, the kinetics of i.v. administered recombinant IL-2 was also examined. During regular time intervals after the vaccination or administration of recombinant IL-2, samples of serum and peritoneal fluid were collected and examined, using CTLL bioassay or its MTT modification. After i.p. administration of irradiated IL-2-producing plasmacytoma (X63-m-IL-2) vaccine, the levels of IL-2 were substantially higher in the peritoneal fluid than in the serum. Both in the peritoneal fluid and in the serum, the IL-2 level was increasing up to 60 min after administration and then it gradually decreased. The last time point when IL-2 was still detectable both in the peritoneal fluid and in the serum was 30 h. Almost identical results were obtained when the IL-2 levels were detected by the conventional CTLL assay, in which DNA synthesis was monitored by 3H-thymidine labeling, and by the isotope-free MTT modification of the CTLL assay, in which the DNA synthesis was monitored by staining. The MTT modification has the advantage of an isotope-free method. Comparison of two different IL-2-producing vaccines, a murine plasmacytoma X63-m-IL-2, with high IL-2 production, and murine sarcoma MC12-IL-2, with low IL-2 production, revealed that whereas after i.p. administration of the high producers, the peak of IL-2 was reached both in the peritoneal fluid and in the serum after 1 h, the administration of low producers gave the peak level of IL-2 later, 5 h after i.p. administration. Comparison of IL-2 levels obtained after i.p. administration of live and irradiated X63-m-IL-2 vaccine revealed that the irradiated vaccine produced both in vitro and in vivo higher amounts of IL-2. As compared to i.p. administration, the kinetics after i.v. administration of the X63-m-IL-2 vaccine was different. The maximum level of recombinant IL-2 was reached 10 min after administration and IL-2 was undetectable after 5 h. When the injections of recombinant IL-2 were repeated, the elimination of IL-2 from the circulation was substantially faster.

Animals↗

PubMed1999

Interleukin 2 gene therapy of surgical minimal residual tumour disease: characterization of cytolytic effector cells from tumour progressors and regressors.

Experiments were designed to characterize cytolytic effector cells from mice with SMRTD treated with IL-2 gene therapy. Mice were inoculated with syngeneic murine MK16 carcinoma cells. When the tumours reached 8-12 mm in diameter, they were excised and the operated mice were randomized into two groups. The first group without any further treatment was designated as operated-only; the second group, vaccinated 3 days after the operation with IL-2-producing tumour vaccine, is referred to as operated-vaccinated. Tumour recurrence rate in the operated-only mice was 90 percent; in the operated-vaccinated group the recurrence rate was 38.5 percent (progressors). The remaining 61.5 percent of mice were permanently protected (regressors). On day 53, the tumour progressors, regressors and healthy controls were sacrificed, and their spleen cells were used for 51Cr microcytotoxicity assay. Splenocytes from any group of mice were not cytolytic when allowed to react with MK16, YAC-1 (NK sensitive) and C1498 (NK resistant) targets. However, when grown for 3 days in IL-2-containing medium, the splenocytes from all groups of mice could develop cytolytic activity. The cytolytic activity of splenocytes from tumour progressors and regressors was substantially lower then that of splenocytes from healthy controls. In addition, significantly lower cytolytic activity was observed with IL-2-activated splenocytes from tumour progressors as compared to that of tumour regressors. Depletion of NK1.1+ cells or CD4+ plus CD8+ cells prevented the induction of significant IL-2-stimulated cytotoxicity directed against MK16 and C1498 targets in spleen cell cultures from tumour progressors, regressors, and healthy control mice, indicating that both, NK1.1+ and CD4+ plus CD8+, cells participate in the antitumour effect of IL-2 gene therapy. This was further supported by the finding that after depletion of CD4+ plus CD8+ cells, a residual cytolytic activity directed exclusively against NK-sensitive YAC-1 cells was observed.

Animals↗

PubMed1999

Granulocyte-macrophage colony-stimulating factor gene-modified vaccines for immunotherapy of cancer.

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Animals↗

PubMed1998

Interleukin-2 gene therapy of surgical minimal residual tumour disease.

Our study was designed to examine the effects of IL-2 gene therapy in a surgical minimal residual tumour disease (SMRTD). Mice were inoculated s.c. with methylcholanthrene (MC)-induced MC12 sarcoma cells. When the tumours reached 8 to 12 mm in diameter, they were excised, either completely ("microscopic SMRTD") or incompletely ("macroscopic SMRTD"). On day 90 after surgery, the tumour recurrence rate in untreated mice with microscopic SMRTD was approximately 30%, whereas in those with macroscopic SMRTD it was 75%. After surgery, experimental mice were treated with 2 types of irradiated, IL-2 gene-modified, IL-2-producing tumour cell vaccine. One type of vaccine was derived from the MC12 sarcoma cells (MC12-1L2/IV-3); the other type was derived from an unrelated X63-Ag8.653 plasmacytoma (X63-m-IL-2). Both types of vaccine failed to cure the macroscopic SMRTD. Whereas the X63-m-IL-2 vaccine was also ineffective in the microscopic SMRTD, the MC12-IL2/IV-3 vaccine was capable of preventing growth in all but one mouse (1164) with microscopic SMRTD when administered 2 to 5 days after surgery. If the vaccination took place 2 days before surgery or later than 5 days after surgery, the therapeutic activity was lost. Vaccination with irradiated parental MC12 cells did not produce any significant benefit compared to the operated-only mice. The protective effect of the MC12-L2/IV-3 vaccine was specific and comparatively long-lasting. Vaccinated mice, which had rejected the MC12 tumour residuum, were capable of rejecting a second inoculum of the MC12 sarcoma cells injected on days 35 to 110 after surgery but succumbed to the growth of 2 other unrelated murine sarcomas carrying different tumour-rejection antigens.

Animals↗