Immunotherapy augments the effect of 5-azacytidine on HPV16-associated tumours with different MHC class I-expression status.

BACKGROUND: Epigenetic mechanisms have important roles in the tumour escape from immune responses, such as in MHC class I downregulation or altered expression of other components involved in antigen presentation. Chemotherapy with DNA methyltransferase inhibitors (DNMTi) can thus influence the tumour cell interactions with the immune system and their sensitivity to immunotherapy. METHODS: We evaluated the therapeutic effects of the DNMTi 5-azacytidine (5AC) against experimental MHC class I-deficient and -positive tumours. The 5AC therapy was combined with immunotherapy, using a murine model for HPV16-associated tumours. RESULTS: We have demonstrated 5AC additive effects against MHC class I-positive and -deficient tumours when combined with unmethylated CpG oligodeoxynucleotides or with IL-12-producing cellular vaccine. The efficacy of the combined chemoimmunotherapy against originally MHC class I-deficient tumours was partially dependent on the CD8(+)-mediated immune responses. Increased cell surface expression of MHC class I cell molecules, associated with upregulation of the antigen-presenting machinery-related genes, as well as of genes encoding selected components of the IFNγ-signalling pathway in tumours explanted from 5AC-treated animals, were observed. CONCLUSION: Our data suggest that chemotherapy of MHC class I-deficient tumours with 5AC combined with immunotherapy is an attractive setting in the treatment of MHC class I-deficient tumours.

Effects of 5-azacytidine and trichostatin A on dendritic cell maturation.

Maturation of dendritic cells (DC) towards functional antigen-presenting cells is a complex process, the regulation of which may also involve epigenetic mechanisms. Thus, it is of interest to investigate how gene expression changes during DC maturation can be influenced with epigenetic agents, such as DNA methyltransferase or histone deacetylase inhibitors. Here, we document the effects of DNA methyltransferase inhibitor 5-azacytidine (5AC) and histone deacetylase inhibitor trichostatin A (TSA) on the murine bone marrow-derived, as well as on the human monocyte-derived DC maturation. The major impact of 5AC and TSA on the DC maturation process consisted in the inhibition of unmethylated CpG oligodeoxynucleotide (CpG ODN) 1826 or LPS-induced activation of pro- and anti-inflammatory cytokine gene expression activation. In the in vitro studies, TSA but not 5AC significantly reduced the capacity of the peptide-pulsed DC to induce total spleen as well as CD8(+) or CD4(+) cell proliferation. IFNγ production by the specific CD4(+) spleen cells co-cultured with TSA- but not with 5AC-treated DC was lower, as compared to the cytokine production after co-cultivation with untreated mature DC. Collectively, these results demonstrate the potential of epigenetic agents, which are under intensive investigation as promising anti-tumour agents, to hamper the immune response induction through their inhibitory effects on DC.

Genetically modified cellular vaccines against human papillomavirus type 16 (HPV16)-associated tumors: adjuvant treatment of minimal residual disease after surgery/chemotherapy.

Local recurrences at the site of tumor resection or after chemotherapy, as well as distant micrometastases represent major problems in oncology. Therapeutic strategies based on insertion of immunostimulatory genes into the genome of tumor cells followed by vaccination with the resulting genetically modified and irradiated cellular vaccines represent a new potential prospect for the treatment of cancer patients. These strategies are based on the presumption that many, if not all tumors, possess cell surface antigens capable of being recognized by defence effectors of the immune system, as well as on the presumption that local treatment of primary tumors can, due to its immunizing potential, result also in the inhibition of distant metastases. Genetically modified cellular vaccines were found to be efficient against cancer both in experimental models and in tumor-bearing patients. It was also shown in various systems that the efficacy of conventional therapeutic modalities can be supported by adjuvant administration of genetically modified vaccines, as well as by depletion of immunosuppressive immunocyte subsets. The purpose of this review was to summarize and evaluate the results obtained with the administration of genetically modified cellular vaccines as well as with depletion of immunosuppressive immunocytes performed as treatment of minimal residual disease after surgery / chemotherapy in the experimental model of murine tumors mimicking human HPV16-associated neoplasms. The prospects and limitations of these adjuvant immunotherapeutic modalities are discussed.

Genetically modified cellular vaccines for therapy of human papilloma virus type 16 (HPV 16)-associated tumours.

Therapeutic strategies based on the insertion of cytokine or other immunostimulatory genes into the genome of tumour cells followed by vaccination with the resulting, genetically modified, cytokine-producing vaccines represent a new potential prospect for the treatment of cancer patients. HPV 16 is the aetiological agent of more than 60 percent human cervical carcinomas (CC). At present, two prophylactic vaccines against HPV 16 are available (GlaxoSmithKline "Cervarix" and Merck "Gardasil"). These vaccines can almost completely protect the immunized individuals against both, persistent HPV 16 infection and HPV 16-related pathological findings in cervical cytology. In contrast, no clinically utilizable therapeutic vaccines against CC are available. During the last decade animal models have substantially contributed to the development of the therapeutic vaccines against HPV 16-associated tumours. It has been demonstrated that the HPV 16 E6/E7 oncoproteins can serve as tumour rejection antigens (TRA) and that the HPV 16-associated tumour cells can be genetically modified with DNA encoding immunostimulatory cytokines (IL-2, IL-12, GM-CSF) or other immunostimulatory molecules, used for vaccination, and inhibit tumour growth. To improve the HPV 16 antigen presentation in tumour-bearing individuals, dendritic cell-based vaccines loaded with HPV 16 E6/E7 DNA or hybrids of the dendritic and tumour cells have also been successfully employed. Unfortunately, when these encouraging approaches used in animal models were translated into clinical trials, the results were less optimistic. The problems that are still to be faced before the therapeutic vaccines against high-risk HPV-associated tumours can be approved for clinical purposes are discussed.

Immunotherapeutic efficacy of vaccines generated by fusion of dendritic cells and HPV16-associated tumour cells.

Utilization of vaccines generated by fusion of dendritic cells and tumour cells is a promising approach to tumour immunotherapy. We have examined the therapeutic efficacy of vaccines generated by fusion of HPV16-associated tumour cells TC-1 with syngeneic and allogeneic dendritic cells. Locally administered hybrid cells generated by fusion of MHC class I+ TC-1 cells and syngeneic DC inhibited the growth of MHC class I+ TC-1 tumours, but not the growth of MHC class I- TC-1/A9-derived tumours. The growth of TC-1 tumours was also inhibited by hybrids generated by fusion of TC-1 cells and allogeneic DC. The therapeutic efficacy was enhanced by co-administration of the vaccine with synthetic immunostimulatory ODN CpG 1826.

Interleukin-2 therapy of cancer.

The aim of this review is to evaluate the results of IL-2 therapy of cancer two decades after the first experiments and to discuss whether and which results of local, systemic and adjuvant IL-2 therapy in preclinical models can be translated into clinics. The attention is also focused on the development and utilization of the IL-2 gene-modified tumour vaccines for therapeutic purposes. The prospects and limitations of both, IL-2 therapy and IL-2 gene therapy are discussed.

Immunogenicity of dendritic cell-based HPV16 E6/E7 peptide vaccines: CTL activation and protective effects.

We have investigated the capacity of cellular vaccines based on dendritic cells loaded with human HPV16 E6/E7 oncoprotein-derived peptides to induce immune responses in vitro and to elicit protective immunity in a murine experimental model mimicking human HPV16-associated carcinomas. Dendritic cells loaded with the HPV16 E6/E7 peptides exhibiting CTL or Th epitopes (E6(41-50), E6(81-90), E6(98-107), E6(130-137), E7(49-57), and E7(44-62)) were able to stimulate in vitro DNA synthesis in syngeneic H-2b spleen cells. The priming capacity of peptide-loaded BMDC and peptide-loaded dendritic cell lines DC2.4 and JAWS II was compared. It has been found that both peptide-loaded BMDC and established dendritic cell lines can activate the syngeneic responder cells, but the priming capacity of BMDC was substantially higher. In the second set of experiments, we have examined the cytolytic activity of syngeneic spleen cells after repeated activation in vitro with BMDC loaded with HPV16 synthetic peptides containing CTL epitopes. Significant cytotoxic responses against HPV16 E6/E7 antigen-expressing TC-1 targets have been found after repeated in vitro activation with all peptides containing the CTL epitopes. In contrast, peptide E7(44-62) harbouring both Th and CTL epitopes induced significant cytotoxic responses already after single in vitro activation. This cytotoxic effect could be enhanced with admixture of the E7(49-57) peptide. Experiments with MHC class I proficient (TC-1, MK16-IFNgamma) and deficient (MK16) target cells revealed that the dendritic cells loaded with the E6/E7 HPV16 peptides activated CTLs in vitro, but not the other cytolytic effector mechanisms. The effectiveness of the peptide-loaded BMDC-based cellular vaccines was also investigated in vivo. C57BL/6 (H-2b) mice were immunized with various peptide-loaded BMDC and subsequently challenged with TC-1 cells. The strongest protective effect was achieved with the BMDC loaded with the peptide E7(44-62) harbouring both CTL and Th epitopes. Mice immunized with the E7(44-62) peptide remained tumour-free after s.c. transplantation of the TC-1 cells and exhibited long-lasting protective immunity, whereas the mice immunized with E6(81-90) and E7(49-57) peptides did not remain tumour-free and exhibited only partial inhibition of tumour growth detectable as depression of the tumour growth curves.

The role of NK1.1+ cells in the protection against MHC class I+ HPV16-associated tumours.

Depletion of NK1.1+ cells by repeated i.p. injections of PK136 antibody significantly enhanced growth of MHC class I+ tumours in syngeneic mice. Depletion starting before tumour transplantation or on the day of transplantation substantially accelerated tumour growth; depletion starting on day 7 or 14 after tumour transplantation was without any effect. These results indicate that the NK1.1+ cells play an important inhibitory role during the early phase of the growth of some MHC class I+ tumours. Since the relevant target for NK cells is a "missing self" signal, absence of the MHC class I molecules, the NK cells cannot be expected to directly inhibit the growth of the MHC class I+ tumours. The results indicate that the effects of non-NK cells or indirect effects mediated by NK cell interactions and release of cytokines were responsible for the results.

Immunotherapy of HPV 16-associated tumours with tumour cell line/dendritic cell line (TC-1/DC2.4) hybrid vaccines.

Hybridization of established dendritic cell lines with tumour cells represents a prospective technology for the construction of antitumour vaccines. Experiments were designed to examine whether administration of cell populations prepared by fusion of HPV 16-associated tumour TC-1 cells with dendritic cell line DC2.4 could be used for treatment of TC-1 tumours growing in syngeneic mice. The therapeutic potency of TC-1/DC2.4 fusion vaccine administered 24 h after fusion and that of TC-1/DC2.4 hybrid cells selected for 3 weeks in HAT-containing medium was tested. It has been found that administration of both types of fusion vaccines at the site of growing TC-1 tumour transplants significantly inhibited tumour growth with regard to the percentage of tumour-bearing mice and to the size of the transplanted tumours. Peritumoral administration of the DC2.4 cells alone also reduced the size of growing TC-1 tumours, but not the percentage of the tumour-bearing mice. Although in the groups of mice treated with fusion vaccines the size of the tumours was reproducibly smaller than that in the mice treated with parental DC2.4 cells, the difference was not statistically significant.

Prophylactic, therapeutic and anti-metastatic effects of BMDC and DC lines in mice carrying HPV 16-associated tumours.

Oncogenic, moderately immunogenic MK16/1/IIIABC (MK16) cells were previously established by co-transfection of HPV 16 E6/E7 and activated H-ras oncogene DNA into C57BL/6 kidney cells. Subcutaneous transplantation of the MK16 cells produced progressively growing neoplasms which metastasized spontaneously to lungs. In this communication we report that prophylactic administration of bone marrow-derived dendritic cells (BMDC) as well as dendritic cell (DC) lines DC2.4 and JAWS II at the site of subsequent MK16 tumour transplants inhibited tumour growth and reduced the number of lung metastases. Similarly, in therapeutic experiments, administration of BMDC and DC lines at the site of the growing MK16 tumours or at the site of MK16 tumour residua after surgery inhibited tumour growth. Both BMDC-based vaccines and vaccines based on DC lines had also an antimetastatic effect. These results indicate that the DC line-based vaccines, which represent a standard, well-characterized and more homogeneous material, technically easier to prepare than the fresh BMDC-based vaccines, can be utilized for therapy of surgical minimal residual disease in HPV 16-associated neoplasms and are prospective for relevant clinical trials.

Local IFN-gamma therapy of HPV16-associated tumours.

We have examined whether peritumoral administration of IFN-gamma can inhibit growth of HPV16-associated, MHC class I- tumour MK16/1/IIIABC (MK16) transplanted in syngeneic mice. It has been found that peritumoral administration of recombinant IFN-gamma performed on days 0-11 after tumour challenge inhibited growth of MK16 s.c. tumour transplants. If the therapy with IFN-gamma was started when the tumours had already reached a palpable size, the IFN-gamma administration was without any effect. To investigate the antitumour effects of IFN-gamma in a clinically more relevant setting, surgical minimal residual tumour disease was utilized. Subcutaneously growing MK16 carcinomas, 8-12 mm in diameter, were removed and the operated mice were injected with IFN-gamma on days 3-14 after the operation at the site of surgery. Treatment with IFN-gamma resulted in a moderate, reproducible, but statistically insignificant inhibition of tumour recurrences. In the next experiments we have addressed the question whether the tumour-inhibitory effect of IFN-gamma was due to the upregulation of MHC class I molecule expression on MK16 tumour cells. IFN-gamma-treated and control mice were sacrificed, their tumours were explanted, and the expression of MHC class I molecules on the MK16 tumour cells was examined. As presumed, the MHC class I expression on the cells of IFN-gamma-treated tumours, as well as on their lung metastases, was upregulated. However, an unexpected moderate upregulation of the MHC class I expression was also observed on MK16 tumours from the control, exogenous IFN-gamma-uninjected mice. Cytofluorometric analysis of the in vivo transplanted MK16 tumours from both groups has excluded that the increased percentage of the MHC class I molecules on the tumour cell populations could be due to the infiltration of the tumours with MHC class I+ leukocytes, since no expression of MHC class II, CD11b, CD80/CD86, and CD11c molecules in the MK16 cell population was observed.

Adjuvant cytokine treatment of minimal residual disease after surgical therapy in mice carrying HPV16-associated tumours: cytolytic activity of spleen cells from tumour regressors.

It has been found previously that IL-2, IFNgamma and GM-CSF were capable of reducing the recurrence rate of HPV 16-associated tumours in mice with SMRTD. We were interested whether the therapeutic effect of the surgery and adjuvant cytokine treatment was accompanied by cytolytic activity of spleen cells and whether the activity of the spleen cells was different in mice that had rejected tumour residua after surgery and adjuvant therapy with cytokines (tumour regressors) as compared to those that had not rejected the tumour residua (tumour progressors). We have examined the cytolytic activity of spleen cells from MHC class I+ TC-1 tumour regressors and progressors after treatment of TC-1 SMRTD with GM-CSF, and the activity of spleen cells from MHC class I- MK16 tumour regressors and progressors after treatment of MK16 SMRTD with IL-2 and IFNgamma. It has been found that irrespective of the tumour type and adjuvant treatment, the spleen cells from tumour regressors after surgery were regularly more cytolytic when allowed to react with target cells from HPV 16-associated tumours than the spleen cells from tumour progressors. No substantial differences between the cytolytic activity of spleen cells from the operated-only and operated plus cytokine (GM-CSF, IL-2, IFNgamma) adjuvant treated groups were observed. The cytolytic activity of spleen cells from mice with SMRTD allowed to react with MHC class I+ , MHC class I-, NK-sensitive and NK-resistant targets is compatible with the interpretation that in the mice with MHC class I+ TC-1 tumours, primarily cytotoxic T lymphocytes (CTL) were efficient, whereas in the mice with MHC class I- MK16 tumours, both NK and non-lymphocytic effector cells were involved.

MHC class I+ and class I- HPV16-associated tumours expressing the E7 oncoprotein do not cross-react in immunization/challenge experiments.

It has been demonstrated repeatedly that a high proportion of tumours derived from MHC class I+ precursors are MHC class I-. Since a major task in immunotherapy strategies for treatment of malignancies is to develop polyvalent tumour vaccines efficient against a broad spectrum of tumours, we have examined whether MHC class I+ cell-based tumour vaccines can cross-protect against homologous MHC class I- tumour challenge and vice versa. For these purposes, we have used two oncogenic cell lines induced independently by co-transfection of murine H-2b cells with E61E7 HPV16 and activated Ha-ras oncogenes, the tumours TC-1 (MHC class I+, HPV16 E7+) and E7+). Surprisingly, it was found that these two tumours do not cross-react, although both of them contain the crucial HPV16-coded tumour rejection antigen E7. Preimmunization with the MHC class I+ tumour did not protect against a subsequent challenge with the MHC class I- tumour and vice versa; however, immunization with the TC-1 tumour could protect syngeneic mice against the TC-1 tumour challenge and, similarly, immunization with the MK16/1/IIIABC tumour could protect mice against the MK16/1/IIIABC tumour challenge. If this finding can also be confirmed as a more general phenomenon with other MHC class I+ and class 1- tumours, it could have serious implications for design of immunotherapeutic vaccines and protocols.