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.

Expression of mouse embryonic epitope TEC-2 on human carcinoma-derived cell lines and characterization of its glycoprotein carriers.

Monoclonal antibody TEC-02, raised against mouse embryonal carcinoma cells, has been shown to react with murine preimplantation embryos and with a very limited number of adult mouse tissues. The target epitope, TEC-2, is a carbohydrate carried in mouse embryonal carcinoma cells by large glycoprotein-bound glycan. We report here the expression of TEC-2 epitope on human carcinoma-derived cell lines, HeLa and HS, and the properties of its carbohydrate carriers. Immunolabeling of Nonidet P-40 lysates of HeLa cells separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that TEC-2 antigens are polydispersed glycoconjugates of high molecular weight (mostly above 100,000). TEC-2 antigens detected by the two-site sandwich assay, in which the antigen is immobilized and detected with the same antibody, had a slightly higher molecular weight than those detected by the solid-phase assay. This suggests heterogeneity in the number of TEC-2 epitopes per carrier molecule. When the cells were lysed by Triton X-114 and the detergent and aqueous phases were separated by warming and centrifugation, most of the TEC-2 antigenic activity was found in the aqueous phase. TEC-2 antigens isolated by indirect precipitation from [3H]galactose-labeled HeLa cells were degraded by extensive pronase digestion or mild alkaline treatment to glycopeptides or oligosaccharides of low molecular weight. Thus, TEC-2 epitope in human HeLa cells is carried by carbohydrates of only several monosaccharide units. TEC-02 antibody was also found to bind to Tamm-Horsfall glycoprotein isolated from human urine and its binding was enhanced by desialylation. Combined data indicate that TEC-02 antibody recognizes the GalNAc beta 1----4Gal beta 1----4 structure which may be carried on different types of molecule, according to the site of their synthesis.

Cytokine gene-modified vaccines in the therapy of cancer.

Therapeutic strategies based on the insertion of cytokine genes into the genome of tumour cells, followed by vaccination with the resulting genetically modified, cytokine-producing cells, represent a new potential prospect for treatment of cancer patients. In this review, the concept of cytokine gene-modified cancer vaccines is discussed; the discussion is focused on the rationale, characterization, progress in the development, preclinical testing, and first clinical trials. An effort is made to analyse and integrate the results obtained in different experimental model systems in order to determine the needed approaches and directions for further research.

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.

Local cytokine treatment of HPV16-associated tumours results in inhibition of their lung metastases.

Experiments were designed to examine whether local cytokine therapy of subcutaneous (s.c.) tumours results in inhibition of their lung metastases. Moderately immunogenic, major histocompatibility complex (MHC) class I and II negative. B7 negative, metastasizing murine carcinoma MK16 transplantable in syngeneic mice was obtained by co-transfection of human papilloma virus type 16 (HPV 16) E6/E7 and activated H-ras oncogene plasmid DNA into C57BL/6 kidney cells. After s.c. transplantation of the malignantly converted MK16 cells, the majority of the transplanted mice developed lung metastases; the number and size of the lung metastases increased with the increasing size of the s.c. tumour. Therapy of 5-day MK16 tumours by peritumoral administration of recombinant interleukin-2 (IL-2) and recombinant interleukin-12 (IL-12) inhibited growth of the s.c. MK16 tumour transplants and reduced the number of MK16 lung metastases. To investigate the antimetastatic effect of IL-2 and IL- 12 in a clinically more relevant setting, surgical minimal residual tumour disease was utilized. Subcutaneously growing MK16 carcinomas, 8-12 mm in diameter, were removed on day 30 and the operated mice were injected with IL-2 or IL- 12 on days 35-39 and 42-46 at the site of the operation. Treatment with IL-2 significantly reduced the percentage of MK16 tumour recurrences as well as the number of lung metastases, whereas the effect of IL- 12 was substantially weaker and statistically insignificant.

Use of cryopreserved lymphocytes for assessment of the immunological effects of interferon therapy in renal cell carcinoma patients.

Experiments were designed to assess whether cryopreserved PBL could be used to monitor the immunological effects of IFN-alpha therapy in renal cell carcinoma (RCC) patients. It was found that programmed freezing and thawing of peripheral blood lymphocytes (PBL) from normal blood donors did not substantially change lymphocyte subset proportions and that cryopreserved PBL were able to proliferate in response to IL-2. It was also possible to activate the cytolytic activity of frozen PBL, and the frozen leukocytes did not lose their ability to secrete IFN-gamma after PHA activation. We have used these findings to investigate the immunological effects of IFN-alpha therapy in RCC patients. Cryopreservation of PBL samples collected from various patients over a period of 9-14 months enabled us to compare the in vitro reactivity of PBL from individual RCC patients repeatedly and under standard conditions. It was found that IL-2 induced proliferative responses of PBL from IFN-alpha non-responders, collected prior to IFN-alpha therapy, were significantly decreased as compared to those from normal blood donors. The proliferative responses of PBL from IFN-alpha responders, collected prior to IFN-alpha therapy, did not substantially differ from normal controls. Culture of PBL from IFN-alpha responders for 3 days in IFN-alpha-containing medium increased their lytic activity towards RCC targets, whereas no such increase was observed with non-RCC targets or using PBL from IFN-alpha non-responders or PBL from normal-blood donors. Enzyme-linked immunospot (ELISPOT) assays performed with cryopreserved lymphocytes from IFN-alpha non-responding RCC patients, collected prior to IFN-alpha therapy, revealed a substantially decreased ability to secrete IFN-gamma, as compared to IFN-gamma secretion of PBL from IFN-alpha responders or normal blood donors.

Macrophage electrophoretic mobility test as sensitive probe of transplantation immunity in mice.

Transplantation immunity was examined in mouse H-2- and non-H-2-disparate strain combinations by the macrophage electrophoretic mobility (MEM) assay. Lymph node cells from the recipients rejecting histoincompatible skin grafts were incubated in the presence of solubilized histocompatibility antigens prepared from spleens of the grafts donors. Product(s) of lymphocyte-antigen interaction in appropriate combinations (sensitized lymphocytes with the sensitizing antigen) significantly diminished the electrophoretic mobility of guinea pig macrophages in all of the histoincompatible strain combinations examined. The data indicate that transplantation immunity in mice can be detected by the MEM assay.

Attempts to compare the effectiveness of blocking factors and enhancing antibodies in vivo and in vitro.

Sera from rats carrying tolerated skin allografts were tested for the presence of blocking activity in vitro. Sera with blocking activity had no effect on transplantation tolerance induction in newborn animals. Immunological enhancement of tumor growth was procured by passive transfer of serum from tolerant animals bearing skin allografts. It made no difference whether or not the serum contained blocking activity in vitro. These results suggest that there is no relationship between blocking factors and enhancing activity in vivo.

Cancer immunotherapy using local interleukin 2 administration.

Peri-tumoural administration of human recombinant interleukin-2 (RIL-2) into C57BL/10ScSnPh (B10) mice carrying subcutaneous transplants of syngeneic methylcholanthrene (MC)-induced sarcomas substantially inhibited tumour growth. Experiments were designed to compare the tumour-inhibitory effect of highly purified RIL-2 with that of unpurified human and rat lymphoid interleukin-2 (IL-2) preparations. It was found that the effect of RIL-2 was significantly lower than that of the lymphoid IL-2 preparations. These findings indicate that other lymphokines may participate in the positive results of local IL-2 immunotherapy using unpurified lymphoid IL-2 preparations. However, the admixture of human recombinant interleukin-1 (RIL-1) did not potentiate the immunotherapeutic effects of RIL-2. Sensitivity of MC-induced sarcomas to local RIL-2 immunotherapy was a general phenomenon. The growth of approximately eighty percent (5/6) of the MC-induced sarcomas could be inhibited with local RIL-2 administration. Moreover, direct correlation between the sensitivity of tumours to the tumour-inhibitory effect of RIL-2 in vivo and their susceptibility to the cytolytic effect of RIL-2-activated syngeneic killer spleen (LAK) cells in vitro was observed. This correlation indicates that LAK cells represent the effector cell mechanism responsible for the anti-tumour efficacy of local RIL-2 immunotherapy and that in vitro testing of sensitivity to the LAK cell-mediated cytolysis may be used to detect tumours responding to the local RIL-2 immunotherapy in vivo.

Immunotherapy of cancer using local administration of lymphoid cells transformed by IL-2 cDNA and constitutively producing IL-2.

Peritumoral administration of X63-m-IL-2 cells transformed by IL-2 cDNA and constitutively producing large quantities of recombinant IL-2 mediated regression of X63-Ag8.653 plasmocytoma and MC14 sarcoma transplanted in syngeneic mice. Injections of the IL-2-producing cells containing an inserted, modified IL-2 gene effectively inhibited tumour growth also in allogeneic recipients. Activation of murine spleen cells in vitro by co-cultivation with X63-m-IL-2 cells gave rise to lymphokine-activated killer (LAK) cell populations cytotoxic for the X63-Ag8.653 plasmocytoma and MC14 sarcoma. The results suggest that peritumoral administration of lymphoid cells transformed with IL-2 cDNA and constitutively producing IL-2 in the immediate tumour vicinity is sufficient for the effective activation of local IL-2-dependent tumour defence mechanisms and, therefore, can be considered a novel, genetic approach to the immunotherapy of cancer.

Defect in lectin-induced interleukin 2 production by peripheral blood lymphocytes of patients with invasive urinary bladder carcinoma.

The production of interleukin 2 (IL-2) by phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) from 21 patients with transitional cell carcinoma of the urinary bladder (BTCC) and 16 control blood donors was measured with a solid phase enzyme immunoassay based on the dual antibody immunometric sandwich principle. PBMC from patients with invasive BTCC (grade III-IV) showed a defect in the production of IL-2. The concentration of IL-2 in the supernatants of PBMC cultures from these patients was substantially lower (0.4 +/- 0.1 U/ml) than that observed in the supernatants of PBMC cultures from patients with non-invasive BTCC, grade II (1.5 +/- 0.7 U/ml), and from tumour-free controls (1.4 +/- 0.8 U/ml). These results suggest an immune dysfunction based on quantitatively impaired IL-2 production in patients with invasive BTCC and indicate that exogenous IL-2 could be used as an immunological response modifier for the treatment of these patients.

Thy-1 epitopes are expressed on murine myeloid leukemia and reticulum sarcoma cells.

Expression of Thy-1.2 specificities in cells from 29 primary spontaneous leukemias of random-bred ICR Swiss mice was examined by cell membrane and cytoplasmic immunofluorescence with monoclonal HO-13-4 antibody [1]. The Thy-1.2 epitopes were detected in all thymic lymphomas and were absent in the lymphomas of non-thymic origin. Unexpectedly, the Thy-1.2 epitopes were also detected in 71% (5/7) of myeloid leukemias and 40% (4/10) of reticulum cell sarcomas examined.

Recombinant interleukin-2 inhibits growth of human tumor xenografts in congenitally athymic mice.

Administration of human recombinant interleukin-2 (RIL-2) into congenitally athymic (nu/nu) mice carrying subcutaneous transplants of HeLa, HU 609T and T24B human carcinoma cells partially inhibited growth of the human tumor xenografts. In vitro activation of nu/nu spleen cells with human RIL-2 resulted in generation of killer cells showing in the 51Cr cytotoxicity assay similar levels of cytolysis as RIL-2-activated spleen cells from heterozygous (nu/+) mice. The RIL-2-activated (LAK) cells were cytotoxic for a variety of mouse and human tumors, reaching the peak of their cytotoxic activity after 3 days of cultivation in the RIL-2-containing medium. The cytotoxic activity of activated nu/nu spleen cells was significantly reduced by treatment with antibody against glycolipid asialo GM1, the differentiation antigen of natural killer (NK) cells. This finding suggests that in addition to the conventional, asialo GM1- LAK cells, asialo GM1+ activated NK cells participated in the cytotoxicity displayed by the IL-2-activated nu/nu killer spleen cells.

Lymphokine-activated killer (LAK) cells: I. Age-dependent decline of LAK cell-mediated cytotoxicity.

Experiments were designed to assess age-related changes in generation of lymphokine-activated killer (LAK) cells and to test whether these changes can be modified by diets differing in the proportion of polyunsaturated to saturated fatty acids (P/S). Ficoll-Hypaque-isolated spleen lymphocytes of rodent chow-fed, 6-85-week-old C57BL/6 (H-2b), 8-81-week-old C57BL/10 (H-2b) and 6-62-week-old SJL (H-2s) mice were cultured in IL-2-containing medium and examined in 51Cr cytotoxicity assay. Similarly, Ficoll-Hypaque-isolated spleen lymphocytes of 6-36-week-old SJL mice fed diets which differed in the ratio of polyunsaturated/saturated fatty acids were cultured in IL-2-containing medium and assayed for cytotoxicity. Age-related decline of LAK cell-mediated cytolysis was observed in mice of both H-2b and H-2s haplotype. The age-related decline of LAK cell-mediated cytolysis was the consequence of age-related decrease in the rate of LAK cell precursor maturation. SJL mice fed from birth with diets differing in P/S did not differ in LAK cell-mediated cytolysis.

Local administration of cells containing an inserted IL-2 gene and producing IL-2 inhibits growth of human tumours in nu/nu mice.

We have prepared a retroviral expression construct, pPS-IL-2, in which human IL-2 cDNA has been inserted into the polylinker region, and have used the retroviral vector to introduce the functional IL-2 gene into a fibroblast cell line, RAT-1. Peritumoral administration of IL-2-producing RAT-1 cells into congenitally athymic (nu/nu) mice carrying subcutaneous transplants of human carcinoma cells inhibited the growth of the human tumour xenografts.

Use of IL-2 gene transfer in local immunotherapy of cancer.

Insertion of functional interleukin-2 (IL-2) gene into a plasmacytoma cell line X63-Ag8.653 substantially reduced tumorigenicity of the resulting cloned cells, designated as X63-m-IL-2. Peritumoral administration of the X63-m-IL-2 cells, producing constitutively large quantities of IL-2, resulted in regressions of established X63-Ag8.653 plasmacytomas growing in the peritoneal cavity of syngeneic mice. In vitro activation of BALB/c spleen cells by co-culture with X63-m-IL-2 cells or their supernatants gave rise to cytotoxic lymphocytes with lymphokine-activated killer (LAK) activity against syngeneic X63-Ag8.653 plasmacytoma and other tumor targets. In contrast, peritumoral administration of X63-Ag8.653 cells carrying an inserted interleukin-4 (IL-4) gene (designated X63-m-IL-4 cells) and producing constitutively large quantities of IL-4 did not result in a therapeutic effect. Moreover, the admixture of the X63-m-IL-4 and X63-m-IL-2 cells substantially diminished the X63-m-IL-2 cell-mediated therapeutic effect. Similarly, IL-4-containing supernatants generated from X63-m-IL-4 cell cultures substantially diminished LAK activation by X63-m-IL-2 cell produced supernatants.

Animal models for development of therapeutic HPV16 vaccines (review).

In several types of human tumours a close association with oncogenic viruses has been established. Animal models mimicking these tumours with regard to their aetiology and expression of virus-related molecules have been developed. The rationale for development of the animal models was to create experimental systems allowing us to compare the efficacy of immunological and gene therapy protocols, with the final aim to optimize these therapeutic procedures prior to clinical trials. Anogenital carcinomas and particularly cervical carcinomas (CC) represent one of the aforementioned human oncogenic virus-associated tumour types. High-risk human papilloma viruses (HPV), particularly HPV16, 18, 31, 33, 45, and 56, are associated with nearly one hundred percent of CC. The only HPV proteins expressed in CC and representing the potential therapeutic targets are the non-structural viral gene products E6 and E7. The E6/E7 oncoproteins inactivate p53 and pRb tumour suppressor genes, they are required for maintenance of the malignant phenotype and their expression correlates with the transforming potential of HPV. Since the various HPV types do not cross-react with regard to the induction of both, humoral and cell-mediated immunity, the research of therapeutic vaccines is focused primarily on the HPV type 16, which is expressed in the majority, nearly 60%, of human squamous CC, and on the HPV16 E6/E7 oncogene products, which represent a target of choice for the therapeutic vaccination. The purpose of this review is to discuss the animal models of HPV-associated human tumours and the results obtained in these models with therapeutic vaccination against HPV-associated tumours. The prospects and limitations of the vaccination against HPV-associated tumours are also addressed.