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.

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.

Tumour-inhibitory effects of dendritic cells administered at the site of HPV 16-induced neoplasms.

Experiments were designed to examine whether administration of APC at the site of HPV 16-associated tumours can inhibit tumour growth and whether the efficacy of established dendritic cell lines is comparable to that of fresh BMDC populations. Mice were inoculated s.c. with APC, either bone marrow-derived dendritic cells differentiated in medium supplemented with GM-CSF and IL-4 (BMDC), or with established dendritic cell lines DC2.4 or JAWS II. The pretreated mice, together with untreated controls, were challenged with syngeneic HPV 16-transformed cells MK16 at the site of APC administration. It has been found that both BMDC and dendritic cell lines can inhibit tumour growth and that the efficacy of the established dendritic cell lines DC2.4 and JAWS II was comparable to that of fresh BMDC populations. In vitro induction of proliferative spleen cell responses by co-cultivation with MK16 antigen-pulsed BMDC or MK16 antigen-pulsed dendritic cell lines revealed that both types of APC populations can prime immune reactions directed against syngeneic HPV 16-associated neoplasms. Taken together, the results suggest that local increase in the number of dendritic cells at the site of HPV 16-associated tumours can inhibit progression of the tumours and that the dendritic cell lines which are efficient in this respect can be considered and should be tested in both, preclinical and human systems for delivery of therapeutic vaccines against HPV 16-associated neoplasms.

Chemoimmunotherapy of cancer: potentiated effectiveness of granulocyte-macrophage colony-stimulating factor and ifosfamide derivative CBM-4A.

The effectiveness of combined chemoimmunotherapy with ifosfamide derivative CBM-4A and granulocyte-macrophage colony-stimulating factor (GM-CSF) was investigated in two experimental tumor models, 3MC-induced MHC class I+ sarcoma Mc12 and HPV16 E6/E7 oncogene-induced MHC class I- carcinoma MK16, transplanted in syngeneic mice. Treatment of Mc12 and MK16 tumor-bearing mice with GM-CSF or CBM-4A alone produced moderate anti-tumor effects. However, when the tumor-bearing mice were first treated i.p. with a single dose of CBM-4A (150 mg/kg) and three days later peritumorally with five daily doses of GM-CSF (100 ng/day), substantially stronger tumor-inhibitory effects were observed. The results indicate that in both, MHC class I+ and MHC class I- tumors, the combined chemoimmunotherapy can inhibit tumor progression more effectively than GM-CSF therapy or chemotherapy alone, and they suggest that GM-CSF should be considered as adjuvant to chemotherapy in clinical trials with HPV 16-associated neoplasms.

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.