Milan Reiniš

Glycopeptide dendrimers, part III: a review. Use of glycopeptide dendrimers in immunotherapy and diagnosis of cancer and viral diseases.

Glycopeptide dendrimers containing different types of tumor associated‐carbohydrate antigens (TN, TF, sialyl‐TN, sialyl‐TF, sialyl‐Lex, sialyl‐Lea etc.) were used in diagnosis and therapy of different sorts of cancer. These dendrimeric structures with incorporated T‐cell epitopes and adjuvants can be used as antitumor vaccines. Best results were obtained with multiantigenic vaccines, containing, e.g. five or six different TAAs. The topic of TAAs and their dendrimeric forms at molecular level are reviewed, including structure, syntheses, and biological activities. Use of glycopeptide dendrimers as antiviral vaccines against HIV and influenza is also described. Their syntheses, physico‐chemical properties, and biological activities are given with many examples. Copyright

Induction of MHC class I molecule cell surface expression and epigenetic activation of antigen-processing machinery components in a murine model for human papilloma virus 16-associated tumours.

Epigenetic events play an important role in tumour progression and also contribute to escape of the tumour from immune surveillance. In this study, we investigated the up‐regulation of major histocompatibility complex (MHC) class I surface expression on tumour cells by epigenetic mechanisms using a murine tumour cell line expressing human E6 and E7 human papilloma virus 16 (HPV16) oncogenes and deficient in MHC class I expression, as a result of impaired antigen‐presenting machinery (APM). Treatment of the cells with the histone deacetylase inhibitor Trichostatin A, either alone or in combination with the DNA demethylating agent 5‐azacytidine, induced surface re‐expression of MHC class I molecules. Consequently, the treated cells became susceptible to lysis by specific cytotoxic T lymphocytes. Further analysis revealed that epigenetic induction of MHC class I surface expression was associated with the up‐regulation of APM genes [transporter associated with antigen processing 1 (TAP‐1), TAP‐2, low‐molecular‐mass protein 2 (LMP‐2) and LMP‐7]. The results demonstrate that expression of the genes involved in APM are modulated by epigenetic mechanisms and suggest that agents modifying DNA methylation and/or histone acetylation have the potential to change the effectiveness of antitumour immune responses and therapeutically may have an impact on immunological output.

DNA demethylating agent 5-azacytidine inhibits myeloid-derived suppressor cells induced by tumor growth and cyclophosphamide treatment

MDSCs represent one of the key players mediating immunosuppression. These cells accumulate in the TME, lymphoid organs, and blood during tumor growth. Their mobilization was also reported after CY therapy. DNMTi 5AC has been intensively studied as an antitumor agent. In this study, we examined, using two different murine tumor models, the modulatory effects of 5AC on TU‐MDSCs and CY‐MDSCs tumor growth and CY therapy. Indeed, the percentage of MDSCs in the TME and spleens of 5AC‐treated mice bearing TRAMP‐C2 or TC‐1/A9 tumors was found decreased. The changes in the MDSC percentage were accompanied by a decrease in the Arg‐1 gene expression, both in the TME and spleens. CY treatment of the tumors resulted in additional MDSC accumulation in the TME and spleens. This accumulation was subsequently inhibited by 5AC treatment. A combination of CY with 5AC led to the highest tumor growth inhibition. Furthermore, in vitro cultivation of spleen MDSCs in the presence of 5AC reduced the percentage of MDSCs. This reduction was associated with an increased percentage of CD11c+ and CD86+/MHCII+ cells. The observed modulatory effect on MDSCs correlated with a reduction of the Arg‐1 gene expression, VEGF production, and loss of suppressive capacity. Similar, albeit weaker effects were observed when MDSCs from the spleens of tumor‐bearing animals were cultivated with 5AC. Our findings indicate that beside the direct antitumor effect, 5AC can reduce the percentage of MDSCs accumulating in the TME and spleens during tumor growth and CY chemotherapy, which can be beneficial for the outcome of cancer therapy.

Dendritic cells pulsed with tumor cells killed by high hydrostatic pressure induce strong immune responses and display therapeutic effects both in murine TC-1 and TRAMP-C2 tumors when combined with docetaxel chemotherapy

High hydrostatic pressure (HHP) has been shown to induce immunogenic cell death of cancer cells, facilitating their uptake by dendritic cells (DC) and subsequent presentation of tumor antigens. In the present study, we demonstrated immunogenicity of the HHP-treated tumor cells in mice. HHP was able to induce immunogenic cell death of both TC-1 and TRAMP-C2 tumor cells, representing murine models for human papilloma virus-associated tumors and prostate cancer, respectively. HHP-treated cells induced stronger immune responses in mice immunized with these tumor cells, documented by higher spleen cell cytotoxicity and increased IFNγ production as compared to irradiated tumor cells, accompanied by suppression of tumor growth in vivo in the case of TC-1 tumors, but not TRAMP-C2 tumors. Furthermore, HHP-treated cells were used for DC-based vaccine antigen pulsing. DC co-cultured with HHP-treated tumor cells and matured by a TLR 9 agonist exhibited higher cell surface expression of maturation markers and production of IL-12 and other cytokines, as compared to the DC pulsed with irradiated tumor cells. Immunization with DC cell-based vaccines pulsed with HHP-treated tumor cells induced high immune responses, detected by increased spleen cell cytotoxicity and elevated IFNγ production. The DC-based vaccine pulsed with HHP-treated tumor cells combined with docetaxel chemotherapy significantly inhibited growth of both TC-1 and TRAMP-C2 tumors. Our results indicate that DC-based vaccines pulsed with HHP-inactivated tumor cells can be a suitable tool for chemoimmunotherapy, particularly with regard to the findings that poorly immunogenic TRAMP-C2 tumors were susceptible to this treatment modality.

Biomedical applications of peptide-, glyco- and glycopeptide dendrimers, and analogous dendrimeric structures

Introduction.- Chemistry and struture to dendrimers: Definition of terms and nomenclature.- Sugar code (glyco code).- Classes of peptide-, glyco, and glycopeptide dendrimers.- The dendritic state and dendritic effects.- Synthesis of dendrimers: convergent and divergent approaches.- Purification and characterization of dendrimers.- Dendrimeric libraries.- Dendrimers in catalysis.-Dendrimers and their biological and therapeutic applications: Dendrimers and solubility.- Biocompability and toxicity of dendrimers.- Dendrimers in nanoscience and nanotechnology.- Dendrimers in drug delivery.- Dendrimers in gene delivery.-Dendrimers and bacteria.- Dendrimers and viruses.- Dendrimers and parasites.- Cancer.- Diagnostics, lectin detection and cell-cell interactions.- Dendrimers as biosensors and imaging tools.- Dendrimers regulating in intracellular signaling pathways.- Vaccines and immunomodulation.- Dendrimers in neurodegenerative diseases.- Conclusions and persperctives.

Inhibitory effects of unmethylated CpG oligodeoxynucleotides on MHC class I-deficient and -proficient HPV16-associated tumours

Unmethylated oligodeoxynucleotides containing guanine–cytidine dimers (CpG ODN) have been described as potent inducers of selected antitumour immune responses and the immunotherapeutic efficacy of CpG ODN has been examined either alone or as a vaccine adjuvant. We hypothesized that CpG ODN therapy could be an effective tool for immunotherapy of not only conventional MHC class I+ tumours but also of those tumours that have lost MHC class I expression during their progression. To address this hypothesis, we employed the animal model resembling MHC class I‐proficient and ‐deficient human papilloma virus (HPV) 16‐associated tumours. A cell line transformed with HPV16 E6 and E7 oncogenes, TC‐1, as a prototype of MHC class I‐positive line, and its MHC class I‐deficient sublines TC‐1/A9 and TC‐1/P3C10 were injected into syngeneic C57BL/6 mice and the growing tumours were subjected to immunotherapy with CpG ODN 1826. The therapy started either 1 day after the challenge with the tumour cells or later, when the tumours had reached a palpable size. In both settings, CpG ODN 1826 significantly reduced the growth of MHC class I‐proficient and ‐deficient tumours. Furthermore, we demonstrated that CpG ODN 1585, whose mechanism of action preferably involves indirect activation of the natural killer cells, induced regression of the MHC class I‐deficient tumours TC1/A9 but not of the MHC class I‐proficient tumours TC‐1. This study infers that synthetic CpG ODN have a potential for the therapy of both MHC class I‐proficient and ‐deficient tumours and thus could be also used against tumours that tend to down‐regulate their MHC class I expression.

Genetically modified tumour vaccines producing IL-12 augment chemotherapy of HPV16-associated tumours with gemcitabine

Genetically modified tumour cells producing cytokines such as interleukin 12 (IL-12) are potent activators of the antitumour immune responses and represent a promising therapeutic modality when combined with chemotherapy. The objective of this study was to examine whether IL-12-producing cellular vaccines can augment chemotherapy of human papilloma virus (HPV) 16-associated murine tumours with the cytostatic agent gemcitabine (GEM). We found that peritumoral administration of IL-12-producing tumour vaccines enhanced the effect of cytoreductive therapy with GEM both in non-metastasizing murine carcinoma TC-1 and in metastasizing murine carcinoma MK16. The percentage of mice with MK16 metastases and the number of lung metastatic nodules was substantially decreased. In another clinically relevant setting, surgical minimal residual tumour disease, the administration of IL-12-producing tumour vaccine and GEM after the MK16 tumour surgery reduced the percentage of mice with tumour recurrences; similarly, the percentage of metastasis-bearing mice and the number of metastatic nodules was decreased. Tumour inhibitory effects exerted by GEM plus IL-12 were associated with high production of interferon-γ (IFNγ) by splenocytes. Our results suggest that the IL-12-producing vaccine can enhance the effect of GEM chemotherapy in some HPV16-associated murine tumour models.

Therapy for minimal residual tumor disease: β-galactosylceramide inhibits the growth of recurrent HPV16-associated neoplasms after surgery and chemotherapy

Natural killer T (NKT) cells are potent modulators of antitumor immunity. Their protective effects can be achieved upon their activation by glycolipid ligands presented in the context of the CD1d molecule. These CD1d‐binding glycolipid antigens have been described as potent therapeutic agents against tumors, infections, as well as autoimmune diseases. Immunoregulatory and therapeutic effects of glycolipid ligands depend on their structure and modes of administration. Therefore, more studies are needed for optimization of the particular therapeutic settings. This study was focused on the tumor‐inhibitory effects of 12 carbon acyl chain β‐galactosyl ceramide (C12 β‐D‐Galactosyl Ceramide; β‐GalCer(C12)) on the growth of human papillomavirus type 16 (HPV16)‐associated neoplasms transplanted in syngeneic mice. Treatment of tumor‐bearing mice with β‐GalCer(C12) 3–14 days after tumor cell transplantation significantly inhibited the growth of the major histocompatability complex (MHC) Class I‐positive (TC‐1), as well as MHC Class I‐deficient (TC‐1/A9) HPV16‐asssociated tumors. Moreover, administration of β‐GalCer(C12) after surgical removal of TC‐1 tumors inhibited the growth of tumor recurrences. Similar results were obtained in the treatment of tumors after chemotherapy. β‐GalCer(C12) treatment turned out to be also synergistic with immunotherapy based on administration of IL‐12‐producing cellular vaccines. These results suggest that β‐GalCer(C12), whose antitumor effects have so far not been studied in detail, can be effective for the treatment of minimal residual tumor disease as well as an adjuvant for cancer immunotherapy.

Dynamic alterations of bone marrow cytokine landscape of myelodysplastic syndromes patients treated with 5-azacytidine.

ABSTRACT Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal stem cell disorders characterized by ineffective hematopoiesis frequently progressing into acute myeloid leukemia (AML), with emerging evidence implicating aberrant bone marrow (BM) microenvironment and inflammation-related changes. 5-azacytidine (5-AC) represents standard MDS treatment. Besides inhibiting DNA/RNA methylation, 5-AC has been shown to induce DNA damage and apoptosis in vitro. To provide insights into in vivo effects, we assessed the proinflammatory cytokines alterations during MDS progression, cytokine changes after 5-AC, and contribution of inflammatory comorbidities to the cytokine changes in MDS patients. We found that IL8, IP10/CXCL10, MCP1/CCL2 and IL27 were significantly elevated and IL12p70 decreased in BM of MDS low-risk, high-risk and AML patients compared to healthy donors. Repeated sampling of the high-risk MDS patients undergoing 5-AC therapy revealed that the levels of IL8, IL27 and MCP1 in BM plasma were progressively increasing in agreement with in vitro experiments using several cancer cell lines. Moreover, the presence of inflammatory diseases correlated with higher levels of IL8 and MCP1 in low-risk but not in high-risk MDS. Overall, all forms of MDS feature a deregulated proinflammatory cytokine landscape in the BM and such alterations are further augmented by therapy of MDS patients with 5-AC.

Tumor growth accelerated by chemotherapy-induced senescent cells is suppressed by treatment with IL-12 producing cellular vaccines.

Standard-of-care chemo- or radio-therapy can induce, besides tumor cell death, also tumor cell senescence. While senescence is considered to be a principal barrier against tumorigenesis, senescent cells can survive in the organism for protracted periods of time and they can promote tumor development. Based on this emerging concept, we hypothesized that elimination of such potentially cancer-promoting senescent cells could offer a therapeutic benefit. To assess this possibility, here we first show that tumor growth of proliferating mouse TC-1 HPV-16-associated cancer cells in syngeneic mice becomes accelerated by co-administration of TC-1 or TRAMP-C2 prostate cancer cells made senescent by pre-treatment with the anti-cancer drug docetaxel, or lethally irradiated. Phenotypic analyses of tumor-explanted cells indicated that the observed acceleration of tumor growth was attributable to a protumorigenic environment created by the co-injected senescent and proliferating cancer cells rather than to escape of the docetaxel-treated cells from senescence. Notably, accelerated tumor growth was effectively inhibited by cell immunotherapy using irradiated TC-1 cells engineered to produce interleukin IL-12. Collectively, our data document that immunotherapy, such as the IL-12 treatment, can provide an effective strategy for elimination of the detrimental effects caused by bystander senescent tumor cells in vivo.