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X-Ray-induced photodynamic therapy represents a suitable modality for the treatment of various malignancies. It is based on the production of reactive oxygen species by radiosensitizing nanoparticles activated by X-rays. Hence, it allows overcoming the depth-penetration limitations of conventional photodynamic therapy and, at the same time, reducing the dose needed to eradicate cancer in the frame of radiotherapy treatment. The direct production of singlet oxygen by octahedral molybdenum cluster complexes upon X-ray irradiation is a promising avenue in order to simplify the architecture of radiosensitizing systems. One such complex was utilized to prepare water-stable nanoparticles using the solvent displacement method. The nanoparticles displayed intense red luminescence in aqueous media, efficiently quenched by oxygen to produce singlet oxygen, resulting in a substantial photodynamic effect under blue light irradiation. A robust radiosensitizing effect of the nanoparticles was demonstrated

The exceptionally high cellular uptake of gold nanorods (GNRs) bearing cationic surfactants makes them a promising tool for biomedical applications . Given the known specific toxic and stress effects of some preparations of cationic nanoparticles, the purpose of this study was to evaluate, in an in vitro and in vivo in mouse, the potential harmful effects of GNRs coated with (16-mercaptohexadecyl)trimethylammonium bromide (MTABGNRs). Interestingly, even after cellular accumulation of high amounts of MTAB GNRs sufficient for induction of photothermal effect, no genotoxicity (even after longer-term accumulation), induction of autophagy, destabilization of lysosomes (dominant organelles of their cellular destination), alterations of actin cytoskeleton , or in cell migration could be detected in vitro. In vivo , after intravenous administration , the majority of GNRs accumulated in mouse spleen followed by lungs and liver. Microscopic examination of the blood and spleen showed that GNRs interacted with white blood cells (mononuclear and polymorphonuclear leukocytes) and thrombocytes, and were delivered to the spleen red pulp mainly as GNR-thrombocyte complexes. Importantly, no acute toxic effects of MTABGNRs administered as 10 or 50 μg of gold per mice, as well as no pathological changes after their high accumulation in the spleen were observed, indicating good tolerance of MTABGNRs by living systems.

// Jana Simova 1 , Olena Sapega 1 , Terezie Imrichova 2 , Ivan Stepanek 1 , Lenka Kyjacova 2 , Romana Mikyskova 1 , Marie Indrova 1 , Jana Bieblova 1 , Jan Bubenik 3 , Jiri Bartek 2, 4, 5 , Zdenek Hodny 2 , Milan Reinis 1 1 Immunology Unit, Czech Centre for Phenogenomics, BIOCEV and Department of Transgenic Models of Diseases, Institute of Molecular Genetics of the ASCR, v.v.i., Prague 14220, Czech Republic 2 Department of Genome Integrity, Institute of Molecular Genetics, v.v.i., Academy of Sciences of the Czech Republic, Prague 14220, Czech Republic 3 First Faculty of Medicine, Charles University in Prague, Prague 12000, Czech Republic 4 Danish Cancer Society Research Center, Copenhagen DK-2100, Denmark 5 Department of Medical Biochemistry and Biophysics, Science For Life Laboratory, Division of Translational Medicine and Chemical Biology, Karolinska Institute, 17121 Solna, Sweden Correspondence to: Zdenek Hodny, email: hodny@img.cas.cz Milan Reinis, email: reinis@img.cas.cz Keywords: cellular senescence, cancer chemotherapy, docetaxel, IL-12, cell therapy Received: April 18, 2016 Accepted: May 29, 2016 Published: July 19, 2016 ABSTRACT 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 .

SO-C101 (RLI-15) is a superagonist fusion protein of interleukin (IL)-15 and the IL-15 receptor α (IL-15Rα) sushi+ domain designed to bypass the need of endogenous IL-15Rα, thereby leveraging the activity of IL-15 in vivo on target immune cells and reducing the toxicity of IL-15 as such. SO-C101 was previously shown to exhibit a potent anti-metastatic activity in Renca, B16F10 melanoma and delayed tumor growth in T cell-based mouse tumor models (CT26, MC38). Here we investigated the anti-tumor efficacy in predominantly natural killer (NK)-cell based mouse tumor models TC-1 and TRAMP-C2. We showed that SO-C101 monotherapy was effective in the treatment of established TC-1 tumors, which was dependent on the presence of both NK and CD8+ T cells, but not CD4+ T cells. In an early treatment setting SO-C101 significantly decreased the rate of tumor development also in dependence on NK and CD8+ T cells. SO-C101 effectively reduced tumor growth in TRAMP-C2 mice in early and advanced treatment settings. However, only in combination with anti-PD-1 antibody treatment the tumor development was prevented in majority of mice. This effect was durable, and the new tumor development was further significantly delayed after a tumor cell re-challenge, which suggests the involvement of memory T cells despite an important NK cell role in anti-tumor efficacy in these models. The efficacy of SO-C101 and anti-PD-1 treatment was not dependent on CD4+ T cells, but mainly on NK and CD8+ T cells. Interestingly, SO-C101 and anti-PD-1 treatment in double NK/CD8+ T cell-depleted mice decreased tumor growth which suggests an involvement of other immune cell populations in the anti-tumor efficacy. SO-C101 stimulated the proliferation and the cytotoxic activity of NK cells and memory CD8+ T cells without significant expansion of regulatory T cells. These data show the importance of various immune cell populations during SO-C101 monotherapy and the treatment in combination with anti-PD-1 antibodies, and set a base for further complex analysis of SO-C101 behavior. The therapeutic potential of SO-C101 is currently being tested in an ongoing Phase I clinical study in cancer patients. Citation Format: Irena Adkins, Romana Mikyskova, Nada Hradilova, Guy de Martynoff, David Bechard, Ulrich Moebius, Milan Reinis, Radek Spisek. SO-C101 displays strong anti-tumor effect in TC-1 and TRAMP-C2 tumor mice and in combination with PD-1 blockade prevents tumor development in a NK and CD8+ T cells dependent manner [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6686.

Photodynamic therapy (PDT) has attracted great attention in cancer treatment. Porphyrin derivative with ethylene glycol chains linked to meta position of meso-tetraphenylporphyrin, mTPP(EG)4, is one of assumed compounds that can be used for treatment of cancer in photodynamic therapy (PDT). The aim of the study is to demonstrate the photodynamic efficacy in vitro and in vivo as well as to analyze its therapeutic mechanism. The cell viability, cell surface exposure of immunogenic molecules, maturation and phagocytic capacity of PDT-treated tumor cells were monitored by flow cytometry. The kinetics of key components of ER stress-mediated apoptotic pathway was analyzed by Western blotting. Our first results indicate a new and attractive possibility of immunogenic cell death induction. Initiation of immunogenic cell death by PDT was successfully demonstrated in vitro on human and mouse tumor cell lines. The effects of mTPP(EG)4 were compared with the effects of mTPP(EG)4 without subsequent photoactivation, hypericin-mediated PDT, and high hydrostatic pressure. In vivo assays evaluated immunogenicity of mTPP(EG)4 in mice and assessed the ability of PDT treated cells to induce effective immunity against transplanted syngeneic tumors (mouse TRAMP-C2, TC-1 and B16). The results obtained in this study will provide a basis for possible further preclinical experiments with mTPP(EG)4 or other derivatives, testing their potential in PDT and development of new antitumor therapies. Citation Format: Sarka Vosahlikova, Irena Kusova Moserova, Jarmila Kralova, Milan Reinis, Romana Mikyskova, Jitka Palich Fucikova. Studies on photo-sensitivity of a glycol porphyrin derivative and its anti-tumor efficacy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B35.

In some settings, cancer cells responding to treatment undergo an immunogenic form of cell death that is associated with the abundant emission of danger signals in the form of damage-associated molecular patterns (DAMPs). Accumulating preclinical and clinical evidence indicates that danger signals play a crucial role in the (re-)activation of antitumor immune responses in vivo, thus having a major impact on patient prognosis. We have previously demonstrated that the presence of calreticulin (CRT) on the surface of malignant blasts is a positive prognostic biomarker for patients with acute myeloid leukemia (AML). CRT exposure not only correlated with enhanced T cell-dependent antitumor immunity in this setting but also affected the number of circulating NK cells upon restoration of normal hematopoiesis. Here, we report that CRT exposure on malignant blasts is associated with enhanced NK cell cytotoxic and secretory functions, both in AML patients and in vivo in mice. The ability of CRT to stimulate NK cells relies on CD11c+CD14high cells that, upon exposure to CRT, display higher levels of IL-15Rα, maturation markers (CD86 and HLA-DR), and CCR7. CRT exposure on malignant blasts also correlates with the upregulation of genes coding for type I interferons (IFNs). This suggests that CD11c+CD14high cells have increased capacity to migrate to secondary lymphoid organs, where can efficiently deliver stimulatory signals (IL-15Rα/IL-15) to NK cells. These findings delineate a multipronged, clinically relevant mechanism whereby surface-exposed CRT favors NK cell activation in AML patients. Citation Format: Iva Truxova, Lenka Kasikova, Cyril Salek, Michal Hensler, Daniel Lysak, Peter Holicek, Pavla Bilkova, Monika Holubova, Xiufen Chen, Romana Mikyskova, Milan Reinis, Marek Kovar, Barbora Tomalova, Justin Kline, Lorenzo Galluzzi, Radek Spisek, Jitka Fucikova. Calreticulin exposure on malignant blasts correlates with improved NK cell-mediated cytotoxicity in AML patients [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B96.

Cellular senescence is the process of the permanent proliferative arrest of cells in response to various inducers. It is accompanied by typical morphological changes, in addition to the secretion of bioactive molecules, including proinflammatory cytokines and chemokines [known as the senescence-associated secretory phenotype (SASP)]. Thus, senescent cells may affect their local environment and induce a so-called ‘bystander’ senescence through the state of SASP. The phenotypes of senescent cells are determined by the type of agent inducing cellular stress and the cell lineages. To characterise the phenotypes of senescent cancer cells, two murine cell lines were employed in the present study: TC-1 and B16F10 (B16) cells. Two distinct senescence inductors were used: Chemotherapeutic agent docetaxel (DTX) and a combination of immunomodulatory cytokines, including interferon γ (IFNγ) and tumour necrosis factor α (TNFα). It was demonstrated that DTX induced senescence in TC-1 and B16 tumour cell lines, which was demonstrated by growth arrest, positive β-galactosidase staining, increased p21Waf1 (p21) expression and the typical SASP capable of inducing a ‘bystander’ senescence. By contrast, treatment with a combination of T helper cell 1 cytokines, IFNγ and TNFα, induced proliferation arrest only in B16 cells. Despite the presence of certain characteristic features resembling senescent cells (proliferation arrest, morphological changes and increased p21 expression), these cells were able to form tumours in vivo and started to proliferate upon cytokine withdrawal. In addition, B16 cells were not able to induce a ‘bystander’ senescence. In summary, the present study described cell line- and treatment- associated differences in the phenotypes of senescent cells that may be relevant in optimization of cancer chemo- and immunotherapy.

RLI-15, a superagonist fusion protein of interleukin (IL)-15 and the IL-15 receptor α (IL-15Rα) sushi+ domain represents a promising candidate for the induction of anti-tumor immunity. RLI-15 was designed to bypass the need of endogenous IL-15Rα, thereby leveraging the activity of IL-15 in vivo on target immune cells. RLI-15 stimulates the proliferation and the cytotoxic activity of natural killer (NK) cells and memory CD8+ T cells with no significant expansion and activation of regulatory T cell compartment. RLI-15 was previously shown to exhibit a potent anti-metastatic activity in B16F10 melanoma and Renca renal cell carcinoma mouse models. RLI-15 also significantly delayed tumor growth and prolonged survival when combined with anti-PD1 therapy in CT26 and MC38 colon carcinoma models. Here, we report that the combination treatment with clinical-grade RLI-15 and an anti-PD1 antibody leads to a significant anti-tumor efficacy in a TRAMP-C2 prostate cancer mouse model with 70 % of mice remaining tumor free after the treatment. We evaluated the optimal schedule of such combination therapy to set the basis for the design of upcoming clinical trials. We further tested how the administration schedule affects the pharmacodynamics properties of clinical-grade RLI-15 and translates into the anti-tumor efficacy in metastatic Renca and CT26 mouse models. In cynomolgous monkeys, various schedules of administration of RLI-15 showed a dose-dependent expansion of peripheral blood lymphocytes, predominantly of NK cell and memory CD8+ T cell compartments. The toxicity in mice and cynomolgous monkeys was evaluated to determine the maximal tolerated dose of RLI-15. Furthermore, the activity of clinical-grade RLI-15 was tested in vitro on human PBMCs and the superiority over IL-2 and IL-15 stimulatory capacity has been confirmed. The complex analysis of RLI-15 behavior and of the induced anti-tumor immune response will be explored in the design of a planned Phase I clinical study in patients with both solid tumors and hematological malignancies. Citation Format: Irena Adkins, Lenka Sadilkova, Nada Hradilova, Jakub Tomala, Barbora Tomalova, Marek Kovar, Romana Mikyskova, Milan Reinis, Guy de Martynoff, David Bechard, Ulrich Moebius, Radek Spisek. Use of RLI-15 a clinical grade fusion protein with IL-15 superagonistic activity for the activation of anti-tumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3775.

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.

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.

Myeloid-derived suppressor cells (MDSC) play an important role in tumor escape from antitumor immunity. MDSC accumulate in the lymphoid organs and blood during tumor growth and their mobilization was also reported after cyclophosphamide (CY) administration. In this communication, spleen MDSC accumulating after CY therapy (CY-MDSC) were compared with those expanded in mice bearing human papilloma viruses 16-associated TC-1 carcinoma (TU-MDSC). Although both CY-MDSC and TU-MDSC accelerated growth of TC-1 tumors in vivo, their phenotype and immunosuppressive function differed. CY-MDSC consisted of higher percentage of monocyte-like subpopulation and this was accompanied by lower relative expression of immunosuppressive genes and lower suppression of T-cell proliferation. After interferon-γ stimulation, the expression of immunosuppressive genes increased, but the suppressive ability of CY-MDSC did not reach that of TU-MDSC. The phenotype and function of MDSC obtained from mice bearing TC-1 tumors treated with CY was, in general, found to lie between CY-MDSC and TU-MDSC. After in vitro cultivation of MDSC in the presence of interleukin 12 (IL-12), the percentage of CD11b+/Gr-1+ cells decreased and was accompanied by an increase in the percentage of CD86+/MHCII+ cells. The strongest modulatory effect was noticed in the group of CY-MDSC. The susceptibility of CY-MDSC to all-trans-retinoic acid (ATRA) was also evaluated. In vitro cultivation with ATRA resulted in MDSC differentiation, and ATRA inhibited MDSC accumulation induced by CY administration. Our findings identified differences between CY-MDSC and TU-MDSC and supported the rationale for utilization of ATRA or IL-12 to alter MDSC accumulation after CY chemotherapy with the aim to improve its antitumor effect.

Peptide, glyco, and glycopeptide dendrimers, capable of binding bacterial polysaccharides, represent attractive tools both for the therapy and for the diagnostics of the diseases of bacterial etiology. Finding new antibacterial drugs, as well as diagnostic tools, is nowadays of particular importance due to enhanced resistance of bacteria toward common antimicrobial agents. Indeed, the resistant microorganisms caused severe diseases with dramatic impact on mortality, disability, and economics. Attractive targets are bacterial lectins; some of them initiate infection by their interaction with the host cell glycoconjugates. Multipresentation and oligomerization of lectin domains on bacterial surface ensure multivalent binding of dendrimers. Owing to a great amount of different sorts of bateria, we limited our attention to only some of them: Escherichia coli, Vibrio cholerae, Pseudomonas aeruginosa, Haemophilus influenzae and Mycobacterium tuberculosis.

The vast majority of signal transduction processes in most living organisms are caused by four sorts of biomolecules: nucleic acids, proteins, glycoconjugates and lipids. The sequence and structure of nucleic acids, as well as, peptides and proteins have been extensively studied, including different sorts of interactions and functions. Genomics, proteomics, glycomics and lipomics represent four logically, chemically and biologically interconnected areas of research approaches to living organisms. The development in glycomics, in comparison with genomics and proteomics, was more demanding owing to the monumental growth of possible isomers and structural variations. Carbohydrates are unbeatable in information potential, compared with proteins and nucleic acids. This sort of coding (language) has been named glycocode resp. sugar code. It represents the complex information pool that carbohydrate structures are able to express. Monosaccharides as building blocks for oligo- and polysaccharide synthesis, represent therefore high-capacity information-storing and coding units, creating the third alphabet of life. The amount of information carried by glycopeptide dendrimers or glycodendrimers, in comparison with peptide dendrimers, is therefore much higher in all parameters, including structural variability, complexity, spectrum of biological activities, etc. The topics of sugar code, glycodendrimers and different sorts of nanoparticles partly overlap. Especially, carbohydrate-mediated molecular recognitions using nano-vehicles have a deep impact on medicine and open a new area of biomedical applications both in vitro and in vivo.

It is a difficult task to create a simple, universal, clear, unambiguous, logical, and generally applicable classification or even nomenclature of different sorts of dendrimers. In general, dendrimers contain a core, branches, and surface functional groups. Sometimes the core is missing (e.g., MAPs), or the branches are missing (RAFTs). The classification can stress either the shape of the core, branches, or surface groups (linear, cyclic) or the chemical nature of the core (fullerene, porphyrin, calixarene, cyclodextrin, RAFT, etc.) or branches (peptide, carbohydrate, PEG, PAMAM, etc.)

The terms of toxicity and biocompatibility of dendrimers are closely related. For biomedical applications, dendrimers should be devoid of toxicity and immunogenicity. The first concept—“toxicity”—is mainly applied by pharmacompanies for description of adverse effects to cells, organs, or patients. The second concept—“biocompatibility”—belongs to the field of biomedical materials as an extent of their compatibility with the studied system. One can say that biocompatibility and toxicity are inversely proportional, i.e., the more biocompatible the less toxic it is. Since 1986, biocompatibility is defined as the capability of a material to act with a proper host response in a specific application. Therefore, a biocompatibility of material can be described only if the precise context of material usage is known. Without knowledge of the exact usage of dendrimer, any chemistry cannot be defined as nontoxic or biocompatible. Clinical experience with dendrimers is still in its infancy; therefore, it is impossible to specify any chemistry insistingly biocompatible or toxic. Coating of nanomaterials with glycodendrimers results in formation of biocompatible nanoparticles such as solid lipid nanoparticles, CNTs, fullerene C60, lanthanide (Ln3 +)-doped nanoparticles, and CdSe quantum dots. These nanoparticles are not only biocompatible but also recognize specific lectins such as Con A, PNA, and PTA. The topic of dendrimer toxicity and biocompatibility will be discussed in detail with many representative examples.

Solubility is a key property influencing behavior of compounds in nature, science, biomedical applications, etc. Hydrophobicity and poor solubility of drugs and other bioactive compounds limit their possible applications in drug delivery and formulation development. Therefore approximately 40 % of newly developed drugs are rejected by the pharmaceutical industry and will never benefit a patient owing to poor bioavailability due to low water solubility and/or cell membrane permeability. In most cases, this problem can be solved by dendrimers. Many examples of dendrimers with different cores, branches, structures, hydrophilic surface groups, and hydrophobic pockets are described, including PEGylated dendrimers. The application of dendrimers as solubility enhancers for many drugs, such as anticancer drugs, antidepressants, antifungal drugs, antihemorrhagic drugs, antihistaminics, anti-inflammatory drugs, and antimicrobial drugs, is discussed. Solubility is in close relation with delivery, bioavailability, and/or cell membrane permeability; therefore see also these chapters.

Due to a strong anti-amyloidogenic activity, dendrimers are powerful agents with potential application for treatment of Alzheimer’s, Parkinson’s and prion diseases. Moreover, phosphorus containing dendrimer are used for sterilization of medical instruments in order to prevent transmission of prion diseases.

Basic terms and nomenclature of dendrimers are discussed. Till now, there is not one simple, unambiguous, generally applicable and worldwide acceptable dendrimer nomenclature, like IUPAC-IUB rules, which must be respected in every journal. This chapter explains basic terms of dendrimeric field: dendriplexes, dendrisomes, dendrigraft, glycotope, glycocluster, glycocluster–cluster, cluster effect, multivalency, cluster glycoside effect, glycoside cluster effect, multivalent effect, multivalent glycotope and clustering effect. The most common term is cluster effect and therefore we will use it in this book. Another discussed terms are macromolecular effect, sugar ball, and “smart” glycodendrimers. Many terms, e.g. glycotope, sugar epitope, carbohydrate epitope, glycopeptide epitope, polyvalent glycotope, glycocluster, glycodomain, etc. more or less coincide. We will use the term glycotope.

Dendrimers can be prepared by both convergent and divergent approaches. In the convergent approach, first suitable building blocks are prepared and then connected by lego chemistry, ligations or click chemistry. In the divergent approach, the synthesis is done stepwise. The advantage of convergent synthesis is greater difference between product and side products. The purification is therefore easier. On the other hand, the synthesis of big building blocks is complicated.

Dendrimers can serve also as antiparasitic tools. They can serve for vaccination, as well as, for specific delivery of toxic cargos. As examples, applications of dendrimers for treatment of various diseases caused by Schistosoma mansoni, Fasciola gigantica, Plasmodium falciparum, and Leishmania spp. are briefly mentioned.

The Possible biological applications of dendrimers stem from their capacity to influence the cell–cell interactions. These interactions are mediated by branched glycoconjugates forming a structure called glycocalyx present on the cell surface. Glycopeptide dendrimers, which can mimic these structures, thus can be used for detection of important players involved in carbohydrate-mediated cell–cell interactions or signaling. Moreover, they can be used as artificial modulators of glycoconjugate-mediated signaling and thus influence important processes, such as immune response, inflammation or fertilization. These types of glycodendrimers are also under investigation as antitumor or antimicrobial therapeutic agents.

Thanks to variable structures and properties, dendrimers are attractive for catalytic applications. They can act as catalytically active species, as well as, soluble supports onto which catalytically active species can be attached. Different sorts of dendrimer-encapsulated nanoparticles are used both in homogeneous and in heterogeneous catalysis. Examples of dendrimeric catalysts and their applications in many sorts of reactions, including chiral dendrimer catalysts and enantioselectivity in catalysis are given. Chiral dendrimer catalysts with a high level of molecular monodispersity, well-defined catalytic sites, and structural regularity, can be prepared either by attachment of achiral complexes to chiral dendrimer structures or by immobilization of chiral catalysts to achiral dendrimers.

Dendrimers, due to their unique chemical and biological properties, are considered to be highly suitable tools for regulation of immunity. They are endowed with unique capacity to expose artificial multivalent carbohydrate, glycoprotein or peptide antigens as highly defined three dimensional structures. A number of experimental vaccines has been developed against both infectious diseases and cancer. Notably, dendrimers can also be designed to be preferentially targeted to antigen-presenting cells and to activate them. As nucleic acid carriers, they can be utilized as carriers in DNA vaccines.

Before 1984, three classes of macromolecular architecture (i.e. linear, cross-linked, and branched) were widely accepted for construction of relatively polydisperse products of different molecular weights. Since that year, the “dendritic state” is acknowledged as a new, fourth class of polymer architecture. It can be divided to five subclasses: random hyperbranched polymers, dendrigrafts, dendrons, dendrimers, and tecto(dendrimers) or megamers. Due to the presence of a large number of terminal groups and the limitations or a complete lack of interpenetration, the physicochemical properties of dendrimers differ from those of classical polymers. On the other site, dendrimers and hyperbranched polymers are partly overlapping groups, which differ in regularity of structure, synthetic availability, and chemical variability. In comparison with hyperbranched polymers, dendrimers are more regular and better defined, but more difficult to prepare. The fundamental properties (both chemical and biological) of dendrimers are described. Dendritic chain reaction (DCR), a signal amplification technique that uses the disassembly properties of self-immolative dendrimers is also mentioned.

Delivery of nucleic acids to target cells can be achieved by both viral and nonviral delivery systems. Both of these methods have distinct advantages and disadvantages. In contrast to viral vectors, the nonviral ones provide the advantage of safety and flexibility. The disadvantage of nonviral carriers is their low efficiency. Inherent properties of dendritic nanoscale devices provide an ideal tool for development of synthetic (nonviral) carriers for delivery of nucleic acids. Among these properties, the defined architecture, high ratio of surface groups to molecular volume, and difference of hydropathic properties of interior and exterior are the most important for drug and gene delivery. Dendrimeric structures have been suggested to be used in gene therapy as potent carriers capable to, sometimes specifically, deliver DNA or RNA into the cells. Dendrimers are able to form complexes with many types of nucleic acids, such as plasmid DNA, antisense oligonucleotides, and RNA. Due to their defined architecture and a high ratio of multivalent surface moieties to molecular volume, they have become very important for the development of synthetic vectors for nucleic acid delivery. Indeed, dendrimer-based transfection agents have become routine tools for many molecular and cell biologists but the therapeutic delivery of nucleic acids complexed with dendrimer should be further investigated. The cationic dendrimers interact with the anionic backbone of nucleic acids mainly electrostatically. Complexed DNA is protected against degradation and the net positive charge of the dendrimer nucleic acid complex determines the transfection efficiency. However, highly cationic complexes are cytotoxic. Their properties can be tuned by many factors such as concentration of dendrimer amines and nucleic acid phosphates, salt concentration, stoichiometry, bulk solvent properties like pH, buffer strength, etc. For a development of dendriplex complexes with nucleic acids which can be used in gene therapy or, in general, as transfection reagents, knowledge of their transfection efficiencies and cytotoxicities is crucial.

Drugs are delivered by dendrimers by simple encapsulation, electrostatic interaction, or covalent conjugation. The application of dendrimer–drug conjugates by oral, intravenous, intraperitoneal, intratumoral, transdermal, ocular, etc. administration is discussed. Drug–dendrimer conjugates have two advantages over drug–dendrimer complexes and traditional drug dosing: prolonged lifetime and more stable level of the active substance. Many examples of different dendrimeric structures are given together with examples of their application in drug delivery. Dendrimers are also supposed to become an important tool for tissue-specific drug targeting. The topics of drug delivery, dendrimer solubility (both dendrimers as such and their influence to drug solubility), dendrimer toxicity and biocompatibility, are closely related and influence each other, therefore see also Chaps. 10 and 11.

Dendrimers, as hyperbranched synthetic well-defined molecules, have been suggested as potential antivirals from several reasons, such as their small sizes (nanometers) and their ease of preparation and functionalization. Importantly they can be potent inhibitors of virus–cell interactions due to their multivalency and capacity to display multiple copies of surface groups (cluster effect) required for processes such as initial virus–cell interactions.From many sorts of viruses, we focused to influenza and HIV virus. Other viruses, like human papilloma virus, swine fever virus, human cytomegalovirus, and herpes simplex viruses are mentioned only briefly. Influenza represents a major health problem, as the pathogenicity of influenza virus is very high and there is still a lack of an effective drug treatment. Almost a quarter of million patients with flu are hospitalized every year in the USA. Death toll of “Spanish” flu was ca 20 ×106. About 20 million people globally have died in the last two decades due to AIDS related causes, millions of boys and girls, young men and women are infected and living with the virus. The number of people living with HIV/AIDS in 2010 was 34 million and AIDS deaths in 2010 were 1.8 million.

Carbohydrates are expressed on majority of mammalian cell surfaces. They are bound to glycoproteins, proteins, and glycolipids, or to different cellular constituents, e.g. proteoglycans that are part of the cell membrane in clustered and multiantennary structures. These oligosaccharides represent signal transductors between extra- and intracellular media. Glyco- and glycopeptide dendrimers are excellent tools, which can be used for studies of inter- and intracellular signaling and also for their modulation. The capability of dendrimers to modulate intracellular signaling is strongly associated with their biological and therapeutic applications. In this chapter, several examples, how dendrimeric structures can influence intracellular enzymes (e.g. kinases or GTPases) that represent crucial components of intracellular signaling are presented.

A potential to serve as nanocarriers for various probes that can be conjugated on their external parts producing unique and defined three dimensional structures justifies dendrimers as candidates to be used as sensors or imaging tools. Typical examples, documenting their utilization as biosensors, are glycopolymers designed to bind specific lectins and endowed with intrinsic fluorescence. Dendrimers are used also for quantitative detection of different compounds, e.g. glucose, avidin, cDNA, dopamine, penicillamine, IgG, BSA, thrombin and many others. Other possibilities are conjugates with enzymes providing electrochemical signaling. Dendrimers are also under intensive studies as probes for magnetic resonance imaging.

Various methods of dendrimers characterization have been elaborated such as NMR, electron paramagnetic resonance, mass spectrometry, UV–VIS spectrometry, time-resolved and nonlinear optical spectroscopy, laser light scattering, optical rotation, circular dichroism, synchrotron radiation-based circular dichroism, IR, Raman, fluorescence, X-ray diffraction, small-angle X-ray scattering, small-angle neutron scattering, atomic force microscopy, scanning tunneling microscopy, optical tweezers, transmission electron microscopy, isothermal titration calorimetry, different chromatographic and electromigration methods (electrophoresis, capillary electrophoresis), dielectric spectroscopy, differential scanning calorimetry. Hence, this chapter is focused only on some ways, which are important for dendrimer characterization. Their con and pros are clearly discussed. Dendrimers can be characterized and purified by separation methods, as well as, they can be employed as stationary phase and/or selection factors improving the efficacy of separation techniques. The two main classes of separation techniques are chromatographic and electromigration methods. Even combination of two complementary techniques is not sufficient for removal of all by-products differing by only a single modification or deletion (the negligible difference such as missing one amino acid in molecules with mass above 7 kDa). A battery of complementary methods must be used to ensure at least some degree of reliability and even then the X + 1 method can show some impurities. By other words, the purity depends on the purification processes and analytical methods used.

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.

Receptor-mediated endocytosis was employed for a highly efficient transport of oligodeoxynucleotides into hepatoma cell line PLC/PRF/5. The oligodeoxynucleotides were bound to the asialofetuin-poly-L-lysine conjugate and this complex was internalized by the cells via asialoglycoprotein receptor, an endocytic receptor unique for hepatocytes. Binding of the oligodeoxynucleotides to the complex dramatically increased their cellular uptake more than 20-fold. Chloroquine, a lysosomatropic agent, further increased the transport of the complex but not of the free oligodeoxynucleotides.

Loss or downregulation of MHC class I molecules on tumour cells is a common mechanism by which tumours can escape T-cell mediated immune responses. In this study, we examined the role of different immune cell lineages in the development of immunity against tumours of the same aetiology but with different MHC class I expression. In vivo depletion of CD8+ cells, but not of CD4+ or NK1.1+ cells in the immunization period resulted in complete elimination of the protective effects of immunization with irradiated TC-1 cells (MHC class I-positive cell line) against the TC-1 tumour challenge. After immunization with irradiated TC-1/A9 or with MK16 tumour cells (MHC class I-deficient sublines) a remarkable dependence on the presence of NK1.1+ cells was observed, while the tumour growth inhibition after CD4+ or CD8+ depletion was not efficient. Cytotoxic activity induced by TC-1 cell immunization was significantly abrogated in the CD8+ and CD4+ but not NK1.1+ cell-depleted mice, as compared to the immunized only controls. After MK16 or TC-1/A9 cell immunization, NK1.1+ but not CD8+ and CD4+ cell-depleted mice displayed significant reduction of specific cytotoxicity. Mice immunized with TC-1 cells showed similar percentage of IFNγ producing cells in CD8+, CD4+ and NK1.1+ cell populations. On the other hand, the highest proportion of IFNγ producing cells after immunization with TC-1/A9 or MK16 cells was concentrated into the NK1.1-positive spleen cell population. Our data demonstrate that the development of immunity against MHC class I-deficient tumours is highly dependent on the activity NK1.1+ cell population.

MHC class I downregulation is a general mechanism by which tumor cells can escape from T-cell-mediated immunity. This downregulation also represents a serious obstacle to the development of effective antitumor immunotherapy or vaccination. Therefore, successful immunotherapeutic and vaccination protocols should be optimized against tumors with distinct cell surface expression of the MHC class I molecules. Mechanisms leading to protective immunity may vary in different models with respect to the particular tumors (e.g., in their levels of residual expression of the MHC class I molecules on tumor cells or inducibility of MHC class I expression). Notably, both CD8+ cell-mediated immunity and MHC class I-unrestricted mechanisms can take place against MHC class I-deficient tumors. Since MHC class I downregulation is frequently reversible by cytokines and also by the activation of epigenetically silenced genes, an attractive strategy is to elicit specific cell-mediated immunity combined with restoration of MHC class I expression on tumor cells.

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 beta-galactosyl ceramide (C12 beta-D-Galactosyl Ceramide; beta-GalCer(C12)) on the growth of human papillomavirus type 16 (HPV16)-associated neoplasms transplanted in syngeneic mice. Treatment of tumor-bearing mice with beta-GalCer(C12) 3-14 days after tumor cell transplantation significantly inhibited the growth of the major histocompatibility complex (MHC) Class I-positive (TC-1), as well as MHC Class I-deficient (TC-1/A9) HPV16-associated tumors. Moreover, administration of beta-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. beta-GalCer(C12) treatment turned out to be also synergistic with immunotherapy based on administration of IL-12-producing cellular vaccines. These results suggest that beta-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.

We have established animal models of HPV16-associated tumours with distinct levels of MHC class I expression. This model was used for examination of immune responses, production of cytokines and kinetics of immune cell subsets after IL-12 therapy of minimal residual tumour disease induced by CBA-4A (cyclophosphamide derivative) treatment. Upregulation of cytokine production was detected, compared to control animals without tumours. No differences in Th1/Th2 polarization of the immune responses after immunotherapy in animals bearing tumours with different surface expression of MHC class I molecules were observed. In the spleens of TC-1 (MHC class I+) but not of TC-1/A9 (MHC class I-) treated tumour-bearing animals, the cytotoxic CD8+ cells detectable in 51Cr microcytotoxicity assay, were found. In the spleens of TC-1/A9 but not of TC-1 tumour-treated animals, the NK activity measured as the lysis of NK-sensitive YAC-1 targets was detected. Down-regulation of the CD4+ and CD8+ subpopulations in spleens of tumour-bearing animals were not restored after therapy. The percentage of CD25+/CD4+ T regulatory (Treg) cells in lymph nodes remained unchanged. The cytoreductive chemotherapy led to strong upregulation and accumulation of immunosuppressive immature myeloid Gr-1+/CD11b+ cells (IMC) in the spleens of treated animals. The accumulation of Gr-1+/CD11b+ cells was significantly decreased after subsequent IL-12 immunotherapy. These data suggest that elimination of IMC after IL-12 immunotherapy may be responsible for the improvement of antitumour responses after adjuvant IL-12 vaccination for the treatment of CMRTD.

As a part of cellular pathogen defense, IFNγ triggers induction of NADPH oxidase NOX2, which produces superoxide into phagosomes of immune cells. Recent data show that a similar mechanism can also operate in IFNγ-mediated anticancer control. IFNγ is capable of inducing expression of constitutively active NADPH oxidase NOX4 in tumor cells leading to generation of reactive oxygen species (ROS) damaging DNA, activation of DNA damage response and cell cycle arrest/premature cellular senescence.

It is generally accepted that T regulatory cells (T reg CD4 + CD25 + Foxp3 + ) play an important role in the suppression of tumour immunity. We examined the impact of T reg cell depletion with anti-CD25 antibody as adjuvant therapy in the treatment of minimal residual disease after excision of murine HPV16-associated tumours. We found that the depletion of T reg cells inhibited growth of the recurrences after surgery of HPV16-associated MHC class I + as well as MHC class I-deficient tumours transplanted in syngeneic mice. These results demonstrate that depletion of CD25 + CD4 + Tree cells can be used as an efficient adjuvant treatment improving the results of surgery in the experimental systems mimicking human MHC class I + and MHC class I-deficient, HPV16-associated neoplasms. Therefore, this therapeutic modality is worth being examined in patients with minimal residual HPV16-associated tumour disease after surgery.

Moderately immunogenic HPV 16-associated murine tumour cell line mimicking human HPV 16-associated neoplasms TC-1 (MHC class I + ) and its variants, TC-1/P3C10 and TC-1/A9, with a marked down-regulation of MHC I molecules, were used to examine the effect of local interleukin 12 (IL-12) gene therapy for the treatment of early tumour transplants and minimal residual tumour disease obtained after cytoreductive chemotherapy (CMRTD). Experiments were designed to examine whether down-regulation of MHC class I molecules plays a role during chemotherapy and gene therapy of early tumour transplants. It was found that peritumoral administration of IL-12-producing tumour cell vaccines (single dose, day 8 after tumour cell administration) inhibited the growth of both TC-1 (MHC class I positive) tumours and their MHC class I-deficient variants. To investigate the antitumour effects in a clinically relevant setting, IL-12 gene therapy was utilised for the treatment of minimal residual tumour disease after cytoreductive chemotherapy. Intra-peritoneal treatment of tumour-bearing mice with ifosfamide derivative, CBM-4A, produced a significant tumour-inhibitory effect. This treatment was followed by peritumoral s.c. administration of genetically modified TC-1 (MHC class I positive) or MK16/I/IIIABC (MHC class I negative) vaccines producing IL-12 (single dose, day 7 after chemotherapy) or with recombinant interleukin 12 (rIL-12) in two cycles of 5 daily doses (days 8-19) after chemotherapy. This combined therapy significantly inhibited the growth of TC-1 and TC-1/A9 (MHC class I-) tumours. When the combined therapy of TC-1 (MHC class I positive) tumours was followed by peritumoral administration of bone marrow dendritic cell (BMDC) vaccines, the IL-12-mediated inhibitory effect was significantly boosted. In the next set of experiments, the impacts of chemotherapy and IL-12 adjuvant therapy on MHC class I surface expression were assessed. Chemotherapy and gene therapy of tumours led to the up-regulation of MHC I expression on MHC class I-deficient tumours (TC-1/A9 and TC-1/P3C10) and to down-regulation on MHC I-proficient tumours (TC-1). These findings indicate that the MHC I phenotype is not stable during tumour progression and treatment. Collectively, these results illustrate the efficacy of IL-12 gene therapy in combination with chemotherapy on HPV-associated tumours regardless of the level of MHC class I expression on the tumour cells.