[Immunotherapy - The New Era of Oncology].

1. Introduction

Especially in the last years, the field of immunotherapy was determined by significant progress although the idea to use the endogenous immune system for fighting against cancer was not new. Already in 1891, William Coley stimulated the immune system of sarcoma patients with bacterial fragments and achieved a short-term tumor reduction in some of his patients 1 . So why could this initial immunotherapeutic approach not prevail in the beginning? The reasons are manifold: the immune system is a very complexly regulated and balanced system, which on one hand may respond to pathogens due to stimulating and inhibiting components, on the other hand, however, it avoids excessive reaction and thus does not attack the own body. Furthermore, tumors are very heterogenic since they develop individually, and their properties depend on the individual patient and the tissue of origin. This situation is even aggravated by the fact that the original tissue of the tumor is not exogenous, and so important mechanisms of immune response, as they might work with the identification of exogenous pathogens, do not apply. Since Coley could only describe an unspecific reaction that was not directed against tumor antigens, the therapeutic effect was only temporary. Those mentioned aspects are the reason for initial difficulties and deceiving results oncological immunotherapy had and has to cope with. But what has finally changed? Why are there currently such high investments and efforts undertaken in the development of new therapeutic modalities with regard to tumor immunology? One crucial step was certainly the possibility to intervene specifically in the tumor development on the molecular level with new monoclonal antibodies (mAb). For many years, efforts were made to develop immunotherapies in the sense of immune activation; however, for some time now it has become obvious that antagonizing or influencing immunological blockades, checkpoints, and immunosuppressive mechanisms are of even higher importance. This was first achieved in the context of malignant melanoma by applying cytotoxic T lymphocyte-associated protein 4 (CTLA4) 2 and programmed cell death 1 (PD1) specific antibodies 3 . The results were convincing so that Science ennobled this type of immunotherapy as breakthrough of the year 4 . In addition, the scientific progress allows focusing the endogenous immune components on specific (tumor) antigens as it is the case for example with adoptive T cell transfer or in the context of vaccinations. Many of those strategies are relevant and innovative, however, they are at the very beginning of their (further) development. In the following, the chances and risks of immunotherapy will be discussed. For this purpose, first immunological basics of tumor interaction with the immune system will be explained in order to present different therapeutic approaches afterwards. This includes an overview of already existing therapeutic modalities as well as an outlook to future developments.

2. Tumor-immunological basics

Based on history and function, the immune system can be divided into 2 branches: the innate (native) immunity is the first front of immune defense and identifies, fights, and removes – mostly successfully – foreign pathogens in a rapid and effective way. However, the innate immunity is neither antigen-specific nor capable of learning (adaptive). Those properties belong to the so-called acquired (adaptive) immunity. It adapts to specific antigens and may thus generate a long-lasting, specifically adapted immune response. Both arms are not autonomous but interact intensively. Additionally, it becomes more and more obvious that the distinction between the innate and the adaptive immune system is not entirely clear.

2.1. Innate immune response

The innate immune response includes physiological barriers such as humoral and cellular components. The cellular parts are characterized mainly by their ability to migrate into the tissue and to initiate the immune response there and at the same time to attract further components of the immune system. Many cells of the innate immune response have the ability of phagocytosis, i. e., they actively take in pathogens, process them, and present – according to the cell type – parts of them on their surface on molecules of the major histocompatibility complex II (MHC II; Fig. 1 ). The cellular components of the innate immune response include granulocytes, macrophages, dendritic cells (DC), and the natural killer cells (NK cells). Macrophages are able to process antigens absorbed by phagocytosis and present them efficiently to other cells via MHC II in order to trigger an antigen-specific response. So they are an important interface between the innate and the adaptive immune response. Depending on the environment, generally 2 phenotypes of macrophages are differentiated, the M1 and the M2 phenotype. Whereas the M1 phenotype is typically activated by interferon γ (IFNγ) or parts of bacteria such as lipopolysaccharides (LPS), the M2 phenotype results mainly from a stimulation by the anti-inflammatory interleukin (IL) 4. M1 macrophages are polarized by IFNγ, LPS, GM-CSF, and TNF; they produce mainly pro-inflammatory cytokines such as IL1, IL6, IL12, IL23, and TNFα and induce in this way a T helper cell response that is directed against the tumor (T H 1 response). M2 macrophages are mainly polarized by IL4, IL10, and IL13, they produce themselves much IL19 and TGFβ but less IL1, IL6, IL12, and TNF and thus cause T H 1 suppression, T H 2 activation as well as immunosuppression and promote wound healing and tissue regeneration. Due to immunosuppressive influences in the tumor environment, tumor-associated macrophages (TAM) mostly polarize in direction of the M2 phenotype 5 . Their number in the tumor often correlates with angiogenesis, the development of metastases, and tumor progression 6 . Natural killer cells (NK cells) identify pathologically changed cells and may directly lyse them. For this purpose, they dispose of different receptors such as NKG2 (natural killer group 2) and KIR (killer cell immunoglobulin-like receptors). They interact with ligands on the tumor cells and send stimulating or inhibitory signals. NK cells do not need to be activated, however, their activity can only be increased by cytokines such as IL12, IFNα, and IFNβ. NK cells produce themselves IFNγ and initiate in this way a direct stimulation of the components in the tumor environment contributing to tumor defense. Even if the innate immune response plays a major role especially in the detection of foreign pathogens, its effect – in particular of the cellular components – on tumor development and progress is increasingly in the focus of research.

2.2. Adaptive immune response

The adaptive immune response completes the innate immune response and allows the development of persisting and antigen-specific immune reaction. The principle is based on antigen presentation. Antibodies represent the humoral components of adaptive immune response. They act by opsonization, induction of antibody-related cytotoxicity, neutralization, activation of the complement system, and agglutination of the pathogens. Antibodies are produced and secreted by B cells. In addition to B cells, the T cells are responsible for the adaptive immune response. Cytotoxic T cells (CD8+) require different signals for activation: first, the specific T cell receptor (TCR) has to be connected to an according antigen ( Fig. 2 ). Second, co-stimulating receptors of the T cells have to be activated in order to achieve proliferation as well as cytotoxic cell activity. Because of their ability to act highly specifically in a cytotoxic way, CD8+ T cells are in the particular focus of new oncological therapy approaches: on the one hand in the field of adaptive T cell therapy where tumor antigen-specific T cells are multiplied and (re-)applied to the patient 7 , on the other hand in the field of checkpoint inhibitors where specific inhibitory receptors of the T cells are blocked in order to impede T cell anergy 8 . T helper cells (CD4+) contribute in particular to regulatory processes of immune defense. They mostly do not dispose of own cytotoxic effects but transmit them via partner cells, e. g., cytotoxic T cells or NK cells. The antigen presentation is performed via MHC II molecules. In contrast to CD8+ T cells, CD4+ T cells need more frequent antigen contact to get stimulated. Even with regard to CD4+ cells, different phenotypes are known: the T H 1 phenotype secretes IL2 as well as IFNγ and stimulates the antitumor immune response whereas T H 2 cells produce IL4 and IL10 that have an inhibitory effect on the immune system in the tumor environment. The consideration of those basic phenotypes and their impact on the tumor environment is crucial for immunotherapy.

2.3. Tumor development and tumor evasion

In the middle of the 20 th century, Burnet and Thomas established the hypothesis of immuno-surveillance 9 . It consists of the assumption that during lifetime the incidence of non-hereditary, genetic changes in the cells increases. Since this phenomenon fosters the development of malignancy, a surveillance mechanism has to exist with an immunological background in order to eliminate or activate those mutated cells 10 . The hypothesis of immuno-surveillance has meanwhile been extended to the model of “Cancer Immuno-Editing”. Chronological aspects of tumor development are considered and different phases of the balance between defense and progress are described ( Fig. 3 ) as “elimination”, “equilibrium”, and “escape” phases 11 . In the elimination phase, the adaptive and the innate immune system can identify and destroy tumor cells. The equilibrium phase represents the transition between elimination and escape when the immune system controls the tumor cells. It is an equilibrium between the interleukins IL12 (immuno-stimulation) and IL23 (immunosuppression) 12 . In the escape phase, the immune system loses control over the tumor resulting in tumor progress: tumor cells achieve progress by mechanisms that reduce their identification by the immune system 13 14 15 16 17 18 , that lead to an increased resistance of the tumor cells 19 20 , and that cause inactivation of the antitumor effector cells 21 . In addition, the tumor environment is influenced in favor of immunosuppressive signaling pathways.

2.4. Tumor environment

The progress in the cancer treatment of the last decades is mainly due to a more profound knowledge of the interacting influences in the tumor environment. It is clear that tumor cells are influenced by the environment and vice versa influence the tumor environment. It becomes more and more obvious that the interactions between involved cells is highly complex and that a differentiation between the origin and the effect of the tumor environment is an important scientific challenge. So, an exact assessment of the involved (cellular) structures and signaling pathways is strongly required. In the tumor environment, tumor cells encounter cells and structures of the surrounding tissue. In addition to the cells of the immune system, they contain components of the extracellular matrix, vascular structures, stroma cells, and fibroblasts. The tumor cells interact with many, partly changing partners. Because of the heterogenic composition, the tumor environment is different from patient to patient. In addition to the inter-individual variation, there is an intra-individual dynamism. This factor is often still neglected in the analysis of tumor/patient specimens.

2.4.1. Extracellular matrix

The extracellular matrix (ECM) describes the space outside the cellular plasma membranes and is formed by interstitial macromolecules. Those macromolecules include glycoproteins and polysaccharides. Additionally, the ECM contains mainly water, electrolytes, and nutrients. It is influenced by surrounding cells. So the composition of the ECM is not unchangeable but subject to changing processes of metabolism and production as well as degradation of macromolecules. Beside important influences on the tissue properties of shape, elasticity, and the contents of water, the ECM also plays a major role in the induction of immune reactions and wound healing as well as signal transduction and binding of signal receptors. In this way, the intracellular gene expression may be influenced which has an effect on the adhesion, migration, and proliferation of the surrounding cells. All this explains the important impact of the ECM on the surrounding tissue. Even in tumors, the ECM acts on the tumor environment and the tumor development 22 . In this context, the deregulation of the ECM plays a crucial role. The increased collagen implementation in the ECM may induce an integrin-mediated cell proliferation 23 . Due to anti-apoptotic effects 24 and support of oncogenic cell transformations 25 , the ECM may be the basis for tumor progress. Additionally, tumor progress may be caused by an inhibiting impact of the ECM on the immune system 26 . The proliferation of T cells may be impeded, for example by binding LAIR (leukocyte-associated Ig-like receptor) by means of collagen 27 or by compromising antigen-presenting cells 28 .

2.4.2. Vascular supply

Such as all metabolically active tissues, especially tumor cells with their highly active cell division and the significantly activated metabolism depend on the supply with nutrients. Also the oxygen consumption and the transportation of metabolites are important reasons for the dependence of tumors on an adequate vascular supply. Thus, angiogenesis in the tumor environment is important for tumor growth, invasive and metastatic behavior 29 30 . Angiogenesis describes the process of new vascular formation developing from the already existing vascular bed. Among others, angiogenesis is induced by hypoxia, a low pH value, hypoglycemia, stress, and inflammation. In this context, endothelial cells are activated by growth factors that migrate and proliferate. Those growth factors are the vascular endothelial growth factors (VEGF), fibroblast growth factors (FGF), platelet derived growth factors (PDGF), hepatocyte growth factors (HGF), and angiopoietin 1 and 2 30 . The receptors of those growth factors, mainly tyrosine kinase receptors, can not only be stimulated by their ligands but also by hormones, neurotransmitters, and lymphokines. Especially the latter play a major role in angiogenesis because the involved messenger substances also contribute to inflammation and immune cell migration 29 . For example, mast cells and macrophages can be recruited by tumor cells and stimulated to secrete angiogenetic cytokines.

2.4.3. Fibroblasts

Fibroblasts have a mesenchymal origin and are involved in the development of ECM as cells of the connective tissue. They secrete in particular collagen, fibronectin, and growth factors. At the same time, however, they contribute to the re-structuring of the ECM and can produce matrix metalloproteinases (MMP) that solve the peptide connections of ECM structures and thus play an important role in angiogenesis, wound healing, and tumor growth. As cells with mesenchymal origin, they are also part of the tumor environment of solid tumors. In the tumor environment, mesenchymal stroma cells may differentiate to so-called cancer-associated fibroblasts (CAF). They are characterized by a clearly higher activity compared to other fibroblasts 31 . They secrete TGFβ and other growth factors that promote tumor growth 32 . Even metabolic processes in tumor cells can be supported by CAFs 33 . Additionally, the MMPs secreted by CAF are associated with increased invasiveness and metastasis 32 34 . CAF have an effect on the immune system by chronic cytokine secretion that is one reason of chronic inflammatory reaction in the tumor environment. The mobilized immune cells, as for example macrophages, are converted by chronic stimulation of the CAF and other immuno-modulating influences of the tumor environment in the context of chronic inflammatory reaction into the tumor promoting phenotype (M2) 35 36 .

2.4.4. Suppressive immune cells

In addition to the initially described cellular parts of the immune system, there are further immune cells that are present in the tumor environment: regulatory T cells (Treg), tumor-associated macrophages (TAM), and myeloid-derived suppressor cells (MDSC).

2.4.1 Treg Treg are mainly a subpopulation of CD4+ T cells that are responsible for the maintenance of self-tolerance as so-called suppressor cells. This is achieved by inhibiting activated T cells. Treg is characterized by the expression of CD4 and CD25 (sub-unit of the IL2 receptor) and the transcription factor FOXP3 (forkhead box protein 3). Up to now, more than 4 different CD4+ subpopulations of regulatory cells have been described. The different subpopulations may possibly be responsible for the partly contradictory research results. Nonetheless, there is a broad consensus that Treg have immunosuppressive properties in the tumor environment 37 . They do not only have this effect due to secretion of specific interleukins and other cytokines but they need direct cell-to-cell contact for part of their functions 38 . The involved cytokines are mainly IL4, IL10, IL35, and TGFβ. Fig. 4 shows an overview of cytokine influence on CD4+ T cells. In the direct cell-to-cell contact, Treg transmit immunosuppressive properties via the expression of so-called checkpoint molecules, in particular CTLA4 and LAG3 (lymphocyte activation gene 3) as well as surface-bound enzymes such as CD39. The checkpoint molecules work via competitive binding in the cell-to-cell contact with co-stimulating molecules of antigen presenting cells and thus impede effective antigen presentation. Additionally, the maturation of the antigen presenting cells, especially of dendritic cells, is prevented. The enzymatic function of CD39, an ectonucleotidase, plays a significant role in the processing of ATP to AMP. In this way, the pro-inflammatory properties of extracellular ATP are inhibited. Beside dendritic cells, Treg have an inhibitory effect on activated CD4+ and CD8+ T cells. They significantly contribute to the development of T cell anergy 39 . Under anergic conditions, T cells can no longer be stimulated and do not respond to new antigen presentation. Even the proliferation of T cells is prevented via an inhibitory effect on the cell cycle 39 . Furthermore, Treg compete with other T cells regarding IL2, which is required for the proliferation and activation of Treg as well as other T cells in the tumor environment. An inhibitory effect of Treg could also be confirmed on NK cells. The secretion of TGFβ causes a reduced expression of NKG2D, an activating NK cell receptor 40 . The abilities of Treg to influence the immune response are manifold and appear significantly in the tumor environment 41 .

2.4.4.2. MDSC MDSC is used as an umbrella term for a subgroup of suppressor cells of myeloid origin. They are defined by the expression of specific surface molecules (CD11b+, CD33+, and CD34+) and could be identified in most solid tumors. They are characterized mainly by protumoral differentiation. In contrast to mature dendritic cells, MDSC suppress the immune response in the tumor environment ( Fig. 5 ). MDSC are stimulated by pro-inflammatory signals and so they are relevant for controlling the immune response in the tumor environment 42 . Similar to Treg, MDSC exercise their immunosuppressing effect in many areas of adaptive and innate immune response. For this purpose, they use different mechanisms: The antigen detection of T cells is disturbed by nitrading the T cell receptor. Furthermore, they inhibit T cell activation by the consumption of cysteine, which is an essential amino acid for T cells 43 . Also the proliferation of T cells is controlled via inhibition of IL2 production. The production of arginase and oxygen radicals impedes the antigen detection and T cell activation 44 . Additionally, the increased differentiation to MDSC from progenitor cells of myeloid origin influences the adaptive immune response via a reduction of antigen presenting cells in favor of MDSC. Further, MDSC support the development of Tregs by increasingly producing IL10, TGFβ, and arginase 42 . Parts of the innate immune response are inhibited by MDSC, in particular NK cells and M1 macrophages that are disturbed in their functionality by an increased production of IL10 and a reduction of the IL12 secretion 45 . The development of MDSC is influenced by different cytokines, among others VEGF, granulocyte monocyte colony stimulating factor (GM-CSF), IL6, IL1β, PGE2 (prostaglandin E2), and complement C5a.

2.4.4.3 TAM Due to their versatility, mobility, and the fact that they belong to the innate immune system, macrophages are active in many pathways of the immune defense – including wound healing and inflammatory processes. This also includes the tumor environment where macrophages are involved in angiogenesis, leukocyte infiltration, mutation of ECM, and immunosuppression 46 . With regard to the tumor environment, a heterogenic group of macrophages has been described in the last years that is defined as tumor-associated macrophages. It must be taken into account that some authors describe mainly the M2 phenotype with regard to tumors, others, however, differentiate even in TAM between M1 and M2 phenotypes because of the high plasticity of the macrophages ( Fig. 6 ). In the majority of the tumors (except colon cancer) TAM correlate with a poor prognosis 47 . They may represent up to one third of the cellular components of the tumor environment 48 . TAM are recruited into the tumor environment via chemokines (e. g., chemokine ligand 2, CCL2, and CCL5) that are mostly secreted by tumor or stroma cells. In addition, VEGF, PDGF, M-CSF, and TGFβ are involved in the macrophages recruiting process 49 . M1 macrophages are polarized by IFNγ, LPS, GM-CSF, and TNF; they produce much IL1, IL6, IL12, IL23, TNF, and lower levels of IL19 and trigger a T H 1 response, tissue destruction, and immune stimulation. M2 macrophages are mainly polarized by IL4, IL10, and IL13, they produce much IL10, TGFβ, and few IL1, IL6, IL12, TNF and cause T H 1 suppression, T H 2 activation, immunosuppression and promote wound healing as well as tissue regeneration. Since macrophages have a high plasticity and strongly depend on influences of the tumor environment, TAM are rather allotted to the M2 phenotype 5 . So in the tumor they correlate with angiogenesis, the development of metastases, and tumor progress 6 .

2.4.5. Cancer stem cells

Cancer stem cells (CSC) are tumor cells with stem cell properties. Those properties include the self-renewal as well as the potential of differentiation 50 . The hypothesis of cancer stem cells is controversially discussed 51 , however, there are more and more hints that different tumor entities contain tumor cells with stem cell characteristics 52 . Cancer stem cells are responsible for example for the development of therapy resistances 53 . In connection with chemotherapy, a conversion of glioblastoma cells into cancer stem cells could be confirmed 54 . Another important property of cancer stem cells is the low immunogenicity 55 . This ability to avoid the immune system is based on different properties. On the one hand, CSC express inhibitory ligands such as for example FasL and inhibitory NK ligands, on the other hand anti-apoptotic molecules such as Bcl2 and survivin. Furthermore, they secrete the classic immunosuppressing cytokines like TGFβ, IL4, IL6, IL10, and PGE2. Additionally, CSC inhibit the T cell proliferation in a STAT3-mediated way 56 .

3. Immunotherapies

The objective of different immunotherapies is to possibly target the activity of the immune system against the tumor, to inhibit protumoral effects, and to allow tumor elimination ( Fig. 7 ). The basics of pro- and antitumoral processes and the involved cellular and non-cellular structures have been described in the previous chapters. In the following, single therapeutic approaches will be discussed more in detail regarding their modes of action.

3.1. Cytokine therapies

Cytokines are proteins, which regulate the activity, migration, and differentiation of cells. In the tumor environment, the cytokine secretion is a relevant communication pathway between tumor and immune cells. The significance of the cytokine environment becomes clear considering the fact that for example the differentiation – and thus the function – of CD4+ T cells is mainly controlled by cytokines. They influence if a T H 1 cell with anti-tumoral activity develops or a Treg cell with immunosuppressive function 57 . The group of cytokines includes interleukins, chemokines, tumor necrosis factors (TNF), interferons, and the colony stimulating factors (CSF).

Interleukins are peptide hormones belonging to the cytokines that influence cell growth and differentiation and that are generated by all cells of the immune system. They may have a stimulating as well as inhibiting effect on growth, division, and especially differentiation of other immune cells. Because of their mostly pleiotropic effects, interleukins influence several phenotypic characteristics. Meanwhile, more than 40 interleukins with different physiological functions are known that are numbered chronologically according to their discovery. In the tumor environment, interleukins mostly work in a paracrine way.

Due to their function, interleukins are appropriate for therapeutic use in order to influence the communication and the control of the immune system. During the last decades, several immunotherapeutic approaches were developed on the basis of interleukin therapies 58 . They include the interleukins IL2, IL7, IL12, IL18, and IL21 57 . One of the most important interleukins applied in therapeutic approaches is IL2. As already mentioned, it plays a major role in the activation and proliferation of T cells. It is the first interleukin that has been applied as anticancer drug in humans and that could achieve therapeutic success. Historically, IL2 as T cell growth factor was discovered in 1976 59 ; in 1994 it was approved as drug for treatment of metastatic renal cell carcinoma and in 1998 in the USA for metastatic malignant melanoma. The systemic application of IL2 especially for metastatic tumors bears the disadvantage of significant side effects. Under certain circumstances, IL2 may lead to the so-called vascular leak syndrome, i. e., the permeability of the vascular walls is strongly increased because of endothelial cell mediated hyperpermeability. This leads to extravasation that might limit treatment 60 . For a small part (8%; 33 of 409) of the treated patients, however, a complete long-lasting (median observation interval>7 years) remission of metastatic renal cell carcinomas (9.3%) and melanomas (6.6%) could be achieved 61 . Further interleukins are currently investigated in clinical trials 62 .

Chemokines have a chemotactic effect on other cells and are increasingly released in cases of inflammation or other acute events. Among others, chemokines seem to be associated with increased angiogenesis 63 . Interestingly, the chemokine receptor expression in tumor-infiltrating lymphocytes is down-regulated which is explained by an increased internalization of the receptors due to the strong concentration in the tumor environment 64 . Clinical trials also investigated monoclonal antibodies against chemokine receptors in patients with T cell malignoma 65 . In addition, a current trial deals with the chemokine modulation in patients with colon cancer ( NCT01545141 ).

Tumor necrosis factors (TNFα and TNFβ) contribute to the proliferation, differentiation, apoptosis, necrosis, angiogenesis, and activation of the immune system. TNF further influence the fat metabolism; insulin resistance, and endothelial cells work systemically via fever and may cause cachexia (TNFα was formerly called cachexin). In cancer therapy, TNFα is applied for malignant melanoma and soft tissue sarcomas 66 . In the tumor itself, it induces hyperpermeability of the vessels with resulting hemorrhagic necrosis and destruction of the vascular structures 67 . However, the initial expectations to broadly apply TNFα-based cancer therapy could not be confirmed. This is sometimes explained by the pleiotropic characteristic of TNFα and the different mode of action depending on the timely course.

Interferons are pleiotropic cytokines that are generated from stroma cells as well as immune cells and that have a broad immuno-stimulating effect by activating transcription proteins (Jak-STAT signaling pathway) and an increased expression of components of antigen presentation (e. g., MHC molecules). Physiologically, the interferon generation occurs mainly after activation by viral or bacterial antigens. In humans, IFNα, IFNβ (type I interferon), and IFNγ (type II interferon) are distinguished. IFNα and IFNβ enhance the MHC I expression, activate dendritic cells, T cells and NK cells 68 and inhibit the development of immunosuppressive Treg and MDSC 69 70 . Additionally, they have an effect on tumor cells and lead to increased differentiation and increased tumor antigen presentation 71 . IFNγ mainly activates CD8+ T cells and enhances the MHC II expression as well as the development of macrophages of the M1 phenotype 68 . On the other hand, also an interferon-dependent immunosuppressive effect in the tumor environment was observed in the last years 72 . It is based on an increased expression of a checkpoint receptor ligand (programmed death receptor ligand 1; PDL1) after interferon stimulation. Thus, also inhibiting effects are increasingly observed 73 . In this context, the duration of the stimulation may play an important role and make the difference between an acute inflammatory reaction with tumor inhibiting properties and a chronic inflammatory reaction with tumor progressing effect. This observation has significant consequences for the clinical application. The immune activating properties of interferons are applied for therapy in order to treat for example viral hepatitis (IFNα). But also in cancer therapy, the treatment with interferons is applied against specific tumor entities (e. g., special lymphomas, leukemia, Kaposi sarcomas, or malignant melanoma). Also hereby, it must be taken into account that partly severe side effects may limit the treatment. Many efforts are undertaken to optimize the effect and at the same time reduce the side effects 74 . In the context of malignant melanoma, a marginally significant impact on the progression-free survival after resection of stage III melanoma could be confirmed in comparison to clinical observation strategy 75 . The application of interferon in cancer therapy depends from the tumor entity, the spectrum of side effects, and the associated patients’ compliance so that this treatment has to the discussed individually with the patient.

Colony stimulating factors are glycoproteins that influence the proliferation and differentiation of cells originating from the hematopoietic system. Several CSF are classified: G-CSF (granulocytes), M-CSF (monocytes), GM-CSF (granulocytes and monocytes), Meg-CSF (megakaryocytes), and the SCF (stem cell factor). Furthermore, some interleukins, e. g., IL2 as well as erythropoietin, range among CSF because they also act on the proliferation and differentiation of cells of the hematopoietic system. The generation and secretion are performed in the bone marrow, the stroma, and in immune cells (B and T cells, macrophages etc.). In oncology, CSF are applied as adjuvant agents in order to achieve restitution of the hematopoietic cell lines after suppression. However, CSF are also investigated in active tumor treatment. As already described, the number of intratumoral TAM is associated with a poor prognosis. Efforts are undertaken to block the receptor of M-CSF to impede the survival of macrophages and thus reduce the number of TAM. Phase I and II studies show a limited to moderate effect in the monotherapy with moderate side effect profile 76 77 . GM-CSF is also applied in oncological immunotherapy because it could be shown that differentiation of the dendritic cells, suppression of the MDSC and development of the M1 phenotype were induced 78 79 .

3.2. Toll-like receptor stimulation

Pattern recognition receptors (PRR) recognize molecules that are associated with pathogens such as for example viruses and bacteria (pathogen-associated molecular patterns, PAMP). Those receptors initiate a defense reaction and belong to the epigenetically oldest components of immune response. Toll-like receptors (TLR), a main group of PRR, are widespread within the different species. They were discovered in the middle of the 1990ies for the first time in drosophila melanogaster. Since then, 10 different TLR subtypes were identified in humans. The activation of TLR leads to an intracellular signaling cascade ( Fig. 8 ) that causes mainly a differentiation of cells of myeloid origin and stimulates their maturation and proliferation. In addition, also cells of adaptive defense are activated. The immunologically activating effect of single TLR antagonists was examined in different cancer entities and led to the approval of single immunotherapeutics of this substance class. Among those are the TLR2/4 agonists Bacillus Calmette-Guérin (BCG, inactivated mycobacterium bovis) for bladder cancer, the TLR4 agonist Picibanil for head and neck cancer 80 , or the TLR7 agonist Imiquimod for basal cell cancer. Resiquimod, which is a potent TLR7 and TLR8 agonist, is currently clinically developed as successor product. Another TLR8 agonist that is currently under clinical investigation, is Motolimod for head and neck cancer and other solid tumors ( NCT01836029 ). Furthermore, TLR agonists are meanwhile frequently combined with vaccinations because of the promotion of maturing and differentiating antigen presenting cells in order to support antigen presentation 81 .

3.3. Oncolytic viruses

Oncolytic viruses may directly or indirectly destroy tumor cells. In a narrow sense, oncolytic viruses infect tumor cells and lyse them. However, there are other possibilities to attack tumor cells by means of virus-based methods. Among those, there is the inclusion of tumor suppressor genes and toxins into the tumor cell or the generation of an immune response with subsequent tumor cell destruction. So oncolytic viruses are applied that may directly cause tumor cell death as well as stimulate the systemic immune response 82 . Trials analyzing the application of oncolytic viruses, are already performed in larger phase II and III studies. A phase III study in patients with advanced malignant melanoma could reveal improved response rates with herpes simplex based viral therapy (talimogene laherparepvec, T-VEC) 83 . Furthermore, trials are currently performed with regard to hepatocellular carcinomas (phase II, pexastimogene devacirepvec, Posavec, NCT01387555 ) and to head and neck cancer (phase III, pelareorep, Reolysin combined with chemotherapy, NCT01166542 ) 84 .

3.4. Monoclonal antibodies

In 1975, Kohler and Milstein developed a technology allowing the production of nearly unlimited quantities of a single, epitope-specific antibody 85 . By merging an antibody-producing B cell with an immortal “myeloma cell” a ‘hybridoma’ results that produces an antibody of an original B cell: it is then called a monoclonal antibody. In 1984, Kohler and Milstein were awarded the Nobel Prize in Physiology or Medicine together with Niels Jerne for their achievements in this context. This award honored in particular the application options to produce highly specific antibodies in theoretically unlimited quantities. This was the breakthrough for the application of antibodies in research and clinic because now antibodies were available in sufficient quantities in order to be able to influence cellular signaling pathways on a molecular level.

In the beginning, the therapeutic application of monoclonal antibodies was suitable only to a limited extent. Since they were generally harvested in mice, they were “foreign” for humans and induced an immune response called HAMA reaction (human antibodies against mouse antibodies). Meanwhile, antibodies can be produced that are partly or even completely of human origin. The terminology of therapeutic antibodies provides information about the origin and composition. The suffix -omab defines antibodies of murine origin; -imab means antibodies with origin of primate species; -ximab describes chimeric antibodies (variable percentage of murine origin, remaining part of human origin); -zumab defines humanized antibodies (murine antigen binding, remaining part of human origin), and -umab describes completely human antibodies ( Fig. 9 ).

The therapeutic spectrum where mAb are meanwhile applied is very broad. It ranges from autoimmune diseases such as rheumatoid arthritis via hematological diseases such as hemophilia up to oncological diseases such as malignant melanoma, bronchial cancer, or even head and neck cancer 86 . Oncology is the main focus of development, research, and application of monoclonal antibodies that are mostly targeted against tumor antigens or checkpoint receptors.

3.4.1. mAb against tumor antigens

Tumor antigens are protein structures that are produced by tumor cells and may trigger an immune response. Tumor-specific antigens (TSA) and tumor-associated antigens (TAA) are currently known. TSA are produced exclusively by tumors and develop for example based on mutations in a protein-coding gene. In contrast, TAA are also present on healthy cells but they are expressed to a clearly higher amount on tumor cells. Examples in this context are TAA of the ErbB family, a receptor family of 4 tyrosine kinase receptors. Two members of this family, ErbB-1 (EGFR) and ErbB-2 (Her2) are of high relevance in oncology. EGFR is overexpressed in most head and neck cancers, non-small-cell lung cancer (NSCLC), and colorectal carcinomas, Her2 in breast and ovarian carcinomas 87 . Antibodies against EGFR and Her2 have been applied for several years in clinical routine (Cetuximab, Trastuzumab, Table 1 ) 88 . Unfortunately, the response rates to those therapies are significantly below the expression rates so the new developments focus on combined therapies in order to avoid tumor cell resistances more effectively.

3.4.2. mAb against checkpoint molecules

A new possibility to intervene specifically into the signaling pathways of the immune system results from the introduction of mAb that are able to stimulate or block checkpoint molecules. Checkpoint molecules are mainly receptors on cells of the immune system, mostly lymphocytes, that have a stimulating or inhibiting effect.

Especially the molecules CTLA4 and PD1 have to be emphasized among the group of checkpoint molecules because in the last years significant therapeutic success could be achieved in different tumor entities with therapeutic antibodies that are targeted against those molecules ( Table 2 ). PD1 is a receptor for the ligands of PDL1 and PDL2. Especially binding PDL1 leads to T cell anergy ( Fig. 10 ), i. e., the IFNγ secretion and proliferation of T cells are suppressed. In the tumor environment an expansion of activated, tumor-reactive T lymphocytes is inhibited 8 . Interestingly, PD1 is also strongly expressed on Treg, but here it leads to an increased functionality 89 . PD1 blockage with the mAb Nivolumab was first applied successfully in 2010 in a clinical phase I study 90 . Since then, long-term success could be achieved with even sometimes complete regression of malignant melanoma, renal cell carcinoma, or colorectal cancer over 3 years 91 .

CTLA4 is expressed on T cells and induces immunosuppression via 2 relevant mechanisms. On the one hand it acts as competitive binding partner for the surface molecules CD80 and CD86 on antigen presenting cells because of its structural similarity to the co-stimulating molecule CD28 and thus impedes the activation of T cells ( Fig. 11 ). On the other hand the binding of CTLA4 directly inhibits T cells, also by inactivating the T cell receptors. Anti CTLA4 mAb were the first immune checkpoint mAb that were clinically tested 92 and that achieved significant, long-lasting therapeutic success 2 .

2.4.2.1. Immuno-stimulating checkpoint molecules

The immuno-stimulating checkpoint molecules include CD27, CD28, CD40, CD122, CD134 (OX49), CD137, CD278 (ICOS) and GITR (glucocorticoid-induced TNFR family related gene) ( Table 3 ) 93 . CD 27 is important for the induction of memory T cells and supports the T cell expansion. CD28 is a relevant co-stimulating T cell receptor that is necessary for activation of CD4+ T cells and binds CD80 and CD86 of antigen presenting cells. CD40 is expressed on antigen presenting cells and is activated by its ligand CD40L that is expressed on CD4+ T cells. CD122 is expressed on CD8+ T cells and supports their proliferation. CD134 (OX40) is expressed on CD4+ and CD8+ T cells and also enhances proliferation. In addition, stimulation by means of OX40 inhibits the development of Treg cells. CD137 is expressed on CD8+ T cells, it stimulates the proliferation and inhibits the activation-related induction of apoptosis. CD278 (inducible T cell co-stimulator, ICOS) is expressed on activated T cells and interacts with antigen presenting cells and B cells. GITR stimulates the expansion of T cells and is also stimulated by antigen presenting cells. For many of the stimulating checkpoint receptors, currently clinical trials are conducted in order to generate an enhanced, long-lasting immune response via pharmaceutic ligands.

2.4.2.2. Inhibiting checkpoint molecules The inhibiting checkpoint molecules that could be identified up to now include the receptors A2AR (adenosine A2A receptor), B7-H3, B7-H4, BTLA (B and T lymphocyte attenuator), CTLA4, KIR, LAG3, PD1, TIM3 (T cell immunoglobulin and mucin domain 3), and VISTA (V-domain Ig suppressor of T cell activation) ( Table 4 ). Via adenosine binding in the tumor environment, A2AR mediates a suppressing activity on cells of the immune system 94 . B7-H3 and B7-H4 are expressed on tumor cells, inhibit T cells and promote tumor migration 95 . BTLA is expressed on T cells and leads to inhibition of the T cell activity after binding 96 . KIR are particularly expressed on NK cells and may have an inhibiting effect on the NK cell function 97 . LAG3 acts especially over the MHC II binding on CD4+ T cells in an immunosuppressive way and is an important molecule in the suppressing function of Treg cells 98 . TIM3 is responsible for the activation of macrophages, at the same time, apoptosis of T H 1 cells can be induced by binding galectin-9 99 . VISTA was identified as another immuno-regulatory checkpoint in refractory melanoma patients 100 .

Other mAb are targeted against cytokines or growth factors ( Table 5 ). The intra- and extracellular signaling pathways of many of the described molecules are not yet completely clarified – so that several effective mechanisms are not yet identified. A clear classification can only be made in a carefully restricted way because many signaling pathways in immunology may induce stimulating as well as inhibiting effects. This may not only lead to contradictory pre-clinical findings but also to various therapy responses. So it is even more important to analyze the effect of the signaling pathways already in pre-clinical trials and to critically evaluate and verify the in vitro results with valid in vivo data.

3.5. Vaccination

In oncology, 2 vaccination strategies have to be differentiated: On the one hand, a preventive vaccination before the development of cancer may be applied. This is possible in the context of vaccination against oncogenic viruses and can have a protective effect against virus-associated carcinomas. On the other hand, efforts are undertaken to establish therapeutic vaccination in other, i. e., not exclusively viral, tumors.

Preventive vaccination to avoid infections with oncogenic viruses turned out to be highly effective to avoid virus-associated cancer. The development of vaccinations against high-risk virus subtypes of oncogenic human papilloma viruses (HPV) 16 and 18 could achieve a protection against HPV infections 101 . For his research that led to the discovery of a correlation between HPV infections and cervical cancer, Harald zur Hausen received the Nobel Prize in Physiology or Medicine in 2008 102 . So there is meanwhile the recommendation for young women to vaccinate against the high-risk subtypes, possible before first sexual contact. Meanwhile it is known that oncogenic HPV infections in the western world are not only responsible for almost all cervical carcinomas, but also for more than 90% of anal carcinomas, 70% of oropharyngeal carcinomas, 70% of vagina carcinomas, 40% of vulva carcinomas, and 50% of penile carcinomas 101 . Because of the high incidence of HPV16-positive oropharyngeal cancers, investigations are performed if vaccination might have a protective effect with regard to the incidence of head and neck cancer 103 . So also the vaccination of boys and young men is discussed 104 . The impact on those epidemiologic rates in the context of vaccination of young women that had started in the last years will have to be analyzed in the upcoming years.

In contrast to preventive vaccination that immunize against viral oncogenic structures, also therapeutic vaccinations against tumor cell antigens are currently developed. Unlike the therapy with mAb, the immune system is intended to be activated against tumor antigens in order to mobilize specific T cells, antibodies, and other components especially of the adaptive immune response against tumor cells. This activation is based on the interaction of dendritic cells and T cells. In this context, different techniques exist how dendritic cells are confronted with tumor antigens. Either the DC are charged with tumor lysates, proteins, or peptides or they are transfected with DNA or RNA 105 .

Active oncologically effective vaccinations have not been broadly established in the clinic. Especially for in situ carcinomas, however, clinical studies could achieve substantial success. In cases of intraepithelial neoplasms, e. g., the vulvar intraepithelial neoplasm (VIN), vaccination against HPV16 oncoproteins led to regression of the lesion and the therapy response correlated directly with the T cell response induced by vaccination 106 107 108 . Similar results were revealed by Czerniecki and colleagues for ductal carcinoma in situ (DCIS) with vaccination targeted against Her2/neu 109 . Morse and colleagues confirmed a significantly higher total survival in patients with resected colon carcinoma metastases after vaccination against the tumor antigens CEA and MUC1 110 . In a phase I study about vaccination against the tumor antigen p53, head and neck cancer patients showed a 2-year-survival rate of 88% 111 .

One difficulty that might also influence the effectiveness of vaccination is the immunosuppressive tumor environment. The development of Treg, TAM, and MDSC promotes immunosuppressing signaling pathways that complicate the expansion of tumor antigen specific T cells. To avoid this, Mould and colleagues described for example a therapeutic efficiency enhancement by using multiple locations for vaccination 112 . Other strategies focus on the combination with other immune stimulants such as for example toll-like receptor agonists 113 .

3.6. Adoptive cell transfer

In the context of adoptive cell transfer, lymphocytes (mainly T cells, but also dendritic cells, NK cells and others) are isolated from the patients’ peripheral blood. Afterwards, tumor antigen specific (T) cells may be produced or expanded that are then re-infused to the patient 114 115 ( Fig. 12 ). An advantage of the adoptive cell transfer is that lymphocytes can be influenced and expanded outside the immunosuppressive tumor environment 115 .

First clinical trials on adoptive cell transfer were already conducted in the 1990ies 61 . Hereby, a therapy response could be achieved in about 30% of the melanoma patients. Meanwhile, remission rates of up to 90% of the patients are reported in the context of specific tumor entities, e. g., patients with acute CD19+ leukemia 116 . “Pre-conditioning” contributes relevantly, consisting of a pre-treatment with lymphatic depletion therapy 117 . In this way, (also) suppressive immune cells such as regulatory T cells or MDSC are reduced that would inhibit the effectiveness of re-infused antigen-specific T cells. Antigen-presenting cells are needed for successful antigen-specific expansion of the cells. APC that are used for adoptive cell transfer for antigen presentation include natural dendritic cells, artificial cells, or “beads” charged with antigens. T cells may be gained from peripheral blood or from the tumor. Then expansion of tumor antigen-specific cells is performed ex vivo with different methods to a cell number of more than 10 9 to 10 11 cells 118 . Selection of the tumor antigen-specific T cells is performed after isolation of single T cell lines that are then tested with regard to their reactivity against different tumor antigens. In this way, T cell lines are expanded that reveal the highest reactivity against the presented tumor antigens. Modern methods use a genetically determined antigen specificity of T cells and can thus be adapted more precisely and variably to specific tumor antigens. Beside the advantage to select extracorporal antigen-specific T cells and to expand them in high quantities, this expansion is not influenced by the immunosuppressive tumor environment. T cells can be expanded in a functional stage. In this context, high-quality expansion of the T cells has to be assured in order to preserve the tumor antigen specificity of the T cells. So not only the differentiation of the T cells is important but also the cellular metabolic processes 119 . In order to stimulate the re-infused expanded T cells, cytokines are applied 120 .

The use of a chimeric antigen receptor (CAR) instead of a tumor antigen-specific T cell receptor is called CAR-T cell transfer. The chimeric antigen receptors consist of an antigen-binding component – for example an antibody – as well as another T cell activating, co-stimulating component to increase the effectiveness of the T cell response 121 . This co-stimulating component influences the cytokine secretion and may stimulate T cell proliferation. Regarding CAR-T cells of the third generation, 2 or more immuno-stimulating domains are integrated in the receptor. The applied co-stimulating molecules include CD28, OX-40 (CD134), or 4-1BB (CD137). A particular effectiveness of those therapeutic methods could be revealed for hematological diseases. Especially in B cell malignomas, a good response rate and clinical effectiveness were observed (NCT02345849) 116 122 . In solid tumors, convincing results of this dimension are not yet available, which is mainly due to the fact that the identification of specific tumor antigens is more complicated. Up to now, trials on CAR-T cell therapies were performed for Her2+ tumors, EGFR+ tumors, CEA+ (carcinoembryonic antigen) tumors, and mesothelin+ tumors 123 124 125 126 .

4. Chances and risks

One major task of medical activity is to weigh the potential benefit against the risks of therapeutic measures. This applies especially in the context of oncology because hereby therapeutic measures may strongly impair the patients’ quality of life. So it is important to consider also the side effects and possible risks of different immunotherapeutic procedures.

Since the immunotherapeutic principle is based on stimulating the immune system and on eliminating inhibiting effects of the tumor environment, many side effects arise because of excessive autoimmune processes. Typically, the profile of the side effects depends on the spectrum of the immunotherapeutic approach. The more pluripotent the influenced signaling pathways are, the higher is the risk of possible side effects.

This is especially true for cytokine-based immunotherapies. So IL2 may be associated with side effects like nausea, vomiting, gastrointestinal complaints, severe malaise, increased capillary permeability, cardiac damage, and low blood pressure 61 . Many of those side effects are so severe that they require discontinuation of the therapy. Regarding IL21, similar side effects were described, especially fever, chills, liver damage, and effects on the hematopoietic system with neutropenia and thrombocytopenia 127 . Beside leukopenia, interferons may induce vertigo, anorexia, and depression 68 .

With regard to monoclonal antibodies targeted against tumor antigens, especially 2 aspects influence the development and the profile of side effects: on one hand there is the above-mentioned composition of the antibody. An increasing “human” percentage reduces the foreign body reaction and allergic reaction against medication is avoided. Meanwhile, this may be achieved by molecular biological modification of the antibody. On the other hand, it is also important to know if the target molecule is present on healthy cells. The more unspecific the tumor antigen is, the severer are the observed side effects. Additionally, a high tumor load may lead to an uncontrolled release of cytokines by cytolysis (cytokine release syndrome). mAb acting against tumor antigen may cause fever, chills, respiratory complaints, and rashes. In rare cases, treatment with Rituximab (anti-CD20 mAb) may lead to progressive multifocal leukencephalopathy (PML) 128 . One typical side effect that may be caused by the anti-EGFR mAb Cetuximab is rashes, of which the etiology is still not completely clarified 129 . The anti-Her2 mAb Trastuzumab may cause in particular cardiac side effects that require regular controls of the cardiac function during and after therapy 130 . Bevacizumab, however, often causes gastrointestinal complaints as well as arterial hypertension and proteinuria 131 .

mAb that are directed against checkpoint molecules increase the immune reaction. This might lead to inflammatory reactions in the body. Nivolumab, an anti-PD1 mAb, may also cause arthritis, colitis, and in particular pneumonitis. Ipilimumab, an anti-CTLA4 mAb, is associated with side effects that concern among others skin, liver, eyes, gut, and the pituitary gland.

Also vaccination therapies may lead to vaccination reactions and other side effects. Even the specific T cell receptor selection in the context of the adoptive cell transfer may cause antigen-related severe side effects. So currently methods are being developed that reduce the probability of such complications. Kunert and colleagues suggest specific methods to allow a risk assessment with regard to the toxicity by means of TCR selection 132 . Among others, this includes the analysis of comprehensive genomic databases regarding the observation if target antigens also appear in other healthy tissues and organs. Another approach is the development of combined therapies that allow a synergistic therapeutic effect with at the same time reduction of the single dose and associated dose-related side effects.

5. Current development

Especially the checkpoint inhibitors achieved an outstanding clinical relevance in the context of immunotherapy in the last years. Up to now, CTLA4, PD1, and PDL1 inhibitors are approved for oncological therapy. The anti-CTLA4 mAb Ipilimumab (Yervoy®, Bristol-Myers Squibb) has been approved in Europe for the treatment of advanced metastatic or non-resectable malignant melanoma since 2011. Since 2015, PD1 inhibitors are available for several tumor entities for treatment of advanced metastatic or non-resectable malignant melanoma. As second-line therapy, it is additionally approved for the treatment of advanced non-small-cell bronchial carcinomas, advanced renal cell carcinomas, and advanced Hodgkin lymphomas. Furthermore, refractory head and neck cancer and urothelial carcinomas wait for approval as indication of anti-PD1 therapy. The already approved anti-PD1 mAb include Nivolumab (Opdivo®, Bristol-Myers Squibb) and Pembrolizumab (Keytruda®, Merck Sharp & Dohme). In March 2017, the FDA approved the anti-PDL1 mAb Avelumab (Bavenico®, Merck KGaA, Pfizer and Eli Lilly and Company) for the treatment of metastatic Merkel cell carcinoma in the USA. Atezolizumab (Tecentriq®, Genentech/Roche), also a PDL1 mAb, has been approved in the USA since 2016 for treatment of advanced or metastatic urothelial carcinomas and pre-treated metastatic non-small-cell lung carcinomas. The number of clinical studies conducted with regard to checkpoint modulators is tremendous. For head and neck cancer alone, more than 45 clinical phase I–III studies have been performed since 2010 133 . The majority of the studies investigates medications that influence the PD1/PDL1 signaling pathway. But also other immuno-stimulating and inhibiting checkpoint molecules are in the focus of research. In a phase I study, one mAb for stimulation of OX40 revealed regression of metastatic findings in one third of the patients ( NCT01644968 , 134 ). Also other stimulatory molecules such as CD137 or inhibitory molecules such as LAG3 are clinically investigated ( NCT02658981 ).

5.1. Biomarkers

Not all patients benefit from newly established therapies; the reasons are mostly unknown. The already mentioned side effects and high therapy costs require the identification of immunotherapeutic predictive biomarkers that allow prognosis regarding the therapeutic response and thus therapy selection. However, investigations show a very heterogenic picture. Especially PDL1 expression was evaluated in different trials with partly contradictory results. This is explained by the fact that different standards for PDL1 determination exist. Additionally, different limit values are set for positivity. Furthermore, the PDL1 expression as biomarker depends on the tumor entity. The PDL1 expression is established as biomarker for non-small-cell bronchial carcinomas rather than for head and neck cancer 135 . A therapy response after inhibition of the PD1/PDL1 signaling pathway was observed also in PDL1 negative patients 136 . Up to now, reliable biomarkers are missing in the field of checkpoint immunotherapy. Recently published investigations of patients with head and neck cancer indicate that the level of PD1 expression of CD8+ cells is associated with the cellular functionality and the overall survival of the patients 137 .

The response rates of immunotherapy do not only depend on the medication and the tumor entities but also on the individual patient. Since the response rates vary and amount to less than 50% in cases of monotherapies, combined therapies are increasingly applied in order to achieve therapeutic success in as many patients as possible. On one hand, this increases the chance that patient-specifically relevant signaling pathways can be influenced; on the other hand, potentially synergistic therapeutic effects may be achieved.

5.2. Combination therapies

For combination, especially checkpoint receptor therapies are very interesting because they directly approach antitumor-specific cells and block a mechanism that possibly impedes the effectiveness of other therapeutic procedures. This is true for classic therapeutic procedures such as radiotherapy that induces PDL1 expression 138 as well as for other immunotherapeutic approaches. The checkpoint receptor therapy is also applied in combination with cytokine therapies and vaccination strategies.

Pre-clinical studies investigated for example the combination of a GM-CSF modified prostate carcinoma vaccine with a PDL1 blockade 139 because the effectiveness of current vaccination therapies had been suppressed by PDL1 induction. In this murine trial a strong increase of CD4+ and CD8+ T cells with enhanced IFNγ secretion could be observed, which was associated with tumor regression. A similar result could be achieved with the oncolytic virus Onkovex in combination with GM-CSF and anti-CTLA4 140 . In this model, a systemic effect due to treatment success in not directly injected tumors (50%) could be observed as well as a locally higher effectiveness (100%) in directly injected tumors. In addition to direct oncolytic therapy in the tumor, CD8+ T cell reaction was observed in the non-injected tumor.

In a clinical study, Hodi and colleagues investigated the combination of GM-CSF (Sargramostim) with the anti-CTLA4 mAb Ipilimumab in patients with non-resectable malignant melanoma stage III or IV 141 and compared this therapy with Ipilimumab monotherapy. In the context of this study, no significant difference could be observed with regard to the progression-free survival but a significant increase of the overall survival and a reduction of the side effects due to combination therapy.

Currently the expectations of adoptive T cell therapy together with checkpoint molecules are very high. Based on promising results, the first CAR-T cell therapy was approved in August 2017 in the USA. CTL019 (Tisagenlecleucel®, Novartis) is applied in children and adolescents for therapy of acute refractory B cell leukemia. For the future, a significant increase of approvals and the associated indications is expected. A combination of adoptive T cell therapy intervening in the PD1 signaling pathway of the CAR-T cells is currently investigated in a phase I study in patients with metastatic non-small-cell bronchial carcinoma ( NCT02793856 ). By means of modern technique of the CRISPR/Cas method, the genome of the T cells is specifically and exactly modified and the PD1 expression is eliminated. T cells modified in this way are then re-infused after selection and proliferation.

6. Summary and outlook

The interactions in the tumor environment are highly complex and very challenging regarding oncological therapy approaches. Nevertheless, oncological immunotherapy could achieve relevant progress in the last years. For some tumor entities successful results could be observed with long-term therapy response rates, sometimes even with complete remission. But not all patients benefit from those developments. For many tumor entities the response rates are limited in the context of monotherapeutic approaches. However, progress in basic and clinical research is the main precondition for an improved understanding of the interaction between tumor and immune system. This allows new therapeutic combinations in order to use synergistic effects. Especially the development of checkpoint-specific antibodies eliminating the blockade of immune inhibiting signaling pathways seems to be very promising and allows combinations with other therapeutic strategies that up to now have not been successful because of this inhibition. At the same time, immuno-stimulating signaling pathways may enhance cells that have been suppressed by the tumor environment leading to an improved immune response. Establishing predictive markers and thus improving the patient selection for different therapeutic modalities will gain in importance in the future. Because immune therapy in oncology has such a potential, critical and reflected assessment of the study results and therapeutic options is essential in order to implement their current and future importance in clinical routine.

Annotation : The figures in this contribution were created with Microsoft Powerpoint 2016© (Microsoft Corporation, Redmond, USA) and Science Slides© (VisiScience Inc., Chapel Hill, USA).

Danksagung

Wir danken Herrn Prof. Dr. Sven Brandau und Herrn Prof. Dr. Reinhard Zeidler für das kritische Korrekturlesen und die wertvollen Kommentare bei der Erstellung dieses Beitrages.

Acknowledgements

We express our thankfulness to Prof. Dr. Sven Brandau and Prof. Dr. Reinhard Zeidler for their critical corrections and valuable comments on this article.

Interessenkonflikt Die Autoren geben an, dass kein Interessenkonflikt besteht.

Literatur

1 Coley W B II Contribution to the Knowledge of Sarcoma Annals of surgery 1891 14 199 220 10.1097/00000658-189112000-00015 PMC1428624 17859590

2 Hodi F S O'Day S J McDermott D F Improved survival with ipilimumab in patients with metastatic melanoma N Engl J Med 2010 363 711 723 20525992 10.1056/NEJMoa1003466 PMC3549297

3 Topalian S L Hodi F S Brahmer J R Safety, activity, and immune correlates of anti-PD-1 antibody in cancer N Engl J Med 2012 366 2443 2454 22658127 10.1056/NEJMoa1200690 PMC3544539

4 Couzin-Frankel J Breakthrough of the year 2013. Cancer immunotherapy Science (New York, NY) 2013 342 1432 1433 10.1126/science.342.6165.1432 24357284

5 Mantovani A Sozzani S Locati M Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes Trends in immunology 2002 23 549 555 12401408 10.1016/s1471-4906(02)02302-5

6 Komohara Y Jinushi M Takeya M Clinical significance of macrophage heterogeneity in human malignant tumors Cancer science 2014 105 1 8 24168081 10.1111/cas.12314 PMC4317877

7 Rosenberg S A Restifo N P Adoptive cell transfer as personalized immunotherapy for human cancer Science (New York, NY) 2015 348 62 68 10.1126/science.aaa4967 PMC6295668 25838374

8 Keir M E Butte M J Freeman G J PD-1 and its ligands in tolerance and immunity Annual review of immunology 2008 26 677 704 10.1146/annurev.immunol.26.021607.090331 PMC10637733 18173375

9 Dunn G P Bruce A T Ikeda H Cancer immunoediting: from immunosurveillance to tumor escape Nature immunology 2002 3 991 998 12407406 10.1038/ni1102-991

10 Burnet F M The concept of immunological surveillance Progress in experimental tumor research 1970 13 1 27 4921480 10.1159/000386035

11 Mittal D Gubin M M Schreiber R D New insights into cancer immunoediting and its three component phases — elimination, equilibrium and escape Current opinion in immunology 2014 27 16 25 24531241 10.1016/j.coi.2014.01.004 PMC4388310

12 Teng M W Vesely M D Duret H Opposing roles for IL-23 and IL-12 in maintaining occult cancer in an equilibrium state Cancer Res 2012 72 3987 3996 22869585 10.1158/0008-5472.CAN-12-1337 PMC4384890

13 Grandis J R Falkner D M Melhem M F Human leukocyte antigen class I allelic and haplotype loss in squamous cell carcinoma of the head and neck: clinical and immunogenetic consequences Clin Cancer Res 2000 6 2794 2802 10914726

14 Mizukami Y Kono K Maruyama T Downregulation of HLA Class I molecules in the tumour is associated with a poor prognosis in patients with oesophageal squamous cell carcinoma Br J Cancer 2008 99 1462 1467 18841157 10.1038/sj.bjc.6604715 PMC2579690

15 Ogino T Shigyo H Ishii H HLA class I antigen down-regulation in primary laryngeal squamous cell carcinoma lesions as a poor prognostic marker Cancer Res 2006 66 9281 9289 16982773 10.1158/0008-5472.CAN-06-0488

16 Goeppert B Frauenschuh L Zucknick M Major histocompatibility complex class I expression impacts on patient survival and type and density of immune cells in biliary tract cancer Br J Cancer 2015 113 1343 1349 26461054 10.1038/bjc.2015.337 PMC4815783

17 Zhang J Xu Z Zhou X Loss of expression of MHC class I-related chain A (MICA) is a frequent event and predicts poor survival in patients with hepatocellular carcinoma International journal of clinical and experimental pathology 2014 7 3123 3131 25031731 PMC4097291

18 Watson N F Ramage J M Madjd Z Immunosurveillance is active in colorectal cancer as downregulation but not complete loss of MHC class I expression correlates with a poor prognosis Int J Cancer 2006 118 6 10 16003753 10.1002/ijc.21303

19 Wang J H Bi X W Li P F Overexpression of MYC and BCL2 Predicts Poor Prognosis in Patients with Extranodal NK/T-cell Lymphoma, Nasal Type Journal of Cancer 2017 8 793 800 28382141 10.7150/jca.17766 PMC5381167

20 Wang T Niu G Kortylewski M Regulation of the innate and adaptive immune responses by Stat-3 signaling in tumor cells Nature medicine 2004 10 48 54 10.1038/nm976 14702634

21 Gastman B R Atarshi Y Reichert T E Fas ligand is expressed on human squamous cell carcinomas of the head and neck, and it promotes apoptosis of T lymphocytes Cancer Res 1999 59 5356 5364 10537320

22 Lu P Weaver V M Werb Z The extracellular matrix: a dynamic niche in cancer progression The Journal of cell biology 2012 196 395 406 22351925 10.1083/jcb.201102147 PMC3283993

23 Paszek M J Zahir N Johnson K R Tensional homeostasis and the malignant phenotype Cancer Cell 2005 8 241 254 16169468 10.1016/j.ccr.2005.08.010

24 Mott J D Werb Z Regulation of matrix biology by matrix metalloproteinases Current opinion in cell biology 2004 16 558 564 15363807 10.1016/j.ceb.2004.07.010 PMC2775446

25 Levental K R Yu H Kass L Matrix crosslinking forces tumor progression by enhancing integrin signaling Cell 2009 139 891 906 19931152 10.1016/j.cell.2009.10.027 PMC2788004

26 Pickup M W Mouw J K Weaver V M The extracellular matrix modulates the hallmarks of cancer EMBO reports 2014 15 1243 1253 25381661 10.15252/embr.201439246 PMC4264927

27 Meyaard L The inhibitory collagen receptor LAIR-1 (CD305) Journal of leukocyte biology 2008 83 799 803 18063695 10.1189/jlb.0907609

28 Vesely M D Kershaw M H Schreiber R D Natural innate and adaptive immunity to cancer Annual review of immunology 2011 29 235 271 10.1146/annurev-immunol-031210-101324 21219185

29 Fox S B Gasparini G Harris A L Angiogenesis: pathological, prognostic, and growth-factor pathways and their link to trial design and anticancer drugs The Lancet Oncology 2001 2 278 289 11905783 10.1016/S1470-2045(00)00323-5

30 Fayette J Soria J C Armand J P Use of angiogenesis inhibitors in tumour treatment Eur J Cancer 2005 41 1109 1116 15911233 10.1016/j.ejca.2005.02.017

31 Li H Fan X Houghton J Tumor microenvironment: the role of the tumor stroma in cancer J Cell Biochem 2007 101 805 815 17226777 10.1002/jcb.21159

32 Xing F Saidou J Watabe K Cancer associated fibroblasts (CAFs) in tumor microenvironment Frontiers in bioscience: a journal and virtual library 15 166 179 10.2741/3613 PMC2905156 20036813

33 Koukourakis M I Giatromanolaki A Harris A L Comparison of metabolic pathways between cancer cells and stromal cells in colorectal carcinomas: a metabolic survival role for tumor-associated stroma Cancer Res 2006 66 632 637 16423989 10.1158/0008-5472.CAN-05-3260

34 Thomasset N Lochter A Sympson C J Expression of autoactivated stromelysin-1 in mammary glands of transgenic mice leads to a reactive stroma during early development Am J Pathol 1998 153 457 467 9708806 10.1016/S0002-9440(10)65589-7 PMC1852990

35 Herrera M Herrera A Dominguez G Cancer-associated fibroblast and M2 macrophage markers together predict outcome in colorectal cancer patients Cancer science 2013 104 437 444 23298232 10.1111/cas.12096 PMC7657228

36 Comito G Giannoni E Segura C P Cancer-associated fibroblasts and M2-polarized macrophages synergize during prostate carcinoma progression Oncogene 2014 33 2423 2431 23728338 10.1038/onc.2013.191

37 Chaudhary B, Elkord E. Regulatory T Cells in the Tumor Microenvironment and Cancer Progression: Role and Therapeutic Targeting. Vaccines 2016; 4: 10.3390/vaccines4030028 PMC5041022 27509527

38 Shevach E M Mechanisms of foxp3+ T regulatory cell-mediated suppression Immunity 2009 30 636 645 19464986 10.1016/j.immuni.2009.04.010

39 Elkord E Alcantar-Orozco E M Dovedi S J T regulatory cells in cancer: recent advances and therapeutic potential Expert opinion on biological therapy 2010 10 1573 1586 20955112 10.1517/14712598.2010.529126

40 Ghiringhelli F Menard C Terme M CD4+CD25+ regulatory T cells inhibit natural killer cell functions in a transforming growth factor-beta-dependent manner J Exp Med 2005 202 1075 1085 16230475 10.1084/jem.20051511 PMC2213209

41 Jie H B Gildener-Leapman N Li J Intratumoral regulatory T cells upregulate immunosuppressive molecules in head and neck cancer patients Br J Cancer 2013 109 2629 2635 24169351 10.1038/bjc.2013.645 PMC3833228

42 Ostrand-Rosenberg S Sinha P Myeloid-derived suppressor cells: linking inflammation and cancer J Immunol 2009 182 4499 4506 19342621 10.4049/jimmunol.0802740 PMC2810498

43 Srivastava M K Sinha P Clements V K Myeloid-derived Suppressor Cells Inhibit T Cell Activation by Depleting Cystine and Cysteine Cancer Res 2010 70 68 77 20028852 10.1158/0008-5472.CAN-09-2587 PMC2805057

44 Rodriguez P C Quiceno D G Zabaleta J Arginase I production in the tumor microenvironment by mature myeloid cells inhibits T-cell receptor expression and antigen-specific T-cell responses Cancer Res 2004 64 5839 5849 15313928 10.1158/0008-5472.CAN-04-0465

45 Barnie P A Zhang P Lv H Myeloid-derived suppressor cells and myeloid regulatory cells in cancer and autoimmune disorders Experimental and therapeutic medicine 2017 13 378 388 28352304 10.3892/etm.2016.4018 PMC5348694

46 Wynn T A Chawla A Pollard J W Macrophage biology in development, homeostasis and disease Nature 2013 496 445 455 23619691 10.1038/nature12034 PMC3725458

47 Quail D F Joyce J A Molecular Pathways: Deciphering Mechanisms of Resistance to Macrophage-Targeted Therapies Clin Cancer Res 2017 23 876 884 27895033 10.1158/1078-0432.CCR-16-0133 PMC5453188

48 Solinas G Germano G Mantovani A Tumor-associated macrophages (TAM) as major players of the cancer-related inflammation Journal of leukocyte biology 2009 86 1065 1073 19741157 10.1189/jlb.0609385

49 Allavena P Sica A Solinas G The inflammatory micro-environment in tumor progression: the role of tumor-associated macrophages Critical reviews in oncology/hematology 2008 66 1 9 17913510 10.1016/j.critrevonc.2007.07.004

50 Reya T Morrison S J Clarke M F Stem cells, cancer, and cancer stem cells Nature 2001 414 105 111 11689955 10.1038/35102167

51 Yoo M H Hatfield D L The cancer stem cell theory: is it correct? Molecules and cells 2008 26 514 516 18711315 PMC2708939

52 Chen J Li Y Yu T S A restricted cell population propagates glioblastoma growth after chemotherapy Nature 2012 488 522 526 22854781 10.1038/nature11287 PMC3427400

53 Tang C Ang B T Pervaiz S Cancer stem cell: target for anti-cancer therapy FASEB journal: official publication of the Federation of American Societies for Experimental Biology 2007 21 3777 3785 17625071 10.1096/fj.07-8560rev

54 Auffinger B Tobias A L Han Y Conversion of differentiated cancer cells into cancer stem-like cells in a glioblastoma model after primary chemotherapy Cell Death Differ 2014 21 1119 1131 24608791 10.1038/cdd.2014.31 PMC4207480

55 Bhatia A Kumar Y Cancer stem cells and tumor immunoediting: putting two and two together Expert Rev Clin Immunol 2016 12 605 607 26919116 10.1586/1744666X.2016.1159133

56 Wei J Barr J Kong L Y Glioblastoma cancer-initiating cells inhibit T-cell proliferation and effector responses by the signal transducers and activators of transcription 3 pathway Molecular cancer therapeutics 2010 9 67 78 20053772 10.1158/1535-7163.MCT-09-0734 PMC2939737

57 Anestakis D Petanidis S Kalyvas S Mechanisms and Αpplications of Ιnterleukins in Cancer Immunotherapy International journal of molecular sciences 2015 16 1691 1710 25590298 10.3390/ijms16011691 PMC4307328

58 Amedei A Prisco D MM D E The use of cytokines and chemokines in the cancer immunotherapy Recent patents on anti-cancer drug discovery 2013 8 126 142 22894642

59 Morgan D A Ruscetti F W Gallo R Selective in vitro growth of T lymphocytes from normal human bone marrows Science (New York, NY) 1976 193 1007 1008 10.1126/science.181845 181845

60 Baluna R Vitetta E S Vascular leak syndrome: a side effect of immunotherapy Immunopharmacology 1997 37 117 132 9403331 10.1016/s0162-3109(97)00041-6

61 Rosenberg S A Yang J C White D E Durability of complete responses in patients with metastatic cancer treated with high-dose interleukin-2: identification of the antigens mediating response Annals of surgery 1998 228 307 319 9742914 10.1097/00000658-199809000-00004 PMC1191483

62 Sim G C Radvanyi L The IL-2 cytokine family in cancer immunotherapy Cytokine & growth factor reviews 2014 25 377 390 25200249 10.1016/j.cytogfr.2014.07.018

63 Belperio J A Keane M P Arenberg D A CXC chemokines in angiogenesis Journal of leukocyte biology 2000 68 1 8 10914483

64 Balkwill F Cancer and the chemokine network Nature reviews Cancer 2004 4 540 550 15229479 10.1038/nrc1388

65 Yoshie O Matsushima K CCR4 and its ligands: from bench to bedside International immunology 2015 27 11 20 25087232 10.1093/intimm/dxu079

66 Taeger G Grabellus F Podleska L E Effectiveness of regional chemotherapy with TNF-alpha/melphalan in advanced soft tissue sarcoma of the extremities International journal of hyperthermia: the official journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group 2008 24 193 203 10.1080/02656730701868387 18392998

67 van Horssen R Ten Hagen T L Eggermont A M TNF-alpha in cancer treatment: molecular insights, antitumor effects, and clinical utility The oncologist 2006 11 397 408 16614236 10.1634/theoncologist.11-4-397

68 Parker B S Rautela J Hertzog P J Antitumour actions of interferons: implications for cancer therapy Nature reviews Cancer 2016 16 131 144 26911188 10.1038/nrc.2016.14

69 Srivastava S Koch M A Pepper M Type I interferons directly inhibit regulatory T cells to allow optimal antiviral T cell responses during acute LCMV infection J Exp Med 2014 211 961 974 24711580 10.1084/jem.20131556 PMC4010906

70 Zoglmeier C Bauer H Norenberg D CpG blocks immunosuppression by myeloid-derived suppressor cells in tumor-bearing mice Clin Cancer Res 2011 17 1765 1775 21233400 10.1158/1078-0432.CCR-10-2672

71 Greiner J W Hand P H Noguchi P Enhanced expression of surface tumor-associated antigens on human breast and colon tumor cells after recombinant human leukocyte alpha-interferon treatment Cancer Res 1984 44 3208 3214 6744259

72 Abiko K Matsumura N Hamanishi J IFN-gamma from lymphocytes induces PD-L1 expression and promotes progression of ovarian cancer Br J Cancer 2015 112 1501 1509 25867264 10.1038/bjc.2015.101 PMC4453666

73 Zaidi M R Merlino G The two faces of interferon-gamma in cancer Clin Cancer Res 2011 17 6118 6124 21705455 10.1158/1078-0432.CCR-11-0482 PMC3186825

74 Rozati S Naef L Levesque M P Real-life experience with pegylated interferon and conventional interferon in adjuvant melanoma therapy Journal of immunotherapy (Hagerstown, Md: 1997) 2013 36 52 56 10.1097/CJI.0b013e31827809c7 23211621

75 Eggermont A M Suciu S Testori A Long-term results of the randomized phase III trial EORTC 18991 of adjuvant therapy with pegylated interferon alfa-2b versus observation in resected stage III melanoma J Clin Oncol 2012 30 3810 3818 23008300 10.1200/JCO.2011.41.3799

76 von Tresckow B Morschhauser F Ribrag V An Open-Label, Multicenter, Phase I/II Study of JNJ-40346527, a CSF-1 R Inhibitor, in Patients with Relapsed or Refractory Hodgkin Lymphoma Clin Cancer Res 2015 21 1843 1850 25628399 10.1158/1078-0432.CCR-14-1845

77 Ries C H Cannarile M A Hoves S Targeting tumor-associated macrophages with anti-CSF-1 R antibody reveals a strategy for cancer therapy Cancer Cell 2014 25 846 859 24898549 10.1016/j.ccr.2014.05.016

78 Waller E K The role of sargramostim (rhGM-CSF) as immunotherapy The oncologist 2007 12 02 22 26 10.1634/theoncologist.12-S2-22 18039636

79 Yan W L Shen K Y Tien C Y Recent progress in GM-CSF-based cancer immunotherapy Immunotherapy 2017 9 347 360 28303764 10.2217/imt-2016-0141

80 Okamoto M Oshikawa T Tano T Mechanism of anticancer host response induced by OK-432, a streptococcal preparation, mediated by phagocytosis and Toll-like receptor 4 signaling Journal of immunotherapy (Hagerstown, Md: 1997) 2006 29 78 86 10.1097/01.cji.0000192106.32206.30 16365603

81 Goldinger S M Dummer R Baumgaertner P Nano-particle vaccination combined with TLR-7 and -9 ligands triggers memory and effector CD8(+) T-cell responses in melanoma patients European journal of immunology 2012 42 3049 3061 22806397 10.1002/eji.201142361 PMC3549564

82 Lichty B D Breitbach C J Stojdl D F Going viral with cancer immunotherapy Nature reviews Cancer 2014 14 559 567 24990523 10.1038/nrc3770

83 Andtbacka R H Kaufman H L Collichio F Talimogene Laherparepvec Improves Durable Response Rate in Patients With Advanced Melanoma J Clin Oncol 2015 33 2780 2788 26014293 10.1200/JCO.2014.58.3377

84 Kaufman H L Andtbacka RH I Collichio F A Durable response rate as an endpoint in cancer immunotherapy: insights from oncolytic virus clinical trials Journal for immunotherapy of cancer 2017 5 72 28923101 10.1186/s40425-017-0276-8 PMC5604502

85 Kohler G Milstein C Continuous cultures of fused cells secreting antibody of predefined specificity Nature 1975 256 495 497 1172191 10.1038/256495a0

86 Ecker D M Jones S D Levine H L The therapeutic monoclonal antibody market mAbs 2015 7 9 14 25529996 10.4161/19420862.2015.989042 PMC4622599

87

88 Wang Z ErbB Receptors and Cancer Methods in molecular biology (Clifton, NJ) 2017 1652 3 35 10.1007/978-1-4939-7219-7_1 28791631

89 Francisco L M Salinas V H Brown K E PD-L1 regulates the development, maintenance, and function of induced regulatory T cells J Exp Med 2009 206 3015 3029 20008522 10.1084/jem.20090847 PMC2806460

90 Brahmer J R Drake C G Wollner I Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates J Clin Oncol 2010 28 3167 3175 20516446 10.1200/JCO.2009.26.7609 PMC4834717

91 Lipson E J Sharfman W H Drake C G Durable cancer regression off-treatment and effective reinduction therapy with an anti-PD-1 antibody Clin Cancer Res 2013 19 462 468 23169436 10.1158/1078-0432.CCR-12-2625 PMC3548952

92 Hodi F S Mihm M C Soiffer R J Biologic activity of cytotoxic T lymphocyte-associated antigen 4 antibody blockade in previously vaccinated metastatic melanoma and ovarian carcinoma patients Proc Natl Acad Sci U S A 2003 100 4712 4717 12682289 10.1073/pnas.0830997100 PMC153621

93 Giuroiu I Weber J Novel Checkpoints and Cosignaling Molecules in Cancer Immunotherapy Cancer journal (Sudbury, Mass) 2017 23 23 31 10.1097/PPO.0000000000000241 28114251

94 Ma S R Deng W W Liu J F Blockade of adenosine A2A receptor enhances CD8+ T cells response and decreases regulatory T cells in head and neck squamous cell carcinoma Molecular cancer 2017 16 99 28592285 10.1186/s12943-017-0665-0 PMC5461710

95 Chen Y W Tekle C Fodstad O The immunoregulatory protein human B7H3 is a tumor-associated antigen that regulates tumor cell migration and invasion Current cancer drug targets 2008 8 404 413 18690846 10.2174/156800908785133141

96 Spodzieja M Lach S Iwaszkiewicz J Design of short peptides to block BTLA/HVEM interactions for promoting anticancer T-cell responses PLoS One 2017 12 e0179201 28594868 10.1371/journal.pone.0179201 PMC5464627

97 Muntasell A Ochoa M C Cordeiro L Targeting NK-cell checkpoints for cancer immunotherapy Current opinion in immunology 2017 45 73 81 28236750 10.1016/j.coi.2017.01.003

98 Andrews L P Marciscano A E Drake C G LAG3 (CD223) as a cancer immunotherapy target Immunological reviews 2017 276 80 96 28258692 10.1111/imr.12519 PMC5338468

99 Zhu C Anderson A C Kuchroo V K TIM-3 and its regulatory role in immune responses Current topics in microbiology and immunology 2011 350 1 15 20700701 10.1007/82_2010_84

100 Kakavand H Jackett L A Menzies A M Negative immune checkpoint regulation by VISTA: a mechanism of acquired resistance to anti-PD-1 therapy in metastatic melanoma patients Modern pathology: an official journal of the United States and Canadian Academy of Pathology, Inc 2017 10.1038/modpathol.2017.89 28776578

101 Lee L Y Garland S M Human papillomavirus vaccination: the population impact F1000Research 2017 6 866 28663791 10.12688/f1000research.10691.1 PMC5473416

102 Hampton T Nobel Prize honors HIV, HPV discoveries Jama 2008 300 2109 19001616 10.1001/jama.2008.616

103 Ramqvist T Dalianis T Oropharyngeal Cancer Epidemic and Human Papillomavirus Emerging Infectious Diseases 2010 16 1671 1677 21029523 10.3201/eid1611.100452 PMC3294514

104 Osazuwa-Peters N Human papillomavirus (HPV), HPV-associated oropharyngeal cancer, and HPV vaccine in the United States – do we need a broader vaccine policy? Vaccine 2013 31 5500 5505 24095883 10.1016/j.vaccine.2013.09.031

105 van der Burg S H Arens R Ossendorp F Vaccines for established cancer: overcoming the challenges posed by immune evasion Nature reviews Cancer 2016 16 219 233 26965076 10.1038/nrc.2016.16

106 Kenter G G Welters M J Valentijn A R Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia N Engl J Med 2009 361 1838 1847 19890126 10.1056/NEJMoa0810097

107 van Poelgeest M I Welters M J Vermeij R Vaccination against Oncoproteins of HPV16 for Noninvasive Vulvar/Vaginal Lesions: Lesion Clearance Is Related to the Strength of the T-Cell Response Clin Cancer Res 2016 22 2342 2350 26813357 10.1158/1078-0432.CCR-15-2594

108 Welters M J Kenter G G de Vos van Steenwijk P J Success or failure of vaccination for HPV16-positive vulvar lesions correlates with kinetics and phenotype of induced T-cell responses Proc Natl Acad Sci U S A 2010 107 11895 11899 20547850 10.1073/pnas.1006500107 PMC2900675

109 Czerniecki B J Koski G K Koldovsky U Targeting HER-2/neu in early breast cancer development using dendritic cells with staged interleukin-12 burst secretion Cancer Res 2007 67 1842 1852 17293384 10.1158/0008-5472.CAN-06-4038

110 Morse M A Niedzwiecki D Marshall J L A randomized phase II study of immunization with dendritic cells modified with poxvectors encoding CEA and MUC1 compared with the same poxvectors plus GM-CSF for resected metastatic colorectal cancer Annals of surgery 2013 258 879 886 23657083 10.1097/SLA.0b013e318292919e PMC3812363

111 Schuler P J Harasymczuk M Visus C Phase I dendritic cell p53 peptide vaccine for head and neck cancer Clinical cancer research: an official journal of the American Association for Cancer Research 2014 20 2433 2444 24583792 10.1158/1078-0432.CCR-13-2617 PMC4017234

112

113 Mehrotra S Britten C D Chin S Vaccination with poly(IC:LC) and peptide-pulsed autologous dendritic cells in patients with pancreatic cancer Journal of hematology & oncology 2017 10 82 28388966 10.1186/s13045-017-0459-2 PMC5384142

114 Krishnadas D K Wang Y Sundaram K Expansion of cancer germline antigen-specific cytotoxic T lymphocytes for immunotherapy Tumour biology: the journal of the International Society for Oncodevelopmental Biology and Medicine 2017 39 1.010428317701309E15 10.1177/1010428317701309 28677424

115 Dudley M E Rosenberg S A Adoptive-cell-transfer therapy for the treatment of patients with cancer Nature reviews Cancer 2003 3 666 675 12951585 10.1038/nrc1167 PMC2305722

116 Maude S L Frey N Shaw P A Chimeric Antigen Receptor T Cells for Sustained Remissions in Leukemia N Engl J Med 2014 371 1507 1517 25317870 10.1056/NEJMoa1407222 PMC4267531

117 Dudley M E Gross C A Langhan M M CD8+ enriched "young" tumor infiltrating lymphocytes can mediate regression of metastatic melanoma Clin Cancer Res 2010 16 6122 6131 20668005 10.1158/1078-0432.CCR-10-1297 PMC2978753

118 Neal L R Bailey S R Wyatt M M The Basics of Artificial Antigen Presenting Cells in T Cell-Based Cancer Immunotherapies Journal of immunology research and therapy 2017 2 68 79 28825053 PMC5560309

119 O'Sullivan D Pearce E L Targeting T cell metabolism for therapy Trends in immunology 2015 36 71 80 25601541 10.1016/j.it.2014.12.004 PMC4323623

120 Dudley M E Rosenberg S A Adoptive Cell Transfer Therapy Semin Oncol 2007 34 524 531 18083376 10.1053/j.seminoncol.2007.09.002 PMC2198934

121 Sadelain M Brentjens R Riviere I The basic principles of chimeric antigen receptor design Cancer Discov 2013 3 388 398 23550147 10.1158/2159-8290.CD-12-0548 PMC3667586

122 Kochenderfer J N Somerville RP T Lu T Long-Duration Complete Remissions of Diffuse Large B Cell Lymphoma after Anti-CD19 Chimeric Antigen Receptor T Cell Therapy Molecular therapy: the journal of the American Society of Gene Therapy 2017 10.1016/j.ymthe.2017.07.004 PMC5628864 28803861

123 Morgan R A Yang J C Kitano M Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2 Molecular therapy: the journal of the American Society of Gene Therapy 2010 18 843 851 20179677 10.1038/mt.2010.24 PMC2862534

124 Feng K Guo Y Dai H Chimeric antigen receptor-modified T cells for the immunotherapy of patients with EGFR-expressing advanced relapsed/refractory non-small cell lung cancer Science China Life sciences 2016 59 468 479 26968708 10.1007/s11427-016-5023-8

125 Katz S C Burga R A McCormack E Phase I Hepatic Immunotherapy for Metastases Study of Intra-Arterial Chimeric Antigen Receptor-Modified T-cell Therapy for CEA+ Liver Metastases Clin Cancer Res 2015 21 3149 3159 25850950 10.1158/1078-0432.CCR-14-1421 PMC4506253

126 Beatty G L Haas A R Maus M V Mesothelin-specific chimeric antigen receptor mRNA-engineered T cells induce anti-tumor activity in solid malignancies Cancer immunology research 2014 2 112 120 24579088 10.1158/2326-6066.CIR-13-0170 PMC3932715

127 Davis I D Skrumsager B K Cebon J An open-label, two-arm, phase I trial of recombinant human interleukin-21 in patients with metastatic melanoma Clin Cancer Res 2007 13 3630 3636 17575227 10.1158/1078-0432.CCR-07-0410

128 Punch C Schofield C Harris P Rituximab-Associated Inflammatory Progressive Multifocal Leukoencephalopathy Case reports in infectious diseases 2016 2016 8.915047E6 10.1155/2016/8915047 PMC5124683 27965904

129 Hasheminasab S M Tzvetkov M V Schumann C High-throughput screening identified inherited genetic variations in the EGFR pathway contributing to skin toxicity of EGFR inhibitors Pharmacogenomics 2015 16 1605 1619 26419366 10.2217/pgs.15.97

130 Jain D Ahmad T Cairo M Cardiotoxicity of cancer chemotherapy: identification, prevention and treatment Annals of translational medicine 2017 5 348 28936442 10.21037/atm.2017.06.35 PMC5599271

131 Morris K A Golding J F Blesing C Toxicity profile of bevacizumab in the UK Neurofibromatosis type 2 cohort Journal of neuro-oncology 2017 131 117 124 27796735 10.1007/s11060-016-2276-9

132 Kunert A Obenaus M Lamers C H T cell receptors for clinical therapy: in vitro assessment of toxicity risk Clin Cancer Res 2017 10.1158/1078-0432.ccr-17-1012 28645940

133 Dogan V Rieckmann T Munscher A Current studies of immunotherapy in head and neck cancer Clinical otolaryngology: official journal of ENT-UK; official journal of Netherlands Society for Oto-Rhino-Laryngology & Cervico-Facial Surgery 2017 10.1111/coa.12895 28464441

134 Curti B D Kovacsovics-Bankowski M Morris N OX40 is a potent immune-stimulating target in late-stage cancer patients Cancer Res 2013 73 7189 7198 24177180 10.1158/0008-5472.CAN-12-4174 PMC3922072

135 Takada K Okamoto T Toyokawa G The expression of PD-L1 protein as a prognostic factor in lung squamous cell carcinoma Lung cancer (Amsterdam, Netherlands) 2017 104 7 15 10.1016/j.lungcan.2016.12.006 28213003

136 Daud A I Wolchok J D Robert C Programmed Death-Ligand 1 Expression and Response to the Anti-Programmed Death 1 Antibody Pembrolizumab in Melanoma J Clin Oncol 2016 34 4102 4109 27863197 10.1200/JCO.2016.67.2477 PMC5562434

137 Kansy B A Concha-Benavente F Srivastava R M PD-1 status in CD8+ T cells associates with survival and anti-PD-1 therapeutic outcomes in head and neck cancer Cancer Res 2017 10.1158/0008-5472.can-16-3167 PMC5690836 28904066

138 Adams D L Adams D K He J Sequential Tracking of PD-L1 Expression and RAD50 Induction in Circulating Tumor and Stromal Cells of Lung Cancer Patients Undergoing Radiotherapy Clin Cancer Res 2017 10.1158/1078-0432.ccr-17-0802 28679765

139 Shi X Zhang X Li J PD-1/PD-L1 blockade enhances the efficacy of SA-GM-CSF surface-modified tumor vaccine in prostate cancer Cancer Lett 2017 406 27 35 28797844 10.1016/j.canlet.2017.07.029

140 Moesta A K Cooke K Piasecki J Local Delivery of OncoVEXmGM-CSF Generates Systemic Anti-Tumor Immune Responses Enhanced by Cytotoxic T-Lymphocyte-Associated Protein Blockade Clin Cancer Res 2017 10.1158/1078-0432.ccr-17-0681 28706012

141 Hodi F S Lee S McDermott D F Ipilimumab plus sargramostim vs ipilimumab alone for treatment of metastatic melanoma: a randomized clinical trial Jama 2014 312 1744 1753 25369488 10.1001/jama.2014.13943 PMC4336189