0. Tumor immunology and immunotherapy

Transworld Research Network 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Review Article

Recent Res. Devel. Cancer, 10(2013): ISBN: 978-81-7895-592-6

0. Tumor immunology and immunotherapy E.V.Svirshchevskaya1, A.V.Prokhorov1, A.A.Zubareva1 and 2N.P.Berkova 1

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Russian Federation 2 INRA; Agrocampus Ouest, UMR 1253, STLO, Rennes, France

Abstract. Immunotherapy of tumors is based on the functional properties of immune cells and thus should be considered as a part on the normal immunity. Immune responses to tumor antigens inevitably mean autoimmune ones as all tumor associated antigens (TAA) originate from normal tissue antigens while their expression can change dramatically and embryonic antigens, normally absent in the developed tissues, can appear in tumors. However, total expression of TAA in normal body tissues often overcomes local expression in tumors making it difficult to target tumors without damaging healthy organs. Induction of antitumor response by vaccination means autoimmune response and the level of normal tissue damage depends on the type of TAA. An important problem in the initiation of artificial antitumor immune response by vaccination is the need to break immune tolerance to TAA, which serves to prevent any pathological autoimmune responses to self antigens. It is due to the tolerance functioning, that all types of vaccinations against pooled tumor antigens mostly fail. Modern more or less successful approaches to tumor immunotherapy are based on the usage of antibodies to different tumor targets. These antibodies are not the result of tolerance break but are developed in Correspondence/Reprint request: Dr. E.V.Svirshchevskaya, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Russian Federation. E-mail: [email protected]

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mice and then humanized. Further perspectives for antitumor immunotherapy is the search for a narrow “window” between immune response against TAA, which will be therapeutic against tumors and not pathogenic against normal antigens.

Immunosurveillance, immunoediting, or homeostasis? There are two concepts on the existence of immunosurveillance of tumors which determine possible pathways of research. Many oncologists consider that immune system controls normal cells and eliminates cells ongoing malignant transformation [1-3]. From this point of view, appearance of tumors is a result of immunity failure and one should enhance natural immunity to prevent tumor formation or remove tumors. However, many experimental data demonstrate that immune system also can promote tumor growth; this is referred to as cancer immunoediting [4-7]. Immunoediting means that immune system takes part in normal body homeostasis directed to the clearance of healthy or diseased tissues from dead or infected cells and produces factors stimulating tissue repair. There are no direct evidences showing that this activity of immune system relates to the control of malignant transformation of cells. To understand whether immune system can control the tumor appearance we should consider major immune functions.

Evolution of the immune system As well as any other, evolution of the immune system is determined by gene transfer. The selection of genes responsible for different types of innate and adaptive immunity in mammals was fulfilled under the high pressure of environmental pathogens. The major challenge for the immune system was to protect the host from all types of pathogens able to invade the body. The first line of defense was to develop effective barriers such as skin, gastrointestinal mucosa and bronchoalveolar epithelium. These barriers play an important role in the innate immune defense against various pathogens by sensing the signal from the external environment. Pathogenic microorganisms have evolved different mechanisms to survive in the host environment; however epithelial cells recognize microorganisms and initiate appropriate signaling which contributes to the endocytosis of microorganisms and the attraction of innate immunity cells. Thus, barrier epithelium provides various fast mechanisms as front-line defence in the protection of the body surfaces from colonization and infection. This includes mucociliary clearance, bactericidal and fungicidal molecules such as defensins, surfactants, mannose-binding lectin, fikolin etcetera [8-9].

Tumor immunology and immunotherapy

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Cellular responses mediated by macrophages, neutrophils and dendritic cells, serve as the second line of innate defence against pathogens invading the body. Local infection can be controlled by phagocytes while disseminated ones, such as induced by viruses, require the activation of adaptive immune responses, which result in long-lasting memory formation. In humans accumulation of immune memory cells continues during childhood and adolescence. Selection of good responders to various infections meant survival and reproduction. The causes of malignant transformation of cells are not well understood while genomic instability during cell division is likely to play a major role in various disturbances such as point mutations, chromosome copy numbers, gene translocations and others, leading to tumor formation [10-12]. It is difficult to conclude that good immune response to pathogens and elimination of transformed cells should correlate as these two processes require different mechanisms of protection. The danger of cell transformation arises much later in life than the death from infections. More likely that genetic predisposition to genomic instability did not affect the evolution of immunity. Immunosurveillance by itself means the control for the antigenic content of host cells. Recognition of some cells as “wrong” means their removal. However, we know many examples of self-recognition demonstrated by autoimmune diseases. Contrary to immunosurveillance, evolution of immune system led to the development of mechanisms of central and peripheral tolerance leading to immune ignorance of host cells. Most self aggressive T-cells are eliminated during negative selection in the thymus and thus are absent in the periphery making it difficult to induce antitumor response [13-14]. T-regulatory cells, which are involved in the functioning of peripheral tolerance, are shown to damper effector cell responses and prevent tumor recognition [15]. It is obvious that autoimmune aggression was as dangerous as pathogenic invasion early in ontogenesis and thus served as a selection factor during the evolution of the immune system. Thus, both major immune mechanisms: recognition of pathogens and ignorance of self antigens serve to protect the body from external invasion and to keep untouched uninfected autologous cells meaning ignorance of cells undergoing internal transformation. The only possible exclusion is virus induced transformations where virus antigens can serve as an immunity inducing agent.

Who can be responsible for antitumor immunosurveillance? Till now there are no clear evidences that any subset of immune cells is responsible for antitumor immunosurveillance or immunoediting. Immune

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system includes innate and adaptive parts which function separately but in a coordinated manner. Polymorphonuclear neutrophils (PMN), eosinophils, basophils, macrophages, dendritic cells, and natural killer (NK) cells belong to the innate immunity part. These cells do not replicate in the periphery and have a limited capacity for pathogen killing however they demonstrate wide specificity to pathogens and recognize pathogen associated patterns by eleven Toll-like receptors [16]. Among innate immunity cells, PMN, eosinophils and basophils are rarely found in non-inflamed tissues showing that their role is to localize the infection and to signal its presence to other immune cells. For the exception of NK cells, these cells are not able to recognize protein antigens and thus can not control malignant transformation of host cells. However the role of PMN is shown during antibody therapy of tumors [17]; and sometimes a correlation of a better prognosis in cancer patients with PMN numbers in the peripheral blood is found [18]. Immunosurveillance must be a physical process when some type of immune cells screens antigenic content of all body tissues. Early stages of malignant transformation occur without signs of inflammation and thus do not induce activation of adhesion molecules by vessel endothelium. This means that screening cells must enter organs through non activated vessel endothelium and reside within tissue like resident macrophages or dendritic cells. Resident macrophages. After emigration from the bone marrow into the peripheral blood, monocytes enter tissues and differentiate into macrophages. Monocytes/macrophages have many roles in immune regulation, angiogenesis, and tumor metastasis and invasion. In addition, studies have revealed that these cells are essential to tumor progression [19] and thus cannot serve immunosurveillance purposes. Natural killer cells. Among innate immunity cells, NK subsets possess the ability to recognize in a limited manner self antigens and thus can be a good candidate for antitumor immunosurveillance sentinel. NK cells is under the fine-tuning control of cell surface receptors that display either inhibitory or activating function and, in healthy condition, mediate self-tolerance [20]. Inhibitory receptors are extremely sensitive to down-regulation or loss of MHC class I surface expression that are induced in autologous cells upon viral infection. Occasionally a decreased expression of MHC class I molecules is also observed in tumor cells due to the high level of cell proliferation. This alteration of the MHC class I expression weakens the strength of the inhibitory receptor-induced interaction, thus resulting in a triggering of NK cell perforin mediated killing function.


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To display immunosurveillance function NK cell must have free excess to the tissues. Few data are available regarding the recirculation of NK cells among human organs. Earlier studies have been often impaired by the use of markers then proved to be either not sufficiently specific for NK cells (e.g., CD57, CD56) or expressed only by subsets of NK cells (e.g., CD16). At the present, available data have confirmed that human NK cells populate blood, lymphoid organs, lung, liver, kidney, breast, gut and other organs under pathophysiological conditions [21-23]. Moreover, it has been demonstrated that, upon activation, NK cells can undergo differentiation in the periphery converting CD56brightCD16neg NK cells into effectors CD56dimCD16+ NK cells [24]. Experimental data suggest that up-regulation of killer immunoglobulin-like receptors (KIR) on CD56brightCD16neg perforin negative NK cells can occur also in vivo, most likely because of a proinflammatory cytokine microenvironment due to local immune reactions at the tumor site [25]. The data presented above demonstrate that NK, as well as NK subsets such as invariant NK (iNK), CD3 expressing NK (NKT) and lymphokineactivated NK (LAK) cells, can be considered as tumor recognizing cells. In mouse models of tumor growth a role of NK and LAK cells was demonstrated by many groups [26-29]. The most convincing results of the NK cell role in tumor control are obtained in NOD/SCID/γnull (NSG) mice which lack T, B, and NK cells [30]. The direct role of NK cells in tumor growth and metastasis was demonstrated by comparing NSG to conventional SCID mice. NSG mice inoculated with breast cancer cells formed large tumors more efficiently than SCID controls. Injection of activated NK cells inhibited tumor formation and organ metastasis, suggesting that NK cells are responsible for inhibiting the growth of tumors in SCID mice [31]. There is a large pool of experimental data showing that antitumor effect of NK cells can be related not to killing of tumor cells by NK cells per ce, but due to their ability to produce interferons (IFN). In the same manner as in NSG mice, in mice deficient in IFN, induced by gene silencing of IFN regulatory factor Irf7, metastasis was accelerated, indicating that suppression of metastasis depended on IFN signaling to host immune cells [32]. Data showed that functional NK cells and CD8+ lymphocytes were both necessary for Irf7-induced and IFN-dependent immune activation to confer protection against metastasis suggesting that tumor immunosurveillance does not regulate the initiation of primary breast tumors. The role of NK cells was also studied in several clinical trials. A Phase I/II clinical trial demonstrated the efficacy of allogeneic cytokine activated (both CD3 and NK (LAK)) cell treatment for patients with haematological malignancies who relapsed after allogeneic haemopoietic stem cell transplantation [33]. Allogeneic or autologous LAK cells were generated in

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vitro and 55 infusions were done for 16 patients at doses ranging from 10 to 200 million cells/kg. Response attributable to LAK cell infusion was observed in five patients with two who had a response sustained for more than 2 years. Another example of clinical benefit after NKT treatment was demonstrated by Yamasaki et al. who ex vivo expanded Vα24 NKT cells and α-galactosylceramide-pulsed antigen-presenting cells to investigate the efficacy and induction of NKT cell-specific immune responses in 10 patients with locally recurrent and operable head and neck squamous cell carcinoma [34]. Five cases achieved objective tumor regression. There are relatively many demonstrations of some NK activity against tumors, however overall clinical benefit was minimal twenty years ago [3536] and still is not sufficient now [37-39]. Recently it was identified that about half of the NK cells did not kill any target cells at all, while a minority of NK cells was responsible for a majority of the target cell deaths [40] meaning that more studies are needed to identify the population of NK cells able to kill tumor cells. It is of interest to mention that mice with different immunodeficiencies such as Rag-/-, SCID, Ifng-/-, STAT1-/- and others often have higher incidence of spontaneous tumors [41]. However these mice are highly susceptible to pathogens due to the absence of major immune components. Treatment with antibiotics suppressed tumor formation showing that uncontrolled infections can initiate some processes leading to tumor formation [41]. This hypothesis also explains high incidence of tumors in HIV, transplant and immunodepressed patients. Again the major role in this case can be attributed to IFN. Taken collectively it can be concluded that there two major points to be resolved when studding NK role in tumor immunosurveillance: i) the need to separate the in vivo effects of NK cells, inducing direct killing of tumor cells, and the effect of IFN production by NK cells, which indirectly leads to tumor control; and ii) to characterize more precisely the minor population of NK cells able to kill tumor cells. Until these issues are not resolved it is very difficult to develop effective tumor treatment protocols based on NK cell therapy. Adaptive immunity and lymphocytes. Effective adaptive immune responses to pathogens require several coordinated steps. In most cases humoral and cellular innate immunity controls the replication of many pathogens. When it fails, tissue resident macrophages and dendritic cells, overloaded with engulfed pathogens, become activated signaling the presence of infections. However to initiate adaptive immune response, some activated phagocytes must migrate back to the lymph nodes where they can assemble the immunological synapse. Indeed, this process is shown at least for


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dendritic cells which upon activation undergo maturation process and become antigen-presenting cells (APCs). During maturation and antigen processing, dendritic cells start expression of fascin-1, an actin-bundling protein not expressed in other primary blood cells [42]. Fascin-1 plays critical roles in maturation-associated dendritic cell functions such as assembly of veil-like membrane protrusions, disassembly of podosomes, migration to lymph nodes, and formation of germinal centers [42]. APC initiated immunological synapse in the lymph node, draining the site of infection, is absolutely essential for adaptive response priming as APCs have to capture naïve T and B lymphocytes from the draining lymph. APC recognition of invading microorganisms relies on evolutionarily conserved, germline-encoded pattern-recognition receptors (PRRs) such as TLRs, intracellular Nod-like receptors and the membrane-associated C-type lectins. Upon binding the pathogen, PRRs initiate specific signalling events that modulate the production of inflammatory cytokines, phagocytosis, intracellular routing of antigen, release of oxidative species, and APC maturation leading to the subsequent development of adaptive immunity [43]. PRRs binding serves for APCs as a danger signal. The duration and magnitude of danger signals play a crucial role in generating the immune response. Damage-associated molecular pattern from necrotic and apoptotic host cells and the activation of innate immunity cells also serve as danger signals for APCs. However antigen capture by tissue resident dendritic cells in the absence of danger signals induces tolerance [44]. Besides danger receptors, activating cells, dendritic cells also express inhibitory receptors called the leucocyte immunoglobulin-like receptors (LILRs). These LILRs exert powerful inhibitory effects on APC phenotype and subsequent T-cell responses, and constrain the effects of PRRs signaling [45]. In particular LILRs bind HLA-G molecule which is expressed in pregnancy to suppress immunity against fetus [46]. Moreover, recent data demonstrate the functional plasticity of APCs, which are able to display a tolerogenic function eliciting the differentiation of T suppressor (Ts) and regulatory T cells (Treg) instead of effector helper CD4 and cytotoxic CD8 cells. This tolerogenic state of APCs is characterized by low costimulatory potential and high expression of LILRs [47]. APCs overexpressing the member of LILR family immunoglobulin-like transcript 3, display lower phosphorylation levels of NF-kappaB and fail to stimulate the full program of T helper proliferation and maturation eliciting instead the differentiation of Ts and Treg cells. In contrast, immunoglobulin-like transcript knockdown APCs have robust cytokine and chemokine production, and are able to trigger stronger T-cell responses to viral antigens or alloantigens [48].

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Malignant transformation of host cells is not associated with the production of danger signals while on the contrary, autologous cells express MHC class I presented self peptides able to bind LILRs on resident phagocytes by this mean preventing self recognition, APC maturation, migration and germinal center formation. In the absence of immunological synapse, specific to TAA, in the lymph nodes, T and B cells are not activated and thus cannot function as immunosurveillance control or tumor effectors. Antitumor vaccination was the first immunotherapy of tumors tried as an alternative to chemotherapy. Most clinical trials attempting to treat cancer patients by immunization with whole tumor homogenates demonstrated that vaccination was inefficient [49-50]. Pilot antitumor vaccine contained autologous tumor homogenates and adjuvant which was routinely used for the production of vaccines against various infections. Such vaccination effectively induces germinal center formation due to the danger signals (for example, BCG) included in the adjuvant. However, effective in the induction of immune response against pathogens, potent adjuvant was unable to stimulate antitumor T cell response. The reasons for this failure are evident and connected to the elimination of T-cells able to recognize self antigens, including TAA, from the periphery during negative selection. Tolerogenic signals via LILRs, expressed by T-cells, also prevented the activation of T-cells, which were not eliminated during central selection. Thus, the major failure in vaccination against TAA is not inability to initiate specific germinal center but the lack of T-cell with needed TAA oriented specificity eliminated during negative selection process or controlled by peripheral tolerance mechanisms. In other words, it is impossible to catch a “black cat in a dark room when there is no black cat there”. Adoptive cell transfer (ACT) therapy tries to exploit the same methodology as vaccination by generating immunological synapse in vitro using patients’ APCs maturated ex vitro, loading them with various TAA, propagating NK, T-cells, Vγ9Vδ2 T-cells, Treg and others using a panel of cytokines [51-54]. First ACT clinical trials were conducted for melanoma patients. It is known that melanoma contains more tumor infiltrating cells than other solid tumors, and thus is considered more immunogeneic. Peripheral T lymphocytes obtained from a patient melanoma were expanded in vitro using IL-2 and then injected back with or without high dose IL-2. Objective response to ACT treatment was found in 30-50% patients in both groups [55-56]. Therapy was efficient also for patients who earlier failed to respond to high-dose IL-2 therapy. In attempts to improve melanoma ACT therapy, a more sophisticated approach was used in the phase I study [57], where reactive to melanoma antigens T-cells were cloned, expanded and infused


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back with or without IL-2. In this case immunoablative therapy was applied to empty “space” for newly generated T-clones. All these very expansive and labor consuming efforts resulted in no clinical effect [57]. The next step was to use T-cells specific to individual melanoma antigens such as MART1/Melan-A, gp100, NY-ESO-1. To do this APC were produced in vitro, loaded with melanoma antigen peptides and used to stimulate T-cells in a specific manner. Melanoma specific T-cells were cloned, expanded, and injected back to a patient. Specific responses were registered in 80% of patients persisting for up to 20 months [58]. More recent phase II clinical study used the same method of cloned CD8 T-cells stimulated with dendritic cells loaded with Melan-A antigen with the only difference – patients received instead of intravenous IL-2 subcutaneous IL-2 or IFN-α [59]. Of the 14 patients treated, six (43%) experienced an objective response with longterm complete remission for two patients (for 5 years and 28 months). ACT treatment for melanoma is still not approved by FDA. There are other examples in the same direction. Hundreds of different protein antigens, peptides, combined or not with cytokines, as well as DNA vectors, were studied. Every clinical trial shows some small “promising” results. It can be speculated that low efficacy of ACT is not because of poor activation of APCs in vivo or in vitro but because of the absence of TAA specific clones in T-cell repertoire. Due to MHC restrictions, T-cells for ACT are obtained from the patient’s blood and thus TAA specific T-cells cannot be generated from a population in which they are not present. Generation of sufficient amount of tumor specific T-cells can be done only in vitro as it was demonstrated for melanoma specific ACT, and these specific T-cells do not destroy tumors and even objective responses are found not in all patients. ACT using gene-modified T-cells is another variant of ACT inspired by the failure of very serious attempts to stimulate self TAA-specific T cells. The idea was to generate T-cell receptors (TCRs) highly reactive to melanoma/melanocyte antigens in mice or select from human clones and transfer the genes encoding these TCRs into retroviral vectors. CD3stimulated T-cells from melanoma patients were transduced by vectors coding MART-1 peptide 27-35 specific TCRs, either human or mouse, and then administered to 36 patients with metastatic melanoma [60]. Objective cancer regressions were seen in 30% and 19% of patients who received the human or mouse TCR, respectively. However, patients exhibited destruction of normal melanocytes in the skin, eye, and ear, and sometimes required local steroid administration to treat uveitis and hearing loss. TCR-based ACT therapy demonstrated a better efficacy however also more aggressive autoimmune potential for normal cells. There are many problems to be solved before it can come to clinics [61].

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Another approach dedicated to the production of artificial T-cells recognizing tumor is based on the transgenic chimeric antigen receptor (CAR) of T cells. CAR consists of an antibody-derived targeting domain fused with T-cell signaling domains that, when expressed by a T-cell, endows the T-cell with antigen specificity determined by the targeting domain of the CAR [62-64]. Proof-of-principle studies demonstrate the potency of CAR Tcells to interact both with tumor and normal host cells [64]. The effect of CARs specific to ganglioside GD2 was studied in neuroblastoma patients [65]. CAR was expressed in EBV-specific cytotoxic T lymphocytes and activated T cells. Three of 11 patients with active disease achieved complete remission, and persistence of CAR cells beyond 6 weeks was associated with superior clinical outcome. Thus, GD2-CAR T cells can induce complete tumor responses in patients with active neuroblastoma; these CAR T cells may have extended, low-level persistence in patients, and such persistence was associated with longer survival. The question left unanswered in the trial: if CAR T cells can bypass brain-blood barrier, do they harm normal brain tissue where expression of GD2 is high?

Concluding remarks Evolution of the immune system was driven by two major factors: necessity to combat environmental invaders and to protect the host from pathological auto-aggression. This was done by the development of highly sophisticated, multi-functional innate and adaptive mechanisms, which are tightly controlled by a balance between activation and inhibition signals able to focus immune response to pathogens, sometimes at the expense of healthy host cells. Elimination of the most potent auto-aggressive immune population, T-cells, through negative selection, results in physical or functional holes in the T-lymphocyte antigen receptor repertoires [66]. The nature and size of these gaps in our immune defenses must be balanced against the necessity of mounting rapid immune responses to foreign pathogens. It is not known what fraction of self-reactive clones, physically eliminated from the repertoire, is specific to TAA. Many self-reactive cells are retained in the periphery under control of flexible restraints such as inhibitory molecules like KIR, LILRs and others; inability to pass some barriers (high endothelium, blood-brain barrier); short living time and others. Disturbances in the balance between tolerance and immunity are manifested as the susceptibility to autoimmune disease, infections, or tumors. Modern approaches permit the generation of genetically modified T-cells with TAA specificity (TCR modified, CAR T cells). Possibly, early or later it will lead to the development of tumor destroying treatment. Of great importance is the


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selection of TAA for this therapy as we have to keep in mind that all TAA are self antigens and can be found on normal tissues. Careful study of gene modified T-cell auto-aggressive potential should be done much in advance in mouse models.

Acknowledgments This work was supported by the Russian Foundation for Basic Research, projects #12-03-31803; #12-04-32094; Scientific and Educational Human Resources of Innovative Russia for 2009-2013”, grant #14.132.21.1671; RAS Fundamental Research Programs “Molecular and Cellular Biology” and “Basic research on nanothechnology and nanomaterials”.

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