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    Tumor Immunology and Immunotherapy
     
     
    Ahmed M Malki1,2* and Ahmed M ElSharkawy2
     
    1Biomedical Program, Health Sciences Department, College of Arts and Sciences, Qatar University, Doha, Qatar
    2Biochemistry department, Faculty of Science, Alexandria University, Alexandria, Egypt
     
    *Corresponding author: Ahmed M Malki, Biomedical Program, Health Sciences Department, College of Arts and Sciences, Qatar University, Doha, Qatar, Tel: 203-3922918; E-mail: [email protected]
     
    1. Introduction

    The hypothesis that the immune system plays a role in the antitumor response and can be manipulated for the treatment of cancer was advanced as early as the 1890s, when William Coley used bacterial extracts to treat patients with sarcoma. It was thought that the ensuing inflammatory response successfully induced regression of large tumors by activating the immune system and inducing immune cells to attack the tumor. Since then, nonspecific forms of immunotherapy have been used with varying degrees of success for the treatment of a limited number of malignancies– (i) BCG adjuvant for superficial bladder cancer and high-dose IL-2 for metastatic melanoma [1], and (ii) donor lymphocyte infusions for leukemic relapse after allogeneic stem cell transplant [2]. However, these strategies were often accompanied by serious and potentially life-threatening toxicities. Advances in immunology, in the understanding of the requirements for T-cell activation and tolerance, and the development of novel technologies to analyze and augment immune response, now provide tumor immunologists with the opportunity to translate more broadly applicable principles in immunology to practice of treating patients with cancer in a more specific and effective manner. This chapter on tumor immunology begins with a description of the endogenous immune response, followed by a discussion of the components involved, [including T cells, Dendritic Cells (DCs)], and finally a summary of immunotherapeutic strategies arising from an understanding of the antitumor immune response.

    2. Immune System and Cancer

    The ability of the immune system to effectively respond to tumors is dependent on the following assumptions:

    a) Tumor cells differ from normal cells.
    b) The immune system can recognize these differences.
    c) The immune system is in an active state and capable in generating an effective and protective immune response. These prerequisites indicate that cancer immune editing is a dynamic process that involves both the tumor as well as the immunocompetent effector system. The efficient eradication of tumors in a living organism requires crosstalk between leukocytes of the innate and adaptive arms of the immune system, which reside in different immunological compartments. It has been shown that the cytokine interferon-gamma (IFN-γ), and the cytolytic effector molecules perforin and granzyme are secreted by cells of the innate and adaptive immune system, which contribute to the host’s immune defense against cancer. Following uptake into tumor cells, intracellular located granzyme B initiates apoptosis via the activation of procaspases 3, 7, 10 inactive cytosolic inhibitor of Caspase-Activated Dnase (ICAD), and the disruption of the membrane potential of mitochondria, which causes the release of cytochrome c into the cytosol. The situation of the host’s immune defense is complicated by the fact that throughout evolution, tumors have adopted strategies to interfere with and to overcome the immune system. These immune escape mechanisms involve the downregulation of Major Histocompatibility Complex class I (MHC I) and costimulatory molecules, the loss of tumor-specific antigens, the stimulation of inhibitory receptors expressed on effector cells, the stimulation of the growth of inhibitory CD4/CD25 double-positive regulatory T cells (Tregs), and the secretion of inhibitory molecules such as serpin- protease inhibitors, which interfere with the apoptosis cascade. For example, about 60% of metastases express significantly reduced levels of MHC class I on their cell surfaces. These findings indicate that a better understanding of the interaction between immune cells, tumor cells, and the tumor microenvironment and their consequences will guide the development of more effective approaches for controlling and successfully treating cancer.

    2.1 Immune surveillance

    In early attempts to demonstrate immunity to tumours, tumours were transplanted from one animal to another. The transplants were rejected and this was taken as evidence of immunity to the tumour. Later it was recognized that these experiments demonstrated transplantation immunity directed against MHC antigens. When genetically homogenous inbred animals (mice) became available it became possible to investigate tumour immunity and it was shown that if an animal was immunized with a tumour and was challenged with a graft of the same tumour, the graft was rejected. A graft of a different tumour was not, showing that the animal was specifically immune to the immunizing tumour. At about the same time Burnett (1973) [3] and later Thomas (1982) [4] put forward the theory of immune surveillance against tumours. They proposed that tumours arose frequently and that the majority are eliminated by the immune system. Burnett summarized his view of immune surveillance as follows:

    (1) Most malignant cells have antigenic qualities distinct from those of the cell type from which they derive, (2) such antigenic differences can be recognized by T cells and provoke an immune response. If this view is correct it follows that, (3) the incidence of malignant disease should be greatest in periods of relative immunological inefficiency, particularly in the perinatal period and old age, (4) immunosuppression whether genetic or induced by drugs, radiation, infection, or other causes should increase the incidence of cancer, (5) spontaneous regression of tumours may occur and evidence of an immune response should be apparent in these cases, and (6) large-scale histological examination of common sites of cancer should reveal a higher proportion of tumours than become clinically apparent. Burnett also suggested ways in which the theory of surveillance might be tested experimentally. Thus, immunosuppressive agents should facilitate the transfer of tumours, or damage to the T cell immune response produced by surgical removal of the thymus might lead to increased tumor incidence.

    At first sight, a variety of clinical and experimental data do seem to be in accord with the surveillance theory. In man, some tumours show a higher incidence in the first few years of life than in early adulthood and the incidence, but of different tumour types, and then rise progressively with increasing age. The incidence of tumours is also greatly increased in immunosuppressed individuals who are treated with immunosuppressive drugs to prevent rejection of the grafted kidney [5]. However, a closer examination does not support the surveillance hypothesis. The age incidence of tumours is as well explained by many other theories of cancer causation as by immunosurveillance. Tumours are caused by genetic changes in their cells of origin. These changes might be expected to occur either as errors during periods of rapid cell division (early life) or when external causes (carcinogens) have had time to take effect as in later life. The data derived from immunosuppressed individuals are similarly less straightforward to interpret when examined more closely. Although there does seem to be a slight increase in the frequency of most tumours, there is a disproportionate increase in a few tumour types (Table 1). The relative risk of suffering from some rare tumour types may be increased more than 1000- fold in immunosuppressed compared to normal individuals. An experiment in mice provided a possible explanation for these results.
     

    Tumour type

    Relative risk

    Virus involved

    Kaposi’s sarcoma

    >1000

    HHV8

    Lymphoma Of the brain
    Non-Hodgkin’s lymphoma

    >1000
    7.4

    EBV
    EBV

    Endocrine tumours

    320

    ­­------

    Skin carcinoma

    40

    Papillomaviruses

    Liver carcinoma

    30

    Hepatitis B

    Leukaemia

    6.4

    HTLV 1

    Cervix carcinoma

    4.2

    Papillomaviruses

    Digestive system carcinoma

    2.6

    ------

    Respiratory system carcinoma

    2.1

    ------

    Breast carcinoma

    1.1

    ------

     
    Table 1: Immunosuppression and tumours.
     
    When a large group of mice were treated from birth with anti-lymphocyte serum (raised in rabbits), their T cell immunity was greatly depressed. The mice did not develop large numbers of spontaneous tumours but when they inadvertently became infected with polyomavirus a number of them developed multiple tumours of a type characteristically caused by this virus [6]. Similarly, the lymphoid tumours seen in transplant recipients (Table 1) contain DNA and proteins of the Epstein–Barr virus (EBV), a herpes virus implicated in the cause of Burkitt’s lymphoma (a B-cell tumor seen in parts of Africa) and nasopharyngeal carcinoma. The virus can also immortalize normal human B lymphocytes in vitro. These findings suggest, therefore, that the most important role of the immune system in tumor protection may be in preventing the spread of potentially oncogenic viruses. This view agrees with experimental and clinical data on EBV, an ubiquitous infectious agent in human populations. Following infection the virus is carried lifelong and the individual also has lifelong immunity. Under normal circumstances, immune CD8 cytotoxic T lymphocytes can be demonstrated in vitro and there is thus a balance between virus production and the immune response, while in immunosuppressed individuals the immune system is unable to prevent virus spread.

    The T cells of such individuals cannot kill EBV-transformed B cells in vitro, and virus can often be isolated from body tissues and secretions such as saliva. Several other viruses have now been implicated in causing human tumours and the risk of acquiring these tumours is generally increased in immunosuppressed patients (Table 2). Although worldwide virally induced tumours are a major cause of cancer, since hepatitis B virus and papillomaviruses infect millions of individuals, there are also many cancers where no viral involvement can be detected. If it is accepted that immune surveillance operates principally against oncogenic viruses, what is the role of the immune system in relation to other tumours? Evidence from experimental animals (suggests that there are immune responses to many tumours and the slight increase of relative risk for tumours with no known viral involvement would support this (Table 1). However, the fact that most tumours grow and kill the host, suggests that the immune response is probably a late event and in most cases is unable to prevent tumor outgrowth. Nevertheless, that there is an immune response suggests that tumours do contain antigens recognized as foreign by the immune system. If this is the case it should be possible to boost immunity to them by deliberate immunization.

    Although the relative risks of Kaposi’s sarcoma and brain lymphoma are very high, the majority of immunosuppressed patients do not get these tumours since they are very rare in non-suppressed individuals. In contrast, most transplant patients eventually acquire skin tumours since, although the relative risk is lower, these are much commoner tumours in normal individuals. HTLV 1 causes adult T-cell leukaemia only.
     

     

    Immat-ure DC

    Mature DC

    Activation signals for DC
    maturation

    Morphology

     

    Increased “veil” and
    dendrite
    appearance

    Bacterial products:

    Phagocytosis

    +++

    -

    LPS (lipopolysaccharide)

    Costimulatory molecules
     HLA-DR (*MHC CLASS II)
         CD40
         CD80 (B7-1)
         CD83
         CD86(B7-2)

     

    +/-

     

    +++

     

    Teichoic acid
    CpG DNA
    Viral products: dsRNA

     

    CD40 ligand

    Chemokine-receptors
         CCR7
         CCR2
         CCR6

     

    -
    ++

     

    ++
    +

     

    TNFα, PGE2

     
    Table 2: Immature versus mature DCs – surface and functional phenotypic differences.
     
    3. Effector Cells in Tumor Immunity

    Effectors of adaptive immunity can be ascribed to a “humoral” and a “cellular” arm represented, respectively, by B cells that mediate effects through production of antibodies, and T cells that interact directly with target cells through the T-Cell Receptor (TCR). In humoral immunity, antibodies binding surface proteins on tumor cells can kill via complement activation or by bridging targets with cytotoxic cells through a process known as ADCC (Antibody-Dependent Cell-Mediated Cytotoxicity). In this process, the Fc portion of antibody couples with receptors on macrophages or NK cells that then effect cell killing. Although antibodies are highly effective in vitro, convincing evidence that antibody responses elicited in vivo play a critical role in antitumor immunity is weak. However, the significance of humoral responses with respect to tumor immunity has been supported by the identification of serum antibodies to potentially immunogenic tumor antigens and the successful therapy of patients using monoclonal antibodies.

    3.1 Dendritic cells

    Dendritic cells are specialized or “professional” APCs that are activated during the innate immune response and are uniquely equipped to take up and present antigen to effector cells of the adaptive immune system – the antigen specific CD4 and CD8 T cells. DCs are so-named because of pseudopods or “dendrites” which are processes that extend from the cell to facilitate antigen presentation. In vivo, the induction of an antitumor immune response may occur by tumor cells presenting antigen directly to T cells, but it is believed that a more common and robust pathway for tumor-specific T-cell activation in vivo is by cross-presentation. This is a process by which antigens released by necrotic, dying, or apoptotic tumors are taken up by DCs and represented to T cells under more favorable stimulatory conditions in the tumor-draining lymph node.

    Dendritic cells can be characterized in their immature or mature forms based on contrasting surface and functional phenotype (Table 2). In their immature form, DCs are well equipped to capture antigens through surface receptors such as the C-type lectins (e.g. DEC-205, mannose receptors), αvβ5 integrins, or CD36 for internal processing and presentation. DC activation via “danger signals” mediated through some of these abovementioned receptors and other surface receptors (e.g. the TLRs), leads to DC maturation [7] in the presence of bacterial or viral products, TNF- α, or prostaglandins. It is also thought that in addition to DC activation by these receptors, cooperation of CD4 helper T cells is required to “license” DCs with the capacity to stimulate CD8 T cells through interaction of the CD40 ligand on CD4 T cells with CD40 on DCs. Upon maturation, further antigen uptake by DCs is downregulated, and, in preparation for optimizing T-cell activation, DCs upregulate surface expression of the T-cell costimulatory molecules (CD80, CD83, and CD86), and secrete cytokines such as IL-7 and IL-12 that facilitate full T-cell activation. In the case of tumor immunity, DCs circulate through the blood and accumulate at tissue sites in response to chemokines arising from the site of tumor necrosis or inflammation. As immature DCs, antigen is collected and processed for presentation on the surface in the context of MHC molecules. Following maturation and upregulation of surface costimulatory molecules, lymphokines, and the chemokine receptor CCR7, DCs traffic to lymph Nodes where T-cell activation can occur.

    3.2. T cells

    3.2.1 Antigen presentation and T-cell stimulation: In contrast to B cells that provide “humoral” immunity through the production of soluble antibodies, T cells mediate “cellular” immunity by interacting directly with their target cells. T cells achieve specificity for cells expressing the target antigen through the surface TCR that recognizes fragments of antigen (usually peptide fragments) presented by the Major Histocompatibility Complex (MHC) through either a Class I or Class II processing pathway (Figure 1).
    Since proper antigen processing and presentation by the MHC is critical to antigen-specific immunity, a brief description of the MHC complex is presented.

    The MHC is encoded by highly polymorphic genes that cluster on chromosome 6 in humans and are codominantly expressed. Human MHC Molecules are called Human Leukocyte Antigens (HLAs) and are divided into Class I and Class II HLA or MHC that present peptides to CD8 and CD4 T cells, respectively. For the most part, Class I MHC molecules are represented in humans by HLA-A, -B, and -C family of alleles, and Class II MHC by HLA-DR, -DQ, and –DP families. The Class I MHC complex comprises three parts: the MHC-encoded heavy α chain, a non-MHC-encoded β2-microglobulin chain, and a 8–11-mer peptide sitting in a groove formed by the polymorphic region of the α chain. The Class II MHC complex comprises three parts, the polymorphic α and β chains and a 10– 30+ mer peptide sitting in a groove formed by the two chains. These parts are assembled in cytosolic compartments together with peptides derived from tumor proteins that have undergone proteasome-mediated cleavage to peptides of the appropriate length. The peptide –MHC complex is then transported to the surface where the immunogenic peptide sitting in the MHC groove is presented to the TCR. The T cell recognizes the peptide only in the context of the MHC complex; therefore, mutations affecting any component of the antigen-presenting machinery can abrogate specific T-cell recognition and killing of tumor cells.
     
     
    Figure 1: Antigen presentation. In the Class I pathway (➀–➄), cytosolic proteins are processed by proteasomes ➁ into peptide fragments, transported through the endoplasmic reticulum (ER, ➂), complexed with Class I MHC ➃ and β2-microglobulin, and brought to the surface ➄ where they are presented to CD8 T cells. In the Class II pathway, extracellular protein antigens are endocytosed (1), degraded into peptide fragments (2), combined with MHC (3), and presented to the surface (4) where they are presented to CD4 T cells (5).
     

    Box 1: T-cell-APC interaction

    Tumor-derived peptides are processed within the APC and presented by the peptide MHC complex to their cognate TCR. For CD4 T cells, optimal T-cell activation requires the ligation of costimulatory molecules such as CD28 with B7. Upregulation of CD40 ligand following TCR engagement delivers a “licensing” signal to APC that result in increased B7 expression and production of T-cell modulatory cytokines such as IL-12. T-cell activation is downregulated by the inducible inhibitory receptor, CTLA-4, which blocks CD28 mediated signals and competes for B7 binding. For CD8 T cells, activation following TCR engagement with the Class I MHC complex may be enhanced through costimulatory signals delivered by 4-1BB and other counter receptors. IL-2 and other cytokines produced by activated CD4 T cells provide growth signals to cognate CD8 T cells.

     
    3.2.2 Costimulatory and inhibitory T-cell signals: In addition to the interaction of the TCR with the peptide –MHC complex, the activation of T cells can be modulated by the engagement of surface costimulatory or accessory molecules by their respective ligands on antigen-presenting cells (see Box 1). The most prominent of these are the signals provided by CD28 upon binding to B7-1 (CD80) or B7-2 (CD86) on APC. B7–CD28 interaction mediates signals that can fully activate an antigen-driven T-cell response, enhance T-cell survival by up-regulation of anti-apoptotic Proteins such as BCL-x L and drive proliferation. Absence of B7 has been associated with T-cell anergy while engineered expression of B7 in potentially immunogenic tumor cells can induce tumor rejection in murine models. While B7– CD28 interaction appears to be critical to the generation or priming of an effective antitumor response, it does not influence the effector or killing phase of T cells. Hence, T cells generated with a B7-transduced tumor vaccine can eradicate B7-negative tumor. Other costimulatory molecules that deliver a positive signal to T cells include ICOS (inducible costimulator), OX40, 4- 1BB, and other B7 family members (e.g. B7-H3). Accessory/adhesion molecules, such as ICAM-1 and LFA-1, are also critical to T-cell recognition. These molecules converge in and reinforce the TCR– peptide –MHC synapse, by forming in aggregate with other molecules, a supra-molecular activation complex to facilitate delivery of a longer-lasting, more potent T-cell signal. Cytotoxic T Lymphocyte Associated Antigen-4 (CTLA-4) delivers a negative regulatory signal to activated T cells and competes with CD28 for binding to B7 on target cells(see Box 1). CTLA-4 is an inducible receptor with a greater affinity for B7 than CD28; however, in contrast to CD28, its surface expression is non-constitutive and relatively short lived. CTLA-4 is believed to provide an immunologic “brake” to prevent overly robust and potentially damaging overstimulation by suppressing T-cell proliferation through IL-2 inhibition and down-regulation of cell cycle activity.

    CTLA-4-deficient mice develop splenomegaly and a lympho-proliferative pathology. Since many tumor target antigens are also normal self proteins, eliminating CTLA-4 inhibition may provide a means of breaking tolerance to self-antigens and augment an otherwise muted T-cell response to tumor. Administration of anti-CTLA-4 antibody in some murine Models results in organ autoimmunity but can also lead to rejection of previously non-immunogenic tumors. In clinical trials, administration of anti-CTLA-4 antibody has produced signs of autoimmune toxicity as well as tumor regression in individuals receiving a tumor-specific vaccine.

    3.2.3 T lymphocytes: T cells can generally be divided into helper CD4 T cells and cytotoxic or killer CD8 T cells. Helper CD4 T cells recognize antigen in the context of MHC Class II and can be further differentiated into Th1 and Th2 subsets on the basis of distinct cytokine and receptor profiles. Th1 CD4 T cells produce IL-2 and interferon-γ, express IL-12 and IL-18 receptors, and regulate T-cell immunity, while Th2 T cells produce IL-14, IL-15, and IL-13, and regulate B cell immunity. It is believed that a Th1- type response would be beneficial in antitumor immunity since it mobilizes a T-cell-mediated response. Cytotoxic CD8 T cells recognize antigen in the context of MHC Class I and, when activated, release perforin and toxic granules that mediate direct cell killing by punching holes in the cell membrane to facilitate entry of enzymatic packets (granzymes A and B). Although most studies have weighed in on a greater role for the cytotoxic CD8+ T lymphocyte (CTL) in tumor eradication, the helper CD4 T lymphocyte has also been shown to be a vital component in the induction and maintenance of a competent antitumor immune response. Not only have tumor antigen-specific responses been identified for CD4 T cells but the presence of CD4 T cells may be required for CTL responsiveness. Acting in concert, both CD4 and CD8 T cells provide for synergistic mechanisms of tumor killing. CD8 T cells kill tumor cells through the release of perforin and granzymes A and B, or through engagement of the death receptor, Fas, through Fas Ligand (FasL) expressed on activated T cells. FasL – Fas interaction leads to a form of cell death known as apoptosis. In contrast to necrosis or death due to cell injury, apoptosis or programmed cell death involves a stepwise cascade of events initiated by receptor engagement at the cell surface (in this case, Fas), leading to DNA fragmentation. CD4 T cells can kill tumor cells directly by FasL – Fas engagement, as well as through indirect mechanisms that involve the recruitment of nonspecific effectors, such as macrophages and eosinophils, that can act even on MHC-negative tumors (Figure 2)
     
     
    Figure2: Effector mechanisms of T cells. Activated CD8 T cells deliver a “death” signal to tumor cells through FasL – Fas interaction ➀. CD8 T cells may also kill tumor cells directly through perforin and granzymes released upon engagement of the TCR➁. Perforin exocytosed in CTL granules form spores in the tumor cell membrane. Granzymes enter tumor cells through poresand induce tumor cell death➂. CD4 T cells can mediate tumor death through Fas interaction. Activated CD4 T cells may also mediate cytotoxicity indirectly through the release of interferon-γ and IL-5 to recruit tumoricidal macrophages (m φ) and eosinophils (Eos) ➃.

    3.2.4 NK cells: Natural Killer cells are activated during the innate response by the inflammatory milieu that is established by invading tumor cells. These effector cells are not antigen specific and do not express a TCR but do kill tumor through Killer Activating Receptors (KARs) expressed on their surface. Engagement of KARs with tumor derived ligands, such as MICA and MICB, which are upregulated in infected or “stressed” cells, such as tumor cells, leads to NK cell activation and tumor cell death. NK cells also engage self- MHC Class I molecules on target cells through inhibitory receptors (Killer Inhibitory Receptors – KIRs), perhaps, as a means of preventing auto reactivity. The loss of MHC expression on tumor cells, a process that can develop during carcinogenesis and immune-selection lends itself to preferential NK cell activation. The contribution of NK cells to the endogenous antitumor response in vivo may be best exemplified in a thymic nude mouse that have no T cells, but retain a population of functional NK cells that appears to be sufficient to mediate tumor resistance. In humans, NK-type cells can be expanded in vitro with high doses of IL-2 for adoptive transfer; however, in this setting, their efficacy isles s well defined and treatment is often accompanied by serious toxicity. The in vivo augmentation of NK-type cells may also be one mechanism by which high-dose IL-2 therapy has shown some clinical effect in the treatment of patients with metastatic melanoma or renal cancer.

    3.2.5 Regulatory T cells: A population of T cells with regulatory properties that control autoimmune and antitumor responses was postulated as early as1975; however, convincing evidence for their existence has been elusive. Recently, a population of CD4+, CD25+ T cells that possess immunosuppressive function has been identified. This discovery has led to a renewed understanding of the role of regulatory cells. CD4+ regulatory T cells (Tregs) are represented by two subsets – naturally occurring T regs representing 5-10% of peripheral T cells and induced Tregs that develop from conventional CD4+ CD25- T cells. Naturally occurring T regs mediate their suppressive properties through cell-to-cell contact by an unknown mechanism. Although activation is dependent on TCR engagement, their suppressive effects are nonspecific. They are known to express Glucocorticoid-Induced TNF Receptor (GITR) and CTLA-4, a known T-cell inhibitor of T-cell costimulation. However, the role of CTLA-4 and GITR in mediating the suppressive effects of naturally occurring Tregs is not well defined. Induced or adaptive Tregs can be generated from conventional CD4+ CD25 T cells following in vitro exposure to antigen and IL-10 and the induced Treg cells themselves appear to mediate their inhibitory properties through the production of IL-10 and TGF-β. Tregs have been found to be fundamental for the control of autoimmune responses in several murine models, such as inflammatory bowel disease, and depletion of CD25+ T cells has been shown to mediate immune rejection of various murine tumors in vivo, presumably through the release of suppressive effects on T cells targeting shared self-tumor antigens [8]. Elevated frequencies of CD4 Treg cells have also been described in cancer patients, leading to the design of clinical trials involving the administration of anti-CD25 antibodies to augment an endogenous antitumor immune response.

    4. Immunotherapy: From Preclinical Limitations into Actions

    4.1 Problems facing immunotherapy

    4.1.1 Introduction: Immunotherapy is treatment by immunological means. In active immunotherapy the tumour bearer’s own immune system is stimulated to respond to the tumour while in passive immunotherapy, immune cells or their products are given. Immunotherapy may also be specific or nonspecific. Specific active immunotherapy aims to stimulate only a response to the tumour or deliver passively agents such as monoclonal antibodies that target the tumour. Non-specific therapy boosts all immune responses, for example, through the use of cytokines or Lymphokine Activated Killer (LAK) cells. This section will deal mainly with the principles underlying and problems facing, attempts to use active immunization. Experimental evidence suggests that CD4 and CD8 T cells and antibody may all play a role in effective immunotherapy in different animal tumour models. However, generally T cells appear to play a major role in immunity to solid tumours, while antibodies are more effective against leukaemia or lymphoma. Most active immunization has therefore sought to induce strong cellular immune responses, which have been shown to be capable of eliminating large tumour masses in experimental animals and humans [9,10]. An additional reason for doing so is that the target antigens need not be cell surface molecules, since processed peptide epitopes reach the cell surface to be recognized by T cells (Figure 3). 4.1.2 Induction of anti-tumour immune responses: antigens and adjuvants: For immunization against tumours, what antigen to use is the first problem to be faced. Although increasing numbers of tumour antigens are being defined by SEREX (recombinant tumor cDNA expression libraries), using T cell clones or by sequencing peptides eluted from tumour MHC antigens, individual tumours vary in antigen expression. This means that for any tumour type it would be sensible if possible, to immunize against several antigens (as is the case for most vaccines against microorganisms). As yet this is rarely possible in humans so that in practice most human experiments have taken one of two approaches. Either immunization is with antigens known to be well expressed on most tumours of a particular type, including CEA for colon cancer, PEM in breast cancer, or MAGE in melanoma. Alternatively, whole tumour cells are used. Irrespective of the antigen used, the aims of immunization will be to induce a strong CD4 and CD8 -cell response against the chosen antigen(s). One problem that particularly applies to immunization against tumours rather than microorganisms is the possibility that damaging responses to self-antigens might be induced. As discussed above, most tumour-associated molecules are unaltered self-molecules, often expressed, though usually at a lower level, in normal tissues as well as tumours. That this is a real problem is shown by experiments in which mice undergoing successful immunotherapy against a melanoma became de-pigmented [11] and patients have exhibited vitiligo (de-pigmentation) while undergoing anti-melanoma immunotherapy [12]. This particular side effect is not life-threatening but autoimmune responses to other antigens might be. Selection of antigens as target for immunotherapy should therefore take into account the tissue distribution of the antigens. It is a disadvantage of the use of whole tumour cells as antigen that it is impossible to control which antigens the host responds to. The recognition that ‘danger signals’ were essential for initiation of immune responses has provided an explanation for why an immune response might be a late event in the evolution of a tumour. This and recognition that tumour cells lacked co-stimuli such as CD80 or CD40 led to experiments in which tumour cells weretransduced with genes coding for costimuli or cytokines (effectively internal ‘danger signals’).
     
     
    Figure 3: Interactions in an immune response. Antigen is taken up by DCs, which migrate to the draining lymph node. Soluble antigen also reaches the node in the afferent lymph. In the node, DCs present antigen to CD4 and CD8 T cells and B cells encounter soluble antigen. Clonal expansion of T and B cells takes place and Cytotoxic T Lymphocyte (CTL) and T-helper effector and memory cells are generated. B cells develop into Plasma Cells (PCs). Effector cells leave the node and migrate via lymph and blood to the original site of antigenic stimulation.

    However, evidence that immune responses to tumours are induced following uptake of tumour antigens by APC, suggests that optimal strategies for induction of antitumour responses should target tumour antigens to APCs. This has led to immunotherapy based on the use of antigen-loaded and activated DCs [13]. For this it is necessary that the DCs are MHC-matched with the patient, in practice usually autologous, and obtaining sufficient DCs is laborious, technically demanding, and expensive. Alternative means of targeting DC in vivo are being explored. Interestingly, giving antigen plus Granulocyte Monocyte Colony Stimulating Factor (GM-CSF), a cytokine that is chemo attractant for DCs, has been shown to be an effective means of immunization [14]. In effect, such a strategy mimics the release of cytokines, induced at a site of inflammation by ‘danger signals’, that leads to accumulation of inflammatory k cells. A similar effect can be achieved by the use of adjuvants. These are substances that potentiate immune responses in several ways. They provide ‘danger signals’, often delivered by incorporated bacterial products, they often provide a slow release depot of antigen and their physicochemical properties, for example, particulate materials, may promote entry of antigen into the cytosol and endogenous antigen processing. At present very few adjuvants are licensed for human use and the most well-established, aluminium salts, favour Th2 responses. However, new adjuvants are becoming available [15] and new immunization strategies are being developed to induce strong and long-lasting cellular (CD4 and CD8) responses. To date, the so-called prime/boost regimes appear to be one of the most effective [16]. In this method the antigen is first presented in one form, often as DNA, and the subjects are boosted with antigen presented in a different form, often in recombinant vaccine or adenoviruses. These induce inflammation (‘danger signals’) and a large secondary immune response is induced and effector cells produced. Whatever the target antigen and means of immunization are, it is important that both CD4 and CD8 responses are induced concurrently, as recent evidence has shown that CD8 memory cells, induced in the absence of CD4 help, do not respond to secondary stimulation [17]. This dictates that the antigen should contain epitopes able to stimulate both sorts of T cells. Whole recombinant proteins or tumour cells are therefore more likely to be effective than CD8 target epitopes alone, which have been used in some human experiments.

    4.1.3 Escape mechanisms: Mechanisms for escaping from immune responses are not confined to tumours. Almost, if not all, microorganisms have escape mechanisms and these are often similar to those found in tumours. Microorganisms and tumours may be immunosuppressive and these effects may be general or local. Many tumour-bearing patients show depressed immune responses and defects in signalling through the TCR and its associated CD3 complex have been demonstrated [18]. The cause of this effect is unclear but may be due to cytokines such as Transforming Growth Factor-b (TGF-b) and Vascular Endothelial Growth Factor (VEGF), often produced by tumours, which have been shown to have suppressive effects on lymphocytes. Furthermore, there is a complex relationship between tumours, their microenvironment, and the immune system, that may facilitate tumour growth and metastasis as much as preventing it [19]. A major escape mechanism of tumours, also found in microorganisms, is interference in antigen presentation. More than half of all tumours show abnormalities in MHC class-I expression, ranging from downregulation of a single allele to loss of all class-I molecules, and diverse molecular mechanisms for this have been demonstrated [20]. Clearly, however effective an antitumour immunization regime may be, it will be ineffective if the target epitopes can no longer be presented to effector T cells by the tumour cells. The common finding of loss of some HLA alleles in tumours suggests that antigen binding to as many different HLA molecules as possible should be used for immunization. Since HLA loss increases with tumour progression, active immunization is likely to be most effective if instituted as early as possible in the course of the disease. As yet this is seldom possible since conventional surgery, radiotherapy, or chemotherapy usually takes precedence over unproven modalities such as immunotherapy.

    4.2 Clinical use of mAbs

    4.2.1 Naked Antibodies: More than 200 mAbs have been tested in clinical studies, but the number of clinically relevant antibodies remains limited (Table 3). The first mAb that received US Food and Drug Administration (FDA) approval is rituximab, which is a chimeric antibody directed against the surface antigen CD20 on B lymphocytes, expressed on most B-cell NHL and subtypes of Acute Lymphatic Leukemias (ALL). In combination with polychemotherapy, rituximab is used for primary therapy of follicular NHL and diffuse large B-cell NHL as well as for maintenance therapy in recurrent follicular B-NHL after successful induction chemotherapy. Chemoimmunotherapy with rituximab is standard in therapy of primary and recurrent mantle cell lymphoma [21,22]. Rituximab might also be successful in combination with chemotherapy in CLL and in Burkitt’s lymphoma, improving progression-free and overall survival. Alemtuzumab is a humanized antibody directed against CD52 on B and T lymphocytes, and monocytes, macrophages, eosinophilic granulocytes, and NK cells. It is approved for clinical application in fludarabine-refractory CLL. In those patients, remission rates of 40% can be achieved. Interestingly, alemtuzumab has been shown to be especially effective for bone marrow manifestations of CLL. The role of alemtuzumab in primary therapy of CLL is not yet clear. Further studies will evaluate whether the efficacy of alemtuzumab in recurrences can be enhanced. Promising results were seen with alemtuzumab-chemoimmunotherapy in periphery T-cell lymphoma [23]. In contrast to rituximab, therapy with alemtuzumab is accompanied by heavier infusion-associated complications such as fever, shivering, dyspnea, or exanthema, and a higher rate of infectious complications. Metastasized Human Epidermal Growth Factor Receptor 2 (HER2)-expressing breast cancer treatment was the first indication for trastuzumab, a HER2-specific humanized monoclonal antibody. HER2 is a receptor tyrosine kinase of the EGFR family that is overexpressed in 25-30% of all breast cancer patients. Overexpression of HER2 leads to enhanced cell proliferation. A phase III study combining trastuzumab with first-line chemotherapy showed prolonged progression-free and overall survival [24]. It has also been approved as monotherapy for chemotherapy refractory metastasized breast cancer [25]. In addition, efficacy of adjuvant chemotherapy can be [26]. The chimeric mAb cetuximab is directed against EGFR. EGFR plays an important role in pathogenesis and progression of solid tumors such as colorectal cancer, NSCLC, and head and neck tumors. Binding of cetuximab to EGFR hinders the activation of intracellular tyrosine kinases and the following signal transduction pathway. The antibody also induces direct lysis of the tumor cells. A multicenter phase II study (BOND-1) was able to show that combination of irinotecan with cetuximab could overcome irinotecan resistance. In 23% of the patients, tumor remission, and in 30% stable disease was reached [27]. Cetuximab is now used for therapy of metastasized colorectal carcinoma in combination with irinotecan after progression with irinotecan monotherapy. In a phase III study of locally advanced head and neck tumors, the combination of cetuximab with radiotherapy significantly prolonged survival [28]. In metastasized NSCLC, a phase II study showed that combination of cisplatin, vinorelbin, and cetuximab leads to a significant survival benefit compared with chemotherapy with cisplatin and vinorelbin alone [29]. Bevacizumab is a VEGF-specific humanized mAb. Binding to VEGF inhibits tumor angiogenesis. It is proved in combination with irinotecan and 5-FU for first-line therapy of metastasized colorectal carcinoma. Patients with contraindications for irinotecan can be successfully treated with 5-FU and bevacizumab.
     

    Generic name

    Target antigen

    Structure

    Application

    Rituximab

    CD20

    Chimeric IgG-1κ

    B-NHL
    Mantle cell lymphoma
    CLL
    B-precursor ALL

    Alemtuzumab

    CD52

    Humanized IgG-1 κ

    CLL
    Peripheral T-cell lymphomas

    Trastuzumab

    HER2

    Humanized IgG-1 κ

    Breast cancer

    Cetuximab

    EGFR

    Chimeric IgG-1 κ

    Head and neck cancer
    Colorectal carcinoma
    NSCLC

    Bevacizumab

    VEGF

    Humanized IgG-1 κ

    Colorectal carcinoma
    NSCLC

     
    Table 3: mAbs in clinical use.
     
    In primary therapy of advanced NSCLC, the addition of bevacizumab to carboplatin and paclitaxel leads to enhanced progression-free and overall survival [30,31]. Contraindications are squamous cell histology and brain metastases because of enhanced risk of heavy bleeding.

    4.2.2 Radioimmunoconjugates: With the help of immunoconjugates, cytotoxic substances such as radioisotopes, cytokines, enzymes, or toxins can specifically be targeted to the tumor cells by the monoclonal antibody. Only two radioimmunoconjugates have approval for therapy, 90Y-ibritumomab tiuxetan and 131I-tositumomab. Both are directed against CD20 and are used for recurrent or refractory follicular B-NHL after therapy with rituximab. The radioimmunoconjugates might also be successful in therapy of transformed follicular NHL and primary diffuse large cell B-NHL.

    5. Conclusion and Future Directions

    The immune response to tumor cells involves a complex interplay of antigen-presenting cells, effector cells, cytokines, and chemokines that evolves over time and space. An understanding of basic immunologic principles can lead to insights into the reasons for failure of the endogenous antitumor immune response and an opportunity to manipulate components of the immune system to augment an antigen-specific effect. The identification of tumor antigens capable of eliciting immunity was one of the first steps toward achieving this goal. Now, an understanding of the mechanisms of T-cell recognition and co-stimulation has led to the possibility of vaccinating patients using DCs and the development of methods to isolate and expand antigen-specific CD4 and CD8 T cells ex vivo for adoptive transfer. The role of cytokines and chemokines in bringing together many of these effectors of innate and adaptive immunity yields yet another opportunity to augment the antitumor immune response. The use of monoclonal antibodies, which are already in clinical use, foreshadows the evolution of immunotherapy as a more broadly applied modality for the treatment of patients with cancer. The potential synergy of combining immunotherapy with chemotherapy, cytokines and chemokines with a tumor-specific vaccine, or antiangiogenic antibodies with adoptive T-cell therapy, may provide additional weapons in the anticancer armamentarium. The application of immunotherapy at earlier stages of malignancy may provide the opportunity for more complete and durable response in patients for whom more conventional therapy would be ineffective. However, many questions remain unanswered. For example, what is the significance of regulatory T cells in tumor immunity? What is the best strategy for optimal vaccination? What phenotypic qualities are desired of effector cells for adoptive therapy? How can cytokines and chemokines be integrated into the use of vaccines in delivering immunogens to the site of activation and augmenting the ensuing response? What triggers cells down the path of immunologic memory to ensure long-term immunoprotection? How can we identify and address obstacles of immune escape? With more precise immunologic tools and preclinical models at our disposal, it is hoped that many of these questions can be answered.
     
     
     
    References































     
     
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