Immunology and immunotherapy of human cancer: present concepts and clinical developments

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1 Critical Reviews in Oncology/Hematology 34 (2000) Immunology and immunotherapy of human cancer: present concepts and clinical developments Andreas J.A. Bremers, Giorgio Parmiani* Unit of Immunotherapy of Human Tumours, Istituto Nazionale per lo Studio e la Cura dei Tumori, Via Venezian 1, Milan, Italy Accepted 24 November 1999 Contents 1. Introduction Recognition of tumour cells by the immune system Tumour antigens T Lymphocytes Dendritic cells Natural killer cells Antibodies Evidence of tumour protection in vivo Tumour escape mechanisms Recognition and selection Downregulation of the immune response Tolerance induction Immunodeficiency in cancer patients Immunotherapy: background and rationale Non specific immunostimulation Cytokines Vaccination with molecularly defined antigens Defined antigens and antigen selection Peptide based vaccines Recombinant viral and bacterial vaccines DNA vaccination Idiotype antibody vaccination Vaccination with unidentified antigens Dendritic cells mediated vaccination Tumour cell based vaccination Heat shock protein vaccination The role of adjuvants Abbre iations: Ab, antibody; ADCC, antibody dependent cellular cytotoxicity; AJCC, American Joint Committee Cancer classification; APC, antigen presenting cell; BCG, Bacillus Calmette-Guérin; CEA, carcino embryonic antigen; CTL, cytotoxic T-lymphocytes; DC, dendritic cells; DTH, delayed type hypersensitivity; ECOG, Eastern Cooperative Oncology Group; EORTC, European Organisation for Research and Treatment of Cancer; FAC, 5 FU, adriamycin and cyclophosphamide; FasL, Fas ligand; 5 FU, 5 fluorouracil; G-CSF, granulocyte-colony stimulating factor; GM-CSF, granulocyte macrophage-colony stimulating factor; HLA, human leukocyte antigen; HPV, human papilloma virus; HSP, heat shock proteins; IFN, interferon; IL, interleukin; LAK, lymphokine activated killer; MCG, Melanoma Cooperative Group; MHC, major histocompatibility complex; NK, natural killer; PBMC, peripheral blood mononuclear cells; TAA, tumour associated antigen; TCR, T cell receptor; TIL, tumour infiltrating lymphocytes; TNF, tumour necrosis factor. * Corresponding author. Tel.: ; fax: address: parmiani@istitutotumori.mi.it (G. Parmiani) /00/$ - see front matter 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S (99)

2 2 A.J.A. Bremers, G. Parmiani / Critical Re iews in Oncology/Hematology 34 (2000) Adoptive transfer Antibodies and bispecific antibodies Results of clinical studies Immune stimulants as single agents Bacillus Calmette-Guérin (BCG) Levamisole Other immunostimulating agents Cytokines Interleukin 2 (IL-2) Interferon (IFN) Other cytokines Combinations including cytokines Cytokines in isolated perfusion Vaccination Vaccination with defined antigens Peptide vaccination Idiotypic antibodies Recombinant vaccines Cell based vaccination Cell lysates Whole cells Gene-modified cells Allogeneic or accessory cell vaccines Heat shock protein (HSP) vaccination Dendritic cells based vaccination Adoptive transfer LAK cells Tumour infiltrating lymphocytes Antibodies Biochemotherapy Conclusion: immunotherapy, present and future Reviewers Acknowledgements References Biographies Abstract Immunotherapy of cancer is entering into a new phase of active investigation both at the pre-clinical and clinical level. This is due to the exciting developments in basic immunology and tumour biology that have allowed a tremendous increase in our understanding of mechanisms of interactions between the immune system and tumour cells. This review briefly summarizes the state of the art in basic tumour immunology before discussing the clinical applications of the new concepts in the clinical setting. Clinical approaches are diverse but can now be based on strong scientific rationales. The analysis of the available clinical results suggests that, despite some disappointments, there is room for optimism that both active immunotherapy (vaccination) and adoptive immunotherapy may soon become part of the therapeutic arsenal to combat cancer in a more efficient way Elsevier Science Ireland Ltd. All rights reserved. Keywords: Immunology; Immunotherapy; Human cancer 1. Introduction At the end of last century medical research was profoundly influenced by the impressive progress that had been made with the discovery of agents that cause infectious diseases, and the protective effect of the (humoral) immune system against these, as induced by vaccination. It is from this background of terminology and principles that the very first theories emerged, postulating the existence of a protective immune response against malignancies and the possibility to induce or augment such a response. In the 1890s Coley attempted to treat cancer patients with bacterial extracts that were supposed to diffusely boost the immune

3 A.J.A. Bremers, G. Parmiani / Critical Re iews in Oncology/Hematology 34 (2000) system [1]. As early as 1908, Paul Ehrlich proposed the concept of cellular immunity directed against malignancies, and successfully carried out vaccination with tumour antigens in animals [2]. Yet, today s tumour immunology was developed through enormous efforts in a history that was paved with disappointments rather than success [3]. Research activity in this field by generations of basic scientists, surgeons, and physicians, revealed that the immune system is a complex defence system that evolved to protect against microorganisms and not necessarily against tumours. To protect the body from self destruction by autoimmune disease, an unavoidable side-effect of the beneficial immune response, a number of safeguards turned out to exist that also shield tumours from the reaction of the immune system. In this article we will first analyse the possible mechanisms of tumour antigen presentation and the existing evidence that the immune system does play a role in the control of neoplastic diseases. Subsequently we will discuss the tumour escape mechanisms from the immune system. This information constitutes the basis for strategies that are judged to be potentially able to overcome the shortcomings of the immune system that are held responsible for the development of the neoplasm into a clinically detectable tumour. Finally, todays clinical achievements of immunotherapy will be summarised. 2. Recognition of tumour cells by the immune system Table 1 Categories of tumour associated antigen recognised by T lymphocytes in solid tumours Category of antigen Unique antigens Cancer testis antigens Tissue of origin related antigens, differentiation antigens Oncogene products Mutated tumour suppressor proteins Viral antigens Antigen (example of malignancy in which presence is common) Mutated catenin, CDK4, caspase 8, mutated HLA-A2 (melanoma, H&N, kidney) MAGE, BAGE and GAGE families, NY-ESO-1 (breast, melanoma, bladder, H&N) CEA (colon, breast, pancreas and others); gp100, TRP-2 and tyrosinase (melanoma) Mutated Ras (pancreas); Her2/neu (breast, ovary) p53 (breast, colon and others), APC (FAP syndrome), BRCA1 & 2 (familial breast cancer) Human papilloma virus (cervical cancer), hepatitis B virus (hepatocellular cancer) The immune system consists of a number of components. The traditional division is between humoral (antibody mediated) and cellular immune responses. One can also distinguish between an innate immunity and adaptive immunity. The innate immunity can discriminate between normal tissues belonging to the organism ( self ) and newly encountered non-self proteins or living cells. Thus, anything non-self, whether originating from viral infection, neoplastic transformation, or because it originates from another individual (as in transplantation), is recognised as such in a non-specific way, and attacked alike by non-specific effector cells like macrophages and natural killer (NK) cells. Adaptive immunity is the more complex system, aimed at the eradication of intracellular pathogens. To do this, antigens derived from such pathogens that are often new to the host organism, need to be recognised by receptor-bearing specialised immune cells which respond to a complex system of stimulatory and costimulatory signals. Both innate and adaptive immunity can contribute to a response to malignancies. For today s immunotherapeutical strategies the following components are of interest Tumour antigens For the adapti e immunity against tumours, antigens are needed to trigger a specific immune response. Ideal antigens should be able to elicit specific B or T cell responses or both, with T cell responses including recognition of HLA class II-restricted epitopes. Tumour associated antigens (TAA) that can be recognised on tumour cells by T lymphocytes form a heterogenous group (Table 1). They are generally divided into three categories [4,5]. The first consists of unique antigens that are specific for each single tumour, usually derived from mutated proteins, and can be recognised either by HLA class I or HLA Class II restricted T lymphocytes, according to their length and aminoacid sequence [4 6]. TAA shared with other tumours but predominantly expressed by malignancies form a second category; these are usually transcriptionally reactivated genes that are not normally expressed (e.g. genes from the MAGE family in various tumours and testicular tissue), previously known as embryonic antigens and recently referred to as cancer testis (CT) antigens. A third category is that of differentiation antigens derived from the tissue of origin of the tumour (e.g. gp100, Melan-A, and tyrosinase in melanomas). Surprisingly enough, these normal antigens turned out to include some of the TAA most frequently recognised by the immune system [4]. Yet other antigens, have been considered for immunotherapy of cancer. Examples of these are overexpressed antigens that are also present in normal tissues (e.g. CEA) [7]. Moreover, oncogene and tumour suppressor gene products that play an essential role in the development of malignancies, can generate epitopes able to induce immune

4 4 A.J.A. Bremers, G. Parmiani / Critical Re iews in Oncology/Hematology 34 (2000) T Lymphocytes Fig. 1. Endogenous processing of antigen and interactions between the cytotoxic T lymphocyte and the target cells. Protein is processed to become peptide fragments; peptides bind to empty MHC molecules, and this complex is transported to the cell surface, where it can be recognised by the appropriate T-cell receptor. Costimulatory molecule interactions are depicted on the left side. Most efforts put into the development of tumour immunology have focused on cytotoxic T lymphocytes (CTL) mediated immunity, since these cells are regarded as the most important effector cells against tumours. Recognition of tumour antigens by CTL is the result of a complex process (Fig. 1). In this system an antigen is processed inside the target or antigen presenting cell (APC) into small peptide fragments that are then presented at the cell surface within the groove of the Major Histocompatibility Complex (MHC, HLA in humans) Class I molecules. This complex is then recognised by a T cell receptor (TCR) present on CTL. This TCR must be fitting to both the HLA allele present on the cell, and to the peptide presented [11]. Though underestimated during the last few years, MHC Class II restricted recognition is also very important for the induction of antitumour CTL which are activated through a (CD4 mediated) T helper response [6]. The outcome of antigen recognition though, is not necessarily tumour cell killing. Inflammation or destruction, occurring during infection, come with cytokine-mediated signals (e.g. TNF- and GM-CSF), resulting in activation of T-lymphocytes. Lack of these signals may result in tolerance, as is usually the case for the majority of antigens physiologically present in the body Dendritic cells Fig. 2. Tumour escape mechanisms, and DC antigen presentation. (A) Tumour cell lacking MHC molecules; (B) tumour cell lacking antigen processing; (C) tumour cell lacking costimulatory molecule; and (D) DC featuring processing of tumour cell fragments, expressing epitope presenting surface MHC along with all the costimulatory molecules responses (e.g. mutated RAS, p53 [4 6]). Since some tumour histotypes, like pancreatic and lung cancers, bear such mutated genes with a high frequency, such antigens may represent interesting targets. Finally, viral products in virus associated tumours can deliver a potent and effective stimulus to the immune system (hepatitis B virus [8], Human papilloma virus 16 [9]). It is of note that only recently HLA class II-restricted epitopes have been clearly defined and that most of them belong to the unique group of antigens that derive by point mutation from already known proteins [10]. Costimulatory signals can be delivered by cytokines (see above) or by specific costimulatory molecules (e.g. B7.1, B7.2). The latter are typically, but not exclusively, present on APC like macrophages, monocytes, B-cells and dendritic cells (DC). Presentation of antigen to naive T-lymphocytes without co-stimulation may lead to T cell tolerance. In fact, the adequate antigen presentation to a naive immune system occurs through these APC [12]. With the possible exception of B-cell lymphoma, tumour cells seldom express costimulatory molecules. The most potent in terms of anti-tumour CTL induction is the APC subset known as DC [13]. Antigen expression on DC comes from endocytosis and processing of antigenic substances, ranging from soluble antigen to apoptotic cells. Subsequently the antigens are endogenously processed by the DC. This results in epitopes presented on the DC cell surface in the context of the autologous HLA alleles and costimulatory molecules (Fig. 2d). DC feature high amounts of the MHC molecules that are essential to CTL recognition. The vast expression of adhesion- and costimulatorymolecules, along with the production of T cell specific chemokines [14] are of essential importance for the

5 A.J.A. Bremers, G. Parmiani / Critical Re iews in Oncology/Hematology 34 (2000) microenvironment in which an effective immune response can be initiated: tumour cells that by themselves induce tolerance do evoke a potent immune response once fused with DC [15]. This essential difference caused by presentation through DC is also confirmed by in vivo experiments [16]. To activate this process of antigen presentation by the DC, the antigen has to be present, along with signals of tissue damage, the so-called danger signals. Heat shock proteins (HSP) that chaperone the important epitopes of a cell, can be regarded as such a signal, and from the available evidence it may be concluded that HSP constitute an important source of antigen for processing and presentation by DC under natural conditions [17]. Apart from their role in presentation of antigen to CTL, DC are also of importance in the induction of CD4 (T helper) and natural killer cell responses. This makes DC a central pivot of anti-tumour response, and implies great promises for clinical application Natural killer cells The cytolytic capacity of NK cells is well established. Research in recent years has revealed various mechanisms of these cells function that led to the conclusion that NK cells are part of the innate as well as of adaptive immunity. As a part of the innate immune system, these cells are licensed to kill malignant tissues or virus infected cells or HLA incompatible grafted cells that are recognised as being different from the normal self tissue. To obtain this selective effect, NK activity is usually inhibited by the autologous MHC class I allele, expressed on self tissue, through specialised receptors [18], though activating forms of receptors (mainly associated with HLA-C alleles) have also been identified. Many malignancies as well as virus infected cells however, also feature a downregulation of MHC class I [19,20], which then allows triggering of NK subsets, resulting in the killing of these targets. Of more recent date is the appreciation of the NK cell regulatory function within the adaptive immunity system. NK cells produce a wide array of cytokines, including IFN-, TNF-, GM-CSF, M-CSF, IL-2, IL- 3, IL-5 and IL-8. This cytokine profile of activated NK cells skews the helper T lymphocyte response and activates macrophages, and thus influences the development of the adaptive immune response [21]. NK cells have aso been shown to induce antibody production by B-cells and even function as antigen presenting cells to specific T cell clones in a MHC Class II restricted manner [22]. Moreover, absence of NK cells prevents the induction of CTL [21]. Thus, NK cells appear to play an important role in modulation of B- as well as in T-lymphocyte mediated immunity Antibodies Antibody dependent cellular cytotoxicity (ADCC) is an important tool in the eradication of intracellular pathogens and tumour cells. In such a case the antigens are expressed on the cell surface, usually in the form of a trans-membrane protein, and recognised by the antigen specific part of the antibodies. The tail of the antibody then binds to the Fc receptor on cells like NK cells and macrophages, resulting in a signal of activation that can cause the lysis of target cells. Naturally occurring antibodies with clinical relevance have been detected against tumour suppressor gene and oncogene products like p53 [23], K-Ras, Her2/neu [24], as well as other tumour antigens like CEA and EpCAM (own unpublished data). Some of the naturally occurring antibodies appear to be correlated with better prognosis, like SK-1 antibodies [25]and antibodies against TA90 [26], while those against EpCAM and p53 [23] can be associated with bad prognosis. 3. Evidence of tumour protection in vivo The immune system does undertake action against neoplastic disease. It has long been known that animals cured of their primary tumour by excision, appeared to be protected against it in subsequent tumour challenge experiments [27]. Moreover, patients with drug-induced prolonged immunosuppression after organ transplantation or due to severe immunodeficiency syndromes develop a higher frequency of several tumours. The immunologically best studied human malignancies are probably melanoma and renal cell carcinoma. MHC class I expression, allowing recognition of the tumour by the immune system, resulted in better clinical prognosis in melanoma [28], and loss of MHC class I or its defective function [29], in a worse prognosis [28]. Moreover, brisk infiltrate of primary melanoma is an independent prognostic factor [30], while the development of vitiligo as a consequence of an immune reaction to melanoma differentiation antigens has been reported to correlate with clinical response [31]. However, other tumours of which the immunogenicity is far less accepted, also appear to be able to provoke such protective responses. In colorectal cancer, CTL infiltration of tumours is associated with much better prognosis [32], and a correlation was shown between the T cell profile (impaired CD4 + counts) and progression of the disease [33]. Moreover, HLA class II expression by tumour cells, essential for the induction of T helper responses, appears to be associated with improved prognosis, at least in colorectal cancer [34].

6 6 A.J.A. Bremers, G. Parmiani / Critical Re iews in Oncology/Hematology 34 (2000) Tumour escape mechanisms According to the immune surveillance hypothesis, the expression of tumour antigens during neoplastic transformation would induce an immune response that can control tumour growth [35]. However, there is evidence from immunocrippled patients to support such a mechanism for only a fraction of tumours. In patients that underwent organ transplantation, and received long term immunosuppressive therapy, there is a high incidence of non-hodgkin lymphoma, renal cell cancer, Kaposi sarcoma, and uncommon carcinomas like lip, vulva, squamous skin carcinoma [36], all of which are of suspected viral origin. A modest but apparently significant increase was also found for melanoma and certain lung cancers. There is, however, no indication for immune surveillance for several of the major human neoplasms like colon or breast cancers. Several mechanisms have now been recognised that allow tumours to escape from an intact and otherwise competent immune system Recognition and selection Naturally occurring tumours are not monoclonal. An effective tumour recognition and cytotoxicity therefore constitutes a selective pressure towards cells that escape from the immune response. Thus, tumours have been found to develop in such a way that previously recognised antigens are no longer expressed, so-called antigen-loss variants [37,38]. Alternatively, selective survival may result in a tumour with defective antigen processing due to mutation in gene coding for molecules known to be crucial for antigen presentation and peptide transport to ER, like TAP (transporter associated with antigen presentation). The antigen is still present, but the recognised epitope will no longer appear at the cell surface in the MHC molecules [39] (Fig. 2b), resulting in disease progression [40]. Yet another escape route used is downregulation of MHC expression itself (Fig. 2a), thus inhibiting recognition by T-lymphocytes altogether [20] Downregulation of the immune response Under normal physiological conditions certain tissues like liver, eyes and testis can downregulate the immune response directed against these essential organs. This effect is obtained through local release of inhibitory molecules, e.g.tgf [41] and by expression of Fas Ligand (FasL) on the cell surface [42]. Interaction between these and the respective receptors or Fas molecule on the T-lymphocytes result in apoptosis of these effector cells. The expression of FasL has also been demonstrated on malignancies like melanoma [42], astrocytoma and glioblastoma [43], colon carcinoma [44], lung carcinoma [45], ovarian cancer [46] and oesophageal cancer [47], and thus may protect these tumours against activated lymphocytes. Alternatively, half of the tumours like lung and colon cancers were recently found to defend themselves against apoptosis by producing a soluble decoy receptor that binds to FasL on effector cells [48]. However, how effective this mechanism is during in vivo tumour growth is still to be established clearly. In fact, melanoma-specific T cell clones were found to be resistant to the FasL/Fas interaction [49] Tolerance induction Tumours are not only capable of reducing the impact of a cytotoxic reaction as outlined above, but can also activate mechanisms that cause immune recognition to result in tolerance. As described in previous sections, the presence of appropriate costimulatory molecules is obligatory if recognition is to result in cytotoxicity instead of anergy. Absence of costimulatory molecules on tumours can even prevent cytotoxic activation of CTL previously present [50,51], and may involve multiple factors [52]. Furthermore, evidence exists that anergy may emerge among CTL previously capable of effective recognition [53]. Finally, tumours may fail to provide the optimal danger signalling microenvironment and associated cytokines, which optimise the function of immune effector cells, as the primary process is neoplasm and not inflammation. One other factor of importance may be the localisation of APC. Basically, these should be able to process antigen from the tumour irrespective of the localisation, but it has been postulated that sensitisation of naive lymphocytes will only take place within the lymph node [54]; this is supported by clinical observation of generation of protective immune responses mounted only after lymph node metastasis occurred (Bremers et al., in preparation) Immunodeficiency in cancer patients All previous mentioned factors in tumour escape from the immune system may play a role at the tumour site. Other mechanisms of generalised diminished immunocompetence appear to be involved in cancer patients [55,56], which might have to do with malnutrition, immunosuppressive therapies, but also with other, not yet identified, factors. 5. Immunotherapy: background and rationale Many strategies have emerged in the course of development of cancer immunotherapy. The oldest approach

7 A.J.A. Bremers, G. Parmiani / Critical Re iews in Oncology/Hematology 34 (2000) was an effort to boost non-specifically the general function of the immune system (acti e non-specific immunostimulation), as initiated by Coley. He injected patients with bacterial toxins and obtained regression of solid neoplasms [1], probably through activation of cytokines. Some of these agents are in clinical use today. Most effort has been put in immunotherapy through the cellular immune system. Three different approaches have been devised. First, one can try to improve conditions for the function of the immune system in general, without actually altering the target or specific effector cells themselves. Examples of these are the administration of cytokines like interleukin-2 (IL-2) and interferon and (IFN-, IFN- ), though their effectivenes may in part be due to other, non-immunological, mechanisms too (see below). A second approach aims at the activation of the immune system by optimising the presentation of antigen: accination or acti e specific immunotherapy. Examples range from the application of synthetic peptides that represent single epitopes, to vaccination with viable whole tumour cells, the antigens of which can then be endogenously processed by professional APC. The third group of therapies, known as adopti e immunotherapy, employs the infusion of effector cells that have been stimulated to act against the tumour and/or expanded in vitro, like anti-tumour CTL or allogeneic lymphocytes after bone marrow transplantation. But even lymphocytes stimulated in vitro with IL-2 in the absence of tumour antigens (lymphokine activeated killer (LAK) cells) were used. This implies that adoptive transfer can be antigen specific (anti tumour CTL) or not (LAK) Non specific immunostimulation Agents belonging to this group have been evaluated for over a century now, and have, in some instances, been shown effective as a sole treatment or valuable as an adjuvant. Most of the agents originate from Coley s idea of stimulating the immune system as a whole by introducing bacterial agents, like BCG, Corynebacterium par um and others. Material from viral origin [57] and various chemical substances [58] have also been applied. With the exception of BCG that is also used as a single agent against superficial bladder cancer, these agents have been shown to be ineffective by themselves and are now employed as adjuvants with other forms of immunotherapy or chemotherapy Cytokines All cellular components necessary for an effective immune response against cancer are essentially present in individuals with a healthy immune system. Cytokines are transmitters that influence essential steps in the induction of the immune response in either qualitative or quantitative terms. This means that new reactions may occur, or weakly present ones may be augmented to become of significant importance. Many of the known human cytokines are now readily available at reasonable cost thanks to recombinant technology. Since application and evaluation are essentially identical to any pharmaceutical preparation, and much less labour intensive in clinical use compared to most other immunotherapeutical options, cytokines are attractive agents for clinical research and implementation. The number of trials with cytokines in cancer patients largely outnumbers any other form of immunotherapy, even though the studied malignancies are often relatively rare, e.g. renal cancer and melanoma. Cytokines are also used in combined strategies together with other treatment modalities. Insufficient concentration of appropriate cytokines is supposedly one of the reasons of failure of the immune system in tumour recognition and eradication. Adoptive transfer of effector cells or tumour vaccination alone is therefore thought to meet the same bottleneck. This is the rationale behind combinations of cytokine treatment (even through insertion of the appropriate gene in tumor cells) and vaccination or adoptive transfer. Though systemic toxicity is a problem with many cytokines [59 62] activity is mainly locoregional. This implies that a locoregional application allows much higher tissue concentrations in the treated tissue resulting in better outcome in clinical settings (see Section 6 for clinical results). A recent report has provided new insights in the possible mechanism by which cytokines like TNF- and IFN- may cause tumour-restricted disruption of tumour vasculature in isolated limb perfusion, via a reduced activation of the V 3 integrin that is expressed selectively on the proliferating tumour endothelium [63]. This form the rationale of isolated perfusions of limbs and liver [64,65], one of the important applications of cytokines, though biological systems to obtain local secretion of cytokines have been developed too [65]. Functions, as well as origins, of cytokines are generally complex. Frequently, the effect involves several other cytokines too. Most frequently tested in a clinical setting are IL-2, IFN-, IFN-, TNF-, and GM-CSF. Though excess IL-2 boosts mainly aspecific effector cell (LAK) activity, IL-2 is known to have a strong stimulating effect on T lymphocytes as well as NK cells. Interferons, like IFN- and IFN- play an important role in up- and down-regulation of oncogenes and suppression of cell replication, as well as having an anti-angiogenesis effect. IFN-, amongst others, is known to upregulate MHC expression and may increase vascular permeability.

8 8 A.J.A. Bremers, G. Parmiani / Critical Re iews in Oncology/Hematology 34 (2000) 1 25 TNF- has a depressing effect on CTL and NK cells but increases the function of monocyte and T-helper cells. It increases the permeability of the vascular bed and tumour infiltration [63,66]. GM-CSF is known to be involved with the maturation of various hematopoietic stem cells as well as the activity of mature effector cells, but most important is its essential role in DC maturation [67] Vaccination with molecularly defined antigens The essential task of vaccination is to activate the host s immune system. This should be achieved by offering the antigens that T lymphocytes can recognise in an optimally effective way, i.e. adequately presented and in the context of appropriate costimulatory signals Defined antigens and antigen selection In the development of tumour vaccines two different principles can be followed. One is to select antigens that are present on the tumour that is to be treated. Much effort has been put in identifying antigens and their epitopes with this objective in mind; in some instances, as in melanoma, a large [4] but as yet incomplete [68] number of TAA could be identified. Some TAA represent physiologically important proteins that are generally (over)expressed in malignancies; therefore, these would be ideal targets for vaccines that could be used on a large scale. This approach has many advantages, also in respect to cost, and safety of production. It may result in perseverant effectiveness in spite of ongoing selective pressure if the antigens (proteins) represent an essential determinant in carcinogenesis, e.g. RAS, and p53. However, when vaccinating with normal proteins, one risk is to generate a cross reactivity against normal tissues, and hence, auto-immune disease. As discussed above, tumours tend to be individual also in terms of antigen expression [4]. This has led to the introduction of vaccination strategies in which the individual (autologous) tumour cells are used to prepare a vaccine. This however is a very laborious and demanding procedure, requiring specialised laboratory facilities. One good example of a TAA suitable for the first type of vaccine is wild type p53, which is prevalent among a great variety of tumours, while being expressed in normal tissues too. Selective tumour eradication may be obtained through targetting of such proteins in malignancies. Instead, mutant p53 and the mutated RAS require a vaccine the antigens of which match with the mutation present in the individual tumour. A more restricted specificity can be obtained by using antigens that represent proteins expressed only in certain tissues or groups of tissues. Examples of these are the CEA for colorectal and other epithelial tumours, and her-2/neu for breast and ovary cancers. In these examples too, tumour specificity depends on overexpression rather than expression of a mutated protein. As mentioned before, TAA have been identified that are restricted in their expression to certain malignancies (Table 1). If a viral agent is involved in a crucial step of carcinogenesis, it may constitute a valuable source of target antigens of non-self origin. Thus, tumours like hepatocellular carcinoma and cervical carcinoma, could be controlled by vaccination with hepatitis B virus and Human Papilloma Virus (HPV) respectively Peptide based accines The ideal vaccine should present no more antigens than necessary to obtain the desired immunological reaction. The most pure antigen containing vaccine would be multiple synthetic peptides that may contain only oligopeptide epitopes of TAA which can be recognized by the patient s immune system in an HLA-restricted way. To cover a wide range of HLA types, the vaccine should be either HLA type matched or contain a cocktail of epitopes for the various HLA alleles. The goal is to load these peptides onto MHC molecules of APC in vivo; more specifically, binding to MHC on other normal tissue was shown to result in tolerance [69] that, in turn, can cause a better tumour outgrowth [16]. In the context of the right costimulation, as it occurs in the presence of DC, this can be avoided [16]. Peptide vaccination is proven to be effective against viral antigens [70] and in tumour challenge experiments in animals [71]. Clinical investigations have shown interesting results (Section ). However, some problems exist in tumour escape through selection at short notice [72] and the discrepancy between the occurrence of peptide specific CTL and clinical outcome [9,55] Recombinant iral and bacterial accines Recombinant techniques give the opportunity to introduce antigens or just epitopes (eventually along with costimulatory molecules and cytokines) into viral vectors. Viral infection and resulting tissue damage should, according to the danger theory, attract professional APC necessary for adequate antigen presentation. Direct infection of APC could result in endogenous processing. Improved presentation in Class I [73] or Class II [74] HLA, and effective introduction of costimulatory molecules [75] have all been shown to improve antigen presentation. Though circulating cross reacting neutralising antibodies recognising the viral vector constitute a major problem at the moment, particularly in adenovirus vectors, this approach seems very promising. A number of bacterial strains suitable for recombinant engineering like Salmonella [76], BCG [77] and Listeria Monocytogenes [78], have two characteristics that seem very attractive for vaccination: enteric route

9 A.J.A. Bremers, G. Parmiani / Critical Re iews in Oncology/Hematology 34 (2000) of application, and endogenous processing through infection of APC. Results in animal models are promising [79] DNA accination Naked DNA vaccines encoding tumour antigens have been shown to result in some degree of systemic tumour protection in animal experiments [80], but they lack the amplification and generation of danger signals that can be elicited with virus vaccines. Yet, some degree of inflammation and attraction of APC [81] and presentation by such cells [82] have been reported. Mechanisms involved in this type of immunisation remain largely unclear Idiotype antibody accination The idiotype is the variable binding part of an antibody, and fits like a mould to the antigen. When vaccinating with TAA specific antibody, this will give rise to the formation of autologous antibodies against the vaccine. The variable part of these induced antibodies fit to the mould, and therefore strongly resemble the TAA. Thus, the TAA (mimic) is available for recognition by the immune system in a completely different environment. This system has two advantages. It allows vaccination without the need for significant quantities of the purified antigen. Second, but also of great practical importance, it allows the induction of a response against non-protein antigens [83].This strategy has now reached the stage of clinical trials Vaccination with unidentified antigens The less defined the antigenic substances to be recognised by the immune system, the larger is the portion of the tumour cell needed tot include sufficient relevant antigens in the vaccine. In fact, many vaccines involve whole tumour cells. The use of malignant tissue as the crude base from which the vaccine is produced can be expected to increase the risk of side effects. Synthetic preparation of such a vaccine is impossible, and likely to remain so in the future. Cost of vaccination can be expected to grow along with the complexity of the vaccine. The use of such autologous vaccines in the form of whole tumour cells, lysates, apoptotic cells, or heat shock protein extracts has one major advantage: the vaccine represents the whole spectrum of unique and shared antigens expressed by the individual tumour. Thus, the possibility to induce an immune response with a variety of antigens expressed also on the target is increased, and the risk of tumour escape is theoretically reduced Dendritic cells mediated accination For reasons outlined above, DC are now believed to play a central role as APC in tumour immunology. Several methods have been devised, and found successfull, to load the MHC molecules of the DC with appropriate epitopes, often employing the endogenous antigen processing pathway of these APC. These methods range from pulsing with peptides [84], protein [85], cell lysates [86], through cross priming with apoptotic cells [87], fusion with whole tumour cells [15], RNA [88] or transfection with viral vectors [89]. The distinct advantages of using peptides, implying a restricted numbers of antigens, have been discussed above. Using the whole antigenic repertoire of the tumour may lead to stimulation of different T lymphocyte precursors, resulting in a larger repertoire of effector lymphocytes, both CD8 and CD4. Moreover, the application of a larger array of antigens theoretically reduces the chance of selection and tumour escape. Several unanswered questions need to be answered in order to optimise this type of treatment strategies. What are known as DC are a heterogeneous set of cells sharing some morphologic, functional, and surface antigen features [90], but subsets might have considerable differences in respect to other characteristics and functions. Endocytosis of antigens by bone marrow derived APC is followed by endogenous processing and subsequent presentation by both MHC Class II and I [12,87]. Introduction or augmentation of antigen production by tumour cells, e.g. by gene transduction, can lead to increased antigen presentation by the DC through processing of the tumour cell (fragments). Alternatively, one may choose to load the MHC on APC with antigens (peptide, soluble antigen) exogenously. It is also not clear which of the several antigen loading tactics is preferable, in vivo or in vitro. Considerable differences between methods used to obtain DC (bone marrow or peripheral blood derived, the employment of leukapheresis, G-CSF or GM-CSF+IL-4 stimulation, either in vivo or in vitro) call for comparison in respect to effects and efficacy. Results so far are certainly encouraging further exploration in the field of DC based strategies, since clinical trials have shown both antigen specific and clinical responses in, e.g. B-cell lymphoma [85] and melanoma [91] (Section for details) Tumour cell based accination Whole cells, either live irradiated transduced with different genes, dead, or lysed have been employed in vaccines. Of course these may be obstructed in their effect by the very reasons why the tumour itself did not provoke an adequate immune response in the first place. Attempts to overcome this by adding adjuvants, like BCG, to tumour cell based vaccines, as explained

10 10 A.J.A. Bremers, G. Parmiani / Critical Re iews in Oncology/Hematology 34 (2000) 1 25 below, turned out to be effective at least with immunogenic tumours. One reason may be that the used irradiated tumour cells go into apoptosis and are subsequently presented by autologous DC through cross priming. BCG was shown to play an additional role by stimulating maturation of DC [92]. More sophisticated approaches came with genetic modification technologies, that allowed, both in mice and humans, to introduce allogeneic MHC genes into the tumour cells [93], even in vivo [94], in an attempt to enhance immunogenicity. The latest in this field is the introduction of cytokine genes [95] or costimulatory molecules (e.g. B7) [96] into the genome of the vaccine tumour cells. This should then modify micro environmental conditions in the area of tumour cells in such a way that anergy is overcome and tumour lysis takes place [95,97]. One promising example of this is GM-CSF [98]. This is known to contribute to the maturation of DC, and can therefore enhance antigen presentation by the tumour cells also in vivo Heat shock protein accination HSP like hsp70 and 96 are naturally occurring intracellular substances that are supposed to chaperone a wide [99], possibly the full array, of antigenic proteins present in the cell, channelling these into both the MHC Class I and II processing pathway even in APC [100]. HSP are also related to signals associated with tissue damage, signalling danger and thus triggering DC mediated antigen presentation. Isolation of these immunological adjuvants from the tumour [99] thus obviates the need for antigen identification in a particular patient or of composing a cocktail of epitopes from TAA the individual tumour might be carrying, without the need for a vaccine composed of viable cells. Evidence exists for receptor mediated endocytosis of HSP by macrophages and DC. Vaccination with these HSP in animal models can thus induce CTL mediated systemic antitumour immunity [101]. The practical importance of HSP is supported by the observation that immunogenicity of tumour cells is related to HSP 70 they release [102]. Reviewing the existing evidence, one can even conclude that HSP are the predominant source of antigen in the natural setting [16] The role of adju ants Not only the choice of peptides, but also that of the adjuvant seems of crucial importance; amongst others the adjuvant used appears to be able to skew the induced immune response into one direction or another. Few tumour cell based vaccines have reached the phase III trial stage. Generally, these studies are combined with an adjuvant of bacterial nature (immune stimulants) to boost the specific immune response [103,104], though other mechanisms, like improved DC maturation may be involved here. Another promising strategy in adjuvant composition is aimed at a transient block of the inhibition of the T-cell response. The best studied example is the CTLA- 4, known to cause inhibition of T cell function to help terminate the immune reaction, thus interfering with T-cell and lymphokine activation [105]. By blocking such molecule one can potentiate cancer vaccines [106] Adopti e transfer In adoptive transfer selected effector cells are infused into the patient. This can be done systemically, or locally into the tumour. The effector cells have been expanded in vitro, as to evade mechanisms that inhibit expansion in vivo. The effector cells may be antigen specific (CTL) or non-specific LAK cells. Several trials have been conducted employing these strategies, as reviewed below. The source of the effectors can be the tumour, as in tumour infiltrating lymphocytes (TIL) or peripheral blood mononuclear cells (PBMC). PBMC are easier to obtain, but tumour-specific lymphocyte precursor frequency can be expected to be much lower in the blood than at the tumour site. Generally, autologous lymphocytes have to be used, since allogeneic cells can be rapidly rejected by the host. Moreover, allogeneic cells will attack normal tissues, inducing a graft versus host type of reaction. However, it has also been demonstrated that the transplanted allogeneic immune cells can recognize malignant cells as being non-self and mount a therapeutic response known as graft-versus-disease effect. By depletion of subsets of cells, the graft-versus-disease effect remains intact, whereas no graft-versus-host effect occurs [107,108]. This principle of preferential malignancy killing by certain subsets of allogeneic bone marrow cells is now being further evaluated for its use in solid tumours Antibodies and bispecific antibodies Monoclonal antibodies against antigens strongly expressed on tumour cells are capable of inducing tumour infiltration by lymphocytes [109] and Fc receptor mediated cytotoxicity [110]. This effect has been evaluated in some trials, demonstrating in vivo efficacy, as outlined below. By the use of Her2/neu transgenic mice, i.p. injection of an anti-her2 antibody resulted in the inhibition of tumour growth in 50% of animals [111]. Bispecific antibodies, with specificity for both target antigens and antigens on effector cells, can be used as a guide to the target for the effector cells [112].

11 A.J.A. Bremers, G. Parmiani / Critical Re iews in Oncology/Hematology 34 (2000) Results of clinical studies Several hundreds of immunotherapy trials have been reported on, the majority dating from the latest decade. Many of these have been phase I and phase II trials. At the first glance, most of the reports appear disappointing, featuring limited clinical responses (10 20%), often at the cost of considerable toxicity (Table 2). However, the majority of studies have been carried out in patients with advanced and often disseminated disease, where no sensible alternative treatment was available. This casts a shadow on the majority of the available studies since, as outlined above, disseminated disease causes immunosuppression, and advanced disease implies a long period of selective pressure in the direction of tumour escape mechanisms. In fact, some phase III studies, that were able to include earlier stages of disease, show results that can match or improve those of much more toxic chemotherapy, as demonstrated below (Table 3) Immune stimulants as single agents These constitute a heterogeneous group of agents that were found capable of modulating immune responses in an unspecific way, and generally by unknown mechanisms. This however does not detract to the fact that some were found usefull in the therapy of cancer Bacillus Calmette-Guérin (BCG) BCG is undoubtebly the best known agent from this group. BCG injection was found to result in cytokine secretion and activation of DC, which may explain for its possible antitumour effect. BCG as a single agent therapy is probably best known from its widespread use in superficial bladder cancer. Phase III studies have shown that BCG after surgery reduces the recurrence risk by 45%, similar or superior to mitomycin C [113]. In treatment of recurrences, BCG improved 5 year disease free survival from 17 to 37% in infiltrating tumours and from 18 to 45% in carcinoma in situ [114]. In many other malignancies however, including melanoma, it proved to have no Table 2 Highest degree of toxicity regularly encountered during various forms of immunotherapy No/minor toxicity Moderate toxicity Severe toxicity BCG, levamisole, OK432, OK42, GM-CSF, cytokines in isolated perfusion, peptide vaccination, tumour cell vaccination, heat shock protein vaccination (preliminary), DC vaccination Interferons, low dose IL-2, IL-6, IL-12 High dose IL-2, TNF-, LAK therapy, TIL therapy, antibodies (rare) effect, as demonstrated in prospective randomised phase III trials [115]. In malignant melanoma, adjuvant BCG after surgery is not beneficial in stage I and III (AJCC), whereas the effect in stage II remains controversial in several studies [116]; oral BCG monotherapy and intralesional BCG are effective, mainly in cutaneous metastasis, but without impact on the overall survival [116] Le amisole Another widely used agent belonging to this group is Levamisole. Though its application application in Dukes C colorectal carcinoma adjuvant treatment was ineffective, the combination with 5 FU reduced the risk of recurrence by 41% and overall death rate by 33% (at a rather short median follow up of 3 years) [117]. These figures compare favourably with the combination of 5 FU and folinic acid (FA) [118]. Toxicity was identical to that of 5 FU alone [117]. Based on these results the NIH recommended this treatment as standard choice of therapy in the adjuvant treatment of colon cancer [119], though subsequent studies did not completely confirm these results and some modifications of this regimen have been proposed based on 5FU and FA, particularly in Dukes C patients [120]. No antitumour activity of levamisole was found in melanoma patients Other immunostimulating agents As stated before, other immunostimulating agents of various origins have been described and used. Among the most studied are OK 432. Picibanil or OK-432 is a lyophilised preparation of inactivated Streptococcus Pyogenes, that is known to augment non-specific T cell cytotoxicity, LAK activity [121] and tumoricidal activation of macrophages. In the compiled results of two phase III studies from the same investigators, an with two arms and a total of 137 patients and another with three arms (immunotherapy, chemotherapy, surgery only) for a total of 330 gastric cancer patients, the addition of OK-432 to standard surgery and chemotherapy almost doubled 5 year survival time, compared to surgery (24%) and surgery plus chemotherapy (29%), to become 45% [56]. However, a more detailed evaluation of this treatment at 5-year casts doubts on its clinical impact since the same authors reported a survival of only 6% higher than the control group [122]. The influences on cytokine levels responsible for the effect of OK-432 were investigated only recently in melanoma patients, and were found to involve IL-1, TNF and IFN- [123]. In a placebo controlled prospective randomised trial in 64 patients with high risk non metastatic breast cancer, sodium dithio carbonate, applied as an adjuvant treatment in conjunction with the FAC regimen after modified radical mastectomy (vs FAC+operation