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Keywords:

  • T-cells;
  • tumor immunity;
  • cancer vaccines;
  • adoptive cell transfer therapy;
  • immune monitoring;
  • adjuvants

Summary

  1. Top of page
  2. Summary
  3. Advances in identification of melanoma-associated T cell-defined antigens
  4. Melanoma stem cells as novel targets for immunotherapy
  5. Recent progress in antigen-defined vaccines
  6. Adoptive T cell transfer therapy
  7. Correlates of T cell mediated protection from disease
  8. Coping with immune tolerance and immune suppression by the tumours
  9. Concluding remarks
  10. Acknowledgements
  11. References

Worldwide incidence of malignant melanoma has been constantly increasing during the last years. Surgical excision is effective when primary tumours are thin. At later disease stages patients often succumb, due to failure of metastasis control. Therefore, great efforts have been made to develop improved strategies to treat metastatic melanoma patients. In the search for novel treatments during the last two decades, immunotherapy has occupied a prominent place. Numerous early phase immunotherapy clinical trials, generally involving small numbers of patients each time, have been reported: significant tumour-specific immune responses could often be measured in patients upon treatments. However, clinical responses remain at a dismal low rate. In some anecdotal cases, objective clinical benefit was more frequently observed among immune responders than immune non-responders. This clearly calls for a better understanding of protective immunity against tumours as well as the cross talk taking place between tumours and the immune system. Here we discuss advances and limitations of specific immunotherapy against human melanoma in the light of the literature from the last 5 yr.


Advances in identification of melanoma-associated T cell-defined antigens

  1. Top of page
  2. Summary
  3. Advances in identification of melanoma-associated T cell-defined antigens
  4. Melanoma stem cells as novel targets for immunotherapy
  5. Recent progress in antigen-defined vaccines
  6. Adoptive T cell transfer therapy
  7. Correlates of T cell mediated protection from disease
  8. Coping with immune tolerance and immune suppression by the tumours
  9. Concluding remarks
  10. Acknowledgements
  11. References

Initial studies in human metastatic melanoma revealed the existence of cell mediated immunity in patients with advanced tumours. The identification and cloning of the melanoma-associated tumour antigens specifically expressed by tumour cells promoted the rational development of specific immunotherapy in patients (Van Der Bruggen et al., 1991). The list of characterized major histocompatibility complex (MHC)-I and MHC-II restricted melanoma-associated tumour antigens has been constantly growing over the past 15 yr, and a carefully curated catalogue of these antigens is available online under http://www.cancerimmunity.org/peptidedatabase/Tcellepitopes.htm (last update of July 2009) and will therefore not be discussed here in detail.

Briefly, new epitopes within already known melanoma-associated tumour antigens (e.g. Gp100, Melan-A, Tyrosinase, MAGEs, SSX-2, SSX-4, NY-ESO-1 and others) continue to be identified. In one report, CD8 T cells from HLA-B7+ melanoma patients were found to efficiently recognize a novel, 13 aa. long, naturally processed and presented NY-ESO-1 epitope (Ebert et al., 2009). The crystal structure of this unusual antigenic complex showed the middle portion of the peptide bulging out the peptide binding site. Interestingly, NY-ESO-160–72 specific, HLA-B7 restricted CD8 T cells were undetectable in the patients’ peripheral circulation during the natural course of disease, but significantly expanded after vaccination with NY-ESO-1 ISCOMATRIX (Ebert et al., 2009). In another recent report, a novel gp100 epitope, presented by HLA-A32, was shown to be generated by an unusual mechanism of antigen processing in tumour cells (Vigneron et al., 2004). Autologous tumour reactive CD8 T cells recognize a nonameric epitope derived from the melanocytic differentiation antigen gp100. The antigenic peptide is generated in the proteasome through splicing and fusion of two non-contiguous peptide fragments. This is the third example of protein splicing in mammalian cells, together with another tumour antigen and a minor histocompatibility antigen (Hanada et al., 2004; Warren et al., 2006). Concerning identification of new tumour antigens, a study of tumour infiltrating lymphocytes (TILs) from an HLA-A2+ melanoma patient revealed the presence of tumour reactive lymphocytes specific for an antigen overexpressed in a high proportion of melanoma cell lines (MELOE-1) and encoded by an unusual anti-sense transcript originating from an intronic DNA stretch and containing multiple orphan open reading frames (Godet et al., 2008). Interestingly, an infusion of TILs containing MELOE-1 specific CD8 T cells correlated with relapse prevention.

While most tumour antigens identified thus far are targeted by bona fide effector T cells, two recent studies report on the characterization of peptides derived from the tumour-associated antigens LAGE-1 and ARTC-1 which are preferentially recognized by CD4 regulatory T cells (Tregs), in the context of HLA-DR13 and HLA-DR1, respectively (Wang et al., 2004, 2005). Amplification of Tregs specific for those peptides might inhibit successful anti-tumour immune responses. Finally, a first report appeared on efficient MHC-II restricted recognition of epitopes derived from phosphopeptides, exclusively expressed by melanoma cells (Depontieu et al., 2009). HLA-DR1 restricted phospho-Melan-A specific CD4 T cells present in two distinct melanoma reactive T cell lines, were able to recognize both phosphopeptide-pulsed APCs as well as tumour cells. In this respect, new technology has recently been developed for the sensitive identification of naturally processed phosphopeptides associated with tumour MHC-I and MHC-II molecules (Meyer et al., 2009).

These few highlighted recent reports and multiple others reveal the high number of T-cell defined tumour antigens which are generated through unusual, poorly known cellular mechanisms. It might be that central tolerance efficiently deletes most of T cells specific for more conventionally derived tumour antigenic peptides. A better understanding of the generation and recognition of MHC-I and MHC-II restricted tumour-associated epitopes, in combination with already known immunogenic peptides, may allow for optimized targeting in melanoma vaccine development.

Melanoma stem cells as novel targets for immunotherapy

  1. Top of page
  2. Summary
  3. Advances in identification of melanoma-associated T cell-defined antigens
  4. Melanoma stem cells as novel targets for immunotherapy
  5. Recent progress in antigen-defined vaccines
  6. Adoptive T cell transfer therapy
  7. Correlates of T cell mediated protection from disease
  8. Coping with immune tolerance and immune suppression by the tumours
  9. Concluding remarks
  10. Acknowledgements
  11. References

A very active area of investigation with important implications for the development of effective immunotherapy strategies consists in the identification and characterization of melanoma stem-cells. Tumour-initiating cells with self-renewal potential, high tumourigenicity and potential to differentiate into various lineages have been identified in several human cancers, including tumours of the blood, brain, breast, pancreas, colon and sarcomas (Al-Hajj et al., 2003; Collins et al., 2005; Galli et al., 2004; Hermann et al., 2007; Lapidot et al., 1994; O’Brien et al., 2007; Singh et al., 2004; Suva et al., 2009). In malignant melanoma, recent evidence suggests four major candidates defining melanoma stem-cells. Using embryonic stem cell-based medium, cells derived from dissociated tumour lesions growing as spheres were shown to possess a 10-fold higher tumourigenic potential than adherent cells grown on standard culture medium. Remarkably, cells with multipotent differentiation properties were enriched in the subpopulation of in vitro established melanoma cell lines defined by expression of the B-cell marker CD20. Also of interest, CD20+ melanoma cells could be directly identified in a fifth of human melanoma specimens (Fang et al., 2005). Another marker commonly used to distinguish stem-cells is the membrane glycoprotein CD133. Several independent studies report that distinct subsets of cultured melanoma cells are positive for CD133 (Frank et al., 2005; Klein et al., 2007; Monzani et al., 2007). In addition, increased aggressiveness and capacity of de novo induction of tumours was only observed in the CD133+ population. Another flow cytometry-based technique has been used to isolate tumour stem-cells according to their peculiar efflux capacities, that seem to be mediated through the expression of ABC transporters. This approach allowed to isolate side populations with stem-cell features and expressing ABC transporters, e.g. ABCG2 and more recently ABCB5 were specifically identified on putative melanoma stem-cells. However further analyses are needed to establish the different constituents of side populations, and if and which ABC transporter(s) better identify stem-cells in human malignant melanoma (Grichnik et al., 2006; Hadnagy et al., 2006; Schatton et al., 2008). Even if still in the early phase, research on melanoma stem-cells might provide tools for identification of useful clinical prognostic factors and/or novel attractive targets for newly designed therapies. Recent results suggest that a subpopulation of clonogenic melanoma cells expressing CD133 may also express, at least in melanoma cell lines, increased levels of shared tumour specific antigens (Gedye et al., 2009). Confirmation of these exciting results in primary tumour tissue would suggest that specific immunotherapy directed against shared tumour specific antigens may also target tumour stem cells. It is expected that selective targeting of stem cells would be efficient in curbing tumour relapse following response to conventional therapeutic options.

Recent progress in antigen-defined vaccines

  1. Top of page
  2. Summary
  3. Advances in identification of melanoma-associated T cell-defined antigens
  4. Melanoma stem cells as novel targets for immunotherapy
  5. Recent progress in antigen-defined vaccines
  6. Adoptive T cell transfer therapy
  7. Correlates of T cell mediated protection from disease
  8. Coping with immune tolerance and immune suppression by the tumours
  9. Concluding remarks
  10. Acknowledgements
  11. References

Experiments in animal models have been the basis for the development of clinical trials of vaccination in cancer patients. Anti-melanoma vaccines have been evolving during the last years, and they considerably vary in terms of antigenic composition, delivery systems, adjuvants, injection frequencies and administration modalities.

Peptide/protein based immunotherapy

Administration of free natural antigenic peptides, emulsified in some of the adjuvants available for human use (IFA, Alum, ASO2B) in melanoma patients resulted in detectable, though limited immunological responses, that correlated with local skin reactions, but did not confer significant clinical benefit (Lienard et al., 2004). Recent improvements of peptide vaccination strategies included the use of analogue peptides displaying increased immunogenicity, simultaneous injection of multiple tumour-associated peptides, addition of immunostimulatory adjuvants, represented by cytokines or toll-like receptor agonists (e.g. CpG-ODNs, Imiquimod), or inclusion of MHC-II restricted epitopes from well known tumour-associated antigens. A non-exhaustive list of recently reported clinical studies is compiled in Table 1. Generally, weak toxicity and few side effects were recorded. In some cases brisk expansion of circulating and/or tumour infiltrating antigen-specific CD8 T cells has been recorded. Noteworthy, the first evidence of clinical benefit in a prospective randomized multicentric phase III clinical trial was reported recently. This trial included 185 melanoma patients treated either with gp-100209–217 analogue peptide and high dose IL-2 or with high dose IL-2 alone. Response rates as well as progression free survival were significantly superior in patients immunized with peptide plus IL-2, compared to the IL-2 only patients’ arm (Schwartzentruber et al., 2009). These positive results suggest that further improvement of peptide-based immunotherapy, with use of multiple peptides combined with more potent adjuvants might be the right way to obtain clinically relevant results.

Table 1.   Peptide-based immunotherapy in metastatic melanoma patients
AntigensAdjuvantsSpecific T cell responsesaReferences
  1. aA wide array of specific T cell assays has been used to evaluate vaccine-specific T cell responses in immunotherapy trials. These assays include direct ex-vivo visualization of specific T cells using pMHC-I/pMHC-II multimers and/or IFN-γ ELISPOT; or in vitro expansion of T cells using peptides prior to assessment of specific T cell frequencies by pMHC-I/pMHC-II multimers and/or IFN-γ ELISPOT and/or intracellular cytokine staining and/or DTH. Analyses were performed on peripheral blood lymphocytes, tumour-infiltrated lymph nodes and/or lymph nodes draining the immunization sites, and/or lymphocytes recruited to DTH skin sites. Therefore, the results from all these trials are not comparable. In fact, the variability in immunomonitoring techniques constitutes one of the current hurdles in cancer immunotherapy.

  2. Ratios represent number of responding over total of analysed patients. When specified in the publications, frequencies of detected CD4 and CD8 T cell responses are reported separately.

  3. n.d., not declared.

Natural peptideIFA7/14(Parkhurst et al., 2004)
Natural peptiden.d.; 1/7; 1/1(Germeau et al., 2005; Lonchay et al., 2004; Lurquin et al., 2005)
Natural peptideIFA, CpG6/6(Speiser et al., 2008)
Natural helper peptideAS02B, IFA3/3(Wong et al., 2004)
Analogue peptideIFA5/5; 33/35(Powell and Rosenberg, 2004; Walker et al., 2004)
Analogue peptideIFA, GM-CSF, IL-125/5(Chiong et al., 2004)
Analogue peptideIFA, CpG3/8; 8/8(Fourcade et al., 2008; Speiser et al., 2005)
Analogue peptideIFA, low-dose IL-21/26(Roberts et al., 2006)
Analogue peptideIFA, high-dose IL-2n.d.(Sosman et al., 2008)
Multipeptides1/2(Francois et al., 2009)
MultipeptidesIFA4/4; 50–85/95(Baumgaertner et al., 2006; Rosenberg et al., 2005)
MultipeptidesLow-dose IL-2, GM-CSF28/40(Slingluff et al., 2004)
MultipeptidesGM-CSF, IFA25/25; 9/23; 9/25; 51/51(Chianese-Bullock et al., 2005; Hersey et al., 2005; Markovic et al., 2006; Slingluff et al., 2007)
MultipeptidesIFA, Alum, IL-12, GM-CSF38/60(Hamid et al., 2007)
Helper multipeptidesGM-CSF, IFA32/39(Slingluff et al., 2008)

One of the limitations in the use of peptides as immunogens is the need for patients’ selection according to expression of defined HLA molecules. In addition, often exclusively MHC-I restricted epitopes have been included in immunization protocols, precluding targeting of tumour-specific helper CD4 T cells. In this regard, vaccination using recombinant proteins has the potential to simultaneously target and prime both CD8 and CD4 T cells specific for a variety of epitopes of a tumour-associated antigen, across any HLA barrier. Recombinant protein vaccines corresponding to tumour-specific antigens from the cancer/testis family have been tested in Phase I/II clinical trials in combination with several adjuvants. Induction of both humoural and cellular responses could be shown, as well as some clinical responses. A summary of protein-based immunotherapy trials is shown in Table 2. Of interest, vaccination of metastatic melanoma patients with a recombinant MAGE-A3 fusion protein administered with an adjuvant containing monophosphoryl lipid A, an oil in water emulsion, QS-21 and liposomal CpG-ODNs proved far superior in terms of both clinical and immune responses than immunization with the same protein in an adjuvant in which the CpG-ODNs component had been left out (Kruit et al., 2008). A large phase III trial using the better vaccine formulation identified in patients with melanoma is underway in resected non-small cell lung carcinoma patients (Brichard and Lejeune, 2008).

Table 2.   Protein-based immunotherapy in metastatic melanoma patients
ProteinAdjuvantsSpecific T cell responsesaReferences
  1. aSee footnote a to Table 1.

  2. ICS, intracellular cytokine staining.

NY-ESO-1IFA, CpG17/18 CD4, 9/18 CD8 T cells(Valmori et al., 2007)
NY-ESO-1Imiquimod3/9(Adams et al., 2008)
NY-ESO-1ISCOMATRIX2/9 by tetramer staining, 6/8 by ICS; 10/25 CD4, 17/25 CD8 T cells(Davis et al., 2004; Nicholaou et al., 2009)
NY-ESO-1CHP1/1; 7/9(Tsuji et al., 2008; Uenaka et al., 2007)
MAGE-A31/6; 1/5(Kruit et al., 2005; Zhang et al., 2005b)
MAGE-A3AS02B or AS-156/6; 39/72(Francois et al., 2009; Kruit et al., 2008)
Hsp gp-96GM-CSF, IFNα10/17(Pilla et al., 2006)

Despite the obvious advantages of recombinant proteins, some shortcomings to their application as vaccines have also become clear. In contrast to viral vectors, proteins are generally poor agents to cross prime MHC-I restricted CD8 T cell responses because they are delivered to the cells via the endocytic compartment. Moreover, from a practical stand point, the generation of clinical grade batches of recombinant proteins may be prohibitively expensive and even prove elusive for certain highly hydrophobic proteins. An interesting compromise between short peptides and full length recombinant proteins might be the use of long synthetic peptides, usually 20–30 amino acid long. Recent encouraging clinical results following vaccination of vulvar intraepithelial neoplasia patients with HPV-16 E6/E7 long synthetic peptides have been reported (Melief and Van Der Burg, 2008).

Dendritic cell based vaccines

One of the key insights in modern immunology is the identification of dendritic cells as the only cell type able to initiate de novo antigen specific T cell responses (Steinman, 2008). Attempts have therefore been made to generate suitable dendritic cells (DCs) loaded with peptides, proteins, whole tumour cell lysates or mRNA to be used in clinical trials. A summary of recently reported vaccination trials using DCs is shown in Table 3. Although immunological activity upon DC immunotherapy has been documented in several patients, clinical benefit remains scarce. The first randomized phase III clinical trial using DCs came recently to end. The endpoint of this study was to compare dacarbazine (DTIC) treatment versus vaccination with autologous peptide-pulsed DCs in a total of 108 metastatic melanoma patients. The results failed to demonstrate superiority of the DCs-based immunotherapy compared to DTIC chemotherapy (Schadendorf et al., 2006). For DCs-based vaccinations to be improved, better knowledge on several parameters concerning DCs are needed, in particular regarding the type and maturation stage of DCs to be injected, the optimal epitope density on the cell surface, and the most promising protocols for DCs in vitro generation (Aarntzen et al., 2008) An alternative approach to ex vivo handling of DCs is to target antigens directly to DCs in vivo, e.g. via the use of antibodies directed against surface molecules selectively expressed in DCs, as shown by encouraging results in preclinical models of vaccination (Tacken et al., 2007).

Table 3.   DC-based immunotherapy in metastatic melanoma patients
DC+AdjuvantsSpecific T cell responsesaReferences
  1. aSee footnote a to Table 1.

Peptide8–12/12; 3/6; 2/2; 3/8; 9/13; 4/7; 1/1(Linette et al., 2005; Lonchay et al., 2004; Schultz et al., 2004; Trakatelli et al., 2006; Tuettenberg et al., 2006; Vonderheide et al., 2004; Zhang et al., 2005a)
Multipeptides7/8; 1/1; 1/6; 13/20; 3/6(Banchereau et al., 2005; Carrasco et al., 2008; Davis et al., 2006; Fay et al., 2006; Francois et al., 2009)
MultipeptidesImiquimod7/16(Shackleton et al., 2004)
Tumour cells16/40; 3/13; 4/12; 2/5(Bercovici et al., 2008; Palucka et al., 2006; Salcedo et al., 2006; Von Euw et al., 2008)

Viral vectors as antigen delivery vehicles and DNA vaccines

Recombinant viral vectors encoding tumour-associated antigens represent an immunization strategy potentially leading to preferential priming or boosting of specific CD8 T cell responses. Indeed, immunological activity could be triggered, and in some cases favourable effect of vaccination on disease evolution could be observed (Jager et al., 2006; Lonchay et al., 2004; Smith et al., 2005a; Spaner et al., 2006; Van Baren et al., 2005). An important draw back of viral vectors, such as vaccinia, is the immunodominance of viral antigens that may out compete tumour antigen-specific immunity (Smith et al., 2005b). This may be overcome by the use of naked DNA as immunogen. Plasmid DNA has been demonstrated to direct antigen expression in muscle cells and dendritic cells in vivo thus providing a rationale to directly use it as an antigen delivery vehicle. At least three exploratory clinical trials have been reported using administration of plasmid DNA either by direct intranodal (Weber et al., 2008) or intramuscular injection (Yuan et al., 2009) or by gene gun delivery into the skin (Cassaday et al., 2007). It has also been reported that established immunosuppressive mechanisms might not be successfully overcome through this vaccination procedure (Gnjatic et al., 2009). Recently, electroporation of plasmid DNA to muscle has been tested in the clinical setting and may hold promise to enhance DNA immunogenicity (Low et al., 2009). Finally, heterologous prime boost regimens may contribute to optimize cancer vaccine development and, in this case, offer a win-win solution by associating plasmid DNA electroporation for priming to boosting with recombinant viral vectors (Lu, 2009). In addition to DNA, interestingly, a phase I trial of intradermal vaccination with total tumour mRNA and GM-CSF was reported recently as feasible and immunogenic (Weide et al., 2008).

Adoptive T cell transfer therapy

  1. Top of page
  2. Summary
  3. Advances in identification of melanoma-associated T cell-defined antigens
  4. Melanoma stem cells as novel targets for immunotherapy
  5. Recent progress in antigen-defined vaccines
  6. Adoptive T cell transfer therapy
  7. Correlates of T cell mediated protection from disease
  8. Coping with immune tolerance and immune suppression by the tumours
  9. Concluding remarks
  10. Acknowledgements
  11. References

Recent technological advances have provided the means to evaluate an alternative immunotherapy strategy, consisting in the ex vivo expansion of large numbers of autologous tumour reactive lymphocytes (isolated from TILs or peripheral blood of the patients), to be subsequently re-infused in the patients’ circulation as immunologically active effector T cells. Encouraging results were reported in malignant melanoma patients treated first with lymphodepleting, non-myeloablative chemotherapy (e.g. fludarabine and cyclophosphamide) (Dudley et al., 2005) or with this same regimen plus 2 or 12 Gy total body irradiation (Dudley et al., 2008), followed by transfer of high numbers of in vitro expanded autologous TILs and high dose IL-2. These approaches resulted in objective clinical response rates ranging between 50 and 70%. Other groups have tested adoptive transfer of large numbers of tumour antigen specific cloned T cells. Infusion of Melan-A specific CTL clones in HLA-A2 metastatic patients resulted in objective clinical responses in 43% of patients. Clinical benefit correlated with detectable expansion of the endogenous Melan-A specific CD8 T cell repertoire (Khammari et al., 2009). Fludarabine conditioning had a modest but sizeable effect of the relative persistence of adoptively transferred tumour reactive CTL clones in a cohort of 10 advanced melanoma patients (Wallen et al., 2009).

In a recent case report, a durable remission took place after infusion of cloned autologous CD4 T cells specific for NY-ESO-1. Again, detectable T cell responses specific for the same antigen could be measured in the post-adoptive transfer period (Hunder et al., 2008).

The need for labour intensive in vitro expansion of isolated or enriched tumour reactive lymphocytes may be obviated by gene transfer of T cell receptor (TCR) genes of controlled specificity into blood lymphocytes (Uckert and Schumacher, 2009). In a trial that enrolled 15 melanoma patients, which were infused with T cells transduced with retroviruses expressing tumour specific TCRs, two patients experienced objective tumour regression (Morgan et al., 2006). These are clearly promising results. However, this approach needs optimization on various fronts such as on the trafficking, survival and persistence of expanded and re-infused cells. Moreover, controlling appropriate levels of TCR transgene expression is proving very challenging (Jorritsma et al., 2007; Kuball et al., 2009; Yang et al., 2008). Either transduced TCRs are out competed by the endogenous ones (Heemskerk et al., 2007), or mispairing of TCR chains often generates T cells with up to four TCRs, some of which might cause immune pathology (Bendle et al., 2009). Finally, it is also noteworthy that chimeric antigen receptors, whereby the antigen recognition module of tumour cell surface antigen specific antibodies is spliced to one or multiple signalling domains, are also very actively developed. This approach would allow to genetically reprogram T cells to acquire the ability to recognize cell surface antigens independently of MHC restriction. In this setting, optimization of signalling from the chimeric receptor to the T cell expressing it involves selected combinations of signalling domains from both TCRs and costimulatory receptors (Sadelain et al., 2009).

An innovative approach using gene transfer to autologous circulating lymphocytes is to transduce tumour specific antigen to be used as vaccine delivery system. This application is based on previous observations in the adoptive transfer setting showing that HSV thymidine kinase transduced lymphocytes, as a suicide gene for safety reasons, are strongly immunogenic in the recipients. In the first clinical trial testing this approach, 10 metastatic melanoma patients received multiple infusions of MAGE-A3 genetically modified autologous lymphocytes which were well tolerated and specific immunity could be detected in three patients (Fontana et al., 2009).

Correlates of T cell mediated protection from disease

  1. Top of page
  2. Summary
  3. Advances in identification of melanoma-associated T cell-defined antigens
  4. Melanoma stem cells as novel targets for immunotherapy
  5. Recent progress in antigen-defined vaccines
  6. Adoptive T cell transfer therapy
  7. Correlates of T cell mediated protection from disease
  8. Coping with immune tolerance and immune suppression by the tumours
  9. Concluding remarks
  10. Acknowledgements
  11. References

In order to protect from tumour progression, tumour antigen specific CD8 T cells need to be activated in a robust manner (Pardoll, 2003). Extended analysis of biological effects of immunotherapy revealed that tumour specific human T cell responses were often insufficient, making it impossible to draw firm conclusions on the role of T cells in clinical responses. Even in animal models, it remains difficult to identify correlates of T cell mediated protection from tumour progression. In infectious diseases, several T cell criteria have been established as correlates of protection. First, the capacity of T cells to efficiently interact with cognate antigen (Alexander-Miller et al., 1996; Bennett et al., 2007; Derby et al., 2001; Gallimore et al., 1998; Messaoudi et al., 2002; Sedlik et al., 2000; Speiser et al., 1992; Yee et al., 1999; Zeh et al., 1999), a property which is often termed as ‘functional avidity’ and which is primarily controlled by T cell receptors (TCRs). However, a survey of oligo or monoclonal CD8 T cell responses to MAGE-A3 vaccination in melanoma patients who had objective tumour regression failed to show high avidity of antigen recognition (Connerotte et al., 2008). Second, a central role is played by the precursor frequency of specific T cells (Moon et al., 2007; Obar et al., 2008). Finally, protection depends on the functional capacity of T cells, likely dependent on functional avidity and many more parameters. Recent attempts have focused on direct ex vivo functional profiling of T cells, in order to identify multiple functional properties of effective T cells that are operational in vivo (Almeida et al., 2007; Betts et al., 2006; Daucher et al., 2008; Lichterfeld et al., 2007; Rehr et al., 2008). There is increasing consensus that T cells must be multifunctional (Appay et al., 2008; Seder et al., 2008). Mouse models suggest that for tumour immunity, similar principles may apply as for infectious diseases. Thus, functional avidity appears to be essential, including the capacity of T cells to recognize naturally expressed and processed tumour antigen (Kochenderfer and Gress, 2007). High avidity CD4 T cells would also be critical in determining tumour rejection mediated by CD8 T cells (Brandmaier et al., 2009). Precursor frequency of appropriate T cells plays also a key role (Rizzuto et al., 2009). Finally, it is important that T cells are fully functional, with competent responsiveness to antigen, proliferation, T cell survival, homing, effector function and generation of immunological memory (Appay et al., 2008; Klebanoff et al., 2006; Wherry et al., 2003). In the adoptive transfer setting, it is increasingly clear that T cells with the ability to persist for prolonged periods of time in vivo correlate with clinical efficacy (Berger et al., 2008; Heemskerk et al., 2008; Shen et al., 2007; Zhou et al., 2005). Improvement of immunotherapy may depend on careful optimization towards the generation of T cells fulfilling these criteria. Results from small scale phase I/II clinical studies provide the rational basis to select the most promising treatment modalities for subsequent costly phase III trials assessing clinical efficacy.

Coping with immune tolerance and immune suppression by the tumours

  1. Top of page
  2. Summary
  3. Advances in identification of melanoma-associated T cell-defined antigens
  4. Melanoma stem cells as novel targets for immunotherapy
  5. Recent progress in antigen-defined vaccines
  6. Adoptive T cell transfer therapy
  7. Correlates of T cell mediated protection from disease
  8. Coping with immune tolerance and immune suppression by the tumours
  9. Concluding remarks
  10. Acknowledgements
  11. References

Consensus has emerged that the modest clinical efficacy of specific immunotherapy is explained not only because of suboptimal vaccine development but also, and perhaps to a great extent, because of limited access to tumour that prevents efficient infiltration by specific T cells and/or of strong tolerogenic conditions prevailing at the tumour sites. The former has been clearly shown in experimental tumour models. Indeed, tumour related angiogenesis has been shown to prevent migration of immune effectors into established tumour tissue. Regulator of G-protein signalling 5 (Rgs5) was recently shown to be a master gene responsible for the abnormal tumour vascular morphology in mice and its deletion resulted in pericyte maturation, vascular normalization and consequent marked reductions in tumour hypoxia and vessel leakiness. Noteworthy, these vascular and intratumoural changes enhanced influx of immune effector cells into tumour parenchyma and markedly prolonged survival of tumour-bearing mice (Hamzah et al., 2008). In another model, the overexpression of endothelin receptor B (ETBR), leading to a shift in the balance of the ETAR/ETBR ratio, leads to increased angiogenesis and, at the same time, suppression of ICAM-1 expression on endothelial cells, and inhibition of transendothelial migration and homing, an effect largely mediated by nitric oxide (NO). ETBR blockade, as well as NO antagonists and TNF-α promote endothelial cell expression of ICAM-1 and increased binding and transendothelial migration and tumour homing of T cells (Kandalaft et al., 2009). In patients, a global gene expression study of 44 melanoma biopsies revealed that lack of critical chemokines in a subset of melanoma metastases may limit the migration of activated T cells, which in turn could limit the effectiveness of antitumour immunity (Harlin et al., 2009).

Evidence for mechanisms rendering T cells hypo functional and unresponsive came from studies in mouse models, and more recently also from analyses of tumour and immune cells in human cancers. First, systemic and locally elevated frequencies of Tregs in cancer patients, including melanoma patients have been reported, and have been shown to interfere with anti-tumour activity of effector cells (Ahmadzadeh et al., 2008; Jandus et al., 2008; Kryczek et al., 2009; Nicholaou et al., 2009; Viguier et al., 2004; Wang et al., 2004, 2005). In this regard, tumour-infiltrating specific CD8 T cells are often blunted in their functions (Appay et al., 2006; Harlin et al., 2006; Mortarini et al., 2003; Zippelius et al., 2004). Second, inhibitory molecules expressed on the surface of CD8 T cells have been proposed to mediate impaired effector capacities (Ahmadzadeh et al., 2009; Demotte et al., 2008; Fourcade et al., 2009; Wang et al., 2009). In addition, tumour cells themselves can evolve during disease progression to escape from immune attacks, e.g. by down-regulation of immune key molecules, like MHCs (Cabrera et al., 2007; Kageshita et al., 2005; Voelter et al., 2008; Yamshchikov et al., 2005), or by production and release of inhibitory factors (Ilkovitch and Lopez, 2008). Evidence for the presence of these suppressive networks affecting T cell competence has been shown in several types of human cancers, including malignant melanoma, and could even be in some cases correlated with poor patients’ survival (Brody et al., 2009; Bronte and Zanovello, 2005; Ekmekcioglu et al., 2006; Johansson et al., 2009; Rubinstein et al., 2004; Zou, 2005). Finally, local infiltration of the tumour mass by immune cells other than T cells [e.g. myeloid derived suppressor cells (MDSCs)] might also interfere with generation of efficient anti-tumour T cell responses (Gabrilovich and Nagaraj, 2009; Mandruzzato et al., 2009).

In view of these complex host-tumour interactions, successful immunotherapies in the future may be identified among multimodal treatment strategies. Blockade of co-inhibitory receptors on activated T cells has shown promise in mouse models. Moreover, the clinical development of humanized anti-CTLA-4 antibody might find approval by regulatory authorities in the not so distant future. Melanoma patients treated with anti-CTLA-4 as single agent have an objective tumour response rate around 17% (Attia et al., 2005) but significantly larger tumour benefit is apparent when clinical response criteria are adjusted in a controlled fashion (Saenger and Wolchok, 2008). Immunomonitoring of responding patients clearly suggests increased tumour specific immunity mediated by both antibodies and specific T cells (Hodi et al., 2008; Klein et al., 2009; Liakou et al., 2008; Yuan et al., 2008). Other potentially useful immunologically active compounds include cytokines (Davis et al., 2009; Kim-Schulze et al., 2009; Li et al., 2009; Mueller et al., 2008; Shinozaki et al., 2009; Sikora et al., 2009), modulators of key signalling pathways such as STAT3 (Kong et al., 2009), chemotherapy (Nistico et al., 2009) or costimulatory agonists (Gray et al., 2008) such as 4-1BB (Kim et al., 2008), LAG-3 (Li et al., 2008), CD40 (Advani et al., 2009; Vonderheide et al., 2007) or chemokines (Loeffler et al., 2009). Reduction, depletion or induction of differentiation of various immunoregulatory cell types would also be synergistic with specific immunotherapy. The major targets in this regard are Tregs (Morse et al., 2008; Powell et al., 2008), MDSCs (Ko et al., 2009; Ugel et al., 2009) and M2 macrophages (Allavena et al., 2008). Tumour antigen based vaccination has been recently shown to either be associated with appearance of vaccine specific Tregs (Francois et al., 2009), or with a significant reduction of specific Tregs (Jandus et al., 2009). In the latter study, the same Melan-A peptide could be recognized by HLA-A2-restricted CD8 T cells and by HLA-DQ6-restricted CD4 T cells. Thus, vaccination of melanoma patients appears to not only result in significant increases of specific CD8 T cells but also in marked reduction of Tregs with concomitant increase of Th1 CD4 T cells specific for the same immunizing peptide.

Concluding remarks

  1. Top of page
  2. Summary
  3. Advances in identification of melanoma-associated T cell-defined antigens
  4. Melanoma stem cells as novel targets for immunotherapy
  5. Recent progress in antigen-defined vaccines
  6. Adoptive T cell transfer therapy
  7. Correlates of T cell mediated protection from disease
  8. Coping with immune tolerance and immune suppression by the tumours
  9. Concluding remarks
  10. Acknowledgements
  11. References

Human melanoma has been and continues to be a paradigm in the understanding of interactions between the immune system and developing metastatic tumours. Rapid progress continues to be made fueled by novel insights derived from both mouse models and the detailed analysis of patients included in phase I-II clinical trials of tumour antigen-based vaccines. The field is currently focused on the dissection of innate and adaptive immune response mechanisms operating in situ, in the tumour stroma. Local immune tolerance appears to be the dominant outcome of tumour-host interactions. Thus, for immunotherapy to reach significant clinical efficacy, novel combinations including vaccination and appropriately tumour targeted immune modulating compounds remain to be identified and optimized. To achieve this goal continued cross-fertilization between basic immunology and translational research is essential. Moreover, innovation in the legal regulatory landscape is urgently needed as to provide incentives for the cooperation between industry and academic institutions. These should also foster much needed industrial partnerships to cooperatively develop combinations of compounds currently in the pipelines of numerous companies. This implies to pursue the transition from monotherapies, sponsored by the owner companies, to combinatorial therapies requiring companies to join forces and share both resources and risk.

References

  1. Top of page
  2. Summary
  3. Advances in identification of melanoma-associated T cell-defined antigens
  4. Melanoma stem cells as novel targets for immunotherapy
  5. Recent progress in antigen-defined vaccines
  6. Adoptive T cell transfer therapy
  7. Correlates of T cell mediated protection from disease
  8. Coping with immune tolerance and immune suppression by the tumours
  9. Concluding remarks
  10. Acknowledgements
  11. References
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