Immunotherapy of melanoma - Wiley Online Library

REVIEW ARTICLE

Immunology 2001 104 1±7

Immunotherapy of melanoma CAROLINE SMITH & VINCENZO CERUNDOLO Institute of Molecular Medicine, Nuf®eld Department of Medicine, University of Oxford, Oxford, UK

INTRODUCTION

with an ever-increasing number of possible therapeutic strategies. Melanoma has been the tumour type particularly studied by tumour immunologists, as melanoma cell lines can be generated relatively easily for assays of cytolytic activity. Several laboratories have demonstrated the presence of melanoma-speci®c CTL in tumour in®ltrated lymph nodes, peripheral blood lymphocytes and skin metastases,6±8 raising hopes for immunotherapeutic strategies for melanoma. However, although these results are consistent with an active tumour-speci®c immune response in melanoma patients, it remains unclear whether melanoma-speci®c CTL are capable of slowing tumour progression. Three main approaches have been used to identify tumour antigens. The `genetic' approach, by which the ®rst human tumour antigen was recognized, is based on isolating cytotoxic T-cell clones with speci®city for autologous tumour cells and identifying the genes coding for the T-cell-recognized epitopes by cDNA expression cloning.9 Although the genetic approach has mainly been used to characterize HLA class I restricted epitopes, a similar protocol has recently been used to identify tumour antigens recognized by CD4+ cells in the context of HLA class II molecules.10 This novel approach involves cloning the tumour-derived cDNA library down-stream of a gene fragment encoding the ®rst 80 amino acids of the invariant chain, and channelling tumour antigens through the HLA class II presentation pathway. More recently, serological analysis of recombination expression libraries (SEREX) has been shown to be a powerful method to identify tumour antigens. SEREX is based on the recognition by cancer patients' autologous sera of tumour antigens expressed by a l-phage library.11,12 A large collection of tumour antigens recognized by antibodies, as detected by the SEREX technology, is now available in the database of the Ludwig Institute for Cancer Research (http://www.licr.org/ SEREX.htm). The fact that many of the antigens isolated using SEREX correspond to those identi®ed in melanoma patients using CTL-based techniques suggests that antibody responses against tumour antigens may be closely associated with CTL responses. Hence the new antigens de®ned by SEREX may also sensitize tumours for lysis by CTL. Representational difference analysis (RDA) is a PCR-based subtractive hybridization technique which can effectively isolate differentially expressed genes from a given cDNA population (tester) compared to another (driver). New

Over the past decade, tumour immunology has progressed enormously, as a result of the application of a range of new technologies. Screening of tumour gene expression libraries using cancer patients' sera and cancer-speci®c T lymphocytes has de®ned a large number of tumour-speci®c antigenic proteins. The repertoire of known tumour antigens is rapidly expanding as a consequence of these advances. In addition, over the last few years the analysis of human T-cell responses speci®c for several tumour antigens in cancer patients has been made possible by the use of novel staining reagents (i.e. tetramers). This has allowed the characterization and monitoring of speci®c tumour responses, and has provided an opportunity to greatly accelerate the development of new anticancer vaccines. Recent results have demonstrated that the immune system can detect the presence of malignant cells in cancer patients, and have raised hopes for the use of antigenic cancer proteins as vaccines to induce tumour-speci®c cell-mediated immunity. Tumour immunology is now on solid technical and conceptual footing. However, the challenge remains to apply to patients the results obtained in the laboratories. TUMOUR ANTIGENS Pioneering work carried out by Thierry Boon and colleagues in carcinogen-induced mouse tumours showed that T cells are capable of recognizing tumour cells containing unique genetic point mutations.1 Subsequent work by many laboratories has led to an ever-increasing list of known tumour-speci®c antigens in spontaneously arising tumours (reviewed by Renkvist et al.2). To date, identi®cation of tumour antigens has been focused almost exclusively on antigens recognized by tumour-speci®c HLA class I restricted cytotoxic T lymphocytes (CTL). Recently, the role of HLA class II restricted CD4+ T cells has been increasingly recognized, and the list of known HLA class II epitopes is now rapidly expanding.3±5 HLA class I and class II epitopes are the tools with which tumour-speci®c immunotherapies can be designed, and as our understanding of the complexity of the immune system expands we are provided Received 31 May 2001; accepted 15 June 2001. Correspondence: Vincenzo Cerundolo, Institute of Molecular Medicine, Headley Way, Headington, Oxford OX3 9DS, UK. E-mail: [email protected] #

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tumour antigens have also been identi®ed by searching DNA databases for homologous gene sequences.13 These latter two approaches have been used to identify new tumour antigens beyond those that are isolated on the basis of their immunogenicity. The use of DNA micro arrays is also likely to increase the number of known genes over-expressed on tumour cells. The `biochemical' approach involves acid-elution of peptides bound to the HLA class I molecules of tumour cells, and fractionation of the peptides with reverse-phase highperformance liquid chromatography. Peptide fractions are then tested for their ability to sensitize HLA class I matched target cells for lysis by tumour-speci®c CTL. In some cases the amount of peptide in the positive fraction is suf®cient to obtain its sequence by Edman degradation, but in most instances the amount of peptide is too low and the sequence can only be obtained by mass spectroscopy. The peptide sequence can then be used to search databases in order to ®nd the gene encoding the antigenic peptide.14 The `reverse immunology' approach is the reverse of the ®rst two strategies. The starting point is a protein that is known to be over-expressed or mutated in tumour cells. Peptides from within the protein are selected for their high binding af®nity to a given HLA class I molecule, and are loaded on to antigenpresenting cells, to stimulate lymphocytes in vitro. To demonstrate that the peptide is a genuine tumour antigen epitope, these CTL must then be demonstrated to kill tumour cells expressing the putative tumour antigen and the relevant class I molecule. There are now several computer algorithms, available on the Internet, for predicting peptide sequences that are likely to bind to a given HLA molecule (http://bimas.dcrt. nih.gov/molbio/hla_bind/). More recently, an algorithm has been developed that will predict ef®ciency of peptide presentation at the cell surface based on what is known about preferred proteasomal cleavage sites (http://www.paproc.de). The number of known tumour antigens expressed in tumour types other than melanoma is now expanding with the development of these new technologies. Renkvist et al. have recently published an exhaustive list of tumour antigens that have been shown to be recognized by T cells.2 They have listed all HLA class I and class II restricted T-cell epitopes encoded by tumour antigens published by 31 July 2000. The use of the techniques described above has led to the identi®cation of a large number of melanoma antigenic proteins, which can be divided into three main categories. 1. Cancer-testis antigens These genes are expressed on a variety of tumour types as well as melanoma, and are not expressed on normal tissues, with the exception of spermatogonia, which do not express HLA class I molecules and do not represent targets for class I restricted immune response. The mechanisms responsible for the expression of this group of antigens in tumours and testis remain unclear. One possibility is that their expression results from de-methylation of genes that are normally silenced in nongerm cells.15 Since many of these antigens occur in a wide variety of tumours, they offer the prospect of `broad spectrum' anticancer vaccines aimed at inducing CTL attack.

Cancer-testis antigens include the MAGE family (with now over 20 members),13,16,17 as well as BAGE,18 GAGE19 and NY-ESO-1.20 NY-ESO-1 is one of the most promising of the cancer-testis antigens. It is expressed in a high proportion of breast (30%), prostate (25%), and ovarian (25%) cancer, as well as melanoma (45%), but not in normal tissues.20 NY-ESO-1 appears to be the most immunogenic of cancer-testis antigens known to date, and combined NY-ESO-1-speci®c T-cell and antibody responses are seen in a high percentage of patients with advanced NY-ESO-1-expressing tumours.21,22 2. Differentiation antigens This group of proteins includes antigens that are expressed in melanomas and normal melanocytes. An immune response speci®c to these antigens may cross-react between melanomas and normal melanocytes. This group of antigens includes melan-A protein (whose function is unknown), tyrosinase, glycoprotein 100 (gp100), gp75/tyrosinase related protein 1 (TRP-1) and TRP-2, which are melanosomal enzymes involved in the melanin biosynthetic pathway. Amongst the known melanoma antigens recognized by CTL, melan-A is probably the best studied. It has been established that, in a signi®cant proportion of HLA-A2 positive melanoma patients and healthy controls, CTL speci®c for the melan-A epitope 26±35 can be detected ex vivo by tetramer staining.6,8,23 This work has also shown that CTLs detected in healthy individuals and many melanoma patients are of a naõÈve phenotype, whereas activated cells tend to be seen in patients with more advanced disease.8,23 3. Tumour-speci®c antigens Tumour antigens can arise from point mutations in normal genes as a result of the inherent genetic instability of malignant transformation. These antigens are expressed only in the individual tumour where they were identi®ed, since it is unlikely that the same mutation will occur in two different tumours, unless the mutation results in an obligatory step in malignant transformation. In mouse models, these unique antigens have been shown to be more immunogenic than the other groups of antigens. This group includes some of the most speci®c targets for immunotherapy, but this potential advantage must be balanced against the impracticality for clinical use if they can only be used against the original tumour in which they were found. Recent studies have shown that the immune system can recognize products from alternative transcripts, including those from cryptic start-sites and alternative reading frames.24 IMMUNE MONITORING The standard chromium release assay remains one of the assays most widely used, but it is only semiquantitative and relies on the measurement of cytolytic function of a population of cells. The most quantitative method used until recently was the limiting dilution analysis (LDA). This protocol, which is based on cloning in multiple microcultures, requires the CTL precursors (CTLp) to undergo several cycles of replication prior to determining CTL activity by chromium release for individual culture wells. The LDA approach suffers from major limitations such as high variability and failure to measure #

2001 Blackwell Science Ltd, Immunology, 104, 1±7

Immunotherapy of melanoma activated CTL, possibly because already activated CTL undergo apoptosis on further stimulation. Several new techniques have been developed that allow quanti®cation of antigen-speci®c T cells. These approaches are simple and allow meaningful comparisons of different clinical trials, as well as an improved understanding of spontaneous immune responses. One new approach is the enumeration of cytokine-releasing lymphocytes. Although this is also indirect and involves the measurement of an effector function, it has certain advantages. Cytokine production can be measured at a single-cell level and so there is no need for antigen-speci®c T cells to be expanded prior to the assay in order to be detected. Two experimental methods have been developed based on this approach. (1) The ELISPOT assay, which measures cytokine secreted from individual cells captured on antibody-coated nitrocellulose membrane.8 Each `spot' represents one cytokinesecreting cell, which appears after labelling with a secondary antibody. (2) Cytokine intracellular staining allows cytokine-producing cells to be identi®ed and counted by ¯ow cytometry.25 This technique requires the stimulation of T cells for a few hours with antigen, prior to their intracellular staining with ¯uorochrome-labelled antibody speci®c for intracellular cytokines. To date, the most sensitive protocol to monitor T-cell responses is based on the use of tetrameric HLA class I/peptide complexes (`tetramers').26 Fluorochrome-labelled tetrameric HLA class I/peptide complexes can be used to stain cells, which are subsequently analysed by ¯ow cytometry, allowing for the ®rst time enumeration of speci®c T cells without an indirect functional assay. This technique allows a detailed phenotypic analysis of speci®c T cells using the large panel of markers already available and has been used to further characterize T-cell responses in melanoma patients.6,8,23,27 Tetramer technology also enables rapid and sensitive separation of homogenous populations of antigen-speci®c T cells by ¯ow cytometry cell sorting. Tetramer-sorted lines or clones provide a unique source of cells for TCR repertoire analysis and, potentially, for antigen-targeted adoptive transfer therapy.7

THERAPEUTIC STRATEGIES Progress in tumour immunotherapy has been hampered by the lack of reliable protocols to measure vaccine-driven T-cell responses. The development of tetramer and ELISPOT assays provides an opportunity to greatly accelerate the development of new anticancer vaccines, as rapid comparison of immunization protocols is now possible. We will attempt to categorize tumour immune-therapeutic interventions according to their therapeutic modality, discussing potential advantages and disadvantages of different therapeutic approaches. This analysis will be focused on antigen-speci®c immunotherapeutic approaches, as only these protocols ensure an accurate analysis of the tumour-speci®c immune responses. #


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T-cell adoptive therapy Early ®ndings that human tumour-in®ltrating lymphocytes (TILs) derived from patients with a variety of types of cancer (including metastatic melanoma, breast cancer, colon cancer and ovarian cancer) can exhibit speci®c tumour lysis or cytokine release in vitro contributed to early optimism about the future role of antitumour immunotherapy. When expanded in vitro with IL-2, TILs were found to maintain antitumour activity, and the adoptive transfer of TILs was demonstrated to treat murine malignancies effectively, leading to early trials of adoptive transfer of TILs plus IL-2 in humans.28±30 In 1994, Rosenberg and colleagues transferred in vitroexpanded TILs and IL-2 in 86 patients with metastatic melanoma.31 The overall response rate was 34%. Although this is similar to response rates achieved with IL-2 alone, there was no difference between patients who had never received IL-2 before and those whose treatment with this cytokine had previously failed, suggesting that IL-2 was not the impacting factor. With the discovery of tumour-speci®c antigen, the opportunity arose for adoptive transfer to evolve into a tumour antigen-speci®c therapy. More recently, Yee et al. have described a patient with metastatic melanoma, treated with the adoptive transfer of melan-A-speci®c clones,32 who developed in¯ammatory lesions around pigmented areas of skin. Analysis of in®ltrating lymphocytes in skin and tumour biopsies by tetramer analysis demonstrated a predominance of melan-A-speci®c T cells. Adoptive transfer therapy has remained limited due to technical dif®culties, although it is being studied in EpsteinBarr virus associated malignancies in transplant recipients.33,34 Active immunotherapy Active immunotherapy is aimed at stimulating an endogenous immune response towards antigen or antigens, resulting in clonal expansion and activation of T and/or B cells. 1. Peptides Clinical trials with antigenic peptides have been undertaken with a view to inducing speci®c immune responses in vivo. This approach is limited by the HLA type of the patient. It has the advantages that peptides are easily produced, stable, free of contaminating material, devoid of oncogenic potential and easy to administer. Many small clinical trials have been performed and, although interpretation and comparison have been limited by the immunoassays used, their results suggest that tumourassociated peptides alone can produce speci®c delayed type hypersensitivity (DTH) and CD8+ T-cell responses. Variable clinical responses and no severe toxicities have been seen. Combinations of peptides with different cytokines and adjuvants, including interleukin 2 (IL-2)35 and granulocytemacrophage colony stimulating factor (GM-CSF),36 have also been used; however, the effect of cytokine alone has rarely been compared in the same trial. Cancer-testis antigens Some of the best clinical responses have been demonstrated by Marchand et al. A clinical response was demonstrated in

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7/25 patients with metastatic melanoma who received three subcutaneous injections of the MAGE-A3 HLA-A1 restricted nonapeptide EVDPIGHLY.37 Responding patients tended to have cutaneous rather than visceral metastases. Three responses were complete and two of these led to a diseasefree state, which lasted for more than 2 years. However, no evidence of CTL response was seen by LDA in the four patients who were analysed (including the two who demonstrated complete clinical responses). JaÈger et al. have also shown mixed clinical responses in 12 patients with metastatic NY-ESO-1-expressing cancers who received intradermal injections of three HLA-A2 binding NY-ESO-1 peptides.38 The patients had different tumour types, including melanoma, ovarian carcinoma and breast carcinoma. Peptides were injected intradermally once weekly for 4 weeks. Patients with no evidence of disease progression on day 50 received further immunizations, combined with subcutaneous GM-CSF. Primary peptide-speci®c CD8+ T-cell responses and DTH responses were generated in 4/7 patients who were antibody-negative at the outset. This was associated with disease stabilization and objective regression of single metastases. NY-ESO-1 antibody-positive patients did not develop signi®cant changes in base-line NY-ESO-1 speci®c T-cell responses; however, stabilization of disease and regression of individual metastases was observed in 3/5 patients. Differentiation antigens There have been several trials with HLA-A2-restricted gp100 peptides in patients with metastatic melanoma. Speci®c CTL responses have been demonstrated for two of the peptides: gp100 209±217 (ITDQVPFSY) and gp100 280±289 (YLEPGPVTA).39 A further study compared these two peptides with modi®ed analogues: IMDQVPFSY (gp100 209 2M) or YLEPGPVTV (gp100 280 9V).40 Post-vaccination speci®c immune responses to gp100 209 2M could be seen in 4/6 patients, measured by ELISPOT. Despite this apparent increased immunogenicity, no clinical responses were seen with modi®ed peptide alone, compared to 1/9 with native peptide alone. A response rate of 13/31 was seen with IL-2 and modi®ed gp100 209, demonstrating the activity of IL-2.35 Adjuvant GM-CSF and IL-12 were shown not to enhance clinical or immune responses.41 The HLA-A2 restricted melan-A peptide (p27±35) AAGIGILTV combined with incomplete Freund adjuvant (IFA) has been shown to result in speci®c immune responses when given to patients with metastatic42 or high-risk resected43 melanoma; however, no clinical responses were seen. Similarly, the HLA-A2 restricted tyrosinase peptide 368±376 (YMDGTMSQV) administered with the adjuvant Quillaja saponin (QS-21) produced speci®c immune responses, as measured by ELISPOT assay, in 2/9 patients with metastatic disease.44 Scheibenbogen and colleagues administered different tyrosinase peptides to 18 patients with metastatic melanoma, according to their HLA type. Tyrosinase-speci®c immune responses as measured by ELISPOT were seen in four patients, and minor clinical responses were also seen.45 The simultaneous injection of a pool of HLA-A2 restricted peptides from melan-A, tyrosinase and gp100 was used to vaccinate six patients with metastatic melanoma. Speci®c immune responses against melan-A (3/6) and the tyrosinase signal sequence (2/6) were demonstrated.46 No major tumour

regressions were seen, however. In a follow-up study, in which systemic GM-CSF was coadministered with the fourth injection, enhanced speci®c immune responses and objective tumour regressions were observed in all three patients.36 JaÈger et al. have since reported the case of a patient who, since 1995, received continued immunization with the above peptides.47 The patient had a partial remission and developed extensive vitiligo, and after eight immunizations developed an increase in CTL activity against the melan-A and tyrosinase peptides. There are now several large multicentre trials underway to further investigate peptide vaccines. The ECOG 1696 trial is examining a polypeptide vaccine, consisting of A2 restricted peptides: melan A 27±35, gp100 209±217 and tyrosinase 368±376. Patients with metastatic melanoma will receive the peptide vaccine, and IFNa, GM-CSF, neither or both. Clinical responses as well as immune responses, measured by tetramers and ELISPOT, will be examined. The Intergroup E4697 trial is using the same vaccine and immune monitoring protocol in patients who are disease free, but who have a >70% chance of disease recurrence. 2. Dendritic cells Dendritic cells (DC) are now recognized to be the pivotal antigen-presenting cells that initiate immune responses. The generation of immature DC requires culturing CD34+ precursor cells or CD14+ monocyte-enriched PBMC in the presence of exogenous GM-CSF and IL-4.48 Additional maturation stimuli of cytokine-derived DC with TNF-a or monocyte-conditioned medium is required to obtain mature DC. Recent published papers have demonstrated that immunostimulatory properties of DC are linked to their maturation state. While injection of mature DC enhances antigen-speci®c T-cell immunity, injection of immature DC results in antigen-speci®c inhibition of effector T-cell function49,50 (reviewed by Roncaolo et al.51). It has also been demonstrated that the cryopreservation of DC does not affect the phenotype or function of the cells, further simplifying the production of cells for vaccination purposes.52 Although the introduction DC into the clinic has been hampered by the enormous resources required to generate DC for clinical use, there is an increasing number of clinical trials being published in the literature, involving a wide range of tumour types. Clinical trials have examined methods of generating DC, the maturation status of injected DC and the route of DC injection. Various methods for `loading' tumour antigens onto DC have also been studied, including peptide `pulsing' of DC,53 loading of DC with tumour cell lysates,54 or the fusion of DC with tumour cells. DC-based immunotherapy has been shown to be safe, and clinical and immunological responses have been reported. Nestle and colleagues treated 16 patients with metastatic melanoma with DC pulsed with HLA-A2 tyrosinase, gp100 and melan-A peptides, HLA-A1 MAGE-A1 and MAGEA-3 peptides; or with tumour lysate, if tumour cells were available.54 They were also pulsed with keyhole limpet haemocyanin (KLH) as an adjuvant. The DC were injected monthly up to a maximum of 10 injections, under ultrasound guidance into lymph node uninvolved with tumour. Objective responses were seen in 5/16 patients, with two complete responses. All patients developed a strong delayed type hypersensitivity #


Immunotherapy of melanoma (DTH) towards KLH, and tumour antigen-speci®c DTH was seen in 11/16 patients. Schuler and colleagues vaccinated HLA-A1 patients with MAGE-A3-expressing metastatic melanoma with ®ve vaccinations of DC pulsed with MAGE-A3 HLA-A1 restricted peptide (three intradermal and two intravenous).53 Sizeable expansions of MAGE-A3 A1 peptide-speci®c CTL precursors were observed in 8/11 patients. Regression of individual metastases was observed in 6/11 patients, and T-cell in®ltrates were observed in a regressing metastasis. 3. Plasmid DNA and recombinant viral vaccines Advances in recombinant gene technology have resulted in the development of new tools with which tumour-speci®c therapies can be developed. Viral vectors or plasmid DNA can be used to express a variety of genes in vivo that encode tumour antigens, cytokines or accessory molecules. Genetic constructs can be modi®ed, allowing improved intracellular traf®cking or processing of antigen, and the tumour antigen sequence itself may be modi®ed to enhance immunogenicity of epitopes. Poly epitope-based vaccines, in which multiple CTL epitopes are expressed as a `string of beads', are also under development. Immunization with plasmid DNA has been shown to result in both antibody and T-cell immune responses, including the generation of antigen-speci®c CD8+ and CD4+ cells in murine models. Pre-clinical studies have demonstrated that DNAbased immunizations against model tumour antigens such as chicken ovalbumin55 or b-galactosidase56 can result in protective immune responses, leading to tumour rejection. Plasmid DNA has the advantages that it is readily deliverable, molecularly de®ned and can readily be constructed and produced in large quantities. It is neither infectious, nor capable of replication. Several phase I trials of plasmid-based vaccines encoding a variety of tumour antigens and/or immunomodulators are now being undertaken in several different tumour types. Viral vectors have the advantage that mimicking natural infections may lead to potent cell-mediated responses. Several attenuated viral vaccines are currently considered for use in clinical trials including recombinant adenoviruses and pox viruses such as fowlpox, canary pox and modi®ed vaccinia ankara (MVA). Combination of DNA priming followed by boosting with a defective recombinant virus (prime-boost protocol) has been shown to result in increased levels of speci®c immunity against infectious agents in murine models,57,58 and this approach has obvious applicability to cancer vaccines. REFERENCES 1 Lurquin C, Van Pel A, Mariame B, De Plaen E, Szikora JP, Janssens C, Reddehase MJ, Lejeune J, Boon T. Structure of the gene of tum-transplantation antigen P91A: the mutated exon encodes a peptide recognized with Ld by cytolytic T cells. Cell 1989; 58:293±303. 2 Renkvist N, Castelli C, Robbins PF, Parmiani GA. Listing of human tumor antigens recognized by T cells. Cancer Immunol Immunother 2001; 50:3±15. 3 Chaux P, Vantomme V, Stroobant V, Thielemans K, Corthals J, Luiten R, Eggermont AM, Boon T, van der Bruggen P. #


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