Recombinant Modified Vaccinia Ankara Primes Functionally Activated CTL Specific for a Melanoma Tumor Antigen Epitope in Melanoma Patients with a High Risk of Disease Recurrence Caroline L. Smith 1 , P. Rod Dunbar 1 , Fareed Mirza 1 , Michael J. Palmowski 1 , Dawn Shepherd 1 , Sarah C. Gilbert 2 , Pierre Coulie 3 , Joerg Schneider 4 , Eric Hoffman 5 , Robert Hawkins 6 , Adrian L. Harris 7 and Vincenzo Cerundolo 1 * 1 Tumour Immunology Unit, Weatherall Institute of Molecular Medicine, Nuffield Department of Clinical Medicine, Oxford University, Oxford, United Kingdom 2 Wellcome Trust Centre for Human Genetics, Oxford, United Kingdom 3 Unite ´ de Ge ´ne ´tique Cellulaire (GECE) Unversite ´ de Louvain, Brussels, Belgium 4 Oxxon Pharmaccines, Ltd., Oxford, United Kingdom 5 Ludwig Institute for Cancer Research, New York, NY, USA 6 Christie Cancer Research UK Research Centre, Manchester, United Kingdom 7 Cancer Research UK Medical Oncology Unit, Oxford, United Kingdom Recombinant plasmid DNA and attenuated poxviruses are under development as cancer and infectious disease vaccines. We present the results of a phase I clinical trial of recombinant plasmid DNA and modified vaccinia Ankara (MVA), both encoding 7 melanoma tumor antigen cytotoxic T lymphocyte (CTL) epitopes. HLA- A*0201-positive patients with surgically treated melanoma re- ceived either a “prime-boost” DNA/MVA or a homologous MVA- only regimen. Ex vivo tetramer analysis, performed at multiple time points, provided detailed kinetics of vaccine-driven CTL responses specific for the high-affinity melan-A 26 –35 analogue epitope. Melan-A26-35-specific CTL were generated in 2/6 pa- tients who received DNA/MVA (detectable only after the first MVA injection) and 4/7 patients who received MVA only. Ex vivo ELISPOT analysis and in vitro proliferation assays confirmed the effector function of these CTL. Responses were seen in smallpox- vaccinated as well as vaccinia-naı ¨ve patients, as defined by anti- vaccinia antibody responses demonstrated by ELISA assay. The observations that 1) CTL responses were generated to only 1 of the recombinant epitopes and 2) that the magnitude of these responses (0.029 – 0.19% CD8  T cells) was below the levels usually seen in acute viral infections suggest that to ensure high numbers of CTL specific for multiple recombinant epitopes, a deeper understand- ing of the interplay between CTL responses specific for the viral vector and recombinant epitopes is required. Key words: vaccine; immunotherapy; cytotoxic T lymphocyte (CTL); tumor antigen; poxvirus The characterization of tumor antigens recognized by cytotoxic T lymphocytes (CTL) has led to large numbers of human trials of vaccines designed to stimulate anti-tumor CTL responses. A va- riety of approaches have been employed, including peptides, 1–5 whole proteins and dendritic cells, 6–8 many of which have shown promising clinical and immunological responses. However, vac- cine-driven CTL responses have not always correlated with clin- ical response. The expression of tumor antigen on tumor cells within an individual is known to be heterogeneous, 9 and it has been proposed that this is a result of selection pressure by tumor antigen-specific CTL. 10,11 Vaccination strategies against multiple tumor antigens are therefore aimed at counteracting this tumor escape mechanism. Recently, recombinant gene technology has led to the development of viral and plasmid DNA vaccine delivery systems, and a recombinant “polyepitope” approach, involving a “string” of known tumor antigen epitopes inserted into a vector delivery system, allows multiple epitopes to be delivered simulta- neously. The Poxviridae family of viruses has proven to be particularly suited to the development of recombinant vaccines, and large numbers of animal studies have shown them to be efficient induc- ers of CTL responses specific for recombinant gene products. 12 MVA, a highly attenuated vaccinia virus, incapable of replication in human and most other mammalian cells, has been shown to be capable of inducing strong CTL and antibody responses against recombinant antigens, leading to protective immunity in preclini- cal animal models. 13–16 Preclinical studies have also demonstrated that some of the most successful vaccination protocols are heter- ologous “prime-boost” regimens, involving sequential injections of different vectors encoding the same recombinant antigen. 17 These regimens are designed to focus the CTL response on the recombinant antigen, which contains the only CTL epitopes shared by the different delivery vectors. 18 A “prime-boost” combination of plasmid DNA and MVA has been shown to be particularly effective in animal models. 15,19 –21 Although McConkey et al. 5 have recently published results from human trials demonstrating effective generation of anti-malaria T cell responses using DNA/ MVA “prime-boost” protocols, evidence of the immunogenicity of recombinant plasmid DNA in humans remains controversial. 22–24 We have engineered 2 recombinant vaccine constructs: plasmid DNA (DNA.Mel3) and MVA (MVA.Mel3), both encoding the same polyepitope string (Mel3) of 7 HLA-A*0201- and HLA- A*01-restricted CTL epitopes from 5 well-defined melanoma tu- mor antigens (melan-A, NY-ESO-1, MAGE-1, MAGE-3 and ty- rosinase), 15 (Fig. 1). We have previously shown that each of the epitopes is properly processed and presented to epitope-specific CTL clones. 15 We have also demonstrated in HLA-A2-transgenic mice that a heterologous “prime-boost” protocol of DNA.Mel3 and MVA.Mel3 generated up to 100-fold higher CTL expansions than sequential injections of the same vaccine vector. 15 To assess the safety and immunogenicity of DNA.Mel3 and MVA.Mel3, we performed a phase I clinical trial in HLA-A*0201- positive patients with resected melanoma and a high risk of disease recurrence. We examined 2 vaccination regimens: a heterologous “prime-boost” DNA/MVA regimen and a homologous MVA-only regimen. We performed detailed immunomonitoring, including ex vivo tetramer and ELISPOT analyses and in vitro proliferation assays of CTL responses specific for the recombinant tumor anti- gen epitopes in the Mel3 polyepitope string. We also measured antibody responses generated against the MVA viral vector. Our Abbreviations: CMV, cytomegalovirus; CTL, cytotoxic T lymphocyte; EBV, Epstein Barr virus; flu, Influenza; IFN-, Interferon-gamma; MVA, modified vaccinia Ankara; PBMC, Peripheral Blood Mononuclear Cell. Trial sponsor: The Ludwig Institute for Cancer Research; Trial sponsor: Oxxon Pharmaccines, Ltd.; Trial sponsor: Cancer Research UK (C399/ A2291). *Correspondence to: Vincenzo Cerundolo, Tumour Immunology Unit, Weatherall Institute of Molecular Medicine, Nuffield Department of Clin- ical Medicine, Oxford University, Oxford, OX3 9DS, U.K. Fax +44-1865-222502. E-mail: vincenzo.cerundolo@imm.ox.ac.uk Received 4 May 2004; Accepted 16 June 2004 DOI 10.1002/ijc.20569 Published online 13 September 2004 in Wiley InterScience (www. interscience.wiley.com). Int. J. Cancer: 113, 259 –266 (2005) © 2004 Wiley-Liss, Inc. Publication of the International Union Against Cancer