- Email: [email protected]
Vaccines and Melanoma
Vac c i n e s a n d Me l a n o m a Patrick A. Ott, MD, PhDa,b,c,d,*, Edward F. Fritsch, Catherine J. Wu, MDa,d,f, Glenn Dranoff, MDa,d,f
PhD
a,e
,
KEYWORDS Melanoma Vaccine Immunotherapy Neoantigen KEY POINTS The potential for therapeutic efficacy of a melanoma vaccine has been evident preclinically for many years. In patients with melanoma, vaccines have resulted in the induction of immune responses, although clinical benefit has not been clearly documented. The recent achievements with immune-checkpoint blockade, such as anti–CTLA-4 and anti–PD-1/PD-L1 in melanoma and other cancers, have illustrated that immunotherapy can be a powerful tool in cancer therapy. With increased understanding of tumor immunity, the limitations of many previous cancer vaccination approaches have become evident. Rapid progress in technologies that enable better vaccine design raise the expectation that these limitations can be overcome, thus leading to a clinically effective melanoma vaccine in the near future.
INTRODUCTION
Vaccination against melanoma represents an effort to stimulate an antitumor immune response that is directed either against an established tumor in patients with unresectable metastatic disease, or against micrometastatic disease in patients who are at high risk for recurrence after surgical resection. In both situations, the host has already failed the task of cancer immunosurveillance. The reasons for this failure are either the complete lack of an antimelanoma immune response or tumor-mediated immune evasion, caused by numerous mechanisms that cancers can elaborate. A successful antitumor vaccine therefore will induce a de novo immune response or boost an ineffective existing response, reprogramming the (failed) immune response by providing it a
Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA; b Melanoma Disease Center, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA; c Center for Immuno-Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA; d Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02215, USA; e Broad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge, MA 02142, USA; f Cancer Vaccine Center, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA * Corresponding author. Melanoma Disease Center, Center for Immuno-Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 450 Brookline Avenue, Boston, MA 02215-5450. E-mail address: [email protected] Hematol Oncol Clin N Am - (2014) -–http://dx.doi.org/10.1016/j.hoc.2014.02.008 hemonc.theclinics.com 0889-8588/14/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved.
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with new targets and stronger stimulation (Fig. 1). Differences among strategies relate to the vehicle used to deliver the antigenic target, the antigenic target itself, the number of targets provided, and the appropriate immune-stimulating context (adjuvant). Checkpoint blockade, an antigen-independent immunotherapy, is designed to overcome 1 or more mechanisms of immune evasion, and provides exciting synergistic opportunities to strengthen vaccine therapy. Vaccination strategies offer the capacity for potentiated adaptive immune activation, antigen(s)/target specificity, multiarm immune engagement, and the realization of durable, effective immunologic memory, a hallmark of effective immunity. No other tumor has been more thoroughly investigated with different vaccine approaches than melanoma. This article first summarizes prior efforts undertaken over the last 20 years, which demonstrated promise but were lacking in broad clinical activity. Further described are exciting new approaches that leverage recent technological advances with important biological insights to yield potential advances in this field. PREVIOUS VACCINE APPROACHES IN MELANOMA: SOME PROMISE, BUT LIMITED CLINICAL ACTIVITY Immunogens
Most vaccine approaches to date have used whole proteins or peptide fragments as the immunogen, which must be delivered to professional antigen-presenting cells
Fig. 1. Important features of an effective vaccine. (1) An immunogen that is capable of inducing tumor-specific T cells, rather than tolerance to “self.” (2) The appropriate immune adjuvant that will provide the necessary inflammatory context, leading to activation of professional antigen presenting cells such as dendritic cells. (3) Reversal of immunosuppressive mechanisms such as checkpoint blockade (anti–CTLA-4, anti–PD-1/PD-L1), deletion/suppression of regulatory T cells (Treg), inhibition of immunosuppressive factors (anti-VEGF, antiIDO, and so forth). CTL, cytotoxic T lymphocyte; CTLA-4, cytotoxic T-lymphocyte antigen-4; GM-CSF, granulocyte-macrophage colony-stimulating factor; IDO, indoleamine 2,3-dioxygenase; IL, interleukin; PD1, programmed death 1; PDL1, programmed death 1 ligand; TGF, transforming growth factor; VEGF, vascular endothelial growth factor.
Vaccines and Melanoma
(APCs) such as dendritic cells (DCs), usually in conjunction with an immunestimulating adjuvant to serve as an effective vaccine. The objective is antigen uptake, processing, and cross-presentation by APCs to effect priming of naı¨ve T cells or stimulation of memory T cells. The appropriate immune adjuvant will provide the danger signal, promoting immune pathways analogous to those activated in response to bacterial or viral infection. Key considerations when designing an effective vaccine include format (ie, whole protein vs peptide), route of delivery, and choice of adjuvant. Regarding the former, factors include cost (advantage: peptide), in vivo stability (advantage: peptide), and breadth of antigenic selection (advantage: protein). A significant limitation to peptide vaccines is their restriction to specific human leukocyte antigen (HLA) haplotypes. Most vaccine developers to date have focused on peptides that bind to the predominant HLA allele (HLA-A2) to capture the largest target population possible with a single vaccine. Unfortunately, this strategy excludes a vast pool of potentially immunogenic peptides and a considerable proportion of melanoma patients. This drawback could partially be addressed by using longer peptides (containing more than 1 epitope) or the use of multiple peptides, but as yet no such research has been conducted. Tumor-associated antigens (TAA) are developmental, differentiation, or growthpromoting proteins that are frequently overexpressed or somewhat specifically expressed by tumors. Much of cancer vaccinology for the past 20 years has focused on inducing or identifying T cells that recognize such proteins. The first TAA for melanoma, MAGE-1, was uncovered in 1991, and the capacity to generate cytotoxic T lymphocytes that recognize the protein was simultaneously demonstrated.1 Following its discovery, vaccines specifically targeting MAGE-1, in addition to other subsequently identified antigenic targets, rapidly emerged.2,3 The first clinical trial targeting a TAA focused on the differentiation antigen glycoprotein 100 (gp100).4 Popular melanoma targets have since included additional gp100 epitopes,5 MART-1,6 MAGE-1,1 MAGE-3,7 tyrosinase,8 and NY-ESO-1.9 Tumor-specific antigens (TSAs), derived from viral proteins or tumor-specific genetic mutations found in tumors, provide a recent opportunity to overcome many of the aforementioned immunogen issues, and will be discussed in more detail. Clinical trials with TAA vaccines
No peptide or protein vaccine has demonstrated a clear improvement in overall survival in melanoma patients to date. For the most part, melanoma vaccines have targeted TAAs such as described above. Small, single-arm studies At the National Cancer Institute surgery branch 323 subjects, almost all with metastatic melanoma, received peptide vaccines on a variety of different protocols. The peptides were derived from various TAAs including MART-1, gp100, tyrosinase, TRP-2, NY-ESO-1, MAGE-1/3, Her2/neu, or telomerase proteins.10 Except for 15 patients who received peptides pulsed on DCs, peptides were emulsified in incomplete Freund adjuvant (IFA). Only 9 of these 323 subjects had a partial response and 2 had a complete response, resulting in an overall objective response rate of 2.9%. Of note, most of the responders had metastatic disease confined to skin and lymph nodes. Peptide vaccine studies in melanoma patients using multiple different epitopes, single peptide and multipeptide strategies, and different adjuvants such as granulocyte-macrophage colony-stimulating factor (GMCSF), and low-dose interleukin (IL)-2 have produced similar results: low objective tumor response rates, generally from lower than 5% up to 10%, with variable effects on the immune response.11,12
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Larger, comparative studies In a phase 2 cooperative group trial, 121 previously treated metastatic melanoma patients received 3 HLA-A2 restricted peptides (MART-1, gp100M, and tyrosinase) either alone or in combination with GM-CSF or interferon (IFN)-a, respectively. Six objective responses were observed, and a T-cell response measured by IFN-g ELISPOT against at least 1 of the 3 peptides was seen in 26 of 75 (35%) patients with serial samples available.13 Patients with immune responses had improved overall survival (21.3 vs 13.4 months; P 5 .046). In a phase 3 trial, stage IV melanoma patients were treated with a modified gp100 peptide with increased binding affinity to HLA-A2 (gp100M) in combination with high-dose IL-2 versus IL-2 alone.14 The objective tumor response rate was higher in the IL-2/gp100M combination arm in comparison with IL-2 alone (16 vs 6%, P 5 .03), and the progression-free survival was longer (2.2 vs 1.6 months, P 5 .008). There was also a trend toward improved overall survival (17.8 vs 11.1 months; P 5 .06). Noteworthy is that the objective response rate of 6% in the IL-2 arm is markedly lower than the 16% reported historically with this drug.15 Only a few patients in the gp100M 1 IL-2 arm developed gp100-specific T cells in the peripheral blood, and there was no correlation between gp100-specific peripheral T cells and clinical responses. The lack of correlation of clinical responses and documented immune responses targeting the vaccine suggest lack of vaccine efficacy. In a series of 3 phase 2 studies conducted by the Cytokine Working Group, 132 HLAA2–positive advanced melanoma patients received the gp100M peptide combined with high-dose IL-2 given on variable schedules.16 Given the strikingly similar response rates with gp100 plus IL-2 in the phase 2 and 3 trials and historical data with IL-2 alone (w15%), it appears that the clinical benefit seen with gp100 plus IL-2 is mostly driven by the high-dose IL-2 and not an effect of the peptide vaccine.15 This finding is also in line with the phase 3 experience using the anti–cytotoxic T-lymphocyte antigen 4 (CTLA-4) antibody ipilimumab and gp100 in previously treated metastatic melanoma patients.17 In this trial, no difference in response rate or overall survival was seen between ipilimumab 1 gp100 when compared with ipilimumab alone. Together, these results suggest that gp100 is an ineffective vaccine as currently administered. Full-length proteins may provide a more comprehensive spectrum of tumor epitopes for presentation to DCs. MAGE-A3 is a cancer testis antigen overexpressed in approximately 65% of melanomas. A recombinant MAGE-A3 vaccine consists of full-length MAGE-A3 protein fused to protein D (a lipoprotein on the surface of Haemophilus influenzae B) and a polyhistidine tail. This vaccine, given with the immune adjuvant ASO2B, consisting of a saponin/lipid-A emulsion combined with TLR4 and TLR9 agonists, is currently being tested in a prospective, randomized phase 3 study in patients with high-risk resected, MAGE-A3 positive melanoma. The coprimary end point of disease-free survival (DFS) was not met according to a recent announcement by the study’s sponsor, Glaxo Smith Kline. Recombinant Viral Vector–Based Vaccines
Viral vectors encoding tumor antigens take advantage of immune responses against viral components, resulting in an adjuvant effect that can augment the antitumordirected T-cell response. In melanoma, a viral-based vaccine approach that has been tested in phase 2 and 3 clinical trials consists of a second-generation herpes virus engineered to selectively replicate in and lyse tumor cells and to express GM-CSF, thereby attracting DCs into the tumor microenvironment (OncoVexGM CSF; BioVex, Cambridge, MA). Based on promising clinical activity in a multi-institutional phase 2 study in patients with unresectable stage IIIC or IV melanoma (8 complete and 5 partial responses in 50 patients), 436 patients with unresectable stage IIIB, IIIC,
Vaccines and Melanoma
and IV melanoma were randomized 2:1 to receive either intratumoral OncoVexGM CSF or GM-CSF given subcutaneously.18,19 In an interim analysis reported at the annual meeting of the American Society of Clinical Oncology 2013, the primary end point of increased durable response rate of OncoVexGM CSF over GM-CSF was met, and a trend toward improved overall survival in patients treated with vaccine was observed.19 Whole Cell–Based Vaccines
A theoretical advantage of using whole tumor cells for vaccination is the broad spectrum of TAAs and mutated antigens potentially available for recognition and attack by immune cells. Vaccination with autologous tumors appears to be most suitable for this approach; a limitation is the difficulty of harvesting and preparing tumor tissue from individual patients. Irradiated melanoma cells harvested from metastatic lesions and engineered to secrete GM-CSF induced tumor necrosis and infiltration with T lymphocytes and plasma cells in metastatic sites of melanoma patients. Vaccination sites showed infiltration with T cells, DCs, and macrophages.20 In a follow-up study, patients with advanced melanoma previously vaccinated with irradiated autologous GM-CSF–secreting melanoma cells received ipilimumab after an interval of several years. Of note, metastatic melanoma lesions in 3 of 3 melanoma patients with previous vaccine exhibited extensive tumor necrosis with lymphocyte and granulocyte infiltrates, whereas no tumor necrosis was seen in 4 of 4 melanoma patients previously immunized with defined melanosomal antigens.21 A separate study, in which patients with metastatic melanoma were treated with ipilimumab 1 to 4 months after treatment with irradiated autologous GM-CSF–secreting melanoma cells, showed a linear relationship between the extent of tumor necrosis in posttreatment biopsies and the ratio of intratumoral CD81 T cells and FoxP31 Tregs.22 M-Vax (AVAX Technologies, Philadelphia, PA) is an autologous whole-cell melanoma vaccine consisting of irradiated tumor cells treated with the hapten dinitrofluorobenzene (DNP). Six of 97 patients with advanced melanoma who received M-Vax after a low dose of cyclophosphamide in a phase 2 trial had a complete or partial response; 5 patients had a mixed tumor response. The median overall survival was 21.4 months in responders and 8.7 months in nonresponders (P 5 .010).23 Based on these data, a phase 3 trial was initiated randomizing M-Vax versus placebo (2:1) given with cyclophosphamide, low-dose IL-2, and bacillus Calmette-Gue´rin (BCG). Allogeneic whole-cell vaccines derived from tumor cell lines or individual tumors from other patients have the advantage that they can be produced for off-the-shelf use. A prominent example is the allogeneic GM-CSF–secreting prostate carcinoma cell vaccine termed GVAX (Cell Genesys, South San Francisco, CA), which showed encouraging tumor activity in phase 1/2 trials,24,25 but ultimately failed because of lack of clinical efficacy in 2 phase 3 trials.26 Similarly, an allogeneic whole tumor cell vaccine comprising 3 melanoma cell lines known as Canvaxin appeared to show an overall survival benefit in a nonrandomized phase 2 trial, in which 150 completely resected stage IV melanoma patients vaccinated with Canvaxin lived longer than 113 matched, nonvaccinated controls. Nevertheless, a prospective phase 3 trial randomizing Canvaxin plus BCG versus BCG alone, enrolling more than 1500 patients with fully resected stage III or stage IV melanoma, was stopped early because of a low likelihood of demonstrating an overall survival benefit in the vaccine arm. NEW STRATEGIES
Several features, lacking in most melanoma vaccines to date, are critical for the induction of effective clinical antitumor responses: (1) an antigen and delivery system that is
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highly specific to the tumor with no cross-reactivity to self-antigens, resulting in maximal immunogenicity and avoiding autoimmunity; (2) an effective immune adjuvant that provides a stimulatory immune context, thereby activating APCs such as DCs; and (3) a strategy to counteract immunosuppressive mechanisms (see Fig. 1). Technological advances such as massively parallel sequencing and novel biomaterials, in addition to the emergence of new agents such as immune-checkpoint blocking antibodies, have enabled the design of tumor vaccines that incorporate these features and are more physiologically relevant. The combination of these new reagents promises to generate effective responses that will build on the already encouraging signal that has been seen with checkpoint inhibition alone. The following sections describe 2 such novel approaches that are currently under clinical trial at the authors’ institution: (1) NeoVax in patients with high-risk melanoma, and (2) WDVAX in patients with unresectable metastatic melanoma. NeoVax: A New Concept for Antigen Selection Coupled with an Effective Immune Adjuvant
Most antigens used for vaccination against tumors are either overexpressed or selectively expressed tumor-associated antigens. The immunogenicity of these types of antigens is limited by the downmodulating effects of central tolerance through thymic deletion, in addition to peripheral tolerance mechanisms. Recent advances in cancer genomics and immunology have provided new tools to overcome these limitations. NeoVax is a novel approach to personalized vaccine that may overcome some of the shortcomings of previous melanoma vaccines. Choice of antigen: neoantigens
Massively parallel sequencing technology allows sequencing of the entire genome or exome of a tumor and identification of all mutations by comparing the information with matched normal tissue cells. Intense and ever more comprehensive tumorsequencing efforts have demonstrated that individual tumors contain a host of patient-specific mutations that alter the protein-coding content of many genes,27 enabling the identification and use of a new class of immunogens termed neoantigens.28,29 These mutations range from single amino acid changes (caused by missense mutations) to addition of long regions of novel amino acid sequence owing to frame shifts, read-through of termination codons, or translation of intron regions (novel open reading frame mutations; neoORFs). Such altered proteins are, to the immune system, distinct from self and analogous to foreign proteins, rendering them less sensitive to the immune-dampening effects of self-tolerance (Fig. 2). Furthermore, these targets are exquisitely tumor specific, being found exclusively in tumor cells. Several studies in both animals and humans have demonstrated that mutated gene products can encode for epitopes effective in inducing an immune response.30,31 Importantly, spontaneous tumor regression or long-term survival were found to correlate in small numbers of patients with CD81 T-cell responses to mutated epitopes.32,33 Furthermore, escape from host immunosurveillance (immunoediting) can be tracked in some cases to alterations in expression of dominant mutated antigens.34,35 Choice of delivery: long peptides
Most peptide vaccines have nearly exclusively consisted of short peptides, based on the minimum length of peptides (usually 8–10 amino acids) needed to bind the major histocompatibility complex I molecule. Recent work has shown that such peptides can be tolerogenic rather than stimulatory,36 because they are capable of direct binding to the HLA molecule on the surface of nonprofessional APCs, including B and T cells, resulting in tolerance. Long peptides, which are approximately 20 to 30 amino acids
Vaccines and Melanoma
Fig. 2. Neoantigens are a novel class of antigens encoded by the unique mutations specific to each patient’s tumor. Tumor neoantigens (right, blue) are specific to tumor cells and are not subject to central tolerance (ie, deletion of their cognate antigen-specific T cells in the thymus). The result of this should be optimal immunogenicity and tumor specificity of the induced T-cell response, potentially comparable with the T-cell response to a foreign antigen such as from a virus or bacteria. By contrast, native antigens such as overexpressed or selectively expressed antigens commonly used in cancer vaccines are at the other end of the spectrum of tumor specificity, resulting in a higher likelihood of self-tolerance and autoimmunity (left, red).
in length, have been shown to produce more robust and more durable immune responses.37,38 Long peptides require internalization, processing, and crosspresentation to bind to HLA molecules, all of which only occur in professional APCs such as DCs. Long peptides therefore necessitate presentation of the peptide by professional APCs, a critical step for the induction of a strong antitumor immune response. Choice of adjuvant: the TLR-3 agonist poly-ICLC
Toll-like receptors (TLRs) belong to the family of pattern recognition receptors (PRRs), which recognize conserved motifs shared by many microorganisms, termed pathogen-associated molecular patterns (PAMPS). Recognition of PAMPS leads to the activation of the innate and adaptive immune systems. Key functions of DCs, including upregulation of costimulation markers, lymph node trafficking, phagocytosis, and cytokine production, are enhanced on activation by TLR stimulation. The TLR-3 agonist poly-ICLC is a synthetically prepared double-stranded RNA consisting of polyI and polyC stabilized by the addition of polylysine and carboxymethylcellulose. Poly-ICLC activates TLR3 and MDA5, complementary pathways leading to DC and natural killer (NK) cell activation, and production of a “natural mix” of type I interferons, cytokines, and chemokines. Preclinical studies in rodents and nonhuman primates demonstrated that poly-ICLC enhances virus, tumor, and autoantigenspecific T-cell responses, emphasizing its effectiveness as a vaccine adjuvant. A placebo-controlled study in healthy human volunteers recently showed similar transcriptional expression profiles of signal transduction canonical pathways, including
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those of the innate immune system, after either treatment with GMP-grade poly-ICLC (Hiltonol; Oncovir, Washington, DC) or vaccination with the highly effective yellow fever vaccine YF17D.39 WDVAX: A Scaffold Vaccine Incorporating Autologous Whole Tumor Cells, GM-CSF, and CpG into a Unique Delivery System
Novel biomaterials provide an opportunity to deliver the critical components of a tumor vaccine in a spatiotemporally controlled fashion, establishing an environment that is maximally supportive for the induction of an effective tumor immune response. WDVAX takes advantage of this technology. Choice of antigen: autologous tumor cells
The authors have previously shown that vaccination with lethally irradiated autologous tumor cells can induce strong immune responses. Specifically, this vaccine elicited dense DC, macrophage, granulocyte, and lymphocyte infiltrates at the injection sites in 19 of 26 evaluable patients with melanoma, and 18 of 25 evaluable patients with lung cancer. Metastatic lesions resected after vaccination showed brisk, T-lymphocyte and plasma-cell infiltrates with tumor necrosis in 10 of 16 patients with melanoma and in 3 of 6 patients with lung cancer. Choice of adjuvant: tumor cell–secreted GM-CSF in combination with the TLR-9 agonist oligodeoxynucleotide containing unmethylated cytosine and guanine
Vaccination with autologous tumor cells established safety, feasibility, and biologic activity, but antitumor activity in melanoma patients was low. One explanation for the insufficient clinical efficacy is the dual role of GM-CSF during the generation of an immune response identified in the authors’ previous studies (autologous tumor cells are engineered by adenovirus-mediated gene transfer to secrete GM-CSF), which found that the homeostatic function of GM-CSF is to maintain immune tolerance through supporting the activities of regulatory T cells (Tregs).40 The concurrent provision of a second signal, such as the TLR-9 agonist oligodeoxynucleotide containing unmethylated cytosine and guanine (CpG), downregulates the GM-CSF tolerance pathway and switches GM-CSF activity toward immune stimulation, with the induction of T-helper (Th)1, Th17, and CD81 T cells. Hence, codelivery of GM-CSF with CpG might lead to the sustained induction of cytotoxic T cells while dampening the generation of Tregs, thereby enhancing tumor destruction. Choice of delivery: a novel material engineered scaffold
To capitalize on the improved understanding of GM-CSF biology, in collaboration with colleagues at the Wyss Institute for Biologically Inspired Engineering at Harvard University, the authors have developed a novel material engineered scaffold, consisting of a macroporous polylactide-coglycolide (PLG) matrix polymer. This scaffold allows delivery of immunostimulatory agents in vivo with precise spatial and temporal control. In addition to serving as a drug-delivery device, PLG represents a physical, antigen-presenting structure to which DCs (in addition to other immune cells and soluble factors) may be recruited and activated. In murine tumor models, the subcutaneous implantation of PLG scaffolds incorporating GM-CSF protein, CpG, and tumor-cell lysates creates a microenvironment in which the appropriate signals for dendritic cell antigen presentation and the induction of antitumor T cells can be maintained for at least 2 weeks. Under these conditions, robust levels of antitumor immunity were achieved, resulting in the regression of established B16 melanomas.41,42
Vaccines and Melanoma
SUMMARY AND OUTLOOK
Despite decades of intense investigation, a melanoma vaccine that induces effective cytotoxic T-cell responses and is capable of controlling or eradicating melanoma has been elusive. However, increased understanding of tumor immunity has led to the recognition that most of the approaches taken in the past were limited through weak immune adjuvants, poor choice of tumor antigen, or delivery of the vaccine. The recent successes with immune checkpoint blockade in melanoma and other cancers, leading to durable tumor responses in a considerable proportion of patients, have highlighted the potential of immunotherapy in melanoma. These insights and advances, coupled with the rapid progress in many areas relevant to effective vaccine design such as sequencing technology, biomaterials, and immune adjuvants, provide new opportunities for the successful development of a clinically effective melanoma vaccine. NeoVax, featuring a novel class of immunogens based on individual tumor mutations, and WDVAX, providing a unique delivery system by taking advantage of a novel material engineered scaffold, are examples of innovative vaccine design in melanoma. Both technologies are currently under clinical investigation in patients with high-risk (NeoVax) and advanced melanoma (WDVAX) at the authors’ institution. These new strategies, either alone or in combination with immune checkpoint blockade or other synergistic immune stimulants will, it is hoped, lead the way toward future successful melanoma vaccines. REFERENCES
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11. Slingluff CL Jr, Petroni GR, Yamshchikov GV, et al. Clinical and immunologic results of a randomized phase II trial of vaccination using four melanoma peptides either administered in granulocyte-macrophage colony-stimulating factor in adjuvant or pulsed on dendritic cells. J Clin Oncol 2003;21: 4016–26. 12. Slingluff CL Jr, Petroni GR, Olson WC, et al. Effect of granulocyte/macrophage colony-stimulating factor on circulating CD81 and CD41 T-cell responses to a multipeptide melanoma vaccine: outcome of a multicenter randomized trial. Clin Cancer Res 2009;15:7036–44. 13. Kirkwood JM, Lee S, Moschos SJ, et al. Immunogenicity and antitumor effects of vaccination with peptide vaccine1/-granulocyte-monocyte colony-stimulating factor and/or IFN-alpha2b in advanced metastatic melanoma: Eastern Cooperative Oncology Group Phase II Trial E1696. Clin Cancer Res 2009;15:1443–51. 14. Schwartzentruber DJ, Lawson DH, Richards JM, et al. gp100 peptide vaccine and interleukin-2 in patients with advanced melanoma. N Engl J Med 2011; 364:2119–27. 15. Atkins MB, Lotze MT, Dutcher JP, et al. High-dose recombinant interleukin 2 therapy for patients with metastatic melanoma: analysis of 270 patients treated between 1985 and 1993. J Clin Oncol 1999;17:2105–16. 16. Sosman JA, Carrillo C, Urba WJ, et al. Three phase II cytokine working group trials of gp100 (210M) peptide plus high-dose interleukin-2 in patients with HLA-A2positive advanced melanoma. J Clin Oncol 2008;26:2292–8. 17. Hodi FS, O’Day SJ, McDermott DF, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 2010;363:711–23. 18. Senzer NN, Kaufman HL, Amatruda T, et al. Phase II clinical trial of a granulocytemacrophage colony-stimulating factor-encoding, second-generation oncolytic herpesvirus in patients with unresectable metastatic melanoma. J Clin Oncol 2009;27:5763–71. 19. Andtbacka RH, CF, Amatruda T, et al. OPTiM: a randomized phase III trial of talimogene laherparepvec (T-VEC) versus subcutaneous (SC) granulocyte-macrophage colony-stimulating factor (GM-CSF) for the treatment (tx) of unresected stage IIIB/C and IV melanoma. J Clin Oncol 2013;31(Suppl): [Abstract LBA9008]. 20. Soiffer R, Lynch T, Mihm M, et al. Vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte-macrophage colony-stimulating factor generates potent antitumor immunity in patients with metastatic melanoma. Proc Natl Acad Sci U S A 1998;95:13141–6. 21. Hodi FS, Schmollinger JC, Soiffer RJ, et al. ATP6S1 elicits potent humoral responses associated with immune-mediated tumor destruction. Proc Natl Acad Sci U S A 2002;99:6919–24. 22. Hodi FS, Butler M, Oble DA, et al. Immunologic and clinical effects of antibody blockade of cytotoxic T lymphocyte-associated antigen 4 in previously vaccinated cancer patients. Proc Natl Acad Sci U S A 2008;105:3005–10. 23. Berd D, Sato T, Cohn H, et al. Treatment of metastatic melanoma with autologous, hapten-modified melanoma vaccine: regression of pulmonary metastases. Int J Cancer 2001;94:531–9. 24. Small EJ, Sacks N, Nemunaitis J, et al. Granulocyte macrophage colonystimulating factor–secreting allogeneic cellular immunotherapy for hormonerefractory prostate cancer. Clin Cancer Res 2007;13:3883–91. 25. Higano CS, Corman JM, Smith DC, et al. Phase 1/2 dose-escalation study of a GM-CSF-secreting, allogeneic, cellular immunotherapy for metastatic hormonerefractory prostate cancer. Cancer 2008;113:975–84.
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26. Copier J, Dalgleish A. Whole-cell vaccines: a failure or a success waiting to happen? Curr Opin Mol Ther 2010;12:14–20. 27. Wood LD, Parsons DW, Jones S, et al. The genomic landscapes of human breast and colorectal cancers. Science 2007;318:1108–13. 28. Heemskerk B, Kvistborg P, Schumacher TN. The cancer antigenome. EMBO J 2013;32:194–203. 29. Hacohen N, Fritsch EF, Carter TA, et al. Getting Personal with Neoantigen-Based Therapeutic Cancer Vaccines. Cancer Immunol Res 2013;1:OF10–4. 30. Buckwalter MR, Srivastava PK. “It is the antigen(s), stupid” and other lessons from over a decade of vaccitherapy of human cancer. Semin Immunol 2008;20: 296–300. 31. Sensi M, Anichini A. Unique tumor antigens: evidence for immune control of genome integrity and immunogenic targets for T cell-mediated patient-specific immunotherapy. Clin Cancer Res 2006;12:5023–32. 32. Karanikas V, Colau D, Baurain JF, et al. High frequency of cytolytic T lymphocytes directed against a tumor-specific mutated antigen detectable with HLA tetramers in the blood of a lung carcinoma patient with long survival. Cancer Res 2001;61: 3718–24. 33. van Rooij N, van Buuren MM, Philips D, et al. Tumor exome analysis reveals neoantigen-specific T-cell reactivity in an ipilimumab-responsive melanoma. J Clin Oncol 2013;31:e439–42. 34. Matsushita H, Vesely MD, Koboldt DC, et al. Cancer exome analysis reveals a T-cell-dependent mechanism of cancer immunoediting. Nature 2012;482:400–4. 35. DuPage M, Mazumdar C, Schmidt LM, et al. Expression of tumour-specific antigens underlies cancer immunoediting. Nature 2012;482:405–9. 36. Bijker MS, van den Eeden SJ, Franken KL, et al. CD81 CTL priming by exact peptide epitopes in incomplete Freund’s adjuvant induces a vanishing CTL response, whereas long peptides induce sustained CTL reactivity. J Immunol 2007;179:5033–40. 37. Welters MJ, Kenter GG, Piersma SJ, et al. Induction of tumor-specific CD41 and CD81 T-cell immunity in cervical cancer patients by a human papillomavirus type 16 E6 and E7 long peptides vaccine. Clin Cancer Res 2008;14:178–87. 38. Kenter GG, Welters MJ, Valentijn AR, et al. Phase I immunotherapeutic trial with long peptides spanning the E6 and E7 sequences of high-risk human papillomavirus 16 in end-stage cervical cancer patients shows low toxicity and robust immunogenicity. Clin Cancer Res 2008;14:169–77. 39. Gaucher D, Therrien R, Kettaf N, et al. Yellow fever vaccine induces integrated multilineage and polyfunctional immune responses. J Exp Med 2008;205: 3119–31. 40. Jinushi M, Nakazaki Y, Dougan M, et al. MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF. J Clin Invest 2007;117:1902–13. 41. Ali OA, Huebsch N, Cao L, et al. Infection-mimicking materials to program dendritic cells in situ. Nat Mater 2009;8:151–8. 42. Ali OA, Emerich D, Dranoff G, et al. In situ regulation of DC subsets and T cells mediates tumor regression in mice. Sci Transl Med 2009;1:8ra19.
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KEYWORDS Melanoma Vaccine Immunotherapy Neoantigen KEY POINTS The potential for therapeutic efficacy of a melanoma vaccine has been evident preclinically for many years. In patients with melanoma, vaccines have resulted in the induction of immune responses, although clinical benefit has not been clearly documented. The recent achievements with immune-checkpoint blockade, such as anti–CTLA-4 and anti–PD-1/PD-L1 in melanoma and other cancers, have illustrated that immunotherapy can be a powerful tool in cancer therapy. With increased understanding of tumor immunity, the limitations of many previous cancer vaccination approaches have become evident. Rapid progress in technologies that enable better vaccine design raise the expectation that these limitations can be overcome, thus leading to a clinically effective melanoma vaccine in the near future.
INTRODUCTION
Vaccination against melanoma represents an effort to stimulate an antitumor immune response that is directed either against an established tumor in patients with unresectable metastatic disease, or against micrometastatic disease in patients who are at high risk for recurrence after surgical resection. In both situations, the host has already failed the task of cancer immunosurveillance. The reasons for this failure are either the complete lack of an antimelanoma immune response or tumor-mediated immune evasion, caused by numerous mechanisms that cancers can elaborate. A successful antitumor vaccine therefore will induce a de novo immune response or boost an ineffective existing response, reprogramming the (failed) immune response by providing it a
Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA; b Melanoma Disease Center, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA; c Center for Immuno-Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA; d Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02215, USA; e Broad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge, MA 02142, USA; f Cancer Vaccine Center, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA * Corresponding author. Melanoma Disease Center, Center for Immuno-Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 450 Brookline Avenue, Boston, MA 02215-5450. E-mail address: [email protected] Hematol Oncol Clin N Am - (2014) -–http://dx.doi.org/10.1016/j.hoc.2014.02.008 hemonc.theclinics.com 0889-8588/14/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved.
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with new targets and stronger stimulation (Fig. 1). Differences among strategies relate to the vehicle used to deliver the antigenic target, the antigenic target itself, the number of targets provided, and the appropriate immune-stimulating context (adjuvant). Checkpoint blockade, an antigen-independent immunotherapy, is designed to overcome 1 or more mechanisms of immune evasion, and provides exciting synergistic opportunities to strengthen vaccine therapy. Vaccination strategies offer the capacity for potentiated adaptive immune activation, antigen(s)/target specificity, multiarm immune engagement, and the realization of durable, effective immunologic memory, a hallmark of effective immunity. No other tumor has been more thoroughly investigated with different vaccine approaches than melanoma. This article first summarizes prior efforts undertaken over the last 20 years, which demonstrated promise but were lacking in broad clinical activity. Further described are exciting new approaches that leverage recent technological advances with important biological insights to yield potential advances in this field. PREVIOUS VACCINE APPROACHES IN MELANOMA: SOME PROMISE, BUT LIMITED CLINICAL ACTIVITY Immunogens
Most vaccine approaches to date have used whole proteins or peptide fragments as the immunogen, which must be delivered to professional antigen-presenting cells
Fig. 1. Important features of an effective vaccine. (1) An immunogen that is capable of inducing tumor-specific T cells, rather than tolerance to “self.” (2) The appropriate immune adjuvant that will provide the necessary inflammatory context, leading to activation of professional antigen presenting cells such as dendritic cells. (3) Reversal of immunosuppressive mechanisms such as checkpoint blockade (anti–CTLA-4, anti–PD-1/PD-L1), deletion/suppression of regulatory T cells (Treg), inhibition of immunosuppressive factors (anti-VEGF, antiIDO, and so forth). CTL, cytotoxic T lymphocyte; CTLA-4, cytotoxic T-lymphocyte antigen-4; GM-CSF, granulocyte-macrophage colony-stimulating factor; IDO, indoleamine 2,3-dioxygenase; IL, interleukin; PD1, programmed death 1; PDL1, programmed death 1 ligand; TGF, transforming growth factor; VEGF, vascular endothelial growth factor.
Vaccines and Melanoma
(APCs) such as dendritic cells (DCs), usually in conjunction with an immunestimulating adjuvant to serve as an effective vaccine. The objective is antigen uptake, processing, and cross-presentation by APCs to effect priming of naı¨ve T cells or stimulation of memory T cells. The appropriate immune adjuvant will provide the danger signal, promoting immune pathways analogous to those activated in response to bacterial or viral infection. Key considerations when designing an effective vaccine include format (ie, whole protein vs peptide), route of delivery, and choice of adjuvant. Regarding the former, factors include cost (advantage: peptide), in vivo stability (advantage: peptide), and breadth of antigenic selection (advantage: protein). A significant limitation to peptide vaccines is their restriction to specific human leukocyte antigen (HLA) haplotypes. Most vaccine developers to date have focused on peptides that bind to the predominant HLA allele (HLA-A2) to capture the largest target population possible with a single vaccine. Unfortunately, this strategy excludes a vast pool of potentially immunogenic peptides and a considerable proportion of melanoma patients. This drawback could partially be addressed by using longer peptides (containing more than 1 epitope) or the use of multiple peptides, but as yet no such research has been conducted. Tumor-associated antigens (TAA) are developmental, differentiation, or growthpromoting proteins that are frequently overexpressed or somewhat specifically expressed by tumors. Much of cancer vaccinology for the past 20 years has focused on inducing or identifying T cells that recognize such proteins. The first TAA for melanoma, MAGE-1, was uncovered in 1991, and the capacity to generate cytotoxic T lymphocytes that recognize the protein was simultaneously demonstrated.1 Following its discovery, vaccines specifically targeting MAGE-1, in addition to other subsequently identified antigenic targets, rapidly emerged.2,3 The first clinical trial targeting a TAA focused on the differentiation antigen glycoprotein 100 (gp100).4 Popular melanoma targets have since included additional gp100 epitopes,5 MART-1,6 MAGE-1,1 MAGE-3,7 tyrosinase,8 and NY-ESO-1.9 Tumor-specific antigens (TSAs), derived from viral proteins or tumor-specific genetic mutations found in tumors, provide a recent opportunity to overcome many of the aforementioned immunogen issues, and will be discussed in more detail. Clinical trials with TAA vaccines
No peptide or protein vaccine has demonstrated a clear improvement in overall survival in melanoma patients to date. For the most part, melanoma vaccines have targeted TAAs such as described above. Small, single-arm studies At the National Cancer Institute surgery branch 323 subjects, almost all with metastatic melanoma, received peptide vaccines on a variety of different protocols. The peptides were derived from various TAAs including MART-1, gp100, tyrosinase, TRP-2, NY-ESO-1, MAGE-1/3, Her2/neu, or telomerase proteins.10 Except for 15 patients who received peptides pulsed on DCs, peptides were emulsified in incomplete Freund adjuvant (IFA). Only 9 of these 323 subjects had a partial response and 2 had a complete response, resulting in an overall objective response rate of 2.9%. Of note, most of the responders had metastatic disease confined to skin and lymph nodes. Peptide vaccine studies in melanoma patients using multiple different epitopes, single peptide and multipeptide strategies, and different adjuvants such as granulocyte-macrophage colony-stimulating factor (GMCSF), and low-dose interleukin (IL)-2 have produced similar results: low objective tumor response rates, generally from lower than 5% up to 10%, with variable effects on the immune response.11,12
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Larger, comparative studies In a phase 2 cooperative group trial, 121 previously treated metastatic melanoma patients received 3 HLA-A2 restricted peptides (MART-1, gp100M, and tyrosinase) either alone or in combination with GM-CSF or interferon (IFN)-a, respectively. Six objective responses were observed, and a T-cell response measured by IFN-g ELISPOT against at least 1 of the 3 peptides was seen in 26 of 75 (35%) patients with serial samples available.13 Patients with immune responses had improved overall survival (21.3 vs 13.4 months; P 5 .046). In a phase 3 trial, stage IV melanoma patients were treated with a modified gp100 peptide with increased binding affinity to HLA-A2 (gp100M) in combination with high-dose IL-2 versus IL-2 alone.14 The objective tumor response rate was higher in the IL-2/gp100M combination arm in comparison with IL-2 alone (16 vs 6%, P 5 .03), and the progression-free survival was longer (2.2 vs 1.6 months, P 5 .008). There was also a trend toward improved overall survival (17.8 vs 11.1 months; P 5 .06). Noteworthy is that the objective response rate of 6% in the IL-2 arm is markedly lower than the 16% reported historically with this drug.15 Only a few patients in the gp100M 1 IL-2 arm developed gp100-specific T cells in the peripheral blood, and there was no correlation between gp100-specific peripheral T cells and clinical responses. The lack of correlation of clinical responses and documented immune responses targeting the vaccine suggest lack of vaccine efficacy. In a series of 3 phase 2 studies conducted by the Cytokine Working Group, 132 HLAA2–positive advanced melanoma patients received the gp100M peptide combined with high-dose IL-2 given on variable schedules.16 Given the strikingly similar response rates with gp100 plus IL-2 in the phase 2 and 3 trials and historical data with IL-2 alone (w15%), it appears that the clinical benefit seen with gp100 plus IL-2 is mostly driven by the high-dose IL-2 and not an effect of the peptide vaccine.15 This finding is also in line with the phase 3 experience using the anti–cytotoxic T-lymphocyte antigen 4 (CTLA-4) antibody ipilimumab and gp100 in previously treated metastatic melanoma patients.17 In this trial, no difference in response rate or overall survival was seen between ipilimumab 1 gp100 when compared with ipilimumab alone. Together, these results suggest that gp100 is an ineffective vaccine as currently administered. Full-length proteins may provide a more comprehensive spectrum of tumor epitopes for presentation to DCs. MAGE-A3 is a cancer testis antigen overexpressed in approximately 65% of melanomas. A recombinant MAGE-A3 vaccine consists of full-length MAGE-A3 protein fused to protein D (a lipoprotein on the surface of Haemophilus influenzae B) and a polyhistidine tail. This vaccine, given with the immune adjuvant ASO2B, consisting of a saponin/lipid-A emulsion combined with TLR4 and TLR9 agonists, is currently being tested in a prospective, randomized phase 3 study in patients with high-risk resected, MAGE-A3 positive melanoma. The coprimary end point of disease-free survival (DFS) was not met according to a recent announcement by the study’s sponsor, Glaxo Smith Kline. Recombinant Viral Vector–Based Vaccines
Viral vectors encoding tumor antigens take advantage of immune responses against viral components, resulting in an adjuvant effect that can augment the antitumordirected T-cell response. In melanoma, a viral-based vaccine approach that has been tested in phase 2 and 3 clinical trials consists of a second-generation herpes virus engineered to selectively replicate in and lyse tumor cells and to express GM-CSF, thereby attracting DCs into the tumor microenvironment (OncoVexGM CSF; BioVex, Cambridge, MA). Based on promising clinical activity in a multi-institutional phase 2 study in patients with unresectable stage IIIC or IV melanoma (8 complete and 5 partial responses in 50 patients), 436 patients with unresectable stage IIIB, IIIC,
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and IV melanoma were randomized 2:1 to receive either intratumoral OncoVexGM CSF or GM-CSF given subcutaneously.18,19 In an interim analysis reported at the annual meeting of the American Society of Clinical Oncology 2013, the primary end point of increased durable response rate of OncoVexGM CSF over GM-CSF was met, and a trend toward improved overall survival in patients treated with vaccine was observed.19 Whole Cell–Based Vaccines
A theoretical advantage of using whole tumor cells for vaccination is the broad spectrum of TAAs and mutated antigens potentially available for recognition and attack by immune cells. Vaccination with autologous tumors appears to be most suitable for this approach; a limitation is the difficulty of harvesting and preparing tumor tissue from individual patients. Irradiated melanoma cells harvested from metastatic lesions and engineered to secrete GM-CSF induced tumor necrosis and infiltration with T lymphocytes and plasma cells in metastatic sites of melanoma patients. Vaccination sites showed infiltration with T cells, DCs, and macrophages.20 In a follow-up study, patients with advanced melanoma previously vaccinated with irradiated autologous GM-CSF–secreting melanoma cells received ipilimumab after an interval of several years. Of note, metastatic melanoma lesions in 3 of 3 melanoma patients with previous vaccine exhibited extensive tumor necrosis with lymphocyte and granulocyte infiltrates, whereas no tumor necrosis was seen in 4 of 4 melanoma patients previously immunized with defined melanosomal antigens.21 A separate study, in which patients with metastatic melanoma were treated with ipilimumab 1 to 4 months after treatment with irradiated autologous GM-CSF–secreting melanoma cells, showed a linear relationship between the extent of tumor necrosis in posttreatment biopsies and the ratio of intratumoral CD81 T cells and FoxP31 Tregs.22 M-Vax (AVAX Technologies, Philadelphia, PA) is an autologous whole-cell melanoma vaccine consisting of irradiated tumor cells treated with the hapten dinitrofluorobenzene (DNP). Six of 97 patients with advanced melanoma who received M-Vax after a low dose of cyclophosphamide in a phase 2 trial had a complete or partial response; 5 patients had a mixed tumor response. The median overall survival was 21.4 months in responders and 8.7 months in nonresponders (P 5 .010).23 Based on these data, a phase 3 trial was initiated randomizing M-Vax versus placebo (2:1) given with cyclophosphamide, low-dose IL-2, and bacillus Calmette-Gue´rin (BCG). Allogeneic whole-cell vaccines derived from tumor cell lines or individual tumors from other patients have the advantage that they can be produced for off-the-shelf use. A prominent example is the allogeneic GM-CSF–secreting prostate carcinoma cell vaccine termed GVAX (Cell Genesys, South San Francisco, CA), which showed encouraging tumor activity in phase 1/2 trials,24,25 but ultimately failed because of lack of clinical efficacy in 2 phase 3 trials.26 Similarly, an allogeneic whole tumor cell vaccine comprising 3 melanoma cell lines known as Canvaxin appeared to show an overall survival benefit in a nonrandomized phase 2 trial, in which 150 completely resected stage IV melanoma patients vaccinated with Canvaxin lived longer than 113 matched, nonvaccinated controls. Nevertheless, a prospective phase 3 trial randomizing Canvaxin plus BCG versus BCG alone, enrolling more than 1500 patients with fully resected stage III or stage IV melanoma, was stopped early because of a low likelihood of demonstrating an overall survival benefit in the vaccine arm. NEW STRATEGIES
Several features, lacking in most melanoma vaccines to date, are critical for the induction of effective clinical antitumor responses: (1) an antigen and delivery system that is
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highly specific to the tumor with no cross-reactivity to self-antigens, resulting in maximal immunogenicity and avoiding autoimmunity; (2) an effective immune adjuvant that provides a stimulatory immune context, thereby activating APCs such as DCs; and (3) a strategy to counteract immunosuppressive mechanisms (see Fig. 1). Technological advances such as massively parallel sequencing and novel biomaterials, in addition to the emergence of new agents such as immune-checkpoint blocking antibodies, have enabled the design of tumor vaccines that incorporate these features and are more physiologically relevant. The combination of these new reagents promises to generate effective responses that will build on the already encouraging signal that has been seen with checkpoint inhibition alone. The following sections describe 2 such novel approaches that are currently under clinical trial at the authors’ institution: (1) NeoVax in patients with high-risk melanoma, and (2) WDVAX in patients with unresectable metastatic melanoma. NeoVax: A New Concept for Antigen Selection Coupled with an Effective Immune Adjuvant
Most antigens used for vaccination against tumors are either overexpressed or selectively expressed tumor-associated antigens. The immunogenicity of these types of antigens is limited by the downmodulating effects of central tolerance through thymic deletion, in addition to peripheral tolerance mechanisms. Recent advances in cancer genomics and immunology have provided new tools to overcome these limitations. NeoVax is a novel approach to personalized vaccine that may overcome some of the shortcomings of previous melanoma vaccines. Choice of antigen: neoantigens
Massively parallel sequencing technology allows sequencing of the entire genome or exome of a tumor and identification of all mutations by comparing the information with matched normal tissue cells. Intense and ever more comprehensive tumorsequencing efforts have demonstrated that individual tumors contain a host of patient-specific mutations that alter the protein-coding content of many genes,27 enabling the identification and use of a new class of immunogens termed neoantigens.28,29 These mutations range from single amino acid changes (caused by missense mutations) to addition of long regions of novel amino acid sequence owing to frame shifts, read-through of termination codons, or translation of intron regions (novel open reading frame mutations; neoORFs). Such altered proteins are, to the immune system, distinct from self and analogous to foreign proteins, rendering them less sensitive to the immune-dampening effects of self-tolerance (Fig. 2). Furthermore, these targets are exquisitely tumor specific, being found exclusively in tumor cells. Several studies in both animals and humans have demonstrated that mutated gene products can encode for epitopes effective in inducing an immune response.30,31 Importantly, spontaneous tumor regression or long-term survival were found to correlate in small numbers of patients with CD81 T-cell responses to mutated epitopes.32,33 Furthermore, escape from host immunosurveillance (immunoediting) can be tracked in some cases to alterations in expression of dominant mutated antigens.34,35 Choice of delivery: long peptides
Most peptide vaccines have nearly exclusively consisted of short peptides, based on the minimum length of peptides (usually 8–10 amino acids) needed to bind the major histocompatibility complex I molecule. Recent work has shown that such peptides can be tolerogenic rather than stimulatory,36 because they are capable of direct binding to the HLA molecule on the surface of nonprofessional APCs, including B and T cells, resulting in tolerance. Long peptides, which are approximately 20 to 30 amino acids
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Fig. 2. Neoantigens are a novel class of antigens encoded by the unique mutations specific to each patient’s tumor. Tumor neoantigens (right, blue) are specific to tumor cells and are not subject to central tolerance (ie, deletion of their cognate antigen-specific T cells in the thymus). The result of this should be optimal immunogenicity and tumor specificity of the induced T-cell response, potentially comparable with the T-cell response to a foreign antigen such as from a virus or bacteria. By contrast, native antigens such as overexpressed or selectively expressed antigens commonly used in cancer vaccines are at the other end of the spectrum of tumor specificity, resulting in a higher likelihood of self-tolerance and autoimmunity (left, red).
in length, have been shown to produce more robust and more durable immune responses.37,38 Long peptides require internalization, processing, and crosspresentation to bind to HLA molecules, all of which only occur in professional APCs such as DCs. Long peptides therefore necessitate presentation of the peptide by professional APCs, a critical step for the induction of a strong antitumor immune response. Choice of adjuvant: the TLR-3 agonist poly-ICLC
Toll-like receptors (TLRs) belong to the family of pattern recognition receptors (PRRs), which recognize conserved motifs shared by many microorganisms, termed pathogen-associated molecular patterns (PAMPS). Recognition of PAMPS leads to the activation of the innate and adaptive immune systems. Key functions of DCs, including upregulation of costimulation markers, lymph node trafficking, phagocytosis, and cytokine production, are enhanced on activation by TLR stimulation. The TLR-3 agonist poly-ICLC is a synthetically prepared double-stranded RNA consisting of polyI and polyC stabilized by the addition of polylysine and carboxymethylcellulose. Poly-ICLC activates TLR3 and MDA5, complementary pathways leading to DC and natural killer (NK) cell activation, and production of a “natural mix” of type I interferons, cytokines, and chemokines. Preclinical studies in rodents and nonhuman primates demonstrated that poly-ICLC enhances virus, tumor, and autoantigenspecific T-cell responses, emphasizing its effectiveness as a vaccine adjuvant. A placebo-controlled study in healthy human volunteers recently showed similar transcriptional expression profiles of signal transduction canonical pathways, including
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those of the innate immune system, after either treatment with GMP-grade poly-ICLC (Hiltonol; Oncovir, Washington, DC) or vaccination with the highly effective yellow fever vaccine YF17D.39 WDVAX: A Scaffold Vaccine Incorporating Autologous Whole Tumor Cells, GM-CSF, and CpG into a Unique Delivery System
Novel biomaterials provide an opportunity to deliver the critical components of a tumor vaccine in a spatiotemporally controlled fashion, establishing an environment that is maximally supportive for the induction of an effective tumor immune response. WDVAX takes advantage of this technology. Choice of antigen: autologous tumor cells
The authors have previously shown that vaccination with lethally irradiated autologous tumor cells can induce strong immune responses. Specifically, this vaccine elicited dense DC, macrophage, granulocyte, and lymphocyte infiltrates at the injection sites in 19 of 26 evaluable patients with melanoma, and 18 of 25 evaluable patients with lung cancer. Metastatic lesions resected after vaccination showed brisk, T-lymphocyte and plasma-cell infiltrates with tumor necrosis in 10 of 16 patients with melanoma and in 3 of 6 patients with lung cancer. Choice of adjuvant: tumor cell–secreted GM-CSF in combination with the TLR-9 agonist oligodeoxynucleotide containing unmethylated cytosine and guanine
Vaccination with autologous tumor cells established safety, feasibility, and biologic activity, but antitumor activity in melanoma patients was low. One explanation for the insufficient clinical efficacy is the dual role of GM-CSF during the generation of an immune response identified in the authors’ previous studies (autologous tumor cells are engineered by adenovirus-mediated gene transfer to secrete GM-CSF), which found that the homeostatic function of GM-CSF is to maintain immune tolerance through supporting the activities of regulatory T cells (Tregs).40 The concurrent provision of a second signal, such as the TLR-9 agonist oligodeoxynucleotide containing unmethylated cytosine and guanine (CpG), downregulates the GM-CSF tolerance pathway and switches GM-CSF activity toward immune stimulation, with the induction of T-helper (Th)1, Th17, and CD81 T cells. Hence, codelivery of GM-CSF with CpG might lead to the sustained induction of cytotoxic T cells while dampening the generation of Tregs, thereby enhancing tumor destruction. Choice of delivery: a novel material engineered scaffold
To capitalize on the improved understanding of GM-CSF biology, in collaboration with colleagues at the Wyss Institute for Biologically Inspired Engineering at Harvard University, the authors have developed a novel material engineered scaffold, consisting of a macroporous polylactide-coglycolide (PLG) matrix polymer. This scaffold allows delivery of immunostimulatory agents in vivo with precise spatial and temporal control. In addition to serving as a drug-delivery device, PLG represents a physical, antigen-presenting structure to which DCs (in addition to other immune cells and soluble factors) may be recruited and activated. In murine tumor models, the subcutaneous implantation of PLG scaffolds incorporating GM-CSF protein, CpG, and tumor-cell lysates creates a microenvironment in which the appropriate signals for dendritic cell antigen presentation and the induction of antitumor T cells can be maintained for at least 2 weeks. Under these conditions, robust levels of antitumor immunity were achieved, resulting in the regression of established B16 melanomas.41,42
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SUMMARY AND OUTLOOK
Despite decades of intense investigation, a melanoma vaccine that induces effective cytotoxic T-cell responses and is capable of controlling or eradicating melanoma has been elusive. However, increased understanding of tumor immunity has led to the recognition that most of the approaches taken in the past were limited through weak immune adjuvants, poor choice of tumor antigen, or delivery of the vaccine. The recent successes with immune checkpoint blockade in melanoma and other cancers, leading to durable tumor responses in a considerable proportion of patients, have highlighted the potential of immunotherapy in melanoma. These insights and advances, coupled with the rapid progress in many areas relevant to effective vaccine design such as sequencing technology, biomaterials, and immune adjuvants, provide new opportunities for the successful development of a clinically effective melanoma vaccine. NeoVax, featuring a novel class of immunogens based on individual tumor mutations, and WDVAX, providing a unique delivery system by taking advantage of a novel material engineered scaffold, are examples of innovative vaccine design in melanoma. Both technologies are currently under clinical investigation in patients with high-risk (NeoVax) and advanced melanoma (WDVAX) at the authors’ institution. These new strategies, either alone or in combination with immune checkpoint blockade or other synergistic immune stimulants will, it is hoped, lead the way toward future successful melanoma vaccines. REFERENCES
1. van der Bruggen P, Traversari C, Chomez P, et al. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 1991; 254:1643–7. 2. Cormier JN, Salgaller ML, Prevette T, et al. Enhancement of cellular immunity in melanoma patients immunized with a peptide from MART-1/Melan A. Cancer J Sci Am 1997;3:37–44. 3. Marchand M, van Baren N, Weynants P, et al. Tumor regressions observed in patients with metastatic melanoma treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA-A1. Int J Cancer 1999;80:219–30. 4. Rosenberg SA, Yang JC, Schwartzentruber DJ, et al. Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nat Med 1998;4:321–7. 5. Kawakami Y, Eliyahu S, Delgado CH, et al. Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection. Proc Natl Acad Sci U S A 1994;91:6458–62. 6. Kawakami Y, Eliyahu S, Delgado CH, et al. Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor. Proc Natl Acad Sci U S A 1994;91:3515–9. 7. Chaux P, Vantomme V, Stroobant V, et al. Identification of MAGE-3 epitopes presented by HLA-DR molecules to CD4(1) T lymphocytes. J Exp Med 1999;189: 767–78. 8. Brichard V, Van Pel A, Wolfel T, et al. The tyrosinase gene codes for an antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J Exp Med 1993;178:489–95. 9. Zeng G, Touloukian CE, Wang X, et al. Identification of CD41 T cell epitopes from NY-ESO-1 presented by HLA-DR molecules. J Immunol 2000;165:1153–9. 10. Rosenberg SA, Yang JC, Restifo NP. Cancer immunotherapy: moving beyond current vaccines. Nat Med 2004;10:909–15.
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