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Adjuvants for peptide-based cancer vaccines

Adjuvants for peptide-based cancer vaccines Cancer therapies based on T cells have shown impressive clinical benefit. In particular, immune checkpoint blockade therapies with anti-CTLA-4 and anti-PD-1/PD-L1 are causing dramatic tumor shrinkage and prolonged patient survival in a variety of cancers. However, many patients do not benefit, possibly due to insufficient spontaneous T cell reactivity against their tumors and/or lacking immune cell infiltration to tumor site. Such tumor-specific T cell responses could be induced through anti-cancer vaccination; but despite great success in animal models, only a few of many cancer vaccine trials have demonstrated robust clinical benefit. One reason for this difference may be the use of potent, effective vaccine adjuvants in animal models, vs. the use of safe, but very weak, vaccine adjuvants in clinical trials. As vaccine adjuvants dictate the type and magnitude of the T cell response after vaccination, it is critical to understand how they work to design safe, but also effective, cancer vaccines for clinical use. Here we discuss current insights into the mechanism of action and practical application of vaccine adjuvants, with a focus on peptide-based cancer vaccines. Keywords: Cancer vaccine, Peptide, T cells, Checkpoint, Adjuvant Abbreviations: APC, Antigen presenting cells; cDC, Conventional dendritic cells; CDN, Cyclic dinucleotide; CFA, Complete Freund’s adjuvant; cGAS, Cyclic GMP-AMP synthase; CTL, Cytotoxic T lymphocytes; CTLA-4, Cytotoxic T-lymphocyte-associated protein 4; DAMP, Damage associated molecular patterns; GM-CSF, Granulocyte macrophage colony- stimulating factor; HBV, Hepatitis C virus; HPV, Human papilloma virus; IFA, Incomplete Freund’s adjuvant; IFN, Interferons; IL, Interleukin; IRF, Interferon response factors; MPL, Monophospholipid A; NK, Natural killer cells; Mɸ, Macrophage; PAMP, Pathogen associated molecular patterns; PD-1, Programmed cell death protein 1; pDC, Plasmacytoid dendritic cells; PLA, Poly(lactic acid); PLG, Poly(lactide-co-glycolide); PLGA, Poly(lactic-co-glycolic acid); poly IC, Polyinosine-polycytidylic acid; PPR, Pattern recognition receptors; RLR, C-type lectin receptors and retinoic acid inducible gene (RIG)-I-like receptors; STING, Stimulator of interferon genes; Th (1,2,17), Helper T cells (type 1, 2, 17); TLR, Toll-like receptors; TNF, Tumor necrosis factors; Treg, Regulatory T cells; TRIF, TIR-domain-containing adapter- inducing interferon-β; VdLN, Vaccination site-draining lymph node Background antigen (Ag, signal 1) by regulating its persistence, loca- The goal of a therapeutic cancer vaccine is to induce the tion, concentration and presentation by antigen presenting activation and proliferation of T cells, in particular cyto- cells (APC). Second, it must enhance the immune res- toxic T lymphocytes (CTL), which specifically recognize ponse by inducing the expression of co-stimulatory and kill cancer cells leading to improved therapeutic molecules (signal 2) and cytokines (signal 3) by APC [1]. outcome for the patient. To maximize CTL responses, Suboptimal delivery of any of these signals can result in an ideal vaccine adjuvant must fulfill two major func- poor T cell numbers and/or function. tions. First, it must provide optimal availability of the Antigen delivery systems Antigen delivery systems facilitate signal 1 by different * Correspondence: woverwijk@mdanderson.org Department of Melanoma Medical Oncology, University of Texas - MD mechanisms. First, they extend Ag presentation time by Anderson Cancer Center, South Campus Research Building 1, 1515 protecting Ag from degradation by cell-associated serum Holcombe Blvd, Houston, TX 77030, USA 2 proteases and peptidases [2]. Second, they enhance the Immunology program – University of Texas - Graduate School of Biomedical Sciences at Houston, 6767 Bertner Ave, Houston, TX 77030, USA uptake of tiny antigenic peptides by APC by forming © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Khong and Overwijk Journal for ImmunoTherapy of Cancer (2016) 4:56 Page 2 of 11 them into particles of a size similar to that of pathogens Incomplete Freund’s adjuvant (IFA) (micrometer or submicrometer size) [3]. Third, some IFA is a water-in-oil emulsion, identical to Complete delivery systems can promote the localization of Ag to Freund’s Adjuvant (CFA) but without the heat-killed peripheral draining lymph nodes which increases the Mycobacteria tuberculosis to avoid acute granulomatous chance of encountering draining lymph node-resident lesions at vaccine sites. It has previously been shown APC, resulting in increased Ag-presentation to T cells [4]. that IFA promotes long-term retention and slow release Collectively, these mechanisms enhance T cells response of emulsified antigen at the inoculation site [7, 8]. Likely number by extending Ag presentation time to be optimal as a result of this, IFA induces strong humoral and cellu- for T cell clonal expansion, effector function and/or mem- lar immune responses. Clinical-grade IFA (Montanide™ ory formation [5, 6] (Fig. 1). Mode of action, the types of oil series, SEPPIC Corp.) has been widely used clinically responses, and advantage/disadvantages of selected anti- in experimental peptide and protein-based cancer vac- gen delivery systems are shown in Table 1. Of notice, cines [9]. Recently, our group showed that IFA-based vaccination can also allow for the delivery of immunodo- peptide vaccines can induce potent cytotoxic CD8 T cell minant or neoantigen epitopes, resulting in enhanced responses in mice, followed by T cell retention, exhaustion anti-tumor efficacy. and deletion at the vaccination site, due to excessively Besides signal 1, antigen delivery systems can also de- long-term peptide Ag retention and chronic release by the liver signal 2 and 3 by activating the innate immune cells. poorly biodegradable IFA emulsion [10]. Mechanistically, Aluminum, PLG and polystyrene particles were shown to the long-term antigen presentation and consequent T cell activate the inflammasome complex in a phagocytosis- recognition and cytokine release at the vaccination site dependent manner while carbon nanotubes trigger the induced chronic tissue inflammation and chemokine pro- complement system (see below). Adjuvants vary in the duction that attracted and retained effector T cells, pre- quality and quantity of signals 1, 2 and 3 they deliver to venting them from reaching the tumor site. Eventually, T cells. These attributes of adjuvants become especially persistent antigen stimulation at the vaccination site re- important when using them to vaccinate with antigens sulted in T cell exhaustion and Fas/FasL-mediated T cell that possess very little, if any, inherent adjuvant activity, apoptosis. Of notice, this observation was obtained using such as the minimally defined peptide epitopes typically vaccines based on minimal epitope-sized short peptides used in peptide vaccines. Here we discuss some adjuvants which can be presented by any MHC Class I-positive, that are commonly used in peptide-based cancer vaccines. nonprofessional APC [11]. In contrast, longer peptides Fig. 1 Mechanisms of action of vaccine adjuvant. Left, some adjuvants can function as antigen delivery systems to affect the geographical availability of the antigen (signal 1). Right, adjuvants also commonly stimulate antigen presenting cells (APC) and induce them to upregulate co-stimulatory molecules such as CD80/CD86 (signal 2) and/or produce cytokines such as IL-12 (signal 3). VdLN: vaccination site-draining lymph node Khong and Overwijk Journal for ImmunoTherapy of Cancer (2016) 4:56 Page 3 of 11 Table l Examples of class I adjuvants (delivery systems) Mode of action(s) Types of response Pros Cons IFA and Depot Ab, Th1, Th2 Widely used for vaccines May not suitable for Montanide when antibody production is therapeutic vaccine when formulations desired [123]. cellular response is desired as extended depot will attract CTL to vaccine sites [10]. Aluminum Depot, inflammasome activation Ab, Th2 Safety characters are well Needs to be combined with defined as it is the most other adjuvants to induce CTL widely used adjuvant [124]. response in therapeutic vaccines. Micro/nano Varies, depending on Not well defined but size of articles Reduce Ag dose, cellular and Rapid clearance in blood and particles particlenature: increase Ag half- may contribute to types of biological characters are well accumulation in filtering life (via encapsulation, sustained response: size of 40–50 nm induces defined, versatile to be organs such as liver and spleen release) delivery Ag to target stronger T cell response than 20nm combined with other [24]. cells/organs, cellular and or 2000 nm particles . adjuvants [121]. Need to be combined with Inflammation induction (see text immunopotentiators. for detail) require trimming by DC-specific enzymes to allow effi- by IFN-γ [18]. When combined with MPL (a detoxified cient binding to MHC Class I molecules, and hence they form of lipopolysaccharide, LPS), a TLR4 agonist, such are presented exclusively by the relatively small population as in the AS04 adjuvant system (Glaxo SmithKline), of DC in the context of optimal co-stimulatory molecules alum-based vaccines induce Th1 responses with produc- and cytokines for efficient T cell priming [12]. Indeed, long tion of IFN-γ and IgG2a. In 2008, alum adjuvants were peptides emulsified in IFA induced minimal T cell traf- found to activate the NALP3 inflammasome in DC [19]. ficking to vaccine sites and greatly reduced contraction Inflammasome activation leads to the production of pro- of T cell levels [10]. It is also proposed that long pep- inflammatory cytokines including IL-1β and IL-18 which tides which contain helper T cell epitopes will induce promote the adaptive cellular (Th1/Th17/Th2) and Th response to further enhance the CTL response [12]. humoral responses [20]. IL-1β promotes Th1 and Th17 However, in 2 separate clinical trials using IFA, separate while IL-18 serves as coactivator for other cytokines. In Th epitopes mixed with short CTL epitopes failed to the presence of IL-12 and IL-15, IL-18 contributes to improve CTL response in patients with metastatic mel- Th1 response via promoting IFN-γ production. In the anoma [13, 14]. This might be due to a difference in absence of IL-12, IL-18 induces IL-4 which drives Th2 the nature of the antigens: virus-derived long peptides response [21]. Thus, adjuvants that activate the inflam- containing both Th and CTL epitopes vs. melanocyte masome, including alum, can induce different types of T self antigen-derived short CTL epitope peptides mixed cell response, depending on tissue- or adjuvant-driven with short Th epitope peptides. Given the clear benefit cytokine context. of CD4+ T cell responses in the generation and intratu- moral function of CD8+ T cells [15, 16], further studies Micro/nano particles are needed to reconcile this discrepancy. Nevertheless, Micro- and nano-particles are attractive antigen/drug de- our preclinical data suggest that prolonged Ag presen- livery systems because they can combine several desired tation (signal 1), even in the presence of signal 2 and 3, characteristics. First, the particles protect their cargo from can induce T cell retention, exhaustion and deletion. serum/tissue peptidases/proteases and other degrading fac- tors, thus increasing the half-life of encapsulated Ag and Aluminum adjuvants immunomodulators in vivo. Second, particles can be engi- Generally referred to as alum, both aluminum hydroxide neered to target specific cell types or organs (such as (Alhydrogel™) and aluminum phosphate (Adjut-phos™) lymph node) [22, 23]. These features help reduce both the adjuvants are widely used in human vaccines such as drug dose and off-target side effect. For example, it has those against influenza, tetanus, diphtheria, pertussis, been shown that Ag encapsulated in poly(lactic-co-glycolic poliomyelitis, and HPV [17]. During vaccine preparation, acid) (PLGA) particles induce similar T cell response with antigens are adsorbed to preformed aluminum adju- a 1000-fold lower dose compared to free Ag [24]. vants, hence their name aluminum-adsorbed vaccines. There are two basic ways to engineer particles for en- Aluminum adjuvants are known to promote Th2 re- hanced uptake by APC. Passive targeting relies on the sponses which make them less suitable for vaccines size, charge and rigidity of the particle while active tar- against intracellular bacteria such as M. tuberculosis, geting is based on added ligands on the particle surface. which require a Th1-type immune response dominated Vaccine particles with size range from 500 to 2000 nm Khong and Overwijk Journal for ImmunoTherapy of Cancer (2016) 4:56 Page 4 of 11 are preferentially trapped by tissue APC at the injection localization, and survival of vaccination-induced, tumor- site (which may then traffic to LN), while 20 to 200 nm specific T cells [10]. However, extremely short antigen particles drain passively to LN where they are taken up presentation (such as after injection of minimal epitope by resident APC. Beside their role as Ag/drug carrier, in- peptides in saline), especially in the absence of adjuvants creasing signal 1, micro and nanoparticles can also en- to induce signals 2 and 3, can likewise lead to subopti- hances signals 2 and 3. PLG and polystyrene particles mal or even abortive/tolerogenic T cell activation. We are thought to participate in inflammasome activation by speculate that in successful, natural immune responses, enhancing the IL-1β secretion by DC in a phagocytosis- such as those against acute viral infections that are rap- dependent manner [25]. Carbon nanotube particles, on idly and completely cleared, the bulk of specific antigen the other hand, activate the complement system and sub- persists for a moderate duration, in the order of a few sequent inflammatory responses via binding to C1q [26]. days [31]. While there is clear evidence that small amounts Materials used to make micro and nanoparticles include of antigens can be retained much longer in APC, the initial liposomes, syntheticpolymerssuch aspolystyrene, large wave of antigen that primes the acutewave of T cell poly(lactide-co-glycolide) PLG, poly(lactic acid) PLA, effectors that follows within days of acute pathogen expos- PLGA or natural polymers such as gelatin, collagen ure is typically gone within a week. By analogy, cancer vac- and chitosan. The choice of material depends on the cines with similar kinetics of antigen availability have the desired biocompatibility, half-life, hydrophobicity and po- best chance of priming a massive wave of tumor-specific larity. For example, liposome particles are very versatile, CTL. Indeed, we have observed such a bell-shaped curve allowing combination of Ag and cytokines like IL-2 or for T cell response and function after different duration of GM-CSF, into a single particle to provide better immune antigen presentation in vivo (Khong et al., manuscript in response and protection [27]. However, major drawbacks preparation). It will be interesting to see whether this is a are the rapid clearing from the blood and accumulation in common principle, and whether this can be harnessed to the liver. Coating a liposome with polyethylene glycol increase the potency and efficacy of peptide-based cancer (PEG) or other biocompatible polymers can reduce rapid vaccines. systemic clearing and thus extend its half-life in vivo [28]. To improve the accumulation of a liposome to tar- The immunopotentiators geted tissue or organ, its surface can be decorated When vaccinologists moved from whole pathogen vac- with receptors (e.g. antibodies) for target cell/tissue cines (live, attenuated or dead pathogens) to recombinant ligands and such modified liposomes are called immuno- subunit vaccines for reasons of safety and manufacturing, liposomes. Micro- and nanoparticles such as hydro- they learned that these vaccines typically evoked weaker philic poly(DL-lactide-co-glycolide) microspheres and immunity and protection. The discovery of how our body poly(propylene sulfide) nanoparticles have been de- senses pathogens via a family of highly conserved pattern signed to target the DC in draining LN [22, 23]. A recognition receptors (PRR) called Toll-like receptors different approach is to attract DC to the site of vaccine (TLR) [32–34] heralded the era of the specific receptor- injection. Recent reports showed that incorporating GM- mediated activation of innate immunity. Since then, other CSF, CpG and tumor antigens in PLG matrices efficiently innate immune receptors have been discovered including attracted and stimulated both conventional DC (CD11c + NOD-like receptors (NLR), C-type lectin receptors and CD11b + and CD11c + CD8a+) and plasmacytoid DC, retinoic acid inducible gene (RIG)-I-like receptors (RLR) resulting in superior immune responses (Th1 and CTL) and most recently cyclic GMP-AMP synthase (cGAS). against B16 melanomas in mice [29, 30]. A very high con- Within the last decades, numerous adjuvants have been centration of GM-CSF (3000 ng) prolonged the DC reten- developed to target these innate receptors. Signaling tion in situ, resulting in suboptimal DC trafficking to mechanisms of these receptors have been thoroughly draining LN and the subsequent inferior T cell priming discussed elsewhere [35–38];herewe focus on thead- and protection against tumor. This observation suggests juvants that target these receptors, in particular those that delivery systems that stimulate the attraction of DC that have entered clinical trials of cancer vaccines. can promote T cell responses, but only if they do not pre- Some notable examples of immunopotentiators and vent the DC from ultimately reaching the LN where T cell their stages of development are listed in Table 2. priming typically occurs. Adjuvants targeting toll-like receptors The antigen depot: what duration of antigen presentation TLR2 agonists is optimal? TLR2 is expressed on the surface of different immune Our preclinical work with IFA as a vaccine adjuvant sug- cells like DC, macrophages and lymphocytes and recog- gests that prolonged antigen presentation has multiple nizes bacterial lipopeptides. Upon engaging its ligands, detrimental effects on the effector function, tumor TLR2 activates NF-kB via the MYD88 signaling pathway. Khong and Overwijk Journal for ImmunoTherapy of Cancer (2016) 4:56 Page 5 of 11 Table 2 Examples of class 2 adjuvants (immunopotentiators) Receptor Target cells Stage of development (not comprehensive) in cancer vaccine “stepping on the gas” Pam CSK TLR2 DC, M$, lymphocytes Preclinical 3 4 Poly-ICLC TLR3 cDC, M$, epithelial cells Several clinical trials for different cancers. MPLA TLR4 cDC, M$, epithelial cells, Clinical trial phase 2 fibroblasts Imiquimod TLR7/8 pDC, B cells, M$, Clinically approved for treating basal cell carcinoma. Multiple clinical trials in combination with monocytes vaccine for different cancers. CpG TLR9 pDc, B cells Multiple clinical trials IL-2 IL-2Ra/ T, B and NK cells Clinically approved for treating renal carcinoma and melanoma. Multiple clinical trials in p/y combination with vaccine for different cancers. GM-CSF GM- many Multiple clinical trials in combination with vaccine and checkpoint blockades for different cancers. CSFR IFNs IFNR many Multiple clinical trials CDNs STING many Preclinical “releasing the brake” a-PD1 Ab PD-1 T, B and NK cells Clinically approved for different cancers. a-CTLA4 Ab CTLA-4 T cells Clinically approved for melanoma, under multiple clinical trials for different cancers. There are two common strategies to engage TLR-2 (IFNs) and the subsequent upregulation of costimulatory through vaccines: conjugating the antigen to bacterial molecules [44]. lipopeptides or to palmitic acid. Bacterial lipopeptide Poly I:C can enhance antigen cross-presentation by MALP-2 and its synthetic analogues like Pam Cys and DC to CD8 T cells. Because of its rapid degradation by Pam Cys are most frequently used. The peptide- serum nucleases in primates, poly I:C has limited anti- lipopeptide construct were shown to induce DC mat- tumor efficacy in humans [39]. Therefore, more stable uration, pro-inflammatory cytokine (IL-12, TNF-α, derivatives of poly I:C were made, including poly ICLC IFN-γ) secretion, B cell activation and enhanced CTL (known as Hiltonol) and poly I:C U [45]. In a phase 1 responses [39]. Most current clinical trials of TLR-2 ovarian cancer trial, addition of poly ICLC to a vaccine based adjuvants are for vaccination against infectious consisting of NY-ESO1 long overlapping peptides in IFA diseases such as HIV, HBV and Lyme disease. In 2014, dramatically induced rapid and efficient CD4 and CD8 T vaccine using TLR-2 ligand (Pam CSK )conjugated cell responses, compared to the vaccine alone [46]. A re- 3 4 with long synthetic peptide showed very promising results cent study in monkeys showed that poly ICLC in com- in a preclinical melanoma model [40]. Interestingly, bination with agonistic CD40 antibody significantly Pam CSK -peptide conjugate, but not the mixture of enhanced both CD4 and CD8 responses compared to 3 4 Pam CSK with peptide, induced robust T cell response either adjuvant alone [47]. This is some of the first pri- 3 4 and protection against tumor. This is in line with the cis- mate data confirming the multitude of mouse studies activation model showed by Desch et al. [41], which essen- that indicated strong synergy when different classes of tially posits that signal 1 and 2 should be delivered by immunopotentiators are used together in vaccine adjuvants same APC for optimal T cell priming. [10, 48, 49]. I:C U and poly ICLC have entered clinical trials for other cancer including glioma, melanoma, carcin- oma (poly ICLC) and HER-2 positive breast cancer [39]. TLR3 agonists TLR3 is expressed in the endosomal compartment of con- ventional dendritic cells (cDC), macrophages and on the TLR4 agonists surface membrane of non-immune cells like epithelial TLR4 is expressed on the surface of immune cells in- cells [42]. TLR3 is activated by double-stranded RNA or cluding cDC and macrophages as well as non-immune its synthetic analog polyinosine-polycytidylic acid (poly cells such as fibroblasts and epithelial cells. Triggering I:C) [43]. TLR3 does not use the MyD88 signaling path- TLR4 will activate both MyD88 and TRIF dependent way but triggers TRIF signaling leading to activation of pathways leading to NF-kB and IRF3/7 activation. TLR4 NF-kB, MAP kinases and IRF3, which in turn induce the activation strongly promotes Th1 response through IL- production of inflammatory cytokines, type 1 interferons 12p70 induction [50]. Due to its high toxicity, LPS has Khong and Overwijk Journal for ImmunoTherapy of Cancer (2016) 4:56 Page 6 of 11 been replaced by the less toxic derivative, monopho- and is a potent pDC activator and IFNα inducer [65]. CpG sphoryl lipid A (MPLA), as vaccine adjuvant. The adju- B only has phosphorothioate backbone. CpG B strongly ac- vanticity of MPLA has been studied extensively in tivates B cells and promotes pDC and monocyte matur- several clinical trials [39]. MPLA is used in combination ation [66]. CpG C is a hybrid of the two above [67]. CpG with aluminum (AS04) to skew the typical Th2 response has been used in clinical trials of therapeutic cancer vac- induced by alum to a Th1 response [51]. MPL as a vac- cines against melanoma, breast/lung/ovarian cancers, sar- cine adjuvant, in combination with tumor antigens, has coma and glioblastoma [68–72]. Overall, the vaccines entered into several clinical trials for melanoma, lung, induced both humoral and cellular responses, but clinical and prostate cancer [52–54]. benefit remained uncommon. TLR7/8 agonists STING agonist Localizing within the endosomal compartments, both In 2006, TLR-independent antiviral responses (i.e. type 1 TLR7 and 8 can recognize single stranded (ss) RNA as interferon induction) were shown to be induced by they are structurally related [42]. In human, TLR7 is pre- double stranded (ds) DNA in the cytosol [73]. Later, dominately expressed in plasmacytoid dendritic cells dsDNA was found to activate the transcription factor (pDC) and to a lesser extent in B cells and monocytes/ NF-kB and IRF3 via an endoplasmic reticulum adaptor macrophages while TLR8 is mainly expressed in mono- called STING (stimulator of interferon genes) [74]. In cytes/macrophages and cDC [55]. TLR7/8 signal through 2013, the receptor for cytosolic DNA, the cylic GMP- the MyD88 pathway leading to upregulation of co- AMP synthase or cGAS, was discovered [75]. Upon stimulatory molecules (CD80/86, CD40), production of binding to cytosolic DNA, cGAS catalyzes the synthesis cytokines (IFN-α, TNF-α, IL-12) and migration of DC of cGAMP which in turns binds to and activates the from skin to lymph nodes. TLR8 is expressed, while adaptor protein STING. Recent results indicate that TLR7 is not, on the important BDCA3+ cDC subset that spontaneous T cell priming against tumor antigen re- is most potently responsible for cross-priming of CD8+ quires STING-dependent type I IFN induction [76]. Very T cells [56], and thus preferential TLR7 agonists may promising results from preclinical studies with STING exert weaker adjuvant activity than TLR8 or TLR7/8 ag- agonists injected directly into tumors in the aggressive onists when used in CD8+ T cell-inducing vaccines. B16 melanoma model had led to high excitement for TLR7/8 can also activate B cells to produce antibody their application in clinical trials [77]. Recent results also and cytokines such as IL-6 and TNF-α, and T cells to indicate that STING agonists can function as adjuvant proliferate and produce cytokines including IFN-γ and in a setting of whole-cell tumor cell vaccine [78]. It will IL-2 [57, 58]. TLR7/8 can be activated by synthetic imi- be interesting to see how STING agonists compare to dazoquinolines including imiquimod (mostly acts on TLR agonists as adjuvants for peptide vaccines in animal TLR7) and resiquimod (TLR7 and 8). Imiquimod models and clinical trials, and whether their combined (Aldara cream) has been approved to treat basal cell car- use offers additional benefit, given their different intra- cinoma and genital warts [59, 60]. Several clinical trials cellular signaling pathways. of imiquimod as vaccine adjuvant in different cancers in- cluding chronic myeloid leukemia (CML), vulval intrae- Cytokines as adjuvants pithelial neoplasia (VIN), prostate cancer and melanoma IL-2 have been conducted [61–64]. Overall, all vaccines in- The most notable cytokine which has been extensively duced both humoral and cellular responses in a major used for immunotherapy is IL-2. IL-2 was initially des- fraction of patients. In vaccinated patients with VIN, cribed as a T cell growth factor (TCGF) responsible for infiltration of both CD4 and CD8 T cells into tumor the clonal expansion, differentiation and survival of T cells sites was shown to correlate with tumor clearance [62]. [79], and later of activated B cells and natural killer (NK) cells as well [80, 81]. Although CD4 T cells are the major TLR9 agonists source of IL-2 in vivo, CD8 T cells, NK cells and DC can TLR9 is expressed by human B cells and pDC and localizes also produce IL-2 [82–85]. IL-2 was FDA-approved for in endo-lysosomal compartment [42]. Its role is to detect the therapy of metastatic renal cell carcinoma in 1992 and unmethylated CpG motifs which are often found in bacter- metastatic melanoma in 1998 [86, 87]. IL-2 mediates anti- ial, but not host cell DNA. Upon activation, TLR9 induces tumor activity by activating tumor-specific T cells and NK production of pro-inflammatory and Th1 cytokines (such cells. In mice, addition of IL-2 to experimental cancer vac- as IL-12) by APC. There are 3 classes of synthetic CpG oli- cines can greatly increase the therapeutic efficacy [10, 48]. gonucleotides (ODN) being used in preclinical and clinical IL-15 signals through the same IL-2 Rβγ complex also studies. CpG A is a mix of phosphodiester/phosphorothio- used by IL-2, and can also promote peptide-induced T cell ate backbone with palindromic sequences and poly G tail, proliferation, especially in T cells with low-affinity TCRs Khong and Overwijk Journal for ImmunoTherapy of Cancer (2016) 4:56 Page 7 of 11 [88]. In patients with melanoma, addition of an experi- however due to the lack of a vaccine arm without GM- mental gp100 peptide/IFA vaccine to IL-2 gave a higher CSF, its exact impact on clinical outcome remains un- clinical response rate than observed in patients receiving known [104, 105]. IL-2 alone, and also higher than previously observed for gp100 peptide vaccine alone, suggesting IL-2 can also Interferons (IFNs) function as a vaccine adjuvant in humans [89]. However, IFNs are of great interest for adjuvant development, IL-2 can also expand immunosuppressive regulatory T owing to their pleiotropic effect on different immune cells cells (Treg) which may dampen the immune response or such as DC, B cells and T cells as well as non-immune anti-tumor efficacy [90]. Because Treg express both IL- cells. IFN-α and IFN-β promote DC maturation, including 2Rα and IL-2Rβγ whileCTL express onlythe latter, the up-regulation of MHC and costimulatory molecules. In blocking IL-2Rα when using IL-2 preferentially expands virus-infected cells, type I IFNs prevent virus replication by CTL [91]. Recently, a mutant form of IL-2 (IL-2 mutein) halting transcriptional and translational machineries, was reported to have higher antitumor efficacy with re- accelerating RNA degradation by inducing RNase L and duced proliferation induction on Treg, possibly thanks to inducing apoptosis [106]. IFN-α and pegylated IFN-α preferential binding to IL-2Rβγ but not IL-2Rα [92]. Simi- have been approved for advanced renal cell carcinoma larly, IL-2 pre-complexed with IL-2-specific antibodies, and chronic hepatitis C treatment, respectively, and and IL-2 covalently modified with polyethylene glycol both are given after surgical resection of primary mel- have shown selective binding to IL-2Rβγ but not IL-2Rα, anoma to reduce the chance of recurrence [107]. Pre- favoring selective effects on CD8+ T cells [93, 94]. If these clinical studies showed direct adjuvant efficacy of type I modifications also lower the toxicity of IL-2, which may IFN in a peptide-based anti-melanoma vaccine, where it be partly mediated by IL-2Rα, these IL-2-based com- promoted T cell numbers, longevity and effector function, pounds may make a comeback in cancer immunotherapy, resulting in improved tumor control [108]. In contrast to including as vaccine adjuvants [91]. type I IFN, IFN-γ (the soletypeIIIFN)istypically only produced by specialized immune cells including T cells, NK cells and NKT cells [109]. Recombinant IFN-γ (or gen- Granulocyte-macrophage colony stimulating factor etically engineered IFN-γ1b) is approved to treat chronic (GM-CSF) granulomatous disease [110]. In cancer immunotherapy, a GM-CSF is a cytokine used as a cancer vaccine adjuvant, phase III clinical trial combining chemotherapy with IFN-γ sometimes with success. GM-CSF can be produced by for patients with advanced ovarian and peritoneal carcino- many cell types including myeloid cells, lymphocytes, mas was terminated due to serious adverse effects [111]. fibroblast, endothelial/epithelial/mesothelial cells and cer- tain tumor cells [95]. The production of GM-CSF is in- Lessons learned from a few successful peptide-based cancer duced by bacterial toxin and inflammatory cytokines such vaccine clinical trials as IL-1, IL-6, and TNF-α [96]. GM-CSF receptor is found A vaccine comprised of long peptide from HPV-16 viral on myeloid cells and non-hematopoietic cells such as oncoproteins E6 and E7 emulsified in IFA was shown to endothelial cells. In vaccine settings, GM-CSF has been be very effective in treating vulvar intraepithelial neopla- shown to initiate the recruitment and maturation of DC sia, a precancerous condition in HPV-16 positive women as well as activation of macrophages, neutrophils, and NK [112]. The overall clinical response was 79 % while cells, indicating that it is a potential vaccine adjuvant [97, complete response was 47 %, after 2 years of follow-up. 98]. Combination of GVAX (irradiated tumor cell express- This remarkable result with an IFA-based peptide vac- ing GM-CSF) with anti-CTLA-4 and anti-PD-1 check- cine was consistent with our findings that long peptides point blockade was very promising in preclinical studies, did not cause severe sequestration of T cells at the leading to the first clinical trials of checkpoint blockade in vaccination site as discussed above. In a phase 3 trial for patients with cancer. Recombinant GM-CSF has been patients with advanced melanoma, combination of IL-2 used in peptide vaccine trials in mouse and man, where it with short gp100 (209–217) peptide emulsified in IFA has had varying success in raising T cell responses. This resulted in a modest but significant improvement of may be partially due to a balance between pro- and anti- overall clinical responses, progression-free survival and inflammatory properties of GM-CSF depending on its overall survival, compared to IL-2 treatment alone [89]. dose [29]. In addition, there appear to be complex interac- Based on some of the preclinical results with IFA dis- tions between GM-CSF and other factors in the tumor- cussed above, a less persistent, and therefore less T cell conditioned microenvironment that influence its abil- sequestering, vaccine formulation might result in more ity to either enhance or reduce vaccine-induced T cell dramatic synergy with IL-2. Indeed, given new insights responses [99–102]. Several positive peptide/protein into the nature of tumor antigens (short vs. long pep- vaccine trials have incorporated GM-CSF [13, 103]; tides, as well as mutated vs. non-mutated antigens) and Khong and Overwijk Journal for ImmunoTherapy of Cancer (2016) 4:56 Page 8 of 11 adjuvants, there is ample opportunity to design new, [118]. We found that adding IL-2 to a TLR7 agonist/CD40 more effective cancer vaccines. A clinical trials in renal agonist combination further enhanced CD8 T cell peak cell cancer with the multiple peptide-based, GM-CSF- effector and memory response, and anti-tumor efficacy adjuvanted, water-formulated IMA091 vaccine showed [10]. Second, some adjuvants may possess both desired that the breadth of CTL response significantly associated and undesired adjuvant properties. By combining with with clinical benefit, perhaps by limiting antigen-loss other adjuvants, the immune response can be skewed to- escape mechanisms [104, 113]. Taken together, results ward favorable one, as in the above-mentioned example of from these clinical studies underscore the importance of alum combined with MPL which is used in HPV vaccine the nature and delivery of target antigens, and the and HBV vaccine to promote Th1 response [119]. A major provision of the right adjuvant. obstacle to successful translation of these long-known pre- clinical findings is the observable paucity of clinical trials Cancer vaccines and T cell checkpoint blockade where multiple pharmaceutical companies combine their While blockade of CTLA-4 and PD-1 T cell checkpoints respective promising, potent agents to create a truly shows strong activity in a variety of cancers, many patients powerful cancer vaccine. This limitation is slowly begin- do not respond, likely due to insufficient spontaneous ning to be addressed by the initiation of co-development anti-tumor T cell immunity (a lack of tumor reactive T agreements between companies, as well as by the develop- cells and/or poor T cell infiltration into the tumor). ment of multiple synergistic adjuvants within single com- Vaccination can enhance tumor-specific immunity, and panies. Thus, clinical trials of cancer vaccines consisting of vaccination is therefore a prime candidate for combination multiple antigens formulated in adjuvants consisting of with checkpoint blockade therapy. Interestingly, the 676- short-lived depots with multiple classes of synergistic patient study that led to FDA approval of anti-CTLA-4 immunostimulatory molecules may finally become a revealed that concurrent vaccination with gp100 peptide reality. vaccine in IFA did not enhance therapeutic efficacy, and in fact modestly but significantly decreased overall res- Adjuvant-free peptide vaccine ponse rate and disease control rate through an unknown There is an emerging new trend of adjuvant-free vaccine mechanism [114]. This has led to uncertainty about that uses self- assembling peptides. Such peptides were whether and how to combine vaccination with checkpoint constructed to have a domain which helps them assemble blockade, hampering efforts to improve overall response into nanofiber structure [120]. Preclinical studies using rates in melanoma and especially in other, less immuno- mouse model showed self-assembling peptides could elicit genic cancers. When modeled in mice, we indeed observe humoral as well as cellular responses [120–122]. The that gp100/IFA vaccination does not synergize with humoral response was shown to be T cell independent, CTLA-4 or PD-1, and that this effect is due to T cell possibly due to cross-liking of repetitive epitopes of nano- entrapment, even of anti-CTLA-4 therapy-induced T fiber peptides to B cell receptors. Yet, mechanisms of how cells, at the gp100/IFA vaccination site. Nevertheless, self-assembling peptides can trigger cellular responses re- by choosing different vaccine formulations, great syn- main undefined. Nevertheless, we anticipate that while ergy between peptide vaccine and checkpoint blockade self-assembling peptide cancer vaccines can possibly by- can be achieved (unpublished results). Other preclinical pass the need for a separate antigen delivery system, they work also indicates synergy between checkpoint blockade will still needs immunopotentiators to optimally activate T and other classes of non-persistent vaccines, opening the cells as well as protecting them from tumor suppressive possibility that vaccines that do not induce excessive T cell mechanisms to ultimately maximize therapeutic vaccine sequestration may combine well with checkpoint blockade efficacy. therapy [78, 99]. Conclusion The need for combining different adjuvants into a single Cancer vaccines are attracting new interest as combin- vaccine ation partners with other immunotherapies, in particular Much preclinical work suggests that combining different T cell checkpoint blockade approaches. A detailed un- adjuvants is needed to induce a strong anti-tumor immune derstanding of the mechanism of action of anti-cancer response [115]. Accumulated evidence has shown that vaccination is critical for the design of potent vaccine CD40 signaling synergizes with almost all TLR ligand indu- approaches that induce robust T cell responses. Vaccine cing far better cellular and humoral responses than that of adjuvants are a major, required component of successful each individual adjuvant [116, 117]. Several groups have vaccines, and several novel adjuvants are now making shown that almost all TLR agonists synergize with CD40 their appearance in the clinic, bridging the wide gap signaling to enhance CTL expansion and function, in part between preclinical and clinical cancer vaccine formula- by inducing the co-stimulatory molecule CD70 on DC tions. This translational effort is further guided by early Khong and Overwijk Journal for ImmunoTherapy of Cancer (2016) 4:56 Page 9 of 11 signs of success in a few clinical trials. The hope is that 14. Phan GQ, et al. 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Adjuvants for peptide-based cancer vaccines

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Springer Journals
Copyright
Copyright © 2016 by The Author(s).
Subject
Medicine & Public Health; Oncology; Immunology
eISSN
2051-1426
DOI
10.1186/s40425-016-0160-y
pmid
27660710
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Abstract

Cancer therapies based on T cells have shown impressive clinical benefit. In particular, immune checkpoint blockade therapies with anti-CTLA-4 and anti-PD-1/PD-L1 are causing dramatic tumor shrinkage and prolonged patient survival in a variety of cancers. However, many patients do not benefit, possibly due to insufficient spontaneous T cell reactivity against their tumors and/or lacking immune cell infiltration to tumor site. Such tumor-specific T cell responses could be induced through anti-cancer vaccination; but despite great success in animal models, only a few of many cancer vaccine trials have demonstrated robust clinical benefit. One reason for this difference may be the use of potent, effective vaccine adjuvants in animal models, vs. the use of safe, but very weak, vaccine adjuvants in clinical trials. As vaccine adjuvants dictate the type and magnitude of the T cell response after vaccination, it is critical to understand how they work to design safe, but also effective, cancer vaccines for clinical use. Here we discuss current insights into the mechanism of action and practical application of vaccine adjuvants, with a focus on peptide-based cancer vaccines. Keywords: Cancer vaccine, Peptide, T cells, Checkpoint, Adjuvant Abbreviations: APC, Antigen presenting cells; cDC, Conventional dendritic cells; CDN, Cyclic dinucleotide; CFA, Complete Freund’s adjuvant; cGAS, Cyclic GMP-AMP synthase; CTL, Cytotoxic T lymphocytes; CTLA-4, Cytotoxic T-lymphocyte-associated protein 4; DAMP, Damage associated molecular patterns; GM-CSF, Granulocyte macrophage colony- stimulating factor; HBV, Hepatitis C virus; HPV, Human papilloma virus; IFA, Incomplete Freund’s adjuvant; IFN, Interferons; IL, Interleukin; IRF, Interferon response factors; MPL, Monophospholipid A; NK, Natural killer cells; Mɸ, Macrophage; PAMP, Pathogen associated molecular patterns; PD-1, Programmed cell death protein 1; pDC, Plasmacytoid dendritic cells; PLA, Poly(lactic acid); PLG, Poly(lactide-co-glycolide); PLGA, Poly(lactic-co-glycolic acid); poly IC, Polyinosine-polycytidylic acid; PPR, Pattern recognition receptors; RLR, C-type lectin receptors and retinoic acid inducible gene (RIG)-I-like receptors; STING, Stimulator of interferon genes; Th (1,2,17), Helper T cells (type 1, 2, 17); TLR, Toll-like receptors; TNF, Tumor necrosis factors; Treg, Regulatory T cells; TRIF, TIR-domain-containing adapter- inducing interferon-β; VdLN, Vaccination site-draining lymph node Background antigen (Ag, signal 1) by regulating its persistence, loca- The goal of a therapeutic cancer vaccine is to induce the tion, concentration and presentation by antigen presenting activation and proliferation of T cells, in particular cyto- cells (APC). Second, it must enhance the immune res- toxic T lymphocytes (CTL), which specifically recognize ponse by inducing the expression of co-stimulatory and kill cancer cells leading to improved therapeutic molecules (signal 2) and cytokines (signal 3) by APC [1]. outcome for the patient. To maximize CTL responses, Suboptimal delivery of any of these signals can result in an ideal vaccine adjuvant must fulfill two major func- poor T cell numbers and/or function. tions. First, it must provide optimal availability of the Antigen delivery systems Antigen delivery systems facilitate signal 1 by different * Correspondence: woverwijk@mdanderson.org Department of Melanoma Medical Oncology, University of Texas - MD mechanisms. First, they extend Ag presentation time by Anderson Cancer Center, South Campus Research Building 1, 1515 protecting Ag from degradation by cell-associated serum Holcombe Blvd, Houston, TX 77030, USA 2 proteases and peptidases [2]. Second, they enhance the Immunology program – University of Texas - Graduate School of Biomedical Sciences at Houston, 6767 Bertner Ave, Houston, TX 77030, USA uptake of tiny antigenic peptides by APC by forming © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Khong and Overwijk Journal for ImmunoTherapy of Cancer (2016) 4:56 Page 2 of 11 them into particles of a size similar to that of pathogens Incomplete Freund’s adjuvant (IFA) (micrometer or submicrometer size) [3]. Third, some IFA is a water-in-oil emulsion, identical to Complete delivery systems can promote the localization of Ag to Freund’s Adjuvant (CFA) but without the heat-killed peripheral draining lymph nodes which increases the Mycobacteria tuberculosis to avoid acute granulomatous chance of encountering draining lymph node-resident lesions at vaccine sites. It has previously been shown APC, resulting in increased Ag-presentation to T cells [4]. that IFA promotes long-term retention and slow release Collectively, these mechanisms enhance T cells response of emulsified antigen at the inoculation site [7, 8]. Likely number by extending Ag presentation time to be optimal as a result of this, IFA induces strong humoral and cellu- for T cell clonal expansion, effector function and/or mem- lar immune responses. Clinical-grade IFA (Montanide™ ory formation [5, 6] (Fig. 1). Mode of action, the types of oil series, SEPPIC Corp.) has been widely used clinically responses, and advantage/disadvantages of selected anti- in experimental peptide and protein-based cancer vac- gen delivery systems are shown in Table 1. Of notice, cines [9]. Recently, our group showed that IFA-based vaccination can also allow for the delivery of immunodo- peptide vaccines can induce potent cytotoxic CD8 T cell minant or neoantigen epitopes, resulting in enhanced responses in mice, followed by T cell retention, exhaustion anti-tumor efficacy. and deletion at the vaccination site, due to excessively Besides signal 1, antigen delivery systems can also de- long-term peptide Ag retention and chronic release by the liver signal 2 and 3 by activating the innate immune cells. poorly biodegradable IFA emulsion [10]. Mechanistically, Aluminum, PLG and polystyrene particles were shown to the long-term antigen presentation and consequent T cell activate the inflammasome complex in a phagocytosis- recognition and cytokine release at the vaccination site dependent manner while carbon nanotubes trigger the induced chronic tissue inflammation and chemokine pro- complement system (see below). Adjuvants vary in the duction that attracted and retained effector T cells, pre- quality and quantity of signals 1, 2 and 3 they deliver to venting them from reaching the tumor site. Eventually, T cells. These attributes of adjuvants become especially persistent antigen stimulation at the vaccination site re- important when using them to vaccinate with antigens sulted in T cell exhaustion and Fas/FasL-mediated T cell that possess very little, if any, inherent adjuvant activity, apoptosis. Of notice, this observation was obtained using such as the minimally defined peptide epitopes typically vaccines based on minimal epitope-sized short peptides used in peptide vaccines. Here we discuss some adjuvants which can be presented by any MHC Class I-positive, that are commonly used in peptide-based cancer vaccines. nonprofessional APC [11]. In contrast, longer peptides Fig. 1 Mechanisms of action of vaccine adjuvant. Left, some adjuvants can function as antigen delivery systems to affect the geographical availability of the antigen (signal 1). Right, adjuvants also commonly stimulate antigen presenting cells (APC) and induce them to upregulate co-stimulatory molecules such as CD80/CD86 (signal 2) and/or produce cytokines such as IL-12 (signal 3). VdLN: vaccination site-draining lymph node Khong and Overwijk Journal for ImmunoTherapy of Cancer (2016) 4:56 Page 3 of 11 Table l Examples of class I adjuvants (delivery systems) Mode of action(s) Types of response Pros Cons IFA and Depot Ab, Th1, Th2 Widely used for vaccines May not suitable for Montanide when antibody production is therapeutic vaccine when formulations desired [123]. cellular response is desired as extended depot will attract CTL to vaccine sites [10]. Aluminum Depot, inflammasome activation Ab, Th2 Safety characters are well Needs to be combined with defined as it is the most other adjuvants to induce CTL widely used adjuvant [124]. response in therapeutic vaccines. Micro/nano Varies, depending on Not well defined but size of articles Reduce Ag dose, cellular and Rapid clearance in blood and particles particlenature: increase Ag half- may contribute to types of biological characters are well accumulation in filtering life (via encapsulation, sustained response: size of 40–50 nm induces defined, versatile to be organs such as liver and spleen release) delivery Ag to target stronger T cell response than 20nm combined with other [24]. cells/organs, cellular and or 2000 nm particles . adjuvants [121]. Need to be combined with Inflammation induction (see text immunopotentiators. for detail) require trimming by DC-specific enzymes to allow effi- by IFN-γ [18]. When combined with MPL (a detoxified cient binding to MHC Class I molecules, and hence they form of lipopolysaccharide, LPS), a TLR4 agonist, such are presented exclusively by the relatively small population as in the AS04 adjuvant system (Glaxo SmithKline), of DC in the context of optimal co-stimulatory molecules alum-based vaccines induce Th1 responses with produc- and cytokines for efficient T cell priming [12]. Indeed, long tion of IFN-γ and IgG2a. In 2008, alum adjuvants were peptides emulsified in IFA induced minimal T cell traf- found to activate the NALP3 inflammasome in DC [19]. ficking to vaccine sites and greatly reduced contraction Inflammasome activation leads to the production of pro- of T cell levels [10]. It is also proposed that long pep- inflammatory cytokines including IL-1β and IL-18 which tides which contain helper T cell epitopes will induce promote the adaptive cellular (Th1/Th17/Th2) and Th response to further enhance the CTL response [12]. humoral responses [20]. IL-1β promotes Th1 and Th17 However, in 2 separate clinical trials using IFA, separate while IL-18 serves as coactivator for other cytokines. In Th epitopes mixed with short CTL epitopes failed to the presence of IL-12 and IL-15, IL-18 contributes to improve CTL response in patients with metastatic mel- Th1 response via promoting IFN-γ production. In the anoma [13, 14]. This might be due to a difference in absence of IL-12, IL-18 induces IL-4 which drives Th2 the nature of the antigens: virus-derived long peptides response [21]. Thus, adjuvants that activate the inflam- containing both Th and CTL epitopes vs. melanocyte masome, including alum, can induce different types of T self antigen-derived short CTL epitope peptides mixed cell response, depending on tissue- or adjuvant-driven with short Th epitope peptides. Given the clear benefit cytokine context. of CD4+ T cell responses in the generation and intratu- moral function of CD8+ T cells [15, 16], further studies Micro/nano particles are needed to reconcile this discrepancy. Nevertheless, Micro- and nano-particles are attractive antigen/drug de- our preclinical data suggest that prolonged Ag presen- livery systems because they can combine several desired tation (signal 1), even in the presence of signal 2 and 3, characteristics. First, the particles protect their cargo from can induce T cell retention, exhaustion and deletion. serum/tissue peptidases/proteases and other degrading fac- tors, thus increasing the half-life of encapsulated Ag and Aluminum adjuvants immunomodulators in vivo. Second, particles can be engi- Generally referred to as alum, both aluminum hydroxide neered to target specific cell types or organs (such as (Alhydrogel™) and aluminum phosphate (Adjut-phos™) lymph node) [22, 23]. These features help reduce both the adjuvants are widely used in human vaccines such as drug dose and off-target side effect. For example, it has those against influenza, tetanus, diphtheria, pertussis, been shown that Ag encapsulated in poly(lactic-co-glycolic poliomyelitis, and HPV [17]. During vaccine preparation, acid) (PLGA) particles induce similar T cell response with antigens are adsorbed to preformed aluminum adju- a 1000-fold lower dose compared to free Ag [24]. vants, hence their name aluminum-adsorbed vaccines. There are two basic ways to engineer particles for en- Aluminum adjuvants are known to promote Th2 re- hanced uptake by APC. Passive targeting relies on the sponses which make them less suitable for vaccines size, charge and rigidity of the particle while active tar- against intracellular bacteria such as M. tuberculosis, geting is based on added ligands on the particle surface. which require a Th1-type immune response dominated Vaccine particles with size range from 500 to 2000 nm Khong and Overwijk Journal for ImmunoTherapy of Cancer (2016) 4:56 Page 4 of 11 are preferentially trapped by tissue APC at the injection localization, and survival of vaccination-induced, tumor- site (which may then traffic to LN), while 20 to 200 nm specific T cells [10]. However, extremely short antigen particles drain passively to LN where they are taken up presentation (such as after injection of minimal epitope by resident APC. Beside their role as Ag/drug carrier, in- peptides in saline), especially in the absence of adjuvants creasing signal 1, micro and nanoparticles can also en- to induce signals 2 and 3, can likewise lead to subopti- hances signals 2 and 3. PLG and polystyrene particles mal or even abortive/tolerogenic T cell activation. We are thought to participate in inflammasome activation by speculate that in successful, natural immune responses, enhancing the IL-1β secretion by DC in a phagocytosis- such as those against acute viral infections that are rap- dependent manner [25]. Carbon nanotube particles, on idly and completely cleared, the bulk of specific antigen the other hand, activate the complement system and sub- persists for a moderate duration, in the order of a few sequent inflammatory responses via binding to C1q [26]. days [31]. While there is clear evidence that small amounts Materials used to make micro and nanoparticles include of antigens can be retained much longer in APC, the initial liposomes, syntheticpolymerssuch aspolystyrene, large wave of antigen that primes the acutewave of T cell poly(lactide-co-glycolide) PLG, poly(lactic acid) PLA, effectors that follows within days of acute pathogen expos- PLGA or natural polymers such as gelatin, collagen ure is typically gone within a week. By analogy, cancer vac- and chitosan. The choice of material depends on the cines with similar kinetics of antigen availability have the desired biocompatibility, half-life, hydrophobicity and po- best chance of priming a massive wave of tumor-specific larity. For example, liposome particles are very versatile, CTL. Indeed, we have observed such a bell-shaped curve allowing combination of Ag and cytokines like IL-2 or for T cell response and function after different duration of GM-CSF, into a single particle to provide better immune antigen presentation in vivo (Khong et al., manuscript in response and protection [27]. However, major drawbacks preparation). It will be interesting to see whether this is a are the rapid clearing from the blood and accumulation in common principle, and whether this can be harnessed to the liver. Coating a liposome with polyethylene glycol increase the potency and efficacy of peptide-based cancer (PEG) or other biocompatible polymers can reduce rapid vaccines. systemic clearing and thus extend its half-life in vivo [28]. To improve the accumulation of a liposome to tar- The immunopotentiators geted tissue or organ, its surface can be decorated When vaccinologists moved from whole pathogen vac- with receptors (e.g. antibodies) for target cell/tissue cines (live, attenuated or dead pathogens) to recombinant ligands and such modified liposomes are called immuno- subunit vaccines for reasons of safety and manufacturing, liposomes. Micro- and nanoparticles such as hydro- they learned that these vaccines typically evoked weaker philic poly(DL-lactide-co-glycolide) microspheres and immunity and protection. The discovery of how our body poly(propylene sulfide) nanoparticles have been de- senses pathogens via a family of highly conserved pattern signed to target the DC in draining LN [22, 23]. A recognition receptors (PRR) called Toll-like receptors different approach is to attract DC to the site of vaccine (TLR) [32–34] heralded the era of the specific receptor- injection. Recent reports showed that incorporating GM- mediated activation of innate immunity. Since then, other CSF, CpG and tumor antigens in PLG matrices efficiently innate immune receptors have been discovered including attracted and stimulated both conventional DC (CD11c + NOD-like receptors (NLR), C-type lectin receptors and CD11b + and CD11c + CD8a+) and plasmacytoid DC, retinoic acid inducible gene (RIG)-I-like receptors (RLR) resulting in superior immune responses (Th1 and CTL) and most recently cyclic GMP-AMP synthase (cGAS). against B16 melanomas in mice [29, 30]. A very high con- Within the last decades, numerous adjuvants have been centration of GM-CSF (3000 ng) prolonged the DC reten- developed to target these innate receptors. Signaling tion in situ, resulting in suboptimal DC trafficking to mechanisms of these receptors have been thoroughly draining LN and the subsequent inferior T cell priming discussed elsewhere [35–38];herewe focus on thead- and protection against tumor. This observation suggests juvants that target these receptors, in particular those that delivery systems that stimulate the attraction of DC that have entered clinical trials of cancer vaccines. can promote T cell responses, but only if they do not pre- Some notable examples of immunopotentiators and vent the DC from ultimately reaching the LN where T cell their stages of development are listed in Table 2. priming typically occurs. Adjuvants targeting toll-like receptors The antigen depot: what duration of antigen presentation TLR2 agonists is optimal? TLR2 is expressed on the surface of different immune Our preclinical work with IFA as a vaccine adjuvant sug- cells like DC, macrophages and lymphocytes and recog- gests that prolonged antigen presentation has multiple nizes bacterial lipopeptides. Upon engaging its ligands, detrimental effects on the effector function, tumor TLR2 activates NF-kB via the MYD88 signaling pathway. Khong and Overwijk Journal for ImmunoTherapy of Cancer (2016) 4:56 Page 5 of 11 Table 2 Examples of class 2 adjuvants (immunopotentiators) Receptor Target cells Stage of development (not comprehensive) in cancer vaccine “stepping on the gas” Pam CSK TLR2 DC, M$, lymphocytes Preclinical 3 4 Poly-ICLC TLR3 cDC, M$, epithelial cells Several clinical trials for different cancers. MPLA TLR4 cDC, M$, epithelial cells, Clinical trial phase 2 fibroblasts Imiquimod TLR7/8 pDC, B cells, M$, Clinically approved for treating basal cell carcinoma. Multiple clinical trials in combination with monocytes vaccine for different cancers. CpG TLR9 pDc, B cells Multiple clinical trials IL-2 IL-2Ra/ T, B and NK cells Clinically approved for treating renal carcinoma and melanoma. Multiple clinical trials in p/y combination with vaccine for different cancers. GM-CSF GM- many Multiple clinical trials in combination with vaccine and checkpoint blockades for different cancers. CSFR IFNs IFNR many Multiple clinical trials CDNs STING many Preclinical “releasing the brake” a-PD1 Ab PD-1 T, B and NK cells Clinically approved for different cancers. a-CTLA4 Ab CTLA-4 T cells Clinically approved for melanoma, under multiple clinical trials for different cancers. There are two common strategies to engage TLR-2 (IFNs) and the subsequent upregulation of costimulatory through vaccines: conjugating the antigen to bacterial molecules [44]. lipopeptides or to palmitic acid. Bacterial lipopeptide Poly I:C can enhance antigen cross-presentation by MALP-2 and its synthetic analogues like Pam Cys and DC to CD8 T cells. Because of its rapid degradation by Pam Cys are most frequently used. The peptide- serum nucleases in primates, poly I:C has limited anti- lipopeptide construct were shown to induce DC mat- tumor efficacy in humans [39]. Therefore, more stable uration, pro-inflammatory cytokine (IL-12, TNF-α, derivatives of poly I:C were made, including poly ICLC IFN-γ) secretion, B cell activation and enhanced CTL (known as Hiltonol) and poly I:C U [45]. In a phase 1 responses [39]. Most current clinical trials of TLR-2 ovarian cancer trial, addition of poly ICLC to a vaccine based adjuvants are for vaccination against infectious consisting of NY-ESO1 long overlapping peptides in IFA diseases such as HIV, HBV and Lyme disease. In 2014, dramatically induced rapid and efficient CD4 and CD8 T vaccine using TLR-2 ligand (Pam CSK )conjugated cell responses, compared to the vaccine alone [46]. A re- 3 4 with long synthetic peptide showed very promising results cent study in monkeys showed that poly ICLC in com- in a preclinical melanoma model [40]. Interestingly, bination with agonistic CD40 antibody significantly Pam CSK -peptide conjugate, but not the mixture of enhanced both CD4 and CD8 responses compared to 3 4 Pam CSK with peptide, induced robust T cell response either adjuvant alone [47]. This is some of the first pri- 3 4 and protection against tumor. This is in line with the cis- mate data confirming the multitude of mouse studies activation model showed by Desch et al. [41], which essen- that indicated strong synergy when different classes of tially posits that signal 1 and 2 should be delivered by immunopotentiators are used together in vaccine adjuvants same APC for optimal T cell priming. [10, 48, 49]. I:C U and poly ICLC have entered clinical trials for other cancer including glioma, melanoma, carcin- oma (poly ICLC) and HER-2 positive breast cancer [39]. TLR3 agonists TLR3 is expressed in the endosomal compartment of con- ventional dendritic cells (cDC), macrophages and on the TLR4 agonists surface membrane of non-immune cells like epithelial TLR4 is expressed on the surface of immune cells in- cells [42]. TLR3 is activated by double-stranded RNA or cluding cDC and macrophages as well as non-immune its synthetic analog polyinosine-polycytidylic acid (poly cells such as fibroblasts and epithelial cells. Triggering I:C) [43]. TLR3 does not use the MyD88 signaling path- TLR4 will activate both MyD88 and TRIF dependent way but triggers TRIF signaling leading to activation of pathways leading to NF-kB and IRF3/7 activation. TLR4 NF-kB, MAP kinases and IRF3, which in turn induce the activation strongly promotes Th1 response through IL- production of inflammatory cytokines, type 1 interferons 12p70 induction [50]. Due to its high toxicity, LPS has Khong and Overwijk Journal for ImmunoTherapy of Cancer (2016) 4:56 Page 6 of 11 been replaced by the less toxic derivative, monopho- and is a potent pDC activator and IFNα inducer [65]. CpG sphoryl lipid A (MPLA), as vaccine adjuvant. The adju- B only has phosphorothioate backbone. CpG B strongly ac- vanticity of MPLA has been studied extensively in tivates B cells and promotes pDC and monocyte matur- several clinical trials [39]. MPLA is used in combination ation [66]. CpG C is a hybrid of the two above [67]. CpG with aluminum (AS04) to skew the typical Th2 response has been used in clinical trials of therapeutic cancer vac- induced by alum to a Th1 response [51]. MPL as a vac- cines against melanoma, breast/lung/ovarian cancers, sar- cine adjuvant, in combination with tumor antigens, has coma and glioblastoma [68–72]. Overall, the vaccines entered into several clinical trials for melanoma, lung, induced both humoral and cellular responses, but clinical and prostate cancer [52–54]. benefit remained uncommon. TLR7/8 agonists STING agonist Localizing within the endosomal compartments, both In 2006, TLR-independent antiviral responses (i.e. type 1 TLR7 and 8 can recognize single stranded (ss) RNA as interferon induction) were shown to be induced by they are structurally related [42]. In human, TLR7 is pre- double stranded (ds) DNA in the cytosol [73]. Later, dominately expressed in plasmacytoid dendritic cells dsDNA was found to activate the transcription factor (pDC) and to a lesser extent in B cells and monocytes/ NF-kB and IRF3 via an endoplasmic reticulum adaptor macrophages while TLR8 is mainly expressed in mono- called STING (stimulator of interferon genes) [74]. In cytes/macrophages and cDC [55]. TLR7/8 signal through 2013, the receptor for cytosolic DNA, the cylic GMP- the MyD88 pathway leading to upregulation of co- AMP synthase or cGAS, was discovered [75]. Upon stimulatory molecules (CD80/86, CD40), production of binding to cytosolic DNA, cGAS catalyzes the synthesis cytokines (IFN-α, TNF-α, IL-12) and migration of DC of cGAMP which in turns binds to and activates the from skin to lymph nodes. TLR8 is expressed, while adaptor protein STING. Recent results indicate that TLR7 is not, on the important BDCA3+ cDC subset that spontaneous T cell priming against tumor antigen re- is most potently responsible for cross-priming of CD8+ quires STING-dependent type I IFN induction [76]. Very T cells [56], and thus preferential TLR7 agonists may promising results from preclinical studies with STING exert weaker adjuvant activity than TLR8 or TLR7/8 ag- agonists injected directly into tumors in the aggressive onists when used in CD8+ T cell-inducing vaccines. B16 melanoma model had led to high excitement for TLR7/8 can also activate B cells to produce antibody their application in clinical trials [77]. Recent results also and cytokines such as IL-6 and TNF-α, and T cells to indicate that STING agonists can function as adjuvant proliferate and produce cytokines including IFN-γ and in a setting of whole-cell tumor cell vaccine [78]. It will IL-2 [57, 58]. TLR7/8 can be activated by synthetic imi- be interesting to see how STING agonists compare to dazoquinolines including imiquimod (mostly acts on TLR agonists as adjuvants for peptide vaccines in animal TLR7) and resiquimod (TLR7 and 8). Imiquimod models and clinical trials, and whether their combined (Aldara cream) has been approved to treat basal cell car- use offers additional benefit, given their different intra- cinoma and genital warts [59, 60]. Several clinical trials cellular signaling pathways. of imiquimod as vaccine adjuvant in different cancers in- cluding chronic myeloid leukemia (CML), vulval intrae- Cytokines as adjuvants pithelial neoplasia (VIN), prostate cancer and melanoma IL-2 have been conducted [61–64]. Overall, all vaccines in- The most notable cytokine which has been extensively duced both humoral and cellular responses in a major used for immunotherapy is IL-2. IL-2 was initially des- fraction of patients. In vaccinated patients with VIN, cribed as a T cell growth factor (TCGF) responsible for infiltration of both CD4 and CD8 T cells into tumor the clonal expansion, differentiation and survival of T cells sites was shown to correlate with tumor clearance [62]. [79], and later of activated B cells and natural killer (NK) cells as well [80, 81]. Although CD4 T cells are the major TLR9 agonists source of IL-2 in vivo, CD8 T cells, NK cells and DC can TLR9 is expressed by human B cells and pDC and localizes also produce IL-2 [82–85]. IL-2 was FDA-approved for in endo-lysosomal compartment [42]. Its role is to detect the therapy of metastatic renal cell carcinoma in 1992 and unmethylated CpG motifs which are often found in bacter- metastatic melanoma in 1998 [86, 87]. IL-2 mediates anti- ial, but not host cell DNA. Upon activation, TLR9 induces tumor activity by activating tumor-specific T cells and NK production of pro-inflammatory and Th1 cytokines (such cells. In mice, addition of IL-2 to experimental cancer vac- as IL-12) by APC. There are 3 classes of synthetic CpG oli- cines can greatly increase the therapeutic efficacy [10, 48]. gonucleotides (ODN) being used in preclinical and clinical IL-15 signals through the same IL-2 Rβγ complex also studies. CpG A is a mix of phosphodiester/phosphorothio- used by IL-2, and can also promote peptide-induced T cell ate backbone with palindromic sequences and poly G tail, proliferation, especially in T cells with low-affinity TCRs Khong and Overwijk Journal for ImmunoTherapy of Cancer (2016) 4:56 Page 7 of 11 [88]. In patients with melanoma, addition of an experi- however due to the lack of a vaccine arm without GM- mental gp100 peptide/IFA vaccine to IL-2 gave a higher CSF, its exact impact on clinical outcome remains un- clinical response rate than observed in patients receiving known [104, 105]. IL-2 alone, and also higher than previously observed for gp100 peptide vaccine alone, suggesting IL-2 can also Interferons (IFNs) function as a vaccine adjuvant in humans [89]. However, IFNs are of great interest for adjuvant development, IL-2 can also expand immunosuppressive regulatory T owing to their pleiotropic effect on different immune cells cells (Treg) which may dampen the immune response or such as DC, B cells and T cells as well as non-immune anti-tumor efficacy [90]. Because Treg express both IL- cells. IFN-α and IFN-β promote DC maturation, including 2Rα and IL-2Rβγ whileCTL express onlythe latter, the up-regulation of MHC and costimulatory molecules. In blocking IL-2Rα when using IL-2 preferentially expands virus-infected cells, type I IFNs prevent virus replication by CTL [91]. Recently, a mutant form of IL-2 (IL-2 mutein) halting transcriptional and translational machineries, was reported to have higher antitumor efficacy with re- accelerating RNA degradation by inducing RNase L and duced proliferation induction on Treg, possibly thanks to inducing apoptosis [106]. IFN-α and pegylated IFN-α preferential binding to IL-2Rβγ but not IL-2Rα [92]. Simi- have been approved for advanced renal cell carcinoma larly, IL-2 pre-complexed with IL-2-specific antibodies, and chronic hepatitis C treatment, respectively, and and IL-2 covalently modified with polyethylene glycol both are given after surgical resection of primary mel- have shown selective binding to IL-2Rβγ but not IL-2Rα, anoma to reduce the chance of recurrence [107]. Pre- favoring selective effects on CD8+ T cells [93, 94]. If these clinical studies showed direct adjuvant efficacy of type I modifications also lower the toxicity of IL-2, which may IFN in a peptide-based anti-melanoma vaccine, where it be partly mediated by IL-2Rα, these IL-2-based com- promoted T cell numbers, longevity and effector function, pounds may make a comeback in cancer immunotherapy, resulting in improved tumor control [108]. In contrast to including as vaccine adjuvants [91]. type I IFN, IFN-γ (the soletypeIIIFN)istypically only produced by specialized immune cells including T cells, NK cells and NKT cells [109]. Recombinant IFN-γ (or gen- Granulocyte-macrophage colony stimulating factor etically engineered IFN-γ1b) is approved to treat chronic (GM-CSF) granulomatous disease [110]. In cancer immunotherapy, a GM-CSF is a cytokine used as a cancer vaccine adjuvant, phase III clinical trial combining chemotherapy with IFN-γ sometimes with success. GM-CSF can be produced by for patients with advanced ovarian and peritoneal carcino- many cell types including myeloid cells, lymphocytes, mas was terminated due to serious adverse effects [111]. fibroblast, endothelial/epithelial/mesothelial cells and cer- tain tumor cells [95]. The production of GM-CSF is in- Lessons learned from a few successful peptide-based cancer duced by bacterial toxin and inflammatory cytokines such vaccine clinical trials as IL-1, IL-6, and TNF-α [96]. GM-CSF receptor is found A vaccine comprised of long peptide from HPV-16 viral on myeloid cells and non-hematopoietic cells such as oncoproteins E6 and E7 emulsified in IFA was shown to endothelial cells. In vaccine settings, GM-CSF has been be very effective in treating vulvar intraepithelial neopla- shown to initiate the recruitment and maturation of DC sia, a precancerous condition in HPV-16 positive women as well as activation of macrophages, neutrophils, and NK [112]. The overall clinical response was 79 % while cells, indicating that it is a potential vaccine adjuvant [97, complete response was 47 %, after 2 years of follow-up. 98]. Combination of GVAX (irradiated tumor cell express- This remarkable result with an IFA-based peptide vac- ing GM-CSF) with anti-CTLA-4 and anti-PD-1 check- cine was consistent with our findings that long peptides point blockade was very promising in preclinical studies, did not cause severe sequestration of T cells at the leading to the first clinical trials of checkpoint blockade in vaccination site as discussed above. In a phase 3 trial for patients with cancer. Recombinant GM-CSF has been patients with advanced melanoma, combination of IL-2 used in peptide vaccine trials in mouse and man, where it with short gp100 (209–217) peptide emulsified in IFA has had varying success in raising T cell responses. This resulted in a modest but significant improvement of may be partially due to a balance between pro- and anti- overall clinical responses, progression-free survival and inflammatory properties of GM-CSF depending on its overall survival, compared to IL-2 treatment alone [89]. dose [29]. In addition, there appear to be complex interac- Based on some of the preclinical results with IFA dis- tions between GM-CSF and other factors in the tumor- cussed above, a less persistent, and therefore less T cell conditioned microenvironment that influence its abil- sequestering, vaccine formulation might result in more ity to either enhance or reduce vaccine-induced T cell dramatic synergy with IL-2. Indeed, given new insights responses [99–102]. Several positive peptide/protein into the nature of tumor antigens (short vs. long pep- vaccine trials have incorporated GM-CSF [13, 103]; tides, as well as mutated vs. non-mutated antigens) and Khong and Overwijk Journal for ImmunoTherapy of Cancer (2016) 4:56 Page 8 of 11 adjuvants, there is ample opportunity to design new, [118]. We found that adding IL-2 to a TLR7 agonist/CD40 more effective cancer vaccines. A clinical trials in renal agonist combination further enhanced CD8 T cell peak cell cancer with the multiple peptide-based, GM-CSF- effector and memory response, and anti-tumor efficacy adjuvanted, water-formulated IMA091 vaccine showed [10]. Second, some adjuvants may possess both desired that the breadth of CTL response significantly associated and undesired adjuvant properties. By combining with with clinical benefit, perhaps by limiting antigen-loss other adjuvants, the immune response can be skewed to- escape mechanisms [104, 113]. Taken together, results ward favorable one, as in the above-mentioned example of from these clinical studies underscore the importance of alum combined with MPL which is used in HPV vaccine the nature and delivery of target antigens, and the and HBV vaccine to promote Th1 response [119]. A major provision of the right adjuvant. obstacle to successful translation of these long-known pre- clinical findings is the observable paucity of clinical trials Cancer vaccines and T cell checkpoint blockade where multiple pharmaceutical companies combine their While blockade of CTLA-4 and PD-1 T cell checkpoints respective promising, potent agents to create a truly shows strong activity in a variety of cancers, many patients powerful cancer vaccine. This limitation is slowly begin- do not respond, likely due to insufficient spontaneous ning to be addressed by the initiation of co-development anti-tumor T cell immunity (a lack of tumor reactive T agreements between companies, as well as by the develop- cells and/or poor T cell infiltration into the tumor). ment of multiple synergistic adjuvants within single com- Vaccination can enhance tumor-specific immunity, and panies. Thus, clinical trials of cancer vaccines consisting of vaccination is therefore a prime candidate for combination multiple antigens formulated in adjuvants consisting of with checkpoint blockade therapy. Interestingly, the 676- short-lived depots with multiple classes of synergistic patient study that led to FDA approval of anti-CTLA-4 immunostimulatory molecules may finally become a revealed that concurrent vaccination with gp100 peptide reality. vaccine in IFA did not enhance therapeutic efficacy, and in fact modestly but significantly decreased overall res- Adjuvant-free peptide vaccine ponse rate and disease control rate through an unknown There is an emerging new trend of adjuvant-free vaccine mechanism [114]. This has led to uncertainty about that uses self- assembling peptides. Such peptides were whether and how to combine vaccination with checkpoint constructed to have a domain which helps them assemble blockade, hampering efforts to improve overall response into nanofiber structure [120]. Preclinical studies using rates in melanoma and especially in other, less immuno- mouse model showed self-assembling peptides could elicit genic cancers. When modeled in mice, we indeed observe humoral as well as cellular responses [120–122]. The that gp100/IFA vaccination does not synergize with humoral response was shown to be T cell independent, CTLA-4 or PD-1, and that this effect is due to T cell possibly due to cross-liking of repetitive epitopes of nano- entrapment, even of anti-CTLA-4 therapy-induced T fiber peptides to B cell receptors. Yet, mechanisms of how cells, at the gp100/IFA vaccination site. Nevertheless, self-assembling peptides can trigger cellular responses re- by choosing different vaccine formulations, great syn- main undefined. Nevertheless, we anticipate that while ergy between peptide vaccine and checkpoint blockade self-assembling peptide cancer vaccines can possibly by- can be achieved (unpublished results). Other preclinical pass the need for a separate antigen delivery system, they work also indicates synergy between checkpoint blockade will still needs immunopotentiators to optimally activate T and other classes of non-persistent vaccines, opening the cells as well as protecting them from tumor suppressive possibility that vaccines that do not induce excessive T cell mechanisms to ultimately maximize therapeutic vaccine sequestration may combine well with checkpoint blockade efficacy. therapy [78, 99]. Conclusion The need for combining different adjuvants into a single Cancer vaccines are attracting new interest as combin- vaccine ation partners with other immunotherapies, in particular Much preclinical work suggests that combining different T cell checkpoint blockade approaches. A detailed un- adjuvants is needed to induce a strong anti-tumor immune derstanding of the mechanism of action of anti-cancer response [115]. Accumulated evidence has shown that vaccination is critical for the design of potent vaccine CD40 signaling synergizes with almost all TLR ligand indu- approaches that induce robust T cell responses. Vaccine cing far better cellular and humoral responses than that of adjuvants are a major, required component of successful each individual adjuvant [116, 117]. Several groups have vaccines, and several novel adjuvants are now making shown that almost all TLR agonists synergize with CD40 their appearance in the clinic, bridging the wide gap signaling to enhance CTL expansion and function, in part between preclinical and clinical cancer vaccine formula- by inducing the co-stimulatory molecule CD70 on DC tions. This translational effort is further guided by early Khong and Overwijk Journal for ImmunoTherapy of Cancer (2016) 4:56 Page 9 of 11 signs of success in a few clinical trials. The hope is that 14. Phan GQ, et al. 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