CNS Drugs (2018) 32:401–410 https://doi.org/10.1007/s40263-018-0518-4 LEADING ARTICLE Beyond the Magic Bullet: Current Progress of Therapeutic Vaccination in Multiple Sclerosis 1,2 2 Barbara Willekens Nathalie Cools Published online: 14 May 2018 The Author(s) 2018 Abstract Multiple sclerosis (MS) is a chronic immune- vaccination. Failures, successes and future directions are mediated disease of the central nervous system (CNS) discussed. characterized by neuroinﬂammation, neurodegeneration and impaired repair mechanisms that lead to neurological disability. The crux of MS is the patient’s own immune Key Points cells attacking self-antigens in the CNS, namely the myelin sheath that protects nerve cells of the brain and spinal cord. Theoretically, antigen-speciﬁc therapeutic Restoring antigen-speciﬁc tolerance via therapeutic vacci- vaccination is designed to speciﬁcally restore nation is an innovative and exciting approach in MS ther- tolerance to self. In doing so, disease-associated apy. Indeed, leveraging the body’s attempt to prevent pathways are accurately targeted without causing autoimmunity, i.e., tolerization, focuses on the underlying general immunosuppression. cause of the disease and could be the key to solving neu- Several experimental approaches have reached the roinﬂammation. In this perspective, antigen-speciﬁc vac- clinical development phase. Safety and feasibility cination targets only the detrimental and aberrant immune have been demonstrated in several phase I/II trials. response against the speciﬁc disease-associated anti- gen(s) involved while retaining the capacity of the immune It can be envisaged that antigen-speciﬁc therapeutic system to respond to unrelated antigens. We review the vaccination will prove to be highly relevant, experimental approaches of tolerance-inducing vaccination especially early in the disease when epitope in relapsing and progressive forms of MS that have reached spreading has not yet occurred. the clinical development phase, including vaccination with autologous T cells, autologous tolerogenic dendritic cells, T cell receptor peptide vaccination, altered peptide ligand, ATX-MS-1467, cluster of differentiation (CD)-206-tar- geted liposomal myelin basic protein peptides and DNA 1 Introduction Multiple sclerosis (MS) is a chronic inﬂammatory disease of the central nervous system (CNS) driven by immune- & Nathalie Cools mediated damage of the myelin sheath. This results in Nathalie.firstname.lastname@example.org axonal loss and neurodegeneration that lead to neurological Department of Neurology, Antwerp University Hospital, disability [1–3]. In recent years, the continuous develop- 2650 Edegem, Belgium ment of more selective disease-modifying therapies Laboratory of Experimental Hematology, Vaccine and (DMTs) to treat MS has dramatically changed the land- Infectious Disease Institute (VAXINFECTIO), Faculty of scape. In general, DMTs are therapeutic interventions that Medicine and Health Sciences, University of Antwerp, 2610 aim to modulate the underlying pathophysiology of the Wilrijk, Belgium 402 B. Willekens, N. Cools disease to improve the disease course. In MS, this can be 2 Tolerance-Inducing Therapeutics Under achieved by neuroprotective, neurorestorative and/or Investigation immunomodulatory strategies. Here, we focus on the latter. Several immunomodulatory agents have demonstrated 2.1 Peptide-Based Tolerance-Inducing Vaccination beneﬁcial clinical effects in different forms of MS. Nev- ertheless, several issues regarding these treatments remain, To date, induction of in vivo antigen-speciﬁc tolerance by including tolerability problems, compliance and adherence subcutaneous or oral administration of peptides (i.e., pep- difﬁculties, and potentially severe treatment-related side tide vaccination) has proven to be a well-tolerated and effects such as opportunistic infections, secondary successful therapy for allergies [16–18]. Given the success autoimmunity and an increased risk of malignancies [4, 5]. in allergies, the possibility of treating MS with peptide While more than ten marketed drugs are currently available vaccination is being investigated. Some of the most that have shown variable efﬁcacy in the treatment of promising results were described by Jurynczyk et al.  relapsing–remitting MS (RRMS), there remains a signiﬁ- and Walczak et al.  in two phase I/II studies in which the cant and unmet need for safer and highly efﬁcacious investigators transdermally applied peptides derived from treatments that are well tolerated. The need for treatments MOG, MBP and PLP. They demonstrated induction of that can stop or slow progression or improve disability in immunological tolerance by activation of Langerhans cells progressive forms of MS is even higher; to date, only one and subsequent induction of interleukin (IL)-10-secreting T drug has been approved for the treatment of primary pro- cells . Moreover, the immunological effect was clini- gressive MS (PPMS). cally translated in a placebo-controlled trial that demon- Given this, more selective immunotherapies designed to strated a signiﬁcant reduction in annualized relapse rate restore self-tolerance, thereby reinstating the immune bal- and magnetic resonance imaging (MRI)-deﬁned measure- ance without causing general immune suppression, may ments of the disease . Interestingly, lower peptide con- hold promise for the treatment of autoimmunity, including centrations following intramuscular  and transdermal MS. Since, theoretically, antigen-speciﬁc therapies com-  administration achieved an even better clinical out- bine maximal efﬁcacy with minimal side effects, these come, underscoring the importance of dosage to achieve strategies are especially appealing [6, 7]. Nevertheless, for tolerance. Nevertheless, whereas peptide vaccination is many autoimmune diseases, the primary target antigen known to induce tolerance in steady-state conditions, remains to be identiﬁed. Also in MS, the target antigen(s) is unexpected adverse events can be anticipated following (are) not known, although proteins within the myelin administration in a pro-inﬂammatory environment. Indeed, sheath, such as myelin basic protein (MBP), myelin three patients in a phase II clinical trial investigating vac- oligodendrocyte glycoprotein (MOG) and proteolipid pro- cination with a MBP-derived APL, in which amino-acid tein (PLP), are important targets of the autoreactive substitutions were incorporated at T-cell receptor (TCR) immune response [4, 8–11]. Hence, attempts to induce contact positions, demonstrated disease exacerbations fol- antigen-speciﬁc tolerance in MS include oral administra- lowing treatment. A clear association with the vaccination tion of myelin proteins, intravenous injection of MBP  strategy was demonstrated in two of the patients, even after or altered peptide ligand (APL) , transdermal [6, 7]or the dose was lowered, and the trial was halted [8, 19]. intradermal  administration of myelin-derived peptides, Alternatively, soluble synthetic peptides were designed and intramuscular injection of plasmids expressing MBP to mimic the naturally processed epitopes. These so-called . Cell-based vaccination strategies, such as T cells, apitopes induce antigen-speciﬁc expansion of regulatory T apoptotic lymphocytes covalently bound with multiple cells, capable of ‘‘switching-off’’ pathogenic T cells, which peptides from different myelin-derived proteins [14, 15], or produce pro-inﬂammatory cytokines and are responsible tolerance-inducing dendritic cells (DCs), have also been for myelin damage in the CNS. In this context, two clinical pursued and appear to reﬂect more selective therapies for trials have recently completed evaluation of the safety and MS. biological disease parameters of ATX-MS-1467, a mixture We review the experimental approaches of tolerance- of four short peptides derived from MBP, i.e., ATX-MS1 inducing vaccination in relapsing and progressive forms of (MBP ), ATX-MS4 (MBP ), ATX-MS6 (MBP ), 30-44 131-145 140-154 MS that have reached the clinical development phase and ATX-MS7 (MBP ). ATX-MS-1467 is administered 83-99 (Table 1) and discuss failures, successes and future intradermally every 2 weeks for 20 weeks. Patients ini- directions. tially receive a dose titration of 50 and 200 lg for 4 weeks, then a dose of 800 lg every 2 weeks for 16 weeks. A phase I open-label dose-escalating study demonstrated that ATX- MS-1467 was safe and well-tolerated in a group of six Tolerance-Inducing Vaccination in MS 403 Table 1 Overview of tolerance-inducing therapeutic approaches that entered the clinic for treatment of multiple sclerosis Vaccination strategy Developmental Administration Clinical outcomes Mode of action References progress route Peptide-based tolerance-inducing vaccination MOG, MBP, PLP Phase I/II Transdermal Reduction in ARR; reduction in Activation of Langerhans [6, 7] peptides MRI measurement of disease cells; generation of IL-10- secreting cells Altered peptide Halted Subcutaneous 62% increase in number of  ligand active lesions; two pts demonstrated disease exacerbations associated with vaccination strategy Apitopes (ATX-MS- Phase IIa Intradermal Safe; 79% decrease in new Gd- Expansion of Treg [20, 21] 1467) enhancing lesions Mannosylated Phase I Subcutaneous Safe Decrease of CCL2, CCL4, [22, 23] liposomes IL-7 and IL-2 at study containing MBP completion peptides TCR peptide Phase I Both Safe Generation of IL-1-secreting [25–31] vaccination intradermal TCR peptide-speciﬁc T (Neurovax) and cells; reduction of MBP- intramuscular speciﬁc T cells DNA vaccination MBP-encoding DNA Phase II Intramuscular Safe and well tolerated; Reduction of IFN-c- [13, 34] vaccine reduction in number of active producing myelin-reactive lesions; decrease in clinical T cells; decrease of myelin- relapse rate speciﬁc auto-antibody titers in the CSF Cell-based tolerance-inducing vaccination Irradiated autologous Phase I Subcutaneous Safe and feasible; 40% Generation of a cytotoxic  T cells reduction in relapse rate; T-cell response against stabilization of disease myelin-reactive cells; progression and lesion depletion of myelin- activity on MRI reactive T cells Mixture of Phase IIb Subcutaneous Clinical endpoints not met , attenuated myelin- (details not published) NCT01684761 reactive T cells (Tcelna) Autologous PBMC Phase I Intravenous Safe and feasible; stabilization Decrease in myelin-speciﬁc  chemically coupled of clinical and MRI T-cell reactivity in pts with a mixture of parameters of disease activity receiving highest dose of myelin-derived at study completion cells ([ 1910 ) peptides tolDC pulsed with Phase I Intradermal Ongoing Ongoing NCT02618902 myelin-derived peptides tolDC pulsed with Phase I Intranodal Ongoing Ongoing NCT02903537 myelin-derived peptides tolDC pulsed with Phase I Intravenous Ongoing Ongoing NCT02283671 myelin-derived peptides ARR annualized relapse rate, CSF cerebrospinal ﬂuid, Gd gadolinium, IFN interferon, IL interleukin, MBP myelin basic protein, MOG myelin oligodendrocyte glycoprotein, MRI magnetic resonance imaging, PBMC peripheral blood mononuclear cells, PLP proteolipid protein, pts patients, TCR T-cell receptor, tolDC tolerogenic dendritic cells, Treg regulatory T cell 404 B. Willekens, N. Cools patients with secondary-progressive MS (SPMS), up to a investigated the administration of incremental doses of dose of 800 lg. A recent multicenter, open-label, TCR Vb5.2 and Vb6.1 peptides. Intradermal injection of single-arm, baseline-controlled phase IIa clinical trial synthetic TCR Vb5.2 peptides resulted in clinical (NCT01973491) evaluated the clinical and biological improvement paralleled by beneﬁcial immunological effects of ATX-MS-1467 in 19 patients with relapsing MS effects, such as the generation of TCR peptide-speciﬁc T (RMS). No treatment-related serious adverse events were cells and reduction of MBP-speciﬁc T cells, in a double- observed, and the adverse event proﬁle was mild, with \ blind placebo-controlled trial in 22 patients with progres- 50% of patients experiencing local injection site reactions. sive MS . Repeated intramuscular injections of Although there was no placebo group with which to TCRVb6 peptide also resulted in immunoregulatory compare results, a review of MRI data showed that treat- effects, warranting further exploration of this approach in ment with ATX-MS-1467 led to a 78% decrease in new T1 the treatment of MS . Administration of both peptides Gadolinium-enhancing lesions as compared with baseline was safe and did not worsen the disease course following . both administration routes . Moreover, a peptide- To engage T cells speciﬁc for the naturally processed speciﬁc immune response was induced in 50–60% of antigen and to serve as a tolerogen, peptides must reach the patients with MS following intradermal injection of TCR resident antigen-presenting cells in vivo. This process can Vb5.2 peptides, whereas 90% of patients with MS be facilitated by targeting speciﬁc markers expressed on demonstrated measurable T-cell immunity towards the the surface of antigen-presenting cells. For instance, the Vb6 peptides upon intramuscular injection in inactivated mannose receptor cluster of differentiation (CD)-206 is a Freund’s adjuvant (IFA). For this, it was hypothesized that C-type lectin primarily present on the membrane of mac- a vaccine consisting of three TCR peptides (BV5S2, rophages and immature DCs. In this context, encapsulation BV6S5, and BV13S1) emulsiﬁed in IFA would be more of selected immunodominant MBP peptides into manno- immunogenic than the three peptides in saline alone. The sylated liposomes signiﬁcantly enhanced the uptake of the trivalent peptide TCR vaccine, now called Neurovax (Im- peptides by DCs via the CD206 receptor. This resulted in mune Response BioPharma, Atlantic City, NJ, USA), was immune tolerance towards the myelin-derived antigens. investigated in several clinical trials and found to be safe CD206-targeted liposomal delivery of co-encapsulated and to induce a surge of proliferating IL-1-secreting TCR immunodominant MBP sequences MBP , MBP peptide-speciﬁc T cells [28–31]. A phase IIb study in 46–62 124–139 TM and MBP (Xemys , JSC Pharmsynthez, Moscow, patients with SPMS (clinical trials.gov identiﬁer 147–170 Russia) was investigated in a phase I, multicenter, open- NCT02057159) to investigate the efﬁcacy and safety of the label, dose-escalating safety and proof-of-concept study in vaccine is yet to start. patients with RRMS or SPMS with relapses for whom ﬁrst- line DMTs had failed. Patients received six weekly sub- 2.2 DNA Vaccination cutaneous injections with incremental doses from 50 to 900 lg. After the last injection, patients were followed-up BHT-3009 is a DNA vaccine that is made of genetically for 12 weeks. No dose-limiting toxicities were observed engineered DNA that encodes the full-length human during treatment. Local injection site reactions were the MBP [32, 33]. The plasmid backbone has been modiﬁed in most common adverse event . Interestingly, a statisti- such a way that it could lead to favorable immunological cally signiﬁcant decrease compared with baseline was changes in patients with MS (reduction in the number of observed in serum CCL2, CCL4, IL-7, and IL-2 levels at immunostimulatory CpG motifs and increase in the number study completion (week 18) . of immunoinhibitory GpG motifs). Its purpose is to restore A completely different approach is effectuated by TCR tolerance to self, leaving protective immunity against peptide vaccination. Hereto, short amino acid sequences infectious and tumor antigens intact. BHT-3009 was ﬁrst derived from the TCR of pathogenic T cell clones are investigated in a randomized placebo-controlled phase I/II administered in an attempt to induce T-cell-mediated trial in patients with RRMS or SPMS and was shown to be immunoregulation directed at T cells expressing those safe and well tolerated. Moreover, a reduction in contrast- TCRs. The repertoire of TCR peptide-reactive T cells is enhancing lesions on MRI was accompanied by reduced positively selected in the thymus after depletion of nega- proliferation of interferon-c-producing myelin-reactive T tively selected clonotypes, and it has been hypothesized cells and decreased titers of myelin-speciﬁc autoantibodies that TCR-speciﬁc T cells might represent a subset of the in the cerebrospinal ﬂuid . Next, a phase II randomized naturally induced regulatory T cells. In patients with MS, placebo-controlled trial comparing two doses of BHT-3009 the Vb repertoire of activated T cells has been reported to was conducted in 289 patients with RRMS. Remarkably, be derived predominantly from the Vb5.2 and Vb6.1 the high dose of 1.5 mg was ineffective, but the low dose families . Hence, several clinical trials have of 0.5 mg showed a trend towards a 50–61% decrease in Tolerance-Inducing Vaccination in MS 405 the number of new enhancing lesions as compared with identiﬁer: NCT01684761) following previous selection of placebo (p = 0.07). In addition, a profound reduction in the appropriate dose regimen . Patients with SPMS myelin-speciﬁc auto-antibody titers was seen, indicative of (n = 183) who presented T-cell reactivity against at least induction of antigen-speciﬁc immune tolerance. Never- one of the myelin-derived peptides used received two theless, no beneﬁcial effects on disease course were vaccination cycles of ﬁve subcutaneous injections with observed , and whether the vaccine will enter phase III Tcelna per year . Nevertheless, Tcelna did not meet clinical trials remains to be seen. its primary or secondary endpoints, i.e. reduction in brain volume change and reduction in the rate of sustained dis- 2.3 Cell-Based Tolerance-Inducing Vaccination ease progression, respectively. However, the promising results observed in another, albeit small, placebo-con- 2.3.1 T-Cell Vaccination trolled clinical trial in 26 patients with relapsing-progres- sive MS  may underscore the importance of careful Autologous T-cell vaccination has been suggested to patient selection and clinical trial design. deplete or regulate the pathogenic myelin-reactive T cells that maintain autoimmune processes within the CNS of 2.3.2 Autologous Leukocytes Chemically Coupled patients with MS [35, 36]. The vaccine consists of a with Multiple Myelin-Derived Peptides patient’s own myelin-speciﬁc T cells isolated from peripheral blood that are inactivated by irradiation. Fol- To simultaneously target autoreactive T cells speciﬁc for lowing administration of the autologous T-cell vaccine, an multiple myelin epitopes, a mixture of myelin-derived immune response is elicited to eliminate other pathogenic peptides could be used. Bielekova et al.  previously T cells in the circulation of the patient without affecting the identiﬁed six myelin-derived peptides (MBP , 13–32 rest of the immune system . Stinissen et al.  and MBP , MBP , PLP ,MOG , and 111–129 154–170 139–154 1–20 others [39, 40] demonstrated that, apart from local reac- MOG ) that were immunodominant for high-avidity T 35–55 tions due to injection of the product, autologous T-cell cells and could discriminate between patients with MS and vaccination was safe and feasible in patients with MS. healthy controls. A seventh immunodominant peptide, Administration of irradiated, autologous MBP-speciﬁc MBP , was identiﬁed in several other studies [10, 48], ? 83–99 T-cell clones resulted in the induction of a cytotoxic CD8 including a phase IIa clinical trial testing an APL of T-cell response directed against the MBP-reactive T cells MBP in which worsening of brain MRI activity and 83–99 used for vaccination. Consequently, circulating MBP-re- MS disease was observed . Grau-Lo´pez et al.  active T cells were also recognized and destroyed in conﬁrmed the relevance of this cocktail of myelin peptides patients with MS receiving the autologous T-cell vaccine in MS pathogenesis, demonstrating a positive T-cell pro- . Furthermore, the depletion of MBP-reactive T cells liferative response to this peptide mix in 74% of patients following three consecutive injections at 6- to 8-week with RRMS compared with 30% of healthy controls. Lut- intervals with autologous T-cell vaccination correlated terotti et al.  demonstrated the feasibility and safety of with a 40% reduction in relapse rate over a period of this selected pool of peptides in an antigen-speciﬁc and 12–24 months after the ﬁrst injection as compared with cell-based tolerization approach in vivo. They performed a baseline. In addition, disease progression stabilized, dose-escalation study in nine patients with MS receiving a including lesion activity on MRI [41, 42]. Nevertheless, single infusion of autologous peripheral blood mononu- acceleration in progression rate 12 months after the last clear cells pulsed with these seven myelin-derived peptides injection suggested a reduced efﬁcacy over time of autol- and chemically ﬁxed with the cross linker 1-ethyl-3-(3- ogous T-cell vaccination, necessitating repetitive injections dimethylaminopropyl)-carbodiimide (EDC). The authors . Indeed, reappearing myelin-reactive T-cell clones concluded that the antigen-coupled cells were well toler- could be effectively depleted by additional vaccinations ated and had a favorable safety proﬁle . A multicenter . phase IIa trial assessing efﬁcacy and safety in patients with It was hypothesized that the immune potential of early RRMS is currently in preparation. autologous T-cell vaccination could be increased by using more than one myelin-derived peptide for T-cell selection 2.3.3 Tolerogenic Dendritic Cell Vaccination , so a T-cell vaccine consisting of attenuated myelin- reactive T cells (MRTCs) selected with multiple peptides DCs, professional antigen-presenting cells of the innate derived from MBP, PLP, and MOG was developed. This immune system, fulﬁl a central role in the polarization of vaccine, Tcelna (imilecleucel-T, formerly known as naive T cells into different effector T cells. In doing so, Tovaxin ), was evaluated in a randomized, double-blind, DCs are of key importance in keeping the balance between placebo-controlled phase IIb study (clinicaltrials.gov 406 B. Willekens, N. Cools immunity and tolerance, as reviewed extensively by Van 3 Discussion Brussel et al. . Several mechanisms by which DCs maintain peripheral tolerance have been delineated. Indeed, To enter a new era for the development of novel MS steady-state and tolerance-inducing or tolerogenic DCs treatment strategies, speciﬁc targeting of only those path- (tolDCs) display reduced expression levels of costimula- ways that contribute to the disease pathogenesis should be tory markers, resulting in T-cell anergy or deletion. In aimed for. As outlined, much effort has been put into addition, tolDCs express so-called negative membrane- precisely silencing only those immune responses that are bound costimulatory molecules, such as immunoglobulin- deleterious in the disease. So far, results from initial trials like transcript (ILT)-3 and programmed death ligand (PD- involving the induction of antigen-speciﬁc tolerance have L)-1, and immunosuppressive soluble factors such as IL- been promising, albeit mainly in patients with RRMS. 10, that can induce and/or expand regulatory T cells, Furthermore, in MS, a wide spectrum of myelin-derived thereby initiating a process called ‘‘infectious tolerance’’ antigens is targeted by a large diversity of T and B cells. In [50–52]. These specialized features of DCs have driven the addition, the progression of MS and the occurrence of development of DC-based therapies to generate antigen- relapses are suggested to be associated with epitope speciﬁc tolerance, restoring the immunological imbalance spreading, a process characterized by loss of tolerance in autoimmune disorders, including MS. against endogenous antigens released during an inﬂam- To date, several biological and pharmacological agents matory or auto-immune exacerbation. Hence, although it have been evaluated to generate tolDCs in vitro. We  seems logical to pursue antigen-speciﬁc tolerance early in and others [54–56] have shown that in vitro treatment of the disease when epitope spreading is limited, it should be monocyte-derived DCs with anti-inﬂammatory biologicals, noted that tolerance-inducing vaccination strategies can including vitamin D , resulted in a maturation-resistant induce so-called infectious tolerance (Fig. 1). Indeed, fol- phenotype of DCs from both healthy controls and patients lowing treatment with BHT-3009, a DNA vaccine encod- with MS. Vitamin D -treated tolDCs induced myelin- ing full-length MBP, the induction of immune tolerance speciﬁc T-cell hyporesponsiveness, whereas the tolDC- that extended beyond MBP to other myelin-derived anti- stimulated T cells retained their capacity to respond to an gens, such as PLP, MOG, and ab-crystallin was observed unrelated antigen. This hyporesponsiveness was robust, as . Several other tolerance-inducing vaccination strate- T cells were not reactivated after rechallenge with gies aim to counteract epitope spreading by including immunostimulatory DCs . Furthermore, treatment of multiple myelin-derived epitopes, thereby targeting mye- experimental autoimmune encephalomyelitis (EAE), the lin-reactive T cells with multiple speciﬁcities. Nonetheless, animal model of MS, with MOG -pulsed bone marrow- numerous questions remain, including dose, route, and 40–55 derived vitamin D -treated tolDCs signiﬁcantly reduced frequency of administration, before tolerance-inducing EAE incidence when administered preventively and vaccination strategies become widely available to a vast resulted in clinical improvement when applied after EAE range of patients. induction [57, 58]. Of note, repeated injections with Although the optimal dose for tolerance-inducing ther- MOG -pulsed tolDCs were necessary to maintain the apeutic vaccination has yet to be determined, it has been 40–55 favorable effect on the disease course. shown that lower peptide concentrations following intra- Four phase I studies investigating the safety and feasi- muscular  as well as transdermal  administration bility of tolDC therapy for autoimmune diseases were achieved a better clinical outcome than higher doses for the completed recently [59–64]. Overall, these clinical studies induction of tolerance. Likewise, in an animal model of revealed that tolDC therapy was well tolerated and safe in rheumatoid arthritis, it was shown that the disease score the patient populations investigated. No discernible adverse ameliorated in mice receiving lower doses of tolDCs but events or toxicities were demonstrated in these studies. worsened in mice receiving higher doses . In contrast, Hence, these reassuring results open the way for larger Lutterotti et al.  demonstrated a reduction in myelin- studies investigating efﬁcacy as well as for implementation speciﬁc T-cell reactivity only when the highest dose of of the use of tolDCs in other autoimmune diseases, cells chemically coupled with a mixture of myelin-derived including MS. To date, three open-label, single-center, peptides was used. However, less is known about the phase I clinical trials evaluating the safety and tolerability minimal dose necessary for a therapeutic effect. Addi- of myelin-derived peptide-pulsed tolDCs administered tionally, to ensure that the ability to regulate the autoim- intradermally, intranodally, or intravenously are ongoing mune response is permanent or at least lasts for years (clinicaltrials.gov identiﬁers NCT02618902, following intervention, a number of repetitive injections NCT02903537, and NCT02283671). with the tolerance-inducing agent may be required. In particular, for cell-based tolerance-inducing strategies, Tolerance-Inducing Vaccination in MS 407 Fig. 1 Possible modes of action of tolerance-inducing therapeutic antigens, such as peptides, apitopes, or encoded by a DNA vaccine, approaches in multiple sclerosis (MS). (1) Although the exact cause are engulfed, processed, and presented by antigen-presenting cells, of MS remains unknown, proteins within the axon-surrounding including Langerhans cells and DCs. (3) Presentation of myelin- myelin sheath, such as myelin oligodendrocyte protein (MOG), derived antigen by DCs in the absence of costimulatory molecules, myelin basic protein (MBP), and proteolipid protein (PLP), are may result in the deletion of myelin-reactive T cells. (4) In addition, important targets of the autoreactive immune response. Furthermore, tolerance-inducing therapeutic approaches can induce so-called the progression of MS and the occurrence of relapses are associated infectious tolerance by antigen-speciﬁc expansion of regulatory T with ‘‘epitope spreading,’’ a process characterized by loss of tolerance cells (Treg) and are capable of counteracting epitope spreading against endogenous antigens released during an inﬂammatory or auto- (Adapted from Neuron Hand-tuned by Quasar Jarosz/CC BY-SA 3.0) immune exacerbation. (2) Following administration, myelin-derived ready-to-use aliquots for clinical applications using cry- therapeutic agent to the draining lymph node or from opreservation is needed. We recently demonstrated that, where cell-based tolerance-inducing treatments can following a freeze–thaw cycle, tolDCs maintained their directly ﬁnd a way to the draining lymph node. It has phenotypic and functional properties as compared with been shown that migration towards lymph nodes is much freshly prepared DCs . These ﬁndings support and lower after subcutaneous injection than after intradermal facilitate the widely applicable clinical use of cell-based injection, whereas the migration of intravenously injected tolerance-inducing vaccination. cells has so far not been monitored in humans [66–68]. When considering the route of delivery of tolerance- Nevertheless, in vivo studies in patients with cancer have inducing therapeutic vaccination, one should consider that shown that, after intradermal injection, only 2–4% of the different routes lead to different sites of accumulation of DCs migrate to draining lymph nodes . Our recent the administered product, whereas for the effective ﬁndings demonstrate that the migratory capacity of these induction of tolerance it is necessary to interact with cells could be optimized by introducing messenger RNA autoreactive T cells, which mainly takes place in the (mRNA) encoding chemokine receptors. In doing so, we lymph nodes. However, direct injection into the lymph were able to endow tolDCs with the capacity to migrate node is technically very difﬁcult and could damage the through the blood–brain barrier by introducing de novo lymph node structure. Moreover, this tissue damage could C–C chemokine receptor (CCR)-5 protein expression. evoke an undesired pro-inﬂammatory microenvironment. Active shuttling of cells across the blood–brain barrier For this reason, most tolerance-inducing products are would allow for targeted in situ down-modulation of administered in the skin comprising an armamentarium of autoimmune responses in MS . immune-competent cells capable of shuttling the 408 B. Willekens, N. Cools References 4 Conclusion 1. Dendrou CA, Fugger L, Friese MA. Immunopathology of mul- Although current DMTs have demonstrated clear efﬁcacy, tiple sclerosis. Nat Rev Immunol. 2015;15(9):545–58. they come with signiﬁcant, sometimes life-threatening, 2. Grigoriadis N, van Pesch V. A basic overview of multiple scle- side effects. Furthermore, current therapies generally delay rosis immunopathology. Eur J Neurol. 2015;22(Suppl 2):3–13. but do not prevent disease progression, which means that 3. Nuyts AH, Lee WP, Bashir-Dar R, Berneman ZN, Cools N. Dendritic cells in multiple sclerosis: key players in the many patients will still develop progressive MS at some immunopathogenesis, key players for new cellular immunother- point. It can be envisaged that the development of new apies? Multiple sclerosis (Houndmills, Basingstoke, England). antigen-speciﬁc immunomodulatory strategies, as outlined 2013;19(8):995–1002. 4. Elong Ngono A, Lepetit M, Reindl M, Garcia A, Guillot F, Genty here, will prove to be highly relevant, especially early in A, et al. Decreased frequency of circulating myelin oligoden- the disease when epitope spreading has not yet occurred. drocyte glycoprotein B lymphocytes in patients with relapsing- Several phase I/II trials have demonstrated the safety and remitting multiple sclerosis. J Immunol Res. 2015;2015:673503. feasibility of therapeutic vaccination strategies for MS. 5. Freedman MS, Bar-Or A, Oger J, Traboulsee A, Patry D, Young However, while German Nobel Laureate Paul Ehrlich C, et al. A phase III study evaluating the efﬁcacy and safety of MBP8298 in secondary progressive MS. Neurology. imagined an ideal therapy for disease as far back as the 2011;77(16):1551–60. early 1900s, a magic bullet precisely targeted to an inva- 6. Jurynczyk M, Walczak A, Jurewicz A, Jesionek-Kupnicka D, der—a one-size-ﬁts-all approach—might not deliver the Szczepanik M, Selmaj K. Immune regulation of multiple sclerosis solution because of the high patient-to-patient variability in by transdermally applied myelin peptides. Ann Neurol. 2010;68(5):593–601. antigen reactivity . To date, this can be circumvented 7. Walczak A, Siger M, Ciach A, Szczepanik M, Selmaj K. by recruiting only patients who demonstrate reactivity Transdermal application of myelin peptides in multiple sclerosis towards the myelin-derived antigens of interest in the treatment. JAMA neurology. 2013;70(9):1105–9. respective therapeutic vaccination strategies, although the 8. Bielekova B, Goodwin B, Richert N, Cortese I, Kondo T, Afshar G, et al. Encephalitogenic potential of the myelin basic protein ultimate dream would be to have an auto-antigen blueprint peptide (amino acids 83–99) in multiple sclerosis: results of a for each patient, for which a personalized vaccine could be phase II clinical trial with an altered peptide ligand. Nat Med. tailor-made. Until then, future strategies should aim at 2000;6(10):1167–75. inducing tolerance to several myelin-derived antigens early 9. Bielekova B, Sung MH, Kadom N, Simon R, McFarland H, Martin R. Expansion and functional relevance of high-avidity in the disease when little to no epitope spreading has myelin-speciﬁc CD4 ? T cells in multiple sclerosis. Journal of occurred. immunology (Baltimore, Md: 1950). 2004;172(6):3893–904. 10. Wallstrom E, Khademi M, Andersson M, Weissert R, Linington Compliance with Ethical Standards C, Olsson T. Increased reactivity to myelin oligodendrocyte glycoprotein peptides and epitope mapping in HLA Funding Open access publication is funded by an article processing DR2(15) ? multiple sclerosis. Eur J Immunol. charge paid by the IWT-TBM140191 grant. This work was supported 1998;28(10):3329–35. by the Methusalem Funding Program from the University of Antwerp, 11. Grau-Lopez L, Raich D, Ramo-Tello C, Naranjo-Gomez M, by an applied biomedical research project of the Institute for the Davalos A, Pujol-Borrell R, et al. Speciﬁc T-cell proliferation to Promotion of Innovation by Science and Technology in Flanders myelin peptides in relapsing-remitting multiple sclerosis. Eur J (IWT-TBM 140191), and by the Belgian Charcot Foundation. Fur- Neurol. 2011;18(8):1101–4. thermore, the authors received funding from the European Union’s 12. Wraith DC. Therapeutic peptide vaccines for treatment of Horizon 2020 research and innovation program under grant agree- autoimmune diseases. Immunol Lett. 2009;122(2):134–6. ment. Dr. Willekens is a neurologist at the Antwerp University 13. Garren H, Robinson WH, Krasulova E, Havrdova E, Nadj C, Hospital supported by a research fellowship (2016–2018) of the Selmaj K, et al. Phase 2 trial of a DNA vaccine encoding myelin University of Antwerp to work on this project. basic protein for multiple sclerosis. Ann Neurol. 2008;63(5):611–20. Conﬂicts of interest Barbara Willekens and Nathalie Cools have no 14. Lutterotti A, Sospedra M, Martin R. Antigen-speciﬁc therapies in conﬂicts of interest. MS—current concepts and novel approaches. J Neurol Sci. 2008;274(1–2):18–22. Open Access This article is distributed under the terms of the 15. Turley DM, Miller SD. Prospects for antigen-speciﬁc tolerance Creative Commons Attribution-NonCommercial 4.0 International based therapies for the treatment of multiple sclerosis. Results License (http://creativecommons.org/licenses/by-nc/4.0/), which per- Probl Cell Differ. 2010;51:217–35. mits any noncommercial use, distribution, and reproduction in any 16. Akdis CA, Akdis M, Blesken T, Wymann D, Alkan SS, Muller U, medium, provided you give appropriate credit to the original et al. Epitope-speciﬁc T cell tolerance to phospholipase A2 in bee author(s) and the source, provide a link to the Creative Commons venom immunotherapy and recovery by IL-2 and IL-15 in vitro. license, and indicate if changes were made. J Clin Investig. 1996;98(7):1676–83. 17. Skripak JM, Nash SD, Rowley H, Brereton NH, Oh S, Hamilton RG, et al. A randomized, double-blind, placebo-controlled study of milk oral immunotherapy for cow’s milk allergy. J Allergy Clin Immunol. 2008;122(6):1154–60. Tolerance-Inducing Vaccination in MS 409 18. Jones SM, Pons L, Roberts JL, Scurlock AM, Perry TT, Kulis M, 32. Fissolo N, Montalban X, Comabella M. DNA-based vaccines for et al. Clinical efﬁcacy and immune regulation with peanut oral multiple sclerosis: current status and future directions. Clin immunotherapy. The Journal of allergy and clinical immunology. Immunol. 2012;142(1):76–83. 2009;124(2):292–300, e1–e97. 33. Stuve O, Cravens PD, Eagar TN. DNA-based vaccines: the future 19. Kappos L, Comi G, Panitch H, Oger J, Antel J, Conlon P, et al. of multiple sclerosis therapy? Expert Rev Neurother. Induction of a non-encephalitogenic type 2 T helper-cell 2008;8(3):351–60. autoimmune response in multiple sclerosis after administration of 34. Bar-Or A, Vollmer T, Antel J, Arnold DL, Bodner CA, Cam- an altered peptide ligand in a placebo-controlled, randomized pagnolo D, et al. Induction of antigen-speciﬁc tolerance in mul- phase II trial. The Altered Peptide Ligand in Relapsing MS Study tiple sclerosis after immunization with DNA encoding myelin Group. Nat Med. 2000;6(10):1176–82. basic protein in a randomized, placebo-controlled phase 1/2 trial. 20. Streeter HB, Rigden R, Martin KF, Scolding NJ, Wraith DC. Arch Neurol. 2007;64(10):1407–15. Preclinical development and ﬁrst-in-human study of ATX-MS- 35. Hellings N, Raus J, Stinissen P. T-cell-based immunotherapy in 1467 for immunotherapy of MS. Neurology(R) Neuroimmunol multiple sclerosis: induction of regulatory immune networks by Neuroinﬂamm. 2015;2(3):e93. T-cell vaccination. Expert Rev Clin Immunol. 2006;2(5):705–16. 21. Chataway J, Martin K, Barrell K, Sharrack B, Stolt P, Wraith DC. 36. Vandenbark AA, Abulaﬁa-Lapid R. Autologous T-cell vaccina- Effects of ATX-MS-1467 immunotherapy over 16 weeks in tion for multiple sclerosis: a perspective on progress. BioDrugs relapsing multiple sclerosis. Neurology. 2018;90(11):e955–62. Clin Immunother Biopharm Gene Ther. 2008;22(4):265–73. 22. Belogurov A Jr, Zakharov K, Lomakin Y, Surkov K, Avtushenko 37. Achiron A, Mandel M. T-cell vaccination in multiple sclerosis. S, Kruglyakov P, et al. CD206-targeted liposomal myelin basic Autoimmun Rev. 2004;3(1):25–32. protein peptides in patients with multiple sclerosis resistant to 38. Stinissen P, Medaer R, Raus J. Preliminary data of an extended ﬁrst-line disease-modifying therapies: a ﬁrst-in-human, proof-of- open label phase I study of T cell vaccination in multiple scle- concept dose-escalation study. Neurotherapeutics. rosis. J Neuroimmunol 90(1):99. 2016;13(4):895–904. 39. Zhang J, Raus J. T cell vaccination in multiple sclerosis: hopes 23. Lomakin Y, Belogurov A Jr, Glagoleva I, Stepanov A, Zakharov and facts. Acta Neurol Belg. 1994;94(2):112–5. K, Okunola J, et al. Administration of myelin basic protein 40. Medaer R, Stinissen P, Truyen L, Raus J, Zhang J. Depletion of peptides encapsulated in mannosylated liposomes normalizes myelin-basic-protein autoreactive T cells by T-cell vaccination: level of serum TNF-alpha and IL-2 and chemoattractants CCL2 pilot trial in multiple sclerosis. Lancet (London, England). and CCL4 in multiple sclerosis patients. Mediat Inﬂamm. 1995;346(8978):807–8. 2016;2016:2847232. 41. Zhang JZ, Rivera VM, Tejada-Simon MV, Yang D, Hong J, Li S, 24. Wilson DB, Golding AB, Smith RA, Dafashy T, Nelson J, Smith et al. T cell vaccination in multiple sclerosis: results of a pre- L, et al. Results of a phase I clinical trial of a T-cell receptor liminary study. J Neurol. 2002;249(2):212–8. peptide vaccine in patients with multiple sclerosis. I. Analysis of 42. Van der Aa A, Hellings N, Medaer R, Gelin G, Palmers Y, Raus T-cell receptor utilization in CSF cell populations. J Neuroim- J, et al. T cell vaccination in multiple sclerosis patients with munol. 1997;76(1–2):15–28. autologous CSF-derived activated T cells: results from a pilot 25. Vandenbark AA, Chou YK, Whitham R, Mass M, Buenafe A, study. Clin Exp Immunol. 2003;131(1):155–68. Liefeld D, et al. Treatment of multiple sclerosis with T-cell 43. Hermans G, Medaer R, Raus J, Stinissen P. Myelin reactive T receptor peptides: results of a double-blind pilot trial. Nat Med. cells after T cell vaccination in multiple sclerosis: cytokine pro- 1996;2(10):1109–15. ﬁle and depletion by additional immunizations. J Neuroimmunol. 26. Gold DP, Smith RA, Golding AB, Morgan EE, Dafashy T, 2000;102(1):79–84. Nelson J, et al. Results of a phase I clinical trial of a T-cell 44. Achiron A, Lavie G, Kishner I, Stern Y, Sarova-Pinhas I, Ben- receptor vaccine in patients with multiple sclerosis. II. Compar- Aharon T, et al. T cell vaccination in multiple sclerosis relapsing- ative analysis of TCR utilization in CSF T-cell populations before remitting nonresponders patients. Clinical immunology (Orlando, and after vaccination with a TCRV beta 6 CDR2 peptide. Fla). 2004;113(2):155–60. J Neuroimmunol. 1997;76(1–2):29–38. 45. Loftus B, Newsom B, Montgomery M, Von Gynz-Rekowski K, 27. Bourdette DN, Whitham RH, Chou YK, Morrison WJ, Atherton Riser M, Inman S, et al. Autologous attenuated T-cell vaccine J, Kenny C, et al. Immunity to TCR peptides in multiple sclerosis. (Tovaxin) dose escalation in multiple sclerosis relapsing-remit- I. Successful immunization of patients with synthetic V beta 5.2 ting and secondary progressive patients nonresponsive to and V beta 6.1 CDR2 peptides. J Immunol (Baltimore, Md: approved immunomodulatory therapies. Clinical immunology 1950). 1994;152(5):2510–9. (Orlando, Fla). 2009;131(2):202–15. 28. Bourdette DN, Edmonds E, Smith C, Bowen JD, Guttmann CR, 46. Press Release. Opexa Therapeutics. 28 October 2016. http:// Nagy ZP, et al. A highly immunogenic trivalent T cell receptor www.evaluategroup.com/Universal/View.aspx?type=Story&id= peptide vaccine for multiple sclerosis. Mult Scler (Houndmills, 679265. Accessed 13 Nov 2017. Basingstoke, England). 2005;11(5):552–61. 47. Karussis D, Shor H, Yachnin J, Lanxner N, Amiel M, Baruch K, 29. Vandenbark AA. TCR peptide vaccination in multiple sclerosis: et al. T cell vaccination beneﬁts relapsing progressive multiple boosting a deﬁcient natural regulatory network that may involve sclerosis patients: a randomized, double-blind clinical trial. PLoS TCR-speciﬁc CD4 ? CD25 ? Treg cells. Curr Drug Targets One. 2012;7(12):e50478. Inﬂamm Allergy. 2005;4(2):217–29. 48. Jingwu Z, Medaer R, Hashim GA, Chin Y, van den Berg-Loonen 30. Wu Y, Borde M, Heissmeyer V, Feuerer M, Lapan AD, Stroud E, Raus JC. Myelin basic protein-speciﬁc T lymphocytes in JC, et al. FOXP3 controls regulatory T cell function through multiple sclerosis and controls: precursor frequency, ﬁne speci- cooperation with NFAT. Cell. 2006;126(2):375–87. ﬁcity, and cytotoxicity. Ann Neurol. 1992;32(3):330–8. 31. Vandenbark AA, Culbertson NE, Bartholomew RM, Huan J, 49. Lutterotti A, Yousef S, Sputtek A, Sturner KH, Stellmann JP, Agotsch M, LaTocha D, et al. Therapeutic vaccination with a Breiden P, et al. Antigen-speciﬁc tolerance by autologous myelin trivalent T-cell receptor (TCR) peptide vaccine restores deﬁcient peptide-coupled cells: a phase 1 trial in multiple sclerosis. Sci FoxP3 expression and TCR recognition in subjects with multiple Transl Med. 2013;5(188):188ra75. sclerosis. Immunology. 2008;123(1):66–78. 50. Van Brussel I, Lee WP, Rombouts M, Nuyts AH, Heylen M, De Winter BY, et al. Tolerogenic dendritic cell vaccines to treat 410 B. Willekens, N. Cools autoimmune diseases: can the unattainable dream turn into real- 62. Bell GM, Anderson AE, Diboll J, Reece R, Eltherington O, ity? Autoimmun Rev. 2014;13(2):138–50. Harry RA, et al. Autologous tolerogenic dendritic cells for 51. Raich-Regue D, Glancy M, Thomson AW. Regulatory dendritic rheumatoid and inﬂammatory arthritis. Ann Rheum Dis. cell therapy: from rodents to clinical application. Immunol Lett. 2017;76(1):227–34. 2014;161(2):216–21. 63. Jauregui-Amezaga A, Cabezon R, Ramirez-Morros A, Espana C, 52. Schmidt SV, Nino-Castro AC, Schultze JL. Regulatory dendritic Rimola J, Bru C, et al. Intraperitoneal administration of autolo- cells: there is more than just immune activation. Front Immunol. gous tolerogenic dendritic cells for refractory crohn’s disease: a 2012;3:274. phase I study. J Crohn’s Colitis. 2015;9(12):1071–8. 53. Lee W-P, Willekens B, Cras P, Goossens H, Martı´nez-Ca´ceres E, 64. Thomas R, Street S, Ramnoruth N, Pahau H, Law S, Brunck M, Berneman ZN, et al. Immunomodulatory effects of 1, 25-dihy- Hyde C, O’Sullivan B, Capini C, Tran A, Ng J, Paul S. Feasi- droxyvitamin D3 on dendritic cells promote induction of T cell bility, safety and clinical effects of a single intradermal admin- hyporesponsiveness to myelin-derived antigens. J Immunol Res. istration of autologous tolerising dendritic cells exposed to 2016;2016:5392623. citrullinated peptides in patients with rheumatoid arthritis. 54. Bartosik-Psujek H, Tabarkiewicz J, Pocinska K, Stelmasiak Z, Arthritis Rheum. 2011;63(S10):2430. Rolinski J. Immunomodulatory effects of vitamin D on mono- 65. Lim DS, Kang MS, Jeong JA, Bae YS. Semi-mature DC are cyte-derived dendritic cells in multiple sclerosis. Mult Scler immunogenic and not tolerogenic when inoculated at a high dose (Houndmills, Basingstoke, England). 2010;16(12):1513–6. in collagen-induced arthritis mice. Eur J Immunol. 55. Huang YM, Stoyanova N, Jin YP, Teleshova N, Hussien Y, Xiao 2009;39(5):1334–43. BG, et al. Altered phenotype and function of blood dendritic cells 66. De Vries IJ, Krooshoop DJ, Scharenborg NM, Lesterhuis WJ, in multiple sclerosis are modulated by IFN-beta and IL-10. Clin Diepstra JH, Van Muijen GN, et al. Effective migration of anti- Exp Immunol. 2001;124(2):306–14. gen-pulsed dendritic cells to lymph nodes in melanoma patients is 56. Hussien Y, Sanna A, Soderstrom M, Link H, Huang YM. determined by their maturation state. Can Res. 2003;63(1):12–7. Glatiramer acetate and IFN-beta act on dendritic cells in multiple 67. Ridolﬁ R, Riccobon A, Galassi R, Giorgetti G, Petrini M, sclerosis. J Neuroimmunol. 2001;121(1–2):102–10. Fiammenghi L, et al. Evaluation of in vivo labelled dendritic cell 57. Mansilla MJ, Selles-Moreno C, Fabregas-Puig S, Amoedo J, migration in cancer patients. J Transl Med. 2004;2(1):27. Navarro-Barriuso J, Teniente-Serra A, et al. Beneﬁcial effect of 68. Morse MA, Coleman RE, Akabani G, Niehaus N, Coleman D, tolerogenic dendritic cells pulsed with MOG autoantigen in Lyerly HK. Migration of human dendritic cells after injection in experimental autoimmune encephalomyelitis. CNS Neurosci patients with metastatic malignancies. Can Res. Ther. 2015;21(3):222–30. 1999;59(1):56–8. 58. Mansilla MJ, Contreras-Cardone R, Navarro-Barriuso J, Cools N, 69. Verdijk P, Aarntzen EH, Lesterhuis WJ, Boullart AC, Kok E, van Berneman Z, Ramo-Tello C, et al. Cryopreserved vitamin D3- Rossum MM, et al. Limited amounts of dendritic cells migrate tolerogenic dendritic cells pulsed with autoantigens as a potential into the T-cell area of lymph nodes but have high immune acti- therapy for multiple sclerosis patients. J Neuroinﬂamm. vating potential in melanoma patients. Clin Cancer Res. 2016;13(1):113. 2009;15(7):2531–40. 59. Benham H, Nel HJ, Law SC, Mehdi AM, Street S, Ramnoruth N, 70. De Laere M, Derdelinckx J, Hassi M, Kerosalo M, Oravama¨ki H, et al. Citrullinated peptide dendritic cell immunotherapy in HLA Van den Bergh J, et al. Shuttling tolerogenic dendritic cells across risk genotype-positive rheumatoid arthritis patients. Sci Transl the blood-brain barrier in vitro via the introduction of de novo Med. 2015;7(290):290ra87. C-C chemokine receptor 5 expression using messenger RNA 60. Hilkens CM, Isaacs JD. Tolerogenic dendritic cell therapy for electroporation. Front Immunol. 2018;8:1964. rheumatoid arthritis: where are we now? Clin Exp Immunol. 71. Derfuss T. Personalized medicine in multiple sclerosis: hope or 2013;172(2):148–57. reality? BMC medicine. 2012;10:116. 61. Giannoukakis N, Phillips B, Finegold D, Harnaha J, Trucco M. Phase I (safety) study of autologous tolerogenic dendritic cells in type 1 diabetic patients. Diabetes Care. 2011;34(9):2026–32.
CNS Drugs – Springer Journals
Published: May 14, 2018
It’s your single place to instantly
discover and read the research
that matters to you.
Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.
All for just $49/month
Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly
Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.
Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.
Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.
All the latest content is available, no embargo periods.
“Hi guys, I cannot tell you how much I love this resource. Incredible. I really believe you've hit the nail on the head with this site in regards to solving the research-purchase issue.”Daniel C.
“Whoa! It’s like Spotify but for academic articles.”@Phil_Robichaud
“I must say, @deepdyve is a fabulous solution to the independent researcher's problem of #access to #information.”@deepthiw
“My last article couldn't be possible without the platform @deepdyve that makes journal papers cheaper.”@JoseServera