Dcs x Ca - Dendríticqas versus Câncer

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A aplicação clínica da imunoterapia do câncer com base nas células dendríticas naturalmente circulantes
Kalijn F. Bol † ,Gerty Schreibelt † ,Katrin Rabold ,Stefanie K. Wculek ,Julia Katharina Schwarze ,Andrzej Dzionek ,Alvaro Teijeira ,Lana E. Kandalaft ,Pedro Romero ,George Coukos ,Bart Neyns ,David Sancho ,Ignacio Melero eI. Jolanda M. de VriesAutor de email
† Contribuiu igualmente


Jornal para ImmunoTherapy of Cancer 2019 7 : 109
https://doi.org/10.1186/s40425-019-0580-6© Autor (es). 2019
Recebido: 11 de fevereiro de 2019Aceito: 26 de março de 2019Publicado em: 18 de abril de 2019


Abstrato
As células dendríticas (DCs) podem iniciar e direcionar respostas imunes adaptativas. Esta capacidade é explorável em estratégias de vacinação DC, em que DCs são educados ex vivo para apresentar antígenos de tumor e são administrados no paciente com o objetivo de induzir uma resposta imune específica do tumor. A vacinação DC continua sendo uma abordagem promissora, com potencial para melhorar ainda mais a imunoterapia do câncer, com pouca ou nenhuma evidência de toxicidade limitadora do tratamento. No entanto, a evidência da atividade antitumoral clínica objetiva da vacina DC é atualmente limitada, dificultando a implementação clínica. Uma possível explicação para isso é que as DCs derivadas de monócitos mais comumente usadas podem não ser a melhor fonte de imunoterapia baseada em DC. A nova abordagem para usar DCs naturalmente circulantes pode ser uma alternativa atraente. Ao contrário das DCs derivadas de monócitos, DCs naturalmente circulantes são relativamente escassas, mas não requerem longos períodos de cultura. Assim, suas capacidades funcionais são preservadas, a reprodutibilidade das aplicações clínicas é aumentada e as células não são disfuncionais antes da injeção. No sangue humano, pelo menos três subconjuntos de DC podem ser distinguidos, DCs plasmocitoides, CD141+ e DC CD1c + mieloide / convencional, cada uma com características funcionais distintas. Em ensaios clínicos completos, DCs mieloides CD1c + ou DC plasmocitóides foram administradas e mostraram resultados imunológicos e clínicos encorajadores. Atualmente, também a combinação de DCs mieloides CD1c + e plasmocitoides bem como o uso intratumoral de DCs mieloides CD1c + está sob investigação na clínica. Estratégias de isolamento e cultura para DCs mieloides CD141 + estão sendo desenvolvidas. Aqui, resumimos e discutimos desenvolvimentos clínicos recentes e as perspectivas futuras da imunoterapia baseada em DC natural.

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Em 1973, Cohn e Steinman descobriram um novo tipo de célula imune, a célula dendrítica (DC) [ 1 ], que desempenha um papel importante na indução de imunidade específica. As CDs são sentinelas do sistema imunológico, pois são implantadas em todo o corpo e monitoram seus arredores quanto a antígenos e sinais de perigo derivados de patógenos ou danos nos tecidos. Eles são as células apresentadoras de antígenos mais potentes, capazes de iniciar e modular respostas imunológicas específicas.

Em seu estado imaturo, as CDs residem principalmente em tecidos linfóides e periféricos, onde reconhecem e capturam antígenos. Ao receber um estímulo ativador na presença de sinais inflamatórios, as DCs sofrem maturação e migram para os órgãos linfóides. A maturação da DC está associada a alterações funcionais e morfológicas, um processo essencial para a ativação de células T. O fenótipo imaturo das DCs é caracterizado principalmente por uma baixa expressão superficial de moléculas de MHC I e II e moléculas co-estimulatórias e uma alta capacidade de fagocitose que medeia a amostragem de antígenos [ 2]. DCs ativadas pelos chamados “sinais de perigo” tornam-se altamente móveis, seus receptores endocíticos e fagocíticos são modulados negativamente, e receptores de quimiocinas que estimulam a migração para órgãos linfóides são supra-regulados. Além disso, a expressão da superfície celular de moléculas de MHC e moléculas de adesão / co-estimuladoras, tais como CD40, CD54, CD80, CD83 e CD86 é regulada positivamente e a produção de citocinas específicas é induzida [ 3 ]. Nos órgãos linfóides, DCs maduras apresentam peptídeos exógenos processados ​​para células T CD4 + na- ves via MHC de classe II e peptídeos endógenos para células T CD8 + via MHC de classe I. Além disso, algumas DCs têm uma capacidade superior de apresentar resultados exógenos. antigénios em células T de MHC classe I a CD8 + [ 2], que é importante para a indução de respostas de células T citotóxicas contra células tumorais. O priming efetivo de células T nos tecidos linfóides requer três sinais entre as DCs e as células T: apresentação do antígeno via complexo MHC-peptídeo (sinal 1), estimulação via moléculas coestimuladoras da DC para a célula T (sinal 2) e citocinas imunoestimuladoras no microambiente (sinal 3) [ 3 ].

A capacidade das DCs de iniciar e direcionar respostas imunes adaptativas é explorada para a imunoterapia do câncer, especialmente na vacinação DC. Com vacinação DC, DCs maduras carregadas com antígenos tumorais ex vivo são injetadas em pacientes com câncer para induzir células T efetoras específicas de tumores que visam reconhecer e eliminar células cancerígenas e induzir memória imunológica para controlar o crescimento do tumor [ 4 ]. Na maioria dos ensaios clínicos de DC realizados até agora, as DCs diferenciaram ex vivo de monócitos ou CD34 +progenitores têm sido utilizados, uma vez que as DC circulantes naturalmente (nDCs) estão presentes no sangue, mas constituem apenas cerca de 1% das células mononucleares do sangue. No entanto, através do desenvolvimento de técnicas de isolamento eficientes, o uso de nDCs tornou-se recentemente viável. Nesta revisão, resumimos e discutimos desenvolvimentos clínicos recentes de imunoterapia baseada em DC com subgrupos nDC, compreendendo estudos clínicos concluídos e em andamento.

Lições da vacinação DC com moDCs
Provocados por excelentes resultados contra tumores de camundongos transplantados com culturas de DC derivadas de medula óssea, os primeiros testes de vacinação DC foram realizados no final dos anos noventa. O efeito de vários parâmetros de vacinação DC no resultado imunológico e clínico da vacinação tem sido estudado em numerosos ensaios clínicos de fase pequena I / II em pacientes com câncer. A maioria desses estudos foi realizada com DCs derivadas de monócitos (moDCs), devido ao seu fácil protocolo de diferenciação in vitro.

Maturação de moDCs
MoDCs are mostly HLA-DR+/MHC-II+ CD11c+ BDCA3− and frequently express CD16, CD14 and DC-SIGN, due to their monocytic origin [5]. Their functions and appearance are very divers, likely due to the inflammatory context they are differentiating in and the variety of cytokine cocktails that are used for their activation ex vivo. From the first clinical studies it became evident that proper activation of the DCs is of major importance for DC vaccination of cancer patients, otherwise antigen-specific tolerance is induced rather than antitumor immunity [6, 7, 8]. Besides inducing expression of molecules important for T-cell activation, maturation of DCs leads to upregulation of chemokine receptors which promotes the migration of injected DCs to the lymph nodes and is thus of importance for vaccination efficacy [9].

In vivo, DC maturation is triggered by pathogens or tissue injury. In vitro, this can be mimicked by incubation with pathogen recognition receptor agonists or a cocktail of proinflammatory cytokines. A cytokine cocktail consisting of tumor necrosis factor (TNF)α, interleukin (IL)-1β, IL-6 and prostaglandin E2 (PGE2), or monocyte-conditioned medium with TNFα and PGE2 are the most widely used methods for moDC maturation [10, 11]. Whether this is the best cocktail to induce maturation remains controversial since PGE2 may confer immunosuppressive effects [12, 13]. To further induce DC activation, mimicking viral infection, type I interferons have been added to the cocktail [14]. More recently, the use of Toll-like receptor (TLR) ligands [15, 16] or electroporation with mRNA-encoding proteins that induce DC maturation [17] has been explored. The latter methods yield DCs that produce higher levels of IL-12, which favors the differentiation of T helper 1 (Th1) cells and promotes activation of potent CD8+ effector T-cells.

Antigen loading and administration of moDCs
To induce a tumor-specific immune response in cancer patients, DCs should be loaded with relevant tumor antigens. The most widely used techniques for antigen loading of DCs vaccines are pulsing DCs with MHC-binding peptides of tumor-associated antigens (TAA), corresponding long peptides or proteins, TAA-encoding mRNA, or tumor lysate. All antigen-loading techniques have their advantages and disadvantages; none has proven to be superior to the others thus far, however, loading with both MHC class I and class II epitopes seems beneficial for the quality of the induced immune response [18].

For DC vaccination, it is crucial that DCs migrate to the T-cell areas of the lymph nodes after administration. In murine models it was shown that intravenously injected DCs mostly accumulate in highly vascularized organs like spleen, lungs, kidneys and liver, rather than lymph nodes and fail to induce skin-homing T-cells [19, 20]. Migration studies with labeled DCs demonstrated that after intradermal injection, only 2–4% of the injected cells migrate to draining lymph nodes, whereas most of the injected cells die at the dermal injection site and were cleared by macrophages [21, 22, 23]. After intranodal injection, injected cells accumulate in the injected node and subsequent draining lymph nodes [23, 24]. By leaving the DCs directly at the site of interaction with T-cells, this route of administration obviates the need for DCs to migrate. However, cells need to be injected under ultrasound guidance.

Comparison of induced immune responses after DC vaccination via different routes of administration showed variable results [23, 25, 26]. Intradermal injection seems to yield superior T-cell responses in terms of tumor recognition and cytokine production [23], which might in part be explained by the fact that, after intradermal migration, only the most mature and most potent DCs reach the lymph nodes, in contrast to intranodal injection, where also nonviable and less mature DCs are directly delivered into the lymph nodes.

moDCs in clinical trials: the outcome
Thus far, numerous phase I/II clinical trials with moDC vaccines have been performed in cancer patients. Side-effects were minimal and included grade 1–2 flu-like symptoms, fever and local injection site reactions. Grade 3–4 toxicity is very uncommon after DC vaccination but can occur with more potent moDC formulations [15, 27, 28, 29]. Thus, DC vaccination can be concluded to be safe when used as monotherapy.

Although safe and able to induce anticancer immunity, so far objective clinical responses have only been achieved in a minority of patients after moDC vaccination, usually around 5–15% of metastatic cancer patients. However, despite the lack of clear benefit in objective clinical responses, a trend to survival benefit was reported in most studies [28]. This is often seen with immunotherapy, as it takes time until the full potential of the anti-tumor response is reached and sometimes delayed objective clinical responses occur, or only stable disease is achieved that nevertheless can be highly durable. This dissociation between objective response and overall survival (OS) is hampering the clinical implementation of DC vaccination as larger randomized clinical trials would be required when survival rather than tumor response is used as a primary endpoint. Furthermore, most trials were conducted with widespread metastatic patients in which tumor-induced immune suppression is probably too strong to overcome with DC vaccination alone. Still, numerous small trials improved the quality of the DC vaccines over the years and moDC vaccination still holds promise for clinical application. Combination of DCs with other forms of anticancer treatment might be a solution to overcome tumor-induced immune suppression. For example, the combination of moDCs with anti-CTLA4 blockade in advanced melanoma patients showed an encouraging response rate of 38%, with all complete responders (n = 7) still free from progression and off-therapy more than 5 years after the initiation of DC therapy [30]. Another option to obtain more robust antitumor responses, might be adjuvant DC vaccination, when only minimal tumor load in present [31, 32]. Data from phase III clinical trials are needed to substantiate the results of the successful smaller trials. Recently, OS data of a randomized phase III clinical trial in glioblastoma patients treated with a moDC vaccine in combination with chemotherapy and radiotherapy were published [33]. The median OS seems promising compared to literature, but due to the cross-over trial design groups within the trial cannot be properly compared and progression-free survival (PFS) data is awaited.

Células dendríticas naturalmente circulantes
MoDCs may not be the best DC source for immunotherapy, since they have been described to have decreased migratory capacities towards the site of T-cell interaction by exhaustion of the cells [34], probably due to the artificial differentiation by cytokines and extensive ex vivo culture periods. nDCs may be a potent alternative for moDCs, as the brief ex vivo exposure of nDCs might preserve the functional capabilities of the cells and prevent exhaustion. Although direct comparison of nDCs and moDCs in clinical trials have not been performed (yet) to validate the in vitro data. In addition, The Cancer Genome Atlas reveals that specific nDC subsets, rather than moDCs, are associated with improved survival in diverse cancer types [35, 36, 37]. Although the isolation of monocytes from the blood has a much higher yield, direct isolation of nDCs is now feasible and facilitates robust standardization for use in multicenter trials and, eventually, standard care.

nDCs comprise a heterogeneous population of cells. Functional, transcriptomic and proteomic reports identified the major circulating DC subsets, which are distinguished by distinct surface markers [38, 39, 40]. Human DCs can be subdivided in two main subsets (Fig. 1); plasmacytoid DCs (pDCs) and myeloid/conventional DCs (mDCs). These subsets differ in function, localization, and phenotype [41]. pDCs have a plasma cell-like shape and are specialized in viral antigen recognition, they largely lack expression of extracellular TLRs and are the main producers of type I interferons (IFNs) [42, 43]. They are mainly localized in T-cell areas of lymph nodes and express BDCA2 and BDCA4 [41, 44]. pDCs appear to be predominantly tolerogenic in the context of cancer and correlate with poor prognosis [44]. However, when properly activated, they have the ability of cross-presentation and may therefore be potent inducers of antitumor responses [45, 46, 47]. Activation of pDCs induces upregulation of MHC molecules and costimulatory molecules, allowing efficient priming of CD4+ and CD8+ T-cells. The secretion of large amounts of type I IFNs can induce Th1 polarization as well as activation of innate immune cells, such as macrophages and natural killer cells [45, 48, 49, 50]. Type I IFNs produced by pDCs are also beneficial for antigen cross-presentation by mDCs [51]. mDCs are mainly localized in the marginal zone of lymph nodes and express MHC II and CD11c [48, 52, 53]. They express extracellular TLRs (TLR1, TLR2, TLR4–6) and endosomal TLRs (TLR3 and TLR8), which are responsible for the ability of mDCs to secrete the Th1 skewing cytokine IL-12 upon activation [43]. The mDC population can be further subdivided into two classes based on surface expression into CD1c(BDCA1)+ DCs (cDC2s) and CD141(BDCA3)+ DCs (cDC1s), with the CD1c+ mDCs being the most potent T-cell stimulators of these subpopulations [43, 54, 55]. CD1c+ mDCs also consist of two subsets, both populations stimulate T-cell proliferation but differ in their potential for cytokine secretion [56]. CD1c+ mDCs seem specialized in immunity against bacteria and fungi, whereas CD141+ mDCs are specialized in detection and uptake of necrotic cell debris of virally infected cells or tumor cells and cross-presentation of derived antigens to CD8+ T-cells [57, 58, 59]. Especially a subset of BDCA3+XCR1+CLEC9A+ cells seems to be a superior cross-priming DC subset in humans [57, 58, 59, 60, 61]. They can migrate from peripheral organs to lymph nodes and efficiently cross-present cell-associated antigens to induce CD8+ T-cells [39, 40, 60, 62, 63]. Most recently, myeloid DC have also found to be of pivotal importance in “relicensing” the antitumor activity of cytotoxic T-cells within the tumor microenvironment [35, 37]. Furthermore, a subset of CD16+ ‘non-classical’ monocytes with DC-like characteristics is found in human blood [64].
Figura 1
Fig. 1
Dendritic cell subsets. Dendritic cells can be differentiated from monocytes (moDC), which are often used in clinical trials because of their high yield. The naturally circulating dendritic cells can now also be enriched by immunomagnetic isolation. The naturally circulating dendritic cells can further be divided in myeloid (CD141+ and CD1c+ mDC) and plasmacytoid dendritic cells (pDC). The subsets differ in function, localization, phenotype and cytokine production

In animal models, it was shown that mature antigen-loaded pDCs or mDCs can prime tumor-specific T-cell responses that result in tumor eradication [65, 66, 67]. In a murine glioma model, vaccination with mDCs was superior to pDCs in survival benefit [67]. Recent studies suggest that pDCs and mDCs cooperate and act synergistically. In mice, pDCs were shown to induce tumor-specific CD8+ T-cell responses and enhance the ability of mDCs to present tumor antigens to T-cells [68, 69]. In human, mDCs and pDCs were shown to activate each other after specific stimulation of one of the subsets with TLR ligands in vitro [53]. Combining the two subsets in one vaccine might thus exploit their functions simultaneously and increase their immunotherapeutic potential [70].

Isolation of nDCs for clinical use
Production of DC vaccines is a labor-intensive process comprising numerous open handling steps such as density gradient cell processing, cell washing steps, cell labeling/separation, cell culture, final product formulation and cryopreservation. The complexity of the process makes it prone to failure and requires experienced personnel and complex protocol development under GMP guidelines. Therefore, manufacturing of clinical grade DC vaccines can be performed only in highly specialized institutions and existing manufacturing processes are hardly transferable, which in turn limits patient’s access to this kind of therapy. To overcome these limitations, there is a growing effort in the field to develop standardized, robust and reproducible protocols for production of DC vaccines. In this regard, automation of such processes is a major step forward as it limits operator-dependent variance and thereby reduces deviations not only between individual production runs but also between productions that are performed at different clinical centers. Miltenyi’s CliniMACS Prodigy® platform consists of an integrated device, clinical grade buffers and reagents and a single-use tubing set that allows for temperature and atmosphere-controlled cell culture. It has been designed to automatically perform all cell handling steps in a closed system with minimal user interaction for highest reproducibility [71]. In addition, the closed system reduces the need for complex class A clean-room resources as it can be operated in a class C GMP-environment. The production of nDC vaccines consists of positive selection to enrich pDCs and/or mDCs using magnetic antibody-coupled beads, optionally preceded by depletion of monocytes and B cells (Fig. 2). Currently, two additional processes are under development aiming for the isolation of cross-presenting CD141+ mDC and panDC (pDC + CD1c+ mDC + CD141+ mDC). Addition of CD141+ mDCs may further improve nDC vaccines, since this mDC subtype is highly efficient in antigen cross-presentation and able to secrete IFNλ and IL-12 upon activation [57, 58, 59, 72].
Figura 2
Fig. 2
Production protocols for naturally circulating dendritic cells. Schematic overview of the (a) CD1c+ myeloid dendritic cell (mDC) and (b) plasmacytoid dendritic cell (pDC) production protocols and vaccination strategy of the various clinical trials

nDCs in clinical trials: the vaccines
Currently, 9 clinical trials with autologous nDC vaccination are performed in cancer patients, of which three are completed [73, 74, 75], one was terminated (ACTRN12607000450415), and 5 are ongoing (NCT02574377, NCT02692976, NCT02993315) of which 2 are still recruiting patients (NCT03707808, NCT03747744). The trials included patients with either melanoma, prostate cancer or any solid tumor. Three trials focus solely on CD1c+ mDC vaccination and one trial solely on pDC vaccination, whereas in the remaining trials that are being performed the combination of both nDC cell types is studied or CD1c+ mDC vaccination is combined with other immunotherapeutics (Table 1). All trials performed isolation of DCs using the immunomagnetic CliniMACS® isolation system. Another method to obtain DCs from an apheresis product is the enrichment of DCs by density centrifugation. This method was mainly performed before the emergence of magnetic separation. Magnetic separation is a faster, less labor-intensive method and results in a purer population of cells with a sufficient yield compared to density centrifugation. As density centrifugation does not result in pure populations, studies performing density gradient isolation were not included in this review. Among these are studies using the clinically approved sipuleucel-T for metastatic castration-resistant prostate cancer [76], which only contains a small fraction of CD54+ DCs, i.e. stimulated DCs, among T-cells, natural killer cells, monocytes, and B-cells. In essence, the vaccination product is the result of peripheral blood mononuclear cells (PBMCs) obtained from apheresis activated by a fusion protein between prostate acid phosphatase and granulocyte-macrophage colony-stimulating factor (GM-CSF) [77]. The contribution of the diverse cell types to the activity of the intravenously administered vaccination product remains unclear.
Table 1
Clinical trials with natural DC vaccination

aStage IV melanoma patients or irresectable stage III melanoma patients

bAnticipated capture of tumor antigens and maturation in vivo

cmDC vaccination in combination with T-VEC intratumorally or anti-CTLA4 and anti-PDL1 intratumorally and anti-PD1 intravenously

dAll mDCs obtained from the leukapheresis are injected

Abbreviations: FSME, Frühsommer-meningoencephalitis; GM-CSF, granulocyte-macrophage colony-stimulating factor; mDC, myeloid dendritic cell; pDC, plasmacytoid dendritic cell; T-VEC, Talimogene laherparepvec. (source: clinicaltrials.gov and anzctr.org.au)

mDC vaccines
The first clinical trial with mDCs, in 2007, was conducted in hormone-refractory metastatic prostate cancer patients (mDC-PROS1). Isolation of mDCs was performed by positive selection for CMRF-56, an early DC activation/differentiation antigen expressed by mDCs after in vitro culture for 6 h [78, 79]. This single-step isolation procedure showed higher yields as well as less variable purity compared to the isolation by density gradients performed in the clinical setting until then, but the product still contained about 30% CD14+ and CD19+ cells [80]. Therefore, this study was terminated and as the study results are not published, no conclusions can be drawn concerning the isolation and culture method.

In subsequent trials, the CD1c+ mDCs were selected by depletion of B cells (CD19+) followed by positive selection of CD1c+ cells. In the trial with metastatic melanoma patients (mDC-MEL1), initiated in 2010, this procedure resulted in an average purity of 93% and a yield between 27 × 106 and 96 × 106. The isolated cells were stimulated by GM-CSF, resulting in semi-mature mDCs that are HLA-ABC/DQ/DR+ CD86+ and showed variable CD83 and CD80 expression [74]. A trial conducted with mDCs in metastatic prostate cancer patients (mDC-PROS2) used the same selection technique and obtained a similar purity (median 82%) and yield (28-101 × 106). However, no stimulation was added in this trial and phenotyping established that all CD1c+ cells within the vaccines had a semi-mature phenotype (CD86+ CD40− CD80− CD83−) [75].

In 4 trials, to obtain CD1c+ mDCs, cells expressing the monocytic marker CD14 were also depleted in addition to CD19+ cells, since CD1c+ CD14+ cells were shown to suppress CD4+ T-cells and may severely hamper DC vaccine efficacy [81]. In two trials with completed patient accrual, stage III melanoma patients (combiDC-MEL1) or metastatic prostate cancer patients (combiDC-PROS1) were either vaccinated with mDCs, pDCs or the combination of both. In the two ongoing trials, the mDCs are neither fully matured nor loaded with antigen ex vivo, but injected intratumorally in combination with Talimogene Laherparepvec (T-VEC; mDC-MEL-TV), an oncolytic virus approved for non-visceral melanoma metastasis [82], or the immune checkpoint inhibitors anti-CTLA4 and anti-PDL1 intratumorally and anti-PD1 intravenously (mDC-SOLID-ICI). It is hypothesized that the semi-mature mDC capture tumor antigens and mature in vivo after intratumoral exposure to the co-injected T-VEC virus or immune checkpoint inhibitors, which have the potential to elicit antigen-dependent cellular cytotoxicity and complement-dependent cytotoxicity, thereby creating an inflamed tumor microenvironment.

To stimulate the DCs in the combination trials, DCs are activated with protamine/mRNA which can induce maturation of both pDCs and CD1c+ mDCs [47]. Although the two DC subsets do not express an overlapping repertoire of TLRs, single-stranded RNA is a suitable maturation stimulus as it is a ligand for TLR7 on pDCs and TLR8 on mDCs, inducing IFNα and IL-12 production, respectively. Furthermore, stimulation with protamine/mRNA was shown to result in an increase in the expression of MHC class I and CD86, and a variable expression of CD80 on both mDCs and pDCs. Consequently, the stimulated DCs were able to induce T-cell proliferation and activation [47]. This characteristic of protamine/mRNA to activate both CD1c+ mDCs and pDCs provides more flexibility in combining the two subsets in one vaccine. Preliminary data from these two trials shows a similar phenotype as in the preclinical study (unpublished data).

pDC vaccines
The pDCs, used in the combination trials and a trial with a single pDC vaccine in melanoma patients (pDC-MEL1), are isolated by BDCA4 bead-coupled antibodies, without any prior depletion step. Compared to mDCs, pDCs have a lower average purity of 75% and a yield between 13 × 106 and 33 × 106 cells [73]. Activation of the autologous pDCs is either performed with Frühsommer-meningoencephalitis (FSME) vaccine (pDC-MEL1) or protamine/mRNA (combination trials). In contrast to mDCs, GM-CSF is not suitable as stimulus for pDCs, as it does not efficiently activate pDCs to produce type I IFNs [74]. In contrast, FSME is a suitable maturation stimulus for pDCs as it triggers TLR7, leading to cytokine production and maturation [83].

Although outside the scope of this review, there is one trial with a pDC vaccine in melanoma patients (NCT01863108) using pDCs from an allogeneic cell line. This cell line is derived from a malignant leukemic pDC and cells are irradiated prior to administration to prevent further proliferation of pDCs in the patient [84]. The advantage of an allogeneic cell line lies in the abolishment of the limited cell yield and obviates the need for a leukapheresis. Although vaccination with allogeneic DCs will induce an allogeneic immune response, these responses may even be beneficial for the antitumor response [85]. Preclinical studies showed that using allogeneic DCs was safe and effective, however, the allogeneic DCs and the patient must share a HLA antigen, in this case HLA-A*02:01, to enable antigen presentation. Furthermore, there is a chance to develop immune reactions against the DCs itself. In the case of repetitive vaccination this would lead to unwanted killing of the allogeneic DCs by the immune system.

Administration and antigen loading
In all but two trials a cycle of three vaccinations with a 2 to 4-week interval was administered. In the 4 trials performed in Nijmegen, maximally two maintenance cycles were given in the absence of progressive disease. In the phase I trials combining mDCs with T-VEC or immune checkpoint inhibition intratumorally, currently only a single vaccination is given. Concerning the administration of the vaccine, the trials differ in the number of cells that are administered, the administration schedule, and the route of administration (Table 1). In all trials using antigen loading, peptide pulsing was performed. As electroporation procedures comes with moderate cell toxicity, this is a serious drawback for the scarce nDCs. The tumor antigens used differ between the trials, mainly due to the different tumor types expressing different antigens. The number of cells mainly depend on the yield of nDC isolation and ranges from 1 × 106 to 1 × 107 cells per vaccination. There is little evidence about the most effective cell number per vaccination, even for moDCs. In most trials intranodal injection was performed, again due to the scarcity of the cells.

nDCs in clinical trials: the outcome
Monitoring immune responses
In all phase I/II trials, the primary and secondary endpoints were safety and immunological outcome. For immunomonitoring purposes, DCs were loaded with a control antigen in all trials performing antigen loading. Both trials with CD1c+ mDCs (mDC-MEL1, mDC-PROS2) used keyhole limpet hemocyanin (KLH) as a control antigen and as a source for T helper epitopes. In the mDC-PROS2 trial, KLH-specific antibodies could be detected by ELISA in the peripheral blood after vaccination in 4 of 12 patients, of which one patient already had KLH-specific antibodies prior to vaccination [75]. In the mDC-MEL1 trial, KLH-specific antibodies could be found in a similar percentage of patients (4 of 13 patients), with detectable KLH-specific antibodies prior to vaccination in 2 patients but with increased levels after vaccination. Also, T-cell proliferation upon stimulation with KLH was shown in 11 of 13 patients after the first round of vaccinations [74]. However, previously no correlation with survival and a strong KLH-specific T-cell response could be found in a cohort of 91 patients [86].

As pDCs do not have the capacity to take up soluble KLH [87], KLH cannot be used for immunomonitoring in patients vaccinated with pDCs. In the pDC-MEL1 trial, the FSME vaccine was used as a maturation stimulus and served as a control antigen. In this study, peripheral blood showed T-cell proliferation upon stimulation with FMSE in 10 of 14 patients tested, while FMSE-specific antibodies were present in 12 of 15 patients [73]. The data on immune responses against the control antigens indicate that nDC vaccination can effectively induce de novo immune responses in cancer patients. As different control antigens were used, no direct comparison of the efficacy of the induction of the novo immune responses between mDCs and pDCs can be made.

All published trials also analyzed the presence of tumor antigen-specific T-cells, either by FACS or ELIspot assays. In the mDC-MEL1 trial, tumor antigen-specific T-cells were detected in PBMCs of 4 of 12 patients [74]. In the mDC-PROS2 trial no tumor antigen-specific T-cells could be detected [88]. In the pDC-MEL1 trial, tetramer stainings were negative, however, after in vitro restimulation with antigenic peptides, an increase in tumor antigen-specific T-cells after vaccination could be detected in 7 of 15 patients [73]. This in vitro restimulation was not performed in the other trials.

Furthermore, delayed-type hypersensitivity (DTH) skin tests were performed in all 3 trials after (each round of) 3 vaccinations. In the mDC-PROS2 trial, DTH skin test were conducted with prostate-specific peptides. No skin reactions, pain/itching or erythema was observed to KLH or prostate-specific peptide. However, skin reactions were present against a control peptide (FMP) in 4 patients [75]. Unfortunately, no skin biopsies were taken for further analyses, although swelling/erythema of a DTH skin test does not correlate with the presence of tumor antigen-specific T-cells [89] but the presence of tumor-specific T-cells correlates with clinical outcome [86, 89]. Therefore, in both trials performed in Nijmegen biopsies were taken irrespective of induration of the DTH injection sites. Tumor-specific CD8+ T-cells were detected with tetramer stainings in 4 of 13 patients (mDC-MEL1) and 2 of 15 patients tested (pDC-MEL1) [73, 74]. Despite the small number of patients in the mDC-MEL1 trial, the correlation between the presence of tumor-specific T-cells and survival could be observed [74].

Toxicity and survival
Toxicity data for nDC vaccination thus far is limited, but the toxicity seems to be similar or even more favorable than with moDC (Table 2). To date, little can be concluded on the clinical efficacy of nDC vaccination. In both trials with metastatic melanoma patients, PFS was short in most patients. However, in the trial with CD1c+ mDCs, PFS was longer in patients with tumor-specific T-cells compared to patients without tumor-specific T-cells. Furthermore, despite the short PFS in most patients, OS seems to be relatively long. The median OS was 13 (mDC-MEL1) and 22 months (pDC-MEL1) [73, 74]. However, OS might be biased by subsequent treatments. In the mDC-PROS2 trial, the asymptomatic hormone-refractory metastatic prostate cancer patients showed a median OS of 18 months, including one patient alive over 5 years after enrollment [75]. Of the more recent trials, highly preliminary data of the intratumoral mDC vaccination in combination with immune checkpoint inhibitors (mDC-SOLID-ICI) showed a durable partial response (> 8 months) in a melanoma patient who previously progressed on immune checkpoint inhibition [90]. More robust survival data should be obtained from the first phase III trial with nDCs (combiDC-MEL2). This trial started in 2016 and studies vaccination with the combination of pDCs and CD1c+ mDCs compared to placebo in the adjuvant setting in stage III melanoma patients. Due to the recent approval of anti-PD1 antibodies and combined BRAF/MEK inhibition as adjuvant treatment, the inclusion had to be stopped before completing planned accrual. However, the circa 150 enrolled patients will provide valuable results in the near future.
Table 2
Baseline characteristics and outcome measures after natural dendritic cell vaccination

pDC-MEL1

mDC-MEL1

mDC-PROS2

Cancer type

Melanoma

Melanoma

Prostate

Patients

Male/female

10/5

10/4

12/0

Age, years

Median (range)

52 (35–69)

50 (31–73)

69.5 (52–78)

Disease stage

M1a

1

4a

2

M1b

8

2

5

M1c

6

9

3

Unknown

0

0

2

Line of systemic treatment

1st line

14

14

0

2nd line or later

1

0

12b

Number of vaccines received

2 or 3

12

9

12

6

2

2

–

9

1

3

–

Vaccine-specific toxicity

Grade 1

6

4

n.a.

Grade 2

0

1

n.a.

Immunological responses

Control antigenc (blood)

T-cell: 10/14

Ab: 12/15

T-cell. 11/13

Ab: 4/13

T-cell: n.t.

Ab: 4/12

Control antigen (DTH)

n.t.

n.t.

4/12d

Tumor antigen (blood)

7/15e

4/12

0/12

Tumor antigen (DTH)

2/15

4/13

0/12d

Progression-free survival

Median (range; months)

4.0 (< 4–20)

2.8 (< 4–67+)

n.a.

Overall survival

Median (range; months)

22.3 (< 4–64)

13.3 (< 4–67+)

18 (6–40+)

aIncluding 1 irresectable stage III melanoma patient

bAll patients received 2–4 lines of hormonal treatment. Four patients received prior chemotherapy

cT-cell proliferation upon stimulation with the control antigen (T-cell) and control antigen-specific antibodies (Ab) are shown

dSkin reaction tested only

eNo tumor-specific T-cells were detected prior to restimulation

Abbreviations: n.a., not available; n.t., not tested

Future perspectives: CD141+ mDCs, neoantigens and in vivo targeting of nDCs
Of the different nDC subsets, the CD141+ mDCs are the only subset that has not been explored in a clinical trial yet. The isolation of these cells is even more challenging because of their extreme scarcity in peripheral blood (0.2–0.3% of total mononuclear leukocytes). With recently developed isolation kits, the cells can be isolated with a purity of 70–85% after positive selection for CD141 and 3-6 × 106 cells can be obtained from one leukapheresis. This subset is truly specialized in cross-presentation and a similar subset in mice, Batf3-dependent CD8α+ lymphoid or CD103+ DCs, were shown to be crucial for the induction of antitumor T-cell responses and tumor control [35, 37, 59]. These mouse equivalents of human CD141+ mDCs are also essential for recruitment of T-cells within the tumor and effective checkpoint antibody therapy [91, 92]. Moreover, there is a strong link of mDC infiltration in the tumor with increased survival in several cancers in The Cancer Genome Atlas [36, 37, 93]. Therefore, vaccination with this subset of CD141+ mDCs is postulated to result in superior antitumor immune responses in cancer patients and is being optimized for clinical application.

A different path to improve DC vaccines might the use of neoantigens. Neoantigens are generated by somatic mutations in the tumor. Exploiting neoantigens requires sequencing of the tumor of the patients and prediction of their MHC molecule binding capacity. Although labor-intensive and time-consuming, it is feasible and might be the future for antigen loading of DCs [94, 95, 96]. Alternatively, intratumoral injection of DCs is currently under evaluation for its potential to capture unknown neoantigens in vivo.

Another recent approach to exploit DCs for cancer immunotherapy is to target DCs subsets in vivo, by antibodies with activating agents and antigens [97]. Antigen bound to antibodies directed against surface receptors of DCs that are implicated in endocytosis, can lead to uptake of the antigen, loading on MHC, and subsequent induction of immune responses [98]. However, if these antibody-antigen conjugates are not accompanied by adjuvant to stimulate the immune system, tolerance rather than immunity might occur. The adjuvant can be given systemically, locally or specifically targeted to nDCs by antibody-coated (nano)particles loaded with both antigen and adjuvant [99]. A vantagem da última abordagem é que os adjuvantes apenas ativam as DCs que são direcionadas pelos anticorpos, impedindo assim a ativação sistêmica e a toxicidade, e, inversamente, que as DCs carregadas com antígenos também são estimuladas e amadurecidas com adjuvante, portanto nenhuma DC imatura é carregada com antígenos tumorais [ 100 ]. A principal vantagem das estratégias de segmentação in vivo é o desenvolvimento de um produto de prateleira. No entanto, mais pesquisas são necessárias antes que os ensaios clínicos possam ser iniciados.

Conclusão
Based on all the in vitro data, nDCs may be a potent and more practical alternative to moDCs. Currently, with immunomagnetic isolation the scarce nDCs can be obtained for DC vaccination. The advantage of nDCs lies in the rapid and highly standardized, automated production of the vaccines, which can improve the quality of the DC vaccines and enables multicenter trials. Furthermore, as nDCs are not artificially differentiated and only undergo a short ex vivo culture period, it is hypothesized that they retain their functional capabilities and avert exhaustion. The results from the few completed trials with nDCs show promising results with very limited toxicity. Subsequent trials as well as data from ongoing trials will have to substantiate the role of nDCs in DC-based immunotherapy as data is currently too limited to draw firm conclusions regarding nDCs and their comparison to moDCs. It will be interesting to investigate what DC vaccines can offer and whether their therapeutic effects can enhance those of checkpoint inhibitors when used in combination.

Notas
Kalijn F. Bol e Gerty Schreibelt são Autores contribuídos igualmente

Abreviaturas
DC:
Células dendríticas

DTH:
Hipersensibilidade do tipo retardado

FMSE:
Meningoencefalite

GM-CSF:
Fator estimulante de colônias de granulócitos e macrófagos

IFN:
Interferon

IL:
Interleucina

KLH:
Hemocianina de lapa

mDC:
Células dendríticas mielóides

MHC:
Complexo principal de histocompatibilidade

moDC:
Células dendríticas derivadas de monócitos

nDC:
Células dendríticas naturalmente circulantes

SO:
Sobrevida global

PBMC:
Células mononucleares do sangue periférico

pDC:
Células dendríticas plasmocitóides

PFS:
Sobrevida livre de progressão

PGE2:
Prostaglandina E2

TAA:
Antígeno associado ao tumor

Th1:
T ajudante 1

TLR:
Receptor Toll-like

TNF:
Fator de necrose tumoral

T-VEC:
Talimogene Laherparepvec

Declarações
Agradecimentos
Não aplicável.

Disponibilidade de dados e material
Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

Funding
This work was supported by EU grant PROCROP (635122).

Authors’ contributions
KB, GS and KR wrote the manuscript. All authors edited, read and approved the final manuscript.

Ethics approval and consent to participate
Not applicable.

Consent for publication
Not applicable.

Competing interests
AD is an employee of Miltenyi Biotec. All other authors declare no conflict of interest.

Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Acesso Aberto
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