Method for in vitro differentiation and maturation of dendritic cells for therapeutic use

Abstract

The present invention relates to an accelerated method for generating high yields of dendritic cells loaded with type 1 polarized mRNA, which is used for immunotherapy, especially for cancer vaccination.

Description

Field of the Invention

[0001] The present invention relates to an accelerated method for generating high yields of type 1 polarized mRNA-loaded dendritic cells for use in immunotherapy, particularly for cancer vaccination. Background Art

[0002] Since the discovery of dendritic cells more than 40 years ago, translating the unique biological properties of these cells into medical applications has been challenging. Most efforts have focused on introducing DCs into the clinic in the form of anti-cancer vaccines. This is based on the ability of DCs to initiate T cell responses against tumor antigens, leading to the prevention of tumor development and even the eradication of established tumors, as demonstrated by numerous preclinical models.

[0003] Based on this role, there is a unique set of biological properties that are summarized by the "4-signal" concept: (1) presentation of large amounts of processed antigen on major histocompatibility (MHC) molecules, (2) upregulation of a large number of T cell co-stimulatory molecules on the cell surface, (3) release of cytokines that drive the correct polarization of T cell responses, and (4) provision of additional signals that program the tissue homing patterns of the induced T cell effectors. In the specific context of anti-tumor immunity, DCs can pick up dead cells through specialized receptors (such as DNGR-1), leading to MHC I cross-presentation and the initiation of antigen-specific cytotoxic T cells. High expression of the T cell co-stimulatory molecule CD40 increases the magnitude of CD4+ and CD8+ T cell expansion, leading to enhanced tumor protection and the conversion from tolerance to immunity, while upregulation of CD70 is crucial for generating strong and long-lasting memory cytotoxic T cell responses. In contrast, the expression of T cell inhibitory receptors or checkpoint ligands (such as programmed death ligand 1 (PD-L1)) on the DC surface should be minimal.

[0004] Next, the ability to secrete sufficient amounts of bioactive IL-12 upon T cell contact is crucial for driving the type 1 polarized response necessary for optimal tumor control, while also supporting NK cell effector function. Additionally, the pattern of chemokines released by DCs determines which type of T cells will be recruited, i.e., in the case of anti-tumor immunity, preferentially type 1 polarized effectors rather than T-helper (Th) 2 cells (which have tumor-supporting potential) or immunosuppressive regulatory T cells (T-regs).

[0005] Based on this knowledge, it is clear that designing an ideal DC-based cancer vaccine requires maximizing control and optimization of all these key parameters. The correct DC activation or maturation state is crucial in determining the outcome of T cell responses, as immature DCs (iDCs) are very ineffective in stimulating T cell responses and can even enhance T cell tolerance.

[0006] Therefore, careful consideration is required when selecting strong activating stimuli to generate fully effective mature DCs while avoiding the phenomenon of DC "exhaustion". Toll-like receptor (TLR) ligands are among the strongest triggers for DC maturation and can be exogenous (i.e., pathogen-derived) or endogenous (danger-associated molecules from tissue damage or cell death). Despite this knowledge, one of the most commonly used maturation strategies is to expose monocyte-derived DCs to a combination of inflammatory mediators including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin 6 (IL-6), and prostaglandin E2 (PGE2), as first described by Jonuleit et al. The value of adding PGE2 lies in the observation that it can further enhance DC yield, maturity, and migratory capacity (Jonuleit et al., 1997). However, PGE2 has also been shown to impair the ability of DCs to secrete bioactive IL-12p70 and shift the polarization of T helper cells towards Th2 rather than Th1 development (Kalinski et al., 1998).

[0007] Since then, many alternative strategies have been explored to maximize the ability of DCs to induce a type 1 polarization response. Mailliard et al. developed a protocol in which DCs are matured in the presence of the pro-inflammatory cytokines TNF-α, IL-1β, IFN-γ, and interferon-α (IFN-α), as well as the TLR3 agonist poly(I:C) (US7972847; US8691570). Compared to "standard" TNF-α, IL-1β, IL-6, and PGE2-matured DCs, these type 1 polarized DCs (αDC1) produce higher levels of IL-12p70 and induce a more robust expansion of long-lived cytolytic T cells (CTLs) specific for melanoma-associated antigens (Mailliard et al., 2004). Although these αDC1 cells have been used in clinical trials for patients with recurrent malignant glioma (Okada et al. 2011), the complexity of the maturation "cocktail" poses a significant challenge to the implementation of a good manufacturing practice (GMP)-compatible production process.

[0008] A simpler alternative involves combining TLR4 ligands with interferon-gamma (IFN-γ). Lipopolysaccharide (LPS) is one of the strongest innate stimulants of DC maturation and triggers the production of large amounts of immunostimulatory cytokines such as IL-12. However, when LPS-stimulated DCs subsequently engage in cognate interactions with T cells in vivo, they become refractory to further IL-12 release. This "exhaustion" phenomenon can be counteracted by co-exposing DCs with IFN-γ, resulting in a "second burst" of IL-12 production following T cell contact or triggering by artificial CD40 ligation (Paustian et al., 2011). Due to its toxicity and lack of GMP formulation, the use of LPS remains problematic in large-scale cell therapy applications. However, since acid hydrolysis of LPS retains immunostimulatory properties but significantly reduces the toxicity level, LPS-derived monophosphoryl lipid A (MPLA) has been approved for clinical use (Boccaccio et al., 1997). MPLA is an essential component of the adjuvant formulation of currently mass-produced vaccines. It has been reported that in terms of the secretion of IL-12p70 and the chemotactic factor attracting effector T cells, as well as the ability to prime CD4+ and CD8+ T cells, MPLA / IFN-γ DCs and type α1 polarized DCs are equally superior compared to DCs matured with TNF-α, IL-1β, IL-6, and PGE2 (Flansen et al., 2013). The group of Ten Brinke et al. further explored the method of MPLA / IFN-γ DC maturation, in which monocytes cultured for 8 days in the presence of GM-CSF and IL-4 obtained enhanced maturity in the last 2 days of culture. After harvest, the obtained DCs demonstrated the ability to induce de novo Th1 polarization and prime antigen-specific CD8+ T cells with high cytolytic activity (Ten Brinke et al., 2007; WO2007 / 078196; ten Brinke et al., 2010), while retaining the ability to migrate towards CCR7 ligands. This DC culture protocol has been further investigated for possible clinical implementation, and additional studies have shown the adverse effects of human serum on DC maturation and migration in this context (Kolanowski et al., 2014).

[0009] After the correct type of maturation stimulus, the antigen loading method is an important determinant of clinical applicability. Passive loading of DCs with immunogenic peptides (such as those commonly used for functional testing in the studies described above) implies existing knowledge of the immunodominant epitopes for each candidate antigen under consideration and imposes specific restrictions with respect to the human leukocyte antigen (HLA) type of eligible patients. An alternative approach to exploit the high antigen uptake capacity of immature DCs is incubation with tumor lysates. However, this requires sufficient amounts of patient tumor material, which again limits feasibility in metastatic disease, where usually only small biopsy or cytology samples are available. Loading DCs with full-length mRNA encoding tumor antigens has now been widely recognized as an elegant way to induce presentation of a large number of potential epitopes. It also offers the opportunity to co-introduce RNA constructs that can optimize the immunogenic power of DCs (Van Lint et al., 2014). This is usually achieved by cell electroporation, the flip side of which is the risk of substantial cell loss, thus compromising the possibility of administering sufficient vaccine doses to patients (Tuyaerts et al. 2003; Ponsaerts et al. 2003; Bonehill et al. 2004).

[0010] An important additional aspect in terms of vaccine production is the duration of cell culture. For monocyte-derived DCs, this duration has traditionally been in the range of 7 to 8 days, which implies repeated replenishment of the culture with fresh medium and cytokines. In the demanding environment of a GMP production setting, this translates into increased costs in terms of consumables as well as operator intervention. Several groups have demonstrated that it is possible to distinguish fully functional DCs from monocytes using accelerated culture protocols (Jarnjak-Jankovic et al., 2007; Dauer et al., 2003; Kvistborg et al., 2009; Massa et al., 2013; Truxova et al., 2014; EP2829600).

[0011] A final practical consideration is the choice to use a closed-system cell culture: in terms of GMP requirements, this constitutes another advantage and allows transfer of the production process to commercially available automated cell culture equipment.

[0012] Taking these considerations into account, the aim of the present invention is to develop a method for the production of a cancer vaccine based on clinical-grade DCs, thereby recombining several key assets for the first time in one and the same production method: accelerating the culture time and exploiting a GMP-compatible type 1 polarization maturation mixture, combined with antigen loading by mRNA electroporation. Furthermore, it has been demonstrated that this can be achieved under serum-free conditions, using a closed culture system in a GMP-compatible cell culture bag and maximizing the use of GMP-certified or drug-grade components.

[0013] The performance of the method according to the invention was compared with that of the widely established "standard" 8-day cultured monocyte-derived DCs matured with a combination of TNF-α and PGE2. This maturation mixture is well known to those skilled in the art and is a simplified version of the original classical single DC maturation mixture containing TNF-α, PGE2, IL-1b and IL-6 described by Jonuleit et al.

[0014] Importantly, contrary to many previous reports and to closely mimic the real-life vaccination setting, all functional assays with electroporated DCs in the present invention were performed after cryopreservation and thawing, rather than using freshly processed cells. The method according to the invention achieves a higher yield of DCs that are superior both phenotypically and functionally compared to the standard protocol. Surprisingly, we found that the expression of the T cell inhibitory checkpoint ligand PD-L1 on DCs generated according to the invention was significantly reduced compared to DCs obtained by the classical protocol. Moreover, after thawing of cryopreserved aliquots, the expression on classical DCs further increased, which was not the case for thawed DC aliquots obtained by the method of the present invention. This is a crucial observation for cells that will actually be injected into patients, where the expression of such immunosuppressive ligands should be as low as possible. Summary of the Invention

[0016] The present invention relates to an in vitro method for generating mature, preferably autologous, clinical-grade dendritic cells in a closed system (such as a culture with one or more sterile connections), and the method is suitable for vaccination of, for example, cancer patients. In one embodiment, the method mainly comprises differentiating monocytes obtained by leukapheresis by using clinical-grade cytokines, preferably GM-CSF and IL-4, and combining with further maturation of the DCs, thereby generating (such as in a closed system) mature clinical-grade dendritic cells, wherein the further maturation of the DCs is obtained by additional exposure to a combination of a maturation factor, preferably clinical-grade IFN-γ and the endotoxin detoxified derivative MPLA.

[0017] The obtained product is then loaded with one or more antigens, especially tumor-associated antigens (TAAs), and the product contains mature dendritic cells generated / produced within a total in vitro culture time of about 4 days (preferably within a culture of about 3 days to about 5 days). This rapid method according to the invention is capable of generating a large number of DCs from leukapheresis products preferably in a closed system using serum-free medium.

[0018] In one embodiment, the method according to the invention comprises the following steps:

[0019] - obtaining a monocyte leukapheresis product from a patient,

[0020] - Isolate monocytes from said leukapheresis,

[0021] - Incubate said monocytes with clinical-grade cytokines (preferably appropriate amounts of GM-CSF and IL-4) for differentiation into dendritic cells,

[0022] Add maturation factors MPLA and IFN-γ for the final maturation of monocyte-derived dendritic cells, and

[0023] - Recover the obtained cells and transfect them with a nucleic acid sequence (especially mRNA) encoding one or several antigens or epitopes.

[0024] In one embodiment, the monocytes are contacted with GM-CSF and IL-4 for about 1 to 4 days, preferably 1 to 3 days, more preferably 2 to 3 days (24 hours per day), during which the DC precursors differentiate into immature dendritic cells. In a further embodiment, the maturation time in the presence of IFN-γ and MPLA is 1 to 3 days, preferably 1 to 2 days, more preferably about 2 days (24 hours). The culture conditions are suitable for the maturation of immature DCs to form a population of mature DCs.

[0025] In a further embodiment, the present invention provides mature (and transfected) dendritic cells or a population of mature (and transfected) dendritic cells obtainable by the methods provided herein. The present invention also provides compositions, kits, clinical-grade bags or cryovials comprising dendritic cells obtained by the methods of the present invention.

[0026] The transfected dendritic cells are particularly useful for preparing compositions for immunotherapy, especially their use in immunotherapy, more particularly their use in the treatment of cancer. Accordingly, the present invention also provides methods for immunotherapy, tumor therapy or for activating T cells, which comprise administering transfected dendritic cells obtained by the methods provided herein to a subject. Brief Description of the Drawings

[0028] Specific reference is made to the accompanying drawings, and it should be noted that the details shown are by way of example and for the purpose of illustrative discussion of different embodiments of the present invention. They are presented to provide what is considered to be the most useful and easily described content regarding the principles and conceptual aspects of the present invention. In this regard, no attempt is made to show the structural details of the present invention in more detail than is necessary for a basic understanding of the present invention. The description in conjunction with the drawings enables those skilled in the art to clearly understand how to implement several forms of the present invention in practice.

[0029] Figure 1 . Characteristics of 4-day cultured moDCs at harvest: (A) After debris exclusion, CD11c 高 HLA-DR高 Flow cytometric purity of DC; (B) Morphology under light microscopy after preparation of cytocentrifuge smears and May-Grunwald Giemsa staining; (C) Viability and monocyte-to-DC conversion rate (flow cytometry) (n = 33) (box plots show median and 95% C.I.); (D) Cell surface expression of phenotypic and maturation markers, including representative open histograms (relative to gray background staining) and summary box plots (median and 95% C.I.; n = 33), showing relative MFI (geometric mean ratio of positive fluorescence signal to background fluorescence), both gated on live CD11c 高 HLA-DR 高 Gated in DCs.

[0030] Figure 2 . DC characteristics at harvest in comparison between 4-day moDCs and 8-day moDCs (n = 10): (A) Viability and monocyte-to-DC conversion rate (flow cytometry); (B) Comparison of cell surface expression of phenotypic and maturation markers, calculated as relative MFI (ratio of geometric mean of positive fluorescence signal to background fluorescence, gated on live CD11c 高 HLA-DR 高 DCs). Statistics: Wilcoxon paired signed-rank test.

[0031] Figure 3 . Relative contribution of MPLA, IFN-γ, or both to induction of maturation profile in 4-day moDCs at harvest (n = 3). Relative MFI of DC maturation markers, shown as bar graphs. Statistics: Kruskal-Wallis combined with Dunn's multiple comparison test.

[0032] Figure 4 . Combined effect of MPLA and IFN-γ on 4-day moDCs in terms of naive T helper cell polarization potential (n = 6 to 12 replicate samples pooled from repeated experiments, with 2 different DC donors and 3 different allogeneic T cell donors). (A) Schematic of the experimental timeline for allogeneic naive T helper cell polarization assay. (B) Representative dot plots showing CD4+ T cell IFN-γ / IL-10 cytokine production within CD4+ T cells after co-culture with immature or fully mature allogeneic DCs for 14 days. (C) Relative contribution of MPLA, IFN-γ, or their combination to DC-mediated naive T helper cell polarization: Bar graphs represent the percentage of CD4+ cells showing intracellular expression of IFN-γ, IL-10, IL-4, and IL-17.

[0033] Figure 5. (A) Representative dot plots of 4-day moDCs pulsed with the vector (MOCK-EP) or eGFP mRNA (1 μg mRNA / 10e6 DC) EP, showing viable CD11c 高 HLA-DR 高 expression levels of eGFP in DCs. (B) Intensity of eGFP expression levels over time, expressed as the percentage of viable CD11c 高 HLA-DR 高 DCs and relative MFI. The geometric mean of MOCK-EP DCs was used as background staining. The following time points were included in the assay: 4 hours after EP (n = 9), immediately after thawing (n = 9), and 24 hours later in the absence of cytokines (n = 3). (C) Viability (trypan blue) and recovery rate of 4-day moDCs after electroporation with eGFP-mRNA (n = 17). The recovery rate was calculated as the number of live DCs (trypan blue) after electroporation divided by the number of live DCs before electroporation. (D) Comparison of viability (trypan blue) and recovery rate between 4-day and 8-day moDCs after EP with eGFP mRNA (n = 8). (B-C) Statistics: Combined Kruskal-Wallis with Dunn's multiple comparison test; (D) Wilcoxon paired signed-rank test.

[0034] Figure 6 . (A) Cytokine and chemokine secretomes of cryopreserved 4-day (n = 5) and 8-day (n = 2) eGFP mRNA-EP DCs after incubation in cytokine-free medium for 24 hours, as measured using Luminex assays. Statistics: Unpaired t-test. (B) Timeline of co-culture of cryopreserved EP-DCs with allogeneic T helper cells. (C) T cell polarization properties of electroporated DCs after cryopreservation and thawing (light gray bars). Allogeneic naive CD4+ T cells without DCs were used as negative controls (white bars). (n = 3 to 6 replicate samples pooled from repeated experiments, with 2 different DC donors and 1 allogeneic T cell donor). Data show the percentage of CD4+ T cells expressing cytokines. Statistics: Mann-Whitney test.

[0035] Figure 7. (A) Autologous 4d-moDCs electroporated with the designated mRNA or pulsed with the A2-restricted peptide of MART-1, AAAGIGILTV, were used to stimulate MACS-purified CD8+ T cells from HLA-A2-positive donors twice. Representative dot plots show the expansion of tetramer-positive CD8+ T cells. The DCs used in all assays were cryopreserved and thawed. (B) Data summary of CD8+ T cells stimulated with different HLA-A2+ donors and DCs pulsed with MOCK, DCs pulsed with eGFP-mRNA-EP, DCs pulsed with MART-1 mRNA-EP, and DCs pulsed with MART-1 peptide (pooled n = 4 to 8 replicate samples from repeated experiments, with 2 different HLA-A2-positive donors). (C) Levels of intracellular IFN-γ and granzyme B in MART-1-specific CD8+ T cells stimulated with the indicated DC conditions. (B-C) Statistics: Kruskal-Wallis with Dunn's multiple comparison test.

[0036] Figure 8 . (A) Schematic of antigen-specific cytotoxicity assay after autologous DC:CD8 T cell co-culture using HLA-A2+ donors and MART-1 as a model antigen. After stimulation with bi-weekly rounds of autologous 4d-moDCs, cytolytic CD8+ T cells were co-cultured with T2 target cells without, irrelevant peptide-pulsed T2 target cells (influenza peptide), or MART-1 peptide-pulsed T2 target cells. DC counterparts included negative control DCs (MOCK-pulsed DCs (not shown) and DCs pulsed with eGFP mRNA-EP), DCs pulsed with MART-1 mRNA-EP, and positive control DCs (pulsed with the peptide AAAGIGILTV from MART-1 (not shown)). The cytolytic activity of CD8+ T cells was characterized by the simultaneous upregulation of the degranulation marker CD107a and the activation marker CD137, along with the secretion of granzyme B and IFN-γ. (B) Representative dot plots of CD107a / CD137 expression after co-culture of CD8+ T cells pre-stimulated with the indicated DC conditions with T2 cells loaded with MART-1 peptide. (C) Cytotoxic activity of autologous CD8+ T cells (CD107a / CD137 expression) according to the type of previous DC stimulation and T2 target cells (pooled n = 4 to 8 replicate samples from repeated experiments, with 2 different HLA-A2-positive donors). Statistics: Two-way ANOVA with Tukey's multiple comparison test.

[0037] Figure 9. Comparison of DC phenotypes between 8-day TNF-α / PGE2 / IL-1β / IL-6 matured moDCs and 8-day TNF-α / PGE2 matured moDCs (n = 3), as determined at different time points. Any relevant time points in the assay included "at harvest", "4 hours after EP", "immediately after thawing", and "24 hours after cytokine withdrawal". (A) Monocyte-to-DC conversion rate (trypan blue) at harvest; (B) Viability over time (trypan blue); (C) Comparison of cell surface expression of phenotypic and maturation markers over time, calculated as relative MFI (ratio of geometric mean of positive fluorescence signal to background fluorescence, gated on live CD11c 高 HLA-DR 高 DCs); (D) Intensity of eGFP expression level over time, described as percentage of eGFP+ cells and relative MFI in live CD11c 高 HLA-DR 高 DC cells. Geometric mean of MOCK-EP DCs was used as background staining. Statistics: Bar graphs represent median with 95% C.I.

[0038] Figure 10 . Expression levels of the T cell co-inhibitory molecule PD-L1 before and after cryopreservation in comparison between 4-day MPLA / IFN-γ and "classical" moDC protocols. Level of surface PD-L1 expression was calculated as relative MFI (ratio of geometric mean of positive fluorescence signal to background fluorescence, gated within live CD11c 高 HLA-DR 高 DCs). Time points of "at harvest (n = 2) (day 4 or day 8 respectively)" and "immediately after thawing (n = 4)" were included in the assay. In both DC cultures, each donor was divided into two electroporation conditions (i.e., eGFP mRNA and MART-1 mRNA-EP) at harvest for subsequent cryopreservation and thawing.

[0039] Figure 11 . DC viability was evaluated 4 hours after electroporation (or further incubation under non-electroporated conditions), and after freezing and thawing. Short DC culture: 3 days GM-CSF / IL-4; 24 hours MPLA (2,5 μg / mL) and IFN-γ (1000 U / mL). At harvest, DCs were divided into the following electroporation groups:

[0040] · No electroporation (No-EP);

[0041] · Exponential pulse (EXP-EP): 300 V; 150 μF; 200 μl; Ω; + / -5×10E6 DCs / cuvette;

[0042] Square wave pulse (SQW-EP): 500 V; 0.5 ms; 200 μl; 1 pulse; + / - 5×10E6 DCs / cuvette. eGFP mRNA was used at 0.5 μg / 10E6 cells.

[0043] Figure 12 . (A) Flow cytometry analysis of viability and eGFP expression; (B) Stability of DCs after thawing of cryopreserved aliquots, as evaluated by viability of live DCs relative to pre-freeze and efficient recovery. Monocyte-derived DCs were generated from 2 separate donor leukaphereses. At harvest, DCs were electroporated with square wave pulses with the following settings, using 0.5 μg eGFP mRNA / 10E6 cells: 500 V; 1.0 ms; 200 μl; 1 pulse; 50×10E6 DCs / cuvette.

[0044] Figure 13 . Monocyte-derived dendritic cells were generated according to the protocol described in the present invention (“MIDRIX DCs”) or the alt-2 protocol described in Massa et al., 2013 (“Massa DCs”). DCs were harvested at various time points and electroporated with mRNA encoding eGFP. Data are from 6 different donors. (A) Viability and absolute cell yield of live CD11c+ HLA-DR+ dendritic cells obtained with the two protocols at harvest. (B) Expression of the monocyte marker CD14 relative to the DC differentiation marker CD83. (C) Expression of the DC maturation markers CD40, CD70, CD86, and CCR7. (D) Expression of the T cell co-inhibitory receptor PD-L1. (E) Electroporation efficiency, expressed as the level of translated protein (relative mean fluorescence intensity of the eGFP signal) and the proportion of cells in which eGFP-mRNA was successfully translated (percentage of eGFP+ DCs), as measured 4 hours after electroporation. Detailed Description

[0045] The present invention will now be further described. In the following paragraphs, different aspects of the present invention are defined in more detail. Unless explicitly indicated to the contrary, each aspect so defined can be combined with any other aspect. As used in the specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, "compound" refers to one compound or more than one compound. Throughout the specification and claims of this application, the word "comprise" and other forms of this word, such as "comprising" and "comprises", mean including but not limited to, and are not intended to exclude, for example, other additives, components, integers or steps. The terms described above and other terms used in the specification are well known to those skilled in the art. All references cited in this specification, and the specific teachings referred to, are hereby incorporated by reference in their entirety.

[0046] The present invention relates to dendritic cell vaccines, and in particular to the preparation of autologous monocyte-dendritic cell vaccines. Of particular interest is that the method of the present invention can be carried out using a clinically grade fully closed system. A gas-permeable culture bag or container provides the advantages of a closed fluid path culture system, whereby a cell suspension can be added to the culture bag via a sterile connection port. Ideally, the entire cell collection and preselection (if required) is carried out in a closed fluid path system, which is then aseptically connected to a gas-permeable bag to transfer the cells into the bag. Then, the culture medium can be continuously perfused through the bag via a sterile connection port and a sterile piping system, or updated periodically. The cell culture within the gas-permeable bag can be maintained in a gas-regulated atmosphere of an incubator without being exposed to environmental hazards, such as microorganisms, which might be introduced into the culture when the cells are initially introduced into the bag or container, when the culture medium is updated, or when new culture medium is added. During the entire culture period, samples of the cultured cells can be aseptically removed from the bag via a sterile connection port for analysis. Similarly, when the DC culture is ready for harvest, the cells can be aseptically removed for closed system washing and / or further processing. The closed system also opens up the possibility of carrying out cell culture in a clean room environment with a lower strictness in terms of dust count (e.g., a Class C clean room environment). This has advantages in terms of the working conditions of the operator, reduces costs, and also provides the possibility of easily transferring the cell differentiation process to a commercially available automated culture system (e.g., CliniMACS System, Miltenyi Biotec GmBH, Bergisch Gladbach, Germany).

[0047] Accordingly, as used herein, the term "closed system" refers to a combination of components, each component being closed off from the surrounding environment and each component being provided with means for effecting a sterile connection between the components. In one embodiment, the closed system comprises a leukapheresis product, and the differentiation and maturation components provided herein. An example of a GMP-certified gas-permeable culture bag system is the GMP Cell Culture Bag (Miltenyi Biotec GmBH, Bergisch Gladbach, Germany). As used herein, "GMP-certified" refers to Good Manufacturing Practice and describes the minimum standards that pharmaceutical manufacturers must meet during their production processes. The European Medicines Agency (EMA), for example, coordinates inspections to verify compliance with these standards and plays a key role in coordinating GMP activities at the European Union (EU) level.

[0048] In one embodiment, the method of the present invention includes the step of isolating and / or providing a population of dendritic cell precursors. Generally, as used herein, "dendritic cell precursor" is a (human) peripheral blood mononuclear cell, monocyte or another bone marrow progenitor cell. As used herein, "monocyte" refers to a CD14+ mononuclear leukocyte capable of differentiating into dendritic cells. Monocytes can be from any mammal, but are preferably human monocytes. Monocytes can be provided and incubated in a composition such as, but not limited to, blood, blood fractions (e.g., white blood cells (WBC), buffy coat, peripheral blood mononuclear cells (PBMC), monocyte leukapheresis product, and incubated in a composition further enriched in monocytes. In a preferred embodiment, monocytes are provided together with other peripheral blood mononuclear cells (PBMC), such as a monocyte apheresis product. Methods for isolating enriched populations of dendritic cell precursors such as monocytes and conventional dendritic cells from various sources including blood and bone marrow are known in the art. For example, monocytes and conventional dendritic cells can be isolated by collecting heparinized blood, by apheresis or leukapheresis, by preparing buffy coat, rosetting, centrifugation, density gradient centrifugation, differential lysis of cells, filtration, elutriation, fluorescence-activated cell sorting or immunomagnetic separation. In a preferred embodiment, monocytes are isolated from monocyte leukapheresis. Methods of leukapheresis are known in the art. Leukapheresis is a procedure for removing white blood cells from a subject's blood and then transfusing the remainder back to the subject. Leukapheresis products are typically blood fractions rich in PBMC with low levels of contaminating red blood cells, granulocytes and platelets. Methods and devices for performing leukapheresis are well known in the art. Monocytic dendritic cell precursors and / or differentiated conventional dendritic cells can be isolated from healthy subjects or from subjects in need of immune stimulation, such as, for example, cancer patients or other subjects for whom cellular immune stimulation may be beneficial or desired (i.e., subjects with bacterial or viral infections, etc.). Dendritic cell precursors and / or immature dendritic cells can also be obtained from HLA-matched healthy individuals for administration to HLA-matched subjects in need of immune stimulation.

[0049] In one embodiment, monocytes are enriched prior to the differentiation step. The manipulation can be performed on monocytes or PBMCs, etc., including, for example, centrifugation, elutriation, tangential flow filtration, Ficoll density gradient, diluted Ficoll density gradient centrifugation, diluted Percoll density gradient centrifugation, antibody panning, magnetic cell sorting, positive or negative immunomagnetic selection, etc. Additionally, once isolated from a subject, monocytes (e.g., purified monocytes, enriched monocytes, PBMCs containing monocytes, etc.) can optionally be incubated, for example, maintained at a temperature of 1°C - 34°C for a period of time, such as approximately 1 to 96 hours after the time they are isolated from the subject.

[0050] In a particular embodiment, monocyte progenitors are obtained by immunomagnetic separation from leukapheresis. Even more particularly, the population of viable monocyte DC precursors is highly purified, for example, with a purity exceeding 90%, 95% or even 99%, as determined by flow cytometry using monocyte marker CD14 and viability staining.

[0051] Thus, the first step of the method disclosed herein includes providing isolated (autologous) monocyte DC precursors, particularly using the closed system provided herein. Generally, the density of DC precursor cells in the culture bag or container at the start of cell culture is 0.5×10E6 to 2×10E6 cells / ml, preferably about 1×10E6 cells / ml, as determined by methods known in the art.

[0052] After isolation, purification and / or enrichment, DC precursors are induced to differentiate into dendritic cells. Thus, in a further embodiment, the method of the present invention includes a culture and / or differentiation step to obtain immature DCs, for example, culturing the precursor cells in the presence of at least granulocyte - macrophage colony - stimulating factor (GM - CSF) and interleukin - 4 (IL - 4) (referred to as the differentiation medium), and this is continued for about 48 to 96 hours, more particularly 48 to 84 hours, even more particularly up to 80, 75, 74, 73, 72, 71, 70 hours or less, more particularly up to at least 48 hours and up to 72 hours. Limits of + / - 4 hours or + / - 2 hours are acceptable and may be necessary in view of practical constraints. In a specific embodiment, the isolated DC precursors are transferred through a closed system into a gas - permeable culture bag containing (serum - free) differentiation medium.

[0053] The use concentrations of GM-CSF and IL-4 are about 100 U / ml to 5000 U / ml, preferably 500 U / ml to 2500 U / ml, more preferably 500 U / ml to 1500 U / ml or about 500 to 1000 U / ml for each cytokine. In particular, the use concentration of GM-CSF can be 500 U / ml to 2500 U / ml, preferably 1000 to 1500 U / ml, and more preferably about 1000 U / ml. More specifically, the use concentration of IL-4 can be 500 U / ml to 2500 U / ml, preferably 500 to 1500 U / ml, more preferably 500 to 1000 U / ml, and even more preferably about 500 U / ml.

[0054] After monocytes differentiate into immature dendritic cells, the immature dendritic cells can mature into mature dendritic cells. Thus, in one embodiment, the method of the present invention includes a maturation step, such as adding interferon gamma (IFN-γ) and monophosphoryl lipid A (MPLA) (referred to as maturation stimulants or mixtures) to (differentiated) immature DCs, and this process lasts for at least 30 hours, preferably at least 24 hours. In particular, during the cell culture in the last 24 hours + / - 4 hours (especially + / - 2 hours) before harvesting and / or transfection, the maturation stimulants IFN-γ and MPLA are added to the culture medium.

[0055] The use concentration of IFN-γ is 500 U / ml to 2000 U / ml, preferably 500 U / ml to 1500 U / ml, even more preferably 500 U / ml to 1000 U / ml, and in a particular embodiment is about 1000 U / ml. The use concentration of MPLA is 1 to 20 μg / ml, more particularly 1 to 10 μg / ml, even more particularly 1 to 5 μg / ml. In a particular embodiment, MPLA is used at a concentration of about 2.5 μg / ml. In a further embodiment, IFN-γ is pharmaceutical grade or GMP-certified recombinant human IFN-γ. As used herein, a "pharmaceutical grade" compound refers to any active or inactive drug, biologic, or reagent for which a chemical purity standard has been established by a recognized national or regional pharmacopoeia.

[0056] Thus, according to the present invention, precursors and / or immature dendritic cells are cultured with a combination of (at least) the above factors, i.e., differentiation and / or maturation factors. This can be done by adding the factors to the culture medium. Alternatively, the culture medium in which precursor cells and / or immature dendritic cells have been grown is replaced with a culture medium that already contains the factors. In a further embodiment, the above substances are added or can be part of a composition added to the culture medium of the cells. The culture medium can be of any suitable type, i.e., it can be supplemented or not with any other supplements such as proteins, amino acids or antibiotics. In a particular embodiment, the culture medium is produced and used under GMP conditions. Even more particularly, the culture medium is serum-free, such as serum-free GMP (CG) medium (CellGenix GmBH, Freiburg, Germany). In an embodiment of a fully closed system, precursor cells can be transferred to a culture bag containing DC differentiation medium as provided herein. In a second step, approximately 48 hours (plus or minus 4 hours) later, the maturation stimulants IFN-γ and MPLA are added to the medium and cells in the culture bag.

[0057] Furthermore, it is an object of the present invention to provide an "accelerated" in vitro cell differentiation method for generating clinical-grade dendritic cells (DCs) with strong Th1 polarization ability and combined with efficient presentation of nucleic acid-encoded antigens. Generally, the duration of the DC culture protocol of the present invention is limited to about 4 days, rather than the "standard" protocol of 8 days.

[0058] When evaluated on a series of different donors, both the DC viability and the monocyte-to-DC conversion rate using the method of the present invention are significantly higher compared to the standard protocol (e.g., regarding the conversion rate: method of the present invention: about 45%, standard method: about 25%).

[0059] Phenotypically, the cells obtained by the method provided herein exhibit the basic characteristics of dendritic cells, including:

[0060] - A typical dendritic cell morphology evaluated by light microscopy,

[0061] - Uniform expression of the DC differentiation markers CD11c, class II MHC (HLA-DR) and CD83,

[0062] Uniform downregulation of the monocyte marker CD14.

[0063] Regarding the state of maturity of DCs, this is evaluated by measuring the expression of specific cell surface markers, where preferably T cell co-stimulatory molecules among them are analyzed by flow cytometry. In that case, the expression levels are given as relative mean fluorescence intensity (MFI) (the ratio of the geometric mean of the positive fluorescence signal to the background fluorescence), as determined by commonly known methods. T cell co-stimulatory molecules are generally evaluated as maturity markers, and the expression on the surface of DCs of such maturity markers should be as high as possible. Conversely, efforts are made to keep the expression of T cell co-inhibitory molecules on the final DC product as low as possible.

[0064] DCs obtained by the method according to the invention demonstrate that:

[0065] - a uniform upregulation of T cell co-stimulatory molecules (CD40, CD70, CD86), and

[0066] - a uniform expression of the lymphoid tissue homing chemokine receptor CCR7.

[0067] The median levels of CD40, CD70, and CCR7 are higher and statistically significant (two-tailed p-value < 0.05) compared to those shown by DCs generated using a "classical" protocol.

[0068] In one embodiment of the invention, the expression levels of cell surface markers on the DCs generated herein are compared with the expression levels of cell surface markers on DCs generated using a "classical" method that uses PGE2 and TNF-α as maturation stimulants, thereby obtaining mature dendritic cells after 8 days.

[0069] For example, for the T cell co-inhibitory ligand PD-L1, the cell surface level expressed as relative mean fluorescence intensity (relMFI) (for a description of the analysis method used, see the "Materials and Methods" section under "Examples") is below 400, 350, 320, 310, 300, 250, 200, especially below 150. Additionally, on DCs produced according to the invention, the expression of surface PD-L1 after electroporation, cryopreservation, and cell thawing (i.e., representing the time of product administration to the patient) is below 500, 470, 450, especially below 400.

[0070] Thus, at the time of cell harvest (directly after DC maturation), the PD-L1 expression level of DCs generated by the method according to the invention is at least 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold lower, especially at least 10-fold lower, than that of DCs generated using a "classical" method that uses PGE2 and TNF-α as maturation stimulants, thereby obtaining mature dendritic cells after 8 days (see Examples Figure 10)。Therefore, upon thawing of the cells, the PD-L1 expression level of the DCs generated by the method according to the invention is at least 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, and particularly at least 10-fold lower than that of the DCs generated by using the "classical" method, which uses PGE2 and TNF-α as maturation stimulants, whereby mature dendritic cells are obtained after 8 days (see Figure 10 )).

[0071] Functionally, after cryopreservation and thawing, during the extended incubation in cytokine-free medium, the cells retain the ability to secrete type 1 polarized cytokines (IL-12, IFN-γ) and chemokines that attract Th1, CD8, and NK cells. In particular, compared to the DCs obtained by the above "classical" method, the DCs produced by the method according to the invention secrete statistically significantly higher levels of the CXCR3 ligands CXCL9 (MIG30) and CXCL10 (IP-10), as well as the CCR5 ligands CCL3 (MIP-1α), CCL4 (MIR-1β), and CCL5 (RANTES), and the level of CCL17 is undetectably low. In one embodiment, the cytokine and chemokine secretion levels are expressed in relative terms, i.e., when compared to the secretion levels of the resp cytokines or chemokines of the DCs generated by the "classical" method, which uses PGE2 and TNF-α as maturation stimulants and thereby obtains mature dendritic cells after 8 days. The secretion levels can be determined by using standard protein measurement methods, such as ELISA or the method provided herein.

[0072] For example, for the prototype type 1 T cell polarization and the chemokine IL-12 that supports NK cells, after thawing and further culturing under the above conditions, the levels released in the dendritic cell supernatant range from:

[0073] · For DCs generated by the method according to the invention: 50 to 250 pg / ml; particularly 60 to 200 pg / ml; more particularly 70 to 150 pg / ml;

[0074] · For DCs generated according to the "standard" 8-day protocol: 0 to 35 pg / ml but less than 50 pg / ml.

[0075] For the chemokine CXCL10 (which is important in recruiting type 1 polarized T cells and NK cells), after thawing and further culturing under the above conditions, the levels released in the dendritic cell supernatant range from:

[0076] For DC generated according to the method of the invention: 200 to 2000 pg / ml; in particular 250 to 1800 pg / ml; more particularly 280 to 1600 pg / ml;

[0077] For DC produced according to the "standard" 8-day protocol: 0 to 5 pg / ml but less than 10 pg / ml.

[0078] Thus, for CXCL10, the secretion level by DC generated according to the method of the invention is at least 50-fold higher than the secretion level by DC generated using the "classical" method. Similar advantages of DC obtained by the method of the invention are observed with additional cytokines and chemokines that promote a type 1 polarized inflammatory response required for anti-cancer immunity, among which are IFN-γ, CCL3, CCL4, CCL5, and CXCL9.

[0079] In contrast, for the chemokine CCL17 (involved in the recruitment of regulatory T cells and type 2 polarized T cells, both of which are detrimental to the anti-cancer immune response), the release by DC generated according to the method of the invention is at least 3-fold lower than the secretion by DC generated using the "classical" method.

[0080] Thus, DC obtained by the method of the invention drives the differentiation of naive T helper cells towards a type 1 polarized phenotype, which is characterized by high IFN-γ secretion, as required for example in active cancer immunotherapy. Furthermore, the cells can present immunogenic epitopes derived from transfected mRNA and subsequently drive the expansion of autologous tumor antigen-specific CD8+ T cells that express IFN-γ and the cytotoxic molecule granzyme B, as already mentioned.

[0081] In a further embodiment, the method of the invention comprises loading or transfecting mature DCs with antigen-encoding nucleic acids, in particular RNA, more particularly mRNA. As used herein, "antigen" is not limited to the invention. In one embodiment, the antigen is selected from the group consisting of: tumor antigens, tumor-associated antigens, testicular cancer antigens, antigens from mutant pools, (oncogenic) viral antigens, bacterial antigens, yeast antigens, parasitic antigens, and fungal antigens. The antigen can be autologous to the subject and can be used to prepare an autologous DC vaccine loaded with the antigen for administration to the subject. Autologous to the subject means that the antigen (or its sequence) is obtained from or derived from the same subject. As a non-limiting example, the antigen can be from cancer cells or tumor tissue obtained from the subject. The cancer antigen can be loaded into dendritic cells as cancer cells, cancer cell or tissue lysates, cancer cell or tissue extracts, purified or cloned components of cancer cells or tissues, total RNA or total mRNA, or selected RNA or mRNA from these cells or tissues (whether present in the extract, purified, amplified, in vitro translated, etc.). Alternatively, the antigen can be obtained from or derived from a pathogen present in the subject or a cell infected with the pathogen. The term "nucleic acid" refers to single-stranded, double-stranded and triple-helical molecules, genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. More specifically, the dendritic cells are transfected in vitro with one or more mRNAs encoding the antigen. Optionally and in alternative embodiments, after completion of the maturation phase, the DCs can be first harvested before further processing (e.g., transfection), whereby the cells are collected, centrifuged, and / or the cytokines are washed out.

[0082] Taking into account the accelerated culture protocol, transfection of mature DCs may occur at about 72 to 96 hours, particularly 86 hours + / - 4 hours, after monocyte isolation or addition of the differentiation stimulant to the precursor DCs.

[0083] In the context of the present invention, transfection methods include, but are not limited to, electroporation, photoporation, lipofection, viral vector systems, incubation with naked nucleic acids, or fusion of DCs with infected cells or tumor cells. These standard methods are well known in the art and are feasible and introduce nucleic acids, such as antigen-encoding plasmids, their RNA or DNA, into DCs. There may also be other antigen combinations with the original MHC molecules, such as membrane fragments or exosomes that can conceivably serve as antigen sources of any kind. In a specific embodiment, mature DCs are transfected by electroporation. Three different types of pulses can be used for electroporation, the three different types of pulses being, for example, exponentially decaying pulses, square wave pulses, and time constants. In a particular embodiment of the invention, the electroporation consists of square wave pulses. Typically, 1 or 2 pulses are induced to complete the transfection.

[0084] In one embodiment of the invention, antigen is loaded by electroporating dendritic cells with nucleic acid, preferably mRNA. Preferably, the dendritic cells are transfected with about 0.25 to 4 μg RNA per 10E6 dendritic cells, and most preferably with about 1 to 3 μg RNA per 10E6 dendritic cells. In one embodiment, 1-2 μg of antigen RNA per million DC is used per transfection.

[0085] It is demonstrated herein that cells obtained by the method of the invention express the protein derived from the transfected mRNA uniformly and stably (see Examples). In addition, the cells can present immunogenic epitopes derived from the transfected mRNA and subsequently drive the expansion of autologous tumor antigen-specific CD8+ T cells with cytotoxic characteristics, as required for, for example, active cancer immunotherapy.

[0086] In the context of the present invention, it has been found that stimulation of immature dendritic cells as provided herein can generate mature dendritic cells with improved viability, function, and / or immunostimulatory activity at a shortened incubation time (e.g., within about 3 days) compared to mDCs prepared by an 8-day "classical" protocol.

[0087] As used herein, the term "immunostimulatory activity" refers to the ability of mature dendritic cells or a population of mature dendritic cells to produce and / or secrete sufficient amounts of specific cytokines and chemokines (in particular IL-12 and CXCL10), which mediate the correct differentiation and mobilization of type 1-polarized effector T cells and NK cells, as required for immunity against cancer and specific pathogens.

[0088] In one embodiment of the invention, the loaded / transfected dendritic cells can be frozen in a composition containing a cryoprotectant. Many cryoprotectants and methods for freezing DCs are known to those skilled in the art. For example, the dendritic cells are cooled using a controlled-rate freezer and transferred to the vapor phase of a liquid nitrogen container for cryopreservation and storage. In particular, the dendritic cells are resuspended in a suitable cryopreservation medium in 100 μL volume aliquots at 20 - 70×10E6 viable cells / mL, more specifically about 40 - 60×10E6 viable cells / mL, and even more specifically about 50×10E6 viable cells / mL. In a further step, the thawed dendritic cell vaccine is ready to be administered to a subject at any time (usually up to about 4 hours) after thawing.

[0089] In one embodiment, the invention provides cryovials containing cryopreserved, mature, transfected, particularly electroporated DCs as provided herein, particularly in an amount of about 5×10E6 cells per 100 μL, as measured before cryopreservation.

[0090] The present invention further provides a method for administering a dendritic cell vaccine loaded with an antigen, the method comprising thawing cryopreserved live dendritic cells prepared according to the method provided herein and administering them to a subject.

[0091] The present invention also provides the use of antigen-loaded dendritic cells obtained by the methods disclosed herein as a medicament, in particular for the preparation of a medicament or a pharmaceutical composition. The present invention provides the DCs or compositions described herein for use in immunotherapy, in particular for the treatment or prevention of cancer or pathogen infection.

[0092] In a further aspect, the present invention encompasses a pharmaceutical composition comprising mature dendritic cells according to the present invention and a pharmaceutically acceptable carrier and / or excipient. Furthermore, the present invention also relates to mature dendritic cells or populations of mature dendritic cells of the present invention for use in a method of treating a disease selected from the group consisting of: malignant disorders (cancer), certain non-malignant disorders (such as LAM lung disease (lymphangioleiomyomatosis)), and infectious diseases (such as those caused by viruses, bacteria, intracellular bacteria or fungi). Furthermore, the present invention relates to a method for treating a patient suffering from a neoplastic disease (such as cancer) or an infectious disease, wherein an effective amount of mature dendritic cells of the present invention is administered to the patient.

[0093] The antigen-loaded dendritic cells of the present invention can be used as a vaccine for the treatment or prevention of diseases or for the activation of T cells. For example, antigen-loaded dendritic cells can be used to elicit an immune response against an antigen. They can be used as a vaccine to prevent future infections or diseases ("prophylactic vaccination"), or to activate the immune system to treat an ongoing disease ("therapeutic vaccination"), such as but not limited to pathogen infection or cancer. The antigen-loaded dendritic cells prepared as herein can be formulated to be used as a vaccine or a pharmaceutical composition with a suitable carrier such as a physiological buffer or other injectable liquid. The vaccine or pharmaceutical composition is administered in a therapeutically effective amount sufficient to elicit an immune response.

[0094] As used herein, the terms "treatment" and "treating" generally refer to obtaining the desired pharmacological and / or physiological effect, and encompass the treatment of any disease in mammals, particularly humans, including:

[0095] (1) preventing a disease or symptom from occurring in a subject who may be predisposed to the disease or symptom but has not yet been diagnosed as having the disease or symptom;

[0096] (2) inhibiting the disease symptom, i.e., preventing its development; or

[0097] (3) alleviating the disease symptom, i.e., causing the regression of the disease or symptom.

[0098] With respect to the complete or partial prevention of a disease or its symptoms, the effect can be prophylactic, and / or with respect to the partial or complete stabilization or cure of a disease and / or side effects attributable to the disease, the effect can be therapeutic. In addition, in addition to the primary or initial therapy, the vaccine can also be used as an "adjuvant therapy" to maximize its effectiveness in a therapeutic setting, or as a "maintenance" or "consolidative" therapy after the initial therapy to maximize disease control and delay disease recurrence.

[0099] In the context of the present invention, the term "cancer" refers to any kind of disease caused by a malignant tumor. As used herein, the term "infectious disease" refers to any kind of clinically apparent disease caused by the presence of a pathogenic microorganism agent (including pathogenic viruses, pathogenic bacteria, fungi, protozoa or multicellular parasites).

[0100] Methods for formulating dendritic cell vaccines are known to those skilled in the art. Suitable formulations for administration can include aqueous isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostatic agents and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, wherein the suspensions can include suspending agents, solubilizing agents, thickening agents, stabilizing agents, preservatives, immunostimulating agents, cytokines and adjuvants.

[0101] Dendritic cell compositions / vaccines can be administered by a variety of methods, such as but not limited to injection (e.g., subcutaneous, intradermal, intravenous, intralymphatic, intra-articular, intramuscular, intraperitoneal), by continuous infusion, slow release from implants, etc. DC vaccines can be administered at specific intervals. In one embodiment, DCs are administered at intervals of 2 to 4 weeks, especially at two-week intervals. Dendritic cell vaccines can be administered together with physiologically acceptable carriers, buffers, diluents, adjuvants, immunomodulators, etc. Preferably, the dendritic cell vaccine is autologous to the patient to whom it is administered, or is HLA-matched to the greatest extent possible.

[0102] The cell dose administered to the subject is an effective amount that may one day effectively achieve a desired beneficial therapeutic response in the subject, or inhibit the growth of cancer cells, or inhibit an infection, while maintaining a favorable tolerance profile (minimal toxicity). The amount sufficient to accomplish this effect is defined as a "therapeutically effective dose". This dose will be determined by the biological and / or clinical activity of the dendritic cells generated and optionally the condition of the patient. The magnitude of this dose will also be determined by the presence, nature, and extent of any adverse side effects attendant to the administration of the specific cells in a particular patient. When determining the effective amount of cells for treating or preventing a disease such as cancer (e.g., metastatic melanoma, prostate cancer, etc.), the physician (or investigator) needs to evaluate the immune response against the targets included in the vaccine (i.e., immune monitoring), as well as evaluate tumor clinical progression using measurable parameters (radiological tumor burden by conventional or immune-related RECIST criteria, tumor markers, circulating tumor cells, plasma circulating tumor DNA, or other surrogate markers of disease burden or disease activity).

[0103] As is well known to those skilled in the art, there is no evidence that a preferred dose of DCs to be administered achieves a specific level of biological and / or clinical effect. Similarly, no clear dose-limiting toxicity (DLT) has been observed, and thus no maximum tolerated dose (MTD) has been observed. The most frequently administered dose depends on the yield of DCs obtained from a single leukapheresis and the number of subsequent vaccinations required. In one embodiment, the dose falls within 5 - 100×10E6 DCs per vaccination round, repeated 2 to 8 times, particularly 2 to 6 times, more particularly 2 to 4 times. Similarly, there is no relationship between the number of cells injected and toxicity. The toxicity of DC vaccination is generally low and is related to the route of administration (the intravenous route has greater acute side effects compared to the intradermal route). The injections can be repeated, for example, 2, 3, 4, 5, or 6 times at intervals of 1, 2, or 3 weeks and should be administered by: intravenous injection or by intradermal or subcutaneous injection near a lymph node or directly into a lymph node. A booster injection can be given after a pause of, for example, 1 to several months.

[0104] Optionally, a biological response modifier is added for treatment with the DCs or activated T cells of the present invention. For example, optionally the cells are administered together with an adjuvant or cytokines such as GM-CSF, IL-12, IFN-α, or IL-2.

[0105] All features (including any appended claims, abstract, and drawings) described herein and / or all steps of any method or process so disclosed may be combined in any combination with any of the above aspects, unless there is at least some combination of such features and / or steps that are mutually exclusive.

[0106] The present invention will be further described by the following figures, tables and examples, which are not intended to limit the scope of protection defined in the claims. The methods and experiments described in the examples mainly relate to the preclinical development using anonymized donor buffy coats as starting materials.

[0107] Embodiments

[0108] Materials and Methods

[0109] Monocyte-derived dendritic cell culture

[0110] Buffy coats were obtained from local blood transfusion centers and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-paque density gradient centrifugation (GE Healthcare Life Science, Chicago, Illinois, USA). Monocytes were immunomagnetically purified using human anti-CD14 immunomagnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol. Purity of >90% was consistently obtained as assessed by flow cytometry (data not shown).

[0111] The monocyte-depleted fraction (peripheral blood lymphocytes (PBLs)) was frozen in RPMI-GlutaMAX medium (Invitrogen by Life Technologies, California, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, Missouri, USA), 100 U / ml penicillin / streptomycin (P / S) (Gibco, Life Technologies, California, USA) and 10% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Missouri, USA).

[0112] For our accelerated (i.e., 4-day) DC culture protocol, monocytes were cultured at a density of 2×10E6 cells / ml in serum-free GMP CellGro (CG) medium (CellGenix GmBH, Freiburg, Germany) in 30 ml GMP cell differentiation bags (Miltenyi Biotec, Bergisch Gladbach, Germany), where the medium contained 1000 U / ml pharmaceutical-grade granulocyte macrophage colony-stimulating factor (GM-CSF) (Leukine (Berlex), Bayer Healthcare Pharmaceuticals, New Jersey, USA), 1000 U / ml GMP-certified recombinant human interleukin 4 (huIL-4) (Miltenyi Biotec, Bergisch Gladbach, Germany), and 100 U / ml P / S (Gibco, Life Technologies, California, USA). On day 3, 2.5 μg / ml synthetic MPLA (Invivogen, California, USA) and 1000 U / ml pharmaceutical-grade IFN-γ (Immukine, Boehringer Ingelheim BV, Ingelheim, Germany) were added to the medium and incubated for an additional 24 hours. Mature DCs (mDCs) were harvested on day 4.

[0113] For the “classical” (8-day) protocol, monocytes were cultured at a density of 1×10E6 in the same complete medium in polystyrene culture flasks (Nunc, Thermo Fisher Scientific, Massachusetts, USA), except that the concentration of recombinant huIL-4 (250 U / ml; Miltenyi Biotec, Bergisch Gladbach, Germany) was lower and 1% pooled human AB serum (huAB serum) (Sigma-Aldrich, Missouri, USA) was added. On day 3 or 4, fresh medium containing GM-CSF and IL-4 was added. On day 6, 20 ng / ml recombinant human TNF-α (Miltenyi Biotec, Bergisch Gladbach, Germany) and 2.5 μg / ml pharmaceutical-grade PGE2 (Prostin E2, Pfizer, New York, USA) were added to the medium and incubated for an additional 48 hours. Mature DCs were harvested on day 8.

[0114] DC Phenotype Analysis

[0115] For surface staining, cells were first washed and then resuspended in phosphate-buffered saline (PBS) (Invitrogen by Life Technologies, California, USA), and then incubated with a combination of FcR blocker (Miltenyi Biotec, Bergisch Gladbach, Germany) and fixable viability dye eFluor 506 (eBioscience by ThermoFisher Scientific, Massachusetts, USA) for 20 minutes at 4°C to stain dead cells.

[0116] Next, the cells were washed with FACS buffer, which consisted of: PBS (Invitrogen by Life Technologies, California, USA) supplemented with 0.5 mM ethylenediaminetetraacetic acid (EDTA); 0.25% bovine serum albumin (BSA); and 0.05% NaN3 (all from Sigma-Aldrich, Missouri, USA), and then surface antibodies (Abs) were added for 30 minutes at 4°C. The following fluorescent dye-conjugated monoclonal antibodies were used: anti-CD40 FITC; anti-HLA-ABC FITC; anti-CCR7 APC; anti-CD11c Alexa Fluor 700; anti-HLA-DR APC-Cy7 (eBioscience by Thermo Fisher Scientific, Massachusetts, USA); anti-HLA-A2 FITC; anti-DNGR-1 PE; anti-CD86 PE Texas Red; anti-CD83 PE-Cy7; anti-PD-L1 Pacific Blue (BD Biosciences, New Jersey, USA); anti-CD70 PE; and anti-CD14 Pacific Blue (Miltenyi Biotec, Bergisch Gladbach, Germany).

[0117] Samples were acquired on an LSR Fortessa analytical flow cytometer (BD Biosciences, New Jersey, USA) and analyzed using FlowJo software (version 9.9.4; BD Biosciences, New Jersey, USA). The expression levels of phenotypic and maturation markers were shown as relative mean fluorescence intensity (MFI) (the ratio of the geometric mean of the positive fluorescence signal to the background fluorescence, gated on live CD11c 高 HLA-DR 高 DCs).

[0118] Luminex assay

[0119] Frozen aliquots of 4-day and 8-day monocyte-derived DCs (moDCs) were thawed and cultured in serum-free and cytokine-free CG medium (CellGenix GmBH, Freiburg, Germany) supplemented with 100 U / ml P / S (Gibco, Life Technologies, California, USA). DC culture supernatants were collected and analyzed using a Luminex assay (R&D Systems, Minneapolis, USA), which was customized to include the following human cytokines and chemokines: IL-12p70; IFN-γ; IL-10; CCL3; CCL4; CCL5; CXCL9; CXCL10; CCL17; CCL20; and CXCL12. The Luminex assay was analyzed on a Bio-Plex (Bio-Rad, California, USA) reader.

[0120] mRNA electroporation of DCs

[0121] After harvesting, on day 4 or day 8 respectively, the DCs were electroporated and then cryopreserved in Plasma-Lyte A (Baxter, Illinois, USA), which was enriched with 3.5% human serum albumin (Sanquin, Amsterdam, The Netherlands); 6.25% hydroxyethyl starch (HES) (Grifols, Barcelona, Spain); and 6.25% DMSO (Sigma-Aldrich, Missouri, USA). The eGFP mRNA was derived from the pST1-eGFP2 plasmid, which was kindly provided by Professor K. Thielemans of the Laboratory of Molecular and Cellular Therapy (LMCT), Free University of Brussels. The plasmid was first linearized using the Sapl restriction enzyme (New England Biolab, Massachusetts, USA) and then in vitro transcribed into mRNA using the mMESSAGE mMACHINE T7 Ultra kit (Ambion by Thermo Fisher Scientific, Massachusetts, USA). The MART-1 mRNA was also donated by the LMCT. As described by Bonehill et al. (2004), the open reading frame of MART-1 was fused with the HLA class II targeting sequence of the lysosomal protein DC-LAMP1. 4 to 16×10E6 DCs were resuspended in 170 μl of serum-free CG medium (CellGenix GmBH, Freiburg, Germany), which was supplemented with 30 μl of mRNA dissolved in nuclease-free water at a dose of 1 μg mRNA / 10E6 DCs, and transferred to a 4 mm gap cuvette (Bio-Rad, California, USA). Electroporation using exponential wave pulses was performed using the Gene Pulser Xcell electroporation system (Bio-Rad, California, USA) with the following parameters: capacitance 150 μF; voltage 300 V; resistance. Immediately after EP, the DCs were placed on an ultra-low attachment plate (Corning, New York, USA) at 37 °C and 5% CO 2They were then allowed to recover for 4 hours in CG medium (CellGenix GmBH, Freiburg, Germany) supplemented with 1000 U / ml GM-CSF (Leukine (Berlex), Bayer Healthcare Pharmaceuticals, New Jersey, USA), recombinant huIL-4 (1000 U / ml or 250 U / ml, depending on the DC type; Miltenyi Biotec, Bergisch Gladbach, Germany) and 100 U / ml P / S (Gibco, Technologies, California, USA). MOCK-EP DCs were electroporated with the same pulse settings in CG medium without mRNA (CellGenix GmBH, Freiburg, Germany). Electroporation using square wave pulse electroporation (SQW-EP) was performed using the same electroporation system with the following parameters: voltage 500 V; 0.5 ms; 200 μl; 1 pulse; 5×10E6 or 50×10E6 DCs / well.

[0122] Allogeneic T helper cell polarization assay

[0123] The electroporated and cryopreserved DCs were thawed and allowed to equilibrate at 37 °C and 5% CO in warm RPMI-GlutaMAX medium (Invitrogen by Life Technologies, California, USA). 2Recover for at least 1 hour, and the medium is supplemented with 10% huAB serum (Invitrogen by Life Technologies, California, USA) and 100 U / ml P / S (Gibco, Life Technologies, California, USA) and used as a stimulator. As responders, naive CD4+ T cell isolation kit II (both from Miltenyi Biotec, Bergisch Gladbach, Germany) was used on an AutoMACS cell separator to enrich CD45RO-negative T helper cells from allogeneic PBL. DCs and T cells were co-cultured in RPMI-GlutaMAX medium (Invitrogen by Life Technologies, California, USA) at a 1:5 DC:T cell ratio for 14 days, and the medium was supplemented with 10% huAB serum (Sigma-Aldrich, Missouri, USA) and 100 U / ml P / S (Gibco, Life Technologies, California, USA). 10 ng / ml recombinant human IL-2 (R&D Systems, Minneapolis, USA) was added on day 7 of co-culture, and additionally, for control conditions without DCs, it was added on days 3 and 10.

[0124] At the end of allogeneic co-culture, 50 ng / ml phorbol 12-myristate 13-acetate (PMA); 1 μg / ml ionomycin (iono) and 10 μg / ml brefeldin A (BFA) (all from Sigma-Aldrich, Missouri, USA) were added at 37 °C and 5% CO 2Incubate for 5 hours continuously, then harvest the cells for flow cytometry staining. Antibodies for detecting surface T cell markers include anti-CD3 PerCP-Cy5.5; anti-CD8a PE-Cy7 (BioLegend, California, USA); anti-CD4 APC-Cy7 (BD Biosciences, New Jersey, USA); and anti-CD45RO PE-Cy7 (eBioscience by Thermo Fisher Scientific, Massachusetts, USA). For intracellular (IC) staining, after surface staining, wash the cells with FACS buffer and treat with Cytofix / Cytoperm (BD Biosciences, New Jersey, USA) according to the manufacturer's operating procedures, then incubate with the following antibodies at 4 °C for 30 minutes: anti-IL-4 FITC (BD Biosciences, New Jersey, USA); anti-IL-10 PE; anti-IL-17A APC; and anti-IFN-γ Pacific Blue (eBioscience, Thermo Fisher Scientific, Massachusetts, USA).

[0125] Expansion of antigen-specific autologous CTL

[0126] Buffy coats from HLA-A2+ donors were used to generate 4-day MPLA / IFN-γ matured DCs, which were harvested and frozen 4 hours later (i.e., non-EP Dc), or electroporated with either a vector (i.e., eGFP mRNA-EP DC) or antigen MART-1 mRNA (i.e., MART-1 mRNA-EP DC) before cryopreservation. For more details on DC culture and manipulation, please refer to the "Monocyte-derived dendritic cell culture" and "mRNA electroporation of DCs" sections in the Materials and Methods above.

[0127] After thawing, non-EP DCs; eGFP mRNA-EP DCs; and MART-1 mRNA-EP DCs were cultured in RPMI-GlutaMAX medium (Invitrogen by Life Technologies, California, USA) at 37 °C and 5% CO 2Recover for at least 1 hour at, and the medium is supplemented with 10% huAB serum (Invitrogen by Life Technologies, California, USA) and 100 U / ml P / S (Gibco, Life Technologies, California, USA). Thereafter, half of the non-EP DCs are pulsed with 10 μM of an optimized immunodominant HLA-A*201-restricted peptide from MART-1 (AAAGIGILTV; SEQ ID NO 1) (Genscript, New Jersey, USA) (Valmori D. et al. 1998) as a positive control condition. Half of the non-EP DCs are pulsed with only the vector and used as a negative control condition (MOCK-pulsed DCs). After incubation at 37 °C and 5% CO 2 for at least 1 hour, unbound peptides are washed away using the same medium as above.

[0128] CD8+ T cells were purified from cryopreserved autologous CD14-negative fractions using a positive immunomagnetic selection kit (Miltenyi Biotec, Bergisch Gladbach, Germany). DCs and T cells were co-cultured at a 1:10 ratio in RPMI-GlutaMAX medium (Invitrogen by Life Technologies, Califotnia, USA) supplemented with 10% huAB serum (Invitrogen by Life Technologies, California, USA) and 100 U / ml P / S (Gibco, Life Technologies, California, USA) for 14 days. Recombinant human IL-2 (20 ng / ml; R&D Systems, Minneapolis, USA) was added on days 3 and 10. Culture wells containing autologous CD8+ T cells without DCs were included as additional controls. On day 7 of co-culture, autologous CD8+ T cells were restimulated with the corresponding DCs (i.e., MOCK-pulsed DCs, eGFP mRNA-EP DCs, MART-1 mRNA DCs, and MART-1 peptide-pulsed DCs). At the end of co-culture, cells were incubated with PMA / iono / brefA for 5 h and harvested as described above for surface staining using PE-conjugated A*02:01 / human MART-1 MHC tetramers (Sanquin, Amsterdam, The Netherlands), as well as for intracellular staining using the following markers: anti-IFN-γ FITC (BioLegend, California, USA); and anti-granzyme B Pacific Blue (BD Biosciences, New Jersey, USA).

[0129] Evaluation of DC-induced antigen-specific cytolytic activity

[0130] As described above, effector T cells were harvested on day 14 of the autologous DC:T cell co-culture setup. Target cells consisted of TAP2-deficient T2 cells loaded with the same peptide from MART-1 as described above (Genscript, New Jersey, USA), or an irrelevant A2-restricted peptide from influenza matrix protein with the sequence GILGFVFTL (AnaSpec, California, USA; SEQ ID NO 2) as a control, both used at a quantity of 10 μg / ml. The T2 cells were pulsed for 3 hours and washed thoroughly to remove unbound peptide. Co-culture was performed at an E:T ratio of 10:1 for 14 hours in the presence of monensin (Golgistop, BD Biosciences, New Jersey, USA) and anti-CD107a Pacific Blue antibody (Miltenyi Biotec, Bergisch Gladbach, Germany). At the end of the co-culture, the cells were stained with surface anti-CD3, anti-CD8, and anti-CD137 (eBioscience, Thermo Fisher Scientific, Massachusetts, USA).

[0131] Statistics

[0132] Statistical analysis was performed using GraphPad Prism (version 7.02, GraphPad Software, California, USA). Normal distribution was first tested using the D'Agostino-Pearson omnibus normality test. Normally distributed data were analyzed using unpaired or paired t-tests for 2 groups or ANOVA test combined with Tukey's multiple comparison test for 3 groups or more. For non-normally distributed data, non-parametric tests were used, namely the Mann-Witney test for unpaired datasets and the Wilcoxon matched-pairs signed-rank test for paired datasets of two groups. For more than 2 groups, the non-parametric Kruskal-Wallis test was combined with Dunn's multiple comparison test. Statistical significance levels were coded with asterisk symbols as follows: p-value 0.01 - 0.05 (*), p-value 0.001 - 0.01 (**), p-value < 0.001 (***), and p-value < 0.0001 (****).

[0133] Results

[0134] High yields of fully differentiated mature dendritic cells can be obtained by shortening the monocyte culture protocol, including maturation with TLR4-ligand and IFN-γ.

[0135] By using a large-scale series of small-scale cultures starting from buffy coats, the feasibility of generating DCs by combining a greatly reduced monocyte culture duration and maturation using an established combination of type 1 polarizing factors was evaluated. Cell culture media, cytokines, and closed-system containers were selected to translate directly into our GMP production environment.

[0136] To reduce the need for operator intervention, our goal was to shorten the standard 8-day DC culture duration to a total of 4 days. This consisted of culturing for 3 days in a GMP-compliant serum-free medium supplemented with GM-CSF / IL-4, followed by exposure to a combination of MPLA and IFN-γ for an additional 24 hours before harvest. This protocol yielded a CD11c 高 HLA-DR 高 monocyte population with a median purity of 94.6% [95% Cl: 93.7 - 96.9]( Figure 1 A), which showed a characteristic dendritic morphology by light microscopy( Figure 1 B). At harvest, the median monocyte-to-DC conversion rate was 41.5% [95% Cl: 30.7 - 51.7], and the median viability (by flow cytometry) was 95.7% [95% Cl: 92.7 - 96.4]( Figure 1 C).

[0137] The phenotype of the cells was consistent with that of fully differentiated mature DCs, with a profound downregulation of the monocyte marker CD14, while the upregulation of CD83 was accompanied by high surface expression of the T cell co-stimulatory markers CD40, CD70, and CD86, as well as high levels of HLA class I and II antigen-presenting molecules. Additionally, the observation that the molecule DNGR-1 could be detected at high levels indicated the potential to capture and cross-present exogenous cell-bound antigens. CCR7 was induced on mature DCs, indicating the ability to migrate to secondary lymphoid organs. The T cell checkpoint molecule PD-L1 was also upregulated, reflecting the overall activation state of moDCs( Figure 1 D).

[0138] Then, in terms of several key parameters relevant to vaccine production, we compared this 4-day moDC differentiation protocol with an established "classical" clinical-grade 8-day DC culture. 8-day moDCs were generated in a medium supplemented with GM-CSF / IL-4 and matured by the addition of TNF-α and PGE2 in the last two days. Although the original maturation mixture first described by Jonuleit et al. (1997) consisted of TNF-α, PGE2, IL-1β, and IL-6, we and others have observed that the absence of IL-1β and IL-6 has no adverse effect on the viability, differentiation, and maturation of the resulting DCs(Figure 9 ), and has no negative impact on DC function (Van Driessche et al., 2009).

[0139] First, we consistently observed that at harvest, the viability of 4-day moDCs was significantly higher than that of 8-day moDCs (p-value 0.0010). The median viability of 4-day moDCs (by flow cytometry) was 96.3% [95% CI: 92.7 - 98], while the median viability of 8-day moDCs was 58% [95% CI: 45.1 - 69.1]. 4-day moDCs also had the highest median monocyte-to-DC conversion rate (46.9% [95% CI: 27.2 - 63.2] vs 26.8% [95% CI: 14.1 - 36.2]), reaching statistical significance (p-value 0.0195) ( Figure 2 A).

[0140] Next, we looked at the differences in phenotypic characteristics at harvest. 4-day moDCs showed significantly higher levels of CD40, CD70, and HLA-ABC (MFI) compared to standard 8-day moDCs. Unexpectedly, CCR7 was also expressed at higher levels on MPLA / IFN-γ - matured 4-day moDCs despite not being exposed to PGE2. In contrast, the expression of CD86 was higher in 8-day moDCs ( Figure 2 B and Table 1). In both DC culture protocols, the expression of CD83, HLA-DR, and DNGR-1 did not show statistically significant differences. Unexpectedly, in standard 8-day moDCs, the expression of PD-L1 was consistently higher (on average four-fold) than in 4-day moDCs and even further increased after thawing cryopreserved DC aliquots ( Figure 10 ).

[0141] Table 1.

[0142]

[0143] * Background signal: live CD11c 高 HLA-DR 高 Geometric mean of DCs

[0144] From these data, we conclude that reducing the monocyte culture duration by half and combining the activating factors MPLA and IFN-γ produces fully differentiated mature DCs with higher conversion yields, higher cell viability, and no detrimental effects on the expression levels of costimulatory molecules.

[0145] To our knowledge, only one report described the integration of MPLA+IFN-γ as a maturation cocktail in an accelerated DC differentiation protocol with a monocyte-to-DC differentiation period of only 24-36 hours (Massa et al., 2013). However, no results of DC vaccine activity were evaluated for the said alternative DC. For comparison, monocyte-derived dendritic cells were generated according to the protocol described previously herein (“MIDRIX DC”) or according to the alt-2 protocol described by Massa et al. (“Massa DC”). As described previously, CD14+ monocytes were isolated from buffy coats. DC were harvested at various time points and electroporated with mRNA encoding eGFP. Data were derived from 6 different donors and the following aspects were evaluated:

[0146] (A) Viability and absolute cell yield at harvest of viable CD11c+HLA-DR+ dendritic cells obtained by the two methods.

[0147] (B) Expression of the monocyte marker CD14 relative to the DC differentiation marker CD83;

[0148] (C) Expression of the DC maturation markers CD40, CD70, CD86 and CCR7;

[0149] (D) Expression of the T cell co-inhibitory receptor PD-L1;

[0150] (E) Electroporation efficiency, expressed as the level of the translated protein (relative mean fluorescence intensity of the eGFP signal) and the proportion of cells with successful translation of the electroporated eGFP-mRNA (percentage of eGFP+ DC), measured 4 hours after electroporation.

[0151] As Figure 13 shown, a differentiation step duration of 24 to 36 hours was not sufficient to achieve full differentiation of monocytes into DC and generate a mature phenotypic profile associated with T cell stimulatory capacity. Importantly, the absolute yield of viable DC using the protocol described by Massa et al. was significantly lower compared to the method of the present invention, which is considered to significantly impair the possibility of further processing these cells by electroporation and cryopreservation, thus having an impact on DC vaccines. “Massa DC” was not readily electroporated with mRNA encoding full-length proteins, while DC generated according to the present invention showed high electroporation efficiency.

[0152] In addition, "Massa DC" showed less downregulation of the monocyte marker CD14, less upregulation of the DC differentiation marker CD83, and lower levels of the DC maturation / T cell co-stimulatory receptors CD40, CD70, and CD86. The level of CCR7 required for migration to the T cell zone of lymphoid tissue was also less upregulated on D1 DC. Moreover, the PD-L1 level showed a tendency of higher expression on "MassaDC".

[0153] Both MPLA and IFN-γ are necessary for conferring a fully mature phenotype on short-term cultured DCs and for inducing de novo T helper cell 1 polarization.

[0154] Next, we dissected the relative contributions of MPLA, IFN-γ, or their combination to the phenotypic maturation status, as well as to the functional impact in terms of the T helper cell polarization capacity of 4-day-cultured moDCs.

[0155] We found that both maturation stimuli were required to maximize the surface expression levels of the T cell co-stimulatory molecules CD40, CD70, CD86, as well as CD83 and CCR7 ( Figure 3 ). No such effect was observed regarding the expression of HLA-DR or DNGR-1, the latter remaining stable relative to immature DCs. Notably, the induction of PD-L1 on moDCs was mainly caused by MPLA exposure rather than IFN-γ exposure.

[0156] At the functional level, the maximal induction of IFN-γ secretion by naive allogeneic CD4+ T cells was achieved only by pre-exposing DCs to both MPLA and IFN-γ. Immature DCs induced a limited amount of IL-10 production in naive T helper cells, which could be further inhibited in the presence of DCs pre-exposed to MPLA, regardless of prior IFN-γ exposure. The production of T helper cell IL-4 was induced only at low levels, as was the case for IL-17, which showed a slight increase in the presence of MPLA / IFN-γ matured DCs ( Figure 4 ).

[0157] Therefore, it is necessary to expose short-term differentiated moDCs to both MPLA and IFN-γ to obtain a fully mature phenotype and to endow these cells with the ability to induce a robust de novo type-1 polarized T helper cell response.

[0158] Short-term cultured DCs showed excellent resilience to electroporation and high mRNA translation efficiency.

[0159] In addition to phenotypic maturation and type 1 immune polarization potential, sufficient amounts of DCs should be recovered after the stresses of electroporation and cryopreservation for implementation in clinical practice.

[0160] We first evaluated the ability of mature, short-term cultured MPLA / IFN-γ DCs to successfully and stably express protein antigens derived from electroporated antigen-encoding mRNAs. Using mRNA encoding eGFP as a marker of electroporation efficiency, we observed eGFP expression 4 hours after electroporation / before cryopreservation, immediately after cell thawing, and 24 hours after cell thawing (further incubation in cytokine-free medium).

[0161] The median percentage of eGFP-positive DCs by exponential pulse electroporation evolved from 64.8 [95% CI: 55.2 - 87.7] before cryopreservation to 80.2 [95% CI: 73.1 - 87.7] immediately after thawing and remained stable over the next 24 hours (86.3 [95% CI: 75.2 - 86.4), with no significant change in the expression intensity (MFI) during this period ( Figure 5 A, B).

[0162] Exponential pulse electroporation resulted in an average 17.3% decrease in viability (trypan blue) in 4-day moDCs. Combining the net cell loss induced by electroporation, this translated to a median percentage of viable DC recovery of 51.4% [95% CI: 36 - 67%] (viable cell recovery after electroporation relative to before electroporation) ( Figure 5 C). Using a separate series of donors, we compared the electroporation resilience of 4-day MPLA / IFN-γ moDCs with standard 8-day moDCs. We observed that the viability (trypan blue) of 4-day DCs after EP was significantly higher than that of 8-day DCs, with median viabilities of 67.3% [95% CI: 18.2 - 93.5] and 16.5% [95% CI: 2.8 - 57.8], respectively. After eGFP mRNA EP, 8-day moDCs were also more susceptible to net cell loss, with an average viable cell recovery rate of 24.6% [95% CI: 3.7 - 47.5], while that of 4-day moDCs was 41.5% [95% CI: 12.8 - 83.8] ( Figure 5 D).

[0163] In further tests, we evaluated the results after square wave pulse electroporation. Using small-scale runs with DCs generated at a range of cytokine concentrations, we found that cell viability after electroporation and after cryopreservation was consistently higher using square wave pulses compared to the exponential pulse protocol ( Figure 11 ).

[0164] Further evaluation of square wave pulses was performed on a full-scale DC production run in a GMP environment. In terms of % eGFP-positive cells, flow cytometry analysis of eGFP expression in DCs electroporated with square wave pulses was non-inferior compared to exponential pulses (representative data asFigure 12 as shown in A). Electroporation with square-wave pulses can result in >80% DC recovery immediately after thawing, and cryopreserved DCs have >75% viability ( Figure 12 B).

[0165] The formation of visible cell aggregates was not observed after square-wave pulse electroporation, which greatly facilitated further cell processing and improved the overall cell recovery rate (results not shown).

[0166] Electroporation and cryopreservation do not impair the ability of short-term cultured DCs to selectively promote type 1 polarized T cell responses

[0167] After electroporation and cryopreservation stress, the key DC property that should remain intact is the potential to selectively mobilize type 1 polarized and cytolytic T cells when administered to patients. To provide an assessment of this function, we analyzed the cytokine and chemokine secretomes of electroporated and cryopreserved 4-day moDCs and standard 8-day DCs after incubation in cytokine-free medium for 24 hours ( Figure 6 A). We found that 4-day moDCs were still able to secrete bioactive IL-12 and IFN-γ, while the production of these cytokines by 8-day moDCs was below the detection limit. No difference in IL-10 production was observed between the two DC types. More surprisingly, we found that only MPLA / IFN-γ matured 4-day moDCs were able to produce substantial amounts of chemokines that are involved in attracting type 1 polarized T helper cells, cytolytic T cells, and NK cells (Colantonio et al., 2002), with no detectable secretion from standard 8-day MoDCs. This includes high levels of the CXCR3 ligands CXCL9 (MIG30) and CXCL10 (IP-10) (Groom et al 2011), as well as the CCR5 ligands CCL3 (MIP-1α), CCL4 (MIR-1β), and CCL5 (RANTES) (Samson et al., 1997). The secretion of the CXCR3 ligand CXCL11 (Groom et al., 2011) was below the detection limit. In contrast, the secretion of the chemokine CCL17 (TARC) that mobilizes T-reg and Th2 (Yoshie et al., 2015) was five-fold higher in standard 8-day moDCs. There was a trend for high release of the chemokine CCL20 that attracts Th17 and T-reg by 4-day moDCs (Yamazaki et al 2008), while the production of the CXCR4 ligand CXCL12 (SDF-1α) that attracts T-reg (Colantonio et al., 2002) did not differ between the two DC culture protocols (data not shown).

[0168] We also investigated whether electroporation and cryopreservation affect the ability of 4-day moDCs to induce de novo T helper cell type 1 polarization responses ( Figure 6 C). Co-culture of allogeneic naive CD4 T cells with thawed 4-day moDCs led to high levels of IFN-γ production, which were comparable to those obtained with co-culture with freshly harvested, non-electroporated 4-day moDCs ( Figure 4 ). In this context, induction of IL-10 production was very low ( Figure 6 C), consistent with results obtained with fresh DCs ( Figure 4 ).

[0169] DCs cultured for a short term can effectively initiate and expand tumor antigen-specific CD8+ T cells with cytolytic activity

[0170] Having established the superiority of short-term cultured moDCs in terms of yield, phenotype, recovery, and ability to promote type 1 polarized T cell responses after electroporation / cryopreservation, we next tested the ability of these cells to present immunogenic epitopes from electroporated tumor antigen-encoding mRNAs. Again, to reflect the implementation of DC vaccines in the clinical settings of real life, we performed all assays with cryopreserved rather than fresh mRNA-EP DCs. Considering the possibility of using tetramers to detect MART-1-specific CD8+ T cells in HLA-A2-positive healthy donors, MART-1 / Melan-A was used as a model tumor-associated antigen.

[0171] We observed that a total of every two-week stimulation rounds with MART-1-mRNA-EP DCs were sufficient to induce more than 30-fold expansion of antigen-specific (tetramer-positive) CD8+ T cells compared to stimulation with DCs loaded with an irrelevant antigen (eGFP) (median 0.43% [95% Cl: 0.22 - 0.53]) vs 13.2% [95% Cl: 1.21 - 37.6]). No differences were observed in terms of viability and recovery rate after electroporation, regardless of whether 4-day moDCs were electroporated with MART-1 mRNA or eGFP mRNA (data not shown). The expansion of MART-1-specific CD8+ T cells was in the same order of magnitude as that obtained with DCs pulsed with MART-1 peptide (positive control) (median 18.9% [95% Cl: 5.75 - 28.8]). These results indicate that MPLA / IFN-γ-matured 4-day moDCs are able to extract immunogenic epitopes from electroporated mRNA encoding MART1 to efficiently present to Ag-specific autologous CD8+ T cells ( Figure 7 A - B).

[0172] To evaluate the effector potential of stimulated CD8+ T cells, we combined tetramer assays with IC staining for IFN-γ and granzyme B. Compared to negative control conditions (i.e., stimulation with MOCK pulsed or eGFP mRNA-EP-DC, or no DC stimulation), we found that MART-1-mRNA-EP 4-day moDCs induced the highest numbers of antigen-specific CD8+ T cells producing IFN-γ and granzyme B( Figure 7 C).

[0173] To further evaluate the cytolytic capacity of 4-day moDC-stimulated CD8+ T cells, we used TAP-deficient HLA-A2+ T2 cells as targets passively loaded with A2-restricted MART-1 peptide and an irrelevant (Flu) peptide( Figure 8 experimental setup shown in A). As previously described (Bonehill et al. 2009), flow cytometric analysis focusing on the dual expression of the T cell activation marker CD137 / 4-1BB and the cytolytic degranulation marker CD107a was used to detect target engagement and killing activity. We observed that, over a 2-week period, only CD8+ T cells stimulated with 4-day moDCs loaded with MART-1-mRNA and pulsed with MART-1 peptide upregulated CD137 / CD107a after contact with MART-1-peptide-loaded T2 cells (representative dot plots are shown in Figure 8 B). This signal was detected in most donors and was specific, as engagement of an irrelevant target (Flu-peptide-loaded T2 cells) did not induce the expression of cytolytic markers, nor did prior stimulation of effector CD8+ T cells with MOCK pulsed or eGFP-mRNA electroporated DCs( Figure 8 C).

[0174] Discussion

[0175] To our knowledge, this is the first description of an accelerated in vitro cell differentiation method that allows the generation of clinical-grade DCs with strong Th1-polarizing capacity and the efficient presentation of mRNA-encoded tumor antigens introduced by electroporation.

[0176] The feasibility of shortening the classical 7 - 8 day in vitro culture to generate fully mature DCs has been previously described by other groups. Commonly referred to as "rapid DCs", these are cells obtained by a monocyte - to - DC differentiation time of 24 (Dauer et al. 2003; Kvistborg et al. 2009; Jarnjak - Jankovic et al. 2007) to 72 hours (Truxova et al. 2014) in the presence of GM - CSF and IL - 4, followed by a 24 - hour maturation period using a standard mixture of inflammatory cytokines TNF - α, IL - 1β, IL - 6, PGE2 or TLR ligands (Truxova et al. 2014). In terms of maturation characteristics and function, they perform comparably to classical long - term DC cultures. Only one report described the integration of MPLA + IFN - γ as a maturation mixture in an accelerated DC differentiation protocol as part of a comparative study that used 4 different maturation strategies after a 24 - 36 - hour monocyte - to - DC differentiation period (Massa et al., 2013). Compared to DCs matured with the classical mixture of TNF - α + IL - 1β + IL - 6 + PGE2 or alternative options TNF - α + IL - 1β + IFN - α + IFN - γ + poly(I:C) or TNF - α + IL - 1β + IFN - γ + CL097, MPLA + IFN - γ - matured DCs expressed the highest levels of co - stimulatory molecule expression and produced the best IL - 12p70 / IL - 10 release ratio.

[0177] Studies conducted by Ten Brinke et al. (2007; 2010) also documented the use of MPLA / IFN - γ in terms of type - 1 polarization potency, although a 6 - 7 - day culture time was used. The present invention demonstrates that MPLA / IFN - γ can also drive the full maturation of DCs when applied to an accelerated culture protocol ( Figure 1 D; 2B). Additionally, we demonstrate that the combination of the two reagents is necessary for inducing maximal expression of key T - cell co - stimulatory molecules (such as CD86, CD40 and CD70 as well as the lymph node - homing chemokine receptor CCR7) ( Figure 3 ). Among them, the up - regulation of CD40 and CD70 was consistently higher than that obtained with 8 - day DCs matured using complex inflammatory mixtures. Sufficient levels of both molecules are crucial for the anti - tumor immune response: CD40 is important in promoting T - helper cell - DC activation, allowing optimal downstream stimulation of CD8+ cytotoxic T lymphocytes, while CD70 is key in driving Th1 rather than T - reg or Th17 T - cell differentiation and for endowing CD8+ T cells with effector and memory properties. Therefore, exploring the potential of the CD40 / CD40L and CD70 / CD27 axes has been successfully developed as a strategy to increase the immunogenicity of DCs for clinical cancer vaccine applications.

[0178] We surprisingly detected lower levels of the T cell co-inhibitory receptor PD-L1 on MPLA / IFN-γ matured DCs relative to TNF-α / PGE2 DCs( Figure 2 B). Strikingly, after cryopreservation / thawing, the difference in PD-L1 expression levels at harvest was further increased( Figure 10 ), i.e., a biologic agent that will be effectively administered to patients, in which the expression of this immunosuppressive ligand should be as low as possible. Although type 2 interferon is the prototypical inducer of PD-L1 expression on many cell types (Gato-Canas et al. 2017), PGE2 has been described as a powerful driver of PD-L1 upregulation on myeloid cells, as shown in tumor-associated myeloid cells with immunosuppressive capabilities (Prima et al. 2017). The use of PGE2 in DC culture protocols is usually motivated by its ability to induce optimal expression of CCR7 on DCs, thus maximizing the efficiency of migration to T cell-dependent areas of lymphoid tissue. However, in the present invention, 4-day MPLA / IFN-γ DCs express at least as much CCR7 as TNF-α / PGE2 matured DCs. Combining with the IL-12 inhibition seen in our TNF-α / PGE2 matured DCs( Figure 6 A), altogether, these findings strongly support the shift from classical DC maturation cocktails to next-generation DC-based cancer vaccines.

[0179] The chemokine signature released after cryopreservation, thawing and further culturing for 24 hours in cytokine-free medium further confirmed the ability of 4-day MPLA / IFN-γ matured DCs to support a type 1 polarized immune response( Figure 6A). Compared to DCs matured with TNF-α / PGE2 for 8 days, only DCs matured with MPLA / IFN-γ for 4 days secrete high levels of Th1-attracting chemokines CCL3, CCL4, CCL5, CXCL9, and CXCL10. In vivo, the interaction between CXCL10 secreted by DCs and the expression of CXCR3 receptor on CD4+ T cells is shown to ensure the formation of stable contacts between these cell types in lymph nodes. This stable cell contact, combined with placing these CD4+ T cells in a potential niche with high IFN-γ production, can further promote Th1-differentiation. Additionally, our experiments show that the T-reg and Th2-mobilizing chemokine CCL17 is mainly released by DCs matured with TNF-α / PGE2 for 8 days, likely as a result of PGE2 pre-treatment. Although statistical significance has not been reached, DCs matured with MPLA / IFN-γ for 4 days still tend to release more Th17- and T-reg-attracting chemokine CCL20. The fact that the choice of DC maturation stimulant defines Th1- or Th2-T cell mobilization characteristics has been documented by Lebre et al. (2005). In their experiments, the chemokine production of freshly harvested mature DCs in response to CD40 ligation was evaluated. DCs matured in the presence of LPS and IFN-γ showed mainly the release of Th1-attracting chemokines, while the expression level of the Th2-related chemokine CCL22 increased significantly when PGE2 was present in the maturation mixture. Contrary to our findings, the expression pattern of CCL17 in the paper by Lebre et al. was independent of the DC type.

[0180] An additional factor potentially affecting the achieved level of DC maturation is the physical properties of the culture vessel used. By the method of the present invention, it is feasible to differentiate and activate cells in a breathable bag that constitutes a closed system compatible with a clinically certified immunomagnetic separation system. Our results contradict early studies that showed DCs generated in clinical-grade bags had impaired maturation programs, downregulation of co-stimulatory molecule expression, chemokine, and IL-12 secretion (Rouas et al. 2010). Surprisingly, we show that all these characteristics are induced in our DCs and are even intact after cryopreservation, thawing, and further culture in cytokine-free basal medium. Similar studies by the groups of G. Gaudernack et al. and G. Kvalheim et al. (Kyte et al. 2005; Mu et al. 2003) strengthened the idea that clinically grade DCs with intact immunogenic properties can indeed be generated in bags. Culturing in cell differentiation bags will also allow us to easily transfer our method to commercially available, fully automated closed cell culture systems. This option will be able to further reduce operator intervention, lower the risk of contamination, and improve overall reproducibility.

[0181] The present invention differentiates itself further from early reports focusing on alternative culture durations and / or maturation protocols by selecting mRNA electroporation as the means of loading DCs with antigen. The advantages of this technique are the flexibility in synthesizing custom sequences encoding tumor-associated antigens or new epitopes of mutant origin, and the option to incorporate sequences to optimize both MHC I and MHC II presentation. Additionally, contrary to previous studies where DCs were passively loaded with selected HLA-restricted peptides, electroporation with full-length mRNA ensures the processing and potential presentation of a broad repertoire of epitopes without any patient pre-selection in terms of HLA type. Moreover, the half-life of the translated protein in DCs ensures the extended generation of MHC I-epitope complexes, while passively loaded exogenous peptides only transiently bind to surface HLA molecules or are depleted by internalization. The ability of DCs electroporated with mRNA to induce T cell responses is as robust as that of peptide-loaded DCs, as has been demonstrated previously. Here we show that 4-day cultured MPLA / IFN-γ-matured DCs electroporated with a model tumor-associated antigen can induce a vigorous expansion of rare antigen-specific CD8+ T cells equipped with the essential anti-tumor toolkit (e.g., high expression of IFN-γ and perforin), which is reflected by efficient and highly specific cytotoxic activity. Importantly, we evaluated this fundamental DC property after cryopreservation and thawing, which reflects a realistic vaccination scenario.

[0182] In summary, the present invention demonstrates that 4-day MPLA / IFN-γ-matured monocyte-derived DCs are superior to "classical" 8-day TNF-α / PGE2-matured DCs in terms of cell yield, phenotype, and type 1 polarization characteristics. Shortening the culture time using GMP-compliant materials and serum-free medium in a closed system, electroporation and cryopreservation do not impair the ability of short-term cultured MPLA / IFN-γ-DCs to induce cytolytic tumor-derived antigen-specific CD8+ T cell responses, which further emphasizes the robustness of this production method for clinical implementation.

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Claims

1. An in vitro method for preparing an autologous dendritic cell vaccine, the method comprising the following steps: - providing isolated monocyte dendritic cell precursors; - culturing the precursors for 48 to 96 hours in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4; - contacting the immature dendritic cells with interferon gamma and monophosphoryl lipid A for the last 24 hours under culture conditions suitable for maturing the immature dendritic cells to form a population of mature dendritic cells; and - transfecting the mature dendritic cells with mRNA encoding an antigen.

2. The method according to claim 1, wherein the dendritic cells are differentiated and matured in a clinical-grade fully enclosed system.

3. The method according to claim 1 or 2, wherein the monocyte dendritic cell precursors are provided in a culture bag.

4. The method according to claim 3, wherein the cell density in the culture bag at the start of cell culture is 0.5 to 2×10E6 cells / mL.

5. The method according to claim 1 or 2, wherein the concentrations of the granulocyte-macrophage colony-stimulating factor, interleukin-4, and interferon gamma are 500 to 2500 U / ml.

6. The method according to claim 1 or 2, wherein the concentration of the monophosphoryl lipid A is 1 to 10 μg / ml.

7. The method according to claim 1 or 2, wherein the transfection is carried out by electroporation.

8. The method according to claim 7, wherein the transfection is carried out by electroporation using square wave pulses.

9. The method according to claim 1 or 2, wherein, the transfected dendritic cells are further resuspended in a cryopreservation medium and stored in the gas phase of a liquid nitrogen container.

10. The method according to claim 1 or 2, wherein the antigen is selected from the group consisting of tumor-associated antigens, viral antigens, bacterial antigens, parasitic antigens, and fungal antigens.

11. The method according to claim 1 or 2, wherein the antigen is a tumor antigen.

12. The method according to claim 1 or 2, wherein the antigen is a testicular cancer antigen.

13. The method according to claim 1 or 2, wherein the antigen is an oncogenic viral antigen.

14. The method according to claim 1 or 2, wherein the antigen is an antigen from a mutant group source.

15. The method according to claim 1 or 2, wherein the antigen is a yeast antigen.

16. Dendritic cells obtainable by the method according to any one of claims 1 to 15.

17. A pharmaceutical composition or vaccine comprising mature transfected dendritic cells obtainable by the method according to any one of claims 1 to 15.

18. The pharmaceutical composition or vaccine according to claim 17, wherein the dendritic cells are cryopreserved.

19. Transfected dendritic cells obtainable by the method according to any one of claims 1 to 15, for use in active immunotherapy.

20. A population of mature, mRNA-electroporated, cryopreserved, and thawed dendritic cells, the dendritic cells being dendritic cells obtainable by the method according to any one of claims 1 to 15.

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