Dendritic cells (DCs) are the orchestrators of the immune system and are frequently used in clinical trials in order to boost the immune system in cancer patients. Among several available techniques for DC modification, mRNA electroporation is an interesting technique due to the favorable characteristics of mRNA. Antigen expression level and duration can be increased by multiple optimizations of an antigen-encoding mRNA template. Here, we describe different molecular modifications to a WT1-encoding mRNA construct in order to increase antigen expression and the subsequent introduction of mRNA into DCs.

Cancer immunotherapy has been proposed as a powerful treatment modality. Active immunotherapy aspires to stimulate the patient's immune system, particularly T cells. These cells can recognize and kill cancer cells and can form an immunological memory. Dendritic cells (DCs) are the professional antigen-presenting cells of our immune system. They take up and process antigens to present them to T cells. Consequently, DCs have been investigated as a means to stimulate cancer-specific T-cell responses. An efficient strategy to program DCs is the use of mRNA, a well-defined and safe molecule that can be easily generated at high purity. Importantly, vaccines consisting of mRNA-modified DCs showed promising results in clinical trials. Therefore, we will introduce cancer immunotherapy and DCs and give a detailed overview on the application of mRNA to generate cancer-fighting DC vaccines.

The aim of the present study was to enhance the efficiency of leukemia immunotherapy by increasing the antigen-specific cytotoxic T lymphocyte-inducing ability of leukemia cells. The leukemic plasmacytoid dendritic cell line PMDC05 containing the HLA-A02/24 antigen, which was previously established in our laboratory (Laboratory of Hematology and Oncology, Graduate School of Health Sciences, Niigata University, Niigata, Japan), was used in the present study. It exhibited higher expression levels of CD80 following transduction with lentiviruses encoding the CD80 gene. This CD80-expressing PMDC05 was named PMDC11. In order to establish a more potent antigen-presenting cell for cellular immunotherapy of tumors or severe infections, PMDC11 cells were transduced with a constitutively active (ca) toll-like receptor 4 (TLR4) gene using the Tet-On system (caTLR4-PMDC11). CD8(+) T cells from healthy donors with HLA-A02 were co-cultured with mutant WT1 peptide-pulsed PMDC11, lipopolysaccharide (LPS)-stimulated PMDC11 or caTLR4-PMDC11 cells. Interleukin (IL)-2 (50 IU/ml) and IL-7 (10 ng/ml) were added on day three of culture. Priming with mutant WT1 peptide-pulsed PMDC11, LPS-stimulated PMDC11 or caTLR4-PMDC11 cells was conducted once per week and two thirds of the IL-2/IL-7 containing medium was replenished every 3-4 days. Immediately prior to the priming with these various PMDC11 cells, the cultured cells were analyzed for the secretion of interferon (IFN)-γ in addition to the percentage and number of CD8(+)/WT1 tetramer(+) T cells using flow cytometry. caTLR4-PMDC11 cells were observed to possess greater antigen-presenting abilities compared with those of PMDC11 or LPS-stimulated PMDC11 cells in a mixed leukocyte culture. CD8 T cells positive for the WT1 tetramer were generated following 3-4 weeks of culture and CD8(+)/WT1 tetramer+ T cells were markedly increased in caTLR4-PMDC11-primed CD8(+) T cell culture compared with PMDC11 or LPS-stimulated PMDC11-primed CD8(+) T cell culture. These CD8(+) T cells co-cultured with caTLR4-PMDC11 cells were demonstrated to secrete IFN-γ and to be cytotoxic to WT1-expressing target cells. These data suggested that the antigen-specific cytotoxic T lymphocyte (CTL)-inducing ability of PMDC11 was potentiated via transduction of the caTLR4 gene. The present study also suggested that caTLR4-PMDC11 cells may be applied as potent antigen-presenting cells for generating antigen-specific CTLs in adoptive cellular immunotherapy against tumors and severe viral infections.

Vaccination strategies to improve outcome of hematopoietic stem cell transplant in leukemia patients: early evidence and future prospects Naoki Hosen, Tetsuo Maeda, Yoshiko Hashii, Akihiro Tsuboi, Sumiyuki Nishida, Jun Nakata, Yoshiki Nakae, Satoshi Takashima, Yusuke Oji, Yoshihiro Oka, Atsushi Kumanogoh & Haruo Sugiyama October 2014, Vol. 7, No. 5, Pages 671-681 (doi:10.1586/17474086.2014.953925)

PMDC05, a leukemic plasmacytoid dendritic cell (pDC) line with HLA-A*02/24, which was established in our laboratory, acquired a higher expression of CD80 after transduction with lentiviruses encoding the CD80 gene. CD80-expressing PMDC05 was named PMDC11. In order to establish a more potent antigen presenting cells for cellular immunotherapy in tumors or severe infections, PMDC11 cells were transduced with a constitutively active (ca) TLR4 gene by using the Tet-On system (caTLR4-PMDC11). CD8+ T cells from healthy donors with HLA-A*02:01 were co-cultured with mutant WT1 peptide-pulsed PMDC11, LPS-stimulated PMDC11 or caTLR4-PMDC11 cells. IL-2 (50 IU/ml) and IL-7 (10 ng/ml) were added on day 3 of culture. Priming with mutant WT1 peptide-pulsed PMDC11, LPS-stimulated PMDC11 or caTLR4-PMDC11 was performed every week and the cultured cells were analyzed for the secretion of IFN-γ or the percentage and number of CD8+/WT1 tetramer+ T cells immediately before the priming with these various PMDC11 cells. caTLR4-PMDC11 cells possessed more potent antigen presenting ability compared with PMDC11 or LPS-stimulated PMDC11 cells in mixed leukocyte culture. CD8 T cells positive for WT1 tetramer were generated after 3-4 weeks of culture and CD8+/WT1 tetramer+ T cells were much increased in caTLR4-PMDC11-primed CD8+ T cell culture compared with PMDC11 or LPS-stimulated PMDC11-primed CD8+ T cell culture. These CD8+ T cells co-cultured with caTLR4-PMDC11 cells were demonstrated to secrete IFN-γ and be cytotoxic to WT1-expressing target cells. These data revealed that the antigen-specific CTL inducing ability of PMDC11 is potentiated by transducing caTLR4 gene, and that caTLR4-PMDC11 cells could be applied as potent antigen presenting cells for generating antigen-specific CTLs in adoptive cellular immunotherapy against tumors and severe viral infections.

Melanoma patients with a high risk of recurrence may benefit from immunotherapy with mRNA-electroporated autologous monocyte-derived dendritic cells (DCs). Further benefit may be found in combining DC-therapy with interferon alfa-2b.

Dendritic cells (DC) are key players in several types of cancer vaccines. Large numbers of DC can easily be generated in closed systems from the monocyte fraction of the peripheral blood. They are the professional antigen-presenting cells, and electroporation of mRNA-encoding tumor antigens is a very efficient and a relatively simple way to load the DC with antigen. The co-electroporation of a tumor antigen of choice and the combination of 3 costimulatory molecules, including CD70, caTLR4, and CD40L (TriMix-DC), leads to fully potent antigen-presenting DC able to generate a broad immune response.Here we describe the in vitro transcription of the mRNA and the subsequent generation and electroporation of autologous DC used for the treatment of melanoma patients.

Since decades, the main goal of tumor immunologists has been to increase the capacity of the immune system to mediate tumor regression. In this regard, one of the major focuses of cancer immunotherapy has been the design of vaccines promoting strong tumor-specific cytotoxic T lymphocyte responses in cancer patients. Here, dendritic cells (DCs) play a pivotal role as they are regarded as nature’s adjuvant and as such have become the natural agents for antigen delivery in order to finally elicit strong T cell responses (Villadangos and Schnorrer in Nat Rev Immunol 7:543–555, 2007; Melief in Immunity 29:372–383, 2008; Palucka and Banchereau in Nat Rev Cancer 12:265–277, 2012; Vacchelli et al. in Oncoimmunology 2:e25771, 2013; Galluzzi et al. in Oncoimmunology 1:1111–1134, 2012). Therefore, many investigators are actively pursuing the use of DCs as an efficient way of inducing anticancer immune responses. Nowadays, DCs can be generated at a large scale in closed systems, yielding sufficient numbers of cells for clinical application. In addition, with the identification of tumor-associated antigens, which are either selectively or preferentially expressed by tumors, a whole range of strategies using DCs for immunotherapy have been designed and tested in clinical studies. Despite the evidence that DCs loaded with tumor-associated antigens can elicit immune responses in vivo, clinical responses remained disappointingly low. Therefore, optimization of the cellular product and route of administration was urgently needed. Here, we review the path we have followed in the development of TriMixDC-MEL, a potent DC-based cellular therapy, discussing its development as well as further modifications and applications.

Leukemic plasmacytoid dendritic cell (pDC) line with HLA-A02/24, PMDC05, which was established in our laboratory, acquired a higher antigen presenting ability by being transduced with lentiviruses encoding the CD80 gene. CD80 gene-transduced PMDC05 was named PMDC11. In order to establish a more potent antigen presenting cells for cellular immunotherapy in tumors, PMDC11 cells were transduced with a constitutively active (ca) TLR4 gene by using the Tet-On system (caTLR4-PMDC11). CD8+ T cells from healthy donors with HLA-A24 were co-cultured with WT1 peptide-pulsed caTLR4-PMDC11 cells. Priming with WT1 peptide-pulsed caTLR4-PMDC11 cells was performed every week. Immediately before the priming with caTLR4-PMDC11 cells, cultured cells were analyzed for the percentage and number of CD8+/WT1 tetramer+ T cells. CD8+/WT1 tetramer+ T cells were generated after 2-3 weeks of culture and much increased in caTLR4-PMDC11-primed CD8+ T cell culture compared with parental PMDC11-primed CD8+ T cell culture. These CD8+ T cells co-cultured with caTLR4-PMDC11 cells were demonstrated to be cytotoxic to WT1-expressing HLA-A24+ target cells. These data revealed that the antigen-specific cytotoxic T lymphocyte (CTL) inducing ability of PMDC11 was enhanced by being transduced with caTLR4 gene, and that caTLR4-PMDC11 cells could be applied as potent antigen presenting cells for generating antigen-specific CTLs in adoptive cellular immunotherapy against tumors and severe viral infection.

Treatment of melanoma patients with mRNA electroporated dendritic cells (TriMixDC-MEL) stimulates T-cell responses against the presented tumor-associated antigens (TAAs). In the current clinical trials, melanoma patients with systemic metastases are treated, requiring priming and/or expansion of preexisting TAA-specific T cells that are able to migrate to both the skin and internal organs. We monitored the presence of TAA-specific CD8(+) T cells infiltrating the skin at sites of intradermal TriMixDC-MEL injection (SKILs) and within the circulation of melanoma patients treated in two clinical trials. In 10 out of fourteen (71%) patients screened, CD8(+) T cells recognizing any of the four TAA presented by TriMixDC-MEL cellular vaccine were found in both compartments. In total, 30 TAA-specific T-cell responses were detected among the SKILs and 29 among peripheral blood T cells, of which 24 in common. A detailed characterization of the antigen specificity of CD8(+) T-cell populations in four patients indicates that the majority of the epitopes detected were only recognized by CD8(+) T cells derived from either skin biopsies or peripheral blood, indicating that some compartmentalization occurs after TriMix-DC therapy. To conclude, functional TAA-specific CD8(+) T cells distribute both to the skin and peripheral blood of patients after TriMixDC-MEL therapy.

Regulatory T cells (Tregs) counteract anticancer immune responses through a number of mechanisms, limiting dendritic cell (DC)-based anticancer immunotherapy. In this study, we investigated the influence of various DC activation stimuli on the Treg functionality. We compared DCs activated by electroporation with mRNA encoding constitutively active TLR4 (caTLR4) and CD40 ligand (DiMix-DCs), or these factors together with mRNA encoding the costimulatory molecule CD70 (TriMix-DCs) with DCs maturated in the presence of a mixture of inflammatory cytokines (DCs maturated with a combination of the cytokines IL-1β, IL-6, TNF-α, and PGE2) for their ability to counteract Tregs on different levels. We first demonstrated that there was no difference in the extent of Treg induction starting from CD4(+)CD25(-) T cells under the influence of the different DC maturation stimuli. Second, we showed that both DiMix- and TriMix-DCs could partly alleviate Treg inhibition of CD8(+) T cells. Third, we observed that CD8(+) T cells that had been precultured with DiMix-DCs or TriMix-DCs were partially protected against subsequent Treg suppression. Finally, we showed that Tregs cocultured in the presence of TriMix-DCs, but not DiMix-DCs, partially lost their suppressive capacity. This was accompanied by a decrease in CD27 and CD25 expression on Tregs, as well as an increase in the expression of T-bet and secretion of IFN-γ, TNF-α, and IL-10, suggesting a shift of the Treg phenotype toward a Th1 phenotype. In conclusion, these data suggest that TriMix-DCs are not only able to suppress Treg functions, but moreover could be able to reprogram Tregs to Th1 cells under certain circumstances.

Tumor antigen-encoding mRNA for dendritic cell (DC)-based vaccination has gained increasing popularity in recent years. Within this context, two main strategies have entered the clinical trial stage: the use of mRNA for ex vivo antigen loading of DCs and the direct application of mRNA as a source of antigen for DCs in vivo. DCs transfected with mRNA-encoding Wilms' tumor 1 (WT1) protein have shown promising clinical results. Using a stepwise approach, we re-engineered a WT1 cDNA-carrying transcription vector to improve the translational characteristics and immunogenicity of the transcribed mRNA. Different modifications were performed: (i) the WT1 sequence was flanked by the lysosomal targeting sequence of dendritic cell lysosomal-associated membrane protein to enhance cytoplasmic expression; (ii) the nuclear localization sequence (NLS) of WT1 was deleted to promote shuttling from the nucleus to the cytoplasm; (iii) the WT1 DNA sequence was optimized in silico to improve translational efficiency; and (iv) this WT1 sequence was cloned into an optimized RNA transcription vector. DCs electroporated with this optimized mRNA showed an improved ability to stimulate WT1-specific T-cell immunity. Furthermore, in a murine model, we were able to show the safety, immunogenicity, and therapeutic activity of this optimized mRNA. This work is relevant for the future development of improved mRNA-based vaccine strategies K.Molecular Therapy-Nucleic Acids (2013) 2, e134; doi:10.1038/mtna.2013.54; published online 19 November 2013.

Dendritic cell therapy has been optimized a lot aiming to induce a strong and broad immune response in terms of the recognized epitopes by both CD8+ and CD4+ T cells and the use of the patients' complete unique set of HLA molecules. We here give an overview of our approach for antigen loading and maturation of dendritic cells and describe the consequences to evaluate the immune response after treatment as well as the Brussels experience in clinical trials.

The efficacy of cancer immunotherapy can be improved by treatment with full-length tumor antigen and by combining several antigens. This approach allows the induction of a broad immune response irrespective of the patient's HLA type which at the same time challenges immune monitoring. Also, the number of available lymphocytes is most often limited and minimal in vitro restimulations of the lymphocytes should maintain information about the actual in vivo situation. To overcome these hurdles, we developed a method to measure the CD8+ and CD4+ T-cell responses directly ex vivo. Skin biopsies taken from dendritic cell (DC)-induced DTH reactions from melanoma patients participating in a DC-clinical trial served as lymphocyte source. Antigen-specificity of skin infiltrating lymphocytes was investigated by coculture with antigen-presenting autologous B cells and assessed for CD137 upregulation and enhanced cytokine secretion. Using this approach we could detect treatment-specific CD8+ T-cells without restimulation in vitro. Upregulation of the activation marker CD137 correlated with the upregulation of the lytic marker CD107a. CD137 upregulation by treatment-specific CD4+ lymphocytes however was more pronounced after antigen-specific in vitro restimulation. Both CD8+ and CD4+ lymphocytes could be further expanded using the same B cells as for screening allowing characterization of the recognized antigenic region. In addition, this technique can be extended to detect a broader array of T-cell functions and to monitor a large cohort of patients. We believe that this approach of direct ex vivo monitoring, irrespective of the patient's HLA-type or the recognized peptide, and using a limited number of lymphocytes is a valuable tool in the immune monitoring of current cellular immunotherapies.

Antitumor cytolytic T lymphocytes (CTLs) recognize peptides derived from cellular proteins and presented on MHC class I. One category of peptides recognized by these CTLs is derived from proteins encoded by “cancer‐germline” genes, which are specifically expressed in tumors, and therefore represent optimal targets for cancer immunotherapy. Here, we identify an antigenic peptide, which is derived from the MAGE‐A1‐encoded protein (160‐169) and presented to CTLs by HLA‐B*44:02. Although this peptide is encoded by MAGE‐A1, processed endogenously and presented by tumor cells, the corresponding synthetic peptide is hardly able to sensitize target cells to CTL recognition when pulsed exogenously. Endogenous processing and presentation of this peptide is strictly dependent on the presence of tapasin, which is believed to help peptide loading by stabilizing a peptide‐receptive form of HLA‐B*44:02. Exogenous loading of the peptide can be dramatically improved by paraformaldehyde fixation of surface molecules or by peptide loading at acidic pH. Either strategy allows efficient exogenous loading of the peptide, presumably by generating or stabilizing a peptide‐receptive, empty conformation of the HLA. Altogether, our results indicate a potential drawback of short peptide‐based vaccination strategies and offer possible solutions regarding the use of problematic epitopes such as the one described here.

Antitumor cytolytic T lymphocytes (CTLs) recognize peptides derived from cellular proteins and presented on MHC class I. One category of peptides recognized by these CTLs is derived from proteins encoded by "cancer-germline" genes, which are specifically expressed in tumors, and therefore represent optimal targets for cancer immunotherapy. Here, we identify an antigenic peptide, which is derived from the MAGE-A1-encoded protein (160-169) and presented to CTLs by HLA-B*44:02. Although this peptide is encoded by MAGE-A1, processed endogenously and presented by tumor cells, the corresponding synthetic peptide is hardly able to sensitize target cells to CTL recognition when pulsed exogenously. Endogenous processing and presentation of this peptide is strictly dependent on the presence of tapasin, which is believed to help peptide loading by stabilizing a peptide-receptive form of HLA-B*44:02. Exogenous loading of the peptide can be dramatically improved by paraformaldehyde fixation of surface molecules or by peptide loading at acidic pH. Either strategy allows efficient exogenous loading of the peptide, presumably by generating or stabilizing a peptide-receptive, empty conformation of the HLA. Altogether, our results indicate a potential drawback of short peptide-based vaccination strategies and offer possible solutions regarding the use of problematic epitopes such as the one described here.