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Modulating the activity of tumor-infiltrating dendritic cells (TiDC) provides opportunities for novel cancer interventions. In this article, we report on our study of the uptake of mRNA by CD8α+ cross-presenting TiDCs upon its intratumoral (i.t.) delivery. We exploited this property to deliver mRNA encoding the costimulatory molecule CD70, the activation stimuli CD40 ligand, and constitutively active Toll-like receptor 4, referred to as TriMix mRNA. We show that TiDCs are reprogrammed to mature antigen-presenting cells that migrate to tumor-draining lymph nodes (TDLN). TriMix stimulated antitumor T-cell responses to spontaneously engulfed cancer antigens, including a neoepitope. We show in various mouse cancer models that i.t. delivery of TriMix mRNA results in systemic therapeutic antitumor immunity. Finally, we show that the induction of antitumor responses critically depends on TiDCs, whereas it only partially depends on TDLNs. As such, we provide a platform and a mechanistic rationale for the clinical testing of i.t. administration of TriMix mRNA. Cancer Immunol Res; 4(2); 146–56. ©2015 AACR.

Supplementary Fig. S1. Delivery of TriMix mRNA to the tumor reprograms TiDCs. Supplementary Fig. S2. Delivery of TriMix mRNA to tumors induces tumor-specific CTLs.

PDF file, 1359K, Vaccine characteristics of first cycle of all patients. (A) Expression of HLA-ABC, HLA-DR/DP, HLA-DQ, CD80, CD86, CD83, and CCR7 was analyzed by flow cytometry. (B) Tumor antigen expression by DCs 2-4 hrs after electroporation with mRNA encoding gp100 and tyrosinase. Data are shown as percentage of positive DCs used for the first vaccination. The graphs represent mean � SD of all patients.

PDF file 98K, Influence of GM-CT-01 on T cell and melanoma cell growth

PDF file, 96K, Detection of tumor-specific T cells in SKIL cultures does not require challenge with defined epitopes. Tetramer analysis of SKILs cultured from biopsies of DTH reactions to DCs pulsed with tumor peptides (upper plots) or DCs electroporated with mRNA encoding tumor proteins (lower plots) of patients II-E-09 (A), II-E-03 (B), and II-E-10 (C). This figure shows that sampling DTH biopsy cultures with tetramers yields similar results when the patient is challenged with intradermal injections with DC pulsed with defined epitopes or DC electroporated with mRNA encoding the full tumor antigen.

PDF file 231K, Effect of GM-CT-01 on the capacity of CD8+ TIL to degranulate

Purpose: Electroporation of dendritic cells (DC) with mRNA encoding tumor-associated antigens (TAA) has multiple advantages compared to peptide loading. We investigated the immunologic and clinical responses to vaccination with mRNA-electroporated DC in stage III and IV melanoma patients.Experimental design: Twenty-six stage III HLA*02:01 melanoma patients scheduled for radical lymph node dissection (stage III) and 19 melanoma patients with irresectable locoregional or distant metastatic disease (referred to as stage IV) were included. Monocyte-derived DC, electroporated with mRNA encoding gp100 and tyrosinase, were pulsed with keyhole limpet hemocyanin and administered intranodally. TAA-specific T-cell responses were monitored in blood and skin-test infiltrating lymphocyte (SKIL) cultures.Results: Comparable numbers of vaccine-induced CD8+ and/or CD4+ TAA-specific T-cell responses were detected in SKIL cultures; 17/26 stage III patients and 11/19 stage IV patients. Strikingly, in this population, TAA-specific CD8+ T cells that recognize multiple epitopes and produce elevated levels of IFNγ upon antigenic challenge in vitro, were significantly more often observed in stage III patients; 15/17 versus 3/11 stage IV patients, P = 0.0033. In stage IV patients, one mixed and one partial response were documented. The presence or absence of IFNγ-producing TAA-specific CD8+ T cells in stage IV patients was associated with marked difference in median overall survival of 24.1 months versus 11.0 months, respectively.Conclusion: Vaccination with mRNA-electroporated DC induces a broad repertoire of IFNγ producing TAA-specific CD8+ and CD4+ T-cell responses, particularly in stage III melanoma patients. Clin Cancer Res; 18(19); 5460–70. ©2012 AACR.

PDF file, 149K, CONSORT flow chart.

PDF file 156K, Efficacy of different galactomannans for boosting TIL functions

PDF file 105K, GM-CT-01 boosts secretion of various cytokines by TIL

PDF file 114K, In contrast to LacNAc, GM-CT-01 does not detach galectin-1 and galectin-3 from melanoma cell surface

Purpose: Several galectins are released by tumor cells and macrophages and accumulate in the tumor microenvironment. Galectin-1 and -3 were found to bind to glycosylated receptors at the surface of tumor-infiltrating lymphocytes (TIL), forming glycoprotein–galectin lattices that could reduce the motility and therefore the functionality of surface molecules. In contrast to blood T cells, human TIL show defective IFN-γ secretion upon ex vivo stimulation. We have previously shown that extracellular galectin-3 participates in the impairment of TIL functions. Indeed, disruption of glycoprotein–galectin-3 lattices using anti-galectin-3 antibodies, or N-acetyllactosamine as a competing sugar, boosted cytokine secretion by TIL. Here we have tested a clinical grade galectin antagonist: GM-CT-01, a galactomannan obtained from guar gum reported to be safe in more than 50 patients with cancer.Experimental Design: TIL were isolated from human tumor ascites, treated for 2 to 20 hours with galectin antagonists and tested for function.Results: We found that GM-CT-01 boosts cytotoxicity of CD8+ TIL and their IFN-γ secretion in a dose-dependent manner. Treating TIL obtained from patients with various cancers, during a few hours, resulted in an increased IFN-γ secretion in up to 80% of the samples.Conclusions: These observations pave the way for investigating the potential benefit of this galectin antagonist in patients with cancer, alone or combined with cancer vaccination, in order to correct in vivo impaired functions of TIL. Clin Cancer Res; 20(7); 1823–33. ©2014 AACR.

Melanoma patients were injected with various vaccines containing a MAGE-A3 peptide presented by HLA-DP4. Anti–MAGE-A3.DP4 T cells were not detectable in the blood before vaccination, but their frequencies after vaccination ranged from 2 × 10−6 to 2 × 10−3 among the CD4+ blood T lymphocytes of the patients. The CD4+ blood T lymphocytes that stained ex vivo with HLA-DP4 tetramers folded with the MAGE-A3 peptide were selected by flow cytometry and amplified under clonal conditions. About 5% of the CD4+ T-cell clones that recognized the MAGE-A3.DP4 antigen had a CD25+ phenotype in the resting state. These CD25+ clones had a high capacity to suppress the proliferation of another T-cell clone after peptide stimulation in vitro. Most of them had high FOXP3 expression in the resting state and an unmethylated FOXP3 intron 1. They produced active transforming growth factor-β but none of cytokines IFN-γ, interleukin-2 (IL-2), IL-4, IL-5, and IL-10. About 20% of CD25− clones had a significant but lower suppressive activity. Most of the CD25− clonal populations contained cells that expressed FOXP3 in the resting state, but FOXP3 demethylation was not observed. We conclude that MAGE-A3.DP4 vaccination can produce CD4+ T cells that may exert regulatory T-cell function in vivo. [Cancer Res 2009;69(10):4335–45]

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Supplementary Table 1 from A Galectin-3 Ligand Corrects the Impaired Function of Human CD4 and CD8 Tumor-Infiltrating Lymphocytes and Favors Tumor Rejection in Mice

Supplementary Figure 4 from The CD4+ T-Cell Response of Melanoma Patients to a MAGE-A3 Peptide Vaccine Involves Potential Regulatory T Cells

PDF file - 117K, Uptake of LV-tSMAC transduced dying human melanoma cells enables dendritic cells to activate tumor-infiltrating lymphocytes.

Human CD8+ tumor-infiltrating T lymphocytes (TIL), in contrast with CD8+ blood cells, show impaired IFN-γ secretion on ex vivo restimulation. We have attributed the impaired IFN-γ secretion to a decreased mobility of T-cell receptors on trapping in a lattice of glycoproteins clustered by extracellular galectin-3. Indeed, we have previously shown that treatment with N-acetyllactosamine, a galectin ligand, restored this secretion. We strengthened this hypothesis here by showing that CD8+ TIL treated with an anti–galectin-3 antibody had an increased IFN-γ secretion. Moreover, we found that GCS-100, a polysaccharide in clinical development, detached galectin-3 from TIL and boosted cytotoxicity and secretion of different cytokines. Importantly, we observed that not only CD8+ TIL but also CD4+ TIL treated with GCS-100 secreted more IFN-γ on ex vivo restimulation. In tumor-bearing mice vaccinated with a tumor antigen, injections of GCS-100 led to tumor rejection in half of the mice, whereas all control mice died. In nonvaccinated mice, GCS-100 had no effect by itself. These results suggest that a combination of galectin-3 ligands and therapeutic vaccination may induce more tumor regressions in cancer patients than vaccination alone. Cancer Res; 70(19); 7476–88. ©2010 AACR.

Supplementary Figure 3 from The CD4+ T-Cell Response of Melanoma Patients to a MAGE-A3 Peptide Vaccine Involves Potential Regulatory T Cells

Supplementary Table 1 from The CD4+ T-Cell Response of Melanoma Patients to a MAGE-A3 Peptide Vaccine Involves Potential Regulatory T Cells

PDF file - 184K, The therapeutic effect of LV-tSMAC involves effector T-cells.

Supplementary Figure Legends 1-6 from The CD4+ T-Cell Response of Melanoma Patients to a MAGE-A3 Peptide Vaccine Involves Potential Regulatory T Cells

Supplementary Figure 5 from The CD4+ T-Cell Response of Melanoma Patients to a MAGE-A3 Peptide Vaccine Involves Potential Regulatory T Cells

Supplementary Methods from A Galectin-3 Ligand Corrects the Impaired Function of Human CD4 and CD8 Tumor-Infiltrating Lymphocytes and Favors Tumor Rejection in Mice

Supplementary References from A Galectin-3 Ligand Corrects the Impaired Function of Human CD4 and CD8 Tumor-Infiltrating Lymphocytes and Favors Tumor Rejection in Mice

Supplementary Figures 1-3 from A Galectin-3 Ligand Corrects the Impaired Function of Human CD4 and CD8 Tumor-Infiltrating Lymphocytes and Favors Tumor Rejection in Mice

Supplementary Figure Legends 1-3 from A Galectin-3 Ligand Corrects the Impaired Function of Human CD4 and CD8 Tumor-Infiltrating Lymphocytes and Favors Tumor Rejection in Mice

Supplementary Figure 6 from The CD4+ T-Cell Response of Melanoma Patients to a MAGE-A3 Peptide Vaccine Involves Potential Regulatory T Cells

Supplementary Figure 2 from The CD4+ T-Cell Response of Melanoma Patients to a MAGE-A3 Peptide Vaccine Involves Potential Regulatory T Cells

Modulating the activity of tumor-infiltrating dendritic cells (TiDC) provides opportunities for novel cancer interventions. In this article, we report on our study of the uptake of mRNA by CD8α+ cross-presenting TiDCs upon its intratumoral (i.t.) delivery. We exploited this property to deliver mRNA encoding the costimulatory molecule CD70, the activation stimuli CD40 ligand, and constitutively active Toll-like receptor 4, referred to as TriMix mRNA. We show that TiDCs are reprogrammed to mature antigen-presenting cells that migrate to tumor-draining lymph nodes (TDLN). TriMix stimulated antitumor T-cell responses to spontaneously engulfed cancer antigens, including a neoepitope. We show in various mouse cancer models that i.t. delivery of TriMix mRNA results in systemic therapeutic antitumor immunity. Finally, we show that the induction of antitumor responses critically depends on TiDCs, whereas it only partially depends on TDLNs. As such, we provide a platform and a mechanistic rationale for the clinical testing of i.t. administration of TriMix mRNA. Cancer Immunol Res; 4(2); 146–56. ©2015 AACR.

Background: Treatment of triple negative breast cancer (TNBC) is hampered by lack of established therapeutic targets like hormone receptors or HER-2. Surgery, chemotherapy and radiotherapy are the standard of care yet cure rates in patients with TNBC remain inferior compared to other BC subtypes. Approaches tailored to the patient’s individual tumor signature may lead to improvement. The “Mutanome Engineered RNA Immuno-Therapy (MERIT)” consortium is validating an innovative individualized mRNA-based vaccine for TNBC treatment. MERIT is a collaboration of five European partners (academia and industry) dedicated to realizing a personalized approach for TNBC treatment. The consortium set up a clinical research program covering drug development from target discovery and validation to GMP manufacturing and drug release for each individual patient (MUTANOME). Moreover, the consortium also established a pre-synthesized mRNA vaccine warehouse containing the most frequently shared tumor-associated antigens (TAA) in TNBC for drug supply (WAREHOUSE). Methods: A Phase I trial (NCT02316457) in two European countries is assessing the feasibility, safety and vaccine-induced immune response of this personalized immunotherapy. TNBC patients (pT1cN0M0 - TxNxM0) after surgery and (neo-)adjuvant chemotherapy are allocated to one of three study arms. Patients in ARM1 receive eight WAREHOUSE vaccinations with personalized TAA combinations corresponding to the patient’s tumor antigen-expression profile. Patients in ARM2 are first treated with the WAREHOUSE vaccine (optional) followed by eight vaccination cycles of an on-demand manufactured MUTANOME vaccine encoding the unique mutation signature of the individual patient identified by next-generation sequencing. Patients in ARM3 receive eight WAREHOUSE vaccinations including an additional helper RNA. The treatment of twelve patients in ARM1 is completed, while treatment of patients for ARM2 and ARM3 is ongoing. The mRNAs are administered intravenously as a RNA-lipoplex formulation, which BioNTech has previously shown is capable of protecting RNA from degradation, activating innate immunity, and transfecting APCs. Four clinical sites are open for recruitment; >30 patients have been screened and vaccination with WAREHOUSE or MUTANOME RNA has started. Biomarker analysis is ongoing while the trial is continuing as planned. Preliminary data on the assessment of feasibility of WAREHOUSE and MUTANOME approaches will be given. We will give insights into features of the established process and upcoming adaptations in study design. Citation Format: Ludwig Heesen, Katrin Frenzel, Stefanie Bolte, Valesca Bukur, Mustafa Diken, Evelyna Derhovanessian, Sebastian Kreiter, Andreas N. Kuhn, Klaus Kühlcke, Martin Löwer, Henrik Lindman, Steve Pascolo, Marcus Schmidt, Andreas Schneeweiss, Tobias Sjöblom, Kris Thielemans, Laurence Zitvogel, Özlem Türeci, Ugur Sahin. Mutanome engineered RNA immuno-therapy (MERIT) for patients with triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT221.

Purpose: The purpose of this study was to investigate expression and epigenetic regulation of CD9 in multiple myeloma (MM) cells during disease progression. Experimental Design: CD9 expression was retrospectively analyzed on bone marrow myeloma samples from 81 patients by immunophenotyping. CD9 expression by murine 5TMM cells was detected by flow cytometric staining and quantitative PCR. The methylation status of the CD9 promoter was determined by bisulfite PCR sequencing. Results: Primary plasma cells in the majority of MM patients with nonactive disease (n = 28) showed CD9 expression, whereas most cases with active disease (n = 53) were CD9 negative. CD9 expression in diagnostic bone marrow samples (n = 74) correlated with survival. Moreover, CD9 expression on murine 5T33 and 5T2MM cells was significantly down-regulated during disease development. Treatment of CD9-nonexpressing 5T33MMvt cells with the clinically relevant histone deacetylase inhibitor LBH589 resulted in a significant increase in CD9 expression. In contrast, cells treated with the demethylation agent 5-aza-2′deoxycytidine barely showed any increase. A combination study with both compounds resulted in a strong synergistic reactivation of CD9. CD9-expressing 5T33MMvv cells and 5T33MMvt cells stably transduced with a mCD9 lentiviral transferplasmid were shown to be more susceptible to natural killer cell–mediated cytolysis than CD9-negative 5T33MMvt cells. Conclusions: CD9 expression correlates with disease status and survival of MM patients. In the murine 5T33MM model, we show that histone modifications, and to a lesser extent CpG methylation, are key epigenetic events in CD9 down-regulation. Furthermore, as CD9 expression becomes down-regulated, 5T33MM cells become less susceptible to natural killer cell–mediated cytolysis.

Tumor regressions have been observed in a small proportion of melanoma patients vaccinated with a MAGE-A3 peptide presented by HLA-A1, administered as peptide, ALVAC canarypox virus containing a MAGE-A3 minigene, or peptide-pulsed dendritic cells (DC). There was a correlation between tumor regression and the detection of anti–MAGE-3.A1 CTL responses. These responses were monoclonal and often of a very low magnitude after vaccination with peptide or ALVAC, and usually polyclonal and of a higher magnitude after DC vaccination. These results suggested that, at least in some patients, surprisingly few anti–MAGE-3.A1 T-cells could initiate a tumor regression process. To understand the role of these T cells, we carried out a functional analysis of anti–MAGE-3.A1 CTL clones derived from vaccinated patients who displayed tumor regression. The functional avidities of these CTL clones, evaluated in lysis assays, were surprisingly low, suggesting that high avidity was not part of the putative capability of these CTL to trigger tumor rejection. Most anti–MAGE-3.A1 CTL clones obtained after DC vaccination, but not after peptide or ALVAC vaccination, produced interleukin 10. Transcript profiling confirmed these results and indicated that approximately 20 genes, including CD40L, prostaglandin D2 synthase, granzyme K, and granzyme H, were highly differentially expressed between the anti–MAGE-3.A1 CTL clones derived from patients vaccinated with either peptide-ALVAC or peptide-pulsed DC. These results indicate that the modality of vaccination with a tumor-specific antigen influences the differentiation pathway of the antivaccine CD8 T-cells, which may have an effect on their capacity to trigger a tumor rejection response. [Cancer Res 2008;68(10):3931–40]

Autologous monocyte-derived synthetic mRNA electroporated dendritic cells (TriMixDC-MEL) are immunogenic and can induce anti-tumor activity in pretreated advanced melanoma patients when administered by the intradermal (id) and intravenous (iv) route. We investigated the safety and activity of TriMixDC-MEL in stage III/IV melanoma patients who are disease free following the local treatment of macrometastases. We performed a randomized, controlled, non-comparative phase II clinical trial where TriMixDC-MEL was administered iv (20.106 DCs) and id (4.106 DCs) at 2 separate sites of the body every 2 weeks for a total of 4 administrations and a fifth administration after 16 weeks in patients randomized to the treatment arm. Patients on the control arm did not receive treatment but were allowed to “cross-over” and be treated with TriMixDC-MEL at the time of non-salvageable recurrence. Tumor evaluations on both arms were performed by total-body 18-FDG-PET/CT every 12 weeks. The primary endpoint was the percentage of patients who were alive and free from melanoma macrometastases at 1 year following randomization. Patients were allowed to undergo local salvage treatments for loco-regional melanoma recurrences during the study (week 0-52). If local salvage treatment during the study resulted in a disease-free status that was confirmed at any of the planned assessment, but not earlier than 16 weeks after the salvage treatment, these patients were considered not to have reached the primary endpoint of the study. Between November 2012 and November 2014, 41 eligible patients were randomized between the TriMixDC-MEL treatment arm (n = 21) and control-arm (n = 20). Baseline characteristics (22M/19F; median age 57.5y (range 24-81); AJCC stage III/IV-M1a/-M1b/-M1c: 33/1/6/1 patients, ulcerated primary melanoma: 12 patients) were well balanced between both groups. After a median follow-up of 28 months (range 15 to 40 months) 21 patients experienced a non-salvageable melanoma recurrence (7 on the DC- and 14 on the control-arm). The rate of patients who were disease-free at 1 year (evaluable population = 41 patients) was higher in the TriMixDC-MEL treated group (68% [95%CI 46-86] vs. 35% [14-55]). The time-to-non-salvageable melanoma recurrence was significantly lower in the TriMixDC-MEL treated group (log-rank p = .021). Seven patients in the TriMixDC-MEL group and 3 patients in the control arm were offered local salvage therapy at first recurrence (surgery: 9 patients; RT: 1pt). TriMixDC-MEL was well tolerated (there were no grade ≥3 AE related to TriMixDC-MEL). AE were limited to: local skin injection reactions (grade 1-2; 17/21 patients), flu-like symptoms (grade 1-2; 4/21 patients), and post-infusion chills (grade 1-2; 4 [19%] patients). The results of this non-comparative randomized controlled phase II clinical trial of TriMixDC-MEL id/iv versus observation support the further evaluation of TriMixDC-MEL as a well-tolerated adjuvant therapy for melanoma patients following the resection of macrometastases. Citation Format: Bart Neyns, Yanina Jansen, Jurgen Corthals, Sofie Wilgenhof, Max Schreuer, Carlo Heirman, Kris Thielemans. A randomized controlled phase II clinical trial on mRNA electroporated autologous dendritic cells for stage III/IV melanoma patients who are disease-free following the local treatment of macrometastases [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B003.

The majority of metastatic cancers remain incurable since the current methods of treatment often fail to target the heterogeneous nature of each individual patient's tumor. Personalized approaches targeting each individual patient's tumor may therefore bring significant improvements. The Mutanome Engineered RNA Immuno-Therapy (MERIT) consortium will clinically validate a pioneering RNA-based immunotherapy concept for the treatment of triple negative breast cancer (TNBC) by targeting shared tumor antigens and individual neo-antigens in TNBC patients. MERIT combines two personalized treatment concepts: (i) treatment with vaccines containing “off-the-shelf” mRNAs selected from a pre-synthesized mRNA vaccine warehouse (MERIT WAREHOUSE) that encode shared breast cancer tumor antigens expressed in the respective patient's tumor; (ii) treatment with mRNAs engineered on-demand that encode patient-specific mutated neo-antigens identified by next generation sequencing (NGS) and ranked according to the predicted immunogenicity (MERIT MUTANOME). The mRNAs are administered intravenously as a nanoparticulate lipoplex formulation, which specifically targets APCs and consequently induces antigen-specific T cell responses. MERIT is a multi-center phase I trial (NCT02316457) conducted in four European countries to assess the feasibility, safety and biological efficacy of this personalized immunotherapy. TNBC patients (pT1cN0M0 - anyTanyNM0) after surgery and adjuvant chemotherapy will be allocated to one of two study arms. Patients in ARM1 will receive eight vaccination cycles with a personalized set of shared tumor antigens selected from the WAREHOUSE that correspond to the patient tumor's antigen-expression profile. Patients in ARM2 will be first treated with the personalized WAREHOUSE vaccine approach followed by six vaccination cycles of on-demand manufactured MUTANOME vaccine targeting the unique mutation signature of the individual patient. During the clinical trial, patients will receive the individualized combination of the RNAs in parallel to standard radiotherapy. The clinical trial is approved and the study start is planned for Q1 2016. The consortium has built a multi-disciplinary clinical workflow and trial design tailored to this unique therapeutic concept, which covers the whole individualized drug development cycle from target discovery, validation to GMP manufacturing and drug release for each individual patient. We will present the therapeutic concept and study protocol as well as the methodologies required for this highly innovative phase I trial. The personalized immunotherapy overcomes the current limitations of fixed, off-the-shelf therapeutics and thus might increase the clinical benefit for TNBC patients. This project is a collaborative effort of five partners from academia and industry funded by the European Commission's FP7 and led by BioNTech AG. Citation Format: Sandra Heesch, Valesca Bukur, Janina Buck, Jan Diekmann, Mustafa Diken, Kerstin Ewen, Heinrich Haas, Alexandra Kemmer-Brueck, Björn-Philipp Kloke, Sebastian Kreiter, Andreas N. Kuhn, Klaus Kuehlcke, Martin Loewer, Anna Paruzynski, Kathrin Schultheiß, Doreen Schwarck, Marcus Schmidt, Fabrice André, Jacques De Greve, Thomas Kuendig, Henrik Lindman, Steve Pascolo, Tobias Sjöblom, Kris Thielemans, Laurence Zitvogel, Özlem Türeci, Ugur Sahin. MERIT: introducing individualized cancer vaccines for the treatment of TNBC - a phase I trial. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT020.

Modification of dendritic cells (DC) with mRNA allows the loading of these cells with tumor antigens and the functional modification of the cellular vaccine. To reprogram immature DC towards potent and immunostimulatory antigen presenting cells, we provide three different molecular adjuvants to DCs with mRNA coding for caTLR4, mimicking TLR-activation, CD40L, mimicking the ‘licensing’ of DCs by Th cells, and CD70 to provide an extra stimulus for the priming of CD8+ T cells. The mixture of these three mRNA's (caTLR4, CD40L and CD70) is referred to as ‘TriMix’ The programming of DC with mRNA can be performed either in vitro, by electroporation, or even in vivo after injection of mRNA into lymph nodes or at the tumor site. At our institution, clinical trials in advanced melanoma patients are being performed using ex vivo modified DCs. These patients are treated with TriMixDC-MEL, a mixture of TriMix-DC co-electroporated with mRNA encoding a fusion of DC.LAMP and 1 of 4 melanoma associated antigens (TAA) (gp100, tyrosinase, MAGE-C2 or MAGE-A3). In a first pilot clinical trial, 24.106 TriMixDC-MEL cells were administrated solely by the intradermal (ID) route. Subsequently, a phase IB was conducted to investigate the safety of administrating TriMixDC-MEL by the intravenous (IV) and ID-route. ID administration of TriMixDC-MEL was found to be feasible, safe, effectively stimulating CD8+ T-cell responses, but did not result in objective tumor responses. In contrast, the combined ID/IV administration is associated with distinct but manageable side-effects and has seemingly superior clinical activity as compared to DC administered solely ID in patients with pretreated advanced melanoma. We also investigated the safety and activity of TriMixDC-MEL combined with check-point blockade (ipilimumab). Thirty nine AJCC stage IIIc/IV melanoma pts initiated study treatment). Following DC-administration, gr2 skin injection site reactions were observed in all pts, post-infusion chills (< gr2) in 15 (38%), and transient flu-like symptoms (< gr2) in 33 pts (85%). Most frequent grade 3/4 adverse events of special interest were: dermatitis, diarrhea/colitis, hypophysitis, hepatitis, and pneumonitis. Systemic corticotherapy was used to treat irAE in 18 pts (46%). Best overall tumor response by irRC: 8 CR, and 7 PR (BORR 38%), 6 SD and 18 PD. All CR, and 3 PR are ongoing after a median duration of 19 mths (range 3-29 mths). The 6-mths DCR by irRC is 50% (95% CI 34-66). Median PFS and OS are respectively 6.2 (95% CI 3-9), and 14.4 mths (95%CI 10-18). These encouraging clinical responses indicate that further clinical development of TriMixDC-MEL in combination with immune checkpoint modulators is warranted. Furthermore, more recent pre-clinical studies indicate that this cellular therapy with ex vivo modified DCs could evolve towards a less complex approach via the direct in vivo modification of DCs. We evaluated whether intranodal (IN) and intratumoral (IT) delivery of TriMix mRNA together or not with TAA mRNA, results in the in situ modification and maturation of DCs hence priming of TAA-specific T-cells. Upon IN and IT delivery of mRNA, we could demonstrate the selective uptake and translation of mRNA by lymph node- and tumor-resident CD11c+ cells, respectively. This process was hampered by co-delivery of classical maturation stimuli but not by TriMix mRNA. Importantly, injection of TriMix mRNA induced a T-cell attracting and stimulatory environment. Enhanced induction of antigen-specific CD4+ and CD8+ T-cells was demonstrated using TriMix mRNA and mRNA's several antigens upon IN immunization. Intranodal mRNA immunization was shown to be as efficient in induction of CTLs and in therapy as vaccination with ex vivo mRNA electroporated DCs in several mouse tumor models. Upon IT injection of TriMix mRNA, the tumor-resident DCs become activated, migrate to lymph nodes and activate CD8+ cytotoxic T lymphocyte responses against several cancer-antigens, including a cancer-testis antigen and a neoepitope. Since no TAA encoding mRNA was added, these results indicate that the tumor-resident DCs are loaded with tumor antigens, can be activated in situ and can be tuned into an auto-vaccine. Intratumoral injection of TriMix mRNA only showed curative potential against disseminated tumors. In conclusion, IN and IT administration of TAA mRNA together with mRNA encoding DC modulating molecules is a promising vaccination strategy that could evolve towards an off-the-shelf anti-cancer vaccine. Citation Format: Kris Thielemans. mRNA and dendritic cell based immunotherapy. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B36.

The Mutanome Engineered RNA Immuno-Therapy (MERIT) consortium will clinically and industrially validate a pioneering RNA-based immunotherapy concept that targets individual tumor antigens and tumor-specific mutations in triple negative breast cancer (TNBC) patients. This biomarker-guided, personalized therapy is a collaborative effort of five partners from academia and industry and is funded by the European Commission's FP7 and led by BioNTech AG. TNBC is an aggressive, molecularly heterogeneous cancer that accounts for 20% of all breast cancer patients. The 5-year survival rate is less than 80%. The molecular heterogeneity across TNBCs results in a lack of common targetable molecular alterations, and thus targeted therapies frequently fail to provide clinical benefit. The MERIT concept attempts to address this unmet medical need. The personalized treatment consists in (i) injecting vaccines containing “off the shelf” mRNAs selected from a pre-synthesized mRNA vaccine warehouse (MERIT WAREHOUSE) that encode tumor specific antigens expressed in the respective patient's tumor; and (ii) thereafter mRNAs engineered on-demand that encode patient-specific sequence stretches incorporating non-synonymous mutations identified by next generation sequencing (NGS) and ranked by predicted immunogenicity (MERIT MUTANOME). The mRNAs are administered intravenously as a nanoparticulate lipoplex formulation and are selectively delivered to splenic APCs. The encoded antigens are translated into proteins that are rapidly processed. Subsequent peptide presentation on the surface of APCs induces antigen-specific T cell responses. The central part of the MERIT project, a multi-center first in human trial, will assess the feasibility, safety and biological efficacy of this innovative personalized immunotherapy in TNBC patients. After discussing the regulatory challenges with the German national regulatory agency (PEI), a phase I study is now in preparation. The trial will start in Q2 2015 in five academic centers in Europe and will recruit thirty TNBC patients. Furthermore, the project includes a comprehensive T-cell immunomonitoring and biomarker program. Moreover, an extensive research program will address the optimization of algorithms for improved prediction of immunogenic mutations. Additionally, compounds to enhance vaccine efficacy will be developed and improved to support further clinical development. We have established a RNA delivery platform as well as a MERIT WAREHOUSE containing mRNAs coding for a selection of TNBC specific antigens. Additionally, we have built a multi-disciplinary clinical workflow and trial design tailored to this unique therapeutic concept. We will describe the therapeutic concept and the critical skills, and methodologies required for this project, including cancer genomics, NGS, bioinformatics, tumor immunomics, industrial drug development, GMP manufacturing, clinical immunotherapy and immunological monitoring. Citation Format: Sandra Heesch, Cedrik M. Britten, Valesca Bukur, Janina Buck, John Castle, Jan Diekmann, Mustafa Diken, Katrin Frenzel, Sebastian Kreiter, Andreas N. Kuhn, Klaus Kuehlcke, Martin Loewer, Heinrich Haas, Alexandra Kemmer-Brueck, Bjoern-Philipp Kloke, Burkhard Otte, Anna Paruzynski, Sebastian Petri, Doreen Schwarck-Kokarakis, Marcus Schmidt, Fabrice André, Jacques De Greve, Thomas Kuendig, Henrik Lindman, Steve Pascolo, Tobias Sjöblom, Kris Thielemans, Laurence Zitvogel, Oezlem Tuereci, Ugur Sahin. The Mutanome Engineered RNA Immuno-Therapy (MERIT) project. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT201. doi:10.1158/1538-7445.AM2015-CT201