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Ex vivo ELISPOT and multimer staining are well-established tests for the assessment of antigen-specific T cells. Many laboratories are now using a period of in vitro stimulation (IVS) to enhance detection. Here, we report the findings of a multi-centre panel organised by the Association for Cancer Immunotherapy Immunoguiding Program to investigate the impact of IVS protocols on the detection of antigen-specific T cells of varying ex vivo frequency. Five centres performed ELISPOT and multimer staining on centrally prepared PBMCs from 3 donors, both ex vivo and following IVS. A harmonised IVS protocol was designed based on the best-performing protocol(s), which was then evaluated in a second phase on 2 donors by 6 centres. All centres were able to reliably detect antigen-specific T cells of high/intermediate frequency both ex vivo (Phase I) and post-IVS (Phase I and II). The highest frequencies of antigen-specific T cells ex vivo were mirrored in the frequencies following IVS and in the detection rates. However, antigen-specific T cells of a low/undetectable frequency ex vivo were not reproducibly detected post-IVS. Harmonisation of the IVS protocol reduced the inter-laboratory variation observed for ELISPOT and multimer analyses by approximately 20 %. We further demonstrate that results from ELISPOT and multimer staining correlated after (P

With the recent results of promising cancer vaccines and immunotherapy 1 – 5 , immune monitoring has become increasingly relevant for measuring treatment-induced effects on T cells, and an essential tool for shedding light on the mechanisms responsible for a successful treatment. Flow cytometry is the canonical multi-parameter assay for the fine characterization of single cells in solution, and is ubiquitously used in pre-clinical tumor immunology and in cancer immunotherapy trials. Current state-of-the-art polychromatic flow cytometry involves multi-step, multi-reagent assays followed by sample acquisition on sophisticated instruments capable of capturing up to 20 parameters per cell at a rate of tens of thousands of cells per second. Given the complexity of flow cytometry assays, reproducibility is a major concern, especially for multi-center studies. A promising approach for improving reproducibility is the use of automated analysis borrowing from statistics, machine learning and information visualization 21 – 23 , as these methods directly address the subjectivity, operator-dependence, labor-intensive and low fidelity of manual analysis. However, it is quite time-consuming to investigate and test new automated analysis techniques on large data sets without some centralized information management system. For large-scale automated analysis to be practical, the presence of consistent and high-quality data linked to the raw FCS files is indispensable. In particular, the use of machine-readable standard vocabularies to characterize channel metadata is essential when constructing analytic pipelines to avoid errors in processing, analysis and interpretation of results. For automation, this high-quality metadata needs to be programmatically accessible, implying the need for a consistent Application Programming Interface (API). In this manuscript, we propose that upfront time spent normalizing flow cytometry data to conform to carefully designed data models enables automated analysis, potentially saving time in the long run. The ReFlow informatics framework was developed to address these data management challenges.

Robust and sensitive ELISPOT protocols are commonly applied concomitant with the development of new immunotherapeutics. Despite the knowledge that individual serum batches differ in their composition and may change properties over time, serum is still commonly used in immunologic assays. Commercially available serum batches are expensive, limited in quantity and need to be pretested for suitability in immunologic assays, which is a laborious process. The aim of this study was to test whether serum-free freezing media can lead to high cell viability and favorable performance across multiple ELISPOT assay protocols. Thirty-one laboratories from ten countries participated in a proficiency panel organized by the Cancer Immunotherapy Immunoguiding Program to test the influence of different freezing media on cell quality and immunologic function. Each center received peripheral blood mononuclear cells which were frozen in three different media. The participants were asked to quantify antigen-specific CD8+ T-cell responses against model antigens using their locally established IFN-gamma ELISPOT protocols. Self-made and commercially available serum-free freezing media led to higher cell viability and similar cell recovery after thawing and resting compared to freezing media supplemented with human serum. Furthermore, the test performance as determined by (1) background spot production, (2) replicate variation, (3) frequency of detected antigen-specific spots and (4) response detection rate was similar for serum and serum-free conditions. We conclude that defined and accessible serum-free freezing media should be recommended for freezing cells stored for subsequent ELISPOT analysis. Electronic supplementary material The online version of this article (doi:10.1007/s00262-012-1359-5) contains supplementary material, which is available to authorized users.

One of the hallmarks of cancer is the inherent instability of the genome leading to multiple genomic alterations and epigenetic changes that ultimately drive carcinogenesis. These processes lead to a unique molecular profile of every given tumor and to substantial intratumoral heterogeneity of cancer tissues. Recently, a series of independent reports revealed that pre-formed neoantigen specific T-cell responses are of crucial relevance for the clinical efficacy of immune checkpoint inhibitors. However, spontaneous immune recognition of neoantigens seems to be a rare event with only less than 1% of mutations inducing a T-cell response in the tumor-bearing patient. Accordingly, only patients with a high burden of mutations profit from currently approved therapies. To overcome this restriction, the IVAC® MUTANOME-project harnesses the individual patient's mutation profile by manufacturing highly potent neoantigen-coding RNA vaccines. To this end, the individual mutation repertoire is identified by next-generation-sequencing, potentially immunogenic mutations are selected and incorporated into a poly-epitopic RNA vaccine that is tailored to activate and expand the individual patient's neoantigen-specific CD4+ and CD8+ T cells. A phase I study to test this novel concept of an active individualized cancer vaccine for the treatment of malignant melanoma was initiated in 2013 (NCT02035956). Notably, BioNTech RNA Pharmaceutical's IVAC® MUTANOME trial is the first-in-human trial that introduces a fully personalized RNA vaccine for the treatment of malignant melanoma. The objective of this clinical trial is to study the feasibility, safety, tolerability, immunogenicity and the potential clinical activity of the IVAC® MUTANOME approach. The recruitment of a patient into the trial triggers the multi-step IVAC® MUTANOME process covering (i) the receipt and processing of tumor and blood sample specimens, (ii) the identification, prioritization and confirmation of mutations, (iii) testing of pre-existing immunity against identified tumor mutations, (iv) the selection of mutant neoantigen sequences as vaccine targets, (v) design, production of a DNA lead structure, (vi) GMP manufacturing and release of the patient-specific mRNA, (vii) shipment to the clinical trial site and (viii) the administration of the IMP to patients. Detailed information on the trial, the recruitment and treatment status as well as data on the assessment of vaccine induced immune responses will be presented. Citation Format: Matthias Miller, Carmen Loquai, Björn-Philipp Kloke, Sebastian Attig, Nicole Bidmon, Stefanie Bolte, Valesca Bukur, Evelyna Derhovanessian, Jan Diekmann, Angela Filbry, Sandra Heesch, Christoph Hoeller, Klaus Kuehlcke, David Langer, Martin Loewer, Felicitas Mueller, Inga Ortseifer, Burkhard Otte, Anna Paruzynski, Richard Rae, Barbara Schroers, Christine Seck, Kristina Spiess, Arbel D. Tadmor, Isabel Vogler, Mathias Vormehr, Alexandra Kemmer-Brueck, Andreas N. Kuhn, Ulrich Luxemburger, Sebastian Kreiter, Jochen Utikal, Stephan Grabbe, Oezlem Tuereci, Ugur Sahin. IVAC® MUTANOME - A first-in-human phase I clinical trial targeting individual mutant neoantigens for the treatment of melanoma. [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 CT022.

Immunotherapeutic approaches have evolved as promising and valid alternatives to available conventional cancer treatments. Amongst others, vaccination with tumor antigen-encoding RNAs by local administration is currently successfully employed in various clinical trials. To allow for a more efficient targeting of antigen-presenting cells (APCs) and to overcome potential technical challenges associated with local administration, we have developed a novel RNA immunotherapeutic for systemic application based on a fixed set of four liposome complexed RNA drug products (RNA(LIP)), each encoding one shared melanoma-associated antigen. The novel RNA(LIP) formulation was engineered (i) to protect RNA from degradation by plasma RNases and (ii) to enable directed in vivo targeting of APCs in lymphoid compartments, thus (iii) allowing for intravenous administration of multiple RNA products advancing from local to systemic targeting of APCs. Here, RNA(LIP) products trigger a Toll-like receptor (TLR)-mediated Interferon-α (IFN-α) release from plasmacytoid dendritic cells (DCs) and macrophages stimulating DC maturation and hence inducing innate immune mechanisms as well as potent vaccine antigen-specific immune responses. Notably, BioNTech RNA Pharmaceuticals′ RNA(LIP) formulation is a universally applicable potent novel vaccine class for intravenous APC targeting and the induction of potent synchronized adaptive and type-I interferon-mediated innate immune responses for cancer immunotherapy. Similar to other liposomal drugs, the ready-to-use RNA(LIP) products are prepared individually in a straight-forward manner directly prior to use from three components, namely solutions containing RNA drug product, NaCl diluent, and liposome excipient, that are provided as a kit. A multi-center phase I/II trial to clinically validate this pioneering RNA(LIP) formulation for the treatment of malignant melanoma was initiated in 2015 (NCT02410733). The objective of the clinical trial is to study the feasibility, safety, tolerability, immunogenicity and evaluate potential clinical activity of the RNA(LIP) immunotherapy concept. Detailed information on the ongoing trial, the recruitment and treatment status as well as data on the assessment of vaccine-induced immune responses will be presented. Citation Format: Robert A. Jabulowsky, Carmen Loquai, Mustafa Diken, Lena M. Kranz, Heinrich Haas, Sebastian Attig, Nicole Bidmon, Janina Buck, Evelyna Derhovanessian, Jan Diekmann, Daniel Fritz, Veronika Jahndel, Alexandra Kemmer-Brueck, Klaus Kuehlcke, Andreas N. Kuhn, Peter Langguth, Ulrich Luxemburger, Martin Meng, Felicitas Mueller, Richard Rae, Fatih Sari, Doreen Schwarck-Kokarakis, Christine Seck, Kristina Spieß, Meike Witt, Jessica C. Hassel, Jochen Utikal, Roland Kaufmann, Sebastian Kreiter, Christoph Huber, Oezlem Tuereci, Ugur Sahin. A first-in-human phase I/II clinical trial assessing novel mRNA-lipoplex nanoparticles for potent cancer immunotherapy in patients with malignant melanoma. [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 CT032.

Cancer arises from the accumulation of genomic alterations and epigenetic changes that constitute a hallmark of cancer. Owing to the molecular heterogeneity in cancer, only a minor fraction of patients profit from approved therapies. Available targeted therapies can only address alterations common to a particular type of cancer and induce transient effects due to the generation of resistant sub-clones. In contrast, the IVAC MUTANOME project aims to immunologically target multiple cancer mutations uniquely expressed in a given patient's tumor. The IVAC MUTANOME approach should be applicable to the majority of patients irrespective of the tumor entity and offers the potential to exploit the whole tumor mutanome of a given patient using a multi-target approach. The IVAC approach is supported by (i) the availability of technologies that allow fast discovery and validation of individual mutations based on sequencing of whole exome and (ii) an innovative vaccine platform based on RNA-technology supporting fast manufacturing and release of patient-specific vaccines targeting multiple immunogenic mutations within weeks. The phase I study to test the individualized cancer immunotherapeutics for the treatment of malignant melanoma was approved and initiated in 2013 (NCT02035956). With that, the IVAC MUTANOME trial is the first trial in Europe that introduces a fully personalized mutanome vaccine for cancer. The objectives of the clinical trial are to study the feasibility, safety, tolerability and immunogenicity of the IVAC MUTANOME approach for malignant melanoma. Feasibility will be shown by the proven ability to provide the fully personalized IVAC MUANOME vaccine to patients. Recruitment of a patient in the trial repetitively triggers the IVAC MUTANOME process covering (i) the receipt of tumor and blood sample specimens, (ii) the identification, prioritization and confirmation of mutations, (iii) testing of pre-existing immunity against private tumor mutations, (iv) the final selection of mutated sequences, (iv) design, production of a DNA lead structure, (v) GMP manufacturing and release of the patient-specific mRNA, (vi) shipment to the clinical trial site, and (vii) the administration of the IMP to patients. The IVAC MUTANOME recruitment status, manufacturing experiences and treatment status of this first-in-class clinical trial as well as novel data on the immune assessment incl. vaccine-induced mutation-specific T cell responses of the first patients treated will be presented. Citation Format: Bjoern-Philipp Kloke, Cedrik M. Britten, Carmen Loquai, Martin Löwer, Sebastian Attig, Valesca Bukur, Nicole Bidmon, Evelyna Derhovanessian, Jan Diekmann, Mustafa Diken, Angela Filbry, Stephan Grabbe, Sandra Heesch, Christoph Hoeller, David Langer, Uli Luxemburger, Matthias Miller, Felicitas Mueller, Tina Mueller-Brenne, Inga Ortseifer, Burkhard Otte, Anna Paruzynski, Sebastian Petri, Richard Rae, Christine Seck, Kristina Spieß, Arbel D. Tadmor, Jochen Utikal, Klaus Kuehlke, John Castle, Alexandra Kemmer-Brueck, Isabel Vogler, Andreas N. Kuhn, Sebastian Kreiter, Oezlem Tuereci, Ugur Sahin. IVAC MUTANOME: Individualized vaccines for the treatment of cancer. [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 CT202. doi:10.1158/1538-7445.AM2015-CT202