The use of clonal mRNA as an antigenic format for the detection of antigen-specific T lymphocytes in IFN-gamma ELISPOT assays.

Abstract Most assay systems for the quantification of antigen-specific T-cell responses in infectious, malignant and autoimmune disease depend on the peptide antigen format and are therefore restricted to known epitopes and their presenting HLA molecules. Here we tested in ELISPOT assays the application of in vitro-transcribed clonal mRNA as an alternative antigen format covering all potential epitopes of a given antigen. As model antigens, we chose pp65 of human cytomegalovirus (HCMV) and human tyrosinase (hTyr). Antigen-presenting cells (APC) were K562 cells stably transfected with single HLA class I alleles and autologous dendritic cells (DC). As effectors, we applied in vitro-generated …

Hierarchical modeling for rare event detection and cell subset alignment across flow cytometry samples.

Flow cytometry is the prototypical assay for multi-parameter single cell analysis, and is essential in vaccine and biomarker research for the enumeration of antigen-specific lymphocytes that are often found in extremely low frequencies (0.1% or less). Standard analysis of flow cytometry data relies on visual identification of cell subsets by experts, a process that is subjective and often difficult to reproduce. An alternative and more objective approach is the use of statistical models to identify cell subsets of interest in an automated fashion. Two specific challenges for automated analysis are to detect extremely low frequency event subsets without biasing the estimate by pre-processing…

Response Determination Criteria for ELISPOT: Toward a Standard that Can Be Applied Across Laboratories

ELISPOT assay readout is often dichomized as positive or negative responses according to prespecified criteria. However, these criteria can vary widely across institutions. The adoption of a common response criterion is a key step toward cross-laboratory comparability. This chapter describes the two main approaches to response determination, identifying the strengths and limitations of each. Nonparametric statistical tests and consideration of data quality are recommended and instructions provided for their ready implementation by nonstatisticians and statisticians alike.

The European regulatory environment of rna-based vaccines

A variety of different mRNA-based drugs are currently in development. This became possible, since major breakthroughs in RNA research during the last decades allowed impressive improvements of translation, stability and delivery of mRNA. This article focuses on antigen-encoding RNA-based vaccines that are either directed against tumors or pathogens. mRNA-encoded vaccines are developed both for preventive or therapeutic purposes. Most mRNA-based vaccines are directly administered to patients. Alternatively, primary autologous cells from cancer patients are modified ex vivo by the use of mRNA and then are adoptively transferred to patients. In the EU no regulatory guidelines presently exist t…

Abstract CT202: IVAC MUTANOME: Individualized vaccines for the treatment of cancer

Abstract 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 expl…

The Impact of Harmonization on ELISPOT Assay Performance

During more than 25 years of application in immunological sciences, ELISPOT has been established as a routine, robust, versatile, and reliable assay. From basic research to clinical immune monitoring, ELISPOT is being used to address the quantification and (to a lesser extent) functional characterization of immune cells secreting different molecules in the context of health and disease, immune intervention, and therapy in humans and other species [Kalyuzhny (Ed.) (2005) Handbook of Elispot: methods and protocols, Vol. 302, Humana Press Inc., Totowa, NJ]. Over the last decade, ELISPOT assays have been increasingly implemented as an immune-monitoring tool in clinical trials [Schmittel et al. …

Meeting Report from the Second “Minimum Information for Biological and Biomedical Investigations” (MIBBI) workshop

This report summarizes the proceedings of the second workshop of the ‘Minimum Information for Biological and Biomedical Investigations’ (MIBBI) consortium held on Dec 1–2, 2010 in Rudesheim, Germany through the sponsorship of the Beilstein-Institute. MIBBI is an umbrella organization uniting communities developing Minimum Information (MI) checklists to standardize the description of data sets, the workflows by which they were generated and the scientific context for the work. This workshop brought together representatives of more than twenty communities to present the status of their MI checklists and plans for future development. Shared challenges and solutions were identified and the role…

Prophylactic transfer of CD8-depleted donor lymphocytes after T-cell–depleted reduced-intensity transplantation

AbstractAllogeneic hematopoietic stem cell transplantation (SCT) regimens incorporating the lymphocytotoxic CD52 antibody alemtuzumab demonstrate efficient engraftment and reduced graft-versus-host disease (GVHD). However, these protocols substantially impair posttransplantation antiviral and antitumor immunity. To accelerate immune reconstitution after alemtuzumab-based reduced-intensity SCT, we administered prophylactic CD8-depleted donor lymphocyte infusions (DLIs) starting on days 60 and 120 after transplantation. DLIs were processed in an immunomagnetic good manufacturing practice depletion procedure resulting in a 2.5- to 6-log reduction in CD8 T cells. Of 23 high-risk patients with h…

Managing Multi-center Flow Cytometry Data for Immune Monitoring.

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…

T cell assays and MIATA: the essential minimum for maximum impact.

The Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA*Correspondence: cedrik.britten@tron-mainz.dehttp://dx.doi.org/10.1016/j.immuni.2012.07.010The field of immunology has recentlyexperienced enormous advances fromwhich most have so far not been incorpo-rated into standard medical practice (Da-vis, 2008). One approach to fully exploitthe existing wealth of knowledge is toimplement a systematic strategy to eval-uate the immune system. The potentialbenefit of such an approach is that itmay lead to results that can be translatedinto the rational development of diagnos-tics and therapeutics (Hoos et al., 2011).Two prerequisites for its application ar…

A methodological framework to enhance the clinical success of cancer immunotherapy.

The role of the reporting framework MIATA within current efforts to advance immune monitoring

The use of HLA-A*0201-transfected K562 as standard antigen-presenting cells for CD8+ T lymphocytes in IFN-γ ELISPOT assays

ELISPOT assays are increasingly used for a direct detection and quantification of single antigen-specific T cells in freshly isolated peripheral blood mononuclear cells (PBMC). They are particularly attractive for the monitoring of specific T lymphocyte responses in clinical trials assessing antigen-specific immunizations in patients with cancer or chronic viral infections. However, one major limitation for the broad clinical implementation of ELISPOT assays is the lack of an inexhaustible source of suitable HLA-matched antigen-presenting cells (APC). Currently available allogeneic or xenogeneic APC (such as the human lymphoid hybrid T2 or HLA-transfected insect cells) can either lead to st…

A neoepitope generated by an FLT3 internal tandem duplication (FLT3-ITD) is recognized by leukemia-reactive autologous CD8+ T cells.

Abstract The FLT3 receptor tyrosine kinase is expressed in more than 90% of acute myelogeneous leukemias (AMLs), up to 30% of which carry an internal tandem duplication (ITD) within the FLT3 gene. Although varying duplication sites exist, most FLT3-ITDs affect a single protein domain. We analyzed the FLT3-ITD of an AML patient for encoding HLA class I–restricted immunogenic peptides. One of the tested peptides (YVDFREYEYY) induced in vitro autologous T-cell responses restricted by HLA-A*0101 that were also detectable ex vivo. These peptide-reactive T cells recognized targets transfected with the patient's FLT3-ITD, but not wild-type FLT3, and recognized the patient's AML cells. Our results …

Flow Cytometry in Cancer Immunotherapy: Applications, Quality Assurance, and Future

Cancer immunotherapy seeks to elicit or augment the antitumor immune response in a patient in order to enlist the help of the patient’s own immune system for tumor control. In this context, immune monitoring provides evidence of immunogenicity, guides the choice and dosage of antigens, assesses the effects of immune modulators and therapy combinations, and has the potential to reveal early biomarkers of clinical efficacy. In view of their role in the anticancer immune response, the quantity and quality of tumor antigen-specific effector CD4+ and CD8+ T cells are of particular interest, and characterization of regulatory T cells and myeloid-derived suppressor cells is increasingly relevant. …

The regulatory landscape for actively personalized cancer immunotherapies

Campath-1H-Based Reduced-Intensity Conditioning Followed by Allogeneic Blood Stem-Cell Transplantation and Preemptive CD8-Depleted Donor Lymphocyte Infusions.

Abstract Recent reports have demonstrated the feasibility of using the anti-CD52 antibody Campath-1H for in vivo T-cell depletion (TCD) in the context of a fludarabine/melphalan-based reduced-intensity conditioning regimen (Kottaridis et al. Blood 2000, 96:2419). Major disadvantage of this protocol is the severe immunosuppression which results in an increased rate of opportunistic infections and disease relapses. Donor lymphocyte infusions (DLI) are frequently used to overcome this limitation. The application of DLI, however, is associated with a profound risk of GvHD. Depletion of CD8+ lymphocytes from either the allotransplant or from DLI has proven feasible to reduce the incidence of GvH…

Development of an RNA-based kit for easy generation of TCR-engineered lymphocytes to control T-cell assay performance.

Cell-based assays to monitor antigen-specific T-cell responses are characterized by their high complexity and should be conducted under controlled conditions to lower multiple possible sources of assay variation. However, the lack of standard reagents makes it difficult to directly compare results generated in one lab over time and across institutions. Therefore TCR-engineered reference samples (TERS) that contain a defined number of antigen-specific T cells and continuously deliver stable results are urgently needed. We successfully established a simple and robust TERS technology that constitutes a useful tool to overcome this issue for commonly used T-cell immuno-assays. To enable users t…

Cryopreservation of MHC Multimers: Recommendations for Quality Assurance in Detection of Antigen Specific T Cells

Fluorescence-labeled peptide-MHC class I multimers serve as ideal tools for the detection of antigen-specific T cells by flow cytometry, enabling functional and phenotypical characterization of specific T cells at the single cell level. While this technique offers a number of unique advantages, MHC multimer reagents can be difficult to handle in terms of stability and quality assurance. The stability of a given fluorescence-labeled MHC multimer complex depends on both the stability of the peptide-MHC complex itself and the stability of the fluorochrome. Consequently, stability is difficult to predict and long-term storage is generally not recommended. We investigated here the possibility of…

Generation of TCR-Engineered T Cells and Their Use To Control the Performance of T Cell Assays

Abstract The systematic assessment of the human immune system bears huge potential to guide rational development of novel immunotherapies and clinical decision making. Multiple assays to monitor the quantity, phenotype, and function of Ag-specific T cells are commonly used to unravel patients’ immune signatures in various disease settings and during therapeutic interventions. When compared with tests measuring soluble analytes, cellular immune assays have a higher variation, which is a major technical factor limiting their broad adoption in clinical immunology. The key solution may arise from continuous control of assay performance using TCR-engineered reference samples. We developed a simp…

Immunoguiding, the Final Frontier in the Immunotherapy of Cancer

T cells play an important role in cancer. This notion is strongly supported by an enormous number of trials on the clinical impact of tumor-infiltrating T cells and studies consequently showing that therapeutic interventions which are based on transfer, activation and expansion, or de-blocking of tumor-specific T cells, which have met with clinical success. To optimally profit from the flow of newly developed immune-based therapeutics aiming to reinforce the systemic and local tumor-specific T-cell response, it will be required to identify biomarkers that provide the rationale to use a particular therapy, that measure the effect of intervention, and that can predict the outcome of a therape…

“MIATA”—Minimal Information about T Cell Assays

Immunotherapy, especially therapeutic vaccination, has a great deal of potential in the treatment of cancer and certain infectious diseases such as HIV (Allison et al., 2006; Fauci et al., 2008; Feldmann and Steinman, 2005). Numerous vaccine candidates have been tested in patients with a variety of tumor types and chronic viral diseases. Often, the best way to assess the clinical potential of these vaccines is to monitor the induced T cell response, and yet there are currently no standards for reporting these results. This letter is an effort to address this problem.

Translation of genomics-guided RNA-based personalised cancer vaccines: towards the bedside

Cancer is a disease caused by DNA mutations. Cancer therapies targeting defined functional mutations have shown clinical benefit. However, as 95% of the mutations in a tumour are unique to that single patient and only a small number of mutations are shared between patients, the addressed medical need is modest. A rapidly determined patient-specific tumour mutation pattern combined with a flexible mutation-targeting drug platform could generate a mutation-targeting individualised therapy, which would benefit each single patient. Next-generation sequencing enables the rapid identification of somatic mutations in individual tumours (the mutanome). Immunoinformatics enables predictions of mutat…

Abstract B041: A novel nanoparticular formulated tetravalent RNA cancer vaccine for treatment of patients with malignant melanoma

Abstract 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) 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. Similar to other liposomal drugs, the four injectable RNA(LIP) products constituting the investigational medicinal product will be prepared …

Recommendations from the iSBTc-SITC/FDA/NCI Workshop on Immunotherapy Biomarkers

Abstract Purpose: To facilitate development of innovative immunotherapy approaches, especially for treatment concepts exploiting the potential benefits of personalized therapy, there is a need to develop and validate tools to identify patients who can benefit from immunotherapy. Despite substantial effort, we do not yet know which parameters of antitumor immunity to measure and which assays are optimal for those measurements. Experimental Design: The iSBTc-SITC (International Society for Biological Therapy of Cancer-Society for Immunotherapy of Cancer), FDA (Food and Drug Administration), and NCI (National Cancer Institute) partnered to address these issues for immunotherapy of cancer. Here…

Functional TCR Retrieval from Single Antigen-Specific Human T Cells Reveals Multiple Novel Epitopes

Abstract The determination of the epitope specificity of disease-associated T-cell responses is relevant for the development of biomarkers and targeted immunotherapies against cancer, autoimmune, and infectious diseases. The lack of known T-cell epitopes and corresponding T-cell receptors (TCR) for novel antigens hinders the efficient development and monitoring of new therapies. We developed an integrated approach for the systematic retrieval and functional characterization of TCRs from single antigen-reactive T cells that includes the identification of epitope specificity. This is accomplished through the rapid cloning of full-length TCR-α and TCR-β chains directly from single antigen-spec…

Rapid molecular dissection of viral and bacterial immunomes

The development of preventive or therapeutic recombinant vaccines and the generation of serodiagnostic assays for infectious diseases depend essentially on the availability of molecularly defined antigens. A major bottleneck for the identification of suitable target antigens for many pathogens is the isolation of sufficient amounts of material for subsequent genomic or proteomic screening. Applying a highly efficient expression cloning strategy to the human pathogens vaccinia virus (VV) and Chlamydia pneumoniae (CP), we demonstrate that sub-nanogram amounts of isolated nucleic acids can be utilized to determine comprehensive sets of immunodominant antigens. Remarkably, the approach not only…

Immunotherapy of Malignant Melanoma

For the field of cancer immunotherapy, malignant melanoma has a very special role. It has certainly critically influenced the past and the present of the field. Research performed in malignant melanoma has started to shape the field of cancer immunotherapy more than 20 years ago with the discovery of the first tumor-associated antigens documented to be recognized by antigen-specific T cells in patients. As discussed in the following sections, the privileged role of malignant melanoma has not been restricted to the pioneering phase of initial antigen discovery but has continued to exist through more than 20 years as documented by the clinical successes reported for immunotherapies such as cy…

Targeting the activation-induced antigen CD137 can selectively deplete alloreactive T cells from antileukemic and antitumor donor T-cell lines.

AbstractIn HLA-incompatible hematopoietic stem cell transplantation, alloreactive donor T cells recognizing recipient mismatch HLA cause severe graft-versus-host disease (GVHD). Strategies allowing the selective depletion of alloreactive T cells as well as the enhancement of graft-versus-malignancy immunity would be beneficial. We generated donor CD8 T-cell lines in vitro using allogeneic recipient cells mismatched at a single HLA class I allele or haplotype as stimulators. Recipient cells were obtained from acute myeloid leukemias, renal-cell carcinomas, and CD40L-induced B lymphoblasts. Resulting alloreactive T cells were activated by incubating day 21 T-cell cultures with HLA-mismatch tr…

Evolution of the Regulatory Landscape for Immunomodulatory Compounds and Personalized Therapeutic Cancer Vaccines

The term cancer immunotherapy covers a huge range of different therapeutic interventions based on highly diverse medicinal products. Some immunotherapy products such as monoclonal antibodies have successfully been developed during the last years and contributed to major advances in cancer therapy. Development of cancer vaccines seemed to be more challenging. Nevertheless, some promising candidates are currently in late-stage clinical development, and one product (sipuleucel-T) was recently approved in the United States (USA) and in the European Union (EU). Additionally, cell therapy approaches based on adoptive lymphocyte transfer have recently come into focus due to surprisingly efficient …

Preemptive Transfer of GMP-Grade CD8-Depleted Donor Lymphocytes after T-Cell Depleted Reduced-Intensity Transplantation.

Abstract The combination of fludarabin (150mg/m2) / melphalan (140mg/m2) based reduced-intensity allo-transplantation (RIT) with in vivo T-cell depletion (TCD) by the anti-CD52 antibody alemtuzumab (100mg) has demonstrated efficient engraftment and reduced graft-versus-host disease (GVHD) (Kottaridis et al., Blood 2000). However, the slow lymphocyte recovery following this protocol is associated with reduced anti-infectious and antitumor immunity. In an attempt to improve immune-reconstitution after TCD / RIT, we investigated the early preemptive use of CD8-depleted donor lymphocyte infusions (DLIs). For CD8 depletion of donor leukaphereses, a new depletion protocol using clinical grade CD8…

Abstract CT201: The Mutanome Engineered RNA Immuno-Therapy (MERIT) project

Abstract 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 molecu…

Identification of T cell epitopes by the use of rapidly generated mRNA fragments.

Abstract Although the number of defined T cell epitopes of clinically relevant antigens is constantly increasing, there is still an enormous need to identify further peptides, processed from new antigens or presented by rare HLA molecules, respectively. Here we introduce a novel two-step approach for the rapid identification of T cell epitopes. It was established in the CMV infection model. From the peripheral blood of healthy donors sharing HLA-A1 according to HLA serotyping we isolated CD8 + T lymphocytes and generated dendritic cells (DCs). DCs were electroporated with CMV pp65 mRNA and tested for recognition by autologous CD8 + T lymphocytes in IFN-γ ELISPOT assays. In all 10 CMV-seropo…

Harmonization of Immune Biomarker Assays for Clinical Studies

Assays that measure a patient's immune response play an increasingly important role in the development of immunotherapies. The inherent complexity of these assays and independent protocol development between laboratories result in high data variability and poor reproducibility. Quality control through harmonization--based on integration of laboratory-specific protocols with standard operating procedures and assay performance benchmarks--is one way to overcome these limitations. Harmonization guidelines can be widely implemented to address assay performance variables. This process enables objective interpretation and comparison of data across clinical trial sites and also facilitates the ide…

Selective uptake of naked vaccine RNA by dendritic cells is driven by macropinocytosis and abrogated upon DC maturation.

Even though it is known for more than one decade that antigen-encoding RNA can deliver antigenic information to induce antigen-specific immunity against cancer, the nature and mechanism of RNA uptake have remained enigmatic. In this study, we investigated the pharmacokinetics of naked RNA administered into the lymph node. We observed that RNA is rapidly and selectively uptaken by lymph node dendritic cells (DCs). Furthermore, in vitro and in vivo studies revealed that the efficient internalization of RNA by human and murine DCs is primarily driven by macropinocytosis. Selective inhibition of macropinocytosis by compounds or as a consequence of DC maturation abrogated RNA internalization and…

Graft-Versus-Host Disease or Infection: Rapid Detection of HLA Mismatch-Reactive T Cells Ex Vivo Can Facilitate Diagnosis and Guide Therapy after Allogeneic Transplantation.

Abstract Diagnosis of graft-versus-host disease (GVHD) is mainly based on clinical features and on tissue biopsies. However, clinicians and pathologists are well aware of cases, in which GVHD cannot be distinguished from infections arising from severe immunodeficiency after allogeneic stem-cell transplantation (SCT). This may pose a deep therapeutic dilemma of whether to modify immunosuppressive treatment or to use donor lymphocyte infusion (DLI) for promoting anti-microbial immunity. We observed a 68-year-old patient with myelodysplastic syndrome who developed acute GVHD grade II of skin and gut at d+16 after T-cell depleted reduced-intensity SCT (Fig. 1). GVHD was confirmed by histology a…

Confidence-based Somatic Mutation Evaluation and Prioritization

Next generation sequencing (NGS) has enabled high throughput discovery of somatic mutations. Detection depends on experimental design, lab platforms, parameters and analysis algorithms. However, NGS-based somatic mutation detection is prone to erroneous calls, with reported validation rates near 54% and congruence between algorithms less than 50%. Here, we developed an algorithm to assign a single statistic, a false discovery rate (FDR), to each somatic mutation identified by NGS. This FDR confidence value accurately discriminates true mutations from erroneous calls. Using sequencing data generated from triplicate exome profiling of C57BL/6 mice and B16-F10 melanoma cells, we used the exist…