2015

Vormehr M, Diken M, Boegel S, Kreiter S, Türeci Ö, Sahin U: Mutanome directed cancer immunotherapy. Current opinion in immunology 2015, 39:14-22.
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Somatic mutations are important drivers of cancer development. Accumulating evidence suggests that a significant subset of mutations result in neo-epitopes recognized by autologous T cells and thus may constitute the Achilles’ heel of tumor cells. T cells directed against mutations have been shown to have a key role in clinical efficacy of potent cancer immunotherapy modalities, such as adoptive transfer of autologous tumor infiltrating lymphocytes and immune checkpoint inhibitors. Whereas these findings strengthen the idea of a prominent role of neo-epitopes in tumor rejection, the systematic therapeutic exploitation of mutations was hampered until recently by the uniqueness of the repertoire of mutations (‘the mutanome’) in every patient’s tumor. This review highlights insights into immune recognition of neo-epitopes and novel concepts for comprehensive identification and immunotherapeutic exploitation of individual mutations.

Vormehr M, Schrörs B, Boegel S, Löwer M, Türeci Ö, Sahin U: Mutanome Engineered RNA Immunotherapy: Towards Patient-Centered Tumor Vaccination. Journal of Immunology Research 2015, 2015:1-6.
DOI

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Advances in nucleic acid sequencing technologies have revolutionized the field of genomics, allowing the efficient targeting of mutated neoantigens for personalized cancer vaccination. Due to their absence during negative selection of T cells and their lack of expression in healthy tissue, tumor mutations are considered as optimal targets for cancer immunotherapy. Preclinical and early clinical data suggest that synthetic mRNA can serve as potent drug format allowing the cost efficient production of highly efficient vaccines in a timely manner. In this review, we describe a process, which integrates next generation sequencing based cancer mutanome mapping, in silico target selection and prioritization approaches, and mRNA vaccine manufacturing and delivery into a process we refer to as MERIT (mutanome engineered RNA immunotherapy).

Scholtalbers J, Boegel S, Bukur T, Byl M, Goerges S, Sorn P, Loewer M, Sahin U, Castle JC: TCLP: an online cancer cell line catalogue integrating HLA type, predicted neo-epitopes, virus and gene expression. Genome medicine 2015, 7:118.
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Human cancer cell lines are an important resource for research and drug development. However, the available annotations of cell lines are sparse, incomplete, and distributed in multiple repositories. Re-analyzing publicly available raw RNA-Seq data, we determined the human leukocyte antigen (HLA) type and abundance, identified expressed viruses and calculated gene expression of 1,082 cancer cell lines. Using the determined HLA types, public databases of cell line mutations, and existing HLA binding prediction algorithms, we predicted antigenic mutations in each cell line. We integrated the results into a comprehensive knowledgebase. Using the Django web framework, we provide an interactive user interface with advanced search capabilities to find and explore cell lines and an application programming interface to extract cell line information. The portal is available at http://celllines.tron-mainz.de.

Stadler CR, Bähr-Mahmud H, Plum LM, Schmoldt K, Kölsch AC, Türeci Ö, Sahin U: Characterization of the first-in-class T-cell-engaging bispecific single-chain antibody for targeted immunotherapy of solid tumors expressing the oncofetal protein claudin 6. OncoImmunology 2015, 5:e1091555.
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The fetal tight junction molecule claudin 6 (CLDN6) is virtually absent from any normal tissue, whereas it is aberrantly and frequently expressed in various cancers of high medical need.We engineered 6PHU3, a T-cell-engaging bispecific single chain molecule (bi-(scFv)2) with anti-CD3/anti-CLDN6 specificities, and characterized its pharmacodynamic properties.Our data show that upon engagement by 6PHU3, T cells strongly upregulate cytotoxicity and activation markers, proliferate and acquire an effector phenotype. 6PHU3 exerts potent killing of cancer cells in vitro with EC50 values in the pg/mL range. Subcutaneous xenograft tumors in NSG mice engrafted with human PBMCs are eradicated by 6PHU3 treatment and survival of mice is significantly prolonged. Tumors of 6PHU3-treated mice are strongly infiltrated with activated CD4+, CD8+ T cells and TEM type cells but not Tregs and display a general activation of a mostly inflammatory phenotype.
These effects are only observed upon bispecific but not monospecific engagement of 6PHU3. Together with the exceptionally cancer cell selective expression of the oncofetal tumor marker CLDN6, this provides a safeguard with regard to toxicity.
In summary, our data shows that the concept of T-cell redirection combined with that of highly selective targeting of CLDN6-positive solid tumors is effective. Thus, exploring 6PHU3 for clinical therapy is warranted.

Poleganov MA, Eminli S, Beissert T, Herz S, Moon J, Goldmann J, Beyer A, Heck R, Burkhart I, Barea Roldan D, Türeci Ö, Yi K, Hamilton B, Sahin U: Efficient Reprogramming of Human Fibroblasts and Blood-Derived Endothelial Progenitor Cells Using Nonmodified RNA for Reprogramming and Immune Evasion. Human Gene Therapy 2015, 26:751-766.
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mRNA reprogramming results in the generation of genetically stable induced pluripotent stem (iPS) cells while avoiding the risks of genomic integration. Previously published mRNA reprogramming protocols have proven to be inconsistent and time-consuming and mainly restricted to fibroblasts, thereby demonstrating the need for a simple but reproducible protocol applicable to various cell types. So far there have been no published reports using mRNA to reprogram any cell type derived from human blood. Nonmodified synthetic mRNAs are immunogenic and activate cellular defense mechanisms, which can lead to cell death and inhibit mRNA translation upon repetitive transfection. Hence, to overcome RNA-related toxicity we combined nonmodified reprogramming mRNAs (OCT4, SOX2, KLF4, cMYC, NANOG, and LIN28 [OSKMNL]) with immune evasion mRNAs (E3, K3, and B18R [EKB]) from vaccinia virus. Additionally, we included mature, double-stranded microRNAs (miRNAs) from the 302/367 cluster, which are known to enhance the reprogramming process, to develop a robust reprogramming protocol for the generation of stable iPS cell lines from both human fibroblasts and human blood-outgrowth endothelial progenitor cells (EPCs). Our novel combination of RNAs enables the cell to tolerate repetitive transfections for the generation of stable iPS cell colonies from human fibroblasts within 11 days while requiring only four transfections. Moreover, our method resulted in the first known mRNA-vectored reprogramming of human blood-derived EPCs within 10 days while requiring only eight daily transfections.

Malczyk AH, Kupke A, Prüfer S, Scheuplein VA, Hutzler S, Kreuz D, Beissert T, Bauer S, Hubich-Rau S, Tondera C, Eldin HS, Schmidt J, Vergara-Alert J, Süzer Y, Seifried J, Hanschmann K, Kalinke U, Herold S, Sahin U, Cichutek K, Waibler Z, Eickmann M, Becker S, Mühlebach MD: A Highly Immunogenic and Protective Middle East Respiratory Syndrome Coronavirus Vaccine Based on a Recombinant Measles Virus Vaccine Platform. Journal of virology 2015, 89:11654-11667.
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In 2012, the first cases of infection with the Middle East respiratory syndrome coronavirus (MERS-CoV) were identified. Since then, more than 1,000 cases of MERS-CoV infection have been confirmed; infection is typically associated with considerable morbidity and, in approximately 30% of cases, mortality. Currently, there is no protective vaccine available. Replication-competent recombinant measles virus (MV) expressing foreign antigens constitutes a promising tool to induce protective immunity against corresponding pathogens. Therefore, we generated MVs expressing the spike glycoprotein of MERS-CoV in its full-length (MERS-S) or a truncated, soluble variant of MERS-S (MERS-solS). The genes encoding MERS-S and MERS-solS were cloned into the vaccine strain MVvac2 genome, and the respective viruses were rescued (MVvac2-CoV-S and MVvac2-CoV-solS). These recombinant MVs were amplified and characterized at passages 3 and 10. The replication of MVvac2-CoV-S in Vero cells turned out to be comparable to that of the control virus MVvac2-GFP (encoding green fluorescent protein), while titers of MVvac2-CoV-solS were impaired approximately 3-fold. The genomic stability and expression of the inserted antigens were confirmed via sequencing of viral cDNA and immunoblot analysis. In vivo, immunization of type I interferon receptor-deficient (IFNAR−/−)-CD46Ge mice with 2 × 105 50% tissue culture infective doses of MVvac2-CoV-S(H) or MVvac2-CoV-solS(H) in a prime-boost regimen induced robust levels of both MV- and MERS-CoV-neutralizing antibodies. Additionally, induction of specific T cells was demonstrated by T cell proliferation, antigen-specific T cell cytotoxicity, and gamma interferon secretion after stimulation of splenocytes with MERS-CoV-S presented by murine dendritic cells. MERS-CoV challenge experiments indicated the protective capacity of these immune responses in vaccinated mice.

Diken M, Kreiter S, Kloke B, Sahin U: Current Developments in Actively Personalized Cancer Vaccination with a Focus on RNA as the Drug Format. Progress in tumor research 2015, 42:44-54.
PubMed

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Developments in sequencing technologies have not only led to a rapid generation of genomic and transcriptional data from cancer patients, but also revealed the vast diversity of cancer-specific changes in patient tumors. Among these, mutation changes in the protein sequence can result in novel epitopes recognized by the immune system and, therefore, can be employed in the development of personalized vaccines. Thanks to its easy design and scalable GMP production, vaccines based on mRNAs coding for mutated epitopes have emerged as a reliable strategy for the exploitation of the potential of patient-specific genomic data. In this review, we provide an overview of recent developments in actively personalized vaccinations, with a special focus on the promise of mRNA vaccines.

Jiménez Calvente C, Sehgal A, Popov Y, Kim YO, Zevallos V, Sahin U, Diken M, Schuppan D: Specific hepatic delivery of procollagen α1(I) small interfering RNA in lipid-like nanoparticles resolves liver fibrosis. Hepatology (Baltimore, Md.) 2015, 62:1285-1297.
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Fibrosis accompanies the wound-healing response to chronic liver injury and is characterized by excessive hepatic collagen accumulation dominated by collagen type I. Fibrosis often progresses to cirrhosis. Here we present in vivo evidence of an up to 90% suppression of procollagen α1(I) expression, a reduction of septa formation, and a 40%-60% decrease of collagen deposition in mice with progressive and advanced liver fibrosis that received cationic lipid nanoparticles loaded with small interfering RNA to the procollagen α1(I) gene. After intravenous injection, up to 90% of lipid nanoparticles loaded with small interfering RNA to the procollagen α1(I) gene were retained in the liver of fibrotic mice and accumulated in nonparenchymal more than parenchymal cells for prolonged periods, significantly ameliorating progression and accelerating regression of fibrosis. Conclusion: Our lipid nanoparticles loaded with small interfering RNA to the procollagen α1(I) gene specifically reduce total hepatic collagen content without detectable side effects, potentially qualifying as a therapy for fibrotic liver diseases.

Paret C, Simon P, Vormbrock K, Bender C, Kölsch A, Breitkreuz A, Yildiz Ö, Omokoko T, Hubich-Rau S, Hartmann C, Häcker S, Wagner M, Roldan DB, Selmi A, Türeci Ö, Sahin U: CXorf61 is a target for T cell based immunotherapy of triple-negative breast cancer. Oncotarget 2015, 6:25356-25367.
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Triple-negative breast cancer (TNBC) is a high medical need disease with limited treatment options. CD8+ T cell-mediated immunotherapy may represent an attractive approach to address TNBC. The objectives of this study were to assess the expression of CXorf61 in TNBCs and healthy tissues and to evaluate its capability to induce T cell responses. We show by transcriptional profiling of a broad comprehensive set of normal human tissue that CXorf61 expression is strictly restricted to testis. 53% of TNBC patients express this antigen in at least 30% of their tumor cells. In CXorf61-negative breast cancer cell lines CXorf61 expression is activated by treatment with the hypomethylating agent 5-aza-2′-deoxycytidine.By vaccination of HLA-A*02-transgenic mice with CXorf61 encoding RNA we obtained high frequencies of CXorf61-specific T cells. Cloning and characterization of T cell receptors (TCRs) from responding T cells resulted in the identification of the two HLA-A*0201-restricted T cell epitopes CXorf6166–74 and CXorf6179–87. Furthermore, by in vitro priming of human CD8+ T cells derived from a healthy donor recognizing CXorf6166–74 we were able to induce a strong antigen-specific immune response and clone a human TCR recognizing this epitope. In summary, our data confirms this antigen as promising target for T cell based therapies.

Boegel S, Scholtalbers J, Löwer M, Sahin U, Castle JC: In Silico HLA Typing Using Standard RNA-Seq Sequence Reads. Methods in molecular biology (Clifton, N.J.) 2015, 1310:247-258.
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Next-generation sequencing (NGS) enables high-throughput transcriptome profiling using the RNA-Seq assay, resulting in billions of short sequence reads. Worldwide adoption has been rapid: many laboratories worldwide generate transcriptome sequence reads daily. Here, we describe methods for obtaining a sample’s human leukocyte antigen (HLA) class I and II types and HLA expression using standard NGS RNA-Seq sequence reads. We demonstrate the application using our algorithm, seq2HLA, and a publicly available RNA-Seq dataset from the Burkitt lymphoma cell line Raji.

Vallazza B, Petri S, Poleganov MA, Eberle F, Kuhn AN, Sahin U: Recombinant messenger RNA technology and its application in cancer immunotherapy, transcript replacement therapies, pluripotent stem cell induction, and beyond. Wiley interdisciplinary reviews. RNA 2015, 6:471-499.
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In recent years, the interest in using messenger RNA (mRNA) as a therapeutic means to tackle different diseases has enormously increased. This holds true not only for numerous preclinical studies, but mRNA has also entered the clinic to fight cancer. The advantages of using mRNA compared to DNA were recognized very early on, e.g., the lack of risk for genomic integration, or the expression of the encoded protein in the cytoplasm without the need to cross the nuclear membrane. However, it was generally assumed that mRNA is just not stable enough to give rise to sufficient expression of the encoded protein. Yet, an initially small group of mRNA aficionados could demonstrate that the stability of mRNA and the efficiency, by which the encoded protein is translated, can be significantly increased by selecting the right set of cis-acting structural elements (including the 5′-cap, 5′- and 3′-untranslated regions, poly(A)-tail, and modified building blocks). In parallel, significant advances in RNA packaging and delivery have been made, extending the potential for this molecule. This paved the way for further work to prove mRNA as a promising therapeutic for multiple diseases. Here, we review the developments to optimize mRNA regarding stability, translational efficiency, and immune-modulating properties to enhance its functionality and efficacy as a therapeutic. Furthermore, we summarize the current status of preclinical and clinical studies that use mRNA for cancer immunotherapy, for the expression of functional proteins as so-called transcript (or protein) replacement therapy, as well as for induction of pluripotent stem cells.

Maaß F, Wüstehube-Lausch J, Dickgießer S, Valldorf B, Reinwarth M, Schmoldt H, Daneschdar M, Avrutina O, Sahin U, Kolmar H: Cystine-knot peptides targeting cancer-relevant human cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). Journal of peptide science: an official publication of the European Peptide Society 2015, 21:651-660.
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Cystine-knot peptides sharing a common fold but displaying a notably large diversity within the primary structure of flanking loops have shown great potential as scaffolds for the development of therapeutic and diagnostic agents. In this study, we demonstrated that the cystine-knot peptide MCoTI-II, a trypsin inhibitor from Momordica cochinchinensis, can be engineered to bind to cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), an inhibitory receptor expressed by T lymphocytes, that has emerged as a target for the treatment of metastatic melanoma. Directed evolution was used to convert a cystine-knot trypsin inhibitor into a CTLA-4 binder by screening a library of variants using yeast surface display. A set of cystine-knot peptides possessing dissociation constants in the micromolar range was obtained; the most potent variant was synthesized chemically. Successive conjugation with neutravidin, fusion to antibody Fc domain or the oligomerization domain of C4b binding protein resulted in oligovalent variants that possessed enhanced (up to 400-fold) dissociation constants in the nanomolar range. Our data indicate that display of multiple knottin peptides on an oligomeric scaffold protein is a valid strategy to improve their functional affinity with ramifications for applications in diagnostics and therapy.

Bidmon N, Attig S, Rae R, Schröder H, Omokoko TA, Simon P, Kuhn AN, Kreiter S, Sahin U, Gouttefangeas C, van der Burg, Sjoerd H, Britten CM: Generation of TCR-engineered T cells and their use to control the performance of T cell assays. Journal of immunology (Baltimore, Md.: 1950) 2015, 194:6177-6189.
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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 simple, stable, robust, and scalable technology to generate reference samples that contain defined numbers of functional Ag-specific T cells. First, we show that RNA-engineered lymphocytes, equipped with selected TCRs, can repetitively deliver functional readouts of a controlled size across multiple assay platforms. We further describe a concept for the application of TCR-engineered reference samples to keep assay performance within or across institutions under tight control. Finally, we provide evidence that these novel control reagents can sensitively detect assay variation resulting from typical sources of error, such as low cell quality, loss of reagent stability, suboptimal hardware settings, or inaccurate gating.

Kang B, Okwieka P, Schöttler S, Seifert O, Kontermann RE, Pfizenmaier K, Musyanovych A, Meyer R, Diken M, Sahin U, Mailänder V, Wurm FR, Landfester K: Tailoring the stealth properties of biocompatible polysaccharide nanocontainers. Biomaterials 2015, 49:125-134.
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Fundamental development of a biocompatible and degradable nanocarrier platform based on hydroxyethyl starch (HES) is reported. HES is a derivative of starch and possesses both high biocompatibility and improved stability against enzymatic degradation; it is used to prepare nanocapsules via the polyaddition reaction at the interface of water nanodroplets dispersed in an organic miniemulsion. The synthesized hollow nanocapsules can be loaded with hydrophilic guests in its aqueous core, tuned in size, chemically functionalized in various pathways, and show high shelf life stability. The surface of the HES nanocapsules is further functionalized with poly(ethylene glycol) via different chemistries, which substantially enhanced blood half-life time. Importantly, methods for precise and reliable quantification of the degree of functionalization are also introduced, which enable the precise control of the chemistry on the capsules’ surface. The stealth properties of these capsules is studied both in-vitro and in-vivo. The functionalized nanocapsules serve as a modular platform for specific cell targeting, as they show no unspecific up-taken by different cell types and show very long circulating time in blood (up to 72 h).

Kreiter S, Vormehr M, van de Roemer, Niels, Diken M, Löwer M, Diekmann J, Boegel S, Schrörs B, Vascotto F, Castle JC, Tadmor AD, Schoenberger SP, Huber C, Türeci Ö, Sahin U: Mutant MHC class II epitopes drive therapeutic immune responses to cancer. Nature 2015, 520:692-696.
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Tumour-specific mutations are ideal targets for cancer immunotherapy as they lack expression in healthy tissues and can potentially be recognized as neo-antigens by the mature T-cell repertoire. Their systematic targeting by vaccine approaches, however, has been hampered by the fact that every patient’s tumour possesses a unique set of mutations (‘the mutanome’) that must first be identified. Recently, we proposed a personalized immunotherapy approach to target the full spectrum of a patient’s individual tumour-specific mutations1. Here we show in three independent murine tumour models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that, unexpectedly, the majority of the immunogenic mutanome is recognized by CD4+ T cells. Vaccination with such CD4+ immunogenic mutations confers strong antitumour activity. Encouraged by these findings, we established a process by which mutations identified by exome sequencing could be selected as vaccine targets solely through bioinformatic prioritization on the basis of their expression levels and major histocompatibility complex (MHC) class II-binding capacity for rapid production as synthetic poly-neo-epitope messenger RNA vaccines. We show that vaccination with such polytope mRNA vaccines induces potent tumour control and complete rejection of established aggressively growing tumours in mice. Moreover, we demonstrate that CD4+ T cell neo-epitope vaccination reshapes the tumour microenvironment and induces cytotoxic T lymphocyte responses against an independent immunodominant antigen in mice, indicating orchestration of antigen spread. Finally, we demonstrate an abundance of mutations predicted to bind to MHC class II in human cancers as well by employing the same predictive algorithm on corresponding human cancer types. Thus, the tailored immunotherapy approach introduced here may be regarded as a universally applicable blueprint for comprehensive exploitation of the substantial neo-epitope target repertoire of cancers, enabling the effective targeting of every patient’s tumour with vaccines produced ‘just in time’.

Foerster F, Strobl S, Kaps L, Weng S, Kim YO, Bros M, Diken M, Boegel S, Castle J, Bockamp E, Schuppan D: P0311. Balb/c and C57/Bl6 mice exhibit differences in their susceptibility and anti-tumor response to B16F10 melanoma liver metastasis. Journal of Hepatology 2015, 62:S425.
DOI

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While progress has been made in increasing survival rates in neoplasms detected at early stages, the prognosis of metastasized tumors remains dismal. Liver metastases in particular are survival-limiting in a broad range of malignancies. Therefore, a better understanding of the mechanisms that underlie (liver) metastasis is essential for improving cancer patients’ outcome. The B16F10 melanoma cell line is a well described cancer cell line with high metastatic potential that has been successfully used for human anti-cancer drug development. Since the B16F10 cell line has very low expression of MHC class I molecules, it is predestined for studying the innate anti-tumor immune response. We administered B16F10 cells stably transfected with a luciferase-expressing lentivirus via intrasplenic injection to induce liver metastasis in two different strains of wild-type mice. Bioluminescence was recorded 7 and 14 days after injection using an IVIS in vivo imaging system. 2 weeks after injection, mice were sacrificed and their metastatic burden measured. Liver probes were analysed with regard to immune cell populations and markers of M1-/M2-polarization by FACS, RT-qPCR and IHC. Whole transcriptome sequencing was performed on selected samples and their differential gene expression analysed. Within 2 weeks, mice developed severe metastatic disease. Notably, the allogeneic mouse strain (Balb/c) was more susceptible to B16F10 liver metastasis than the syngeneic strain (C57/Bl6). Metastasized livers showed a pattern of predominantly innate immunity with macrophages representing the major immune cell population. Interestingly, Balb/c mice exhibited a pattern of M1-, while C57/Bl6 mice showed a pattern of M2-macrophage polarization. Complete RNA sequencing of metastasized liver samples demonstrated that interferon-g and its downstream pathway (which is usually associated with an anti-tumor response) was significantly overexpressed in Balb/c mice, whereas the Tgfbeta pathway (which is considered immunosuppressive) was significantly upregulated in C57/Bl6 mice.The B16F10 melanoma liver metastasis model allows to study the innate immune response to invading cancer cells in the liver. The two wild-type mouse strains, Balb/c and C57/Bl6, appear to differ dramatically in their immunological anti-tumor responses, which is currently further explored.

Omokoko T, Simon P, Türeci Ö, Sahin U: Retrieval of functional TCRs from single antigen-specific T cells: Toward individualized TCR-engineered therapies. Oncoimmunology 2015, 4:e1005523.
DOI; PubMed

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We have developed a highly versatile platform for the systematic retrieval of T-cell receptors (TCRs) from single-antigen-reactive T cells and for characterization of their function and specificity. This approach enables rapid extraction of multiple TCRs from repertoires in individuals and not only broadens the diversity of TCRs suitable for clinical use, but also sets the stage for actively personalized immunotherapeutic strategies.