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Systemic pan-tumor analyses may reveal the significance of common features implicated in cancer immunogenicity and patient survival. Here, we provide a comprehensive multi-omics data set for 32 patients across 25 tumor types for proteogenomic-based discovery of neoantigens. By using an optimized computational approach, we discover a large number of tumor-specific and tumor-associated antigens. To create a pipeline for the identification of neoantigens in our cohort, we combine DNA and RNA sequencing with MS-based immunopeptidomics of tumor specimens, followed by the assessment of their immunogenicity and an in-depth validation process. We detect a broad variety of non-canonical HLA-binding peptides in the majority of patients demonstrating partially immunogenicity. Our validation process allows for the selection of 32 potential neoantigen candidates. The majority of neoantigen candidates originates from variants identified in the RNA data set, illustrating the relevance of RNA as a still understudied source of cancer antigens. This study underlines the importance of RNA-centered variant detection for the identification of shared biomarkers and potentially relevant neoantigen candidates., (© 2023. The Author(s).)

Background: BCMA-directed CAR T-cell therapy (CAR-T) has altered the treatment landscape of relapsed/refractory (r/r) multiple myeloma, but is hampered by unique side effects that can lengthen hospital stays and increase morbidity. Hematological toxicity (e.g. profound and prolonged cytopenias) represents the most common grade ≥ 3 toxicity and can predispose for severe infectious complications. Here, we examined the utility of the CAR-HEMATOTOX (HT) score to predict toxicity and survival outcomes in patients receiving standard-of-care idecabtagene vicleucel and ciltacabtagene autoleucel., Methods: Data were retrospectively collected from 113 r/r multiple myeloma patients treated between April 2021 and July 2022 across six international CAR-T centers. The HT score-composed of factors related to hematopoietic reserve and baseline inflammatory state-was determined prior to lymphodepleting chemotherapy., Results: At lymphodepletion, 63 patients were HT low (score 0-1) and 50 patients were HT high (score ≥ 2). Compared to their HT low counterparts, HT high patients displayed prolonged severe neutropenia (median 9 vs. 3 days, p < 0.001), an increased severe infection rate (40% vs. 5%, p < 0.001), and more severe ICANS (grade ≥ 3: 16% vs. 0%, p < 0.001). One-year non-relapse mortality was higher in the HT high group (13% vs. 2%, p = 0.019) and was predominantly attributable to fatal infections. Response rates according to IMWG criteria were higher in HT low patients (≥ VGPR: 70% vs. 44%, p = 0.01). Conversely, HT high patients exhibited inferior progression-free (median 5 vs. 15 months, p < 0.001) and overall survival (median 10.5 months vs. not reached, p < 0.001)., Conclusions: These data highlight the prognostic utility of the CAR-HEMATOTOX score for both toxicity and treatment response in multiple myeloma patients receiving BCMA-directed CAR-T. The score may guide toxicity management (e.g. anti-infective prophylaxis, early G-CSF, stem cell boost) and help to identify suitable CAR-T candidates., (© 2023. The Author(s).)

Adoptive cell therapy (ACT) has seen a steep rise of new therapeutic approaches in its immune-oncology pipeline over the last years. This is in great part due to the recent approvals of chimeric antigen receptor (CAR)-T cell therapies and their remarkable efficacy in certain soluble tumors. A big focus of ACT lies on T cells and how to genetically modify them to target and kill tumor cells. Genetically modified T cells that are currently utilized are either equipped with an engineered CAR or a T cell receptor (TCR) for this purpose. Both strategies have their advantages and limitations. While CAR-T cell therapies are already used in the clinic, these therapies face challenges when it comes to the treatment of solid tumors. New designs of next-generation CAR-T cells might be able to overcome these hurdles. Moreover, CARs are restricted to surface antigens. Genetically engineered TCR-T cells targeting intracellular antigens might provide necessary qualities for the treatment of solid tumors. In this review, we will summarize the major advancements of the CAR-T and TCR-T cell technology. Moreover, we will cover ongoing clinical trials, discuss current challenges, and provide an assessment of future directions within the field., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hiltensperger and Krackhardt.)

T cells are important players in the antitumor immune response. Over the past few years, the adoptive transfer of genetically modified, autologous T cells-specifically redirected toward the tumor by expressing either a T cell receptor (TCR) or a chimeric antigen receptor (CAR)-has been adopted for use in the clinic. At the moment, the therapeutic application of CD19- and, increasingly, BCMA-targeting-engineered CAR-T cells have been approved and have yielded partly impressive results in hematologic malignancies. However, employing transgenic T cells for the treatment of solid tumors remains more troublesome, and numerous hurdles within the highly immunosuppressive tumor microenvironment (TME) need to be overcome to achieve tumor control. In this review, we focused on the challenges that these therapies must face on three different levels: infiltrating the tumor, exerting efficient antitumor activity, and overcoming T cell exhaustion and dysfunction. We aimed to discuss different options to pave the way for potent transgenic T cell-mediated tumor rejection by engineering either the TME or the transgenic T cell itself, which responds to the environment.

In the last decades, our understanding of the role of the immune system in cancer has significantly improved and led to the discovery of new immunotherapeutic targets and tools, which boosted the advances in cancer immunotherapy to fight a growing number of malignancies. Approved immunotherapeutic approaches are currently mainly based on immune checkpoint inhibitors, antibody-derived targeted therapies, or cell-based immunotherapies. In essence, these therapies induce or enhance the infiltration and function of tumor-reactive T cells within the tumors, ideally resulting in complete tumor eradication. While the clinical application of immunotherapies has shown great promise, these therapies are often accompanied either by a variety of side effects as well as partial or complete unresponsiveness of a number of patients. Since different stages of disease progression elicit different local and systemic immune responses, the ability to longitudinally interrogate the migration and expansion of immune cells, especially T cells, throughout the whole body might greatly facilitate disease characterization and understanding. Furthermore, it can serve as a tool to guide development as well as selection of appropriate treatment regiments. This review provides an overview about a variety of immune-imaging tools available to characterize and study T-cell responses induced by anti-cancer immunotherapy. Moreover, challenges are discussed that must be taken into account and overcome to use immune-imaging tools as predictive and surrogate markers to enhance assessment and successful application of immunotherapies., (© 2022. The Author(s).)

Despite the substantial improvement of therapeutic approaches, multiple myeloma (MM) remains mostly incurable. However, immunotherapeutic and especially T cell-based approaches pioneered the therapeutic landscape for relapsed and refractory disease recently. Targeting B-cell maturation antigen (BCMA) on myeloma cells has been demonstrated to be highly effective not only by antibody-derived constructs but also by adoptive cellular therapies. Chimeric antigen receptor (CAR)-transgenic T cells lead to deep, albeit mostly not durable responses with manageable side-effects in intensively pretreated patients. The spectrum of adoptive T cell-transfer covers synthetic CARs with diverse specificities as well as currently less well-established T cell receptor (TCR)-based personalized strategies. In this review, we want to focus on treatment characteristics including efficacy and safety of CAR- and TCR-transgenic T cells in MM as well as the future potential these novel therapies may have. ACT with transgenic T cells has only entered clinical trials and various engineering strategies for optimization of T cell responses are necessary to overcome therapy resistance mechanisms. We want to outline the current success in engineering CAR- and TCR-T cells, but also discuss challenges including resistance mechanisms of MM for evading T cell therapy and point out possible novel strategies.

Background: Neoantigens derived from somatic mutations correlate with therapeutic responses mediated by treatment with immune checkpoint inhibitors. Neoantigens are therefore highly attractive targets for the development of therapeutic approaches in personalized medicine, although many aspects of their quality and associated immune responses are not yet well understood. In a case study of metastatic malignant melanoma, we aimed to perform an in-depth characterization of neoantigens and respective T-cell responses in the context of immune checkpoint modulation., Methods: Three neoantigens, which we identified either by immunopeptidomics or in silico prediction, were investigated using binding affinity analyses and structural simulations. We isolated seven T-cell receptors (TCRs) from the patient's immune repertoire recognizing these antigens. TCRs were compared in vitro by multiparametric analyses including functional avidity, multicytokine secretion, and cross-reactivity screenings. A xenograft mouse model served to study in vivo functionality of selected TCRs. We investigated the patient's TCR repertoire in blood and different tumor-related tissues over 3 years using TCR beta deep sequencing., Results: Selected mutated peptide ligands with proven immunogenicity showed similar binding affinities to the human leukocyte antigen complex and comparable disparity to their wild-type counterparts in molecular dynamic simulations. Nevertheless, isolated TCRs recognizing these antigens demonstrated distinct patterns in functionality and frequency. TCRs with lower functional avidity showed at least equal antitumor immune responses in vivo. Moreover, they occurred at high frequencies and particularly demonstrated long-term persistence within tumor tissues, lymph nodes and various blood samples associated with a reduced activation pattern on primary in vitro stimulation., Conclusions: We performed a so far unique fine characterization of neoantigen-specific T-cell responses revealing defined reactivity patterns of neoantigen-specific TCRs. Our data highlight qualitative differences of these TCRs associated with function and longevity of respective T cells. Such features need to be considered for further optimization of neoantigen targeting including adoptive T-cell therapies using TCR-transgenic T cells., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Blocking the interaction of the immune checkpoint molecule programmed cell death protein-1 and its ligand, PD-L1, using specific antibodies has been a major breakthrough for immune oncology. Whole-body PD-L1 expression PET imaging may potentially allow for a better prediction of response to programmed cell death protein-1-targeted therapies. Imaging of PD-L1 expression is feasible by PET with the adnectin protein 18 F-BMS-986192. However, radiofluorination of proteins such as BMS-986192 remains complex and labeling yields are low. The goal of this study was therefore the development and preclinical evaluation of a 68 Ga-labeled adnectin protein ( 68 Ga-BMS-986192) to facilitate clinical trials. Methods: 68 Ga labeling of DOTA-conjugated adnectin (BXA-206362) was performed in NaOAc-buffer at pH 5.5 (50°C, 15 min). In vitro stability in human serum at 37°C was analyzed using radio-thin layer chromatography and radio-high-performance liquid chromatography. PD-L1 binding assays were performed using the transduced PD-L1-expressing lymphoma cell line U-698-M and wild-type U-698-M cells as a negative control. Immunohistochemical staining studies, biodistribution studies, and small-animal PET studies of 68 Ga-BMS-986192 were performed using PD-L1-positive and PD-L1-negative U-698-M-bearing NSG mice. Results: 68 Ga-BMS-986192 was obtained with quantitative radiochemical yields of more than 97% and with high radiochemical purity. In vitro stability in human serum was at least 95% after 4 h of incubation. High and specific binding of 68 Ga-BMS-986192 to human PD-L1-expressing cancer cells was confirmed, which closely correlates with the respective PD-L1 expression level determined by flow cytometry and immunohistochemistry staining. In vivo, 68 Ga-BMS-986192 uptake was high at 1 h after injection in PD-L1-positive tumors (9.0 ± 2.1 percentage injected dose [%ID]/g) and kidneys (56.9 ± 9.2 %ID/g), with negligible uptake in other tissues. PD-L1-negative tumors demonstrated only background uptake of radioactivity (0.6 ± 0.1 %ID/g). Coinjection of an excess of unlabeled adnectin reduced tumor uptake of PD-L1 by more than 80%. Conclusion: 68 Ga-BMS-986192 enables easy radiosynthesis and shows excellent in vitro and in vivo PD-L1-targeting characteristics. The high tumor uptake combined with low background accumulation at early imaging time points demonstrates the feasibility of 68 Ga-BMS-986192 for imaging of PD-L1 expression in tumors and is encouraging for further clinical applications of PD-L1 ligands., (© 2021 by the Society of Nuclear Medicine and Molecular Imaging.)

Characterizing the human leukocyte antigen (HLA) bound ligandome by mass spectrometry (MS) holds great promise for developing vaccines and drugs for immune-oncology. Still, the identification of non-tryptic peptides presents substantial computational challenges. To address these, we synthesized and analyzed >300,000 peptides by multi-modal LC-MS/MS within the ProteomeTools project representing HLA class I & II ligands and products of the proteases AspN and LysN. The resulting data enabled training of a single model using the deep learning framework Prosit, allowing the accurate prediction of fragment ion spectra for tryptic and non-tryptic peptides. Applying Prosit demonstrates that the identification of HLA peptides can be improved up to 7-fold, that 87% of the proposed proteasomally spliced HLA peptides may be incorrect and that dozens of additional immunogenic neo-epitopes can be identified from patient tumors in published data. Together, the provided peptides, spectra and computational tools substantially expand the analytical depth of immunopeptidomics workflows.

Cancer immunotherapies have made major advancements in recent years and are becoming the prevalent treatment options for numerous tumor entities. However, substantial response rates have only been observed in specific subsets of patients since pre-existing factors determine the susceptibility of a tumor to these therapies. The development of approaches that can actively induce an anti-tumor immune response, such as adoptive cell transfer and oncolytic virotherapy, have shown clinical success in the treatment of leukemia and melanoma, respectively. Based on the immune-stimulatory capacity of oncolytic VSV-NDV virotherapy, we envisioned a combination approach to synergize with adoptive T cell transfer, in order to enhance tumor cell killing. Using the immune-competent B16 melanoma model, we demonstrate that combination treatment has beneficial effects on the suppressive microenvironment through upregulation of MHC-I and maintaining low expression levels of PD-L1 on tumor cells. The approach led to additive cytotoxic effects and improved the recruitment of T cells to virus-infected tumor cells in vitro and in vivo. We observed substantial delays in tumor growth and evidence of abscopal effects, as well as prolongation of overall survival time when administered at clinically relevant dosing conditions. Our results indicate that treatment with oncolytic VSV-NDV, combined with adoptive T cell therapy, induces multi-mechanistic and synergistic tumor responses, which supports the further development of this promising translational approach.

Adoptive transfer of T cells transgenic for tumor-reactive T-cell receptors (TCR) is an attractive immunotherapeutic approach. However, clinical translation is so far limited due to challenges in the identification of suitable target antigens as well as TCRs that are concurrent safe and efficient. Definition of key characteristics relevant for effective and specific tumor rejection is essential to improve current TCR-based adoptive T-cell immunotherapies. We here characterized in-depth two TCRs derived from the human leukocyte antigen (HLA)-mismatched allogeneic repertoire targeting two different myeloperoxidase (MPO)-derived peptides presented by the same HLA-restriction element side by side comprising state of the art biochemical and cellular in vitro, in vivo , and in silico experiments. In vitro experiments reveal comparable functional avidities, off-rates, and cytotoxic activities for both TCRs. However, we observed differences especially with respect to cytokine secretion and cross-reactivity as well as in vivo activity. Biochemical and in silico analyses demonstrate different binding qualities of MPO-peptides to the HLA-complex determining TCR qualities. We conclude from our biochemical and in silico analyses of peptide-HLA-binding that rigid and high-affinity binding of peptides is one of the most important factors for isolation of TCRs with high specificity and tumor rejection capacity from the MHC-mismatched repertoire. Based on our results, we developed a workflow for selection of such TCRs with high potency and safety profile suitable for clinical translation.

Adoptive transfer of TCR transgenic T cells holds great promise for treating various cancers. So far, mainly semi-randomly integrating vectors have been used to genetically modify T cells. These carry the risk of insertional mutagenesis, and the sole addition of an exogenous TCR potentially results in the mispairing of TCR chains with endogenous ones. Established approaches using nonviral vectors, such as transposons, already reduce the risk of insertional mutagenesis but have not accomplished site-specific integration. Here, we used CRISPR-Cas9 RNPs and adeno-associated virus 6 for gene targeting to deliver an engineered TCR gene specifically to the TCR alpha constant locus, thus placing it under endogenous transcriptional control. Our data demonstrate that this approach replaces the endogenous TCR, functionally redirects the edited T cells' specificity in vitro , and facilitates potent tumor rejection in an in vivo xenograft model ., (© 2019 Albers et al.)

Cancer immunotherapy has proven high efficacy in treating diverse cancer entities by immune checkpoint modulation and adoptive T-cell transfer. However, patterns of treatment response differ substantially from conventional therapies, and reliable surrogate markers are missing for early detection of responders versus non-responders. Current imaging techniques using 18 F-fluorodeoxyglucose-positron-emmission-tomograpy ( 18 F-FDG-PET) cannot discriminate, at early treatment times, between tumor progression and inflammation. Therefore, direct imaging of T cells at the tumor site represents a highly attractive tool to evaluate effective tumor rejection or evasion. Moreover, such markers may be suitable for theranostic imaging. Methods: We mainly investigated the potential of two novel pan T-cell markers, CD2 and CD7, for T-cell tracking by immuno-PET imaging. Respective antibody- and F(ab´) 2 fragment-based tracers were produced and characterized, focusing on functional in vitro and in vivo T-cell analyses to exclude any impact of T-cell targeting on cell survival and antitumor efficacy. Results: T cells incubated with anti-CD2 and anti-CD7 F(ab´) 2 showed no major modulation of functionality in vitro , and PET imaging provided a distinct and strong signal at the tumor site using the respective zirconium-89-labeled radiotracers. However, while T-cell tracking by anti-CD7 F(ab´) 2 had no long-term impact on T-cell functionality in vivo , anti-CD2 F(ab´) 2 caused severe T-cell depletion and failure of tumor rejection. Conclusion: This study stresses the importance of extended functional T-cell assays for T-cell tracer development in cancer immunotherapy imaging and proposes CD7 as a highly suitable target for T-cell immuno-PET imaging., Competing Interests: Competing Interests: M.S. received a commercial research grant from Siemens Medical Research and speakers´ bureau honoraria from Siemens Lunch Symposium, and he has ownership interest (including patents) in Siemens. The other authors have no competing interest to declare.

Background: Cell separators are routinely used to collect CD34 + blood stem cells in the context of customized stem cell transplantation procedures. The Spectra Optia (Terumo BCT) is a novel development of the precursor instrument, the Cobe Spectra (Terumo BCT)., Study Design and Methods: In this report, 146 autologous and 42 allogeneic donors undergoing apheresis on the Cobe Spectra using the mononuclear cell (MNC) program 4.7 or on the Spectra Optia using the new continuous mononuclear cell (cMNC) program 11.2 are compared., Results: Viability of cells and collection efficacy within the apheresis products was comparable for autologous and allogeneic products collected with the MNC or cMNC method. However, we found a reduced duration of the apheresis procedure and lower hematocrit within the apheresis products when using the cMNC in autologous and allogeneic donors. Moreover, allogeneic donors collected substantially more CD34 + cells per kilogram of body weight when using the cMNC method. Differences in platelets before and after apheresis were substantially smaller in this cohort when compared to the cohort collected with the MNC method. Neutrophil and platelet engraftment after autologous or allogeneic transplantation with a product collected with the MNC procedure was comparable to a transplantation with a product processed according to the cMNC method., Conclusion: Comparison of the MNC (Cobe Spectra) and the cMNC (Spectra Optia) methods demonstrated an equal performance and outcome. However, advantages were present using the cMNC method with respect to apheresis duration and hematocrit within the apheresis product (autologous/allogeneic donors) and numbers of CD34 + cells collected, especially in allogeneic donors., (© 2018 AABB.)

Adoptive transfer of T cells genetically modified by TCRs or CARs represents a highly attractive novel therapeutic strategy to treat malignant diseases. Various approaches for the development of such gene therapy medicinal products (GTMPs) have been initiated by scientists in recent years. To date, however, the number of clinical trials commenced in Germany and Europe is still low. Several hurdles may contribute to the delay in clinical translation of these therapeutic innovations including the significant complexity of manufacture and non-clinical testing of these novel medicinal products, the limited knowledge about the intricate regulatory requirements of the academic developers as well as limitations of funds for clinical testing. A suitable good manufacturing practice (GMP) environment is a key prerequisite and platform for the development, validation, and manufacture of such cell-based therapies, but may also represent a bottleneck for clinical translation. The German Cancer Consortium (DKTK) and the Paul-Ehrlich-Institut (PEI) have initiated joint efforts of researchers and regulators to facilitate and advance early phase, academia-driven clinical trials. Starting with a workshop held in 2016, stakeholders from academia and regulatory authorities in Germany have entered into continuing discussions on a diversity of scientific, manufacturing, and regulatory aspects, as well as the benefits and risks of clinical application of CAR/TCR-based cell therapies. This review summarizes the current state of discussions of this cooperative approach providing a basis for further policy-making and suitable modification of processes.

Immunotherapies have been traditionally applied in malignant melanoma, which represent one of the most immunogenic tumours. Recently, immune checkpoint modulation has shown high therapeutic efficacy and may provide long-term survival in a significant proportion of affected patients. T cells are the major players in tumour rejection and recognize tumour cells predominantly in an MHC-dependent way. The immunopeptidome comprises the peptide repertoire presented by MHC class I and II molecules on the surface of the body's cells including tumour cells. To understand characteristics of suitable rejection antigens as well as respective effective T-cell responses, determination of the immunopeptidome is of utmost importance. Suitable rejection antigens need to be further characterized and validated not only to systematically improve current therapeutic approaches, but also to develop individualized treatment options. In this review, we report on current tools to explore the immunopeptidome in human melanoma and discuss current understanding and future developments to specifically detect and select those antigens that may be most relevant and promising for effective tumour rejection., (© 2017 John Wiley & Sons Ltd.)

Cancer immunotherapy has recently emerged as a powerful tool for the treatment of diverse advanced malignancies. In particular, therapeutic application of immune checkpoint modulators, such as anti-CTLA4 or anti-PD-1/PD-L1 antibodies, have shown efficacy in a broad range of malignant diseases. Although pharmacodynamics of these immune modulators are complex, recent studies strongly support the notion that altered peptide ligands presented on tumor cells representing neoantigens may play an essential role in tumor rejection by T cells activated by anti-CTLA4 and anti-PD-1 antibodies. Neoantigens may have diverse sources as viral and mutated proteins. Moreover, posttranslational modifications and altered antigen processing may also contribute to the neoantigenic peptide ligand landscape. Different approaches of target identification are currently applied in combination with subsequent characterization of autologous and non-self T-cell responses against such neoantigens. Additional efforts are required to elucidate key characteristics and interdependences of neoantigens, immunodominance, respective T-cell responses, and the tumor microenvironment in order to define decisive determinants involved in effective T-cell-mediated tumor rejection. This review focuses on our current knowledge of identification and characterization of such neoantigens as well as respective T-cell responses. It closes with challenges to be addressed in future relevant for further improvement of immunotherapeutic strategies in malignant diseases.

T-cell therapy of chronic hepatitis B is a novel approach to restore antiviral T-cell immunity and cure the infection. We aimed at identifying T-cell receptors (TCR) with high functional avidity that have the potential to be used for adoptive T-cell therapy. To this end, we cloned HLA-A*02-restricted, hepatitis B virus (HBV)-specific T cells from patients with acute or resolved HBV infection. We isolated 11 envelope- or core-specific TCRs and evaluated them in comprehensive functional analyses. T cells were genetically modified by retroviral transduction to express HBV-specific TCRs. CD8+ as well as CD4+ T cells became effector T cells recognizing even picomolar concentrations of cognate peptide. TCR-transduced T cells were polyfunctional, secreting the cytokines interferon gamma, tumor necrosis factor alpha and interleukin-2, and effectively killed hepatoma cells replicating HBV. Notably, our collection of HBV-specific TCRs recognized peptides derived from HBV genotypes A, B, C and D presented on different HLA-A*02 subtypes common in areas with high HBV prevalence. When co-cultured with HBV-infected cells, TCR-transduced T cells rapidly reduced viral markers within two days. Our unique set of HBV-specific TCRs with different affinities represents an interesting tool for elucidating mechanisms of TCR-MHC interaction and dissecting specific anti-HBV mechanisms exerted by T cells. TCRs with high functional avidity might be suited to redirect T cells for adoptive T-cell therapy of chronic hepatitis B and HBV-induced hepatocellular carcinoma.

A number of different technologies have been developed to monitor in vivo the distribution of gene-modified T cells used in immunotherapy. Nevertheless, in-depth characterization of novel approaches with respect to sensitivity and clinical applicability are so far missing. We have previously described a novel method to track engineered human T cells in tumors using 89 Zr-Df-aTCRmu-F(ab') 2 targeting the murinized part of the TCR beta domain (TCRmu) of a transgenic TCR. Here, we performed an in-depth in vitro characterization of the tracer in terms of antigen affinity, immunoreactivity, influence on T-cell functionality and stability in vitro and in vivo . Of particular interest, we have developed diverse experimental settings to quantify TCR-transgenic T cells in vivo . Local application of 89 Zr-Df-aTCRmu-F(ab') 2 -labeled T cells in a spot-assay revealed signal detection down to approximately 1.8x10 4 cells. In a more clinically relevant model, NSG mice were intravenously injected with different numbers of transgenic T cells, followed by injection of the 89 Zr-Df-aTCRmu-F(ab') 2 tracer, PET/CT imaging and subsequent ex vivo T-cell quantification in the tumor. Using this setting, we defined a comparable detection limit of 1.0x10 4 T cells. PET signals correlated well to total numbers of transgenic T cells detected ex vivo independently of the engraftment rates observed in different individual experiments. Thus, these findings confirm the high sensitivity of our novel PET/CT T-cell tracking method and provide critical information about the quantity of transgenic T cells in the tumor environment suggesting our technology being highly suitable for further clinical translation., Competing Interests: Competing Interests: A.M. Krackhardt and R. Klar are involved in a patent application currently ongoing for the defined MPO peptide and sequences of TCR2.5D6. M. Schwaiger received a commercial research grant from Siemens Medical Research, received speakers' bureau honoraria from Siemens Lunch Symposium, and has ownership interest (including patents) in Siemens. No potential conflicts of interest were disclosed by the other authors.

Background: The anti-programmed cell death-1 (PD-1) inhibitors pembrolizumab and nivolumab alone or in combination with ipilimumab have shown improved objective response rates and progression-free survival compared to ipilimumab only in advanced melanoma patients. Anti-PD-1 therapy demonstrated nearly equal clinical efficacy in patients who had progressed after ipilimumab or were treatment-naïve. However, only limited evidence exists regarding the efficacy of ipilimumab alone or in combination with nivolumab after treatment failure to anti-PD-therapy., Patients and Methods: A multicenter retrospective study in advanced melanoma patients who were treated with nivolumab (1 or 3 mg/kg) and ipilimumab (1 mg or 3 mg/kg) or ipilimumab (3 mg/kg) alone after treatment failure to anti-PD-1 therapy was performed. Patient, tumour, pre- and post-treatment characteristics were analysed., Results: In total, 47 patients were treated with ipilimumab (ipi-group) and 37 patients with ipilimumab and nivolumab (combination-group) after treatment failure to anti-PD-1 therapy. Overall response rates for the ipi- and the combination-group were 16% and 21%, respectively. Disease control rate was 42% for the ipi-group and 33% for the combination-group. One-year overall survival rates for the ipi- and the combination-group were 54% and 55%, respectively., Conclusions: Ipilimumab should be considered as a viable treatment option for patients with failure to prior anti-PD-1 therapy, including those with progressive disease as best response to prior anti-PD-1. In contrast, the combination of ipilimumab and nivolumab appears significantly less effective in this setting compared to treatment-naïve patients., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The membrane protein expression repertoire of cells changes in course of activation. In equine recurrent uveitis (ERU), a spontaneous autoimmune disease in horses with relapsing and ultimately blinding inner eye inflammation, CD4+ T lymphocytes are the crucial pathogenic cells activated in the periphery directly prior to an inflammatory episode. In order to find relevant changes in the membrane proteome associated to disease, we sorted CD4+ lymphocytes and compared protein abundance from the generated proteome datasets of both healthy horses and ERU cases. We detected formin like 1, a key player in actin dependent cellular processes such as phagocytosis, cell adhesion and cell migration, with significantly higher abundance in the CD4+ cell membrane proteome of horses with ERU. In transmigration experiments, we demonstrated higher migration rate of cells originating from diseased animals connecting formin like 1 to the migratory ability of cells. These findings are the first description of formin like 1 in association to processes involved in migration of inflammatory CD4+ T cells across the blood-retinal barrier in a spontaneous ocular autoimmune disease and suggest formin like 1 to play a role in the molecular mechanisms of ERU disease pathogenesis. Data are available via ProteomeXchange with identifier PXD005384., Biological Significance: This comparative proteomic study of membrane proteins of CD4+ T cells identified a novel protein, formin like 1, with expression on the plasma cell membrane of equine CD4+ T cells and a significant change of abundance on CD4+ T cells of horses with a spontaneous autoimmune disease. Functional studies in a cell culture model for transmigration at the blood-retinal barrier (BRB) unraveled a strong impact of formin like 1 on migratory processes of CD4+ T cells across the BRB, a key event in uveitis pathogenesis. These findings provide novel knowledge about changes in the CD4+ immune cell membrane proteome in a spontaneously and naturally occurring autoimmune disease in horses with high relevance for veterinary medicine. Additionally, this model has proven translational quality for human medicine and provides novel proteomic information on autoimmune uveitis in man., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) represents a treatment option for a diversity of advanced hematopoietic malignancies providing hope for long-term responses especially due to immunogenic effects associated with the treatment modality. Despite respectable progress in the field, relapses and/or opportunistic infections are major reasons for the high treatment-related mortality. However, a number of novel immunotherapeutic approaches using defined cell populations have been developed to directly target residual malignant cells as well as defined infectious diseases. We here provide an overview of current adoptive cellular immunotherapies in the context of allo-HSCT and close with an outlook on new directions within the field., (© 2017 S. Karger GmbH, Freiburg.)

Background: Plerixafor is predominantly used for patients mobilizing inadequate stem cell numbers for autologous transplantation after stimulation with granulocyte-colony-stimulating factor (G-CSF)., Study Design and Methods: We here report on 300 patients undergoing stem cell mobilization with G-CSF, among them 36 poor mobilizers (CD34+ cell counts < 50 × 10 6 /L blood) receiving G-CSF alone and 49 receiving G-CSF in combination with plerixafor for rescue intervention. Mobilization efficacy and short-time outcome after autologous stem cell transplantation were analyzed and compared in the respective subgroups., Results: Out of 49 patients treated with plerixafor and G-CSF, 46 (94%) collected sufficient hematopoietic stem cell numbers although the number was clearly inferior in poor mobilizers. Compared to good mobilizers, viability of CD34+ cells analyzed after collection was slightly reduced in poor mobilizers independent of the application of plerixafor. A total of 232 patients underwent autologous stem cell transplantation, among them 26 poor mobilizers who received only G-CSF and 31 patients who received G-CSF in combination with plerixafor. Time until neutrophil engraftment was in median 1 day later in poor mobilizers irrespective of the application of plerixafor. Platelet engraftment was in median 2 days delayed in patients mobilized with G-CSF and plerixafor compared to 1 day in poor mobilizers treated with G-CSF only. Frequency of detected CD38+ CD138+ CD45- CD56+ plasma cells in the apheresis products of myeloma patients was comparable for all groups., Conclusion: Our data demonstrate that plerixafor is highly effective as rescue measurement after mobilization failure with G-CSF alone and short-term clinical outcome after stem cell transplantation is comparable., (© 2016 AABB.)

Although mutations may represent attractive targets for immunotherapy, direct identification of mutated peptide ligands isolated from human leucocyte antigens (HLA) on the surface of native tumour tissue has so far not been successful. Using advanced mass spectrometry (MS) analysis, we survey the melanoma-associated immunopeptidome to a depth of 95,500 patient-presented peptides. We thereby discover a large spectrum of attractive target antigen candidates including cancer testis antigens and phosphopeptides. Most importantly, we identify peptide ligands presented on native tumour tissue samples harbouring somatic mutations. Four of eleven mutated ligands prove to be immunogenic by neoantigen-specific T-cell responses. Moreover, tumour-reactive T cells with specificity for selected neoantigens identified by MS are detected in the patient's tumour and peripheral blood. We conclude that direct identification of mutated peptide ligands from primary tumour material by MS is possible and yields true neoepitopes with high relevance for immunotherapeutic strategies in cancer.

Sensitive in vivo imaging technologies applicable to the clinical setting are still lacking for adoptive T-cell-based immunotherapies, an important gap to fill if mechanisms of tumor rejection or escape are to be understood. Here, we propose a highly sensitive imaging technology to track human TCR-transgenic T cells in vivo by directly targeting the murinized constant TCR beta domain (TCRmu) with a zirconium-89 ((89)Zr)-labeled anti-TCRmu-F(ab')2 fragment. Binding of the labeled or unlabeled F(ab')2 fragment did not impair functionality of transgenic T cells in vitro and in vivo Using a murine xenograft model of human myeloid sarcoma, we monitored by Immuno-PET imaging human central memory T cells (TCM), which were transgenic for a myeloid peroxidase (MPO)-specific TCR. Diverse T-cell distribution patterns were detected by PET/CT imaging, depending on the tumor size and rejection phase. Results were confirmed by IHC and semiquantitative evaluation of T-cell infiltration within the tumor corresponding to the PET/CT images. Overall, these findings offer a preclinical proof of concept for an imaging approach that is readily tractable for clinical translation. Cancer Res; 76(14); 4113-23. ©2016 AACR., (©2016 American Association for Cancer Research.)

Background: Anti-programmed cell death receptor-1 (PD-1) antibodies represent an effective treatment option for metastatic melanoma as well as for other cancer entities. They act via blockade of the PD-1 receptor, an inhibitor of the T-cell effector mechanisms that limit immune responses against tumours. As reported for ipilimumab, the anti-PD-1 antibodies pembrolizumab and nivolumab can induce immune-related adverse events (irAEs). These side-effects affect skin, gastrointestinal tract, liver, endocrine system and other organ systems. Since life-threatening and fatal irAEs have been reported, adequate diagnosis and management are essential., Methods and Findings: In total, 496 patients with metastatic melanoma from 15 skin cancer centers were treated with pembrolizumab or nivolumab; 242 side-effects were described in 138 patients. In 116 of the 138 patients, side-effects affected the skin, gastrointestinal tract, liver, endocrine, and renal system. Rare side-effects included diabetes mellitus, lichen planus, and pancreas insufficiency due to pancreatitis., Conclusion: Anti-PD1 antibodies can induce a plethora of irAEs. The knowledge of them will allow prompt diagnosis and improve the management resulting in decreased morbidity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Anti-programmed cell death 1 (PD-1) antibodies represent an effective treatment option for metastatic melanoma and other cancer entities. They act via blockade of the PD-1 receptor, an inhibitor of the T-cell effector mechanisms that limit immune responses against tumours. As reported for ipilimumab, the anti-PD-1 antibodies pembrolizumab and nivolumab can induce immune-related adverse events (irAEs). These side-effects can involve skin, gastrointestinal tract, liver, the endocrine system and other organ systems. Since life-threatening and fatal irAEs have been reported, adequate diagnosis and management are essential., Methods and Findings: In total, 496 patients with metastatic melanoma from 15 skin cancer centres were treated with pembrolizumab or nivolumab. Two hundred forty two side-effects in 138 patients have been analysed. In 77 of the 138 patients side-effects affected the nervous system, respiratory tract, musculoskeletal system, heart, blood and eyes. Not yet reported side-effects such as meningo-(radiculitis), polyradiculitis, cardiac arrhythmia, asystolia, and paresis have been observed. Rare and difficult to manage side-effects such as myasthenia gravis are described in detail., Conclusion: Anti-PD-1 antibodies can induce a plethora of irAEs. The knowledge of them will allow prompt diagnosis and improve the management resulting in decreased morbidity., (Copyright © 2016. Published by Elsevier Ltd.)

Pediatric cancers, including Ewing sarcoma (ES), are only weakly immunogenic and the tumor-patients' immune system often is devoid of effector T cells for tumor elimination. Based on expression profiling technology, targetable tumor-associated antigens (TAA) are identified and exploited for engineered T-cell therapy. Here, the specific recognition and lytic potential of transgenic allo-restricted CD8(+) T cells, directed against the ES-associated antigen 6-transmembrane epithelial antigen of the prostate 1 (STEAP1), was examined. Following repetitive STEAP1(130) peptide-driven stimulations with HLA-A*02:01(+) dendritic cells (DC), allo-restricted HLA-A*02:01(-) CD8(+) T cells were sorted with HLA-A*02:01/peptide multimers and expanded by limiting dilution. After functional analysis of suitable T cell clones via ELISpot, flow cytometry and xCELLigence assay, T cell receptors' (TCR) α- and β-chains were identified, cloned into retroviral vectors, codon optimized, transfected into HLA-A*02:01(-) primary T cell populations and tested again for specificity and lytic capacity in vitro and in a Rag2(-/-)γc(-/-) mouse model. Initially generated transgenic T cells specifically recognized STEAP1(130)-pulsed or transfected cells in the context of HLA-A*02:01 with minimal cross-reactivity as determined by specific interferon-γ (IFNγ) release, lysed cells and inhibited growth of HLA-A*02:01(+) ES lines more effectively than HLA-A*02:01(-) ES lines. In vivo tumor growth was inhibited more effectively with transgenic STEAP1(130)-specific T cells than with unspecific T cells. Our results identify TCRs capable of recognizing and inhibiting growth of STEAP1-expressing HLA-A*02:01(+) ES cells in vitro and in vivo in a highly restricted manner. As STEAP1 is overexpressed in a wide variety of cancers, we anticipate these STEAP1-specific TCRs to be potentially useful for immunotherapy of other STEAP1-expressing tumors.

Lentiviral vectors (LV) are widely used to successfully transduce cells for research and clinical applications. This optimized LV infection protocol includes a nontoxic poloxamer-based adjuvant combined with antibody-retargeted lentiviral particles. The novel poloxamer P338 demonstrates superior characteristics for enhancing lentiviral transduction over the best-in-class polybrene-assisted transduction. Poloxamer P338 exhibited dual benefits of low toxicity and high efficiency of lentiviral gene delivery into a range of different primary cell cultures. One of the major advantages of P338 is its availability in pharma grade and applicability as cell culture medium additive in clinical protocols. Lentiviral vectors pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G) can be produced to high titers and mediate high transduction efficiencies in vitro. For clinical applications the need for optimized transduction protocols, especially for transduction of primary T and stem cells, is high. The successful use of retronectin, the second lentivirus enhancer available as GMP material, requires the application of specific coating protocols not applicable in all processes, and results in the need of a relatively high multiplicity of infection (MOI) to achieve effective transduction efficiencies for hematopoietic cells (e.g., CD34+ hematopoietic stem cells). Cell specificity of lentiviral vectors was successfully increased by displaying different ratios of scFv-fused VSV-G glycoproteins on the viral envelope. The system has been validated with human CD30+ lymphoma cells, resulting in preferential gene delivery to CD30+ cells, which was increased fourfold in mixed cell cultures, by presenting scFv antibody fragments binding to respective surface markers. A combination of spinoculation and poloxamer-based chemical adjuvant increases the transduction of primary T-cells by greater than twofold. The combination of poloxamer-based and scFv-retargeted LVs increased transduction of CD30+ lymphoma cells more than tenfold, and has the potential to improve clinical protocols.

Autologous and allogeneic stem cell transplantation (SCT) represents a therapeutic option widely used for hematopoietic malignancies. One important milestone in the development of this treatment strategy was the development of effective cryopreservation technologies resulting in a high quality with respect to cell viability as well as lack of contamination of the graft.Stem cell preparations have been initially performed within standard laboratories as it is routinely still the case in many countries. With the emergence of cleanrooms, manufacturing of stem cell preparations within these facilities has become a new standard mandatory in Europe. However, due to high costs and laborious procedures, novel developments recently emerged using closed bag systems as reliable alternatives to conventional cleanrooms. Several hurdles needed to be overcome including the addition of the cryoprotectant dimethylsulfoxide (DMSO) as a relevant manipulation. As a result of the development, closed bag systems proved to be comparable in terms of product quality and patient outcome to cleanroom products. They also comply with the strict regulations of good manufacturing practice.With closed systems being available, costs and efforts of a cleanroom facility may be substantially reduced in the future. The process can be easily extended for other cell preparations requiring minor modifications as donor lymphocyte preparations. Moreover, novel developments may provide solutions for the production of advanced-therapy medicinal products in closed systems.

Background: In several European countries, preparation of cellular products with open manufacturing systems as used for cryopreservation of peripheral blood stem cells (PBSCs) needs to be performed in a clean-room facility. However, this form of manufacturing is highly expensive and laborious. Thus, safe techniques providing improved efficacy regarding time and material, which are in accordance with legal requirements are highly desirable., Study Design and Methods: We have developed, validated, and applied a simple method for cryopreservation of PBSCs within a functionally closed-bag system using the closed cryo freeze prep set. This process fulfills good manufacturing practice requirements and allows for the cryopreservation of PBSCs without a clean-room facility. In addition to cryopreservation of PBSCs, we have recently successfully modified our system for processing, portioning, and cryopreservation of allogeneic donor lymphocytes., Results: Since 2010, cryopreservation of PBSCs using a closed-bag system has been performed in our facility on a routine basis and 210 patients and healthy donors have been included in this analysis. No significant reduction in viability of CD34+ cells and no process-related contamination were observed. Outcome of hematopoietic stem cell transplantation regarding time of engraftment and infectious complications is comparable to products manufactured in conventional clean-room facilities., Conclusion: Our data confirm that cryopreservation of PBSCs within a functionally closed-bag system is safe, effective, and economical. Furthermore, the system has the potential to be extended to other manufacturing processes of cellular products., (© 2015 AABB.)

Background & Aims: Cancer therapies are being developed based on our ability to direct T cells against tumor antigens. Glypican-3 (GPC3) is expressed by 75% of all hepatocellular carcinomas (HCC), but not in healthy liver tissue or other organs. We aimed to generate T cells with GPC3-specific receptors that recognize HCC and used them to eliminate GPC3-expressing xenograft tumors grown from human HCC cells in mice., Methods: We used mass spectrometry to obtain a comprehensive peptidome from GPC3-expressing hepatoma cells after immune-affinity purification of human leukocyte antigen (HLA)-A2 and bioinformatics to identify immunodominant peptides. To circumvent GPC3 tolerance resulting from fetal expression, dendritic cells from HLA-A2-negative donors were cotransfected with GPC3 and HLA-A2 RNA to stimulate and expand antigen-specific T cells., Results: Peptide GPC3367 was identified as a predominant peptide on HLA-A2. We used A2-GPC3367 multimers to detect, select for, and clone GPC3-specific T cells. These clones bound the A2-GPC3367 multimer and secreted interferon-γ when cultured with GPC3367, but not with control peptide-loaded cells. By genomic sequencing of these T-cell clones, we identified a gene encoding a dominant T-cell receptor. The gene was cloned and the sequence was codon optimized and expressed from a retroviral vector. Primary CD8(+) T cells that expressed the transgenic T-cell receptor specifically bound GPC3367 on HLA-A2. These T cells killed GPC3-expressing hepatoma cells in culture and slowed growth of HCC xenograft tumors in mice., Conclusions: We identified a GPC3367-specific T-cell receptor. Expression of this receptor by T cells allows them to recognize and kill GPC3-positive hepatoma cells. This finding could be used to advance development of adoptive T-cell therapy for HCC., (Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.)

T-cell receptors (TCR) play an important role in the adaptive immune system as they recognize pathogen- or cancer-based epitopes and thus initiate the cell-mediated immune response. Therefore there exists a growing interest in the optimization of TCRs for medical purposes like adoptive T-cell therapy. However, the molecular mechanisms behind T-cell signaling are still predominantly unknown. For small sets of TCRs it was observed that the angle between their Vα- and Vβ-domains, which bind the epitope, can vary and might be important for epitope recognition. Here we present a comprehensive, quantitative study of the variation in the Vα/Vβ interdomain-angle and its influence on epitope recognition, performing a systematic bioinformatics analysis based on a representative set of experimental TCR structures. For this purpose we developed a new, cuboid-based superpositioning method, which allows a unique, quantitative analysis of the Vα/Vβ-angles. Angle-based clustering led to six significantly different clusters. Analysis of these clusters revealed the unexpected result that the angle is predominantly influenced by the TCR-clonotype, whereas the bound epitope has only a minor influence. Furthermore we could identify a previously unknown center of rotation (CoR), which is shared by all TCRs. All TCR geometries can be obtained by rotation around this center, rendering it a new, common TCR feature with the potential of improving the accuracy of TCR structure prediction considerably. The importance of Vα/Vβ rotation for signaling was confirmed as we observed larger variances in the Vα/Vβ-angles in unbound TCRs compared to epitope-bound TCRs. Our results strongly support a two-step mechanism for TCR-epitope: First, preformation of a flexible TCR geometry in the unbound state and second, locking of the Vα/Vβ-angle in a TCR-type specific geometry upon epitope-MHC association, the latter being driven by rotation around the unique center of rotation.

Background: Nivolumab, a fully human IgG4 PD-1 immune checkpoint inhibitor antibody, can result in durable responses in patients with melanoma who have progressed after ipilimumab and BRAF inhibitors. We assessed the efficacy and safety of nivolumab compared with investigator's choice of chemotherapy (ICC) as a second-line or later-line treatment in patients with advanced melanoma., Methods: In this randomised, controlled, open-label, phase 3 trial, we recruited patients at 90 sites in 14 countries. Eligible patients were 18 years or older, had unresectable or metastatic melanoma, and progressed after ipilimumab, or ipilimumab and a BRAF inhibitor if they were BRAF(V 600) mutation-positive. Participating investigators randomly assigned (with an interactive voice response system) patients 2:1 to receive an intravenous infusion of nivolumab 3 mg/kg every 2 weeks or ICC (dacarbazine 1000 mg/m(2) every 3 weeks or paclitaxel 175 mg/m(2) combined with carboplatin area under the curve 6 every 3 weeks) until progression or unacceptable toxic effects. We stratified randomisation by BRAF mutation status, tumour expression of PD-L1, and previous best overall response to ipilimumab. We used permuted blocks (block size of six) within each stratum. Primary endpoints were the proportion of patients who had an objective response and overall survival. Treatment was given open-label, but those doing tumour assessments were masked to treatment assignment. We assessed objective responses per-protocol after 120 patients had been treated with nivolumab and had a minimum follow-up of 24 weeks, and safety in all patients who had had at least one dose of treatment. The trial is closed and this is the first interim analysis, reporting the objective response primary endpoint. This study is registered with ClinicalTrials.gov, number NCT01721746., Findings: Between Dec 21, 2012, and Jan 10, 2014, we screened 631 patients, randomly allocating 272 patients to nivolumab and 133 to ICC. Confirmed objective responses were reported in 38 (31·7%, 95% CI 23·5-40·8) of the first 120 patients in the nivolumab group versus five (10·6%, 3·5-23·1) of 47 patients in the ICC group. Grade 3-4 adverse events related to nivolumab included increased lipase (three [1%] of 268 patients), increased alanine aminotransferase, anaemia, and fatigue (two [1%] each); for ICC, these included neutropenia (14 [14%] of 102), thrombocytopenia (six [6%]), and anaemia (five [5%]). We noted grade 3-4 drug-related serious adverse events in 12 (5%) nivolumab-treated patients and nine (9%) patients in the ICC group. No treatment-related deaths occurred., Interpretation: Nivolumab led to a greater proportion of patients achieving an objective response and fewer toxic effects than with alternative available chemotherapy regimens for patients with advanced melanoma that has progressed after ipilimumab or ipilimumab and a BRAF inhibitor. Nivolumab represents a new treatment option with clinically meaningful durable objective responses in a population of high unmet need., Funding: Bristol-Myers Squibb., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

T cells have been proven to be therapeutically effective in patients with relapsed leukemias, although target antigens on leukemic cells as well as T-cell receptors (TCRs), potentially recognizing those antigens, are mostly unknown. We have applied an immunopeptidomic approach and isolated human leukocyte antigen (HLA) ligands from primary leukemia cells. We identified a number of ligands derived from different genes that are restrictedly expressed in the hematopoietic system. We exemplarily selected myeloperoxidase (MPO) as a potential target and isolated a high-avidity TCR with specificity for a HLA-B*07:02-(HLA-B7)-restricted epitope of MPO in the single HLA-mismatched setting. T cells transgenic for this TCR demonstrated high peptide and antigen specificity as well as leukemia reactivity in vitro and in vivo. In contrast, no significant on- and off-target toxicity could be observed. In conclusion, we here demonstrate, exemplarily for MPO, that leukemia-derived HLA ligands can be selected for specific effector tool development to redirect T cells to be used for graft manipulation or adoptive T-cell therapies in diverse transplant settings. This approach can be extended to other HLA ligands and HLA molecules in order to provide better treatment options for this life-threatening disease.

Formin-like 1 (FMNL1) is a formin-related protein highly expressed in hematopoietic cells and overexpressed in leukemias as well as diverse transformed cell lines. It has been described to play a role in diverse functions of hematopoietic cells such as phagocytosis of macrophages as well as polarization and cytotoxicity of T cells. However, the specific role of FMNL1 in these processes has not been clarified yet and regulation by interaction partners in primary hematopoietic cells has never been investigated. We performed a proteomic screen for investigation of the interactome of FMNL1 in primary hematopoietic cells resulting in the identification of a number of interaction partners. Bioinformatic analysis considering semantic similarity suggested the giant protein AHNAK1 to be an essential interaction partner of FMNL1. We confirmed AHNAK1 as a general binding partner for FMNL1 in diverse hematopoietic cells and demonstrate that the N-terminal part of FMNL1 binds to the C-terminus of AHNAK1. Moreover, we show that the constitutively activated form of FMNL1 (FMNL1γ) induces localization of AHNAK1 to the cell membrane. Finally, we provide evidence that overexpression or knock down of FMNL1 has an impact on the capacitative calcium influx after ionomycin-mediated activation of diverse cell lines and primary cells., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Adoptive transfer of T cells genetically modified with tumour-specific T-cell receptors (TCR) is a promising novel approach in the treatment of cancer. We have previously isolated an allorestricted MHC class I-restricted TCR with specificity for Formin-like protein 1 (FMNL1) with potent activity against chronic lymphocytic leukaemia cells. CD4(+) T cells have been described to be highly important for tumour elimination although TCR derived from CD4(+) T cells with anti-tumour reactivity have been only rarely described. In this study we aimed to isolate MHC class-II-restricted CD4(+) T cells and TCR with specificity for leukaemia antigens. We used professional antigen-presenting cells pulsed with the leukaemia-associated and tumour-associated antigen FMNL1 for stimulation of autologous T cells in vitro. We isolated two CD4(+) HLA-DR-restricted T-cell clones and T-cell-derived TCR with so far unknown specificity but high reactivity against lymphoma cells and native malignant cells derived from HLA-matched patients with diverse leukaemias. Moreover, characterization of the TCR after TCR gene transfer revealed that specific characteristics of isolated TCR as reactivity in response to Toll-like receptors were transferable on effector cells. Our results have a major impact on the development of novel immunotherapies. They demonstrate that TCR with potent HLA-DR-restricted anti-leukaemic reactivity against so far undefined self-restricted antigens can be isolated from the healthy autorestricted CD4(+) T-cell repertoire and these TCR are highly interesting candidate tools for novel immunotherapies., (© 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.)

Background: Although lentiviral transduction methods are widely used, their broader application is dependent upon the optimization of lentiviral transduction efficiency for a broad range of cell types. In the present study, we focus on the evaluation of two chemical classes with respect to their ability to increase lentiviral transduction without cytotoxicity., Methods: We compared the activity of adjuvants that are already used for lentivirus delivery with that of novel adjuvants selected on the basis of their chemical and physical characteristics., Results: The novel poloxamer synperonic F108 demonstrated superior characteristics for enhancing lentiviral transduction over the best-in-class polybrene-assisted transduction. The results revealed that poloxamer synperonic F108 exhibited the dual benefits of low toxicity and a high efficiency of lentiviral gene delivery into a range of different primary cell cultures. In the presence of poloxamer synperonic F108, cells showed an increased propidium dye influx indicating a re-organization of membrane microstructures accompanying lentivirus uptake. The administration of a mixture of poloxamer synperonic F108 with polybrene further enhanced lentiviral transduction rates., Conclusions: The results obtained in the present study indicate that a contribution to efficiency is made by each adjuvant, with polybrene acting as a charge protector and poloxamer synperonic F108 as a membrane modulator. Therefore, poloxamer synperonic F108, either alone or in combination, can lead to the optimization of large-scale lentiviral transduction approaches., (Copyright © 2012 John Wiley & Sons, Ltd.)

T cells can recognize tumor cells specifically by their TCR and the transfer of TCR-engineered T cells is a promising novel tool in anticancer therapies. We isolated and characterized four allorestricted TCRs with specificity for the HER2/neu-derived peptide 369 (HER2(369)) demonstrating high peptide specificity. PBMCs transduced with especially one TCR, HER2-1, mediated specific tumor reactivity after TCR optimization suggesting that this TCR represents a potential candidate for targeting HER2 by TCR-transduced effector cells. Another TCR showed high-peptide specificity without tumor reactivity. However, the TCR alpha-chain of this TCR specifically recognized HER2(369) not only in combination with the original beta-chain but also with four other beta-chains of the same variable family deriving from TCRs with diverse specificities. Pairing with one beta-chain derived from another HER2(369)-specific TCR potentiated the chimeric TCRs in regard to functional avidity, CD8 independency, and tumor reactivity. Although the frequency of such TCR single chains with dominant peptide recognition is currently unknown, they may represent interesting tools for TCR optimization resulting in enhanced functionality when paired to novel partner chains. However, undirected mispairing with novel partner chains may also result in enhanced cross-reactivity and self-reactivity. These results may have an important impact on the further design of strategies for adoptive transfer using TCR-transduced T cells.

The formin protein formin-like 1 (FMNL1) is highly restrictedly expressed in hematopoietic lineage-derived cells and has been previously identified as a tumor-associated antigen. However, function and regulation of FMNL1 are not well defined. We have identified a novel splice variant (FMNL1gamma) containing an intron retention at the C terminus affecting the diaphanous autoinhibitory domain (DAD). FMNL1gamma is specifically located at the cell membrane and cortex in diverse cell lines. Similar localization of FMNL1 was observed for a mutant lacking the DAD domain (FMNL1DeltaDAD), indicating that deregulation of autoinhibition is effective in FMNL1gamma. Expression of both FMNL1gamma and FMNL1DeltaDAD induces polarized nonapoptotic blebbing that is dependent on N-terminal myristoylation of FMNL1 but independent of Src and ROCK activity. Thus, our results describe N-myristoylation as a regulative mechanism of FMNL1 responsible for membrane trafficking potentially involved in a diversity of polarized processes of hematopoietic lineage-derived cells.

Cell-based immunotherapy in settings of allogeneic stem cell transplantation or donor leukocyte infusion has curative potential, especially in hematologic malignancies. However, this approach is severely restricted due to graft-versus-host disease (GvHD). This limitation may be overcome if target antigens are molecularly defined and effector cells are specifically selected. We chose formin-related protein in leukocytes 1 (FMNL1) as a target antigen after intensive investigation of its expression profile at the mRNA and protein levels. Here, we confirm restricted expression in peripheral blood mononuclear cells (PBMCs) from healthy donors but also observe overexpression in different leukemias and aberrant expression in transformed cell lines derived from solid tumors. We isolated allorestricted T-cell clones expressing a single defined TCR recognizing a particular HLA-A2-presented peptide derived from FMNL1. This T-cell clone showed potent antitumor activity against lymphoma and renal cell carcinoma cell lines, Epstein-Barr virus (EBV)-transformed B cells, and primary tumor samples derived from patients with chronic lymphocytic leukemia (CLL), whereas nontransformed cells with the exception of activated B cells were only marginally recognized. Allorestricted TCRs with specificity for naturally presented FMNL1-derived epitopes may represent promising reagents for the development of adoptive therapies in lymphoma and other malignant diseases.

Chronic lymphocytic leukemia (CLL) is associated with a variety of immunologic disturbances. Hypogammaglobulinemia and autoimmune phenomena are both often present in this disease. In contrast, humoral or cellular antitumor responses are rarely observed. It has been previously shown that antigens detected in patients with malignant diseases can provide information regarding intracellular molecules engaged in the transformation process and can identify tumor antigens that may be useful for development of immunotherapeutic strategies. Serologic identification by recombinant expression cloning (SEREX) has been demonstrated to be a useful method to detect tumor and tumor-associated antigens in a variety of malignancies. Although this approach is complicated in CLL, we used a modified SEREX approach and identified 14 antigens (KW-1 to KW-14) using this methodology. Several clones showed a restricted expression pattern in normal tissues. Moreover, distinctive expression of splice variants and aberrant gene expression in malignant tissue were detected. In this study, 6 antigens were detected exclusively in patients with CLL. Eight antigens were detected also in lymphoma patients. Healthy donors showed antibody responses against only 3 of the identified antigens. T cells with specific cytotoxicity against peptides derived from the 2 antigens tested could be generated from healthy donors. These findings demonstrate that humoral and cellular immune responses against CLL-associated antigens can be detected. Ongoing experiments investigate their potential for the development of immunotherapeutic strategies.

Chronic lymphocytic leukemia (CLL) cells are ineffective antigen-presenting cells (APCs) although CD40-activated CLL cells can stimulate proliferation of autologous and allogeneic T cells. We examined the antigen-presenting capacity of CD40-activated CLL cells as well as dendritic cells pulsed with apoptotic bodies of CLL cells to generate autologous and allogeneic immune responses against CLL cells. Both APC types were capable of generating T-cell lines that proliferate specifically in response to unstimulated CLL cells. Whereas cytotoxic responses against stimulated and unstimulated CLL cells could be repeatedly generated by allogeneic healthy donors, autologous cytotoxic immune responses against CD40-activated and native CLL cells were rarely detected. However, T cells isolated from patients with CLL could recognize and lyse allogeneic stimulated and unstimulated CLL cells, demonstrating that cytotoxic T cells from these tumor-bearing patients are functionally intact.

Cytotoxic T-lymphocyte (CTL) responses can be generated against peptides derived from the immunoglobulin (Ig) V region in some but not all patients. The main reason for this appears to be the low peptide-binding affinity of Ig-derived peptides to major histocompatibility complex (MHC) class I molecules and their resulting low immunogenicity. This might be improved by conservative amino acid modifications at the MHC-binding residues of the peptides (heteroclitic peptides). In this study, it was found that in 18 Ig-derived peptides, that heteroclitic peptides from the Ig gene with improved binding to human leukocyte antigen (HLA)-A*0201 can be used to improve CTL responses. Amino acid substitution substantially increased predicted binding affinity, and there was a strong correlation between predicted and actual binding to HLA-A*0201. CTLs generated against the heteroclitic peptide had not only enhanced cytotoxicity against the heteroclitic peptide but also increased killing of antigen-presenting cells pulsed with the native peptide. Surprisingly, no difference was observed in the frequency of T cells detected by MHC class I peptide tetramers after stimulation with the heteroclitic peptide compared with the native peptide. CTLs generated against heteroclitic peptides could kill patients' tumor cells, showing that Ig-derived peptides can be presented by the tumor cell and that the failure to mount an immune response (among other reasons) likely results from the low immunogenicity of the native Ig-derived peptide. These results suggest that heteroclitic Ig-derived peptides can enhance immunogenicity, thereby eliciting immune responses, and that they might be useful tools for enhancing immunotherapy approaches to treating B-cell malignant diseases.