Search

In multiple sclerosis (MS) patients pathogenic, autoreactive effector T cells (Teffs) provoke demyelination and central nerve system damage. To impede those harmful immune reactions, the adoptive transfer of regulatory T cells (Tregs) emerged as a promising therapeutic strategy. Several preclinical mouse models confirm an inferior functionality of polyclonal compared to antigen-specific Treg cells. However, isolation and expansion of Tregs with a desired antigen-specificity proves to be highly time-consuming and labor-intensive. To overcome these hurdles, we armed polyclonal Tregs isolated from MS patients with a universal chimeric antigen receptor (UniCAR) construct. This innovative technology allows a site-specific redirection of cells against any desired surface structure, as cross-linkage to target cells is mediated by a separate, antigen-binding targeting module (TM). Highly pure CD4+CD25highCD127dimCD45RA+ MS-Tregs could be genetically modified to stably express the UniCAR 4-1BB/ζ construct. UniCAR-endowed Tregs strongly expand and show phenotypic stability also upon pro-inflammatory challenge. By adding a TM in the presence of target cells, UniCAR-engrafted Tregs are antigen-specifically activated demonstrated by CD69 and LAP upregulation. Most importantly, upon TM-stimulation UniCAR-armed Tregs efficiently suppress pre-activated, patient-derived Teffs. Taken together, the UniCAR system holds an enormous therapeutic potential for MS, as it not only allows a site-specific and precisely regulated Treg activation but also confers strong suppressive capacity to Tregs from MS patients. Thereby, this innovate technology might broaden current treatment strategies to overcome impaired functionality of Tregs as well as resistance of pathogenic Teffs to Treg suppression reported for MS patients.

Antibodies (Abs) and their recombinant derivatives play an increasing role in tumor therapy and diagnostic imaging. The theranostic target modules (TMs) recently developed by us can be used for individualized and precise cancer immunotherapy and at the same time as radiotracers for tumor imaging. The TMs consist of an antigen binding moiety that binds to the prostate stem cell antigen (PSCA) on the tumor cell surface and present the peptide epitope E5B9. Via E5B9 the TMs can retarget human T cells that are genetically modified with universal anti-E5B9 chimeric antigen receptors (UniCARs) towards the tumor cells. The cross-linkage of UniCAR T cells and tumor cells via the TM results in T cell activation and tumor cell killing. This immunotherapeutic approach was shown as an inducible CAR platform technology that can be used for the treatment of leukemic as well as solid cancers. This technic has an intrinsic emergency brake: The short half-life of the TMs in vivo allows a fast switch off of the UniCAR system. In detail, UniCAR T cells per se are inactive because their UniCARs do not recognize an antigen on the cell surface. Only in the presence of tumor-bound TMs UniCAR T cells can be redirected to tumor cells and turned on. But they are switched off when the application of TMs is stopped and their concentration is too low to activate UniCAR T cells what also becomes particularly relevant in case of side effects occur in the patients. TMs cannot only be used as a therapeutic drug but also as a diagnostic tool for tumor imaging. For this purpose TMs can be modified with a bifunctional chelator. After conjugation they can be labeled with radiometals like 64Cu2+ or 68Ga3+ and used as an imaging tracer for PET analysis. Usually our TMs contain a tag of six histidine residues (His6-tag) at the C-terminus. The His6-tag is used for convenient purification by immobilized metal ion affinity chromatography and detection of the TMs. However the His6-tag may affect PK and biodistribution of the TMs. In particular with respect to clinical application of the TMs the His6-tag may induce immunologic reactions. Furthermore the purification of His6-tagged TMs by immobilized metal ion affinity chromatography might cause metal ions in the TMs which have influence on their radiolabeling and can be critical for patients’ health. To address these concerns we have generated an anti-PSCA TM without His6-tag (PSCA TM) and compared its theranostic properties to the corresponding TM with the His6-tag (PSCA-His TM). The PSCA TMs were successfully prepared by digestion with TEV protease recognizing a strict seven amino acid cleavage recognition site which was introduced upstream from the His6-tag in the PSCA-TEV-His TM construct. Both TMs with or without His6-tag showed similar high binding affinities to PSCA on PC3-PSCA prostate cancer cells whereas the Kd values were in the nM range. As shown by chromium release assays both target modules were able to redirect UniCAR T cells to efficiently kill PSCA-presenting tumor cells in a strictly target-dependent and target-specific manner. In vitro tumor cell killing of PSCA TM was less efficient than PSCA-His TM but still in pM range. Most importantly in NMRI-FOX1nu/nu mice the PSCA TM redirected UniCAR T cells to eliminate luciferase/PSCA-double positive tumor cells as efficient as the PSCA-His TM. PET analysis of the 64Cu-labeled TMs in these mice showed higher accumulation of the PSCA TM in the PSCA-positive tumors and less nonspecific binding in normal tissues (like blood and heart) after 2 h. At the same time the blood clearance of PSCA TM was distinctly faster than of the PSCA-His TM resulting in better contrast of the tumor for the PSCA TM in the PET images after 2 h. On the other hand, the PSCA-His TMs was slower cleared from the blood resulting in a higher PSCA-His TM concentration in the tumor later on after one day. To summarize we have established two PSCA TMs one with and the other without a C-terminal His6-tag which differ in their theranostic properties. Both TMs efficiently redirect UniCAR T cells to kill tumor cells. The PSCA TMs have a higher tumor accumulation and lower nonspecific binding in the normal tissues and blood resulting in a very short half-life that may allow an even faster emergency shut down of the UniCAR system if necessary and excellent short time imaging. The PSCA-His TMs are characterized by a larger nonspecific binding, slower blood-clearance and are apparently better suited for long-term imaging. Both TMs are applicable for the UniCAR based immunotherapy and imaging, complement each other and so they are suitable for the individualized, precise human theranostic.

Regulatory T cells (Tregs) possess a central role in impeding harmful immune reactions and represent important immunomodulatory players in the pathogenesis of inflammatory diseases. For that reason, Tregs are intensively studied as an innovative cell product for the treatment of autoimmunity e.g. multiple sclerosis (MS) or Graft-versus-Host disease. In recent years, several preclinical mouse model clearly demonstrate a superior suppressive capacity of antigen-specific Tregs compared to polyclonal cells. On the downside, isolation of Tregs with a distinct antigen-specificity is a highly time-consuming and laborious process. To overcome these challenges, we armed polyclonal Tregs isolated from healthy donors or MS patients with a universal chimeric antigen receptor (UniCAR) construct. As T cells and target cells are indirectly cross-linked by a separate, antigen-binding targeting module (TM), this innovative technology enables side-specific redirection of Tregs to any desired surface structure. Moreover, UniCAR armed Tregs are silenced in the absence of the TM allowing for a finely tuned regulation of Treg activity between an “on” and “off” status. Here, we demonstrate that highly pure CD4+CD25highCD127dimCD45RA+ Tregs isolated from both healthy donors or MS patients stably express a UniCAR construct with an intracellular 4-1BB/ζ signaling domain. UniCAR-engrafted Tregs vigorously expand and maintain their phenotype even under pro-inflammatory conditions. Most importantly, upon TM-activation UniCAR-endowed Tregs significantly hamper autologous T effector cells both in vitro and in vivo. Taken together, our results underline the enormous therapeutic potential of the UniCAR system for treatment of inflammatory diseases including MS, as it facilitates an antigen-specific and precisely controlled Treg activation at the side of inflammation. Moreover, this innovative technology allows redirection of Tregs against a wide range of surface structures simply by exchanging the TM and might thereby broaden current treatment modalities.

Adoptive transfer of chimeric antigen receptor (CAR) T cells represents one of the fastest growing areas in cancer immunotherapy. Albeit gene-modified cells have demonstrated unparalleled antitumor efficiency in B cell malignancies, highly potent CAR T cells can cause severe and partly life-threatening side effects including cytokine release syndrome, neurological toxicity and off-target effects. Hence, there is an increasing demand for developing effective approaches to selectively ablate gene-modified cells in vivo. Previously, we described an epitope tag (E-tag) derived from the human nuclear protein La that is incorporated into the extracellular domain of CARs and accessible by an anti-La monoclonal antibody (mAb). Based on this mAb, we generated a novel CAR construct for specific binding and depletion of E-tag-expressing CAR T cells. We demonstrate that anti-E-tag-redirected T cells selectively eliminate CAR T cells that extracellularly express the E-tag whilst CAR T cells lacking this tag are not attacked. Interestingly, T cell killing is reciprocal and occurs in dependence of an intracellular signaling domain. Our studies further indicate that T cells expressing high CAR levels are more efficiently depleted than T cells with low CAR expression. Besides, CD4+ and CD8+ target cells are equally well eliminated by both CD4+ and CD8+ effector T cells. Overall, we provide an approach for specific and efficient depletion of overactive CAR T cells in case patients experience severe side effects. The E-tag can be incorporated into all CARs irrespective of the targeted tumor antigen and represents a promising tool to improve safety of cell-based immunotherapies.

Objectives Local, abdominal fat depots may be related to alterations in cardiac function and morphology due to a metabolic linkage. Thus, we aimed to determine their association with subtle cardiac changes and the potential interaction with hyperglycemic metabolic states. Methods Subjects from the general population and without history of cardiovascular disease were drawn from the Cooperative Health Research in the Region of Augsburg FF4 cohort and underwent 3 T cardiac and body MRI. Measures of abdominal adiposity such as hepatic proton-density fat fraction [PDFFhepatic], subcutaneous (SAT) and visceral abdominal fat (VAT) as well as established cardiac left-ventricular (LV) measures including LV remodeling index (LVCI) were derived. Associations were determined using linear regression analysis based on standard deviation normalized predictors. Results Among a total of 374 subjects (56.2 ± 9.1 years, 58% males), 49 subjects had diabetes, 99 subjects had prediabetes and 226 represented normal controls. Only subtle cardiac alterations were observed (e.g. LVCI: 1.13 ± 0.30). While SAT was not associated, increasing VAT and increasing PDFFhepatic were independently associated with increasing LVCI (β = 0.11 and 0.06, respectively), decreasing LV end-diastolic volume (β = − 6.70 and 3.23, respectively), and decreasing LV stroke volume (β = − 3.91 and − 2.20, respectively). Hyperglycemic state did not modify the associations between VAT or PDFF and LV measures (interaction term: all p ≥ 0.29). Conclusion In a healthy population, VAT but also PDFFhepatic were associated with subclinical measures of LV remodeling without evidence for a modifying effect of hyperglycemic state.

Background: Vaginal/uterine rhabdomyosarcoma (VU RMS) is one of the most favorable RMS sites. To determine the optimal therapy, the experience of four cooperative groups (Children's Oncology Group [COG], International Society of Pediatric Oncology (SIOP) Malignant Mesenchymal Tumor Group [MMT], Italian Cooperative Soft Tissue Sarcoma Group [ICG], and European pediatric Soft tissue sarcoma Study Group [EpSSG]) was analyzed. Procedure: From 1981 to 2009, 237 patients were identified. Median age (years) at diagnosis differed by tumor location; it was 1.9 for vagina (n = 160), 2.7 for uterus corpus (n = 26), and 13.5 for uterus cervix (n = 51). Twenty-eight percent of patients received radiation therapy (RT) as part of primary therapy (23% COG, 27% MMT, 46% ICG, and 42% EpSSG), with significant differences in the use of brachytherapy between the cooperative groups (23% COG, 76% MMT, 64% ICG, and 88% EpSSG). Results: Ten-year event-free (EFS) and overall survival (OS) were 74% (95% CI, 67–79%) and 92% (95% CI, 88–96%), respectively. In univariate analysis, OS was inferior for patients with uterine RMS and for those with regional lymph node involvement. Although EFS was slightly lower in patients without initial RT (71% without RT vs. 81% with RT; P = 0.08), there was no difference in OS (94% without RT vs. 89% with RT; P = 0.18). Local control using brachytherapy was excellent (93%). Fifty-one (51.5%) of the 99 survivors with known primary therapy and treatment for relapse were cured with chemotherapy with or without conservative surgery. Conclusions: About half of all patients with VU RMS can be cured without systematic RT or radical surgery. When RT is indicated, modalities that limit sequelae should be considered, such as brachytherapy.

Introduction: Radiation is a common therapy for solid tumors. Unfortunately there is a high risk for the outgrowth of radioresistant tumor cells against which only limited treatment options exist. We challenged the idea whether or not chimeric antigen receptor (CAR) engineered T lymphocytes could be used as an adjuvant immunotherapy in combination with radiotherapy. Recently, we have established switchable universal CARs (UniCARs) that bind to a short peptide epitope (E5B9) which does not exist on the surface of living cells. UniCAR T cells are exclusively redirected to tumor cells in the presence of a target module (TM) that exhibits the E5B9 epitope and binds to a tumor associated antigen (TAA) on the tumor cell surface. Materials and Methods: We used different radioresistant sublines of the head and neck cancer cell line Cal33. Gene expression data for certain TAAs were confirmed by flow cytometry. TMs against potential targets were created from the variable domains of monoclonal antibodies, cloned in lentiviral vectors and purified from supernatants of permanently TM producing 3T3 cells. UniCAR T cells were generated by lentiviral transduction. Results: Radioresistant Cal33 cell lines expressed PSCA, EGFR and CD98. UniCAR TMs were created against these TAAs. Armed with these TMs UniCAR T cells efficiently killed radioresistant Cal33 cells in vitro and in vivo. Conclusions: Radioresistant tumor cells can efficiently be killed by redirecting UniCAR T cells against PSCA, CD98 and EGFR and thus resistance to radiotherapy can be overcome by immunotherapy based on the UniCAR technology to these targets.

Background: High-grade osteosarcoma is a primary malignant bone tumour mainly affecting children and young adults. The European and American Osteosarcoma Study (EURAMOS)-1 is a collaboration of four study groups aiming to improve outcomes of this rare disease by facilitating randomised controlled trials.Methods: Patients eligible for EURAMOS-1 were aged 2000 patients registered to EURAMOS-1 demonstrated survival rates in concordance with institution-or group-level osteosarcoma trials. Further efforts are required to drive improvements for patients who can be identified to be at higher risk of adverse outcome. This trial reaffirms known prognostic factors, and owing to the large numbers of patients registered, it sheds light on some additional factors to consider. (C) 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Radiation therapy represents a commonly applied treatment regimen for solid tumors. Unfortunately, it is often accompanied by a high risk for the outgrowth of radioresistant cancer cells against which treatment options are limited. We challenged the idea whether or not chimeric antigen receptor (CAR)-modified T cells could be exploited as an adjuvant immunotherapy in combination with standard radiotherapy. Over the past several years, we have established switchable universal CAR constructs (UniCARs) that recognize a short peptide epitope (E5B9) which does not exist on the surface of living cells. UniCAR T cells are redirected to malignant cells exclusively in the presence of a target module (TM) that contains the epitope E5B9 and specifically binds to a tumor-associated antigen (TAA) on the tumor cell surface. For providing a rationale for the combination of CAR and radiation therapy, we used different radioresistant sublines of the head and neck cancer cell line Cal33. Expression of various TAAs including of PSCA, EGFR and CD98 was confirmed by flow cytometry analysis. Subsequently, TMs recognizing these potential targets were generated from the variable domains of monoclonal antibodies, cloned into lentiviral vectors and purified from cell culture supernatants of TM-producing stable cell lines. In parallel, T cells isolated from healthy donors were engrafted with UniCARs by lentiviral transduction. Armed with our anti-TAA TMs, UniCAR T cells efficiently lysed radioresistant Cal33 tumor cells both in vitro and in vivo. Taken together, we could demonstrate that radioresistant cancer cells can effectively be killed by retargeting UniCAR T cells against PSCA, CD98 and EGFR. Thus, resistance to standard of care radiotherapy can be overcome by concomitant or subsequent immunotherapy using the flexible UniCAR technology.

Immunotherapy with CAR-modified T cells has recently entered into the clinical routine. Nonetheless, until now most concerning side effects associated with CAR T cell therapies are cytokine release syndrome and “on-target, off-tumor” reactions. In order to improve CAR technology regarding safety, we developed a novel switchable platform termed UniCAR. It relies on the separation of the functional domains of conventional CARs. Thus, the UniCAR system is composed of (I) T cells modified to express an universal CAR (UniCAR) and (II) tumor-specific target modules (TM). UniCAR T cell activity can be easily controlled: While they are inert in the absence of TMs, their anti-tumor reactivity can be only switched on in the presence of TMs. For redirection of UniCAR T cells to EGFR+ epithelial tumors, we recently established a monovalent nanobody-based α-EGFR TM, either expressed in bacterial or eukaryotic cells. In spite of the identical primary sequence the eukaryotic α-EGFR TM showed a reduced killing capability and affinity. This observation encouraged us, to elucidate whether TM functionality can be further improved by an increase in affinity. Consequently, we here constructed a novel bivalent α-EGFR-EGFR TM, expressed it in eukaryotic cells and compared its anti-tumor reactivity and pharmacokinetic properties with the monovalent α-EGFR TM. As expected, the avidity of the bivalent TM is higher than that of its monovalent counterpart. By raising the number of binding sites, the resulting bivalent α-EGFR-EGFR TM shows also an improved killing efficacy and capability in vitro and in vivo. While the monovalent α-EGFR TM could only mediate the killing of tumor cells expressing high levels of EGFR, the bivalent α-EGFR-EGFR TM could also redirect UniCAR T cells to tumor cells expressing lower levels of EGFR. According to in vivo PET experiments, the increased molecular weight of the bivalent α-EGFR-EGFR TM delays its elimination and thereby improves the enrichment at the tumor site. Consequently, the bivalent TM seems to be more suitable for PET imaging approaches and tumor eradication.

Enteric methane (CH) production from cattle contributes to global greenhouse gas emissions. Measurement of enteric CH is complex, expensive, and impractical at large scales; therefore, models are commonly used to predict CH production. However, building robust prediction models requires extensive data from animals under different management systems worldwide. The objectives of this study were to (1) collate a global database of enteric CH production from individual lactating dairy cattle; (2) determine the availability of key variables for predicting enteric CH production (g/day per cow), yield [g/kg dry matter intake (DMI)], and intensity (g/kg energy corrected milk) and their respective relationships; (3) develop intercontinental and regional models and cross-validate their performance; and (4) assess the trade-off between availability of on-farm inputs and CH prediction accuracy. The intercontinental database covered Europe (EU), the United States (US), and Australia (AU). A sequential approach was taken by incrementally adding key variables to develop models with increasing complexity. Methane emissions were predicted by fitting linear mixed models. Within model categories, an intercontinental model with the most available independent variables performed best with root mean square prediction error (RMSPE) as a percentage of mean observed value of 16.6%, 14.7%, and 19.8% for intercontinental, EU, and United States regions, respectively. Less complex models requiring only DMI had predictive ability comparable to complex models. Enteric CH production, yield, and intensity prediction models developed on an intercontinental basis had similar performance across regions, however, intercepts and slopes were different with implications for prediction. Revised CH emission conversion factors for specific regions are required to improve CH production estimates in national inventories. In conclusion, information on DMI is required for good prediction, and other factors such as dietary neutral detergent fiber (NDF) concentration, improve the prediction. For enteric CH yield and intensity prediction, information on milk yield and composition is required for better estimation., This study is part of the Joint Programming Initiative on Agriculture, Food Security and Climate Change (FACCE‐JPI)'s “GLOBAL NETWORK” project and the “Feeding and Nutrition Network” (http://animalscience.psu.edu/fnn) of the Livestock Research Group within the Global Research Alliance for Agricultural Greenhouse Gases (www.globalresearchalliance.org). Authors gratefully acknowledge funding for this project from: USDA National Institute of Food and Agriculture Grant no. 2014‐67003‐21979) University of California, Davis Sesnon Endowed Chair Program, USDA, and Austin Eugene Lyons Fellowship (University of California, Davis); Funding from USDA National Institute of Food and Agriculture Federal Appropriations under Project PEN 04539 and Accession number 1000803, DSM Nutritional Products (Basel, Switzerland), Pennsylvania Soybean Board (Harrisburg, PA, USA), Northeast Sustainable Agriculture Research and Education (Burlington, VT, USA), and PMI Nutritional Additives (Shoreview, MN, USA); the Ministry of Economic Affairs (the Netherlands; project BO‐20‐007‐006; Global Research Alliance on Agricultural Greenhouse Gases), the Product Board Animal Feed (Zoetermeer, the Netherlands) and the Dutch Dairy Board (Zoetermeer, the Netherlands); USDA National Institute of Food and Agriculture (Hatch Multistate NC‐1042 Project Number NH00616‐R; Project Accession Number 1001855) and the New Hampshire Agricultural Experiment Station (Durham, NH); French National Research Agency through the FACCE‐JPI program (ANR‐13‐JFAC‐0003‐01), Agricultural GHG Research Initiative for Ireland (AGRI‐I), Academy of Finland (No. 281337), Helsinki, Finland; Swiss Federal Office of Agriculture, Berne, Switzerland; the Department for Environment, Food and Rural Affairs (Defra; UK); Defra, the Scottish Government, DARD, and the Welsh Government as part of the UK's Agricultural GHG Research Platform projects (www.ghgplatform.org.uk); INIA (Spain, project MIT01‐GLOBALNET‐EEZ); German Federal Ministry of Food and Agriculture (BMBL) through the Federal Office for Agriculture and Food (BLE); Swedish Infrastructure for Ecosystem Science (SITES) at Röbäcksdalen Research Station; Comisión Nacional de Investigación Científica y Tecnológica, Fondo Nacional de Desarrollo Científico y Tecnológico (Grant Nos. 11110410 and 1151355) and Fondo Regional de Tecnología Agropecuaria (FTG/RF‐1028‐RG); European Commission through SMEthane (FP7‐SME‐262270). The authors are thankful to all colleagues who contributed data to the GLOBAL NETWORK project and especially thank Luis Moraes, Ranga Appuhamy, Henk van Lingen, James Fadel, and Roberto Sainz for their support on data analysis. All authors read and approved the final manuscript. The authors declare that they have no competing interests.

CAR-modified T cells show impressive results in clinical trials. However, cytokine release syndrome and “on-target, off-tumor” reactions represent most concerning side effects. To improve the safety of CAR-T cell therapy, we established a switchable CAR platform termed UniCAR system consisting of two components: UniCAR-modified T cells and tumor-specific target modules (TM). For treatment of EGFR+ epithelial tumors, we recently described a monovalent nanobody-based α-EGFR TM, either expressed in bacteria or eukaryotic cells. In spite of the identical primary sequence the eukaryotic showed a reduced killing capability and affinity. Here we describe a novel bivalent α-EGFR-EGFR TM. As expected, the avidity of the bivalent TM is higher than that of its monovalent counterpart. Binding of neither the monovalent α-EGFR TM nor the bivalent α-EGFR-EGFR TM to EGFR effected the EGF-mediated signaling. While the monovalent α-EGFR TM could only mediate the killing of tumor cells expressing high levels of EGFR, the bivalent α-EGFR-EGFR TM could redirect UniCAR T cells to tumor cells expressing low levels of EGFR. According to PET experiments in vivo, the increased avidity of the bivalent α-EGFR-EGFR TM improves the enrichment at the tumor site and its use for PET imaging.

Recently, chimeric antigen receptor (CAR) expressing T cells have shown tremendous clinic effects in several cancer patients. However once those genetically modified T cells are adoptively transferred in a patient their reactivity cannot be controlled in case of life-threatening side effects or tumor alterations including antigen loss occur. These limitations encouraged us to develop an on/off switchable universal CAR (UniCAR) platform. As an optimization of conventional CARs, UniCARs do not bind to a cell surface antigen. In contrast their extracellular single-chain fragment variable (scFv) is redirected to the short peptide epitope E5B9 that is physiologically not presented on the surface of living cells. Consequently the UniCAR T cells are inert. Only in the presence of a target module, that exhibits the E5B9 and binds to a tumor surface target, the UniCAR T cells can be cross-linked to tumor cells and thus get activated to kill them. Recently, we have produced a series of monospecific and bispecific target modules against a series of tumor associated antigens including PSCA, PSMA, CD33, CD123, GD2, and EGFR. Here we demonstrate in vitro as well as in experimental mice that all these target modules are able to efficiently redirect UniCAR T cells against tumor cells in a strictly target-dependent and target-specific manner. Tumor cell killing occurred at pM target module concentrations and the killing efficacy of UniCAR T cells was comparable to conventional CAR T cells. As measured by ELISA and/or flow cytometry-based multiplex assays redirected UniCAR T cells released pro-inflammatory cytokines including for example TNF, IL-2 and IFN-γ but not IL-6. Bispecific tumor targeting mediated superior tumor cell killing effects than the usage of monospecific target modules whereas the amount of released pro-inflammatory cytokines were not increased. Finally, we have proven that redirected UniCAR T cells can kill luciferase-positive tumor cells in immunodeficient mice. In agreement with the UniCAR concept, target modules showed a very short half-life in peripheral blood, could accumulate in established tumors and were released from UniCAR-target module-complexes in a concentration-dependent manner as measured by dynamic PET analysis in mice. In summary, we established a controllable UniCAR platform for tumor immunotherapy. The reactivity of UniCAR armed T cells can be switched on and off in the presence or absence of target modules and can be regulated in a dose-dependent manner providing an improved safety of the CAR technology. Moreover a variety of different target modules against a series of different tumor targets can be introduced in the UniCAR platform supporting its high flexibility.

Regulatory T cells (Tregs) play a fundamental role in preventing inflammatory diseases and, therefore, their adoptive transfer emerged as a promising therapeutic strategy for the treatment of autoimmunity, graft rejection and Graft-versus-Host disease. However, preclinical animal models have already substantiated that the application of antigen-specific instead of polyclonal Tregs results in a far more efficient suppression of pathogenic immune reactions. Due to their low frequency in human peripheral blood, the isolation of Tregs with defined antigen specificity is a highly time-consuming and labour-intensive process that does not yet provide therapeutically relevant cell numbers. To overcome this obstacle, we equipped polyclonal Tregs with a novel modular chimeric antigen receptor (CAR) technology called UniCAR. Unlike conventional CARs, the UniCAR binding domain does not directly recognize a target cell antigen but a small peptide epitope, which is a subunit of a separate target module (TM) providing antigen specificity. Hence, UniCAR-armed Tregs are silenced until they encounter the TM that mediates their cross-linkage with target cells. Therefore, this novel CAR technology not only allows for precise regulation of Treg activity between an “on” and “off” status but also enables their specific retargeting towards any desired antigen simply by replacing the TM. To additionally compare the influence of different costimulatory signals on Treg properties and functionality, UniCARs were generated and introduced comprising either a CD3ζ, CD28-CD3ζ or CD137-CD3ζ signaling domain. For generation of UniCAR-expressing Tregs the CD45RA+ subpopulation was used, as these cells show the highest capacity in preserving Treg phenotype and functionality ex vivo. Thus, highly pure, sorted CD4+CD25+CD127lowCD45RA+ Tregs were genetically modified by using a lentiviral gene transfer system resulting in an average of 80 % UniCAR+ Tregs. These UniCAR-armed Tregs maintain their phenotype (≥ 93 % FOXP3+) and expand approximately 150- to 200-fold already after 8 days of in vitro culture. In addition, in the presence of target cells and a respective TM the genetically modified Tregs are activated antigen-specifically as shown by CD69 and LAP upregulation as well as an increased FOXP3 expression level. Most importantly, upon TM-mediated restimulation via the UniCAR, Tregs efficiently suppress proliferation and overall expansion of bead-activated autologous T effector cells. Taken together, our results underline the enormous clinical potential of UniCAR-armed Tregs as this technology facilitates an antigen-specific activation of polyclonal Tregs at the side of inflammation where they subsequently exert their suppressive capacity. In addition, the UniCAR system enables a precise control over Treg activity. Moreover, the UniCAR-equipped Tregs can be applied for treatment of a wide range of inflammation-related diseases as their antigen specificity can be easily modified just by exchanging the TM.

Recent treatments of leukemias with T cells expressing chimeric antigen receptors (CARs) underline their impressive therapeutic potential but also their risk of severe side effects including cytokine release storms and tumor lysis syndrome. In case of cross-reactivities, CAR T cells may also attack healthy tissues. To overcome these limitations, we previously established a switchable CAR platform technology termed UniCAR. UniCARs are not directed against typical tumor-associated antigens (TAAs) but instead against a unique peptide epitope: Fusion of this peptide epitope to a recombinant antibody domain results in a target module (TM). TMs can cross-link UniCAR T cells with tumor cells and thereby lead to their destruction. So far, we constructed TMs with a short half-life. The fast turnover of such a TM allows to rapidly interrupt the treatment in case severe side effects occur. After elimination of most of the tumor cells, however, longer lasting TMs which have not to be applied via continous infusion would be more convenient for the patient. Here we describe and characterize a TM for retargeting UniCAR T cells to CD19 positive tumor cells. Moreover, we show that the TM can efficiently be produced in vivo from producer cells housed in a sponge-like biomimetic cryogel and, thereby, serving as an in vivo TM factory for an extended retargeting of UniCAR T cells to CD19 positive leukemic cells.

In recent years, adoptively transferred autologous human T cells that are genetically modified with chimeric antigen receptors (CARs) have been very successfully used for treatment of different hematological malignancies. However, immunotherapy of solid tumors seems to be more challenging. Especially for this application it is of great interest to enhance the very promising CAR technology. Conventional CARs consist of (I) an extracellular single-chain fragment variable (scFv) redirected to a tumor-associated antigen (TAA), (II) a transmembrane region and (III) intracellular activating motifs. Although these CAR-armed T cells showed impressive therapeutic effects in leukemia patients, some limitations have appeared. Most importantly, CAR-armed T cells can cause life-threatening side effects as a consequence of immoderate on-target, on-tumor reactions or aggressive on-target, off-tumor attack against healthy tissues. Furthermore, under the pressure of a monospecific CAR therapy the targeted antigen can be reduced on tumor cells. To overcome these problems we have recently described a novel modular universal CAR (UniCAR) platform that consists of two separate arms: (I) the universal effector arm and (II) the individual targeting arm. T cells that are genetically modified with UniCARs are redirected to the short peptide epitope E5B9 that is physiologically not presented on the surface of living cells. E5B9 is coupled to the anti-TAA scFv of the target module (TM). Consequently, UniCAR T cells can be cross-linked to tumor cells via the TM which results in antigen-specific tumor cell killing. We recently described a series of monospecific and bispecific TMs against TAAs including PSCA, PSMA, CD33, CD123, and EGFR (1-3). Here we summarize both in vitro and in experimental mice that all these TMs can efficiently redirect UniCAR T cells against tumor cells in a strictly target-dependent and target-specific manner. Killing occurred at pM TM concentrations. The killing efficacy of UniCAR T cells was comparable to conventional CAR T cells. Redirected UniCAR T cells released pro-inflammatory cytokines as measured by ELISA and/or flow cytometry-based multiplex assays including for example TNF, IL-2 and IFN-γ but not IL-6. In agreement with the UniCAR concept, TMs were released from UniCAR TM complexes in a concentration-dependent manner as measured by dynamic PET analysis. In summary, we demonstrate that the reactivity of UniCAR-armed T cells can be switched on and off in the presence or absence of a variety of TMs against a series of different TAAs and thus supporting its high flexibility. Moreover, UniCAR activity can be regulated in a dose-dependent manner and thus improve the safety of the CAR technology. 1_A. Feldmann, C. Arndt, R. Bergmann, S. Loff, M. Cartellieri, D. Bachmann, R. Aliperta, M. Hetzenecker, F. Ludwig, S. Albert, P. Ziller-Walter, A. Kegler, S. Koristka, S. Gärtner, M. Schmitz, A. Ehninger, G. Ehninger, J. Pietzsch, J. Steinbach and M. Bachmann. Retargeting of T lymphocytes to PSCA- or PSMA positive prostate cancer cells using the novel modular chimeric antigen receptor platform technology “UniCAR”. Oncotarget 2017, in press. 2_S. Albert, C. Arndt, A. Feldmann, R. Bergmann, D. Bachmann, S. Koristka, F. Ludwig, P. Ziller-Walter, A. Kegler, S. Gärtner, M. Schmitz, A. Ehninger, M. Cartellieri, G. Ehninger, H.-J. Pietzsch, J. Pietzsch, J. Steinbach and M. Bachmann. A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform. OncoImmunology 2017, in press. 3_M. Cartellieri, A. Feldmann, S. Koristka, C. Arndt, S. Loff, A. Ehninger, M. von Bonin, EP Bejestani, G Ehninger and MP Bachmann. Switching CAR T cells on and off: a novel modular platform for retargeting of T cells to AML blasts. Blood Cancer J. 2016 Aug 12;6(8):e458. doi: 10.1038/bcj.2016.61.

Aim: Advances in molecular engineering have led to the development of a multiplicity of antibody fragments with variations in molecular size. With respect to tumor targeting, the molecular size evidently determines the tumor uptake and pharmacokinetics. Consequently, they are proposed for different applications: small radiolabeled antibody fragments, such as single-chain variable fragments (scFv, 25-35 kDa) for tumor imaging and large full-size monoclonal antibodies (mAbs, 150 kDa) for radioimmunotherapy. Here, mAbs and thereof derived scFvs were produced that are directed against the prostate stem cell antigen (PSCA). Due to its overexpression on the surface of various tumor types, including prostate cancer and its metastases, it is proposed as a promising tumor target structure. Both antibody-based targeting molecules might provide a combinatory tool for theranostics of PSCA-positive prostate cancer. Methods: In this study, two different anti-PSCA mAb clones, RD1 and RD2, as well as their respective anti-PSCA scFvs were produced and compared with regard to their binding properties towards PSCA, using flow cytometry analysis. The anti-PSCA mAbs were conjugated with the chelating agent p-SCN-CHX-A’’-DTPA, measured by MALDI-TOF, and subsequently radiolabeled with lutetium-177, whereas the scFvs were radiolabeled with technetium-99m on their histidine-tag. Thereafter, all radiolabeled conjugates were characterized by thin-layer chromatography, and regarding binding properties on PC3-PSCA cells in vitro. Results: The non-radiolabeled anti-PSCA mAbs RD1 and RD2 showed a high affinity, with dissociation constants of 10 and 6 nM, respectively. The corresponding scFvs of RD1 and RD2 exhibit a lower affinity, with Kd-values of 170 and 98 nM. Both full mAbs were conjugated with about three CHX-A’’-DTPA. This conjugation had no influence on binding affinity towards the PSCA. Subsequent radiolabeling of the mAb-conjugates and scFvs could be performed with high radiochemical purity (> 95%) with preserving their binding properties to the PSCA. Conclusion: Full-size mAbs and scFvs that target the tumor antigen PSCA were successfully produced and radiolabeled. The in vitro characterization showed promising results to proceed with studies on tumor mouse models.

As the expression of a tumor associated antigen (TAA) is commonly not restricted to tumor cells adoptively transferred T cells modified to express a conventional chimeric antigen receptor (CAR) might not only destroy the tumor cells but also attack target-positive healthy tissues. Furthermore, CAR T cells in patients with large tumor bulks will unpredictably proliferate and put the patients at high risk of adverse side effects including cytokine storms and tumor lysis syndrome. To overcome these problems, we previously established a modular CAR technology termed UniCAR: UniCAR T cells can repeatedly be turned on and off via dosing of a target module (TM). TMs are bispecific molecules which cross-link UniCAR T cells with target cells. After elimination of the respective TM, UniCAR T cells automatically turn off. Here we describe novel TMs against the disialoganglioside GD2 which is overexpressed in neuroectodermal but also many other tumors. In the presence of GD2-specific TMs, we see a highly efficient target-specific and -dependent activation of UniCAR T cells, secretion of pro-inflammatory cytokines, and tumor cell lysis both in vitro and experimental mice. According to PET-imaging anti-GD2 TM enrich at the tumor site and are rapidly eliminated thus fulfilling all prerequisites of a UniCAR TM.

In general, adoptive transfer of chimeric antigen receptor (CAR)-expressing T cells has an impressive immunotherapeutic potential. However, due to the time-consuming establishment of new CAR constructs, the risk of life-threatening side effects, and the lack of control mechanisms once infused into patients, we developed a switchable modular CAR platform technology termed UniCAR. The UniCAR system is composed of two individual components, the universal signal-transducing UniCAR and an exchangeable target module (TM). In contrast to conventional CARs, the single-chain fragment variable (scFv) of the UniCAR binds to a small peptide epitope, which is physiologically not accessible on intact cells. The cross-linkage to tumor cells is mediated via TMs comprising the antigen-specifity and the epitope recognized by the UniCAR. Consequently, UniCAR-engrafted T cells are inert in the absence of redirecting TMs and only switched on in their presence. In addition to the increased safety, the modular structure enables a flexible targeting of different tissue antigens. New TMs can be easily constructed just by fusing the UniCAR epitope to a targeting entity. So far we produced a series of functional scFv-based TMs against different tumor-associated antigens like PSCA, PSMA, GD2 and CD33. Here we demonstrate that TMs can alternatively contain a nanobody (nb) domain instead of an scFv. Nbs are derived from camelid heavy-chain antibodies, consist of a single variable domain and form the smallest known antigen binding fragments. For redirection of UniCAR T cells to epithelial tumors the frequently overexpressed EGFR is a suitable target antigen. Thus, we generated a nb-based -EGFR TM. As shown by in vitro assays with EGFR+ tumor cell lines, the novel TM efficiently activates UniCAR T cells in a strictly target-specific manner and induces the release of pro-inflammatory cytokines. Furthermore, the data reveal that the -EGFR TM triggers a highly potent tumor lysis at low pM concentrations and redirects UniCAR-engrafted T cells to tumor cells in immunodeficient mice. Using dynamic PET analysis we observed a short half-life of the TM and could confirm its release from UniCAR-TM-complexes. Thereby, it is possible to precisely dose the TM concentration and to rapidly switch the system off in case of adverse side effects. Finally, the -EGFR TM also offers the possibility to image the tumor during therapy. For an increased anti-tumor response we additionally generated a bivalent -EGFR-EGFR TM that shows improved in vitro and in vivo functionality compared to the monovalent construct. In summary, we established a novel mono- and bivalent nb-based TM for EGFR-specific recruitment of UniCAR T cells which results in an efficient, target-specific and -dependent killing of EGFR+ tumor cells. Thus, we could prove that instead of scFvs also other binding moieties can be used and confirmed the high flexibility of the modular UniCAR platform.

New treatment options especially of solid tumors including for metastasized prostate cancer (PCa) are urgently needed. Recent treatments of leukemias with chimeric antigen receptors (CARs) underline their impressive therapeutic potential. However CARs currently applied in the clinics cannot be repeatedly turned on and off potentially leading to severe life threatening side effects. To overcome these problems, we recently described a modular CAR technology termed UniCAR: UniCAR T cells are inert but can be turned on by application of one or multiple target modules (TMs). Here we present preclinical data summarizing the retargeting of UniCAR T cells to PCa cells using TMs directed to prostate stem cell-(PSCA) or/and prostate membrane antigen (PSMA). In the presence of the respective TM(s), we see a highly efficient target-specific and target-dependent activation of UniCAR T cells, secretion of proinflammatory cytokines, and PCa cell lysis both in vitro and experimental mice.

Objective: This study evaluates the relevance of transfusion guidelines for clinical transfusion practice. Background: There is little data available on current practice related to appropriate use of blood products. Recent data suggest incorrect use and over transfusion in Europe. In Germany, indications for transfusion and administration of blood products are strict and detailed. However, in clinical practice, alignment of transfusion guidelines to clinical situations seems to be difficult for physiciansespecially in complex diseases and severity such as in critical care. We hypothesized that significant practice variability exists with regard to guidelines adherence in critically ill. Materials and methods: Data sets of transfused patients from a surgical intensive care unit of a university center retrospectively were analyzed over a 12 month period. Indications for blood products were compared with guidelines focusing on numbers of ordered and administered units as well as transfusion triggers. Results: In total, during the study period, 450 packed red cells (PRC, 249 orders), 454 fresh frozen plasma (FFP) (201) and 43 platelet units (PC) (29) were given to 89 patients. The mean number of administered PRC was 5.8+/-6.6 (mean +/- SD) units. Double units were administered in 57.4%. Discordant to actual guidelines, 75 (30.12%) PRC`s, 79 (39.30%) FFP's and 9 (31.03%) of PC orders were given without indication. Fresh frozen plasma was administered in ineffective dosage in 83.6% (mean 7.3 ml/kg body weight), platelet transfusion despite intact platelet function in 30.2% and beyond platelet counts of 100 000/μl in 60%. Conclusion: Detailed guidelines for transfusion of blood products were not followed in daily practice in a third of all applications. The reasons for malcompliance are key for improvement.

Chimeric antigen receptor (CAR)-modified T cells are intensively studied for their application in cancer patients and already proved incredible success in clinical trials. However, the choice of the intracellular signaling domain integrated into the CAR architecture can largely influence T cell function and fate, as already shown in vitro and in vivo. Moreover, especially within solid tumors regulatory T cells (Tregs) play an important role in establishing an anti-inflammatory milieu and suppressing effector cells. Consequently, endogenous Tregs might impair CAR-engrafted T cells and thereby affect treatment outcome of cancer patients. Therefore, it is of large interest to investigate the responsiveness of T cells comprising CARs with different intracellular signaling domains on Treg suppression. To address this question, we isolated CD4+CD25- conventional T cells (Tconv) and genetically modified them to express a universal CAR (UniCAR) construct as part of our previously developed UniCAR platform technology. In contrast to conventional CARs, UniCARs are indirectly linked to their target cells via a separate antigen-specificity providing target module (TM), which allows a flexible application of UniCAR-engrafted T cells against a wide range of tumor-associated antigens. It also enables a modulation of T cell activity between an “on” and “off” status. To compare the influence of different intracellular costimulatory signals, we designed UniCARs containing either a CD3ζ, CD28-CD3ζ or CD137-CD3ζ domain. By using a lentiviral gene transfer system for genetic modification, transduction rates of more than 80 % were achieved. Upon TM-mediated activation via the UniCAR, Tconvs containing UniCAR28/ζ produced significantly higher amounts of the pro-inflammatory cytokine TNF and the growth-related cytokine IL 2 than UniCAR137/ζ- or UniCARζ-engrafted cells. To investigate the impact of Tregs, Tconvs containing the individual UniCAR constructs were cultured in the presence of expanded, autologous CD4+CD25+CD127lowCD45RA+ Tregs for 96h. On the one hand, Tregs were pre-stimulated with anti-CD3/CD28 beads to mimic polyclonal activation via the endogenous TCR. On the other hand, an antigen-specific stimulation was achieved by engrafting Tregs with UniCARs. In both cases, UniCAR-armed Tconvs showed a distinct responsiveness on Treg suppression in dependence on the intracellular signaling domain. We observed, that in contrast to UniCAR28/ζ-armed Tconvs, UniCAR137/ζ- and UniCARζ-engrafted cells could be substantially repressed by Tregs. In summary, we could demonstrate that Tconvs containing UniCARs with different intracellular signaling domains display not only a distinct cytokine secretion profile but also a disparate resistance against Treg suppression. These data indicate, that the chosen costimulatory signal has an impact on both the efficacy and the safety of a cancer treatment conducted with genetically modified CAR T cells.

Ziel: Das Prostata-Stammzellantigen (PSCA) wird auf der Zelloberfläche von über 80% der Prostatatumore (PCa) und deren Knochenmetastasen exprimiert. Aus diesem Grund wird es häufig als Zielstruktur sowohl für die Radioimmuntherapie (RIT) als auch für molekulare Bildgebungstechniken von PCa in der Nuklearmedizin (Positronen-Emissions-Tomographie (PET) und Einzelphotonen-Emissionscomputertomographie (SPECT)) verwendet. Für die jeweiligen Anwendungsgebiete wurden Radioimmunkonjugate basierend auf monoklonalen anti-PSCA-Antikörpern (mAk, 150 kDa) und von diesen abgeleitete Einzelkettenantikörperfragmente („single-chain Fragment variable“ (scFv), 35 kDa) hergestellt. In Kombination sollen diese Radioimmunkonjugaten als therapeutisches und diagnostisches Instrument für PSCA-positive PCa Anwendung finden. Methoden und Ergebnisse: Zwei unterschiedliche anti-PSCA mAk-Klone, RD1 und RD2 genannt, sowie davon abgeleitete scFvs wurden hergestellt, über Affinitätschromatographie gereinigt und deren Bindungseigenschaften an PSCA-positiven PC3-Zellen mittels Durchflusszytometrie ermittelt. Die unmarkierten mAk-Klone RD1 und RD2 zeigten hohe Affinitäten, mit Dissoziationskonstanten von 10 und 6 nM. Für die scFvs von RD1 und RD2 wurden geringere Affinitäten von 170 und 98 nM bestimmt. Die beiden mAk-Klone wurden anschließend mit dem bifunktionellen Chelator p-SCN-Bn-CHX-A’’-DTPA, die scFv-Antikörper hingegen mit p-SCN-Bn-NOTA konjugiert. Für alle Konstrukte wurde mittels MALDI-TOF-Massenspektrometrie eine durchschnittliche Anzahl von drei Chelator-Einheiten je Antikörpermolekül gemessen. Anschließend wurden die mAk-Konjugate für eine mögliche RIT-Anwendung mit Lutetium-177 und die scFv-Konjugate für eine mögliche PET-Bildgebung mit Kupfer-64 radiomarkiert. Des Weiteren wurden scFv-Antikörper auch direkt mit Technetium-99m mit Hilfe eines Tricarbonyl-Präkursor an ihrem Hexahistidin-Tag für eine mögliche SPECT-Bildgebung markiert. Für alle radiomarkierten Konjugate wurde eine radiochemische Reinheit von über 95% (radio-Dünnschichtchromatographie) erzielt. Nachfolgende In-vitro-Studien an PC3-PSCA-Zellen zeigten, dass die Bindungseigenschaften zum PSCA erhalten bleiben. Ausblick: An entsprechenden Tumormaus-Modellen wird gegenwärtig geprüft, ob die positiven in-vitro Resultate für die hergestellten Radioimmunkonjugate bestätigt werden können.