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Background: Initial treatment (ventilator settings, rescue therapy, supportive measures), and prevention of critical events improve survival in ARDS patients, but little data exists on its effect on health-related quality of life (HRQOL) and return to work (RtW) in survivors. We analyzed the association of the intensity of treatment at ARDS onset and the incidence of critical events on HRQOL and RtW a year after ICU discharge. Methods: In a prospective multi-centre cohort study, the intensity of treatment and the incidence of critical events were determined at 61 ICUs in Germany. At 3, 6, and 12 months, 396 survivors reported their HRQOL (Short-Form 12) and RtW. The parameters of the intensity of acute management (lung protective ventilation, prone position, hemodynamic stabilization, neuromuscular blocking agents), and critical events (hypoxemia, hypoglycemia, hypotension) were associated with HRQOL and RtW. Results: Patients ventilated at ARDS onset with a low tidal volume (VT≤7 ml/kg) had higher arterial carbon dioxide levels (PaCO2=57.5±17 mmHg) compared to patients ventilated with VT>7ml/kg (45.7±12, p=0.001). In a multivariate adjusted dichotomized analysis, a better mental 3-month SF-12 was observed in the higher VT-group (mean 43.1±12) compared to the lower VT-group (39.5±9, p=0.042), while a dichotomized analysis for driving pressures (≤14 mbar vs >14 mbar) did not show any difference neither in PaCO2 levels nor in HRQOL parameters. A decrease in the mental (6-month: 40.0±11 vs 44.8±13, p=0.038) and physical SF-12 (12-month: 38.3±11 vs 43.0±13, p=0.015) was reported from patients with hypoglycemia (blood glucose, Hintergrund: Die Qualität der aktuellen Behandlungsstrategie (Beatmungseinstellung, rescue-Maßnahmen, unterstützende Intensivtherapie) sowie die Vermeidung kritischer Ereignisse verbessern das Überleben von Patienten mit schwerem akuten Lungenversagen (ARDS). Es ist bisher wenig bekannt darüber, ob eine solche Behandlungsqualität auch einen Einfluss auf die gesundheitsbezogene Lebensqualität (health-related quality of life, HRQOL) und die Rückkehr in das Arbeitsleben (return to work, RtW) der Überlebenden hat. Wir untersuchten eine mögliche Beziehung zwischen akuter Behandlungsqualität, HRQOL und RtW ein Jahr nach Überleben eines ARDS. Methoden: Die prospektiv-multizentrische Studie wurde in 61 Intensivstationen (ICUs) in Deutschland durchgeführt. Drei, sechs und zwölf Monate nach Verlegung von der ICU gaben 396 Überlebende über ihre HRQOL (Short-Form-12) und RtW Auskunft. Die Parameter der akuten Behandlungsqualität (lungenprotektive Beatmungseinstellung, Gradient zwischen endexpiratorischem und inspiratorischem Beatmungsdruck (driving pressure), Einsatz von Bauchlagerung und/oder Muskelrelaxantien, hämodynamische Stabilisierung) und die Häufigkeit kritischer Ereignisse (Hypoxämie, Hypoglykämie, Hypotension) wurden mit HRQOL und RtW assoziiert. Ergebnisse: Patienten, die zu Beginn des ARDS mit einem niedrigen Tidalvolumen (VT≤7 ml/kg) beatmet wurden, wiesen höhere arterielle Karbondioxid-Werte auf (PaCO2=57.5±17 mmHg) im Vergleich zu Patienten mit höherem VT>7 ml/kg (PaCO2 45.7±12, p=0.001). In einer multivariaten adjustierten dichotomisierten Analyse wurde eine bessere 3-Monats-Lebensqualität in der mentalen Domäne des SF-12 in der Gruppe mit einem höheren VT gefunden (Mittelwert 43.1±12) im Vergleich zu den mit niedrigerem VT beatmeten Patienten (39.5±9, p=0.042); für eine dichotomisierte Analyse des driving pressure (≤14 mbar vs >14 mbar) wurde weder für PaCO2 noch für HRQOL ein Unterschied gefunden. Eine Einschränkung der mentalen (6 Monate: 40.0±11 vs 44.8±13, p=0.038) und körperlichen SF-12 Domäne (12 Monate: 38.3±11 vs 43.0±13, p=0.015) wurde für Patienten berichtet, die während der akuten Behandlung eine Hypoglykämie (Blutglukose-Wert, GMS German Medical Science; 18:Doc01

Digital, Recently, we established the controllable modular UniCAR platform technology to advance the efficacy and safety of CAR T cell therapy. The UniCAR system is composed of (i) target modules (TMs) and (ii) UniCAR armed T cells. TMs are bispecific molecules that are able to bind to the tumor cell surface and simultaneously to UniCAR T cells. For interaction with UniCAR T cells, TMs contain a peptide epitope sequence which is recognised by UniCAR T cells. So far, a series of TMs against a variety of tumor targets including against the prostate stem cell antigen (PSCA) were constructed and functionally characterised. In order to facilitate their purification all these TMs are expressed as recombinant proteins equipped with an oligo-His-tag. The aim of the here presented manuscript was to learn whether or not the oligo-His-tag of the TM influences the UniCAR system. For this purpose, we constructed TMs against PSCA equipped with or lacking an oligo-His-tag. Both TMs were compared side by side including for functionality and biodistribution. According to our data, an oligo-His-tag of a UniCAR TM has only little if any effect on its binding affinity, in vitro and in vivo killing capability and in vivo biodistribution., 1 ed.

This dissertation draws on concepts from political science, sociology, media studies, organisational studies, and psychoanalysis to describe and explain the manifestations and process of politicisation of the South African Broadcasting Corporation (SABC) after the end of apartheid (1993-2013) through the lens of organisational culture. It is based on ethnographic fieldwork conducted between 2005 and 2011, including six months participant observation in seven SABC newsrooms and 117 in-depth interviews with journalists and managers. The study links an intra-organisational analysis of journalistic practice with the macro-level of the political field and the micro-level of individual actors. It explores in detail the unconscious processes inscribed in the SABC's culture and subcultures, as well as their interplay, before approaching the understudied problem of agency in newsrooms, in particular agency-as-resistance to politicisation. The thesis makes three main contributions to the literature: (1) The most in-depth empirical account of the SABC's politicisation to date, and an example of an African public broadcaster in a post-authoritarian environment. Findings challenge conventional explanations for the failures of public broadcasting transformations by showing that politicisation is far more than a top-down phenomenon, and that organisational culture shapes susceptibility to it profoundly. (2) A systematic and chronological analysis of drivers, enablers and inhibitors of politicisation, highlighting the multi-causality and multi-directionality of the process. This contests the over-reliance on structural explanations in political science by emphasising culture and subjectivity, e.g. by introducing the concept of 'anticipatory politicisation'. (3) A conceptual framework that integrates group relations theories with Bionian concepts and Lacanian discourse theory to shed light on individual agency in the context of collective unconscious processes. Findings are of interest to scholars working on the SABC or media in transition, on politicised organisations, organisational culture, or agency.

Although CAR T cell therapy has demonstrated tremendous clinical efficacy especially in hematological malignancies, severe treatment-associated toxicities still compromise the widespread application of this innovative technology. Therefore, developing novel approaches to abrogate CAR T cell-mediated side effects is of great relevance. Several promising strategies pursue the selective antibody-based depletion of adoptively transferred T cells via elimination markers. However, given the limited half-life and tissue penetration, dependence on the patients’ immune system and on-target/off-side effects of proposed monoclonal antibodies, we sought to exploit αCAR-engineered T cells to efficiently eliminate CAR T cells. For comprehensive and specific recognition, a small peptide epitope (E-tag) was incorporated into the extracellular spacer region of CAR constructs. We provide first proof-of-concept for targeting this epitope by αE-tag CAR T cells, allowing an effective killing of autologous E-tagged CAR T cells both in vitro and in vivo whilst sparing cells lacking the E-tag. In addition to CAR T cell cytotoxicity, the αE-tag-specific T cells can be empowered with cancer fighting ability in case of relapse, hence, have versatile utility. Our proposed methodology can most probably be implemented in CAR T cell therapies regardless of the targeted tumor antigen aiding in improving overall safety and survival control of highly potent gene-modified cells.

T-cells armed with conventional CARs (cCARs) are highly effective especially in hematological malignancies. However, they often fail against solid tumors, induce tumor escape variants and cause life-threatening side effects. The safety of cCAR therapy should be improved by on/off-switchable CARs. Additionally, gated targeting strategies could increase the CAR specificity, minimize on-target/off-tumor toxicities and reduce tumor escape variants. For AND gate targeting, the signaling and costimulatory motifs are split from one onto two separate CARs of different specificities. Dual-CAR-T-cells, expressing both CARs, get activated only after recognizing both antigens. However, the application of such pairs of cCARs is very challenging as the affinity and signal strength of both CARs have to be adapted accordingly. Furthermore, the cCAR size limits the number of specificities that can be simultaneously transduced into a T-cell. Therefore, our idea was to replace the extracellular scFv domain of cCARs with a small peptide epitope. Resulting RevCARs have a small size, avoid unspecific binding and tonic signaling caused by scFv dimerization. RevCAR-T-cells can be redirected against tumors only via bispecific target modules. Such RevTMs can be used as on/off-switch of RevCAR-T-cells and flexibly replaced for targeting of any antigens. For proof of concept, two RevCARs with different peptide epitopes and a series of respective RevTMs were constructed and functionally proven for targeting of leukemic as well as solid cancers. Moreover, for gated targeting, two RevCARs were expressed in the same T-cell that differ in their extracellular peptide epitope, transmembrane and intracellular signaling domains to separately transmit isolated activation and costimulatory signals. For efficient activation of Dual-RevCAR-T-cells, both RevCARs must be engaged by respective RevTMs recognizing different epitopes and antigens. In summary, we established a novel switchable, modular and adaptable RevCAR system characterized by small size, improved safety, easy controllability, and high flexibility allowing gated targeting strategies.

Background Induction chemotherapy is currently the standard of care for treatment of acute myeloid leukemia (AML) with 5-year disease-free survival of 33%. Given the large proportion of non-responders and relapsed patients, novel adjuvant drugs are urgently needed. Especially, targeted therapies including small molecules and T cell based immunotherapies are under intensive preclinical and clinical investigation. The tyrosine kinase inhibitor Midostaurin recently received approval for treatment of FLT3-positive AML. In addition to chemotherapy, it significantly deepens remission rates and improves overall survival of patients. In light of future combinatorial approaches, simultaneous application of different targeted therapies should theoretically augment anti-tumor effects. Aims Therefore, we questioned whether Midostaurin could strengthen cytotoxic effector mechanisms of redirected switchable UniCAR T cells or bispecific antibody-redirected T cells against primary AML cells. Methods/Results By performing in vitro co-cultivation assays with patient-derived AML cells, it was shown that Midostaurin concentrations ≥ 1 µM significantly impair the activation, proliferation, cytokine production and cytotoxicity of autologous and allogeneic T cells after engagement via bsAb or the UniCAR system. Data could be also verified in a solid tumor model. Summary/Conclusion At concentrations ranging between 0.1 and 10 M, it was shown that Midostaurin and its metabolites are indeed able to inhibit several components of the TCR signaling pathway including LcK, Zeta-chain-associated protein kinase 70 (ZAP-70), mitogen-activated protein kinase (MAPK) and Protein kinase C (PKC) in vitro. Therefore, we argue that the observed T cell inhibition by Midostaurin in our studies is caused by the inhibition of several of these kinases. This hypothesis is supported by the work of two individual research groups that were able to show synergistic effects by combining FLT3 selective TKIs with different T cell-based immunotherapies. Because Midostaurin through concentrations above ≥ 1 µM have been observed in earlier performed dose finding studies, we speculate that current standard Midostaurin therapy will inhibit T cell function in vivo. In summary, our data underline that combination of Midostaurin and T cell-based immunotherapies in FLT3-positive AML patients is not recommended due to the suppressive effect of Midostaurin on T cells. Therefore, more selective TKI or other small molecules should be chosen to avoid impairment of T cell functions.

The common acute lymphoblastic leukemia antigen CD10 is a marker for several hematological malignancies, including acute lymphoblastic leukemia as well as T and B cell lymphomas, Burkitt lymphomas, and some solid tumors like renal cell carcinomas, pancreatic tumors and melanomas. Because of its tumor related expression pattern, CD10 is an attractive target for adoptively transferred T cells that are genetically modified to express chimeric antigen receptors (CARs). Recently, conventional CAR T cell therapy targeting CD19-positive hematological malignancies was clinically approved because of its impressive effectiveness in patients. However, CAR T cells can also cause severe side effects like on-target, off-tumor reactions, tumor lysis syndrome and cytokine release syndrome. Most critically, activity of conventional CAR T cells cannot be controlled, once they are applied in patients. As CD10 is also widely expressed on normal tissues, CAR T cell reactivity has to be controllable in order to stop CAR T cell therapy in case of on-target, off-tumor toxicities occur. Especially for this purpose, we have recently established a switchable, modular and universal CAR platform technology, named UniCAR system, which can be repeatedly turned on and off. In contrast to conventional CARs, that directly recognize a tumor-associated antigen (TAA) on the tumor cell surface via their extracellular single-chain variable fragment (scFv), the UniCAR system is structured in a modular manner of two components. The first component are T cells genetically engineered to express UniCARs and the second component are target modules (TMs). Most importantly, UniCARs cannot directly bind to a TAA because their extracellular scFv is directed against the peptide epitope E5B9 which is not present on the surface of living cells. Consequently, UniCAR armed T cells are per se inert. They can be redirected towards tumor cells only via a TM. TMs consist of a scFv targeting a TAA and the epitope E5B9 recognized by UniCARs allowing a cross-linkage of UniCAR T cells with tumor cells which results in T cell activation. As TMs have a very short half-life, UniCAR T cell activity can be controlled by dosing of the TM. Once the TM is administered, UniCAR T cells can be switched on, but once the TM injection is stopped and the TM is eliminated, UniCAR T cells are switched off immediately. Here, we show proof of concept for functionality of the UniCAR system targeting CD10-positive malignancies. Therefor, a novel anti-CD10 TM was constructed which is able to redirect UniCAR T cells to eliminate CD10-expressing tumor cells. In summary, we have established a universal, switchable, modular UniCAR platform technology that can be used to target CD10-positive malignancies.

Since epithelial growth factor receptor (EGFR) mutations or overexpression is linked with variety of malignancies, including lung, breast, stomach, colorectal, head and neck, and pancreatic carcinomas as well as glioblastomas it is an attractive target for tailored treatment of solid cancer. Thus over the last twenty years many strategies targeting EGFR were developed and even clinically approved, including disrupting intracellular signalling involving tyrosine kinase inhibitors (TKIs) or the inhibition of ligand binding using therapeutic monoclonal antibodies like e.g. Cetuximab, Panitumumab or Necitumumab. Unfortunately, cancers treated with these targeted drugs commonly become resistant to them. These limitations justify the need of more efficient therapy options. As chimeric antigen receptor (CAR) engineered T cells highly effectively eliminate hematological malignancies already in the clinics, one idea is to redirect CAR T cells also against EGFR expressing solid cancers. However, CAR T cell therapy can lead to severe even life-threatening side effects and its effectiveness against solid tumors is still limited. Particular worrying is that EGFR is a widespread antigen commonly expressed also on healthy tissues bearing a high risk of severe on-target/off-tumor side effects due to EGFR-targeted therapies, which cannot be controlled in patients. In order to overcome these challenges our UniCAR technology might be an appropriate answer combining high anti-tumor effectiveness, tumor specificity, flexibility, and safety control mechanisms. In contrast to conventional CARs, UniCAR T cells are per se inert because UniCARs are directed against a small peptide epitope, which is not present on living cells. The redirection of UniCAR T cells to tumor cells occurs only in the presence of a tumor specific targeting module (TM). TMs, on one hand carry the specificity for a certain tumor antigen and on the other hand contain the UniCAR peptide epitope recognized by UniCARs mediating the cross-linkage of UniCAR T cells and antigen presenting tumor cells. As TMs have a very short half-life in vivo they can be used as a switch to control UniCAR T cell activity on demand in patients. In detail, UniCAR T cells are only switched on in the presence of antigen specific TMs realized by permanent TM infusion, but could be rapidly switched off when the application of the TM is stopped and the TM is eliminated. Meanwhile we successfully generated a series of different TMs against different tumor antigens and entities. Interestingly, TMs can be made of different molecules showing various structures and can flexible be exchanged in order to target any tumor antigen and overcome tumor escape variants. Commonly our TMs consist of a humanized single-chain variable fragment (scFv) derived from the variable heavy and light chain domains of a murine monoclonal antibody. In addition, we successfully generated TMs based on different monovalent and bivalent antibody derivatives, nanobodies derived from one variable camelid antibody domain, affibodies and even small peptide molecules. Recently we demonstrated proof-of-concept for the redirection of UniCAR T cells to EGFR expressing tumor cells by a nanobody based αEGFR TM derived from the camelid αEGFR antibody 7C12. Considering that the affinity and anti-tumor efficiency of the eucaryotically expressed αEGFR nanobody based TM was limited, we therefore asked the question, whether we could further improve the therapeutic effect against EGFR positive tumor cells using the UniCAR technology. In order to answer this question, we generated a novel TM based on a scFv derived from the clinically used chimeric monoclonal antibody Cetuximab (IMC C-225). In detail, we designed a murine and humanized αEGFR scFv TM, successfully expressed them in mammalian cell lines and compared their functionality with the eucaryotic αEGFR nanobody-based TM in vitro and in vivo. In principle, we observed that both TM formats, the αEGFR nanobody as well as the scFv-based TM, are able to redirect UniCAR T cells eliminate EGFR-expressing tumor cells in an antigen-specific and TM-dependent manner. As both the murine and humanized scFv TM variants worked equally well, obviously humanization of the αEGFR scFv does not affect its functionality. However most interestingly, the tumor killing efficiency of the αEGFR scFv TM was significantly superior in comparison to the αEGFR nanobody based TM. Here, the half maximal effective TM concentration (EC50) value of scFv based TM was improved 1000-fold, from nM to pM range. Consequently, UniCAR T cells in combination with the scFv based TM efficiently eliminate also target cells expressing a low EGFR density level, while UniCAR T cells redirected by the nb based TM clearly attack only highly EGFR expressing tumor cells. Furthermore, the high anti-tumor efficacy of the αEGFR scFv TM over nb TM was manifested in experimental mice. In summary, we successfully established different αEGFR TM formats that are able to redirect UniCAR T cells to eliminate EGFR-positive tumor cells. However, the analysed αEGFR TM formats differ with respect to their anti-tumor efficiency, which might decide whether UniCAR T cells attack target cells showing different EGFR density levels.

Chimeric antigen receptors (CARs) are highly efficient tools for T‐cell‐based cancer immunotherapy. Nonetheless, this approach is associated with mild to severe toxicities including cytokine release syndrome and on‐target/off‐tumor effects. To increase clinical safety while maintaining the efficacy of CAR T‐cell therapy, we developed a novel modular universal CAR (UniCAR) platform. UniCAR T‐cells are exclusively activated via a target module (TM) that establishes the cross‐link between UniCAR T‐cells and cancer cells. Given the small size of such molecules, they are rapidly eliminated and thus, have to be administrated via continuous infusion. Consequently, activation and possible side effects of UniCAR T‐cells can be easily controlled by TM dosing. Regulation of CAR T‐cell activity is mainly important during onset of therapy when tumor burden and the risk for severe side effects are high. Therefore, TMs with extended half‐life may improve eradication of residual tumor cells in late phase of treatment and further expedite clinical application. In this line of thought, we developed both short‐lived and longer lasting TMs directed against several tumor‐associated antigens. Short‐lived TMs are composed of a tumor‐specific binding moiety and the E5B9 peptide epitope which is recognized by UniCAR T‐cells. In order to generate extended half‐life TMs, these two components are fused to the human IgG4 Fc domain. Both short‐lived and longer lasting TMs efficiently redirect UniCAR T‐cells to cancer cells in a highly target‐specific manner, thereby promoting the secretion of pro‐inflammatory cytokines and tumor cell lysis in vitro and in vivo. As demonstrated by PET‐imaging, all TMs specifically enriched at the tumor site presenting either short or prolonged serum half‐lives. Taken together, combination of different short‐lived and extended half‐life TMs provides a highly promising and customized tool for retargeting of UniCAR T‐cells in a flexible, individualized and safe manner at different phases of tumor therapy.

Although chimeric antigen receptor (CAR) T cells have demonstrated outstanding therapeutic efficacy, current design of clinically approved CAR constructs renders therapies monospecific, inflexible and imperiled to severe side effects. We therefore refined the CAR approach and established a switchable platform technology termed UniCAR. Instead of directly interacting with tumor cells, UniCAR‐engrafted T cells recognize the short peptide epitope E5B9. Their on/off switch is mediated by E5B9‐comprising targeting modules (TMs), facilitating indirect cross‐linkage to antigen‐expressing cancer cells. As all TMs described to date contain an antibody‐derived binding moiety, we aimed to develop a ligand‐based low‐molecular weight agent that enables not only UniCAR T cell retargeting, but also monitoring of the therapeutic response by non‐invasive PET imaging. For proof of concept of this novel theranostic approach, we successfully converted the clinically approved radiotracer PSMA‐11 into a TM by fusion with E5B9 without affecting its diagnostic properties in mice and humans. Moreover, the chemically synthesized, ligand‐based compound efficiently mediated PSMA‐expressing tumor cell lysis by UniCAR T cells both in vitro and in vivo. Taken together, the PSMA‐ligand TM represents a novel theranostic agent that is an attractive candidate for immunotherapy of prostate cancer as well as for initial diagnosis and follow‐up treatment.

With the first approvals of chimeric antigen receptor (CAR) T cell therapies by the FDA the use of genetically modified T cells in the immunotherapy of tumors has recently become a very promising approach. CAR T cells are able to recognize tumor-associated antigens (TAAs) via specific single-chain variable fragments (scFvs) in a major histocompatibility-complex (MHC)-independent manner. Although highly efficient, the inability to regulate the activity of CAR T cells can cause severe even life-threatening side effects such as cytokine-release syndrome (CRS) and on-target, off-tumor toxicities. Modular CAR systems may overcome these limitations allowing to switch the activity of CAR T cells repeatedly “ON” and “OFF”. Alternatively or in addition, the safety of CAR T cells could also be improved by “gated” targeting strategies e.g. by splitting the signaling and costimulatory motifs to independent CARs of different specificities. Theoretically, the idea of gated targeting could be extended to include further e.g. inhibitory signals. However, the size of current CARs limit the number of specificities that can be simultaneously transduced into a T cell. We therefore developed a novel switchable modular universal artificial receptor having a minimal size. The platform was termed RevCAR system. In order to reduce the size of the artificial receptor the original idea was to replace the extracellular scFv domain of a conventional CAR with a small peptide epitope and to engage the resulting RevCAR T cell via a bispecific target module which we termed RevTM. For proof of concept two small peptide epitopes were selected and the respective RevCARs constructed. In addition, a series of different RevTMs were constructed. On the one hand the RevTM recognized one of the two peptide epitopes on the other hand the RevTM was directed to a potential tumor associated antigen (TAA). Until now a series of such pairs of RevTMs were constructed and functionally analyzed. RevCAR T cells armed via the respective RevTM were able to specifically lyse their respective target cell in a peptide epitope specific and target specific as well as target dependent manner. These data are supported by analysis of cytokine secretion. We only observed a specific cytokine release from RevCAR T cells in the presence of both target cells and the respective RevTM. Released cytokines detected were IFN-gamma, GM-CSF, TNF, and IL-2. Taken together these results demonstrate the high anti-tumor efficiency of the novel RevCAR platform which is characterized by a small size, an improved safety, easy controllability as well as high flexibility.

Long-term survival of adoptively transferred chimeric antigen receptor (CAR) T cells is often limited. Transplantation of hematopoietic stem cells (HSCs) transduced to express CARs could help to overcome this problem as CAR-armed HSCs can continuously deliver CAR+ multi-cell lineages (e.g. T cells, NK cells). In dependence on the CAR construct a variable extent of tonic signaling in CAR T cells was reported, thus, effects of CAR-mediated tonic signaling on the hematopoiesis of CAR-armed HSCs is unclear. To assess effects of tonic signaling two CAR constructs were established and analyzed: (i) A signaling CAR inducing a solid antigen-independent tonic signaling termed CAR-28/, and (ii) a non-stimulating control CAR construct lacking intracellular signaling domains termed CAR-Stop. Bone marrow (BM) cells from immunocompetent mice were isolated, purified for HSC-containing Lin-cKit+ (LK) cells or the LK Sca-1+ subpopulation (Lin Sca 1+cKit+, LSK) and transduced with both CAR constructs. Subsequently, modified BM cells were transferred into irradiated mice where they successfully engrafted and differentiated into hematopoietic progenitors. HSCs expressing the CAR-Stop sustained normal hematopoiesis. In contrast, expression of the CAR-28/ led to elimination of mature CAR+ T and B cells suggesting that the CAR-mediated tonic signaling mimics autorecognition via the newly recombined immune receptors in the developing lymphocytes.

More than 100 years ago Paul Ehrlich postulated that our immun system should be able to eliminate tumor cells. Just recently, the development of check point inhibitors, bispecific antibodies, and T cells genetically modified to express chimeric antigen receptors (CARs) underlines the true power of our immune system. T cells genetically modified with CARs can lead to complete remission of malignant hematologic diseases. However, they can also cause life-threatening side effects. In case of cytokine release syndrome, tumor lysis syndrome, or deadly side effects on the central nervous system, an emergency shut down of CAR T cells is needed. Targeting of tumor-associated antigens that are also expressed on vital tissues require a possibility to repeatedly switch the activity of CAR T cells on and off on demand and to follow the treatment by imaging. Theranostic, modular CARs such as the UniCAR system may help to overcome these problems.

Hematological malignancies are successfully treated with chimeric antigen receptor (CAR) armed T cells. Despite the clinical success, CAR T cell therapy struggles still with some problems including the selection of tumor escape variants and on-target, off-tumor side reactions as well as massive cytokine release and uncontrollability of CAR T cell activity in the patients. In order to enable controllability of CAR T cells and to avoid unspecific side effects, we established a novel switchable, split and adaptable CAR platform technology, termed RevCAR system. The novel RevCARs lack the single chain variable fragment (scFv) commonly used as extracellular domain in conventional CARs. Instead of the scFv, RevCARs contain only a small peptide epitope as extracellular portion. This design reduces the CAR size, avoids unspecific antigen binding and prevents antigen independent tonic signaling caused by scFv dimerization. As RevCAR T cells do not recognize anything, they are per se inert. Only in the presence of a corresponding bispecific antibody based target module (RevTM) they can be specifically redirected to tumor cells. Therefor RevTMs consist of two scFvs. One recognizes the RevCAR peptide epitope and the other one simultaneously binds to a tumor associated antigen (TAA). By dosing of the RevTM, which has a very short half-life, the reactivity of RevCAR T cells can be switched on and off reversibly. Another advantage is that the RevCAR system can be flexibly adapted to any tumor antigen simply by exchanging the RevTM. Furthermore, the small RevCAR size is favorable for inserting more than one RevCAR in the same T cell thus facilitating the mode of gated targeting which is a highly attractive approach to minimize the risk for on-target, off-tumor toxicities against healthy tissues and to increase tumor specificity of conventional CAR T cells. For 'AND' gate targeting via the RevCAR system, two different RevCARs were constructed and expressed simultaneously in the same T cell. The two RevCARs differed with respect to the extracellular peptide epitope and the intracellular signaling domain. Moreover, the respective transmembrane domain was selected to isolate the respective RevCAR signal. The first RevCAR is designed to transmit the activation signal, the second RevCAR to deliver a costimulatory signal. For efficient RevCAR T cell activation, both RevCARs must be engaged via their respective RevTM which on the one hand binds to one of the two RevCAR epitopes and on the other hand to one of two TAAs expressed on the same target cell. Here, we present two RevCAR/RevTM systems for retargeting of AML cells as well as solid tumor cells including via gated targeting. In summary, we show proof of concept for a novel switchable RevCAR system that can be used for retargeting of AML cells as well as solid tumors. The novel modular RevCAR platform is characterized by small size, lacks unwanted tonic signaling effects, allows the control of RevCAR T cell activity, enables gated targeting strategies, and can be adapted to any tumor antigen and tumor type.

Since epithelial growth factor receptor (EGFR) mutations or overexpression is linked with variety of malignancies, including lung, breast, stomach, colorectal, head and neck carcinomas as well as glioblastomas, it is an attractive target for tailored treatment. As chimeric antigen receptor (CAR) engineered T cells highly effectively eliminate hematological malignancies already in the clinics, one idea is to redirect CAR T cells also against EGFR expressing solid cancers. However, CAR T cell therapy can lead to severe, even life‐threatening side effects with high risk of on‐target/off‐tumor activity. To overcome these challenges our UniCAR technology might be an appropriate answer combining high anti‐tumor effectiveness, tumor specificity, flexibility, and safety control mechanisms. In contrast to conventional CARs, UniCAR T cells are per se inert because UniCARs are directed against a small peptide epitope, which is not present on living cells. The redirection of UniCAR T cells toward target cells occurs only in the presence of a tumor specific targeting molecule (TM) – bifunctional moieties carrying the specificity for a certain tumor antigen and contain the UniCAR peptide epitope recognized by UniCARs. TMs can be made of different molecules showing various structures. Here we are presenting the comparison in functionality, in vitro and in vivo, of two TM formats: nanobody based αEGFR TM derived from the camelid αEGFR antibody 7C12 and scFv derived TM from the clinically used chimeric monoclonal antibody Cetuximab (IMC C‐225). In principle, we observed that both TM formats are able to redirect UniCAR T cells to eliminate EGFR‐expressing tumor cells in an antigen‐specific and TM‐dependent manner. Most interestingly, the tumor killing efficiency of the αEGFR scFv TM was significantly superior in comparison to the nanobody based TM, what might decide whether UniCAR T cells attack target cells showing different EGFR density level.

1. Introduction Recently the use of chimeric antigen receptor (CAR) modified T cells in the immunotherapy of tumors has become a promising approach. CAR T cells are able to recognize tumor-associated antigens (TAAs) in a major histocompatibility-complex (MHC)-independent manner. Although highly efficient, the inability to regulate the activity of CAR T cells can cause severe side effects and thus needs to be considered in future developments. Here, we introduce the RevCAR system – a novel switchable modular universal CAR system having a minimal size to overcome the obstacles of conventional CAR therapy. 2. Objectives In order to improve the controllability of CAR T cells a modular CAR system, which allows switching the activity of CAR T cells repeatedly “ON” and “OFF”, was generated. Furthermore, to avoid unspecific side effects and minimize tonic signaling of conventional CAR T cells, the extracellular single chain variable fragment (scFv) was removed. Thus, resulting RevCARs have a smaller size allowing “gated” targeting strategies, e.g. by facilitating simultaneous transduction of two independent CARs with different specificities and split motifs, which could further improve the safety of CAR T cells. 3. Materials & methods In order to reduce the size of the artificial receptor the original idea was to replace the extracellular scFv domain of a conventional CAR with a small peptide epitope and to engage the resulting RevCAR T cell via a bispecific target module which we termed RevTM. For proof of concept two small peptide epitopes were selected and the respective RevCARs constructed. In addition, a series of different RevTMs was generated. On the one hand the RevTM recognizes one of the two peptide epitopes on the other hand the RevTM can be directed to any potential TAA. 4. Results Until now a series of RevTMs was constructed and functionally analyzed. RevCAR T cells armed via the respective RevTM were able to efficiently lyse their respective target cells in a peptide epitope and target specific, as well as target module dependent manner. These data are supported by the analysis of cytokine secretion from RevCAR T cells which was only observed in the presence of both target cells and the respective RevTM. 5. Conclusion Taken together these results demonstrate the high anti-tumor efficiency of the novel RevCAR platform which is characterized by a small size, an improved safety, easy controllability as well as high flexibility.

Background: Chimeric antigen receptor (CAR) T cell therapy has demonstrated impressive clinical efficiency, but can also cause moderate to severe adverse effects that might be even fatal. Thus, preventing or managing CAR T cell toxicity is still an important issue for successful treatment of tumor patients. In order to provide a novel CAR technology platform with an improved safety profile, we established the switchable UniCAR system. This platform consists of (I) universal CAR (UniCAR) T cells that are per se inactive. Their anti-tumor activity can be specifically and repeatedly turned on/off in dependence of soluble tumor-binding target modules (TM).e.g.1-4 TMs are constructed by fusing an antigen-specific binding moiety with the E5B9 peptide epitope recognized by UniCARs. As these molecules are rapidly eliminated, UniCAR T cells can be easily controlled by TM dosing. Aims: As the risk for CAR T cell-related toxicities will also decrease with reduction of tumor burden, we intended to develop TMs with prolonged half-life that might ease clinical application and improve elimination of residual tumor cells in late phase of tumor therapy. Methods: We constructed a set of novel, longer lasting TMs by fusion of different tumor-specific single-chain fragment variables (scFv) and the E5B9 peptide epitope to the Fc domain of human IgG4 antibodies. The resulting IgG4-based TMs were functionally compared with smaller, scFv-based TMs in vitro and characterized for their pharmacokinetic properties in experimental mice. Results: The novel IgG4-based TMs are able to efficiently activate UniCAR T cells for killing of various tumor cell lines. In comparison to short-lived TMs, they are characterized by a comparable or increased efficiency at low TM concentrations. Pharmacokinetic studies in tumor-bearing mice further revealed that IgG4-based TM have a prolonged plasma half-life and enhanced bioavailability. Summary: Our data demonstrate that IgG4-based TMs in combination with smaller TMs are highly promising tools for redirection of UniCAR T cells to various cancer cells. Once the tumor burden is reduced, UniCAR T cells can be combined with IgG4-based TMs instead of small TMs. This is more convenient for patients as IgG4-based TM have not to be continuously infused due to their prolonged serum half-lives. Overall, the combination of UniCAR T cells with TMs of different size and specificity should allow a more convenient, individualized and safe treatment regimen of cancer patients.

The clinical efficacy of CAR T cell therapies has been widely recognized, particularly in the treatment of hematologic malignancies. Nevertheless, CAR T cells also have the capability to elicit undesired effects such as on-target/off-tumor recognition and cytokine release syndrome. To increase clinical safety of CAR T cell therapy, a novel modular universal CAR platform termed UniCAR was developed by our group. In the UniCAR system, antigen-binding specificity and signaling features are two distinct moieties, in which the antigen specificity is provided by targeting modules (TMs) to redirect UniCAR T cells in an individualized time- and target-dependent manner. In this way, UniCAR T-cells acquire killing potential only in the presence of a tumour-specific TM. Given the reduced size of such molecules, they are rapidly eliminated and therefore, need to be continuously infused. Thus, possible side effects and activation of UniCAR T cells can be easily monitored and controlled by TM dosing. During the onset of therapy, tumor burden and the risk for severe side effects are high and regulation of CAR T cell activity is particularly important at this stage. For this reason, TMs with extended half-life may play an important role by improving eradication of residual tumor cells in late phases of treatment and further expedite clinical application. In this line of thought, a set of novel short-lived and longer-lasting TMs directed against several tumor-associated antigens was developed. Short-lived TMs are composed of a tumor-specific binding moiety fused to the La peptide epitope (E5B9) which is recognized by UniCAR T cells. In order to generate extended half-life TMs, these two components are fused via an Fc domain derived from the human IgG4 molecule. In vitro and in vivo assays have shown that both short-lived and longer-lasting TMs efficiently redirect UniCAR T cells to cancer cells in a highly target-specific manner, thereby promoting the secretion of pro-inflammatory cytokines and tumor cell lysis. Further assays using PET-imaging, demonstrated that all TM formats specifically enriched at the tumor site presenting either short or prolonged serum half-lives. From a clinical point of view, after the initial reduction of tumor burden promoted by the small TMs, IgG4-based TMs could be subsequently administrated allowing a more convenient and personalized treatment of the patients avoiding the continuous infusion of the short-lived TMs. Furthermore, the specific accumulation of such IgG4-based TMs at the tumor site sets these molecules as attractive candidates for in vivo imaging and endoradiotherapy. Taken together, combination of switchable UniCAR T cells and TMs with different sizes, specificities and half-lives represent a flexible and individualized approach at different stages of cancer treatment.