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Chimeric antigen receptor T cells (CAR-T) targeting CD19 or B cell maturation antigen (BCMA) are highly effective against B cell malignancies. However, application of CAR-T to less differentially expressed targets remains a challenge due to lack of tumor-specific antigens and CAR-T controllability. CD123, a highly promising leukemia target, is expressed not only by leukemic and leukemia-initiating cells, but also by myeloid, hematopoietic progenitor, and certain endothelial cells. Thus, CAR-T lacking fine-tuned control mechanisms pose a high toxicity risk. To extend the CAR-T target landscape and widen the therapeutic window, we adapted our rapidly switchable universal CAR-T platform (UniCAR) to target CD123. UniCAR-T efficiently eradicated CD123+ leukemia in vitro and in vivo. Activation, cytolytic response, and cytokine release were strictly dependent on the presence of the CD123-specific targeting module (TM123) with comparable efficacy to CD123-specific CAR-T in vitro. We further demonstrated a pre-clinical proof of concept for the safety-switch mechanism using a hematotoxicity mouse model wherein TM123-redirected UniCAR-T showed reversible toxicity toward hematopoietic cells compared to CD123 CAR-T. In conclusion, UniCAR-T maintain full anti-leukemic efficacy, while ensuring rapid controllability to improve safety and versatility of CD123-directed immunotherapy. The safety and efficacy of UniCAR-T in combination with TM123 will now be assessed in a phase I clinical trial (ClinicalTrials.gov: NCT04230265)., Graphical Abstract, CAR-T cell treatment of acute leukemia beyond CD19 remains challenging. Loff et al. demonstrate that rapidly switchable universal CAR-T cells in combination with soluble CD123-specific adaptors achieve robust anti-leukemic responses, while hematotoxicity is rapidly reversible, enabling safe targeting of such less differentially expressed target antigens and broadening of the therapeutic window.

Despite remarkable therapeutic success using chimeric antigen receptor modified T-cells (CAR-T) in hematologic malignancies, targeting solid tumors still remains challenging, given their heterogeneity and immunosuppressive milieu, including the expression of inhibitory immune checkpoints such as PD-1/PD-L1. Preclinical models demonstrate that combining CAR-T therapy with checkpoint inhibitors targeting the PD-1/PD-L1 axis could be a promising strategy to enhance anti-cancer activity of CAR-T in solid tumors and on-going phase I trials are clinically evaluating this strategy. However, primary and acquired resistance to checkpoint blockade is frequently observed limiting its broad applicability and efficacy. In addition, lack of specificity results in frequent occurrence of immune-related adverse events affecting several organs. Alternatively, expression of PD-L1 by solid tumor cells could be an attractive target for CAR-T therapy, especially in order to prevent or reverse tumor re-growth and relapse after successful initial tumor cell lysis. The development of a rapidly switchable universal CAR-T platform (UniCAR) with enhanced safety features allowed us to explore the possibility to directly attack PD-L1 expressing solid tumor cells. An inherent key feature of the UniCAR-T platform is a rapid and reversible turn off mechanism (less than 4 hours) determined by the short pharmacokinetic half-life of soluble targeting modules (TMs) and fast internalization of cell-bound TMs. TMs provide the antigen-specificity for UniCAR gene-modified T-cells (UniCAR-T) and consist of an antigen-binding moiety, e.g. an scFv, linked to a small peptide motif recognized by the UniCAR. For redirecting UniCAR-T against the PD-1/PD-L1 axis, a PD-L1 specific TM (TM-PD-L1) was developed and its functionality confirmed in binding assays to human and murine PD-L1 using surface plasmon resonance as well as in cell-based cytotoxicity assays. Half maximal killing efficacy (EC50) mediated by TM-PD-L1 redirected UniCAR-T was in the low picomolar range. As PD-L1 is also expressed on healthy tissue, toxicity studies were performed in a humanized mouse model with human UniCAR-T. Humanized mice treated with TM-PD-L1 twice a day for ten consecutive days did not reveal any signs of toxicity. In a prostate cancer (PCa) xenograft model efficient suppression of tumor growth could be demonstrated by administration of TM-PD-L1. Our data are the first to demonstrate safety and feasibility of active targeting of PD-L1 expressed by solid tumors with CAR-T and that this approach is capable of inducing a significant anti-tumor response. Citation Format: Josephine Dietrich, Simon Loff, Johannes Spehr, Julia Riewaldt, Cordula Gründer, Maria Schreiber, Michael Bachmann, Gerhard Ehninger, Michael Pehl, Marc Cartellieri, Armin Ehninger. Rapidly switchable universal CAR-T cells with improved safety profile allow for active targeting of PD-L1 expressing solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2209.

CAR-T cell therapy holds great promise for treating a wide range of malignancies. Nevertheless, the CAR-T approach faces multiple challenges, including lack of suitable targets, insufficient tumor penetration and a microenvironment hostile to CAR-T cells. Here we provide pre-clinical evidence for using a low-molecular-weight, chemically synthesized compound to re-target CAR-T cells against solid tumors in conjunction with the CD28 co-stimulatory domain to address several of these challenges. PSMA is a validated target for treatment of advanced and metastatic prostate cancer (PCa), but also broadly expressed on tumor neo-vasculature beyond PCa. PSMA is also known to be expressed on a number of normal tissues, albeit to a much lower extent, which necessitates additional safety mechanisms for CAR-T therapy. Therefore, a rapidly switchable universal CAR-T platform (UniCAR) was developed. In this system, antigen-specificity is provided by soluble adaptors termed targeting modules (TM), which redirect T-cells engineered to express a universal CAR in an antigen-specific manner against tumors. The TM explored in the present study incorporates a clinically well-characterized radiotracer peptide motif binding to the enzymatic groove of PSMA. The small-sized TM has favorable pharmacokinetic and pharmacodynamics properties (short half-life of < 30 min; rapid internalization in < 1h), which allows a fast silencing and excellent controllability of UniCAR-T reactivity (< 4 h). Furthermore, the small molecule TM efficiently penetrates and rapidly accumulates within solid tumors as clinically demonstrated by related radiotracers. Results from our pre-clinical in vivo model demonstrate potent recruitment of UniCAR-T into tumor tissue by the TM and an efficient anti-tumor response. Of note, it was recently shown that the chosen PCa model suppresses CAR-T response by secreting high levels of transforming growth factor beta (TGF-β), creating an immunosuppressive milieu. We could overcome immunosuppression utilizing CD28 compared to 4-1-BB in the intracellular signaling domain of the UniCAR. Administered CD28/zeta UniCAR efficiently suppressed tumor growth in our in vivo model and could be re-activated by consecutive cycles of TM administration even after several weeks. In contrast to CARs with fixed binding moieties, which continuously signal and thereby induce T- cell exhaustion, the discontinuous activation of UniCAR keeps a balanced mix of T-effector and T-memory cells. In summary, our rapidly switchable universal CAR-T platform in combination with a low-molecular-weight TM allows to target less differentially expressed solid tumor antigens. Furthermore, incorporation of the CD28 costimulatory domain overcomes the immunosuppressive tumor microenvironment while discontinuous activation of UniCAR-T prevents exhaustion. Citation Format: Marc Cartellieri, Simon Loff, Johannes Spehr, Mridula Swayampakula, Julia Riewaldt, Cordula Gründer, Maria Schreiber, Michael Bachmann, Anja Feldmann, Michael Pehl, Gerhard Ehninger, Armin Ehninger. Using a PSMA-specific low-molecular-weight compound for prostate cancer treatment with rapidly switchable universal CAR-T cells: Overcoming the challenges of cellular immunotherapies in solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2176.

Chimeric antigen receptor T-cell (CAR-T) therapy targeting CD19 or BCMA is tremendously effective in treating hematological malignancies. However, the application of CAR-T beyond certain B-cell malignancies such as ALL, MM, lymphoma and CLL remains challenging due to the lack of tumor-specific antigens and the limited ability to control acute and potential long term CAR-T reactivity in patients. Therefore, a rapidly switchable universal CAR-T platform (UniCAR) was developed. In this system, antigen-specificity is provided by soluble adaptors of short pharmacokinetic half-life, termed targeting modules (TM), which redirect T-cells engineered to express a universal CAR against tumors, allowing for excellent controllability of CAR-T reactivity while maintaining the high anti-tumor activity of CAR-T cells. Here we present results from late stage pre-clinical characterization of UniCAR-T targeting CD123 (UniCAR-T-CD123) for treatment of acute leukemia. Pre-clinical characterization confirmed specificity and safety of the approach. UniCAR-T proofed to be per se inert and safe in clinically relevant in vivo toxicity models. Activation of UniCAR-T occurs solely in the presence of the CD123-specific TM (TM123) and upon cross-linkage to CD123 expressing leukemic cells. Specific lysis of leukemic target cell lines and primary patient material is induced in a dose-dependent manner at pico-molar TM123 concentrations. Notably, induction of cytokine release occurs at higher TM doses than on-set of target cell lysis, opening a therapeutic window for clinical application. In vivo efficacy of UniCAR-T re-directed against CD123 was proven in CDX and PDX mouse models. Interestingly, anti-leukemic responses of switch-controllable UniCAR-T were demonstrated to be comparable to conventional CD123-specific CAR-T in vitro at comparable doses. In contrast to conventional CD123 CAR-T, CD123-specific toxicity of TM-activated UniCAR-T towards hematopoietic progenitors was reversible and could be abrogated by withdrawal of TM, allowing for normal development of human hematopoiesis in a xenograft model. In summary, in vitro and in vivo evidence suggests that UniCAR-T-CD123 could provide strong efficacy against CD123 expressing hematological malignancies while providing excellent control and ensuring recovery of normal hematopoiesis post treatment. A phase IA dose-finding study is ongoing. Citation Format: Simon Loff, Jan-Erik Meyer, Johannes Spehr, Julia Riewaldt, Cordula Gründer, Maria Schreiber, Michael Bachmann, Michael Pehl, Gerhard Ehninger, Armin Ehninger, Marc Cartellieri. More than a bridging therapy: Targeting CD123 with rapidly switchable universal CAR-T cells for treatment of acute leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4232.

International audience; Curli are functional amyloids that form a major part of the biofilm produced by many enterobacteriaceae. A multiprotein system around the outer membrane protein CsgG is in charge of the export and controlled propagation of the main Curli subunits, CsgA and CsgB. CsgF is essential for the linkage of the main amyloid-forming proteins to the cell surface. Here, we present a profound biochemical and biophysical characterization of recombinant CsgF, highlighted by a solution NMR structure of CsgF in the presence of dihexanoylphosphocholine micelles. Interestingly, CsgF contains large unstructured domains and does not show a globular fold. The data presented shed new light on the molecular mechanism of Curli amyloid surface attachment.

Stable mammalian production cell lines in suspension culture enable the reproducible expression of target genes in any desired scale using bioreactor technology. Targeted integration methods have been developed to cut down timelines for the generation of stable producer cell lines. The powerful Flp recombinase mediated cassette exchange (RMCE) technique allows fast integration of target genes in preselected and optimized high expression loci in so called master cell lines. Up to now, these cells only enable the expression from a single locus. Here, we describe the set-up required for the generation of multiple tagged master cell lines on the example of a binary RMCE expression system in the glycosylation mutant CHO Lec3.2.8.1 cell line. We show how this technology is used for the expression of proteins from multiple loci by generating a binary RMCE expression system. The tools and strategy for the construction of binary master cell lines with different combinations of expression level are described in detail. The binary production cell lines show independent expression of the individual exchange loci of the producer cell lines. The expression level for the model protein tdTomato is the cumulative expression for the chosen combination of the expression loci of the master cell line. This binary RMCE expression system can be further developed to a multi RMCE expression system for co-expression of protein complex subunits with predetermined expression ratio of each individual exchange locus.

The universal modular chimeric antigen receptor (UniCAR) platform redirects CAR-T cells using a separated, soluble targeting module with a short half-life. This segregation allows precise controllability and flexibility. Herein we show that the UniCAR platform can be used to efficiently target solid cancers in vitro and in vivo using a pre-clinical prostate cancer model which overexpresses prostate stem cell antigen (PSCA). Short-term administration of the targeting module to tumor bearing immunocompromised mice engrafted with human UniCAR-T cells significantly delayed tumor growth and prolonged survival of recipient mice both in a low and high tumor burden model. In addition, we analyzed phenotypic and functional changes of cancer cells and UniCAR-T cells in association with the administration of the targeting module to reveal potential immunoevasive mechanisms. Most notably, UniCAR-T cell activation induced upregulation of immune-inhibitory molecules such as programmed death ligands. In conclusion, this work illustrates that the UniCAR platform mediates potent anti-tumor activity in a relevant in vitro and in vivo solid tumor model.

Application of autologous T cells genetically engineered to express CD19-specific chimeric antigen receptors (CAR-T) is highly effective in the treatment of B cell malignancies. To this date, application of CAR-T therapy beyond CD19 remains challenging due to the inability to control CAR-T reactivity in patients and the lack of tumor-associated antigens exclusively expressed by malignant cells. The interleukin-3 receptor alpha chain (CD123) is a promising immunotherapeutic target and associated with leukemia-initiating compartments in myeloid- or lymphoid derived diseases. However, in contrast to CD19, CD123 is a precarious target due to its prevalent expression on healthy hematopoietic stem and progenitor cells (HSPC) as well as endothelial cells. Thus, CAR-T lacking any fine-tuned control mechanisms are at risk to cause life threatening toxicities or can only act as bridging therapy to an allogeneic stem cell transplantation. To extend application of CAR-T therapy and safely redirect CAR-engineered T cells to challenging targets such as CD123, a switch-controllable universal CAR-T platform (UniCAR) was recently introduced. The UniCAR system consists of two components: (1) a non-reactive inducible second generation CAR with CD28/CD3ζ stimulation for an inert manipulation of T cells (UniCAR-T) and (2) soluble targeting modules (TM) enabling UniCAR-T reactivity in an antigen-specific manner. Here we provide late stage pre-clinical data for UniCAR-T in combination with a CD123-specific TM (TM123) for treatment of acute leukemia. Primary patient-derived CD123-positive leukemic blasts were efficiently eradicated by TM123-redirected clinical-grade manufactured UniCAR-T in vitro and in vivo. Activation, cytolytic responses and cytokine release were proven to be strictly switch-controlled. Moreover, anti-leukemic responses of UniCAR-T were demonstrated to be comparable to conventional CD123-specific CAR-T in vitro. In contrast to conventional CD123 CAR-T, TM123-redirected UniCAR-T discriminate between CD123high malignant cells and CD123low healthy cells with negligible toxicity towards HSPC in vivo. As 4-1BB mediated co-stimulation is known to enhance CAR-T activity in vivo, a novel CD123-specific targeting module bearing a covalently bound trimeric 4-1BB ligand (4-1BBL) was developed and characterized for co-stimulation at the leukemic site in trans. Specific binding of TM123-4-1BBL was demonstrated against native 4-1BB as well as CD123-positive leukemic blasts. In long-term tumor eradication models, TM123-4-1BBL ameliorated the killing capability of UniCAR-T in vitro. Additionally, the increased hydrodynamic radius of trimeric 4-1BBL-coupled TM123 prolonged plasma half-life and enhanced bioavailability in vivo. In conclusion, UniCAR-T maintain high anti-leukemic efficacy, while adding a sophisticated mechanism for immediate control to improve safety and versatility of CD123-directed CAR-T therapy. Moreover, switching between several TMs from short to moderate plasma half-life allows for an individualized treatment of various leukemic settings while minimizing potential adverse effects. Disclosures Loff: GEMoaB Monoclonals GmbH: Employment. Meyer:Cellex Patient Treatment GmbH: Employment. Dietrich:Cellex Patient Treatment GmbH: Employment. Spehr:Cellex Patient Treatment GmbH: Employment. Julia:Cellex Patient Treatment GmbH: Employment. Gründer:GEMoaB Monoclonals GmbH: Employment. Franke:GEMoaB Monoclonals GmbH: Employment. Bachmann:GEMoaB Monoclonals GmbH: Equity Ownership. Ehninger:Bayer: Research Funding; GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership. Ehninger:GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Cellex Gesellschaft fuer Zellgewinnung mbH: Equity Ownership. Cartellieri:Cellex Patient Treatment GmbH: Employment.

International audience; The controlled formation of filamentous protein complexes plays a crucial role in many biological systems and represents an emerging paradigm in signal transduction. The mitochondrial antiviral signaling protein (MAVS) is a central signal transduction hub in innate immunity that is activated by a receptor-induced conversion into helical superstructures (filaments) assembled from its globular caspase activation and recruitment domain. Solid-state NMR (ssNMR) spectroscopy has become one of the most powerful techniques for atomic resolution structures of protein fibrils. However, for helical filaments, the determination of the correct symmetry parameters has remained a significant hurdle for any structural technique and could thus far not be precisely derived from ssNMR data. Here, we solved the atomic resolution structure of helical MAVS(CARD) filaments exclusively from ssNMR data. We present a generally applicable approach that systematically explores the helical symmetry space by efficient modeling of the helical structure restrained by interprotomer ssNMR distance restraints. Together with classical automated NMR structure calculation, this allowed us to faithfully determine the symmetry that defines the entire assembly. To validate our structure, we probed the protomer arrangement by solvent paramagnetic resonance enhancement, analysis of chemical shift differences relative to the solution NMR structure of the monomer, and mutagenesis. We provide detailed information on the atomic contacts that determine filament stability and describe mechanistic details on the formation of signaling-competent MAVS filaments from inactive monomers.

The Baculovirus Expression Vector System (BEVS) is widely used to produce high amounts of recombinant proteins. Nevertheless, generating recombinant baculovirus in high quality is rather time-consuming and labor-intensive. Alternatively, virus-free expression in insect cells did not achieve similar expression levels for most proteins so far. The transactivation method is a promising approach for protein expression in Sf21 cells. It combines advantages of BEVS and plasmid-based expression by activating strong virus-dependent promoters on a transfected plasmid by baculoviral coinfection. Here, we identified expression elements required for transactivation. Therefore, we designed several vectors comprising different viral promoters or promoter combinations and tested them for eGFP expression using the automated BioLector microcultivation system. Remarkably, only the combination of the very late promoter p10 together with the homologous region 5 (hr5) could boost expression during transactivation. Other elements, like p10 alone or the late viral promoter polH, did not respond to transactivation. A new combination of hr5 and p10 with the strongest immediate early OpMNPV viral promoter OpIE2 improved the yield of eGFP by ~25% in comparison to the previous applied hr5-IE1-p10 expression cassette. Furthermore, we observed a strong influence of the transcription termination sequence and vector backbone on the level of expression. Finally, the expression levels for transactivation, BEVS and solely plasmid-based expression were compared for the marker protein eGFP, underlining the potential of transactivation for fast recombinant protein expression in Sf21 cells. In conclusion, essential elements for transactivation could be identified. The optimal elements were applied to generate an improved vector applicable in virus-free plasmid-based expression, transactivation and BEVS.

During the past years promising clinical outcomes have been reported using immunotherapy for treatment of solid tumors and malignancies of the hematopoietic system. Since T cells act as key players in immune surveillance, an encouraging approach is to genetically engineer T cells with lenti- or retroviral vectors, in order to express artificial chimeric antigen receptors (CARs), directing them against cancer cells and leading to a distinct cytotoxic response. CARs are synthetic fusion proteins consisting of an antigen-binding moiety, commonly a tumor-associated antigen (TAA)-specific single-chain fragment variable (scFv), combined with activating signaling domains like the CD3-zeta chain. Signaling can be enhanced by adding co-stimulatory domains, such as the intracellular regions of CD28 or CD137. Although, CAR T cells showed promising results in patients with B cell malignancies, a major drawback of conventional CARs, bearing a tumor-antigen-binding moiety, is the uncontrollable continuous T cell activity. Hence, CAR T cells can achieve a sustained anti-tumoral response. However, reactivity against any healthy tissues expressing the target antigen might cause severe adverse effects. A chance to gain safety on CAR T cell approaches by retaining efficacy is offered through a novel universal binary CAR system (UniCAR). The system is based on a second generation CAR with an extracellular scFv binding domain, which is directed against a short non-immunogenic peptide motif instead of a cell surface antigen. Thus, engineered T cells are inert per se, remaining in a “sleeping mode” after re-infusion. Small protein links (targeting modules, TMs), consisting e.g. of TAA-specific scFvs harboring the small peptide epitope recognized by the UniCAR are used to redirect UniCAR expressing T cells in an antigen-specific manner. Primary results demonstrate that UniCAR driven T cell activation and cytotoxic effects against tumor cells are strictly dependent on the presence of the antigen-specific TMs. UniCAR T cells show high anti-tumor efficacy even at picomolar TM concentrations and low effector to target cell (e:t) ratios in vitro. After those proof-of-concept studies we disclose positive evidence of high efficacy UniCAR T cells with reactivity against CD123-positive acute myeloid leukemia (AML) and CD19-bearing acute lymphoblastic leukemia (ALL) cells. UniCAR T cell anti-tumor efficacy proofed to be superior when compared to conventional CAR T cells at low e:t ratios adjusted to the situation usually found in patients. Furthermore, UniCAR T cells pre-decorated with CD123-specific TMs in vitro are capable of prohibiting tumor engraftment of CD123-positive AML in vivo and persist in immunodeficient NSG mice. Ex vivo isolated UniCAR T cells retained cytotoxic potential against MOLM-13 cells for at least four month post transplantation fortifying the therapeutic potential in vitro and in vivo. The tremendous benefit of the novel UniCAR platform is the precise control of CAR T cell activity by regulating TM administration and dosage, preventing off side-toxicities arising from conventional CAR approaches. Moreover, the modular UniCAR system also enables combinatorial targeting strategies against several TAAs to increase therapy performance and circumvent the occurrence of tumor resistance mechanisms due to selectable compositions of target structures. Leukemia-related target antigens like CD19, CD20, CD22, CD33 or CD123 are conceivable therapy settings, offering advantages compared to monotherapeutic CAR-based therapies. Taken together, the modular composition of the UniCAR platform maintains the high anti-tumor potential of CAR-engrafted T cells, while introducing tight control mechanisms and unparalleled target flexibility. These features will allow a more sophisticated application of CAR technology and a reduction of adverse events in the clinical setting. Citation Format: Simon Loff, Malte v. Bonin, Josephine Dietrich, Jan-Erik Meyer, Anja Feldmann, Claudia Arndt, Johannes Spehr, Cordula Gründer, Gerhard Ehninger, Michael P. Bachmann, Armin Ehninger, Marc Cartellieri. The UniCAR system: Inducible CAR T cells for precise reactivity and high efficacy against hematopoietic malignancies [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B099.