Co-stimulation via receptors of the tumor necrosis factor superfamily (TNFSF) emerges as promising strategy to support antitumor immune responses. Targeted strategies with antibody-fusion proteins composed of a tumor-directed antibody part and the extracellular domain of a co-stimulatory ligand of the TNFSF constitute an attractive option to focus the co-stimulatory activity to the tumor site. Since TNFSF members intrinsically form functional units of non-covalently linked homotrimers, the protein engineering of suitable antibody-fusion proteins is challenging. Aiming for molecules of simple and stable configuration, we used TNFSF ligands in a single-chain format (scTNFSF), i.e., three units of the ectodomain connected by polypeptide linkers, folding into an intramolecular trimer. By fusing tumordirected scFv antibody fragments directed against EpCAM or FAP to co-stimulatory scTNFSF molecules (sc4-1BBL, scOX40L, scGITRL or scLIGHT), a set of monomeric scFv-scTNFSF fusion proteins was generated. In comparison to the scFv-TNFSF format, defined by intermolecular homotrimerization via the TNFSF part, scFv-scTNFSF showed equal or enhanced co-stimulatory activity despite reduced avidity in antibody binding. In addition, enhanced serum stability and improved bioavailability in mice were observed. We show that the scFv-scTNFSF format can be applied to various members of the TNFSF, presenting targeting-dependent co-stimulatory activity. Hence, this format exhibits favorable properties that make it a promising choice for further therapeutic fusion protein development.Abbreviations: EpCAM, epithelial cell adhesion molecule; ED-A, fibronectin extradomain A; FAP, fibroblast activation protein; GITRL, glucocorticoid-induced tumor necrosis factor receptor (GITR) ligand; LIGHT, homologous to lymphotoxins, shows inducible expression and competes with herpes simplex virus glycoprotein D for herpesvirus entry mediator (HVEM), a receptor expressed by T lymphocytes; mAb, monoclonal antibody; scFv, single-chain fragment variable; TNF, tumor necrosis factor; TNFSF, tumor necrosis factor superfamily; TRAIL, TNF-related apoptosis inducing ligand
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 CD123specific 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).
Chimeric antigen receptor T cells (CAR-T) targeting CD19 have achieved significant success in patients with B cell malignancies. To date, implementation of CAR-T in other indications remains challenging due to the lack of truly tumor-specific antigens as well as control of CAR-T activity in patients. CD123 is highly expressed in acute myeloid leukemia (AML) blasts including leukemia-initiating cells making it an attractive immunotherapeutic target. However, CD123 expression in normal hematopoietic progenitor cells and endothelia bears the risk of severe toxicities and may limit CAR-T applications lacking fine-tuned control mechanisms. Therefore, we recently developed a rapidly switchable universal CAR-T platform (UniCAR), in which CAR-T activity depends on the presence of a soluble adapter called targeting module (TM), and confirmed clinical proof-of-concept for targeting CD123 in AML with improved safety. As costimulation via 4-1BB ligand (4-1BBL) can enhance CAR-T expansion, persistence, and effector functions, a novel CD123-specific TM variant (TM123-4-1BBL) comprising trimeric single-chain 4-1BBL was developed for transient costimulation of UniCAR-T cells (UniCAR-T) at the leukemic site in trans. TM123-4-1BBL-directed UniCAR-T efficiently eradicated CD123-positive AML cells in vitro and in a CDX in vivo model. Moreover, additional costimulation via TM123-4-1BBL enabled enhanced expansion and persistence with a modulated UniCAR-T phenotype. In addition, the increased hydrodynamic volume of TM123-4-1BBL prolonged terminal plasma half-life and ensured a high total drug exposure in vivo. In conclusion, expanding the soluble adapter optionality for CD123-directed UniCAR-T maintains the platforms high anti-leukemic efficacy and immediate control mechanism for a flexible, safe, and individualized CAR-T therapy of AML patients.
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.
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.
Approximately 80% of AML patients express CD123 on their leukemic blasts and CD123 is frequently expressed on other hematologic and lymphatic malignancies. Chimeric antigen receptor (CAR) T-cell therapy has demonstrated significant efficacy in B cell malignancies. Breakthrough of conventional CAR-T technology in AML has been hampered by expression of suitable target antigens on normal hematopoietic progenitor cells posing a risk for continued aplasia. Thus, innovative approaches putting the power of CAR-T technology under the control of reliable and fast-acting on/off switches to avoid and/or abrogate acute and long-term side effects are required. We have recently reported clinical proof-of-concept for autologous switchable CAR-T cell therapy in AML (Wermke et al. Blood 2021). However, manufacturing of autologous products is very costly and rrAML patients are often in urgent need for a product and multiple prior lines of treatment may be detrimental for product quality. While current clinical allogeneic approaches successfully prevent graft-versus-host disease, observed persistence has been limited due to rejection of the grafted T cells by host immune cells. Here, we report preclinical development of a donor-derived allogeneic switchable CAR-T therapy for CD123-positive hematologic and lymphatic malignancies (AVC-201). The reverse universal chimeric antigen receptor platform (RevCAR) is a 2-component CAR-T platform. The first component is a universal CAR-T cell. Its binding domain is a short, non-immunogenic peptide that by itself does not recognize any human cell surface antigen but is specifically bound by an scFv included in the second component, a soluble adaptor called targeting module (TM). To target CD123, a TM with a short half-life was selected (R-TM123), enabling a rapid switch-off of the RevCAR system by TM withdrawal to avoid acute and long-term toxicity usually associated with continuous activation of CAR-T cells. The allogeneic cells are generated by three edits with CRISPR-Cas9, which is meant to fully overcome graft-versus-host disease as well as graft rejection by host T and NK cells. Therefore, we expect a persistent T cell product that can be re-expanded with additional TM cycles. A summary of preclinical characterization of AVC-201 will be presented at the meeting including in vitro and in vivo pharmacology and toxicology experiments. Activation of Allo-RevCAR-T cells is strictly dependent on the presence of CD123-positive target cells and R-TM123. Allo-RevCAR-T cells redirected by R-TM123 efficiently lyse CD123-positive AML cells in vitro and in vivo, and in vitro EC50 values are in the low picomolar range of R-TM123 concentrations. In conclusion, preclinical data support the clinical exploration of AVC-201 in a first in human study. Citation Format: Armin Ehninger, Johannes Spehr, Simon Loff, Anika Langer, Julia Riewaldt, Julia Reinhardt, Jan-Erik Meyer, Josephine Dietrich, Gabriel Jurado, Reynald Lescarbeau, Marc Cartellieri. Preclinical characterization of switchable allogeneic chimeric antigen receptor T cells to support first in human clinical study in CD123-positive hematologic and lymphatic malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4095.
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.
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