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Chimeric antigen receptor (CAR) T cells are efficacious in patients with B-cell malignancies, while their activity is limited in patients with solid tumors. We developed a novel heterodimeric TCR-like CAR (TCAR) designed to achieve optimal chain pairing and integration into the T-cell CD3 signaling complex. The TCAR mediated high antigen sensitivity and potent antigen-specific T-cell effector functions in short-term in vitro assays. Both persistence and functionality of TCAR T cells were augmented by provision of costimulatory signals, which improved proliferation in vitro and in vivo . Combination with a nanoparticulate RNA vaccine, developed for in vivo expansion of CAR T cells, promoted tightly controlled expansion, survival, and antitumor efficacy of TCAR T cells in vivo ., Significance: A novel TCAR is tightly controlled by RNA vaccine-mediated costimulation and may provide an alternative to second-generation CARs for the treatment of solid tumors., Competing Interests: M. Birtel reports other from BioNTech Cell and Gene Therapies and grants from BMBF during the conduct of the study; other from BioNTech Cell and Gene Therapy outside the submitted work; in addition, M. Birtel has a patent to PCT/EP2015/073156 pending and issued and a patent to PCT/EP2019/086950 pending. R.-H. Voss reports grants from German Research Foundation (DFG) and Federal Ministry of Education and Research (BMBF) during the conduct of the study; in addition, R.-H. Voss has a licensed patent (WO 2017/059900-A1). K. Reinhard reports grants from BioNTech during the conduct of the study; other from BioNTech outside the submitted work; in addition, K. Reinhard has a patent to PCT/EP2015/060356 pending and issued and a patent to PCT/EP2019/086950 pending. B. Rengstl reports other from BioNTech SE during the conduct of the study; other from BioNTech SE outside the submitted work; in addition, B. Rengstl has a patent to PCT/EP2019/086950 pending. K. Michel reports other from Biontech SE during the conduct of the study; other from Biontech SE outside the submitted work; in addition, K. Michel has a patent to PCT/EP2019/086950 pending. N. Hayduk reports other from Biontech Cell & Gene Therapy during the conduct of the study; other from Biontech Cell & Gene Therapy outside the submitted work; in addition, N. Hayduk has a patent to PCT/EP2019/086950 pending. M. Theobald reports grants from DFG during the conduct of the study; in addition, M. Theobald has a patent to WO2017059900 issued and licensed. P. Oehm reports grants from BMBF and personal fees from BioNTech Cell & Gene Therapies during the conduct of the study; other from BioNTech SE outside the submitted work; in addition, P. Oehm has a patent to PCT/EP2015/056899 pending and issued, a patent to PCT/EP2015/060356 pending and issued, a patent to PCT/EP2015/073156 pending and issued, and a patent to PCT/EP2019/086950 pending. O. Tüereci reports other from BioNTech SE and grants from BMBF during the conduct of the study; other from BioNTech SE outside the submitted work; in addition, O. Tuereci has a patent to PCT/EP2015/056899 pending and issued and a patent to PCT/EP2015/060357 pending and issued; and Managing board members BioNTech SE. U. Sahin reports other from BioNTech SE and grants from BMBF during the conduct of the study; other from BioNTech SE outside the submitted work; in addition, U. Sahin has a patent to PCT/EP2015/056899 pending and issued, a patent to PCT/EP2015/060357 pending and issued, a patent to PCT/EP2015/060356 pending and issued, a patent to PCT/EP2015/073156 pending and issued, and a patent to PCT/EP2019/086950 pending; and Management board member BioNTech SE. No other disclosures were reported., (© 2022 The Authors; Published by the American Association for Cancer Research.)

Chimeric antigen receptor (CAR)-T cells have shown efficacy in patients with B cell malignancies. Yet, their application for solid tumors has challenges that include limited cancer-specific targets and nonpersistence of adoptively transferred CAR-T cells. Here, we introduce the developmentally regulated tight junction protein claudin 6 (CLDN6) as a CAR target in solid tumors and a strategy to overcome inefficient CAR-T cell stimulation in vivo. We demonstrate that a nanoparticulate RNA vaccine, designed for body-wide delivery of the CAR antigen into lymphoid compartments, stimulates adoptively transferred CAR-T cells. Presentation of the natively folded target on resident antigen-presenting cells promotes cognate and selective expansion of CAR-T cells. Improved engraftment of CAR-T cells and regression of large tumors in difficult-to-treat mouse models was achieved at subtherapeutic CAR-T cell doses., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The endosteal bone marrow niche and vascular endothelial cells provide sanctuaries for leukemic cells. In murine chronic myeloid leukemia (CML) CD44 on leukemia cells and E-selectin on bone marrow endothelium are essential mediators for the engraftment of leukemic stem cells. We hypothesized that non-adhesion of CML-initiating cells to E-selectin on the bone marrow endothelium may lead to superior eradication of leukemic stem cells in CML after treatment with imatinib than imatinib alone. Indeed, here we show that treatment with the E-selectin inhibitor GMI-1271 in combination with imatinib prolongs survival of mice with CML via decreased contact time of leukemia cells with bone marrow endothelium. Non-adhesion of BCR-ABL1 + cells leads to an increase of cell cycle progression and an increase of expression of the hematopoietic transcription factor and proto-oncogene Scl/Tal1 in leukemia-initiating cells. We implicate SCL/TAL1 as an indirect phosphorylation target of BCR-ABL1 and as a negative transcriptional regulator of CD44 expression. We show that increased SCL/TAL1 expression is associated with improved outcome in human CML. These data demonstrate the BCR-ABL1-specific, cell-intrinsic pathways leading to altered interactions with the vascular niche via the modulation of adhesion molecules - which could be exploited therapeutically in the future., (Copyright© 2020 Ferrata Storti Foundation.)

Targeting mRNA to eukaryotic cells is an emerging technology for basic research and provides broad applications in cancer immunotherapy, vaccine development, protein replacement, and in vivo genome editing. Although a plethora of nanoparticles for efficient mRNA delivery exists, in vivo mRNA targeting to specific organs, tissue compartments, and cells remains a major challenge. For this reason, methods for reporting the in vivo targeting specificity of different mRNA nanoparticle formats will be crucial. Here, we describe a straightforward method for monitoring the in vivo targeting efficiency of mRNA-loaded nanoparticles in mice. To achieve accurate mRNA delivery readouts, we loaded lipoplex nanoparticles with Cre-recombinase-encoding mRNA and injected these into commonly used Cre reporter mouse strains. Our results show that this approach provides readouts that accurately report the targeting efficacy of mRNA into organs, tissue structures, and single cells as a function of the used mRNA delivery system. The method described here establishes a versatile basis for determining in vivo mRNA targeting profiles and can be systematically applied for testing and improving mRNA packaging formats.