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Although chimeric antigen receptor (CAR) T cells have altered the treatment landscape for B cell malignancies, the risk of on-target, off-tumour toxicity has hampered their development for solid tumours because most target antigens are shared with normal cells 1,2 . Researchers have attempted to apply Boolean-logic gating to CAR T cells to prevent toxicity 3-5 ; however, a truly safe and effective logic-gated CAR has remained elusive 6 . Here we describe an approach to CAR engineering in which we replace traditional CD3ζ domains with intracellular proximal T cell signalling molecules. We show that certain proximal signalling CARs, such as a ZAP-70 CAR, can activate T cells and eradicate tumours in vivo while bypassing upstream signalling proteins, including CD3ζ. The primary role of ZAP-70 is to phosphorylate LAT and SLP-76, which form a scaffold for signal propagation. We exploited the cooperative role of LAT and SLP-76 to engineer logic-gated intracellular network (LINK) CAR, a rapid and reversible Boolean-logic AND-gated CAR T cell platform that outperforms other systems in both efficacy and prevention of on-target, off-tumour toxicity. LINK CAR will expand the range of molecules that can be targeted with CAR T cells, and will enable these powerful therapeutic agents to be used for solid tumours and diverse diseases such as autoimmunity 7 and fibrosis 8 . In addition, this work shows that the internal signalling machinery of cells can be repurposed into surface receptors, which could open new avenues for cellular engineering., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

The remarkable clinical activity of chimeric antigen receptor (CAR) therapies in B cell and plasma cell malignancies has validated the use of this therapeutic class for liquid cancers, but resistance and limited access remain as barriers to broader application. Here we review the immunobiology and design principles of current prototype CARs and present emerging platforms that are anticipated to drive future clinical advances. The field is witnessing a rapid expansion of next-generation CAR immune cell technologies designed to enhance efficacy, safety and access. Substantial progress has been made in augmenting immune cell fitness, activating endogenous immunity, arming cells to resist suppression via the tumour microenvironment and developing approaches to modulate antigen density thresholds. Increasingly sophisticated multispecific, logic-gated and regulatable CARs display the potential to overcome resistance and increase safety. Early signs of progress with stealth, virus-free and in vivo gene delivery platforms provide potential paths for reduced costs and increased access of cell therapies in the future. The continuing clinical success of CAR T cells in liquid cancers is driving the development of increasingly sophisticated immune cell therapies that are poised to translate to treatments for solid cancers and non-malignant diseases in the coming years., (© 2023. Springer Nature Limited.)

Regulatable CAR platforms could circumvent toxicities associated with CAR-T therapy, but existing systems have shortcomings including leakiness and attenuated activity. Here, we present SNIP CARs, a protease-based platform for regulating CAR activity using an FDA-approved small molecule. Design iterations yielded CAR-T cells that manifest full functional capacity with drug and no leaky activity in the absence of drug. In numerous models, SNIP CAR-T cells were more potent than constitutive CAR-T cells and showed diminished T cell exhaustion and greater stemness. In a ROR1-based CAR lethality model, drug cessation following toxicity onset reversed toxicity, thereby credentialing the platform as a safety switch. In the same model, reduced drug dosing opened a therapeutic window that resulted in tumor eradication in the absence of toxicity. SNIP CARs enable remote tuning of CAR activity, which provides solutions to safety and efficacy barriers that are currently limiting progress in using CAR-T cells to treat solid tumors., Competing Interests: Declaration of interests L.L., R.G.M., M.Z.L., and C.L.M. are coinventors on a patent related to this work. C.L.M. is a cofounder of Lyell Immunopharma, Syncopation Life Sciences, and Link Cell Therapies, which are developing CAR-based therapies, and consults for Lyell, NeoImmune Tech, Apricity, Nektar, Immatics, Ensoma, Mammoth, Glaxo Smith Kline, and Bristol Myers Squibb. L.L., R.G.M., E.S., and E.W.W. are consultants for and hold equity in Lyell Immunopharma. L.L. is a cofounder of, consults for, and holds equity in Syncopation Life Sciences. R.G.M. is a cofounder of, consults for, and holds equity in Syncopation Life Sciences and Link Cell Therapies. R.G.M. is a consultant for Illumina Radiopharmaceuticals, NKarta, ImmunAI, Arovella Therapeutics, Zai Lab, and Aptorum Group. R.G.M. serves on the Data and Safety Monitoring Board for Fate Therapeutics. J.T. is a consultant for Dorian Therapeutics. E.W.W. consults for and holds equity in VISTAN Health. A.T.S. is a founder of Immunai and Cartography Biosciences and receives research funding from Arsenal Biosciences, Allogene Therapeutics, and 10x Genomics. K.R.P. is a cofounder and employee of Cartography Biosciences. H.Y.C. is a cofounder of Accent Therapeutics, Boundless Bio, and Cartography Biosciences and is an advisor to 10x Genomics, Arsenal Biosciences, and Spring Discovery. J.R.C. is a cofounder and equity holder of Trapeze Therapeutics, Combangio, and Virsti Therapeutics; he has financial interests in Aravive, Xyence Therapeutics, and Syncopation Life Sciences; and he is a member of the Board of Directors of Ligand Pharmaceuticals and Revel Pharmaceuticals. S.A.Y.-H. is a consultant for Trapeze Therapeutics and Xyence Therapeutics., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Adoptive cell therapy (ACT) has proven to be highly effective in treating blood cancers, but traditional approaches to ACT are poorly effective in treating solid tumors observed clinically. Novel delivery methods for therapeutic cells have shown promise for treatment of solid tumors when compared with standard intravenous administration methods, but the few reported approaches leverage biomaterials that are complex to manufacture and have primarily demonstrated applicability following tumor resection or in immune-privileged tissues. Here, we engineer simple-to-implement injectable hydrogels for the controlled co-delivery of CAR-T cells and stimulatory cytokines that improve treatment of solid tumors. The unique architecture of this material simultaneously inhibits passive diffusion of entrapped cytokines and permits active motility of entrapped cells to enable long-term retention, viability, and activation of CAR-T cells. The generation of a transient inflammatory niche following administration affords sustained exposure of CAR-T cells, induces a tumor-reactive CAR-T phenotype, and improves efficacy of treatment.

T cell exhaustion limits immune responses against cancer and is a major cause of resistance to chimeric antigen receptor (CAR)-T cell therapeutics. Using murine xenograft models and an in vitro model wherein tonic CAR signaling induces hallmark features of exhaustion, we tested the effect of transient cessation of receptor signaling, or rest, on the development and maintenance of exhaustion. Induction of rest through enforced down-regulation of the CAR protein using a drug-regulatable system or treatment with the multikinase inhibitor dasatinib resulted in the acquisition of a memory-like phenotype, global transcriptional and epigenetic reprogramming, and restored antitumor functionality in exhausted CAR-T cells. This work demonstrates that rest can enhance CAR-T cell efficacy by preventing or reversing exhaustion, and it challenges the notion that exhaustion is an epigenetically fixed state., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Insufficient reactivity against cells with low antigen density has emerged as an important cause of chimeric antigen receptor (CAR) T-cell resistance. Little is known about factors that modulate the threshold for antigen recognition. We demonstrate that CD19 CAR activity is dependent upon antigen density and that the CAR construct in axicabtagene ciloleucel (CD19-CD28ζ) outperforms that in tisagenlecleucel (CD19-4-1BBζ) against antigen-low tumors. Enhancing signal strength by including additional immunoreceptor tyrosine-based activation motifs (ITAM) in the CAR enables recognition of low-antigen-density cells, whereas ITAM deletions blunt signal and increase the antigen density threshold. Furthermore, replacement of the CD8 hinge-transmembrane (H/T) region of a 4-1BBζ CAR with a CD28-H/T lowers the threshold for CAR reactivity despite identical signaling molecules. CARs incorporating a CD28-H/T demonstrate a more stable and efficient immunologic synapse. Precise design of CARs can tune the threshold for antigen recognition and endow 4-1BBζ-CARs with enhanced capacity to recognize antigen-low targets while retaining a superior capacity for persistence. SIGNIFICANCE: Optimal CAR T-cell activity is dependent on antigen density, which is variable in many cancers, including lymphoma and solid tumors. CD28ζ-CARs outperform 4-1BBζ-CARs when antigen density is low. However, 4-1BBζ-CARs can be reengineered to enhance activity against low-antigen-density tumors while maintaining their unique capacity for persistence. This article is highlighted in the In This Issue feature, p. 627 ., (©2020 American Association for Cancer Research.)

Purpose: Patients with relapsed pediatric solid tumors and CNS malignancies have few therapeutic options and frequently die of their disease. Chimeric antigen receptor (CAR) T cells have shown tremendous success in treating relapsed pediatric acute lymphoblastic leukemia, but this has not yet translated to treating solid tumors. This is partially due to a paucity of differentially expressed cell surface molecules on solid tumors that can be safely targeted. Here, we present B7-H3 (CD276) as a putative target for CAR T-cell therapy of pediatric solid tumors, including those arising in the central nervous system., Experimental Design: We developed a novel B7-H3 CAR whose binder is derived from a mAb that has been shown to preferentially bind tumor tissues and has been safely used in humans in early-phase clinical trials. We tested B7-H3 CAR T cells in a variety of pediatric cancer models., Results: B7-H3 CAR T cells mediate significant antitumor activity in vivo , causing regression of established solid tumors in xenograft models including osteosarcoma, medulloblastoma, and Ewing sarcoma. We demonstrate that B7-H3 CAR T-cell efficacy is largely dependent upon high surface target antigen density on tumor tissues and that activity is greatly diminished against target cells that express low levels of antigen, thus providing a possible therapeutic window despite low-level normal tissue expression of B7-H3., Conclusions: B7-H3 CAR T cells could represent an exciting therapeutic option for patients with certain lethal relapsed or refractory pediatric malignancies, and should be tested in carefully designed clinical trials., (©2019 American Association for Cancer Research.)