Enhanced safety and efficacy of protease-regulated CAR-T cell receptors.

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.

Engineered CD47 protects T cells for enhanced antitumour immunity.

Adoptively transferred T cells and agents designed to block the CD47-SIRPα axis are promising cancer therapeutics that activate distinct arms of the immune system1,2. Here we administered anti-CD47 antibodies in combination with adoptively transferred T cells with the goal of enhancing antitumour efficacy but observed abrogated therapeutic benefit due to rapid macrophage-mediated clearance of T cells expressing chimeric antigen receptors (CARs) or engineered T cell receptors. Anti-CD47-antibody-mediated CAR T cell clearance was potent and rapid enough to serve as an effective safety switch. To overcome this challenge, we engineered the CD47 variant CD47(Q31P) (47E), which engages SIRPα and provides a 'don't eat me' signal that is not blocked by anti-CD47 antibodies. TCR or CAR T cells expressing 47E are resistant to clearance by macrophages after treatment with anti-CD47 antibodies, and mediate substantial, sustained macrophage recruitment to the tumour microenvironment. Although many of the recruited macrophages manifested an M2-like profile3, the combined therapy synergistically enhanced antitumour efficacy. Our study identifies macrophages as major regulators of T cell persistence and illustrates the fundamental challenge of combining T-cell-directed therapeutics with those designed to activate macrophages. It delivers a therapeutic approach that is capable of simultaneously harnessing the antitumour effects of T cells and macrophages, offering enhanced potency against solid tumours.

Co-opting signalling molecules enables logic-gated control of CAR T cells.

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 cells1,2. Researchers have attempted to apply Boolean-logic gating to CAR T cells to prevent toxicity3-5; however, a truly safe and effective logic-gated CAR has remained elusive6. 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 autoimmunity7 and fibrosis8. 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.

Latent human herpesvirus 6 is reactivated in CAR T cells.

Cell therapies have yielded durable clinical benefits for patients with cancer, but the risks associated with the development of therapies from manipulated human cells are understudied. For example, we lack a comprehensive understanding of the mechanisms of toxicities observed in patients receiving T cell therapies, including recent reports of encephalitis caused by reactivation of human herpesvirus 6 (HHV-6)1. Here, through petabase-scale viral genomics mining, we examine the landscape of human latent viral reactivation and demonstrate that HHV-6B can become reactivated in cultures of human CD4+ T cells. Using single-cell sequencing, we identify a rare population of HHV-6 'super-expressors' (about 1 in 300-10,000 cells) that possess high viral transcriptional activity, among research-grade allogeneic chimeric antigen receptor (CAR) T cells. By analysing single-cell sequencing data from patients receiving cell therapy products that are approved by the US Food and Drug Administration2 or are in clinical studies3-5, we identify the presence of HHV-6-super-expressor CAR T cells in patients in vivo. Together, the findings of our study demonstrate the utility of comprehensive genomics analyses in implicating cell therapy products as a potential source contributing to the lytic HHV-6 infection that has been reported in clinical trials1,6-8 and may influence the design and production of autologous and allogeneic cell therapies.

GD2-CAR T cell therapy for H3K27M-mutated diffuse midline gliomas.

Diffuse intrinsic pontine glioma (DIPG) and other H3K27M-mutated diffuse midline gliomas (DMGs) are universally lethal paediatric tumours of the central nervous system1. We have previously shown that the disialoganglioside GD2 is highly expressed on H3K27M-mutated glioma cells and have demonstrated promising preclinical efficacy of GD2-directed chimeric antigen receptor (CAR) T cells2, providing the rationale for a first-in-human phase I clinical trial (NCT04196413). Because CAR T cell-induced brainstem inflammation can result in obstructive hydrocephalus, increased intracranial pressure and dangerous tissue shifts, neurocritical care precautions were incorporated. Here we present the clinical experience from the first four patients with H3K27M-mutated DIPG or spinal cord DMG treated with GD2-CAR T cells at dose level 1 (1 × 106 GD2-CAR T cells per kg administered intravenously). Patients who exhibited clinical benefit were eligible for subsequent GD2-CAR T cell infusions administered intracerebroventricularly3. Toxicity was largely related to the location of the tumour and was reversible with intensive supportive care. On-target, off-tumour toxicity was not observed. Three of four patients exhibited clinical and radiographic improvement. Pro-inflammatory cytokine levels were increased in the plasma and cerebrospinal fluid. Transcriptomic analyses of 65,598 single cells from CAR T cell products and cerebrospinal fluid elucidate heterogeneity in response between participants and administration routes. These early results underscore the promise of this therapeutic approach for patients with H3K27M-mutated DIPG or spinal cord DMG.

Content-aware frame interpolation (CAFI): deep learning-based temporal super-resolution for fast bioimaging.

The development of high-resolution microscopes has made it possible to investigate cellular processes in 3D and over time. However, observing fast cellular dynamics remains challenging because of photobleaching and phototoxicity. Here we report the implementation of two content-aware frame interpolation (CAFI) deep learning networks, Zooming SlowMo and Depth-Aware Video Frame Interpolation, that are highly suited for accurately predicting images in between image pairs, therefore improving the temporal resolution of image series post-acquisition. We show that CAFI is capable of understanding the motion context of biological structures and can perform better than standard interpolation methods. We benchmark CAFI's performance on 12 different datasets, obtained from four different microscopy modalities, and demonstrate its capabilities for single-particle tracking and nuclear segmentation. CAFI potentially allows for reduced light exposure and phototoxicity on the sample for improved long-term live-cell imaging. The models and the training and testing data are available via the ZeroCostDL4Mic platform.

Immune receptor inhibition through enforced phosphatase recruitment.

Antibodies that antagonize extracellular receptor-ligand interactions are used as therapeutic agents for many diseases to inhibit signalling by cell-surface receptors1. However, this approach does not directly prevent intracellular signalling, such as through tonic or sustained signalling after ligand engagement. Here we present an alternative approach for attenuating cell-surface receptor signalling, termed receptor inhibition by phosphatase recruitment (RIPR). This approach compels cis-ligation of cell-surface receptors containing ITAM, ITIM or ITSM tyrosine phosphorylation motifs to the promiscuous cell-surface phosphatase CD452,3, which results in the direct intracellular dephosphorylation of tyrosine residues on the receptor target. As an example, we found that tonic signalling by the programmed cell death-1 receptor (PD-1) results in residual suppression of T cell activation, but is not inhibited by ligand-antagonist antibodies. We engineered a PD-1 molecule, which we denote RIPR-PD1, that induces cross-linking of PD-1 to CD45 and inhibits both tonic and ligand-activated signalling. RIPR-PD1 demonstrated enhanced inhibition of checkpoint blockade compared with ligand blocking by anti-PD1 antibodies, and increased therapeutic efficacy over anti-PD1 in mouse tumour models. We also show that the RIPR strategy extends to other immune-receptor targets that contain activating or inhibitory ITIM, ITSM or ITAM motifs; for example, inhibition of the macrophage SIRPα 'don't eat me' signal with a SIRPα-CD45 RIPR molecule potentiates antibody-dependent cellular phagocytosis beyond that of SIRPα blockade alone. RIPR represents a general strategy for direct attenuation of signalling by kinase-activated cell-surface receptors.

c-Jun overexpression in CAR T cells induces exhaustion resistance.

Chimeric antigen receptor (CAR) T cells mediate anti-tumour effects in a small subset of patients with cancer1-3, but dysfunction due to T cell exhaustion is an important barrier to progress4-6. To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system with a tonically signaling CAR, which induces hallmark features of exhaustion6. Exhaustion was associated with a profound defect in the production of IL-2, along with increased chromatin accessibility of AP-1 transcription factor motifs and overexpression of the bZIP and IRF transcription factors that have been implicated in mediating dysfunction in exhausted T cells7-10. Here we show that CAR T cells engineered to overexpress the canonical AP-1 factor c-Jun have enhanced expansion potential, increased functional capacity, diminished terminal differentiation and improved anti-tumour potency in five different mouse tumour models in vivo. We conclude that a functional deficiency in c-Jun mediates dysfunction in exhausted human T cells, and that engineering CAR T cells to overexpress c-Jun renders them resistant to exhaustion, thereby addressing a major barrier to progress for this emerging class of therapeutic agents.

In vivo imaging of nanoparticle-labeled CAR T cells.

Metastatic osteosarcoma has a poor prognosis with a 2-y, event-free survival rate of ∼15 to 20%, highlighting the need for the advancement of efficacious therapeutics. Chimeric antigen receptor (CAR) T-cell therapy is a potent strategy for eliminating tumors by harnessing the immune system. However, clinical trials with CAR T cells in solid tumors have encountered significant challenges and have not yet demonstrated convincing evidence of efficacy for a large number of patients. A major bottleneck for the success of CAR T-cell therapy is our inability to monitor the accumulation of the CAR T cells in the tumor with clinical-imaging techniques. To address this, we developed a clinically translatable approach for labeling CAR T cells with iron oxide nanoparticles, which enabled the noninvasive detection of the iron-labeled T cells with magnetic resonance imaging (MRI), photoacoustic imaging (PAT), and magnetic particle imaging (MPI). Using a custom-made microfluidics device for T-cell labeling by mechanoporation, we achieved significant nanoparticle uptake in the CAR T cells, while preserving T-cell proliferation, viability, and function. Multimodal MRI, PAT, and MPI demonstrated homing of the T cells to osteosarcomas and off-target sites in animals administered with T cells labeled with the iron oxide nanoparticles, while T cells were not visualized in animals infused with unlabeled cells. This study details the successful labeling of CAR T cells with ferumoxytol, thereby paving the way for monitoring CAR T cells in solid tumors.

Novel NanoLuc substrates enable bright two-population bioluminescence imaging in animals.

Sensitive detection of two biological events in vivo has long been a goal in bioluminescence imaging. Antares, a fusion of the luciferase NanoLuc to the orange fluorescent protein CyOFP, has emerged as a bright bioluminescent reporter with orthogonal substrate specificity to firefly luciferase (FLuc) and its derivatives such as AkaLuc. However, the brightness of Antares in mice is limited by the poor solubility and bioavailability of the NanoLuc substrate furimazine. Here, we report a new substrate, hydrofurimazine, whose enhanced aqueous solubility allows delivery of higher doses to mice. In the liver, Antares with hydrofurimazine exhibited similar brightness to AkaLuc with its substrate AkaLumine. Further chemical exploration generated a second substrate, fluorofurimazine, with even higher brightness in vivo. We used Antares with fluorofurimazine to track tumor size and AkaLuc with AkaLumine to visualize CAR-T cells within the same mice, demonstrating the ability to perform two-population imaging with these two luciferase systems.

Detection of Aberrant CD58 Expression in a Wide Spectrum of Lymphoma Subtypes: Implications for Treatment Resistance.

CD58 or lymphocyte function-associated antigen-3, is a ligand for CD2 receptors on T and NK cells and is required for their activation and target cell killing. We recently showed a trend toward higher frequency of CD58 aberrations in patients with diffuse large B-cell lymphoma (DLBCL) who progressed on chimeric antigen receptor-T-cell treatment compared with those who responded. Given that CD58 status may be an important measure of T-cell-mediated therapy failure, we developed a CD58 immunohistochemical assay and evaluated CD58 status in 748 lymphomas. Our results show that CD58 protein expression is downregulated in a significant proportion of all subtypes of B-, T-, and NK-cell lymphomas. CD58 loss is significantly related to poor prognostic indicators in DLBCL and to ALK and DUSP22 rearrangements in anaplastic large-cell lymphoma. However, it is not associated with overall or progression-free survival in any of the lymphoma subtypes. As eligibility for chimeric antigen receptor-T-cell therapy is being extended to a broader spectrum of lymphomas, mechanisms of resistance, such as target downregulation and CD58 loss, may limit therapeutic success. CD58 status is therefore an important biomarker in lymphoma patients who may benefit from next-generation T-cell-mediated therapies or other novel approaches that mitigate immune escape.

How to stop using gadolinium chelates for magnetic resonance imaging: clinical-translational experiences with ferumoxytol.

Gadolinium chelates have been used as standard contrast agents for clinical MRI for several decades. However, several investigators recently reported that rare Earth metals such as gadolinium are deposited in the brain for months or years. This is particularly concerning for children, whose developing brain is more vulnerable to exogenous toxins compared to adults. Therefore, a search is under way for alternative MR imaging biomarkers. The United States Food and Drug Administration (FDA)-approved iron supplement ferumoxytol can solve this unmet clinical need: ferumoxytol consists of iron oxide nanoparticles that can be detected with MRI and provide significant T1- and T2-signal enhancement of vessels and soft tissues. Several investigators including our research group have started to use ferumoxytol off-label as a new contrast agent for MRI. This article reviews the existing literature on the biodistribution of ferumoxytol in children and compares the diagnostic accuracy of ferumoxytol- and gadolinium-chelate-enhanced MRI. Iron oxide nanoparticles represent a promising new class of contrast agents for pediatric MRI that can be metabolized and are not deposited in the brain.

Charting a path for prioritization of novel agents for clinical trials in osteosarcoma: A report from the Children's Oncology Group New Agents for Osteosarcoma Task Force.

Osteosarcoma is the most common bone tumor in children and young adults. Metastatic and relapsed disease confer poor prognosis, and there have been no improvements in outcomes for several decades. The disease's biological complexity, lack of drugs developed specifically for osteosarcoma, imperfect preclinical models, and limits of existing clinical trial designs have contributed to lack of progress. The Children's Oncology Group Bone Tumor Committee established the New Agents for Osteosarcoma Task Force to identify and prioritize agents for inclusion in clinical trials. The group identified multitargeted tyrosine kinase inhibitors, immunotherapies targeting B7-H3, CD47-SIRPα inhibitors, telaglenastat, and epigenetic modifiers as the top agents of interest. Only multitargeted tyrosine kinase inhibitors met all criteria for frontline evaluation and have already been incorporated into an upcoming phase III study concept. The task force will continue to reassess identified agents of interest as new data become available and evaluate novel agents using this method.

New developments in immunotherapy for pediatric solid tumors.

PURPOSE OF REVIEW: Building upon preclinical advances, we are uncovering immunotherapy strategies that are translating into improved outcomes in tumor subsets. Advanced pediatric solid tumors carry poor prognoses and resultant robust efforts to apply immunotherapy advances to pediatric solid tumors are in progress. Here, we discuss recent developments in the field using mAb and mAb-based therapies including checkpoint blockade and chimeric antigen receptors (CARs). RECENT FINDINGS: The pediatric solid tumor mAb experience targeting the diganglioside, GD2, for patients with neuroblastoma has been the most compelling to date. GD2 and alternative antigen-specific mAbs are now being incorporated into antibody-drug conjugates, bispecific antibodies and CARs for treatment of solid tumors. CARs in pediatric solid tumors have not yet achieved comparative responses to the hematologic CAR experience; however, novel strategies such as bispecific targeting, intratumoral administration and improved understanding of T-cell biology may yield enhanced CAR-efficacy. Therapeutic effect using single-agent checkpoint blocking antibodies in pediatric solid tumors also remains limited to date. Combinatorial strategies continue to hold promise and the clinical effect in tumor subsets with high antigenic burden is being explored. SUMMARY: Pediatric immunotherapy remains at early stages of translation, yet we anticipate that with advanced technology, we will achieve widespread, efficacious use of immunotherapy for pediatric solid tumors.

Tumor Antigen and Receptor Densities Regulate Efficacy of a Chimeric Antigen Receptor Targeting Anaplastic Lymphoma Kinase.

We explored the utility of targeting anaplastic lymphoma kinase (ALK), a cell surface receptor overexpressed on pediatric solid tumors, using chimeric antigen receptor (CAR)-based immunotherapy. T cells expressing a CAR incorporating the single-chain variable fragment sequence of the ALK48 mAb linked to a 4-1BB-CD3ζ signaling domain lysed ALK-expressing tumor lines and produced interferon-gamma upon antigen stimulation but had limited anti-tumor efficacy in two xenograft models of human neuroblastoma. Further exploration demonstrated that cytokine production was highly dependent upon ALK target density and that target density of ALK on neuroblastoma cell lines was insufficient for maximal activation of CAR T cells. In addition, ALK CAR T cells demonstrated rapid and complete antigen-induced loss of receptor from the T cell surface via internalization. Using a model that simultaneously modulated antigen density and CAR expression, we demonstrated that CAR functionality is regulated by target antigen and CAR density and that low expression of either contributes to limited anti-tumor efficacy of the ALK CAR. These data suggest that stoichiometric relationships between CAR receptors and target antigens may significantly impact the anti-tumor efficacy of CAR T cells and that manipulation of these parameters could allow precise tuning of CAR T cell activity.

Post-Transplantation Cyclophosphamide after Bone Marrow Transplantation Is Not Associated with an Increased Risk of Donor-Derived Malignancy.

Post-transplantation cyclophosphamide (PTCy) can be used for graft-versus-host disease (GVHD) prophylaxis alone or in combination with other agents and is associated with excellent rates of engraftment and acute and chronic GVHD, as well as absence of post-transplantation lymphoproliferative disease. No study has previously evaluated the risk for developing donor-derived malignancy (DDM) in patients who receive PTCy. Giving chemotherapy in the immediate post-transplantation period carries with it a theoretic risk of disturbing the graft at a time of increased hematopoietic stress and causing or accelerating the development of malignancy. From 2000 to 2011, 789 patients underwent allogeneic transplantation and received PTCy at the Johns Hopkins Hospital. There were 4 cases of DDM identified among this large population, which is similar to or below the rate of DDM published in the literature. We found that the estimated cumulative incidence by competing risk analysis of DDM is 1.4% (SE, 1.02%). The use of PTCy does not appear to increase the risk of DDM.

Assessment of programmed death-ligand 1 expression and tumor-associated immune cells in pediatric cancer tissues.

BACKGROUND: Programmed death 1 (PD-1) signaling in the tumor microenvironment dampens immune responses to cancer, and blocking this axis induces antitumor effects in several malignancies. Clinical studies of PD-1 blockade are only now being initiated in pediatric patients, and little is known regarding programmed death-ligand 1 (PD-L1) expression in common childhood cancers. The authors characterized PD-L1 expression and tumor-associated immune cells (TAICs) (lymphocytes and macrophages) in common pediatric cancers. METHODS: Whole slide sections and tissue microarrays were evaluated by immunohistochemistry for PD-L1 expression and for the presence of TAICs. TAICs were also screened for PD-L1 expression. RESULTS: Thirty-nine of 451 evaluable tumors (9%) expressed PD-L1 in at least 1% of tumor cells. The highest frequency histotypes comprised Burkitt lymphoma (80%; 8 of 10 tumors), glioblastoma multiforme (36%; 5 of 14 tumors), and neuroblastoma (14%; 17 of 118 tumors). PD-L1 staining was associated with inferior survival among patients with neuroblastoma (P = .004). Seventy-four percent of tumors contained lymphocytes and/or macrophages. Macrophages were significantly more likely to be identified in PD-L1-positive versus PD-L1-negative tumors (P < .001). CONCLUSIONS: A subset of diagnostic pediatric cancers exhibit PD-L1 expression, whereas a much larger fraction demonstrates infiltration with tumor-associated lymphocytes. PD-L1 expression may be a biomarker for poor outcome in neuroblastoma. Further preclinical and clinical investigation will define the predictive nature of PD-L1 expression in childhood cancers both at diagnosis and after exposure to chemoradiotherapy. Cancer 2017;123:3807-3815. © 2017 American Cancer Society.

INSPIRED Symposium Part 5: Expanding the Use of CAR T Cells in Children and Young Adults.

Chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable efficacy in relapsed/refractory (r/r) B cell malignancies, including in pediatric patients with acute lymphoblastic leukemia (ALL). Expanding this success to other hematologic and solid malignancies is an area of active research and, although challenges remain, novel solutions have led to significant progress over the past decade. Ongoing clinical trials for CAR T cell therapy for T cell malignancies and acute myeloid leukemia (AML) have highlighted challenges, including antigen specificity with off-tumor toxicity and persistence concerns. In T cell malignancies, notable challenges include CAR T cell fratricide and prolonged T cell aplasia, which are being addressed with strategies such as gene editing and suicide switch technologies. In AML, antigen identification remains a significant barrier, due to shared antigens across healthy hematopoietic progenitor cells and myeloid blasts. Strategies to limit persistence and circumvent the immunosuppressive tumor microenvironment (TME) created by AML are also being explored. CAR T cell therapies for central nervous system and solid tumors have several challenges, including tumor antigen heterogeneity, immunosuppressive and hypoxic TME, and potential for off-target toxicity. Numerous CAR T cell products have been designed to overcome these challenges, including "armored" CARs and CAR/T cell receptor (TCR) hybrids. Strategies to enhance CAR T cell delivery, augment CAR T cell performance in the TME, and ensure the safety of these products have shown promising results. In this manuscript, we will review the available evidence for CAR T cell use in T cell malignancies, AML, central nervous system (CNS), and non-CNS solid tumor malignancies, and recommend areas for future research.

Immunotherapy-related cognitive impairment after CAR T cell therapy in mice.

Persistent central nervous system (CNS) immune dysregulation and consequent dysfunction of multiple neural cell types is central to the neurobiological underpinnings of a cognitive impairment syndrome that can occur following traditional cancer therapies or certain infections. Immunotherapies have revolutionized cancer care for many tumor types, but the potential long-term cognitive sequelae are incompletely understood. Here, we demonstrate in mouse models that chimeric antigen receptor (CAR) T cell therapy for both CNS and non-CNS cancers can impair cognitive function and induce a persistent CNS immune response characterized by white matter microglial reactivity and elevated cerebrospinal fluid (CSF) cytokines and chemokines. Consequently, oligodendroglial homeostasis and hippocampal neurogenesis are disrupted. Microglial depletion rescues oligodendroglial deficits and cognitive performance in a behavioral test of attention and short-term memory function. Taken together, these findings illustrate similar mechanisms underlying immunotherapy-related cognitive impairment (IRCI) and cognitive impairment following traditional cancer therapies and other immune challenges.

Overcoming on-target, off-tumour toxicity of CAR T cell therapy for solid tumours.

Therapies with genetically modified T cells that express chimeric antigen receptors (CARs) specific for CD19 or B cell maturation antigen (BCMA) are approved to treat certain B cell malignancies. However, translating these successes into treatments for patients with solid tumours presents various challenges, including the risk of clinically serious on-target, off-tumour toxicity (OTOT) owing to CAR T cell-mediated cytotoxicity against non-malignant tissues expressing the target antigen. Indeed, severe OTOT has been observed in various CAR T cell clinical trials involving patients with solid tumours, highlighting the importance of establishing strategies to predict, mitigate and control the onset of this effect. In this Review, we summarize current clinical evidence of OTOT with CAR T cells in the treatment of solid tumours and discuss the utility of preclinical mouse models in predicting clinical OTOT. We then describe novel strategies being developed to improve the specificity of CAR T cells in solid tumours, particularly the role of affinity tuning of target binders, logic circuits and synthetic biology. Furthermore, we highlight control strategies that can be used to mitigate clinical OTOT following cell infusion such as regulating or eliminating CAR T cell activity, exogenous control of CAR expression, and local administration of CAR T cells.