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Regulatory T cells (Treg) are important players in the tumor microenvironment. However, the mechanisms behind their immunosuppressive effects are poorly understood. We found that CCR6-CCL20 activity in tumor-infiltrating Tregs is associated with greater glycolytic activity and ablation of Ccr6 reduced glycolysis and lactic acid production while increasing compensatory glutamine metabolism. Immunosuppressive activity toward CD8+ T cells was abrogated in Ccr6-/- Tregs due to reduction in activation-induced glycolysis. Furthermore, Ccr6-/- mice exhibited improved survival across multiple tumor models compared to wild-type mice and Treg and CD8+ T-cell depletion abrogated the improvement. In addition, Ccr6 ablation further promoted the efficacy of anti-PD-1 therapy in a preclinical glioma model. Follow-up knockdown of Ccl20 with siRNA also demonstrated improvement in antitumor efficacy. Our results unveil CCR6 as a marker and regulator of Treg-induced immunosuppression and identify approaches to target the metabolic determinants of Treg immunosuppressive activity., (©2024 American Association for Cancer Research.)

The intensive nutrient requirements needed to sustain T cell activation and proliferation, combined with competition for nutrients within the tumor microenvironment, raise the prospect that glucose availability may limit CAR-T cell function. Here, we seek to test the hypothesis that stable overexpression (OE) of the glucose transporter GLUT1 in primary human CAR-T cells would improve their function and antitumor potency. We observe that GLUT1OE in CAR-T cells increases glucose consumption, glycolysis, glycolytic reserve, and oxidative phosphorylation, and these effects are associated with decreased T cell exhaustion and increased Th 17 differentiation. GLUT1OE also induces broad metabolic reprogramming associated with increased glutathione-mediated resistance to reactive oxygen species, and increased inosine accumulation. When challenged with tumors, GLUT1OE CAR-T cells secrete more proinflammatory cytokines and show enhanced cytotoxicity in vitro, and demonstrate superior tumor control and persistence in mouse models. Our collective findings support a paradigm wherein glucose availability is rate limiting for effector CAR-T cell function and demonstrate that enhancing glucose availability via GLUT1OE could augment antitumor immune function., (© 2024. The Author(s).)

Tumor-infiltrating lymphocyte (TIL) hypofunction contributes to the progression of advanced cancers and is a frequent target of immunotherapy. Emerging evidence indicates that metabolic insufficiency drives T cell hypofunction during tonic stimulation, but the signals that initiate metabolic reprogramming in this context are largely unknown. Here, we found that Meteorin-like (METRNL), a metabolically active cytokine secreted by immune cells in the tumor microenvironment (TME), induced bioenergetic failure of CD8 + T cells. METRNL was secreted by CD8 + T cells during repeated stimulation and acted via both autocrine and paracrine signaling. Mechanistically, METRNL increased E2F-peroxisome proliferator-activated receptor delta (PPARδ) activity, causing mitochondrial depolarization and decreased oxidative phosphorylation, which triggered a compensatory bioenergetic shift to glycolysis. Metrnl ablation or downregulation improved the metabolic fitness of CD8 + T cells and enhanced tumor control in several tumor models, demonstrating the translational potential of targeting the METRNL-E2F-PPARδ pathway to support bioenergetic fitness of CD8 + TILs., Competing Interests: Declaration of interests C.M.J. is a consultant for Egret Therapeutics with equity interests in the company. He is an inventor on a patent filed by Johns Hopkins University for using immune checkpoint agonists to treat cerebrovascular disorders. He receives research support from Biohaven, InCephalo, and Grifols. Johns Hopkins University has filed a provisional patent on METRNL blockade for cancer treatment on which C.M.J., M.L., A.P., H.B., and J.C. are inventors. E.S. consults for Lepton Pharmaceuticals and Galaria and consults for and holds equity in Lyell Immunopharma. C.B. is a consultant for Depuy-Synthes and Bionaut Labs. C.L.M. has multiple patents in the field of CAR T cell therapy; is an equity holder, director, and consultant for Syncopation Life Sciences and Link Cell Therapies; and is an equity holder and consultant for Lyell Immunopharma, Mammoth Biosciences, Ensoma Therapeutics, and Apricity Bio. She also consults for Immatics, Glaxo Smith Kline, and Bristol Myers Squibb. H.B. is a consultant for Perosphere, AsclepiX Therapeutics, StemGen, Accelerating Combination Therapies, Catalio Nexus Fund II. LLC, LikeMinds, Inc. Acuity Bio Corp, InSightec, Galen Robotics, and Nurami Medical. M.L. has received research support from Arbor, BMS, Accuray, Tocagen, Biohaven, Kyrin-Kyowa, and Biohaven; has been a consultant to Tocagen, VBI, InCephalo Therapeutics, Pyramid Bio, Merck, BMS, Insightec, Biohaven, Sanianoia, Hemispherian, Black Diamond Therapeutics, and Novocure; is a shareholder of Egret Therapeutics; and has patents for focused radiation + checkpoint inhibitors, local chemotherapy + checkpoint inhibitors, and checkpoint agonists for neuro-inflammation., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cancer immunotherapy with chimeric antigen receptor (CAR) T cells can cause immune effector cell-associated neurotoxicity syndrome (ICANS). However, the molecular mechanisms leading to ICANS are not well understood. Here we examined the role of microglia using mouse models and cohorts of individuals with ICANS. CD19-directed CAR (CAR19) T cell transfer in B cell lymphoma-bearing mice caused microglia activation and neurocognitive deficits. The TGFβ-activated kinase-1 (TAK1)-NF-κB-p38 MAPK pathway was activated in microglia after CAR19 T cell transfer. Pharmacological TAK1 inhibition or genetic Tak1 deletion in microglia using Cx3cr1 CreER :Tak1 fl/fl mice resulted in reduced microglia activation and improved neurocognitive activity. TAK1 inhibition allowed for potent CAR19-induced antilymphoma effects. Individuals with ICANS exhibited microglia activation in vivo when studied by translocator protein positron emission tomography, and imaging mass cytometry revealed a shift from resting to activated microglia. In summary, we prove a role for microglia in ICANS pathophysiology, identify the TAK1-NF-κB-p38 MAPK axis as a pathogenic signaling pathway and provide a rationale to test TAK1 inhibition in a clinical trial for ICANS prevention after CAR19 T cell-based cancer immunotherapy., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Outcomes are poor for patients with large B-cell lymphoma who relapse after CD19-directed chimeric antigen receptor (CAR) T-cell therapy (CAR19). CD22 is a nearly universally expressed B-cell surface antigen and the efficacy of a CD22-directed CAR T-cell therapy (CAR22) in large B-cell lymphoma is unknown, which was what we aimed to examine in this study., Methods: In this single centre, open-label, dose-escalation phase 1 trial, we intravenously administered CAR22 at two dose levels (1 million and 3 million CAR22-positive T cells per kg of bodyweight) to adult patients (aged ≥18 years) who relapsed after CAR19 or had CD19-negative large B-cell lymphoma. The primary endpoints were manufacturing feasibility, safety measured by the incidence and severity of adverse events and dose-limiting toxicities, and identification of the maximum tolerated dose (ie, the recommended phase 2 dose). This study is registered with ClinicalTrials.gov (NCT04088890) and is active, but closed for enrolment., Findings: From Oct 17, 2019, to Oct 19, 2022, a total of 41 patients were assessed for eligibility; however, one patient withdrew. 40 patients underwent leukapheresis and 38 (95%) had CAR T-cell products manufactured successfully. The median age was 65 years (range 25-84), 17 (45%) were women, 32 (84%) had elevated pretreatment lactate dehydrogenase, 11 (29%) had refractory disease to all previous therapies, and patients had received a median of four lines of previous therapy (range 3-8). Of the 38 patients treated, 37 (97%) had relapsed after previous CAR19. The identified maximum tolerated dose was 1 million CAR T cells per kg. Of 29 patients who received the maximum tolerated dose, no patients developed a dose-limiting toxicity or grade 3 or higher cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, or immune effector cell-associated haemophagocytic lymphohistiocytosis-like syndrome., Interpretation: This trial identifies CD22 as an immunotherapeutic target in large B-cell lymphoma and demonstrates the durable clinical activity of CAR22 in patients with disease progression after CAR19 therapy. Although these findings are promising, it is essential to recognise that this is a phase 1 dose-finding study. Further investigations are warranted to establish the long-term efficacy and to delineate the patient subgroups that will derive the most benefit from this therapeutic approach., Funding: National Cancer Institute, National Institutes of Health, Stanford Cancer Institute, Leukemia & Lymphoma Society, Parker Institute for Cancer Immunotherapy, Lymph & Co, and the European Hematology Association., Competing Interests: Declaration of interests ARR received research support from Pharmacyclics and AbbVie; one-time ad hoc scientific advisory board work for Nohla Therapeutics and Kaleido; and expert witness work for the US Department of Justice. CLM is the founder, has equity in, consults for, and is a Director of Cargo Therapeutics and Link Cell Therapies; has equity in Lyell Immunopharma; and receives royalties from the National Institutes of Health for CAR22 consulting for Immatics, Ensoma, Mammoth, Adaptimmune, and Bristol Myers Squibb. DBM consults for Kite Pharma-Gilead, Juno Therapeutics-Celgene, Novartis, Janssen, and Pharmacyclics; and receives research support from Kite Pharma-Gilead, Allogene, Cargo Therapeutics, Pharmacyclics, Miltenyi Biotec, and Adaptive Biotechnologies. JHB consults for Kite Pharma-Gilead; and receives research support from Kite Pharma-Gilead, Genentech-Roche, Regeneron Pharmaceuticals, and Cellular Biomedicine Group. MJF consults for Kite Pharma-Gilead, Adaptative Biotechnologies, and Cargo Therapeutics; receives research support from Kite-Pharma-Gilead, Allogene Therapeutics, Cargo Therapeutics, and Adaptative Biotechnologies; and is on the data safety monitoring board for Fate Therapeutics. LM receives research support from Adaptive Biotechnologies and Servier Laboratories; and consults for Amgen and Pfizer. JYS consults for Kite Pharma-Gilead and ImmPACT Bio. PS receives research support from Kite Pharma-Gilead. SS consults for Janssen. SAF consults for Lonza PerMed, Gradalis, Obsidian, and Samsara BioCapital. MPH is on the Kite Pharma-Gilead advisory board. All other authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.)

Increasing numbers of cell and gene therapies (CGTs) are emerging to treat and cure pediatric diseases. However, small market sizes limit the potential return on investment within the traditional biopharmaceutical drug development model, leading to a market failure. In this Perspective, we discuss major factors contributing to this failure, including high manufacturing costs, regulatory challenges, and licensing practices that do not incorporate pediatric development milestones, as well as potential solutions. We propose the creation of a new entity, the Pediatric Advanced Medicines Biotech, to lead late-stage development and commercialize pediatric CGTs outside the traditional biopharmaceutical model in the United States-where organized efforts to solve this problem have been lacking. The Pediatric Advanced Medicines Biotech would partner with the academic ecosystem, manufacture products in academic good manufacturing practice facilities and work closely with regulatory bodies, to ferry CGTs across the drug development 'valley of death' and, ultimately, increase access to lifesaving treatments for children in need., (© 2024. Springer Nature America, Inc.)

Abstract: Chimeric antigen receptor (CAR) T cells directed against CD19 (CAR19) are a revolutionary treatment for B-cell lymphomas (BCLs). CAR19 cell expansion is necessary for CAR19 function but is also associated with toxicity. To define the impact of CAR19 expansion on patient outcomes, we prospectively followed a cohort of 236 patients treated with CAR19 (brexucabtagene autoleucel or axicabtagene ciloleucel) for mantle cell lymphoma (MCL), follicular lymphoma, and large BCL (LBCL) over the course of 5 years and obtained CAR19 expansion data using peripheral blood immunophenotyping for 188 of these patients. CAR19 expansion was higher in patients with MCL than other lymphoma histologic subtypes. Notably, patients with MCL had increased toxicity and required fourfold higher cumulative steroid doses than patients with LBCL. CAR19 expansion was associated with the development of cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, and the requirement for granulocyte colony-stimulating factor 14 days after infusion. Younger patients and those with elevated lactate dehydrogenase (LDH) had significantly higher CAR19 expansion. In general, no association between CAR19 expansion and LBCL treatment response was observed. However, when controlling for tumor burden, we found that lower CAR19 expansion in conjunction with low LDH was associated with improved outcomes in LBCL. In sum, this study finds CAR19 expansion principally associates with CAR-related toxicity. Additionally, CAR19 expansion as measured by peripheral blood immunophenotyping may be dispensable to favorable outcomes in LBCL., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: The risk of second tumors after chimeric antigen receptor (CAR) T-cell therapy, especially the risk of T-cell neoplasms related to viral vector integration, is an emerging concern., Methods: We reviewed our clinical experience with adoptive cellular CAR T-cell therapy at our institution since 2016 and ascertained the occurrence of second tumors. In one case of secondary T-cell lymphoma, a broad array of molecular, genetic, and cellular techniques were used to interrogate the tumor, the CAR T cells, and the normal hematopoietic cells in the patient., Results: A total of 724 patients who had received T-cell therapies at our center were included in the study. A lethal T-cell lymphoma was identified in a patient who had received axicabtagene ciloleucel therapy for diffuse large B-cell lymphoma, and both lymphomas were deeply profiled. Each lymphoma had molecularly distinct immunophenotypes and genomic profiles, but both were positive for Epstein-Barr virus and were associated with DNMT3A and TET2 mutant clonal hematopoiesis. No evidence of oncogenic retroviral integration was found with the use of multiple techniques., Conclusions: Our results highlight the rarity of second tumors and provide a framework for defining clonal relationships and viral vector monitoring. (Funded by the National Cancer Institute and others.)., (Copyright © 2024 Massachusetts Medical Society.)

Adoptively transferred T cells and agents designed to block the CD47-SIRPα axis are promising cancer therapeutics that activate distinct arms of the immune system 1,2 . 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) (47 E ), 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 47 E 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 profile 3 , 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., (© 2024. The Author(s).)

Chimeric antigen receptor (CAR) T cells targeting CD22 (CD22-CAR) provide a therapeutic option for patients with CD22 + malignancies with progression after CD19-directed therapies. Using on-site, automated, closed-loop manufacturing, we conducted parallel Phase 1b clinical trials investigating a humanized CD22-CAR with 41BB costimulatory domain in children and adults with heavily treated, relapsed/refractory (r/r) B-ALL. Of 19 patients enrolled, 18 had successful CD22-CAR manufacturing, and 16 patients were infused. High grade (3-4) cytokine release syndrome (CRS) and immune effector-cell-associated neurotoxicity syndrome (ICANS) each occurred in only one patient; however, three patients experienced immune-effector-cell-associated hemophagocytic lymphohistiocytosis-like syndrome (IEC-HS). Twelve of 16 patients (75%) achieved CR with an overall 56% MRD-negative CR rate. Duration of response was overall limited (median 77 days), and CD22 expression was downregulated in 4/12 (33%) available samples at relapse. In summary, we demonstrate that closed-loop manufacturing of CD22-CAR T cells is feasible and is associated with a favorable safety profile and high CR rates in pediatric and adult r/r B-ALL, a cohort with limited CD22-CAR reporting., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

A major limitation of chimeric antigen receptor (CAR) T cell therapies is the poor persistence of these cells in vivo 1 . The expression of memory-associated genes in CAR T cells is linked to their long-term persistence in patients and clinical efficacy 2-6 , suggesting that memory programs may underpin durable CAR T cell function. Here we show that the transcription factor FOXO1 is responsible for promoting memory and restraining exhaustion in human CAR T cells. Pharmacological inhibition or gene editing of endogenous FOXO1 diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype and impaired the antitumour activity of CAR T cells. Overexpression of FOXO1 induced a gene-expression program consistent with T cell memory and increased chromatin accessibility at FOXO1-binding motifs. CAR T cells that overexpressed FOXO1 retained their function, memory potential and metabolic fitness in settings of chronic stimulation, and exhibited enhanced persistence and tumour control in vivo. By contrast, overexpression of TCF1 (encoded by TCF7) did not enforce canonical memory programs or enhance the potency of CAR T cells. Notably, FOXO1 activity correlated with positive clinical outcomes of patients treated with CAR T cells or tumour-infiltrating lymphocytes, underscoring the clinical relevance of FOXO1 in cancer immunotherapy. Our results show that overexpressing FOXO1 can increase the antitumour activity of human CAR T cells, and highlight memory reprogramming as a broadly applicable approach for optimizing therapeutic T cell states., (© 2024. The Author(s).)

Lysosome-targeting chimeras (LYTACs) are a promising therapeutic modality to drive the degradation of extracellular proteins. However, early versions of LYTAC contain synthetic glycopeptides that cannot be genetically encoded. Here, we present our designs for a fully genetically encodable LYTAC (GELYTAC), making our tool compatible with integration into therapeutic cells for targeted delivery at diseased sites. To achieve this, we replaced the glycopeptide portion of LYTACs with the protein insulin-like growth factor 2 (IGF2). After showing initial efficacy with wild-type IGF2, we increased the potency of GELYTAC using directed evolution. Subsequently, we demonstrated that our engineered GELYTAC construct not only secretes from HEK293T cells but also from human primary T-cells to drive the uptake of various targets into receiver cells. Immune cells engineered to secrete GELYTAC thus represent a promising avenue for spatially selective targeted protein degradation., Competing Interests: Competing interests statement:C.R.B. is a co-founder and scientific advisory board member of Lycia Therapeutics, Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent), OliLux Bio, InterVenn Bio, GanNA Bio, Firefly Bio, Neuravid, and Valora Therapeutics. S.A.Y.-H. is a consultant for Quince Therapeutics. L.L. is a cofounder of, consults for, and holds equity in CARGO Therapeutics. E.S. is a consultant for Lepton Pharmaceuticals and Galaria, and holds equity in Lyell Immunopharma. C.L.M. is a cofounder of Lyell Immunopharma, CARGO Therapeutics, and Link Cell Therapies, which are developing CAR-based therapies, and consults for CARGO, Link Immatics, Ensoma and Red Tree Capital. A.Y.T. is a scientific advisor to Third Rock Ventures and Nereid Therapeutics. The remaining authors declare no competing interest. C.R.B. owns founders shares of Lycia Therapeutics, which is developing medicines based on targeted extracellular protein degradation.

CRISPR technologies have begun to revolutionize T cell therapies; however, conventional CRISPR-Cas9 genome-editing tools are limited in their safety, efficacy, and scope. To address these challenges, we developed multiplexed effector guide arrays (MEGA), a platform for programmable and scalable regulation of the T cell transcriptome using the RNA-guided, RNA-targeting activity of CRISPR-Cas13d. MEGA enables quantitative, reversible, and massively multiplexed gene knockdown in primary human T cells without targeting or cutting genomic DNA. Applying MEGA to a model of CAR T cell exhaustion, we robustly suppressed inhibitory receptor upregulation and uncovered paired regulators of T cell function through combinatorial CRISPR screening. We additionally implemented druggable regulation of MEGA to control CAR activation in a receptor-independent manner. Lastly, MEGA enabled multiplexed disruption of immunoregulatory metabolic pathways to enhance CAR T cell fitness and anti-tumor activity in vitro and in vivo. MEGA offers a versatile synthetic toolkit for applications in cancer immunotherapy and beyond., Competing Interests: Declaration of interests The authors have filed a patent related to this work. E.S. consults for Lyell Immunopharma, Lepton Pharmaceuticals, and Galaria. C.L.M. is a cofounder of Lyell Immunopharma, CARGO Therapeutics, and Link Cell Therapies and consults for Lyell, CARGO, Link, Apricity, Nektar, Immatics, Ensoma, Mammoth, Glaxo Smith Kline, Bristol Myers Squibb, and RedTree Capital. L.S.Q. is a founder of Epic Bio and scientific advisor of Laboratory of Genomic Research and Kytopen Corp., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8 + CAR-T cells express CD39 and CD73, which mediate proximal steps in Ado generation. Here, we sought to enhance CAR-T cell potency by knocking out CD39, CD73, or adenosine receptor 2a (A2aR) but observed only modest effects. In contrast, overexpression of Ado deaminase (ADA-OE), which metabolizes Ado to inosine (INO), induced stemness and enhanced CAR-T functionality. Similarly, CAR-T cell exposure to INO augmented function and induced features of stemness. INO induced profound metabolic reprogramming, diminishing glycolysis, increasing mitochondrial and glycolytic capacity, glutaminolysis and polyamine synthesis, and reprogrammed the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR-T cell products meeting criteria for clinical dosing. These results identify INO as a potent modulator of CAR-T cell metabolism and epigenetic stemness programming and deliver an enhanced potency platform for cell manufacturing., Competing Interests: Declaration of interests D.D.K, S.A.F., and C.L.M. are co-inventors on a pending patent application for inosine media supplementation during cell manufacturing. D.D.K and C.L.M. are inventors on a patent application for the use of T cells overexpressing ADA1/2 for cancer immunotherapy. C.L.M. holds equity in and receives research funding from Lyell Immunopharma, holds equity in and consults for Link Cell Therapies and C.L.M., and L.L. hold equity and consult for CARGO Therapeutics. L.L. and E.S. hold equity in Lyell Immunopharma. E.S consults for Lepton Pharmaceuticals and Galaria. S.A.F. serves on the Scientific Advisory Boards for Alaunos Therapeutics and Fresh Wind Biotech and has equity interest in both; S.A.F. receives research funding from CARGO and Tune Therapeutics. S.P. is a current employee of and holds equity in CARGO. C.L.M. consults for Immatics, Mammoth, and Ensoma. A.T.S. is a cofounder of Immunai and Cartography Biosciences. A.T.S. receives research funding from Allogene Therapeutics and Merck Research Laboratories., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent developments in cancer immunotherapy have highlighted the potential of harnessing natural killer (NK) cells in the treatment of neoplastic malignancies. Of these, bispecific antibodies, and NK cell engager (NKCE) protein therapeutics in particular, have been of interest. Here, we used phage display and yeast surface display to engineer RLN131, a unique cross-reactive antibody that binds to human, mouse, and cynomolgus NKp46, an activating receptor found on NK cells. RLN131 induced proliferation and activation of primary NK cells, and was used to create bispecific NKCE constructs of varying configurations and valency. All NKCEs were able to promote greater NK cell cytotoxicity against tumor cells than an unmodified anti-CD20 monoclonal antibody, and activity was observed irrespective of whether the constructs contained a functional Fc domain. Competition binding and fine epitope mapping studies were used to demonstrate that RLN131 binds to a conserved epitope on NKp46, underlying its species cross-reactivity., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Chimeric antigen receptor T cells (CAR-Ts) have remarkable efficacy in liquid tumors, but limited responses in solid tumors. We conducted a Phase I trial (NCT02107963) of GD2 CAR-Ts (GD2-CAR.OX40.28.z.iC9), demonstrating feasibility and safety of administration in children and young adults with osteosarcoma and neuroblastoma. Since CAR-T efficacy requires adequate CAR-T expansion, patients were grouped into good or poor expanders across dose levels. Patient samples were evaluated by multi-dimensional proteomic, transcriptomic, and epigenetic analyses. T cell assessments identified naive T cells in pre-treatment apheresis associated with good expansion, and exhausted T cells in CAR-T products with poor expansion. Myeloid cell assessment identified CXCR3 + monocytes in pre-treatment apheresis associated with good expansion. Longitudinal analysis of post-treatment samples identified increased CXCR3 - classical monocytes in all groups as CAR-T numbers waned. Together, our data uncover mediators of CAR-T biology and correlates of expansion that could be utilized to advance immunotherapies for solid tumor patients., Competing Interests: Declaration of interests C.J.W. receives research funding from Pharmacyclics and hold equity in BioNTech, Inc. F.M. is a cofounder of and has equity in Harbinger Health, has equity in Zephyr AI, and serves as a consultant for Harbinger Health, Zephyr AI, and Red Cell Partners and Exscientia. F.M. declares that none of these relationships are directly or indirectly related to the content of this manuscript. E.S. consults for and holds equity in Lyell Immunopharma and consults for Lepton Pharmaceuticals and Galaria. M.S.M. is currently employed at Normunity and holds stock in AstraZeneca; her contributions to this work were made prior to these industry positions which are not relevant to the content of this manuscript. C.L.M. is an inventor on numerous patents and patents pending related to CAR-T cell therapies. C.L.M. holds equity in and receives research funding from Lyell Immunopharma and holds equity in and consults for CARGO Therapeutics and Link Cell Therapies. C.L.M. consults for Immatics, Mammoth, Ensoma, and Red Tree Venture Capital., (Published by Elsevier Inc.)

The central role of target antigen density on chimeric antigen receptor T cell potency highlights the need for accurate measurement of antigen levels on clinical tumor samples. Here, we present a protocol for quantifying antigen density for six cell-surface antigens on neuroblastoma cells metastatic to bone marrow. We describe steps for patient sample acquisition, flow cytometry panel development, instrument setup, and compensation and detail procedures for running clinical samples and data analysis. For complete details on the use and execution of this protocol, please refer to Heitzeneder et al. (2022). 1 ., Competing Interests: Declaration of interests S.H. and C.L.M. are co-inventors on patents relevant to CAR T cells targeting GPC2. C.L.M. has multiple patents pertinent to CAR T cells and is a co-founder of Lyell Immunopharma and CARGO Therapeutics, formerly Syncopation Life Sciences, which develop CAR-based therapies, and consults for Lyell, NeoImmune Tech, Apricity, Nektar, and Immatics. S.J.B. and M.M. are employees of BD Biosciences., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Tisagenlecleucel was approved by the Food and Drug Administration (FDA) in 2017 for refractory B-cell acute lymphoblastic leukemia (B-ALL) and B-ALL in ≥2nd relapse. Outcomes of patients receiving commercial tisagenlecleucel upon 1st relapse have yet to be established. We aimed to report real-world tisagenlecleucel utilisation patterns and outcomes across indications, specifically including patients treated in 1st relapse, an indication omitted from formal FDA approval., Methods: We conducted a retrospective analysis of real-world tisagenlecleucel utilisation patterns across 185 children and young adults treated between August 30, 2017 and March 6, 2020 from centres participating in the Pediatric Real-World CAR Consortium (PRWCC), within the United States. We described definitions of refractory B-ALL used in the real-world setting and categorised patients by reported Chimeric Antigen Receptor (CAR) T-cell indication, including refractory, 1st relapse and ≥2nd relapse B-ALL. We analysed baseline patient characteristics and post-tisagenlecleucel outcomes across defined cohorts., Findings: Thirty-six percent (n = 67) of our cohort received tisagenlecleucel following 1st relapse. Of 66 evaluable patients, 56 (85%, 95% CI 74-92%) achieved morphologic complete response. Overall-survival (OS) and event-free survival (EFS) at 1-year were 69%, (95% CI 58-82%) and 49%, (95% CI 37-64%), respectively, with survival outcomes statistically comparable to remaining patients (OS; p = 0.14 , EFS; p = 0.39 ). Notably, toxicity was increased in this cohort, warranting further study. Interestingly, of 30 patients treated for upfront refractory disease, 23 (77%, 95% CI 58-90%) had flow cytometry and/or next-generation sequencing (NGS) minimum residual disease (MRD)-only disease at the end of induction, not meeting the historic morphologic definition of refractory., Interpretation: Our findings suggested that tisagenlecleucel response and survival rates overlap across patients treated with upfront refractory B-ALL, B-ALL ≥2nd relapse and B-ALL in 1st relapse. We additionally highlighted that definitions of refractory B-ALL are evolving beyond morphologic measures of residual disease., Funding: St. Baldrick's/Stand Up 2 Cancer, Parker Institute for Cancer Immunotherapy, Virginia and D.K. Ludwig Fund for Cancer Research., Competing Interests: V.B. serves on the boards of ArsenalBio and Umoja Biopharma and consults or holds stock in Zafrens and Treeline Biosciences which are developing therapies for cancer treatment and Illumina, Invitae, Pacific Biosciences, and Guardant who are developing oncology NGS tests. C.L.M. is an inventor on several patents related to CAR T-cell therapies. C.L.M. is a cofounder of Lyell Immunopharma, CARGO Therapeutics and Link Cell Therapies, which are developing CAR-based therapies, and consults for Lyell, CARGO, Link, Ensoma, Mammoth, Immatics, Apricity, Glaxo Smith Klein, Nektar, Legend and Bristol Myers Squibb. C.L.M receives royalties for CD-22 CAR licensing from NIH, has had grant/contract funding from St. Baldrick’s Foundation, NIH, CIRM, Parker, Tune therapeutics, Lyell Immunopharma, Ludwig Institute, Emerson Collective, Department of Defense and Goldhirsh-Yellin Foundation. She is a member of the Board of Directors of CARGO Therapeutics and Link Cell Therapies and owns stocks in Lyell Immunopharma, CARGO Therapeutics, Link Cell Therapies, Ensoma, Mammoth and Apricity. T.W.L. served on advisory boards or consults for Novartis, Bayer, Aptitude Health, Jumo Health, Massive Bio, Medscape, AI Therapeutics, Jazz Pharmaceuticals, GentiBio, Menarini, Pyramid Biosciences, Targeted Oncology, Treeline Biosciences. He owns stocks/other ownership interest in advanced microbubbles. T.W.L. received research funding from Lily, Roche/Genentech, Taiho Oncology, Advanced Accelerator Applications/Novartis, Bristol-Myers Squibb, BioAtla, Pfizer, Bayer and Turning Point Therapeutics. G.D.M. received funding for medical writing from Novartis. C.L.P. served on an advisory board for Novartis. L.S. served on an advisory board for Novartis. H.S. served on an advisory board for Novartis. M.H. served on editorial advisory board for Novartis and Sobi Pharmaceuticals and is the Vice Chair for COG NHL committee and COG NHL Biology Committee. V.F. consulted for Adaptimmune. S.P. is supported by the UCSF-Stanford CERSI grant UOI FD005978 from the FDA. P.S. served on advisory board for Sobi Pharmaceuticals. A.K. received COG support for meeting attendance. K.J.C. received grant support for an investigator-initiated trial and sat on advisory boards for Novartis and Atara Biotherapeutics. M.R.V. consults for Novartis, Sanofi, Qihan, Forge, Takada and Equillium. M.R.V. has a provisional patent describing methods of producing and using immunotherapy for cancer. M.R.V.participates on the safety monitoring/advisory board for FBX-101 and owns stocks/options for Fate therapeutics., (© 2023 The Authors.)

Pediatric patients with relapsed or refractory rhabdomyosarcoma (RMS) have dismal cure rates, and effective therapy is urgently needed. The oncogenic receptor tyrosine kinase fibroblast growth factor receptor 4 (FGFR4) is highly expressed in RMS and lowly expressed in healthy tissues. Here, we describe a second-generation FGFR4-targeting chimeric antigen receptor (CAR), based on an anti-human FGFR4-specific murine monoclonal antibody 3A11, as an adoptive T cell treatment for RMS. The 3A11 CAR T cells induced robust cytokine production and cytotoxicity against RMS cell lines in vitro. In contrast, a panel of healthy human primary cells failed to activate 3A11 CAR T cells, confirming the selectivity of 3A11 CAR T cells against tumors with high FGFR4 expression. Finally, we demonstrate that 3A11 CAR T cells are persistent in vivo and can effectively eliminate RMS tumors in two metastatic and two orthotopic models. Therefore, our study credentials CAR T cell therapy targeting FGFR4 to treat patients with RMS., Competing Interests: Declaration of interests J. Khan, R.J.O., D.S.D., and A.T.C. are inventors on international patent application no. PCT/US2016/052496. The 3A11 CAR sequence is in this patent application (see https://patents.justia.com/patent/11078286) filed on September 19, 2016, titled “Monoclonal antibodies specific for fibroblast growth factor receptor 4 (FGFR4) and methods of their use.”, (Published by Elsevier Inc.)

Introduction: Antigen downregulation and early chimeric antigen receptor (CAR) T-cell loss have emerged as two major challenges threatening outcomes following CD19-specific CAR T-cell therapy for children and young adults with B-cell acute lymphoblastic leukemia (B-ALL). In addressing the future of CAR T-cell therapy for B-ALL, innovative strategies to avert antigen downregulation and enhance CAR persistence warrant prioritized focus., Areas Covered: We describe promising engineering strategies to refine CAR constructs to reverse exhaustion, develop regulatable CARs, optimize manufacturing, enrich for immune memory, and disrupt immune inhibition. We additionally focus on alternative targeting to CD19-monospecific targeting and contextualize possibilities for expanded CAR utilization., Expert Opinion: We describe research advances as they are independently reported, however, anticipate an integrative strategy incorporating complementary modifications will be required to effectively address CAR loss, overcome antigen downregulation, and enhance reliability and durability of CAR T-cell responses for B-ALL.

Chimeric antigen receptor-associated hemophagocytic lymphohistiocytosis (HLH)-like toxicities (LTs) involving hyperferritinemia, multiorgan dysfunction, coagulopathy, and/or hemophagocytosis are described as occurring in a subset of patients with cytokine release syndrome (CRS). Case series report poor outcomes for those with B-cell acute lymphoblastic leukemia (B-ALL) who develop HLH-LTs, although larger outcomes analyses of children and young adults (CAYAs) with B-ALL who develop these toxicities after the administration of commercially available tisagenlecleucel are not described. Using a multi-institutional database of 185 CAYAs with B-ALL, we conducted a retrospective cohort study including groups that developed HLH-LTs, high-grade (HG) CRS without HLH-LTs, or no to low-grade (NLG) CRS without HLH-LTs. Primary objectives included characterizing the incidence, outcomes, and preinfusion factors associated with HLH-LTs. Among 185 CAYAs infused with tisagenlecleucel, 26 (14.1%) met the criteria for HLH-LTs. One-year overall survival and relapse-free survival were 25.7% and 4.7%, respectively, in those with HLH-LTs compared with 80.1% and 57.6%, respectively, in those without. In multivariable analysis for death, meeting criteria for HLH-LTs carried a hazard ratio of 4.61 (95% confidence interval, 2.41-8.83), controlling for disease burden, age, and sex. Patients who developed HLH-LTs had higher pretisagenlecleucel disease burden, ferritin, and C-reactive protein levels and lower platelet and absolute neutrophil counts than patients with HG- or NLG-CRS without HLH-LTs. Overall, CAYAs with B-ALL who developed HLH-LTs after tisagenlecleucel experienced high rates of relapse and nonrelapse mortality, indicating the urgent need for further investigations into prevention and optimal management of patients who develop HLH-LTs after tisagenlecleucel., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: Chimeric Antigen Receptor (CAR) T cells are now standard of care (SOC) for some patients with B cell and plasma cell malignancies and could disrupt the therapeutic landscape of solid tumors. However, access to CAR-T cells is not adequate to meet clinical needs, in part due to high cost and long lead times for manufacturing clinical grade virus. Non-viral site directed CAR integration can be accomplished using CRISPR/Cas9 and double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) via homology-directed repair (HDR), however yields with this approach have been limiting for clinical application (dsDNA) or access to large yields sufficient to meet the manufacturing demands outside early phase clinical trials is limited (ssDNA)., Methods: We applied homology-independent targeted insertion (HITI) or HDR using CRISPR/Cas9 and nanoplasmid DNA to insert an anti-GD2 CAR into the T cell receptor alpha constant (TRAC) locus and compared both targeted insertion strategies in our system. Next, we optimized post-HITI CRISPR EnrichMENT (CEMENT) to seamlessly integrate it into a 14-day process and compared our knock-in with viral transduced anti-GD2 CAR-T cells. Finally, we explored the off-target genomic toxicity of our genomic engineering approach., Results: Here, we show that site directed CAR integration utilizing nanoplasmid DNA delivered via HITI provides high cell yields and highly functional cells. CEMENT enriched CAR T cells to approximately 80% purity, resulting in therapeutically relevant dose ranges of 5.5 × 10 8 -3.6 × 10 9 CAR + T cells. CRISPR knock-in CAR-T cells were functionally comparable with viral transduced anti-GD2 CAR-T cells and did not show any evidence of off-target genomic toxicity., Conclusions: Our work provides a novel platform to perform guided CAR insertion into primary human T-cells using nanoplasmid DNA and holds the potential to increase access to CAR-T cell therapies., (© 2023. The Author(s).)

Regulatory T cell (Treg) therapy is a promising approach to improve outcomes in transplantation and autoimmunity. In conventional T cell therapy, chronic stimulation can result in poor in vivo function, a phenomenon termed exhaustion. Whether or not Tregs are also susceptible to exhaustion, and if so, if this would limit their therapeutic effect, was unknown. To "benchmark" exhaustion in human Tregs, we used a method known to induce exhaustion in conventional T cells: expression of a tonic-signaling chimeric antigen receptor (TS-CAR). We found that TS-CAR-expressing Tregs rapidly acquired a phenotype that resembled exhaustion and had major changes in their transcriptome, metabolism, and epigenome. Similar to conventional T cells, TS-CAR Tregs upregulated expression of inhibitory receptors and transcription factors such as PD-1, TIM3, TOX and BLIMP1, and displayed a global increase in chromatin accessibility-enriched AP-1 family transcription factor binding sites. However, they also displayed Treg-specific changes such as high expression of 4-1BB, LAP, and GARP. DNA methylation analysis and comparison to a CD8 + T cell-based multipotency index showed that Tregs naturally exist in a relatively differentiated state, with further TS-CAR-induced changes. Functionally, TS-CAR Tregs remained stable and suppressive in vitro but were nonfunctional in vivo, as tested in a model of xenogeneic graft-versus-host disease. These data are the first comprehensive investigation of exhaustion in Tregs and reveal key similarities and differences with exhausted conventional T cells. The finding that human Tregs are susceptible to chronic stimulation-driven dysfunction has important implications for the design of CAR Treg adoptive immunotherapy strategies.

Cancer immunotherapies have unique toxicities. Establishment of grading scales and standardized grade-based treatment algorithms for toxicity syndromes can improve the safety of these treatments, as observed for cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS) in patients with B cell malignancies treated with chimeric antigen receptor (CAR) T cell therapy. We have observed a toxicity syndrome, distinct from CRS and ICANS, in patients treated with cell therapies for tumors in the central nervous system (CNS), which we term tumor inflammation-associated neurotoxicity (TIAN). Encompassing the concept of 'pseudoprogression,' but broader than inflammation-induced edema alone, TIAN is relevant not only to cellular therapies, but also to other immunotherapies for CNS tumors. To facilitate the safe administration of cell therapies for patients with CNS tumors, we define TIAN, propose a toxicity grading scale for TIAN syndrome and discuss the potential management of this entity, with the goal of standardizing both reporting and management., (© 2023. Springer Nature America, Inc.)

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.)

Remarkable complete response rates have been shown with tisagenlecleucel, a chimeric antigen receptor (CAR) T-cell therapy targeting CD19, in patients up to age 26 years with refractory/relapsed B-cell acute lymphoblastic leukemia; it is US Food and Drug Administration approved for this indication. Currently, patients receive a single dose of tisagenlecleucel across a wide dose range of 0.2 to 5.0 × 106 and 0.1 to 2.5 × 108 CAR T cells per kg for patients ≤50 and >50 kg, respectively. The effect of cell dose on survival and remission is not yet well established. Our primary goal was to determine if CAR T-cell dose affects overall survival (OS), event-free survival (EFS), or relapse-free-survival (RFS) in tisagenlecleucel recipients. Retrospective data were collected from Pediatric Real World CAR Consortium member institutions and included 185 patients infused with commercial tisagenlecleucel. The median dose of viable transduced CAR T cells was 1.7 × 106 CAR T cells per kg. To assess the impact of cell dose, we divided responders into dose quartiles: 0.134 to 1.300 × 106 (n = 48 [27%]), 1.301 to 1.700 × 106 (n = 46 [26%]), 1.701 to 2.400 × 106 (n = 43 [24%]), and 2.401 to 5.100 × 106 (n = 43 [24%]). OS, EFS, and RFS were improved in patients who received higher doses of tisagenlecleucel (P = .031, .0079, and .0045, respectively). Higher doses of tisagenlecleucel were not associated with increased toxicity. Because the current tisagenlecleucel package insert dose range remains broad, this work has implications in regard to targeting higher cell doses, within the approved dose range, to optimize patients' potential for long-standing remission., (Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: Chimeric antigen receptor (CAR) T cell therapy is an effective salvage therapy for pediatric relapsed B-cell acute lymphoblastic leukemia (B-ALL), yet is challenged by high rates of post-CAR relapse. Literature describing specific relapse patterns and extramedullary (EM) sites of involvement in the post-CAR setting remains limited, and a clinical standard for post-CAR disease surveillance has yet to be established. We highlight the importance of integrating peripheral blood minimal residual disease (MRD) testing and radiologic imaging into surveillance strategies, to effectively characterize and capture post-CAR relapse., Main Body: Here, we describe the case of a child with multiply relapsed B-ALL who relapsed in the post-CAR setting with gross non-contiguous medullary and EM disease. Interestingly, her relapse was identified first from peripheral blood flow cytometry MRD surveillance, in context of a negative bone marrow aspirate (MRD <0.01%). Positron emission tomography with 18F-fluorodeoxyglucose revealed diffuse leukemia with innumerable bone and lymph node lesions, interestingly sparing her sacrum, the site of her bone marrow aspirate sampling., Conclusions: We highlight this case as both peripheral blood MRD and 18F-fluorodeoxyglucose positron emission tomography imaging were more sensitive than standard bone marrow aspirate testing in detecting this patient's post-CAR relapse. Clinical/Biologic Insight: In the multiply relapsed B-ALL setting, where relapse patterns may include patchy medullary and/or EM disease, peripheral blood MRD and/or whole body imaging, may carry increased sensitivity at detecting relapse in patient subsets, as compared with standard bone marrow sampling., Competing Interests: Competing interests: CM is an inventor on multiple patents for CAR T cells. CM is a cofounder and holds equity in Lyell Immunopharma and Syncopation Life Sciences, which are developing CAR-based therapies, and consults for Lyell, NeoImmune Tech, Apricity, Nektar and Immatics, Ensome and Mammoth., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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.)

Purpose: Nonresponse and relapse after CD19-chimeric antigen receptor (CAR) T-cell therapy continue to challenge survival outcomes. Phase II landmark data from the ELIANA trial demonstrated nonresponse and relapse rates of 14.5% and 28%, respectively, whereas use in the real-world setting showed nonresponse and relapse rates of 15% and 37%. Outcome analyses describing fate after post-CAR nonresponse and relapse remain limited. Here, we aim to establish survival outcomes after nonresponse and both CD19+ and CD19- relapses and explore treatment variables associated with inferior survival., Methods: We conducted a retrospective multi-institutional study of 80 children and young adults with B-cell acute lymphoblastic leukemia experiencing nonresponse (n = 23) or relapse (n = 57) after tisagenlecleucel. We analyze associations between baseline characteristics and these outcomes and establish survival rates and salvage approaches., Results: The overall survival (OS) at 12 months was 19% across nonresponders (n = 23; 95% CI, 7 to 50). Ninety-five percent of patients with nonresponse had high preinfusion disease burden. Among 156 morphologic responders, the cumulative incidence of relapse was 37% (95% CI, 30 to 47) at 12 months (CD19+; 21% [15 to 29], CD19-; 16% [11 to 24], median follow-up; 380 days). Across 57 patients experiencing relapse, the OS was 52% (95% CI, 38 to 71) at 12 months after time of relapse. Notably, CD19- relapse was associated with significantly decreased OS as compared with patients who relapsed with conserved CD19 expression (CD19- 12-month OS; 30% [14 to 66], CD19+ 12-month OS; 68% [49 to 92], P = .0068). Inotuzumab, CAR reinfusion, and chemotherapy were used as postrelapse salvage therapy with greatest frequency, yet high variability in treatment sequencing and responses limits efficacy analysis across salvage approaches., Conclusion: We describe poor survival across patients experiencing nonresponse to tisagenlecleucel. In the post-tisagenlecleucel relapse setting, patients can be salvaged; however, CD19- relapse is distinctly associated with decreased survival outcomes.

Most relapsed/refractory large B cell lymphoma (r/rLBCL) patients receiving anti-CD19 chimeric antigen receptor (CAR19) T cells relapse. To characterize determinants of resistance, we profiled over 700 longitudinal specimens from two independent cohorts (n = 65 and n = 73) of r/rLBCL patients treated with axicabtagene ciloleucel. A method for simultaneous profiling of circulating tumor DNA (ctDNA), cell-free CAR19 (cfCAR19) retroviral fragments, and cell-free T cell receptor rearrangements (cfTCR) enabled integration of tumor and both engineered and non-engineered T cell effector-mediated factors for assessing treatment failure and predicting outcomes. Alterations in multiple classes of genes are associated with resistance, including B cell identity (PAX5 and IRF8), immune checkpoints (CD274), and those affecting the microenvironment (TMEM30A). Somatic tumor alterations affect CAR19 therapy at multiple levels, including CAR19 T cell expansion, persistence, and tumor microenvironment. Further, CAR19 T cells play a reciprocal role in shaping tumor genotype and phenotype. We envision these findings will facilitate improved chimeric antigen receptor (CAR) T cells and personalized therapeutic approaches., Competing Interests: Declaration of interests B.J.S. reports consultancy for Foresight Diagnostics. D.M.K. reports consultancy for Roche, Adaptive Biotechnologies, and Genentech and equity ownership interest in Foresight Diagnostics S.K.A. reports speaker honoraria from Takeda. M.J.F. reports consultancy and research funding from Adaptive Biotechnologies, research funding from Kite/Gilead, stock options from Allogene Therapeutics, and equity in Roche/Genentech. M.S.E. reports consultancy for Foresight Diagnostics. J.H.B. reports research funding from Kite Pharma. S.B. reports employment and stock ownership at Kite-a Gilead company. J.W. has research funding from Kite/Gilead, BMS, Novartis, Genentech/Roche, Morphosys/Incyte, AstraZeneca, and ADC Therapeutics, and consulting funding for Kite/Gilead, BMS, Novartis, Genentech/Roche, Morphosys/Incyte, AstraZeneca, ADC Therapeutics, Merck, MonteRosa, Umoja, and Ikusda. M.S.K. reports research funding from CRISPR Therapeutics and Nutcracker Therapeutics, and advisory committee membership for Myeloid Therapeutics and Daiichi Sankyo. Y.N. reports consulting for Leica Biosystems and Roche, and research funding from Kite Pharma. C.L.M. holds several patents focused on CAR T cells therapies; is a co-founder and holds equity in Lyell Immunopharma, CARGO Therapeutics, and Link Cell Therapies, which are developing CAR-based therapies; and consults for Lyell, CARGO, Link, Apricity, Nektar, Immatics, Mammoth, and Ensoma. R.G.M. is a co-founder of and holds equity in Link Cell Therapies and Syncopation Life Sciences. R.G.M. is a consultant for Lyell Immunopharma, NKarta, Arovella Pharmaceuticals, Innervate Radiopharmaceuticals, GammaDelta Therapeutics, Aptorum Group, Zai Labs, ImmunAI, Gadeta, FATE Therapeutics (DSMB), and Waypoint Bio. M.D. reports research funding from AstraZeneca, Genentech, Varian Medical Systems, and Illumina; ownership interest in CiberMed and Foresight Diagnostics; and consultancy from AstraZeneca, Boehringer Ingelheim, Bristol Myers Squibb, Genentech, Gritstone Oncology, Illumina, Novartis, and Roche. D.B.M. holds a patent with Pharmacyclics supporting ibrutinib for chronic graft-versus-host disease and receives consulting or research fees or serves as an advisor for Pharmacyclics, Kite Pharma, Adaptive Biotechnologies, Novartis, BMS, Janssen Pharmaceuticals, Roche, Genentech, Precision Bioscience, Allogene, Miltenyi Biotec, Fate Therapeutics, 2Seventy, and Adicet. A.A.A. reports consultancy for Celgene, Chugai, Genentech, Gilead, Janssen, Pharmacyclics, and Roche; scientific advisory board membership in the Lymphoma Research Foundation; professional affiliations with the American Society of Hematology, American Society of Clinical Oncology, American Society of Clinical Investigation, and Leukemia & Lymphoma Society; research funding from the National Cancer Institute, National Heart, Lung, and Blood Institute, National Institutes of Health, Celgene, Bristol Myers Squibb, and Pfizer; patent filings, including patent issued, licensed, and with royalties paid from FortySeven, a patent pending and Licensed to Foresight, a patent pending relating to MARIA, a patent issued and licensed to CiberMed, a patent issued, a patent pending to CiberMed, a patent issued to Idio-type Vaccines, and a patent issued, licensed, and with royalties paid From Roche; and equity ownership interests in CiberMed Inc., Foresight Diagnostics, FortySeven Inc., and CARGO Therapeutics. B.J.S., D.M.K., M.S.E., M.D., and A.A.A. also report patent filings related to cancer biomarkers. The remaining authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Cancer outcomes with chemotherapy are inferior in patients of minority racial/ethnic groups and those with obesity. Chimeric antigen receptor (CAR) T-cell therapy has transformed outcomes for relapsed/refractory hematologic malignancies, but whether its benefits extend commensurately to racial/ethnic minorities and patients with obesity is poorly understood. With a primary focus on patients with B-cell acute lymphoblastic leukemia (B-ALL), we retrospectively evaluated the impact of demographics and obesity on CAR T-cell therapy outcomes in adult and pediatric patients with hematologic malignancies treated with CAR T-cell therapy across 5 phase 1 clinical trials at the National Cancer Institute from 2012 to 2021. Among 139 B-ALL CAR T-cell infusions, 28.8% of patients were Hispanic, 3.6% were Black, and 29.5% were overweight/obese. No significant associations were found between race, ethnicity, or body mass index (BMI) and complete remission rates, neurotoxicity, or overall survival. Hispanic patients were more likely to experience severe cytokine release syndrome compared with White non-Hispanic patients even after adjusting for leukemia disease burden and age (odds ratio, 4.5; P = .001). A descriptive analysis of patients with multiple myeloma (n = 24) and non-Hodgkin lymphoma (n = 23) displayed a similar pattern to the B-ALL cohort. Our findings suggest CAR T-cell therapy may provide substantial benefit across a range of demographics characteristics, including for those populations who are at higher risk for chemotherapy resistance and relapse. However, toxicity profiles may vary. Therefore, efforts to improve access to CAR therapy for underrepresented populations and elucidate mechanisms of differential toxicity among demographic groups should be prioritized., (Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Purpose: In many cancers, nivolumab in combination with ipilimumab improves response rates compared with either agent alone, but the combination has not been evaluated in childhood cancer. We conducted a phase I/II trial of nivolumab plus ipilimumab in children and young adults with recurrent/refractory solid tumors., Patients and Methods: ADVL1412, Part C assessed safety of nivolumab plus ipilimumab at two dose levels (DL): DL1 1 mg/kg of each drug and DL2 3 mg/kg nivolumab plus 1 mg/kg ipilimumab. Part D evaluated response at the recommended phase II dose (RP2D) in Ewing sarcoma, rhabdomyosarcoma, and osteosarcoma. Part E tested DL3 (1 mg/kg nivolumab plus 3 mg/kg ipilimumab) in Ewing sarcoma and rhabdomyosarcoma. Tumor response was measured using RECIST v1.1. Pharmacokinetics and PD-L1 expression on archival tissues were assessed., Results: Fifty-five eligible patients enrolled. Based on safety, tolerability, and similar drug exposure to the same doses administered in adults, DL2 was defined as the pediatric RP2D. Among 41 patients treated at the RP2D, 2 patients experienced dose-limiting toxicities during cycle 1, and 4 patients experienced toxicities beyond that period. Two patients had clinically significant sustained partial responses (1 rhabdomyosarcoma, 1 Ewing sarcoma) and 4 had stable disease. Among 8 patients treated at DL3, 3 dose-limiting toxicities (DLT) occurred, all immune-related adverse events; no objective responses were observed., Conclusions: The RP2D of nivolumab (3 mg/kg) plus ipilimumab (1 mg/kg) is well tolerated in children and young adults with solid tumors and shows some clinical activity. Increased dose of ipilimumab (3 mg/kg) plus nivolumab (1 mg/kg) was associated with increased toxicity without clinical benefit., (©2022 American Association for Cancer Research.)

T cells are the major arm of the immune system responsible for controlling and regressing cancers. To identify genes limiting T cell function, we conducted genome-wide CRISPR knockout screens in human chimeric antigen receptor (CAR) T cells. Top hits were MED12 and CCNC , components of the Mediator kinase module. Targeted MED12 deletion enhanced antitumor activity and sustained the effector phenotype in CAR- and T cell receptor-engineered T cells, and inhibition of CDK8/19 kinase activity increased expansion of nonengineered T cells. MED12 -deficient T cells manifested increased core Meditator chromatin occupancy at transcriptionally active enhancers-most notably for STAT and AP-1 transcription factors-and increased IL2RA expression and interleukin-2 sensitivity. These results implicate Mediator in T cell effector programming and identify the kinase module as a target for enhancing potency of antitumor T cell responses.

Approximately 60% of patients with large B cell lymphoma treated with chimeric antigen receptor (CAR) T cell therapies targeting CD19 experience disease progression, and neurotoxicity remains a challenge. Biomarkers associated with resistance and toxicity are limited. In this study, single-cell proteomic profiling of circulating CAR T cells in 32 patients treated with CD19-CAR identified that CD4 + Helios + CAR T cells on day 7 after infusion are associated with progressive disease and less severe neurotoxicity. Deep profiling demonstrated that this population is non-clonal and manifests hallmark features of T regulatory (T Reg ) cells. Validation cohort analysis upheld the link between higher CAR T Reg cells with clinical progression and less severe neurotoxicity. A model combining expansion of this subset with lactate dehydrogenase levels, as a surrogate for tumor burden, was superior for predicting durable clinical response compared to models relying on each feature alone. These data credential CAR T Reg cell expansion as a novel biomarker of response and toxicity after CAR T cell therapy and raise the prospect that this subset may regulate CAR T cell responses in humans., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Neurotoxicity is an established toxicity of CD19 CAR T-cell therapy; however, there is little information on neurotoxicity in children, adolescents, and young adults (CAYA) receiving CD19/CD28ζ CAR T-cells for B-cell malignancies., Methods: We analyzed neurotoxicity of CD19/CD28ζ CAR T-cells in CAYA treated on a phase I study (NCT01593696). Assessments included daily inpatient monitoring, caregiver-based neuro-symptom checklist (NSC), exploratory neurocognitive assessments, clinically-indicated imaging, CSF analysis, and systematic cytokine profiling, outcomes of which were associated with cytokine release syndrome (CRS) and treatment response postinfusion. Patients with active CNS leukemia were included., Results: Amongst 52 patients treated, 13 patients had active CNS leukemia at infusion. Neurotoxicity was seen in 11/52 (21.2%) patients, with an incidence of 29.7% (11/37) in patients with CRS. Neurotoxicity was associated with the presence and severity of CRS. Those with neurotoxicity had higher levels of peak serum IL-6, IFNγ, and IL-15. Additionally, CNS leukemia was effectively eradicated in most patients with CRS. Pilot neurocognitive testing demonstrated stable-to-improved neurocognitive test scores in most patients, albeit limited by small patient numbers. The NSC enabled caregiver input into the patient experience., Conclusions: This is the first systematic analysis of neurotoxicity utilizing a CD19/CD28ζ CAR construct in CAYA, including in those with active CNS involvement. The experience demonstrates that the neurotoxicity profile was acceptable and reversible, with evidence of anti-leukemia response and CNS trafficking of CAR T-cells. Additionally, neurocognitive testing, while exploratory, provides an opportunity for future studies to employ systematic evaluations into neurotoxicity assessments and validation is needed in future studies., (Published by Oxford University Press on behalf of the Society for Neuro-Oncology 2022.)

Remission durability following single-antigen targeted chimeric antigen receptor (CAR) T-cells is limited by antigen modulation, which may be overcome with combinatorial targeting. Building upon our experiences targeting CD19 and CD22 in B-cell acute lymphoblastic leukemia (B-ALL), we report on our phase 1 dose-escalation study of a novel murine stem cell virus (MSCV)-CD19/CD22-4-1BB bivalent CAR T-cell (CD19.22.BBζ) for children and young adults (CAYA) with B-cell malignancies. Primary objectives included toxicity and dose finding. Secondary objectives included response rates and relapse-free survival (RFS). Biologic correlatives included laboratory investigations, CAR T-cell expansion and cytokine profiling. Twenty patients, ages 5.4 to 34.6 years, with B-ALL received CD19.22.BBζ. The complete response (CR) rate was 60% (12 of 20) in the full cohort and 71.4% (10 of 14) in CAR-naïve patients. Ten (50%) developed cytokine release syndrome (CRS), with 3 (15%) having ≥ grade 3 CRS and only 1 experiencing neurotoxicity (grade 3). The 6- and 12-month RFS in those achieving CR was 80.8% (95% confidence interval [CI]: 42.4%-94.9%) and 57.7% (95% CI: 22.1%-81.9%), respectively. Limited CAR T-cell expansion and persistence of MSCV-CD19.22.BBζ compared with EF1α-CD22.BBζ prompted laboratory investigations comparing EF1α vs MSCV promoters, which did not reveal major differences. Limited CD22 targeting with CD19.22.BBζ, as evaluated by ex vivo cytokine secretion and leukemia eradication in humanized mice, led to development of a novel bicistronic CD19.28ζ/CD22.BBζ construct with enhanced cytokine production against CD22. With demonstrated safety and efficacy of CD19.22.BBζ in a heavily pretreated CAYA B-ALL cohort, further optimization of combinatorial antigen targeting serves to overcome identified limitations (www.clinicaltrials.gov #NCT03448393)., (© 2022 by The American Society of Hematology.)

Infants with B-cell acute lymphoblastic leukemia (B-ALL) have poor outcomes because of chemotherapy resistance leading to high relapse rates. Tisagenlecleucel, a CD19-directed chimeric antigen receptor T-cell (CART) therapy, is US Food and Drug Administration approved for relapsed or refractory B-ALL in patients ≤25 years; however, the safety and efficacy of this therapy in young patients is largely unknown because children <3 years of age were excluded from licensing studies. We retrospectively evaluated data from the Pediatric Real-World CAR Consortium to examine outcomes of patients with infant B-ALL who received tisagenlecleucel between 2017 and 2020 (n = 14). Sixty-four percent of patients (n = 9) achieved minimal residual disease-negative remission after CART and 50% of patients remain in remission at last follow-up. All patients with high disease burden at time of CART infusion (>M1 marrow) were refractory to this therapy (n = 5). Overall, tisagenlecleucel was tolerable in this population, with only 3 patients experiencing ≥grade 3 cytokine release syndrome. No neurotoxicity was reported. This is the largest report of tisagenlecleucel use in infant B-ALL and shows that this therapy is safe and can be effective in this population. Incorporating this novel immunotherapy into the treatment of infant B-ALL offers a promising therapy for a highly aggressive leukemia., (Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

T cell exhaustion limits antitumor immunity, but the molecular determinants of this process remain poorly understood. Using a chronic stimulation assay, we performed genome-wide CRISPR-Cas9 screens to systematically discover regulators of T cell exhaustion, which identified an enrichment of epigenetic factors. In vivo CRISPR screens in murine and human tumor models demonstrated that perturbation of the INO80 and BAF chromatin remodeling complexes improved T cell persistence in tumors. In vivo Perturb-seq revealed distinct transcriptional roles of each complex and that depletion of canonical BAF complex members, including Arid1a, resulted in the maintenance of an effector program and downregulation of exhaustion-related genes in tumor-infiltrating T cells. Finally, Arid1a depletion limited the acquisition of exhaustion-associated chromatin accessibility and led to improved antitumor immunity. In summary, we provide an atlas of the genetic regulators of T cell exhaustion and demonstrate that modulation of epigenetic state can improve T cell responses in cancer immunotherapy., Competing Interests: Declaration of interests A.T.S. is a scientific founder of Immunai and founder of Cartography Biosciences and receives research funding from Arsenal Biosciences, Allogene Therapeutics, and Merck Research Laboratories. J.A.B. is a consultant to Immunai. S.A.V. is an advisor to Immunai. K.E.Y. is a consultant to Cartography Biosciences. C.L.M. is a co-founder of Lyell Immunopharma and Syncopation Life Sciences, and consults for Lyell, Syncopation, NeoImmune Tech, Apricity, Nektar, Immatics, Mammoth, and Ensoma. A.A. is a co-founder of Tango Therapeutics, Azkarra Therapeutics, Ovibio Corporation, and Kytarro; a consultant for SPARC, Bluestar, Pro-Lynx, Earli, Cura, GenVivo, Ambagon, Phoenix Molecular Designs, and GlaxoSmithKline (GSK); a member of the Scientific Advisory Board of Genentech, GLAdiator, Circle and Cambridge Science Corporation; receives research support from SPARC and AstraZeneca; holds patents on the use of poly (ADP-ribose) polymerase (PARP) inhibitors held jointly with AstraZeneca. A.M. is a co-founder of Spotlight Therapeutics, Arsenal Biosciences, and Survey Genomics; a member of the Scientific Advisory Board of NewLimit; owns stock in Arsenal Biosciences, Spotlight Therapeutics, NewLimit, Survey Genomics, PACT Pharma, and Merck; has received fees from 23andMe, PACT Pharma, Juno Therapeutics, Trizell, Vertex, Merck, Amgen, Genentech, AlphaSights, Rupert Case Management, Bernstein, and ALDA; is an investor in and informal advisor to Offline Ventures; and is a client of EPIQ. The Marson lab has received research support from Juno Therapeutics, Epinomics, Sanofi, GSK, Gilead, and Anthem. K.A.F., E.S., J.C., A.A., A.M., and C.L.M. hold patents in the arena of CAR-T cell therapeutics. J.A.B. and A.T.S. have filed a patent related to the contents of this study., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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.)