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Discusses research on the enhancement of immune reconstitution after stem cell transplants with cytokines. Potential of interleukin-2 for immunorestoration; Information on interleukin-7 treatment in T-cell; Pathways of immune reconstitution following T-cell depletion.

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).)

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.)

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).)

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.

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.)

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.)

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.)

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.)

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.)

Chimeric antigen receptor (CAR) T cells provide a therapeutic option in hematologic malignancies. However, treatment failure after initial response approaches 50%. In allogeneic hematopoietic cell transplantation, optimal fludarabine exposure improves immune reconstitution, resulting in lower nonrelapse mortality and increased survival. We hypothesized that optimal fludarabine exposure in lymphodepleting chemotherapy before CAR T-cell therapy would improve outcomes. In a retrospective analysis of patients with relapsed/refractory B-cell acute lymphoblastic leukemia undergoing CAR T-cell (tisagenlecleucel) infusion after cyclophosphamide/fludarabine lymphodepleting chemotherapy, we estimated fludarabine exposure as area under the curve (AUC; mg × h/L) using a validated population pharmacokinetic (PK) model. Fludarabine exposure was related to overall survival (OS), cumulative incidence of relapse (CIR), and a composite end point (loss of B-cell aplasia [BCA] or relapse). Eligible patients (n = 152) had a median age of 12.5 years (range, <1 to 26), response rate of 86% (n = 131 of 152), 12-month OS of 75.1% (95% confidence interval [CI], 67.6% to 82.6%), and 12-month CIR of 36.4% (95% CI, 27.5% to 45.2%). Optimal fludarabine exposure was determined as AUC ≥13.8 mg × h/L. In multivariable analyses, patients with AUC <13.8 mg × h/L had a 2.5-fold higher CIR (hazard ratio [HR], 2.45; 95% CI, 1.34-4.48; P = .005) and twofold higher risk of relapse or loss of BCA (HR, 1.96; 95% CI, 1.19-3.23; P = .01) compared with those with optimal fludarabine exposure. High preinfusion disease burden was also associated with increased risk of relapse (HR, 2.66; 95% CI, 1.45-4.87; P = .001) and death (HR, 4.77; 95% CI, 2.10-10.9; P < .001). Personalized PK-directed dosing to achieve optimal fludarabine exposure should be tested in prospective trials and, based on this analysis, may reduce disease relapse after CAR T-cell therapy., (© 2022 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.)

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

Diffuse intrinsic pontine glioma (DIPG) and other H3K27M-mutated diffuse midline gliomas (DMGs) are universally lethal paediatric tumours of the central nervous system 1 . 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 cells 2 , 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 × 10 6 GD2-CAR T cells per kg administered intravenously). Patients who exhibited clinical benefit were eligible for subsequent GD2-CAR T cell infusions administered intracerebroventricularly 3 . 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., (© 2022. The Author(s).)

The disialoganglioside GD2 is overexpressed on several solid tumors, and monoclonal antibodies targeting GD2 have substantially improved outcomes for children with high-risk neuroblastoma. However, approximately 40% of patients with neuroblastoma still relapse, and anti-GD2 has not mediated significant clinical activity in any other GD2 + malignancy. Macrophages are important mediators of anti-tumor immunity, but tumors resist macrophage phagocytosis through expression of the checkpoint molecule CD47, a so-called 'Don't eat me' signal. In this study, we establish potent synergy for the combination of anti-GD2 and anti-CD47 in syngeneic and xenograft mouse models of neuroblastoma, where the combination eradicates tumors, as well as osteosarcoma and small-cell lung cancer, where the combination significantly reduces tumor burden and extends survival. This synergy is driven by two GD2-specific factors that reorient the balance of macrophage activity. Ligation of GD2 on tumor cells (a) causes upregulation of surface calreticulin, a pro-phagocytic 'Eat me' signal that primes cells for removal and (b) interrupts the interaction of GD2 with its newly identified ligand, the inhibitory immunoreceptor Siglec-7. This work credentials the combination of anti-GD2 and anti-CD47 for clinical translation and suggests that CD47 blockade will be most efficacious in combination with monoclonal antibodies that alter additional pro- and anti-phagocytic signals within the tumor microenvironment., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Chimeric antigen receptor (CAR) T cells have transformed the therapeutic options for relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia. Data for CAR therapy in extramedullary (EM) involvement are limited. Retrospective data were abstracted from the Pediatric Real World CAR Consortium (PRWCC) of 184 infused patients from 15 US institutions. Response (complete response) rate, overall survival (OS), relapse-free survival (RFS), and duration of B-cell aplasia (BCA) in patients referred for tisagenlecleucel with EM disease (both central nervous system (CNS)3 and non-CNS EM) were compared with bone marrow (BM) only. Patients with CNS disease were further stratified for comparison. Outcomes are reported on 55 patients with EM disease before CAR therapy (CNS3, n = 40; non-CNS EM, n = 15). The median age at infusion in the CNS cohort was 10 years (range, <1-25 years), and in the non-CNS EM cohort it was 13 years (range, 2-26 years). In patients with CNS disease, 88% (35 of 40) achieved a complete response vs only 66% (10 of 15) with non-CNS EM disease. Patients with CNS disease (both with and without BM involvement) had 24-month OS outcomes comparable to those of non-CNS EM or BM only (P = .41). There was no difference in 12-month RFS between CNS, non-CNS EM, or BM-only patients (P = .92). No increased toxicity was seen with CNS or non-CNS EM disease (P = .3). Active CNS disease at time of infusion did not affect outcomes. Isolated CNS disease trended toward improved OS compared with combined CNS and BM (P = .12). R/R EM disease can be effectively treated with tisagenlecleucel; toxicity, relapse, and survival rates are comparable to those of patients with BM-only disease. Outcomes for isolated CNS relapse are encouraging., (© 2022 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.)

Pediatric cancers often mimic fetal tissues and express proteins normally silenced postnatally that could serve as immune targets. We developed T cells expressing chimeric antigen receptors (CARs) targeting glypican-2 (GPC2), a fetal antigen expressed on neuroblastoma (NB) and several other solid tumors. CARs engineered using standard designs control NBs with transgenic GPC2 overexpression, but not those expressing clinically relevant GPC2 site density (∼5,000 molecules/cell, range 1-6 × 10 3 ). Iterative engineering of transmembrane (TM) and co-stimulatory domains plus overexpression of c-Jun lowered the GPC2-CAR antigen density threshold, enabling potent and durable eradication of NBs expressing clinically relevant GPC2 antigen density, without toxicity. These studies highlight the critical interplay between CAR design and antigen density threshold, demonstrate potent efficacy and safety of a lead GPC2-CAR candidate suitable for clinical testing, and credential oncofetal antigens as a promising class of targets for CAR T cell therapy of solid tumors., Competing Interests: Declaration of interest C.L.M., S.H., J.M.M., K.R.B., R.G.M., D.S.D., and Z.Z. are co-inventors on patents related to this work. C.L.M. (and others) have multiple patents pertinent to CAR T cells. C.L.M. is a co-founder of Lyell Immunopharma and Syncopation Life Sciences, which develop CAR-based therapies, and consults for Lyell, NeoImmune Tech, Apricity, Nektar, and Immatics. K.R.B. and J.M.M. receive research funding from Tmunity for research on GPC2-directed immunotherapies. D.Z., Z.Z., D.S.D., J.M.M., and K.R.B. receive royalties from Tmunity for licensing of GPC2-related IP. R.G.M. and E.S. are consultants for and hold equity in Lyell Immunopharma. R.G.M. consults for GammaDelta Therapeutics, Aptorum Group, Zai Lab, and Illumina Radiopharmaceuticals and J.T. for Dorian Therapeutics. S.J.B. is an employee of BD Biosciences. A.T.S. is a founder of Immunai and Cartography Biosciences and receives research funding from Arsenal Biosciences and 10× Genomics. K.R.P. is a co-founder and employee of Cartography Biosciences. H.Y.C. is a co-founder of Accent Therapeutics and Boundless Bio and is an advisor to 10× Genomics, Arsenal Bio, and Spring Discovery., (Copyright © 2021. Published by Elsevier Inc.)

Hematopoietic stem cell transplantation (HSCT) may be used to consolidate chimeric antigen receptor (CAR) T cell therapy-induced remissions for patients with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL), but little is known about the factors impacting overall survival (OS) and event-free survival (EFS) for post-CAR hematopoietic stem cell transplantation (HSCT). The present study's primary objective was to identify factors associated with OS and EFS for consolidative HSCT following CAR-induced complete remission (CR) in transplantation-naïve patients. Secondary objectives included evaluation of OS/EFS, relapse-free survival and cumulative incidence of relapse for all patients who proceeded to HSCT, stratified by first and second HSCT, as well as the tolerability of HSCT following CAR-induced remission. This was a retrospective review of children and young adults enrolled on 1 of 3 CAR T cell trials at the National Cancer Institute targeting CD19, CD22, and CD19/22 (ClinicalTrials.gov identifiers NCT01593696, NCT02315612, and NCT03448393) who proceeded directly to HSCT following CAR T cell therapy. Between July 2012 and February 2021, 46 children and young adults with pre-B ALL went directly to HSCT following CAR therapy. Of these patients, 34 (74%) proceeded to a first HSCT, with a median follow-up of 50.8 months. Transplantation-naïve patients were heavily pretreated prior to CAR T cell therapy (median, 3.5 lines of therapy; range, 1 to 12) with significant prior immunotherapy exposure (blinatumomab, inotuzumab, and/or CAR T cell therapy in patients receiving CD22 or CD19/22 constructs (88%; 15 of /17)). Twelve patients (35%) had primary refractory disease, and the median time from CAR T cell infusion to HSCT Day 0 was 54.5 days (range, 42 to 127 days). The median OS following first HSCT was 72.2 months (95% confidence interval [CI], 16.9 months to not estimable [NE]), with a median EFS of 36.9 months (95% CI, 5.2 months to NE). At 12 and 24 months, the OS was 76.0% (95% CI, 57.6% to 87.2%) and 60.7% (95% CI, 40.8% to 75.8%), respectively, and EFS was 64.6% (95% CI, 46.1% to 78.1%) and 50.9% (95% CI, 32.6% to 66.6%), respectively. The individual factors associated with both decreased OS and EFS in univariate analyses for post-CAR consolidative HSCT in transplantation-naïve patients included ≥5 prior lines of therapy (not reached [NR] versus 12.4 months, P = .014; NR versus 4.8 months, P = .063), prior blinatumomab therapy (NR versus 16.9 months, P = .0038; NR versus 4.4 months, P = .0025), prior inotuzumab therapy (NR versus 11.5 months, P = .044; 36.9 months versus 2.7 months, P = .0054) and ≥5% blasts (M2/M3 marrow) pre-CAR T cell therapy (NR versus 17 months, P = .019; NR versus 12.2 months, P = .035). Primary refractory disease was associated with improved OS/EFS post-HSCT (NR versus 21.9 months, P = .075; NR versus 12.2 months, P = .024). Extensive prior therapy, particularly immunotherapy, and high disease burden each individually adversely impacted OS/EFS following post-CAR T cell consolidative HSCT in transplantation-naïve patients, owing primarily to relapse. Despite this, HSCT remains an important treatment modality in long-term cure. Earlier implementation of HSCT before multiply relapsed disease and incorporation of post-HSCT risk mitigation strategies in patients identified to be at high-risk of post-HSCT relapse may improve outcomes., (Published by Elsevier Inc.)

Infections are a known complication of chimeric antigen receptor (CAR) T-cell therapy with data largely emerging from CD19 CAR T-cell targeting. As CAR T-cell therapy continues to evolve, infection risks and management thereof will become increasingly important to optimize outcomes across the spectrum of antigens and disease targeted. We retrospectively characterized infectious complications occurring in 162 children and adults treated among 5 phase 1 CAR T-cell clinical trials. Trials included targeting of CD19, CD22, disialoganglioside (GD2) or B-cell maturation antigen (BCMA). Fifty-three patients (32.7%) had 76 infections between lymphocyte depleting (LD) chemotherapy and day 30 (D30); with the majority of infections (61, 80.3%) occurring between day 0 (D0) and D30. By trial, the highest proportion of infections was seen with CD22 CAR T cells (n = 23/53; 43.4%), followed by BCMA CAR T cells (n = 9/24; 37.5%). By disease, patients with multiple myeloma had the highest proportion of infections (9/24; 37.5%) followed by acute lymphoblastic leukemia (36/102; 35.3%). Grade 4 infections were rare (n = 4; 2.5%). Between D0 and D30, bacteremia and bacterial site infections were the most common infection type. In univariate analysis, increasing prior lines of therapy, recent infection within 100 days of LD chemotherapy, corticosteroid or tocilizumab use, and fever and neutropenia were associated with a higher risk of infection. In a multivariable analysis, only prior lines of therapy and recent infection were associated with higher risk of infection. In conclusion, we provide a broad overview of infection risk within the first 30 days post infusion across a host of multiple targets and diseases, elucidating both unique characteristics and commonalities highlighting aspects important to improving patient outcomes.

Adoptive T cell therapies have shown impressive signals of activity, but their clinical impact could be enhanced by technologies to increase T cell potency and diminish the cost and labor involved in manufacturing these products. Gene editing platforms are under study in this arena to (1) enhance immune cell potency by knocking out molecules that inhibit immune responses; (2) deliver genetic payloads into precise genomic locations and thereby enhance safety and/or improve the gene expression profile by leveraging physiologic promoters, enhancers, and repressors; and (3) enable off-the-shelf therapies by preventing alloreactivity and immune rejection. This review discusses gene editing approaches that have been the best studied in the context of human T cells and adoptive T cell therapies, summarizing their current status and near-term potential for translation., Competing Interests: Declaration of interests C.L.M. has multiple patents pertinent to CAR T cells, is a cofounder of Lyell Immunopharma and Syncopation Life Sciences, which are developing CAR-based therapies, and consults for Lyell, NeoImmune Tech, Apricity, Nektar, Immatics, Mammoth Biosciences, and Ensoma. T.M. declares no competing interests., (Copyright © 2021 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Purpose: The development of allogeneic chimeric antigen receptor (CAR) T-cell therapies for off-the-shelf use is a major goal that faces two main immunologic challenges, namely the risk of graft-versus-host disease (GvHD) induction by the transferred cells and the rejection by the host immune system limiting their persistence. In this work we assessed the direct and indirect antitumor effect of allogeneic CAR-engineered invariant natural killer T (iNKT) cells, a cell population without GvHD-induction potential that displays immunomodulatory properties., Experimental Design: After assessing murine CAR iNKT cells direct antitumor effects in vitro and in vivo , we employed an immunocompetent mouse model of B-cell lymphoma to assess the interaction between allogeneic CAR iNKT cells and endogenous immune cells., Results: We demonstrate that allogeneic CAR iNKT cells exerted potent direct and indirect antitumor activity when administered across major MHC barriers by inducing tumor-specific antitumor immunity through host CD8 T-cell cross-priming., Conclusions: In addition to their known direct cytotoxic effect, allogeneic CAR iNKT cells induce host CD8 T-cell antitumor responses, resulting in a potent antitumor effect lasting longer than the physical persistence of the allogeneic cells. The utilization of off-the-shelf allogeneic CAR iNKT cells could meet significant unmet needs in the clinic., (©2021 The Authors; Published by the American Association for Cancer Research.)

Alterations of the gut microbiota after allogeneic hematopoietic cell transplantation (allo-HCT) are a key factor in the development of transplant-related complications such as graft-versus-host disease (GVHD). Interventions that preserve the gut microbiome hold promise to improve HCT-associated morbidity and mortality. Murine models demonstrate that prebiotics such as fructo-oligosaccharides (FOSs) may increase gut levels of short-chain fatty acids (SCFAs) such as butyrate and consequently induce proliferation of immunomodulatory FOXP3 + CD4 + regulatory T cells (Tregs), which impact GVHD risk. We conducted a pilot phase I trial to investigate the maximum tolerated dose of FOS in patients undergoing reduced-intensity allo-HCT (n = 15) compared with concurrent controls (n = 16). We administered the FOS starting at pretransplant conditioning and continuing for a total of 21 days. We characterized the gut microbiome using shotgun metagenomic sequencing, measured stool short-chain fatty acids (SCFAs) using liquid chromatography-mass spectrometry, and determined peripheral T cell concentrations using cytometry by time-of-flight. We found that FOS was safe and well-tolerated at 10 g/d without significant adverse effects in patients undergoing allo-HCT. Community-level gut microbiota composition differed significantly on the day of transplant (day 0) between patients receiving FOS and concurrent controls; however, FOS-associated alterations of the gut microbiota were not sustained after transplant. Although the impact of FOS was fleeting, transplantation itself impacted a substantial number of taxa over time. In our small pilot trial, no significant differences were observed in gut microbial metabolic pathways, stool SCFAs, or peripheral Tregs, although Tregs trended higher in those patients who received FOS. A marker of CD4 + T cell activation (namely, CTLA4 + ) was significantly higher in patients receiving FOS, whereas a non-significant trend existed for FOP3 + CD4 + Treg cells, which were higher in those receiving FOS compared with controls. FOS is well tolerated at 10 g/d in patients undergoing reduced-intensity allo-HCT. Although the alterations in gut microbiota and peripheral immune cell composition in those receiving FOS are intriguing, additional studies are required to investigate the use of prebiotics in HCT recipients., (Copyright © 2021 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Chimeric antigen receptor (CAR) T cells hold promise for the treatment of acute myeloid leukemia (AML), but optimal targets remain to be defined. We demonstrate that CD93 CAR T cells engineered from a novel humanized CD93-specific binder potently kill AML in vitro and in vivo but spare hematopoietic stem and progenitor cells (HSPC). No toxicity is seen in murine models, but CD93 is expressed on human endothelial cells, and CD93 CAR T cells recognize and kill endothelial cell lines. We identify other AML CAR T-cell targets with overlapping expression on endothelial cells, especially in the context of proinflammatory cytokines. To address the challenge of endothelial-specific cross-reactivity, we provide proof of concept for NOT-gated CD93 CAR T cells that circumvent endothelial cell toxicity in a relevant model system. We also identify candidates for combinatorial targeting by profiling the transcriptome of AML and endothelial cells at baseline and after exposure to proinflammatory cytokines., Significance: CD93 CAR T cells eliminate AML and spare HSPCs but exert on-target, off-tumor toxicity to endothelial cells. We show coexpression of other AML targets on endothelial cells, introduce a novel NOT-gated strategy to mitigate endothelial toxicity, and demonstrate use of high-dimensional transcriptomic profiling for rational design of combinatorial immunotherapies. See related commentary by Velasquez and Gottschalk, p. 559 . This article is highlighted in the In This Issue feature, p. 549 ., (©2021 The Authors; Published by the American Association for Cancer Research.)