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Abstract Chimeric antigen receptor-modified T cell (CAR-T) immunotherapy has revolutionised blood cancer treatment. Parsing the genetic underpinnings of T cell quality and CAR-T efficacy is challenging. Transcriptomics inform CAR-T state, but the nature of dynamic transcription during activation hinders identification of transiently or minimally expressed genes, such as transcription factors, and over-emphasises effector and metabolism genes. Here we explore whether analyses of transcriptionally repressive and permissive histone methylation marks describe CAR-T cell functional states and therapeutic potential beyond transcriptomic analyses. Histone mark analyses improve identification of differences between naïve, central memory, and effector memory CD8 + T cell subsets of human origin, and CAR-T derived from these subsets. We find important differences between CAR-T manufactured from central memory cells of healthy donors and of patients. By examining CAR-T products from a clinical trial in lymphoma (NCT01865617), we find a novel association between the activity of the transcription factor KLF7 with in vivo CAR-T accumulation in patients and demonstrate that over-expression of KLF7 increases in vitro CAR-T proliferation and IL-2 production. In conclusion, histone marks provide a rich dataset for identification of functionally relevant genes not apparent by transcriptomics.

Chimeric antigen receptor-modified T cell (CAR-T) immunotherapy has revolutionised blood cancer treatment. Parsing the genetic underpinnings of T cell quality and CAR-T efficacy is challenging. Transcriptomics inform CAR-T state, but the nature of dynamic transcription during activation hinders identification of transiently or minimally expressed genes, such as transcription factors, and over-emphasises effector and metabolism genes. Here we explore whether analyses of transcriptionally repressive and permissive histone methylation marks describe CAR-T cell functional states and therapeutic potential beyond transcriptomic analyses. Histone mark analyses improve identification of differences between naïve, central memory, and effector memory CD8 + T cell subsets of human origin, and CAR-T derived from these subsets. We find important differences between CAR-T manufactured from central memory cells of healthy donors and of patients. By examining CAR-T products from a clinical trial in lymphoma (NCT01865617), we find a novel association between the activity of the transcription factor KLF7 with in vivo CAR-T accumulation in patients and demonstrate that over-expression of KLF7 increases in vitro CAR-T proliferation and IL-2 production.  In conclusion, histone marks provide a rich dataset for identification of functionally relevant genes not apparent by transcriptomics., (© 2024. The Author(s).)

Bispecific antibodies (bsAbs) are an emerging therapy in the treatment of large B-cell lymphomas (LBCLs). There is a gap in the research on the safety and efficacy of bsAbs in adults with LBCL, with current research focusing on the wider non-Hodgkin's lymphoma population. To address this research gap, we conducted a systematic review aiming to evaluate the safety and efficacy outcomes of bsAbs in adults with LBCL. A systematized search was conducted in PubMed, EMBASE, and CENTRAL on 10 April 2024. Interventional clinical trials were eligible for inclusion. Observational studies, reviews, and meta-analyses were excluded. According to the Revised Risk of Bias Assessment Tool for Nonrandomized Studies, the included studies were largely of a high quality for safety outcome reporting, but of mixed quality for efficacy outcome reporting. Due to the heterogeneity of the included studies, the results were discussed as a narrative synthesis. Nineteen early phase studies were evaluated in the final analysis, with a pooled sample size of 1332 patients. Nine bsAbs were investigated across the studies as monotherapy (nine studies) or in combination regimes (10 studies). The rates of cytokine release syndrome were variable, with any grade events ranging from 0 to 72.2%. Infection rates were consistently high across the reporting studies (38-60%). Cytopenias were found to be common, in particular, anemia (4.4-62%), thrombocytopenia (3.3-69%), and neutropenia (4.4-70%). Immune effector cell-associated neurotoxicity syndrome (ICANS) and grade ≥3 adverse events were not commonly reported. Promising efficacy outcomes were reported, with median overall response rates of 95-100% in the front-line and 36-91% in terms of relapsed/refractory disease. The results of this systematic review demonstrate that bsAbs are generally well-tolerated and effective in adults with LBCL. BsAbs appear to have superior tolerability, but inferior efficacy to CAR T-cell therapies in adults with LBCL. Future research on safety and efficacy should focus on evaluating adverse event timing and management, the impact on the patient's quality of life, the burden on the healthcare system, and overall survival outcomes.

CD19-targeted chimeric antigen receptor T-cell (CAR-T) immunotherapy has transformed the management of relapsed/refractory large B-cell lymphoma (LBCL), yet durable remissions are observed in less than half of treated patients. The tumor microenvironment (TME) is a key and understudied factor impacting CD19 CAR-T therapy outcomes. Using NanoString nCounter transcriptome profiling ( n  = 24) and multiplex immunohistochemistry (mIHC, n  = 15), we studied the TME in pretreatment biopsies from patients with LBCL undergoing CD19 CAR-T therapy. Patients who achieved complete response (CR) after CAR-T therapy demonstrated higher expression of genes associated with T-cell trafficking and function, whereas those who did not achieve CR had higher expression of genes associated with macrophages and T-cell dysfunction. Distinct patterns of immune infiltration and fibrosis in the TME were associated with CAR-T therapy outcomes, and these findings were corroborated using artificial intelligence-assisted image analyses. Patients who achieved CR had a lower proportion of the biopsy occupied by an interspersed immune infiltrate and a higher proportion of hypocellular/fibrotic regions. Furthermore, mIHC revealed lower density of CD4 + T cells and higher densities of both macrophages and tumor cells expressing PD-L1 in non-CR patients. Spatial analysis revealed that PD-1 + T cells were in close proximity to PD-L1 + macrophages or PD-L1 + tumor cells in patients who did not compared to those who did achieve CR after CAR-T therapy. These findings suggest that morphologic patterns in the TME and engagement of the PD-1/PD-L1 axis in pretreatment biopsies may impact CD19 CAR-T immunotherapy response in patients with LBCL., Competing Interests: Alexandre V. Hirayama has received research funding from Juno Therapeutics, a Bristol Myers Squibb Company, and Nektar Therapeutics; has received honoraria from Bristol Myers Squibb. Kimberly S. Smythe has received consulting/honoraria from Sensei Biotherapeutics. Salvatore Fiorenza reports grants from Bristol Myers Squibb and other support in the form of pending equity from Link Immunotherapeutics outside of the submitted work; has issued patents for PCT/US2021/025255 and PCT/US2021/025248d, and a patent for PCT/US2021/025260 issued, licensed, and with royalties paid from Bristol Myers Squibb. Jordan Gauthier has received research funding from Sobi, Juno Therapeutics, a Bristol Myers Squibb Company, Celgene, and Angiocrine Bioscience; has received consulting/honoraria from Sobi, Legend Biotech, Janssen, Kite Pharma, a Gilead company, and MorphoSys. Cecilia C. S. Yeung has received research funding from OBI, Lonza, Sensei, Signal One, and Pfizer; serves on scientific advisory boards for Twinstrand Biosciences, Abbvie, Eli Lily, and Loxo. David G. Maloney has received research funding from Juno Therapeutics, a Bristol Myers Squibb Company, Celgene, and Kite Pharma, a Gilead company; has served on ad hoc advisory board meetings from Amgen, BMS, Genentech, Gilead, Incyte, Janssen, Legend Biotech, Mustang Bio, MorphoSys, Novartis, Pharmacyclics, and Umoja; has rights to receive royalties from Fred Hutch for patents licensed to Juno Therapeutics; serves on scientific advisory board with stock options and compensations for A2 Biotherapeutics and Navan Technologies. Cameron J. Turtle has received research funding from Juno Therapeutics, a Bristol Myers Squibb Company, NanoString Technologies, and Nektar Therapeutics; has served on scientific and consulting roles in the last 12 months for Caribou Biosciences, T‐CURX, Myeloid Therapeutics, ArsenalBio, Cargo Therapeutics, Differentia Bio, Advesya, eGlint, Celgene/Bristol Myers Squibb Cell Therapy, Novartis, Prescient Therapeutics, Century Therapeutics, IGM Biosciences, and Abbvie; serves on a DSMB for Kyverna; has stock options in Eureka Therapeutics, Caribou Biosciences, Myeloid Therapeutics, Cargo Therapeutics, and ArsenalBio; and has the right to receive payments from Fred Hutchinson Cancer Center as an inventor on patents related to CAR‐T cell therapy. The remaining authors have no conflicts of interest to report related to this work., (© 2024 The Author(s). HemaSphere published by John Wiley & Sons Ltd on behalf of European Hematology Association.)

Current CD33-targeted immunotherapies typically recognize the membrane-distal V-set domain of CD33. Here, we show that decreasing the distance between T cell and leukemia cell membrane increases the efficacy of CD33 chimeric antigen receptor (CAR) T cells. We therefore generated and optimized second-generation CAR constructs containing single-chain variable fragments from antibodies raised against the membrane-proximal C2-set domain, which bind CD33 regardless of whether the V-set domain is present (CD33 PAN antibodies). CD33 PAN CAR T cells resulted in efficient tumor clearance and improved survival of immunodeficient mice bearing human AML cell xenografts and, in an AML model with limited CD33 expression, forced escape of CD33 neg leukemia. Compared to CD33 V-set CAR T cells, CD33 PAN CAR T cells showed greater in vitro and in vivo efficacy against several human AML cell lines with differing levels of CD33 without increased expression of exhaustion markers. CD33 PAN moieties were detected at a higher frequency on human leukemic stem cells, and CD33 PAN CAR T cells had greater in vitro efficacy against primary human AML cells. Together, our studies demonstrate improved efficacy with CAR T cells binding CD33 close to the cell membrane, providing the rationale to investigate CD33 PAN CAR T cells further toward possible clinical application., Competing Interests: S.F., G.S.L., C.J.T., and R.B.W. filed a patent application related to CD33 antibodies and CD33-directed CAR T cell therapy. S.F. has filed patents on optimizing CAR T cell cytotoxicity, received research laboratory grants from Bristol Myers Squibb, and is a consultant for Prescient Therapeutics. E.L.K. received research support from Juno Therapeutics. H.-P.K. received support as the inaugural recipient of the José Carreras/E. Donnall Thomas Endowed Chair for Cancer Research and the Stephanus Family Endowed Chair for Cell and Gene Therapy, and is or was a consultant to and has or had ownership interests with Rocket Pharma, Homology Medicines, VOR, and Ensoma. C.J.T. received research funding from Juno Therapeutics/BMS and Nektar Therapeutics; serves on advisory boards for Caribou Biosciences, T-CURX, Myeloid Therapeutics, ArsenalBio, Cargo Therapeutics, and Celgene/BMS Cell Therapy; has served on ad hoc advisory boards/consulting (last 12 months) for Nektar Therapeutics, Century Therapeutics, Legend Biotech, Allogene, Sobi, Syncopation Life Sciences, Prescient Therapeutics, Orna Therapeutics, and IGM Biosciences; and holds stock options in Eureka Therapeutics, Caribou Biosciences, Myeloid Therapeutics, ArsenalBio, and Cargo Therapeutics. R.B.W. received laboratory research grants and/or clinical trial support from Aptevo, Celgene/Bristol Myers Squibb, ImmunoGen, Janssen, Jazz, Kite, Kura, Pfizer, and VOR, and has been a consultant to Wugen., (© 2024 The Author(s).)

Aqueous film forming foam (AFFF)-impacted asphalt and concrete may serve as potential secondary sources of per- and polyfluoroalkyl substances (PFAS) to the environment through surficial leaching. We aimed to understand the vertical distribution and surficial release of PFAS from AFFF-impacted asphalt and concrete cores collected from various locations (∼10-70 m distance between samples). Among the PFAS analyzed, 6:2 FTS was observed as having the highest concentration in the surface layer (0 - 0.5 cm) of concrete (225 µg kg -1 ) and in the runoff from the concrete (2600 ng L -1 ). PFOS was detected at the highest concentration in the surface layer (0 - 0.5 cm) of asphalt (47 µg kg -1 ) and associated runoff (780 ng L -1 ). The total mass of PFAS released during three rainfall simulations accounts for a fraction of the total mass in the surface layer (0 - 0.5 cm), ranging from 0.10 - 9.8% and 0.078 - 2.4% for asphalt and concrete cores, respectively. Asphalt exhibited a higher release rate than concrete, demonstrated by the higher total release coefficient of PFAS (4 - 16 m -2 ) compared to that of concrete cores (1 - 5 m -2 ). These results suggested that, similar to concrete, AFFF-impacted asphalt may be a secondary source of PFAS to the environment., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Phong Thai reports financial support was provided by ExxonMobil Environmental and Property Solutions Company., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Abstract: More than half of the patients treated with CD19-targeted chimeric antigen receptor (CAR) T-cell immunotherapy for large B-cell lymphoma (LBCL) do not achieve durable remission, which may be partly due to PD-1/PD-L1-associated CAR T-cell dysfunction. We report data from a phase 1 clinical trial (NCT02706405), in which adults with LBCL were treated with autologous CD19 CAR T cells (JCAR014) combined with escalating doses of the anti-PD-L1 monoclonal antibody, durvalumab, starting either before or after CAR T-cell infusion. The addition of durvalumab to JCAR014 was safe and not associated with increased autoimmune or immune effector cell-associated toxicities. Patients who started durvalumab before JCAR014 infusion had later onset and shorter duration of cytokine release syndrome and inferior efficacy, which was associated with slower accumulation of CAR T cells and lower concentrations of inflammatory cytokines in the blood. Initiation of durvalumab before JCAR014 infusion resulted in an early increase in soluble PD-L1 (sPD-L1) levels that coincided with the timing of maximal CAR T-cell accumulation in the blood. In vitro, sPD-L1 induced dose-dependent suppression of CAR T-cell effector function, which could contribute to inferior efficacy observed in patients who received durvalumab before JCAR014. Despite the lack of efficacy improvement and similar CAR T-cell kinetics early after infusion, ongoing durvalumab therapy after JCAR014 was associated with re-expansion of CAR T cells in the blood, late regression of CD19+ and CD19- tumors, and enhanced duration of response. Our results indicate that the timing of initiation of PD-L1 blockade is a key variable that affects outcomes after CD19 CAR T-cell immunotherapy for adults with 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.)