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Patients with large B-cell lymphoma (LBCL) progressing after anti-CD19 CAR T-cell (CAR19) therapy have poor outcomes. Subsequent CAR T-cell therapy shows promise, but the impact of residual CAR19 and early relapse remains unclear. We evaluated 37 CAR19-refractory LBCL patients who received anti-CD22 CAR T-cell (CAR22) in a phase 1b trial (NCT04088890). Residual CAR19 was unquantifiable in 17 of 33 evaluable patients post-CAR22 infusion. Single-cell RNA sequencing revealed minimal CAR19/CAR22 co-transduction. Peak CAR19 transgene levels did not affect CAR22 efficacy or toxicities. CAR22 products from patients undergoing leukapheresis > 6 months post-CAR19 had higher CD4+ naïve T and CD4+/CD8+ T- central memory (TCM) cells, with lower CD4+ T-effector memory cells. High and low percentages of CAR22 TCM and TEM, respectively, were correlated with CAR22 transduction efficiency and achieving complete response. Residual CAR19 and leukapheresis timing did not significantly affect outcomes, while CAR22 product composition was significantly correlated with treatment response.

Glioblastoma (GBM) is a highly aggressive brain tumor with poor prognosis and high recurrence rates. The complex immune microenvironment of GBM is highly infiltrated by tumor-associated microglia and macrophages (TAM). TAMs are known to be heterogeneous in their functional and metabolic states and can transmit either protumoral or antitumoral signals to glioma cells. Here, we performed bulk RNA sequencing and single-cell RNA sequencing on samples from patients with GBM, which revealed increased ATP synthase expression and oxidative phosphorylation activity in TAMs located in the tumor core relative to the tumor periphery. Both in vitro and in vivo models displayed similar trends of augmented TAM mitochondrial activity, along with elevated mitochondrial fission, glucose uptake, mitochondrial membrane potential, and extracellular ATP (eATP) production by TAMs in the presence of GBM cells. Tumor-secreted factors, including GM-CSF, induced the increase in TAM eATP production. Elevated eATP in the GBM microenvironment promoted glioma growth and invasion by activating the P2X purinoceptor 7 (P2X7R) on glioma cells. Inhibition of the eATP-P2X7R axis attenuated tumor cell viability in vitro and reduced tumor size and prolonged survival in glioma-bearing mouse models. Overall, this study revealed elevated TAM-derived eATP in GBM and provided the basis for targeting the eATP-P2X7R signaling axis as a therapeutic strategy in GBM. Significance: Glioblastoma-mediated metabolic reprogramming in tumor-associated microglia increases ATP secretion that supports cancer cell proliferation and invasion by activating P2X7R, which can be inhibited to attenuate tumor growth., (©2024 American Association for Cancer Research.)

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

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.)

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.)

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.)

Purpose: Immunomonitoring of chimeric antigen receptor (CAR) T cells relies primarily on their quantification in the peripheral blood, which inadequately quantifies their biodistribution and activation status in the tissues. Noninvasive molecular imaging of CAR T cells by PET is a promising approach with the ability to provide spatial, temporal, and functional information. Reported strategies rely on the incorporation of reporter transgenes or ex vivo biolabeling, significantly limiting the application of CAR T-cell molecular imaging. In this study, we assessed the ability of antibody-based PET (immunoPET) to noninvasively visualize CAR T cells., Experimental Design: After analyzing human CAR T cells in vitro and ex vivo from patient samples to identify candidate targets for immunoPET, we employed a syngeneic, orthotopic murine tumor model of lymphoma to assess the feasibility of in vivo tracking of CAR T cells by immunoPET using the 89 Zr-DFO-anti-ICOS tracer, which we have previously reported., Results: Analysis of human CD19-CAR T cells during activation identified the Inducible T-cell COStimulator (ICOS) as a potential target for immunoPET. In a preclinical tumor model, 89 Zr-DFO-ICOS mAb PET-CT imaging detected significantly higher signal in specific bone marrow-containing skeletal sites of CAR T-cell-treated mice compared with controls. Importantly, administration of ICOS-targeting antibodies at tracer doses did not interfere with CAR T-cell persistence and function., Conclusions: This study highlights the potential of ICOS-immunoPET imaging for monitoring of CAR T-cell therapy, a strategy readily applicable to both commercially available and investigational CAR T cells. See related commentary by Volpe et al., p. 911 ., (©2020 American Association for Cancer Research.)

Imaging strategies to monitor chimeric antigen receptor (CAR) T-cell biodistribution and proliferation harbor the potential to facilitate clinical translation for the treatment of both liquid and solid tumors. In addition, the potential adverse effects of CAR T cells highlight the need for mechanisms to modulate CAR T-cell activity. The herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene has previously been translated as a PET reporter gene for imaging of T-cell trafficking in patients with brain tumor. The HSV1-TK enzyme can act as a suicide gene of transduced cells through treatment with the prodrug ganciclovir. Here we report the molecular engineering, imaging, and ganciclovir-mediated destruction of B7H3 CAR T cells incorporating a mutated version of the HSV1-tk gene (sr39tk) with improved enzymatic activity for ganciclovir. The sr39tk gene did not affect B7H3 CAR T-cell functionality and in vitro and in vivo studies in osteosarcoma models showed no significant effect on B7H3 CAR T-cell antitumor activity. PET/CT imaging with 9-(4-[ 18 F]-fluoro-3-[hydroxymethyl]butyl)guanine ([ 18 F]FHBG) of B7H3-sr39tk CAR T cells in an orthotopic model of osteosarcoma revealed tumor homing and systemic immune expansion. Bioluminescence and PET imaging of B7H3-sr39tk CAR T cells confirmed complete tumor ablation with intraperitoneal ganciclovir administration. This imaging and suicide ablation system can provide insight into CAR T-cell migration and proliferation during clinical trials while serving as a suicide switch to limit potential toxicities. SIGNIFICANCE: This study showcases the only genetically engineered system capable of serving the dual role both as an effective PET imaging reporter and as a suicide switch for CAR T cells., (©2020 American Association for Cancer Research.)

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

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

Emerging data from chimeric antigen receptor (CAR) T-cell trials in B-cell malignancies demonstrate that a common mechanism of resistance to this novel class of therapeutics is the emergence of tumors with loss or downregulation of the target antigen. Antigen loss or antigen-low escape is likely to emerge as an even greater barrier to success in solid tumors, which manifest greater heterogeneity in target antigen expression. Potential approaches to overcome this challenge include engineering CAR T cells to achieve multispecificity and to respond to lower levels of target antigen and more efficient induction of natural antitumor immune responses as a result of CAR-induced inflammation. In this article, we review the evidence to date for antigen escape and downregulation and discuss approaches currently under study to overcome these obstacles. Significance: Antigen escape and downregulation have emerged as major issues impacting the durability of CAR T-cell therapy. Here, we explore their incidence and ways to overcome these obstacles in order to improve clinical outcomes. Cancer Discov; 8(10); 1219-26. ©2018 AACR., (©2018 American Association for Cancer Research.)

We evaluated the safety and activity of autologous T cells expressing NY-ESO-1 c259 , an affinity-enhanced T-cell receptor (TCR) recognizing an HLA-A2-restricted NY-ESO-1/LAGE1a-derived peptide, in patients with metastatic synovial sarcoma (NY-ESO-1 c259 T cells). Confirmed antitumor responses occurred in 50% of patients (6/12) and were characterized by tumor shrinkage over several months. Circulating NY-ESO-1 c259 T cells were present postinfusion in all patients and persisted for at least 6 months in all responders. Most of the infused NY-ESO-1 c259 T cells exhibited an effector memory phenotype following ex vivo expansion, but the persisting pools comprised largely central memory and stem-cell memory subsets, which remained polyfunctional and showed no evidence of T-cell exhaustion despite persistent tumor burdens. Next-generation sequencing of endogenous TCRs in CD8 + NY-ESO-1 c259 T cells revealed clonal diversity without contraction over time. These data suggest that regenerative pools of NY-ESO-1 c259 T cells produced a continuing supply of effector cells to mediate sustained, clinically meaningful antitumor effects. Significance: Metastatic synovial sarcoma is incurable with standard therapy. We employed engineered T cells targeting NY-ESO-1, and the data suggest that robust, self-regenerating pools of CD8 + NY-ESO-1 c259 T cells produce a continuing supply of effector cells over several months that mediate clinically meaningful antitumor effects despite prolonged exposure to antigen. Cancer Discov; 8(8); 944-57. ©2018 AACR. See related commentary by Keung and Tawbi, p. 914 This article is highlighted in the In This Issue feature, p. 899 ., (©2018 American Association for Cancer Research.)

Central nervous system (CNS) toxicity associated with chimeric antigen receptor-based therapeutics has emerged as a significant cause of morbidity and mortality, and insights into the pathophysiology of this syndrome have been lacking. A new study provides evidence that cytokine-induced CNS endothelial cell activation leading to disruption of the blood-brain barrier plays an early and critical role in this phenomenon. These insights provide new opportunities for targeted therapeutic interventions to modulate endothelial cell activation. Cancer Discov; 7(12); 1371-3. ©2017 AACR See related article by Gust et al., p. 1404 ., (©2017 American Association for Cancer Research.)