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Cancer progression is marked by dysfunctional tumor-infiltrating lymphocytes (TIL) with high inhibitory receptor (IR) expression. Because IR blockade has led to clinical responses in some patients with non–small cell lung cancer (NSCLC), we investigated how IRs influenced CD8+ TIL function from freshly digested early-stage NSCLC tissues using a killing assay and intracellular cytokine staining after in vitro T-cell restimulation. Early-stage lung cancer TIL function was heterogeneous with only about one third of patients showing decrements in cytokine production and lytic function. TIL hypofunction did not correlate with clinical factors, coexisting immune cells (macrophages, neutrophils, or CD4+ T regulatory cells), nor with PD-1, TIGIT, TIM-3, CD39, or CTLA-4 expression. Instead, we found that the presence of the integrin αeβ7 (CD103), characteristic of tissue-resident memory cells (TRM), was positively associated with cytokine production, whereas expression of the transcription factor Eomesodermin (Eomes) was negatively associated with TIL function. These data suggest that the functionality of CD8+ TILs from early-stage NSCLCs may be influenced by competition between an antitumor CD103+ TRM program and an exhaustion program marked by Eomes expression. Understanding the mechanisms of T-cell function in the progression of lung cancer may have clinical implications for immunotherapy.

Supplementary Figures 1-7

Description of Flow Cytometric Methods, and killing assays.

Supplemental Tables 1-6 Supplemental Table 1. Results of some recent trials of chemotherapy in MPM. Supplemental Table 2. Post-Trial Therapies Supplemental Table 3. Characteristics of Patients Selected for Flow Cytometry Analysis Supplemental Table 4. List of Immune Response Genes assayed by Nanostring Supplemental Table 5: Correlation of Antibody Response to MOS or Radiographic Response Supplemental Table 6. Flow Cytometry Results

PDF file - 37K, CAR spleen infiltrating lymphocytes have lower levels of inhibitory receptors than CAR tumor infiltrating lymphocytes.

Chimeric antigen receptor (CAR)–modified adoptive T-cell therapy has been successfully applied to the treatment of hematologic malignancies, but faces many challenges in solid tumors. One major obstacle is the immune-suppressive effects induced in both naturally occurring and genetically modified tumor-infiltrating lymphocytes (TIL) by inhibitory receptors (IR), namely PD1. We hypothesized that interfering with PD1 signaling would augment CAR T-cell activity against solid tumors. To address this possibility, we introduced a genetically engineered switch receptor construct, comprising the truncated extracellular domain of PD1 and the transmembrane and cytoplasmic signaling domains of CD28, into CAR T cells. We tested the effect of this supplement, “PD1CD28,” on human CAR T cells targeting aggressive models of human solid tumors expressing relevant tumor antigens. Treatment of mice bearing large, established solid tumors with PD1CD28 CAR T cells led to significant regression in tumor volume due to enhanced CAR TIL infiltrate, decreased susceptibility to tumor-induced hypofunction, and attenuation of IR expression compared with treatments with CAR T cells alone or PD1 antibodies. Taken together, our findings suggest that the application of PD1CD28 to boost CAR T-cell activity is efficacious against solid tumors via a variety of mechanisms, prompting clinical investigation of this potentially promising treatment modality. Cancer Res; 76(6); 1578–90. ©2016 AACR.

Ex-vivo killing of and cytokine secretion in response to tumor targets by TILs isolated from EMMESO flank tumors.

Supplementary Figures S1-S4 from Expression of a Functional CCR2 Receptor Enhances Tumor Localization and Tumor Eradication by Retargeted Human T cells Expressing a Mesothelin-Specific Chimeric Antibody Receptor

Enhanced in-vitro killing of EMMESO-PDL1 tumor targets by PD1CD28 modification.

PDF file - 38K, CAR TILs upregulate inhibitory receptors.

Expression of inhibitory receptors on tumor vs. spleen infiltrating lymphocytes.

Cell lines and cell culture conditions, antibodies, cytokine production of restimulated T-cells, animals, and stastical analysis.

Purpose: “In situ vaccination” using immunogene therapy has the ability to induce polyclonal antitumor responses directed by the patient's immune system.Experimental Design: Patients with unresectable malignant pleural mesothelioma (MPM) received two intrapleural doses of a replication-defective adenoviral vector containing the human IFNα2b gene (Ad.IFN) concomitant with a 14-day course of celecoxib followed by chemotherapy. Primary outcomes were safety, toxicity, and objective response rate; secondary outcomes included progression-free and overall survival. Biocorrelates on blood and tumor were measured.Results: Forty subjects were treated: 18 received first-line pemetrexed-based chemotherapy, 22 received second-line chemotherapy with pemetrexed (n = 7) or gemcitabine (n = 15). Treatment was generally well tolerated. The overall response rate was 25%, and the disease control rate was 88%. Median overall survival (MOS) for all patients with epithelial histology was 21 months versus 7 months for patients with nonepithelial histology. MOS in the first-line cohort was 12.5 months, whereas MOS for the second-line cohort was 21.5 months, with 32% of patients alive at 2 years. No biologic parameters were found to correlate with response, including numbers of activated blood T cells or NK cells, regulatory T cells in blood, peak levels of IFNα in blood or pleural fluid, induction of antitumor antibodies, nor an immune-gene signature in pretreatment biopsies.Conclusions: The combination of intrapleural Ad.IFN, celecoxib, and chemotherapy proved safe in patients with MPM. OS rate was significantly higher than historical controls in the second-line group. Results of this study support proceeding with a multicenter randomized clinical trial of chemo-immunogene therapy versus standard chemotherapy alone. Clin Cancer Res; 22(15); 3791–800. ©2016 AACR.

Purpose: Immunotherapy using vaccines or adoptively transferred tumor-infiltrating lymphocytes (TIL) is limited by T-cell functional inactivation within the solid tumor microenvironment. The purpose of this study was to determine whether a similar tumor-induced inhibition occurred with genetically modified cytotoxic T cells expressing chimeric antigen receptors (CAR) targeting tumor-associated antigens.Experimental Design: Human T cells expressing CAR targeting mesothelin or fibroblast activation protein and containing CD3ζ and 4–1BB cytoplasmic domains were intravenously injected into immunodeficient mice bearing large, established human mesothelin-expressing flank tumors. CAR TILs were isolated from tumors at various time points and evaluated for effector functions and status of inhibitory pathways.Results: CAR T cells were able to traffic into tumors with varying efficiency and proliferate. They were able to slow tumor growth, but did not cause regressions or cures. The CAR TILs underwent rapid loss of functional activity that limited their therapeutic efficacy. This hypofunction was reversible when the T cells were isolated away from the tumor. The cause of the hypofunction seemed to be multifactorial and was associated with upregulation of intrinsic T-cell inhibitory enzymes (diacylglycerol kinase and SHP-1) and the expression of surface inhibitory receptors (PD1, LAG3, TIM3, and 2B4).Conclusions: Advanced-generation human CAR T cells are reversibly inactivated within the solid tumor microenvironment of some tumors by multiple mechanisms. The model described here will be an important tool for testing T cell–based strategies or systemic approaches to overcome this tumor-induced inhibition. Our results suggest that PD1 pathway antagonism may augment human CAR T-cell function. Clin Cancer Res; 20(16); 4262–73. ©2014 AACR.

Purpose: Tumor-infiltrating lymphocytes (TILs) become hypofunctional, although the mechanisms are not clear. Our goal was to generate a model of human tumor-induced TIL hypofunction to study mechanisms and to test anti-human therapeutics.Experimental Design: We transduced human T cells with a published, optimized T-cell receptor (TCR) that is directed to a peptide within the cancer testis antigen, NY-ESO-1. After demonstrating antigen-specific in vitro activity, these cells were used to target a human lung cancer line that expressed NY-ESO-1 in the appropriate HLA context growing in immunodeficient mice. The ability of anti-PD1 antibody to augment efficacy was tested.Results: Injection of transgenic T cells had some antitumor activity, but did not eliminate the tumors. The injected T cells became profoundly hypofunctional accompanied by upregulation of PD1, Tim3, and Lag3 with coexpression of multiple inhibitory receptors in a high percentage of cells. This model allowed us to test reagents targeted specifically to human T cells. We found that injections of an anti-PD1 antibody in combination with T cells led to decreased TIL hypofunction and augmented the efficacy of the adoptively transferred T cells.Conclusions: This model offers a platform for preclinical testing of adjuvant immunotherapeutics targeted to human T cells prior to transition to the bedside. Because the model employs engineering of human T cells with a TCR clone instead of a CAR, it allows for study of the biology of tumor-reactive TILs that signal through an endogenous TCR. The lessons learned from TCR-engineered TILs can thus be applied to tumor-reactive TILs. Clin Cancer Res; 22(2); 436–47. ©2015 AACR.See related commentary by Yang, p. 275

Schematic of PD1CD28 switch receptor, mutated PD1CD28 switch receptor, and tailless/signal-dead PD1.

Purpose: Adoptive T-cell immunotherapy with tumor infiltrating lymphocytes or genetically-modified T cells has yielded dramatic results in some cancers. However, T cells need to traffic properly into tumors to adequately exert therapeutic effects.Experimental Design: The chemokine CCL2 was highly secreted by malignant pleural mesotheliomas (MPM; a planned tumor target), but the corresponding chemokine receptor (CCR2) was minimally expressed on activated human T cells transduced with a chimeric antibody receptor (CAR) directed to the MPM tumor antigen mesothelin (mesoCAR T cells). The chemokine receptor CCR2b was thus transduced into mesoCAR T cells using a lentiviral vector, and the modified T cells were used to treat established mesothelin-expressing tumors.Results: CCR2b transduction led to CCL2-induced calcium flux and increased transmigration, as well as augmentation of in vitro T-cell killing ability. A single intravenous injection of 20 million mesoCAR + CCR2b T cells into immunodeficient mice bearing large, established tumors (without any adjunct therapy) resulted in a 12.5-fold increase in T-cell tumor infiltration by day 5 compared with mesoCAR T cells. This was associated with significantly increased antitumor activity.Conclusions: CAR T cells bearing a functional chemokine receptor can overcome the inadequate tumor localization that limits conventional CAR targeting strategies and can significantly improve antitumor efficacy in vivo. Clin Cancer Res; 17(14); 4719–30. ©2011 AACR.

Legends for the Supplementary Figures

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Description of cell culture conditions, isolation/activation/transduction/expansion of T cells, generation of target cell lines, flow cytometric conditions, ELISA measurements, statistical analyses, and animal use.

Co-expression of inhibitory receptors on tumor infiltrating lymphocytes.

Comparison of inhibitory receptors on CD8 and CD4 tumor infiltrating lymphocytes.

Effect of tailless/signal-dead PD1 on in-vivo CAR T cell anti-tumor control.

Summary of publications describing tumor induced hypofunction of human tumor infiltrating lymphocytes.

Additional Trial related information and methods

PDF file - 756K, Supplemental Fig 1. EMP and EMMESO mesothelin expression. Supplemental Fig 2. EMMESO flank tumors retain antigen expression in vivo. Supplemental Fig 3. MesoCAR TILs are unable to secrete IL-2 in response to antigen. Supplemental Fig 4. Supplemental IL-2 helps but is not necessary for CAR TIL recovery. Supplemental Fig 5. IFNγ and TNFα secretion by CAR TILs recovers after resting away from tumor.

Schematic of lentiviral and retroviral expression plasmids.

Expression of PDL1 on tumors.

Supplemental Figure 1. Immunoblots of Patient Serum before and after Therapy Supplemental Figure 2. Distribution of 1/Nab Titers for the 40 Patients Supplemental Figure 3. Distribution of the Day 2 serum levels of Interferon-alpha Supplemental Figure 4. Lack of Correlation of lymphocyte or macrophage infiltration with survival. Supplemental Figure 5. Lack of Correlation of PDL1 staining with survival. Supplemental Figure 6. Correlation of the mRNA immunoscore with survival.

Recent clinical trials have shown promise in the use of chimeric antigen receptor (CAR)-transduced T cells; however, augmentation of their activity may broaden their clinical use and improve their efficacy. We hypothesized that because CAR action requires proteins essential for T-cell receptor (TCR) signal transduction, deletion of negative regulators of these signaling pathways would enhance CAR signaling and effector T-cell function. We tested CAR activity and function in T cells that lacked one or both isoforms of diacylglycerol kinase (dgk) expressed highly in T cells, dgkα and dgkζ, enzymes that metabolize the second messenger diacylglycerol (DAG) and limit Ras/ERK activation. We found that primary murine T cells transduced with CARs specific for the human tumor antigen mesothelin showed greatly enhanced cytokine production and cytotoxicity when cocultured with a murine mesothelioma line that stably expresses mesothelin. In addition, we found that dgk-deficient CAR-transduced T cells were more effective in limiting the growth of implanted tumors, both concurrent with and after establishment of tumor. Consistent with our studies in mice, pharmacologic inhibition of dgks also augments function of primary human T cells transduced with CARs. These results suggest that deletion of negative regulators of TCR signaling enhances the activity and function of CAR-expressing T cells and identify dgks as potential targets for improving the clinical potential of CARs. Cancer Res; 73(12); 3566–77. ©2013 AACR.

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PDF file - 52K, MesoCAR-transduced dgkzeta-deficient T cells demonstrate resistance to multiple inhibitory stimuli

PDF file - 103K, Enhanced expression of FasL and TRAIL in dgk-deficient mesoCAR T cells

PDF file - 73K, Similar expression of Granzyme B and perforin in dgk-deficient mesoCAR T cells

Gene-mediated cytotoxic immunotherapy (GMCI) is an immuno-oncology approach involving local delivery of a replication-deficient adenovirus expressing herpes simplex thymidine kinase (AdV-tk) followed by anti-herpetic prodrug activation that promotes immunogenic tumor cell death, antigen-presenting cell activation, and T cell stimulation. This phase I dose-escalation pilot trial assessed bronchoscopic delivery of AdV-tk in patients with suspected lung cancer who were candidates for surgery. A single intra-tumoral AdV-tk injection in three dose cohorts (maximum 1012 viral particles) was performed during diagnostic staging, followed by a 14-day course of the prodrug valacyclovir, and subsequent surgery 1 week later. Twelve patients participated after appropriate informed consent. Vector-related adverse events were minimal. Immune biomarkers were evaluated in tumor and blood before and after GMCI. Significantly increased infiltration of CD8+ T cells was found in resected tumors. Expression of activation, inhibitory, and proliferation markers, such as human leukocyte antigen (HLA)-DR, CD38, Ki67, PD-1, CD39, and CTLA-4, were significantly increased in both the tumor and peripheral CD8+ T cells. Thus, intratumoral AdV-tk injection into non-small-cell lung cancer (NSCLC) proved safe and feasible, and it effectively induced CD8+ T cell activation. These data provide a foundation for additional clinical trials of GMCI for lung cancer patients with potential benefit if combined with other immune therapies., Graphical Abstract, Predina and colleagues describe a phase I dose-escalation trial using bronchoscopic delivery of a replication-deficient adenovirus expressing herpes simplex thymidine kinase (AdV-tk) in lung cancer patients who underwent surgery. AdV-tk injection proved safe, feasible, and effectively induced CD8+ T cell activation in blood and in resected tumor tissues.

Chimeric antigen receptor (CAR)-modified T cells are endowed with novel antigen specificity and are most often administered to patients without an engineered mechanism to control the CAR T cells once infused. “Suicide switches” such as the small molecule-controlled, inducible caspase-9 (iCas9) system afford the ability to selectively eliminate engineered T cells; however, these approaches are designed for all-or-none, irreversible termination of an ongoing immune response. In order to permit reversible and adjustable modulation, we have created a CAR that is capable of on-demand downregulation by fusing the CAR to a previously developed ligand-induced degradation (LID) domain. Addition of a small molecule ligand triggers exposure of a cryptic degron within the LID domain, resulting in proteasomal degradation of the CAR-LID fusion protein and loss of CAR on the surface of T cells. This fusion construct allowed for reversible and “tunable” inhibition of CAR T cell activity in vitro. Delivery of the triggering molecule in CAR-LID-treated tumor-bearing mice temporarily reduced CAR activity through modulation of CAR surface expression. The ability to more flexibly modulate CAR T cell expression through a small molecule provides a platform for controlling possible adverse side effects, as well as preclinical investigations of CAR T cell biology.

The metabolic milieu of solid tumors provides a barrier to chimeric antigen receptor (CAR) T-cell therapies. Excessive lactate or hypoxia suppresses T-cell growth, through mechanisms including NADH buildup and the depletion of oxidized metabolites. NADH is converted into NAD+ by the enzyme Lactobacillus brevis NADH Oxidase (LbNOX), which mimics the oxidative function of the electron transport chain without generating ATP. Here we determine if LbNOX promotes human CAR T-cell metabolic activity and antitumor efficacy. CAR T-cells expressing LbNOX have enhanced oxygen as well as lactate consumption and increased pyruvate production. LbNOX renders CAR T-cells resilient to lactate dehydrogenase inhibition. But in vivo in a model of mesothelioma, CAR T-cell’s expressing LbNOX showed no increased antitumor efficacy over control CAR T-cells. We hypothesize that T cells in hostile environments face dual metabolic stressors of excessive NADH and insufficient ATP production. Accordingly, futile T-cell NADH oxidation by LbNOX is insufficient to promote tumor clearance.

Immune checkpoint inhibitors demonstrate clinical activity in many tumor types, however, only a fraction of patients benefit. Combining CD137 agonists with these inhibitors increases anti-tumor activity preclinically, but attempts to translate these observations to the clinic have been hampered by systemic toxicity. Here we describe a human CD137xPD-L1 bispecific antibody, MCLA-145, identified through functional screening of agonist- and immune checkpoint inhibitor arm combinations. MCLA-145 potently activates T cells at sub-nanomolar concentrations, even under suppressive conditions, and enhances T cell priming, differentiation and memory recall responses. In vivo, MCLA-145 anti-tumor activity is superior to immune checkpoint inhibitor comparators and linked to recruitment and intra-tumor expansion of CD8 + T cells. No graft-versus-host-disease is observed in contrast to other antibodies inhibiting the PD-1 and PD-L1 pathway. Non-human primates treated with 100 mg/kg/week of MCLA-145 show no adverse effects. The conditional activation of CD137 signaling by MCLA-145, triggered by neighboring cells expressing >5000 copies of PD-L1, may provide both safety and potency advantages., The anti-tumour effect of immune checkpoint inhibitors is potentiated by CD137 agonists in preclinical models, but translation of these results to the clinical practice is hampered by toxicity. Authors describe here a human CD137xPD-L1 bispecific antibody with improved anti-cancer activity whilst maintaining low toxicity in non-human primates.

This phase I study investigated the safety and activity of lentiviral-transduced chimeric antigen receptor (CAR)-modified autologous T cells redirected against mesothelin (CART-meso) in patients with malignant pleural mesothelioma, ovarian carcinoma, and pancreatic ductal adenocarcinoma. Fifteen patients with chemotherapy-refractory cancer (n = 5 per indication) were treated with a single CART-meso cell infusion. CART-meso cells were engineered by lentiviral transduction with a construct composed of the anti-mesothelin single-chain variable fragment derived from the mouse monoclonal antibody SS1 fused to intracellular signaling domains of 4-1BB and CD3zeta. Patients received 1–3 × 10(7) or 1–3 × 10(8) CART-meso cells/m(2) with or without 1.5 g/m(2) cyclophosphamide. Lentiviral-transduced CART-meso cells were well tolerated; one dose-limiting toxicity (grade 4, sepsis) occurred at 1–3 × 10(7)/m(2) CART-meso without cyclophosphamide. The best overall response was stable disease (11/15 patients). CART-meso cells expanded in the blood and reached peak levels by days 6–14 but persisted transiently. Cyclophosphamide pre-treatment enhanced CART-meso expansion but did not improve persistence beyond 28 days. CART-meso DNA was detected in 7/10 tumor biopsies. Human anti-chimeric antibodies (HACA) were detected in the blood of 8/14 patients. CART-meso cells were well tolerated and expanded in the blood of all patients but showed limited clinical activity. Studies evaluating a fully human anti-mesothelin CAR are ongoing.

Fibroblast activation protein (FAP), a cell surface serine protease, is upregulated on a subset of activated fibroblasts (often distinct from α-smooth muscle actin-expressing myofibroblasts) associated with matrix remodeling, including fibroblasts in idiopathic pulmonary fibrosis (Acharya PS, Zukas A, Chandan V, Katzenstein AL, Puré E. Hum Pathol 37: 352–360, 2006.). As FAP+fibroblasts could be pivotal in either breakdown and/or production of collagen and other matrix components, the goal of this study was to define the role of FAP+cells in pulmonary fibrosis in two established, but different, mouse models of chronic lung fibrosis: repetitive doses of intratracheal bleomycin and a single dose of an adenoviral vector encoding constitutively active TGF-β1 (Ad-TGFβ). To determine their role in fibrotic remodeling, FAP-expressing cells were depleted by injection of T cells expressing a chimeric antigen receptor specific for murine FAP in mice with established fibrosis. The contribution of FAP to the function of FAP-expressing cells was assessed in FAP knockout mice. Using histological analyses, quantification of soluble collagen content, and flow cytometry, we found that loss of FAP+cells exacerbated fibrosis in the bleomycin model, a phenotype largely recapitulated by the genetic deletion of FAP, indicating that FAP plays a role in this model. In contrast, depletion of FAP+cells or genetic deletion of FAP had little effect in the Ad-TGFβ model highlighting the potential for distinct mechanisms driving fibrosis depending on the initiating insult. The role of FAP in human lung fibrosis will need to be well understood to guide the use of FAP-targeted therapeutics that are being developed.

Cancer progression is marked by dysfunctional tumor-infiltrating lymphocytes (TIL) with high inhibitory receptor (IR) expression. Because IR blockade has led to clinical responses in some patients with non–small cell lung cancer (NSCLC), we investigated how IRs influenced CD8+ TIL function from freshly digested early-stage NSCLC tissues using a killing assay and intracellular cytokine staining after in vitro T-cell restimulation. Early-stage lung cancer TIL function was heterogeneous with only about one third of patients showing decrements in cytokine production and lytic function. TIL hypofunction did not correlate with clinical factors, coexisting immune cells (macrophages, neutrophils, or CD4+ T regulatory cells), nor with PD-1, TIGIT, TIM-3, CD39, or CTLA-4 expression. Instead, we found that the presence of the integrin αeβ7 (CD103), characteristic of tissue-resident memory cells (TRM), was positively associated with cytokine production, whereas expression of the transcription factor Eomesodermin (Eomes) was negatively associated with TIL function. These data suggest that the functionality of CD8+ TILs from early-stage NSCLCs may be influenced by competition between an antitumor CD103+ TRM program and an exhaustion program marked by Eomes expression. Understanding the mechanisms of T-cell function in the progression of lung cancer may have clinical implications for immunotherapy.

PD1 blockade to reinvigorate T cells has become part of standard of care for patients with NSCLC across disease stages. However, the majority of patients still do not respond. One potential mechanism of resistance is increased expression of other checkpoint inhibitory molecules on T cells leading to their suppression; however, this phenomenon has not been well studied in tumor-reactive, human T cells. The purpose of this study was to evaluate this compensatory mechanism in a novel model using human effector T cells infiltrating and reactive against human lung cancer. Immunodeficient mice with flank tumors established from a human lung cancer cell line expressing the NYESO1 antigen were treated with activated human T cells expressing a TCR reactive to NYESO1 (Ly95) with or without anti-PD1 alone and with combinations of anti-PD1 plus anti-TIM3 or anti-TIGIT. A month later, the effect on tumor growth and the phenotype and ex vivo function of the TILs were analyzed. Anti-PD1 and Ly95 T cells led to greater tumor control than Ly95 T cells alone; however, tumors continued to grow. The ex-vivo function of PD1-blocked Ly95 TILs was suppressed and was associated with increased T cell expression of TIM3/TIGIT. Administering combinatorial blockade of PD1+ TIM3 or PD1+ TIGIT with Ly95 T cells led to greater tumor control than blocking PD1 alone. In our model, PD1 blockade was suboptimally therapeutic alone. The effect of TIM3 and TIGIT was upregulated on T cells in response to PD1 blockade and anti-tumor activity could be enhanced when these inhibitory receptors were also blocked with antibodies in combination with anti-PD1 therapy.

Chimeric antigen receptor (CAR) T cell immunotherapy is FDA approved for the treatment of a subset of B cell malignancies but has shown limited clinical success in solid tumor therapy. T cell exhaustion is an important factor involved in treatment failure, and can, in part, result from continuous stimulation of the CAR by tumor cells expressing the cognate antigen. To gain deeper understanding of CAR T cell exhaustion induced by chronic antigen exposure (CAE), we developed and validated a robust in vitro model, in which mesothelin-redirected CAR T cells (M5CAR) were continuously stimulated with mesothelin-expressing AsPC-1 pancreatic tumor cells such that tumor cells were never cleared, and we characterized these CAE CAR T cells by gene expression at population and single-cell levels, and by epigenetic analyses. CAE M5CAR T cells recapitulate the hallmarks of T cell exhaustion, including reduced proliferation, down-modulation of surface CAR, decreased cytokine production, and reduced cytotoxicity. In addition, CAR T cells undergoing CAE have a transcriptional signature and an epigenetic landscape consistent with exhaustion. Further, transcriptomic analysis revealed that CAE M5CAR T cells undergo a transition from T cells to a post-thymic NK-like T cell phenotype. This plasticity was confirmed by TCR lineage tracing and was also detected in CD19 CAR T cells analyzed from post-infusion blood from DLBCL patients and in M5CAR T cells infiltrating relapsed tumors derived from a xenograft NSG/AsPC-1 mouse model. The dysfunctional signature and the NK phenotype were further detected in vivo by transcriptomic analysis in Ly95 TCR-specific TILs infiltrating NY-ESO-1 tumors. Among the genes included in the dysfunctional signature, the transcription factors SOX4 and ID3 were identified as potential regulators of dysfunction by differential gene expression and pathway analyses. To determine their role in the establishment of a dysfunctional phenotype, we generated ID3KO.M5 and SOX4KO.M5 CAR T cells using CRISPR technology in primary human lymphocytes. The cytotoxic potential of the M5 CAR T cell product generated was not modified by the genetic disruption of the transcription factors. However, when CAR T cells were challenged with chronic antigen stimulation, KO CAR T cells showed a significant reduction of the dysfunctional signature and the NK-like T cell gene expression. Importantly, CAE KO CAR T cells exhibit improved cytotoxicity as compared to Mock.M5CAR T cells. In summary, we have developed a robust in vitro model that recapitulates the hallmarks of T cell exhaustion and that facilitated the identification of a gene signature defining CAR dysregulation, a T-to-NK-like-T cell transition as a novel feature of CAR T cell dysfunction and the transcription factors SOX4 and ID3 as key regulators of CAR T cell exhaustion. Citation Format: M. Angela Aznar, Charly R. Good, Shunichiro Kuramitsu, Parisa Samareh, Sangya Agarwal, Greg Donahue, Kenichi Ishiyama, Nils Wellhausen, Austin K. Rennels, Yujie Ma, Lifeng Tian, Sonia Guedan, Katherine A. Alexander, Zhen Zhang, Philipp C. Rommel, Nathan Singh, Karl M. Glastad, Max W. Richardson, Keisuke Watanabe, Janos L. Tanyi, Mark H. O’Hara, Marco Ruella, Simon F. Lacey, Edmund K. Moon, Stephen J. Schuster, Steven M. Albelda, Lewis L. Lanier, Regina M. Young, Shelley L. Berger, Carl H. June. Mechanisms of CAR T cell dysfunction and identification of transcription factors that drive the exhaustion phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3602.

Summary Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable success in hematological malignancies but remains ineffective in solid tumors, due in part to CAR T cell exhaustion in the solid tumor microenvironment. To study dysfunction of mesothelin-redirected CAR T cells in pancreatic cancer, we establish a robust model of continuous antigen exposure that recapitulates hallmark features of T cell exhaustion and discover, both in vitro and in CAR T cell patients, that CAR dysregulation is associated with a CD8+ T-to-NK-like T cell transition. Furthermore, we identify a gene signature defining CAR and TCR dysregulation and transcription factors, including SOX4 and ID3 as key regulators of CAR T cell exhaustion. Our findings shed light on the plasticity of human CAR T cells and demonstrate that genetic downmodulation of ID3 and SOX4 expression can improve the efficacy of CAR T cell therapy in solid tumors by preventing or delaying CAR T cell dysfunction.

Background Airway complications affect roughly 15-20% of lung transplant patients. Airway stents are an attractive therapeutic option; however, no experimental or controlled observational data exists to draw firm conclusions regarding airway stent efficacy and safety in this population. Methods We performed a retrospective cohort study of patients who underwent airway stent placement for post-transplant anastomotic airway complications. The primary outcomes were improvements in FEV1 and reduction in bronchoscopies post-stent. Results We identified 36 patients who underwent airway stenting between October 2012 and October 2017. A total of 47 airways underwent stent placement. Improvement in FEV1 after stent placement was only observed in patients who ultimately were able to undergo stent removal. Patients who expired prior to stent removal had no immediate FEV1 improvement after stent placement. Among subjects who underwent stent removal, there was a statistically significant reduction in number of bronchoscopies per month after stent removal compared to pre-stent placement. Male gender was the only predictor of FEV1 improvement after stent placement while male gender and dehiscence prior to stent placement predicted increased number of bronchoscopies after stent placement. Mucous plugging and granulation tissue formation were the most common stent related complications. Conclusions Only select patients benefit from stent placement for airways stenosis after lung transplant. Complications related to stent placement are common. Patients with airway complications treated with airway stents undergo a high volume of repeat procedures.