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Chimeric antigen receptor (CAR) T-cells directed against CD19 have drastically altered outcomes for children with relapsed and refractory acute lymphoblastic leukemia (r/r ALL). Pediatric patients with r/r ALL treated with CAR-T are at increased risk of both cytokine release syndrome (CRS) and sepsis. We sought to investigate the biologic differences between CRS and sepsis and to develop predictive models which could accurately differentiate CRS from sepsis at the time of critical illness. We identified 23 different cytokines that were significantly different between patients with sepsis and CRS. Using elastic net prediction modeling and tree classification, we identified cytokines that were able to classify subjects as having CRS or sepsis accurately. A markedly elevated interferon γ (IFNγ) or a mildly elevated IFNγ in combination with a low IL1β were associated with CRS. A normal to mildly elevated IFNγ in combination with an elevated IL1β was associated with sepsis. This combination of IFNγ and IL1β was able to categorize subjects as having CRS or sepsis with 97% accuracy. As CAR-T therapies become more common, these data provide important novel information to better manage potential associated toxicities.

Chimeric antigen receptor–engineered T cells targeting CD19 (CART19) provide an effective treatment for pediatric acute lymphoblastic leukemia but are less effective for chronic lymphocytic leukemia (CLL), focusing attention on improving efficacy. CART19 harbor an engineered receptor, which is delivered through lentiviral vector integration, thereby marking cell lineages and modifying the cellular genome by insertional mutagenesis. We recently reported that vector integration within the host TET2 gene was associated with CLL remission. Here, we investigated clonal population structure and therapeutic outcomes in another 39 patients by high-throughput sequencing of vector-integration sites. Genes at integration sites enriched in responders were commonly found in cell-signaling and chromatin modification pathways, suggesting that insertional mutagenesis in these genes promoted therapeutic T cell proliferation. We also developed a multivariate model based on integration-site distributions and found that data from preinfusion products forecasted response in CLL successfully in discovery and validation cohorts and, in day 28 samples, reported responders to CLL therapy with high accuracy. These data clarify how insertional mutagenesis can modulate cell proliferation in CART19 therapy and how data on integration-site distributions can be linked to treatment outcomes.

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.

Adoptive T cell therapies for solid tumors is challenging. We generated metabolically enhanced co-activated-T cells by transducing intracellular co-stimulatory (41BB, ICOS or ICOS-27) and CD3ζ T cell receptor signaling domains followed by arming with bispecific antibodies (BiAbs) to produce armed “Headless CAR T cells” (hCART). Various hCART armed with BiAb directed at CD3ϵ and various tumor associated antigens were tested for: 1) specific cytotoxicity against solid tumors targets; 2) repeated and dual sequential cytotoxicity; 3) survival and cytotoxicity under in vitro hypoxic condition; and 4) cytokine secretion. The 41BBζ transduced hCART (hCART41BBζ) armed with HER2 BiAb (HER2 hCART41BBζ) or armed with EGFR BiAb (EGFR hCART41BBζ) killed multiple tumor lines significantly better than control T cells and secreted Th1 cytokines/chemokines upon tumor engagement at effector to target ratio (E:T) of 2:1 or 1:1. HER2 hCART serially killed tumor targets up to 14 days. Sequential targeting of EGFR or HER2 positive tumors with HER2 hCART41BBζ followed by EGFR hCART41BBζ showed significantly increased cytotoxicity compared single antigen targeting and continue to kill under in vitro hypoxic conditions. In summary, metabolically enhanced headless CAR T cells are effective serial killers of tumor targets, secrete cytokines and chemokines, and continue to kill under in vitro hypoxic condition.

PURPOSE CD19-targeted chimeric antigen receptor (CAR)–modified T cells demonstrate unprecedented responses in B-cell acute lymphoblastic leukemia (B-ALL); however, relapse remains a substantial challenge. Short CAR T-cell persistence contributes to this risk; therefore, strategies to improve persistence are needed. METHODS We conducted a pilot clinical trial of a humanized CD19 CAR T-cell product (huCART19) in children and young adults with relapsed or refractory B-ALL (n = 72) or B-lymphoblastic lymphoma (n = 2), treated in two cohorts: with (retreatment, n = 33) or without (CAR-naive, n = 41) prior CAR exposure. Patients were monitored for toxicity, response, and persistence of huCART19. RESULTS Seventy-four patients 1-29 years of age received huCART19. Cytokine release syndrome developed in 62 (84%) patients and was grade 4 in five (6.8%). Neurologic toxicities were reported in 29 (39%), three (4%) grade 3 or 4, and fully resolved in all cases. The overall response rate at 1 month after infusion was 98% (100% in B-ALL) in the CAR-naive cohort and 64% in the retreatment cohort. At 6 months, the probability of losing huCART19 persistence was 27% (95% CI, 14 to 41) for CAR-naive and 48% (95% CI, 30 to 64) for retreatment patients, whereas the incidence of B-cell recovery was 15% (95% CI, 6 to 28) and 58% (95% CI, 33 to 77), respectively. Relapse-free survival at 12 and 24 months, respectively, was 84% (95% CI, 72 to 97) and 74% (95% CI, 60 to 90) in CAR-naive and 74% (95% CI, 56 to 97) and 58% (95% CI, 37 to 90) in retreatment cohorts. CONCLUSION HuCART19 achieved durable remissions with long-term persistence in children and young adults with relapsed or refractory B-ALL, including after failure of prior CAR T-cell therapy.

INTRODUCTION Anti-CD19 CAR T-cell immunotherapy is promising for patients with relapsed/refractory CLL. Hypogammaglobinemia can result from normal CD19+ B-cell depletion by CAR-T cells. CLL patients are already at risk for infections due to impaired immune function, lymphodepletion prior to CAR-T cells infusion, and immunosuppressive therapies. Intravenous immunoglobulin (IVIG) is used to manage hypogammaglobulinemia, although standard criteria for IVIG administration in this setting has not been established. We studied the incidence of hypogammaglobinemia and report infectious complications, risk factors, IVIG use, and clinical outcomes for CLL patients treated with anti-CD19 CAR-T cells. METHODS Adult CLL patients who received CD19-directed CAR-T therapy in 3 clinical trials (NCT01029366, NCT01747486, NCT02640209) from July 2010 to February 2020 were included. We reviewed demographics, available IgG levels, IVIG use, and clinical outcomes with a particular focus on infectious complications. Hypogammaglobulinemia was defined as IgG RESULTS Records of 71 adult patients with CLL were reviewed; 4 were excluded from further analysis as they did not have IgG levels after CAR-T. The median age at time of CAR-T was 63 years (range 43-78 years). 31 patients (46%) were alive, 31 (46%) were deceased, and 5 (7%) were lost to follow up at time of review. Median follow up for all patients was 33 months (range 2-114 months). Of the 55 patients with an IgG level prior to CAR-T infusion, 24 (44%) had hypogammaglobulinemia at baseline After CAR-T infusion, 54 of 67 patients (81%) developed new or persistent hypogammaglobulinemia, and 40 of these patients (74%) received IVIG (Table 1). Forty-eight patients (72%) received at least one infusion of IVIG after CAR-T. Median time to initiation of IVIG after CAR-T infusion was 2.8 months (range 0.1-71.2 months). IVIG was used in 29 of 35 (83%) responders (defined as PR or CR) vs 19 of 32 (59%) in non-responders (defined as NR or PD) (p=0.056). There was no difference in survival observed based on whether or not patients had hypogammaglobulinemia (Figure). 42 patients (63%) had documented infections not related to chemotherapy-induced neutropenia. Fifteen (22%) had one documented infection, and 27 (40%) had more than one documented infection. There were 13 infections documented in patients who did not have hypogammaglobulinemia; the most common were ENT/sinus infections (5), bacteremia (2), URI (2), and other (2). There were 94 infections documented in patients who developed or had persistent hypogammaglobulinemia; the most common were lower respiratory tract infection/pneumonia (21), URI (20), and ENT/sinus infections (13). Complete and partial responders had more infections compared to non-responders or those who progressed (p = 0.01). Patients with hypogammaglobulinemia after CAR-T had an average of 1.74 (range 0-15) infections vs 1 (range 0-3) in patients without hypogammaglobulinemia. Patients who received IVIG had more infections (p=0.003); without IVIG the average number of infections was 0.58 (range 0-3), and with IVIG, the average number of infections was 2.00 (range 0-15). CONCLUSION Evaluating clinical outcomes with infections after CAR-T and potential strategies to minimize infection risk may improve morbidity and mortality in CLL patients. Use of IVIG was driven by individual practice, with heterogeneity regarding indication, frequency, and duration of treatment, though there was no difference in patient characteristics or response in patients who did or did not develop hypogammaglobulinemia. Patients who responded to CAR-T had more frequent infections, as might be expected in the setting of transient or persistent B- cell aplasia or hypoplasia. Patients who had more infections were more likely to receive IVIG. Further studies to define criteria for IVIG repletion in CLL patients treated with CD19-directed CAR-T cells may be incorporated in a standard clinical management algorithm. Table 1 Disclosures Frey: Amgen: Consultancy, Honoraria; Syntax: Consultancy, Honoraria; Kite Pharma: Consultancy, Honoraria. Davis:Tmunity Therapeutics, Inc.: Consultancy, Patents & Royalties, Research Funding; Cellares Corporation: Membership on an entity's Board of Directors or advisory committees; Novartis Institutes for Biomedical Research: Patents & Royalties. Hexner:Blueprint Medicines Corporation: Other: serves on a data safety monitoring committee, Research Funding; Novartis: Research Funding; Samus Therapeutics: Research Funding; American Board of Internal Medicine: Other: member of the hematology exam committee. Schuster:Novartis, Genentech, Inc./ F. Hoffmann-La Roche: Research Funding; AlloGene, AstraZeneca, BeiGene, Genentech, Inc./ F. Hoffmann-La Roche, Juno/Celgene, Loxo Oncology, Nordic Nanovector, Novartis, Tessa Therapeutics: Consultancy, Honoraria. Gill:Fate: Consultancy; Sensei: Consultancy; Aileron: Consultancy; Tmunity Therapeutics: Research Funding; Carisma Therapeutics: Patents & Royalties, Research Funding; Novartis: Research Funding. Grupp:Servier: Research Funding; Kite/Gilead: Research Funding; Roche: Consultancy; GlaxoSmithKline: Consultancy; Humanigen: Consultancy; CBMG: Consultancy; Jazz: Other: SSC; Adaptimmune: Other: SAB; TCR2: Other: SAB; Cellectis: Other; Juno/BMS: Other; Janssen/JnJ: Consultancy; CRISPR Therapeutics/Vertex Pharmaceuticals: Other; Allogene: Other; Novartis: Consultancy, Other: SSC, Research Funding. Melenhorst:Johnson & Johnson: Consultancy, Other: Speaker; Novartis: Other: Speaker, Research Funding; Kite Pharma: Research Funding; IASO Biotherapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Poseida Therapeutics: Consultancy; Simcere of America: Consultancy. Lacey:Novartis: Patents & Royalties: CAR T cells, Research Funding; Tmunity: Research Funding; Cabaletta: Research Funding; Carisma: Research Funding. Fraietta:Tmunity: Research Funding. Hwang:Novartis: Research Funding; Tmunity Therapeutics: Research Funding. Siegel:Novartis: Patents & Royalties; Tmunity: Patents & Royalties; Poseida: Membership on an entity's Board of Directors or advisory committees; Vetigenics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Levine:Terumo: Consultancy; Novartis: Consultancy, Patents & Royalties: Dr. Levine has a patent Methods for treatment of cancer (US 8906682) (US 8916381)(US 9101584) with royalties paid to University of Pennsylvania, a patent Compositions for treatment of cancer (US 8911993) (US 9102761) (US 9102760) with royalties paid to U; Lilly Asia Ventures: Consultancy; Avectas: Membership on an entity's Board of Directors or advisory committees; Patheon: Membership on an entity's Board of Directors or advisory committees; Immuneel: Membership on an entity's Board of Directors or advisory committees; Incysus: Membership on an entity's Board of Directors or advisory committees; Ori Biotech: Membership on an entity's Board of Directors or advisory committees; Vycellix: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Current equity holder in private company, Research Funding. June:Bluesphere Bio: Membership on an entity's Board of Directors or advisory committees; Cabaletta Bio: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Carisma Therapeutics: Membership on an entity's Board of Directors or advisory committees; Cellares: Membership on an entity's Board of Directors or advisory committees; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; DeCART Therapeutics: Membership on an entity's Board of Directors or advisory committees; Immune Design: Membership on an entity's Board of Directors or advisory committees; Kiadis Pharma: Current equity holder in private company; Novartis: Patents & Royalties, Research Funding; Tmunity Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Ziopharm Oncology: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Porter:Tmunity: Patents & Royalties; Novartis: Honoraria, Other: Advisory board, Patents & Royalties: CAR T cells for CD19+ malignancies, Research Funding; American Board of Internal Medicine: Other: Member, exam writing committee (end date Oct 2019); National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Janssen: Other: Advisory board; Genentech/Roche: Current equity holder in publicly-traded company, Other: Spouse employment (ended Sept 2020); her salary includes stock/options; Glenmark: Other: Advisory board; Adicet bio: Other: Advisory board; Incyte: Other: Advisory board; Kite/Gilead: Other: Advisory board. OffLabel Disclosure: CAR T cells for CLL

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.

Genetically engineered T cells are powerful new medicines, offering hope for curative responses in patients with cancer. Chimeric antigen receptor (CAR) T cells were recently approved by the US Food and Drug Administration and are poised to enter the practice of medicine for leukemia and lymphoma, demonstrating that engineered immune cells can serve as a powerful new class of cancer therapeutics. The emergence of synthetic biology approaches for cellular engineering provides a broadly expanded set of tools for programming immune cells for enhanced function. Advances in T cell engineering, genetic editing, the selection of optimal lymphocytes, and cell manufacturing have the potential to broaden T cell–based therapies and foster new applications beyond oncology, in infectious diseases, organ transplantation, and autoimmunity.

Tisagenlecleucel is a chimeric antigen receptor–T cell therapy that facilitates the killing of CD19+ B cells. A model was developed for the kinetics of tisagenlecleucel and the impact of therapies for treating cytokine release syndrome (tocilizumab and corticosteroids) on expansion. Data from two phase II studies in pediatric and young adult relapsed/refractory B cell acute lymphoblastic leukemia were pooled to evaluate this model and evaluate extrinsic and intrinsic factors that may impact the extent of tisagenlecleucel expansion. The doubling time, initial decline half‐life, and terminal half‐life for tisagenlecleucel were 0.78, 4.3, and 220 days, respectively. No impact of tocilizumab or corticosteroids on the expansion rate was observed. This work represents the first mixed‐effect model‐based analysis of chimeric antigen receptor–T cell therapy and may be clinically impactful as future studies examine prophylactic interventions in patients at risk of higher grade cytokine release syndrome and the effects of these interventions on chimeric antigen receptor–T cell expansion.

Genome engineering methods have advanced greatly with the development of programmable nucleases, but methods for quantifying on- and off-target cleavage sites and associated deletions remain nascent. Here, we report an improvement of the GUIDE-seq method, iGUIDE, which allows filtering of mispriming events to clarify the true cleavage signal. Using iGUIDE, we specify the locations of Cas9-guided cleavage for four guide RNAs, characterize associated deletions, and show that naturally occurring background DNA double-strand breaks are associated with open chromatin, gene dense regions, and chromosomal fragile sites. iGUIDE is available from https://github.com/cnobles/iGUIDE. Electronic supplementary material The online version of this article (10.1186/s13059-019-1625-3) contains supplementary material, which is available to authorized users.

The adoptive transfer of T lymphocytes reprogrammed to target tumor cells has demonstrated significant potential in various malignancies. However, little is known about the long-term potential and the clonal stability of the infused cells. Here, we studied the longest persisting CD19 redirected chimeric antigen receptor (CAR) T cells to date in two chronic lymphocytic leukemia (CLL) patients who achieved a complete remission in 2010. CAR T-cells were still detectable up to 10+ years post-infusion, with sustained remission in both patients. Surprisingly, a prominent, highly activated CD4+ population developed in both patients during the years post-infusion, dominating the CAR T-cell population at the late time points. This transition was reflected in the stabilization of the clonal make-up of CAR T-cells with a repertoire dominated by few clones. Single cell multi-omics profiling via Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) with TCR sequencing of CAR T-cells obtained 9.3 years post-infusion demonstrated that these long-persisting CD4+ CAR T-cells exhibited cytotoxic characteristics along with strong evidence of ongoing functional activation and proliferation. Our data provide novel insight into the CAR T-cell characteristics associated with long-term remission in leukemia.

Autologous chimeric antigen receptor (CAR) T cells targeted to epidermal growth factor receptor variant III (CAR T-EGFRvIII) have been developed and administered experimentally to treat patients with IDH1 wildtype recurrent glioblastoma (rGBM) (NCT02209376). We report the case of a 59-year-old patient who received a single peripheral infusion of CAR T-EGFRvIII cells and survived 36 months after disease recurrence, exceeding expected survival for recurrent glioblastoma. Post-infusion histopathologic analysis of tissue obtained during a second stage surgical resection revealed immunosuppressive adaptive changes in the tumor tissue as well as reduced EGFRvIII expression. Serial brain imaging demonstrated a significant reduction in relative cerebral blood volume (rCBV), a measure strongly associated with tumor proliferative activity, at early time points following CAR T treatment. Notably, CAR T-EGFRvIII cells persisted in her peripheral circulation during 29 months of follow-up, the longest period of CAR T persistence reported in GBM trials to date. These findings in a long-term survivor show that peripherally administered CAR T-EGFRvIII cells can persist for years in the circulation and suggest that this cell therapy approach could be optimized to achieve broader efficacy in recurrent GBM patients.

BackgroundAutologous T cells engineered to express a chimeric antigen receptor (CAR) specific for CD19 molecule have transformed the therapeutic landscape in patients with highly refractory leukemia and lymphoma, and the use of donor-generated allogeneic CAR T is paving the way for further breakthroughs in the treatment of cancer. However, it remains unknown how the intrinsic heterogeneities of these engineered cells mediate therapeutic efficacy and whether allogeneic products match the effectiveness of autologous therapies.MethodsUsing single-cell mRNA sequencing in conjunction with CITE-seq, we performed multiomics characterization of CAR T cells generated from healthy donor and patients with acute lymphoblastic leukemia. CAR T cells used in this study were manufactured at the University of Pennsylvania through lentiviral transduction with a CD19-4-1BB-CD3ζ construct. Besides the baseline condition, we engineered NIH-3T3 cells with human CD19 or mesothelin expression to conduct ex vivo antigen-specific or non-antigen stimulation of CAR T cells through 6-hour coculture at a 1:1 ratio.ResultsWe delineated the global cellular and molecular CAR T landscape and identified that transcriptional CAR tonic signaling was regulated by a mixture of early activation, exhaustion signatures, and cytotoxic activities. On CD19 stimulation, we illuminated the disparities of CAR T cells derived from different origins and found that donor CAR T had more pronounced activation level in correlation with the upregulation of major histocompatibility complex class II genes compared with patient CAR T cells. This finding was independently validated in additional datasets from literature. Furthermore, GM-CSF(CSF2) expression was found to be associated with functional gene productions, but it induced little impact on the CAR T activation.ConclusionsThrough integrated multiomics profiling and unbiased canonical pathway analyses, our results unveil heterogeneities in the transcriptional, phenotypic, functional, and metabolic profiles of donor and patient CAR T cells, providing mechanistic basis for ameliorating clinical outcomes and developing next-generation ‘off- the-shelf’ allogeneic products.

Chimeric antigen receptor-modified (CAR) T cells targeting CD19 have mediated dramatic responses in relapsed/refractory acute lymphoblastic leukemia (ALL), yet a notable number of patients have CD19-positive relapse within one year of treatment. It remains unclear if the long-term response is associated with the characteristics of CAR T cells in infusion products, hindering the identification of biomarkers to predict therapeutic outcomes prior to treatment. Herein we present 101,326 single cell transcriptomes and surface protein landscape from the CAR T infusion products of 12 pediatric ALL patients upon CAR antigen-specific stimulation in comparison with TCR-mediated activation and controls. We observed substantial heterogeneity in the antigen-specific activation states, among which a deficiency of Th2 function was associated with CD19-positive relapsed patients (median remission 9.6 months) compared with very durable responders (remission>54 months). Proteomic profiles also revealed that the frequency of early memory T cell subsets, rather than activation or co-inhibitory signatures could distinguish CD19-positive relapse. Additionally, a deficit of type 1 helper and cytotoxic effector function and an enrichment for terminally differentiated CD8+ T cells exhibiting low cytokine polyfunctionality was associated with initial non-responders. By contrast, the single-cell transcriptomic data of unstimulated or TCR-activated CAR T cells failed to predict clinical responses. In aggregate, our results dissect the landscape of CAR-specific activation states in infusion products that can identify patients who do not develop a durable response to the therapy, and unveil the molecular mechanisms that may inform strategies to boost specific T cell function to maintain long term remission.

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

BackgroundCAR-T cell therapy has demonstrated remarkable success in hematological malignancies but displays limited efficacy in solid tumors, which comprise most cancer cases. Recent studies suggest that CAR-T cell failure via T cell exhaustion is characterized by decreased surface CAR expression, cytotoxicity, and Th1 cytokine production, leading to reduced antitumor functionality.1 2 3 To address these issues, studies have turned to genetically knocking out or overexpressing targets associated with an exhaustion or effector phenotype, such as PD-1 knockout (KO) and c-Jun overexpression, among other candidates that are typically receptors or transcription factors.4 5 6 However, there are other underexplored factors that mediate various aspects of immune regulation. While genome-wide CRISPR screens may discover such factors, they are unlikely to reveal phenotypes for genes whose function is partially redundant, therefore promising candidates may be missed. Such candidates include post-transcriptional regulators (PTRs) that coordinate immune responses, which are less well-studied in the context of CAR-T cell function. We hypothesized that KO of these PTRs may increase CAR-T cell cytokine activity, phenotype, and persistence, potentially under long-term or exhaustion-inducing conditions, leading to increased tumor control. Ultimately, disruption of negative immune regulators could produce CAR-T cells with enhanced activity and persistence, narrowing the gap between efficacy in hematological and solid tumors.MethodsTo explore whether the disruption of two target PTRs improves solid tumor efficacy, we used CRISPR-Cas9 to genetically delete one or both PTRs in mesothelin-targeting human CAR-T cells and assayed their function in vitro and in vivo in NSG mice.ResultsWe show successful genetic deletion of these genes in post-thymic human T cells and that their disruption does not affect primary expansion (figure 1) or transduction efficiency (figure 2). These KO CAR-T cells display increased expression of co-stimulatory receptors and various cytokines (figure 3). While KO CAR-T cells are functionally similar to WT CAR-T cells in in vitro assays (figure 4), KO CAR-T cells demonstrate superior activity in vivo and can clear large, established tumors compared to WT CAR-T cells at low dose (figure 5).Abstract 131 Figure 1Expansion kinetics of KO CAR-T cellsAbstract 131 Figure 2Transduction efficiency and baseline phenotype of KO CAR-T cellsAbstract 131 Figure 3Costimulatory receptor and cytokine expression of KO CAR-T cellsAbstract 131 Figure 4In vitro cytotoxicity of KO CAR-T cellsAbstract 131 Figure 5In vivo activity of KO CAR-T cellsConclusionsThese results indicate that KO of our target PTRs may improve the potency of CAR-T cells in solid tumors and may have important implications on the development of effective solid-tumor cell therapies.ReferencesJE Wherry and M Kurachi, Molecular and cellular insights into T cell exhaustion, Nature Reviews Immunology 2015;15:486–499.EW Weber, et al. Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling. Science 2021;372:6537.S Kuramitsu et al. Induction of T cell dysfunction and NK-like T cell differentiation in vitro and in patients after CAR T cell treatment. Cell, in revision.BD Choi et al, CRISPR-Cas9 disruption of PD-1 enhances activity of university EGFRvIII CAR T cells in a preclinical model of human glioblastoma. Journal for ImmunoTherapy of Cancer 2019;7:304.RC Lynn et al. c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature 2019;576:293–300.LJ Rupp et al. CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells. Scientific Reports 2017;7:737.

Long-Term Follow-up of CAR T-Cell–Treated Lymphoma Progression-free survival at 5 years was 31% in patients with diffuse large B-cell lymphoma and 43% in patients with follicular lymphoma. A total ...

Chimeric antigen receptor (CAR) T cell therapy has shown promise in hematologic malignancies, but its application to solid tumors has been challenging1–4. Given the unique effector functions of macrophages and their capacity to penetrate tumors5, we genetically engineered human macrophages with CARs to direct their phagocytic activity against tumors. We found that a chimeric adenoviral vector overcame the inherent resistance of primary human macrophages to genetic manipulation and imparted a sustained pro-inflammatory (M1) phenotype. CAR macrophages (CAR-Ms) demonstrated antigen-specific phagocytosis and tumor clearance in vitro. In two solid tumor xenograft mouse models, a single infusion of human CAR-Ms decreased tumor burden and prolonged overall survival. Characterization of CAR-M activity showed that CAR-Ms expressed pro-inflammatory cytokines and chemokines, converted bystander M2 macrophages to M1, upregulated antigen presentation machinery, recruited and presented antigen to T cells and resisted the effects of immunosuppressive cytokines. In humanized mouse models, CAR-Ms were further shown to induce a pro-inflammatory tumor microenvironment and boost anti-tumor T cell activity. Primary macrophages engineered to express chimeric antigen receptors have anti-tumor activity in humanized mice.

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.

Off-tumor targeting of human antigens is difficult to predict in preclinical animal studies and can lead to serious adverse effects in patients. To address this, we developed a mouse model with stable and tunable human Her2 (hHer2) expression on normal hepatic tissue and compared toxicity between affinity-tuned Her2 chimeric antigen receptor T cells (CARTs). In mice with hHer2-high livers, both the high-affinity (HA) and low-affinity (LA) CARTs caused lethal liver damage due to immunotoxicity. In mice with hHer2-low livers, LA-CARTs exhibited less liver damage and lower systemic levels of IFN-γ than HA-CARTs. We then compared affinity-tuned CARTs for their ability to control a hHer2-positive tumor xenograft in our model. Surprisingly, the LA-CARTs outperformed the HA-CARTs with superior antitumor efficacy in vivo. We hypothesized that this was due, in part, to T cell trafficking differences between LA and HA-CARTs and found that the LA-CARTs migrated out of the liver and infiltrated the tumor sooner than the HA-CARTs. These findings highlight the importance of T cell targeting in reducing toxicity of normal tissue and also in preventing off-tumor sequestration of CARTs, which reduces their therapeutic potency. Our model may be useful to evaluate various CARTs that have conditional expression of more than 1 single-chain variable fragment (scFv).

Summary Poor tumor infiltration, development of exhaustion, and antigen insufficiency are common mechanisms that limit chimeric antigen receptor (CAR)-T cell efficacy. Delivery of pattern recognition receptor agonists is one strategy to improve immune function; however, targeting these agonists to immune cells is challenging, and off-target signaling in cancer cells can be detrimental. Here, we engineer CAR-T cells to deliver RN7SL1, an endogenous RNA that activates RIG-I/MDA5 signaling. RN7SL1 promotes expansion and effector-memory differentiation of CAR-T cells. Moreover, RN7SL1 is deployed in extracellular vesicles and selectively transferred to immune cells. Unlike other RNA agonists, transferred RN7SL1 restricts myeloid-derived suppressor cell (MDSC) development, decreases TGFB in myeloid cells, and fosters dendritic cell (DC) subsets with costimulatory features. Consequently, endogenous effector-memory and tumor-specific T cells also expand, allowing rejection of solid tumors with CAR antigen loss. Supported by improved endogenous immunity, CAR-T cells can now co-deploy peptide antigens with RN7SL1 to enhance efficacy, even when heterogenous CAR antigen tumors lack adequate neoantigens.

PURPOSE: Although adoptive cell therapy with chimeric antigen receptor (CAR)-engineered T cells has shown durable clinical efficacy in patients with CD19(+) B-cell malignancies, the application of this approach to solid tumors is challenging. The goal of this proof-of-concept study was to investigate whether loading of CD19-CAR T cells (CART19) with anti-HER2 or anti-EGFR bispecific antibodies (BiAb) will target HER2+/EGFR+ CD19- targets and signal the intracellular domain of CAR without engaging antigen-specific CD19 ScFv of CAR T cells. METHODS: We used CART19 armed with anti-CD3 (OKT3) × anti-HER2 BiAb (HER2Bi) or anti-CD3 (OKT3) × anti-EGFR BiAb (EGFRBi) to evaluate the cytotoxicity directed at HER2 or EGFR expressing cancer cell lines compared with unarmed CART19 measured by short-term (51)Cr release assay and long-term real-time cell analysis using xCelligence. We also determined the differences in exhaustion or effector phenotypes and cytokine profiles during the short- and long-term cytotoxicity assays. RESULTS: Specific cytotoxicity was exhibited by CART19 armed with HER2Bi or EGFRBi against multiple tumor cell lines. Armed CART19 and armed activated T cells (ATC) showed comparable specific cytotoxicity that ranged between 10 and 90% against breast, pancreatic, ovarian, prostate, and lung cancer cell lines at 10:1 E/T ratio. Serial killing (repeated killing) by HER2Bi-armed CART19 ranged between 80 and 100% at 10:1 E/T ratio against MCF-7 cells up to 19 days (up to 4(th) round of repeated killing) measured by a real-time cell analysis without CART19 becoming exhausted. CONCLUSIONS: HER2Bi- or EGFRBi-armed CART19 exhibited specific cytotoxicity against multiple HER2(+)/EGFR+/CD19(−) tumor targets in overnight and long-term serial killing assays. CART19 showed improved survival and were resistant to exhaustion after prolonged repeated exposure to tumor cells.

Chimeric antigen receptor T cell (CAR-T cell) therapies have shown significant efficacy in CD19+ leukemias and lymphomas. There remain many challenges and questions for improving next-generation CAR-T cell therapies, and mathematical modeling of CAR-T cells may play a role in supporting further development. In this review, we introduce a mathematical modeling taxonomy for a set of relatively simple cellular kinetic-pharmacodynamic models that describe the in vivo dynamics of CAR-T cell and their interactions with cancer cells. We then discuss potential extensions of this model to include target binding, tumor distribution, cytokine-release syndrome, immunophenotype differentiation, and genotypic heterogeneity.

Clinical response to chimeric antigen receptor (CAR) T cell therapy is correlated with CAR T cell persistence, especially for CAR T cells that target CD19(+) hematologic malignancies. 4-1BB–costimulated CAR (BBζ) T cells exhibit longer persistence after adoptive transfer than do CD28-costimulated CAR (28ζ) T cells. 4-1BB signaling improves T cell persistence even in the context of 28ζ CAR activation, which indicates distinct prosurvival signals mediated by the 4-1BB cytoplasmic domain. To specifically study signal transduction by CARs, we developed a cell-free, ligand-based activation and ex vivo culture system for CD19-specific CAR T cells. We observed greater ex vivo survival and subsequent expansion of BBζ CAR T cells when compared to 28ζ CAR T cells. We showed that only BBζ CARs activated noncanonical nuclear factor κB (ncNF-κB) signaling in T cells basally and that the anti-CD19 BBζ CAR further enhanced ncNF-κB signaling after ligand engagement. Reducing ncNF-κB signaling reduced the expansion and survival of anti-CD19 BBζ T cells and was associated with a substantial increase in the abundance of the most pro-apoptotic isoforms of Bim. Although our findings do not exclude the importance of other signaling differences between BBζ and 28ζ CARs, they demonstrate the necessary and nonredundant role of ncNF-κB signaling in promoting the survival of BBζ CAR T cells, which likely underlies the engraftment persistence observed with this CAR design.

Introduction. Anti-CD19 chimeric antigen receptor T cells (CART19, CTL019) have led to impressive clinical responses in relapsing or refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL). With the likely imminent approval of CTL019 for patients with B-ALL, increasing numbers of patients will be exposed to this therapy. Last year we reported a rare case of a single patient who relapsed with CD19-negative, CAR19-expressing leukemia, likely due to inadvertent transduction of a leukemic cell with the CAR19 lentivirus during CTL019 manufacturing (Lacey SF, ASH, 2016 #281). The high expression of the CAR in leukemic cells and its absence from normal tissues make it an ideal engineered tumor target. We therefore developed anti-CAR19 CAR T cells with the goal of specifically targeting CAR19+ B-ALL. Methods. We designed two anti-CAR19 CAR using an anti-CAR19 idiotype scFv (clone 136.20.1, original clone kind gift of Dr. Laurence Cooper) testing two light chain orientations for the scFv: light to heavy (L2H) and heavy to light (H2L). We cloned the constructs into our standard backbone containing the CD8 hinge and trans-membrane domains, 4-1BB costimulatory and CD3z signaling domain. The constructs were packaged into a lentiviral vector and used to transduce normal donor T cells. As target cells, we used ex-vivo expanded primary leukemic blasts from the CAR19-expressing relapsed patient (CHP-107), as well as a luciferase+ B-ALL cell line (NALM6) transduced with the full-length CAR19 used in our clinical studies. Of note, the expression of CAR19 in CD19+ NALM6 cells led to apparent loss of CD19 by flow cytometry. We tested CART function in vitro with luciferase-based killing assays and in vivo in human xenograft models using NOD-SCID gamma chain deficient (NSG) mice. Results. Both L2H and H2L anti-CAR19 CAR were efficiently expressed on T cells as detected by flow cytometry. In vitro CART-CAR19 efficiently killed CAR19-expressing B-ALL (NALM6) (L2H orientation: p Conclusions. The acquisition of CAR expression by leukemic cells during CART manufacturing is a rare event that generates a tumor-specific antigen, thereby presenting an opportunity for specific targeting without off-target toxicity. In addition, we hypothesize that anti-CAR19 CART could be used as an "antidote" to CAR19 T cells for those patients who have been in stable deep remission for several years, in order to deplete their CAR T cells and alleviate B cell aplasia. Disclosures Ruella: Novartis: Patents & Royalties, Research Funding. Lacey: Novartis: Research Funding; Genentech: Honoraria. Melenhorst: Novartis: Research Funding. June: Celldex: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Immune Design: Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; WIRB/Copernicus Group: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Novartis: Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding. Gill: Novartis: Patents & Royalties, Research Funding; CARMA Therapeutics: Consultancy, Equity Ownership, Research Funding.

Pancreatic ductal adenocarcinoma (PDAC) is resistant to T-cell mediated immunotherapy. We engineered T cells to transiently express an mRNA encoding a chimeric antigen receptor (CAR) specific for mesothelin—a protein that is over-expressed by PDAC cells. We performed a phase 1 study to evaluate the safety and efficacy of adoptive cell therapy with autologous mesothelin-specific CAR T cells (CARTmeso cells) in 6 patients with chemotherapy-refractory metastatic PDAC. Patients were given intravenous CARTmeso cells 3 times weekly for 3 weeks. None of the patients developed cytokine release syndrome or neurologic symptoms and there were no dose limiting toxicities. Disease stabilized in 2 patients, with progression-free survival times of 3.8 and 5.4 months. We used FDG-positron emission tomography/computed tomography imaging to monitor the metabolic active volume (MAV) of individual tumor lesions. The total MAV remained stable in 3 patients and decreased by 69.2% in 1 patient with biopsy-proven mesothelin expression; in this patient, all liver lesions had a complete reduction in FDG uptake at 1 month compared to baseline, although there was no effect on the primary PDAC. Transient CAR expression was detected in patients’ blood after infusion and led to expansion of new immunoglobulin G proteins. Our results provide evidence for the potential anti-tumor activity of mRNA CARTmeso cells, as well as PDAC resistance to the immune response. Keywords: immune response heterogeneity, immune therapy, anti-tumor immunity, pancreatic cancer treatment

Exposure to replication-competent lentivirus (RCL) is a theoretical safety concern for individuals treated with lentiviral gene therapy. For certain ex vivo gene therapy applications, including cancer immunotherapy trials, RCL detection assays are used to screen the vector product as well as the vector-transduced cells. In this study, we reviewed T cell products screened for RCL using methodology developed in the National Gene Vector Biorepository. All trials utilized third-generation lentiviral vectors produced by transient transfection. Samples from 26 clinical trials totaling 460 transduced cell products from 375 subjects were evaluated. All cell products were negative for RCL. A total of 296 of the clinical trial participants were screened for RCL at least 1 month after infusion of the cell product. No research subject has shown evidence of RCL infection. These findings provide further evidence attesting to the safety of third-generation lentiviral vectors and that testing T cell products for RCL does not provide added value to screening the lentiviral vector product.

CD8+ T lymphocytes mediate potent immune responses against tumor, but the role of human CD4+ T cell subsets in cancer immunotherapy remains ill-defined. Herein, we exhibit that CD26 identifies three T helper subsets with distinct immunological properties in both healthy individuals and cancer patients. Although CD26neg T cells possess a regulatory phenotype, CD26int T cells are mainly naive and CD26high T cells appear terminally differentiated and exhausted. Paradoxically, CD26high T cells persist in and regress multiple solid tumors following adoptive cell transfer. Further analysis revealed that CD26high cells have a rich chemokine receptor profile (including CCR2 and CCR5), profound cytotoxicity (Granzyme B and CD107A), resistance to apoptosis (c-KIT and Bcl2), and enhanced stemness (β-catenin and Lef1). These properties license CD26high T cells with a natural capacity to traffic to, regress and survive in solid tumors. Collectively, these findings identify CD4+ T cell subsets with properties critical for improving cancer immunotherapy., The role of human CD4+ T cell subsets in cancer immunotherapy is still unclear. Here, the authors show that CD26 identifies three CD4+ T cell subsets with distinct immunological properties in both healthy individuals and cancer patients.

Introduction: The most common severe toxicity associated with chimeric antigen receptor T-cells targeting CD19 (CART19) is cytokine release syndrome (CRS; PMID: 29972754). Our group and others have published seminal observations on the biology of CRS through cytokine profiling, measuring a small number of analytes (PMID: 27076371, 33434058). Multiple biomarkers including interferon gamma (IFNG), IL-6, and IL-10 have been associated with the development of severe CRS in previous studies (PMID: 33434058). To date, the only biomarker predictive of the development of CRS prior to infusion has been disease burden. To obtain a more robust understanding of CRS biology, we performed comprehensive secretome profiling to measure more than 1400 serum analytes on serial serum samples collected from patients treated with the 41BB-containing CTL019 on two clinical trials. Methods: Serum from patients enrolled on two clinical trials of the CART19 product CTL019 (NCT01626495 & NCT02906371) were obtained serially from pre-infusion to one month post infusion. Patients were categorised as having "minimal" (no CRS, Grade 1, or Grade 2) or "severe" (Grade 3 or 4) CRS. The serum secretome was profiled using the Olink Explore 1536 Analysis platform (Olink, Upsala, Sweden). 1484 proteins were measured from serum via proximity extension assay (PEA) high-multiplex immunoassay. Differential expression analysis, correlation analyses and receiver operating characteristic (ROC) calculations were performed using R (version 4.0.4) in RStudio. Significance was based on a fold change of greater than 2 or less than -2 and a false discovery rate of less than 0.05 calculated using a Benjamini-Hochberg correction. Results: 26 patients (10 NCT01626495 & 16 NCT02906371) were included comprising 128 unique datapoints from baseline to 35 days post-infusion. Thirteen patients had minimal and 13 had severe CRS. Differentially expressed proteins between minimal and severe CRS at the peak timepoint are shown in (A; green represents IFNG responsive proteins). Not surprisingly, proteins involved in IL-6 and IFNG signalling were increased, including biomarkers of hemophagocytic lymphohistiocytosis (HLH) such as VSIG4, CXCL9, CXCL10, CD163. The IL-18 signalling axis was dysregulated at peak CRS in severe patients with markedly elevated IL18 and IL18BP, despite prior reports suggesting IL-18 up-regulation is unique to the late CRS seen with CART22 (PMID: 32925169). Soluble markers of checkpoint inhibition, including soluble PDL1 (CD274) and LAG3 were also highly elevated. Finally, biomarkers of endothelial damage, such as PLAT, TMSB10 and CALCA were significantly elevated in patients with severe CRS. Pathway analysis revealed significant dysregulation in targetable cytokine, chemokine, and signalling pathways (B). A volcano plot of differentially expressed proteins at pre-infusion (C) identified a single protein, MILR1, as a candidate biomarker that was highly differentially expressed in patients who would subsequently develop severe CRS. MILR1 expression decreased over time (D). An ROC of MILR1 as a predictor for development of severe CRS (E) demonstrated pre-infusion elevated MILR1 could accurately predict development of severe CRS (sensitivity 88%, specificity 97%, AUC=0.977). We identified correlates of MILR1 at pre-infusion and found that MILR1 correlated most highly with soluble FLT3 (R=0.86, p Conclusions: With comprehensive secretome profiling we made multiple novel insights into the biology of CRS after CART19 and identified several potentially targetable proteins and pathways that could mitigate severe CRS. Similar secretome profiling in patients who developed neurotoxicity will also be shown. We identified two novel pre-infusion biomarkers that demonstrate significant capacity to predict the development of severe CRS following CART19 infusion. The inverse relationship apparent between FLT3 and FLT3LG that persists over time is an important finding that implies a potential biological role for FLT3/FLT3 ligand in the development of severe CRS. Mechanistic studies exploring the role of MILR1 and FLT3 in the initiation of CRS are ongoing. Figure 1 Figure 1. Disclosures Lambert: Novartis, shionogi, argenx, Rigel, octapharma: Consultancy; Rigel, Novartis, Sysmex, octapharma: Research Funding. Bassiri: Kriya Therapeutics: Consultancy, Current holder of individual stocks in a privately-held company; Guidepoint Global: Consultancy. Levine: Vycellix: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Other: Co-Founder and equity holder; Ori Biotech: Membership on an entity's Board of Directors or advisory committees; Immusoft: Membership on an entity's Board of Directors or advisory committees; Immuneel: Membership on an entity's Board of Directors or advisory committees; Avectas: Membership on an entity's Board of Directors or advisory committees; Akron: Membership on an entity's Board of Directors or advisory committees; In8bio: Membership on an entity's Board of Directors or advisory committees. Maude: Wugen: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding. June: Tmunity, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Current equity holder in publicly-traded company; Novartis: Patents & Royalties; AC Immune, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Consultancy. Barrett: Tmunity Therapeutics: Current Employment. Grupp: Novartis, Kite, Vertex, and Servier: Research Funding; Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy; Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding. Teachey: Janssen: Consultancy; NeoImmune Tech: Research Funding; Sobi: Consultancy; BEAM Therapeutics: Consultancy, Research Funding.

Background: Chimeric Antigen Receptor (CAR) T cell immunotherapy has revolutionized the treatment of B-cell malignancies. However, a significant subset of these patients either fails to respond or eventually relapses. Moreover, in solid cancers, CAR T immunotherapy has had little to no success in the clinic so far. In recent years, several studies have shown the influence of commensal gut microbes on T cell function, in particular in the setting of checkpoint immunotherapy. Our group has recently demonstrated that modulation of the gut microbiota using antibiotics such as oral vancomycin (vanco) can enhance the efficacy of tumor-specific T cells in animal models. In this study, we sought to study the effect of vanco-induced dysbiosis on CART immunotherapy using murine models and clinical correlates. Methods and Results: We used the CD19+ A20 lymphoma and the B16 melanoma (transduced with CD19) murine models. Lymphoma- and melanoma-bearing mice were randomized to received oral vanco or vehicle alone (CTR), or in combination with either control untransduced murine T cells (UTD) or murine CART19 (4-1BB). Oral vanco or vehicle treatments started on the day of A20 cells injection and throughout the duration of the experiment (40-45 days). A20-bearing mice treated with CART19+vanco showed strikingly improved tumor control compared to either vanco alone or UTD+vanco (day 40 tumor volume in mm 3 (mean ± s.e.m): CTR=1,678.8±279.4, UTD=1,803.2±180, UTD+vanco=1,477±174, CART19=1,219±208, CART19+vanco=439.5±122.5 , CART versus CART+vanco Two Way Anova P Conclusions: These results suggest that the modulation of the gut microbiota using vancomycin affects the outcome of CART therapy in preclinical models with better anti-tumor effect via cross-priming and enhanced CART expansion in tumor samples. In a retrospective cohort of patients with B-ALL receiving vancomycin after CART19 therapy, we observed higher CART expansion and serum inflammatory cytokines. Based on these observations, a clinical trial of oral vanco in patients receiving CD19-directed CAR T cells for B-cell lymphomas is planned. Disclosures Ruella: viTToria biotherapeutics: Research Funding; Tmunity: Patents & Royalties; Novartis: Patents & Royalties; BMS, BAYER, GSK: Consultancy; AbClon: Consultancy, Research Funding. Frey: Novartis: Research Funding; Sana Biotechnology: Consultancy; Kite Pharma: Consultancy; Syndax Pharmaceuticals: Consultancy. June: Tmunity, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Current equity holder in publicly-traded company; AC Immune, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Consultancy; Novartis: Patents & Royalties. Porter: American Society for Transplantation and Cellular Therapy: Honoraria; ASH: Membership on an entity's Board of Directors or advisory committees; DeCart: Membership on an entity's Board of Directors or advisory committees; Genentech: Current Employment, Current equity holder in publicly-traded company; Incyte: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Membership on an entity's Board of Directors or advisory committees; National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Unity: Patents & Royalties; Wiley and Sons Publishing: Honoraria. Schuster: TG Theraputics: Research Funding; Incyte: Research Funding; Adaptive Biotechnologies: Research Funding; Pharmacyclics: Research Funding; Merck: Research Funding; Genentech/Roche: Consultancy, Research Funding; Tessa Theraputics: Consultancy; Loxo Oncology: Consultancy; Juno Theraputics: Consultancy, Research Funding; BeiGene: Consultancy; Alimera Sciences: Consultancy; Acerta Pharma/AstraZeneca: Consultancy; Novartis: Consultancy, Honoraria, Patents & Royalties, Research Funding; Abbvie: Consultancy, Research Funding; Nordic Nanovector: Consultancy; Celgene: Consultancy, Honoraria, Research Funding.

Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable success in hematological malignancies but remains largely ineffective in solid tumors. A major factor leading to the reduced efficacy of CAR T cell therapy is T cell dysfunction, and the mechanisms mediating this dysfunction are under investigation. Here we establish a robust model to study dysfunction of mesothelin-redirected CAR T cells in pancreatic cancer. Continuous antigen exposure results in hallmark features of exhaustion including reduced proliferation capacity and cytotoxicity, and severe defects in cytokine production. Here we identified a transcriptional signature at both population and single-cell levels in CAR T cells after continuous antigen exposure. In addition, TCR lineage tracing revealed a CD8+ T-to-NK-like T cell plasticity that results in reduced tumor cell killing. The transcription factors SOX4 and ID3 are specifically expressed in the dysfunctional CAR NK-like T cells and are predicted to be master regulators of the dysfunction gene expression signature and the post-thymic acquisition of an NK-like T cell fate. Finally, we identified the emergence of NK-like CAR T cells in a subset of patients after infusion of CAR T cells. The findings gleaned from this study shed light on the plasticity of human CAR T cells and suggest new approaches to improve the efficacy of CAR T cell therapy in solid tumors by preventing or revitalizing CAR T cell dysfunction. Citation Format: Charly R. Good, Shunichiro Kuramitsu, Parisa Samareh, Greg Donahue, Kenichi Ishiyama, Yujie Ma, Nils Wellhausen, Lifeng Tian, Sangya Agarwal, Sonia Guedan, M. Angela Aznar, Katherine A. Alexander, Zhen Zhang, Nathan Singh, 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. Induction of T cell dysfunction and NK-like T cell differentiation in vitro and in patients after CAR T cell treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 60.

In this review, we discuss the most recent developments in gene-editing technology and discuss their application to adoptive T cell immunotherapy. Engineered T cell therapies targeting cancer antigens have demonstrated significant efficacy in specific patient populations. Most impressively, CD19-directed chimeric antigen receptor T cells (CART19) have led to impressive responses in patients with B-cell leukemia and lymphoma. CTL019, or KYMRIAH™ (tisagenlecleucel), a CD19 CAR T cell product developed by Novartis and the University of Pennsylvania, was recently approved for clinical use by the Food and Drug Administration, representing a landmark in the application of adoptive T cell therapies. As CART19 enters routine clinical use, improving the efficacy of this exciting platform is the next step in broader application. Novel gene-editing technologies like CRISPR-Cas9 allow facile editing of specific genes within the genome, generating a powerful platform to further optimize the activity of engineered T cells.

As genome editing rapidly progresses toward the realization of its clinical promise, assessing the suitability of current tools and processes used for its benefit-risk assessment is critical. Although current regulations may initially provide an adequate regulatory framework, improvements are recommended to overcome several existing technology-based safety and efficacy issues.

SUMMARY The effects of transgenically encoded human and mouse IL-18 on T cell proliferation and its application in boosting chimeric antigen receptor (CAR) T cells are presented. Robust enhancement of proliferation of IL-18-secreting human T cells occurred in a xenograft model, and this was dependent on TCR and IL-18R signaling. IL-18 augmented IFN-γ secretion and proliferation of T cells activated by the endogenous TCR. TCR-deficient, human IL-18-expressing CD19 CAR T cells exhibited enhanced proliferation and antitumor activity in the xenograft model. Antigen-propelled activation of cytokine helper ensemble (APACHE) CAR T cells displayed inducible expression of IL-18 and enhanced antitumor immunity. In an intact mouse tumor model, CD19-IL-18 CAR T cells induced deeper B cell aplasia, significantly enhanced CAR T cell proliferation, and effectively augmented antitumor effects in mice with B16F10 melanoma. These findings point to a strategy to develop universal CAR T cells for patients with solid tumors., In Brief Hu et al. create IL-18-secreting chimeric antigen receptor T (IL-18-CAR T) cells to significantly boost CAR T cell proliferation and antitumor activity.

The generation of T cells with maximal anti-tumor activities will significantly impact the field of T-cell-based adoptive immunotherapy. In this report, we found that OKT3/IL-2-stimulated T cells were phenotypically more heterogeneous, with enhanced anti-tumor activity in vitro and when locally administered in a solid tumor mouse model. To further improve the OKT3/IL-2-based T cell manufacturing procedure, we developed a novel T cell stimulation and expansion method in which peripheral blood mononuclear cells were electroporated with mRNA encoding a chimeric membrane protein consisting of a single-chain variable fragment against CD3 and the intracellular domains of CD28 and 4-1BB (OKT3-28BB). The expanded T cells were phenotypically and functionally similar to T cells expanded by OKT3/IL-2. Moreover, co-electroporation of CD86 and 4-1BBL could further change the phenotype and enhance the in vivo anti-tumor activity. Although T cells expanded by the co-electroporation of OKT3-28BB with CD86 and 4-1BBL showed an increased central memory phenotype, the T cells still maintained tumor lytic activities as potent as those of OKT3/IL-2 or OKT3-28BB-stimulated T cells. In different tumor mouse models, T cells expanded by OKT3-28BB RNA electroporation showed anti-tumor activities superior to those of OKT3/IL-2 T cells. Hence, T cells with both a less differentiated phenotype and potent tumor killing ability can be generated by RNA electroporation, and this T cell manufacturing procedure can be further optimized by simply co-delivering other splices of RNA, thus providing a simple and cost-effective method for generating high-quality T cells for adoptive immunotherapy. Electronic supplementary material The online version of this article (doi:10.1007/s13238-017-0422-6) contains supplementary material, which is available to authorized users.

Purpose: Using gene-disrupted allogeneic T cells as universal effector cells provides an alternative and potentially improves current chimeric antigen receptor (CAR) T-cell therapy against cancers and infectious diseases. Experimental Design: The CRISPR/Cas9 system has recently emerged as a simple and efficient way for multiplex genome engineering. By combining lentiviral delivery of CAR and electro-transfer of Cas9 mRNA and gRNAs targeting endogenous TCR, β-2 microglobulin (B2M) and PD1 simultaneously, to generate gene-disrupted allogeneic CAR T cells deficient of TCR, HLA class I molecule and PD1. Results: The CRISPR gene–edited CAR T cells showed potent antitumor activities, both in vitro and in animal models and were as potent as non-gene–edited CAR T cells. In addition, the TCR and HLA class I double deficient T cells had reduced alloreactivity and did not cause graft-versus-host disease. Finally, simultaneous triple genome editing by adding the disruption of PD1 led to enhanced in vivo antitumor activity of the gene-disrupted CAR T cells. Conclusions: Gene-disrupted allogeneic CAR and TCR T cells could provide an alternative as a universal donor to autologous T cells, which carry difficulties and high production costs. Gene-disrupted CAR and TCR T cells with disabled checkpoint molecules may be potent effector cells against cancers and infectious diseases. Clin Cancer Res; 23(9); 2255–66. ©2016 AACR.

Antiviral immune responses present a major hurdle to the efficacious use of oncolytic adenoviruses as cancer treatments. Despite the existence of a highly immunosuppressive tumor environment, adenovirus-infected cells can nonetheless be efficiently cleared by infiltrating cytotoxic T lymphocytes (CTL) without compromising tumor burden. In this study, we tested the hypothesis that tumor-infiltrating T cells could be more effectively activated and redirected by oncolytic adenoviruses that were armed with bispecific T-cell–engager (BiTE) antibodies. The oncolytic adenovirus ICOVIR-15K was engineered to express an EGFR-targeting BiTE (cBiTE) antibody under the control of the major late promoter, leading to generation of ICOVIR-15K-cBiTE, which retained its oncolytic properties in vitro. cBiTE expression and secretion was detected in supernatants from ICOVIR-15K-cBiTE–infected cells, and the secreted BiTEs bound specifically to both CD3+ and EGFR+ cells. In cell coculture assays, ICOVIR-15K-cBiTE–mediated oncolysis resulted in robust T-cell activation, proliferation, and bystander cell-mediated cytotoxicity. Notably, intratumoral injection of this cBiTE-expressing adenovirus increased the persistence and accumulation of tumor-infiltrating T cells in vivo, compared with the parental virus lacking such effects. Moreover, in two distinct tumor xenograft models, combined delivery of ICOVIR-15K-cBiTE with peripheral blood mononuclear cells or T cells enhanced the antitumor efficacy achieved by the parental counterpart. Overall, our results show how arming oncolytic adenoviruses with BiTE can overcome key limitations in oncolytic virotherapy. Cancer Res; 77(8); 2052–63. ©2017 AACR.

Cytokine release syndrome (CRS) is a potentially severe systemic toxicity seen after adoptive T-cell therapy and caused by T-cell activation and proliferation and is associated with elevated circulating levels of cytokines such as C-reactive protein, interleukin-6 (IL-6), and interferon-γ and has previously been described as a systemic response in hematologic malignancies. A 52-year-old woman with BRCA 1 mutation positive heavily pretreated advanced recurrent serous ovarian cancer was treated under a compassionate use protocol with autologous mesothelin-redirected chimeric antigen receptor T cells (CART-meso). Autologous T cells were transduced to express a receptor composed of an extracellular antimesothelin single-chain variable fragment fused to 4-1BB and TCR-zeta signaling domain. This patient was infused with 3×10 CART-meso T cells/m without lymphodepletion and developed compartmental CRS confined to the pleural cavities. The compartmental CRS was evidenced by an increase in IL-6 and accumulation of CART-meso T cells in pleural fluid compared with peripheral blood and was successfully treated the anti-IL6 receptor antagonist tocilizumab on D21 after the T-cell infusion. This is the first description of a compartmental CRS in a patient with solid malignancy. This response could be due to malignant pleural fluid creating an environment where T cells could interact with tumor cells and suggests localized on-target CAR-T-cell activation.

// Jiangtao Ren 1 , Xuhua Zhang 1 , Xiaojun Liu 1 , Chongyun Fang 1 , Shuguang Jiang 1 , Carl H. June 1, 2 , Yangbing Zhao 1, 2 1 Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 2 Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Correspondence to: Yangbing Zhao, email: yangbing@upenn.edu Keywords: CRISPR/CAS9, T lymphocytes, chimeric antigen receptors, PD-1, CD95 Received: November 18, 2016 Accepted: January 27, 2017 Published: February 09, 2017 ABSTRACT The therapeutic potential of CRISPR system has already been demonstrated in many instances and begun to overlap with the rapidly expanding field of cancer immunotherapy, especially on the production of genetically modified T cell receptor or chimeric antigen receptor (CAR) T cells. Efficient genomic disruption of multiple gene loci to generate universal donor cells, as well as potent effector T cells resistant to multiple inhibitory pathways such as PD-1 and CTLA4 is an attractive strategy for cell therapy. In this study, we accomplished rapid and efficient multiplex genomic editing, and re-directing T cells with antigen specific CAR via a one-shot CRISPR protocol by incorporation of multiple gRNAs in a CAR lentiviral vector. High efficient double knockout of endogenous TCR and HLA class I could be easily achieved to generate allogeneic universal CAR T cells. We also generated Fas-resistant universal CAR T cells by triple gene disruption. Simultaneous gene editing of four gene loci using the one-shot CRISPR protocol to generate allogeneic universal T cells deficient of both PD1 and CTLA-4 was also attempted.

Chimeric antigen receptor (CAR) Tcells have proven that engineered immune cells can serve as a powerful new class of cancer therapeutics. Clinical experience has helped to define the major challenges that must be met to make engineered Tcells a reliable, safe, and effective platform that can be deployed against a broad range of tumors. The emergence of synthetic biology approaches for cellular engineering is providing us with a broadly expanded set of tools for programming immune cells. We discuss how these tools could be used to design the next generation of smart Tcell precision therapeutics.

Background aims Thymic-derived regulatory T cells (tTreg) are critical regulators of the immune system. Adoptive tTreg transfer is a curative therapy for murine models of autoimmunity, graft rejection, and graft-versus-host disease (GVHD). We previously completed a "first-in-human" clinical trial using in vitro expanded umbilical cord blood (UCB)-derived tTreg to prevent GVHD in patients undergoing UCB hematopoietic stem cell transplantation (HSCT). tTreg were safe and demonstrated clinical efficacy, but low yield prevented further dose escalation. Methods To optimize yield, we investigated the use of KT64/86 artificial antigen presenting cells (aAPCs) to expand tTreg and incorporated a single re-stimulation after day 12 in expansion culture. Results aAPCs increased UCB tTreg expansion greater than eightfold over CD3/28 stimulation. Re-stimulation with aAPCs increased UCB tTreg expansion an additional 20- to 30-fold. Re-stimulated human UCB tTreg ameliorated GVHD disease in a xenogeneic model. Following current Good Manufacturing Practice (cGMP) validation, a trial was conducted with tTreg. tTreg doses up to >30-fold higher compared with that obtained with anti-CD3/28 mAb coated-bead expansion and Foxp3 expression was stable during in vitro expansion and following transfer to patients. Increased expansion did not result in a senescent phenotype and GVHD was significantly reduced. Discussion Expansion culture with cGMP aAPCs and re-stimulation reproducibly generates sufficient numbers of UCB tTreg that exceeds the numbers of T effector cells in an UCB graft. The methodology supports future tTreg banking and is adaptable to tTreg expansion from HSC sources. Furthermore, because human leukocyte antigen matching is not required, allogeneic UCB tTreg may be a useful strategy for prevention of organ rejection and autoimmune disease.

Chimeric Antigen Receptor T cells (CART) have led to unprecedented clinical responses in relapsed or refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL), non-Hodgkin lymphomas (NHL), and multiple myeloma. However, despite these exciting results, most patients treated with CART therapy either do not respond or eventually relapse. Moreover, CART therapy has not yet been proven effective in several hematological malignancies, such as T cell lymphoma and leukemia (T-NHL/T-ALL) and acute myeloid leukemia (AML). Thus, there is a need to enhance currently available CART products and also to develop next-generation CART therapies to successfully treat additional neoplasms like T-NHL /T-ALL and AML. To this goal, we studied the cysteine-rich scavenger receptor CD5, an attractive target for CART immunotherapy because of its dual role in malignant cells and normal T cells. In malignant cells, CD5 is expressed by ~90% of TCL cells, by ~15-20% of AML cells, and also by most cases of chronic lymphocytic leukemia and mantle cell lymphoma (MCL). Of note, promising results using a CD28-based anti-CD5 CART against T-NHL and T-ALL were reported at this meeting in 2019 (LaQuisa C. Hill #199). In T cells, CD5 is highly expressed and inhibits T cell receptor (TCR)-mediated activation through several mediators including SHP-1, CBL, CBL-B, and GRB2. Therefore, we hypothesized that the genetic deletion of CD5 in engineered T cells could potentially enhance their effector functions. First, we designed and screened six 4-1BB-costimulated anti-CD5 lentiviral CAR constructs designed to have high, medium, and low affinity for CD5. We then selected the lead CAR5 construct (high affinity, heavy to light light chain orientation) based on its superior anti-tumor function in vivo in NOD-SCID IL2Rgnull (NSG) mice engrafted with T-cell leukemia (Jurkat). Then, to further improve CART5 activity, we optimized a CD5 short-guide RNA and deleted CD5 in CART5 cells using CRISPR-Cas9. CD5 gene deletion was reproducibly efficient (95-100% by flow cytometry and TIDE) during manufacturing (6 donors). Interestingly, the growth rate of wild type (WT) CART5 was comparable to CD5 KO CART5 and the expression of CD5 in WT CART5 was reduced. However, at the end of manufacturing, CD5 KO CART5 had increased central memory T cells (33.0% vs. 18.4%) and reduced expression of activation/exhaustion markers (PD-1 4.4% vs. 14.8%, LAG3 13.1% vs. 55.9%) compared to WT CART5, potentially indicating that CD5 KO reduces CART5-CART5 fratricide during manufacturing. We then compared wild type (WT) CART5 to CD5 KO CART5 in vitro using several T-NHL/T-ALL, MCL, and AML models, including primary samples (Sezary cells, primary MCL cells, and CD5+ AML cells). Both WT and CD5 KO CART5 were highly effective in killing CD5+ malignant cells, but CD5 KO CART5 showed enhanced proliferation upon activation. In two xenograft models of T-cell leukemia (primary T-ALL and Jurkat), CD5 KO CART5 showed dramatically increased tumor control compared to WT (Fig.1A, median overall survival for WT= 62 days vs. CD5 KO=not reached, p = 0.006, Mantel-Cox). This enhanced anti-tumor effect was associated with increased expansion of CD5 KO CART5 in the peripheral blood (PB) compared to WT CART5. To test the hypothesis that deletion of CD5 could increase the anti-tumor effect of CART targeting antigens other than CD5, we knocked out CD5 in anti-CD19 CART cells and tested their function in a CD19+ B-ALL xenograft model (NALM6). Remarkably, CD5 KO CART19 displayed significantly enhanced anti-leukemia activity and PB expansion compared to WT (Fig.1B,C, p Finally, we aimed to define the mechanisms by which CD5 KO enhances CART anti-tumor efficacy. We analyzed the phosphorylation of multiple targets in T cells after 15 minutes of CAR stimulation. Remarkably, CD5 KO CART5 cells had higher (>2fold) phosphorylation of several signaling proteins, including key regulators of T cell activation, migration, and survival compared to WT CART5 (Fig. 1D). To confirm that the CD5 pathway was indeed the mediator of this effect, we knocked out SHP-1 in CART19 cells using CRISPR/Cas9 and observed increased leukemia killing. In conclusion, we demonstrate that CRISPR-Cas9 KO of CD5 enhances the anti-tumor activity of CAR T cells by enhancement of CAR-mediated activation and proliferation. These findings support the development of CD5 KO CART products in early-phase clinical trials. Disclosures Schuster: Novartis, Genentech, Inc./ F. Hoffmann-La Roche: Research Funding; AlloGene, AstraZeneca, BeiGene, Genentech, Inc./ F. Hoffmann-La Roche, Juno/Celgene, Loxo Oncology, Nordic Nanovector, Novartis, Tessa Therapeutics: Consultancy, Honoraria. Barta:Atara: Honoraria; Monsanto: Consultancy; Seattle Genetics: Honoraria, Research Funding; Janssen: Honoraria; Pfizer: Honoraria. June:Tmunity Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Ziopharm Oncology: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Novartis: Patents & Royalties, Research Funding; Bluesphere Bio: Membership on an entity's Board of Directors or advisory committees; Cabaletta Bio: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Carisma Therapeutics: Membership on an entity's Board of Directors or advisory committees; Cellares: Membership on an entity's Board of Directors or advisory committees; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; DeCART Therapeutics: Membership on an entity's Board of Directors or advisory committees; Immune Design: Membership on an entity's Board of Directors or advisory committees; Kiadis Pharma: Current equity holder in private company. Ruella:Abclon, BMS, NanoString: Consultancy; UPenn/Novartis: Patents & Royalties; Abclon: Consultancy, Research Funding.

Lessons from arthritis and cell therapy in cancer patients point to therapy for severe disease

Chimeric antigen receptor (CAR) gene-engineered T cell therapy holds the potential to make a meaningful difference in the lives of patients with terminal cancers. For decades, cancer therapy was based on biophysical parameters, with surgical resection to debulk, followed by radiation and chemotherapy to target the rapidly growing tumor cells, while mostly sparing quiescent normal tissues. One breakthrough occurred with allogeneic bone-marrow transplant for patients with leukemia, which provided a sometimes curative therapy. The field of adoptive cell therapy for solid tumors was established with the discovery that tumor-infiltrating lymphocytes could be expanded and used to treat and even cure patients with metastatic melanoma. Tumor-specific T-cell receptors (TCRs) were identified and engineered into patient peripheral blood lymphocytes, which were also found to treat tumors. However, these were limited by patient HLA-restriction. Close behind came generation of CAR, combining the exquisite recognition of an antibody with the effector function of a T cell. The advent of CD19-targeted CARs for treating patients with multiple forms of advanced B-cell malignancies met with great success, with up to 95% response rates. Applying CAR treatment to solid tumors, however, has just begun, but already certain factors have been made clear: the tumor target is of utmost importance for clinicians to do no harm; and solid tumors respond differently to CAR therapy compared with hematologic ones. Here we review the state of clinical gene-engineered T cell immunotherapy, its successes, challenges, and future.

While CD19-directed chimeric antigen receptor (CAR) T cells can induce remission in patients with B cell acute lymphoblastic leukemia (ALL), a large subset relapse with CD19- disease. Like CD19, CD22 is broadly expressed by B-lineage cells and thus serves as an alternative immunotherapy target in ALL. Here we present the composite outcomes of two pilot clinical trials ( NCT02588456 and NCT02650414 ) of T cells bearing a 4-1BB-based, CD22-targeting CAR in patients with relapsed or refractory ALL. The primary end point of these studies was to assess safety, and the secondary end point was antileukemic efficacy. We observed unexpectedly low response rates, prompting us to perform detailed interrogation of the responsible CAR biology. We found that shortening of the amino acid linker connecting the variable heavy and light chains of the CAR antigen-binding domain drove receptor homodimerization and antigen-independent signaling. In contrast to CD28-based CARs, autonomously signaling 4-1BB-based CARs demonstrated enhanced immune synapse formation, activation of pro-inflammatory genes and superior effector function. We validated this association between autonomous signaling and enhanced function in several CAR constructs and, on the basis of these observations, designed a new short-linker CD22 single-chain variable fragment for clinical evaluation. Our findings both suggest that tonic 4-1BB-based signaling is beneficial to CAR function and demonstrate the utility of bedside-to-bench-to-bedside translation in the design and implementation of CAR T cell therapies.

Human cancer cells were eradicated by adoptive transfer of T cells transduced with a chimeric antigen receptor (CAR) made from an antibody (237Ab) that is highly specific for the murine Tn-glycosylated podoplanin (Tn-PDPN). The objectives were to determine the specificity of these CAR-transduced T (CART) cells and the mechanism for the absence of relapse. We show that although the 237Ab bound only to cell lines expressing murine Tn-PDPN, the 237Ab-derived 237CART cells lysed multiple different human and murine cancers not predicted by the 237Ab binding. Nevertheless, the 237CART cell reactivities remained cancer specific because all recognitions were dependent on the Tn glycosylation that resulted from COSMC mutations that were not present in normal tissues. While Tn was required for the recognition by 237CART, Tn alone was not sufficient for 237CART cell activation. Activation of 237CART cells required peptide backbone recognition but tolerated substitutions of up to 5 of the 7 amino acid residues in the motif recognized by 237Ab. Together, these findings demonstrate what we believe is a new principle whereby simultaneous recognition of multiple independent Tn-glycopeptide antigens on a cancer cell makes tumor escape due to antigen loss unlikely.

Cellular immunotherapy using T cells engineered to express chimeric antigen receptors targeting CD19 (CART19) leads to long-term remission in patients with B-cell malignancies1–5. Unfortunately, a significant fraction of patients demonstrate primary resistance to CART19 or experience relapse after achieving remission. Beyond loss of target antigen, the molecular pathways governing CART19 failure are unknown. Here we demonstrate that death receptors, cell surface signaling molecules that induce target cell apoptosis, are key mediators of leukemic resistance and CART19 failure. Using a functional CRISPR/Cas9-based genome-wide knockout screen6 in B-cell acute lymphoblastic leukemia (ALL), we identified the death receptor signaling pathway as a central regulator of sensitivity to CART19-induced cell death. In the absence of the pro-apoptotic death receptor signaling molecules BID or FADD, ALL cells were resistant to CART19 cytotoxicity, resulting in rapid disease progression in mice. We found that this initial resistance to cytotoxicity led to persistence of tumor cells, which drove the development of T cell dysfunction that further compromised anti-tumor immunity and permitted tumor outgrowth. We validated these findings using clinical samples collected from patients with ALL treated with CD19-targeted CAR T cells and found that expression of pro-apoptotic death receptor pathway genes in pre-treatment tumor samples correlated with CAR T cell expansion and persistence, as well as patient response and overall survival. Our findings indicate that tumor-intrinsic death receptor signaling directly contributes to CAR T cell failure.