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Chimeric antigen receptor (CAR) therapy has had a transformative effect on the treatment of haematologic malignancies 1 – 6 , but it has shown limited efficacy against solid tumours. Solid tumours may have cell-intrinsic resistance mechanisms to CAR T cell cytotoxicity. Here, to systematically identify potential resistance pathways in an unbiased manner, we conducted a genome-wide CRISPR knockout screen in glioblastoma, a disease in which CAR T cells have had limited efficacy 7 , 8 . We found that the loss of genes in the interferon-γ receptor (IFNγR) signalling pathway ( IFNGR1 , JAK1 or JAK2 ) rendered glioblastoma and other solid tumours more resistant to killing by CAR T cells both in vitro and in vivo. However, loss of this pathway did not render leukaemia or lymphoma cell lines insensitive to CAR T cells. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell-adhesion pathways after exposure to CAR T cells. We found that loss of IFNγR1 in glioblastoma cells reduced overall CAR T cell binding duration and avidity. The critical role of IFNγR signalling in susceptibility of solid tumours to CAR T cells is surprising, given that CAR T cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumours, IFNγR signalling was required for sufficient adhesion of CAR T cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumours differ in their interactions with CAR T cells and suggests that enhancing binding interactions between T cells and tumour cells may yield improved responses in solid tumours. A genome-wide CRISPR knockout screen in a model of glioblastoma shows that killing by chimeric antigen receptor T cells requires interferon-γ receptor-dependent adhesion to tumour cells, but cytotoxicity of liquid tumours does not rely on this pathway.

Chimeric Antigen Receptor (CAR) T cells directed to B cell maturation antigen (BCMA) mediate profound responses in patients with multiple myeloma, but most patients do not achieve long-term complete remissions. In addition, recent evidence suggests that high-affinity binding to BCMA can result in on-target, off-tumor activity in the basal ganglia and can lead to fatal Parkinsonian-like disease. Here we develop CAR T cells against multiple myeloma using a binder to targeting transmembrane activator and CAML interactor (TACI) in mono and dual-specific formats with anti-BCMA. These CARs have robust, antigen-specific activity in vitro and in vivo. We also show that TACI RNA expression is limited in the basal ganglia, which may circumvent some of the toxicities recently reported with BCMA CARs. Thus, single-targeting TACI CARs may have a safer toxicity profile, whereas dual-specific BCMA-TACI CAR T cells have potential to avoid the antigen escape that can occur with single-antigen targeting. Patients with myeloma multiple treated with BCMA CAR T cells often relapse with BCMA-negative disease or antigen escape. Here the authors describe the design of TACI-directed single and dual CAR T cells with in vitro and in vivo activity against multiple myeloma, overcoming BCMA antigen loss.

Claudin 18.2 (CLDN18.2)-targeted CAR-T cell therapies have shown promising clinical efficacy in gastric cancer. However, early-phase trials have reported gastrointestinal adverse events due to on-target off-tumor recognition of CLDN18.2 in the gastric mucosa. By leveraging shared CLDN18.2 epitopes and expression in humans and mice, we establish an in vivo model that replicates the on-target off-tumor toxicity of CLDN18.2 CAR-T. Our findings confirm that this toxicity is independent of the CAR construct’s design, co-stimulatory domain, and tumor model. Additionally, we demonstrate the utility of this model in testing strategies to mitigate on-target toxicity, such as Boolean-logic AND-gate approaches. Our results offer insights into the use of mouse models that recapitulate on-target off-tumor toxicities, with the caveat that although we are often concerned that models will undercall toxicities in humans, they may also overcall the incidence and severity of toxicities, prematurely discarding promising therapeutic agents from further clinical development. Promising clinical activity of Claudin (CLDN) 18.2-directed CAR-T cell therapy in patients with gastric cancer has been recently reported, however gastrointestinal toxicities have also been described. Here the authors recapitulate the on-target off-tumor toxicity of CLDN18.2-directed CAR-T cells due to gastric mucosa damage in preclinical models, suggesting an AND-gate strategy targeting CLDN18.2 and mesothelin to overcome CAR-T cell toxicity

Chimeric antigen receptor (CAR) T cell therapy has transformed cancer treatment but its efficacy remains limited in solid tumors due to antigen heterogeneity, an immunosuppressive microenvironment, and the glycocalyx barrier. The glycocalyx, composed of dense glycoproteins such as MUC1, is markedly expanded in cancers, where it impedes immune cell access and antigen engagement, thereby reducing efficacy. In most adenocarcinomas, Tn antigen, comprising N-acetylgalactosamine linked to serine or threonine, is overexpressed. Tn-MUC1, a truncated form of MUC1 decorated with Tn antigen, is frequently overexpressed in pancreatic cancer. Here, we incorporate a non-signaling glyco-bridge binder recognizing Tn-MUC1 into mesothelin-directed CAR-T cells. This bridge enhances tumor recognition and cytotoxicity by increasing avidity and facilitating CAR activation in a density- and affinity-dependent manner. To broaden its applicability, we design a tandem Helix pomatia agglutinin (HPA) lectin-based bridge that recognizes Tn antigens across cancer types. CAR-T cells with the HPA-bridge exhibit superior cytotoxicity in pancreatic cancer models. Components of the glycocalyx have been shown to impair immune cell functions, including of CAR-T cells. Here the authors show that CAR-T cell mediated cytotoxicity in pancreatic cancer models can be enhanced by incorporating non-signalling binding domains that target the glycocalyx.

BackgroundTumor heterogeneity and antigen escape are mechanisms of resistance to chimeric antigen receptor (CAR)-T cell therapy, especially in solid tumors. Targeting multiple antigens with a unique CAR construct could be a strategy for a better tumor control than monospecific CAR-T cells on heterogeneous models. To overcome tumor heterogeneity, we targeted mesothelin (meso) and Mucin 16 (MUC16), two antigens commonly expressed in solid tumors, using a tandem CAR design.MethodsWe designed a series of tandem CAR constructs based on various anti-meso (SS1) and anti-MUC16 ectodomain (MUC16ecto) (4H11) single-chain variable fragment (scFv) arrangements and G4S linker lengths. Then we determined the best tandem CAR design based on binding of soluble antigens, steric hindrance, avidity and functionality against cell lines expressing one or both antigens in vitro. Finally, we compared the tandem CAR to monospecific CAR-T cells in mixed tumor models in vitro (two-dimensional and three-dimensional models) and in vivo.ResultsWe show that the scFv arrangement and linker length impacted antigen binding and CAR expression in T cells. Tandem CAR configuration (TanCAR1) (with SS1 scFv located distally and one G4S repeat as the linker between scFvs) had the best binding and activation profile in vitro and outperformed SS1 and 4H11 monospecific CAR-T cells in mixed tumor models in vitro and in vivo, showing an antigen-driven killing of tumor cells based on antigen density. Moreover, acoustic force microscopy, using tumor cells with different levels of antigen expression, revealed that TanCAR1-T cells likely bind to one antigen at a time rather than simultaneously.ConclusionsThis is the first time using a tandem CAR design targeting meso and MUC16, and demonstrating a benefit on tumor control over monospecific CAR-T cells. Tandem CAR-T cells targeting meso and MUC16ecto could be employed as a strategy to overcome tumor cell heterogeneity in ovarian and pancreatic tumors, and may help to design therapeutic approaches relying on its one-antigen-at-a-time binding properties and on its antigen-driven killing of tumor cells based on antigen density.

BackgroundAdoptive cell therapy, such as chimeric antigen receptor (CAR)-T cell therapy, has improved patient outcomes for hematological malignancies. Currently, four of the six FDA-approved CAR-T cell products use the FMC63-based αCD19 single-chain variable fragment, derived from a murine monoclonal antibody, as the extracellular binding domain. Clinical studies demonstrate that patients develop humoral and cellular immune responses to the non-self CAR components of autologous CAR-T cells or donor-specific antigens of allogeneic CAR-T cells, which is thought to potentially limit CAR-T cell persistence and the success of repeated dosing.MethodsIn this study, we implemented a one-shot approach to prevent rejection of engineered T cells by simultaneously reducing antigen presentation and the surface expression of both Classes of the major histocompatibility complex (MHC) via expression of the viral inhibitors of transporter associated with antigen processing (TAPi) in combination with a transgene coding for shRNA targeting class II MHC transactivator (CIITA). The optimal combination was screened in vitro by flow cytometric analysis and mixed lymphocyte reaction assays and was validated in vivo in mouse models of leukemia and lymphoma. Functionality was assessed in an autologous setting using patient samples and in an allogeneic setting using an allogeneic mouse model.ResultsThe combination of the Epstein-Barr virus TAPi and an shRNA targeting CIITA was efficient and effective at reducing cell surface MHC classes I and II in αCD19 ‘stealth’ CAR-T cells while retaining in vitro and in vivo antitumor functionality. Mixed lymphocyte reaction assays and IFNγ ELISpot assays performed with T cells from patients previously treated with autologous αCD19 CAR-T cells confirm that CAR T cells expressing the stealth transgenes evade allogeneic and autologous anti-CAR responses, which was further validated in vivo. Importantly, we noted anti-CAR-T cell responses in patients who had received multiple CAR-T cell infusions, and this response was reduced on in vitro restimulation with autologous CARs containing the stealth transgenes.ConclusionsTogether, these data suggest that the proposed stealth transgenes may reduce the immunogenicity of autologous and allogeneic cellular therapeutics. Moreover, patient data indicate that repeated doses of autologous FMC63-based αCD19 CAR-T cells significantly increased the anti-CAR T cell responses in these patients.

BackgroundChimeric antigen receptor (CAR)-T cell therapy has achieved significant success in hematologic malignancies; however, its efficacy in solid tumors remains limited. A major limitation is the difficulty in identifying suitable target antigens that are abundantly expressed on the surface of tumor cells while sparing life-sustaining normal tissues.MethodsWe identified MUC17, a membrane-tethered mucin-type glycoprotein with minimal expression in normal tissues and frequent upregulation in gastric cancers, as a potential target for CAR-T therapy. We developed and validated MUC17-specific CAR-T cells incorporating a 4-1BB/CD3ζ signaling domain. In vitro assays assessed cytotoxicity, cytokine secretion, and T cell phenotypes across multiple gastric cancer cell lines, including CRISPR-mediated MUC17 knockout controls. In vivo efficacy was evaluated using NSG xenograft models.ResultsMUC17 CAR-T cells exhibited potent, antigen-specific cytotoxicity, robust cytokine release, and sustained effector functions characterized by enrichment of central memory phenotypes. In vivo, MUC17 CAR-T cells significantly suppressed tumor growth without signs of toxicity in GSU and ASPC-1 models.ConclusionsThese findings support MUC17 as a promising immunotherapeutic target for gastric cancer and demonstrate how targeting glycocalyx-associated antigens can expand the range of surface proteins amenable to CAR-T cell-based therapies in solid tumors.

BackgroundChimeric Antigen Receptor (CAR) therapy has had a transformative impact on the treatment of hematologic malignancies1–6 but success in solid tumors remains elusive. We hypothesized solid tumors have cell-intrinsic resistance mechanisms to CAR T-cell cytotoxicity.MethodsTo systematically identify resistance pathways, we conducted a genome-wide CRISPR knockout screen in glioblastoma cells, a disease where CAR T-cells have had limited efficacy.7 8 We utilized the glioblastoma cell line U87 and targeted endogenously expressed EGFR with CAR T-cells generated from 6 normal donors for the screen. We validated findings in vitro and in vivo across a variety of human tumors and CAR T-cell antigens.ResultsLoss of genes in the interferon gamma receptor (IFNγR) signaling pathway (IFNγR1, JAK1, JAK2) rendered U87 cells resistant to CAR T-cell killing in vitro. IFNγR1 knockout tumors also showed resistance to CAR T cell treatment in vivo in a second glioblastoma line U251 in an orthotopic model. This phenomenon was irrespective of CAR target as we also observed resistance with IL13Ralpha2 CAR T-cells. In addition, resistance to CAR T-cell cytotoxicity through loss of IFNγR1 applied more broadly to solid tumors as pancreatic cell lines targeted with either Mesothelin or EGFR CAR T-cells also showed resistance. However, loss of IFNγR signaling did not impact sensitivity of liquid tumor lines (leukemia, lymphoma or multiple myeloma) to CAR T-cells in vitro or in an orthotopic model of leukemia treated with CD19 CAR. We isolated the effects of decreased cytotoxicity of IFNγR1 knockout glioblastoma tumors to be cancer-cell intrinsic because CAR T-cells had no observable differences in proliferation, activation (CD69 and LFA-1), or degranulation (CD107a) when exposed to wildtype versus knockout tumors. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell adhesion pathways compared to wildtype glioblastoma cells after exposure to CAR T-cells. We found that loss of IFNγR1 reduced CAR T-cell binding avidity to glioblastoma.ConclusionsThe critical role of IFNγR signaling for susceptibility of solid tumors to CAR T-cells is surprising given that CAR T-cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumors, IFNγR signaling was required for sufficient adhesion of CAR T-cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumors differ in their interactions with CAR T-cells and suggests that enhancing T-cell/tumor interactions may yield improved responses in solid tumors.AcknowledgementsRCL was supported by T32 GM007306, T32 AI007529, and the Richard N. Cross Fund. ML was supported by T32 2T32CA071345-21A1. SRB was supported by T32CA009216-38. NJH was supported by the Landry Cancer Biology Fellowship. JJ is supported by a NIH F31 fellowship (1F31-MH117886). GG was partially funded by the Paul C. Zamecnik Chair in Oncology at the Massachusetts General Hospital Cancer Center and NIH R01CA 252940. MVM and this work is supported by the Damon Runyon Cancer Research Foundation, Stand Up to Cancer, NIH R01CA 252940, R01CA238268, and R01CA249062.ReferencesMaude SL, et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med 2018;378:439–448.Neelapu SS, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med 2017;377:2531–2544.Locke FL, et al. Long-term safety and activity of axicabtagene ciloleucel in refractory large B-cell lymphoma (ZUMA-1): a single-arm, multicentre, phase 1–2 trial. The Lancet Oncology 2019;20:31–42.Schuster SJ, et al. Chimeric antigen receptor T cells in refractory B-cell lymphomas. N Engl J Med 2017;377:2545–2554.Wang M, et al. KTE-X19 CAR T-cell therapy in relapsed or refractory mantle-cell lymphoma. N Engl J Med 2020;382:1331–1342.Cohen AD, et al. B cell maturation antigen-specific CAR T cells are clinically active in multiple myeloma. J Clin Invest 2019;129:2210–2221.Bagley SJ, et al. CAR T-cell therapy for glioblastoma: recent clinical advances and future challenges. Neuro-oncology 2018;20:1429–1438.Choi BD, et al. Engineering chimeric antigen receptor T cells to treat glioblastoma. J Target Ther Cancer 2017;6:22–25.Ethics ApprovalAll human samples were obtained with informed consent and following institutional guidelines under protocols approved by the Institutional Review Boards (IRBs) at the Massachusetts General Hospital (2016P001219). Animal work was performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) (2015N000218 and 2020N000114).

BackgroundCAR-T cell therapy has shown limited efficacy in solid tumors due to factors such as antigen heterogeneity, immunosuppressive microenvironments, and physical barriers.1–7 The glycocalyx, a dense layer of membrane-bound mucins, including MUC1, can physically block CAR-T cell access to target antigens. In pancreatic cancer, the overexpression of truncated MUC1 (Tn-MUC1) and the Tn antigen results in a tumor-specific glycoform. While this glycoform offers a selective target, it also reinforces the mucin barrier and contributes to immune evasion.8–13 MethodsCAR-T cells were engineered to express a non-signaling glycan-binding receptor, referred to here as a glyco-bridge, which binds to glycoproteins or glycans such as Tn-MUC1 and enhances CAR-T cell adhesion and activation. Initially, a Tn-MUC1 scFv was utilized, and we later developed tandem Helix pomatia agglutinin (HPA) lectins as a glyco-bridge to enable broader recognition of the Tn antigen (figure 1). CAR-T cell function was evaluated both in vitro and in vivo using cell lines, the Capan-2 model, and a patient-derived xenograft (PDX) model of pancreatic cancer. Tissues were harvested to assess off-tumor T cell infiltration.ResultsThe Tn-MUC1-based bridge not only facilitates CAR-T cell infiltration by >8-fold but also enhances avidity by 1.3-fold toward target cells. Additionally, it activates CAR signaling in a manner dependent on both the density of bridge-target antigens and the affinity of the antigen-binding domain. HPA-based CAR-T cells exhibited toxicity due to on-target/off-tumor binding. To mitigate this, we next incorporate HPA lectins into the glyco-bridge approach to enable selective targeting of universal Tn antigens on cancer cells. This toxicity was effectively mitigated by placing HPA within a non-signaling glyco-bridge. Using the same donors, glyco-bridge CAR-T cells showed improved tumor control and overall survival in the Capan-2 model, without systemic toxicity. In the PDX pancreatic cancer model, dual HPA glyco-bridge CAR-T cells exhibited significantly enhanced tumor regression (~46%; P = 0.00206) and survival compared to the control bridge.ConclusionsOur study presents a proof-of-concept that equipping CAR-T cells with a Tn-MUC1 binder and a Tn antigen binder targeting the glycocalyx significantly enhances CAR-T efficacy in pancreatic cancer models in vitro and in vivo. To better understand the underlying mechanisms of enhanced CAR-T cell activity, we investigated how the glyco-bridge enhanced CAR-T cell efficacy using in vitro co-culture assays, acoustic force microscopy, and mouse xenograft models. Our data suggest that the glyco-bridge increased cell avidity and improved tumor penetration, offering a novel approach to engage the cancer cell glycocalyx without directly initiating cytotoxicity.AcknowledgementsWe thank the core facilities at the MGH Cancer Center: Flow Cytometry, Histopathology, and Blood Bank. S.P. is a Merck Fellow of the Damon Runyon Cancer Research Foundation (DRG-2529-24). DSB received funding from the CRIS Foundation Out-Back Fellowship Programme (outback2021_6) and from the Spanish Society of Medical Oncology (SEOM). FB received funding from the American-Italian Cancer Foundation (AICF) and the Italian Association for Cancer Research (AIRC). This work was funded by NIH R01 CA238268 (MVM).ReferencesGhasempour S, Freeman SA. The glycocalyx and immune evasion in cancer. FEBS J. 2021 Oct 19.Öhlund D, Elyada E, Tuveson D. Fibroblast heterogeneity in the cancer wound. J Exp Med. 2014 Jul 28;211(8):1503–23.Connor AA, Gallinger S. Pancreatic cancer evolution and heterogeneity: integrating omics and clinical data. Nat Rev Cancer. 2022 Mar;22(3):131–42.Kankeu Fonkoua LA, Sirpilla O, Sakemura R, Siegler EL, Kenderian SS. CAR T cell therapy and the tumor microenvironment: Current challenges and opportunities. Molecular Therapy - Oncolytics. 2022 Jun;25:69–77.Beatson R, Tajadura-Ortega V, Achkova D, Picco G, Tsourouktsoglou TD, Klausing S, et al. The mucin MUC1 modulates the tumor immunological microenvironment through engagement of the lectin Siglec-9. Nat Immunol. 2016 Nov;17(11):1273–81.Park S, Colville MJ, Paek JH, Shurer CR, Singh A, Secor EJ, et al. Immunoengineering can overcome the glycocalyx armour of cancer cells. Nat Mater. 2024 Mar;23(3):429–38.Park S, Choi S, Shimpi AA, Estroff LA, Fischbach C, Paszek MJ. Collagen Mineralization Decreases NK Cell-Mediated Cytotoxicity of Breast Cancer Cells via Increased Glycocalyx Thickness. Advanced Materials. 2024 Feb 11; 2311505.Posey AD, Schwab RD, Boesteanu AC, Steentoft C, Mandel U, Engels B, et al. Engineered CAR T Cells Targeting the Cancer-Associated Tn-Glycoform of the Membrane Mucin MUC1 Control Adenocarcinoma. Immunity. 2016 Jun 21;44(6):1444–54.Supimon K, Sangsuwannukul T, Sujjitjoon J, Phanthaphol N, Chieochansin T, Poungvarin N, et al. Anti-mucin 1 chimeric antigen receptor T cells for adoptive T cell therapy of cholangiocarcinoma. Sci Rep. 2021 Mar 18;11(1):6276.al. MUC1 as a target for CAR-T therapy in head and neck squamous cell carinoma. Cancer Med. 2020 Jan;9(2):640–52.Zhou R, Yazdanifar M, Roy LD, Whilding LM, Gavrill A, Maher J, et al. CAR T Cells Targeting the Tumor MUC1 Glycoprotein Reduce Triple-Negative Breast Cancer Growth. Front Immunol. 2019;10:1149.Yazdanifar M, Zhou R, Grover P, Williams C, Bose M, Moore LJ, et al. Overcoming Immunological Resistance Enhances the Efficacy of A Novel Anti-tMUC1-CAR T Cell Treatment against Pancreatic Ductal Adenocarcinoma. Cells. 2019 Sep 11;8(9):1070.Wilkie S, Picco G, Foster J, Davies DM, Julien S, Cooper L, et al. Retargeting of human T cells to tumor-associated MUC1: the evolution of a chimeric antigen receptor. J Immunol. 2008 Apr 1;180(7):4901–9.Ethics ApprovalNo human subject data was generated in this study; all experiments were conducted using murine models and human immune cells from commercial or de-identified healthy donor sources under approved IACUC protocols (Protocol #: 2020N000114) at Mass General Brigham.Abstract 262 Figure 1Schematic of mesothelin-targeting CAR-T cells with glyco-bridge. The glyco-bridge contains a CD28 hinge and transmembrane domain (H/TM) and an inactive CD28 intracellular signaling domain (ICD)[Image Omitted. See PDF.]

BackgroundCD19-targeted CAR T-cell therapies have shown success in treating B-cell malignancies, with response rates up to 90%. However, up to half of patient’s relapse, often with CD19+ disease, suggesting T-cell intrinsic deficits. Prior studies indicate that CAR T-cell persistence and proliferation are key predictors of efficacy. Genetic modifications that mimic hyperproliferative states, such as TET2 loss or overexpression of BATF, have been explored to enhance CAR T-cell function. Notch1, a key regulator of T-cell development and proliferation, is upregulated post-TCR stimulation and has been implicated in T-cell leukemia via mutations to its PEST domain, which regulates protein degradation. While canonical Notch1 signaling has been shown to improve CAR T-cell metabolism and memory phenotype, its precise modulation in engineered CD8+ T-cells remains understudied.MethodsCD19-BBz CAR T-cells were engineered with CRISPR-Cas9 to knockout Notch1 or truncate the endogenous PEST domain. Lentiviral vectors were used for CAR and Notch1 intracellular domain (N1ICD) overexpression for transgene delivery. T-cell proliferation, cytotoxicity, and activation markers were assessed via flow cytometry, luciferase reporters, cytokine secretion assays, and live cell imaging. CAR T-cell function was evaluated in vitro and in NALM6 xenograft murine models.ResultsNotch1 knockout in CD19-BBz CAR T-cells impaired proliferation, cytokine production, and cytotoxicity, consistent with previous findings in CD8+ T cells. Surprisingly, constitutive overexpression of N1ICD also impaired CAR expression, viability, and function. In contrast, truncation of the Notch1 PEST domain—mimicking T-ALL mutations—prolonged N1ICD stability post-activation, resulting in enhanced proliferation, survival, cytokine secretion, and tumor cell killing. These CAR T-cells persisted better in vivo and improved leukemia clearance and mouse survival.ConclusionsNotch1 signaling must be tightly regulated for optimal CAR T-cell function. Both knockout and overexpression impair effector activity, underscoring the need for precise modulation. Truncating the PEST domain stabilizes N1ICD in an activation-dependent manner, enhancing CAR T-cell proliferation and anti-tumor efficacy. These findings identify Notch1 PEST editing as a promising strategy to boost CAR T-cell function and highlight the broader utility of Cas9-mediated domain editing in cell therapy.