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Plasma cell-free DNA analysis has emerged as a powerful liquid biopsy assay to assess circulating tumour DNA in response to cancer treatments. A new study shows that cell-free DNA can also inform on expansion kinetics and tumour-infiltration patterns in patients receiving chimeric antigen receptor T cells and, together with circulating tumour DNA, provides vivid prognostic insights into intratumoural dynamics.

Despite remarkable success in the treatment of hematological malignancies, CAR T-cell therapies for solid tumors have floundered, in large part due to local immune suppression and the effects of prolonged stimulation leading to T-cell dysfunction and exhaustion. One mechanism by which gliomas and other cancers can hamper CAR T cells is through surface expression of inhibitory ligands such as programmed cell death ligand 1 (PD-L1). Using the CRIPSR-Cas9 system, we created universal CAR T cells resistant to PD-1 inhibition through multiplexed gene disruption of endogenous T-cell receptor (TRAC), beta-2 microglobulin (B2M) and PD-1 (PDCD1). Triple gene-edited CAR T cells demonstrated enhanced activity in preclinical glioma models. Prolonged survival in mice bearing intracranial tumors was achieved after intracerebral, but not intravenous administration. CRISPR-Cas9 gene-editing not only provides a potential source of allogeneic, universal donor cells, but also enables simultaneous disruption of checkpoint signaling that otherwise impedes maximal antitumor functionality.

In addition to remarkable antitumor activity, chimeric antigen receptor (CAR) T-cell therapy is associated with acute toxicities such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Current treatment guidelines for CRS and ICANS include use of tocilizumab, a monoclonal antibody that blocks the interleukin (IL)-6 receptor, and corticosteroids. In patients with refractory CRS, use of several other agents as third-line therapy (including siltuximab, ruxolitinib, anakinra, dasatinib, and cyclophosphamide) has been reported on an anecdotal basis. At our institution, anakinra has become the standard treatment for the management of steroid-refractory ICANS with or without CRS, based on recent animal data demonstrating the role of IL-1 in the pathogenesis of ICANS/CRS. Here, we retrospectively analyzed clinical and laboratory parameters, including serum cytokines, in 14 patients at our center treated with anakinra for steroid-refractory ICANS with or without CRS after standard treatment with tisagenlecleucel (Kymriah) or axicabtagene ciloleucel (Yescarta) CD19-targeting CAR T. We observed statistically significant and rapid reductions in fever, inflammatory cytokines, and biomarkers associated with ICANS/CRS after anakinra treatment. With three daily subcutaneous doses, anakinra did not have a clear, clinically dramatic effect on neurotoxicity, and its use did not result in rapid tapering of corticosteroids; although neutropenia and thrombocytopenia were common at the time of anakinra dosing, there were no clear delays in hematopoietic recovery or infections that were directly attributable to anakinra. Anakinra may be useful adjunct to steroids and tocilizumab in the management of CRS and/or steroid-refractory ICANs resulting from CAR T-cell therapies, but prospective studies are needed to determine its efficacy in these settings.

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.

In this first-in-human, investigator-initiated, open-label study, three participants with recurrent glioblastoma were treated with CARv3-TEAM-E T cells, which are chimeric antigen receptor (CAR) T cells engineered to target the epidermal growth factor receptor (EGFR) variant III tumor-specific antigen, as well as the wild-type EGFR protein, through secretion of a T-cell–engaging antibody molecule (TEAM). Treatment with CARv3-TEAM-E T cells did not result in adverse events greater than grade 3 or dose-limiting toxic effects. Radiographic tumor regression was dramatic and rapid, occurring within days after receipt of a single intraventricular infusion, but the responses were transient in two of the three participants. (Funded by Gateway for Cancer Research and others; INCIPIENT ClinicalTrials.gov number, NCT05660369 .) A novel CAR T-cell therapy directed at EGFRvIII with a secretable EGFR T-cell engager produced rapid responses in three patients with recurrent glioblastoma, but the responses were transient in two of the three.

I had learned that communicating with patients and families is a critical part of patient care. So, facing the communication challenges brought by Covid-19, as our unit worked on technological solutions to enable widespread video calling, we learned to improvise.

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.

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.

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.