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Chimeric antigen receptor (CAR) T cell expansion and persistence represent key factors to achieve complete responses and prevent relapses. These features are typical of early memory T cells, which can be highly enriched through optimized manufacturing protocols. Here, we investigated the efficacy and safety profiles of CAR T cell products generated from preselected naive/stem memory T cells (TN/SCM), as compared with unselected T cells (TBULK). Notwithstanding their reduced effector signature in vitro, limiting CAR TN/SCM doses showed superior antitumor activity and the unique ability to counteract leukemia rechallenge in hematopoietic stem/precursor cell-humanized mice, featuring increased expansion rates and persistence together with an ameliorated exhaustion and memory phenotype. Most relevantly, CAR TN/SCM proved to be intrinsically less prone to inducing severe cytokine release syndrome, independently of the costimulatory endodomain employed. This safer profile was associated with milder T cell activation, which translated into reduced monocyte activation and cytokine release. These data suggest that CAR TN/SCM are endowed with a wider therapeutic index compared with CAR TBULK.

Chimeric antigen receptor (CAR) T cell expansion and persistence emerged as key efficacy determinants in cancer patients. These features are typical of early-memory T cells, which can be enriched with specific manufacturing procedures, providing signal one and signal two in the proper steric conformation and in the presence of homeostatic cytokines. In this project, we exploited our expertise with paramagnetic beads and IL-7/IL-15 to develop an optimized protocol for CAR T cell production based on reagents, including a polymeric nanomatrix, which are compatible with automated manufacturing via the CliniMACS Prodigy. We found that both procedures generate similar CAR T cell products, highly enriched of stem cell memory T cells (TSCM) and equally effective in counteracting tumor growth in xenograft mouse models. Most importantly, the optimized protocol was able to expand CAR TSCM from B-cell acute lymphoblastic leukemia (B-ALL) patients, which in origin were highly enriched of late-memory and exhausted T cells. Notably, CAR T cells derived from B-ALL patients proved to be as efficient as healthy donor-derived CAR T cells in mediating profound and prolonged anti-tumor responses in xenograft mouse models. On the contrary, the protocol failed to expand fully functional CAR TSCM from patients with pancreatic ductal adenocarcinoma, suggesting that patient-specific factors may profoundly affect intrinsic T cell quality. Finally, by retrospective analysis of in vivo data, we observed that the proportion of TSCM in the final CAR T cell product positively correlated with in vivo expansion, which in turn proved to be crucial for achieving long-term remissions. Collectively, our data indicate that next-generation manufacturing protocols can overcome initial T cell defects, resulting in TSCM-enriched CAR T cell products qualitatively equivalent to the ones generated from healthy donors. However, this positive effect may be decreased in specific conditions, for which the development of further improved protocols and novel strategies might be highly beneficial.

Chimeric antigen receptor (CAR)-T cell immunotherapy is at the forefront of innovative cancer therapeutics. However, lack of standardization of cellular products within the same clinical trial and lack of harmonization between different trials have hindered the clear identification of efficacy and safety determinants that should be unveiled in order to advance the field. With the aim of facilitating the isolation and tracking of CAR-T cells, we here propose the inclusion within the CAR molecule of a novel extracellular spacer based on the low-affinity nerve-growth-factor receptor (NGFR). We screened four different spacer designs using as target antigen the CD44 isoform variant 6 (CD44v6). We successfully generated NGFR-spaced CD44v6 CAR-T cells that could be efficiently enriched with clinical-grade immuno-magnetic beads without negative consequences on subsequent expansion, immuno-phenotype, antitumor reactivity, and conditional ablation when co-expressing a suicide gene. Most importantly, these cells could be tracked with anti-NGFR monoclonal antibodies in NSG mice, where they expanded, persisted, and exerted potent antitumor effects against both high leukemia and myeloma burdens. Similar results were obtained with NGFR-enriched CAR-T cells specific for CD19 or CEA, suggesting the universality of this strategy. In conclusion, we have demonstrated that the incorporation of the NGFR marker gene within the CAR sequence allows for a single molecule to simultaneously work as a therapeutic and selection/tracking gene. Looking ahead, NGFR spacer enrichment might allow good manufacturing procedures-manufacturing of standardized CAR-T cell products with high therapeutic potential, which could be harmonized in different clinical trials and used in combination with a suicide gene for future application in the allogeneic setting.

BackgroundTo date, T cells redirected with CD19-specific chimeric antigen receptors (CAR) have gained impressive success in B-cell malignancies. However, treatment failures are common and the occurrence of severe toxicities, such as cytokine release syndrome (CRS), still limits the full exploitation of this approach. Therefore, the development of cell products with improved therapeutic indexes is highly demanded.MethodsIn this project, we investigated how CD4 and CD8 populations cooperate during CD19 CAR-T cell responses and what is their specific role in CRS development. To this aim, we took advantage of immunodeficient mice reconstituted with a human immune system (HuSGM3) and engrafted with the B-cell acute lymphoblastic leukemia cell line NALM-6, a model that allows to thoroughly study efficacy and toxicity profiles of CD19 CAR-T cell products.ResultsCD4 CAR-T cells showed superior proliferation and activation potential, which translated into stronger stimulation of myeloid cells, the main triggers of adverse events. Accordingly, toxicity assessment in HuSGM3 mice identified CD4 CAR-T cells as key contributors to CRS development, revealing a safer profile when they harbor CARs embedded with 4-1BB, rather than CD28. By comparing differentially co-stimulated CD4:CD8 1:1 CAR-T cell formulations, we observed that CD4 cells shape the overall expansion kinetics of the infused product and are crucial for maintaining long-term responses. Interestingly, the combination of CD4.BBz with CD8.28z CAR-T cells resulted in the lowest toxicity, without impacting antitumor efficacy.ConclusionsTaken together, these data point out that the rational design of improved adoptive T-cell therapies should consider the biological features of CD4 CAR-T cells, which emerged as crucial for maintaining long-term responses but also endowed by a higher toxic potential.

BackgroundImmune effector cell-associated neurotoxicity syndrome (ICANS) is an adverse event commonly observed in cancer patients receiving CAR-T cell therapies. Despite the clinical features of ICANS being recognizable, its pathophysiology remains poorly understood, current hypothesis are mainly based on findings in autopsies performed on individuals who suffered fatal neurotoxicity after CAR-T cell therapy and preclinical models are limited. Recently, a syngeneic mouse model of CAR-related neurotoxicity has been described; unfortunately, it does not allow to study ICANS that develops upon interaction of human CAR-T cells with human immune and tumor cells.MethodsHere we proposed a humanized mouse model, previously characterized for its ability to recapitulate CAR-related cytokine release syndrome (CRS) toxicity, in which we observed the development of multifocal brain haemorrhages, reminiscent of neurotoxic manifestations observed in patients with high-grade ICANS.ResultsInterestingly, we found that microhaemorrhages did not occur in mice treated with untransduced T cells, while they were observed in about the 45% of mice treated with human CD19 CAR-T cells. Moreover, we observed that this event is associated with CRS severity, as it occurs in patients, and by assessing the number and dimensions of microhaemorrhages by brain tissue area we found them to be greater in terms of extension and incidence in the cerebellum.By magnetic resonance imaging we observed the onset of hypointense lesions in deep brain structures and noteworthily an extravasation event of the injected contrast agent occurred, suggesting changes in blood vessels permeability. Moreover, according to the most affected mentioned brain areas, we find changes in locomotor abilities likely providing a clinical relevance to the phenomena occurring in the brain.ConclusionsThese findings suggest that our humanized mouse model is promisingly able to recapitulate ICANS-related complications, thus providing us with a preclinical tool to deepen ICANS pathophysiology studying.

BackgroundAdoptive transfer of CAR T cells demonstrated impressive results against B-cell malignancies, but still limited efficacy against solid tumors. In this context, multiple challenges need to be overcome, including poor tumor recognition and strong immunosuppression within the tumor microenvironment (TME). Our Unit has recently reported that pharmacological inhibition of N-glycan synthesis in cancer cells increases CAR T cell efficacy by improving tumor recognition and preventing T cell exhaustion. In this project, we investigated the role of N-glycosylation blockade on TME cells in the context of colorectal cancer (CRC) and pancreatic adenocarcinoma (PDAC)-derived liver metastases and CEA-specific CAR T cell therapy.MethodsTo understand the effect of N-glycosylation blockade on TME cells (both M2-macrophages, M2-M and Hepatic stellate cells, HepSCs), we analyzed the phenotypic and transcriptional profile and we performed in vitro functional assays, such as tripartite co-cultures, suppressive assays and released-cytokines analysis. Moreover, to evaluate the effect of N-glycosylation inhibition on TME cells in vivo, we exploited immunodeficient mice reconstituted with a human immune system (huSGM3), engrafted intra-hepatically with tumor cells and treated with CEA CAR T cells.ResultsIn vitro studies revealed that N-glycosylation inhibition abolishes the ability of both TME cells to restrain T cell proliferation and increases the elimination of cancer cell lines and patient-derived tumor organoids (PDOs from CRC-liver metastases). Interestingly, these effects were associated with profound phenotypic and transcriptional changes in M2-M and HepSCs. In particular, the treatment was able to inhibit M2-polarization in terms of surface markers expression, IL-10 secretion and gene expression profile, and was shown to hinder the activation of HepSCs and inhibit the PD- 1/PDL-1 axis. Interestingly, in the in vivo model, the presence of human immune cells supports CAR T cell responses and helps recreate an immune TME more representative of the human disease. Importantly, using these mice we observed that N-glycosylation inhibition increases CEA CAR T cell antitumor activity, in terms of survival, and this is associated with the downregulation of immunosuppressive genes in tumor-infiltrating human immune cells.ConclusionsOverall, these data suggest that blocking N-glycosylation can help overcome multiple barriers that currently limit CAR T cell efficacy in solid tumors, acting not only on tumor cells, but also on immunosuppressive tumor microenvironment cells.

In the clinic, chimeric antigen receptor–modified T (CAR T) cell therapy is frequently associated with life-threatening cytokine-release syndrome (CRS) and neurotoxicity. Understanding the nature of these pathologies and developing treatments for them are hampered by the lack of appropriate animal models. Herein, we describe a mouse model recapitulating key features of CRS and neurotoxicity. In humanized mice with high leukemia burden, CAR T cell–mediated clearance of cancer triggered high fever and elevated IL-6 levels, which are hallmarks of CRS. Human monocytes were the major source of IL-1 and IL-6 during CRS. Accordingly, the syndrome was prevented by monocyte depletion or by blocking IL-6 receptor with tocilizumab. Nonetheless, tocilizumab failed to protect mice from delayed lethal neurotoxicity, characterized by meningeal inflammation. Instead, the IL-1 receptor antagonist anakinra abolished both CRS and neurotoxicity, resulting in substantially extended leukemia-free survival. These findings offer a therapeutic strategy to tackle neurotoxicity and open new avenues to safer CAR T cell therapies. IL-1 blockade prevents cytokine-release syndrome and neurotoxicity by CAR T cells.