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Background: Immunotherapy with chimeric antigen receptor (CAR)-expressing T cells that target the disialoganglioside GD2 expressed on tumor cells may be a therapeutic option for patients with high-risk neuroblastoma. Methods: In an academic, phase 1-2 clinical trial, we enrolled patients (1 to 25 years of age) with relapsed or refractory, high-risk neuroblastoma in order to test autologous, third-generation GD2-CAR T cells expressing the inducible caspase 9 suicide gene (GD2-CART01). Results: A total of 27 children with heavily pretreated neuroblastoma (12 with refractory disease, 14 with relapsed disease, and 1 with a complete response at the end of first-line therapy) were enrolled and received GD2-CART01. No failure to generate GD2-CART01 was observed. Three dose levels were tested (3-, 6-, and 10×106 CAR-positive T cells per kilogram of body weight) in the phase 1 portion of the trial, and no dose-limiting toxic effects were recorded; the recommended dose for the phase 2 portion of the trial was 10×106 CAR-positive T cells per kilogram. Cytokine release syndrome occurred in 20 of 27 patients (74%) and was mild in 19 of 20 (95%). In 1 patient, the suicide gene was activated, with rapid elimination of GD2-CART01. GD2-targeted CAR T cells expanded in vivo and were detectable in peripheral blood in 26 of 27 patients up to 30 months after infusion (median persistence, 3 months; range, 1 to 30). Seventeen children had a response to the treatment (overall response, 63%); 9 patients had a complete response, and 8 had a partial response. Among the patients who received the recommended dose, the 3-year overall survival and event-free survival were 60% and 36%, respectively. Conclusions: The use of GD2-CART01 was feasible and safe in treating high-risk neuroblastoma. Treatment-related toxic effects developed, and the activation of the suicide gene controlled side effects. GD2-CART01 may have a sustained antitumor effect. (Funded by the Italian Medicines Agency and others

Autologous CD19-directed chimeric antigen receptor (CAR)-T cells have shown unprecedented efficacy in children with relapsed/refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, patients either relapsing after allogeneic hematopoietic stem cell transplantation (allo-HSCT) or displaying profound lymphopenia and/or rapidly progressing disease often cannot access autologous products. These hurdles may be overcome by allogeneic, donor-derived CAR-T cells. We tested donor-derived T cells transduced with a second-generation (4.1BB) CD19-directed CAR for treatment of patients with BCP-ALL in a hospital-exemption setting. Two constructs were tested: a retroviral construct incorporating the suicide gene inducible caspase-9 (CD19-CAR–Retro_ALLO) first and then a lentiviral construct and an automated, Prodigy-based manufacturing process (CD19-CAR–Lenti_ALLO). Thirteen children/young adults received ALLO–CAR-T cells between March 2021 and October 2022. Doses ranged between 1.0 × 106 and 3.0 × 106 CAR-T cells per kg. The toxicity profile was comparable with that of autologous CAR-T cells, characterized mainly by cytopenia, cytokine release syndrome (maximum grade 1), and grade 2 immune-effector cell–associated neurotoxicity syndrome. One case of acute graft-versus-host disease (GVHD) occurred and was rapidly controlled with steroids and ruxolitinib. None of the other patients, including 3 given ALLO–CAR-T cells from an HLA-haploidentical donor, experienced GVHD. Two patients received ALLO–CAR-T cells before HSCT and showed a significant expansion of CAR-T cells without any sign of GVHD. All patients obtained complete remission (CR) with absence of minimal residual disease in the bone marrow. With a median follow-up of 12 months (range, 5-21), 8 of 13 patients maintained CR. Allogeneic anti-CD19 CAR-T cells can effectively treat highly refractory BCP-ALL relapsing after allo-HSCT without showing increased toxicity as compared with autologous CAR-T cells.

Chimeric antigen receptor T-cells (CAR T-cells) for the treatment of relapsing/refractory B-cell precursor acute lymphoblastic leukemia have led to exciting clinical results. However, CAR T-cell approaches revealed a potential risk of CD19-/CAR+ leukemic relapse due to inadvertent transduction of leukemia cells. BackgroundMethods We evaluated the impact of a high percentage of leukemia blast contamination in patient-derived starting material (SM) on CAR T-cell drug product (DP) manufacturing. In vitro as well as in vivo models were employed to identify characteristics of the construct associated with better profile of safety in case of inadvertent B-cell leukemia transduction during CAR T-cell manufacturing. Results The presence of large amounts of CD19+ cells in SM did not affect the transduction level of DPs, as well as the CAR T-cell rate of expansion at the end of standard production of 14 days. DPs were deeply characterized by flow cytometry and molecular biology for Ig-rearrangements, showing that the level of B-cell contamination in DPs did not correlate with the percentage of CD19+ cells in SM, in the studied patient cohort. Moreover, we investigated whether CAR design may affect the control of CAR+ leukemia cells. We provided evidences that CAR.CD19 short linker (SL) prevents complete epitope masking in CD19+CAR+ leukemia cells and we demonstrated in vitro and in vivo that CD19 +CAR(SL)+leukemic cells are killed by CAR.CD19 T-cells. Conclusions Taken together, these data suggest that a VL-VH SL may result in a safe CAR-T product, even when manufacturing starts from biological materials characterized by heavy contamination of leukemia blasts.

Background: Chimeric antigen receptor (CAR) T cells directed against CD19 (CART19) are effective in B-cell malignancies, but little is known about the molecular factors predicting clinical outcome of CART19 therapy. The increasingly recognized relevance of epigenetic changes in cancer immunology prompted us to determine the impact of the DNA methylation profiles of CART19 cells on the clinical course. Methods: We recruited 114 patients with B-cell malignancies, comprising 77 patients with acute lymphoblastic leukemia and 37 patients with non-Hodgkin lymphoma who were treated with CART19 cells. Using a comprehensive DNA methylation microarray, we determined the epigenomic changes that occur in the patient T cells upon transduction of the CAR vector. The effects of the identified DNA methylation sites on clinical response, cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, event-free survival, and overall survival were assessed. All statistical tests were 2-sided. Results: We identified 984 genomic sites with differential DNA methylation between CAR-untransduced and CAR-transduced T cells before infusion into the patient. Eighteen of these distinct epigenetic loci were associated with complete response (CR), adjusting by multiple testing. Using the sites linked to CR, an epigenetic signature, referred to hereafter as the EPICART signature, was established in the initial discovery cohort (n = 79), which was associated with CR (Fisher exact test, P <. 001) and enhanced event-free survival (hazard ratio [HR] = 0.36; 95% confidence interval [CI] = 0.19 to 0.70; P =. 002; log-rank P =. 003) and overall survival (HR = 0.45; 95% CI = 0.20 to 0.99; P =. 047; log-rank P =. 04;). Most important, the EPICART profile maintained its clinical course predictive value in the validation cohort (n = 35), where it was associated with CR (Fisher exact test, P <. 001) and enhanced overall survival (HR = 0.31; 95% CI = 0.11 to 0.84; P =. 02; log-rank P

Chimeric antigen receptor T-cells (CAR T-cells) for the treatment of relapsing/refractory B-cell precursor acute lymphoblastic leukemia have led to exciting clinical results. However, CAR T-cell approaches revealed a potential risk of CD19-/CAR+ leukemic relapse due to inadvertent transduction of leukemia cells. BackgroundMethods We evaluated the impact of a high percentage of leukemia blast contamination in patient-derived starting material (SM) on CAR T-cell drug product (DP) manufacturing. In vitro as well as in vivo models were employed to identify characteristics of the construct associated with better profile of safety in case of inadvertent B-cell leukemia transduction during CAR T-cell manufacturing. Results The presence of large amounts of CD19+ cells in SM did not affect the transduction level of DPs, as well as the CAR T-cell rate of expansion at the end of standard production of 14 days. DPs were deeply characterized by flow cytometry and molecular biology for Ig-rearrangements, showing that the level of B-cell contamination in DPs did not correlate with the percentage of CD19+ cells in SM, in the studied patient cohort. Moreover, we investigated whether CAR design may affect the control of CAR+ leukemia cells. We provided evidences that CAR.CD19 short linker (SL) prevents complete epitope masking in CD19+CAR+ leukemia cells and we demonstrated in vitro and in vivo that CD19 +CAR(SL)+leukemic cells are killed by CAR.CD19 T-cells. Conclusions Taken together, these data suggest that a VL-VH SL may result in a safe CAR-T product, even when manufacturing starts from biological materials characterized by heavy contamination of leukemia blasts.

T cells engineered with chimeric antigen receptor (CAR-T cells) are an effective treatment in patients with relapsed/refractory B-cell precursor acute lymphoblastic leukemia or B-cell non-Hodgkin lymphoma. Despite the reported exciting clinical results, the CAR-T cell approach needs efforts to improve the safety profile, limiting the occurrence of adverse events in patients given this treatment. Besides the most common side effects, such as cytokine release syndrome and CAR-T cell–related encephalopathy syndrome, another potential issue involves the inadvertent transduction of leukemia B cells with the CAR construct during the manufacturing process, thus leading to the possibility of a peculiar mechanism of antigen masking and treatment resistance. In this study, we investigated whether the inclusion of the inducible caspase 9 (iC9) suicide gene in the CAR construct design could be an effective safety switch to control malignant CAR+ B cells, ultimately counteracting this serious adverse event. iC9 is a suicide gene able to be activated through binding with an otherwise inert small biomolecule, known as AP1903. The exposure of iC9.CAR.CD19-DAUDI lymphoma and iC9.CAR.CD19-NALM-6 leukemia cells in vitro to 20 nM of AP1903 resulted into the prompt elimination of CAR+ B-leukemia/lymphoma cell lines. The results obtained in the animal model corroborate in vitro data, since iC9.CAR.CD19+ tumor cells were controlled in vivo by the activation of the suicide gene through administration of AP1903. Altogether, our data indicate that the inclusion of the iC9 suicide gene may result in a safe CAR-T cell product, even when manufacturing starts from biological materials characterized by heavy leukemia blast contamination.

The outcome of patients affected by high-risk or metastatic neuroblastoma (NB) remains grim, with ≥ 50% of the children experiencing relapse or progression of the disease despite multimodal, intensive treatment. In order to identify new strategies to improve the overall survival and the quality of life of these children, we recently developed and optimized a third-generation GD2-specific chimeric antigen receptor (CAR) construct, which is currently under evaluation in our Institution in a phase I/II clinical trial (NCT03373097) enrolling patients with relapsed/refractory NB. We observed that our CAR T-cells are able to induce marked tumor reduction and even achieve complete remission with a higher efficiency than that of other CAR T-cells reported in previous studies. However, often responses are not sustained and relapses occur. Here, we demonstrate for the first time a mechanism of resistance to GD2.CAR T-cell treatment, showing how polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) increase in the peripheral blood (PB) of NB patients after GD2.CAR T-cell treatment in case of relapse and loss of response. In vitro, isolated PMN-MDSC demonstrate to inhibit the anti-tumor cytotoxicity of different generations of GD2.CAR T-cells. Gene-expression profiling of GD2.CAR T-cells “conditioned” with PMN-MDSC shows downregulation of genes involved in cell activation, signal transduction, inflammation and cytokine/chemokine secretion. Analysis of NB gene-expression dataset confirms a correlation between expression of these genes and patient outcome. Moreover, in patients treated with GD2.CAR T-cells, the frequency of circulating PMN-MDSC inversely correlates with the levels of GD2.CAR T-cells, resulting more elevated in patients who did not respond or lost response to the treatment. The presence and the frequency of PMN-MDSC in PB of high-risk and metastatic NB represents a useful prognostic marker to predict the response to GD2.CAR T-cells and other adoptive immun

We developed an innovative and efficient, feeder-free culture method to genetically modify and expand peripheral blood-derived NK cells with high proliferative capacity, while preserving the responsiveness of their native activating receptors. Activated peripheral blood NK cells were efficiently transduced by a retroviral vector, carrying a second-generation CAR targeting CD19. CAR expression was demonstrated across the different NK-cell subsets. CAR.CD19-NK cells display higher antileukemic activity toward CD19+ cell lines and primary blasts obtained from patients with B-cell precursor ALL compared with unmodified NK cells. In vivo animal model data showed that the antileukemia activity of CAR.CD19-NK cell is superimposable to that of CAR-T cells, with a lower xenograft toxicity profile. These data support the feasibility of generating feeder-free expanded, genetically modified peripheral blood NK cells for effective “off-the-shelf” immuno-gene-therapy, while their innate alloreactivity can be safely harnessed to potentiate allogeneic cell therapy.

Medulloblastoma is the most frequent malignant childhood brain tumor with a high morbidity. Identification of new therapeutic targets would be instrumental in improving patient outcomes. We evaluated the expression of the tumor-associated antigen PRAME in biopsies from 60 patients with medulloblastoma. PRAME expression was detectable in 82% of tissues independent of molecular and histopathologic subgroups. High PRAME expression also correlated with worse overall survival. We next investigated the relevance of PRAME as a target for immunotherapy. Medulloblastoma cells were targeted using genetically modified T cells with a PRAME-specific TCR (SLL TCR T cells). SLL TCR T cells efficiently killed medulloblastoma HLA-A-02+ DAOY cells as well as primary HLA-A-02+ medulloblastoma cells. Moreover, SLL TCR T cells controlled tumor growth in an orthotopic mouse model of medulloblastoma. To prevent unexpected T-cell- related toxicity, an inducible caspase-9 (iC9) gene was introduced in frame with the SLL TCR; this safety switch triggered prompt elimination of genetically modified T cells. Altogether, these data indicate that T cells genetically modified with a high-affinity, PRAME-specific TCR and iC9 may represent a promising innovative approach for treating patients with HLA-A-02+ medulloblastoma. Significance: These findings identify PRAME as a medulloblastoma tumor-associated antigen that can be targeted using genetically modified T cells.

Chimeric antigen receptor (CAR) T-cell therapy has been shown to be dramatically effective in the treatment of B-cell malignancies. However, there are still substantial obstacles to overcome, before similar responses can be achieved in patients with solid tumors. We evaluated both in vitro and in a preclinical murine model the efficacy of different 2nd and 3rd generation CAR constructs targeting GD2, a disial-ganglioside expressed on the surface of neuroblastoma (NB) tumor cells. In order to address potential safety concerns regarding clinical application, an inducible safety switch, namely inducible Caspase-9 (iC9), was also included in the vector constructs. Our data indicate that a 3rd generation CAR incorporating CD28.4-1BB costimulatory domains is associated with improved anti-tumor efficacy as compared with a CAR incorporating the combination of CD28.OX40 domains. We demonstrate that the choice of 4-1BB signaling results into significant amelioration of several CAR T-cell characteristics, including: 1) T-cell exhaustion, 2) basal T-cell activation, 3) in vivo tumor control and 4) T-cell persistence. The fine-tuning of T-cell culture conditions obtained using IL7 and IL15 was found to be synergic with the CAR.GD2 design in increasing the anti-tumor activity of CAR T cells. We also demonstrate that activation of the suicide gene iC9, included in our construct without significantly impairing neither CAR expression nor anti-tumor activity, leads to a prompt induction of apoptosis of GD2.CAR T cells. Altogether, these findings are instrumental in optimizing the function of CAR T-cell products to be employed in the treatment of children with NB.

Lo scritto presenta il progetto editoriale, i criteri della scelta dei testi, l'operazione inconsueta di raccogliere oltre che gli scritti a favore anche le stroncature dell'opera di un intellettuale anomalo nel panorama italiano come Alfonso Berardinelli, un critico della cultura ancor prima che un critico letterario con gusti precisi e peculiari tic stilistici, un critico «contro»