Single-Cell RNA Analysis Reveals Cell-Intrinsic Functions of CAR T Cells Correlating with Response in a Phase II Study of Lymphoma Patients.

PURPOSE: Although CD19 chimeric antigen receptor T cells (CAR-T) therapy has shown remarkable success in B-cell malignancies, a substantial fraction of patients do not obtain a long-term clinical response. This could be influenced by the quality of the individual CAR-T infusion product. To shed some light on this, clinical outcome was correlated to characteristics of CAR-T infusion products. PATIENTS AND METHODS: In this phase II study, patients with B-cell lymphoma (n = 23) or leukemia (n = 1) received one or two infusions of third-generation CD19-directed CAR-Ts (2 × 108/m2). The clinical trial was registered at clinicaltrials.gov: NCT03068416. We investigated the transcriptional profile of individual CD19 CAR-T infusion products using targeted single-cell RNA sequencing and multicolor flow cytometry. RESULTS: Two CAR-T infusions were not better than one in the settings used in this study. As for the CAR-T infusion products, we found that effector-like CD8+CAR-Ts with a high polyfunctionality, high cytotoxic and cytokine production profile, and low dysfunctional signature were associated with clinical response. An extended ex vivo expansion time during CAR-T manufacturing negatively influenced the proportion of effector CD8+CAR-Ts in the infusion product. CONCLUSIONS: We identified cell-intrinsic characteristics of effector CD8+CAR-Ts correlating with response that could be used as an indicator for clinical outcome. The results in the study also serve as a guide to CAR-T manufacturing practices.

Whole body FDG PET/MR for progression free and overall survival prediction in patients with relapsed/refractory large B-cell lymphomas undergoing CAR T-cell therapy.

BACKGROUND: To find semi-quantitative and quantitative Positron Emission Tomography/Magnetic Resonance (PET/MR) imaging metrics of both tumor and non-malignant lymphoid tissue (bone marrow and spleen) for Progression Free Survival (PFS) and Overall Survival (OS) prediction in patients with relapsed/refractory (r/r) large B-cell lymphoma (LBCL) undergoing Chimeric Antigen Receptor (CAR) T-cell therapy. METHODS: A single-center prospective study of 16 r/r LBCL patients undergoing CD19-targeted CAR T-cell therapy. Whole body 18F-fluorodeoxyglucose (FDG) PET/MR imaging pre-therapy and 3 weeks post-therapy were followed by manual segmentation of tumors and lymphoid tissues. Semi-quantitative and quantitative metrics were extracted, and the metric-wise rate of change (Δ) between post-therapy and pre-therapy calculated. Tumor metrics included maximum Standardized Uptake Value (SUVmax), mean SUV (SUVmean), Metabolic Tumor Volume (MTV), Tumor Lesion Glycolysis (TLG), structural volume (V), total structural tumor burden (Vtotal) and mean Apparent Diffusion Coefficient (ADCmean). For lymphoid tissues, metrics extracted were SUVmean, mean Fat Fraction (FFmean) and ADCmean for bone marrow, and SUVmean, V and ADCmean for spleen. Univariate Cox regression analysis tested the relationship between extracted metrics and PFS and OS. Survival curves were produced using Kaplan-Meier analysis and compared using the log-rank test, with the median used for dichotomization. Uncorrected p-values < 0.05 were considered statistically significant. Correction for multiple comparisons was performed, with a False Discovery Rate (FDR) < 0.05 considered statistically significant. RESULTS: Pre-therapy (p < 0.05, FDR < 0.05) and Δ (p < 0.05, FDR > 0.05) total tumor burden structural and metabolic metrics were associated with PFS and/or OS. According to Kaplan-Meier analysis, a longer PFS was reached for patients with pre-therapy MTV ≤ 39.5 ml, ΔMTV≤1.35 and ΔTLG≤1.35. ΔSUVmax was associated with PFS (p < 0.05, FDR > 0.05), while ΔADCmean was associated with both PFS and OS (p < 0.05, FDR > 0.05). ΔADCmean > 0.92 gave longer PFS and OS in the Kaplan-Meier analysis. Pre-therapy bone marrow SUVmean was associated with PFS (p < 0.05, FDR < 0.05) and OS (p < 0.05, FDR > 0.05). For bone marrow FDG uptake, patient stratification was possible pre-therapy (SUVmean ≤ 1.8). CONCLUSIONS: MTV, tumor ADCmean and FDG uptake in bone marrow unaffected by tumor infiltration are possible PET/MR parameters for prediction of PFS and OS in r/r LBCL treated with CAR T-cells. TRIAL REGISTRATION: EudraCT 2016-004043-36.

Plasma protein biomarker profiling reveals major differences between acute leukaemia, lymphoma patients and controls.

Aiming to accommodate the unmet need for easily accessible biomarkers with a focus on biological differences between haematological diseases, the diagnostic value of plasma proteins in acute leukaemias and lymphomas was investigated. A multiplex proximity extension assay (PEA) was used to analyze 183 proteins in diagnostic plasma samples from 251 acute leukaemia and lymphoma patients and compared with samples from 60 healthy controls. Multivariate modelling using partial least square discriminant analysis revealed highly significant differences between distinct disease subgroups and controls. The model allowed explicit distinction between leukaemia and lymphoma, with few patients misclassified. Acute leukaemia samples had higher levels of proteins associated with haemostasis, inflammation, cell differentiation and cell-matrix integration, whereas lymphoma samples demonstrated higher levels of proteins known to be associated with tumour microenvironment and lymphoma dissemination. PEA technology can be used to screen for large number of plasma protein biomarkers in low µL sample volumes, enabling the distinction between controls, acute leukaemias and lymphomas. Plasma protein profiling could help gain insights into the pathophysiology of acute leukaemia and lymphoma and the technique may be a valuable tool in the diagnosis of these diseases.

A Phase I/IIa Trial Using CD19-Targeted Third-Generation CAR T Cells for Lymphoma and Leukemia.

PURPOSE: The chimeric antigen receptor (CAR) T-cell therapy has been effective for patients with CD19+ B-cell malignancies. Most studies have investigated the second-generation CARs with either CD28 or 4-1BB costimulatory domains in the CAR receptor. Here, we describe the first clinical phase I/IIa trial using third-generation CAR T cells targeting CD19 to evaluate safety and efficacy. PATIENTS AND METHODS: Fifteen patients with B-cell lymphoma or leukemia were treated with CAR T cells. The patients with lymphoma received chemotherapy during CAR manufacture and 11 of 15 were given low-dose cyclophosphamide and fludarabine conditioning prior to CAR infusion. Peripheral blood was sampled before and at multiple time points after CAR infusion to evaluate the persistence of CAR T cells and for immune profiling, using quantitative PCR, flow cytometry, and a proteomic array. RESULTS: Treatment with third-generation CAR T cells was generally safe with 4 patients requiring hospitalization due to adverse reactions. Six of the 15 patients had initial complete responses [4/11 lymphoma and 2/4 acute lymphoblastic leukemia (ALL)], and 3 of the patients with lymphoma were in remission at 3 months. Two patients are still alive. Best predictor of response was a good immune status prior to CAR infusion with high IL12, DC-Lamp, Fas ligand, and TRAIL. Responding patients had low monocytic myeloid-derived suppressor cells (MDSCs; CD14+CD33+HLA-DR-) and low levels of IL6, IL8, NAP3, sPDL1, and sPDL2. CONCLUSIONS: Third-generation CARs may be efficient in patients with advanced B-cell lymphoproliferative malignancy with only modest toxicity. Immune profiling pre- and posttreatment can be used to find response biomarkers.