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To address antigen escape and loss of T-cell functionality, we report a phase I clinical trial (NCT04007029) evaluating autologous naive and memory T (TN/MEM) cells engineered to express a bispecific anti-CD19/CD20 chimeric antigen receptor (CAR; CART19/20) for patients with relapsed/refractory non-Hodgkin lymphoma (NHL), with safety as the primary endpoint. Ten patients were treated with 36 × 106 to 165 × 106 CART19/20 cells. No patient experienced neurotoxicity of any grade or over grade 1 cytokine release syndrome. One case of dose-limiting toxicity (persistent cytopenia) was observed. Nine of 10 patients achieved objective response [90% overall response rate (ORR)], with seven achieving complete remission [70% complete responses (CR) rate]. One patient relapsed after 18 months in CR but returned to CR after receiving a second dose of CART19/20 cells. Median progression-free survival was 18 months and median overall survival was not reached with a 17-month median follow-up. In conclusion, CART19/20 TN/MEM cells are safe and effective in patients with relapsed/refractory NHL, with durable responses achieved at low dosage levels.Significance:Autologous CD19/CD20 bispecific CAR-T cell therapy generated from TN/MEM cells for patients with NHL is safe (no neurotoxicity, maximum grade 1 cytokine release syndrome) and demonstrates strong efficacy (90% ORR, 70% CR rate) in a first-in-human, phase I dose-escalation trial.This article is highlighted in the In This Issue feature, p. 517

Representativeness of study participants

CART19/20 cell product characteristics. (A) % CD4+ among total T cells and CAR-expressing T cells. (B) Comparison of T-cell subtype frequency among CD4+ vs. CD8+ T cells in cryopreserved CART19/20 cell products. Te/exh: effector/exhausted T cells, CD45RA+/CD45RO–/CD62L–; Tem: effector-memory T cells, CD45RA–/CD45RO+/CD62L–; Tcm: central-memory T cells: CD45RA–/CD45RO+/CD62L+; naïve: CD45RA+/CD45RO–/CD62L+. (C) Phenotype of leukopak content prior to cell isolation. (D) Phenotype of cells obtained after isolation. (E) CD14 and CD25 expression patterns among CD62L+ cells in patient leukopak content prior to cell isolation.

T cell populations become CD8-dominant post CART19/20 cell infusion. % CD8+ among (A) CAR-expressing T cells and (B) all CD3+ T cells was quantified by flow cytometry. Data are shown for CAR-T cells only up to day 90 post infusion as values post day 90 become unreliable due to low CAR+ cell count detected by flow. FP: final product (i.e., cryopreserved CART19/20 cells).

PET scans taken for all patients treated with CART19/20 cell therapy.

CD14/CD20 depletion did not significantly alter regulatory T (Treg) cell content in CART19/20 cell product and in patient peripheral blood post infusion. Intracellular staining of starting (unactivated) TN/MEM population, CART19/20 final product, and patient PBMC collected 14 days post CART19/20 infusion. FOXP3+ frequency is shown as a percentage of CD3+CD4+ T cells.

Flow-cytometry analysis of post-infusion peripheral blood samples. (A) Gating strategy for T-cell analysis in CART19/20 cell products and post-infusion patient peripheral mononuclear blood cells (PBMCs). Two viable gates were set based on side scatter (SSC-A) vs. forward scatter (FSC-A). The more restrictive “Viable” gate includes only viable lymphocytes, to enable more precise identification of T cells. The “Viable (inclusive)” gate includes all viable PBMCs, to enable calculation of CAR-T cells as a fraction of PBMCs. Truncated epidermal growth factor receptor (EGFRt) is co-expressed with the CAR, and EGFRt staining is used for CAR expression detection. Percent CAR+ T cells among viable singlets as shown in main-text Fig. 4b is the EGFRt+ population as a % of grandparent in the viable PBMC branch.Percent CAR expression among T cells as shown in main-text Fig. 4c is the EGFRt+ population as a % of parent in the viable lymphocyte branch. (B) Examples of post-infusion patient sample analysis.

Patient 009 exhibited elevated cytokine, C-reactive protein, and ferritin levels prior to and after CART19/20 cell infusion. (A) Serum levels of various cytokines measured by Luminex multiplex assay. (B) C-reactive protein (CRP) levels for Patient 009 (left-most) and all patients (second from left). Peak and median CRP levels are shown in box-and-whisker plots. (C) Ferritin levels for Patient 009 (left-most) and all patients (second from left). Peak and median ferritin levels are shown in box-and-whisker plots.

Comparison of CART19/20 cell therapy results with reported data from trials evaluating other CD19/CD20 bispecific CAR-T cell therapies and commercially available CD19 CAR-T cell therapies.

To address antigen escape and loss of T-cell functionality, we report a phase I clinical trial (NCT04007029) evaluating autologous naive and memory T (TN/MEM) cells engineered to express a bispecific anti-CD19/CD20 chimeric antigen receptor (CAR; CART19/20) for patients with relapsed/refractory non-Hodgkin lymphoma (NHL), with safety as the primary endpoint. Ten patients were treated with 36 × 106 to 165 × 106 CART19/20 cells. No patient experienced neurotoxicity of any grade or over grade 1 cytokine release syndrome. One case of dose-limiting toxicity (persistent cytopenia) was observed. Nine of 10 patients achieved objective response [90% overall response rate (ORR)], with seven achieving complete remission [70% complete responses (CR) rate]. One patient relapsed after 18 months in CR but returned to CR after receiving a second dose of CART19/20 cells. Median progression-free survival was 18 months and median overall survival was not reached with a 17-month median follow-up. In conclusion, CART19/20 TN/MEM cells are safe and effective in patients with relapsed/refractory NHL, with durable responses achieved at low dosage levels.Significance:Autologous CD19/CD20 bispecific CAR-T cell therapy generated from TN/MEM cells for patients with NHL is safe (no neurotoxicity, maximum grade 1 cytokine release syndrome) and demonstrates strong efficacy (90% ORR, 70% CR rate) in a first-in-human, phase I dose-escalation trial.This article is highlighted in the In This Issue feature, p. 517

To address antigen escape and loss of T-cell functionality, we report a phase-1 clinical trial (NCT04007029) evaluating autologous naïve and memory T (TN/MEM) cells engineered to express a bispecific anti-CD19/CD20 CAR (CART19/20) for patients with relapsed/refractory NHL, with safety as the primary end point. Ten patients were treated with 36–165 × 106 CART19/20 cells. No patient experienced neurotoxicity of any grade, or over grade-1 cytokine release syndrome. One case of dose-limiting toxicity (persistent cytopenia) was observed. Nine of ten patients achieved objective response (90% ORR), with seven achieving complete remission (70% CR rate). One patient relapsed after 18 months in CR, but returned to CR after receiving a second dose of CART19/20 cells. Median progression-free survival and overall survival were not reached with a 17-month median follow-up. In conclusion, CART19/20 TN/MEM cells are safe and effective in patients with relapsed/refractory NHL, with durable responses achieved at low dosage levels.

Background: Single-input anti-CD19 CAR T-cells have demonstrated clinical efficacy for relapsed or refractory (R/R) non-Hodgkin B-cell lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Despite excellent response rates, over 50% of CD19 CAR T-cell recipients relapse. Preclinical data show engineering of bispecific anti-CD19/CD20 CAR T-cells via lentiviral transduction effectively targets tumor cells and overcomes antigen escape (Zah E et al., Cancer Immunol Res, 2016). Based on these promising preclinical results and the limitations of single-input anti-CD19 CARs, we investigated the bispecific anti-CD19/CD20 CAR naïve/memory T-cells in a phase I dose-escalation clinical trial for patients with R/R NHL/CLL (NCT04007029). Methods: This trial includes patients who have measurable disease after 2 lines of therapy for diffuse large B-cell lymphoma (DLBCL) and primary mediastinal B-cell lymphoma (PMBCL), and after 3 lines of therapy for mantle cell lymphoma (MCL), follicular lymphoma (FL), CLL and small lymphocytic leukemia (SLL). Eligible participants received lymphodepleting chemotherapy with fludarabine 30 mg/m2 and cyclophosphamide 500 mg/m2 for three days, followed by anti-CD19/CD20 CAR T-cell infusion. The CAR T-cell infusion will be given with standard "3+3" dose escalation to determine the maximum tolerated dose (MTD), with a dose range of 5 x 107 to 6 x 108 CAR-positive cells per patient. Results: To date, three patients received treatment on cohort 1 with 5 x 107 CD19/CD20 CAR T-cells for R/R MCL, FL and PMBCL, with an average age of 49.3 (range, 29-60) and a mean of 3.7 prior regimens (range, 3-4). All 3 patients' lymphomas were CD19+/CD20+ on tissue biopsy prior to CAR infusion and all 3 received bridging chemotherapy. The infusion was well tolerated and no major infusion reactions occurred. Peak expansion was noted on day 14. No dose limiting toxicities were identified. The maximum grade CRS was 1 and there was no ICANS. At the 6.0-month cutoff date, 2 of the 3 patients remain in ongoing complete remission. Unfortunately, one patient developed progressive disease 0.5 months after CAR infusion, yet remains alive after treatment with immunotherapy. Both of the responders continue to demonstrate ongoing CAR T-cell persistence and B-cell aplasia by 3.0 and 6.0-month follow up, respectively. Conclusions: Here we demonstrate impressive responses in 2 of 3 patients at the 5 x 107 CD19/CD20 CAR T-cell dosages. Bispecific CD19/CD20 CAR T-cell therapy appears to be safe and effective in patients with R/R NHL and CLL and obviates the challenges with the single antigen directed CARs by decreasing risk of target antigen loss and expression downregulation. A longer follow up period is required to determine the impact of modifying naïve/memory T cells and the durability of response. The trial continues to enroll patients and additional clinical and translational data are being collected on the initial patient cohort. Disclosures Timmerman: Corvus: Current equity holder in publicly-traded company; Marker Therapeutics: Current equity holder in publicly-traded company; Bluebird Bio: Current equity holder in publicly-traded company; Immune Design: Honoraria; Celldex Therapeutics: Consultancy; Valor: Research Funding; Merck: Research Funding; Spectrum Pharmaceuticals: Research Funding; BMS: Other: Travel support, Research Funding; Kite, a Gilead Company: Consultancy, Other: Travel support, Research Funding; Genmab: Current equity holder in publicly-traded company. Chen:Kalthera Therapeutics: Other: Co-founder; Notch Therapeutics: Membership on an entity's Board of Directors or advisory committees; Gritstone Oncology: Membership on an entity's Board of Directors or advisory committees. Larson:BMS, Bioline, Celgene, Juno, Janssen: Research Funding; TORL Biotherapeutics: Current equity holder in private company.

50% of patients with relapsed/refractory (R/R) B-cell lymphomas (BCL) who initially respond to CD19 CAR T therapy will relapse either due to poor CAR-T persistence or CD19 antigen escape. Preclinical data demonstrate that engineering of bispecific anti-CD19/CD20 CAR in naïve/memory T-cells (TN/MEM) via lentiviral transduction effectively targets tumor cells, overcomes antigen escape and enhances CART persistence (Zah E et al., Cancer Immunol Res, 2016). We conducted a first-in-human phase 1 clinical trial with bispecific CD19/CD20 CAR T-cells for patients with R/R BCL (NCT04007029). Patients with measurable disease after 2 or more lines of therapy for DLBCL and PMBCL, and 3 or more lines of therapy for MCL, FL and CLL were included. Autologous leukocytes obtained by leukapheresis were sorted for CD14-/CD25-/CD62L+ TN/MEM cells, followed by lentiviral transduction of the bispecific CD19/CD20 CAR. Following Cy-Flu lymphodepletion, patients are infused with CD19/CD20 CAR TN/MEM cells, at doses ranging from 5 x 107 and 2 x 108 CAR positive cells. 5 patients are included in the analysis, 4 in the first cohort and 1 in the second cohort. All patients had CD19/CD20+ B cell malignancy on tissue biopsy prior to CAR-T. Patients had previously received a median of 4 prior lines of therapy, and 4 received bridging therapy. Infusions were well tolerated, and no dose limiting toxicities were identified. All patients had grade 1 CRS, with median onset 8 days (range 7-11) after infusion. No patient had immune effector cell-associated neurotoxicity syndrome (ICANS). Peak expansion was noted on day 14. Median follow up is 11.1 months (95% CI 2.1-14.8), with 4 of patients in ongoing complete remission. The 1 patient who did not respond had early disease progression, with CD19 and CD20 negative mediastinal lymphoma at day 14 after CAR infusion. Median progression-free survival (PFS) and overall survival (OS) were not reached and all responders demonstrate ongoing CAR T-cell persistence and B-cell aplasia by data cutoff. Bispecific CD19/CD20 CAR in naïve/memory T-cells are safe and effective in patients with R/R BCL. To our knowledge, this is the first study that potentially obviates the challenges of the commonest causes of relapse after CAR T-cell therapy, poor CAR-T persistence and antigen escape mechanisms, by means of modifying naïve/memory T cells and dual antigen targeting, respectively. Patient and Treatment CharacteristicsID12345DiagnosisMCLFLPMBCLFLFLSexMMFMFAge6058296235Prior lines of therapy44343Stage44442EECOG01100Bridging therapy?YesYesYesYesNoDosage, x 106 CART50505050200Max CRS/ICANS gradeGrade 1 CRS. No ICANS.Grade 1 CRS. No ICANS.Grade 1 CRS. No ICANS.Grade 1 CRS. No ICANS.Grade 1 CRS. No ICANS.Required treatment for CRS/ICANS?NoNoNoNoNoBest Response to CARTCRCRNone; refractoryCRCRPFS (months)14.811.10.54.82.1OS (months)14.811.16.24.82.1Subsequent Follow-upAlive, ongoing remissionAlive, ongoing remissionNo response to CART; died from infectious complications after subsequent therapy with nivolumab and brentuximab.Alive, ongoing remissionAlive, ongoing remission Citation Format: Sanaz Noelle Ghafouri, Christopher Walthers, Mobina Roshandell, Brenda Ji, Jacqueline Trent, Jia Ming Chen, Jacob Naparstek, Caitlin Harris, Thomas Schweppe, Karla K. Nawaly, Monica Mead, Sven de Vos, Patricia Young, Caspian Oliai, Gary Schiller, John M. Timmerman, Yvonne Y. Chen, Sarah Larson. CD19/CD20 bispecific chimeric antigen receptor (CAR) in naive/memory T-cells for the treatment of relapsed or refractory B-cell lymphomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT007.

Objective In-person lifestyle interventions for obesity treatment yield significant improvements in depression. These improvements may be attributed to the excellent weight losses produced by in-person interventions. In contrast, Internet programs yield more modest weight losses, and their effect on depression is unknown. This study is the first to examine whether Internet-delivered obesity treatment impacts depressive symptoms. Methods Participants (N=136) were randomized to either a community campaign PLUS Internet behavioral weight loss (IBWL) or community campaign alone (Control). IBWL did not include online social support components. A measure of depressive symptoms was administered and weight was objectively assessed. Results Of the total sample, 24% met the clinical cut-off for elevated depression risk at baseline. IBWL participants lost more weight during treatment (p=.005) and experienced significantly greater improvements in depressive symptoms (p=.02). Among participants who met the clinical cut-off for elevated risk for depression at baseline, those assigned to IBWL had greater improvements in depressive symptoms during treatment compared to Controls (p=.033). Consequently, at post-treatment, a smaller percentage of IBWL participants were at elevated risk for depression. Conclusions This study is the first to show that Internet-delivered obesity treatment improves depression risk and depressive symptoms in individuals with overweight or obesity.

Four patients who manifested an association between chronic liver disease and lymphoma are reported. Three of them had nonalcoholic cirrhosis and one had chronic cholangitis. Non-Hodgkin's lymphoma developed long after the presence of liver disease had been established in three of the patients. The fourth patient died of hepatic coma and Hodgkin's disease was discovered incidentally at autopsy. Drugs could not be incriminated in the development of either cirrhosis or lymphoma in any of the patients. A review of the literature is presented, and the view that the association between chronic liver disease and lymphoma is not coincidental is supported.