Chimeric antigen receptor (CAR)-T cell therapy has shown remarkable clinical efficacy against B-cell malignancies, yet marked vulnerability to antigen escape and tumor relapse exists. Here we report the rational design and optimization of bispecific CART cells with robust activity against heterogeneous multiple myeloma (MM) that is resistant to conventional CART cell therapy targeting B-cell maturation antigen (BCMA). We demonstrate that BCMA/CS1 bispecific CART cells exhibit superior CAR expression and function compared to T cells that co-express individual BCMA and CS1 CARs. Combination therapy with anti-PD-1 antibody further accelerates the rate of initial tumor clearance in vivo, while CART cell treatment alone achieves durable tumor-free survival even upon tumor re-challenge. Taken together, the BCMA/CS1 bispecific CAR presents a promising treatment approach to prevent antigen escape in CART cell therapy against MM, and the vertically integrated optimization process can be used to develop robust cell-based therapy against novel disease targets.

ABSTRACTChimeric antigen receptor (CAR)-T cell therapy has shown remarkable clinical efficacy against B-cell malignancies but also demonstrated marked vulnerability to antigen escape and tumor relapse. Here, we report the rational design and systematic optimization of bispecific CAR-T cells with robust activity against multiple myeloma (MM), including heterogeneous MM that is resistant to conventional CAR-T cell therapy targeting B-cell maturation antigen (BCMA). We demonstrate that BCMA/CS1 bispecific CAR-T cells exhibit significantly higher CAR expression levels and greater antigen-stimulated proliferation compared to T cells that co-express individual BCMA and CS1 CARs. Compared to single-input BCMA- or CS1-targeting CAR-T cells, BCMA/CS1 bispecific CAR-T cells significantly prolong the survival of animals bearing heterogeneous MM tumors. Combination therapy with anti–PD-1 antibody further accelerates the rate of initial tumor clearance in vivo, but CAR-T cell treatment alone was able to achieve durable tumor-free survival even upon tumor re-challenge. Taken together, the BCMA/CS1 bispecific CAR presents a promising treatment approach to prevent antigen escape in CAR-T cell therapy against MM, and the vertically integrated optimization process can be used to develop robust cell-based therapy against novel disease targets.

To address antigen escape and loss of T-cell functionality, we report a phase-1 clinical trial (NCT04007029) evaluating autologous naive 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 x 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.

2543 Background: Although chimeric antigen receptor (CAR)-T cells produce impressive outcomes in B-cell malignancies, a substantial fraction of patients with relapsed/refractory B-cell leukemia and lymphoma treated with anti-CD19 CAR-T cell therapy (CART19) either do not respond to treatment or relapse, with poor CAR-T cell persistence or CD19 antigen escape being two key factors that limit durability of response. In order to address these factors, we initiated a clinical trial with naïve/memory T (TN/MEM) cells engineered to express bispecific anti-CD19/CD20 CARs (CART19/20) (NCT04007029). Methods: This trial is a Phase 1, first-in-human, dose-escalation trial enrolling patients with relapsed or refractory follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle-cell lymphoma (MCL) and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Following lymphodepletion chemotherapy with fludarabine and cyclophosphamide, patients received CART19/20 cell doses ranging from 50 x 106 to 200 x 106 CAR-positive cells. The primary endpoint was to evaluate the safety of CART19/20 as measured by adverse events and dose limiting toxicities. Secondary endpoints were efficacy as assessed by disease response, progression-free survival (PFS), overall survival (OS), and CAR transgene persistence. Results: As of February 7, 2022, dose-escalation has been completed with 9 patients enrolled and 8 patients infused (3 FL, 4 DLBCL including 2 transformed follicular and 1 primary mediastinal B cell, and 1 MCL). with CART19/20 cells on this study. The median age at the time of CART19/20 infusion was 59 and median prior lines of therapy was 3.5. All patients had stage IV disease and 7 of 9 patients required bridging therapy. Grade-1 cytokine release syndrome (CRS) occurred in 6 of 8 patients, and no patient experienced immune effector cell-associated neurotoxicity syndrome ( ICANS). Among all patients, only one dose of tocilizumab was administered to one subject, and no steroids were given. With a median follow-up of 12 months from time of CART19/20 infusion (range: 4+ to 24+ months), 7 of 8 of patients remain in a complete remission. Median PFS and OS were not reached, and all patients with a complete remission demonstrate ongoing B-cell aplasia. Conclusions: This study demonstrates that CART19/20 cells are safe and effective in patients with relapsed/refractory NHL and potentially obviates the challenges of the commonest causes of relapse after CAR-T cell therapy by means of modifying TN/MEM cells and dual-antigen targeting, respectively. Given the strong safety and response observed, dose escalation was completed with the second dosing level (DL2) of 200 x 106 CAR-positive cells, and DL1 of 50 x 106 CAR-positive cells was chosen as the therapeutic dose for future trial expansion. Clinical trial information: NCT04007029.

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.