Tri-specific autologous CD19.20.22 chimeric antigen receptor (CAR) T-cells for patients with Relapsed/Refractory B-cell lymphomas.

Abstract Introduction: CD19-directed CAR T cell (CAR-T) therapy has curative potential for patients with relapsed/refractory (R/R) B-cell lymphoma, however, most patients still ultimately relapse (Neelapu, Jacobson et al. 2023). CAR T cell resistance mechanisms include the loss of CD19 expression thus multi-specific CAR-T are a promising strategy to help overcome tumor escape (Schneider, Xiong et al. 2021). We set up a first-in-human study investigating a novel tri-specific duoCAR architecture based on a single lentiviral vector expressing a tandem CAR targeting CD19 and CD20, which is linked to a monoCAR targeting the CD22 antigen. This tri-specific CAR-T contains two novel costimulatory domains, ICOS and OX40. Here we report an interim analysis of the first planned dose level (DL). Methods: This is a single-center, single arm phase I study employing the Bayesian optimal interval design with a 3+3 design run-in. There are three planned dose levels (DL): DL-1 – 0.75x106 cells/kg, DL0 – 1x106 cells/kg, DL+1 – 2.5x106 cells/kg. Main inclusion criteria include diagnosis of R/R B-cell lymphoma, age ≥ 18 years, ≥ two prior lines of therapy, and expression of 2/3 target antigens. Main exclusion criteria include active CNS disease and immunosuppression. Patients previously treated with commercial CAR-T, bi-specific antibodies, and allogeneic stem cell transplantation (allo-SCT) are permitted. In-house manufacturing utilizes the CliniMACS Prodigy platform for T-cell enrichment, activation, transduction, and expansion. Leukapheresis was performed on day -11, product was manufactured from day -10 to day -2, and quality assessment was completed by day 0. Patients received cyclophosphamide 500mg/m2 and fludarabine 30mg/m2 on days -5 to -3. CAR-T were administered intravenously on day 0 starting at DL0. Results: Four patients were enrolled to receive 1x106 CAR-T/kg. One patient had a manufacturing failure, and three patients were successfully treated. Of these three products, median fold increase in T-cells and viability after expansion was 16.1 (range 13.8-28.1) and 90.6% (range 87.9-94.0), respectively. Median transduction efficiency of the CD20.CD19 CAR and CD22 CAR was 20.1% (range 12.9-21.8) and 25.4% (range 15.5-29.6), respectively. Median age was 65 (range 60-68) and 2/3 patients were female. One patient had marginal zone lymphoma (MZL) and two had Diffuse large B-cell lymphoma (DLBCL). All patients had ≥ stage III at initial diagnosis. Prior therapies included BR, RCHOP, commercial CD19 CAR-T therapy, polatuzumab and allo-SCT. Patient 1 was CAR-T-naive, patients 2 and 3 had previously received axi-cel. Patient 3 had received an allo-SCT. 2/3 patients expressed all three target antigens. At DL0 (1x106 cells/kg; n=3) no CRS, ICANS, fever, HLH or infections occurred. None of the patients required anti-cytokine therapy or steroids. The most common adverse event was grade 4 neutropenia in all patients. Two patients developed grade 1 thrombocytopenia, and one patient developed grade 2 hepatotoxicity. All 3 patients treated showed a substantial expansion of CD4+ and CD8+ CAR-T in their blood as measured by flow cytometry after staining with fluorescent target proteins or anti-linker antibody. Peak levels occurred around d+14 post-treatment and in some cases with sufficient follow-up CAR-T persisted for > 2 months. The ORR was 100%. All patients had a PR by Lugano criteria on day +30. Patient 1, who had MZL, had a CR on day +90 and remained in a CR at 6 months. Patients 2 and 3 developed disease progression by day +90. Post-relapse biopsy of patient 2 was negative for CD19 and positive for CD20 and CD22, consistent with the pre-treatment biopsy. At last follow up all three patients were still alive with no toxicities. Discussion: This interim analysis shows that point-of-care manufacturing and treatment with the first-in-human CD19.20.22 CAR-T autologous product containing OX40/ICOS co-stimulatory domains is safe and feasible. No CRS or ICANS was observed. We also report substantial expansion and persistence of CAR-T, evidence of clinically meaningful efficacy with responses in all patients, and a durable response in 1/3 patients. The study has received approval to advance to the next dose level which will hopefully further improved efficacy.