Background: Relapse due to loss of the CD19 targeted epitope presents a therapeutic challenge of CD19 CAR-T therapy. These patients universally have a poor outcome and the unmet medical need is high. CD20 is a proven therapeutic target for B-NHL, supported by approved and widely used monoclonal antibody therapy. C-CAR066 is a novel 2nd generation chimeric antigen receptor T (CAR-T) therapy targeting CD20 antigen. Preclinical studies suggest that C-CAR066 has superior anti-tumor activity compared to CAR-Ts derived from scFVs of Leu16, Rituximab and Obinutuzumab and anti-CD19 BBZ CAR with FMC63. Methods: A phase I clinical trial (NCT04036019) was conducted to evaluate the safety and efficacy of C-CAR066 in subjects with r/r B-NHL who were previously treated with anti-CD19 CAR-T therapy. Patients (≥ 18 years) with r/r DLBCL, r/r FL or r/r MCL, ECOG < 2 were eligible. GMP manufacture of C-CAR066 was in a serum free and fully closed semi-automatic system. A 3-day cyclophosphamide plus fludarabine regimen was followed by a single infusion of C-CAR066. Bridging therapy was allowed. Results: As of Jan 31, 2021, 7 patients (6 DLBCL, 1 tFL) were enrolled and infused with C-CAR066 at dose ranges of 2.0 x 10⁶ to 4.8x10⁶ CAR-T cells/kg. The manufacturing success rate was 100%. The median age was 51 (range, 41-62) years, and 42.9% (3/7) patients were male. The median number of prior lines of therapy was 5 (range, 2-6). One patient (14.3%) underwent autologous stem cell transplant (ASCT) and one patient received bridging therapy. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) were graded according to ASTCT 2019 criteria. All 7 patients experienced CRS and most (85.7%) were grade 1 or 2. One patient had grade 4 CRS and recovered after treatment with tocilizumab and corticosteroids. Median time to onset of CRS was 5 days (range, 1-9), with median duration of 4 days (range, 2-17). There were no episodes of ICANS. Grade ≥3 neutropenia, anemia, thrombocytopenia, and infections were reported in 57.1%, 42.9%, 28.6%, and 14.3% of patients, respectively. At a median follow-up of 7.8 months, the best overall response rate was 100%, with 71.4% (5/7) achieving complete response (CR). Median time to response was 1.0 month (range, 0.9-2.7). Median time to CR was 2.7 months (range, 0.9-2.8). By the cutoff date, 3 patients (2 PR, 1 CR) had disease progression. Median duration of response was not reached. Conclusions: C-CAR066 has shown a favorable safety profile and promising efficacy in patients with r/r B-NHL following failure of CD19 CAR-T therapy. These results show that C-CAR066 has a different mechanism of action compared to anti-CD-19 CAR-T therapy and could provide a solution to address the unmet medical need in B-NHL patients that have failed anti-CD19 CAR-T therapy. Clinical trial information: NCT04036019