Although commercial anti-CD19 chimeric antigen receptor T-cell (CART19) therapies are effective in treating advanced B-cell non-Hodgkin lymphoma (NHL), the majority of patients ultimately relapse. Several factors contribute to treatment failure, such as loss of CD19 expression on tumor cells and functional exhaustion of CAR T cells. All approved CART19 products rely on the FMC63 single-chain variable fragment (scFv), which binds a membrane-distal epitope of CD19 and displays comparatively slow association (on) and dissociation (off) rates. We proposed that an alternative anti-CD19 scFv targeting a distinct membrane-proximal epitope, independent of FMC63 and possessing faster on- and off-rates, could help overcome these limitations and yield improved therapeutic results. An autologous CART19 construct incorporating 4-1BB costimulatory signaling was generated using a novel humanized chicken-derived antibody designated h1218. This antibody specifically recognizes a membrane-proximal region of CD19 and exhibits accelerated binding and release kinetics compared with FMC63. The h1218-CART19 product was assessed in laboratory and animal models designed to mimic resistance to FMC63-based therapies. A first-in-human multicenter phase I study was subsequently initiated to evaluate the clinical performance of AT101, the GMP-grade version of h1218-CART19, in individuals with relapsed or refractory (r/r) NHL. Preclinical experiments showed that h1218-CART19, unlike FMC63-CART19, efficiently eliminated lymphoma cells carrying CD19 point mutations (L174V or R163L) and those co-expressing the FMC63-CAR19 antigen, a phenomenon seen in patients who relapse following FMC63-CART19 treatment. In addition, h1218-CART19 demonstrated stronger cytotoxic activity against B-cell malignancies than FMC63-CART19 in both in vitro and in vivo settings. Mechanistic analyses indicated that the superior performance of h1218-CART19 stemmed from decreased activation-induced cell death (AICD) and greater proliferative capacity, resulting from its faster on- and off-rates. These promising findings prompted a phase I dose-escalation trial of AT101 using a 3 + 3 design across three dose levels (DL). In the 12 enrolled patients (7 DLBCL, 3 FL, 1 MCL, 1 MZL), AT101 displayed an encouraging safety profile, with grade 3 CRS observed in 8.3% (n = 1) and grade 4 ICANS in 8.3% (n = 1). Across the entire cohort, the overall response rate was 91.7%, and the complete response rate was 75.0%, rising to 100% in patients treated at DL-2 and DL-3. Greater AT101 expansion was associated with achievement of complete remission and sustained B-cell aplasia. A new CART19 therapy was developed that safely and potently targets a membrane-proximal epitope of CD19 via an scFv with rapid on- and off-rates. This product exhibited substantial clinical activity and a favorable safety profile in patients with relapsed B-cell NHL.
