To optimize the affinity of 1218 scFv, six complementarity-determining regions (CDR) residues in the heavy and light chains were randomly mutated using NNK degenerate codons with 70% or more of the parent sequence. in patients relapsing after FMC63-CART19. Furthermore, h1218-CART19 exhibited enhanced killing of B-cell malignancies in vitro and in vivo compared with FMC63-CART19. Mechanistically, we found that h1218-CART19 had reduced activation-induced cell death (AICD) and enhanced expansion compared to FMC63-CART19 owing to faster on- and off-rates. Based on these preclinical results, we performed a phase I dose-escalation trial, testing three dose levels (DL) of AT101 (the GMP version of h1218) using a 3 + 3 design. In 12 treated patients (7 DLBCL, 3 FL, 1 MCL, and 1 MZL), AT101 showed a promising safety profile with 8.3% grade 3 CRS (n= 1) and 8.3% grade 4 ICANS (n= 1). In the whole cohort, the overall response rate was 91.7%, with a complete response rate of 75.0%, which improved to 100% in DL-2 and -3. AT101 growth correlates with CR and B-cell aplasia. == Conclusions == We developed a novel, safe, and potent CART19 product that recognizes a membrane-proximal domain name of CD19 with fast on- and off-rates and showed significant efficacy and promising safety in patients with relapsed B-cell NHL. == Trial registration == NCT05338931; Date: 202204-01. == Supplementary Information == The online version contains supplementary material available at 10.1186/s12943-023-01886-9. Keywords:CD19, CAR T cells, Resistance, CD19 mutations, Epitope masking, Lymphoma, Leukemia, Low avidity, Fast on- and off-rate, Membrane-proximal epitope == Background == CD19-directed chimeric antigen receptor T (CART19) cell therapies have shown impressive clinical outcomes in CD19+B-cell malignancies. However, a significant fraction of patients still fails to a Rabbit Polyclonal to FZD6 achieve complete response (CR) or eventually relapse after CART19, with different relapse rates based on specific diseases [1]. The quality of the conversation between CAR and CD19 on tumor cells is usually a critical factor governing CAR T cell function. Currently, all US FDA-approved commercial CART19 products, including tisagenlecleucel, lisocabtagene maraleucel, axicabtagene ciloleucel, and brexucabtagene autoleucel, use single-chain variable fragments (scFv) derived from the murine anti-human CD19 antibody FMC63 [24]. FMC63 recognizes an epitope in CD19 that is located between exons 2 and 4 [5]. Interestingly, several mechanisms of relapse include the loss of the FMC63-acknowledged CD19 epitope either through point mutations or epitope masking [68]; furthermore, ineffective CAR:CD19 conversation can lead to CART dysfunction [911]. CD19 FMC63-epitope loss can occur when CD19 is expressed on the surface of relapsed tumor cells, but the CD19 epitope is not available for CART19 recognition due to multiple causes. CD19 FMC63-epitope loss includes either mutation of the FMC63 epitope [12] or masking of that epitope by CAR19 itself [13]. Several CD19 point mutations have been reported in patients with diffuse large B cell lymphoma (DLBCL) and B-acute lymphoblastic leukemia (B-ALL) who showed resistance to CART19 cell therapy. Some of these mutations affect the FMC63-acknowledged epitope, leading to failure of FMC63-CART19 [8,12,14]. In addition, we previously identified a novel mechanism of resistance in which the CAR19 gene was inadvertently introduced into leukemic B-cell blasts during manufacturing, leading to thein-cisinteraction of CAR19:CD19 leading to epitope masking and subsequent resistance [13]. Therefore, targeting epitopes that differ from FMC63 may TAS 103 2HCl potentially overcome FMC63-CART19-driven epitope loss. Additionally, T cell dysfunction further contributes to FMC63-CART19 failure. While several factors contribute to this phenomenon, such as the baseline T cell quality (differentiation/ exhaustion) and the immunosuppressive microenvironment, the quality of the CAR:CD19 conversation also plays a critical role in determining the efficacy of CAR T immunotherapy [15]. For instance, it was shown that targeting a CD22 epitope that TAS 103 2HCl is closer to the cell membrane leads to improved immune synapse formation and superior in vivo CAR T anti-tumor effect as compared to targeting a membrane-distal epitope [16,17]. Furthermore, we and others have exhibited that modifying the structure of the CAR, for example, by shortening the linker between the variable regions TAS 103 2HCl of the scFv, can improve binding to the target by shortening the distance between CAR T cells and tumor cells, thus enhancing CAR T cell effector function [18]. Moreover, the on- and off-rates of the CAR:target interactions can determine the overall function of CAR T cells. TAS 103 2HCl It has been postulated that CARs with faster off-rates from tumor cells are more effective in providing prolonged CAR T cell function [11,19]. To address the issues of CART19 relapse following FMC63-CART19 treatment, we hypothesized that: 1. a CART19 product targeting a non-FMC63 epitope.