Development and characterization of a low-affinity humanized CD19-chimeric antigen receptor for B cell malignancies

In this study, we aim to develop a humanized CD19 CAR that matches the potency of the FMC63 CAR while potentially reducing the risk of immunogenicity. The murine FMC63 scFv was humanized yielding two lead candidate scFvs, VH4vκ1 and 4D5, which exhibit weaker binding affinity than FMC63 scFv. These humanized CD19-scFvs were incorporated into CAR constructs to generate huCD19R(VH4Vκ1) and huCD19R(4D5) CAR, both containing the 41BB costimulatory domain. The antitumor activity of the CAR T cells was assessed against CD19+ and low-expressing tumors. FMC63 CAR T cells with the same backbone in all studies were used as controls. The results showed that the huCD19R(VH4vκ1) CAR T cells exhibited similar expansion, phenotype, and effector function to the FMC63 CAR upon stimulation with CD19 targets. When the CAR T cells were challenged with CD19-bearing tumors, the huCD19R(VH4vκ1) CAR T cells showed similar proliferation to the FMC63 CAR T cells, while the huCD19R(4D5) CAR T cells essentially failed to proliferate. Moreover, the huCD19R(VH4vκ1) CAR T cells exhibited significantly better in vivo antitumor activity than the huCD19R(4D5) CAR T cells when tested against tumors expressing a range of antigens. Finally, using a hybrid model, we found that the huCD19R(VH4vκ1) T cells had a comparable cytokine secretion profile to FMC63 CAR T cells. Furthermore, the huCD19R(VH4vκ1) CAR T cells exhibited efficacy against both CD19+ and engineered CD19 low-expressing tumors. These findings suggest that huCD19R(VH4vκ1) CAR T cells may offer enhanced persistence and represent a promising candidate for clinical translation as a therapy for CD19-positive tumors.