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 chimeric antigen receptor (CAR) that matches the potency of the FMC63 CAR and potentially reduces the risk of immunogenicity. The murine FMC63 single-chain variable fragment (scFv) was humanized yielding 2 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) CARs, both containing the 41BB costimulatory domain. The antitumor activity of the CAR T cells was assessed against CD19⁺ and CD19 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, whereas 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 CD19 antigens. Finally, using a hybrid model, we found that the huCD19R(VH4vκ1) T cells had a comparable cytokine secretion profile to that of 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⁺ tumors.