Lawrence A. Stern ∗ , Vibhuti Vyas ∗ , Laura Lim , Christian Huynh , Ryan Urak , Ruby Espinosa , Zhiqiang Wang , Michalina Silva Thiel , John C. Williams , Stephen J. Forman , Christine E. Brown † , Xiuli Wang †
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Abstract
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.
摘要 在本研究中，我们旨在开发一种人源化 CD19 嵌合抗原受体（CAR），其效力与 FMC63 CAR 相当，并可能降低免疫原性风险。对小鼠 FMC63 单链可变片段（scFv）进行了人源化处理，得到了两个主要候选 scFv：VH4vκ1 和 4D5，它们的结合亲和力比 FMC63 scFv 弱。这些人源化 CD19-scFv 被整合到 CAR 构建物中，生成了 huCD19R(VH4Vκ1) 和 huCD19R(4D5)CAR，这两种 CAR 都含有 41BB costimulatory domain。评估了 CAR T 细胞对 CD19+ 和 CD19 低表达肿瘤的抗肿瘤活性。所有研究中使用相同骨架的 FMC63 CAR T 细胞作为对照。结果显示，huCD19R(VH4vκ1) CAR T细胞在受到CD19靶点刺激时，表现出与FMC63 CAR相似的扩增、表型和效应功能。当 CAR T 细胞受到 CD19 肿瘤的挑战时，huCD19R(VH4vκ1) CAR T 细胞表现出与 FMC63 CAR T 细胞相似的增殖，而 huCD19R(4D5) CAR T 细胞基本上没有增殖。此外，在对表达一系列 CD19 抗原的肿瘤进行测试时，huCD19R(VH4vκ1) CAR T 细胞的体内抗肿瘤活性明显优于 huCD19R(4D5) CAR T 细胞。最后，我们利用混合模型发现，huCD19R(VH4vκ1) T细胞的细胞因子分泌情况与FMC63 CAR T细胞相当。此外，huCD19R(VH4vκ1) CAR T细胞对CD19+和低表达CD19的工程肿瘤都有疗效。这些研究结果表明，huCD19R(VH4vκ1)CAR T细胞可能具有更强的持久性，有望作为CD19+肿瘤的治疗方法应用于临床。
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