Stern Lawrence A, Vyas Vibhuti, Lim Laura, Huynh Christian, Urak Ryan, Espinosa Ruby, Wang Zhiqiang, Silva Thiel Michalina, Williams John C, Forman Stephen J, Brown Christine E, Wang Xiuli
Department of Hematology and Hematopoietic Cell Transplantation, Cellular Immunotherapy Center, City of Hope, Duarte, CA.
Department of Molecular Medicine, City of Hope National Medical Center, Duarte, CA.
Blood Neoplasia. 2024 Oct 13;1(4):100048. doi: 10.1016/j.bneo.2024.100048. eCollection 2024 Dec.
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.
在本研究中，我们旨在开发一种人源化的CD19嵌合抗原受体（CAR），其效力与FMC63 CAR相当，并有可能降低免疫原性风险。对鼠源FMC63单链可变片段（scFv）进行人源化，得到2个候选先导scFv，即VH4vκ1和4D5，它们的结合亲和力比FMC63 scFv弱。将这些人源化CD19-scFv整合到CAR构建体中，以生成huCD19R(VH4Vκ1)和huCD19R(4D5) CAR，二者均包含4-1BB共刺激结构域。评估CAR T细胞针对CD19和低表达CD19的肿瘤的抗肿瘤活性。在所有研究中，使用具有相同骨架的FMC63 CAR T细胞作为对照。结果显示，在用CD19靶点刺激后，huCD19R(VH4vκ1) CAR T细胞在扩增、表型和效应功能方面与FMC63 CAR相似。当CAR T细胞受到携带CD19的肿瘤攻击时，huCD19R(VH4vκ1) CAR T细胞的增殖情况与FMC63 CAR T细胞相似，而huCD19R(4D5) CAR T细胞基本无法增殖。此外，在针对表达一系列CD19抗原的肿瘤进行测试时，huCD19R(VH4vκ1) CAR T细胞在体内的抗肿瘤活性明显优于huCD19R(4D5) CAR T细胞。最后，使用混合模型，我们发现huCD19R(VH4vκ1) T细胞的细胞因子分泌谱与FMC63 CAR T细胞相当。此外，huCD19R(VH4vκ1) CAR T细胞对CD19和工程化低表达CD19的肿瘤均有效。这些发现表明，huCD19R(VH4vκ1) CAR T细胞可能具有更强的持久性，作为一种针对CD19肿瘤的治疗方法，是临床转化的一个有前景的候选方案。
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