Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital, 0379 Oslo, Norway.
Department of Clinical Cancer Research, Oslo University Hospital, 0424 Oslo, Norway.
Int J Mol Sci. 2024 Jan 2;25(1):586. doi: 10.3390/ijms25010586.
We have developed a chimeric antigen receptor (CAR) against the six-transmembrane epithelial antigen of prostate-1 (STEAP1), which is expressed in prostate cancer, Ewing sarcoma, and other malignancies. In the present study, we investigated the effect of substituting costimulatory domains and spacers in this STEAP1 CAR. We cloned four CAR constructs with either CD28 or 4-1BB costimulatory domains, combined with a CD8a-spacer (sp) or a mutated IgG-spacer. The CAR T-cells were evaluated in short- and long-term in vitro T-cell assays, measuring cytokine production, tumor cell killing, and CAR T-cell expansion and phenotype. A xenograft mouse model of prostate cancer was used for in vivo comparison. All four CAR constructs conferred CD4 and CD8 T cells with STEAP1-specific functionality. A CD8sp_41BBz construct and an IgGsp_CD28z construct were selected for a more extensive comparison. The IgGsp_CD28z CAR gave stronger cytokine responses and killing in overnight caspase assays. However, the 41BB-containing CAR mediated more killing (IncuCyte) over one week. Upon six repeated stimulations, the CD8sp_41BBz CAR T cells showed superior expansion and lower expression of exhaustion markers (PD1, LAG3, TIGIT, TIM3, and CD25). In vivo, both the CAR T variants had comparable anti-tumor activity, but persisting CAR T-cells in tumors were only detected for the 41BBz variant. In conclusion, the CD8sp_41BBz STEAP1 CAR T cells had superior expansion and survival in vitro and in vivo, compared to the IgGsp_CD28z counterpart, and a less exhausted phenotype upon repeated antigen exposure. Such persistence may be important for clinical efficacy.
我们开发了一种针对前列腺六跨膜上皮抗原 1（STEAP1）的嵌合抗原受体（CAR），该抗原在前列腺癌、尤文肉瘤和其他恶性肿瘤中表达。在本研究中，我们研究了替代该 STEAP1 CAR 中的共刺激结构域和间隔物的效果。我们克隆了四种 CAR 构建体，它们分别具有 CD28 或 4-1BB 共刺激结构域，并与 CD8a 间隔子（sp）或突变的 IgG 间隔子结合。在短期和长期体外 T 细胞测定中评估了 CAR T 细胞，测量细胞因子产生、肿瘤细胞杀伤以及 CAR T 细胞扩增和表型。使用前列腺癌异种移植小鼠模型进行体内比较。所有四种 CAR 构建体均赋予 CD4 和 CD8 T 细胞针对 STEAP1 的特异性功能。选择 CD8sp_41BBz 构建体和 IgGsp_CD28z 构建体进行更广泛的比较。IgGsp_CD28z CAR 在过夜半胱天冬酶测定中产生更强的细胞因子反应和杀伤。然而，含有 41BB 的 CAR 在一周以上的时间内介导更多的杀伤（IncuCyte）。经过六次重复刺激，CD8sp_41BBz CAR T 细胞表现出优越的扩增和较低的衰竭标志物（PD1、LAG3、TIGIT、TIM3 和 CD25）表达。在体内，两种 CAR T 变体都具有相当的抗肿瘤活性，但仅在 41BBz 变体中检测到肿瘤中持续存在的 CAR T 细胞。总之，与 IgGsp_CD28z 对应物相比，CD8sp_41BBz STEAP1 CAR T 细胞在体外和体内具有更好的扩增和存活能力，并且在反复抗原暴露时具有较少的衰竭表型。这种持久性对于临床疗效可能很重要。
