State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100021, P. R. China.
Department of Urology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100021, P. R. China.
Cancer Commun (Lond). 2022 Aug;42(8):768-783. doi: 10.1002/cac2.12321. Epub 2022 Jun 15.
The mortality of castration-resistant prostate cancer (CRPC) is high due to lack of an effective treatment. Chimeric antigen receptor (CAR)-based therapy is a promising immunotherapeutic strategy. Here, we aimed to design a novel CAR-natural killer (NK) cells with a clinically significant tumoricidal effect on CRPC.
We constructed novel CAR-NK92MI cells with a CD244-based recombinant lentiviral vector. Different intracellular segments (CD244, NKG2D, or CD3ζ) were screened to identify the best candidate according to cell lysis assay and CD107a expression levels. To enhance the affinity of the CAR to the tumor antigen, we compared an antibody specific for prostate-specific membrane antigen (anti-PSMA) with PSMA-targeted polypeptide (p-PSMA), which was screened by phage display combinatorial library. Then, CAR-NK92MI cells with both a high affinity for PSMA and a strong tumoricidal capacity were generated. In addition, we verified their tumor-killing effect in vitro and in vivo. The release of cytokine by NK92MI cells was compared with that by CAR-NK92MI cells through flow cytometry and enzyme-linked immunosorbent assay. Moreover, ferroptosis-related cell death was explored as a possible underlying mechanism.
Three different CAR intracellular regions CAR1 (CD244), CAR2 (CD244, NKG2D) and CAR3 (CD244, NKG2D, and CD3ζ) were constructed. CAR2 was chosen to confer a stronger tumoricidal ability on CAR-NK92MI cells. Compared with anti-PSMA, p-PSMA exhibited enhanced affinity for the tumor antigen. Thus, p-PSMA-CAR-NK92MI cells, which expressed CAR with a polypeptide-based antigen-binding region, an intracellular CD244 and a NKG2D costimulatory domain, were generated. They could selectively and successfully kill PSMA target cells and exhibited specific lysis rate of 73.19% for PSMA-positive C4-2 cells and 33.04% for PSMA-negative PC3 cells. Additionally, p-PSMA-CAR-NK92MI cells had significantly higher concentrations of IFN-γ, TNF-α and granzyme B than NK92MI cells. In a CRPC cancer xenograft model, p-PSMA-CAR-NK92MI cells significantly inhibited tumor growth and exerted a more consistent killing effect than NK92MI cells. Moreover, ferroptosis is a potential mechanism through which CAR-NK92MI cells attack cancer cells, and is triggered by IFN-γ.
p-PSMA-CAR-NK92MI cells can effectively kill CRPC cells in vitro and in vivo. This strategy may provide additional treatment options for patients with CRPC.
由于缺乏有效的治疗方法，去势抵抗性前列腺癌（CRPC）的死亡率很高。嵌合抗原受体（CAR）为基础的治疗是一种很有前途的免疫治疗策略。在这里，我们旨在设计一种新型的 CAR-自然杀伤（NK）细胞，对 CRPC 具有临床显著的杀瘤作用。
我们构建了基于 CD244 的新型 CAR-NK92MI 细胞的重组慢病毒载体。根据细胞裂解试验和 CD107a 表达水平，筛选不同的细胞内片段（CD244、NKG2D 或 CD3ζ），以确定最佳候选物。为了增强 CAR 对肿瘤抗原的亲和力，我们比较了针对前列腺特异性膜抗原（抗 PSMA）的抗体与通过噬菌体展示组合文库筛选出的 PSMA 靶向多肽（p-PSMA）。然后，生成了具有高亲和力 PSMA 和强大杀瘤能力的 CAR-NK92MI 细胞。此外，我们还在体外和体内验证了它们的杀伤肿瘤效应。通过流式细胞术和酶联免疫吸附试验比较了 NK92MI 细胞和 CAR-NK92MI 细胞的细胞因子释放。此外，还探索了铁死亡相关的细胞死亡作为一种可能的潜在机制。
构建了三种不同的 CAR 细胞内区 CAR1（CD244）、CAR2（CD244、NKG2D）和 CAR3（CD244、NKG2D 和 CD3ζ）。选择 CAR2 赋予 CAR-NK92MI 细胞更强的杀瘤能力。与抗 PSMA 相比，p-PSMA 对肿瘤抗原具有增强的亲和力。因此，生成了表达基于多肽的抗原结合区、CD244 和 NKG2D 共刺激结构域的 CAR 的 p-PSMA-CAR-NK92MI 细胞。它们可以选择性地成功杀伤 PSMA 靶细胞，对 PSMA 阳性 C4-2 细胞的特异性裂解率为 73.19%，对 PSMA 阴性 PC3 细胞的特异性裂解率为 33.04%。此外，p-PSMA-CAR-NK92MI 细胞中 IFN-γ、TNF-α 和颗粒酶 B 的浓度明显高于 NK92MI 细胞。在 CRPC 癌症异种移植模型中，p-PSMA-CAR-NK92MI 细胞显著抑制肿瘤生长，并比 NK92MI 细胞具有更一致的杀伤作用。此外，铁死亡是 CAR-NK92MI 细胞攻击癌细胞的一种潜在机制，是由 IFN-γ触发的。
p-PSMA-CAR-NK92MI 细胞可有效杀伤体外和体内的 CRPC 细胞。该策略可为 CRPC 患者提供额外的治疗选择。
