Tumor Vaccine and Biotechnology Branch, Division of Cell Therapy II, Silver Spring, Maryland, USA.
Cellular and Tissue Therapy Branch, Office of Cellular Therapy & Human Tissues, Office of Therapeutic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, White Oak, Silver Spring, Maryland, USA.
Clin Transl Med. 2024 May;14(5):e1664. doi: 10.1002/ctm2.1664.
Previously, we discovered that human solid tumours, but not normal human tissues, preferentially overexpress interleukin-13Receptor alpha2, a high binding receptor for IL-13. To develop novel anti-cancer approaches, we constructed a chimeric antigen receptor construct using a high binding and codon optimised scFv-IL-13Rα2 fragment fused with CD3ζ and co-stimulatory cytoplasmic domains of CD28 and 4-1BB.
We developed a scFv clone, designated 14-1, by biopanning the bound scFv phages using huIL-13Rα2Fc chimeric protein and compared its binding with our previously published clone 4-1. We performed bioinformatic analyses for complementary determining regions (CDR) framework and residue analyses of the light and heavy chains. This construct was packaged with helper plasmids to produce CAR-lentivirus and transduced human Jurkat T or activated T cells from peripheral blood mononuclear cells (PBMCs) to produce CAR-T cells and tested for their quality attributes in vitro and in vivo. Serum enzymes including body weight from non-tumour bearing mice were tested for assessing general toxicity of CAR-T cells.
The binding of 14-1 clone is to IL-13Rα2Fc-chimeric protein is ∼5 times higher than our previous clone 4-1. The 14-1-CAR-T cells grew exponentially in the presence of cytokines and maintained phenotype and biological attributes such as cell viability, potency, migration and T cell activation. Clone 14-1 migrated to IL-13Rα2Fc and cell free supernatants only from IL-13Rα2+ve confluent glioma tumour cells in a chemotaxis assay. scFv-IL-13Rα2-CAR-T cells specifically killed IL-13Rα2+ve but not IL-13Rα2-ve tumour cells in vitro and selectively caused significant release of IFN-γ only from IL-13Rα2+ve co-cultures. These CAR-T cells regressed IL-13Rα2+ve glioma xenografts in vivo without any general toxicity. In contrast, the IL-13Rα2 gene knocked-down U251 and U87 xenografts failed to respond to the CAR-T therapy.
Taken together, we conclude that the novel scFv-IL-13Rα2 CAR-T cell therapy may offer an effective therapeutic option after designing a careful pre-clinical and clinical study.
此前，我们发现人类实体瘤而非正常人体组织优先过度表达白细胞介素-13 受体 alpha2（IL-13Rα2），这是一种与 IL-13 高亲和力结合的受体。为了开发新的抗癌方法，我们构建了一种嵌合抗原受体构建体，该构建体使用高亲和力和密码子优化的 scFv-IL-13Rα2 片段与 CD3ζ 和 CD28 和 4-1BB 的共刺激细胞质结构域融合。
我们通过使用 huIL-13Rα2Fc 嵌合蛋白对结合的 scFv 噬菌体进行生物淘选，开发了一种 scFv 克隆，命名为 14-1，并比较了其与我们之前发表的克隆 4-1 的结合情况。我们对轻链和重链的互补决定区（CDR）框架和残基进行了生物信息学分析。该构建体与辅助质粒一起包装，产生 CAR-慢病毒，并转导来自外周血单核细胞（PBMC）的人 Jurkat T 或激活 T 细胞，以产生 CAR-T 细胞，并在体外和体内测试其质量属性。来自无肿瘤小鼠的血清酶，包括体重，用于评估 CAR-T 细胞的一般毒性。
14-1 克隆与 IL-13Rα2Fc-嵌合蛋白的结合比我们之前的克隆 4-1 高约 5 倍。14-1-CAR-T 细胞在细胞因子存在下呈指数生长，并保持表型和生物学特性，如细胞活力、效力、迁移和 T 细胞激活。在趋化性测定中，克隆 14-1 仅迁移到 IL-13Rα2Fc 和无细胞上清液来自 IL-13Rα2+ve 汇合的神经胶质瘤肿瘤细胞。scFv-IL-13Rα2-CAR-T 细胞在体外特异性杀伤 IL-13Rα2+ve 但不杀伤 IL-13Rα2-ve 肿瘤细胞，并仅从 IL-13Rα2+ve 共培养物中选择性地引起 IFN-γ 的显著释放。这些 CAR-T 细胞在体内消退了 IL-13Rα2+ve 神经胶质瘤异种移植物，而没有任何一般毒性。相比之下，IL-13Rα2 基因敲低的 U251 和 U87 异种移植物对 CAR-T 治疗无反应。
综上所述，我们得出结论，新型 scFv-IL-13Rα2 CAR-T 细胞疗法在进行仔细的临床前和临床研究后，可能提供一种有效的治疗选择。
