Tumor Vaccines and Biotechnology Branch, Division of Cellular and Gene Therapies, Office of Tissues and Advance Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, MD, 20993, USA.
Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
J Transl Med. 2023 Jun 7;21(1):367. doi: 10.1186/s12967-023-04142-2.
Chimeric antigen receptor (CAR) T cell therapy is an exciting cell-based cancer immunotherapy. Unfortunately, CAR-T cell therapy is associated with serious toxicities such as cytokine release syndrome (CRS) and neurotoxicity. The mechanism of these serious adverse events (SAEs) and how homing, distribution and retention of CAR-T cells contribute to toxicities is not fully understood. Enabling in vitro methods to allow meaningful, sensitive in vivo biodistribution studies is needed to better understand CAR-T cell disposition and its relationship to both effectiveness and safety of these products.
To determine if radiolabelling of CAR-T cells could support positron emission tomography (PET)-based biodistribution studies, we labeled IL-13Rα2 targeting scFv-IL-13Rα2-CAR-T cells (CAR-T cells) with Zirconium-oxine (Zr-oxine) and characterized and compared their product attributes with non-labeled CAR-T cells. The Zr-oxine labeling conditions were optimized for incubation time, temperature, and use of serum for labeling. In addition, T cell subtype characterization and product attributes of radiolabeled CAR-T cells were studied to assess their overall quality including cell viability, proliferation, phenotype markers of T-cell activation and exhaustion, cytolytic activity and release of interferon-γ upon co-culture with IL-13Rα2 expressing glioma cells.
We observed that radiolabeling of CAR-T cells with Zr-oxine is quick, efficient, and radioactivity is retained in the cells for at least 8 days with minimal loss. Also, viability of radiolabeled CAR-T cells and subtypes such as CD4 + , CD8 + and scFV-IL-13Rα2 transgene positive T cell population were characterized and found similar to that of unlabeled cells as determined by TUNEL assay, caspase 3/7 enzyme and granzyme B activity assay. Moreover, there were no significant changes in T cell activation (CD24, CD44, CD69 and IFN-γ) or T cell exhaustion (PD-1, LAG-3 and TIM3) markers expression between radiolabeled and unlabeled CAR-T cells. In chemotaxis assays, migratory capability of radiolabeled CAR-T cells to IL-13Rα2Fc was similar to that of non-labeled cells.
Importantly, radiolabeling has minimal impact on biological product attributes including potency of CAR-T cells towards IL-13Rα2 positive tumor cells but not IL-13Rα2 negative cells as measured by cytolytic activity and release of IFN-γ. Thus, IL-13Rα2 targeting CAR-T cells radiolabeled with Zr-oxine retain critical product attributes and suggest Zr-oxine radiolabeling of CAR-T cells may facilitate biodistribution and tissue trafficking studies in vivo using PET.
嵌合抗原受体 (CAR) T 细胞疗法是一种令人兴奋的基于细胞的癌症免疫疗法。不幸的是，CAR-T 细胞疗法与细胞因子释放综合征 (CRS) 和神经毒性等严重毒性有关。这些严重不良事件 (SAE) 的机制以及 CAR-T 细胞的归巢、分布和保留如何导致毒性尚不完全清楚。需要能够进行体外方法的研究，以支持有意义的、敏感的体内生物分布研究，从而更好地了解 CAR-T 细胞的处置及其与这些产品的有效性和安全性的关系。
为了确定 CAR-T 细胞的放射性标记是否可以支持正电子发射断层扫描 (PET) 为基础的生物分布研究，我们用锆-oxine (Zr-oxine) 标记白细胞介素 13 受体 α2 靶向 scFv-IL-13Rα2-CAR-T 细胞 (CAR-T 细胞)，并对其产品特性进行了表征和比较与非标记的 CAR-T 细胞。优化了 Zr-oxine 标记条件，包括孵育时间、温度和血清的使用。此外，还研究了放射性标记的 CAR-T 细胞的 T 细胞亚型特征和产品特性，以评估其整体质量，包括细胞活力、增殖、T 细胞激活和耗竭的表型标志物、细胞毒性和与表达白细胞介素 13 受体 α2 的神经胶质瘤细胞共培养时干扰素-γ的释放。
我们观察到，用 Zr-oxine 对 CAR-T 细胞进行放射性标记是快速、高效的，并且放射性在细胞中保留至少 8 天，损失最小。此外，通过 TUNEL 测定、半胱天冬酶 3/7 酶和颗粒酶 B 活性测定，对放射性标记的 CAR-T 细胞及其亚型（如 CD4+、CD8+和 scFV-IL-13Rα2 转基因阳性 T 细胞群）的活力进行了表征，发现与未标记的细胞相似。此外，放射性标记的 CAR-T 细胞与未标记的 CAR-T 细胞之间，T 细胞激活（CD24、CD44、CD69 和 IFN-γ）或 T 细胞耗竭（PD-1、LAG-3 和 TIM3）标志物的表达没有显著变化。在趋化性测定中，放射性标记的 CAR-T 细胞向白细胞介素 13 受体 α2Fc 的迁移能力与未标记的细胞相似。
重要的是，放射性标记对包括 CAR-T 细胞对 IL-13Rα2 阳性肿瘤细胞的效力在内的生物产品特性几乎没有影响，但对 IL-13Rα2 阴性细胞没有影响，如细胞毒性和 IFN-γ的释放。因此，用 Zr-oxine 放射性标记的白细胞介素 13 受体 α2 靶向 CAR-T 细胞保留了关键的产品特性，并表明 Zr-oxine 放射性标记的 CAR-T 细胞可能有助于使用 PET 进行体内生物分布和组织分布研究。
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