Mata-Molanes Juan José, Alserawan Leticia, España Carolina, Guijarro Carla, López-Pecino Ana, Calderón Hugo, Altuna Ane, Pérez-Amill Lorena, Klein-González Nela, Fernández de Larrea Carlos, González-Navarro Europa Azucena, Delgado Julio, Juan Manel, Castella Maria
Department of Immunology, CDB, Hospital Clinic de Barcelona (HCB), 08036 Barcelona, Spain.
Immunogenetics and Immunotherapy in Autoinflammatory and Immune Responses Group, Fundació Clínic-IDIBAPS, 08036 Barcelona, Spain.
Pharmaceutics. 2025 Feb 25;17(3):303. doi: 10.3390/pharmaceutics17030303.
: Potency testing of clinical-grade lentiviral vectors (LVVs) is critical to support a drug's commercial approval. Careful consideration should be paid to the development of a suitable potency test during the drug's clinical development. We aimed to develop an affordable, quantitative test for our CAR19-LVV, based on a measure of transgene's functional activity. : Several indicators of functional activity of CAR19-LVV were explored in a co-culture setting of CAR-transduced Jurkat cells and CD19-expressing target cells. The selected assay was further developed and subjected to validation. Assay's adaptability to other CAR-encoding LVV and autologous CAR-T cell products was also investigated. : Measure of CD69 expression on the membrane of Jurkat-CAR-expressing cells is a specific indicator of CAR functionality. Quantification of CD69 in terms of mean fluorescence intensity (MFI), coupled with an intra-assay standard curve calibration, allows for a quantitative assay with high precision, specificity, robustness, linearity and accuracy. The assay has also shown optimal performance for a CARBCMA-LVV product. Importantly, we show that in primary T cells, CD69 expression reflects CAR-T cell cytotoxicity. After adaptation, we have applied a CD69-based potency test, with simultaneous measurement of CAR-T cell cytotoxicity, to autologous CAR-T cell products, demonstrating the assay's specificity also in this context. : We developed a validated, in vitro cell-based potency test, using a quantitative flow-cytometry method, for our CAR19-LVV. The assay is based on the detection of T-cell activation upon CAR binding to antigen, which is a measure of transgene functionality. The assay was easily adapted to another CAR-encoding LVV, targeting a different molecule. Furthermore, the same assay principle can be applied in the context of autologous CAR-T cell products. The quantitative CD69 potency assay shows reduced variability among autologous products compared to the IFNγ assay and allows for simultaneous evaluation of traditional semi-quantitative cytotoxicity, thereby directly evaluating the drug's mechanism of action (MoA) in the same assay.
临床级慢病毒载体（LVV）的效价检测对于支持药物的商业批准至关重要。在药物临床开发过程中，应仔细考虑开发合适的效价检测方法。我们旨在基于转基因功能活性的测量，为我们的CAR19-LVV开发一种经济实惠的定量检测方法。
在CAR转导的Jurkat细胞和表达CD19的靶细胞的共培养环境中探索了CAR19-LVV功能活性的几个指标。对选定的检测方法进行了进一步开发和验证。还研究了该检测方法对其他编码CAR的LVV和自体CAR-T细胞产品的适应性。
表达Jurkat-CAR的细胞表面CD69表达的测量是CAR功能的特异性指标。根据平均荧光强度（MFI）对CD69进行定量，并结合检测内标准曲线校准，可实现高精度、特异性、稳健性、线性和准确性的定量检测。该检测方法对CARBCMA-LVV产品也表现出最佳性能。重要的是，我们表明在原代T细胞中，CD69表达反映了CAR-T细胞的细胞毒性。经过调整后，我们将基于CD69的效价检测方法与CAR-T细胞细胞毒性的同时测量应用于自体CAR-T细胞产品，证明了该检测方法在这种情况下的特异性。
我们为我们的CAR19-LVV开发了一种经过验证的基于体外细胞的效价检测方法，采用定量流式细胞术方法。该检测方法基于检测CAR与抗原结合后T细胞的激活，这是转基因功能的一种测量方法。该检测方法很容易适用于另一种编码CAR的LVV，靶向不同的分子。此外，相同的检测原理可应用于自体CAR-T细胞产品的背景下。与IFNγ检测相比，定量CD69效价检测显示自体产品之间的变异性降低，并允许同时评估传统的半定量细胞毒性，从而在同一检测中直接评估药物的作用机制（MoA）。
