A Quantitative Approach to Potency Testing for Chimeric Antigen Receptor-Encoding Lentiviral Vectors and Autologous CAR-T Cell Products, Using Flow Cytometry.

IF 4.9 3区 医学 Q1 PHARMACOLOGY & PHARMACY
Juan José Mata-Molanes, Leticia Alserawan, Carolina España, Carla Guijarro, Ana López-Pecino, Hugo Calderón, Ane Altuna, Lorena Pérez-Amill, Nela Klein-González, Carlos Fernández de Larrea, Europa Azucena González-Navarro, Julio Delgado, Manel Juan, Maria Castella
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引用次数: 0

Abstract

Background/Objectives: 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. Methods: 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. Results: 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. Conclusions: 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.

用流式细胞术定量检测嵌合抗原受体编码慢病毒载体和自体CAR-T细胞产物的效价。
背景/目的:临床级慢病毒载体(LVVs)的效价检测对于支持药物的商业批准至关重要。在药物的临床开发过程中,应仔细考虑开发合适的效价试验。我们的目标是基于对转基因功能活性的测量,为我们的CAR19-LVV开发一种负担得起的定量测试。方法:在car转导Jurkat细胞和表达cd19的靶细胞共培养环境中,研究CAR19-LVV的几个功能活性指标。所选的测定法进一步发展并进行验证。我们还研究了Assay对其他car -编码LVV和自体CAR-T细胞产物的适应性。结果:检测jurkat -CAR-表达细胞膜上CD69的表达是CAR功能的特异性指标。CD69的平均荧光强度(MFI)定量,加上测定内标准曲线校准,允许定量分析具有高精度,特异性,稳健性,线性和准确性。该分析也显示了CARBCMA-LVV产品的最佳性能。重要的是,我们发现在原代T细胞中,CD69的表达反映了CAR-T细胞的细胞毒性。在适应后,我们将基于cd69的效价测试应用于自体CAR-T细胞产品,同时测量CAR-T细胞的细胞毒性,证明了该分析在这种情况下的特异性。结论:我们开发了一种有效的体外细胞效价测试,使用定量流式细胞术方法,用于我们的CAR19-LVV。该分析是基于检测t细胞活化后的CAR结合抗原,这是转基因功能的一个措施。该试验很容易适用于另一种car编码的LVV,靶向不同的分子。此外,同样的检测原理也适用于自体CAR-T细胞产品。定量CD69效价分析显示,与ifn - γ分析相比,自体产物之间的可变性降低,并允许同时评估传统的半定量细胞毒性,从而在同一分析中直接评估药物的作用机制(MoA)。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Pharmaceutics
Pharmaceutics Pharmacology, Toxicology and Pharmaceutics-Pharmaceutical Science
CiteScore
7.90
自引率
11.10%
发文量
2379
审稿时长
16.41 days
期刊介绍: Pharmaceutics (ISSN 1999-4923) is an open access journal which provides an advanced forum for the science and technology of pharmaceutics and biopharmaceutics. It publishes reviews, regular research papers, communications,  and short notes. Covered topics include pharmacokinetics, toxicokinetics, pharmacodynamics, pharmacogenetics and pharmacogenomics, and pharmaceutical formulation. Our aim is to encourage scientists to publish their experimental and theoretical details in as much detail as possible. There is no restriction on the length of the papers. The full experimental details must be provided so that the results can be reproduced.
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