Chimeric antigen receptor (CAR) development involves extensive empirical characterization of antigen-binding domain (ABD)/CAR constructs for clinical suitability. Here, we present a cost-efficient and rapid method for evaluating CARs in human Jurkat T cells. Using a modular CAR plasmid, a highly efficient ABD cloning strategy, plasmid electroporation, short-term co-culture, and flow-cytometric detection of CD69, this assay (referred to as CAR-J) evaluates sensitivity and specificity for ABDs. Assessing 16 novel anti-CD22 single-chain variable fragments derived from mouse monoclonal antibodies, CAR-J stratified constructs by response magnitude to CD22-expressing target cells. We also characterized 5 novel anti-EGFRvIII CARs for preclinical development, identifying candidates with varying tonic and target-specific activation characteristics. When evaluated in primary human T cells, tonic/auto-activating (without target cells) EGFRvIII-CARs induced target-independent proliferation, differentiation toward an effector phenotype, elevated activity against EGFRvIII-negative cells, and progressive loss of target-specific response upon in vitro re-challenge. These EGFRvIII CAR-T cells also showed anti-tumor activity in xenografted mice. In summary, CAR-J represents a straightforward method for high-throughput assessment of CAR constructs as genuine cell-associated antigen receptors that is particularly useful for generating large specificity datasets as well as potential downstream CAR optimization.

中文翻译：

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一种高通量方法，用于表征Jurkat细胞中的新型嵌合抗原受体。

嵌合抗原受体（CAR）的开发涉及针对临床适用性的抗原结合域（ABD）/ CAR构建体的广泛经验表征。在这里，我们提出了一种经济高效的快速方法来评估人类Jurkat T细胞中的CAR。使用模块化CAR质粒，高效的ABD克隆策略，质粒电穿孔，短期共培养和CD69的流式细胞仪检测，该测定（称为CAR-J）评估了ABD的敏感性和特异性。评估源自小鼠单克隆抗体的16种新型抗CD22单链可变片段，CAR-J通过对表达CD22的靶细胞的反应幅度对构建体进行分层。我们还针对临床前开发对5种新型抗EGFRvIII CAR进行了表征，确定了具有不同补品和靶标特异性激活特征的候选药物。当在原代人T细胞中评估时，补药/自动激活（无靶细胞）EGFRvIII-CARs诱导靶标非依赖性增殖，向效应子表型的分化，针对EGFRvIII阴性细胞的活性升高以及靶标特异性反应逐渐消失体外再挑战。这些EGFRvIII CAR-T细胞在异种移植小鼠中也显示出抗肿瘤活性。总之，CAR-J代表了一种直接方法，可对作为真正的细胞相关抗原受体的CAR构建体进行高通量评估，这对于生成大特异性数据集以及潜在的下游CAR优化特别有用。增强的针对EGFRvIII阴性细胞的活性，并在体外再次攻击后逐渐丧失靶标特异性反应。这些EGFRvIII CAR-T细胞在异种移植小鼠中也显示出抗肿瘤活性。总之，CAR-J代表了一种直接方法，可对作为真正的细胞相关抗原受体的CAR构建体进行高通量评估，这对于生成大特异性数据集以及潜在的下游CAR优化特别有用。增强的针对EGFRvIII阴性细胞的活性，并在体外再次攻击后逐渐丧失靶标特异性反应。这些EGFRvIII CAR-T细胞在异种移植小鼠中也显示出抗肿瘤活性。总之，CAR-J代表了一种直接方法，可对作为真正的细胞相关抗原受体的CAR构建体进行高通量评估，这对于生成大特异性数据集以及潜在的下游CAR优化特别有用。