Lima Ana Julia Ferreira, Hajdu Karina Lobo, Abdo Luiza, Batista-Silva Leonardo Ribeiro, de Oliveira Andrade Clara, Correia Eduardo Mannarino, Aragão Emmanuel Arthur Albuquerque, Bonamino Martín Hernán, Lourenzoni Marcos Roberto
Research Group on Protein Engineering and Health Solutions (GEPeSS), Oswaldo Cruz Foundation Ceará (Fiocruz-CE), São José, Precabura, 61773-270 Eusébio, Ceará, Brazil.
Federal University of Ceará (UFC), Pici campus (Building 873), 60440-970 Fortaleza, Ceará, Brazil.
Comput Struct Biotechnol J. 2024 May 19;23:2375-2387. doi: 10.1016/j.csbj.2024.05.032. eCollection 2024 Dec.
Anti-CD19 CAR-T cell therapy represents a breakthrough in the treatment of B-cell malignancies, and it is expected that this therapy modality will soon cover a range of solid tumors as well. Therefore, a universal cheap and sensitive method to detect CAR expression is of foremost importance. One possibility is the use of epitope tags such as c-Myc, HA or FLAG tags attached to the CAR extracellular domain, however, it is important to determine whether these tags can influence binding of the CAR with its target molecule. Here, we conducted structural modelling of an FMC63-based anti-CD19 single-chain variable fragment (scFv) with and without a c-Myc peptide tag added to the N-terminus portion and performed molecular dynamics simulation of the scFv with the CD19 target. We show that the c-Myc tag presence in the N-terminus portion does not affect the scFv's structural equilibrium and grants more stability to the scFv. However, intermolecular interaction potential (IIP) analysis reveals that the tag can approximate the complementarity-determining regions (CDRs) present in the scFv and cause steric impediment, potentially disturbing interaction with the CD19 protein. We then tested this possibility with CAR-T cells generated from human donors in a Nalm-6 leukemia model, showing that CAR-T cells with the c-Myc tag have overall worse antitumor activity, which was also observed when the tag was added to the C-terminus position. Ultimately, our results suggest that tag addition is an important aspect of CAR design and can influence CAR-T cell function, therefore its use should be carefully considered.
抗CD19嵌合抗原受体T细胞（CAR-T）疗法是B细胞恶性肿瘤治疗领域的一项突破，预计这种治疗方式很快也将覆盖一系列实体瘤。因此，一种通用、廉价且灵敏的检测CAR表达的方法至关重要。一种可能性是使用附着于CAR胞外结构域的表位标签，如c-Myc、HA或FLAG标签，然而，确定这些标签是否会影响CAR与其靶分子的结合非常重要。在此，我们对基于FMC63的抗CD19单链可变片段（scFv）进行了结构建模，该片段在N端部分添加或未添加c-Myc肽标签，并对scFv与CD19靶标进行了分子动力学模拟。我们发现，N端部分存在c-Myc标签不会影响scFv的结构平衡，反而赋予scFv更高的稳定性。然而，分子间相互作用势（IIP）分析表明，该标签会使scFv中存在的互补决定区（CDR）靠近，并造成空间位阻，可能干扰与CD19蛋白的相互作用。然后，我们在Nalm-6白血病模型中用人源供体产生的CAR-T细胞测试了这种可能性，结果表明带有c-Myc标签的CAR-T细胞总体抗肿瘤活性较差，在标签添加到C端位置时也观察到了这一现象。最终，我们的结果表明，添加标签是CAR设计的一个重要方面，会影响CAR-T细胞功能，因此应谨慎考虑其使用。
