Kitidee Kuntida, Amonyingcharoen Sumet, Preedagasamzin Sarinthip, Atjanasuppat Korakot, Sawaisorn Piamsiri, Srimorkun Pornprapa, Petvises Sawang, Chaicumpa Wanpen, Borwornpinyo Suparerk, Anurathapan Usanarat, Hongeng Suradej
Center for Research Innovation and Biomedical Informatics, Faculty of Medical Technology, Mahidol University, Salaya, Nakhon Pathom, Thailand.
Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
PLoS One. 2025 Jun 9;20(6):e0325389. doi: 10.1371/journal.pone.0325389. eCollection 2025.
Cancer immunotherapy, particularly T cell-based therapies, is considered to have strong potential for treating various types of cancer. A promising approach that has emerged is the use of γδ T cell-based strategies for cancer treatment. Neuroblastoma (NB), a solid tumor frequently found in childhood, is one of the more intriguing targets for immunotherapy. In this study, we report an alternative immunotherapy method for treating neuroblastoma by combining bispecific antibody with human Vγ9Vδ2 T cells. Initially, we screened for human scFv against CD3 epsilon using phage panning technology. Human scFv CD3 clone 18 demonstrated the highest ability to bind CD3 epsilon in an indirect ELISA assay. Consequently, we selected human scFv CD3 clone 18 to create a bispecific T cell engager antibody targeting both CD3 and disialoganglioside (GD2), called CD3/GD2 BiTE. This bispecific antibody was composed of human scFv CD3 clone 18 (VH-VL) and mouse scFv GD2 (VL-VH), linked by a flexible peptide linker. The interleukin-2 signal sequence and polyhistidine tag were added at the N- and C-termini for protein secretion and purification, respectively. CD3/GD2 BiTE was transiently produced in a mammalian cell expression system, which provided both high yield and quality. The CD3/GD2 BiTE folded naturally into a compact monomeric structure. Cell-based binding activity assays demonstrated that CD3/GD2 BiTE specifically binds to its target antigens on CD3-positive Jurkat cells and GD2-positive SH-SY5Y cells, but did not react with CD3-negative Raji cells and GD2-negative SK-N-SH cells. In subsequent in vitro experiments, the cytotoxicity of CD3/GD2 BiTE combined with human Vγ9Vδ2 T cells against neuroblastoma cells was evaluated. Human Vγ9Vδ2 T cells were primed with CD3/GD2 BiTE to improve the binding specificity and avidity against neuroblastoma cell lines before adding into SH-SY5Y cells. At concentrations of 180 and 360 nM, the CD3/GD2 BiTE significantly enhanced the killing ability of human Vγ9Vδ2 T cells against SH-SY5Y cells at an E:T ratio of 1:1. Moreover, CD3/GD BiTE armed with human Vγ9Vδ2 T cells enabled the killing of neuroblastoma cells using five- to ten-times fewer effector cells. The combination of CD3/GD2 BiTE and human Vγ9Vδ2 T cells also exhibited cytotoxic activity against a three-dimensional tumor spheroid model of SH-SY5Y GFP at an E:T ratio of 1:1. Consequently, CD3/GD2 BiTE enhances tumor-targeting and cytotoxic abilities of human Vγ9Vδ2 T cells against neuroblastoma cells in both two-dimensional and three-dimensional cell cultures. These results suggest that the combination of CD3/GD2 BiTE and human Vγ9Vδ2 T cells could represent an alternative immunotherapy strategy for treating neuroblastoma patients in the future.
癌症免疫疗法，尤其是基于T细胞的疗法，被认为在治疗各种类型癌症方面具有强大潜力。一种新兴的有前景的方法是使用基于γδT细胞的策略进行癌症治疗。神经母细胞瘤（NB）是儿童期常见的实体瘤，是免疫疗法中更具吸引力的靶点之一。在本研究中，我们报告了一种通过将双特异性抗体与人Vγ9Vδ2 T细胞相结合来治疗神经母细胞瘤的替代免疫疗法。最初，我们使用噬菌体淘选技术筛选针对CD3ε的人单链抗体片段（scFv）。人scFv CD3克隆18在间接酶联免疫吸附测定（ELISA）中表现出与CD3ε结合的最高能力。因此，我们选择人scFv CD3克隆18来创建一种靶向CD3和二唾液酸神经节苷脂（GD2）的双特异性T细胞衔接抗体，称为CD3/GD2双特异性T细胞衔接器（BiTE）。这种双特异性抗体由人scFv CD3克隆18（VH - VL）和小鼠scFv GD2（VL - VH）组成，通过柔性肽接头连接。在N端和C端分别添加白细胞介素 - 2信号序列和多组氨酸标签用于蛋白质分泌和纯化。CD3/GD2 BiTE在哺乳动物细胞表达系统中瞬时表达，该系统可实现高产量和高质量。CD3/GD2 BiTE自然折叠成紧密的单体结构。基于细胞的结合活性测定表明，CD3/GD2 BiTE特异性结合CD3阳性的Jurkat细胞和GD2阳性的SH - SY5Y细胞上的靶抗原，但不与CD3阴性的Raji细胞和GD2阴性的SK - N - SH细胞反应。在随后的体外实验中，评估了CD3/GD2 BiTE与人Vγ9Vδ2 T细胞联合对神经母细胞瘤细胞的细胞毒性。在将人Vγ9Vδ2 T细胞加入SH - SY5Y细胞之前，先用CD3/GD2 BiTE对其进行预处理，以提高其对神经母细胞瘤细胞系的结合特异性和亲和力。在1:1的效应细胞与靶细胞（E:T）比例下，当浓度为180和360 nM时，CD3/GD2 BiTE显著增强了人Vγ9Vδ2 T细胞对SH - SY5Y细胞的杀伤能力。此外，配备人Vγ9Vδ2 T细胞的CD3/GD BiTE能够使用少五到十倍的效应细胞杀伤神经母细胞瘤细胞。CD3/GD2 BiTE与人Vγ9Vδ2 T细胞的组合在1:1的E:T比例下对SH - SY5Y绿色荧光蛋白（GFP）的三维肿瘤球体模型也表现出细胞毒性活性。因此，在二维和三维细胞培养中，CD3/GD2 BiTE增强了人Vγ9Vδ2 T细胞对神经母细胞瘤细胞的肿瘤靶向和细胞毒性能力。这些结果表明，CD3/GD2 BiTE与人Vγ9Vδ2 T细胞的组合可能代表未来治疗神经母细胞瘤患者的一种替代免疫疗法策略。
