Tissues, Cells and Advanced Therapeutics, Scottish National Blood Transfusion Service, Jack Copland Centre, Heriot Watt Research Park, Edinburgh, United Kingdom.
Chemokine Research Group, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, United Kingdom.
Front Immunol. 2024 Jul 1;15:1412211. doi: 10.3389/fimmu.2024.1412211. eCollection 2024.
Adoptive immunotherapy with Epstein-Barr virus (EBV)-specific T cells is an effective treatment for relapsed or refractory EBV-induced post-transplant lymphoproliferative disorders (PTLD) with overall survival rates of up to 69%. EBV-specific T cells have been conventionally made by repeated stimulation with EBV-transformed lymphoblastoid cell lines (LCL), which act as antigen-presenting cells. However, this process is expensive, takes many months, and has practical risks associated with live virus. We have developed a peptide-based, virus-free, serum-free closed system to manufacture a bank of virus-specific T cells (VST) for clinical use. We compared these with standard LCL-derived VST using comprehensive characterization and potency assays to determine differences that might influence clinical benefits. Multi-parameter flow cytometry revealed that peptide-derived VST had an expanded central memory population and less exhaustion marker expression than LCL-derived VST. A quantitative HLA-matched allogeneic cytotoxicity assay demonstrated similar specific killing of EBV-infected targets, though peptide-derived EBV T cells had a significantly higher expression of antiviral cytokines and degranulation markers after antigen recall. High-throughput T cell receptor-beta (TCRβ) sequencing demonstrated oligoclonal repertoires, with more matches to known EBV-binding complementary determining region 3 (CDR3) sequences in peptide-derived EBV T cells. Peptide-derived products showed broader and enhanced specificities to EBV nuclear antigens (EBNAs) in both CD8 and CD4 compartments, which may improve the targeting of highly expressed latency antigens in PTLD. Importantly, peptide-based isolation and expansion allows rapid manufacture and significantly increased product yield over conventional LCL-based approaches.
采用 EBV 特异性 T 细胞过继免疫疗法治疗复发或难治性 EBV 诱导的移植后淋巴增殖性疾病（PTLD）是一种有效的治疗方法，总生存率高达 69%。EBV 特异性 T 细胞通常通过反复刺激 EBV 转化的淋巴母细胞系（LCL）制成，LCL 作为抗原呈递细胞。然而，该过程昂贵，需要数月时间，并且存在与活病毒相关的实际风险。我们已经开发了一种基于肽的、无病毒的、无血清的封闭系统，用于制造用于临床使用的病毒特异性 T 细胞（VST）库。我们使用全面的表征和效力测定来比较这些与标准 LCL 衍生的 VST，以确定可能影响临床益处的差异。多参数流式细胞术显示，与 LCL 衍生的 VST 相比，肽衍生的 VST 具有扩展的中央记忆群体，并且表达的衰竭标志物较少。定量 HLA 匹配的同种异体细胞毒性测定表明，对 EBV 感染靶标的特异性杀伤相似，尽管肽衍生的 EBV T 细胞在抗原召回后表现出更高水平的抗病毒细胞因子和脱颗粒标志物表达。高通量 T 细胞受体-β（TCRβ）测序显示寡克隆库，肽衍生的 EBV T 细胞中与已知 EBV 结合互补决定区 3（CDR3）序列的匹配更多。肽衍生产品在 CD8 和 CD4 区均显示出对 EBV 核抗原（EBNAs）更广泛和增强的特异性，这可能改善了对 PTLD 中高表达潜伏期抗原的靶向。重要的是，基于肽的分离和扩增允许快速制造，并显著提高了与传统 LCL 方法相比的产品产量。
