细胞免疫治疗（ACT）已经在癌症和病毒感染的治疗中显示出了有希望的结果。成功的ACT依赖于体外扩增大量具有强烈细胞毒性和体内持久性的目标T细胞，这是目前ACT策略面临的最大挑战。在本研究中，我们提出了一种新技术，用于体外扩增抗原特异性T细胞；人工抗原呈递脚手架（Ag-scaffold），由葡聚糖多糖骨架、装饰有肽-MHC复合物（pMHC）、细胞因子和共刺激分子的组合构成，实现对抗原特异性T细胞的协调刺激。我们在健康供体和转移性黑色素瘤患者的外周血单个核细胞的体外培养中探究了Ag-scaffold扩增抗原特异性T细胞的能力。结果获得的T细胞产品进行了表型和功能特性评估。我们确定了一种最佳的Ag-scaffold用于ACT的T细胞扩增，携带pMHC、白细胞介素-2（IL-2）和IL-21，从健康供体和患者的外周血中高效扩增了病毒特异性和肿瘤特异性的CD8+ T细胞。所得的T细胞产品具有高频率的抗原特异性细胞，具有高自我更新能力、低疲劳、抗原挑战时具有多功能细胞因子产生和优越的肿瘤杀伤能力的特点。这表明，T细胞特征的获得需要通过优化的TCR结合物（pMHC）和刺激物（细胞因子）的配比提供协调刺激。为了生成对转移性黑色素瘤患者有相关性的“即用型”多靶向Ag-scaffold产品，我们在87名患者中确定了30个最常识别的HLA-A0201限制性黑色素瘤表位。通过将它们组合成Ag-scaffold产品，我们能够从60-70%的黑色素瘤患者中扩增肿瘤特异性T细胞，产生具有目标多靶向性和表型功能改善的T细胞产品。总的来说，Ag-scaffold代表了一种有希望的新技术，用于直接从血液中选择性扩增抗原特异性CD8+ T细胞，产生高度特异性和功能增强的T细胞产品用于ACT。© 2023 作者（或其雇主）。在CC BY-NC许可下被允许重复使用。不允许商业再利用。由BMJ出版。

Adoptive cell therapy (ACT) has shown promising results for the treatment of cancer and viral infections. Successful ACT relies on ex vivo expansion of large numbers of desired T-cells with strong cytotoxic capacity and in vivo persistence, which constitutes the greatest challenge to current ACT strategies. Here, in this study, we present a novel technology for ex vivo expansion of antigen-specific T-cells; artificial antigen-presenting scaffolds (Ag-scaffolds) consisting of a dextran-polysaccharide backbone, decorated with combinations of peptide-Major Histocompatibility Complex (pMHC), cytokines and co-stimulatory molecules, enabling coordinated stimulation of antigen-specific T-cells.The capacity of Ag-scaffolds to expand antigen-specific T-cells was explored in ex vivo cultures with peripheral blood mononuclear cells from healthy donors and patients with metastatic melanoma. The resulting T-cell products were assessed for phenotypic and functional characteristics.We identified an optimal Ag-scaffold for expansion of T-cells for ACT, carrying pMHC and interleukin-2 (IL-2) and IL-21, with which we efficiently expanded both virus-specific and tumor-specific CD8+ T cells from peripheral blood of healthy donors and patients, respectively. The resulting T-cell products were characterized by a high frequency of antigen-specific cells with high self-renewal capacity, low exhaustion, a multifunctional cytokine profile upon antigen-challenge and superior tumor killing capacity. This demonstrates that the coordinated stimuli provided by an optimized stoichiometry of TCR engaging (pMHC) and stimulatory (cytokine) moieties is essential to obtain desired T-cell characteristics. To generate an 'off-the-shelf' multitargeting Ag-scaffold product of relevance to patients with metastatic melanoma, we identified the 30 most frequently recognized shared HLA-A0201-restricted melanoma epitopes in a cohort of 87 patients. By combining these in an Ag-scaffold product, we were able to expand tumor-specific T-cells from 60-70% of patients with melanoma, yielding a multitargeted T-cell product with up to 25% specific and phenotypically and functionally improved T cells.Taken together, the Ag-scaffold represents a promising new technology for selective expansion of antigen-specific CD8+ T cells directly from blood, yielding a highly specific and functionally enhanced T-cell product for ACT.© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.