在恶性肿瘤或HIV-1感染者中，抗原特异性细胞毒性T淋巴细胞（CTL）常常显示出筋疲力尽的表型，导致其消除病魔的能力受到损害。现有的基于细胞的免疫治疗策略往往受到CTL养育转移的限制。我们开发了一种免疫治疗技术，通过疫苗接种在体内产生强大的CTL应答，并利用双特异性疫苗诱导的效应细胞反应重定向器（RoVER）来消除靶细胞。在对51名健康志愿者（NCT04083430）进行黄热病（YF）17D疫苗接种后，利用四聚体染色和多参数流式细胞术量化了单表位YF特异性CTL反应。通过细胞杀伤试验评估了RoVER诱导的YF特异性CTLs重定向杀伤表达抗原的Raji-Env细胞、自体CD19+ B细胞或滋养于离体培养中全长度HIV-1-eGFP感染的CD4+ T细胞。此外，通过介质尺度多重检测分析了分泌的干扰素-γ、破酶B和肿瘤坏死因子-α。YF-17D疫苗接种引发了参与研究者强烈的表位特异性CTL反应。在细胞杀伤试验中，RoVER诱导的YF特异性CTLs对自体CD19+ B细胞或HIV-1感染的CD4+细胞的重定向杀伤率在效应细胞与靶细胞比例为1:1时分别为58%和53%。我们开发了一种免疫治疗技术，能够通过RoVER连接将由疫苗引发的表位特异性CTLs重定向以消灭表达抗原的靶细胞。RoVER技术具有极高的特异性，可以用于识别不同的细胞表面抗原。重要的是，这项技术摒弃了对CTL的养育转移的需求。本研究由诺和诺德基金会（Hallas Møller NNF10OC0054577）资助。版权所有©2023该作者。由Elsevier B.V.出版。保留所有权利。

In individuals with malignancy or HIV-1 infection, antigen-specific cytotoxic T lymphocytes (CTLs) often display an exhausted phenotype with impaired capacity to eliminate the disease. Existing cell-based immunotherapy strategies are often limited by the requirement for adoptive transfer of CTLs. We have developed an immunotherapy technology in which potent CTL responses are generated in vivo by vaccination and redirected to eliminate target cells using a bispecific Redirector of Vaccine-induced Effector Responses (RoVER).Following Yellow fever (YF) 17D vaccination of 51 healthy volunteers (NCT04083430), single-epitope YF-specific CTL responses were quantified by tetramer staining and multi-parameter flow cytometry. RoVER-mediated redirection of YF-specific CTLs to kill antigen-expressing Raji-Env cells, autologous CD19+ B cells or CD4+ T cells infected in vitro with a full-length HIV-1-eGFP was assessed in cell killing assays. Moreover, secreted IFN-γ, granzyme B, and TNF-α were analyzed by mesoscale multiplex assays.YF-17D vaccination induced strong epitope-specific CTL responses in the study participants. In cell killing assays, RoVER-mediated redirection of YF-specific CTLs to autologous CD19+ B cells or HIV-1-infected CD4+ cells resulted in 58% and 53% killing at effector to target ratio 1:1, respectively.We have developed an immunotherapy technology in which epitope-specific CTLs induced by vaccination can be redirected to kill antigen-expressing target cells by RoVER linking. The RoVER technology is highly specific and can be adapted to recognize various cell surface antigens. Importantly, this technology obviates the need for adoptive transfer of CTLs.This work was funded by the Novo Nordisk Foundation (Hallas Møller NNF10OC0054577).Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.