肿瘤坏死因子受体超家族 25 (TNFRSF25) 是一种 T 细胞共刺激受体。其配体 TNF 样细胞因子 1A (TL1A) 在小鼠肿瘤细胞上的表达已被证明可以促进肿瘤消退。本研究旨在开发 TNFRSF25 激动剂（抗体 (Abs) 和 TL1A 蛋白）并研究其潜在的抗肿瘤作用。抗小鼠 TNFRSF25 (mTNFRSF25) Abs 和多聚体 TL1A 蛋白作为 TNFRSF25 激动剂产生。在荧光素酶报告基因和 T 细胞共刺激测定中评估了它们的激动作用，并在同基因小鼠肿瘤模型中评估了它们的抗肿瘤作用。通过流式细胞术评估肿瘤微环境中的 TNFRSF25 表达以及抗 mTNFRSF25 激动性抗体对肿瘤浸润 T 细胞的影响。细胞耗竭测定用于鉴定有助于抗 mTNFRSF25 Ab 抗肿瘤作用的免疫细胞类型。在体内 T 细胞扩增模型和使用 Fc 变体和 FcγR 缺陷小鼠的小鼠肿瘤模型中评估了 TNFRSF25 激动剂的 Fc γ 受体 (FcγR) 依赖性。TNFRSF25 激动剂在同基因小鼠肿瘤模型中表现出抗肿瘤作用，且不会引起观察到的副作用。影响。我们鉴定了一种抗 mTNFRSF25 激动性抗体 1A6-m1，它比亲和力更高的抗 TNFRSF25 Ab 表现出更强的抗肿瘤活性，后者与 1A6-m1 具有重叠表位。 1A6-m1激活CD8 T细胞和抗原特异性T细胞，导致肿瘤消退；它还诱导长期抗肿瘤免疫记忆。尽管 1A6-m1 激活 TNFRSF25 会扩增脾调节 T (Treg) 细胞，但它不会影响瘤内 Treg 细胞。此外，1A6-m1 的抗肿瘤作用需要抑制性 FcγRIIB 和激活性 FcγRIII 的共同作用。用人 IgG2 (h2) 替换 1A6-m1 的 CH1 铰链区可增强抗肿瘤作用。最后，我们还生成了多聚体人和小鼠 TL1A 融合蛋白作为 TNFRSF25 激动剂，即使在没有 Fc-FcγR 相互作用的情况下，它们也会共同刺激 CD8 T 细胞并减少肿瘤生长。我们的数据证明了 Abs 和 FcγR 激活 TNFRSF25 的潜力。用于癌症免疫治疗的多聚体 TL1A 蛋白，并提供对其开发治疗的见解。© 作者（或其雇主）2024。根据 CC BY-NC 允许重复使用。不得商业再利用。请参阅权利和权限。英国医学杂志出版。

Tumor necrosis factor receptor superfamily 25 (TNFRSF25) is a T-cell co-stimulatory receptor. Expression of its ligand, TNF-like cytokine 1A (TL1A), on mouse tumor cells has been shown to promote tumor regression. This study aimed to develop TNFRSF25 agonists (both antibodies (Abs) and TL1A proteins) and to investigate their potential antitumor effects.Anti-mouse TNFRSF25 (mTNFRSF25) Abs and multimeric TL1A proteins were generated as TNFRSF25 agonists. Their agonism was assessed in luciferase reporter and T-cell co-stimulation assays, and their antitumor effects were evaluated in syngeneic mouse tumor models. TNFRSF25 expression within the tumor microenvironment and the effects of an anti-mTNFRSF25 agonistic Ab on tumor-infiltrating T cells were evaluated by flow cytometry. Cell depletion assays were used to identify the immune cell types that contribute to the antitumor effect of the anti-mTNFRSF25 Ab. The Fc gamma receptor (FcγR) dependence of TNFRSF25 agonists was assessed in an in vivo T-cell expansion model and a mouse tumor model using Fc variants and FcγR-deficient mice.TNFRSF25 agonists exhibited antitumor effects in syngeneic mouse tumor models without causing observed side effects. We identified an anti-mTNFRSF25 agonistic Ab, 1A6-m1, which exhibited greater antitumor activity than a higher affinity anti-TNFRSF25 Ab which engages an overlapping epitope with 1A6-m1. 1A6-m1 activated CD8+ T cells and antigen-specific T cells, leading to tumor regression; it also induced long-term antitumor immune memory. Although activating TNFRSF25 by 1A6-m1 expanded splenic regulatory T (Treg) cells, it did not influence intratumoral Treg cells. Moreover, 1A6-m1's antitumor effects required the engagement of both inhibitory FcγRIIB and activating FcγRIII. Replacing 1A6-m1's CH1-hinge region with that of human IgG2 (h2) conferred enhanced antitumor effects. Finally, we also generated multimeric human and mouse TL1A fusion proteins as TNFRSF25 agonists, and they co-stimulated CD8+ T cells and reduced tumor growth, even in the absence of Fc-FcγR interactions.Our data demonstrates the potential of activating TNFRSF25 by Abs and multimeric TL1A proteins for cancer immunotherapy and provides insights into their development astherapeutics.© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.