Pharmaceutical Biotechnology, University Tübingen, Tübingen, Germany.
Cluster of Excellence iFIT (EXC2180) "Image-Guided and Functionally Instructed Tumor Therapies", University of Tübingen, Tübingen, Germany.
Front Immunol. 2024 Oct 15;15:1480091. doi: 10.3389/fimmu.2024.1480091. eCollection 2024.
Human OX40 (hOX40/CD134), a member of the TNF receptor superfamily, is mainly expressed on activated T lymphocytes. Triggered by its ligand OX40L (CD252), it provides costimulatory signals that support the differentiation, proliferation and long-term survival of T cells. Besides being a relevant therapeutic target, hOX40 is also an important biomarker for monitoring the presence or infiltration of activated T cells within the tumor microenvironment (TME), the inflammatory microenvironment (IME) in immune-mediated diseases (IMIDs) and the lymphatic organs. Here, we developed novel single domain antibodies (nanobodies, Nbs) targeting hOX40 to monitor the activation status of T cells by molecular imaging.
Nbs against hOX40 (hOX40-Nbs) were selected from an immunized Nb-library by phage display. The identified hOX40-Nbs were characterized , including determination of their specificity, affinity, stability, epitope recognition and their impact on OX40 signaling and T cell function. A lead candidate was site-specifically conjugated with a fluorophore via sortagging and applied for noninvasive optical imaging (OI) of hOX40-expressing cells in a xenograft mouse model.
Our selection campaign revealed four unique Nbs that exhibit strong binding affinities and high stabilities under physiological conditions. Epitope binning and domain mapping indicated the targeting of at least two different epitopes on hOX40. When analyzing their impact on OX40 signaling, an agonistic effect was excluded for all validated Nbs. Incubation of activated T cells with hOX40-Nbs did not affect cell viability or proliferation patterns, whereas differences in cytokine release were observed. OI with a fluorophore-conjugated lead candidate in experimental mice with hOX40-expressing xenografts demonstrated its specificity and functionality as an imaging probe.
Considering the need for advanced probes for noninvasive monitoring of T cell activation dynamics, we propose, that our hOX40-Nbs have a great potential as imaging probes for noninvasive and longitudinal diagnostics. Quantification of OX40 T cells in TME or IME will provide crucial insights into the activation state of infiltrating T cells, offering a valuable biomarker for assessing immune responses, predicting treatment efficacy, and guiding personalized immunotherapy strategies in patients with cancer or IMIDs.
人 OX40（hOX40/CD134）是肿瘤坏死因子受体超家族的成员，主要表达于活化的 T 淋巴细胞。其配体 OX40L（CD252）与之结合后提供共刺激信号，支持 T 细胞的分化、增殖和长期存活。hOX40 不仅是一个相关的治疗靶点，也是监测肿瘤微环境（TME）、免疫介导疾病（IMIDs）中的炎症微环境（IME）以及淋巴器官中活化 T 细胞存在或浸润的重要生物标志物。在这里，我们开发了针对 hOX40 的新型单域抗体（纳米抗体，Nbs），通过分子成像来监测 T 细胞的激活状态。
通过噬菌体展示从免疫的 Nb 文库中筛选针对 hOX40 的 Nbs。对鉴定出的 hOX40-Nbs 进行了特性分析，包括特异性、亲和力、稳定性、表位识别以及对 OX40 信号和 T 细胞功能的影响。选择一个先导候选物通过 sortagging 特异性地与荧光团偶联，并应用于异种移植小鼠模型中表达 hOX40 的细胞的非侵入性光学成像（OI）。
我们的筛选活动揭示了四个独特的 Nbs，它们在生理条件下具有很强的结合亲和力和高稳定性。表位分组和结构域映射表明，这些 Nbs 靶向 hOX40 上至少两个不同的表位。在分析它们对 OX40 信号的影响时，排除了所有验证的 Nbs 的激动作用。hOX40-Nbs 孵育活化的 T 细胞不会影响细胞活力或增殖模式，但观察到细胞因子释放的差异。在表达 hOX40 的异种移植实验小鼠中，用荧光团偶联的先导候选物进行 OI 证明了其作为成像探针的特异性和功能。
考虑到对用于非侵入性监测 T 细胞激活动力学的先进探针的需求，我们提出，我们的 hOX40-Nbs 具有作为非侵入性和纵向诊断成像探针的巨大潜力。TME 或 IME 中 OX40 T 细胞的定量将提供对浸润 T 细胞激活状态的深入了解，为评估免疫反应、预测治疗效果以及指导癌症或 IMIDs 患者的个性化免疫治疗策略提供有价值的生物标志物。
