Translational Molecular Imaging, Institute for Diagnostic and Interventional Radiology & Clinic for Hematology and Medical Oncology, University Medical Center Göttingen, Göttingen, Germany.
R&D Reagents, Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany.
Front Immunol. 2020 Aug 7;11:1704. doi: 10.3389/fimmu.2020.01704. eCollection 2020.
A domain that is often neglected in the assessment of chimeric antigen receptor (CAR) functionality is the extracellular spacer module. However, several studies have elucidated that membrane proximal epitopes are best targeted through CARs comprising long spacers, while short spacer CARs exhibit highest activity on distal epitopes. This finding can be explained by the requirement to have an optimal distance between the effector T cell and target cell. Commonly used long spacer domains are the CH2-CH3 domains of IgG molecules. However, CARs containing these spacers generally show inferior efficacy in mouse models compared to their observed activity, which is linked to unspecific Fcγ-Receptor binding and can be abolished by mutating the respective regions. Here, we first assessed a CAR therapy targeting membrane proximal CD20 using such a modified long IgG1 spacer. However, despite these mutations, this construct failed to unfold its observed cytotoxic potential in an model, while a shorter but less structured CD8α spacer CAR showed complete tumor clearance. Given the shortage of well-described long spacer domains with a favorable functionality profile, we designed a novel class of CAR spacers with similar attributes to IgG spacers but without unspecific off-target binding, derived from the Sialic acid-binding immunoglobulin-type lectins (Siglecs). Of five constructs tested, a Siglec-4 derived spacer showed highest cytotoxic potential and similar performance to a CD8α spacer in a CD20 specific CAR setting. In a pancreatic ductal adenocarcinoma model, a Siglec-4 spacer CAR targeting a membrane proximal (TSPAN8) epitope was efficiently engaged , while a membrane distal (CD66c) epitope did not activate the T cell. Transfer of the TSPAN8 specific Siglec-4 spacer CAR to an setting maintained the excellent tumor killing characteristics being indistinguishable from a TSPAN8 CD8α spacer CAR while outperforming an IgG4 long spacer CAR and, at the same time, showing an advantageous central memory CAR T cell phenotype with lower release of inflammatory cytokines. In summary, we developed a novel spacer that combines cytotoxic potential with an advantageous T cell and cytokine release phenotype, which make this an interesting candidate for future clinical applications.
在嵌合抗原受体 (CAR) 功能评估中，经常被忽视的一个领域是细胞外间隔模块。然而，有几项研究已经阐明，通过包含长间隔区的 CAR，可以最好地靶向膜近端表位，而短间隔区 CAR 则在远端表位上表现出最高的活性。这一发现可以通过效应 T 细胞和靶细胞之间需要最佳距离来解释。常用的长间隔区结构域是 IgG 分子的 CH2-CH3 结构域。然而，与观察到的活性相比，含有这些间隔区的 CAR 在小鼠模型中通常表现出较差的疗效，这与非特异性 Fcγ 受体结合有关，可以通过突变相应区域来消除。在这里，我们首先使用这种经过修饰的长 IgG1 间隔区评估了一种靶向膜近端 CD20 的 CAR 疗法。然而，尽管有这些突变，这种构建体在模型中未能发挥其观察到的细胞毒性潜力，而较短但结构较少的 CD8α 间隔区 CAR 则显示出完全清除肿瘤。鉴于缺乏具有良好功能特性的描述良好的长间隔区结构域，我们设计了一类新型的 CAR 间隔区，其具有与 IgG 间隔区相似的属性，但没有非特异性的脱靶结合，源自唾液酸结合免疫球蛋白型凝集素 (Siglecs)。在测试的五个构建体中，源自 Siglec-4 的间隔区显示出最高的细胞毒性潜力，并在 CD20 特异性 CAR 环境中表现出与 CD8α 间隔区相似的性能。在胰腺导管腺癌模型中，一种靶向膜近端 (TSPAN8) 表位的 Siglec-4 间隔区 CAR 被有效地募集，而膜远端 (CD66c) 表位则不会激活 T 细胞。将 TSPAN8 特异性 Siglec-4 间隔区 CAR 转移到 模型中，保持了出色的肿瘤杀伤特性，与 TSPAN8 CD8α 间隔区 CAR 无法区分，同时表现出有利的中央记忆 CAR T 细胞表型，炎症细胞因子释放减少。总之，我们开发了一种新型的间隔区，它结合了细胞毒性潜力和有利的 T 细胞和细胞因子释放表型，这使其成为未来临床应用的一个有趣候选者。
