Interdisciplinary Center for Clinical Research Laboratory, Department of Internal Medicine II, University Hospital Würzburg, Würzburg, Germany.
Graduate School of Life Sciences, Würzburg University, Würzburg, Germany.
Front Immunol. 2022 Jun 13;13:888274. doi: 10.3389/fimmu.2022.888274. eCollection 2022.
Tumor necrosis factor (TNF) receptor-2 (TNFR2) has attracted considerable interest as a target for immunotherapy. Indeed, using oligomeric fusion proteins of single chain-encoded TNFR2-specific TNF mutants (scTNF80), expansion of regulatory T cells and therapeutic activity could be demonstrated in various autoinflammatory diseases, including graft-versus-host disease (GvHD), experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA). With the aim to improve the availability of TNFR2-specific TNF fusion proteins, we used here the neonatal Fc receptor (FcRn)-interacting IgG1 molecule as an oligomerizing building block and generated a new TNFR2 agonist with improved serum retention and superior activity.
Single-chain encoded murine TNF80 trimers (sc(mu)TNF80) were fused to the C-terminus of an in mice irrelevant IgG1 molecule carrying the N297A mutation which avoids/minimizes interaction with Fcγ-receptors (FcγRs). The fusion protein obtained (irrIgG1(N297A)-sc(mu)TNF80), termed NewSTAR2 (New selective TNF-based agonist of TNF receptor 2), was analyzed with respect to activity, productivity, serum retention and and activity. STAR2 (TNC-sc(mu)TNF80 or selective TNF-based agonist of TNF receptor 2), a well-established highly active nonameric TNFR2-specific variant, served as benchmark. NewSTAR2 was assessed in various and systems.
STAR2 (TNC-sc(mu)TNF80) and NewSTAR2 (irrIgG1(N297A)-sc(mu)TNF80) revealed comparable activity. The novel domain architecture of NewSTAR2 significantly improved serum retention compared to STAR2, which correlated with efficient binding to FcRn. A single injection of NewSTAR2 enhanced regulatory T cell (Treg) suppressive activity and increased Treg numbers by > 300% 5 days after treatment. Treg numbers remained as high as 200% for about 10 days. Furthermore, a single treatment with NewSTAR2 upregulated the adenosine-regulating ectoenzyme CD39 and other activation markers on Tregs. TNFR2-stimulated Tregs proved to be more suppressive than unstimulated Tregs, reducing conventional T cell (Tcon) proliferation and expression of activation markers . Finally, singular preemptive NewSTAR2 administration five days before allogeneic hematopoietic cell transplantation (allo-HCT) protected mice from acute GvHD.
NewSTAR2 represents a next generation ligand-based TNFR2 agonist, which is efficiently produced, exhibits improved pharmacokinetic properties and high serum retention with superior activity exerting powerful protective effects against acute GvHD.
为了提高 TNFR2 特异性 TNF 融合蛋白的可用性，我们在这里使用新生儿 Fc 受体（FcRn）相互作用的 IgG1 分子作为一个聚集构建块，生成了一种具有改进的血清保留和更高活性的新型 TNFR2 激动剂。
将单链编码的鼠 TNF80 三聚体（sc(mu)TNF80）融合到在小鼠中无关的 IgG1 分子的 C 末端，该分子携带 N297A 突变，该突变可避免/最小化与 Fcγ-受体（FcγRs）的相互作用。获得的融合蛋白（irrIgG1（N297A）-sc（mu）TNF80），称为 NewSTAR2（新型 TNF 受体 2 的选择性 TNF 激动剂），针对活性、生产力、血清保留和活性进行了分析。STAR2（TNC-sc（mu）TNF80 或 TNF 受体 2 的选择性 TNF 激动剂），一种成熟的高度活跃的非九聚体 TNFR2 特异性变体，用作基准。NewSTAR2 在各种体内和体外系统中进行了评估。
STAR2（TNC-sc（mu）TNF80）和 NewSTAR2（irrIgG1（N297A）-sc（mu）TNF80）显示出相当的活性。NewSTAR2 的新型结构域架构与 STAR2 相比显著提高了血清保留率，这与 FcRn 的有效结合相关。单次注射 NewSTAR2 可增强调节性 T 细胞（Treg）的抑制活性，并在治疗后 5 天内使 Treg 数量增加超过 300％。Treg 数量在大约 10 天内仍保持在 200％左右。此外，单次治疗用 NewSTAR2 上调了 Tregs 上的腺苷调节外切酶 CD39 和其他激活标记物。与未刺激的 Tregs 相比，TNFR2 刺激的 Tregs 显示出更强的抑制作用，降低了常规 T 细胞（Tcon）的增殖和激活标记物的表达。最后，在异基因造血细胞移植（allo-HCT）前 5 天进行单次预防性 NewSTAR2 给药可保护小鼠免受急性移植物抗宿主病（GvHD）的侵害。
NewSTAR2 是一种下一代基于配体的 TNFR2 激动剂，它可以高效生产，具有改进的药代动力学特性和更高的血清保留率，具有更高的活性，对急性移植物抗宿主病（GvHD）具有强大的保护作用。
