Advancing targeted co-stimulation with antibody-fusion proteins by introducing TNF superfamily members in a single-chain format
Advancing targeted co-stimulation with antibody-fusion proteins by introducing TNF superfamily members in a single-chain format
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DOI：
10.1080/2162402x.2016.1238540
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发表时间：
2016-01-01
期刊：
影响因子：
7.2
通讯作者：
Mueller, Dafne
中科院分区：
文献类型：
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作者：
Fellermeier, Sina;Beha, Nadine;Mueller, Dafne
通过肿瘤坏死因子超家族（TNFSF）受体的共刺激成为支持抗肿瘤免疫应答的有前途的策略。使用由肿瘤定向抗体部分和TNFSF的共刺激配体的胞外结构域组成的抗体融合蛋白的靶向策略构成了将共刺激活性集中于肿瘤部位的有吸引力的选择。由于TNFSF成员本质上形成非共价连接的同三聚体的功能单元，因此合适的抗体融合蛋白的蛋白质工程具有挑战性。针对简单和稳定构型的分子，我们使用单链形式（scTNFSF）的TNFSF配体，即，由多肽接头连接的胞外域的三个单元，折叠成分子内三聚体。通过将针对EpCAM或FAP的肿瘤定向scFv抗体片段融合至共刺激性scTNFSF分子（sc 4 -1BBL、scOX 40 L、scGITRL或scLIGHT），产生一组单体scFv-scTNFSF融合蛋白。与通过TNFSF部分的分子间同源三聚化定义的scFv-NFSF形式相比，scFv-scTNFSF显示出相等或增强的共刺激活性，尽管抗体结合中的亲合力降低。此外，还观察到小鼠血清稳定性增强和生物利用度提高。我们表明，scFv-scTNFSF格式可以应用于TNFSF的各种成员，呈现靶向依赖性共刺激活性。因此，这种形式表现出有利的特性，使其成为进一步开发治疗性融合蛋白的有希望的选择。
Co-stimulation via receptors of the tumor necrosis factor superfamily (TNFSF) emerges as promising strategy to support antitumor immune responses. Targeted strategies with antibody-fusion proteins composed of a tumor-directed antibody part and the extracellular domain of a co-stimulatory ligand of the TNFSF constitute an attractive option to focus the co-stimulatory activity to the tumor site. Since TNFSF members intrinsically form functional units of non-covalently linked homotrimers, the protein engineering of suitable antibody-fusion proteins is challenging. Aiming for molecules of simple and stable configuration, we used TNFSF ligands in a single-chain format (scTNFSF), i.e., three units of the ectodomain connected by polypeptide linkers, folding into an intramolecular trimer. By fusing tumor-directed scFv antibody fragments directed against EpCAM or FAP to co-stimulatory scTNFSF molecules (sc4-1BBL, scOX40L, scGITRL or scLIGHT), a set of monomeric scFv-scTNFSF fusion proteins was generated. In comparison to the scFv-NFSF format, defined by intermolecular homotrimerization via the TNFSF part, scFv-scTNFSF showed equal or enhanced co-stimulatory activity despite reduced avidity in antibody binding. In addition, enhanced serum stability and improved bioavailability in mice were observed. We show that the scFv-scTNFSF format can be applied to various members of the TNFSF, presenting targeting-dependent co-stimulatory activity. Hence, this format exhibits favorable properties that make it a promising choice for further therapeutic fusion protein development.