Richter Fabian, Liebig Timo, Guenzi Eric, Herrmann Andreas, Scheurich Peter, Pfizenmaier Klaus, Kontermann Roland E
Institut für Zellbiologie und Immunologie, Universität Stuttgart, Stuttgart, Germany.
PLoS One. 2013 Aug 19;8(8):e72156. doi: 10.1371/journal.pone.0072156. eCollection 2013.
Selective inhibition of TNFR1 signaling holds the potential to greatly reduce the pro-inflammatory activity of TNF, while leaving TNFR2 untouched, thus allowing for cell survival and tissue homeostasis. ATROSAB is a humanized antagonistic anti-TNFR1 antibody developed for the treatment of inflammatory diseases.
METHODOLOGY/PRINCIPAL FINDINGS: The epitope of ATROSAB resides in the N-terminal region of TNFR1 covering parts of CRD1 and CRD2. By site-directed mutagenesis, we identified Arg68 and His69 of TNFR1 as important residues for ATROSAB binding. ATROSAB inhibited binding of (125)I-labeled TNF to HT1080 in the subnanomolar range. Furthermore, ATROSAB inhibited release of IL-6 and IL-8 from HeLa and HT1080 cells, respectively, induced by TNF or lymphotoxin alpha (LTα). Different from an agonistic antibody (Htr-9), which binds to a region close to the ATROSAB epitope but elicits strong TNFR1 activation, ATROSAB showed a negligible induction of IL-6 and IL-8 production over a broad concentration range. We further verified that ATROSAB, comprising mutations within the Fc region known to abrogate complement fixation and antibody-mediated cellular effector functions, indeed lacks binding activity for C1q, FcγRI (CD64), FcγRIIB (CD32b), and FcγRIII (CD16) disabling ADCC and CDC.
CONCLUSIONS/SIGNIFICANCE: The data corroborate ATROSAB's unique function as a TNFR1-selective antagonist efficiently blocking both TNF and LTα action. In agreement with recent studies of TNFR1 complex formation and activation, we suggest a model of the underlying mechanism of TNFR1 inhibition by ATROSAB.
选择性抑制TNFR1信号传导有可能极大地降低TNF的促炎活性，同时不影响TNFR2，从而实现细胞存活和组织稳态。ATROSAB是一种开发用于治疗炎症性疾病的人源化抗TNFR1抗体。
方法/主要发现：ATROSAB的表位位于TNFR1的N端区域，覆盖CRD1和CRD2的部分区域。通过定点诱变，我们确定TNFR1的Arg68和His69是ATROSAB结合的重要残基。ATROSAB在亚纳摩尔范围内抑制(125)I标记的TNF与HT1080的结合。此外，ATROSAB分别抑制TNF或淋巴毒素α（LTα）诱导的HeLa和HT1080细胞中IL-6和IL-8的释放。与一种激动性抗体（Htr-9）不同，后者结合在靠近ATROSAB表位的区域但引发强烈的TNFR1激活，ATROSAB在广泛的浓度范围内对IL-6和IL-8产生的诱导作用可忽略不计。我们进一步证实，包含已知可消除补体固定和抗体介导的细胞效应功能的Fc区域内突变的ATROSAB，确实缺乏对C1q、FcγRI（CD64）、FcγRIIB（CD32b）和FcγRIII（CD16）的结合活性，从而使ADCC和CDC失活。
结论/意义：数据证实了ATROSAB作为TNFR1选择性拮抗剂有效阻断TNF和LTα作用的独特功能。与最近关于TNFR1复合物形成和激活的研究一致，我们提出了ATROSAB抑制TNFR1的潜在机制模型。
