Novel inhibitors of histamine-releasing factor suppress food allergy in a murine model.
Novel inhibitors of histamine-releasing factor suppress food allergy in a murine model.
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新型组胺释放因子抑制剂可抑制小鼠模型中的食物过敏。
DOI：
10.1016/j.alit.2021.07.005
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Kawakami,Toshiaki
中科院分区：
文献类型：
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作者：
Kawakami,Yu;Kurosawa,Yasunori;Oltean,Daniela;Espinosa,LisaYuko;Kim,HwanSoo;Lemersal,Ian;Kawakami,Yuko;Okumura,Shigeru;Maruyama,Toshiaki;Kawakami,Toshiaki
组胺释放因子（Histamine-releasing factor， HRF），又称翻译控制肿瘤蛋白和fortilin，是细胞内增殖和存活等基本功能所必需的高度保守蛋白。由于它是在过敏反应中分泌的，因此它与过敏性疾病有关。最近的研究表明，在过敏反应、哮喘和食物过敏的动物模型中，HRF通过促进免疫球蛋白（Ig） e依赖性肥大细胞和嗜碱性粒细胞的激活来放大变应性炎症。HRF可以作为单体和二硫连接的低聚物存在。HRF通过IgE/IgG的Fab部分与HRF内的两个IgG结合位点（即氨基末端19残基（N19）和螺旋结构域H3）相互作用，直接结合IgE和IgG分子的一个子集。研究还表明，融合蛋白谷胱甘肽s -转移酶(GST)-N19和两个半胱氨酸残基被丙氨酸取代的重组单体HRF突变体（HRF- 2ca）可以作为体外HRF- ige相互作用的强竞争性抑制剂，并强烈减少体内过敏性炎症。3,4因此，假设HRF低聚物包括二聚体，而不是单体，与高亲和力受体（FcεRI）结合的IgE分子交联FcεRI分子，导致肥大细胞和嗜碱性细胞的活化。3,4与这一观点和HRF在食物过敏中的致病作用相一致，食物过敏小鼠小肠中HRF低聚物和血液中HRF反应性IgE增加。GST- n19不适合临床应用，因为GST部分可能会导致受体产生抗体。在这项研究中，为了进一步开发临床相关的HRF抑制剂，我们试图产生一种单克隆抗体（mAb）和一种最佳肽抑制剂，这两种抗体都能阻断N19-Ig相互作用。这些HRF抑制剂在食物过敏的卵清蛋白（OVA）致敏/OVA激发模型中进行了疗效测试。用GST-N19免疫家兔。从抗gst - n19血清滴度高的家兔脾脏中生成表达fab的噬菌体展示文库。用固定化GST- n19和固定化GST筛选4轮噬菌体。从79个n19特异性Fab噬菌体中随机选择12个Fab序列，克隆到表达载体pTT5中，在HEK293细胞中表达，通过Ni-NTA琼脂糖柱纯化。这些fab都与固定化的GST-N19结合（数据未显示），并且通过酶联免疫吸附试验（ELISA）证实它们能够抑制HRF反应性IgE C38-2与重组HRF的相互作用。其中，hrf结合能力较强的3个fab （A8-1、C4-4和F7-1）如图1a所示。抑制HRF-IgE相互作用活性最强的Fab F7-1转化为全长IgG kappa (CK1) mAb抗体SPF7-1（图1b）。腹腔注射SPF7-1 mAb（> 85%纯）可减轻ova诱导的低温（图1c）并逆转沉默的身体活动（数据未显示）。GST-N19和HRF- 2ca在体外和体内均表现出较强的抑制HRF功能的活性，而口服合成N19肽的作用较弱。3,4考虑到口服给药需要相当大量的肽，以及肽对消化的弹性和恶劣的pH环境，在氨基端和/或羧基端合成了几种化学修饰（图2a），并在食物过敏中测试了它们的预防效果。这些肽在ELISA中降低了IgE-HRF的相互作用（数据未显示）。然而，不幸的是，这些都没有……
Histamine-releasing factor (HRF), also known as translationally controlled tumor protein and fortilin, is a highly conserved protein required for fundamental intracellular functions such as proliferation and survival. 1 Since it is secreted during allergic reactions, it is implicated in allergic diseases. 2 Recent studies demonstrated that HRF amplifies allergic inflammation by promoting immunoglobulin (Ig) E-dependent activation of mast cells and basophils in animal models of anaphylaxis, asthma and food allergy. 3 e6 HRF can be present as a monomer and disulfidelinked oligomers. HRF directly binds to a subset of IgE and IgG molecules by interactions between the Fab portion of IgE/IgG and two Ig-binding sites within HRF, ie, the amino-terminal 19 residues (N19) and the helical domain H3. 4 It was also shown that a fusion protein glutathione S-transferase (GST)-N19 and a recombinant monomeric HRF mutant with two cysteine residues replaced with alanine (HRF-2CA) work as strong competitive inhibitors for in vitro HRF-IgE interactions and strongly reduce in vivo allergic inflammation. 3, 4 Thus, it is postulated that HRF oligomers including dimers, but not monomers, bind highaffinity receptor (FcεRI)-bound IgE molecules to crosslink FcεRI molecules, leading to the activation of mast cells and basophils. 3, 4 Consistent with this notion and the pathogenic role of HRF in food allergy, HRF oligomers in the small intestine and HRF-reactive IgE in blood were increased in food allergic mice. 3 GST-N19 would not be appropriate for clinical application, as the GST portion will likely cause antibody development in recipients. In this study for future development of a clinically relevant HRF inhibitor, we sought to generate a monoclonal antibody (mAb) and an optimal peptide inhibitor, both of which block the N19-Ig interactions. The efficacy of these HRF inhibitors was tested in the ovalbumin (OVA) sensitization/OVA challenge model of food allergy. Rabbits were immunized with GST-N19. Phage-display libraries expressing Fabs were generated from spleens of rabbits with high serum titers of anti-GST-N19. Phages were selected by 4 rounds of panning with immobilized GST-N19 and against immobilized GST. Twelve Fab sequences were randomly selected out of thus selected seventy-nine N19-specific Fab-displaying phages, cloned into an expression vector pTT5, expressed in HEK293 cells and purified by Ni-NTA agarose column. These Fabs all bound to immobilized GST-N19 (data not shown), and they were confirmed for their ability to inhibit interactions of the HRF-reactive IgE, C38-2, with recombinant HRF by enzyme-linked immunosorbent assays (ELISA). Among them, three Fabs (A8-1, C4-4 and F7-1) with strong HRF-binding ability are shown in Figure 1 A. Fab F7-1 with the strongest activity to inhibit HRF-IgE interactions was converted to a full-length IgG kappa (CK1) mAb SPF7-1 (Fig. 1 B). SPF7-1 mAb (> 85% pure) intraperitoneally (ip) administered mitigated OVA-induced hypothermia (Fig. 1 C) and reversed muted physical activity (data not shown).GST-N19 and HRF-2CA showed strong inhibitory activities of HRF function both in vitro and in vivo unlike weaker effects of synthetic N19 peptide, when administered orally. 3, 4 In consideration of oral administration that requires a fairly large amount of peptide and resilience of the peptides to digestion and harsh pH environments, several chemical modifications at the aminoterminal and/or carboxyl-terminal ends (Fig. 2 A) were synthesized, and their preventative efficacy was tested in food allergy. These peptides reduced IgE-HRF interactions in ELISA (data not shown). However, unfortunately, none of these …