National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
AI115469
米国
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
5F30AI120510-03
米国
引用
ジャーナル: Nat Commun / 年: 2021 タイトル: Directed evolution of and structural insights into antibody-mediated disruption of a stable receptor-ligand complex. 著者: Luke F Pennington / Pascal Gasser / Silke Kleinboelting / Chensong Zhang / Georgios Skiniotis / Alexander Eggel / Theodore S Jardetzky / 要旨: Antibody drugs exert therapeutic effects via a range of mechanisms, including competitive inhibition, allosteric modulation, and immune effector mechanisms. Facilitated dissociation is an additional ...Antibody drugs exert therapeutic effects via a range of mechanisms, including competitive inhibition, allosteric modulation, and immune effector mechanisms. Facilitated dissociation is an additional mechanism where antibody-mediated "disruption" of stable high-affinity macromolecular complexes can potentially enhance therapeutic efficacy. However, this mechanism is not well understood or utilized therapeutically. Here, we investigate and engineer the weak disruptive activity of an existing therapeutic antibody, omalizumab, which targets IgE antibodies to block the allergic response. We develop a yeast display approach to select for and engineer antibody disruptive efficiency and generate potent omalizumab variants that dissociate receptor-bound IgE. We determine a low resolution cryo-EM structure of a transient disruption intermediate containing the IgE-Fc, its partially dissociated receptor and an antibody inhibitor. Our results provide a conceptual framework for engineering disruptive inhibitors for other targets, insights into the failure in clinical trials of the previous high affinity omalizumab HAE variant and anti-IgE antibodies that safely and rapidly disarm allergic effector cells.
A: High affinity immunoglobulin epsilon receptor subunit alpha B: Immunoglobulin heavy constant epsilon D: Immunoglobulin heavy constant epsilon H: clone_7 Variable fragment heavy chain J: clone_7 Variable fragment heavy chain K: clone_7 Variable fragment light chain L: clone_7 Variable fragment light chain ヘテロ分子
名称: Locked complex of IgE-Fc (G335C) and alpha chain of the high affinity IgE receptor (W156C) bound to clone_7 scFV タイプ: COMPLEX / Entity ID: #1-#4 / 由来: RECOMBINANT
分子量
実験値: NO
由来(天然)
生物種: Homo sapiens (ヒト)
由来(組換発現)
生物種: Homo sapiens (ヒト)
緩衝液
pH: 7.5
試料
濃度: 4.5 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES
平均露光時間: 2 sec. / 電子線照射量: 44 e/Å2 / フィルム・検出器のモデル: GATAN K3 (6k x 4k) / 撮影したグリッド数: 1 / 実像数: 523
-
解析
ソフトウェア
名称
バージョン
分類
NB
PHENIX
(1.14_3260:phenix.real_space_refine)
精密化
PDB_EXTRACT
3.25
データ抽出
EMソフトウェア
ID
名称
カテゴリ
4
cryoSPARC
CTF補正
7
PHENIX
モデルフィッティング
12
cryoSPARC
3次元再構成
13
PHENIX
モデル精密化
CTF補正
タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION
3次元再構成
解像度: 7.29 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 24396 / 対称性のタイプ: POINT
原子モデル構築
プロトコル: FLEXIBLE FIT 詳細: Initial local fitting done in Chimera, followed by auto dock, and a single round of real space refinement. Carbohydrates were automatically built in phenix when possible, and a handful of ...詳細: Initial local fitting done in Chimera, followed by auto dock, and a single round of real space refinement. Carbohydrates were automatically built in phenix when possible, and a handful of flagged outliers in geometry were fixed manually in Phenix.