ジャーナル: bioRxiv / 年: 2020 タイトル: Bi-paratopic and multivalent human VH domains neutralize SARS-CoV-2 by targeting distinct epitopes within the ACE2 binding interface of Spike. 著者: Colton J Bracken / Shion A Lim / Paige Solomon / Nicholas J Rettko / Duy P Nguyen / Beth Shoshana Zha / Kaitlin Schaefer / James R Byrnes / Jie Zhou / Irene Lui / Jia Liu / Katarina Pance / ...著者: Colton J Bracken / Shion A Lim / Paige Solomon / Nicholas J Rettko / Duy P Nguyen / Beth Shoshana Zha / Kaitlin Schaefer / James R Byrnes / Jie Zhou / Irene Lui / Jia Liu / Katarina Pance / / Xin X Zhou / Kevin K Leung / James A Wells / 要旨: Neutralizing agents against SARS-CoV-2 are urgently needed for treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) ...Neutralizing agents against SARS-CoV-2 are urgently needed for treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domain binders with high affinity toward neutralizing epitopes without the need for high-resolution structural information. We constructed a VH-phage library and targeted a known neutralizing site, the angiotensin-converting enzyme 2 (ACE2) binding interface of the trimeric SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified 85 unique VH binders to two non-overlapping epitopes within the ACE2 binding site on Spike-RBD. This enabled us to systematically link these VH domains into multivalent and bi-paratopic formats. These multivalent and bi-paratopic VH constructs showed a marked increase in affinity to Spike (up to 600-fold) and neutralization potency (up to 1400-fold) on pseudotyped SARS-CoV-2 virus when compared to the standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with half-minimal inhibitory concentration (IC ) of 4.0 nM (180 ng/mL). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain bound an RBD at the ACE2 binding site, explaining its increased neutralization potency and confirming our original design strategy. Our results demonstrate that targeted selection and engineering campaigns using a VH-phage library can enable rapid assembly of highly avid and potent molecules towards therapeutically important protein interfaces.
フィルム・検出器のモデル: GATAN K3 BIOQUANTUM (6k x 4k) 平均電子線量: 78.0 e/Å2
実験機器
モデル: Titan Krios / 画像提供: FEI Company
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画像解析 #1
初期 角度割当
タイプ: RANDOM ASSIGNMENT
最終 角度割当
タイプ: MAXIMUM LIKELIHOOD
最終 再構成
解像度のタイプ: BY AUTHOR / 解像度: 6.0 Å / 解像度の算出法: OTHER 詳細: This reconstruction is a Gaussian low pass filtered map of a 3.2A gold standard FSC map, to better account for the VH density. The model was fit and relaxed into this map. 使用した粒子像数: 21000
Image processing ID
1
FSC曲線 (解像度の算出)
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画像解析 #2
初期 角度割当
タイプ: RANDOM ASSIGNMENT
最終 角度割当
タイプ: MAXIMUM LIKELIHOOD
最終 再構成
解像度のタイプ: BY AUTHOR / 解像度: 3.2 Å / 解像度の算出法: FSC 0.143 CUT-OFF 詳細: Same reconstruction as above but filtered based on the gold standard FSC to 3.2A. Shows high resolution features but density for the low occupancy VH is poorly resolved. 使用した粒子像数: 21000