Constrained Combinatorial Libraries of Gp2 Proteins Enhance Discovery of PD-L1 Binders.
Constrained Combinatorial Libraries of Gp2 Proteins Enhance Discovery of PD-L1 Binders.
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Gp2 蛋白的受限组合文库增强了 PD-L1 结合物的发现。
DOI：
10.1021/acscombsci.8b00010
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发表时间：
2018
影响因子：
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通讯作者：
Hackel,BenjaminJ
中科院分区：
文献类型：
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作者：
Kruziki,MaxA;Sarma,Vidur;Hackel,BenjaminJ
工程蛋白配体用于分子治疗、诊断和工业生物技术。Gp 2结构域是一个45个氨基酸的支架，通过两个暴露于溶剂的环的多样化，已经进化为特异性的、高亲和力的结合多种靶标。受早期Gp 2发现活动中选择氨基酸（包括半胱氨酸对）的位点富集的启发，我们假设从头Gp 2发现的广度和效率将通过组合文库设计内的位点氨基酸约束来辅助。我们系统地构建了八个文库，并通过酵母展示对一组靶标比较评估了它们对结合剂发现的功效。在第一多样化互补位环末端的半胱氨酸对的保守性使结合剂发现增加16倍（p< 0.001）。然而，在第二个环或环间对内具有保守半胱氨酸对的另外两个文库没有帮助发现，从而指示位点特异性影响。通过酵母展示蛋白酶抗性测定，来自环一半胱氨酸对文库的Gp 2变体比非限制性变体稳定3.3 ± 2.1倍（p= 0.005）。非半胱氨酸残基的定点限制-由先前进化的粘合剂，天然Gp 2同源性，计算的稳定性和结构分析指导-并没有帮助发现。从环1半胱氨酸约束文库中发现了一组程序性死亡配体1（PD-L1）的结合物，PD-L1是癌症免疫治疗中的关键靶标。通过环步移的亲和力成熟导致强的特异性细胞PD-L1亲和力（Kd= 6-9 nM）。
Engineered protein ligands are used for molecular therapy, diagnostics, and industrial biotechnology. The Gp2 domain is a 45-amino acid scaffold that has been evolved for specific, high-affinity binding to multiple targets by diversification of two solvent-exposed loops. Inspired by sitewise enrichment of select amino acids, including cysteine pairs, in earlier Gp2 discovery campaigns, we hypothesized that the breadth and efficiency of de novo Gp2 discovery will be aided by sitewise amino acid constraint within combinatorial library design. We systematically constructed eight libraries and comparatively evaluated their efficacy for binder discovery via yeast display against a panel of targets. Conservation of a cysteine pair at the termini of the first diversified paratope loop increased binder discovery 16-fold (p< 0.001). Yet two other libraries with conserved cysteine pairs, within the second loop or an interloop pair, did not aid discovery thereby indicating site-specific impact. Via a yeast display protease resistance assay, Gp2 variants from the loop one cysteine pair library were 3.3 ± 2.1-fold (p= 0.005) more stable than nonconstrained variants. Sitewise constraint of noncysteine residues—guided by previously evolved binders, natural Gp2 homology, computed stability, and structural analysis—did not aid discovery. A panel of binders to programmed death ligand 1 (PD-L1), a key target in cancer immunotherapy, were discovered from the loop 1 cysteine constraint library. Affinity maturation via loop walking resulted in strong, specific cellular PD-L1 affinity (Kd= 6–9 nM).