Chemical Diversification of Simple Synthetic Antibodies.
Chemical Diversification of Simple Synthetic Antibodies.
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DOI：
10.1021/acschembio.0c00865
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发表时间：
2021-02-19
影响因子：
4
通讯作者：
Van Deventer JA
中科院分区：
文献类型：
--
作者：
Islam M;Kehoe HP;Lissoos JB;Huang M;Ghadban CE;Berumen Sánchez G;Lane HZ;Van Deventer JA
抗体所具有的特性使其成为有价值的治疗、诊断和基础研究工具。然而，抗体化学反应性和共价抗原结合受到规范氨基酸中编码的窄范围的化学物质的限制或甚至阻止。在这项工作中，我们研究了利用酵母展示的抗体中或附近的抗体互补决定区（CDR）含有非典型氨基酸（ncAAs）的化学功能范围扩大的策略。为了能够系统地表征ncAA掺入对抗体功能的影响，我们首先研究了单个抗体环的多样化是否将支持针对来自三个物种的免疫球蛋白的结合克隆的分离。我们构建并筛选了仅在重链的第3互补决定区（CDR-H3）内含有典型氨基酸多样性和环长度多样性的十亿成员文库。分离的克隆表现出中等亲和力（两位数至三位数纳摩尔亲和力），并且在几种情况下表现出单物种特异性，证实抗体特异性可以由单个CDR介导。这种受限的多样性使得能够利用额外的CDR来安装化学反应性和光可交联的ncAA。ncAA取代的抗体的结合研究表明，ncAA掺入是合理的耐受性良好，所观察到的亲和力的变化发生作为ncAA侧链身份，取代位点，和所使用的ncAA掺入机制的函数。多个含叠氮化物的ncAA支持铜催化的叠氮化物-炔环加成（CuAAC）和应变促进的叠氮化物-炔环加成（SPAAC），而不废除结合功能。类似地，几个炔取代促进了CuAAC而没有明显的结合破坏。最后，评价了用可光交联的ncAA取代的抗体在酵母表面上的紫外线介导的交联。基于竞争的测定揭示了位置依赖性共价键，强烈表明成功的交联。使用可溶形式的ncAA取代的克隆证实了关于CuAAC反应和酵母表面上的光交联的关键发现。酵母表面和溶液中发现的一致性表明化学多样化可以纳入酵母展示筛选方法中。两者合计，我们的研究结果突出了整合使用酵母展示和ncAAs在寻找具有“化学增强”结合功能的蛋白质的能力。这包括在结合蛋白结构内系统地引入小分子功能和评估基于蛋白质的共价靶结合的策略。酵母表面抗体的有效制备和化学多样化为高通量发现“药物样”蛋白质先导物开辟了新的可能性。
Antibodies possess properties that make them valuable as therapeutics, diagnostics, and basic research tools. However, antibody chemical reactivity and covalent antigen binding are constrained, or even prevented, by the narrow range of chemistries encoded in canonical amino acids. In this work, we investigate strategies for leveraging an expanded range of chemical functionality using yeast displayed antibodies containing noncanonical amino acids (ncAAs) in or near antibody complementarity determining regions (CDRs). To enable systematic characterization of the effects of ncAA incorporation on antibody function, we first investigated whether diversification of a single antibody loop would support isolation of binding clones against immunoglobulins from three species. We constructed and screened a billion-member library containing canonical amino acid diversity and loop length diversity only within the 3rd complementarity determining region of the heavy chain (CDR-H3). Isolated clones exhibited moderate affinities (double- to triple-digit nanomolar affinities) and in several cases, single-species specificity, confirming that antibody specificity can be mediated by a single CDR. This constrained diversity enabled utilization of additional CDRs for the installation of chemically reactive and photo-crosslinkable ncAAs. Binding studies of ncAA-substituted antibodies revealed that ncAA incorporation is reasonably well tolerated, with observed changes in affinity occurring as a function of ncAA side chain identity, substitution site, and the ncAA incorporation machinery used. Multiple azide-containing ncAAs supported copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC) without abrogation of binding function. Similarly, several alkyne substitutions facilitated CuAAC without apparen disruption of binding. Finally, antibodies substituted with a photo-crosslinkable ncAA were evaluated for ultraviolet-mediated crosslinking on the yeast surface. Competition-based assays revealed position-dependent covalent linkages, strongly suggesting successful crosslinking. Key findings regarding CuAAC reactions and photo-crosslinking on the yeast surface were confirmed using soluble forms of ncAA-substituted clones. The consistency of findings on the yeast surface and in solution suggest that chemical diversification can be incorporated into yeast display screening approaches. Taken together, our results highlight the power of integrating the use of yeast display and ncAAs in search of proteins with “chemically augmented” binding functions. This includes strategies for systematically introducing small molecule functionality within binding protein structures and evaluating protein-based covalent target binding. The efficient preparation and chemical diversification of antibodies on the yeast surface opens up new possibilities for discovering “drug-like” protein leads in high throughput.
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影响因子：
46.9
作者：
通讯作者：
--
DOI：
10.1007/978-1-62703-586-6_9
发表时间：
2014-01-01
期刊：
HUMAN MONOCLONAL ANTIBODIES: METHODS AND PROTOCOLS
影响因子：
--
作者：
Adams, Jarrett J.;Nelson, Bryce;Sidhu, Sachdev S.
通讯作者：
Sidhu, Sachdev S.
影响因子：
4.6
作者：
Cheng AC;Doherty EM;Johnstone S;DiMauro EF;Dao J;Luthra A;Ye J;Tang J;Nixey T;Min X;Tagari P;Miranda LP;Wang Z
通讯作者：
Wang Z
影响因子：
6.8
作者：
Adams, Jarrett J.;Sidhu, Sachdev S.
通讯作者：
Sidhu, Sachdev S.
DOI：
10.1073/pnas.1609375114
发表时间：
2016-12-27
影响因子：
11.1
作者：
Dong Hyun Nam;Rodriguez, Carlos;Ge, Xin
通讯作者：
Ge, Xin