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Semisynthetic Phage Display Library Construction: Generation of Filtered Libraries. 半合成噬菌体展示文库构建：过滤文库的生成。
Cold Spring Harbor protocols Pub Date : 2024-10-16 DOI: 10.1101/pdb.prot108615
Juan C Almagro, Mary Ann Pohl
{"title":"Semisynthetic Phage Display Library Construction: Generation of Filtered Libraries.","authors":"Juan C Almagro, Mary Ann Pohl","doi":"10.1101/pdb.prot108615","DOIUrl":"https://doi.org/10.1101/pdb.prot108615","url":null,"abstract":"<p><p>Display of antibody fragments on the surface of M13 filamentous bacteriophages is a well-established approach for the identification of antibodies binding to a target of interest. Here, we describe the second of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Gold Plus Libraries. The three-step method involves (1) primary library (PL) construction, (2) filtered library (FL) construction, and (3) secondary library construction. The second step, described here, involves display of the PLs as single-chain variable fragment (scFv) fusions to protein pIII of the M13 phage, as well as heat shock treatment and subsequent selection of well-folded and thermostable scFvs via protein L binding, whereas unstable and defective scFvs are removed by washing steps and centrifugation. The quality of the filtration process is assessed by sequencing clones chosen at random from the FLs. These libraries, enriched with thermostable antibodies, are then ready to be used for the third and final step of the process: generation of secondary libraries.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142459808","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Considerations for Using Phage Display Technology in Therapeutic Antibody Drug Discovery. 在治疗性抗体药物研发中使用噬菌体展示技术的注意事项。
Cold Spring Harbor protocols Pub Date : 2024-10-16 DOI: 10.1101/pdb.top107757
Mary Ann Pohl, Juan C Almagro
{"title":"Considerations for Using Phage Display Technology in Therapeutic Antibody Drug Discovery.","authors":"Mary Ann Pohl, Juan C Almagro","doi":"10.1101/pdb.top107757","DOIUrl":"https://doi.org/10.1101/pdb.top107757","url":null,"abstract":"<p><p>Phage display is a versatile and effective platform for the identification and engineering of biologic-based therapeutics. Using standard molecular biology laboratory techniques, one can create a highly diverse and functional antibody phage-displayed library, and rapidly identify antibody fragments that bind to a target of interest with exquisite specificity and high affinity. Here, we discuss key aspects for the development of an antibody discovery strategy to harness the power of phage display technology to obtain molecules that can successfully be developed into therapeutics, including target validation, antibody design goals, and considerations for preparing and executing phage panning campaigns. Careful design and implementation of discovery campaigns-regardless of the target-provides the best chance of identifying desirable antibody fragments for further therapeutic development, so these principles can be applied to any new discovery project.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142459794","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Maize Abiotic Stress Treatments in Controlled Environments. 受控环境中的玉米非生物胁迫处理。
Cold Spring Harbor protocols Pub Date : 2024-10-16 DOI: 10.1101/pdb.prot108620
Katherine M Murphy, Anna L Casto, Leonardo Chavez, Leonardo W Lima, Alejandra Quiñones, Malia A Gehan, Cory D Hirsch
{"title":"Maize Abiotic Stress Treatments in Controlled Environments.","authors":"Katherine M Murphy, Anna L Casto, Leonardo Chavez, Leonardo W Lima, Alejandra Quiñones, Malia A Gehan, Cory D Hirsch","doi":"10.1101/pdb.prot108620","DOIUrl":"https://doi.org/10.1101/pdb.prot108620","url":null,"abstract":"<p><p>Maize (<i>Zea mays</i>) is one of the world's most important crops, providing food for humans and livestock and serving as a bioenergy source. Climate change and the resulting abiotic stressors in the field reduce crop yields, threatening food security and the global economy. Water deficit (i.e., drought), heat, and insufficient nutrients (e.g., nitrogen and phosphorus) are major environmental stressors that affect maize yields, and impact growth and development at all stages of the plant life cycle. Understanding the biological processes underlying these responses in maize has the potential to increase yields in the face of abiotic stress. Optimizing individual or combined abiotic stress treatments in controlled environments reduces potential noise in data collection that can be present under less controlled growth conditions. Here, we describe methods and conditions for controlled abiotic stress treatments and associated controls during early vegetative growth of maize, conducted in greenhouses or growth chambers. This includes the environmental conditions, equipment, soil preparation, and intensity and duration of heat, drought, nitrogen deficiency, and phosphorous deficiency. Controlled experiments at early growth stages are informative for future in-field studies that require greater labor and inputs, saving researchers time and growing space, and thus research funds, before testing plants across later stages of development. We suggest that stress treatments be severe enough to result in a measurable phenotype, but not so severe that all plants die prior to sample collection. This protocol is designed to set important standards for replicable research in maize.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142459803","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Semisynthetic Phage Display Library Construction: Design and Synthesis of Diversified Single-Chain Variable Fragments and Generation of Primary Libraries. 半合成噬菌体展示文库构建：多样化单链可变片段的设计与合成以及初级文库的生成。
Cold Spring Harbor protocols Pub Date : 2024-10-16 DOI: 10.1101/pdb.prot108614
Juan C Almagro, Mary Ann Pohl
{"title":"Semisynthetic Phage Display Library Construction: Design and Synthesis of Diversified Single-Chain Variable Fragments and Generation of Primary Libraries.","authors":"Juan C Almagro, Mary Ann Pohl","doi":"10.1101/pdb.prot108614","DOIUrl":"https://doi.org/10.1101/pdb.prot108614","url":null,"abstract":"<p><p>Display of antibody fragments on the surface of M13 filamentous bacteriophages is a well-established approach for the identification of antibodies binding to a target of interest. Here, we describe the first of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Libraries. The three-step method involves (1) primary library (PL) construction, (2) filtered library construction, and (3) secondary library construction. The first step, described here, entails design, synthesis, and cloning of four PLs. These PLs are designed with specific properties amenable to therapeutic antibody development using one universal variable heavy (V<sub>H</sub>) scaffold and four distinct variable light (V<sub>L</sub>) scaffolds. The scaffolds are diversified in positions that bind both protein and peptide targets identified in antibody-antigen complexes of known structure using the amino acid frequencies found in those positions in known human antibody sequences, avoiding residues that may lead to developability liabilities. The diversified scaffolds are combined with 90 synthetic neutral HCDR3 sequences designed with developable human diversity genes (IGHD) and joining heavy genes (IGHJ) in germline configuration, and assembled as single-chain variable fragments (scFvs) in a V<sub>L</sub>-linker-V<sub>H</sub> orientation. The four designed PLs are synthesized using trinucleotide phosphoramidites (TRIMs) and cloned independently into a phagemid vector for M13 pIII display. Quality control of the cloning of the four PLs is also described, which involves sequencing scFvs in each library.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142459807","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Generation of Antibody Libraries for Phage Display: Library Reamplification. 生成用于噬菌体展示的抗体库：文库再扩增
Cold Spring Harbor protocols Pub Date : 2024-10-15 DOI: 10.1101/pdb.prot108601
Haiyong Peng, Christoph Rader
{"title":"Generation of Antibody Libraries for Phage Display: Library Reamplification.","authors":"Haiyong Peng, Christoph Rader","doi":"10.1101/pdb.prot108601","DOIUrl":"https://doi.org/10.1101/pdb.prot108601","url":null,"abstract":"<p><p>Phage display of Fab libraries enables the de novo discovery and in vitro evolution of monoclonal antibodies. Fab libraries are collections of millions to billions of different antibodies that collectively cover a large antigen or epitope binding space. To preserve the diversity of the Fab library for repeated selection campaigns, it is recommended to use the original phage from the Fab library generation rather than reamplified phage, if practically possible. This is because reamplification will bias the Fab library for clones that are expressed at higher rates. Fab-phage, however, should only be used if they have been prepared on the same day, to avoid proteolytic cleavage of the physical linkage of phenotype (phage-displayed Fab protein) and genotype (phage-encapsulated Fab DNA). Thus, in practice, reamplification of a Fab-phage library cannot usually be avoided. Here, we describe the steps for the reamplification of an original Fab-phage library prior to its selection. The protocol can also be used to reamplify Fab-phage from the third or later panning rounds when enriched clones are unlikely to be lost by reamplification biases.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142459800","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Cloning, Expression, and Purification of Phage Display-Selected Fab for Biophysical and Biological Studies. 用于生物物理和生物学研究的噬菌体展示选择 Fab 的克隆、表达和纯化。
Cold Spring Harbor protocols Pub Date : 2024-10-15 DOI: 10.1101/pdb.prot108604
Matthew G Cyr, Haiyong Peng, Christoph Rader
{"title":"Cloning, Expression, and Purification of Phage Display-Selected Fab for Biophysical and Biological Studies.","authors":"Matthew G Cyr, Haiyong Peng, Christoph Rader","doi":"10.1101/pdb.prot108604","DOIUrl":"https://doi.org/10.1101/pdb.prot108604","url":null,"abstract":"<p><p>The antigen-binding fragment (Fab) is the ∼50-kDa monovalent arm of an antibody molecule. In the laboratory, the Fab can be produced via either enzymatic digestion or recombinant expression, and its use facilitates the accurate assessment of affinity and specificity of monoclonal antibodies. The high melting temperature of the Fab, together with its low tendency to aggregate and ready conversion to natural and nonnatural immunoglobulin (Ig) formats (without affecting antigen binding properties), have made it a preferred format for phage display, as well as a tool for accurate assessment of affinity, specificity, and developability of monoclonal antibodies. Here, we outline a strategy to clone, express, and purify human or chimeric nonhuman/human Fabs that have previously been selected by phage display. Fabs purified using this approach, which results in milligram amounts, enable a variety of downstream biophysical and biological assays that ultimately inform the success of phage display library generation and selection.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142459793","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Generation of Antibody Libraries for Phage Display: Chimeric Rabbit/Human Fab Format. 生成用于噬菌体展示的抗体库：嵌合兔/人Fab格式。
Cold Spring Harbor protocols Pub Date : 2024-10-15 DOI: 10.1101/pdb.prot108598
Haiyong Peng, Christoph Rader
{"title":"Generation of Antibody Libraries for Phage Display: Chimeric Rabbit/Human Fab Format.","authors":"Haiyong Peng, Christoph Rader","doi":"10.1101/pdb.prot108598","DOIUrl":"https://doi.org/10.1101/pdb.prot108598","url":null,"abstract":"<p><p>Rabbit monoclonal antibodies are attractive reagents for research, and have also found use in diagnostic and therapeutic applications. This is owed to their high affinity and specificity, along with their ability to recognize epitopes conserved between mouse and human antigens. Phage display is a powerful method for the de novo generation, affinity maturation, and humanization of rabbit monoclonal antibodies from naive, immune, and synthetic antibody repertoires. Using phagemid family pComb3, a preferred phage display format is chimeric rabbit/human Fab, which consists of rabbit variable domains (V<sub>H</sub>, V<sub>κ</sub>, and V<sub>λ</sub>) fused to human constant domains. The human constant domains, C<sub>H</sub>1 of IgG1 and C<sub>L</sub> (C<sub>κ</sub> or C<sub>λ</sub>), not only provide established purification and detection handles but also facilitate higher expression in <i>Escherichia coli</i> compared to the corresponding rabbit constant domains. Here, we describe the use of a pComb3 derivative, phagemid pC3C, for the generation of chimeric rabbit/human Fab libraries with randomly combined rabbit variable domains of high sequence diversity, starting from the preparation of total RNA from rabbit spleen and bone marrow. Depending on the complexity of the parental antibody repertoire, the protocol can be scaled for yielding a library size of 10<sup>8</sup>-10<sup>11</sup> independent chimeric rabbit/human Fab clones. As such, it can be used, for instance, for the generation of either specialized immune or large naive rabbit antibody libraries.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142459798","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Generation of Antibody Libraries for Phage Display: Preparation of Helper Phage. 生成用于噬菌体展示的抗体库：制备辅助噬菌体
Cold Spring Harbor protocols Pub Date : 2024-10-15 DOI: 10.1101/pdb.prot108600
Haiyong Peng, Christoph Rader
{"title":"Generation of Antibody Libraries for Phage Display: Preparation of Helper Phage.","authors":"Haiyong Peng, Christoph Rader","doi":"10.1101/pdb.prot108600","DOIUrl":"https://doi.org/10.1101/pdb.prot108600","url":null,"abstract":"<p><p>The generation and selection of antibody libraries by phagemid-based phage display requires three components; namely, phagemid library, host bacterial cells, and helper phage. The use of helper phage is necessary for the selection of phagemid libraries by phage display because it provides all genes needed for production of infectious phage particles. Here, we describe the generation of high-titer helper phage preparations suitable for phagemid-based phage display. The approach is based on helper phage VCSM13, which includes a gene for kanamycin resistance and a mutated packaging signal that, in the presence of a phagemid with an unmutated packaging signal, favors the production of infectious phage particles with phagemid phenotype and genotype.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142459802","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Generation and Selection of Phage Display Antibody Libraries in Fab Format. 以 Fab 格式生成和筛选噬菌体展示抗体库。
Cold Spring Harbor protocols Pub Date : 2024-10-15 DOI: 10.1101/pdb.top107764
Christoph Rader
{"title":"Generation and Selection of Phage Display Antibody Libraries in Fab Format.","authors":"Christoph Rader","doi":"10.1101/pdb.top107764","DOIUrl":"https://doi.org/10.1101/pdb.top107764","url":null,"abstract":"<p><p>Monoclonal antibodies (mAbs) have exceptional utility as research reagents and pharmaceuticals. As a complement to both traditional and contemporary single-B-cell cloning technologies, the mining of antibody libraries via display technologies-which mimic and simplify B cells by physically linking phenotype (protein) to genotype (protein-encoding DNA or RNA)-has become an important method for mAb discovery. Among these display technologies, phage display has been particularly successful for the generation of mAbs that bind to a wide variety of antigens with exceptional specificities and affinities. Rather than multivalent whole antibodies, phage display typically uses monovalent antibody fragments, such as \"fragment antigen binding\" (Fab), as the format of choice. The ∼50-kDa Fab format consists of four immunoglobulin (Ig) domains on two polypeptide chains (light chain and shortened heavy chain), and exhibits its antigen binding site in a natural configuration found in bivalent IgG and other multivalent Ig molecules. The Fab fragment has a high melting temperature and a low tendency to aggregate, and can be readily converted to natural and nonnatural Ig formats without affecting antigen binding properties, which has made it a favored format for phage display for more than three decades. Here, I briefly summarize some of the approaches used for the generation and selection of phage display antibody libraries in Fab format, from human and nonhuman antibody repertoires.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142459797","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Phage Display Selection of Antibody Libraries: Screening of Selected Binders. 噬菌体展示选择抗体库：筛选选定的结合剂
Cold Spring Harbor protocols Pub Date : 2024-10-15 DOI: 10.1101/pdb.prot108603
Haiyong Peng, Christoph Rader
{"title":"Phage Display Selection of Antibody Libraries: Screening of Selected Binders.","authors":"Haiyong Peng, Christoph Rader","doi":"10.1101/pdb.prot108603","DOIUrl":"https://doi.org/10.1101/pdb.prot108603","url":null,"abstract":"<p><p>Phage display selection of antibody libraries is a powerful method for generating and evolving monoclonal antibodies. The pComb3 phagemid family of phage display vectors facilitates the mining of antibody libraries in Fab format from human and nonhuman antibody repertoires. Here, we describe the screening for monoclonal Fab binders after selection of a polyclonal pool of Fab binders to an antigen of interest, with the goal of identifying and sequencing monoclonal antibodies that bind the antigen with high affinity and specificity. The screening cascade involves a phage ELISA, followed by a crude Fab ELISA and DNA fingerprinting and sequencing. The protocol outlines phage and crude Fab ELISAs using purified antigen immobilized on microplates, native antigen expressed on eukaryotic cells, or both.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142459806","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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