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Journal ArticleDOI

Generation and analysis of the improved human HAL9/10 antibody phage display libraries.

TL;DR: It is shown that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display, and the highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv phagemid vector design.
Abstract: Antibody phage display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries - in theory - antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody phage display library. The naive antibody gene libraries HAL9 and HAL10, with a combined theoretical diversity of 1.5×1010 independent clones, were constructed from 98 healthy donors using improved phage display vectors. In detail, most common phagemids employed for antibody phage display are using a combined His/Myc tag for detection and purification. We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display. For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate. Deletion of a phenylalanine at the end of the CL linker sequence in our new phagemid design increased scFv production rate and frequency of selected kappa antibodies significantly. The HAL libraries and 834 antibodies selected against 121 targets were analyzed regarding the used germline V-genes, used V-gene combinations and CDR-H3/-L3 length and composition. The amino acid diversity and distribution in the CDR-H3 of the initial library was retrieved in the CDR-H3 of selected antibodies showing that all CDR-H3 amino acids occurring in the human antibody repertoire can be functionally used and is not biased by E. coli expression or phage selection. Further, the data underline the importance of CDR length variations. The highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv phagemid vector design. Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.

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Citations
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Journal ArticleDOI
TL;DR: The first crystal structure of the C-terminal adhesion domain of InvD revealed a distinct Ig-related fold that, apart from the canonical β-sheets, comprises various modifications of and insertions into the Ig-core structure, suggesting that InvD modulates Ig functions in the intestine and affects direct interactions with a subset of cell surface-exposed B-cell receptors.

497 citations


Cites methods from "Generation and analysis of the impr..."

  • ...Two scFv naive universal libraries (HAL10 and HAL9 HAL10) were employed (32)....

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  • ...45 10(9), respectively, were used for panning (32)....

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Journal ArticleDOI
TL;DR: Some opportunities and achievements are summarized, e.g., the generation of antibodies which could not be generated otherwise, and the design of antibody properties by different panning strategies, including the adjustment of kinetic parameters.
Abstract: With six approved products and more than 60 candidates in clinical testing, human monoclonal antibody discovery by phage display is well established as a robust and reliable source for the generation of therapeutic antibodies. While a vast diversity of library generation philosophies and selection strategies have been conceived, the power of molecular design offered by controlling the in vitro selection step is still to be recognized by a broader audience outside of the antibody engineering community. Here, we summarize some opportunities and achievements, e.g., the generation of antibodies which could not be generated otherwise, and the design of antibody properties by different panning strategies, including the adjustment of kinetic parameters.

425 citations

Journal ArticleDOI
14 Jul 2016-mAbs
TL;DR: A comprehensive overview about phage display-derived antibodies that are approved for therapy or in clinical development is provided and a selection of these antibodies is described in more detail to demonstrate different aspects of thephage display technology and its development over the last 25 years.
Abstract: Over the last 3 decades, monoclonal antibodies have become the most important class of therapeutic biologicals on the market. Development of therapeutic antibodies was accelerated by recombinant DNA technologies, which allowed the humanization of murine monoclonal antibodies to make them more similar to those of the human body and suitable for a broad range of chronic diseases like cancer and autoimmune diseases. In the early 1990s in vitro antibody selection technologies were developed that enabled the discovery of “fully” human antibodies with potentially superior clinical efficacy and lowest immunogenicity.Antibody phage display is the first and most widely used of the in vitro selection technologies. It has proven to be a robust, versatile platform technology for the discovery of human antibodies and a powerful engineering tool to improve antibody properties. As of the beginning of 2016, 6 human antibodies discovered or further developed by phage display were approved for therapy. In 2002, ada...

248 citations


Cites background from "Generation and analysis of the impr..."

  • ...The theoretical size of universal libraries is now greater than 10(10) independent clones.(38,47,59,61,68,69) A particular type of antibody library is generated during guided phage display selection of human VH and VL repertoires using the counter chain of a murine monoclonal antibody as template....

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  • ...Examples of na€ıve universal libraries are the human Fab library constructed by de Haard and colleagues at Dyax (now Shire),(40) the scFv libraries from Cambridge Antibody Technology (CAT),(39,61) scFv and Fab libraries from XOMA(59) or the HAL scFv libraries.(37,47)...

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Journal ArticleDOI
TL;DR: The utility of phage display as a powerful platform for therapeutic antibodies discovery is illustrated and all the approved mAbs derived fromphage display are described.
Abstract: Monoclonal antibodies (mAbs) have become one of the most important classes of biopharmaceutical products, and they continue to dominate the universe of biopharmaceutical markets in terms of approval and sales. They are the most profitable single product class, where they represent six of the top ten selling drugs. At the beginning of the 1990s, an in vitro antibody selection technology known as antibody phage display was developed by John McCafferty and Sir. Gregory Winter that enabled the discovery of human antibodies for diverse applications, particularly antibody-based drugs. They created combinatorial antibody libraries on filamentous phage to be utilized for generating antigen specific antibodies in a matter of weeks. Since then, more than 70 phage–derived antibodies entered clinical studies and 14 of them have been approved. These antibodies are indicated for cancer, and non-cancer medical conditions, such as inflammatory, optical, infectious, or immunological diseases. This review will illustrate the utility of phage display as a powerful platform for therapeutic antibodies discovery and describe in detail all the approved mAbs derived from phage display.

116 citations

Journal ArticleDOI
TL;DR: The data demonstrate that the converted, insulin-specific CAR Tregs (cTregs) were functional stable, suppressive and long-lived in vivo, and a proof of concept for both redirection of T cell specificity and conversion of Teffs to cT Regs.

96 citations

References
More filters
Book
15 Jan 2001
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol

215,169 citations

Book
01 Jan 2001
TL;DR: The content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol

25,596 citations

Book
01 Jan 1989
TL;DR: To develop a program to print the barcodes using two commonly uses command sets and hence evaluates their ease of use for such applications, students should be able to program dot matrix printers, by manipulating bit level information and ink jet printers using page description language, such as PCL.
Abstract: 1. OBJECTIVE To develop a program to print the barcodes using two commonly uses command sets and hence evaluates their ease of use for such applications. Upon completion of this experiment, students should be able to program dot matrix printers, by manipulating bit level information and ink jet printers using page description language, such as PCL which uses raster image for graphics output. Students will also become familiar with the encoding and dimensional requirements of barcode symbologies, and the suitability of the selected printers for barcode printing. The Computer Engineering Laboratory is set up with IBM-compatible PC's installed with standard C compiler. The laboratory also has several kinds of printers for this experiment. They are classified into two groups. One group consists of dot-matrix printers and the other group consists of HP ink jet printers. In this experiment, each student will have to program a dot-matrix printer using Epson 9-pin graphics command codes in group A printers and HP PCL graphics commands for the ink jet printer in group B. The assignment of different printers is as follows: 4. INTRODUCTION Barcode labelling is widely implemented in the retail marketplace and is gaining increasing visibility in a broad range of non-retail applications. To read the information contained in a barcode symbol, a scanning device such as a wand can be used. As the scanning device is moved across the symbol, the width pattern of the bars and the spaces is analysed by the reading equipment and the original data is recovered. A number of barcodes has been developed over the years, these include UPS, interleaved 2 of 5, rationalised codabar, code 39, code 128, code 93 and code 49. The American format for article numbering is the Universal Product Code (UPC). The UPC has been successfully employed in the supermarket industry since 1973. It is designed to uniquely identify a product and its manufacturer. Refer to Appendix A for UPC specifications which are necessary for this experiment. Barcode symbols can be printed with various printing technologies and on a variety of substrate labels, tags and papers. In this experiment, plain paper will be used. Hence, the quality of printed barcode will only depend on the printing mechanism. In this experiment, students will learn to program dot matrix printers by manipulating bit level information. A brief summary of the commonly used commands is attached in Appendix B and abstract from the manuals …

8,170 citations


"Generation and analysis of the impr..." refers methods in this paper

  • ...A volume of 180 μL phosphate-buffered 2 × YT-GA in a PP-MTP well was inoculated with 10 μL of the overnight culture and grown at 37°C and 800 rpm for 2 h. Bacteria were harvested by centrifugation for 10 min at 3,220 × g and 180 μL supernatants were removed....

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  • ...Protean II Minigel system (BioRad Inc, München, Germany) according to [37]....

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  • ...After transformation of XL1-Blue MRF’, overnight incubation on 25 cm 2 × YT-GAT plates and resuspension in 25 mL 2 × YT medium, glycerol stocks of each library were made using 800 μL bacteria solution (~1010 bacteria) and 200 μL glycerol (Roth, Karlsruhe) and stored at −80°C....

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  • ...0,5 mL, Greiner, Frickenhausen, Germany) containing 150 μL 2 × YT-GA [37] were inoculated with the bacteria bearing scFv expressing phagemids....

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  • ...Electroporation was performed with 0.1 cm prechilled cuvettes using a MicroPulser electroporator (BioRad, München) at 1.7 kV. Immediately, 1 mL prewarmed 37°C SOC medium pH 7.0 (2% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.05% (w/v) NaCl, 20 mM Mg solution, 20 mM glucose) was added and incubated for 1 h at 600 rpm and 37°C. Transformation was plated out on 25 cm square Petri dishes with 2xYT-GAT agar (1,6% (w/v) Tryptone, 1% (w/v) yeast extract, 0,5% (w/v) NaCl, 100 mM glucose, 100 μg/mL ampiciline, 20 μg/mL tetracycline, 1.5% (w/v) agar-agar) and incubated overnight at 37°C....

    [...]

Journal ArticleDOI
TL;DR: Human antibody fragments with many different binding specificities have been isolated from the same phage repertoire, including haptens, carbohydrates, secreted and cell surface proteins, viral coat proteins, and intracellular antigens from the lumen of the endoplasmic reticulum and the nucleus.
Abstract: Antibody fragments of predetermined binding specificity have recently been constructed from repertoires of antibody V genes, bypassing hybridoma technology and even immunization. The V gene repertoires are harvested from populations of lymphocytes, or assembled in vitro, and cloned for display of associated heavy and light chain variable domains on the surface of filamentous bacteriophage. Rare phage are selected from the repertoire by binding to antigen; soluble antibody fragments are expressed from infected bacteria; and the affinity of binding of selected antibodies is improved by mutation. The process mimics immune selection, and antibodies with many different binding specificities have been isolated from the same phage repertoire. Thus human antibody fragments have been isolated with specificities against both foreign and self antigens, including haptens, carbohydrates, secreted and cell surface proteins, viral coat proteins, and intracellular antigens from the lumen of the endoplasmic reticulum and ...

1,973 citations


"Generation and analysis of the impr..." refers methods in this paper

  • ...antibody gene libraries are used: immune libraries and universal or “single-pot” libraries [24,25]....

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Journal ArticleDOI
24 Jan 1991-Nature
TL;DR: This work has shown that monoclonal antibodies can be genetically engineered and endowed with new properties and could be extended to production of 'in vitro' repertoires of variable domain genes, and obviate the immunization of animals.
Abstract: Monoclonal antibodies can now be genetically engineered and endowed with new properties. In the future, gene technology could enable antigen-binding fragments to be made by exploiting repertoires of variable domain genes derived from immunized animals and expressed in bacteria. How readily can this approach be extended to production of 'in vitro' repertoires of variable domain genes, and obviate the immunization of animals?

1,170 citations