Preface

Chapter 8 **The Development of Single Domain Antibodies for Diagnostic**

Chapter 9 **Use, Applications and Mechanisms of Intracellular Actions of**

Anneleen Steels, Laurence Bertier and Jan Gettemans

Chapter 10 **Structural Diversity Problems and the Solving Method for**

Chapter 11 **Immune-Mediated Skin Reactions Induced by Recombinant Antibodies and Other TNF-Alpha Inhibitors 259**

Chapter 12 **Bioinformatics as a Tool to Identify Infectious Disease**

Chiuan Herng Leow, Qin Cheng, Katja Fischer and James McCarthy

Emi Hifumi, Hiroaki Taguchi, Ryuichi Kato, Mitsue Arakawa, Yoshiki

Karolína Vorčáková, Péč Juraj, Péčová Tatiana and Martinásková

Lavanya Suneetha, Prasanna Marsakatla, Rachel Supriya Suneetha

**Pathogen Peptide Sequences as Targets for Antibody**

**and Therapeutic Applications 175**

**Camelid VHHs 205**

**Antibody Light Chains 231**

Katayama and Taizo Uda

**Engineering 277**

and Sujai Suneetha

Klára

**VI** Contents

Antibodies are most important molecules for therapy, diagnosis, and biological and medical research. For about 40 years, the hybridoma technology is used to generate mouse antibod‐ ies with high affinities. However, antibodies against nonimmunogenic, toxic, self-antigens and human/mouse cross-reactive epitopes cannot be obtained by this classical technique. Additionally, the mouse monoclonal antibodies elicit a strong immune response when ap‐ plied in patients.

A breakthrough to solve these problems was the development of human antibody reper‐ toires and *in vitro* antibody selection methods in the beginning of the nineties. In the follow‐ ing years, it was demonstrated that the generation of specific antibodies by the immune system could be imitated successfully *in vitro*. The development of antibodies, particularly human antibodies, could be dramatically improved. The human antibody repertoires and corresponding *in vitro* selection techniques are now used to generate and modify human an‐ tibodies against virtually every protein and desired epitope or conformation. This is the power and tasks of antibody engineering.

The most frequently *in vitro*–selected recombinant antibody format is the scFv, which can be converted into full-length antibodies or scFv-Fc proteins, both of which have similar charac‐ teristics. Engineering of the Fc region increases the half-life and improves the affinity of Fc domains for their receptors on immune effector cells or to complement. For specific applica‐ tions, different antibody formats such as bispecific antibodies, minibodies, or diabodies can be generated. Furthermore, very stable single domain antibodies comprising only the varia‐ ble domain of the heavy chain can be selected by phage display from camel- or shark-de‐ rived repertoires.

The first approved recombinant therapeutic antibodies were chimeric or humanized variants of mouse hybridoma antibodies. Today, fully human antibodies are selected from human an‐ tibody repertoires mainly against antigens involved in infectious diseases and cancer. How‐ ever, the number of new mAbs validating new therapeutic targets is limited because therapeutic target discovery is very laborious. Affinity maturation of selected clones is impor‐ tant when low-affinity clones are selected from naive or synthetic libraries where the antibody genes have not been subjected to somatic hypermutation in contrast to immune libraries. New promising affinity maturation approaches include mammalian cell surface display of antibod‐ ies coupled with somatic hypermutation mediated by activation-induced cytidine deaminase (AID). Furthermore, affinity maturation based on incorporation of a random repertoire of mi‐ crochip-synthesized CDRs into a known antibody framework has recently been demonstrat‐ ed. However, affinity maturation based on introduction of random or site-directed mutations can be time-consuming because secondary libraries must be built up and screened.

By the end of 2016, over 50 fully human antibodies had been approved by the US Food and Drug Administration (FDA) or European Medicines Agency (EMA). Basically, they were se‐ lected by phage display or from transgenic mice comprising a large part of the human germ line antibody repertoire. Regarding phage display selection, most phase three and approved recombinant antibodies were selected from naïve single pot antibody libraries and only a very few were selected from synthetic or semisynthetic libraries. Besides phage display and yeast display, other *in vitro* display techniques have been established such as mRNA dis‐ play, DNA display, bacterial display, mammalian cell surface display, and ribosomal dis‐ play. In the future, it remains to be seen if the amount of new approved therapeutic antibodies from synthetic libraries as well as alternative *in vitro* display techniques will in‐ crease.

novo synthesis of antibody genes is described in **Chapter 2** by CC Lim et al. **Chapter 3** by LR Tsuruta et al. focuses on all known *in vitro* display techniques and antibody humanization by chain shuffling. **Chapter 4** by P Diebolder and A Krawczyk provides a detailed protocol for the selection of antiviral human antibodies from combinatorial immune phage display libraries. M Kato and Y Hanju compare in **Chapter 5** display screening with colony assays for antibody library screening. **Chapter 6** by T Sawa et al. deals with antibody-based immu‐ notherapy against bacterial infections, particularly the construction of an scFv against PcrV, the cap structure in the translocational needle of the type III secretory apparatus of *P. aerugi‐ nosa.* The aimis to block bacterial toxin translocation. The following **Chapter 7** by A Chakra‐ barti describes purification of monoclonal antibodies by size exclusion chromatography. Two following chapters, **Chapters 8 and 9** by CH Leow et al. and A Steels et al., respective‐ ly, focus on the application and mechanism of single-domain antibodies, the most stable an‐ tibody format. **Chapter 10** by E Hifumi et al. describes isolation of the monomolecular structure of the constant region of a catalytic light chain.**Chapter 11** by V Karolina et al. comprises a review about immune-mediated skin reactions of anti-TNF alpha recombinant antibodies. Finally, **Chapter 12** by L Suneetha et al.demonstrates bioinformatic approaches for identification of pathogen-derived peptides involved in infectious diseases and discuss

Antibody engineering facilitates the generation and modification of human antibodies against therapeutic antigens. Selected single clones can be modified and improved by engineering the variable binding domains and the Fc domains in the case of a full length antibody. New opti‐ mized recombinant antibodies are in development and will have extensive applications in the

I want to express my thanks to the authors for very good collaboration, Ms. Iva Simcic, InTech's Publishing Process Manager for coordination through the entire book processing, my students

**Thomas Böldicke**

Preface IX

Braunschweig, Germany

Helmholtz-Centre for Infection Research Structure and Function of Proteins

for fruitful discussions, and Prof. Wulf Blankenfeldt for supporting my editorial work.

fields of immunology, biotechnology, diagnostics, and therapeutic medicine.

their potential as targets to elicit recombinant antibodies.

In addition to extracellular targeting of antigens, intracellular proteins can be targeted by intracellular antibodies (intrabodies), too. These intrabodies are essential to trace *in vivo* traf‐ ficking of proteins and to knock down proteins for revelation of protein functions. Due to their high specificity, they are able to target posttranslational modifications, interaction re‐ gions, conformers, splice variants, and isoforms without off target effects, which are a major problem of RNAi-mediated gene silencing. Some intrabodies have therapeutic potential against viral infections, brain diseases, or cancer. Anticancer intrabodies have been evaluat‐ ed in xenograft tumor mouse models. Numerous preclinical studies and a smaller number of clinical trials have been investigated using therapeuticmRNA molecules that deliver the genetic information of genes and maybe this technique can be transferred to the delivery of intrabody mRNA in the future.

Antibodies against conformers of a protein are difficult to generate by the hybridoma techni‐ que because maintaining of a specific conformation in an immunized animal is difficult. But it can be done by *in vitro* selection of antibodies. Antibodies recognizing different conform‐ ers of signaling proteins are important to study cell signaling. In addition, for cancer treat‐ ment, antibodies that recognize active cell surface receptor conformers as well as antibodies that have the capability to differentiate cancer cells into nonmalignant cells are very valua‐ ble for the future. Finally, it has to be mentioned that new promising synthetic binders com‐ prising variable non–immunoglobulin-binding sites embedded in a nonimmunoglobulin framework have been selected by the *in vitro* display systems generally used for selection of recombinant antibodies. For example, nonimmunoglobulin scaffolds are ankyrin repeat pro‐ tein (DARPin), monobody, affibody, Kunitz domain, and anticalin. The main advantage is their stability. However, until now, no nonimmunoglobulin binder has been approved for application in patients.

This new antibody engineering book comprises current techniques to select and improve recombinant antibodies. Furthermore, single-domain antibodies, the problems of structural diversity of antibodies, unwanted immune reactions of recombinant antibodies, and bioin‐ formatic approaches are demonstrated and discussed. The topics are presented by experts in the field of antibody engineering and are important for live science researchers and students and particularly for researches working with recombinant antibodies. A description of indi‐ vidual chapters is as follows:

**Chapter 1** by O Backhauscomprises the generation of natural antibody diversity, which helps to understand the strategies of constructing immune, naive, and synthetic libraries. Construction of antibody libraries as well as *in vitro* and *in vivo* affinity maturation and de

novo synthesis of antibody genes is described in **Chapter 2** by CC Lim et al. **Chapter 3** by LR Tsuruta et al. focuses on all known *in vitro* display techniques and antibody humanization by chain shuffling. **Chapter 4** by P Diebolder and A Krawczyk provides a detailed protocol for the selection of antiviral human antibodies from combinatorial immune phage display libraries. M Kato and Y Hanju compare in **Chapter 5** display screening with colony assays for antibody library screening. **Chapter 6** by T Sawa et al. deals with antibody-based immu‐ notherapy against bacterial infections, particularly the construction of an scFv against PcrV, the cap structure in the translocational needle of the type III secretory apparatus of *P. aerugi‐ nosa.* The aimis to block bacterial toxin translocation. The following **Chapter 7** by A Chakra‐ barti describes purification of monoclonal antibodies by size exclusion chromatography. Two following chapters, **Chapters 8 and 9** by CH Leow et al. and A Steels et al., respective‐ ly, focus on the application and mechanism of single-domain antibodies, the most stable an‐ tibody format. **Chapter 10** by E Hifumi et al. describes isolation of the monomolecular structure of the constant region of a catalytic light chain.**Chapter 11** by V Karolina et al. comprises a review about immune-mediated skin reactions of anti-TNF alpha recombinant antibodies. Finally, **Chapter 12** by L Suneetha et al.demonstrates bioinformatic approaches for identification of pathogen-derived peptides involved in infectious diseases and discuss their potential as targets to elicit recombinant antibodies.

By the end of 2016, over 50 fully human antibodies had been approved by the US Food and Drug Administration (FDA) or European Medicines Agency (EMA). Basically, they were se‐ lected by phage display or from transgenic mice comprising a large part of the human germ line antibody repertoire. Regarding phage display selection, most phase three and approved recombinant antibodies were selected from naïve single pot antibody libraries and only a very few were selected from synthetic or semisynthetic libraries. Besides phage display and yeast display, other *in vitro* display techniques have been established such as mRNA dis‐ play, DNA display, bacterial display, mammalian cell surface display, and ribosomal dis‐ play. In the future, it remains to be seen if the amount of new approved therapeutic antibodies from synthetic libraries as well as alternative *in vitro* display techniques will in‐

In addition to extracellular targeting of antigens, intracellular proteins can be targeted by intracellular antibodies (intrabodies), too. These intrabodies are essential to trace *in vivo* traf‐ ficking of proteins and to knock down proteins for revelation of protein functions. Due to their high specificity, they are able to target posttranslational modifications, interaction re‐ gions, conformers, splice variants, and isoforms without off target effects, which are a major problem of RNAi-mediated gene silencing. Some intrabodies have therapeutic potential against viral infections, brain diseases, or cancer. Anticancer intrabodies have been evaluat‐ ed in xenograft tumor mouse models. Numerous preclinical studies and a smaller number of clinical trials have been investigated using therapeuticmRNA molecules that deliver the genetic information of genes and maybe this technique can be transferred to the delivery of

Antibodies against conformers of a protein are difficult to generate by the hybridoma techni‐ que because maintaining of a specific conformation in an immunized animal is difficult. But it can be done by *in vitro* selection of antibodies. Antibodies recognizing different conform‐ ers of signaling proteins are important to study cell signaling. In addition, for cancer treat‐ ment, antibodies that recognize active cell surface receptor conformers as well as antibodies that have the capability to differentiate cancer cells into nonmalignant cells are very valua‐ ble for the future. Finally, it has to be mentioned that new promising synthetic binders com‐ prising variable non–immunoglobulin-binding sites embedded in a nonimmunoglobulin framework have been selected by the *in vitro* display systems generally used for selection of recombinant antibodies. For example, nonimmunoglobulin scaffolds are ankyrin repeat pro‐ tein (DARPin), monobody, affibody, Kunitz domain, and anticalin. The main advantage is their stability. However, until now, no nonimmunoglobulin binder has been approved for

This new antibody engineering book comprises current techniques to select and improve recombinant antibodies. Furthermore, single-domain antibodies, the problems of structural diversity of antibodies, unwanted immune reactions of recombinant antibodies, and bioin‐ formatic approaches are demonstrated and discussed. The topics are presented by experts in the field of antibody engineering and are important for live science researchers and students and particularly for researches working with recombinant antibodies. A description of indi‐

**Chapter 1** by O Backhauscomprises the generation of natural antibody diversity, which helps to understand the strategies of constructing immune, naive, and synthetic libraries. Construction of antibody libraries as well as *in vitro* and *in vivo* affinity maturation and de

crease.

VIII Preface

intrabody mRNA in the future.

application in patients.

vidual chapters is as follows:

Antibody engineering facilitates the generation and modification of human antibodies against therapeutic antigens. Selected single clones can be modified and improved by engineering the variable binding domains and the Fc domains in the case of a full length antibody. New opti‐ mized recombinant antibodies are in development and will have extensive applications in the fields of immunology, biotechnology, diagnostics, and therapeutic medicine.

I want to express my thanks to the authors for very good collaboration, Ms. Iva Simcic, InTech's Publishing Process Manager for coordination through the entire book processing, my students for fruitful discussions, and Prof. Wulf Blankenfeldt for supporting my editorial work.

> **Thomas Böldicke** Helmholtz-Centre for Infection Research Structure and Function of Proteins Braunschweig, Germany

**Chapter 1**

**Provisional chapter**

**Generation of Antibody Diversity**

**Generation of Antibody Diversity**

DOI: 10.5772/intechopen.72818

Because of the huge diversity, the immunoglobulin repertoire cannot be encoded by static genes, which would explode the genomic capacity comprising about 20,000–25,000 human genes. The immunoglobulin repertoire is provided by the process of somatic germ line recombination, which is the only controlled alteration of the genomic DNA after meiosis. It takes place in mammalian B lymphocyte (B cells) precursors in the bone marrow. The genome germ line sequence of undeveloped B cells is organized in gene segments and compromise V (variable), D (diversity), and J (joining) gene segments constituting the variable domain of the heavy chain and only V and J genes for building up the variable domain of the light chain. The rearrangement of the variable region follows a strict order. The following processes that participate in the generation of antibody diversity were summarized—allelic, combinational, and junctional diversity, pairing of IgH and IgL, and receptor editing—which all together produce the primary antigen repertoire (pre-antigen stimulation). When a B cell encounters a foreign antigen, affinity maturation and class switch are induced. Thereby the antibody repertoire increases. The resulting secondary immunoglobulin repertoire reveals in humans at least 1011 specifici-

**Keywords:** antibody diversity, somatic recombination, somatic hypermutation, class-switch recombination, allelic exclusion, B-cell receptor editing, pairing of VH

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

The immune system is a complex system, comprising different organs and many specialized cell types, which are carrying out their development, maturation, and pathogen recognition at various sides in the body. The immune system has two major approaches to recognize and attack pathogens. The first is the innate immunity followed by the delayed adaptive immune response, based on specific antigen recognition receptors. The innate immune system is

http://dx.doi.org/10.5772/intechopen.72818

ties for different antigens.

and VL, germinal center

**1. Introduction**

Oliver Backhaus

**Abstract**

Oliver Backhaus
