**1. Introduction**

The introduction of recombinant antibody technology has revolutionized and improved the way antibodies are being generated for various applications in research, diagnosis and therapy [1–4]. Antibodies have been the cornerstone for many biomedical advances in the past due to its high specificity and affinity to capture target antigens. The key characteristic of antibodies that makes it highly sought after is the defined specificity of the complementarity determining regions (CDR) of the variable domains against a specific target [5]. This specificity is programmed *in vivo* by a series of different molecular mechanisms such as V-D-J

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

recombination of the heavy chain, V-J recombination of the light chain and somatic hypermutation [6, 7]. After primary immune response the VHDHJH and VLJL exons are randomly mutated, mainly in the CDRs, by somatic hypermutation leading to high affinity antibodies (see article of Oliver Backhaus in this book). These molecular processes have a profound effect on the way the genotype is delineated as the gene rearrangements will bring about multiple gene segment combinations. Additional mutagenesis is elicited through incorporation of additional nucleotides between the junctions of the V, D and J gene segment of the heavy chain and V and J gene segment of the light chain. These variations at the genotypic level have a direct influence on the phenotypic variation seen in terms of target specificity and affinity of the generated antibody [8, 9]. **Figure 1** shows the correlation between the genotypic variations and the phenotypic nature of the generated antibodies.

The introduction of recombinant DNA technology and display technologies has allowed recombinant antibodies to be generated at a rapid pace. This is evident with the increase of recombinant antibodies going into clinical trials in the last 3 years [10, 11]. The general concept of display oriented techniques for antibody generation relies upon the ability to harness the natural or synthetic diversity of an antibody library [12]. As with most recombinant DNA approaches, the ability to customize or modify the genotype either at single base or amino acid level was now possible [13]. This opened many new avenues in the field of recombinant antibody technology to allow modification and customization of characteristics of the phenotype. The advent of display technologies allowed for selective isolation of specific phenotypes with their respective genotypic information to be retrieved together [14]. This means that it was now possible to replicate the *in vivo* antibody generation and maturation process *in vitro* [13]. The impact of antibody display technologies combined with affinity maturation strategies in the isolation and identification of high affinity antibodies is monumental in the way

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The first display technology that was applied for the generation of recombinant antibodies was phage display. Although initially the technology was designed to display polypeptides, the robust nature of the method meant that larger proteins could also be displayed by bacteriophages [15, 16]. This allowed the introduction of antibody fragments to be presented on the surface of the phage particles for selection. Phage display takes advantage of the natural replication cycle of bacteriophages to fuse the antibody gene with the gene of a phage coat protein. This design allows the co-expression and translocation of the antibody fused coat protein during the phage packaging process to display the antibody proteins on the surface of mature phage particles. More importantly, this allowed for a physical linkage to be estab-

Since the introduction of phage display, other display systems have been developed. This includes systems like yeast display, bacterial cell surface display, ribosomal display, mRNA display, DNA display and mammalian cell surface display [17]. **Figure 1** shows the alternative display systems used for antibody presentation. **Yeast display** has an additional feature compared to phage display when dealing with mammalian proteins. This is due to the application of the eukaryotic machinery to assimilate the display mechanism. In this system, the antibody gene is fused to the Aga2p agglutinin subunit found on the surface of yeast cells [18, 19]. In a similar fashion, **bacterial cell display** functions by displaying antibodies on the surface of Gram-negative or Gram-positive cells as a fusion to the flagella or outer membrane

**Ribosome display** is a cell-free display approach where polysomes are stalled on mRNA templates and nascent antibody protein remains in complex with the ribosomes. The stalling of the ribosomes is done with the removal of a stop codon and a C-terminal peptide spacer is required to ensure proper folding of the protein [20]. This is critical as steric hindrance

antibodies are made today.

**1.1. Display technologies**

proteins [12, 16].

lished between the genotype and phenotype [12].

**Figure 1.** Illustration highlighting the different *in vitro* display technologies available.

The introduction of recombinant DNA technology and display technologies has allowed recombinant antibodies to be generated at a rapid pace. This is evident with the increase of recombinant antibodies going into clinical trials in the last 3 years [10, 11]. The general concept of display oriented techniques for antibody generation relies upon the ability to harness the natural or synthetic diversity of an antibody library [12]. As with most recombinant DNA approaches, the ability to customize or modify the genotype either at single base or amino acid level was now possible [13]. This opened many new avenues in the field of recombinant antibody technology to allow modification and customization of characteristics of the phenotype. The advent of display technologies allowed for selective isolation of specific phenotypes with their respective genotypic information to be retrieved together [14]. This means that it was now possible to replicate the *in vivo* antibody generation and maturation process *in vitro* [13]. The impact of antibody display technologies combined with affinity maturation strategies in the isolation and identification of high affinity antibodies is monumental in the way antibodies are made today.

#### **1.1. Display technologies**

recombination of the heavy chain, V-J recombination of the light chain and somatic hypermutation [6, 7]. After primary immune response the VHDHJH and VLJL exons are randomly mutated, mainly in the CDRs, by somatic hypermutation leading to high affinity antibodies (see article of Oliver Backhaus in this book). These molecular processes have a profound effect on the way the genotype is delineated as the gene rearrangements will bring about multiple gene segment combinations. Additional mutagenesis is elicited through incorporation of additional nucleotides between the junctions of the V, D and J gene segment of the heavy chain and V and J gene segment of the light chain. These variations at the genotypic level have a direct influence on the phenotypic variation seen in terms of target specificity and affinity of the generated antibody [8, 9]. **Figure 1** shows the correlation between the genotypic variations and the phenotypic nature of the generated

**Figure 1.** Illustration highlighting the different *in vitro* display technologies available.

antibodies.

18 Antibody Engineering

The first display technology that was applied for the generation of recombinant antibodies was phage display. Although initially the technology was designed to display polypeptides, the robust nature of the method meant that larger proteins could also be displayed by bacteriophages [15, 16]. This allowed the introduction of antibody fragments to be presented on the surface of the phage particles for selection. Phage display takes advantage of the natural replication cycle of bacteriophages to fuse the antibody gene with the gene of a phage coat protein. This design allows the co-expression and translocation of the antibody fused coat protein during the phage packaging process to display the antibody proteins on the surface of mature phage particles. More importantly, this allowed for a physical linkage to be established between the genotype and phenotype [12].

Since the introduction of phage display, other display systems have been developed. This includes systems like yeast display, bacterial cell surface display, ribosomal display, mRNA display, DNA display and mammalian cell surface display [17]. **Figure 1** shows the alternative display systems used for antibody presentation. **Yeast display** has an additional feature compared to phage display when dealing with mammalian proteins. This is due to the application of the eukaryotic machinery to assimilate the display mechanism. In this system, the antibody gene is fused to the Aga2p agglutinin subunit found on the surface of yeast cells [18, 19]. In a similar fashion, **bacterial cell display** functions by displaying antibodies on the surface of Gram-negative or Gram-positive cells as a fusion to the flagella or outer membrane proteins [12, 16].

**Ribosome display** is a cell-free display approach where polysomes are stalled on mRNA templates and nascent antibody protein remains in complex with the ribosomes. The stalling of the ribosomes is done with the removal of a stop codon and a C-terminal peptide spacer is required to ensure proper folding of the protein [20]. This is critical as steric hindrance caused by the ribosomal tunnel can alter the folding of the protein leading to lower display efficiency [21]. A somewhat related method to ribosome display is **mRNA display**. In mRNA display systems, the interaction between the template and protein is covalently linked via puromycin. Puromycin functions to mimic the role of amino-acyl tRNA by attaching itself to a DNA primer affixed to the mRNA template. This allows puromycin to attach itself covalently to the nascent antibody protein based on the peptidyl transferase activity of the ribosome [12, 22].

circumstances and has its own brand of unique features that makes some more suitable for a particular set of antigens. Ultimately, all the display systems are capable of isolating and identifying recombinant human monoclonal antibodies using a library of antibody genes [16]. The variation of the antibody sequences in the antibody gene repertoire (the diversity) will have a significant impact on the quality of antibodies generated. The antibody repertoire being presented on the various display platforms is in essence the basic antibody response divulged by the immune response system [32]. The multi-level process of antibody gene generation and maturation of the V-D-J gene segments will finally dictate the antibody characteristic being inherited to the display systems for recombinant antibody generation. This is evident as antibody V-D-J gene segments function as the basic building blocks of antibodies influencing the characteristics of the antibodies of an antibody gene repertoire [33]. Therefore, an understanding of the processes involved in antibody gene repertoire generation is vital to design engi-

High Affinity Maturated Human Antibodies from Naïve and Synthetic Antibody Repertoires

http://dx.doi.org/10.5772/intechopen.71664

21

The human antibody repertoire represents a diverse collection of immunoglobulin gene segments that encodes for heavy (VH) and light chain (VL) domains [34], forming an unique set of antigen-binding sites [35, 36]. The heavy chain (HC) locus is located at chromosome 14, comprises of VH, D, JH and CH gene segments. The kappa light chain locus is found in chromosome 2 with the VK, JK and CK gene segments. The lambda LC locus with the Vλ, Jλ, and Cλ gene

The generation of a natural antibody repertoire is attributed to several natural mechanisms such as somatic recombination that is rearrangements of gene segments to form a single unique antibody gene sequence [38]. The V(D)J recombination process that takes place during B-cell development allows for combinatorial rearrangements of V (variable), D (diversity), and J (joining) gene segments of the heavy chain resulting in the formation of numerous possibilities [35, 39, 40], see also Backhaus O. this book. A similar process (the VL-JL rearrangement of the light chain) occurs at the light chain locus [39], see also Backhaus O. this book. This process is regulated by lymphocyte-specific RAG1 and RAG2 endonucleases that cleaves DNA at the recombination signal sequences (RSSs) resulting in blunt signal ends and hairpin coding ends. The ends are later joined by classical non-homologous end-joining (cNHEJ) pathway to ensure genomic stability [40, 41]. The outcome of recombination is an ordered fashion of V-D-J and VL-J<sup>L</sup> gene assembly that encodes the antibody binding site (variable region). Antibody diversity is further enhanced by junctional diversification, characterized by variability at the junctions due to insertions or/and deletions of few nucleotides during fusion of segments

responsive to unique antigens [42]. This natural repertoire is known as the naïve or primary repertoire, expresses cell surface IgM and has not undergone specialization by antigen

of antibody-producing B-cell clones that are

neering strategies for antibodies with improved affinities.

**1.2. Generation of human antibody repertoires**

segments are found on chromosome 22 [37].

An individual is expected to have at least 108

[38, 40].

**Mammalian display** approaches utilizes mammalian host cells like HEK293T and Chinese hamster ovary (CHO) cells to present a library of antibodies on the cell surface for selection [23]. The approach adapts a similar concept to that of yeast cell display [18]. It capitalizes on the transient expression of antibodies in which antibody encoding DNA introduced into the cells persist over days to consistently express antibodies. Transient expression systems are commonly used for single round selections from immune repertoires. A stable expression system with the integration of DNA to the host genome is inefficient because of multi-gene incorporation per cell making libraries difficult to resolve [24]. A suitable method to allow multi-round selection is by stable episomal vectors derived from viruses. Virus based vectors are used to infect mammalian cells to display the antibodies for selection making it suitable for large sized libraries [25]. A major advantage of mammalian cell systems is the ability to screen using full length IgG [26]. **DNA display** applied for the screening of peptides/proteins was originally based on streptavidin-fused peptides/proteins linked with their encoding DNAs via biotin in emulsion compartments [27]. However this method was not often used for recombinant antibody selection except some promising results with Fabs, and recently with bispecific diabody fragments have been reported [28, 29].

Other alternative DNA display systems are cis-activity based (CIS) and covalent display technology (CDT) display systems. CIS display uses the ability of the bacterial replication initiator protein, RepA to carry out a cis-activity. This means that RepA has the ability to bind the encoding DNA that was utilized. This activity is largely dependent on the presence of two non-coding regions 3′ to bind to the repA sequence. The actual mechanism is unknown but is believed to involve stalling of RNA polymerase during transcription at the CIS element allowing the nascent repA protein to non-covalently attach to its binding site of the template [30]. The covalent display technology (CDT) exploits the properties of the replication initiator protein from *E. coli* bacteriophage P2 [31]. A pool of DNA encoding antibody molecules is generated as a fusion to the P2A coding sequence [12]. The DNA pool is then transcribed and translated using cell free expression systems. The cis-activity of the P2A protein allows the DNA molecule to covalently tag with its own gene product [16, 17]. Ultimately, the recurring trend of all display systems is the ability of the system to allow the translation of antibody genes to produce a collection of antibody molecules that is physically fused to the encoding genes for selection.

Taking advantage of the different display systems, many different forms of libraries can be represented for antibody generation. There is no actual discrimination as to which method is best for antibody generation. All the display approaches highlighted are useful in different circumstances and has its own brand of unique features that makes some more suitable for a particular set of antigens. Ultimately, all the display systems are capable of isolating and identifying recombinant human monoclonal antibodies using a library of antibody genes [16]. The variation of the antibody sequences in the antibody gene repertoire (the diversity) will have a significant impact on the quality of antibodies generated. The antibody repertoire being presented on the various display platforms is in essence the basic antibody response divulged by the immune response system [32]. The multi-level process of antibody gene generation and maturation of the V-D-J gene segments will finally dictate the antibody characteristic being inherited to the display systems for recombinant antibody generation. This is evident as antibody V-D-J gene segments function as the basic building blocks of antibodies influencing the characteristics of the antibodies of an antibody gene repertoire [33]. Therefore, an understanding of the processes involved in antibody gene repertoire generation is vital to design engineering strategies for antibodies with improved affinities.

#### **1.2. Generation of human antibody repertoires**

caused by the ribosomal tunnel can alter the folding of the protein leading to lower display efficiency [21]. A somewhat related method to ribosome display is **mRNA display**. In mRNA display systems, the interaction between the template and protein is covalently linked via puromycin. Puromycin functions to mimic the role of amino-acyl tRNA by attaching itself to a DNA primer affixed to the mRNA template. This allows puromycin to attach itself covalently to the nascent antibody protein based on the peptidyl transferase activity of the ribo-

**Mammalian display** approaches utilizes mammalian host cells like HEK293T and Chinese hamster ovary (CHO) cells to present a library of antibodies on the cell surface for selection [23]. The approach adapts a similar concept to that of yeast cell display [18]. It capitalizes on the transient expression of antibodies in which antibody encoding DNA introduced into the cells persist over days to consistently express antibodies. Transient expression systems are commonly used for single round selections from immune repertoires. A stable expression system with the integration of DNA to the host genome is inefficient because of multi-gene incorporation per cell making libraries difficult to resolve [24]. A suitable method to allow multi-round selection is by stable episomal vectors derived from viruses. Virus based vectors are used to infect mammalian cells to display the antibodies for selection making it suitable for large sized libraries [25]. A major advantage of mammalian cell systems is the ability to screen using full length IgG [26]. **DNA display** applied for the screening of peptides/proteins was originally based on streptavidin-fused peptides/proteins linked with their encoding DNAs via biotin in emulsion compartments [27]. However this method was not often used for recombinant antibody selection except some promising results with Fabs, and recently with

Other alternative DNA display systems are cis-activity based (CIS) and covalent display technology (CDT) display systems. CIS display uses the ability of the bacterial replication initiator protein, RepA to carry out a cis-activity. This means that RepA has the ability to bind the encoding DNA that was utilized. This activity is largely dependent on the presence of two non-coding regions 3′ to bind to the repA sequence. The actual mechanism is unknown but is believed to involve stalling of RNA polymerase during transcription at the CIS element allowing the nascent repA protein to non-covalently attach to its binding site of the template [30]. The covalent display technology (CDT) exploits the properties of the replication initiator protein from *E. coli* bacteriophage P2 [31]. A pool of DNA encoding antibody molecules is generated as a fusion to the P2A coding sequence [12]. The DNA pool is then transcribed and translated using cell free expression systems. The cis-activity of the P2A protein allows the DNA molecule to covalently tag with its own gene product [16, 17]. Ultimately, the recurring trend of all display systems is the ability of the system to allow the translation of antibody genes to produce a collection of antibody molecules that is physically fused to the encoding

Taking advantage of the different display systems, many different forms of libraries can be represented for antibody generation. There is no actual discrimination as to which method is best for antibody generation. All the display approaches highlighted are useful in different

bispecific diabody fragments have been reported [28, 29].

some [12, 22].

20 Antibody Engineering

genes for selection.

The human antibody repertoire represents a diverse collection of immunoglobulin gene segments that encodes for heavy (VH) and light chain (VL) domains [34], forming an unique set of antigen-binding sites [35, 36]. The heavy chain (HC) locus is located at chromosome 14, comprises of VH, D, JH and CH gene segments. The kappa light chain locus is found in chromosome 2 with the VK, JK and CK gene segments. The lambda LC locus with the Vλ, Jλ, and Cλ gene segments are found on chromosome 22 [37].

The generation of a natural antibody repertoire is attributed to several natural mechanisms such as somatic recombination that is rearrangements of gene segments to form a single unique antibody gene sequence [38]. The V(D)J recombination process that takes place during B-cell development allows for combinatorial rearrangements of V (variable), D (diversity), and J (joining) gene segments of the heavy chain resulting in the formation of numerous possibilities [35, 39, 40], see also Backhaus O. this book. A similar process (the VL-JL rearrangement of the light chain) occurs at the light chain locus [39], see also Backhaus O. this book. This process is regulated by lymphocyte-specific RAG1 and RAG2 endonucleases that cleaves DNA at the recombination signal sequences (RSSs) resulting in blunt signal ends and hairpin coding ends. The ends are later joined by classical non-homologous end-joining (cNHEJ) pathway to ensure genomic stability [40, 41]. The outcome of recombination is an ordered fashion of V-D-J and VL-J<sup>L</sup> gene assembly that encodes the antibody binding site (variable region). Antibody diversity is further enhanced by junctional diversification, characterized by variability at the junctions due to insertions or/and deletions of few nucleotides during fusion of segments [38, 40].

An individual is expected to have at least 108 of antibody-producing B-cell clones that are responsive to unique antigens [42]. This natural repertoire is known as the naïve or primary repertoire, expresses cell surface IgM and has not undergone specialization by antigen encounter [37]. The antigen-binding site of an antibody consists of the surrounding framework regions and the complementarity determining regions (CDRs), CDR1, CDR2 and CDR3. CDR3 region are particularly important for antibody-antigen specificity [43]. The V(D)J and V(J) rearrangement of the antibody gene segments and somatic mutations will give rise to higher binding diversities to various antigens [35, 36].

surface of phages, without affecting the infectivity of phages [50]. Moreover, the displayed antibody molecules retains its antigen-antibody binding capabilities [51]. However, the challenge in generating high affinity antibodies is closely related to the quality of the library generated. Even so, the advancement of recombinant DNA technologies has allowed for downstream affinity maturation processes to be carried out for the improvement of antibody

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An antibody library is basically a physical collection of various antibody genes being represented in a single pool. Antibody molecules are divided into two sets of binding domains, the variable domain of the heavy chain (HC) and light chain [54] that either preferentially or concomitantly contributes to the binding affinity of the antibody to the target antigen [42]. Therefore, in order to replicate the diverse repertoire of antibodies afforded by the immune system, a random combinatorial mix of both the HC and LC repertoire is required. The source of the antibody repertoire has a profound influence on the type of antibody libraries being constructed as for example if you amplify the variable antibody genes from immune patients the immune response of different individuals in different health and disease states will have a definite impact on the diversity of the generated antibody repertoire. The diversity of naïve antibody repertoires will be reflected by random variations in the genetic information of the clones generated in the library [55]. This brings to light the different classification of antibody libraries that are essentially defined by the origin of the antibody repertoire. There are generally three different classes of antibody libraries namely the naïve, immune and synthetic

The natural collection of immunoglobulins for antibody library generation is obtained from circulating B-cells in primary and secondary lymphoid tissues and blood. Naïve libraries are constructed from IgM mRNA of B-cells from healthy donors, non-immunized donors, isolated from peripheral blood lymphocytes, spleen, tonsils, and bone marrow. In some cases, the repertoire could also be retrieved from animal sources resulting in antibodies of different origins [57]. The diversity offered by a naïve repertoire is undeniably vast, whereby the antibody fragments are PCR amplified randomly from the antibody cDNA of non-antigen stimulated B-cells as well as those B-cells that have been resided in the immune system due to earlier infections [58, 59]. A single naïve library (also known as single pot library, generated from several donors) can be used to generate antibodies against all types of antigens, peptides, toxins, as well as self-antigens (typically important in the area of cancer and autoimmune disease therapeutics). Some of the antibodies are generated against red cell antigens, haptens, tumor necrosis factor (TNF) [15]. The clonal diversity exhibited by B-cells enables the generation of a range of antibodies against a wide variety of antigens. The characteristics of a naïve repertoire mainly result in modest affinity and polyreactivity antibodies. Due to the polyreactive nature of a naïve library, it is important to generate a larger library

affinities post-selection [52, 53].

antibody libraries applied for antibody display [56].

**2.1. Naïve antibody libraries**

**2. Antibody libraries**

Upon encountering new antigens, naïve B-cells are stimulated and become activated B-cells, undergo proliferation and differentiation. B-cell proliferation is also known as clonal expansion, in which an antibody B-cell clone specific to an antigen is selected and produced in large scale. This process takes place in secondary lymphoid organs such as lymph nodes and spleen, also referred to as germinal centers. The differentiation process generally involves somatic hypermutation (SHM) and class switch recombination (CSR). Somatic hypermutation introduced extensive point mutations in the variable (V) region gene, such as single base substitutions, insertions and deletions. Consequently, the V region exon is further diversified resulting in altered affinities against the target antigen [39, 41]. Class switch recombination replaces the constant region (CH) gene of the HC resulting in class switching from IgM to IgE, IgA and IgG. The type of isotype used determines the methods for elimination of captured antigen by immunoglobulin or the location for antibody accumulation [37, 44, 45]. The combination of both mechanisms offers an improved diversity to the antibodies [46] and enables the selection for high affinity antibody-producing cells against a particular antigen. This process of improved affinity is known as affinity maturation of antibodies.

An essential element that mediates both SHM and CSR is the activation-induced cytidine deaminase [47]. AID is a protein exclusively expressed in activated B-cells in germinal centers but the exact function and mechanism of AID in SHM and CSR are not fully understood. However, several studies have been reported and shown that AID is capable of editing RNA and DNA deamination. AID deaminates cytidine residues to uracil residues on single-stranded DNA (ssDNA) at preferred "hotspots," described as DGYW motif. Such motif favors mutation and is ubiquitous throughout the genome. The maintenance of genome fidelity attempts to correct the "deamination error" by base excision repair and mismatch repair pathways, thereby producing mutations and doublestranded breaks [41, 45, 48].

The natural diversification processes has allowed for highly diverse antibody repertoires to be generated. This natural phenomenon is the basis of the unique ability of the immune system to counter any foreign infection. The ability to replicate or represent the *in vivo* repertoire in the laboratory is the basis of recombinant antibody technology. The need to reproduce this feature is achieved for example by the construction of antibody phage libraries. The robust nature of combinatorial technologies has enabled easy selection of monoclonal antibodies from highly diverse naïve, immune and synthetic repertoires by coupling it with biopanning processes [49].

Phage display enables the sorting and handling of large antibody libraries. Antibody phage libraries consist of a random collection of antibody variable genes being presented as a fusion to phage coat proteins. The antibody fragments can be expressed as a fusion protein on the surface of phages, without affecting the infectivity of phages [50]. Moreover, the displayed antibody molecules retains its antigen-antibody binding capabilities [51]. However, the challenge in generating high affinity antibodies is closely related to the quality of the library generated. Even so, the advancement of recombinant DNA technologies has allowed for downstream affinity maturation processes to be carried out for the improvement of antibody affinities post-selection [52, 53].
