**2. Antibody libraries**

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 bind-

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

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 double-

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

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

ing diversities to various antigens [35, 36].

22 Antibody Engineering

maturation of antibodies.

stranded breaks [41, 45, 48].

processes [49].

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 antibody libraries applied for antibody display [56].

#### **2.1. Naïve antibody libraries**

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 to increase success rates for obtaining high affinity antibodies against multiple antigens by successive rounds of selection. The main advantage of a naïve library is the ability to screen for antibodies against any antigens. This comes with a huge drawback, in which the antibodies are of lower affinities than from immunized clones [56, 60]. However, this issue can be solved and improved by affinity maturation *in vitro* to yield high affinity antibody against a specific antigen. Other shortcomings that can affect the library quality are inconsistent levels of variable antibody gene expression and the limitation of IgM to exhibit diversity, as well as increase chances of cross-reactivity [15]. To improve library quality, one of the method is to randomize the CDR regions of the variable genes while maintaining the original frameworks of the naïve library, this results in further diversification and modifications, becoming a semi-synthetic library [60].

of pre-defined designs of the framework and the degree of randomization of the CDRs. The design pattern is generated based on bioinformatics analysis using existing experimental data on antibody epitopes, antigen-antibody interactions, affinity maturation designs, variable gene segments recombination, and structural predictions on variable regions to yield desirable synthetic repertoires. These studies provide insights to the hypervariable regions on amino acid predominance and variabilities [60]. The hypervariable regions (CDR loops) have been shown to exhibit some amino acid biases. In particular, certain residues (G, P, S, N, H, L, and Y) are predominantly found on CDR loops that are associated with improved antigen binding [64]. Designing CDR sequences that mimic the natural diversity can help circumvent selection of low affinity binders. The two major synthetic antibody libraries available, HuCAL® and n-CoDeR® are based on two separate platforms. Their models will

High Affinity Maturated Human Antibodies from Naïve and Synthetic Antibody Repertoires

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

25

A novel concept of synthetic human library construction, named Human Combinatorial Antibody Library (HuCAL) uses more than one framework sequence to construct the library. The HuCAL construction is based on modular consensus frameworks, consisting of seven VH and seven VL consensus sequences to represent the major germline families, yielding 49 possible combinations of master genes [65]. The master genes are designed such that different frameworks promote different structural diversity of human antibodies while unfavorable residues that cause protein aggregation are removed. Furthermore, HuCAL is characterized by having unique restriction sites flanking all CDRs of the antibodies as well as usage of phage display and unique expression vectors. This allows for a seamless conversion to differ-

In HuCAL, the CDR3 regions are designed to exhibit natural amino acid composition and distribution as well as length variation at each position for each framework. The CDR is synthesized using trinucleotide mixtures (TRIM technology), which offers the elimination of stop codons and redundant amino acid residues in order to optimize CDR design for downstream production of encoded antibodies. TRIM technology uses trinucleotide phosphoramidites to add three bases at a time to a growing single strand of synthetic DNA [68]. The addition of three bases allows for the design and pre-determination of specific codons to be added. In addition to codon optimization for *E. coli*, improved accuracy of antibody design would then be possible. This ultimately improves the functional library size of HuCAL as well as the diversity by having higher number of clones with correct assembly, devoid of frameshifts,

There have been different versions of the HuCAL library being constructed over the years, each with different characteristics. The initial HuCAL focuses on the scFv library construction using 49 master genes, resulting in high expression levels of HuCAL-scFv antibodies (2 × 109 clones) and nanomolar range of affinities to several antigens tested, such as haptens, DNA, peptides, and proteins [69]. HuCAL GOLD® is a synthetic Fab library, generated by diversifying six CDRs that mimics the natural diversity. Affinities of antibodies generated from this

be discussed further as case studies.

stop codons and deletions [69].

*2.3.1. Case study of synthetic antibody libraries: HuCAL®*

ent antibody formats, for instance scFv and Fab [66, 67].

#### **2.2. Immune antibody libraries**

The source of antibody genes for immune library generation is mainly focused on using IgG mRNA from disease-infected individuals or cancer patients. This may include patients with acute infections, recovery stage or patients which have recovered from a particular disease or infection [15]. In addition to that, cancer derived material can also be used as a source [61]. The unique characteristic of an immune repertoire is that the sample material is obtained from activated B-cells, where affinity maturation has taken place during antigen encounter [47]. Thus, it is easier to obtain high affinity binders specific to an antigen from immune libraries in comparison to naïve libraries due to the biased nature of the repertoire post-exposure of the antigen. The size of an immune library need not be as large as naïve libraries per se, but it can also be applied for other targets but may not be suitable for self-antigens [56, 62]. The obvious limitation of an immune library is the possibility of generating immune libraries of human donors against various targets. Therefore, the application of immune libraries from humans is mainly confined to disease-infected individuals [60] or cancer patients [61]. The biased nature of the library repertoire also means that the library is mainly useful against the antigen used for immunization. Therefore new libraries are required when dealing with targets of different diseases [62]. However, it is also possible for immune libraries to successfully enrich antibodies against non-related targets of the disease of origin. This indirectly indicates the influence of B-cell memory during immune responses that provides an extended breath of protection for individuals.

#### **2.3. Synthetic antibody libraries**

The main difference between naïve and immune libraries with synthetic libraries is the source of the repertoire used to build the library. While both naïve and immune libraries are amplified from a natural source, synthetic libraries are designed *in silico* and the repertoire is generated in controlled conditions [49, 56]. The artificial repertoire is generated from the diversity afforded by the randomization of the CDR using synthetic approaches [63]. The basic design of most synthetic libraries is the randomization of various CDRs using degenerated oligonucleotides. The freedom afforded by synthetic libraries is the possibility of pre-defined designs of the framework and the degree of randomization of the CDRs. The design pattern is generated based on bioinformatics analysis using existing experimental data on antibody epitopes, antigen-antibody interactions, affinity maturation designs, variable gene segments recombination, and structural predictions on variable regions to yield desirable synthetic repertoires. These studies provide insights to the hypervariable regions on amino acid predominance and variabilities [60]. The hypervariable regions (CDR loops) have been shown to exhibit some amino acid biases. In particular, certain residues (G, P, S, N, H, L, and Y) are predominantly found on CDR loops that are associated with improved antigen binding [64]. Designing CDR sequences that mimic the natural diversity can help circumvent selection of low affinity binders. The two major synthetic antibody libraries available, HuCAL® and n-CoDeR® are based on two separate platforms. Their models will be discussed further as case studies.

#### *2.3.1. Case study of synthetic antibody libraries: HuCAL®*

to increase success rates for obtaining high affinity antibodies against multiple antigens by successive rounds of selection. The main advantage of a naïve library is the ability to screen for antibodies against any antigens. This comes with a huge drawback, in which the antibodies are of lower affinities than from immunized clones [56, 60]. However, this issue can be solved and improved by affinity maturation *in vitro* to yield high affinity antibody against a specific antigen. Other shortcomings that can affect the library quality are inconsistent levels of variable antibody gene expression and the limitation of IgM to exhibit diversity, as well as increase chances of cross-reactivity [15]. To improve library quality, one of the method is to randomize the CDR regions of the variable genes while maintaining the original frameworks of the naïve library, this results in further diversification and modifications, becoming

The source of antibody genes for immune library generation is mainly focused on using IgG mRNA from disease-infected individuals or cancer patients. This may include patients with acute infections, recovery stage or patients which have recovered from a particular disease or infection [15]. In addition to that, cancer derived material can also be used as a source [61]. The unique characteristic of an immune repertoire is that the sample material is obtained from activated B-cells, where affinity maturation has taken place during antigen encounter [47]. Thus, it is easier to obtain high affinity binders specific to an antigen from immune libraries in comparison to naïve libraries due to the biased nature of the repertoire post-exposure of the antigen. The size of an immune library need not be as large as naïve libraries per se, but it can also be applied for other targets but may not be suitable for self-antigens [56, 62]. The obvious limitation of an immune library is the possibility of generating immune libraries of human donors against various targets. Therefore, the application of immune libraries from humans is mainly confined to disease-infected individuals [60] or cancer patients [61]. The biased nature of the library repertoire also means that the library is mainly useful against the antigen used for immunization. Therefore new libraries are required when dealing with targets of different diseases [62]. However, it is also possible for immune libraries to successfully enrich antibodies against non-related targets of the disease of origin. This indirectly indicates the influence of B-cell memory during immune responses that provides an extended breath of protection

The main difference between naïve and immune libraries with synthetic libraries is the source of the repertoire used to build the library. While both naïve and immune libraries are amplified from a natural source, synthetic libraries are designed *in silico* and the repertoire is generated in controlled conditions [49, 56]. The artificial repertoire is generated from the diversity afforded by the randomization of the CDR using synthetic approaches [63]. The basic design of most synthetic libraries is the randomization of various CDRs using degenerated oligonucleotides. The freedom afforded by synthetic libraries is the possibility

a semi-synthetic library [60].

24 Antibody Engineering

for individuals.

**2.3. Synthetic antibody libraries**

**2.2. Immune antibody libraries**

A novel concept of synthetic human library construction, named Human Combinatorial Antibody Library (HuCAL) uses more than one framework sequence to construct the library. The HuCAL construction is based on modular consensus frameworks, consisting of seven VH and seven VL consensus sequences to represent the major germline families, yielding 49 possible combinations of master genes [65]. The master genes are designed such that different frameworks promote different structural diversity of human antibodies while unfavorable residues that cause protein aggregation are removed. Furthermore, HuCAL is characterized by having unique restriction sites flanking all CDRs of the antibodies as well as usage of phage display and unique expression vectors. This allows for a seamless conversion to different antibody formats, for instance scFv and Fab [66, 67].

In HuCAL, the CDR3 regions are designed to exhibit natural amino acid composition and distribution as well as length variation at each position for each framework. The CDR is synthesized using trinucleotide mixtures (TRIM technology), which offers the elimination of stop codons and redundant amino acid residues in order to optimize CDR design for downstream production of encoded antibodies. TRIM technology uses trinucleotide phosphoramidites to add three bases at a time to a growing single strand of synthetic DNA [68]. The addition of three bases allows for the design and pre-determination of specific codons to be added. In addition to codon optimization for *E. coli*, improved accuracy of antibody design would then be possible. This ultimately improves the functional library size of HuCAL as well as the diversity by having higher number of clones with correct assembly, devoid of frameshifts, stop codons and deletions [69].

There have been different versions of the HuCAL library being constructed over the years, each with different characteristics. The initial HuCAL focuses on the scFv library construction using 49 master genes, resulting in high expression levels of HuCAL-scFv antibodies (2 × 109 clones) and nanomolar range of affinities to several antigens tested, such as haptens, DNA, peptides, and proteins [69]. HuCAL GOLD® is a synthetic Fab library, generated by diversifying six CDRs that mimics the natural diversity. Affinities of antibodies generated from this library are able to achieve picomolar range when tested on different target molecules [63]. The latest optimized version, HuCAL PLATINUM® has a more advantageous design focusing on the optimization of CDR3 sequences in the modular sequence in order to yield antibodies with improved folding and enhanced binding [70]. The optimization includes avoiding N-glycosylation sites and unproductive sequences to maximize the sequence space and availability. In addition, the library is improved to enhance antibody expression in both bacterial and mammalian expression systems. Sequence optimization on nucleotides has been extensively carried out during library construction, therefore Fab fragments and IgG formats can be expressed optimally in both bacterial and mammalian systems, respectively. The resulting library offers higher diversity than the HuCAL GOLD® library [64, 70].

the antibody genes. Antibodies isolated from combinatorial libraries against their respective targets sometimes may not exhibit the desired specificity and affinity. The increased affinity of an antibody is important to enhance its pharmacokinetics, efficacy and safety profile by enhancing the binding strength and function of an antibody [75]. Such optimizations can be

High Affinity Maturated Human Antibodies from Naïve and Synthetic Antibody Repertoires

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

27

There are several strategies that have been used to perform *in vitro* affinity maturation to improve recombinant antibody molecules. Mutagenesis is widely employed to introduce mutations into antibody sequences. Sequences of antibody are diversified by random mutations via methods such as error-prone PCR or through site-directed mutations, where mutations are assigned to specific positions in CDRs or framework regions as well as mutational hot spots by using PCR and degenerate primers [76]. In addition, *de novo* synthesis of DNA offers the most straightforward modification procedure to further diversify the antibody

**Random mutagenesis** is a non-systematic mutagenesis method that can be performed in the absence of information regarding the importance of structures and residues that contribute to antigen-antibody binding as well as affinity maturation of antibody [77]. The method introduces point mutations into antibody genes in a random fashion. The mechanisms involves: (1) transitions, where a purine or pyrimidine is substituted by another purine or pyrimidine, (2) transversions, where a purine is substituted by a pyrimidine, or *vice versa*, (3) deletions of one or more nucleotides from the gene sequence, (4) insertions of one or more nucleotides into a gene sequence, (5) inversions where double-stranded DNA segments of two base pairs or

Error-prone PCR is a universal method used for the introduction of random mutations by capitalizing on the natural error rate of a low fidelity DNA polymerase, for example *Taq* polymerase that lacks 3′ to 5′ proofreading activity. Several parameters during PCR amplification govern the error rate of DNA polymerases in order to create ideal mismatches in the amplified product. The manipulation of the enzyme's fidelity can be performed by varying several parameters like: (1) concentration of *Taq* DNA polymerase, (2) concentration of divalent cations (Mn2+ and Mg2+), (3) concentration of deoxyribonucleoside triphosphates (dNTPs), (4) polymerase extension time and the (5) number of PCR cycles [79, 80]. Upon amplification, the product must be ligated to a suitable plasmid and an additional step is required to recover the transformants that consist of the mutations. Error-prone PCR is a robust technique, whereby it can only introduce limited amount of base substitutions into the gene sequence. Therefore it is very useful to identify amino acid positions that are associated with function, affinity and specificity of antibodies for the method to be applied on [1]. The resulting libraries consist of a large amount of A to G and T to C transitions, thus causing high GC content amplification bias. This limitation can be circumvented by the addition of unbalanced ratios of nucleotides to reduce the amplification bias. A commercial DNA polymerase, Mutazyme® was introduced for error-prone PCR with reduced mutational bias which overcomes the issue of preferential nucleotide base selection by *Taq* DNA polymerase during amplification [78]. Error-prone

achieved either by *in vitro* or *in vivo* affinity maturation strategies.

**3.1.** *In vitro* **approaches toward affinity maturation of antibodies**

sequences as a whole.

longer is rotated at 180° [78].

#### *2.3.2. Case study of synthetic antibody libraries: n-CoDeR®*

The principle of the n-CoDeR® library is based on the recombination of a single framework with multiple CDRs from non-immunized donors to generate functional diversity [71]. This approach allows the retrieval of CDR loops from immunoglobulin genes from different germline origins. All CDR loops are successfully recombined into one single VH-V<sup>L</sup> scaffold, while maintaining reactivity and functionality of the antibody fragments [72]. The underlying concept of constructing the n-CoDeR® library is the amplification of desired CDR loops from immunoglobulin cDNA with overlap extension and assembly being performed to place the CDRs into the single framework [73]. The use of CDR loops originating from the human immune system is said to be remarkable as the sequences obtained have undergone *in vivo* processing, thus such sequences are said to have undergone proof-reading and the functionality has been confirmed [74]. The resulting genetic diversity of this library is remarkably enormous (2 × 109 ), and has the potential to yield diversities equaling the human immune system [56].

This library appears to be a suitable candidate for therapeutic and diagnostic applications as it can generate functional antibody fragments against many types of antigens. Initially the approach of using a single framework to present various types of CDR loops was seemed risky due to the limitation in capacity. It was later proved to be successful with the isolation of antibodies specific to various types of antigens reaching affinities in the sub-nanomolar range. Another benefit afforded by this approach was the ability to select a single framework that can customize desirable characteristics and properties, as well as ensuring that antibodies can be generated which can be produced and folded in good condition [56]. Antibodies harnessed from the n-CoDeR® library are potentially advantageous for therapeutic purpose as they demonstrated a lower number of T-cell epitopes than normal antibodies. It indicates that self-reactivity is therefore circumvented and immunogenicity issues are reduced [72].
