Recombinant human antibody technology has been the cornerstone of the uprising of biologics in the pharmaceutical industry. The introduction of various display technologies like phage, yeast, bacterial, ribosomal, mRNA, DNA display and mammalian cell surface display has allowed improved antibody generation programs. The ability to generate recombinant antibodies from available human antibody libraries by using in vitro display methods pave the way to select recombinant human antibodies against almost every antigen. The libraries are a close representation of the B-cell response elicited by the natural immune system. The introduction of various methods to fine tune the antibody affinities has made recombinant antibody technology highly sought after. The ability to engineer specific characteristics of each antibody by design is possible utilizing advanced in vitro strategies. This chapter will focus on the technologies commonly applied in antibody display technologies to engineer improved affinities.
- naïve- immune- and synthetic- antibody library
- in vitro display systems
- affinity maturation
- de novo antibody gene synthesis
- genome editing techniques
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, 2, 3, 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 . This specificity is programmed
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 . 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 . 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 . This means that it was now possible to replicate the
1.1. Display technologies
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 .
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 . Figure 1 shows the alternative display systems used for antibody presentation.
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 . The covalent display technology (CDT) exploits the properties of the replication initiator protein from
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 . 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 . 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 . 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
The human antibody repertoire represents a diverse collection of immunoglobulin gene segments that encodes for heavy (VH) and light chain (VL) domains , 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 .
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 . 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 , 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-JL 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 . This natural repertoire is known as the naïve or primary repertoire, expresses cell surface IgM and has not undergone specialization by antigen encounter . 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 . 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].
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  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 . 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-stranded 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
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 . Moreover, the displayed antibody molecules retains its antigen-antibody binding capabilities . 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].
2. Antibody libraries
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  that either preferentially or concomitantly contributes to the binding affinity of the antibody to the target antigen . 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 . 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 .
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 . 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) . 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
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 . In addition to that, cancer derived material can also be used as a source . 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 . 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  or cancer patients . 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 . 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
2.3.1. Case study of synthetic antibody libraries: HuCAL®
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 . 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 . The addition of three bases allows for the design and pre-determination of specific codons to be added. In addition to codon optimization for
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 . 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 . 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 . 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].
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 . 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-VL scaffold, while maintaining reactivity and functionality of the antibody fragments . 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 . The use of CDR loops originating from the human immune system is said to be remarkable as the sequences obtained have undergone
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 . 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 .
3. Affinity maturation strategies for recombinant antibodies
Recombinant antibodies obtained via combinatorial library technology from naïve or synthetic libraries have the advantage of increased diversity as a result of the large repertoire of 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 . Such optimizations can be achieved either by
In vitro approaches toward affinity maturation of antibodies
There are several strategies that have been used to perform
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
Recombination provides another approach for gene modification and diversification. Mutational rearrangements are highly advantageous to identify and obtain beneficial mutational combinations otherwise absent in nature. Chain shuffling is a process that serves as a “mix and match” system to increase gene repertoires. Chain shuffling allows for one of the two antibody chains (heavy or light chains) to be paired with a repertoire of partner chains to generate a secondary library in order to produce higher affinity antibodies. The domain shuffling is a useful affinity maturation tool for antibodies as it mimics the
Mutagenesis on a single-stranded DNA template (ssDNA) is labor intensive because the template requires subcloning and ssDNA rescue. Therefore, several commercial kits are available that utilizes double-stranded DNA (dsDNA) as template for site-directed mutagenesis with mutagenic primers. The QuickChange™ system (Stratagene) uses a pair of complementary oligonucleotides (forward and reverse) that consists of the desired mutations to amplify the whole plasmid with high fidelity polymerase, followed by removal of the parental DNA using
Another variant of site-directed mutagenesis is a PCR-driven method termed as overlap extension PCR. This technique employs PCR to generate modified genes from cloned DNA with just a few simple steps. The segments of a target gene are amplified from a DNA template using two flanking master primers and two internal primers. The internal primers consist of the desired mutation and overlapping nucleotide sequences. Two rounds of PCR are carried out, first by amplifying the target genes with their respective pair of primers to create two gene fragments that share some overlapping sequences at the 3′ end. Subsequently, these double-stranded duplexes are denatured and annealed, resulting in two heteroduplexes with each strand consisting of the mutated site. Then DNA polymerase functions to extend the overlapping ends of each heteroduplexes. A second PCR is done with the use of two flanking master primers to amplify the entire modified gene [90, 91]. This method was recently employed by Kitzman et al.  to create massive single amino acid mutagenesis in a parallel fashion coupled with microarray-based DNA synthesis technology. This is particularly useful for assessing and screening of variants in libraries.
The increased understanding of molecular biology and specific functions of molecular biology enzymes has allowed the introduction of different approaches for mutagenesis. The combination of the different function of various enzymes has been utilized successfully to carry out directed evolution of antibody genes. Lambda exonuclease in nature functions to assist the repair of dsDNA breaks of viral DNA. It is a highly processive 5′→3′ dsDNA exonuclease which selectively degrades the phosphorylated chain of a duplex DNA to yield mononucleotides and ssDNA. A strategy that takes advantage of this feature of lambda exonuclease was applied for antibody gene mutagenesis. The formed ssDNA template will function as the template for
The diversification of the antibody repertoire can also be realized by
Typically, the cytidines are targeted at the mutational hotspot motif RGYW and AGY (R = A/G, Y = C/T, W = A/T). This motif is also the preferred region for mutations during
Studies have been carried out to analyze the amino acid diversities in the germline and mature antibody sequences. It was found that the number of germline hotspots decreases in high affinity antibodies, suggesting that hotspot-based somatic mutations occurred via
The diversity associated with the utilization of various sequences either in the CDR or framework is directly related to the affinity of the clones generated [5, 103]. The continuous development in molecular technologies has allowed the introduction of various approaches for gene modification. The design of the framework regions in the antibody gene also plays a contributing role in the improvement of the antibody affinity. This is due to the influence of the framework genes on the stability, solubility and affinity of the antibody . The framework regions mainly in the neighboring regions of the CDRs have been known to also contribute to the binding characteristic of antibody clones .
De novo synthesis of antibody genes
Direct gene synthesis of modified sequences or
Lastly, targeted sequences from natural constructs are sometimes hard to access, therefore synthesis provides a more efficient alternative to retrieve the targeted sequences . Currently, oligos are generated or synthesized automatically, employing solid-phase phosphoramidite chemistry. The principle behind phosphoramidite-based oligo synthesis encompasses a total of four key steps (deprotection, coupling, capping and oxidation) to add one base at a time to a growing oligo chain attached to a solid support.
Synthesis takes place individually in small columns. The purified oligos are then subjected to quality assessments. The automated process can generate up to 100 nmol of oligos at a time with low error rates in the region of one base error in 200 nucleotides [105, 106].
Besides conventional gene synthesis from oligo fragments using column-based synthesized oligos, array-based oligos can be used for gene synthesis as well. An array-based synthesis has the advantage of high throughput synthesis. The polymer array support by Affymetrix is synthesized chemically comprising photolabile protecting groups and photolithography. The photolithographic mask is able to direct UV light over the solid substrate and selectively deprotect and activate the 5′-hydroxyl group in the growing chain, in order for free nucleotides to be incorporated into the chain. The mask is designed for exposing targeted sites on the microarray, where incorporation of nucleotides occurs while masking other non-targeted sites. The oligo fragments are directly synthesized on the support surface, and can be recovered as a heterogenous pool of sequences. Today, several technologies have surpassed the need to use the masking technique. An ink-jet-based printing developed by Agilent allows picolitres of free nucleotides and activator to be spotted on targeted sites on one array. NimbleGen Systems uses the programmed automated micromirror device to activate specific sites on the array. Furthermore, CustomArray (CombiMatrix) utilizes semiconductor-based electrochemical acid production to deprotect desired nucleosides [107, 108].
Nevertheless, the NimbleGen and CustomArray oligo synthesis techniques suffer high error rates when trying to generate longer and multiple oligo strands in parallel. This is due to the side reactions such as depurination and inefficient addition of nucleotides that results in unwanted substitution and indels (insertion/deletion) errors, which greatly affects the overall quality of the synthesized product. Therefore purification steps utilizing polyacrylamide gel electrophoresis and high performance liquid chromatography are essential to remove erroneous sequences upon generating the intended DNA sequences .
The generated oligo fragments obtained after conventional or area-based synthesis are then used as raw substrates to construct larger synthetic fragments (usually few hundreds of base pairs), also known as gene synthesis. Using a ligation-based approach, the complementary overlapping fragments are joined enzymatically by the thermostable DNA ligases, producing larger DNA fragments under high stringency . Another approach, known as polymerase cycling assembly (PCA)-based method, utilizes polymerase to elongate the originated overlapping oligo fragments into double-stranded fragments . Ligation-based synthesis offer higher stringency, therefore error in sequences is less likely to be assembled, but the oligo synthesis are costly due to synthesis of longer fragments. The longer oligonucleotides will allow for better annealing and less steps in comparison to shorter oligonucleotdies. The final step would sometimes involve an additional Polymerase chain reaction (PCR) amplification step to yield more material for cloning. On the contrary, PCA-based methods are more cost-effective as it relies on overlapping short oligo fragments (15–25 nt) per gene synthesis. However, this approach promotes higher error rates due to the lack of error elimination during hybridization . Also, target diversity can be introduced at the regions where the overlapping oligo fragments hybridizes .
Despite the fact that concentrations of individual oligos on the array are quite low and insufficient for priming, as well as the error rates of the oligo pools are higher as the column-based methods, there are successful examples that overcome these challenges. This is done with the use of programmable DNA microchips with an array of oligonucleotides and their selection . To increase the concentration of oligonucleotides for gene synthesis, amplification of oligo fragments before assembly is required. Sequence errors can be detected via hybridization of the synthetized cleaved oligonucleotides to complementary oligonucleotides spanned on a second area. Lastly error-free fragments will be assembled into full-length sequences. However, this method is not feasible for assembling a large pool of oligos because of the risk of cross-hybridization based on the huge diversity. Another approach used selective oligonucleotide pool amplification directed by predesigned barcodes to generate and assemble particular DNA fragments that are required to make a full gene before the barcodes are digested prior to full gene assembly . Recently, this approach was applied to construct few scFv gene libraries with degenerate oligonucleotides synthesized on two DNA microchips in parallel . The humanized anti-ErbB2 antibody (HuA21) was targeted to diversify the CDR regions via a small perturbation mutagenesis method and was validated using deep sequencing by the Illumina platform. Finally, the mutant candidates were screened by phage display to select for high affinity binders [115, 116].
gBlocks gene fragments are readily usable short-to-medium length synthesized DNA fragments that contains particular desired gene modifications. gBlocks are dsDNA blocks that undergo controlled synthesis allowing various applications for antibody and protein engineering. The main application focuses specifically on gene construction and editing. gBlocks are constructed using gene fragment libraries (pools of short DNA fragments that comprise 18 consecutive N bases or K (G,T) bases). The synthesized product is then subjected to various quality control tests such as capillary electrophoresis (fragment length) and mass spectrometry (sequence composition) to verify the final product and reduce potential errors. For gene editing, gBlocks can introduce modifications such as deletion or insertion on relatively short stretches of DNA fragments. The primers are designed to target the region of the gene that is to be edited. Subsequently, the region will be cleaved and replaced by the gBlock . This method has allowed the design and generation of antibody libraries [117, 118].
In vivo approaches towards affinity maturation of antibodies
Bacterial mutator strains, such as
The advancement of genome editing technologies offers a new approach to create sequence diversity. In fact, cells can repair DNA damages intrinsically by joining two ends together or filling the gap with similar sequences. However, cells can also repair the break by using a new piece of DNA that has the desired mutation. This is the basis of
Meganucleases are derived from microbial mobile genetic elements that integrate nuclease and DNA binding domains. ZF nucleases (Cys2His2 bound to a single atom of zinc) are eukaryotic transcription factors that contain the DNA binding domain and is similar to a set of three fingers, with each finger contacting with 3 nucleotides of DNA . TALEs are produced naturally by
While other approaches have their own limitations, the robustness of the CRISPR-Cas9 system sheds some light on direct endogenous genome editing on virtually any organism of choice. Meganuclease lacks target specificity, which is why it is not widely employed. However, ZF domains have a tendency to crosslink with neighboring protein -domains or -complexes resulting in lower binding efficiency towards DNA targets. Although TALEs require only one nucleotide for binding towards target, however the synthesis for novel TALEs is costly due to their repetitive sequences . Nevertheless, these enzymes are constructed in customizable fashion to cater for the need of genome editing, as well as programming the enzymes for multiplex gene targeting . Some model organisms were tested with the genome editing technologies, such as zebrafish, rats, mice,
Naïve and synthetic human antibody repertoires are a very valuable source for the selection of antibodies against nearly any antigen. The role display technologies play in the quest to generate monoclonal antibodies from these libraries is obvious with the increasing number of antibody lead candidates going into clinical trials.
Affinity maturation of selected binders is now possible by expressing for example the AID enzyme during selection of antibodies using antibody mammalian cell surface display or by using a pool of microchip-synthesized CDRs incorporated into an antibody framework. Selection of naïve and synthetic recombinant antibodies combined with
The authors would like to acknowledge the support from the Malaysian Ministry of Higher Education under the Higher Institution Centre of Excellence (HICoE) Grant (Grant no. 311/CIPPM/44001005).
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