Biotechnol Lett DOI 10.1007/s10529-014-1635-x
REVIEW
Principles and application of antibody libraries for infectious diseases Bee Nar Lim • Gee Jun Tye • Yee Siew Choong Eugene Boon Beng Ong • Asma Ismail • Theam Soon Lim
•
Received: 2 June 2014 / Accepted: 11 August 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Antibodies have been used efficiently for the treatment and diagnosis of many diseases. Recombinant antibody technology allows the generation of fully human antibodies. Phage display is the gold standard for the production of human antibodies in vitro. To generate monoclonal antibodies by phage display, the generation of antibody libraries is crucial. Antibody libraries are classified according to the source where the antibody gene sequences were obtained. The most useful library for infectious
B. N. Lim G. J. Tye Y. S. Choong E. B. B. Ong A. Ismail T. S. Lim (&) Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia e-mail:
[email protected] B. N. Lim e-mail:
[email protected] G. J. Tye e-mail:
[email protected] Y. S. Choong e-mail:
[email protected] E. B. B. Ong e-mail:
[email protected] A. Ismail e-mail:
[email protected] T. S. Lim ADAPT Research Cluster, Centre for Research Initiatives Clinical & Health Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia
diseases is the immunized library. Immunized libraries would allow better and selective enrichment of antibodies against disease antigens. The antibodies generated from these libraries can be translated for both diagnostic and therapeutic applications. This review focuses on the generation of immunized antibody libraries and the potential applications of the antibodies derived from these libraries. Keywords Antibody libraries Antibody phage display Immunized antibody libraries Infectious diseases
Introduction Antibodies are a unique product of the immune system and are commonly used for diagnostic or therapeutic applications. Antibodies are highly desirable due to their specificity and affinity to a specific target antigen. However, the production of murine monoclonal antibodies (mAb) and/or antibody fragments is a challenge. The conventional approach to the production of murine mAb is the hybridoma technology by Kohler and Milstein (1975). The process requires the introduction of a specific target antigen to an animal host supplemented with adjuvants to elicit an immune response towards the target antigen. Murine B lymphocytes are fused with myeloma cells to generate hybrid cells called hybridomas. These hybridomas will have the ability to propagate indefinitely in
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culture and secrete murine mAbs in culture. This way, highly specific antibodies are generated as a result of the acquired immune response. The era of biotechnology and recombinant DNA brought about several new display technologies that physically link the antibody genotype and phenotype on a single phage particle (Winter et al. 1994). Of the many display methods available, phage display has been successfully utilized for the display and isolation of desirable antibody molecules against specific target antigens. Phage display, first introduced in 1985, was used to display peptides on the surface of a filamentous phage (Smith 1985). From this, other researchers applied the use of phage display to present antibody fragments for selection (Hammers and Stanley 2014). The robust nature of phage made it very popular for use, as it is able to withstand extreme pHs, temperatures, non-aqueous solutions, DNase, exposure to UV and even proteolytic enzymes. The ability to withstand such extreme conditions could bring about serious contamination issues. However, when handled well, these features permits changes in the selection conditions during the panning process to customize the antibody characteristics required (Schirrmann et al. 2011). The only requirement is the availability of an antibody library for use. Antibody libraries are a collection of unique antibody clones, which are introduced using molecular techniques to a suitable vector for the packaging of phage particles presenting antibody molecules on its surface. To date, there is a foray of strategies used to generate antibody libraries. This resulted in various types of libraries being generated for different applications (Thie et al. 2008). However, the technology is useful for infectious diseases especially when drug-resistant strains have emerged. Discovery of novel antibody based therapies enables the treatment of diseases caused by such potentially life-threatening strains. In this review, we will focus on the application of immunized antibody libraries for the production of therapeutic antibodies for infectious diseases. The description of immunized libraries with different panning strategies and the possible applications will be discussed mainly for infectious diseases.
Generation of immunized antibody libraries Antibody libraries are generally categorized as immune, naı¨ve, semi-synthetic or synthetic libraries
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(Miersch and Sidhu 2012). The antibody library category is dependent mainly on the source of the antibody sequences. Immune and naı¨ve libraries are generated exclusively from naturally-occurring sequences. Here the diversity generated is mainly the product of somatic hypermutation (Di Noia and Neuberger 2007). Hence, the diversity is a representation of the response by the immune system towards an immunological insult. Large naı¨ve libraries can produce many antibodies to a wide array of antigens but with lower affinities in general (Pansri et al. 2009). However, semi-synthetic and synthetic libraries are devoid of any natural immune maturation process whereby synthetic genes are used to generate diversity (Miersch and Sidhu 2012). To further differentiate semi-synthetic from fully synthetic libraries, the former is a combination of natural diversity together with in silico design of framework sequences. Fully synthetic libraries are diversified according to predetermined designs dependent on requirements. As semi-synthetic and synthetic antibody libraries are dependent on synthetic oligonucleotides to generate the diversity required for highly diverse, complementarity-determining regions (CDR), combinatorial mixing is required (Rothe et al. 2008). Natural source libraries, however, are also dependent on a certain degree of combinatorial mixing. The combinatorial mix of naı¨ve and immune libraries are accomplished by chain pairing of heavy and light chains with little preference shown only for germline sequences (Jayaram et al. 2012). The combinatorial mixing process makes antibody library generation very challenging as the diversity of the library is vital to the success of the library. Some of the technical challenges include transformation efficiency and frame shift mutations that result in non-functional antibody sequences. One of the main complications in antibody library generation is the technical difficulties associated with cloning. Conventional approaches using restriction enzyme digestion and ligation are mostly used although they may prove to be ineffective at times. This, in turn has brought about an array of new molecular-based techniques such as PCR-based assembly (Andris-Widhopf et al. 2000), Ligation Independent Cloning (Thieme et al. 2011), temperature cascade assembly (Loh et al. 2012) and other variants of molecular methods for the generation of highly diverse libraries (Sotelo et al. 2012). Although the cloning of a highly diverse antibody library can be
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Fig. 1 Comparison of the different types of antibody based libraries generated for display methods. Naı¨ve libraries are generated using cells from healthy donors whereas immunized
libraries obtained cells from patients or immunized animals. Synthetic and semi-synthetic libraries are designed in silico and synthesized by chemical means
difficult, laborious, costly and time consuming, the return of investments in generating such libraries are multifaceted. Figure 1 shows the differences between each of the antibody library available. Immune antibody libraries are unique in comparison to other forms of libraries as they are derived from immune donors. The donors can either be immunized animals or patients who show protection or have recovered from an infection without the need of treatment (Ayat et al. 2013). By definition, immune antibody libraries are merely antibody cDNA expression libraries obtained from B cell lymphocyte pools. The cDNA can be obtained from any host (human or animal), which has been immunized, infected or exposed (Saggy et al. 2012). As there are so many diseases in the world today, vaccination against common diseases, such as diphtheria, tetanus, rubella, hepatitis and many more, has become normal. Therefore the collection of samples from a naı¨ve healthy donor may not be a true representation of a naı¨ve repertoire due to prior vaccinations. The ability for
memory cells to remember past infections in a donor’s lifetime may likely influence the true naı¨ve nature of the antibody repertoire. Therefore the definition of an immune library as antibody gene repertoires obtained from clinically or recently infected people would be best suited. The collection of antibody genes obtained from these groups are predisposed to a specific group of disease specific antigens, hence the repertoire is obtained from antigen sensitized B cell IgG mRNA. Therefore a bias is expected from the antibody repertoire to a specific group of antigens. Immune libraries are commonly used to discover novel antibodies against infectious diseases, pathogen exposure, toxins, venoms, lost of tolerance in autoimmunity and conditions whereby immune responses have occurred (Chahboun et al. 2011; Klooster et al. 2007; Zhang et al. 2013). Immune libraries are a good representation of the immune response in vivo as they are able to exhibit antibodies against different antigens at different
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reactivity depending on host responses and immunogen. Therefore, the influence of other immune regulatory mechanisms, such as clonal selection, results in the enrichment of antigen specific B cells. Subsequent editing and maturation of binding domains by processes, such as somatic hypermutation and receptor editing, contributes towards further affinity maturation to generate higher affinity antibodies against presented antigens (Beerli and Rader 2010). This feature is important because the exposure of the host to a specific disease allows the immune mechanism to generate disease specific antibodies hence the production of affinity matured antibodies. The skewed repertoire in immune libraries may limit its use for antigens that are not related to the disease. Immune libraries generally offer high quality and high affinity antibodies but a common restriction is the requirement to generate new libraries for different diseases. Immune libraries are naturally by default biased to specific immunogens due to the pre-defined mechanisms discussed earlier. This is in direct contradiction to the naı¨ve antibody library concept. The basis of diversity generation is a combinatorial mix of the heavy and light chain genes in random. As random pairing of heavy and light chain generates antibody libraries, there is a possibility of obtaining non-natural and non-functional pairings. However, a study has shown that in both heterohybridoma and antibody phage display (APD) the same genes for VH and VL are detected suggesting that the mAbs derived from APD accurately represent the antibodies in patients (Hammers and Stanley 2014). This representation is also true for immune antibody libraries, but due to the maturation process, the repertoire is naturally skewed in comparison to the naı¨ve repertoire. Therefore smaller sized libraries are sufficient for immune antibody libraries as opposed to the larger sized libraries required for naı¨ve repertoires. New novel molecular based methods have been introduced to increase diversity or even refine antibody affinities in vitro. Such methods are implemented either at the early stages of library generation or at the later stages, after specific clones are identified. The common strategies involve chain shuffling methods (Lou and Marks 2010), point mutation (Drummond et al. 2005), CDR randomization or editing (Lim et al. 2012). The strategies used for mutagenesis or sequence evolution can be carried out in vivo or in vitro. In vivo mutagenesis involves the use of
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mutator strains (Low et al. 1996) or cre-lox recombination (Sblattero and Bradbury 2000) to allow higher diversity libraries to be generated. In vitro methods are more frequently used as it allows easier manipulation without the hassle of in vivo systems. In vitro methods such as conventional cloning via restriction enzymes (Christensen et al. 2009), error prone PCR (Fujii et al. 2004), exonuclease (Holland et al. 2013) and oligonucleotide based mutagenesis (GonzalezMunoz et al. 2012) are preferred as the enzymes required are commercially available. These methods allow for the physical substitution of either an entire V-gene or segments of a V-gene with a random oligonucleotide. This way, chain shuffling methods whereby substitution of the entire V-gene of either heavy or light can be completed. This allows for the generation of diverse genes for display on phage. A more focus diversification can be carried out at the positions of the CDR to increase affinities without compromising the canonical structure of the antibody. Figure 2 shows the two main strategies to be used for introducing higher diversity to the generated antibody libraries with examples of the methods used to accomplish it. The different forms of directed evolution can generate customizable antibody fragments for various applications. Even so, the mammalian in vivo immune system that has evolved over time is by far the most intricate and efficient method to produce high-affinity antibodies (Kehoe et al. 2014). This is why immune antibody libraries are still applied for the generation of antibodies specifically for conditions such as infectious diseases.
Antibody selection by panning The in vitro selection process of antibodies from antibody libraries based on target affinity is termed as ‘‘panning’’ (Willats 2002). The process is an iterative process whereby the populations of target specific antibodies are enriched relative to the number of panning rounds. In general, the target antigens are coated on the surface of the solid phase for presentation and will be incubated with antibody bearing phage particles from the library. This will be followed by an incubation step to allow binding of antibodies to the antigen. During this time, physical, chemical or biological parameters can be regulated in order to
Biotechnol Lett Fig. 2 Common approaches for antibody chain shuffling and CDR randomization to increase antibody diversity
select for the characteristics of the antibody required. To remove unbound phage particles from bound phage particles, a wash step is required by which stringency can be controlled. Variations in wash strategies can result in the enrichment of clones with varying characteristics such as affinity and avidity. Finally an elution step is carried out either by enzymatic digestion or pH shift (Hairul Bahara et al. 2013). The eluted phage particles will be amplified by infecting E. coli. Here, the phage particles can either be repackaged for use in the subsequent panning round or for final analysis. The identification of bound phage particles will then be carried out after 4 to 6 rounds of panning. Panning experiments can also be carried out in a high-throughput manner using robotic handlers (Turunen et al. 2009). Evaluation can either be in soluble form of antibodies or antibody presenting phage. The most convenient method for evaluation is by ELISA (Fukushi et al. 2012). The positive clones will then be sequenced to obtain the genotypic information pertaining to the positive clone. As the genetic information of the clone is now available, modifications to the antibody can be done and produced in different host depending on the platform where the antibodies will be used. The availability of the genetic information of the antibodies would also facilitate additional modifications in terms of stability and affinity maturation (Wang et al. 2011). As infectious diseases may include diseases derived from bacterial, viral, fungal and parasitic microbial
groups, the types of target proteins would vary tremendously. As such, careful consideration is required when designing panning strategies to generate mAbs. The panning strategies can be customized to select against varying sample matrixes. The different strategies include cell-based, negative enrichment, semi-automated, microtitre plate, immunotube and next generation sequencer integrated panning (Barzon et al. 2011; Glo¨kler et al. 2010; Steiniger et al. 2007). For most cases, target antigens are coated on different solid phases such as nitrocellulose, magnetic beads, agarose columns, monolithic columns, polystyrene tubes and 96 well microtitre plates (Cho et al. 2007; Mu et al. 2010; Jung et al. 2008a). In the context of working with infectious diseases, the adaptability of the method to different sample matrix is more important to ensure successful enrichment of mAbs. The concepts of the unique panning strategies such as cell-based panning, semi-automated panning, negative selection and next generation sequencer integrated panning are discussed in the following sections. Cell-based panning Cell-based panning is an important method used for the generation of antibodies against cell surface antigens for therapeutic applications. Cell-based panning is similar in concept to other panning methods whereby affinity selection is applied. However, cellbased panning involves the introduction of live cells to
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antibody presenting phage particles. Here, the procedure involves the incubation of antibody phage libraries with intact cells in growth conditions. This way the antigens presented are in their native conformation located on the cell surface. This method overcomes the limitation of using recombinant antigens on various solid phase surfaces. It is also an attractive method as some proteins can be difficult to express in their native conformation, rendering the antibodies generated irrelevant as the recombinant protein is different from protein presented on the cell surface (Yoon et al. 2012). Cell-based panning allows the isolation of mAbs to an antigen or receptor that is either unknown or difficult to purify in its native folded conformation (de Kruif et al. 1995). Cell-based panning methods normally include a subtractiveselection or fluorescently activated cell sorting instrument to improve specificity (de Kruif et al. 1995; Siegel et al. 1997). In infectious diseases, this method allows the generation of antibodies against novel epitopes by overexpression or modification to certain cell surface proteins.
various chemical and enzymatic conjugation methods has made the transition from conventional methods to semi-automated platforms easier (Behrens and Liu 2014; Ta et al. 2012). Another main issue related to the use of automation for panning is the effects of cross contamination. Cross contamination could occur during the colony picking with the colony picker picking colonies from plates that are too dense. Plating of the output clones is a difficult step to be automated. The bottleneck with automation is the physical restrictions of multiple antigen screening in parallel. As more antigens are screened, this would mean that more dilutions of each plate would be required making it unpractical. If the dilution is not optimum, the overwhelming growth of colonies would result in small and dense colonies for picking, increasing the risk of cross-contamination. Even so, with a proper set up the cross-contamination problem would not be a significant issue (Turunen et al. 2009).
Semi-automated panning
A common problem with antibody phage panning experiments is the background binding of non-specific clones. Negative selection panning, as the name entails, involves the mechanism of enriching clones that do not bind to the solid phase bound target. Commonly, this selection method is used to reduce background enrichments during panning rounds. However, this form of selection can also be beneficial to identify specific population of polyclonals from the library used for panning. This way, a very specific pool of polyclonals can be enriched for conventional panning without the presence of non-specific or interfering proteins. This is most useful when panning is done with cells (Lou and Marks 2010). As there are plenty of cell surface markers being presented at any given time, the differentiation of cell populations in respect to disease prevalence is challenging. This is because panning with a naı¨ve library would present the possibility of enriching binders against auto-antigens or normal cell surface proteins. Therefore, negative selection is useful in this context to capture binders against normal cells and moving the unbound phage to disease cells for enrichment (Popkov et al. 2004). This way a more concentrated population of phage polyclonals will be presented to the disease cells. This requires negative selection to be followed by positive
Conventional APD panning using solid phases such as microtitre plates and immunotubes requires a longer time due to the longer incubation period and multiple steps involved in phage rescue. Semi-automated panning using magnetic nanoparticles can help to elevate the restrictions of conventional panning methods. The use of magnetic nanoparticles allows the conjugation of target proteins on the surface and separation of complexes by magnetic force (Schwenk et al. 2007). The main advantage of using magnetic nanoparticles for panning is the effective binding of proteins on the surface of nanoparticles compared to a larger solid phase like microtitre plates. The availability of a larger surface area increases accessibility of proteins to the phage particles for binding. Incorporating automation into the panning process ensures it to be more robust, efficient and reproducible. The semi-automated protocol utilizing a magnetic particle processor has allowed successful generation of mAbs against various antigens (Konthur et al. 2010). One of the main problems associated with the conversion from conventional methods to a semi-automated platform is the tedious preparation process required for antigen conjugation. However, the availability of
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Negative selection panning
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selection and then amplification steps to generate specific antibodies. Next generation sequencing (NGS) integrated panning Phage display panning requires repetitive rounds of selection in order to generate a pool of specific binders. However, the identity of the mAbs will only be known after selection at monoclonal level and DNA sequencing of the clones. This does not allow an in depth analysis of antibody recognition patterns from the panning rounds and becomes a problem because the affinity of the clones enriched will increase proportionately with the number of rounds. Hence, to customize antibody selections, the ability to identify patterns of clonal selection throughout the rounds is necessary (Di Niro et al. 2010). NGS provides sequence information of all antibody repertoires and in depth analysis of selection and enrichment steps for antibody binders against the targets (Li and Zhu 2010). Therefore, the integration of NGS to APD panning allows for high throughput selection and in depth analysis of the panning rounds. With NGS integrated in the panning process, it is possible to completely skip numerous rounds of panning and focus only on frequent sequences instead of proceeding with possible clones (Dias-Neto et al. 2009; Ravn et al. 2010; T’Hoen et al. 2012). In the context of immunized libraries, the use of NGS can aid in the identification of selective clones against specific protein markers in comparison to healthy population libraries for example. The patterns of gene usage as well as the somatic mutation processes can be studied by incorporating NGS into the panning process. Conventional panning strategies also have the risk of losing important clones from the process because the feasibility of sequencing a large number of positive clones from intermediary rounds is not possible. In this instance, NGS data can also identify the loss of certain potentially useful clones throughout the rounds. Antibody-antigen interactions are solely dependent on antigen specificity. NGS panning may only provide sequence based information with no clear correlation to binding capabilities. The confirmation would still require standard immunoassays for binding confirmation. However, it is likely that NGS can help guide the screening process as V-gene sorting of the clones would provide branched information based on frequency of
occurrence by each V-gene family. By mapping the evolution of the antibody V-gene sequences over the panning rounds, the information may shed light on the antigen specificity for clones present over the course of the panning rounds (Georgiou et al. 2014). The array of panning platforms greatly contributes to the evergrowing application of APD for the generation of relevant antibodies for various applications. The generation of various antibodies for different applications can be catered with the different panning methods that introduce different stringencies and analysis.
Application of antibody libraries for infectious diseases Infectious diseases are one of the leading causes of human mortality in the world today. This is due to the emergence of new pathogens in addition to the existing pathogens infecting humans. The emergence of multidrug-resistant strains for certain diseases makes tackling the issue a difficult one (Berkelman et al. 1994). The ability to diagnose and treat diseases rapidly is important for the management of infectious diseases. Therefore, alternative treatments such as antibody-based therapies can complement conventional drug based treatments (Ter Meulen 2011). Most diagnostic methods available for infectious diseases are either protein or DNA based (Chin et al. 2011). Protein based diagnostics are highly dependent on the ability to recognize the protein marker specifically with high sensitivity. The best way to capture protein markers is with the use of antibodies. However, the common bottleneck faced in the application of antibodies for the development of new diagnostics is the ability to generate mAbs at a rapid rate (Nelson et al. 2010). To overcome this, antibody libraries in general can generate the desired antibodies against a specific antigen at a faster rate. For example, APD methods are able to generate/identify mAbs at a fraction of the time required by conventional hybridoma methods (Hairul Bahara et al. 2013). As phage display-derived antibodies are devoid of the common restrictions of animal based antibodies, the use of toxic antigens, such as toxins or even live cultures of the disease, does not impede the antibody generation process. The numerous panning strategies discussed earlier can be used to isolate target antigens that are toxic or lethal to the animal host.
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Biotechnol Lett Table 1 List of antibody libraries used to generate monoclonal antibodies against infectious disease antigens Strain
Target protein
Antibody format
Library source
Type
Ref
Mycobacterium tuberculosis
Antigen 85
scFv
Naı¨ve human
Bacteria
Ferrara et al. (2012)
Vibrio parahaemolyticus
Thermolabile hemolysin (TLH)
scFv
Immunized mice
Bacteria
Wang et al. (2012)
Propionibacterium acnes
Native form
scFv
Bacteria
Jung et al. (2008b)
Yersinia pestis
F1 antigen
scFv
In vitro immunized human Naı¨ve human
Bacteria
Lillo et al. (2011)
Cryptosporidium parvum
Surface antigen S16
scFv
Synthetic
Parasite
Boulter-Bitzer et al. (2009)
Toxoplasma gondii
Protein TgMIC2
scFv
Immunized mice
Parasite
Hoe et al. (2005)
Plasmodium falciparum
Merozoite surface protein 3 (MSP-3)
Fab
Immunized human
Parasite
Lundquist et al. (2006)
Hepatitis B virus
Surface antigen (HBsAg)
scFv
Naı¨ve human
Viral
Zhang et al. (2006)
West Nile virus (WNV)
Envelope glycoprotein (E protein domain III)
Fab
Immunized human
Viral
Duan et al. (2009)
Rhabdoviridae
Glycoprotein/envelope protein
Domain antibody
Naı¨ve llama library
Viral
Boruah et al. (2013)
Dengue virus (DENV)
Envelope glycoprotein (E protein domain III)
Fab
Naive human and mouse
Viral
Moreland et al. (2012)
The application of an immunized library for the generation of mAbs for diagnostic applications allows the isolation of affinity rich binders against the disease protein (Kramer et al. 2005). The skewed antibody profile by the immune library will also allow a higher success rate as opposed to the use of naı¨ve libraries. The ability to produce the mAbs using only bacterial culture is also beneficial as large amounts of antibodies can be produced for application. For diagnostic applications, most antibodies are required to be conjugated or fused to a reporter system for measurements (Roda and Guardigli 2012). Recombinant antibodies generated using phage display can be coupled to various different reporter systems allowing a great degree of flexibility. Coupled with a suitable reporter system, antibodies derived by disease immune libraries can assist in rapid development of new diagnostic assays for infectious diseases (Wang et al. 2013). The modifications may also include the possibility of presenting the antibody libraries in different formats such as domain antibodies, single chain fragment-variable (scFv), fragment antigen binding (Fab) including other formats that could stem from being monovalent to polyvalent specificity (Carter 2006). The different formats are critical in the application of mAbs in diagnostics and
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therapeutics as the different formats have different characteristics such as solubility, heat stability, plasma half-life and size (De Souza et al. 2009). Table 1 shows a list of antibody libraries that were developed for the generation of mAbs against infectious disease targets. Serum therapy was one of the first effective treatments for microbial disease in the early 20th century. However, production of serum from animal sources limits its use in humans as it can cause immunological complications (Saylor et al. 2009). Therefore to eliminate the complications from using animal derived antibodies fully human antibodies were introduced. Antibody based therapies have been used successfully in the treatment of cancer and autoimmune diseases (Chan and Carter 2010; Pillay et al. 2011; Weiner et al. 2010). The concept of monoclonal antibody therapy to be applied for the treatment of infectious disease is still at its infancy. Several proposed applications have been reported for HIV, malaria and tuberculosis (Balu et al. 2011; Pleass and Holder 2005; Uchtenhagen et al. 2014). The mechanism of action for therapeutic antibodies is based on the ability to recognize unique epitopes on pathogens to produce a protective response or elimination by the body’s immune system. Antibody-based
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therapies can inhibit infections through toxin neutralization, promoting phagocytosis and prevent attachment to host cell (Berry and Gaudet 2011). An antibody library is a representative of the humoral immune system and has a broad variety of antibodies against all types of pathogens. Thus, disease-specific antibody libraries are suitable for the generation of mAbs against a specific group of disease targets (Zhang et al. 2013). Therapeutic applications of antibodies for infectious disease can either be based on the immunological properties of antibodies in combination with other complement factors or they can also be designed as a carrier molecule for drug or payload delivery to infected cells specifically. This diminishes the harm from a general payload or drug treatment. This is because undirected treatments like radiotherapy will also eliminate normal cells during the process (Litvak-Greenfeld and Benhar 2012). The importance of biological treatment alternatives is even greater with the emergence of resistant strains for certain infections whereby conventional treatments are no longer sufficient. Antibody engineering plays an important role in the development of novel diagnostics and therapeutics. Protein engineering is used to modify antibody properties to improve their application in diagnostics and therapeutics (Nelson and Reichert 2009). The antibodyantigen interaction is largely dependent on the conformation of antibody specificity regions towards its antigen. Besides antibody binding affinity and specificity, in order to improve diagnostic detection and therapeutic treatment, new approaches for the development of antibodies with improved targeting selectivity and delivery efficiency with minimal side effects have been carried out. Antibodies had been modified to have better specificity and affinity by introducing various conjugations (Teicher and Chari 2011). In order to expand antibody applications, modifications of antibodies with different conjugates have been widely explored. Antibody conjugation provides new route in improving antibody efficacy in therapy by incorporating toxins, radioactive isotopes and chemotherapeutic drugs (Litvak-Greenfeld and Benhar 2012). Although most modifications have been used mainly for oncology, the same principles can be applied for infectious diseases. Development of radioactive conjugates normally used in tumor imaging and radioimmunotherapy of tumor cells is also applicable for infected cells. In short, antibodies obtained from
immune libraries can be engineered to expand the applications and activity for diagnostics and therapeutics. This in turn, will help to increase the possibilities of applying antibodies in the diagnosis and treatment of infectious diseases.
Conclusions Phage display technology has emerged as a powerful tool to isolate antibodies for various diagnostic and therapeutic applications. Even so, various antibody selection techniques have also been used successfully to provide rapid and easy antibody generation against various infectious disease target antigens. The basic requirement to carry out antibody discovery with phage display is the generation of a highly diverse antibody library. The application of immunized or infected individuals for the generation of immune libraries is a key alternative to conventional naı¨ve libraries. The skewed preference of a disease immune library provides greater potential to isolate antibodies against targets of that particular disease. Challenges remain in the development of libraries for neglected infectious diseases due to the lack of samples available to generate the antibody libraries. In this regard, the application of immunized libraries for infectious diseases remains difficult due to the unavailability of such libraries for use. However, with the rise in emerging and re-emerging infectious diseases, disease-specific antibody libraries have the potential that is worth developing and exploring as this review has shown. Together with the developments in the field of recombinant DNA technology, a greater influence and application of phage-derived antibodies is expected for the development of diagnostics and therapeutics of infectious diseases. Acknowledgments The authors would like to acknowledge support by the Malaysian Ministry of Science, Technology and Innovation ScienceFund Grant (Grant No. 305/CIPPM/613229) and USM Short Term Research Grant (Grant No. 304/CIPPM/ 6312060).
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