**5.2. Applications of shark VNAR domains**

Evidence that IgNAR is part of the shark adaptive immune response was demonstrated in a work where increasing levels of hen egg lysozyme (HEL) led to the development of specific IgNARs developed in the shark sera after 4–5 months of immunization [25]. The peculiar structure of the shark IgNAR variable domain renders it amenable to create synthetic peptide mimetics to target specific epitopes that are inaccessible to conventional antibodies [118]. Therefore, VNAR may be suitable as new molecular reagents for research, diagnostic and immunotherapeutic applications.

Apical membrane antigen-1 (AMA1) is a highly polymorphic 83 kDa merozoite surface protein that is essential for erythrocyte invasion in malaria parasites [143]. A VNAR isolated from a wobbegong shark showed high-binding affinity to *Plasmodium falciparum* AMA1 through its unique CDR3 region after undergoing affinity maturation [144]. The binding specificity of a monovalent VNAR clone to *P. falciparum* AMA1 was comparable with commercially available

**Table 2.** The applications of camelids VHH against specified antigens from various diseases.

**Target antigens Diseases Applications Reference** VEGF-A<sup>165</sup> Neoangiogenesis Diagnostic and therapeutic [143] HER2 Breast cancer Diagnostic [144, 145] HPV-16 L1 protein Cervical cancer Diagnostic and therapeutic [146, 147] HSP-60 Brucellosis (livestock) Diagnostic and vaccine [23, 148] VCAM1 Atherosclerotic lesions Molecular imaging [149–151] VEGFR2 Angiogenesis Therapeutic [152] TNT Explosive Diagnostic [133, 153] SEB Toxin Sensor and diagnostic [134] Ricin Toxin Sensor and diagnostic [134]

BoNT/A Toxin Sensor and diagnostic [90, 154, 155]

Neurocysticercosis Immunodiagnosis [97]

therapeutic

The Development of Single Domain Antibodies for Diagnostic and Therapeutic Applications

http://dx.doi.org/10.5772/intechopen.73324

187

Poliomyelitis Diagnostic and therapeutic [159, 160]

[141, 156–158]

Brucellosis Vaccination, diagnostic,

Scorpion AahII Toxin Neutralizing and therapeutic [135]

CEA Colon cancer *In vivo* imaging [161–163] RSV protein F Acute lower respiratory tract Therapeutic [164] CD105 Angiogenesis related tumors Diagnostic and therapeutic [165, 166] Ts14 glycoprotein *T. solium* cysticercosis Diagnostic [97]

vWF Thrombosis Therapeutic www.ablynx.com TNFα, IL-6R, IgE Rheumatoid arthritis Therapeutic www.ablynx.com RANKL Bone metastasis Therapeutic www.ablynx.com RSV Bronchiolitis and pneumonia Therapeutic www.ablynx.com DR5 Solid tumors Therapeutic www.ablynx.com Not stated Alzheimer's disease Therapeutic www.ablynx.com

EGFR Tumors Detection and imaging DARC Malaria (by *P. vivax*) Diagnostic or therapeutic

LMM, ES, CSE, TSB, LLGPs,

Heat-killed *B. melitensis* Riv1

Poliovirus type 1 Sabin strain

VF of *T. solium*

lysates

particles


Cysticercosis is a serious tissue infection caused by larval cysts of the pork tapeworm, which is prevalent in many low-income countries [129]. Monoclonal antibodies that are currently deployed in sandwich ELISAs are mainly genus-specific against *Taenia* sp., but poorly specific at a species level to identify *Taenia solium*, the major *Taenia* species threatening human health [130, 131]. To circumvent such limitations, an *in vitro* selection of nanobodies from immunized dromedaries was developed to recognize a specific marker on *T. solium*. After *in vitro* selection, the nanobodies showed no cross-reactivity against other livestock *Taenia* species, while having a very specific response to a specific 14 kDa glycoprotein (Ts14) in *T. solium*. Therefore, nanobodies showed potential as an alternative to genus-species mAb for develop-

Apart from diagnostic reagents for infectious diseases, nanobodies have been identified as alternative binders to analyze the compositions of substances in food and beverages industries. Due to their excellent thermal stability, nanobodies showed superior behavior to classical mouse mAbs in ELISA to measure caffeine concentration in hot and cold beverages [132]. Camelid sdAbs have recently been applied in ELISA methods to detect a wide range of small molecules, including explosive materials (trinitroluene or TNT) [133], agents of bioterrorism (Botulinum A neurotoxin) [90], toxins (ricin, cholera and staphylococcal enterotoxin B) [134], scorpion toxin [135] and viruses (HIV, rotavirus, Vaccinia and Marburg) [136–138]. Owing to the combination of several favorable properties, camelid nanobodies have also been employed as molecules to diagnose diseases. In small molecule development, the advanced features of highly stable and unique conformational structure of nanobodies have permitted overcoming many problems faced by traditional whole antibodies and scFv fragments such as cross-reactivity and nanoparticle agglutination. The development of biosensors coupled with nanobodies (nanoconjugates system) has enabled significant improvement in the performance of a device to identify harmful bacteria (*Staphylococcus aureus*) to a nanometer scale

Nevertheless, mAbs remain the common binding agents to identify and trace tumor-associated proteins for noninvasive *in vivo* imaging. However, limitations, particularly large size (150 kDa) and Fc regions, result in mAbs poorly penetrating into solid tumors [140]. The emergence of nanobodies offers the possibility of resolving such problems, and thereby enables nanobodies to diagnose tumor markers such as EGF receptor [141]. This will enable cancer staging predictions in blood circulation such as prostate-specific antigen [142]. More applications using camelids VHH targeting antigens from various diseases are summarized in **Table 2**.

Evidence that IgNAR is part of the shark adaptive immune response was demonstrated in a work where increasing levels of hen egg lysozyme (HEL) led to the development of specific IgNARs developed in the shark sera after 4–5 months of immunization [25]. The peculiar structure of the shark IgNAR variable domain renders it amenable to create synthetic peptide mimetics to target specific epitopes that are inaccessible to conventional antibodies [118]. Therefore, VNAR may be suitable as new molecular reagents for research, diagnostic and immu-

ing unambiguous ELISA tests for human cysticercosis [97].

within 10 min [139].

186 Antibody Engineering

**5.2. Applications of shark VNAR domains**

notherapeutic applications.

**Table 2.** The applications of camelids VHH against specified antigens from various diseases.

Apical membrane antigen-1 (AMA1) is a highly polymorphic 83 kDa merozoite surface protein that is essential for erythrocyte invasion in malaria parasites [143]. A VNAR isolated from a wobbegong shark showed high-binding affinity to *Plasmodium falciparum* AMA1 through its unique CDR3 region after undergoing affinity maturation [144]. The binding specificity of a monovalent VNAR clone to *P. falciparum* AMA1 was comparable with commercially available binding reagents, derived from conventional polyclonal sera, monoclonal antibodies, small fragments (Fab and scFv) and peptides [145]. Foley and co-workers demonstrated the heat stability of purified recombinant VNAR was superior to that of conventional mAbs by targeting immobilized *P. falciparum* AMA1 in various formats at 45°C, and the refolding property of VNAR was retained when the temperature increased to 80°C. The excellent stability property at extreme pH and resistance to proteolytic cleavage was further evidenced by incubating VNAR with homogenized murine stomach tissues under *in vivo* conditions [9]. From this point of view, it was purposed that VNAR domains have potential for development as alternate binders for malaria diagnostic platforms.

Human periodontal disease is an advanced gingivitis caused by the bacterial pathogen *Porphyromonas gingivalis* [146]. Late treatments often lead to dental loss due to the accumulation of lysine gingipain (KgP). KgP is a high molecular weight polyprotease produced by *P. gingivalis* [147]. This bacterial toxin is responsible for destruction of dental tissue of host by suppressing the secretion of specific lytic enzymes from the immune system [148]. Nuttall and co-workers [149] identified two distinct clones specific to KgP from a naïve wobbegong shark VNAR phage display library with synthetic CDR3 loops. The high stability and binding affinity toward *P. gingivalis* KgP indicated the potential for VNAR sdAbs as a valuable source of single domain binding reagents [149].

In recent studies, shark VNAR domains have been reported to detect markers from viral diseases at greater sensitivity compared to mAbs and scFvs. Ebola virus hemorrhagic fever (EVHF) is a highly lethal disease caused by Bundibugyo virus (BDBV), Sudan virus (SUDV), Tai Forest virus (TAFV) and Zaire Ebolavirus (ZEBOV) [150–152]. Shark VNAR and murine scFv phage display libraries have been generated against specified markers on Zaire Ebolavirus. The results indicated that the sensitivity and thermal stability of shark VNAR sdAbs against viral nucleoprotein (NP) of ZEBOV was superior in comparison to murine mAbs and scFvs. [116].

As in the case with camelids nanobodies, highly diversified shark VNAR libraries have also been used to detect different kind of toxins, including staphylococcal enterotoxin B (SEB), ricin and botulinum toxin A (BoNT/A) complex toxoid [153] and cholera toxin (CT) [113]. In addition, VNAR sdAbs have been reported to recognize immunosilent targets in human, for example, the 70 kDa translocase of outer membrane (Tom70) [154]. Owing to the findings of negligible cross-reactivity with other antigens and superior heat stability, shark VNAR domains may be a potent source of sdAbs with thermal stability over conventional antibodies in diagnostic and biotherapeutic applications [155, 156]. The applications of recombinant shark VNAR sdAbs against specified antigens from various diseases are summarized in **Table 3**.

*marinus*) using hybridoma technology [11]. *Bacillus anthracis* is the causative agent for anthrax and the only pathogenic species in the genus *Bacillus* [158]. Due to their extreme dormancy and durability, anthrax spores have long been considered ideal biological weapons [159–161]. In this work, the recombinant monoclonal VLRs were shown to be capable of identifying bacteria at a genus level, by differentiating the C-terminal domain of BclA *Bacillus anthracis* from non-

**Target antigens Diseases Applications Reference** BclA glycoprotein *B. anthracis* spores (anthrax) Diagnostic [11]

tumor cells

**Table 4.** The applications of lamprey VLRs against specified antigens from various diseases.

GPCR's ion channels Therapeutic www.adalta.com.au Anti-thrombotic drug targets Cardiovacular disease Diagnostic and therapeutic www.adalta.com.au

Myostatin Neurological disease Therapeutic www.ossianix.com

**Target antigens Diseases Applications Reference** Kgp protease (*P. gingivalis*) Periodontal disease Neutralization [173] rhTNFα Pro-inflammatory cytokine Therapeutic [114, 181] AMA1 (*P. falciparum*) Malaria Diagnostic [168, 169]

Ebolavirus Haemorrhagic

HBeAg Hepatitis B virus Immunolocalization and

Cholera toxin Toxin Diagnostic [113] SEB Toxin Sensor and diagnostic [177] Ricin Toxin Sensor and diagnostic [177] BoNT/A Toxin Sensor and diagnostic [177]

Fever

Tom70 Human immunosilent target

processes

**Table 3.** The applications of shark VNAR against specified antigens from various diseases.

Alzheimer's disease Modeling [182]

diagnostic

The Development of Single Domain Antibodies for Diagnostic and Therapeutic Applications

Blood brain barrier Therapeutic www.ossianix.com Gastrointestinal tract Therapeutic www.ossianix.com

Uveitis Therapeutic www.elasmogen.com

Complex protein antigens Affinity determination [39, 189]

Binding interaction [190]

Diagnostic [116]

Detection [178]

[183]

http://dx.doi.org/10.5772/intechopen.73324

189

www.adalta.com.au

coated bacteria of *Bacillus cereus* [11].

C1q and C3 proteins Cytotoxicity for bacteria and

HEL, β-gal, cholera toxin subunit B, R-phycoerythrin, and B-trisaccharide

Nonfibrillar oligomer

Zaire ebolavirus viral nucleoprotein

formation

#### **5.3. Applications of lamprey VLRs**

The variable lymphocyte receptors (VLRs) discovered from jawless fish had recently attracted interests and is leading to the development of new monoclonal antibodies for biomedical applications [11, 36, 157]. Despite possessing an unusual structure, VLRs have been shown to have excellent binding ability to specified targets (**Table 4**).

Cooper and co-workers demonstrated high specificity of recombinant VLRs for BclA, a major anthrax spore coat which could be produced from an immunized sea lamprey (*Petromyzon* 


**Table 3.** The applications of shark VNAR against specified antigens from various diseases.

binding reagents, derived from conventional polyclonal sera, monoclonal antibodies, small fragments (Fab and scFv) and peptides [145]. Foley and co-workers demonstrated the heat stability of purified recombinant VNAR was superior to that of conventional mAbs by targeting immobilized *P. falciparum* AMA1 in various formats at 45°C, and the refolding property of VNAR was retained when the temperature increased to 80°C. The excellent stability property at extreme pH and resistance to proteolytic cleavage was further evidenced by incubating VNAR with homogenized murine stomach tissues under *in vivo* conditions [9]. From this point of view, it was purposed that VNAR domains have potential for development as alternate binders

Human periodontal disease is an advanced gingivitis caused by the bacterial pathogen *Porphyromonas gingivalis* [146]. Late treatments often lead to dental loss due to the accumulation of lysine gingipain (KgP). KgP is a high molecular weight polyprotease produced by *P. gingivalis* [147]. This bacterial toxin is responsible for destruction of dental tissue of host by suppressing the secretion of specific lytic enzymes from the immune system [148]. Nuttall and co-workers [149] identified two distinct clones specific to KgP from a naïve wobbegong shark VNAR phage display library with synthetic CDR3 loops. The high stability and binding affinity toward *P. gingivalis* KgP indicated the potential for VNAR sdAbs as a valuable source of single

In recent studies, shark VNAR domains have been reported to detect markers from viral diseases at greater sensitivity compared to mAbs and scFvs. Ebola virus hemorrhagic fever (EVHF) is a highly lethal disease caused by Bundibugyo virus (BDBV), Sudan virus (SUDV), Tai Forest virus (TAFV) and Zaire Ebolavirus (ZEBOV) [150–152]. Shark VNAR and murine scFv phage display libraries have been generated against specified markers on Zaire Ebolavirus. The results indicated that the sensitivity and thermal stability of shark VNAR sdAbs against viral nucleoprotein (NP) of ZEBOV was superior in comparison to murine

As in the case with camelids nanobodies, highly diversified shark VNAR libraries have also been used to detect different kind of toxins, including staphylococcal enterotoxin B (SEB), ricin and botulinum toxin A (BoNT/A) complex toxoid [153] and cholera toxin (CT) [113]. In addition, VNAR sdAbs have been reported to recognize immunosilent targets in human, for example, the 70 kDa translocase of outer membrane (Tom70) [154]. Owing to the findings of negligible cross-reactivity with other antigens and superior heat stability, shark VNAR domains may be a potent source of sdAbs with thermal stability over conventional antibodies in diagnostic and biotherapeutic applications [155, 156]. The applications of recombinant shark VNAR

The variable lymphocyte receptors (VLRs) discovered from jawless fish had recently attracted interests and is leading to the development of new monoclonal antibodies for biomedical applications [11, 36, 157]. Despite possessing an unusual structure, VLRs have been shown to

Cooper and co-workers demonstrated high specificity of recombinant VLRs for BclA, a major anthrax spore coat which could be produced from an immunized sea lamprey (*Petromyzon* 

sdAbs against specified antigens from various diseases are summarized in **Table 3**.

for malaria diagnostic platforms.

188 Antibody Engineering

domain binding reagents [149].

mAbs and scFvs. [116].

**5.3. Applications of lamprey VLRs**

have excellent binding ability to specified targets (**Table 4**).

*marinus*) using hybridoma technology [11]. *Bacillus anthracis* is the causative agent for anthrax and the only pathogenic species in the genus *Bacillus* [158]. Due to their extreme dormancy and durability, anthrax spores have long been considered ideal biological weapons [159–161]. In this work, the recombinant monoclonal VLRs were shown to be capable of identifying bacteria at a genus level, by differentiating the C-terminal domain of BclA *Bacillus anthracis* from noncoated bacteria of *Bacillus cereus* [11].


**Table 4.** The applications of lamprey VLRs against specified antigens from various diseases.

In another study, a large library of recombinant VLRs was constructed to target lysozyme, β-gal, cholera toxin subunit B, R-phycoerythrin and B-trisaccharide antigens using yeast surface display technologies [36]. This high-throughput technology platform offers the potential of rapid identification and isolation of monoclonal VLRs that specifically bind to target antigens with affinities in the micromolar to nanomolar range [36]. Using such display methods, the specificity of selected VLR antibodies can recognize the target antigen with high binding affinity up to 100-fold compared to conventional mouse mAb [36]. These data indicate that the function of VLRs is comparable or perhaps better than that of mammalian IgG antibodies. Therefore, it is speculated that VLRs may be an alternative reagent for the future development of therapeutic and diagnostic applications.

**Author details**

Penang, Malaysia

**References**

Chiuan Herng Leow<sup>1</sup>

\*Address all correspondence to: herng.leow@usm.my

3 Australian Army Malaria Institute, Brisbane, Australia

4 University of Queensland, Brisbane, Australia

bodies. Eur J Biochem 1981;**118**:429-436

Virol Methods 2006;**134**:164-170

ELISA. Anal Biochem 2009;**386**:36-44

2011;**31**:509-513

2003;**283**:17-25

Antibodies 2013;**2**:66-81

2 QIMR Berghofer Medical Research Institute, Brisbane, Australia

\*, Qin Cheng2,3, Katja Fischer2,4 and James McCarthy2,4

The Development of Single Domain Antibodies for Diagnostic and Therapeutic Applications

http://dx.doi.org/10.5772/intechopen.73324

191

[1] Conroy PJ, Hearty S, Leonard P, O'Kennedy RJ. Antibody production, design and use

[2] Newcombe C, Newcombe AR. Antibody production: polyclonal-derived biotherapeu-

[3] Milstein C. 12th Sir Hans Krebs Lecture. From antibody diversity to monoclonal anti-

[4] Torrance L, Ziegler A, Pittman H, Paterson M, Toth R, Eggleston I. Oriented immobilisation of engineered single-chain antibodies to develop biosensors for virus detection. J

[5] Moon SA, Ki MK, Lee S, Hong ML, Kim M, Kim S, Chung J, Rhee SG, Shim H. Antibodies against non-immunizing antigens derived from a large immune scFv library. Mol Cells

[6] Proba K, Wörn A, Honegger A, Plückthun A. Antibody scFv fragments without disulfide

[7] Dong J, Ihara M, Ueda H. Antibody Fab display system that can perform open-sandwich

[8] Zhang MY, Shu Y, Phogat S, Xiao X, Cham F, Bouma P, Choudhary A, Feng YR, Sanz I, Rybak S, et al. Broadly cross-reactive HIV neutralizing human monoclonal antibody Fab selected by sequential antigen panning of a phage display library. J Immunol Methods

[9] Griffiths K, Dolezal O, Parisi K, Angerosa J, Dogovski, C, Barraclough M, Sanalla A, Casey J, González I, Perugini M, Nuttall S, Foley M. Shark Variable New Antigen Receptor (VNAR) Single Domain Antibody Fragments: Stability and Diagnostic Applications.

for biosensor-based applications. Semin Cell Dev Biol 2009;**20**:10-26

tics. J Chromatogr B Analyt Technol Biomed Life Sci 2007;**848**:2-7

bonds made by molecular evolution. J Mol Biol 1998;**275**:245-253

1 Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia,

#### **6. Conclusion**

The fields of antibody engineering have undergone major advancements in the past few decades. New surface display technologies, in particular phage display and yeast display, are powerful tools that could facilitate the isolation of new antibodies with high specificities for a broad range of targets. Due to their affinity, which often is similar to conventional antibodies and reliable production, recombinant antibodies are becoming increasingly important in the field of diagnosis and therapy for targeting a wide range of diseases such as cancer, inflammatory, autoimmune and viral diseases. In view of natural scaffold design, previous studies showed that the sdAbs repertoires derived from animals such as camelid VHHs, shark VNARs and lamprey VLRs contain several advantages over conventional antibodies. One of the unusual characteristics shared among the sdAbs is that they possess better penetration ability. This feature allows the sdAbs to effectively penetrate into antigen clefts (enzyme active sites, viral capsids and cell surface receptors) which are not easily recognized by the concave surfaces of CDR loops of complex conventional antibodies. To date, due to their ability to target both refractory antigens and immunosilent epitopes, the engineered antibody fragments coupled with latest screening technologies have extensively been used in positron emission tomography and high-sensitivity (nonradioactive, noninvasive) laser technologies for medical imaging. To sum up, it is believed that with rapid progress in antibody engineering technologies, sdAbs will become indispensable as clinical and research reagents in the next decades.

#### **Acknowledgements**

The authors would like to acknowledge support from University of Queensland, QIMR Berghofer Medical Research Institute in Australia and Malaysian Government, including Malaysian Ministry of Higher Education the Higher Institutions Centre of Excellence Program (Grant no.: 311/CIPPM/4401005), RUI Grant no.: RU(1001/CIPPM/811296) and USM Short Term (Grant no.: 304/CIPPM/6313191).
