Application of nanobodies in medical and applied sciences

Anagha Bhaskaran¹, K. Pramod^1,2,*, K.C. Ajithkumar ², U.S. Jijith²

¹College of Pharmaceutical Sciences, Govt. Medical College, Thiruvananthapuram – 695011, Kerala, India.

²College of Pharmaceutical Sciences, Govt. Medical College, Kozhikode – 673008, Kerala, India.

*Corresponding Author E-mail: pramodkphd@yahoo.com

Abstract:

Nanobodies are antigen specific, recombinant, single domain, variable fragments of Camelide heavy chain only antibodies, which are able to bind specific antigen. They are considered as smallest naturally derived antigen –antibody fragment. The supremacy of nanobodies over antibodies is their minimal size, great stability, reversible refolding and high solubility in aqueous solution. Nanobodies, because of small size might be considered as “magic bullets” of molecular imaging in the near future, especially oncologic imaging. Construction of nanobodies involves stages such as immunisation, isolation and cloning and genetic engineering. Nanobodies are mainly used for the molecular imaging application. Apart from molecular imaging it will also used for development of biological sensors and clinical applications in viral infections, atherosclerosis etc. Therapeutic applications of nanobodies includes; inhibition of tumour cell proliferation, binding and intracellular routing of nanobody albumin nanoparticles, nanobullets– nanobody coupled liposomes for treating angiogenesis and tumour, nanobodies against infection, nanobodies against pathogens and cancer. Drug loaded nanobodies are also used as a novel drug delivery system. In the future more targets and their corresponding nanobodies would be identified. Furthermore, future clinical research should investigate the application value of nanobodies. Properties of nanobodies like high resistance to p^Hand heat, refolding capacity, high solubility, high stability etc make them more prominent than antibodies. Considering all these aspects, nanobodies appear to be a promising method of tumour targeting therapy and diagnosis.

KEY WORDS: Antibodies, Antigen, Cancer, Molecular imaging.

1. Introduction:

Since antibodies (Abs) are digested quickly in the gut, blocked from entering in the brain held to periphery of solid tumours. Many illnesses are thus unreachable by monoclones and certain conditions in which mAbs do not work well leads the invention of nanobodies.

After several years of investigation scientists found that half Ab circulating blood [commonly in camels] lack light chain. And also it is surprising that these incomplete antibodies have ability to grasp their targets as normal Ab can do. This is how nanobodies are introduced and now it plays an important role in biological activities.

2. IMPORTANCE OF NANOBODIES

Nanobodies, because of small size might be considered as “magic bullets” of molecular imaging in the near future, especially oncologic imaging^[1]. Apart from molecular imaging it will also used for development of biological sensors and clinical applications in viral infections, atherosclerosis etc^[2]. They are highly stable and highly soluble in aqueous solution. As they were so much smaller than antibodies and not chemically hydrophobes [as domain antibodies], they were more resistant to PH and heat.

Because of its simplicity in chemical composition and shape, it can be encoded by a single gene and was easier to synthesize.

3. ANTIBODY AND NANOBODY

The millions of kinds of human antibodies all shares the same basic structure; two larger [or heavy] protein chain linked with two smaller [or light] chain. The pair of variable segments as the tip of the arms is unique for each model of antibody that help to determine target to which it will bind. While the nanobodies are variable part of a camel antibody that lacks light chain and about one tenth of size of an antibody^[3].

4. GENERATION OF NANOBODIES

Heavy chain antibodies, devoid of light chain can easily produced by Camelidae family^[4]. Among which camels are perfect one. Generally, an animal is immunised with suitable quantity of antigen to raise the H₂ type antibody response. Then heavy chain fragments from lymphocytes are amplified by standard molecular biological techniques such as reverse transcription followed by PCR. Then it is transferred to bacteria using vectors to make VHH library. Then, antigen binding VHH fragments were selected from the library and selection of nanobodies involves enrichment antigen specific binders from VHH library by ELISA test.

5. CONSTRUCTING NANOBODIES

Construction of an effective nanobody was easy and inexpensive than a therapeutic antibody. The immune system of live animal plays the initial “design” of a protein that can latch to target molecule.

STEPS INVOLVED:

Immunisation:

A camel is immunized and produces both normal and heavy chain only antibodies against the target.

Isolation and cloning:

From a blood sample, biologists identify the cells that produce heavy chain only antibodies with high affinity at target. Then they obtain the DNA sequence for the genes that code for the antibody.

Genetic engineering:

Geneticists change the DNA to a part that encodes just a single variable segment-nanobody. These mutated forms of nanobody are tested to identify the one that is most medically useful ^[2].

6. CHARACTERISTICS OF NANOBODIES:

The camelid VHH sequences belong to a single gene family III. The heavy chain variable domain conventional antibodies (VH) in human and the VHH in camelid shows a high degree of homology and researchers found that there are differences between VH and VHH. Four amino acid substitutions were reported in the framework II region of VHH: the V37F or V37Y, the G44E, the L45R and the W47G substitutions. The framework 2 region of VH is highly conservative, supplied with hydrophobic amino acid residues, and is important for binding with a light chain variable domain VL.

In mAbs VH and VL domain form a stable structure through hydrophobic interactions. But VHH amino acid substitution makes it impossible and four substitutions render its surface more hydrophilic and soluble.

There was high degree of similarity between conventional mAbs and nanobodies, but some important differences offers an explanation of better antigen binding capacity of single domain structure. They were composed of four-frame work region and three CDRs. But the CDR1 and CDR3 are longer than those of VH. The length of CDR3 in human and mouse VH is 12 and 9 respectively. While camelid VHH have 16-18 aminoacid length. Although in camelid considerable fraction of VHH seems to have shorter CDR (approximately 6 aminoacids). In addition CDR normally involves 31-35 residue in all VH is enlarged to accommodate residues 27-35 in camelid VHH. There hyper variable residues 27-30 forming the loop connecting the two beta sheets of immunoglobulin fold are exposed to solvent. These residues are contacting the antigen and the larger CDRs of VHH will enlarge the actual surface area antigen-binding site, which might compensate for the absence of antigen binding surface area providing by VL domain.

The antigen-binding site of conventional antibodies, such as Fab and scFV, is a concave or planar structure that can identify only sites on the surface of antigen. For heavy chain antibodies, a long CDR3 can form a stable and large convex ring structure.

The physiochemical and pharmacokinetic properties of nanobodies comply the requirements of many biochemical applications and have many advantages over antibody technology for immune therapy, drug delivery and diagnostics. The affinities of nanobodies are superior over conventional antibody derivatives.

7. APPLICATIONS OF NANOBODIES:

7.1. THERAPEUTIC APPLICATIONS

7.1.1. Inhibition of tumour cell proliferation

Anti proliferative effects of loaded nanobodies were determined by BrdU assay. 14C were seeded in 96 well plates and allowed to adhere overnight. Cells were incubated with nanoparticles in culture medium to different concentration. After incubation of 48h the medium was replaced with BrdU (10µm) containing complete medium and cells were incubated overnight at 37ºC ^[5].

7.1.2. Binding and intracellular routing of nanobody albumin nanoparticles

Binding of NANPs by EGFR expressing 14C tumour cells was investigated using Rho-Lx labelled nanoparticles. After incubation of 1 h at 40ºC, nanoparticles bound nonspecifically to 14C cells, however after PEGylation this nonspecific binding was completely prevented. Nanobody albumin nanoparticles bind to EGa1and the binding increased 5 fold in comparison to bare nanoparticles. At the highest dose EGa1- PEG – NPs bound over 40 fold more efficiently than PEGylated nanoparticles without EGa1. Binding of EGa1 nanoparticles was strongly inhibited by free nanobody.

7.1.3. Nanobullets– Nanobody coupled liposomes for treating angiogenesis and tumour:

Epidermal growth factor receptor is a target for anticancer strategies because, it involves in tumour development and progression and is frequently over expressed on a variety of epithelial cancers including squamous cell carcinoma of head and neck, breast cancer and colorectal cancer. When a ligand binds to EGFR initiates signal transduction pathways and ultimately leads to certain cellular process which contributes to tumour development and progression. Certain antibodies or nanobodies acts as targeting ligands for binding this receptor.

Thus anti-EGFR nanobodies act as targeting ligand and liposomes as drug delivery system. Liposomes are vesicular carriers with an aqueous core, enclosed in one or more phopholipid bilayers. A large variety of therapeutic molecules can be incorporated into liposomes and surface can be modified with targeting ligands. Selection of nanobodies over antibodies is its high stability, high solubility, more resistance to high pH and heat changes. Also it is cheap and easy to produce.

Anti EGFR nanobody is incorporated with liposomes and this multivalent system demonstrated EGFR down regulation in vivo and in vitro. Recently it is coupled to micellar system which demonstrated by specific cell association and uptake. Similar to EGFR, Insulin-Like Growth Factor1-Receptor [IGF-1R] also acts as target for anti cancer strategies. Upregulated expression of IGF-1R has been seen in cancers and many IGF-1R inhibitors are under clinical trials. Recently anti- EGFR coupled liposome with AG538, a potent IGF-1R kinase inhibitor is used as multivalent dual targeted drug delivery system for cancer treatment ^[6].

7.1.4. Nanobodies against infection

Antibodies naturally defence against bacterial, viral and parasite infections. Now nanobodies are also used for curing infections. The strict monomeric nanobodies facilitate their fusion with molecules and, irradicate pathogens through enzymes or toxic substances.

7.1.5. Nanobodies against pathogens and cancer

The application of nanobodies to combat diseases and pathologies focused on the inhibition of ligand receptor interactions. The small size and high stability/folding capacity of nanobodies suggest that they are more applicable than antibodies ^[7]. Results for testing nanobodies against cancer on mice after they were injected with human tumour cells indicates that high doses of nanobody directed chemotherapy, however knocked the tumours into remission.

7.2. DIAGNOSTIC APPLICATIONS:

Due to the small molecule size, nanobodies can attains their targets easily after administration. They have a great potential in tumour by PET (Position Emission Tomography), SPECT (Single Photon Emission Computed Tomography), ultrasound and optical imaging. Gamma emitting radionuclide labelled nanobody needs to be used for SPECT imaging, thus the γ-rays can be easily detected by SPECT instrument. In case of PET it requires radio labelling of nanobody with a suitable positron emitting radionuclide.

7.2.1. Imaging of epidermal growth factor:

EGFR is over expressed in variety of human tumours including non-small cell lung cancer, gastric, colorectal, prostate, bladder, renal, pancreatic and ovarian cancer. Molecular imaging can advantageously measure EGFR expression and SPECT imaging of EGFR expression by an anti EGFR nanobody as the targeting agent was first reported by Huang et al ^[8]. The radiolabelled nanobody exhibit high selectivity and specificity towards EGFR expressing cells and can differentiated tumours with high and moderate EGFR expression. Since the tracer clearance is mainly through kidney, retention of radiolabelled nanobodies in kidney leads to renal toxicity. This toxicity was evaluated by the role of megalin [endocytic receptor in renal proximal tubule] on the renal uptake of nanobody 7C12 and concluded that megalin contributed renal accumulation.

7.2.2. Imaging of human epidermal growth factor receptor 2:

It is also a member of HER kinase family that is over expressed in tumour type such as breast, ovarian, prostatic and breast cancer. There were 38 different nanobodies that could successfully target HER-2 expression. Out of which nanobody 2Rs15dHis6 was an important one. SPECT imaging and biodistribution studies shows high uptake of 99m Tc labelled 2Rs15dHis6 in HER-2 positive receptor ^[9].

7.2.3. Imaging of hepatocyte growth factor

Elevated level of Hepatocyte growth factor (HGF) and C-met (membrane receptor) have been found in most solid tumours and associated with increased aggressiveness of tumours. Therapeutic potential of nanobodies towards HGF is studied in mice by treating with anti-HGF nanobodies. This results in the delayed tumour growth when compared with control group that was injected with saline and non-toxicity to normal organ was observed.

7.2.4. Imaging of vascular cell adhesion molecule

The VCAM-1 protein mediates adhesion of monocytes, lymphocytes, eosinophils and basophils to vascular endothelium and functions in leucocyte endothelial cell signal transduction ^[10]. It is expressed at low level in non atherosclerotic arteries. However, if a person suffers from hypercholesterolemia VCAM-1 is expressed at high level in them. By using anti VCAM-1 nanobodies preclinical imaging of atherosclerotic plaques was determined ^[11].

7.2.5. Nanobodies as probe in biosensors:

Nanobodies have significant applications medical diagnosis, environmental and food analysis as biosensors. Its small size provides surfaces with high binding capacity resulting in higher sensitivity. Moreover, nanobodies directionally immobilised on to solid sensor surface that allow immunoreactions close to sensor–solution interface resulting increased sensitivity.

7.3. OTHER APPLICATIONS:

· Nanobodies as crystallisation chaperones

Nanobodies mimic as s. chaperones to crystallize challenging protein And also these single domain antibody fragment often lock protein in a particular confirmation. The ability of nanobodies to stabilize in a flexible region or shield aggregating surfaces was certain features to allow crystallization process.

· Serum half-life extension:

Specific Nanobodies on compination with the other therapeutic compounds provide a generic approach to extend the plasma half life of this therapeutics.

· Nanobodies against envenoming:

Nanobodies are applicable in the field of envenoming caused by scorpions or snakes. Current antivenoms are immunoglobulin fragment purified from blood of venom immunised horses and sheeps. Intact Camelid IgG antibodies and particular their nanobody derivatives are equally or more potent than in neutralizing the lethal, hemorrhagic and coagulopathic effects of West African viper victims ^[12].

8. ADVANTAGES OF NANOBODIES:

· Molecular size is very small such that it can reach the target within hours.

· Widely used in the field of molecular imaging especially in the case of oncologic imaging.

· Important diagnostic tool in tumour detection, detection of possible reoccurrence of diseases etc.

· Fast blood clearance.

· High solubility and high stability.

· Resistant to high PH and temperature.

· Refolding capacity is high compared to antibodies.

· Gastrointestinal stability and oral bioavailability.

· Tailored half life.

9. DISADVANTAGES OF NANOBODIES:

· Due to rapid clearance from blood the conventional antibodies have dominant role in immunotherapy.

· Nanobodies may sometimes lead to toxicity.

· Selection of nanobodies against a specific target is little difficult.

10. CONCLUSION:

Discovery of nanobodies create interest in researchers on their use in tumour diagnosis and therapy. Numerous specific nanobodies have been screened from library and showed high affinity with their antigen. The small size of nanobodies makes it easy for them to penetrate the membrane or get through the blood brain barrier as solo drug. But the small size is a disadvantage for pharmacokinetics because of their rapid elimination via the kidney. Drug loaded nanobodies are also used as a novel drug delivery system. In the future more targets and their corresponding nanobodies should be identified. Furthermore, future clinical research should investigate the application value of nanobodies. It is now considered as magic bullets in molecular imaging. Properties of nanobodies like high resistance to p^Hand heat, refolding capacity, high solubility, high stability etc make them more prominent than antibodies. In conclusion, nanobodies appear to be a promising method of tumour targeting therapy and diagnosis.

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Received on 26.06.2016 Modified on 16.07.2016

Research J. Science and Tech. 2016; 8(3):150-154.

DOI: 10.5958/2349-2988.2016.00023.1