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=Introduction=

Antibodies are essential components of the immune system that protects humans from disease. The removal or destruction of potentially harmful foreign molecules (antigens) is facilitated by binding interactions between the antibody and antigen at specific, localized molecular sites. Enhancing immune system capabilities by engineering of innovative antibodies and similar therapeutic proteins present new treatment methods for cancer, cardiovascular disorders and inflammatory diseases. One group of novel drug candidates emerging from this field are Nanobodies; a collection of therapeutic proteins derived from the antibodies camels and llamas (camelids). This review provides a comparison of Nanobody and conventional human antibody features as well as consideration of potential therapeutic applications including the preliminary results from clinical drug trials.

Nanobody Structure
Nanobodies are isolated variable domains of heavy chain antibodies. These proteins incorporate several characteristics of parental heavy chain antibodies including an elongated CDR3 amino acid sequence, hydrophilic amino acids in the second framework region (FR2) and a CDR1 with greater variability. Although Nanbodies are noticeably different from conventional antibody fragments, both proteins display several common characteristics. The variable heavy domains of human and camelid antibodies are folded into two β-pleated sheets. Each domain contains three CDR as well as four framework regions. In addition, the framework region sequences display high homology in both species. This reduces the potential for an adverse immunogenic response in humans. Nanobodies are also significantly smaller than conventional antibodies. Variable heavy camelid domains (VHHs) consist of approximately 120 amino acid residues and are about 15kDa in size, whereas a conventional antibody approaches 150kDa.

Nanobody Properties
Nanobodies display characteristics unobserved in conventional antibodies and associated protein fragments that render them potential drug candidates. The elongated CDR3 participates in Nanobody-antigen binding. Unlike conventional antibodies, the CDR3 loop can recognize cryptic epitopes recessed in antigen cavities. This enables VHHs to interact with a greater number and variety of epitopes than human antibodies. In addition, numerous Nanobodies that bind to active enzyme sites have been isolated. De Gent and colleagues demonstrated 75% of single variable domains isolated immunized camels targeted the active antigen site. Evidence suggests that some Nanobodies may function as competitive enzyme inhibitors.



Nanobodies exhibit superior thermal and conformational stability. Although VHHs and conventional antibodies denature at high temperatures, Nanobodies are capable of refolding to their original configuration. As an example, after incubation at 37°C for one week, Nanobodies retain 80% binding efficiency. This is attributed to increased hydrophilicity of the Nanobody. In addition, VHHs are resistant to pH extremes and chaotropic agents. This makes Nanobodies suitable for oral delivery, a desirable pharmaceutical characteristic that generally increases patient compliance. The binding interaction between an antigen and therapeutic compound is also reduced by protein aggregation and insolubility therefore avoiding these processes is advantageous. Substitution of hydrophilic amino acid residues within framework region increases solubility of the Nanobodies and discourages aggregation.

Nanobodies are considerably smaller than conventional antibodies enabling greater tissue penetration. This also reduces the potential of an adverse immune system response. Unfortunately this feature also allows Nanobodies to pass through the renal filter, which is approximately 60kDa in diameter. Consequently, Nanobodies are rapidly cleared from the blood stream. This compromises the efficiency of therapeutic Nanobodies, since persistence of a drug within the body is desirable for treatment of disease. Efforts to minimize Nanobody excretion includes synthesis of bivalent VHHs that can bind to larger albumin proteins in blood serum, or increasing the molecular weight by addition of polyethylene glycol groups.

Another advantage is production of Nanobodies in vectors, such as E. coli and yeasts. Production costs using vectors are significantly lower than isolation of conventional antibodies from mammalian cultures. Vector production methods also reduce the protein development time. Unfortunately, glycosylation and mutation of the VHH has been documented using vectors. This limits the efficiency of the process at this time.

Perspectives
Numerous properties desirable in engineered proteins are expressed in Nanobodies earning them recognition as novel drug candidates. This is evident from current evaluation of several Nanobodies in pre-clinical and clinical trials. A number of the resources consulted to produce this summary are associated with the pharmaceutical company. Ablynx, which patented this technology. Although some of the disadvantages associated with Nanobody engineering and development are presented, the medical contribution of Nanobodies may be overestimated.This may introduce minor biases into the information presented. As an example the cilincal trial results reported by Van Bockstaele and colleagues fail to acknowledge any observed side effects of immpediments. 19 This portrays Nanobodies favourably whereas realistically numerous drug candidates fail to reach public consumption stages. Current obstacles include short blood serum residence times and potential immunogenic responses following repeated exposure. These properties influence the safety and efficiency of therapeutic compounds and cannot be overlooked. Consequently, we believe Nanobodies are superior candidates to many conventional antibody therapeutics, however the safety and potential as a treatment for human diseases remains uncertain.