In Vivo Applications of Single Chain Fv (Variable Domain) (scFv) Fragments

Philippe P. Monnier1,2,3,*, Robin J. Vigouroux1,2 and Nardos G. Tassew1,2 Antibodies 2013, 2, 193-208; doi:10.3390/antib2020193 Definition: scFV Single chain variable domain (Fv) fragments (scFv) are powerful tools in research and clinical settings, owing to better pharmacokinetic properties compared to the parent monoclonal antibodies and the relative ease of producing them in large quantities, at low cost. Though they offer several advantages, they suffer from lower binding affinity and rapid clearance from circulation, which limits their therapeutic potential. However, these fragments can be genetically modified to enhance desirable properties, such as multivalency, high target retention and slower blood clearance, and as such, a variety of scFv formats have been generated. ScFvs can be administered by systemic injection for diagnostic and therapeutic purposes. They can be expressed in vivo through viral vectors in instances where large infection rates and sustenance of high levels of the antibody is required. ScFvs have found applications as tools for in vivo loss-of-function studies and inactivation of specific protein domains, diagnostic imaging, tumor therapy and treatment for neurodegenerative and infectious diseases. This review will focus on their in vivo applications.

Later, advances in antibody engineering have enabled the further reduction of mAbs to single chain Fv fragments (scFvs), monovalent Fabs, diabodies and minibodies [710]. The properties of these fragments have been tailored to optimize their binding affinity, in vivo pharmacokinetics, stability and expression levels [11]. ScFvs have provided an alternative to full-length mAbs in diagnostic and therapeutic applications and account for 35% of antibody fragments in clinical trials [5,12,13]. AnscFv fragment consists of the smallest functional antigen-binding domain of an antibody (~30 kDa), in which the variable heavy (VH) and variable light (VL) chains are joined together by a flexible peptide linker [14] (Figure 1). The first scFv fragments were cloned by two labs in the same year, Bird et al. [15] and Huston et al. [16] in 1988. The length and amino acid composition of the linker are crucial in maintaining the correct folding of these proteins [17]. The linker is typically about 3.5 nm in length and must contain hydrophilic residues together with stretches of Gly and Ser sequences for flexibility, in addition to Glu and Lys for enhanced solubility [18,19]. ScFv fragments are most often produced in bacterial cells [20], although other expression systems, including mammalian cells [21], plants [22]and insect cells [23] have been employed. ScFv fragments retain the binding specificity of the parent antibody and offer several advantages compared to full-length mAbs. They display improved pharmacokinetic properties, such as better tissue penetration and rapid blood clearance, which may be beneficial in radiotherapy and diagnostic applications. For instance, these fragments can penetrate more rapidly into tumors compared to an intact antibody [24,25]. Also, when scFvs are joined to radionuclides in radiotherapy applications, their increased rate of clearance from blood minimizes exposure to healthy tissue [26,27]. Recombinant

antibodies lack the Fc region, leading to low immunogenicity, making them better therapeutic agents compared to the full-length mAbs for many applications. In addition, scFvs can be cloned and expressed in bacterial and mammalian cells, making it possible to produce large quantities easily and cost-effectively.

ZuhaidaAsra Ahmad, SweeKeongYeap, Abdul Manaf Ali, Wan Yong Ho, NoorjahanBanu Mohamed Alitheen, and Muhajir Hamid, scFv Antibody: Principles and Clinical Application, Clinical and Developmental Immunology, vol. 2012, Article ID 980250, 15 pages, 2012. doi:10.1155/2012/980250

Single-Chain Fragment Variable (scFv)

Fv fragment is the smallest unit of immunoglobulin molecule with function in antigen-binding activities. An antibody in scFv (single chain fragment variable) (Figure 1) format consists of variable regions of heavy () and light () chains, which are joined together by a flexible peptide linker that can be easily expressed in functional form in E. coli, allowing protein engineering to improve the properties of scFv (single chain fragment variable) such as increase of affinity and alteration of specificity [30].

The length of the flexible DNA linker used to link both of the domains is critical in yielding the correct folding of the polypeptide chain. Previously, it has been estimated that the peptide linker must span 3.5nm (35) between the carboxy terminus of the variable domain and the amino terminus of the other domain without affecting the ability of the domains to fold and form an intact antigenbinding site [31]. In addition to the linker peptides designed de novo, peptide sequences derived from known protein structure have been applied to provide a compatible length and conformational in bridging the variable domains of a scFv (single-chain fragment variable) without serious steric interference [3234]. Apart from the length of the linker, their amino acid composition also plays an important

role in the design of a viable linker peptide. They must have a hydrophilic sequence in order to avoid intercalation of the peptide within or between the variable domains throughout the protein folding [35]. Nowadays, the most extensively used designs have sequences comprising stretches of Gly and Ser residues which meant for flexibility and or together with the charged residues such as Glu and Lys interspersed to enhance the solubility [36].

4. The Generation of scFv (Single-Chain Fragment Variable) Antibodies

scFv (single chain fragment variable) antibodies have been constructed mainly from hybridoma [16,3739], spleen cells from immunized mice [4042], and B lymphocytes from human [4345]. scFv (single chain fragment variable) is a noncovalent heterodimer comprised of the and domains [12] in which can then be used in the construction of recombinant scFv (single chain fragment variable) antibody. In order to attain them, mRNA is first isolated from hybridoma (or also from the spleen, lymph cells, and bone morrow) followed by reverse transcribed into cDNA to serve as a template for antibody genes amplification (PCR). With this method, large libraries with a diverse range of antibody and genes could be created [43]. One successful approach to recombinant antibody production has been developed by McCafferty and coworkers in which they utilized the phage recombinants that are displaying antibody at their tips together with the new techniques of affinity selection, called biopanning step. The work by McCafferty et al. [14] thus has opened the opportunity for in vitro selection of scFv (single chain fragment variable) from large libraries of variable domains circumventing the traditional hybridoma method. In the scFv (single-chain fragment variable) construction, the order of the domains can be either -linker- or -linker- and both orientations have been applied [4648]. Even though Luo et al. [49] have shown that the expression of scFv (single-chain fragment variable) of Pichiapastoris system is -linker- orientationdependent; most of the scFv (single-chain fragment variable) are constructed in a -linker- orientation. One of the most popular methods used is through PCR assembly [40, 43,50] which was first described by Horton et al. [51]. In this method, it allows the domains of antibody to be cloned without any prior information about the nucleic acid as well as amino acid sequence of the particular antibody [52]. Moreover, the domains of antibody can be combined by in vitrorecombination directly after the PCR of and genes into plasmid [38, 53] or phagemid [54]. Alternatively, scFv (single-chain fragment variable) can also be constructed with sequential cloning [55,56] or combinatorial infection [57]. Numerous scFv (single-chain fragment variable) have been constructed against hapten [55], protein [46,58], carbohydrate [59, 60], receptor [16], tumor antigen [44, 61], and viruses [47]. All these scFv (single chain fragment variable) have good potential for use in many fields such as medical therapies and diagnostic applications