Recombinant Antibody Engineering

The major types of recombinant antibody fragments that are usually expressed in E. coli are named Fv, dsFv, scFv, and Fab (Fig. 3). Fv (fragment variable) fragments consists of only the variable domains of the heavy and light chains (VH and VL) and are the smallest units that retain binding properties to a given antigen (10). VH and VL expressed in E. coli assemble spontaneously into Fv fragments through noncovalent interactions. However, the VH-VL interaction is usually weak, and therefore these dimeric proteins are sometimes prone to dissociation and aggregation, depending on the antibody sequence. Recombinant DNA techniques have been used to either create an interdisulfide bond between VH and VL (leading to the dsFv antibody fragment) or to introduce a short peptide linker between VH and the VL to create a single polypeptide chain folding into the so-called scFv antibody fragment. While the dsFv needs site-directed mutagenesis within the framework and/or Complementarity Determining Region (CDR) region of a given antibody, the creation of scFv is a generally applicable method. It has been shown that shortening the linker between the two variable domains leads to so-called diabodies (11), which pair with the complementary domains of another scFv chain and thereby promote the assembly of dimeric or bispecific molecules with two functional antigen-binding sites.

Recombinant Antibody Engineering

Fig. 2. Specificity ELISA of an antibody (bivalent Fab-dHLX fragment) selected from the HuCAL® library on a phosphorylated peptide with the sequence RKSAPpSTGG-C (P-antigen), coupled to the carrier proteins bovine serum albumin (BSA) and human transferring (TRF) and immobilized on a microtiter well. Prior to selection, the antibody library was incubated with the nonphosphorylated counterpart RKSAPSTGG-C (non-P-antigen), thereby blocking antibody specificities toward the nonphosphorylated form. During the panning, the peptide-carrier conjugate was alternated between TRF in rounds 1 and 3, and BSA in round 2, thereby removing carrier-specific antibodies. One of selected antibodies was purified and tested in ELISA (2 ^g/mL) for binding to a range of immobilized peptides and proteins (5 ^g/mL each). Detection was performed with horseradish peroxidase-conjugated mouse anti-His-tag antibody (1:500 diluted) and Quanta-Blue as substrate. As can be seen in the figure, the antibody recognizes the phosphorylated antigen and does neither bind to the nonphosphorylated counterpart nor to the carrier proteins. In addition, unrelated peptides (with or without a phosphor-serine group) or unrelated proteins are also not bound.

Fig. 2. Specificity ELISA of an antibody (bivalent Fab-dHLX fragment) selected from the HuCAL® library on a phosphorylated peptide with the sequence RKSAPpSTGG-C (P-antigen), coupled to the carrier proteins bovine serum albumin (BSA) and human transferring (TRF) and immobilized on a microtiter well. Prior to selection, the antibody library was incubated with the nonphosphorylated counterpart RKSAPSTGG-C (non-P-antigen), thereby blocking antibody specificities toward the nonphosphorylated form. During the panning, the peptide-carrier conjugate was alternated between TRF in rounds 1 and 3, and BSA in round 2, thereby removing carrier-specific antibodies. One of selected antibodies was purified and tested in ELISA (2 ^g/mL) for binding to a range of immobilized peptides and proteins (5 ^g/mL each). Detection was performed with horseradish peroxidase-conjugated mouse anti-His-tag antibody (1:500 diluted) and Quanta-Blue as substrate. As can be seen in the figure, the antibody recognizes the phosphorylated antigen and does neither bind to the nonphosphorylated counterpart nor to the carrier proteins. In addition, unrelated peptides (with or without a phosphor-serine group) or unrelated proteins are also not bound.

Another commonly used recombinant antibody fragment is the Fab fragment, which is composed of the truncated heavy chain containing the variable and the first constant region, and the entire light chain composed of the variable and constant domain. These two polypeptides are either covalently linked by disulfide bridges at the C-terminus, or are produced in higher yields without those, which nevertheless lead to highly stable H/L heterodimers (8). The Fab fragment is truly monovalent (which is not always the case with scFv fragments), it does not contain an artificial linker sequence which might interfere with the antigen binding site, and well-established anti-Fab detection antibodies can be used.

Scfv Antibody

Fig. 3. Antibody fragments commonly expressed in Escherichia coli. The Fv, dsFv, and Fab formats are expressed as two distinct polypeptide chains and assemble after folding in the periplasm to functional antibody fragments. The Fab format can be either cova-lently linked by a C-terminal disulfide bond or associated by noncovalent forces. The scFv formats are expressed as one polypeptide chain, whereby the two variable domains are connected with a Gly4Ser linker of variable length. Both orientations (VH-linker-VL or VL-linker-VH) have been described.

Fig. 3. Antibody fragments commonly expressed in Escherichia coli. The Fv, dsFv, and Fab formats are expressed as two distinct polypeptide chains and assemble after folding in the periplasm to functional antibody fragments. The Fab format can be either cova-lently linked by a C-terminal disulfide bond or associated by noncovalent forces. The scFv formats are expressed as one polypeptide chain, whereby the two variable domains are connected with a Gly4Ser linker of variable length. Both orientations (VH-linker-VL or VL-linker-VH) have been described.

Recombinant antibodies offer many advantages that are only beginning to be explored. These advantages stem from two properties: the ability to assess the antibody DNA within an E. coli environment that allows the use of well-known genetic engineering methodologies, and the use of antibody fragments rather than intact IgG molecules because of the small size of Fab and scFv fragments, and because of the absence of the Fc domain. The latter eliminates nonspecific binding to cellular Fc receptors and avoids other nonspecific effects caused by the Fc part of intact IgG, which for instance induces cytokine release in functional assays with immunocompetent cells. In addition, nonspecific binding to the Fc region in immunocytochemical applications is eliminated, which often leads to lower background, better signal-to-noise ratio, and increased sensitivity. Moreover, fragments diffuse better through tissue and through cell membranes than intact IgG because of its small size, leading to faster and more efficient staining. The fact that scFv and Fab perform monovalent interactions is important when avidity effects should be avoided, e.g., when the intrinsic binding affinity needs to be determined. In fluorescent or enzyme conjugates, Fab or scFv fragments provide reduced hydrophobicity, reduced steric hindrance (improved stoichiometry because more markers can bind to the presented antigens), and less interference with serum factors and macromolecules (which mostly bind to the Fc region). Cocrystallization of target proteins with antibodies also requires smaller antibody fragments.

Generation of antibodies in vitro enables manipulation of their sequences, for instance by linking desired sequences to the antibody framework regions. Examples are peptide tags for purification, immobilization, and detection, enzymatic activities like alkaline phosphatase for direct detection, modules for multi-merization to create multimeric-binding sites with increased functional affinity (avidity), modules that lead to heterodimerization, thereby creating bispecific antibodies, and, last but not least, toxins for the elimination of tumor cells in therapeutic applications. Typically, such fusions are cloned in-frame at the 3'-end of the antibody gene, leading to a maximum distance in the native fusion protein between the antigen-binding site and the additional functionality. A few such fusions will be highlighted here.

Peptide tags are mostly used for affinity purification purposes. Most tags that have been generally developed for recombinant protein purification will also work for antibodies. A typical example is the His-tag, a series of five to six histidines that bind to affinity media such as Nitrilotriacetic acid (NTA)-agarose or Talon resin, when metal ions (nickel or cobalt) are bound. His-tagged proteins bind with millimolar affinity to the column and are gently eluted with 150-300 mM imidazole. Another such tag is the StrepII-tag, which shows affinity to streptavidin-or streptactin-sepharose (streptactin is a genetically engineered streptavidin with higher affinity to the Strep-tag). Antibodies to both tags are available, so the tags can be used for detection purposes as well. Other detection tags like the V5-tag

(GKPIPNPLLGLDST), which is derived from a small epitope present on the P/V proteins of the paramyxovirus, SV5, or the myc-tag (EQKLISEEDL), which correspond to residues 408-439 of the human p62 c-myc protein are also frequently used because specific high-affinity MAbs are commercially available. The FLAG-tag (DYKDDDDK) is of special interest because a MAb (termed M1) exists that only binds to the tag when the N-terminus is free and not involved in a peptide bond. This has been used to monitor cleavage of signal sequences during E. coli antibody expression (12).

Site-directed immobilization of recombinant antibodies should improve functional activity in array- or bead-based applications because it has been shown that random passive adsorption of whole antibodies on plastic surfaces results in major loss of protein function (13). Indeed, in a comparative study it was shown that specifically oriented Fab fragments that had been immobilized via a C-terminal thiol group had an up to 10-fold higher capture capacity compared with surfaces which randomly oriented capture agents (14). Recombinant antibodies with a C-terminal cysteine residue can easily be engineered.

Antibody fragments usually have the same antigen-binding specificity as the corresponding intact antibody because the complete antigen-binding site is present. However, multivalency is a very general nature of antibodies. IgG contain two binding sites per molecule, which increases the apparent affinity (avidity) compared with a Fab antibody fragment in cases where the antigen contains multiple epitopes or where multiple antigens are bound to a surface. The most noticeable example is IgM, which carries 10 recognition binding sites. For particular applications, bivalency might be advantageous or even required. Thus, bivalency and further multivalency have been also engineering for recombinant antibody fragments (Fig. 4). Dimeric mini-antibodies have been created using small "association domains," which are fused to the C-terminal portion of Fab or scFv antibody fragments (15). For example, the leucine zipper from the yeast transcription factor GCN4 has been shown to be suitable as a dimerization device. Such "mini-antibodies" have been shown to display identical functional affinities as a whole parent antibody. Antibody fragments fused to the small tetramerization domain of p53 can form tetrameric molecules. In general, the expression yields are not reduced with such constructs.

Genetic fusions to enzymes like bacterial alkaline phosphatase (BAP) connect the binding and detection capability into one molecule. Such fusions lead to molecules the size of full-length antibodies to be expressed in E. coli with the same yields as smaller antibody fragments. Because the BAP is a homo-dimer, the resulting molecules are bivalent (see Fig. 4). Many other antibody fusions for various applications have been desribed in the literature, such as core streptavidin for avidity increase, P-lactamase for prodrug activation, or interleukin-8 fusion for neutrophil activation, to name a few. Cleary this field is still in its infancy.

Genetic Engineering Antibody

Fig. 4. Examples of multivalent antibody fragments obtained by genetic engineering. The Fab-dHLX fragment is dimerized by a self-assembling helix-turn-helix motif. The linker between the CH1 domain and the dimerization motif is derived from the mouse IgG3 upper hinge region. The Fab-bacterial alkaline phosphatase (BAP) format is obtained by fusing the bacterial phoA gene to the 3'-end of the heavy chain gene segment. Because the BAP is a homodimeric molecule, the resulting antibodies are bivalent. The tetrameric scFv-p53 format is obtained by using the tetramerization domain of human p53. All these formats can be functionally expressed in Escherichia coli. Usually there are also peptide tags attached to the C-termini of the molecules, which are not shown here.

Fig. 4. Examples of multivalent antibody fragments obtained by genetic engineering. The Fab-dHLX fragment is dimerized by a self-assembling helix-turn-helix motif. The linker between the CH1 domain and the dimerization motif is derived from the mouse IgG3 upper hinge region. The Fab-bacterial alkaline phosphatase (BAP) format is obtained by fusing the bacterial phoA gene to the 3'-end of the heavy chain gene segment. Because the BAP is a homodimeric molecule, the resulting antibodies are bivalent. The tetrameric scFv-p53 format is obtained by using the tetramerization domain of human p53. All these formats can be functionally expressed in Escherichia coli. Usually there are also peptide tags attached to the C-termini of the molecules, which are not shown here.

The following section will describe the use of recombinant antibody Fab fragments and derivatives in typical immunoassays such as Western blot, ELISA, and immunohistochemistry (IHC).

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