B

in intracellular inclusion bodies in separate cultures; in the latter case, they are reconstituted by a refolding reaction using a redox-shuffling buffer system. Several immunotoxins with Fab fragments have been constructed and produced in this way (Pastan et al., 1992; Choe et al., 1994; Vitteta, 1994; Kreitman and Pastan, 1998; Chamow and Ashkenazi, 1999; Kreitman, 1999; Reiter, 2001).

The smallest functional modules of antibodies necessary to maintain antigen binding are Fv fragments. This makes them especially useful for clinical applications, not only for generating recombinant immunotoxins but also for tumor imaging, because their small size improves tumor penetration. Fv fragments are heterodimers of the variable heavy-chain (VH) and the variable light-chain (VL) domains (Figure 5.3). Unlike whole IgG or Fab, in which the heterodimers are held together and stabilized by interchain disulfide bonds, the VH and VL of Fvs are not covalently connected and are consequently unstable; this instability can be overcome by making recombinant Fvs that have the VH and VL covalently connected by a peptide linker that fuses the C-terminus of the VL or VH to the N-terminus of the other domain (Figure 5.2). These molecules are termed single-chain Fvs (scFvs) (Bird et al., 1988; Huston et al., 1988), and some retain the specificity and affinity of the original antibody. The cloning, construction and composition of recombinant Fv fragments of antibodies and of Fv-immunotoxins are described in Figure 5.3.

Many recombinant immunotoxins have been constructed using scFvs, in which the scFv gene is fused to PE38 to generate a potent cytotoxic agent with targeted specificity (Figures 5.1 and 5.4) (Chaudhary et al., 1989; Brinkmann et al., 1991; Batra et al., 1992; Pastan et al., 1992; Vitteta, 1994; Kreitmann et al., 1994; Debinski and Pastan, 1995; Kuan and Pastan, 1996; Reiter et al., 1996; Kreitman and Pastan, 1998; Chamow and Ashkenazi, 1999; Kreitman, 1999; Reiter, 2001).

Until recently, the construction of scFvs was the only general method available to make stable Fvs. However, many scFvs are unstable or have reduced affinity for the antigen compared with the parent antibody or Fab fragment. This is because the linker interferes with binding or because the linker does not sufficiently stabilize the Fv structure, leading to aggregation and loss of activity. This is particularly true at physiological

Figure 5.2 (Continued)

a uracil-containing single-stranded DNA of the scFv construct from the F+origin present in the expression plasmid and cotransfection with M13 helper phage. In addition to the cysteines, cloning sites, ATG translation-initiation codons, and stop codons are introduced at the 5' end and 5' end of the VH and VL genes as shown by site-directed mutagenesis or PCR. The antibody variable genes are subcloned into an expression vector which contains the gene for a truncated form of Pseudomonas exotoxin. This expression vector is controlled by the T7 promoter and upon induction of the T7 RNA polymerase, which is under the control of the lacUV5 promoter, in E. coli BL21 ADE3 by IPTG, large amounts of recombinant protein are produced. (B) Design and composition of recombinant immunotoxins. In PE-derived recombinant Fv-immunotoxins the Fv region of the targeting antibody is fused to the N-terminus of a truncated form of PE which contains the translocation domain (domain II) and enzymatically active ADP-ribosylation domain (domain III). The cell-binding domain of whole PE (domain I) is replaced by the Fv targeting moiety thus, preserving the relative position of the binding domain function to the other functional domains of PE in the dsFv-immunotoxins there are two components. In one the VH or VL domains are fused to the amino terminus of the truncated PE, and the other variable domain is covalently linked by the engineered disulfide bond. DT-derived immunotoxins are fused to the carboxyl terminus due to the inverse arrangement of the functional modules of PE and DT.

Figure 5.3 Recombinant Fv fragments. Fv fragments are heterodimers of the heavy- and light-chain variable domains (VH in green, VL in pink). They are the smallest functional modules of antibodies necessary to maintain antigen binding. Unlike whole IgG or Fab, in which the heterodimer is held together and stabilized by interchain disulfide bonds, the VH and VL are not covalently connected and are consequently unstable (A). In scFv this instability can be overcome by connecting the VH and VL by a peptide linker (in white) that fuses the C terminus of the VH to the N terminus of the VL (B). An alternative strategy to scFvs is to connect the VH and VL domains by an interchain disulfide bond (C, in white) engineered between structurally conserved framework residues of the VH and VL (dsFv). Because the positions at which the cysteine residues are located, in the conserved framework of each VH and VL, this location can be used as a general method to stabilize any Fv without the need for further structural information. (See Color Plate VII.)

temperatures (37 °C). To overcome these problems, an alternative strategy has been developed to generate stable Fvs by connecting the VH and VL domains by an interchain disulfide bond engineered between structurally conserved framework residues of the Fv; these molecules are termed disulfide-stabilized Fv (dsFv) (Glockshuber et al., 1990; Brinkmann et al., 1993; Reiter et al., 1996a; Reiter and Pastan, 1996). The positions at which the cysteine residues are placed were identified by computer-based molecular modeling; as they are located in the framework of each VH and VL, this location can be used as a general method to stabilize all Fvs without the need for further structural information (Figure 5.3).

Figure 5.4 Recombinant scFv-immunotoxin. (A) Pseudomonas exotoxin (PE) is a single-chain 66-kDa molecule secreted by Pseudomonas aeruginosa that irreversibly ADP-ribosylates EF2 and as a consequence, protein synthesis is inhibited and cell death occurs. PE is composed of three major domains. The N-terminal domain I, aa 1-252 (green) mediates binding to the a2 macroglobulin receptor. Domain II, aa 253-364 (yellow) mediates translocation of domain III aa 400-613 (red), the carboxyl-terminal ADP-ribosylating domain, into the cytosol of target cells. Currently, almost all PE-derived recombinant immunotoxins are constructed with PE38 (MW 38 kDa), a PE derivative in which the cell-binding domain (domain I, green) has been deleted and replaced by a targeting Fv molecule. (B) The antibody moiety of the recombinant immunotoxin is responsible for specific targeting of the immunotoxin to the tumor cell, meaning that the usefulness of the immunotoxin depends on the specificity of the antibody or antibody fragment that is connected to the toxin. In scFv PE-based recombinant immunotoxins the targeting scFv fragment consists of the antibody VH (pink) and VL (green) variable domain which are held together by a flexible peptide linker (white). This tumor-specific scFv is fused to a truncated form of Pseudomonas exotoxin (PE38) via a short connector (white) and thus, a single gene encodes the recombinant immunotoxin. The truncated PE38 contains the translocation (yellow) and enzymatically active ADP-ribosylation (red) domains but lacks the cell binding domain (Green in A). (See Color Plate VIII.)

Many dsFvs have been constructed in the past three years (mainly as dsFv-immunotoxins, in which the dsFv is fused to PE38) and they show several advantages over scFvs, (Reiter et al., 1994a,b; 1996a; Mansfield et al., 1997). In addition to their increased stability (owing to a decreased tendency to aggregate), they are often produced in higher yields than scFvs; in several cases, the binding affinity of the dsFv is significantly improved over that of the scFv.

A major advantage of using recombinant Fvs for immunotoxin design is the ability today to select antibodies with unique specificities from very large antibody repertoires that are presented on phage. Phage-display technology is revolutionizing the field of targeted immunotoxin therapy because the antibodies are displayed as recombinant fragments (Fab fragments or scFvs) and once isolated and characterized, they can be used directly to construct a recombinant Fv-immunotoxin.

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