Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli.

Abstract

A biosynthetic antibody binding site, which incorporated the variable domains of anti-digoxin monoclonal antibody 26-10 in a single polypeptide chain (Mr = 26,354), was produced in Escherichia cofi by protein engineering. This variable region fragment (Fv) analogue comprised the 26-10 heavy-and light-chain variable regions (VH and VL) connected by a 15-amino acid linker to form a single-chain Fv (sFv). The sFv was designed as a prolyl-VH-(linker)-VL sequence of 248 amino acids. A 744-base-pair DNA sequence corresponding to this sFv protein was derived by using an E. colt codon preference, and the sFv gene was assembled starting from synthetic oligonucleotides. The sFv polypeptide was expressed as a fusion protein in E. colt, using a leader derived from the trp LE sequence. The sFv protein was obtained by acid cleavage of the unique Asp-Pro peptide bond engineered at the junction of leader and sFv in the fusion protein [(leader)-Asp-Pro-VH-(linker)-VL]. After isolation and renaturation, folded sFv displayed specificity for digoxin and related cardiac glycosides similar to that of natural 26-10 Fab fragments. Binding between afirmity-purified sFv and digoxin exhibited an association constant [Ka = (3.2 ± 0.9) x 107 M -1] that was about a factor of 6 smaller than that found for 26-10 Fab fragments [K. = (1.9 @ 0.2) x 108 M 'I under the same buffer conditions, consisting of 0.01 M sodium acetate, pH 5.5/0.25 M urea.It is known that antigen binding fragments of antibodies (1,2) can be refolded from denatured states with recovery of their specific binding activity (3)(4)(5)(6). The smallest such fragment that contains a complete binding site is termed Fv, consisting of an Mr 25,000 heterodimer of the VH and VL domains (2, 5-11). Givol and coworkers were the first to prepare an Fv by peptic digestion of murine IgA myeloma MOPC 315 (2). However, subsequent development of general cleavage procedures for Fv isolation has met with limited success (7-11). As a result, the Mr 50,000 Fab (1) has remained the only monovalent binding fragment used routinely in biomedical applications.An Fv analogue was constructed in which both heavy-and light-chain variable domains (VH and VL) were part of a single polypeptide chain. Synthetic genes for the 26-10 anti-digoxin VH and VL regions were designed to permit their connection through a linker segment, as well as other manipulations (12,13 MATERIALS AND METHODSModel Antibody. The digoxin binding site of the IgG2a,K monoclonal antibody 26-10 has been analyzed by MudgettHunter and colleagues (14-16). The 26-10 V region sequences were determined from both protein sequencing (17) (14) and has a well-defined specificity profile (15) (Fig. 1).Gene Synthesis. Design of the 744-base sequence for the synthetic sFv gene was derived from the sFv protein sequence by choosing codons preferred by E. coli (25). Synthetic genes encoding the trp promoter-operator, the modified trp LE leader peptide (MLE), and VH were prepared largely as described (26). The gene encoding VH was assembled from 46...

Construction and characterization of DNA vaccines encoding the single-chain variable fragment of the anti-idiotype antibody 1A7 mimicking the tumor-associated antigen disialoganglioside GD2

“…15,16 In scFv constructs, the carboxy terminus is joined to the amino terminus of variable domains by a Ln of appropriate length and flexibility. Therefore, the scFv can be constructed at least in two different configurations, either VHLnVL or VLLnVH.…”

“…Therefore, the scFv can be constructed at least in two different configurations, either VHLnVL or VLLnVH. 15 These two different orientations of domains may distort native conformation of the antigenbinding site of scFv. We have previously shown that for the best antigen mimicry of scFv derived from 3H1 Ð an antiidiotype antibody mimicking carcinoembryonic antigen Ð the VH chain should be in the amino terminus of the scFv; VH and VL should be linked by a 15 -amino acid Ln.…”

“…15,16 In this communication, we report the construction of two antiidiotype DNA vaccines, designated pc1A7VHLnVL and pc1A7VLLnVH, encoding two different configurations of the scFv of 1A7. We present results demonstrating that scFvs of 1A7 encoded by these plasmids are expressed in vitro and in vivo and the expressed protein can bind to the monoclonal Ab1 antibody, 14G2a.…”

Disruption of P‐glycoprotein anticancer drug efflux activity by a small recombinant single‐chain Fv antibody fragment targeted to an extracellular epitope

“…A major outcome of these advances is the ability to generate very small and functional antibody fragments, such as scFvs. [13][14][15] Moreover, using antibody phage display libraries, it is possible to isolate new recombinant scFv antibodies with the desired specificity and affinity directed toward a large array of antigens. 16 Our aim was to explore the possibility of using a small recombinant scFv antibody molecule for the modulation and reversal of the MDR phenotype in tumor cells by direct inhibition of Pgpmediated drug-efflux activity.…”

A recombinant single-chain antibody interleukin-2 fusion protein

“…As a delivery system the smaller single chain antibody (SCA), comprising linked variable heavy (VH) and variable light (VL) chain antibody domains shows great promise (Huston et al, 1988). Where tested SCAs demonstrate good tissue penetration (Yokota et al, 1992), rapid renal clearance of non-localised protein and potentially low immunogenicity (Colcher et al, 1990).…”