Properties of a single-chain antibody containing different linker peptides

Single-chain antibodies were constructed using six differentlinker peptides to join the V_H and V_L domains of an anti-2-phenyloxazolone(Ox) antibody. Four of the linker peptides originated from theinterdomain linker region of the fungal cellulase CBHI and consistedof 28, 11, six and two amino acid residues. The two other linkerpeptides used were the (GGGGS)₃ linker with 15 amino acid residuesand a modified IgG2b hinge peptide with 22 residues. Proteolyticstability and Ox binding properties of the six different scFvderivatives produced in Escherichia coli were investigated andcompared with those of the corresponding Fv fragment containingno joining peptide between the V domains. The hapten bindingproperties of different antibody fragments were studied by ELISAand BIAcore^TM. The interdomain linker peptide improved the haptenbinding properties of the antibody fragment when compared withFv fragment, but slightly increased its susceptibility to proteases.Single-chain antibodies with short CBHI linkers of 11, six andtwo residues had a tendency to form multimers which led to ahigher apparent affinity. The fragments with linkers longerthan 11 residues remained monomeric.     

A33scFv-green fluorescent protein, a recombinant single-chain fusion protein for tumor targeting

Chemical conjugates of monoclonal antibodies with fluorophores or enzymes have long been used for diagnostic purposes and experimental therapeutic approaches. Recombinant technology allows for the design and expression of tailored genuine fusion proteins, providing defined molecules as to size, molar ratios of the functional components and stability. The production of functional protein, however, is often limited or impossible due to refolding and solubility problems. Here, we report on the production of a soluble recombinant fusion construct, A33scFv-green fluorescent protein (A33scFv::GFP) in Pichia pastoris. A33scFv is a single-chain antibody recognizing the A33 antigen, which is expressed by approximately 95% of colorectal carcinomas and has become a focus of pre-clinical and clinical investigation. The fusion partner GFP was selected both as an experimental tool for functional studies of the A33 antigen and as a potential diagnostic for colon cancer detection and therapy planning. Pichia pastoris yeast strains were transformed with A33scFv::GFP cDNA under the methanol-inducible AOX1 promotor. The construct was properly expressed and secreted into culture supernatants as a soluble protein, which was bifunctional without additional renaturation or solubilization steps. The crude protein solution was purified by affinity chromatography. Surface plasmon resonance, flow cytometry and fluorescence microscopy on sections of normal and cancerous colon tissue revealed specific binding and the applicability of this fusion protein for diagnostic purposes. In addition, the biodistribution of A33scFv::GFP was analyzed in mice bearing A33-positive tumor xenografts, confirming specific tumor targeting.     

Effects of humanization by variable domain resurfacing on the antiviral activity of a single-chain antibody against respiratory syncytial virus

HNK20 is a mouse monoclonal IgA that binds to the F glycoproteinof respiratory syncytial virus (RSV) and neutralizes the virus,both in vitro and in vivo. The single-chain antibody fragment(scFv) derived from HNK20 is equally active and has allowedus to assess rapidly the effect of mutations on affinity andantiviral activity. Humanization by variable domain resurfacingrequires that surface residues not normally found in a humanFv be mutated to the expected human amino acid, thereby eliminatingpotentially immunogenic sites. We describe the constructionand characterization of two humanized scFvs, hu7 and hu10, bearing7 and 10 mutations, respectively. Both molecules show unalteredbinding affinities to the RSV antigen (purified F protein) asdetermined by ELISA and surface plasmon resonance measurementsof binding kinetics (K_a 1x10⁹ M^–1). A competition ELISAusing captured whole virus confirmed that the binding affinitiesof the parental scFv and also of hu7 and hu10 scFvs were identical.However, when compared with the original scFv, hu10 scFv wasshown to have significantly decreased antiviral activity bothin vitro and in a mouse model. Our observations suggest thatbinding of the scFv to the viral antigen is not sufficient forneutralization. We speculate that neutralization may involvethe inhibition or induction of conformational changes in thebound antigen, thereby interfering with the F protein-mediatedfusion of virus and cell membranes in the initial steps of infection.     

Lipid-tagged antibodies: bacterial expression and characterization of a lipoprotein--single-chain antibody fusion protein

In order to achieve a stable and functional immobilization ofantibodies, we investigated the possibility of adding hydrophobicmembrane anchors to antibody fragments expressed in Escherichiacoli. The DNA sequence encoding the signal peptide and the nineN-terminal amino add residues of the major lipoprotein of E.coliwas fused to the sequence of an anti-2-phenyloxazolone single-chainFv antibody fragment [Takkinen et al. (1991) Protein Engng,4, 837–841]. The expression of the fusion construct inE.coli resulted in specific accumulation of an immunoreactive28 kDa polypeptide. Unlike the unmodified single-chain Fv fragment,the fusion protein was cell-associated, labelled by [³H]palmitatewhich is indicative of the presence of N-terminal lipid modification,partitioned into the detergent phase upon Triton X-114 phaseseparation and was localized predominantly in the bacterialouter membrane. The fusion antibody displayed specific 2-phenyloxazolone-bindingactivity in the membranebound form and after solubilizationwith non-ionic detergents. Furthermore, upon removal of detergentthe fusion antibody was incorporated into proteoliposomes whichdisplayed specific hapten-binding activity. Our results showthat antibodies can be converted to membrane-bound proteinswith retention of antigen-binding properties by introductionof lipid anchors during biosynthesis. This approach may proveuseful in the design of immunoliposomes and immunosensors.     

An active single-chain antibody containing a cellulase linker domain is secreted by Escherichia coli

Single-chain antibodies consist of the variable, antigen-bindingdomains of antibodies joined to a continuous polypeptide bygenetically engineered peptide linkers. We have used the flexibleinterdomain linker region of a fungal cellulase to link togetherthe variable domains of an anti-2-phenyloxazolone IgGl and showhere that the resulting single-chain antibody is efficientlysecreted and released to the culture medium of Escherichia coli.The yield of affinity-purified single-chain antibody is 1 -2mg/1 of culture medium and its affinity and stability are comparableto those of the corresponding native IgG.     

Effect of linker sequences between the antibody variable domains on the formation, stability and biological activity of a bispecific tandem diabody

Bispecific single-chain Fv antibodies comprise four covalently linked immunoglobulin variable (V(H) and V(L)) domains of two different specificities connected by three linkers. When assembled in the order V(H)(A)-linker(1)-V(L)(B)-linker(2)-V(H)(B)-linker(3)-V(L)(A), the single-chain molecule either folds head-to-tail with the formation of a diabody-like structure, a so-called bispecific single-chain diabody, or forms a homodimer that is twice as large, a so-called tandem diabody. The formation of the tandem diabody is determined by the association of complementary V(H) and V(L) domains located on different polypeptide chains, and depends on the length and probably the amino acid composition of the three linkers joining the variable domains. We generated a number of single-chain constructs using four V(H) and V(L) domains specific either for human CD3, a component of T-cell receptor (TCR) complex, or for CD19, a human B-cell antigen, separated by different rationally designed peptide linkers of 6-27 amino acid residues. The generated bispecific constructs were expressed in bacterial periplasm and their molecular forms, antigen-binding properties, stability, and T-cell proliferative and anti-tumor activities were compared. Using peripheral blood mononuclear cell cultures from patients suffering from B-cell chronic lymphocytic leukemia, we demonstrated that the tandab-mediated activation of autologous T cells and depletion of malignant cells correlates with the stability of the recombinant molecule and with the distance between the CD19 and CD3 binding sites.     

Synthesis, cloning and expression of the single-chain Fv gene of the HPr-specific monoclonal antibody, Jel42. Determination of binding constants with wild-type and mutant HPrs

The monoclonal antibody Jel42 is specific for the Escherichiacoli histidine-containing protein, HPr, which is an 85 aminoacid phosphocarrier protein of the phosphoenolpyruvate:sugarphosphotransferase system. The binding domain (Fv) has beenproduced as a single chain Fv (scFv). The scFv gene was synthesizedin vitro and coded for pelB leader peptide–heavy chain–linker–lightchain–(His)₅ tail. The linker is three repeats from theC-terminal repetitive sequence of eukaryotic RNA polymeraseII. This linker acts as a tag; it is the antigen for the monoclonalantibody Jel352. The codon usage was maximized for E.coli expression,and many unique restriction endonuclease sites were incorporated.The scFv gene incorporated into pT7-7 was highly expressed,yielding 10–30% of the cell protein as the scFv, whichwas found in inclusion bodies with the leader peptide cleaved.Jel42 scFv was purified by denaturation/renaturation yieldingpreparations with K_d values from 20 to 175 nM. However, basedupon an assessment of the amount of active refolded scFv, thebinding dissociation constant was estimated to be 2.7 ±2.0 nM compared with 2.8 ± 1.6 and 3.7 ± 0.3 nMpreviously determined for the Jel42 antibody and Fab fragmentrespectively. The effect of mutation of the antigen HPr on thebinding constant of the scFv was very similar to the propertiesdetermined for the antibody and the Fab fragment. It was concludedthat the small percentage (~6%) of refolded scFv is a true mimicof the Jel42 binding domain and that the incorrectly foldedscFv cannot be detected in the binding assay.     

Direct expression in E.coli of a functionally active protein A-snake toxin fusion protein

We constructed a recombinant expression plasmid encoding a proteinA–neurotoxin fusion protein. The fused toxin is directlyexpressed in the periplasmic space of Escherichia coli and canbe purified in the milligram range by a single immuno-affinitystep. The LD₅₀ values of the fused toxin and native toxin are130 and 20 nmol/kg mouse respectively. The K_d values characterizingtheir binding to the nicotinic acetylcholine receptor (AcChoR)are respectively 4.8 ± 0.8 and 0.07 ± 0.03 nM.In contrast, the fused and native toxins are equally well recognizedby a toxin-specific monoclonal antibody which recognizes theAcChoR binding site. The lower toxicity of the fused toxin mightresult, therefore, from a steric hindrance, due to the presenceof the bulky protein A moiety (mol. wt = 31 kd) rather thanto a direct alteration of the ‘toxic’ site. Thefused toxin is more immunogenic than native toxin, since 1 nmolof hybrid toxin and 14 nmol of native toxin give rise to comparabletiters of antitoxin antibodies which, furthermore, are equallypotent at neutralizing neurotoxicity. The work described inthis paper shows that the use of fused toxins may be of paramountimportance for future development of serotherapy against envenomationby snake bites.     

A novel tri-functional antibody fusion protein with improved pharmacokinetic properties generated by fusing a bispecific single-chain diabody with an albumin-binding domain from streptococcal protein G

The therapeutic application of small recombinant antibody molecules is often limited by a short serum half-life. In order to improve the pharmacokinetic properties, we have investigated a strategy utilizing fusion with an albumin-binding domain (ABD) from streptococcal protein G. This strategy was applied to a bispecific single-chain diabody (scDb CEACD3) developed for the retargeting of cytotoxic T cells to CEA-expressing tumor cells. This novel tri-functional fusion protein (scDb-ABD) was expressed in mammalian cells and recognized both antigens as well as human and mouse serum albumin. scDb-ABD was capable to retarget T cells to CEA-expressing target cells in vitro and to activate the effector cells as measured by stimulation of IL-2 release. Although activity was reduced 3-fold compared with scDb and further reduced 4-fold in the presences of human serum albumin, this assay demonstrated that scDb-ABD is active when exposed to all three antigens. Compared with scDb, the circulation time of scDb-ABD in mice was prolonged 5- to 6-fold similar to a previously described scDb-HSA fusion protein. This strategy, which adds only a small protein domain (46 amino acids) and which utilizes high-affinity, non-covalent albumin interaction, should be broadly applicable to improve serum half-lives of small recombinant antibody molecules.     

Destabilizing interactions between the partners of a bifunctional fusion protein

Hybrid MalE–GVP is a bifunctional protein in vitro sinceit binds maltose as protein MalE of Escherichia coli and sinceit is dimeric and specifically binds single-stranded DNA asprotein GVP of phage M13. The oxidation rate of a unique cysteineresidue was used to compare the stabilities of GVP in its freeand hybrid forms, under conditions where MalE was either foldedor unfolded by a denaturing agent. The results showed that boththe covalent link and tertiary non-covalent interactions betweenMalE and GVP destabilized GVP in MalE–GVP. To test whetherGVP had identical structures in its free and hybrid forms, mutationswere used as local conformational probes. The effects of thesemutations on the capabilities of MalE–GVP to dimerizeand to bind single-stranded DNA were assayed in vitro. Theywere compatible with the effects of the same mutations on theglobal activity of free GVP in vivo and with the effects thatcould be predicted from the known data on free GVP, in particularits crystal structure. Thus, one partner of a hybrid proteincan be destabilized by the other partner while maintaining itsstructural and functional characteristics.     

Bioactive IL7-diphtheria fusion toxin secreted by mammalian cells

A number of targeted cytotoxic agents have been developed that selectively kill malignant or otherwise pathological cells. These engineered proteins consist of a potent cytotoxic element connected to a ligand domain that binds to specific molecules on the surface of the target cell. Several of these agents have shown promise in clinical trials and one is currently administered to patients. A significant technical obstacle that has impeded the development of some of these toxins is the difficulty of preparing certain recombinant proteins in properly folded forms. These fusion proteins have generally been produced in bacteria requiring them to be denatured and renatured in vitro. For some proteins this is an efficient process whereas for others it is not. We describe here a system to produce fusion toxins rapidly and efficiently by engineering mammalian cells to secrete them as properly folded molecules which can be purified in native form from cell culture medium. We have used this system to produce highly active preparations of DAB(389)-IL7, a molecule consisting of the catalytic and transmembrane domains of diphtheria toxin fused to interleukin 7. This system is generalizable and can be used to produce and evaluate rapidly fusion toxins incorporating novel or uncharacterized ligands.     

Cloning, expression and purification of a sarcoplasmic calcium-binding protein from the sandworm Nereis diversicolor via a fusion product with chloramphenicol acetyltransferase

A gene coding for the Nereis sarcoplasmic calcium-binding protein(NSCP) was synthesized and expressed in Escherichia coli. Thesequence of the gene was derived from the protein sequence byreverse translation. It possesses a number of unique, regularlyspaced, restriction endonuclease cleavage sites to facilitatefuture site-directed mutagenesis. For the cloning strategy thegene sequence was divided into four parts. Three parts werecloned by ligation of hybridized oligomers and one part by inversePCR. The protein was expressed as a fusion protein with thebacterial chloramphenicol acetyltransferase (CAT), which couldbe easily purified by affinity chromatography. At the junctionof the CAT and NSCP moieties a recognition site for the proteolyticenzyme factor Xa was built in. However, the distance betweenthe moieties appeared to be crucial to warrant cleavage. A kineticanalysis showed that NSCP prepared from the sandworm and theone expressed by E.coli behaved in the same way. This systemprovides a basis for site-specific mutagenesis studies, in orderto elucidate the molecular mechanism of cation binding and concomitantconformational changes     

Production and properties of a factor X-cellulose-binding domain fusion protein

A fusion protein, FX–CBD_Cex, which comprises factor Xwith a cellulose-binding domain (CBD_Cex) fused to its C-terminus,was produced in BHK cells. It was purified from the culturemedium by affinity chromatography on cellulose. FX–CBD_Cexcould be activated to FXa–CBD_Cex with Russell viper venom.FXa–CBD_Cex was as active as FXa against a chromogenicsubstrate and against proteins containing the Ile–Glu–Gly–Argsequence hydrolysed by FXa. FXa–CBD_Cex retained its activitywhen adsorbed to cellulose.     

A single Fc binding domain-alkaline phosphatase gene fusion expresses a protein with both IgG binding ability and alkaline phosphatase enzymatic activity

A recombinant gene fusion was created and cloned using a previouslyconstructed gene encoding a monodomain IgG Fc binding proteinand the gene coding for bacterial alkaline phosphatase. Theconstruct was able to express and secrete a fusion protein thatexhibited both IgG binding and alkaline phosphatase enzymaticactivities. Greater than 60% of the protein demonstrating bothbiological activities was detected from periplasmic space preparations.Nanogram concentrations of the Fc binding-alkaline phosphatasefusion protein allowed primary IgG antibody detection withoutthe use of conjugated secondary antibodies. Removal of the domaincoding for alkaline phosphatase resulted in decreased resistanceof the protein to proteolytic degradation and the loss of IgGFc binding ability. Using affinity-purified fusion protein,the specificity of binding to IgG, IgM and IgA was examined;binding was strong to IgG and barely detectable against IgMor IgA. Affinity for binding of the fusion protein to IgG (k_d= 6.7 x10^-8 M) was determined to be equal to or greater thanpreviously reported for protein A.     

Disulfide stabilization of antibody Fv: computer predictions and experimental evaluation

Using molecular modeling technology we have recently identifiedpositions in conserved framework regions of Fvs which can beused to stabilize antibody Fvs by an interchain disulfide bondengineered in between the structurally conserved framework positionsof the variable domains of heavy (V_H) and light (V_L) immunoglobulinchains (disulfide-stabilized Fv; dsFv). The computer model indicatedthe existence of other potential sites in the framework regionsthat might be suitable for disulfide bond formation betweenV_H and V_L. The possibility of obtaining dsFvs using these positionsis evaluated here experimentally by constructing dsFv immunotoxinsin which the Fv moiety is fused to a truncated form of Pseudomonasexotoxin. We analyzed the extent of dsFv formation and the activityof the resulting dsFv immunotoxins, and compared various dsFvmolecules with the scFv immunotoxin. Our results demonstratethat position H44-L105 is the only one which gives high productionyields of active dsFv. All other positions gave either low yieldsand activity or completely failed to produce active dsFv. Withone exception, the formation and activities of the dsFvs correspondedto the C-C distance between the VH and VL positions, with anoptimal distance of 5.7 Å producing the best dsFv. Distancesof 6.0–6.9 Å resulted in a' low yield of proteinthat was still capable of binding antigen, whereas distances>7.0 Å resulted in molecules in which dsFv formationwas not obtained.     

Efficient production of the C-terminal domain of secretory leukoprotease inhibitor as a thrombin-cleavable fusion protein in Escherichia coli

We have developed a high-level production system for the C-terminaldomain of secretory leukoprotease inhibitor (SLPI) to investigateits pharmacological activities. A gene for the C-terminal domainof SLPI, (Asn55-AlalO7)SLPI, was constructed from chemicallysynthesized deoxyoligonucleotides. It was fused to a gene forthe N-terminal portion of human growth hormone via a DNA sequenceencoding Leu-Val-Pro-Arg, which can he cleaved by thrombin.The fused gene was expressed in Escherichia coli under the controlof a trp promoter, and the fusion protein was obtained as aninclusion body. After sulfonation of the cysteine residues,the sulfonated fusion protein was cleaved at the desired siteby thrombin. Sulfonated (Asn55-Ala107)SLPI was refolded in Trisbuffer containing reduced and oxidized glutathione. The resulting(Asn55-Ala107) SLPI was purified by cation-exchange chromatographyand reverse-phase high performance liquid chromatography. Thefinal yield was 50 mg/l culture. (Asn55-Ala107)SLPI was as activeagainst elastase as, but had less trypsin inhibitory activitythan, native SLPI. This system is suitable for the large-scaleproduction of the C-terminal domain of SLPI, which is an elastase-specificinhibitor.     

Hydrophobicity engineering of cholera toxin A1 subunit in the strong adjuvant fusion protein CTA1-DD

Protein engineering of the cholera toxin A1 subunit (CTA1) fusedto a dimer of the Ig-binding D-region of Staphylococcus aureusprotein A (DD) was employed to investigate the effect of specificamino acid changes on solubility, stability, enzymatic activityand capacity to act as an adjuvant in vivo. A series of CTA1-DDanalogues were selected by a rational modeling approach, inwhich surface-exposed hydrophobic amino acids of CTA1 were exchangedfor hydrophilic counterparts modeled for best structural fit.Of six different mutants initially produced, two analogues,CTA1Phe132Ser-DD and CTA1Pro185Gln-DD, were demonstrated tohave 50 and 70% increased solubility, respectively, at neutralpH. The double mutant CTA1Phe132Ser/Pro185Gln-DD was at leastthreefold more soluble, demonstrating an additive effect ofthe two mutations. Only the Phe132Ser analogue retained fullbiological activity and stability compared with the native CTA1-DDfusion protein. Two mutants, Pro185Gln and Phe31His mutations,exhibited unaltered ADP-ribosyltransferase activity in vitro,but demonstrated markedly reduced adjuvant function. Since thePro185 and Phe31 amino acids are located in close vicinity onthe distal side of the molecule relative to the enzymaticallyactive cleft, it is conceivable that this region is involvedin mediating a biological function, separate from the enzymaticactivity but intrinsic to the adjuvant activity of CTA1.     

Engineering disulfide-linked single-chain Fv dimers [(sFv')2] with improved solution and targeting properties: anti-digoxin 26-10 (sFv')2 and anti-c-erbB-2 741F8 (sFv')2 made by protein folding and bonded through C-terminal cysteinyl peptides

Single-chain Fv fusions with C-terminal cysteinyl peptides (sFv')have been engineered using model sFv proteins based upon the26-10 anti-digoxin IgG and 741F8 anti-c-erbB-2 IgG monoclonalantibodies. As part of the 741F8 sFv construction process, thePCR-amplified 741F8 V_H gene was modified in an effort to correctpossible primer-induced errors. Genetic replacement of the N-terminalp-strandsequence of 741F8 VH with that from the FR1 of anti-c-erbB-2520C9 VH resulted in a dramatic improvement of sFv folding yields.Folding in urea-glutathione redox buffers produced active sFv'with a protected C-terminal sulfhydryl, presumably as the mixeddisulfide with glutathione. Disulfide-bonded (sFv')₂ homodimerswere made by disulfide interchange or oxidation after reductiveelimination of the blocking group. Both 26–10 (sFv')₂and 741F8 (sFv')₂ existed as stable dimers that were well behavedin solution, whereas 741F8 sFv and sFv' exhibited considerableself-association. The 741F8 sFv binds to the extracellular domain(ECD) of the c-erbB-2 oncogene protein, which is often overexpressedin breast cancer and other adenocarcinomas. The recombinantECD was prepared to facilitate the analysis of 741F8 bindingsite properties; the cloned ECD gene, modified to encode a C-terminalSer-Gly-His₆ peptide, was transfected into Chinese hamster ovarycells using a vector that also expressed dihydrofolate reductaseto facilitate methotrexate amplification. Optimized cell linesexpressed ECD-His₆ at high levels in a cell bioreactor; afterisolation by immobilized metal affinity chromatography, finalECD yields were as high as 47 mg/l. An animal tumor model complementedphysicochemical studies of 741F8 species and indicated increasedtumor localization of the targeted 741F8 (sFv')₂ over othermonovalent 741F8 species.