An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability

The effects of linker length on binding affinity and degreeof aggregation have been examined in the antifluorescein 4-4-20and anticarcinoma CC49 single-chain Fvs. Longer linkers in theantifluorescein sFvs have higher affinities for fluoresceinand aggregate less. A proteolytically susceptible site betweenLys8 and Ser9, in the previously reported 212 linker has beenidentified. A new linker sequence, 218 (GSTSGSGKPGSGEGSTKG)was designed in which a praline was placed at the C-terminalside of the proteolytic clip site in the 212 linker. The CC49sFv containing the 218 linker showed reduced aggregation andwas found to be more stable to proteolysis in vitro, when comparedto the CC49/212 sFv. The CC49 sFv with the longer 218 linkerhad higher affinity than CC49/212 sFv. An aggregated CC49/212sFv sample had higher affinity than CC49/218 sFv. The CC49/218and CC49/212 sFvs had similar blood clearances in mice, whilethe aggregated CC49/212 sFv remained in circulation significantlylonger. In mice bearing LS-174T human colon carcinoma xenografts,the CC49/218 sFv showed higher tumor uptake than the CC49/212sFv and lower tumor uptake than the aggregated CC49/212 sFv.The higher tumor uptake of the CC49/218 is most likely a resultof its higher resistance to proteolysis. The higher affinityand higher tumor uptake of the aggregated CC49/212 sFv are mostlikely due to the repetitive nature of the TAG-72 antigen andthe higher avidity of multivalent aggregates. When the sFvswere radiolabeled with a lutetium-chelate the CC49/218 sFv showeda lower accumulation in the liver and spleen compared to theaggregated CC49/212 sFv.     

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.     

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.     

Multimerization behaviour of single chain Fv variants for the tumour-binding antibody B72.3

A systematic study has been performed on the relationship betweenlinker length, relative orientation of variable domains, multimerizationbehaviour and antigen binding activity for single chain Fvs(scFvs) of the tumour-binding antibody B72.3. Thirteen scFvvariants with linkers comprising up to six repeats of the motifGly-Gly-Gry-Gly-Ser were studied. All these scFvs showed a tendencyto form dimers or higher molecular weight species, and thistendency decreased with increasing linker length. The dimersand higher molecular weight forms may arise from head to tailintermolecular association of V_H and V_L domains. For each linkerlength, scFvs with the organization VL-linker-VH showed greaterbinding activity than those with the organization VH-linker-VL.In fact, for the latter organization only the variant with a30 amino acid linker showed good binding activity, suggestingthat (0 for B72.3 the C-tenninus of VH or the N-tenninus ofVL makes a structural contribution to antigen binding, and (ii)shorter linkers interfere with this contribution. Antigen bindingstudies on scFvs should be interpreted with caution becauseof their tendency to multlmerize. Such multimerization can beminimized by using linkers longer than those in common use     

Linker modification introduces useful molecular instability in a single chain antibody

SCA 4-4-20/212, is a recombinant single chain antibody directedagainst fluorescein (Fl) composed of the variable light (V_L)and variable heavy (V_H) domains of the monoclonal antibody 4-4-20,tethered by a 14 amino acid linker. Binding of SCA 4-4-20/212to Fl quenches its fluorescence, thus enabling the distinctionbetween bound and free Fl. This was used to follow antibodydenaturation which followed a two-step process: rapid selectedand restricted denaturation followed by slow and progressivedenaturation. This two-phase phenomenon might reflect selectivesusceptibility of the CDR loops to denaturation. Furthermore,a new SCA, SCA 4-4-20/9, was constructed by site-directed mutagenesisof SCA 4-4-20/212 using PCR methodology. SCA 4-4-20/9 was similarto SCA 4-4-20/212, but for a nine residue linker. The two SCAswere compared for Fl binding, heat stability, the effect ofdenaturing agents and susceptibility to proteolysis. The modificationof the linker caused a general conformational rearrangementin the SCA molecule, rendering it more sensitive to denaturationand proteolysis. This molecular instability may find utilityin the application of SCAs in analytical systems or as the recognitioncomponent in biosensors.     

scFv multimers of the anti-neuraminidase antibody NC10: length of the linker between VH and VL domains dictates precisely the transition between diabodies and triabodies

Single-chain Fv antibody fragments (scFvs) incorporate a polypeptidelinker to tether the V_H and V_L domains together. An scFv moleculewith a linker 5–12 residues long cannot fold into a functionalFv domain and instead associates with a second scFv moleculeto form a bivalent dimer (diabody). Direct ligation of V_H andV_L domains further restricts association and forces three scFvmolecules to associate into a trivalent trimer (triabody). Wehave defined the effect of linker length on scFv associationby constructing a series of scFvs from anti-neuraminidase antibodyNC10 in which the linker varied from one to four glycine residues.NC10 scFv molecules containing linkers of three and four residuesshowed a strong preference for dimer formation (diabodies),whereas a linker length of one or two glycine residues preventedthe formation of diabodies and directed scFv association intotrimers (triabodies). The data suggest a relatively strict transitionfrom dimer (diabody) to trimer (triabody) upon reduction ofthe linker length from three to two glycine residues. Modellingstudies are consistent with three residues as the minimum linkerlength compatible with diabody formation. Electron microscopeimages of complexes formed between the NC10 scFv multimers andan anti-idiotype Fab' showed that the dimer was bivalent forantigen binding and the trimer was trivalent.     

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.     

Affinity enhancement of a recombinant antibody: formation of complexes with multiple valency by a single-chain Fv fragment-core streptavidin fusion

In antigen–antibody interactions, the high avidity ofantibodies depends on the affinity and number of the individualbinding sites. To develop artificial antibodies with multiplevalency, we have fused the single-chain antibody Fv fragmentsto core streptavidin. The resulting fusion protein, termed scFv::strep,was found after expression in Escherichia coli in periplasmicinclusion bodies. After purification of the recombinant productby immobilized metal affinity chromatography, refolding andsize-exclusion FPLC, tetrameric complexes resembling those ofmature streptavidin were formed. The purified tetrameric scFv::strepcomplexes demonstrated both antigen- and biotin-binding activity,were stable over a wide range of pH and did not dissociate athigh temperatures (up to 70°C). Surface plasmon resonancemeasurements in a BIAlite system showed that the pure scFv::streptetramers bound immobilized antigen very tightly and no dissociationwas measurable. The association rate constant for scFv::streptetramers was higher than those for scFv monomers and dimers.This was also reflected in the apparent constants, which wasfound to be 35 times higher for pure scFv::strep tetramers thanmonomeric singlechain antibodies. We could also show that mostof biotin binding sites were accessible and not blocked by biotinylatedE.coli proteins or free biotin from the medium. These sitesshould therefore facilitate the construction of bispecific multivalentantibodies by the addition of biotinylated ligands.     

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.     

Molecular modelling and site-directed mutagenesis on a bovine anti-testosterone monoclonal antibody

A three-dimensional (3D) molecular model of the antigen-combiningsite of a bovine anti-testosterone monoclonal antibody has beenconstructed. In the model, the CDRs, and a single heavy chainframework region residue (Trp47), associate to form a hydrophobiccavity large enough to accommodate a single molecule of testosterone.Tyr97 of CDR-H3 lies at the bottom of the cavity with its hydroxylgroup exposed to solvent. Using the model and data from bindingstudies, we predicted that the cavity forms the antibody's paratopeand on binding testosterone a hydrogen bond is formed betweenTyr97 of CDR-H3 and the hydroxyl group on the D-ring of testosterone.This prediction has subsequently been tested by site-directedmutagenesis. An antibody with phenylalanine in place of tyrosineat position 97 in CDR-H3 has its affinity reduced by {smalltilde}800 fold. The reduction in binding energy associated withthe reduced affinity has been calculated to be 3.9 kcal/molwhich is within the range (0.5–4.0 kcal/mol) expectedfor the loss of a single hydrogen bond. The model has been usedto suggest ways of increasing the antibody's affinity for testosterone.     

Stabilization of TRAIL, an all-beta-sheet multimeric protein, using computational redesign

Protein thermal stability is important for therapeutic proteins, both influencing the pharmacokinetic and pharmacodynamic properties and for stability during production and shelf-life of the final product. In this paper we show the redesign of a therapeutically interesting trimeric all-beta-sheet protein, the cytokine TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), yielding variants with improved thermal stability. A combination of tumor necrosis factor (TNF) ligand family alignment information and the computational design algorithm PERLA was used to propose several mutants with improved thermal stability. The design was focused on non-conserved residues only, thus reducing the use of computational resources. Several of the proposed mutants showed a significant increase in thermal stability as experimentally monitored by far-UV circular dichroism thermal denaturation. Stabilization of the biologically active trimer was achieved by monomer subunit or monomer-monomer interface modifications. A double mutant showed an increase in apparent T(m) of 8 degrees C in comparison with wild-type TRAIL and remained biologically active after incubation at 73 degrees C for 1 h. To our knowledge, this is the first study that improves the stability of a large multimeric beta-sheet protein structure by computational redesign. A similar approach can be used to alter the characteristics of other multimeric proteins, including other TNF ligand family members.     

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.     

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.     

Binding of the globular domain of linker histones H5/H1 to the nucleosome: a hypothesis

The amino acid sequence of the central globular domain of histoneH1/H5 family members is highly homologous. Twenty-four suchsequences have been compared to establish the conserved andvariable residues. Fitting this to the tertiary structure ofthe H5 globular domain shows which of the conserved and variableresidues are peripheral and which internal. Particular attentionis paid to conserved basic residues on the surface, which wetake to be DNA binding. Variable regions and conserved acidicresidues are assumed not to be sites of contact with DNA. Weconclude that one face of the domain, containing a cluster ofbasic residues, is the principal DNA binding site whilst twoopposing faces, orthogonal to the principal site and also containingconserved basic residues, are subsidiary DNA binding sites.Since the DNA binding surface of the domain covers a full 180°arc, we propose that it contacts a ‘cage’ of threeDNA strands on the 2-fold axis of the chromatosome.     

Expression in COS cells of a mouse--human chimaeric B72.3 antibody

B72.3 is a mouse hybndoma cell-line secreting an IgG1 antibodywhich recognises an epitope on a tumour-associated antigen,TAG-72. This high molecular weight mucin-like molecule is foundon a variety of human neoplasms, including colon, breast andovarian carcinomas. Chimaeric Immunoglobulin genes with theB72.3 specificity have been constructed by joining the mousevariable regions from cDNA clones to human genomic constantregions using recombinant DNA techniques. The chimaeric heavyand light chain Immunoglobulin genes were placed under the controlof a strong viral promoter, and co-transfected into COS-1 cells.SDS–PAGE analysis of the ³⁵S-labelled products demonstratedthat the transiently expressed antibodies were correctly synthesisedand assembled. The specific binding characteristics of the parentB72.3 antibody were retained by the chimaeric antibody in anantigen-based ELISA. This system gave sufficiently high transientexpression of the chlmaerlc antibody molecules to allow rapidphysical and Immunological characterisation of the engineeredgene products.     

Humanization of a mouse anti-human IgE antibody: a potential therapeutic for IgE-mediated allergies

Mouse mAb TES-C21(C21) recognizes an epitope on human IgE and,therefore, has potential as a therapeutic agent in patientswith IgE-mediated allergies such as hay fever, food and drugallergies and extrinsic asthma. The clinical usefulness of mouseantibodies is limited, however, due to their immunogenidty inhumans. Mouse C21 antibody was humanized by complementaritydetermining region (CDR) grafting with the aim of developingan effective and safe therapeutic for the treatment of IgE-mediatedallergies. The CDR-grafted, or reshaped human, C21 variableregions were carefully designed using a specially constructedmolecular model of the mouse C21 variable regions. A key stepin the design of reshaped human variable regions is the selectionof the human framework regions (FRs) to serve as the backbonesof the reshaped human variable regions. Two approaches to theselection of human FRs were tested: (i) selection from humanconsensus sequences and (ii) selection from individual humanantibodies. The reshaped human and mouse C21 antibodies weretested and compared using a biosensor to measure the kineticsof binding to human IgE. Surprisingly, a few of the reshapedhuman C21 antibodies exhibited patterns of binding and affinitiesthat were essentially identical to those of mouse C21 antibody.     

'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization

‘Knobs-into-holes’ was originally proposed by Crickin 1952 as a model for the packing of amino acid side chainsbetween adjacent -helices. ‘Knobs-into-holes’ isdemonstrated here as a novel and effective design strategy forengineering antibody heavy chain homodimers for heterodimerization.In this approach a ‘knob’ variant was first obtainedby replacement of a small amino acid with a larger one in theC_H3 domain of a CD4-IgG immuno-adhesin: T366Y. The knob wasdesigned to insert into a ‘hole’ in the C_H3 domainof a humanized anti-CD3 antibody created by judicious replacementof a large residue with a smaller one: Y407T. The anti-CD3/CD4-IgGhybrid represents up to 92% of the protein A purified proteinpool following co-expression of these two different heavy chainstogether with the anti-CD3 light chain. In contrast, only upto 57% of the anti-CD3/CD4-IgG hybrid is recovered followingco-expression in which heavy chains contained wild-type C_H3domains. Thus knobs-into-holes engineering facilitates the constructionof an antibody/ immunoadhesin hybrid and likely other Fc-containingbifunctional therapeutics including bispecific immuno-adhesinsand bispecific antibodies.     

Humanization of a mouse monoclonal antibody by CDR-grafting: the importance of framework residues on loop conformation

A mouse monoclonal antibody (mAb 425) with therapeutic potentialwas ‘humanized’ in two ways. Firstly the mouse variableregions from mAb 425 were spliced onto human constant regionsto create a chimeric 425 antibody. Secondly, the mouse complementarity–determiningregions (CDRs) from mAb 425 were grafted into human variableregions, which were then joined to human constant regions, tocreate a reshaped human 425 antibody. Using a molecular modelof the mouse mAb 425 variable regions, framework residues (FRs)that might be critical for antigen-binding were identified.To test the importance of these residues, nine versions of thereshaped human 425 heavy chain variable (VH) regions and twoversions of the reshaped human 425 light chain variable (VJregions were designed and constructed. The recombinant DNAscoding for the chimeric and reshaped human light and heavy chainswere coexpressed transiently in COS cells. In antigen-bindingassays and competition-binding assays, the reshaped human antibodieswere compared with mouse 425 antibody and to chimeric 425 antibody.The different versions of 425–reshaped human antibodyshowed a wide range of avidities for antigen, indicating thatsubstitutions at certain positions in the human FRs significantlyinfluenced binding to antigen. Why certain individual FR residuesinfluence antigen-binding is discussed. One version of reshapedhuman 425 antibody bound to antigen with an avidity approachingthat of the mouse 425 antibody.     

Association and dissociation kinetics of bobwhite quail lysozyme with monoclonal antibody HyHEL-5

The anti-hen egg lysozyme monoclonal antibody HyHEL-5 and itscomplexes with various species-variant and mutant lysozymeshave been the subject of considerable experimental and theoreticalinvestigation. The affinity of HyHEL-5 for bobwhite quail lysozyme(BWQL) is over 1000-fold lower than its affinity for the originalantigen, hen egg lysozyme (HEL). This difference is believedto arise almost entirely from the replacement in BWQL of thestructural and energetic epitope residue Arg68 by lysine. Inthis study, the association and dissociation kinetics of BWQLwith HyHEL-5 were investigated under a variety of conditionsand compared with previous results for HEL. HyHEL-5–BWQLassociation follows a bimolecular mechanism and the dissociationof the antibody–antigen complex is a first-order process.Changes in ionic strength (from 27 to 500 mM) and pH (from 6.0to 10.0) produced about a 2-fold change in the association anddissociation rates. The effect of viscosity modifiers on theassociation reaction was also studied. The large differencein the HEL and BWQL affinities for HyHEL-5 is essentially dueto differences in the dissociation rate constant.