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.     

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.     

ScFv multimers of the anti-neuraminidase antibody NC10: shortening of the linker in single-chain Fv fragment assembled in VL to VH orientation drives the formation of dimers, trimers, tetramers and higher molecular mass multimers

Synthetic genes encoding single-chain variable fragments (scFvs)of NC10 anti-neuraminidase antibody were constructed by joiningthe V_L and V_H domains with linkers of fifteen, five, four, three,two, one and zero residues. These V_L–V_H constructs wereexpressed in Escherichia coli and the resulting proteins werecharacterized and compared with the previously characterizedNC10 scFv proteins assembled in V_H–V_L orientation. Size-exclusionchromatography and electron microscope images of complexes formedbetween various NC10 scFvs and anti-idiotype Fab' were usedto analyse the oligomeric status of these scFvs. The resultshowed that as the linker length between V_L and V_H was reduced,different patterns of oligomerization were observed comparedwith those with V_H–V_L isomers. As was the case for V_H–V_Lorientation, the scFv-15 V_L–V_H protein existed mainlyas a monomer whereas dimer (diabody) was a predominant conformationfor the scFv-5, scFv-4 and scFv-3 V_L–V_H proteins. In contrastto the V_H–V_L isomer, direct ligation of V_L to V_H led tothe formation of predominantly a tetramer (tetrabody) ratherthan to an expected trimer (triabody). Furthermore, the transitionbetween dimers and higher order oligomers was not as distinctas for V_H–V_L. Thus reducing the linker length in V_L–V_Hfrom three to two residues did not precisely dictate a transitionbetween dimers and tetramers. Instead, two-residue as well asone-residue linked scFvs formed a mixture of dimers, trimersand tetramers.     

Optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies

Bispecific single-chain diabodies (scDb) consist of the variableheavy and light chain domains of two antibodies connected bythree linkers. The structure of an scDb in the V_H–V_L orientationis V_HA–linkerA–V_LB–linkerM–V_HB–linkerB–V_LA,with linkers A and B routinely chosen to be 5–6 residuesand linker M 15–20 residues. Here, we applied displayof scDb on filamentous phage to analyse the composition of optimallinker sequences. The three linkers were randomized in lengthand sequence using degenerated triplets coding for only sixhydrophilic or aliphatic amino acids (Thr, Ser, Asp, Asn, Gly,Ala). Antigen-binding clones were then isolated by one to tworounds of selection on the two different antigens recognizedby the bispecific scDb. Using an scDb directed against carcinoembryonicantigen (CEA) and ß-galactosidase (Gal), we foundthat monomeric scDb had a preferred length of 15 or more aminoacid residues for the middle linker M and of 3–6 residuesfor the linkers A and B. No obvious bias towards a preferredlinker sequence was observed. Reduction of the middle linkerbelow 13 residues led to the formation of dimeric scDb, whichmost likely results from interchain pairing between all theV_H and V_L domains. Dimeric scDb were also formed by fragmentspossessing a long linker M and linkers A and B of 0 or 1 residue.We assume that these dimeric scDb are formed by intrachain pairingof the central variable domains and interchain pairing of theflanking variable domains. Thus, the latter molecules representa novel format of bispecific and tetravalent molecules. Thedescribed strategy allows for the isolation of both optimizedand minimal linker sequences for the assembly of monomeric ordimeric single-chain diabodies.     

Single-chain Fv fragments of anti-neuraminidase antibody NC10 containing five- and ten-residue linkers form dimers and with zero- residue linker a trimer

Single-chain variable fragments (scFvs) of anti-neuraminidase antibody NC10 were constructed by joining the VH and VL domains with 10-residue (Gly4Ser)2 and five-residue (Gly4Ser) linkers; a zero-residue linker scFv was constructed by joining the C-terminal residue of the VH domain to the N-terminus of the VL domain. The scFv with the 10- and five- residue linkers exclusively formed dimeric antibody fragments (M(r) 52000). These were shown to be bivalent and were able to cross-link two neuraminidase tetramers to form a 'sandwich' type complex; each antigen combining site could also bind an anti-idiotype Fab'. The zero-residue linker scFv (M(r) 70000) was shown to form a trimer with three active antigen combining sites, each binding an anti-idiotype Fab' to yield a complex of M(r) 212000. The orientation of the combining sites in the zero-residue linker scFv, however, was such that it could not cross- link tetramers of neuraminidase. BIAcore biosensor experiments showed that the affinity of each individual antigen combining site in both the 10- and five-residue linker scFv dimers and zero-residue linker scFv trimer was essentially the same when the scFvs were immobilized onto the sensor surface. However, when the scFvs were used as the analyte, the dimeric and trimeric scFvs showed an apparent increase in binding affinity due to the avidity of binding the multivalent scFvs.      

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.     

Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains

We cloned 17 different PCR fragments encoding V_H genes of camel(Camelus dromedarius). These clones were derived from the camelheavy chain immunoglobulins lacking the light chain counterpartof normal immunoglobulins. Insight into the camel V_H sequencesand structure may help the development of single domain antibodies.The most remarkable difference in the camel V_H, consistent withthe absence of the V_L interaction, is the substitution of theconserved Leu45 by an Arg or Cys. Another noteworthy substitutionis the Leu11 to Ser. This amino acid normally interacts withthe C_H1 domain, a domain missing in the camel heavy chain immunoglobulins.The nature of these substitutions agrees with the increasedsolubility behavior of an isolated camel V_H domain. The V_H domainsof the camels are also characterized by a long CDR3, possiblycompensating for the absence of the V_L contacts with the antigen.The CDR3 lacks the salt bridge between Arg94 and Asp101. However,the frequent occurrence of additional Cys residues in both theCDR1 and CDR3 might lead to the formation of a second internaldisulfide bridge, thereby stabilizing the CDR structure as inthe DAW antibody. Within CDRs of the camel V_H domains we observea broad size distribution and a different amino acid patterncompared with the mouse or human V_H. Therefore the camel hypervariableregions might adopt structures which differ substantially fromthe known canonical structures, thereby increasing the repertoireof the camel antigen binding sites within a V_H.     

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.     

Engineering interchain disulfide bonds into conserved framework regions of Fv fragments: improved biochemical characteristics of recombinant immunotoxins containing disulfide-stabilized Fv

Using molecular modeling technology, we have recently identifiedtwo positions in conserved framework regions of antibody Fvfragments (Fvs) that are distant from CDRs, and potentiallycan be used to make recombinant Fv fragments in which the unstableV_H and V_L heterodimer is stabilized by an interchain disulfidebond inserted between structurally conserved framework positions.A disulfide bond has been introduced at one of these positions,V_H44-V_L105, and shown to stabilize various Fvs that retain fullbinding and specificity. Recombinant immunotoxias, e.g. B3(dsFv)-PE38KDELin which this disulfide-stabilized Fv moiety is connected toa truncated form of Pseudomonas exotoxin (PE; PE38KDEL) whichcontains the translocation and ADP ribosylation domains, areindistinguishable in binding and specificity from its single-chainimmunotoxin counterparts. We have now analyzed the alternativeposition, (V_H111-V_L48), predicted by the modeling methodology,for disulfide stabilization of mAb B3(Fv) by producing a recombinantimmunotoxin with such disulfide-stabilized (ds) Fv. This immunotoxinwas also very active and retained full specificity to B3 antigen-positivecells. However, it was 2- to 3-fold less active than the V_H44-V_L105dsFv-molecule. We also tested various biochemical features ofV_H44-V_L105 and V_H111-V_L48 dsFv immunotoxins and compared themwith the corresponding single-chain immunotoxin. We found thedsFv immunotoxins were more stable in human serum and more resistantto thermal and chemical denaturation than the single chain (sc)Fv immunotoxin. Because dsFv immunotoxins and dsFvs have fullactivity and specificity and improved stability, they may bemore useful than scFv immunotoxins as therapeutic and diagnosticagents.     

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.     

Site-specific attachment to recombinant antibodies via introduced surface cysteine residues

Many diagnostic and therapeutic applications of monoclonal antibodiesrequire the covalent linking of effector or reporter moleculesto the immunoglobulin polypeptides. Existing methods generallyinvolve the non-selective modification of amino acid side chains,producing one or more randomly distributed attachment sites.This results in heterogeneous labelling of the antibody moleculesand often to a decrease in antigen-binding due to the modificationof residues close to the antigen-binding site. We report a novelstrategy for site-specifically labelling antibodies throughsurface cysteine residues. Examination of molecular structureswas used to identify amino acids of the C_H1 domain of the IgGheavy chain that were accessible to solvent but not to largermolecules. Site-directed mutagenesis was used to substitutecysteine residues at these positions in the heavy chain of amouse/human chimaeric version of the tumour-binding monoclonalantibody, B72.3. Expression of the modified antibody genes inmammalian cells yielded correctly assembled proteins that hadthiol groups in pre-determined positions and showed no lossof antigen-binding activity. One of the mutants was used todemonstrate the site-specific attachment of a radio-iodinatedligand to the chimaeric B72.3 antibody.     

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.     

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.     

Restructuring an interdomain linker in the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Escherichia coli

The lipoyl, subunit-binding and catalytic domains of the dihydrolipoamideacetyltransferase subunits (E2p) of the Escherichia coli pyruvatedehydrogenase complex are connected by linker sequences whichare characteristically rich in alanine and proline residues.By facilitating domain movement these linkers are thought topromote interactions between the three types of active sitethat participate in the catalytic cycle of the complex. To investigatefunctional constraints associated with linker composition andsequence, the natural linker of an E2p subunit containing onelipoyl domain was replaced by shorter sequences containing:mixtures of alanine plus proline residues; mainly alanine; mainlyproline; and mainly charged residues. Each artificial linkerpossessed a central histidine residue for assessing linker flexibilityby ¹H-NMR spectroscopy. The resultant complexes exhibited 181%(proline), 74–79% (alanine plus proline), 63% (alanine)and 7% (charged residues) of parental activity compared witha value of 75% expected for a complex with a comparably shortenedlinker. The ¹H-NMR spectra showed that the alanine plus prolinelinkers are flexible but the alanine linker and the prolinelinker are relatively inflexible. Substantial variations inlinker sequence and composition were tolerated without lossof function, and the enhanced activity conferred by the prolinelinker was attributed to the combined effects of length andrelative inflexibility.     

Molecular modelling study of antigen binding to oxazolone-specific antibodies: the Ox1 idiotypic IgG and its mature variant with increased affinity to 2-phenyloxazolone

Structural models of the variable domains of the murine anti-2-phenyloxazoloneIgG (Oxl idiotype) and its somatic variant, which has higheraffinity to the hapten 2-phenyloxazolone, were constructed bycomputer-aided model building using known structures of highlyhomologous immunoglobulins as templates. Molecular dynamicssimulations were used to dock the hapten between the V_L andV_H domains. The hapten is predicted to bind to slightly differentsites in the two models. Hypotheses concerning the role of anumber of preferred mutations in anti-oxazolone variants arepresented. These can be tested by mutagenesis and crystallography.In particular, the higher binding affinities of the differentantibody variants are shown to correlate with better complementarityof electrostatics. The molecular dynamic simulations also suggestthat two mobile tryptophans at the mouth of the pocket may playan important role in the binding of hapten.     

The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the detection and purification of a functional Ig Fv fragment

The facile detection and purification of a recombinant proteinwithout detailed knowledge about its individual biochemicalproperties constitutes a problem of general interest in proteinengineering. The use of a novel kind of random peptide libraryfor the stepwise engineering of a C-terminal fusion peptidewhich confers binding activity towards streptavidin is describedin this study. Because of its widespread use as part of a varietyof conjugates and other affinity reagents, streptavidin constitutesthe binding partner of choice both for detection and purificationpurposes. The streptavidin-affinity tag was engineered at theC-terminus of the V_H domain as part of the D1.3 Fv fragmentwhich was functionally expressed in Escherichia coli. Irrespectiveof whether it was displayed by the V_H or the V_L domain, theoptimized version of the affinity peptide termed ‘Strep-tag’allowed the detection of the Fv fragment both on Western blotsand in ELISAs by a streptavidin–alkaline phosphatase conjugate.In addition, the one-step purification of the intact Fv fragmentcarrying a single Strep-tag at the C-terminus of only one ofits domains was achieved by affinity chromatography with streptavidin-agaroseusing very mild elution conditions.     

Humanization of mouse anti-human IL-2 receptor antibody B-B10

Mouse monoclonal anti–human IL–2 receptor antibody(BB10) inhibits EL–2–dependent human T–cellproliferation. It has been used in clinical trials in the transplantationfield and promising results are being accumulated. Mouse B–B10antibody was humanized by grafting all CDRs and some frameworkamino add residues onto human antibodies, KAS for V_H and PAYfor V_x. Nine humanized B–BlOs with differently graftedframework residues were constructed and assessed for their biologicalactivities. One of these humanized B–B10, M5, showed nearlythe same activity as the mouse B–B10. The 49th residueof V_x was demonstrated to play a crucial role in the antigen–antibodyinteraction by 3–D structure analysis with a computermodeling system.     

Directing phage selections towards specific epitopes

It is possible to direct selections from antibody repertoiresdisplayed on filamentous phage towards unique epitopes on proteinantigens by competing with related molecules. A phage displayrepertoire of human single chain Fvs (scFvs) was panned threetimes against foetal haemoglobin (HbF). The selection was dominatedby one clone with a K_d of 10 nM but yielded at least 17 others,all of which bound HbF but crossreacted with adult haemoglobin(HbA). To direct selection towards HbF-specific epitopes, therepertoire was preincubated with HbA in solution before eachpanning. Crossreactive scFvs can form complexes with the solubleHbA and thereby be prevented from binding the immobilized HbF.Four clones with preferential binding to HbF emerged under theseconditions. One of these (Hb-1), with a K_d of 6 µM, hadexquisite specificity for HbF and could distinguish cells expressingHbF from those expressing HbA by immunocytochemistry and flowcytometry. This antibody has an affinity that is 600-fold lowerthan the dominant crossreactive clone, and so only emerged underconditions of ‘competitive deselection’. Thus, competitivedeselection is a viable means for directing selections towardsuseful epitopes. It permits a more effective ‘search’of phage display repertoires and allows the emergence of loweraffinity clones with useful specificities. These clones maybe useful in themselves or may serve as leads for in vitro affinitymaturation.     

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.     

Changes in the specificity of antibodies by site-specific mutagenesis followed by random mutagenesis

The specificity for 11-deoxycortisol (11-DOC) of a monoclonalantibody (mAb), designated SCET, was changed to specificityfor cortisol (CS) by site-specific mutagenesis followed by randommutagenesis. The Fab form of SCET was expressed on the surfaceof a phage. During the first step, mutations were introducedat 14 amino acid positions in three complementarity-determiningregions (CDRs) of the V_H domain that seemed likely to form thesteroid-binding pocket. A clone, DcC16, was isolated from theresultant library with multiple mutations and this clone wasshown to have CS-binding activity but also to retain high 11-DOC-bindingactivity. During the second step, mutations were introducedrandomly into the entire V_H-coding region of the DcC16 cloneby an error-prone polymerase chain reaction, and CS-specificmutant antibodies were selected in the presence of 11-DOC asa competitor. Three representative clones were analyzed withthe BIAcore instrument, and each revealed a large increase inthe binding constant for CS and a decrease in that for 11-DOC.Structural models, constructed by computer simulation, indicatedthe probable molecular basis for these changes in specificity.