THE JOURNAL OF BIOLWICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 1, Issue of January 7, pp. 199-206, 1994 Printed in U.SA.

Construction, Expression, and Activity of a Bivalent Bispecific Single-chain Antibody*

(Received for publication, June 25, 1993, and in revised form, September 7, 1993)

William D. MallenderS and Edward W. Voss, Jr.§ From the Department of Microbiology, University of Illinois, Urbana, Illinois 61801

This report describes the design, construction, and expression of a bivalent bispecific single-chain antibody (SCA) protein in Escherichia coli. The bispecificity of the bivalent protein was based on two previously con- structed monovalent single-chain antibody molecules possessing distinct specificities, SCA 4-4-20 (anti-fluo- rescein) and SCA 04-01 (anti-single-stranded DNA). A flexible linker, modeled after a secreted fungal cellulase protein, was incorporated as the interdomain linker co- valently joining the two active sites. Bivalent bispecific SCA protein that accumulated in bacteria as insoluble inclusion bodies was harvested, denatured, refolded, and affinity-purified in vitro. Affinity-purified bivalent bispecific SCA showed nearly identical ligand binding properties at each site relative to the individual mono- valent single-chain antibody prototype molecules. In both solid and solution phase binding assays, the biva- lent bispecific single-chain antibody simultaneously bound both ligands (fluorescein and (dT)& Construc- tion of a model bivalent bispecific molecule provides a foundation for future assembly of similar molecules de- signed to identify parameters involved in enhanced binding of antibodies due to avidity and dual specificity.

Design, construction, and expression of recombinant single- chain antibody (SCA)’ molecules has facilitated the study of antibody variable domain related structure-function relation- ships, antigen-antibody interactions, and utilization of anti- body derivative fusion proteins for both clinical and medical applications (Bird et al., 1988; Huston et al., 1988; Condra et al., 1990; Glockshuber et al., 1990; Yokota et al., 1992; Wels et al., 1993). Structurally, monovalent single-chain antibodies consist of heavy and light chain variable domains covalently conjugated through a short intradomain polypeptide linker.

* This work was supported in part by a grant from the Biotechnology Research Development Corporation, Peoria, IL. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in ac- cordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by a National Institutes of Health Cell and Molecular Biology Training Grant.

ogy, University of Illinois, 131 Bum11 Hall, 407 S. Goodwin Ave., Ur- 5 To whom correspondence should be addressed: Dept. of Microbiol-

bana, IL 61801. Tel.: 217-333-1738; Fax: 217-244-6697. The abbreviations used are: SCA, single-chain antibody containing

the VLand VH domains covalently linked; BSA, bovine serum albumin; BiSCA, bivalent and bispecific single-chain antibody; CBHl’, modified linker for BiSCA based on fungal cellulase protein linker peptide; DAB, solid phase dual antigen binding assay; (dT),, hexameric deoxythymi- dine; ELISA, solid phase enzyme-linked immunosorbent assay; Fab, antigen binding fragment; HRP, horseradish peroxidase; mAb, mono- clonal antibody; PBS, phosphate-buffered saline; PCR, polymerase

bound ligand; ssDNA, single-stranded DNA, TBS, Tris-buffered saline; chain reaction; Q,,, maximum observed quenching of fluorescence of

TBS-T, Tris-buffered saline with Tween 20; TEBT, TBS-T with EDTA and 1% BSA; VL, antibody light chain variable region; V,, antibody heavy chain variable region.

SCAs have been designed with either VL or V, in the amino- terminal orientation with the assumption that the polylinker does not restrict independent native state folding of the vari- able domains. Achievement of the latter has been verified in various ways (Bedzyk et al., 1990) and was recently confirmed by circular dichroism (CD) and NMR spectroscopy (Tetin et al., 1992; Freund et al., 1993). Monovalent single-chain antibodies approach the minimum necessary structural component for an- tigen binding (Ward et al., 1989) and generally mimic the pa- rental antibody affinity for antigen better than the less stable antibody variable domain fragments, which contain the VL and VH domains non-covalently associated together (Glockshuber et al., 1990; Huston et al., 1991). SCA technology has not only played an important part in the study of antibody active site structure and function (Bedzyk et al., 1990; Denzin et al., 1991; Denzin and Voss, 19921, it has also proven critical in the study of antibody idiotypy and metatypy (Weidner et al., 1990, 1991, 1993).

Bivalent bispecific mAbs and heterodimeric Fab fragments have been generated and characterized (Cheong et al., 1990; Kostelny et al., 1992; Snider et al., 1992; Duncancel et al., 1993; Wu et al., 1993). These proteins have been useful in studies involving cellular targeting of antigen, immune activation, and receptor cross-linking (Snider et al., 1992; MacLean et al., 1993; Wu et al., 19931, indicating the need to genetically develop “single-chain” bivalent bispecific antibodies with the various applications and activities outlined above.

Due to the monovalent nature of SCA molecules relative to the bivalent parental monoclonal antibody (mAb), interactions based on avidity (a measure of the increased apparent binding constant due to entropic effects) (Crothers et al., 1972) are not possible. To intrinsically endow SCA with this property, biva- lent single-chain antibodies have been generated employing chemically mediated linkage via disulfide bonds or by non- covalent domain-domain interactions of engineered SCA or SCA fusion proteins (Cumber et al., 1992; Pack et al., 1992, Holliger et al., 1993). Success of the cited studies indicated the need to examine construction of bivalent SCA by genetically engineering an inter-SCA linker peptide to covalently tether the two antibody active sites.

In this paper we report the construction and characterization of a model bivalent bispecific single-chain antibody (BiSCA) for testing properties such as site-site effects, domain interactions, etc. These studies were performed with the well characterized SCA derivatives of the fluorescein binding antibody 4-4-20 (Bedzyk et al., 1990) and the ssDNA-specific autoantibody BV04-01 (Rumbley et al., 1993), for which the three-dimen- sional structures have been determined (Herron et al., 1989, 1991). In the 4-4-20104-01 construct, the intersite-SCA linker (CBH1’) was based on the linker peptide present in a naturally secreted fungal cellulase protein. The flexible linker peptide of Dichoderma reesi cellobiohydrolase I (CBH1) (Schmuck et al., 1986; Abuja et al., 1988) has been shown to be an effective interdomain linker in an anti-2-phenyloxazolone SCA ex-

199

200 Bivalent Bispecific Single-chain Antibody

A 4420 5': 5' - l T G C f A O T G " - - 3' 442-1' M(: 5' - T G A l " 7 M Z A G C C G G 4 @ 3 X T M Z ~ ~

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tion of the BiSCA gene product. Regions of complementarity with 4-4-20 FIG. 1. A, polynucleotide primers used for amplification and construc-

and 04-01 SCA constructs are underlined. B, diagram of the BiSCA gene. The OmpA signal sequence was added in a separate cloning step to the assembled BiSCA gene construct. C, amino acid sequence of the CBH1' linker peptide.

pressed and secreted in Escherichia coli (Takkinen et al., 1991). Studies presented here show that each active site comprising the model BiSCA molecule possessed similar affinities for the two antigens (fluorescein and (dT),J relative to the respective monovalent native SCAs and functioned as a bivalent bispecific molecule by simultaneously binding both antigens. This report verifies the construction of fully active bivalent bispecific SCA molecules facilitating development of a range of specialized antibody fragments to address various immunochemical prob- lems.

MATERIALS AND METHODS Monoclonal Antibody 4-4-2GmAb 4-4-20 was generated by polyeth-

ylene glycol-mediated fusion of BALB/cV hyperimmune splenocytes with non-secreting Sp2/0-Ag14 myeloma cells as previously described (Kranz and Voss, 1981). mAb 4-4-20 has been extensively characterized with an affinity for fluorescein of 1.7 x 1O1O M - ~ (Kranz and Voss, 1981; Bates et al., 1985; Herron et al., 1991).

Monoclonal Autoantibody BV04-01-mAb BVO4-01 was generated by chemically mediated cell fusion of New Zealand white x New Zealand black (NZB/NZW) F1 splenocytes with non-secreting SpWO-Ag14 my- eloma cells as described (Ballard et al., 1984). mAb 04-01 has been previously characterized with an affinity for oligodeoxythymidine of lo7 M - ~ (Herron et al., 1989; Rumbley et al., 1993; %tin et al., 1993).

Strains, Plasmids, and Media-E. coli strain GX6712 (F galk2 rpsL ~ 1 % ~ ) and plasmid pGX8773 were provided by the Genex Corp. (Gaith- ersburg, MD). Expression vector pGX8773 encodes SCA 4-4-20/212 fused to the OmpA signal sequence and contains the intradomain 212 linker (GSTSGSGKSSEGKG) (Denzin et al., 1991). Expression vector pLY3 encodes the SCA 04-OW212 gene fused to the OmpA signal se- quence with the VL and V, domains tethered by the 212 linker (Rum- bley et al., 1993). All expression vectors utilize a hybrid OJPR A pro- moter with protein expression initiated by a temperature shift from 30 "C to 42 "C in E. coli strain GX6712 (Scandella et al., 1985).

Bivalent Bispecific Single-chain Antibody Construction-Po- lymerase chain reaction (PCR) methodology was used for construction of the specialized bivalent SCA 4-4-20/04-01 from wild type SCA genes. Oligonucleotide primers used in the PCR reactions were synthesized by the phosphoramidite method (Beaucage and Caruthers, 1981) and the primary structures are shown in Fig. 1. Reaction conditions for amplification of DNA fragments with V e n F (New England Biolabs) polymerase were 10 IMI Tris-HC1, pH 8.3, 2.5 m~ MgCI', 50 IMI KCl, 0.01% gelatin (w/v), 0.1% Triton X-100,200 m~ of each dNTP, 5.0 units of polymerase, 10 ng of template DNA, and 30 pmol of primer DNA. Reactions were incubated in a thermal cycler (MJ Research) using the following program: 92 "C for 10 min; 50 "C for 10 min, 72 "C for 10 min; followed by 30 cycles of 92 "C, 1 min; 45 "C, 1 min; and either

61 "C or 62 "C, 1 min (for 4-4-20 3' primer or 04-01 5' primer, respec- tively). SCA 4-4-20 was amplified to contain the 5' end of the inter- SCA linker, and the SCA 04-01 was amplified to contain the 3' end of the linker at the 5' end.

Following amplification, SCA PCR products were purified in low melting temperature agarose (Seaplaque, FMC) and cloned into SnaI- digested pTZ18U (Mead et al., 1986). Correct clones were identified by restriction length analysis and verified by dideoxy sequencing. To con- struct the BiSCA gene, the linker/SCA 04-01 insert gene was digested with SpeI-EamHI and ligated into SCA 4-4-20Ainker digested with the same enzymes. To transfer the BiSCA gene into the pGX expression vector, the pTZ18U/BiSCA gene was partially digested with KpnI- BamHI, yielding a fragment containing part of SCA 4-4-20, the linker region, and all of SCA 04-01. The fragment was ligated into the pBM2 expression vector, containing a mutant SCA 04-01 L34 Arg + His mu- tant, digested withKpnI-EamHI. The final plasmid, pBM3, consisted of the BiSCA gene (SCA 4-4-20 linked to SCA 04-01 by the CBH1' linker) fused to the Om,m,m,m,m,m,m,m,m,m,m,m,m,m,m,m,m,m,rn,m signal sequence.

Sequence Determination-Following cloning, sequences of the PCR products were determined by the dideoxy chain termination procedure using a double-stranded plasmid DNA template (Kraft et al., 1988) and Sequenas& (U. S. Biochemical Corp.).

Large Scale Expression of BiSCA-BiSCA was expressed in E. coli, denatured and refolded (in vitro) as previously described for anti-fluo- rescein (Denzin et al., 1991) and autoanti-ssDNA SCAs (Rumbley et al., 1993).

Purification of BiSCA-Refolded BiSCA was dialyzed extensively against Tris-buffered saline (TBS: 150 IMI NaCI, 50 m~ Tris-HC1, pH 8.0) followed by dialysis against phosphate-buffered saline (PBS: 150 m~ NaC1, 50 m~ PO4, pH 8.0). Precipitates were removed by centrifu- gation, denatured in 6.0 M guanidine-HC1, PBS, pH 8.0, and refolded by dialysis against an equal volume of the following solutions (in order): 3.0 M guanidine-HC1, PBS, pH 8.0; 1.0 M guanidine-HC1, PBS, pH 8.0; and PBS, pH 8.0. Active BiSCA was purified by one of two methods: fluorescein-Sepharose affinity purification or ssDNA-agarose (Life Technologies, Inc.) affinity purification. BiSCA bound through the 4-4-20 site was eluted using either excess free fluorescein (0.1 M fluo- rescein, disodium salt) or 8.0 M urea/PBS followed by dialysis against PBS, pH 8.0. For anti-ssDNAaffinity purification, adsorbed BiSCAwas eluted with 1.0 M NaCl, 50 IMI PO.,, pH 8.0. BiSCApurity was evaluated by SDS-polyacrylamide gel electrophoresis using a 10% gel in the dis- continuous SDS buffer system of Laemmli (1970). Protein bands were visualized with Fast Stain (Zoion Research, Inc.).

Western Blots-Crude and purified BiSCA preparations were ana- lyzed on 10% SDS-polyacrylamide gels. Proteins were blotted to poly- vinylidene difluoride membranes (Immobilon F@, Millipore Corp.) us- ing a Novablot System (Pharmacia LKB Biotechnology Inc.). After blocking the membranes with TEBT (overnight at 4 "C), the blots were stained with the appropriate monoclonal antibody specific for 4-4-20 and/or 04-01 variable domains (1 pg/ml in PBS, pH 8.0). Membranes were washed extensively with TBS-T, and HRP-labeled anti-hamster antibodies were added as the secondary antibody. Binding of HRP- labeled anti-hamster antibodies was visualized using 1-Stepm chloro- naphthol immunoblot solution (Pierce) or using diaminobenzidine/ hydrogen peroxide (Sigma) precipitation as described in the VectastainB ABC kit (Vector Laboratories, Inch

Anti-ssDNA Solid Phase ELISA Binding Assay-For anti-ssDNA binding, the high pHhigh salt (NaC1) method of Lacy and Voss (1989) for ssDNA adsorption to polystyrene wells was used. BiSCA was added to ssDNA-coated wells at varying concentrations and incubated for 2 h at 37 "C. Bound BiSCA was detected by using either hamster anti-4- 4-20 variable domain-specific antibodies or hamster antibodies cross- reactive with both the 44-20 and 04-01 variable domains followed by reaction with horseradish peroxidase (HRPI-labeled anti-hamster (Jackson Immunoresearch Laboratories, Inc.). Enzyme activity was quantitated using 3,3',5,5'-tetramethylbenzidine (l-stepm, Pierce Chemical Co.) and optical density determined using a Dynatech MR 600 automatic 96-well microtiter reader with a 450-nm cut-off filter.

Fluorescein InhibitionlSolid Phase ELISA Binding Assay-For de- termination of anti-fluorescein activity when the BiSCA molecule was bound to adsorbed ssDNA, an inhibition protocol was employed using a modified version of the previously described anti-ssDNA binding assay. After binding BiSCA to the ssDNA-adsorbed polystyrene wells, HRP- labeled 1F4 (a ligand-inhibitable hamster anti-4-4-20-specific antibody) was added in the presence of varying concentrations (10"' M to lo4 M) of free fluorescein. Subsequently, wells were washed three times with TBS-T and bound HRP-1F4 quantitated using the method described above.

Bivalent Bispecific Single-chain Antibody 20 1

(Fc Specific Developmg Reagent) HRP - Prote,in A

I J I & A R R R-m fl

U 1 .Polys(yrene

FIG. 2. Diagram of the dual antigen binding assay used to de-

BiSCA protein tect simultaneous binding of fluorescein and ssDNA by the

Anti-fluorescein Solid Phase ELZSA Binding Assay-To demonstrate anti-fluorescein activity, a solid phase assay similar to that described by Denzin et al. (1993) was employed. ARer addition of BiSCA and proper washing, 50 pl of a 1 pg/ml hamster anti-4-4-20 variable light domain- specific antibody was added and subsequently detected using HRP- labeled antibodies.

DNA Inhibition Solid Phase ELISA Binding Assay-To demonstrate the ability of DNA to inhibit BiSCA from binding to ssDNA adsorbed to polystyrene wells, the following modifications were made in the anti- ssDNA binding protocol. BiSCA was preincubated with increasing con- centrations of calf thymus DNA (4 "C, overnight) before adding to the ssDNA-coated wells. The assay proceeded as described for the anti- ssDNA binding assay. DNA inhibitor concentrations ranged from 0.5 mg/ml to 1.95 pg/ml.

Dual Antigen Binding (DAB) Solid Phase ELZSA Assay-'Ib demon- strate the ability of BiSCA to simultaneously bind both ssDNA and fluorescein, a solid phase assay was designed (Fig. 2). First, ssDNA was adsorbed to polystyrene wells as described above. After washing with TBS-T and masking with TEBT, BiSCA was added to the wells (50 9) at 10 pg/ml concentration. Following extensive washing with TBS-T, fluorescein-BSA (50 pl) was added at varying concentrations (10 pg/ml to pg/ml). After washing, mAb 4-4-20 was added (50 p1 of a 1 pg/ml solution) to the wells to bind to remaining fluorescein epitopes on BSA. Bound mAb 4-4-20 was quantitated using HRP-labeled protein A (Zymed Laboratories, Inc.).

Dissociation Rate Kinetic Assay-Dissociation rates of BiSCA 4-4-20 domains (90% liganded with fluorescein) were determined at 4 "C as described (Herron, 1984). Amnity constants were determined from the dissociation rate using the previously determined association rate of 5 x lo6 sec-l for anti-fluorescyl antibodies to calculate intrinsic af- finities (K, = k l / k z ) (Kranz et al., 1982; Swindlehurst and Voss, 1991). The assay was performed in the presence and absence of (dT)6.

Fluorescein Fluorescence Quenching Assay-Fluorescence quenching measurements of antibody-bound ligand were performed as described by Watt and Voss (1978) using an Aminco-Bowman spectrophotofluo- rometer.

Solution Phase llyptophan Quenching Assay-A solution phase as- say based on quenching of intrinsic tryptophan fluorescence was em- ployed to determine the affinity of mAb, SCA 04-01 and BiSCA for the (dT), ligand (Rumbley et al., 1993; Tetin et al., 1993).

Anti-ssDNA Solid Phase ELISA Competition Assay-To compare the affinity of BiSCA for ssDNA with other 04-01 proteins, the ssDNA competition ELISAdescribed by Rumbley et al. (1993) was used. Briefly, polystyrene wells were coated with ssDNA and blocked as described above. Following washing with TBS-T, HRP-labeled mAb 04-01 (Smith and Ulrich, 1983) was added to wells in the presence of inhibitors (50 pl; mAb 04-01, Fab 04-01, SCA 04-01, SCA 04-01 and SCA 4-4-20, and BiSCA) diluted 1: lO in TBS-T. Inhibitor concentrations ranged from 1 x 10"O to 1 x 10"' mol of active sitedwell. Following incubation a t 37 "C for 2 h, wells were washed with TBS-T and bound HRP-labeled mAb 04-01 detected as described above.

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I

1

1

1

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I

FIG. 3. SDS-polyacrylamide gel electrophoresis (10%) analysis of expressed BiSCA, renatured BiSCAprecipitate, and purified BiSCA. Lane 1, ssDNA-purified BiSCA from renatured precipitate (10 pg); Lane 2, ssDNA-purified BiSCA from crude fraction (10 pg); Lane 3, unpurified renatured BiSCA precipitate (50-pl sample); lane 4, fluores- cein-purified BiSCA (50-pl sample); lane 5, expressed BiSCA (50-pl

subjected to thiol reduction with dithiothreitol and boiled (100 "C) be- sample); lane 6 , protein molecular weight markers. All samples were

fore loading.

RESULTS Construction and Expression of BiSCA-BiSCA gene con-

struction was accomplished using both PCR and conventional cloning techniques (see "Materials and Methods"). Addition of the OmpA signal sequence was achieved by subcloning a frag- ment of the BiSCA gene into an expression vector (SCA 04- OlL34hg) containing the signal sequence and the first 35 amino acids of the 04-01 VL gene with the L34 His residue mutated to Arg (i.e. the first 35 residues were identical to the wild type 4-4-20 VL). From 25 ml of denatured inclusion bodies, with an optical density (at 278 nm) of 15-25 absorbance units, yields ranging from 0.5 to 1 mg of purified BiSCA were obtained. Protein concentrations were calculated from absorption spectra at 240-350 nm (Levine and Federici, 1982) using a Beckman DU-64 spectrophotometer. Extinction coefficients of 2.2 and 1.75 for SCAand BiSCA, respectively, were calculated from chromophore content (Mach et al., 1992).

Polyacrylamide Gel and Western Blot Analysis-BiSCA was affinity-purified using either fluorescein-Sepharose or ssDNA- agarose. SDS-polyacrylamide gel electrophoresis analysis showed the fluorescein-eluted protein contained BiSCA (57 kDa). Similar analysis of ssDNA-purified protein showed BiSCA was >90% pure (Fig. 3). SDS-PAGE and Western blot analyses of protein affinity-purified by both methods described above indicated that proteins of > 28 kDa contained SCA4-4-20 and that the 57-kDa band contained SCA 4-4-20 covalently linked to SCA 04-01 (data not shown). NH2-terminal amino acid analysis of the 57-kDa protein showed that the OmpA signal sequence was properly cleaved from the BiSCA protein.

Anti-ssDNA Activity of BiSCA-BiSCA was analyzed by a direct binding ssDNA ELISA assay to examine the ability of fluorescein-purified and ssDNA-purified BiSCA to bind ssDNA. BiSCA purified by either of the two methods described above was compared to I g G 04-01 for anti-ssDNA activity. Bound mAb 04-01 was detected using HRP-labeled protein A, while BiSCA was detected using hamster anti-4-4-20 and hamster antibod- ies cross-reactive for both 4-4-20 and 04-01 (Weidner et al., 1993). Comparisons with SCA 04-01 could not be made with this assay due to the lack of a detection method for SCA 04-01 (Rumbley et al., 1993). The ligand binding curves for BiSCA

202 Bivalent Bispecific Single-chain Antibody

0.60 I

FIG. 4. Anti-ssDNA activity of BiSCA protein determined in the ELISA. In- dividual points represent mean values of triplicate trials with standard deviations (error bars). Points lacking error bars in- dicate standard deviations smaller than symbol. A, anti-ssDNA activity of fluores- cein-purified BiSCA. Polystyrene wells were coated with ssDNA and incubated with BiSCA (10 pg/ml starting concentra- tion) liganded with fluorescein. BiSCA binding to ssDNA was detected with 1A4 (non-ligand-inhibitable anti-4-4-20 anti- body) (circles) or 1B7 (ligand-inhibitable anti-4-4-20 antibody) (1 pdml) (open tri- angles). BiSCA was compared to mAb 04-01 (crosses; solid line) and mAb 4-4-20 (closed triangles) (1 pg/ml starting con- centration) detected with protein A-HRP. B, anti-ssDNA activity of ssDNA-purified BiSCA (triangles) as compared to equal molar concentrations (1 x lo-? M) mAb 04-01 (crosses; solid line). mAb 4-4-20 was

hibition of mAb 04-01 and BiSCA binding used as a negative control (circles). C , in-

to ssDNA by soluble DNA. Single- stranded DNA-coated polystyrene wells were incubated with equal molar concen- trations(1 x lo" M) of BiSCA (crosses; solid line) and mAb 04-01 (triangles) after preincubation overnight with calf thymus DNA (0.5 m g / d starting concentration). Individual points represent mean values of duplicate trials.

".W

0 1 2 3 4 5 6 7 Log2 D~lut~on

closely resembled that of mAb 04-01 (Fig. 4, A and B ) . Data showed that fluorescein-purified BiSCA retained anti-ssDNA activity similar to that of the DNA-purified BiSCA.

DNA Inhibition Assay-An inhibition assay was performed to confirm binding of ssDNA by BiSCA using free DNA to in- hibit BiSCA from binding ssDNA adsorbed to polystyrene wells. Oligonucleotides did not inhibit either BiSCA or mAb 04-01 from binding even at 100-fold molar excess to antibody active sites. However, free calf thymus DNA specifically inhibited the binding of mAb and BiSCA to the wells (Fig. 4C). BiSCA was inhibited by free DNA to a greater extent than mAb, again due to the higher affinity and avidity of mAb relative to SCA.

Anti-fluorescein Activity of BiSCA-Fluorescein binding by purified BiSCA was examined using a direct binding assay, which compared SCA 4-4-20 to BiSCA. The relative activities of SCA and BiSCA bound to fluorescein-BSA-coated wells were examined using hamster non-ligand-inhibitable antibodies (Weidner et al., 1993). Results showed that crude BiSCA frac- tions and affinity-purified BiSCA (both fluorescein- and ssDNA-purified) possessed levels of activity similar to that of SCA 4-4-20 (Fig. 5, A and B ) .

Bispecificity Analysis of BiSCA-To more accurately charac- terize BiSCA, an assay was designed to determine the presence of active bivalent protein. In this assay, BiSCA bound to ssDNA-adsorbed polystyrene wells was incubated with increas- ing concentrations of free fluorescein. HRP-labeled 1F4, a ham- ster ligand-inhibitable 4-4-20-specific antibody (Weidner et al., 1993), was added to detect 4-4-20 sites not occupied by fluo- rescyl ligand. Thus, fluorescein acted as an inhibitor of the

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Log,0 Dilution

1 2 3 4 5 6 7 8 Loga Dilution ONA

HRP-labeled antibodies, which in turn only detected 4-4-20 variable domains. These domains were only present if they were covalently linked to 04-01 domains bound to ssDNA. Data showed that ssDNA-purified BiSCA bound to DNA-coated wells behaved similarly to SCA 4-4-20-coated wells (Fig. 5C). This indicated that BiSCA, initially purified by ssDNA chromatog- raphy, contained molecules where both active sites displayed near wild type activity with the respective homologous ligand.

Dual Antigen Binding Assay-To quantitatively evaluate the bivalent and bispecific nature of BiSCA, the dual antigen bind- ing assay was developed (Fig. 2). In the assay, purified BiSCA bound to ssDNA-coated wells acted as a presenter of the anti- fluorescein site. Bound BiSCA was incubated with decreasing concentrations of fluorescein-BSA, which was detected using mAb 4-4-20 and HRP-labeled protein A. The quantitation step was designed to detect bound IgG and not SCA. As a positive control, SCA 4-4-20-coated wells were used. Two negative con- trols were also used. The first involved the substitution of BSA for fluorescein-BSA (indicating the requirement for fluores- cein). The second the substitution of SCA 04-01 for BiSCA (in- dicating requirement of a bivalent bispecific molecule). Results indicated that BiSCA bound to ssDNA possessed nearly iden- tical activity as SCA4-4-20-coated wells (Fig. 6). Based on these results, it was assumed that the purified BiSCA examined con- tained a population of molecules with two properly folded, CO-

valently linked, and functional SCA active sites. Spectral Properties of Fluorescein Bound to BiSCA-The

BiSCA and SCA 4-4-20 molecules were analyzed in terms of the spectral properties of bound fluorescein. Anti-fluorescein anti-

BiSCA protein determined in the RG. 5. Anti-fluorescein activity of

ELISA. A, binding of unpurified BiSCAto fluorescein-BSA-coated polystyrene wells (circles) was compared to mAb 4-4-20 (crosses) and mAb 04-01 (closed triangles) (1 pglml starting concentration). Anti- ssDNA activity of the same crude BiSCA protein was monitored for BiSCA (dark crosses) and compared to mAb 04-01 (open circles). Individual points represent mean values of triplicate trials (standard devia- tions not shown). B , binding of ssDNA- purified BiSCA (closed triangles) to fluo- rescein-BSA-coated polystyrene wells as compared to equal molar concentrations (1 x M) of mAb 4-4-20 (crosses; solid line) and mAb 04-01 (circles). Individual points represent mean values of triplicate trials with standard deviations (error bars). Points lacking error bars indicate standard deviations smaller than symbol. C , inhibition of HRP labeled-IF4 (ligand- inhibitable anti-4-4-20 antibody) from binding BiSCA (bound to ssDNA-coated polystyrene wells) by the addition of free fluorescein. DNA-purified BiSCA (crosses; dotted line) and three renatured BiSCA precipitate fractions (10 pglml) (circles, closed triangles, open triangles) were com- pared to mAb 4-4-20-coated wells(light crosses) (10 pdml concentration). Indi- vidual points represent mean values of triplicate trials (standard deviations not shown).

Bivalent Bispecific Single-chain Antibody 203

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1 2 3 4 5 6 7 Log* Diwm

nnn ,

12 11 1 0 9 S 7 6 -Log ,o Molu Car Fl~eacein

0 1 2 3 4 6 6 7 Loolo Olutim FI-BSA

FIG. 6. Dual antigen binding activity of the BiSCA protein. A, comparison of the DAB activity (Fig. 2) for crude BiSCA (crosses; solid line) and two renatured BiSCAprecipitate fractions (10 pg/ml) (open triangles and circles). SCA 4-4-20-coated wells( 10 pglml) (crosses; dashed line) were used as a positive control, while fluorescein-BSA was omitted for a negative control (closed triangles). Individual points represent mean values of triplicate trials (standard deviations not shown). B, DAB activity of ssDNA purified BiSCA (closed friangles) compared to SCA 4-4-20-coated wells (positive control) (crosses; solid line) and SCA 04-01-coated wells (negative control) (open circles). Individual points represent mean values of triplicate trials with standard deviations (error bars). Points lacking error bars indicate standard deviations smaller than symbol.

bodies have been routinely characterized by their individual fluorescein, anti-fluorescein antibodies produce a bathochromic ability to quench (Qm,) the fluorescence of bound fluorescein shift of 10-20 nm in the ligand’s absorption maximum (Watt (Watt and VOSS, 1977). Q,, for BiSCA (83.5 2 0.4%) compared and Voss, 1977; Bates et al., 1985). The bathochromic shift in well with values previously obtained for SCA4-4-20f212 (85.9 2 the absorption of bound fluorescein was identical between 0.4%) (Denzin et al., 1991). In the presence of excess (dTl6, the BiSCA and SCA 4-4-20 (504 n m ) and remained true for BiSCA Q,, remained the same (82 t 1%). with or without excess (dT)6 (Table I).

Along with the ability to quench the fluorescence of bound Afinity Measurements-BiSCA (>go% pure) was incubated

204 Bivalent Bispecific Single-chain Antibody

TABLE I Binding afinity (KJ and spectral properties of 4-4-20 proteins

Protein KO" QmaXb Am-=

x 107 M mAbd 2530 f 540 83.6 * 1.6 nm

SCAd 505

BiSCA 490 f 37 85.9 = 0.4 504 310 f 25 83.5 2 0.4

BiSCA + (dT), 504

270 f 45 82.0 * 1.0 504

%

Reactions monitored at 4 "C with a 5-fold excess of free fluorescein.

Maximum fluorescein fluorescence quenching expressed as a per- Value represents mean of triplicate trials 2 standard deviation.

cent. Value represents mean of triplicate trials = standard deviation. e Absorption wavelength maximum of bound fluorescein.

Previously determined (Denzin and Voss, 1992).

with a &fold excess of fluorescein and analyzed by the disso- ciation-rate fluorescence analysis. BiSCA showed an affinity for fluorescein (3.1 x lo9 M - ~ ) similar to that of wild type SCA 4-4-20 (4.9 x lo9 M - ~ ) . In the presence of saturating concentra- tions of (dT)6, BiSCA showed a modest decrease in affinity (2.7 x lo9 "l). Cumulatively, the binding data suggested that the CBH1' linker caused no significant effect on the activity of 4-4-20 variable domains (Table I).

%ptophan Quenching (dT), Binding Assay-The equilib- rium dissociation constant, &, of BiSCA binding with (dT)6, was determined using the solution phase intrinsic tryptophan quenching assay. Protein samples of fKed concentrations were incubated with increasing concentrations of (dT), ligand and fluorescence emission spectra (310-460 nm) collected. Binding curves were determined based on the area under each titration curve and a single binding curve fitting procedure (Rumbley et al., 1993; Tetin et al., 1993).

While the binding constant for (dT)6 obtained for BiSCA mimicked the value previously determined for SCA 04-01 (3.2 x

M) (Rumbley et al., 1993) and agreed closely with the value previously determined by a gel-shift DNA binding assay (1.0 x

M) (Stevens et al., 1993), the tryptophan Q,, values were not consistent with values obtained with IgG and SCA 04-01 (Table 11).

Anti-ssDNA Solid Phase ELISA Competition Assay-Due to the low Q,, values obtained in the tryptophan quenching assay, the competitive ELISA was employed to qualitatively confirm that BiSCA had similar affinity for ssDNA as SCA 04-01. Fig. 7 compares the inhibition curves of BiSCA relative to SCA 04-01, Fab 04-01, and mAb 04-01. A comparison was also made with SCA04-01 and SCA4-4-20 added to the wells in equal molar active site concentrations. Results demonstrated that BiSCA inhibited HRP-labeled mAb 04-01 similarly to SCA and Fab 04-01. In addition, results showed that BiSCA mim- icked the behavior of monovalent SCA 04-01 in the presence of an equal number of SCA 4-4-20 molecules. These results con- firmed previous data that the affinity of the BiSCA 04-01 do- main for ssDNA was equivalent to that of monovalent SCA 04-01. This suggested that the covalent linking of the SCA molecules caused no significant effects on antibody variable domain activity.

DISCUSSION

As previously stated, single-chain antibodies have played an important role in structure-function and protein folding studies involving antibody active sites. Through genetic engineering, SCA proteins have been fused with toxins and enzymes, dem- onstrating their potential usefulness as in vivo therapeutic and diagnostic agents. The need now exists to develop methods for construction and expression of bivalent bispecific single-chain antibodies that do not rely on low-affinity domain-domain as- sociation or chemically mediated modification for bivalency. This study examined such a construct with development of a

TABLE I1 Binding constants (Kd) and spectral properties of 04-01 proteins

Protein &- Q."mb

PM 0.13 2 0.02 mAb'

%

SCAc 3.2 f 0.9 36.7 2 0.2 76.3 2 6.5

BiSCA 3.2 * 1.0 9.1 2 0.9

a Determined using tryptophan quenching assay using (dT), as de- scribed under "Materials and Methods." Value represents mean of trip- licate trials * standard deviation.

at saturating (dT), concentrations and expressed as a percentage. Value Maximum tryptophan fluorescence quenching values determined

represents mean of triplicate trials 2 standard deviation. Previously determined (Win et al., 1993).

covalently linked bivalent bispecific single-chain antibody model protein. Using the well characterized SCA 4-4-20 (anti- fluorescein) and SCA04-01 (anti-ssDNA) proteins, studies were initiated based on the premise that the unrelated ligand bind- ing properties per site would provide information on the role of introducing a linker peptide covalently joining the two SCA domains and on domain-domain effects.

Due to the high degree of primary structure identity between the two SCA proteins, the BiSCA gene could not be constructed solely by PCR methodology. Therefore, a "cassette" method was employed where the CBH1' linker (primary structure was PG- GNRGTTRPATSGSSPGPTNSHY) was added to the individual SCA by PCR and then one SCMinker gene was cloned into the other SCMinker gene. The cassette design allows for future modification of the BiSCA gene by replacement of either SCA gene or by addition or subtraction of linker sequences. Re- peated rounds of dideoxy sequencing and restriction fragment length analyses were performed, on both amplified PCR prod- ucts as well as cloned BiSCA, to confrm the SCA and inter- active site linker primary structure.

Using the same expression conditions for SCA 4-4-20 and 04-01, refolded protein obtained from E. coli BiSCA inclusion bodies demonstrated both anti-fluorescein and anti-ssDNA ac- tivity (Fig. SA). The presence of bispecific molecules was con- firmed by detection of anti-fluorescein activity from DNA-pu- rified protein and anti-ssDNA activity for fluorescein-purified protein (Figs. 4 and 5). Polyacrylamide gel analyses indicated that the fluorescein-specific affinity-purified fractions con- tained BiSCA plus smaller active protein fragments, while DNA-purified protein contained only BiSCA (Fig. 3). This re- sult may have been due to both proteolytic degradation and incomplete protein synthesis. Purified BiSCA behaved as the respective monovalent prototype molecule in solid phase ELISA assays. In terms of idiotypy and metatypy, the BiSCA 4-4-20 domain showed properties identical to those of SCA 4-4-20 when examined with 4-4-20 variable domain-specific antibodies. These results suggested that the domain conforma- tion of BiSCA 4-4-20 closely approximated the monovalent SCA molecule. Ligand inhibition studies of both domains (Figs. 4C and 5C) confirmed this observation.

To critically examine BiSCA as a bivalent bispecific molecule, a unique dual antigen binding assay was designed (Fig. 2) and introduced in this report. The DAB assay was designed to ex- ploit the dual-specific nature of the molecule, yielding appro- priate results only if the protein was fully active at both sites. Two important features of the assay were the use of a highly substituted fluorescein-BSA molecule and the use of the high affinity 4-4-20 mAb, which was reactive with protein A. Using HRP-labeled protein A as the detector system in the assay, only the amount of fluorescein-BSA bound IgG 4-4-20 was quanti- tated (Fig. 2). By comparing the ssDNA-bound BiSCA to SCA 4-4-20-coated polystyrene wells, a relative measure of the popu- lation of active bispecific molecules was made. Another novel

Bivalent Bispecific Single-chain Antibody 205

100

80

s E 6 0 g 1 40

20

A

100

80

s 5 6 0 f

40

20

B

-17 -16 -15 -14 -13 -12 -11 -10 Moles Idlibitor per Well

-17 -16 -15 -14 -13 -12 -11 -10 Moles lmibitor per Well

co-incubated with HRP-labeled mAb 04-01 and the following inhibitors: A: mAb 04-01 (closed circles); Fab 04-01 (open circles), SCA 04-01 (closed FIG. 7. Inhibition by mAb 04-01 and 04-01 derivative p r ~ t e i n ~ of HRP labeled mAb 04-01. Polystyrene wells were coated with ssDNAand

triangles) and BiSCA (open triangles); B: BiSCA (open circles) and equal molar active site concentration of SCA 04-01 and SCA 4-4-20 (closed circles). Individual uoints reuresent mean value of triulicate trials with standard deviations (error bars). Points lacking error bars indicate standard deviations^srnaller ihan symbol.

feature of the DAB assay was the coupling of a high affinity interaction with a low affinity interaction. Through multiple interactions of BiSCA 4-4-20 domains with fluorescein-BSA molecules, the weaker BiSCA 04-01lssDNA interactions were stabilized by the cross-linkage binding of the high affinity 4-4-20 sites in the BiSCA. This assay demonstrated the de- signed purpose of the BiSCA enhanced antibodylantigen affin- ity through bispecificity and bivalency. Results of the DAB as- say, along with the fluorescein and anti-idiotype antibody inhibition studies, confirmed the presence of active bivalent bispecific SCA molecules (Figs. 5C and 6).

Ligand-binding affinities and ligand-related spectral mea- surements were made to assess BiSCA homology to the mono- valent SCA prototype molecules. For anti-fluorescein antibod- ies, spectral measurements demonstrate the unique characteristics of the antibody active site environment while being relatively independent of affinity (Voss, 1989). The 4-4-20 site in the BiSCA showed almost identical ligand-related spec- tral properties (Q,, and Amax) relative to monovalent SCA 4-4-20. The presence of saturating concentrations of (dTl6 to interact with the 04-01 neighboring site resulted in relatively insignificant changes in these measurements (Table I). These results confirmed that the CBH1’ linker appeared to be a rela- tively inert factor in terms of the BiSCA 4-4-20 domain.

To determine the affinity of the BiSCA 04-01 domain for (dT)6, the intrinsic tryptophan quenching assay of Rumbley et al. (1993) was employed. As stated earlier, the affinity value determined was in good agreement with previously reported values. Interestingly, the total protein tryptophan fluorescence (data not shown) and the Q,, values obtained for the BiSCAin this assay were low (relative to their chromophore content) in comparison with other 04-01 protein derivatives. While a low Q,, value may be explained by the DNA ligand being respon- sible only for 04-01 domain tryptophan quenching, this hypoth- esis offers no explanation for the low overall yield of protein tryptophan fluorescence. Confirmation of BiSCA activity with ssDNA was obtained by the DNA inhibition study (Fig. 4C). Qualitative comparison of the affinity of BiSCA for ssDNA with the various 04-01 proteins was accomplished through use of the competitive ELISA described by Rumbley et al. (1993). Results showed that BiSCA performed equivalently to SCA 04-01 with an equal concentration of SCA 4-4-20 (Fig. 7). Thus, the solu-

tion phase affinity measurement proved valid through a critical comparison of BiSCA with mAb and SCA 04-01 using the solid phase assay.

In summary, these studies confirmed the construction and expression of active bivalent bispecific single-chain antibodies. In addition, the CBH1’ linker (modified fungal cellulase linker peptide) was shown to be an effective inter-SCA linker compat- ible with production of the protein in E. coli. Using both solid phase and solution phase assays, a close simulation of the monovalent SCA properties was confirmed. Characterization of the BiSCA model protein will be continued in order to define the hydrodynamic properties of the molecule. These studies presented indicated that covalent tethering of two SCA mol- ecules and their expression from E. coli is possible without significant alteration of either active site structure. These re- sults clearly demonstrated that novel recombinant, covalently linked antibody fragments with dual specificity and unique properties can be designed and produced for specialized pur- poses. Future studies intend to substantiate the above claim with the development of bivalent bispecific single-chain anti- body constructs specific for two separate and different epitopes on a single antigenic molecule. Such constructs may prove to be important to form stable complexes and would have utility in both diagnostic assay development and antibody therapeutics.

Acknowledgments-We thank Sergey Y. %tin for critical discussions and assistance in performing the tryptophan fluorescence quenching assays. We also acknowledge Myung L. Kim for assistance in perform- ing the fluorescence dissociation rate assays.

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