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Volume 271, Number 10, Issue of March 8, 1996 pp. 5338-5346
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Comparative Properties of the Single Chain Antibody and Fv Derivatives of mAb 4-4-20
RELATIONSHIP BETWEEN INTERDOMAIN INTERACTIONS AND THE HIGH AFFINITY FOR FLUORESCEIN LIGAND (*)

(Received for publication, November 28, 1995; and in revised form, December 28, 1995)

William D. Mallender (§), ,  Jenny Carrero,  Edward W. Voss Jr. (¶)

From the Department of Microbiology, University of Illinois, Urbana, Illinois 61801

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Recombinant Fv derivative of the high affinity murine anti-fluorescein monoclonal antibody 4-4-20 was constructed and expressed in high yields, relative to the single chain antibody (SCA) derivative (2-3-fold), in Escherichia coli. Both variable heavy (V(H)) and variable light (V(L)) domains, that accumulated as insoluble inclusion bodies, were isolated, denatured, mixed, refolded, and affinity-purified to yield active Fv 4-4-20. Affinity-purified Fv 4-4-20 showed identical ligand binding properties compared with the SCA construct, both were slightly lower than the affinities expressed by Fab or IgG 4-4-20. Proper protein folding was shown to be domain-independent by in vitro mixing of individually refolded variable domains to yield functional Fv protein. In solid phase and solution phase assays, Fv 4-4-20 closely approximated the SCA derivative in terms of both idiotype and metatype, confirming identical active site structures and conformations. The equilibrium dissociation constant (K) for the V(L)/V(H) association (1.43 times 10M), which was determined using the change in fluorescein spectral properties upon ligand binding, was relatively low considering the high affinity displayed by the Fv protein for fluorescein (K, 2.9 times 10M). Thus, domain-domain stability in the Fv and SCA 4-4-20 proteins cannot be the sole cause of reduced affinity (2-3-fold) for fluorescein as compared with the Fab or IgG form of 4-4-20. With their identical ligand binding and structural properties, the decreased SCA or Fv affinity for fluorescein must be an ultimate consequence of deletion of the C(H)1 and C(L) constant domains. Collectively, these results verify the importance of constant domain interactions in antibody variable domain structure-function analyses and future antibody engineering endeavors.

INTRODUCTION

Antibody Fv fragments are composed of heavy chain (V(H)) (^1)and light chain (V(L)) variable domains. These two domains associate noncovalently to form the smallest functional antibody protein capable of antigen binding that most closely approximates the Ig molecule(1, 2) . These proteins have been previously found to be less stable in terms of domain-domain association than Fab fragments due to the lack of covalent bonds between the two variable domains(3, 4) . Single chain antibody (SCA) molecules have been produced to diminish this instability by the introduction of an interdomain linker peptide(5, 6) . SCA proteins often mimic the parent antibody active site in terms of antigen binding and structural properties with usually some reduction in affinity for antigen(7, 8, 9) . Recently, Fv proteins have been engineered to possess an interdomain disulfide linkage, effectively disallowing dissociation of the two domains(10) . Due to their small size and amenability to genetic engineering, recombinant Fv proteins have been widely applied in the study of antibody active site structure-function(7, 11, 12, 13) , idiotypy and metatypy(14, 15, 16, 17) , antibody bivalency and bispecificity(18, 19, 20, 21) , and in vivo immunodiagnostics and therapy(10, 22, 23) .

Fv molecules have been efficacious proteins in the study of antibody active site structure-function and protein stability. Studies involving comparative analysis of Fv protein with other immunoglobulin constructs afford unique opportunities for determining domain-domain interactions and the effects these interactions exert upon the intrinsic conformational and antigen binding properties of the variable domains. Being covalently coupled by an interdomain linker, SCA proteins have been suggested to possess greater interdomain stability than their Fv counterparts due to the favorable entropic effect of domain coupling (6, 8) . This would, in turn, suggest that in the appropriate Fv molecule (one with high affinity for antigen), interdomain associative properties would dictate the overall affinity displayed for antigen because only associated V(L)/V(H) proteins would bind antigen. In previous studies, dissociation constants for the V(L)/V(H) association in Fv molecules varied from 10 to >10M(3, 24, 25, 26) . These Fv molecules also displayed similar dissociation constants for their respective antigens (10 to >10M), further supporting some correlation between interdomain and active site/antigen interactions. Further analysis of V(L)/V(H) association constants in relation to antigen affinity would allow identification of components necessary for the production of stable Fv molecules and novel variable domain proteins.

Fv molecules have been especially useful in the study of idiotypy and metatypy. Antibody idiotype and metatype are immunologically resolved markers of active site structural and conformational determinants in the unliganded and liganded state, respectively (review in Refs. 27 and 28). Indeed, the transition between the idiotypic and metatypic states upon ligand binding emphasizes the dynamic properties of antibody proteins(15) . The principle of antibody dynamics is governed by the process of structural fluctuation in both the unliganded and liganded states until the most energetically favorable state is established(29, 30) . Thus, understanding how structural and dynamic features are exerted within antibody variable domains will directly influence our understanding of antibody active site/ligand binding properties. Small differences in antigen binding affinities have been found between certain SCA and IgG despite their structural relatedness(7) . The possibility exists that constant/variable domain interactions results in more than structural rigidity, but are responsible for restricted variable domain conformational dynamics favoring antigen binding. Indeed, studies with MOPC 315, a murine anti-nitrophenyl IgA antibody, have indicated that the C(H)1 domain in Fab fragments influences idiotypic expression of the antibody through a dynamic effect on the variable domain structure(31) . In other antibodies, studies have demonstrated the importance of cooperative interface interactions (cis and trans) between the variable and constant domains regarding variable domain stability and antigen binding(7, 32) . To examine this, Fv and SCA proteins, due to differences in interdomain associative properties with similar ligand binding to IgG, represent critical reagents to study ligand binding properties and variable region structural features as influenced by antibody constant domains.

To study the relation between interdomain association and affinity for antigen, an antibody protein must be available in many derivative forms (e.g. with and without constant domain, with and without an intervariable domain linker). To satisfy these criteria, the Fv derivative of mAb 4-4-20, a high affinity murine anti-fluorescein antibody, has been synthesized. mAb 4-4-20 was a suitable antibody for this study due to its high degree of structural characterization (7, 33, 34) and the previous construction and characterization of SCA 4-4-20(7, 35) . SCA 4-4-20 has been studied extensively in terms of active site environment(36, 37) , antigen binding structure(13) , and thermodynamics(8, 38) . Comparison of SCA with Fab 4-4-20 showed almost identical guanidine-induced denaturation profiles, idiotype and metatype expression, yet a 2-3-fold reduction in affinity for fluorescein(7) . The structural and antigen binding properties required for fluorescein binding and quenching by SCA and mAb 4-4-20 have also been extensively analyzed in relation to the remainder of the 4-4-20 idiotype anti-fluorescein antibody family(11) . Comparison of Fv 4-4-20 with SCA, Fab, and IgG may assist in understanding the basis for this difference in affinity for antigen upon removal of the 4-4-20 constant domains. Studies reported here show that Fv 4-4-20 possessed identical structural, idiotypic, metatypic, and ligand binding properties as SCA 4-4-20. With confirmation of identical ligand binding and structural characteristics between Fv and SCA, the dissociation constant (K) for the V(L)/V(H) association was determined and analyzed in relation to the Fv affinity for fluorescein. Such analyses implicated the necessity of constant domain/variable domain association in the formation of the high affinity liganded state. Overall, results indicated that the reduced affinity of Fv and SCA 4-4-20 did not correlate with reduced variable domain association, but with the absence of antibody constant domains, emphasizing their role in antibody/antigen interactions.

MATERIALS AND METHODS

Monoclonal Antibody 4-4-20

mAb 4-4-20 was generated by polyethylene glycol-mediated fusion of BALB/cV hyperimmune splenocytes with nonsecreting Sp2/O-Ag14 myeloma cells as described previously (39) . mAb 4-4-20 has been extensively characterized with an affinity for fluorescein of 1.7 times 10M(34, 39, 40, 41) . Fab fragments were prepared by papain (Worthington) cleavage of immunoglobulins as described by Oi and Herzenberg (42) and Weidner and Voss(14) . Monoclonal antibodies were obtained from murine ascites fluid as described(43) . Both mAb and Fab proteins were affinity-purified as described previously(44) .

Strains, Plasmids, and Media

Escherichia coli strain GX6712 (F galk2 rpsL cI) and plasmid pGX8773 were provided originally by the Genex Corp. (now Enzon, Inc.). Expression vector pGX8773 encodes SCA 4-4-20/212 fused to the OmpA signal sequence and containing the interdomain 212 linker (GSTSGSGKSSEGKG)(6, 45) . The expression vector utilizes a hybrid O(L)/P(R) promoter with protein expression initiated by temperature shift from 30 °C to 42 °C in E. coli strain GX6712(46) .

Fv 4-4-20 Construction

Polymerase chain reaction (PCR) methodology was used for construction of the V(L) and V(H) expression modules from wild type SCA 4-4-20. Oligonucleotide primers were synthesized by the Genetic Engineering Facility at the University of Illinois (Urbana-Champaign) by the phosphoramidite method(47) , and their respective primary structures are shown in Fig. 1. PCR amplification reactions were performed using both Vent (New England Biolabs) and Taq (Life Technologies, Inc.). Reaction conditions for amplification of DNA fragments with Vent were 10 mM Tris-HCl, pH 8.3, 2.5 mM MgCl(2), 50 mM KCl, 0.01% BSA (w/v), 0.1% Triton X-100, 4 mM of each dNTP, 5.0 units of polymerase, 10 ng of template DNA, and 30 pmol of primer DNA. Reaction conditions for amplification with Taq were 20 mM Tris-HCl, pH 8.4, 2 mM MgCl(2), 50 mM KCl, 4 mM of each dNTP, 2.5 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 5 min, 53-67 °C (depending on primer sequence) for 5 min, 72 °C for 1 min, followed by 30 cycles of 72 °C for 1 min, 92 °C for 1 min, and 53-67 °C for 1 min. The V(L) and V(H) genes were amplified separately to contain the 5` OmpA signal sequence and the 3` terminator sequences necessary for expression from pGX8773.

Figure 1: Polynucleotide primers and amplification strategy used for construction of the V(L) and V(H) gene products. Regions of complementarity with 4-4-20 are underlined. OmpA and terminator complementary sequences are in italics, and vector complementary sequences are roman.


Following amplification, V(L) and V(H) PCR products were purified in low melting temperature agarose (Seaplaque, FMC) and cloned into SmaI digested pTZ18u(48) . Correct clones were identified by restriction length analysis and verified by dideoxy sequencing. To construct the V(L) gene, PCR was used to add the transcription stop codons by 3` primer overhang (Fig. 1). To incorporate the signal sequence in the V(H) gene, PCR was used to amplify the OmpA sequence with the addition of the V(H) 5` sequence to the 3` end of OmpA. The resulting PCR product was then used as the 5` primer to incorporate the signal sequence to the V(H) gene (Fig. 1). Both amplified genes were cloned into pTZ18u to form pJWc2-2 and pJWc1-5, respectively. Following verification of proper primary sequence, the V(L) and V(H) genes were excised using ClaI-BamHI and cloned into pGX8773 for expression.

Sequence Determination

Following cloning, sequences of the PCR products were determined by the dideoxy chain termination procedure using a double-stranded plasmid DNA template (49) and Sequenase® (U. S. Biochemical Corp.).

Large Scale Expression of Fv 4-4-20

Fv 4-4-20 was expressed in E. coli, denatured, and refolded using a modified version of the protocol used in Denzin et al. (45) and Rumbley et. al.(12) . The procedure was modified in two ways: 1) in scale, to accommodate 1 liter of bacterial culture instead of 12 liters, and 2) in molecular weight cut-off size, all concentration and dialysis steps were performed using molecular weight cutoff of 3 kDa. Denatured V(L) and V(H) inclusions bodies were combined in a 1:1 mass ratio in the refolding solution to produce associated active Fv. Concentration of the diluted protein was accomplished using an Amicon Ultrafiltration Cell. Monomer V(L) and V(H) were produced by denaturation and renaturation in the absence of the other protein.

Purification of Fv 4-4-20

Refolded Fv 4-4-20 was dialyzed extensively against Tris-buffered saline (50 mM Tris-HCl, pH 8.0, 150 mM NaCl) followed by dialysis against phosphate-buffered saline (PBS: 50 mM PO(4), pH 8.0, 150 mM NaCl). Precipitates were removed by centrifugation. Active Fv 4-4-20 was purified by fluorescein-Sepharose affinity chromatography. After extensive washing, bound protein was eluted using 8.0 M urea/PBS followed by extensive dialysis against PBS. Individually refolded variable domain proteins were concentrated without purification. Purity of all proteins was evaluated by SDS-polyacrylamide gel electrophoresis using a 15% gel in the discontinuous SDS buffer system of Laemmli(50) . Protein bands were visualized with Fast Stain (Zoion Research, Inc.).

Anti-fluorescein Solid Phase ELISA Binding Assay

To demonstrate anti-fluorescein activity, a solid phase ELISA assay similar to that in Denzin et. al. (45) was performed. After addition of Fv 4-4-20 to fluorescein-BSA-coated wells and extensive washing, 50 µl of 10 µg/ml hamster anti-4-4-20 variable light domain-specific peptide antibody (3A5-1) (63) was added. Bound antibody was detected using horseradish peroxidase-labeled anti-hamster antibodies and 3,3`,5,5`-tetramethylbenzidine (Pierce). Substrate was added and incubated at room temperature for 30 min. Enzyme reactions were terminated with 2 N H(2)SO(4) and optical densities determined using a Dynatech MR500 automatic plate reader.

Anti-metatype Inhibition Solid Phase ELISA Binding Assay

For comparison of the Fv metatypic state with SCA, a solid phase inhibition assay was used(7) . Inhibitors were preincubated with rabbit anti-metatype -globulin (10 ng/ml) (15-16 h, 4 °C) and added to polystyrene wells preadsorbed with affinity-labeled 4-4-20 Fab fragments (see below) (2 µg/ml).

Anti-idiotype Inhibition Solid Phase ELISA Binding Assay

For comparative idiotype analysis between Fv and SCA 4-4-20, a solid phase inhibition assay was performed(7) . Inhibitors were preincubated with horseradish peroxidase-labeled IgG 4-4-20 (10 ng/ml) and added to polystyrene wells preadsorbed with polyclonal anti-idiotype antibodies (1 µg/ml).

Dissociation Rate Kinetic Assay

Ligand dissociation rates of Fv 4-4-20 (90% liganded with fluorescein) were determined at 4 °C as described(51) . Affinity constants were determined from the dissociation rate using the previously determined association rate of 5 times 10^6M s for anti-fluorescyl antibodies to calculate intrinsic affinities (K(a) = k(1)/k(2))(52, 53) . Ligand dissociation rates were also performed in the presence of both polyclonal and monoclonal anti-metatype antibodies(54) . Dissociation assays were performed as above, but liganded Fv 4-4-20 was preincubated with an active site molar excess of anti-metatype antibody active sites for 15-20 min at 4 °C.

Fluorescein Fluorescence Quenching Assay

Fluorescence quenching measurements of antibody-bound ligand were performed as described by Watt and Voss(55) . Fluorescein fluorescence quenching by affinity-purified Fv and by equal optical density amount of preincubated V(L) and V(H) protein was compared with SCA 4-4-20.

Circular Dichroism (CD) Studies

CD spectra of the antibody derivatives were recorded on a Jasco model JA720 instrument. A 0.1-cm cuvette (Hellma) was used for all measurements, and all spectra were averaged five times at room temperature. The bandwidth used was 2 nm, to a resolution of 0.1 nm, in all experiments. The buffer used in all experiments was PBS.

Hydrostatic Pressure Fluorescence Measurements

Fv and SCA 4-4-20 were compared by pressure-induced fluorescein dissociation. Hydrostatic pressure in the range of 1 bar to 2.4 kbar was achieved with a pressure cell as described by Paladini and Weber(56) . Samples were excited at 480 nm (slit width of 8 nm) and emission spectra recorded in the range of 500-600 nm (slit width of 8 nm). The intensity data were collected on an ISS GREG PC photon counting spectrofluorometer (ISS, Champaign, IL), and the intensity at each pressure was acquired by integrating the area under the emission spectra. Temperature was regulated with a circulating water bath and monitored by a thermocouple in direct contact with the stainless steel pressure cell. The temperature of the pressure cell was allowed to equilibrate for 1 h after temperature readings stabilized. Protein samples were prepared in 20 mM Tris-HCl, pH 8.0, and contained 0.127 µM antibody active sites and 0.02 µM fluorescein. The protein pressure samples were allowed to equilibrate for 4 min after each pressure change before spectra measurements were taken.

V(L)/V(H) Interdomain Affinity Measurements

All fluorescence data were collected on an ISS GREG PC photon counting spectrofluorometer (ISS) and all experiments were performed at room temperature in PBS. Anisotropy based domain-domain binding measurements were made using affinity-labeled Fv 4-4-20. Fluorescein was covalently coupled in the Fv 4-4-20 active site using the isothiocyanate derivative of fluorescein (FITC) (Sigma). A 1.1 molar excess of FITC was incubated with Fv 4-4-20 for 4-5 h with agitation at 37 °C. Protein samples were extensively dialyzed against PBS to remove free FITC. The concentration of fluorescein was determined on a Beckman DU-64 spectrophotometer using the absorbance at 492 nm and the extinction coefficient () of 72,000 cmM. The amount of affinity-labeled protein (R) was calculated as the ratio of fluorescein concentration and Fv protein concentration. For anisotropy based measurements, affinity-labeled protein was serially diluted (1:2) over the concentration range of 23.5 µM to 4 nM. Studies were performed using excitation at 480 nm and emission at 525 nm with slit widths at 1 and 2 nm. Steady state fluorescence anisotropy data were analyzed using Delta Graph Professional (Delta Point, Monterey, CA) as described (57) and K(d) values were determined from binding curves. Differences between quantum yield of bound and free fluorescein (due to domain dissociation upon dilution) result in differently weighted fluorescence anisotropy values(58) . In order to correct for this, anisotropy data were analyzed in terms of degree dissociation (alpha) at each protein concentration (56) as calculated from the following equation,

where Q is the ratio between quantum yields of free and bound ligand, r(f) is the anisotropy value for fluorescein-labeled variable domain (0.02), and r(b) is the anisotropy for totally bound fluorescein (0.32). Protein concentrations were corrected for the R value of Fv affinity labeling.

RESULTS

Construction and Expression of Fv 4-4-20

Individual V(L) and V(H) gene constructs were assembled using both PCR and conventional cloning techniques (see ``Materials and Methods''). The 3` stop codons and 3` BamHI restriction site were added to V(L) 4-4-20 by PCR technology. The OmpA signal sequence was added to V(H) 4-4-20 using a modified version of the megaprimer method of mutagenesis(59, 60) . Briefly, the OmpA sequence was amplified from SCA 4-4-20 incorporating a portion of the V(H) 5` sequence to the 3` end of OmpA. This PCR product was then used to amplify the entire V(H) gene, resulting in addition of the signal sequence. Protein yields from 1 liter of E. coli cultures were from 3 to 4 mg of affinity-purified Fv. This represented a 2-3-fold increase of active anti-fluorescein protein as compared with expression yields of SCA 4-4-20. Protein concentrations were calculated from absorption spectra at 240-350 nm (61) using a Beckman DU-64 spectrophotometer. Extinction coefficients (A) of 2.2, 2.1, 1.5 and 2.7 for SCA, Fv, V(L), and V(H) proteins, respectively, were calculated from chromophore content(62) .

Polyacrylamide Gel Analysis

Fv 4-4-20 was purified by affinity chromatography using fluorescein-Sepharose as described (see ``Materials and Methods''). SDS-polyacrylamide gel electrophoresis analysis showed the purified Fv protein consisted of two detectable bands (14.0 and 12.5 kDa) corresponding to V(H) And V(L) proteins. Migration patterns indicated actual molecular weights for the two domains were in good agreement with their calculated values based on amino acid content (data not shown). Additionally, the affinity-purified material was shown to be >90% pure. Similar SDS-polyacrylamide gel electrophoresis analysis on individually refolded domain proteins showed that the V(L) and V(H) proteins were the major detectable band found in their respective samples (data not shown).

Anti-fluorescein Activity of Fv 4-4-20

Fluorescein binding by purified Fv was examined using a solid phase direct binding assay, which compared SCA and mAb 4-4-20 to Fv. Proteins bound to fluorescein-BSA-coated wells were detected using hamster nonligand inhibitable anti-4-4-20 antibodies(16, 54, 63) . Results showed that Fv 4-4-20 possessed similar levels of anti-fluorescein activity as compared with SCA 4-4-20 (Fig. 2A).

Figure 2: Solid phase ELISA analysis of Fv 4-4-20. A, direct binding of IgG, SCA, or Fv 4-4-20 (10 µg/ml starting concentration) to fluorescein-BSA-coated wells. Protein was detected with mAb 3A5-1 (10 µg/ml) and horseradish peroxidase-anti-hamster IgG. B, 4-4-20 proteins (10 to 10M) were used to inhibit anti-metatype antibodies (10 ng/ml) from binding to wells coated with affinity-labeled Fab 4-4-20. C, 4-4-20 proteins (10 to 10M) were used to inhibit horseradish peroxidase-IgG 4-4-20 (10 ng/ml) from binding to wells coated with anti-idiotype antibodies. Individual points represent mean values of triplicate trials with standard deviations (error bars). Points lacking error bars indicate standard deviations smaller than symbol.


Anti-metatype Reactivity

To compare the degree of structural relatedness between the liganded states of SCA, Fab, and Fv 4-4-20, these proteins were used as polyclonal anti-metatype/liganded Fab 4-4-20-soluble inhibitors. All 4-4-20 proteins were affinity-labeled as described previously ((7) ; see ``Materials and Methods''). Fig. 2B compares the inhibition titrations of affinity-labeled Fv with similarly labeled Fab and SCA 4-4-20. Unliganded Fab was also tested to determine the amount of anti-idiotype and anti-constant domain activity present in the anti-metatype reagent. The anti-metatype reagent was not passed over an unliganded IgG 4-4-20 adsorbent to remove such activity prior to this experiment. Results indicated that the Fv 4-4-20 possessed a similar anti-metatype inhibition profile as SCA 4-4-20, implying an overall structural similarity between their liganded states. Comparison of the unliganded and liganded Fab curves suggested the presence of anti-constant domain activity in the anti-metatype reagent, but confirmed specificity for the liganded state of the 4-4-20 active site.

Anti-idiotype Reactivity

In terms of a polyclonal anti-idiotype reagent, comparative inhibition studies revealed identical patterns of SCA and Fv anti-idiotype recognition. Results suggested that SCA and Fv 4-4-20 were idiotypically identical (Fig. 2C). Previous idiotypic analysis of SCA with mAb 4-4-20 indicated that the two were idiotypically identical(7) .

Spectral Properties of Fluorescein Bound to Fv 4-4-20

Anti-fluorescein antibodies have been characterized by their ability to quench (Q(max)) the fluorescence of fluorescein(64) . Q(max) for Fv (87.5 ± 0.9%) compared well with SCA (85.9 ± 0.5%) (Table 1). Identical Q(max) properties were found when affinity-purified Fv was compared with an equal optical density (278 nm) mixture of refolded V(L) and V(H) protein (Fig. 3). This suggested that each individually refolded domain protein had formed a dimerization competent structure in the absence of the other domain protein. Identical Q(max) values confirmed the similar active site environments displayed by SCA and Fv 4-4-20.

Figure 3: Fluorescein fluorescence quenching comparison of purified Fv 4-4-20 protein with mixed V(L) and V(H) protein. Domains were mixed so that the starting concentration of Fv and mixed variable domains would be approximately equivalent. Individual points represent mean values of triplicate trials.


Anti-fluorescein antibodies also produce a characteristic bathochromic shift of 10-20 nm in the ligands absorption maximum ((max)) upon fluorescein binding(40, 64) . The bathochromic shift in bound fluorescein absorption was identical for SCA and Fv 4-4-20 (504 nm) (Table 1).

Affinity Measurements

Affinity-purified Fv 4-4-20 (liganded with fluorescein) was examined by dissociation rate fluorescence analysis. Fv 4-4-20 showed an affinity for fluorescein (3.5 times 10^9M) that was nearly identical to SCA (4.9 times 10^9M) within error limitation of the experiment (Table 1). Similar affinity determinations were performed in the presence of excess polyclonal and monoclonal anti-metatype antibodies (reviewed in (28) and (54) ). These antibodies characteristically delay the fluorescein dissociation rate from the antibody active site against which they were raised. Both polyclonal and monoclonal anti-metatype reagents caused similar changes in the determined affinity values for fluorescein for SCA and Fv 4-4-20 (Table 1). Cumulatively, binding data indicated that the Fv molecule effectively mimics the SCA, proving that the 212 linker peptide was not responsible for the original affinity decrease found in SCA as compared with mAb 4-4-20.

CD Spectra of Fv 4-4-20

Fig. 4shows the CD spectra recovered for Fv and mAb 4-4-20. Results are expressed in terms of mean residue weight ellipticity ([] times 10^3 (degree cm^2 dmol)). Analyses of CD spectra were carried out using previously computed CD spectra for poly-L-lysine containing varying amounts of alpha-helix, beta-sheet, and random coil segments (65) as well as the previously determined CD spectra for SCA and mAb 4-4-20(36) . These analyses enabled estimation of the general secondary structure characteristics as a means of qualitative comparison of Fv with SCA and mAb 4-4-20. Fv 4-4-20 showed the same positive extremum (204 nm) and negative extrema (217 and 230 nm) as reported previously for SCA (Fig. 4). The Fv protein also displayed the slight shift in extrema, as well as the pronounced negative value at 230 nm, that SCA did in comparison with mAb 4-4-20. Similar CD spectra were recorded for samples of refolded V(L) and V(H) proteins (data not shown).

Figure 4: CD spectra of IgG and Fv 4-4-20 (both 0.5 OD units). At the concentration indicated (>5 µM), the Fv sample should be in the associated form.


Pressure-induced Dissociation of Fluorescein from Fv 4-4-20

Further structural comparison of Fv 4-4-20 with SCA was accomplished by measuring their hydrostatic-induced fluorescein dissociation parameters. Hydrostatic pressure has been shown to cause conformational changes (independent of protein tertiary structure) in proteins which can promote ligand dissociation(38, 66) . Fig. 5shows the effect of hydrostatic pressure on liganded SCA and Fv 4-4-20 while monitoring fluorescein fluorescence intensity. Increased fluorescein fluorescence intensity was correlated with structural changes in the active site resulting in alleviated quenching and the ultimate dissociation of fluorescein ligand. Fv and SCA displayed similar fluorescein dissociation profiles as pressure was increased from atmospheric to 2.4 kbar. Results further confirmed the overall similarity in structure between the Fv and SCA 4-4-20 molecule.

Figure 5: Pressure-induced dissociation of fluorescein profiles for Fv and SCA 4-4-20. Equal molar samples (0.127 µM) of protein were subjected to increasing hydrostatic pressure. Total fluorescein fluorescence intensity values were recovered at each pressure and compared with free fluorescein fluorescence intensity values.


V(L)/V(H) Interdomain Affinity Analysis

The variable domain dissociation constant (K(d)) was determined by diluting affinity-labeled Fv 4-4-20 and monitoring for increased fluorescein rotation by steady state anisotropy measurements. Being covalently coupled to the Fv binding pocket, changes in fluorescein anisotropy after dilution were due to domain-domain dissociation and not ligand dissociation. Fluorescence anisotropy-based binding curves were obtained by diluting affinity-labeled Fv protein and plotting degree dissociation values versus liganded protein concentration (see ``Materials and Methods'') (Fig. 6). Fluorescence anisotropy values of fluorescein decreased with affinity liganded Fv concentration, indicating dissociation of the two domains. Final anisotropy values (r = 0.02) were higher than values for free fluorescein (r = 0.008) confirming the linkage of fluorescein to the surface of an individual variable domain, not the static interior of the binding pocket (r = 0.320). In contrast to the high affinity displayed (K(d), 2.9 times 10M) by Fv 4-4-20 for fluorescein, the interdomain K(d) value recovered from Fig. 6was relatively low (1.43 times 10M). Similar experiments performed with affinity-labeled SCA 4-4-20 showed no changes in fluorescein anisotropy over this concentration range (data not shown).

Figure 6: Dissociation of Fv 4-4-20 domains by serial dilution as detected by fluorescein fluorescence. Affinity-liganded protein was serially diluted and fluorescein anisotropy measured at each protein concentration value (domain association being concentration-dependent). Excitation wavelength at 480 nm and emission wavelength at 530 nm. Points represent mean values of triplicate trials. Recovered K = 1.43 (± 0.17) times 10M.


DISCUSSION

In terms of structure-function relationships, recombinant Fv proteins have been invaluable tools for experimental studies of immunoglobulins. More recent endeavors involving these recombinant proteins have included their engineering with specialized effector functions for in vitro and in vivo immunodiagnostic and therapeutic roles. A common characteristic upon production of these diminutive antibody proteins is that their affinity for antigen is often reduced (or abrogated) as compared with the parental IgG. The reduced affinity has been attributed to changes in the active site structure or variable domain associative properties upon removal of the constant domains(3) . If the initial decrease in Fv affinity for antigen was due to decreased domain-domain interactions, the properties governing stable variable domain association in relation to antigen binding must be identified. As such antibody proteins continue to be modified and applied to different systems (reviewed in (67) and (68) ), the nature of this affinity decrease, including how V(L)/V(H) affinity correlates with antigen binding affinity, must be defined and exploited. The well characterized 4-4-20/fluorescein system presented an ideal method to study this phenomenon, based on the fact that SCA 4-4-20 exhibits a slight decrease in affinity for antigen compared with IgG(7) . This study addressed the question by production and characterization of the Fv analogue of the 4-4-20 active site. These studies were based on the premise that comparative analysis provided clarification of the correlation between antibody constant domains, variable domain stability, and affinity for antigen.

Using similar expression conditions for SCA, purified Fv 4-4-20 demonstrated nearly identical anti-fluorescein activity as SCA (Fig. 2A). Polyacrylamide gel analyses confirmed that the purified Fv protein contained only V(L) and V(H) domain proteins (data not shown). In terms of expression yield, E. coli cultures producing V(L) and V(H) protein consistently yielded 2-3-fold more active Fv protein than similar cultures producing SCA upon refolding and affinity purification. The fact that improper disulfide bonds could not form between variable domain proteins during expression and refolding was most likely responsible for this result(4) . In terms of idiotypy and metatypy, Fv 4-4-20 showed properties identical to SCA 4-4-20 when examined with polyclonal 4-4-20 variable domain-specific antibodies (Fig. 2, B and C). These results suggested that despite the dependence on noncovalent interactions for association, Fv 4-4-20 closely approximated the SCA molecule in terms of unliganded and liganded state structure.

Ligand binding affinities and ligand-related spectral measurements were made to assess Fv homology to the SCA molecule (in terms of the initial decrease in affinity for antigen). Such spectral measurements involving fluorescein/anti-fluorescein antibodies are characteristic of the specific anti-fluorescein active site environment which are relatively independent of affinity(69) . Fv 4-4-20 showed almost identical ligand-related spectral properties (Q(max) and (max)) and affinity for antigen relative to SCA (Table 1). Anti-metatype antibodies, both polyclonal and monoclonal, characteristically enhance the affinity for fluorescein displayed by the anti-fluorescein active site for which they are specific(15, 54) . Fluorescein affinity measurements were repeated for Fv and SCA 4-4-20 in the presence of anti-metatype reagents to assess their relationship in terms of ligand binding kinetics and liganded state conformation. Fv and SCA showed similar (proportional) increases in affinity in the presence of anti-metatype antibodies, confirming that both active site structures possess the same conformational perturbations upon ligand binding (Table 1).

In addition, CD analysis suggested identical overall secondary structures for Fv and SCA 4-4-20. Fv 4-4-20 showed the identical positive (204 nm) and negative extremum (217 and 230 nm) as SCA(36) . The negative extrema at 217 nm with a shoulder near 230 nm, found characteristically in immunoglobulin CD spectra, are typical of proteins with beta-sheet structure and a high aromatic content(70, 71) (i.e. SCA 4-4-20). Interestingly, the CD spectra of isolated variable domains consisted of negative extrema at 217 nm, indicative of beta-sheet structure, but also showed negative values at 204 nm, possibly due to a higher degree of random structure (65) (data not shown). The shoulder at 230 nm in the CD spectra of both V(L) and V(H) proteins was reduced compared with the Fv, suggesting a possible re-orientation of tryptophan and tyrosine side chains in their respective environments(37) . This would indicate that isolated domain proteins undergo dynamic secondary structure rearrangement in order to dimerize and form active Fv protein. To support this result, comparative fluorescein quenching studies were performed using affinity-purified Fv and associated V(L) and V(H) proteins. Associated protein showed almost identical fluorescein quenching properties as compared with an equal optical density solution of Fv (Fig. 3). It was also demonstrated that the liganded V(L)/V(H) dimers responded similarly to affinity-purified Fv when affinity measurements were determined in the presence of anti-metatype reagents (data not shown). Collectively, these results indicated that 1) Fv, V(L), and V(H) proteins consisted of mostly beta-sheet structure and some random coil, 2) upon V(L) and V(H) association some conformational changes are necessary for proper dimerization and active site formation, and 3) individually refolded domains maintain a dimerization competent form in the absence of constant domains which can form the proper active site environment for fluorescein binding and quenching.

As previously stated, hydrostatic pressure does not promote changes on the tertiary structure of proteins, but alters regions of secondary structure responsible for global protein conformation(38, 66) . A comparison of the pressure induced dissociation of fluorescein profiles for Fv and SCA would be a definitive evaluation of their dynamic similarity. Identical fluorescein fluorescence profiles were recovered for the two proteins when exposed to increasing hydrostatic pressure (Fig. 5). This indicated that Fv 4-4-20 displayed the same standard volume change (Delta: -50 ml/mol) upon fluorescein dissociation as SCA(38) . Seeing that their structures were apparently identical, this suggested that the Fv 4-4-20 must have increased conformational dynamics relative to the IgG molecule (Delta: -5 ml/mol) as originally postulated for SCA(15, 38, 41) . This indicated that increased dynamics were responsible for the decreased affinity for antigen displayed by Fv and SCA. Determination of the Fv interdomain dissociation constant (1.43 times 10M) showed that despite the relatively low associative affinity, the high affinity fluorescein interaction was unchanged relative to the SCA (Fig. 6). This excluded the possibility that the initial decrease in the affinity for fluorescein upon removal of the constant domains was due to decreased domain-domain stability. The large difference between V(L)/V(H) and Fv/fluorescein K(d) values (400-fold) suggested that in terms of 4-4-20, there was little or no quantitative correlation between interdomain stability and antigen affinity. Seeing that individual variable domain proteins showed no affinity for antigen (data not shown), this confirmed that there was no coupling of fluorescein binding or domain association free energy in the formation of the Fv 4-4-20(72) . Thus, Fv structural characteristics responsible for interdomain association were independent of the structural features necessary for high affinity antigen binding. Collectively, results indicated that the absence of constant domains caused increased dynamic flexibility, not reduced variable domain associative affinity, in Fv and SCA 4-4-20 and resulted in decreased affinity for antigen.

Previous studies have demonstrated that heavy chain isotype (i.e. constant domain structure) influences antibody functional affinity against multivalent antigen(73, 74) . The effect of constant domains reported in these studies, which depended on high multivalent antigen concentrations, suggested that the change in functional affinity was due to change in segmental flexibility of the IgG molecule. Antibody isotype was, however, implicated in the expression of idiotopes on the variable domains of an anti-nitrophenyl antibody MOPC 315(31) . Idiotopes represent structural markers on the antibody active site which are sensitive to conformational fluctuations due to either ligand binding or natural protein dynamics(29, 30, 75) . Such relationships would support the hypothesis that the interaction between the variable and first constant domains are necessary for proper variable domain conformational dynamics and not rigid structural features (Fig. 7). Results presented here support this hypothesis by demonstrating how the absence of constant domains influences active site/antigen interactions. In the case of 4-4-20, the binding of fluorescein can be considered a perturbation of the active site conformation which the constant domains can restrict to maintain the high affinity interaction. Removal of the constant domains from the SCA and Fv constructs resulted in the removal of this ``dynamic buffering'' effect. The ensuing increased domain dynamics translated into an increased dissociation rate of fluorescein from the active site. As studies progress on the re-engineering of antibody proteins, care must be taken to assess the importance of constant domain interactions for proper variable domain function. Methods which can both stabilize the active site structure and maintain wild type conformational dynamics may be necessary to ensure the success in producing recombinant Fv proteins which mimic parental IgG affinities.

Figure 7: Diagram of the domain-domain interactions required for proper variable domain dynamics. Cis interactions represent those interactions which involve contacts and dynamics in the vertical plane. Trans interactions represent those which involve contacts and dynamics in the horizontal plane (both C-C and V-C domain interactions).


FOOTNOTES

*
This work was supported in part by a grant from the Biotechnology Research Development Corp., Peoria, IL. Fluorescence measurements were performed at the Laboratory for Fluorescence Dynamics (LFD) at the University of Illinois at Urbana-Champaign (UIUC). The LFD is supported jointly by Division of Research Resources of the National Institutes of Health Grant RR03155-01 and the UIUC. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported in part by a fellowship from the E. I. duPont de Nemours Company.

To whom correspondence should be addressed. Tel.: 217-333-1738; Fax: 217-244-6697.

(^1)
The abbreviations used are: V(H), heavy chain variable domain; V(L), light chain variable domain; SCA, single chain antibody; mAb, monoclonal antibody; PCR, polymerase chain reaction; BSA, bovine serum albumin; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate.

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