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Multi-disciplinary studies of fluorescein-protein conjugates have led to the generation of a family of antibodies with common idiotypes and affinities for fluorescein ranging over five orders of magnitude. The high affinity 4-4-20 prototype traps the ligand in a highly complementary binding slot, which is lined by multiple aromatic side-chains. An antibody (9-40) of intermediate affinity belongs to the same idiotypic family as 4-4-20 and shares substantial amino acid identities within the VL and VH domains. To establish the structural basis for the affinity differences, we solved the crystal structure of the 9-40 Fab-fluorescein complex at a resolution of 2.3A. Similar to 4-4-20, 9-40 binds fluorescein in a tight aromatic slot with its xanthenonyl ring system accommodated by end-on insertion. However, the combined effects of the amino acid substitutions have resulted in reorganization of the binding site, with the HCDR3 loops showing the greatest differences in conformations. Access to the binding site of 9-40 is substantially more open, leaving the fluorescein's phenylcarboxylate moiety partially exposed to solvent. In addition to the usage of a different D (diversity) mini-gene encoding the HCDR3 loop, the decrease in fluorescein affinity in the 9-40 antibody family appears to be correlated with the substitution of histidine (9-40) for arginine (4-4-20) in position 34 of the antibody light chains.

Eleven individual hyperimmune rabbit polyclonal anti-fluorescein Fab fragment preparations were resolved into heterogeneous subfractions based on differential dissociation times from a specific adsorbent. Four Fab subfractions (i.e., 0.1-, 1.0-, 10-, and 100-day elutions) that differed in affinity were characterized and classified according to the extent of the bathochromic shift in the absorption properties of antibody-bound fluorescein ligand. Absorption maxima of bound fluorescein were shifted in all cases to two distinct narrow ranges, namely, 505 to 507 nm or 518 to 520 nm relative to 491 nm for free fluorescein. There was no direct correlation between the two spectral shift populations and antibody affinity, fluorescence polarization, fluorescence quenching, or fluorescence lifetimes of bound ligand. Fluorescence emission maxima varied with the bathochromic shift range. Bound fluorescein ligand, with absorption maxima of 505 to 507 nm and 518 to 520 nm showed fluorescence emission maxima of 519 to 520 nm and 535 nm, respectively. The two spectral shift ranges differed by approximately 14 to 15 nm and/or energies of approximately 1.5 kcal mol(-1) relative to each other and to the absorption maximum for free fluorescein. Spectral effects on the antibody-bound ligand were discussed relative to solvent-water studies and the atomic structure of a high-affinity liganded anti-fluorescein active site.

Fab fragments derived from ten different IgG populations of hyperimmune rabbit polyclonal anti-fluorescein antibodies were further resolved into subfractions based on differences in time-dependent dissociation from an FITC-adsorbent in the presence of 0.1 M fluorescein at 4 degrees C. Fab fragments separated into subpopulations based on specific dissociation times of 0.1 day, 1.0 day, 10 days and 100 days from the adsorbent. Finally, after the 100 days elution step incubation with 6.0 M guanidine-HCl was included to determine total protein concentration of specific anti-fluorescein Fab fragments. Yields of specifically eluted Fab fragments ranged from 12.7 to 84.1% of the total Fab population originally incubated with the adsorbent. All Fab polyclonal populations and subpopulations analyzed quenched the fluorescence of the bound ligand by 90% or greater. None of the plots of protein concentration versus percent yield of the total specific antibody obtained for each of the five resolved fractions constituting a specific polyclonal population conformed to Gaussian distributions. All resolved Fab subpopulations retained bound fluorescein ligand that exhibited significant bathochromic shifts in absorbancy. Based on the extent of the red-shift the antibodies segregated into one of two general spectral families showing either a peak shift to 505-507 nm or to 518-520 nm. The red-shift to 518-520 nm appeared unique to rabbit anti-fluorescein antibodies, since corresponding large shifts have not been observed with antibodies derived from other species (e.g. mouse, rat, chicken, etc.). K(d) values determined for the resolved fractions confirmed a continuous progression in affinity from the 0.1day through the 100 days elution. Preliminary isoelectric focusing analyses revealed progressive selection for relatively more homogeneous fractions, especially in the 100 days resolved fraction.

Anti-dsDNA autoantibodies and immune complex formation are major factors in SLE pathogenesis. Understanding stable immune complex formation is critical in deciphering mechanisms of autoimmune pathogenesis. Previous studies identified a subpopulation of murine lupus monoclonal autoantibodies that exhibited dual specificity (anti-DNA and anti-IgG2a hinge) and formed stable immune complexes [J. Mol. Rec. 10(1997)225]. Two monoclonal autoantibodies, BV 17-45 and BV 16-13, were extensively studied because of their dual specificity. To quantitatively assess the role of each specificity in the formation of stable immune complexes, studies were performed to determine binding affinities for various sized dsDNA fragments (21, 43, 84, and 114 bp) and the covalent dimer of a nine amino acid hinge peptide. Results characterizing BV 17-45 showed that the affinity for dsDNA directly correlated with increased dsDNA size. Results with BV 16-13 revealed a generally lower affinity for the various dsDNA fragments. Binding inhibition studies, using a covalently linked dimer of a nine amino acid synthetic hinge peptide as an inhibitor of the antibody-43 bp dsDNA interaction, yielded relative affinities for the anti-hinge activity. Binding affinities for the synthetic hinge specificity were lower than affinities measured for the anti-dsDNA activity. Collectively, the binding and inhibition studies provided insight into the correlation between dual specificity and avid immune complex formation. A model was proposed based on the concept that large dsDNA fragments caused localization of the dual-specific antibodies through the anti-dsDNA activity, thereby facilitating subsequent binding and cross-linkage via the anti-hinge specificity. These synergistic interactions resulted in the formation of avid immune complexes.

Extensive documentation shows that macrophage efficiently present antigen to CD 4(+) T-cells in conjunction with the MHC II molecule. Previously, a novel fluorescent probe, FITC-BSA, was developed to analyze intracellular antigen processing and presentation pathways within viable peritoneal murine macrophage. The studies revealed fluorescein's accessibility to antibody binding when associated with peptides bound within the MHC II cleft. To determine if MHC II-fluoresceinated-peptide complexes on the surface of macrophage were also sufficient to stimulate antigen-specific B-cells, nylon wool-purified splenic B-cells from FITC-KLH injected BALB/c mice (H-2(d)) were co-cultured with antigen-pulsed macrophage. B-cell stimulation and antibody production was observed in the presence of FITC-BSA-pulsed macrophage, whereas, macrophage incubated in the presence of unlabeled BSA were not stimulated. Compared with control cells, similar levels of stimulation were detected following depletion of Thy 1.2(+) cells from nylon wool-based spleen cell preparations. Stimulation was inhibited upon preincubation with anti-fluorescein IgG antibodies. Stimulation was not measurable using B-cells derived from the naive mice. The interaction was inhibited upon addition of MHC II specific antibodies and leupeptin, a microbial product that inhibits MHC II-peptide complex formation. Importantly, antibody production was not observed in the presence of antigen-pulsed macrophage from H-2(b) mice. Moreover, B-cell stimulation via this pathway was dependent upon antigen concentration as well as the cell to cell ratio.

In a previous study, we demonstrated that exhaustive exercise suppressed peritoneal macrophage antigen presentation (AP). In this study, we explored the intracellular mechanism(s) responsible for this suppression. Pathogen-free male BALB/c mice (8 +/- 2 wk) were randomly assigned to either home cage control (HCC) or exhaustive exercise stress (Exh, 18-30 m/min for 3 h/day) treatment groups. The mice underwent treatments for a period of 4 days during induced peritoneal thioglycollate inflammation. Elicited macrophages were harvested, purified, and incubated with chicken ovalbumin (C-Ova, 2. 5 and 10 mg/ml) for 18 h. After macrophages were washed, they were cocultured with C-Ova-specific T cells for 48 h at which time the supernates were harvested and analyzed via ELISA for interleukin (IL)-2 as an indication of macrophage AP. There was no significant (P > 0.05) difference in macrophage AP between cells fixed with paraformaldehyde vs. those that remained unfixed, suggesting that Exh did not affect production of soluble factors influencing macrophage AP (i.e., IL-1, IL-4, PGE(2)). The ability of macrophages to generate C-Ova immunogenic peptides was analyzed using FITC-labeled C-Ova, which shows fluorescence only when degraded intracellularly. There was a significant ( approximately 20%, P < 0. 05) suppression in fluorescence in the Exh compared with HCC, indicating a possible defect in the ability of macrophages from Exh to degrade C-Ova into immunogenic peptides. Macrophages were also incubated with C-Ova immunogenic peptide in a manner identical to that for native C-Ova. We found a similar suppression ( approximately 22-38%, P < 0.05) in macrophage AP using a C-Ova peptide when compared with native C-Ova in the Exh group, indicating reduced major histocompatibility complex (MHC) II loading and/or C-Ova-MHC II complex cell surface expression. In conclusion, these data indicate an intracellular defect in the macrophage antigen processing pathway induced by Exh.

Monoclonal anti-DNA autoantibody BV 04-01 catalyzed hydrolysis of DNA in the presence of Mg2+. Catalysis was associated with BV 04-01 IgG, Fab, and single-chain-antibody (SCA) proteins. Cleavage of both ss and dsDNA was observed with efficient hydrolysis of the C-rich region of A7C7ATATAGCGCGT2, as well as a preference for cleaving within CG-rich regions of dsDNA. Data on specificity of ssDNA hydrolysis and kinetic data obtained from wild-type SCA, and two SCA mutants were used to model the catalytically active antibody site using the previously resolved X-ray structure of BV 04-01. The resulting model suggested that the target phosphodiester bond is activated by induction of conformational strain. In addition, the antibody-DNA complex contained a Mg2+ coordination site composed of the L32Tyr and L27dHis side chains and a DNA 3'-phosphodiester group. Induction of strain along with the metal coordination could be part of the mechanism by which this antibody catalyzes DNA hydrolysis. Sequence data for BV 04-01 V(H) and V(L) genes suggested that the proposed catalytic-antibody active site was germline-encoded. This observation suggests that catalytic activity might represent an important-rarely examined-function for some antibody molecules.

A fluorescent antigen, FITC10BSA, that is sensitive to several of the biochemical processes involved in antigen processing was constructed. In combination with both flow cytometry and subcellular fractionation, the unique probe provided new details regarding the kinetics and intracellular pathways involved in antigen processing in murine macrophage. These studies suggested that macrophage utilized multiple vesicles as opposed to a few specific organelles for major histocompatibility complex (MHC) type II-peptide loading and transport. Although newly formed MHC II-peptide complexes were detected in cathepsin D-positive, lysosomal associated membrane glycoprotein (LAMP-1)-positive lysosomes, MHC II-peptide loading also occurred in transferrin receptor-positive endosomes. Interestingly, MHC II-fluoresceinated complexes were only observed in transferrin receptor-positive organelles as opposed to MHC II-unlabelled peptide complexes which were detected in traditional early lysosomal compartments. More importantly, MHC II-peptide complexes were monitored in light transferrin receptor-positive fractions following their initial appearance in dense endosomal/lysosomal fractions. Control experiments suggested that these complexes represented intermediates in the process of migrating to the cell surface through a retrograde pathway within the macrophage.

Binding properties and requirements for internalization of hapten-protein antigens, such as fluorescein-polyderivatized bovine serum albumin (FITC-BSA) and poly-D-lysine (FITC-PDL), by murine macrophage was consistent with surface receptor recognition of fluorescyl moieties (Cherukuri et al., Mol. Immunol. 34, 21-32, 1997; Cherukuri et al., Cytometry 31, 110-124, 1998). Ligand binding properties of the putative macrophage receptor pointed toward specificity for various aromatic moieties including phenylalanine, phenyloxazolone and fluorescein (Cherukuri and Voss, Mol. Immunol. 35, 115-125, 1998). Purification of the hapten-recognizing receptor from J774 macrophage cells involved subcellular fractionation of plasma membrane fractions, and affinity chromatography of solubilized membranes employing a phenyl-Sepharose adsorbent with subsequent specific elution of receptor using fluorescein ligand. The final product was a protein with a molecular mass of approximately 180 kDa. Characterization of the purified receptor involved absorption and fluorescence spectroscopy, circular dichroism, fluorescence quenching analyses, various ligand binding assays and an immunological analysis. Spectroscopic analyses revealed that the receptor possessed aromatic amino acids while circular dichroism suggested significant alpha-helical secondary structure. Binding specificity of the purified receptor was confirmed in a spectrofluorometric assay where the fluorescence of fluorescein ligand was quenched approximately 97%. Finally, specific binding activity of the receptor with FITC-BSA was demonstrated in Western blot analysis under native conditions. Receptor purity was confirmed in amino acid sequencing analysis when the amino-terminal residue was found to be totally blocked. Results are discussed in terms of the possible identity of the isolated macrophage receptor and its biological-immunological role., (Copyright 1999 John Wiley & Sons, Ltd.)

The anti-IgG properties of two dual-specific (anti-dsDNA and anti-IgG) monoclonal NZB/NZW F1-derived autoantibodies, BV 17-45 and BV 16-13, were studied to resolve the location and possible commonality of the IgG epitope. To determine if BV 17-45 and BV 16-13 recognized the same IgG epitope, the relative temperature sensitivity of the conformational IgG epitopes were evaluated using the conformational sensitive immunoassay. Comparison of the temperature sensitivity of the conformational immunoglobulin epitopes over a temperature range of 25-100 degrees C suggested that the epitope recognized by BV 17-45 was the same as the IgG epitope recognized by BV 16-13. Further studies with papain- and pepsin-generated F(ab')2, Fab, and Fc fragments of BV 17-45 and BV 16-13 revealed that the dual-specific autoantibodies BV 17-45 and BV 16-13 both bound an epitope in the hinge region of the IgG molecule. The potential correlation between these studies and the pathogenic nature of dual-specific autoantibodies is discussed.

A receptor possessing specificity for fluorescein was previously identified on murine macrophage. The goal of the present study was to determine if this receptor influenced MHC II-peptide loading and surface expression of a hapten-protein conjugate within murine macrophage. Although inhibition of fluid-phase pinocytosis had no detectable effect, lower levels of intracellular MHC II-peptide complexes were observed upon inhibition of receptor-mediated endocytosis. Moreover, lower levels of surface expressed MHC II-fluoresceinated peptide complexes were also detected. Following subcellular fractionation experiments, it was revealed that the receptor altered the endocytic trafficking of the antigen within the cell. Namely, degraded antigen and MHC II-peptide complexes were not observed in dense transferrin receptor positive, cathepsin D positive, LAMP-1 positive organelles upon inhibition of the receptor. Previous studies also suggested that this receptor enhanced MHC II-peptide loading by concentrating high levels of antigen to endocytic organelles. The implications of these findings on subsequent development of the immune response were also discussed.

Although fluorescein is a widely used fluorescent probe in the biosciences, the effect of solvent environment on its spectral properties is poorly understood. In this paper we explore the use of fluorescein as a probe of the state of hydrogen bonding in its local environment. This application is based on the observation, originally made by Martin (Chem. Phys. Lett. 35, 105-111, 1975), that the absorption maximum of fluorescein undergoes substantial shifts in organic solvents related to the hydrogen bonding power of the solvents. We have extended this work by studying the spectral properties of the dianion form of the probe in solvent-water mixtures. We show that the magnitude of the shift correlates with the alpha and beta parameters of Kamlet and Taft (J. Am. Chem. Soc. 98, 377-383; 2886-2894, 1976), which provide a scale of the hydrogen bond donor acidities and acceptor basicities, respectively, of the solvents. In solvent-water mixtures, these shifts reflect general effects of the solvents on the hydrogen bonding environment of the fluorescein through water-solvent hydrogen bonding and specific effects due to fluorescein-solvent hydrogen bonding. Indeed, both the absorption and fluorescence properties appear to be dominated by these effects indicating that the spectral shifts of the dianion can be used as an indicator of its hydrogen bonding environment. We discuss the application of fluorescein as a probe of hydrogen bonding in the microenvironment immediately surrounding the fluorophore, and we illustrate the effect with reference to the fluorescein-antifluorescein antibody complex where it appears that antibodies selected during the immune response possess binding sites that are increasingly dehydrated and hydrophobic.

Secondary forces have been defined as those interactions between antibody and antigen that occur external to the antibody active site. Previous investigations indicated that non-active-site secondary interactions can modulate immune complex stability and may influence antibody variable domain conformation and/or dynamics. To assess secondary force-induced perturbations of monoclonal antibodies 4-4-20 and 9-40 a series of monofluoresceinated peptides was reacted and the various interactions analyzed by circular dichroism (CD). The mAbs 4-4-20 and 9-40 vary by nearly 1000-fold in their respective affinities for the fluorescein ligand, yet both immunoglobulins are highly related at the primary structural (idiotype) level. Near-UV CD spectra were evaluated as well as the induced optical activity (visible CD) of the antibody-bound fluorescein moiety when covalently attached to various peptide carriers. Comparative spectral studies revealed significant differences in the near-UV CD spectra of mAbs 9-40 and 4-4-20 relative to the various peptide antigens and to one another. CD spectra were interpreted as reflecting differential secondary force-induced perturbations of the antibody variable domains as well as intrinsic differences between the two mAbs.

A novel fluorescent hapten-protein conjugate was constructed to monitor the events required for CD 4+ lymphocyte recognition of antigenic proteins. Previous studies utilizing the probe demonstrated that the hapten-protein was localized to an acidic endocytic compartment within the macrophage and that the hapten-protein was sensitive to multiple intracellular events including enzymatic degradation, acidification, and disulfide bond reduction. More importantly, recent experiments indicated that efficient internalization of the probe was dependent upon specific recognition of the hapten. Therefore, the present report addressed the effect of receptor-mediated endocytosis upon the processing of the hapten-protein within murine peritoneal macrophage. These studies determined that the rate of endocytosis was significantly faster than the rate of exocytosis. Specifically, the rate of exocytosis was estimated to be 3.4 x 10(4)s-1 based on a unimolecular rate constant. Although at higher concentrations, a slightly slower rate was observed (1.9 x 10(4)s-1). This study also represented one of the first efforts to measure the intracellular concentration effect typically associated with receptor-mediated endocytosis. Experiments involving a radioactively labeled hapten-protein conjugate revealed that the probe was at 100-fold higher concentration within the endocytic vesicles when compared to the extracellular media. The intracellular mechanism involved in this phenomenon was discussed as well as the implications of these findings upon MHC II-peptide binding.

A series of fluorescein derivatized poly-D-lysine (FITC-PDL) probes were used to elucidate the role of fluorescein in receptor binding of fluorescein-conjugated macromolecules to J774 murine macrophages. Poly-D-lysine served to eliminate receptor recognition of the carrier due to the biologically inert nature of the D-isomer. This concept enabled the focused investigation of the role played by fluorescein in receptor recognition, binding and internalization. Results revealed dependency of cellular uptake on polymer concentration, hapten density and accessibility. The results differed from those previously obtained with FITC-BSA in that saturating fluorescein densities on the poly-D-lysine polymer resulted in diminished rates of uptake by macrophages. Receptor-mediated endocytosis via clathrin-coated pits was concluded based on results that showed inhibition of FITC-PDL uptake by intracellular K+ depletion but not by the macropinocytosis inhibitor, amiloride. Further, FITC-PDL was found to inhibit the endocytic uptake of FITC-BSA suggesting competition between the two probes at the level of a macrophage receptor. Association rates (kon) for binding to the macrophage surface were measured for the various FITC-PDL probes based on fractional receptor occupancies. Results are discussed on the basis of receptor recognition of fluorescein in J774 macrophages and the requirements for this recognition which include appropriate spacing and accessibility of the hapten moieties to facilitate receptor crosslinking.

The effects of secondary forces on monoclonal IgM anti-fluorescein antibody 18-2-3 reactivity were investigated and the results correlated with similar studies characterizing anti-fluorescein mAbs 4-4-20 and 9-40. mAb 18-2-3 was considered an important model for further elucidation of secondary forces since it possessed ligand binding properties similar to mAb 4-4-20, such as a similar affinity, but due to a very different primary structure it was idiotypically and metatypically distinct. mAb 18-2-3 also possessed cryoglobulin (anti-Ig) and extensive cross-reactive properties (e.g. anti-phenyloxazolone) suggestive of an atypical anti-fluorescein active site. The reactivity of mAb 18-2-3 with model fluorescein-peptides was modulated by secondary forces in a manner that differed from both mAbs 4-4-20 and 9-40. Thus, the effects of secondary forces seemed to vary with each monoclonal antibody even though each of the immunoglobulins studied were specific for the same homologous ligand. Results indicated that secondary forces impacted immune complex stability, variable domain conformation and protein dynamics. Models were postulated to account for secondary effects on the mAb 18-2-3 active site relative to mAbs 4-4-20 and 9-40. Levels of hydration, active site architecture and local amino acid dynamics were among the models cited.

A series of compounds were tested as inhibitors of receptor-binding and uptake of fluorescein-derivatized antigens in primary peritoneal and J774 murine macrophage. Results were analysed with regard to the inhibitory potency of the tested ligands and revealed that the putative cell surface receptor preferentially recognized and bound aromatic structures containing phenyl rings. L-phenylalanine was found to be the most potent inhibitor among the ligands tested. Significant inhibition of FITC10BSA binding to macrophage was observed even at 10(-9) M concentration of specific monovalent ligands tested indicating that these ligands were bound by the putative macrophage receptor with high apparent affinity. Structural comparisons of the various inhibitors employed, demonstrated that accessible, unconjugated phenyl rings were bound by the putative receptor with high apparent affinity whereas non-phenyl derivatives were bound with either low apparent affinity or via non-specific interactions. Therefore, the fluorescein hapten appeared to utilize a receptor with specificity for an essential aromatic amino acid for gaining entry into the endocytic pathway of murine macrophage. Finally, the binding of the hapten was enhanced when polyvalent fluorescein-derivatized antigens were used as a result of receptor crosslinkage on the cell surface.

Monoclonal anti-DNA autoantibody BV 04-01 catalyzed hydrolysis of DNA in the presence of Mg2+ ions. DNA hydrolyzing activity was associated with BV 04-01 IgG, Fab, and SCA 04-01 proteins. Pronounced cleavage specificity for both ss and dsDNA was observed with efficient hydrolysis of the C-rich region of the oligonucleotide A7C7ATATAGCGCGT7 as well as preference for cleavage within CG-rich regions of double-stranded DNA. Data on specificity of ssDNA hydrolysis and kinetic data obtained from wild-type SCA 04-01 and two SCA 04-01 mutants (L32Phe and L27dHis) were used to model the catalytically active antibody site utilizing the previously resolved X-ray structure of (dT)3 liganded Fab 04-01. The resulting model suggested that BV 04-01 activates the target phosphodiester bond by induction of conformational strain. In addition, the antibody-DNA complex contained a potential Mg2+ ion coordination site composed of the L32Tyr and L27dHis amino acid side chains and a DNA 3'-phosphodiester group. Induction of strain and metal coordination could be constituents of a mechanism by which this antibody catalyzed DNA hydrolysis. Sequence data for BV 04-01 VH and VL genes suggested that the proposed catalytic antibody active site was germ-line encoded. This observation suggests the hypothesis that catalytic activity might represent an important but unspecified function of some antibody molecules.

Biochemical interactions that occur external to the antibody active site have been termed secondary forces. Secondary forces are supplemental to interactions within the antibody active site (i.e., primary interactions) and can affect ligand binding efficiency as well as variable domain conformation. The antifluorescein antibody system has been determined to be a superior method for delineating primary from secondary interactive components due to the active site-filling properties of the fluorescyl ligand. To date, all studies of secondary forces within the context of the antifluorescein system have been with the high-affinity monoclonal antibody 4-4-20 (mAb 4-4-20) (Mummert & Voss, 1995, 1996, 1997). In order to determine the generality of experimental observations and proposed models, we investigated the effects of secondary forces on the antifluorescein mAb 9-40. In addition to assessing the results of former studies, mAb 9-40 possesses properties unique from those of mAb 4-4-20, namely, a decreased affinity for fluorescein and increased conformational dynamics relative to mAb 4-4-20 (Carrero & Voss, 1996). Results of fluorescein and intrinsic mAb 9-40 tryptophan quenching as well as differential scanning calorimetric (DSC) studies indicated that secondary forces modulated the conformational (metatypic) state in accordance with previous investigations with mAb 4-4-20. Unlike mAb 4-4-20, mAb 9-40 did not exhibit altered ligand binding efficiency due to the inclusion of secondary interactive components. Models were developed that proposed that the increased malleability of mAb 9-40 variable domains could account for functional differences in properties between mAb 9-40 and mAb 4-4-20.

Since dual specificity at the antibody active-site level involves new principles relative to monospecific antigen-antibody interactions and may be a general property of autoantibodies, it was important to further characterize such antibodies. Four lupus derived autoantibodies were studied to understand parameters and mechanisms involved in the participation of dual-specific antibody molecules in the formation of highly stable immune complexes. Because the dual-specific binding properties of selected lupus-related murine autoantibodies had been previously described using a solid-phase polystyrene-based ELISA, a conformational sensitive membrane based assay (CSI) was used on a comparative basis to further characterize NZB/NZW F1 murine monoclonal anti-DNA autoantibodies BV 04-01 (anti-ssDNA), BV 16-19 (anti-ssDNA), BV 17-45 (anti-dsDNA), and BV 16-13 (anti-dsDNA). All four monoclonal autoantibodies exhibited anti-IgG binding in the solid-phase ELISA. However in the CSI assay, only anti-dsDNA monoclonal autoantibodies BV 17-45 and BV 16-13 demonstrated anti-IgG binding, while anti-ssDNA autoantibodies BV 04-01 and BV 16-19 did not. Upon subjection to time-dependent thermal denaturation, with and without thiol reduction at 100 degrees C in the CSI, the self-binding activities of BV 17-45 and BV 16-13 were abrogated demonstrating that the recognized IgG autoepitope(s) possessed conformational or discontinuous three-dimensional properties. The immunological implications of dual specificity are discussed on a structure-function basis and its correlation with formation of pathogenic immune complexes.

Previous studies indicated that fluorescein derivatized bovine serum albumin was an ideal probe to monitor the time-dependent kinetics of antigen processing in the murine macrophage cell line J774. Whereas previous work focused on fluorescence intensity measurements, the present study relied on fluorescence polarization to dissect the local environment of the fluorescent hapten-protein within the endocytic system of the cell. A steady increase in both fluorescence intensity and fluorescence polarization of the cell population was detected for the first 100 min. However, at 100 min, a plateau in both fluorescence intensity and polarization was observed and was followed by a decrease in fluorescence polarization and a corresponding increase in fluorescence intensity. Western blot analyses revealed that the decrease in fluorescence polarization was due to proteolytic degradation of the probe within the cell. Using a combination of in vitro experiments and an additional fluorescent probe, it was determined that the initial increase in fluorescence polarization was due to movement of the probe through a pH gradient within the cell, suggestive of transport through the endocytic system. By combining fluorescence polarization, flow cytometry, and a unique fluorescent enhancement substrate, these studies represented a novel approach for monitoring intracellular trafficking and processing of proteins within macrophages.

Two-photon fluorescence lifetime imaging microscopy was used noninvasively to monitor a fluorescent antigen during macrophage-mediated endocytosis, intracellular vacuolar encapsulation, and protease-dependent processing. Fluorescein-conjugated bovine serum albumin (FITC-BSA) served as the soluble exogenous antigen. As a relatively nonfluorescent probe in the native state, the antigen was designed to reflect sequential intracellular antigen processing events through time-dependent changes in fluorescence properties. Using two-photon lifetime imaging microscopy, antigen processing events were monitored continuously for several hours. During this time, the initial fluorescein fluorescence lifetime of 0.5 ns increased to approximately 3.0 ns. Control experiments using fluorescein conjugated poly-L-lysine and poly-D-lysine demonstrated that the increase in fluorescence parameters observed with FITC-BSA were due to intracellular proteolysis since addition of the inert D-isomer did not promote an increase in fluorescence lifetime or intensity. Comparisons of intravacuolar and extracellular FITC-dextran concentration suggested active localization of dextran in the vacuoles by the macrophage. In addition, the kinetics of degradation observed using two-photon microscopy were similar to results obtained on the flow cytometer, thus validating the use of flow cytometry for future studies.

Fluorescein-derivatized bovine serum albumin (FITC-BSA) was used as an exogenous antigen and fluorescent probe to measure the kinetics of antigen uptake into the endocytic pathway of murine macrophage, J774, using flow cytometry. Results revealed dependency of the rate of antigen uptake on epitope density (moles FITC/mole BSA) implicating a role for FITC in the endocytosis of the derivatized antigen. In addition, inhibition of clathrin-coated pit formation in macrophage resulted in significantly reduced uptake of differentially labeled FITC BSA probes indicating receptor-mediated endocytosis via clathrin-coated pits. Fluoresceinamine (I) was found to inhibit the endocytic uptake of FITC-BSA at 10(-6) M. Determination of fractional receptor occupancies in macrophage upon binding different FITC BSA probes and calculation of the corresponding association rates (k(on)) for these binding events yielded values of 4.2+/-0.2 x 10(6)/M/min for FITC5BSA and 1.9+/-0.1 x 10(7)/M/min for FITC22BSA, respectively, at 37 degrees C. The five-fold difference in the rates of binding and endocytosis between the two probes was discussed on the basis of receptor cross-linking by a multivalent ligand (FITC22BSA), in contrast to monovalent ligand binding, on the cell surface that would lead to more rapid and efficient internalization of the FITC22BSA antigen.

Biochemical interactions occurring external to the antibody active site or pocket (i.e. secondary forces) that directly effect ligand binding efficiency, and the microenvironment-sensitive spectral properties of bound homologous ligand, residing within the active site of high affinity monoclonal antifluorescyl antibody (mAb) 4-4-20, have been previously reported. This study describes the synthesis and characterization of a series of specially designed and chemically distinct mono-fluoresceinated peptides of equal size (13-mer) as well as the changes in the spectral properties and free energy in the binding of each fluorescein derivatized peptide, upon interaction with mAb 4-4-20. Significant differences in binding efficiency and fluorescence quenching of the ligand, as well as the intrinsic tryptophan fluorescence, were observed for each monofluoresceinated peptide relative to one another and fluorescein ligand. In addition to the effects on the fluorescence quenching of fluorescein and intrinsic tryptophan residues, and the free energy of binding, the conformation of the variable domains of mAb 4-4-20 upon interaction with the fluoresceinated peptides was probed with polyclonal antimetatype (conformational dependent anti-liganded state) antibodies. Studies comparing the results of a solid-phase inhibition assay, with the binding of antimetatype antibodies in solution, suggested that variant metatypic states of mAb 4-4-20 resulted from binding of the various fluorescein derivatized peptides. Depiction of the mAb 4-4-20 active site as a series of thermally averaged substates is proposed as a model and framework to interpret further the results. It was concluded that secondary forces can dictate conformer selection from the various substates. thereby modulating the primary antibody ligand interaction.

Secondary forces, defined as those interactions between the antigen (epitope including the surrounding environment) and areas immediately adjacent to the antibody active site, were investigated using monofluorescein-derivatized synthetic peptides of varying electrostatic properties. Secondary forces were quantitated by measuring the unimolecular rate constants at two different temperatures using the high-affinity anti-fluorescein monoclonal antibody 4-4-20 complexed with fluorescein-derivatized synthetic peptides. Unimolecular rate constants were correlated with transition-state theory to explain secondary effects. An acidic peptide produced a large temperature-dependent effect upon binding including a significant enthalpic factor (+33.28 kcal/mol) relative to the binding of fluorescein ligand (+23.96 kcal/mol). Binding of a basic peptide produced both a relatively smaller temperature effect and enthalpy factor than fluorescein ligand. The antibody-ligand binding results were interpreted invoking the concepts of thermally averaged metatypic (liganded) states of the antibody as well as potential biochemical interactions between the antigen and accessible surface regions of the antibody's complementarity determining regions.

Anti-metatype (anti-Met) antibodies are immunoglobulins that specifically recognize and stabilize antibodies in their liganded or metatypic state, but lack specificity for either the hapten or the unliganded antibody. Autologous anti-Met antibodies were previously observed in vivo, suggesting that a metatypic autoantibody response could play a physiological role in the immune network, e.g. controlling the clearance of immune complexes from circulation. The first elicited anti-Met antibodies were against the fluorescein-liganded high affinity murine anti-fluorescein monoclonal antibody 4-4-20. The fluorescein-hapten system has proved to be an invaluable tool for both the recognition and characterization of the metatypic response by utilization of its spectral properties. In this investigation, hydrostatic pressure measurements, in conjunction with fluorescence spectroscopy, were performed on the recombinant Fv derivative (Fv 4-4-20) of the high affinity anti-fluorescein monoclonal antibody 4-4-20 complexed to anti-Met antibodies to study the influence of anti-Met antibodies of Fv 4-4-20 intervariable domain interactions. Anti-Met antibodies bound to liganded Fv 4-4-20 were observed to cause a change in the fluorescence properties of fluorescein that was not observed when anti-Met antibodies were bound to the liganded parent immunoglobulin. The variation of these spectral properties upon addition of anti-Met antibodies was shown to be correlated with dissociation of the variable domains in Fv 4-4-20 in response to its interaction with the anti-Met antibody. The ability to cause variable domain dissociation was dependent on whether monoclonal or polyclonal anti-Met antibodies were bound to the metatype. A model was proposed that elucidated the interaction of anti-Met antibodies, polyclonal and monoclonal, with variable domains of the primary anti-antigen antibody.

Studies are described regarding generation of anti-hapten antibodies starting with a monoclonal Ig immunogen in the ligand-induced conformation or metatypic state. Liganded monoclonal Ab1 antibodies represent the unique feature of the study since previous reports investigating internal imaging in the original Idiotype Network Hypothesis [Jerne, 1974 (Ann. Immun. 125C, 373-389)] were based on the non-liganded or idiotypic state [as reviewed in: Rodkey, 1980 (Microbiol. Rev. 44, 631-659); Kohler et al., 1979 (In: Methods in Enzymology: Antibodies, Antigens and Molecular Mimicry, pp. 3-35); Greenspan and Bona, 1993 (FASEB J. 7,437-444)]. Affinity-labeled liganded murine monoclonal anti-fluorescein antibodies served as immunogens administered both in the syngenic and xenogenic modes to determine if the metatypic state elicited anti-hapten antibodies through imaging-like mechanisms. Polyclonal and monoclonal anti-Ab1 reagents in various hosts were assayed for anti-fluorescein and/or anti-metatype specificity. Significant anti-fluorescein responses were measured indicating that the metatypic state directly or indirectly stimulates an anti-hapten antibody population.

In a comparative study, the thermodynamic parameter, DeltaV, was obtained using hydrostatic pressure-induced dissociation of fluorescein (Fl) from the active site of monoclonal antibody (mAb) 9-40 and its mutant and native derivatives equilibrated at six pH values (8.0, 7.5, 7.0, 6.5, 6.0, and 5.5) and four temperatures (35, 25, 15, and 5 degrees C). mAb 9-40 and its Fab and single-chain Fv (scFv) derivatives at pH 8.0 were found to have identical Fl dissociation behavior under pressure as a function of temperature. The pressure dissociation at 25 degrees C as a function of pH showed a sigmoidal dependence of DeltaV with a midpoint value at pH 7.4 for mAb 9-40. Comparison of experimental results for scFv 9-40/212 with its mutant scFv 9-40/212Arg-34L indicated that the pH dependence of mAb 9-40 was due to the titration of His-34L in the active site. Iodide quenching of bound Fl showed that the hapten in this active site was solvent accessible. Imperfect packing, which leads to increased conformational dynamics, was determined as a possible cause of the low affinity for mAb 9-40.

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 (VH) and variable light (VL) 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 (Kd) for the VL/VH association (1.43 x 10(-7) M), 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 (Kd, 2.9 x 10(-10) M). 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 CH1 and CL constant domains. Collectively, these results verify the importance of constant domain interactions in antibody variable domain structure-function analyses and future antibody engineering endeavors.

Bovine serum albumin (BSA) highly derivatized with fluorescein isothiocyanate (FITC, isomer I) served as a fluorescent enhancement substrate to measure protease activity. In the native globular BSA structure, the fluorescence of the lysine-conjugated fluorescein moieties was quenched 98%. Proteolytic digestion of highly derivatized BSA with Pronase resulted in fluorescence enhancement of 4300%. Both alpha-chymotrypsin and proteinase K yielded lower but similar fluorescence enhancement values of 2880% and 2800%, respectively. Digestion of the fluorescein-BSA substrate with trypsin, which required basic amino acids for activity, showed fluorescence enhancement of 1480% reflecting the fluorescein-lysine thiocarbamyl linkage. When derivatized substrate was pretreated with a thiol-reducing agent prior to incubation with proteases, a relatively small increase in fluorescence was noted relative to the untreated substrate except in the case of Pronase. The minimum sensitivity of proteolytic activity, based on a comparison of untreated and reduced FITC25BSA was 32 x 10(-6) units for I ng proteinase K, 1 x 10(-3) units for 1 ng alpha-chymotrypsin and 10 x 10(-3) units for Pronase and trypsin (1 ng each). The fluorescence enhancement assay was suited for sensitive intensity measurements or as an endpoint assay to detect protease activity.

To elucidate time-dependent pathways and mechanisms involved in antigen processing, a fluorescent probe suitable to monitor several steps within this pathway was developed. Previous studies utilizing two-photon fluorescence microscopy with time resolved and intensity imaging demonstrated that the probe, fluorescein derivatized BSA, was localized to the endocytic system and degraded over an extended period of time. However, an additional method, flow cytometry, was required to monitor the kinetics of these intracellular events and to better assess the total cell population. Flow cytometric studies indicated that the antigen entered an acidic intracellular environment consistent with the endocytic system of the macrophage. Additional experiments suggested that minimal proteolytic degradation began 10 min after addition of the antigenic probe while extensive enzymatic degradation did not occur until 180-200 min. Inhibitor studies indicated that degradation of the probe was dependent upon both acidic pH and ATP synthesis as well as all four classes of proteases. Experiments involving specific protease inhibitors also revealed that various classes of proteases were active at different time points throughout the processing of the probe. By combining these results with additional kinetic data, a model for the sequence of events involved in the processing of FITC10BSA was proposed. More importantly, these studies represented some of the first time-dependent kinetic measurements of antigen processing in living cells.

General localization of metatypic determinants recognized by polyclonal anti-metatype antibodies relative to the antibody active site of the high-affinity anti-fluorescein monoclonal antibody 4-4-20 was achieved through use of a unique bifluorescent-ligand probe. The fluorescent probe possessed intrinsic energy-transfer properties with the fluorescein hapten serving as the energy acceptor. The donor group 5-(2-iodoacetyl) aminoethylaminonaphthalene-1-sulfonic acid (IAEDANS) proved environmentally sensitive both to binding of the FITC-cys-AEDANS ligand and to subsequent anti-metatype antibody interactions involving the antibody variable domains of 4-4-20. Spectral changes in ligand-conjugated AEDANS upon specific reactivity of the antibody with FITC suggested secondary interactions between AEDANS and the topological protein surface adjacent to the 4-4-20 active site. Results indicated that some anti-metatype antibodies (Fab fragments) within the polyclonal population bound to sites immediately surrounding the liganded active site and perturbed the interactions of AEDANS with topological sites. The results are discussed in terms of the types of interactions that may occur between the AEDANS moiety and the 4-4-20 antibody protein surface and subsequent perturbation of those interactions by anti-metatype antibodies.

Ongoing Ab responses to a T cell-dependent Ag can be suppressed in hyperimmune animals by exogenous, multivalent Ag arrays. The pharmacologic basis for this suppression was studied by varying the molecular mass, ligand valence, and dose of Ag arrays, and then determining their efficacy, pharmacokinetics, and tissue distribution. Arrays ranging in molecular mass from 30 to 500 kDa caused initial clearance of specific serum Abs, but only the smaller arrays caused persistent suppression despite their relatively lower binding avidity and shorter retention in vivo. Suppression by the smaller arrays at lower doses was biphasic, implying two distinct modes of Ab elimination. High affinity IgG was eliminated preferentially, as shown by calibrated variable ligand-density ELISA. Suppressive arrays were localized discretely in the splenic germinal centers of hyperimmune animals. These results indicate that Ag array mass, ligand valence, and dose all play critical roles, and histologic compartmentalization may also be a pertinent parameter, in determining suppressive efficacy in vivo.

Investigations into the intrinsic affinity and reactivity of autoanti-DNA active sites were initiated through the use of purified monoclonal IgG and the synthesis of single-chain Fv derivatives of murine monoclonal anti-DNA autoantibodies BV 04-01 and BV 17-45. Results showed that relative to the respective IgG hybridomas, only the BV 04-01 SCA derivative showed demonstrable reactivity with DNA. The monovalent single-chain derivative of BV 17-45 showed no reactivity with DNA in solution or solid-phase assays, even though the parental IgG had been previously described as high affinity. However, 17-45 displayed reactivity as a bivalent single-chain derivative. In addition, upon concentration, BV 17-45 IgG formed a highly stable, papain-resistant precipitate. Investigations into the nature of the precipitate revealed that BV 17-45 possessed significant, DNA-inhibitable autobinding to its own IgG molecule. BV 04-01 also possessed similar anti-self reactivity. Thus, both monoclonal autoantibodies examined in this study possessed dual binding specificity; anti-DNa and anti-self.

The anti-immunoglobulin reactivity of two monoclonal, dual specific, autoantibodies, BV 17-45 and BV 04-01 was examined. The current study further defined the anti-immunoglobulin autoreactivity of these MoAbs to be Fc-specific. Both BV 17-45 and BV 04-01 bound their own Fc domains in addition to Fc regions of other MoAbs of similar isotype with varying levels of activity. The different anti-Fc reactivity patterns of BV 17-45 and BV 04-01 suggested that these MoAbs recognized distinct epitopes. Neither BV 17-45 nor BV 04-01 bound Fab fragments or single-chain antibody derivatives, which confirmed that the anti-immunoglobulin reactivity of these autoantibodies was Fc-specific. In addition, abrogation of anti-Fc reactivity was observed when affinity-labelled MoAbs were used as coating antigens in solid-phase ELISAs. These results implied that active-site ligand binding induced conformational changes which altered the Fc epitope(s) recognized by BV 17-45 and BV 04-01.

This paper reports the complete V gamma, V kappa, C gamma 1 and C kappa nucleotide and deduced amino acid sequences of two hamster monoclonal anti-metatype antibodies, 3A5-1 and 4A6. These antibodies have been previously characterized in terms of their binding and molecular stabilization properties with liganded murine monoclonal and single-chain antibody 4-4-20 active sites. Also reported are the complete V kappa and C kappa nucleotide and deduced amino acid sequence of hamster monoclonal anti-idiotype antibody 1F4, which is specific for the unliganded 4-4-20 active site. Oligonucleotide primers based on the 5' ends of murine variable genes, along with primers specific for murine IgG C gamma 1 and kappa constant region genes, have been used in cDNA and polymerase chain reactions (PCRs) to amplify IgG cDNA from Armenian hamster/mouse hybridomas. The hamster C gamma 1 and C kappa domain sequences are highly homologous to previously reported murine sequences. The anti-idiotype mAb V kappa gene demonstrated strong similarity to the murine V kappa V gene subgroup while the two anti-metatype mAb V kappa genes approximated more closely to the murine V kappa III gene subgroup. The two anti-metatype mAbs utilized highly homologous V gamma genes, with differing HCDR 3 regions, that appeared similar to the murine V gamma I(a) subgroup. These sequence determinations represent the first primary structures reported for antibodies with anti-metatype activity and are additions to the relatively sparse hamster immunoglobulin genetic database. Results are discussed in terms of 4-4-20 active site specificity and anti-metatype activity, as well as immunoglobulin structural diversity in an anti-Ig immune response.

The crystal complex of fluorescein bound to the high-affinity anti-fluorescein 4-4-20 Fab (Ka = 10(10) M-1 at 2 degrees C) has been determined at 1.85 A. Isomorphous crystals of two isoelectric forms (pI = 7.5 and 7.9) of the anti-fluorescein 4-4-20 Fab, an IgG2A [Gibson et al. (1988) Proteins: Struct. Funct. Genet., 3, 155-160], have been grown. Both complexes crystallize with one molecule in the asymmetric unit in space group P1, with a = 42.75 A, b = 43.87 A, c = 58.17 A, alpha = 95.15 degrees, beta = 86.85 degrees and gamma = 98.01 degrees. The final structure has an R value of 0.188 at 1.85 A resolution. Interactions between bound fluorescein, the complementarity-determining regions (CDRs) of the Fab and the active-site mutants of the 4-4-20 single-chain Fv will be discussed. Differences were found between the structure reported here and the previously reported 2.7 A 4-4-20 Fab structure [Herron et al. (1989) Proteins: Struct. Funct. Genet., 5, 271-280]. Our structure determination was based on 26,328 unique reflections--four times the amount of data used in the previous report. Differences in the two structures could be explained by differences in interpreting the electron density maps at the various resolutions. The r.m.s. deviations between the variable and constant domains of the two structures were 0.77 and 1.54 A, respectively. Four regions of the light chain and four regions of the heavy chain had r.m.s. backbone deviations of > 4 A. The most significant of these was the conformation of the light chain CDR 1.

The ability of antibodies to specifically select and stabilize through binding one or more isomers of highly dynamic ligands remains a relatively unexplored immunochemical problem. The experimental strategy employed in this study was to elicit homogeneous antibodies to polyaromatic fluorescein which exists in one isomeric form. The binding properties of a monoclonal rat antifluorescein antibody specific to a given isomer were quantitatively studied to determine the capacity to bind dynamic analogues of fluorescein which exists in multiple isomers. To generate monoclonal anti-fluorescein antibodies that reacted with specific dynamic analogues of fluorescein possessing unconjugated aromatic ring systems, immune spleenocytes from Lou/M rats immunized with FITC(I)-KLH were fused with Balb/c SP2/0-Ag14 murine myeloma cells forming rat-mouse hybridomas. Cell line P2A12-1-C8 was selected for further characterization from the original 23 stable rat hybrids, since it produced a monoclonal antibody with a binding affinity 2.0 x 10(10)/M for fluorescein based on dissociation rate measurements. P2A12-1-C8 exhibited significant reactivity with HPF and phenol red, which are dynamic structural analogues of the homologous fluorescein ligand. No reactivity was demonstrated with phenolphthalein, which based on relative chemical structures was expected to be more reactive than phenol red. Computer-based molecular modeling and energy minimization studies of fluorescein, HPF, phenol red, and phenolphthalein showed that in terms of the most energetically favorable orientation of the three aromatic rings, phenol red more closely simulated fluorescein than phenolphthalein. The results were analyzed in terms of the mechanisms of dynamic ligand stabilization and binding involving accommodation of specific ligand isomers by energetically permissible conformational states exhibited by an antibody active site. Thus, antibody reactivity of an anti-fluorescein antibody with phenol red and phenolphthalein was dictated more by ligand dynamics and aromatic orientation than by chemical structure similarities.

Stability measurements of SCA 04-01/212 (anti-ssDNA) which possesses a groove-shaped active site were performed by Gdn-HCl-induced unfolding, analyzed assuming a simple two-state equilibrium, and expressed as the free energy of unfolding, delta Gn-u. A delta Gn-u of 1.44 +/- 0.13 kcal/mol was determined experimentally for SCA 04-01/212. In addition, the conformational stabilities of HCDR transplants, hybrid antibody molecules resulting from the transplantation of HCDRs from SCA 4-4-20 (anti-fluorescein) into the corresponding regions of 04-01 in all combinations, were determined using the identical protocol applied to SCA 04-01. On the basis of the results of these stability experiments, the HCDR transplants were categorized into three groups, representing low, intermediate, and high stability. Data were discussed in terms of the relationships between structure-function and conformational stability pertaining to the groove-shaped antibody active site of SCA 04-01/212 and the pocket-shaped active site of SCA 4-4-20/212.

Three-dimensional structures were determined for three crystal forms of the antigen binding fragment (Fab) of anti-fluorescein antibody 4-4-20 in complex with fluorescein. These included 1) a triclinic (P1) form crystallized in 47% (v/v) 2-methyl-2,4-pentanediol (MPD); 2) a triclinic (P1) form crystallized in 16% (w/v) poly(ethylene glycol), molecular weight 3350 (PEG); and 3) a monoclinic (P21) form crystallized in 16% PEG. Solvent molecules were added to the three models and the structures were refined to their diffraction limits (1.75-A, 1.78-A, and 2.49-A resolution for the MPD, triclinic PEG, and monoclinic PEG forms, respectively). Comparisons of these structures were interesting because 4-4-20 exhibited a lower antigen-binding affinity in 47% MPD (Ka = 1.3 x 10(8) M-1) than in either 16% PEG (Ka = 2.9 x 10(9) M-1) or phosphate-buffered saline (Ka = 1.8 x 10(10) M-1). Even though the solution behavior of the antibody was significantly different in MPD and PEG, the crystal structures were remarkably similar. In all three structures, the fluorescein-combining site was an aromatic slot formed by tyrosines L32, H96, and H97 and tryptophans L96 and H33. In addition, several active site constituents formed an electrostatic network with the ligand. These included a salt link between arginine L34 and one of fluorescein's enolate oxygen atoms, a hydrogen bond between histidine L27d and the second enolic group, a hydrogen bond between tyrosine L32 and the phenylcarboxylate group, and two medium range (approximately 5 A) electrostatic interactions with lysine L50 and arginine H52. The only major difference between the triclinic MPD and PEG structures was the degree of hydration of the antigen-combining site. Three water molecules participated in the above electrostatic network in the MPD structure, while eight were involved in the PEG structure. Based on this observation, we believe that 4-4-20 exhibits a lower affinity in MPD due to the depletion of the hydration shell of the antigen-combining site.

Active site structure-function analyses of anti-fluorescein single chain antibody 4-4-20 and anti-single-stranded DNA single chain antibody 04-01 were conducted studying the ligand binding properties of hybrid antibodies resulting from systematic transplantation of heavy chain complementarity regions (HCDRs) from monoclonal antibody 4-4-20 into 04-01. Two prototype monoclonal antibodies were chosen because the primary structures of their respective light chains were nearly identical but the specificities and shape of their active sites were distinctly different. Based on nearly identical light chains, the diverse active site conformations (i.e. 4-4-20 pocket and 04-01 cleft) and subsequent specificities of the two antibodies were likely dictated by heavy chain properties. As a result, specificities of each HCDR transplant were analyzed in terms of binding reactivity with either fluorescein or (dT)8. Results of binding studies, together with idiotypic and secondary structure analyses, were used to determine the relative contribution of each HCDR to the active site conformations and ligand specificities of monoclonal antibodies 4-4-20 and 04-01. Collectively the various analyses led to the conclusion that the intradomain conformational dynamics and cooperativity necessary for the structural integrity of the low affinity cleft-shaped 04-01 anti-single-stranded DNA active site are probably less stringent than those of a high affinity pocket-shaped anti-fluorescein active site.

The solution dynamics of a bivalent bispecific single-chain antibody (BiSCA) specific against fluorescein (Fl) and single-stranded DNA (ssDNA) were investigated. Fluorescence resonance energy transfer (FRET) studies were performed in order to estimate the average distances, R, between the anti-Fl and the anti-ssDNA active sites. In separate experiments, either 2-(dimethylamino)naphthalene-5-sulfonyl chloride coupled to the 5' end of an oligothymidylate polymer of 6 residues length (2,5-DNS-dT6) served as energy donor to Fl or eosin isothiocyanate coupled to the 5' end of an oligothymidylate polymer of 6 residues length (eosin-dT6) served as energy acceptor from Fl. Labeling of dT6 with 2,5-DNS or eosin did not significantly interfere with recognition by the anti-ssDNA binding site. With the 2,5-DNS/Fl energy transfer pair, the calculated values of R(k2 = 2/3), R(min), and R(max) were 44, 37, and 54 A, respectively. With Fl/eosin (opposite direction of FRET), values of 40, 33, and 51 A, respectively, were obtained. Considering the sizes of the two SCA domains and the length of the interdomain polypeptide linker, an R value of approximately 140 A would be expected for the extended molecule. The fact that measured R distances were on average 3-fold shorter than 140 A indicated that BiSCA was not an extended and rigid molecule. The efficiency of energy transfer increased with increasing temperature in the range of 10-30 degrees C, suggesting that conformational fluctuations of the protein resulted in decreased average distance between BiSCA active sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Four hamster monoclonal anti-metatype antibodies were characterized in terms of their binding properties with liganded murine monoclonal single-chain antifluorescein antibody 4-4-20. Based on induced delays in the rate of ligand (fluorescein) dissociation upon the binding of each monoclonal anti-Met antibody, apparent Kd values were determined for monoclonal antibodies 3A5-1, P1E11, 4A6, and 2C3 (3.6 x 10(-8), 3.6 x 10(-8), 5.0 x 10(-8), and 2.6 x 10(-7) M, respectively). The interaction of hamster monoclonal antibody 3A5-1 with liganded SCA 4-4-20 and IgG 4-4-20 was also evaluated on the basis of deuterium oxide exchange to assess the relative ability of each antibody to stabilize the intrinsic dynamics of the variable domains of the single-chain molecule. The results indicated a correlation between the apparent Kd of the monoclonal anti-Met antibody and the degree of delay in the rate of ligand dissociation from the primary antibody.

Hydrostatic pressure was used to promote dissociation of fluorescein (Fl) from single-chain antibody 4-4-20 (SCA 4-4-20). Fl fluorescence intensity was quenched by 97% upon binding to SCA 4-4-20. Increasing pressure to 2.4 kbar enhanced Fl fluorescence from the remaining 3% to 14-17%. The capacity of anti-metatype antibodies (anti-Met), which specifically recognize liganded anti-Fl antibodies, to protect against pressure-induced Fl dissociation was tested. Both polyclonal and monoclonal anti-Met antibodies protected against Fl dissociation, reducing the fluorescence intensity at 2.4 kbar from 14-17% to 6-8%. Additive effects of anti-Met antibodies in protection against pressure-induced Fl dissociation were suggested by the fact that a 2-fold molar excess polyclonal anti-Met reagent promoted additional protection relative to an equimolar amount. On the other hand, combination of different monoclonal anti-Met antibodies did not promote additive protection, suggesting recognition of overlapping metatopes by these monoclonals. The complex formed by SCA 4-4-20 and the Fl analog HPF was more sensitive to pressure than the Fl-SCA 4-4-20 complex. Addition of both polyclonal and monoclonal anti-Met antibodies reduced the Fl fluorescence recovery at 2.4 kbar from 75% to 40-55%. In order to directly study binding of anti-Met antibodies to mAb 4-4-20, monoclonal anti-Met antibody 3A5-1 was labeled with 2-dimethylaminonaphthalene-5-sulfonyl chloride (2,5-Dns-Cl) and Dns fluorescence anisotropy measured. Unliganded mAb 4-4-20 did not bind to 2,5-Dns-3A5-1 as indicated by the absence of measurable changes in Dns fluorescence anisotropy upon increasing mAb concentration. Addition of mAb 4-4-20 bound to Fl produced a sigmoidal increase in Dns anisotropy, compatible with association of the primary immune complex and 3A5-1. An affinity constant, K0.5, of 1.5 x 10(-7) M and a cooperativity coefficient (n) of 3.1 were calculated for formation of the Fl-mAb 4-4-20 complex. The HPF-mAb 4-4-20 complex was also recognized by 2,5-Dns-3A5-1 but with lower affinity, indicating that the monoclonal anti-Met 3A5-1 distinguished between mAb 4-4-20 liganded to different haptens.

Complementarity determining region (CDR) transplant studies were conducted between two monoclonal antibodies of distinctly different specificities (anti-fluorescein monoclonal antibody (mAb) 4-4-20 and anti-single-stranded DNA (ssDNA) mAb 04-01) which possessed nearly identical light chains but dissimilar heavy chains. The variations in binding specificities between the two immunoglobulins suggested that the active-site features of anti-fluorescein antibodies were dictated by characteristics intrinsic to the heavy chain (H-chain). To identify specific regions of the H-chain which influence the structure and function of an anti-fluorescein active site, CDR transplantation was systematically employed to convert the anti-ssDNA 04-01 antibody active site to an active site with anti-fluorescein activity. Each mAb 4-4-20 H-chain CDR (HCDR) was transplanted into the H-chain of a single-chain derivative of the 04-01 molecule. A fluorescence polarization ligand binding assay was utilized to determine the equilibrium dissociation constant, Kd, of hybrid transplant single-chain antibody HCDR1-HCDR2-HCDR3(4-4-20) for fluorescein (3.8 x 10(-7) M, indicating successful conversion of an anti-ssDNA active site to an anti-fluorescein binding site. A similar Kd (6.3 x 10(-7) M) was determined using a fluorescein fluorescence quenching assay. The transplantation results are discussed in terms of the relative contribution of each HCDR to a successful conversion in antibody specificity.

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 constructed monovalent single-chain antibody molecules possessing distinct specificities, SCA 4-4-20 (anti-fluorescein) and SCA 04-01 (anti-single-stranded DNA). A flexible linker, modeled after a secreted fungal cellulase protein, was incorporated as the interdomain linker covalently 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 monovalent single-chain antibody prototype molecules. In both solid and solution phase binding assays, the bivalent bispecific single-chain antibody simultaneously bound both ligands (fluorescein and (dT)6). Construction of a model bivalent bispecific molecule provides a foundation for future assembly of similar molecules designed to identify parameters involved in enhanced binding of antibodies due to avidity and dual specificity.

Hydrostatic pressure was used to dissociate fluorescein (Fl) from the high-affinity anti-Fl single-chain antibody 4-4-20 (SCA 4-4-20). Fl dissociation was monitored by measuring (1) the shift in the Fl absorption peak, (2) the recovery in Fl fluorescence intensity, which is quenched upon SCA binding, or (3) the decrease in Fl fluorescence polarization. Pressure effects were studied at two different Fl:SCA 4-4-20 molar ratios: 1:1, at which Fl fluorescence quenching was ca. 35% at atmospheric pressure, and 1:5, at which quenching reached 95-97% under the same conditions. In both cases, pressure-induced dissociation was favored by concomitant dilution of protein and ligand. Dissociation constants (KD) at each pressure were calculated on the basis of measurements of Fl fluorescence polarization under pressure. The dependence of KD, and consequently of delta G of dissociation, on pressure permitted calculation of the magnitude of the standard volume change (delta V) involved in the dissociation process. According to this study, delta V of dissociation for the Fl-SCA complex is -50 mL/mol, which corresponds to a 10-times higher value than that found for dissociation of Fl from the intact IgG mAb 4-4-20 [Herron, J. N., Kranz, D. M., Jameson, D. M., & Voss, E. W., Jr. (1986) Biochemistry 25, 4602-4609]. This difference is explained in terms of a higher overall flexibility of unliganded SCA and of a less stable binding site in SCA relative to mAb.(ABSTRACT TRUNCATED AT 250 WORDS)

The contribution to high affinity Fl binding by each crystallographically defined Mab 4-4-20 (Ka = 1.7 x 10(10) M-1; Qmax = 90%) ligand contact residue (L27dHis, L32Tyr, L34Arg, L91Ser, L96Trp and H33Trp) has been determined by site-specific mutagenesis studies. All six antigen contact residues were changed to Ala in the single-chain derivative of Mab 4-4-20 and following expression in E. coli, denaturation, refolding and purification, each SCA mutant was characterized in terms of Fl binding affinity, Qmax, lambda max and idiotype. Results demonstrated that Ala substitutions at each ligand contact residue reduced the binding affinities and quenching maxima for all residues except L27d which retained wild type characteristics. The SCA TyrL32Ala, SerL91Ala and TrpH33Ala mutants exhibited binding affinities that were approximately 1000-fold lower than the wild type value and greatly reduced Qmax values. Additionally, other amino acid substitutions were performed at three of the six antigen contact residues (L91Ser, L96Trp and H33Trp) to further evaluate the role of each in Fl binding. Therefore, the following mutations were constructed and characterized: SerL91Asn, TrpL96Tyr, TrpL96Phe, TrpL96Leu, TrpH33Tyr and TrpH33Phe. Results of site-specific mutagenesis studies are discussed in terms of Mab active site structure and suggest that L32Tyr, L91Ser and H33Trp are important for high affinity Fl binding and efficient Fl quenching.

Binding interactions of various synthetic oligohomonucleotides with anti-ssDNA autoantibody BV 04-01 (IgG2b, kappa) and the corresponding single-chain antibody (SCA) 04-01/212 were studied. Oligonucleotide binding to IgG or SCA resulted in quenching of the protein's tryptophan fluorescence permitting direct assessment of ligand binding under equilibrium conditions. The effect of oligothymidylate length, (dT)n, on tryptophan quenching was evaluated. The equilibrium dissociation constants (Kd) for the binding of (dT)6 and (dT)8 were the same [(1.3 +/- 0.02) x 10(-7) M], while decreasing the length of the oligothymidylate to (dT)3 increased the Kd an order of magnitude. To assess base specificity, the comparative binding of other hexahomonucleotides was examined. Neither (dA)6 nor (dC)6 showed measurable binding, while the dissociation constant for (dG)6 was (7.1 +/- 0.3) x 10(-7) M. Fluorescence lifetime quenching data correlated with the steady-state binding results and indicated that the quenching process contains both dynamic and static components. The ability of BV 04-01 to bind (dT)6 and (dG)6 nucleotides was further supported by fluorescence anisotrophy studies with fluorescein-labeled hexadeoxynucleotides. Various levels of tryptophan fluorescence quenching upon titration with oligothymidylates of different length, as well as the similar affinities for (dT)6 and (dG)6, supported the concept that the groove-type binding pocket in BV 04-01 consists of binding subsites that cooperatively adapt for efficient binding of oligonucleotides.