Theoretical considerations of the role of antigen structure in B cell activation

Thymus-independent antigens generally are polymeric molecules with repeating arrays of antigenic determinants. Immunological studies of the activity of haptenated thymus-independent antigens have shown that small changes in hapten density can transform a polymeric antigen from nonimmunogenic to immunogenic, and from immunogenic to tolerogenic. In this paper we compute the equilibrium configuration of a linear flexible, haptenated polymer absorbed to a B cell surface, and correlate configurational features of the molecule with its immunological functioning. A polymeric molecule bound to a cell generally will not lie entirely on the surface; rather there will be sections that form loops extending into solution, separated by tightly bound sections, or trains. Trains link antibody receptors on the B cell surface in a fashion that restricts their mobility. Thus trains cause restrictive cross-linking. Our computations show that there is a critical hapten density below which the polymer does not bind to the surface. At hapten densities slightly above the critical density, the polymer binds weakly to the surface with a configuration dominated by a few, rather long loops. These loops cross-link receptors, but do so without bringing the cross-linked receptors into close proximity and without substantially restricting their motion. Long loops thus cause unrestrictive cross-linking. As the hapten density increases, the average loop length decreases and the average train length increases. Thus cross-linking becomes restrictive. In this density range, immune stimulation is observed. At high hapten densities long trains form, separated by few, very short loops and almost all receptors are cross-linked. Consequently cross-linking may be overly restrictive, freezing receptors into place and generating an abundance of cross-linking or other signals that induce a state of immunological tolerance.     

Theory of equilibrium binding of symmetric bivalent haptens to cell surface antibody: application to histamine release from basophils.

We present a theory of equilibrium binding of symmetric bivalent haptens to cell surface antibody in the presence or absence of monovalent hapten. Bivalent haptens can link together antibodies to form linear chains or rings on cell surfaces. We show how to calculate the amount of any complex of bound bivalent hapten, monovalene fraction of antibody involved in complexes made up of two or more antibodies, i.e., the fraction of antibody that is cross-linked (Xpoly). We treat the case when the antibody on the cell surface, which is specific for the hapten, is homogeneous. For this case we prove a number of general properties about Xpoly: 1) Xpoly approaches zero at both high and low bivalent hapten concentration. 2) Xpoly becomes a maximum when the bivalent hapten concentration equals Amax, where Amax = 1/H + B/2. H is twice the equilibrium constant for the binding of a single hapten site to a single antibody site and B is the monovalent hapten concentration. 3) a plot of Xpoly vs the log of the bivalent hapten concentration is symmetric about the maximum value of Xpoly. We use these and other properties of Xpoly in this paper to clarify the relationship between cross-link formation and histamine release.     

Use of different hapten-protein conjugates immobilized on nitrocellulose to screen monoclonal antibodies to abscisic acid

The dot-immunobinding method for screening antibodies to proteins on sheets of nitrocellulose has been modified to allow monoclonal antibodies (McAb) to the hapten abscisic acid (ABA) to be screened. Several methods for conjugating ABA to proteins using new bifunctional coupling reagents, specific for hapten keto groups, are described. Hybridomas secreting McAb with a defined specificity for the hapten can be identified by screening supernatants against the carrier protein and other hapten-protein conjugates with different conjugation bridges or modified hapten structure. Inhibition of binding to conjugates by free hapten is used to determine the relative avidity of the McAb for free and bound hapten. All of these tests could be done with no more than about 50 microliter of antibody solution. Dot immunobinding is a useful alternative to radioimmunoassay for screening McAb to haptens.     

bisFabs: Tools for rapidly screening hybridoma IgGs for their activities as bispecific antibodies

Bispecific antibodies are a growing class of therapeutic molecules. Many of the current bispecific formats require DNA engineering to convert the parental monoclonal antibodies into the final bispecific molecules. We describe here a method to generate bispecific molecules from hybridoma IgGs in 3–4 d using chemical conjugation of antigen-binding fragments (Fabs) (bisFabs). Proteolytic digestion conditions for each IgG isotype were analyzed to optimize the yield and quality of the final conjugates. The resulting bisFabs showed no significant amounts of homodimers or aggregates. The predictive value of murine bisFabs was tested by comparing the T-cell redirected cytotoxic activity of a panel of antibodies in either the bisFab or full-length IgG formats. A variety of antigens with different structures and expression levels was used to extend the comparison to a wide range of binding geometries and antigen densities. The activity observed for different murine bisFabs correlated with those observed for the full-length IgG format across multiple different antigen targets, supporting the use of bisFabs as a screening tool. Our method may also be used for the screening of bispecific antibodies with other mechanisms of action, allowing for a more rapid selection of lead therapeutic candidates.     

Recognition of Z-RNA and Z-DNA determinants by polyamines in solution: experimental and theoretical studies

Protonated polyamines are among the most efficient cations that induce the left-handed Z-form in certain polynucleotides. It is not known, however, whether these cations bind to specific sites on Z-sequences in solution. We have studied potential polyamine binding sites by measuring the effects of polyamines on the binding of purified immunoglobulins (IgGs) to different regions of the Z-helix and by molecular mechanics modeling. The specific binding of anti-Z-DNA and anti-Z-RNA IgGs to Z-helices was studied as a function of spermidine or spermine concentration. The effect of polyamines on the antibody-nucleic acid interaction was different for IgGs with different specificities for various determinants on the Z-helix. Polyamines inhibit the binding of certain anti-Z IgGs directed against specific sites probably at or near the interface between the major convex surface and the phosphate backbone, most likely by competing with the antibody binding site(s). In contrast, polyamines have no effect on other anti-Z IgGs directed against sites determined by the phosphate backbone. Furthermore, these cations can enhance the binding of anti-Z IgG directed against bulky groups at the C-5 position on the major convex surface of the helix; the enhancement may be related to charge neutralization. Under these conditions, no direct binding of antibodies with polyamines was observed. These data suggest the existence of a specific binding site(s) for polyamines on both Z-DNA and Z-RNA in solution. These binding sites have some similarity to those observed in oligonucleotide crystals by Quigley (in "Molecular Structure and Biological Activity," J.F. Griffin and W.L. Duax, eds., Elsevier, Amsterdam (1982), pp. 317-331). The experimental evidence for specific spermine binding sites on the helical surface was supported by molecular mechanics modeling of the interaction of spermine with the major groove of (dG-dC)5.(dG-dC)5 in both the Z- and B-forms. The crystal coordinates of spermine-containing oligonucleotides in both the B- and Z-forms were used as the starting points for modeling studies. The potential energy of spermine bound to the major convex surface of the Z-form was much less favorable than that of spermine bound to the major groove of the B-form. In the presence of sodium ions, however, the Z-form-spermine complexes were favored over the B-form. Thus, both theoretical and experimental studies indicate that polyamines can specifically recognize Z-helical determinants in solution as well as in crystals.     

Camelid heavy-chain variable domains provide efficient combining sites to haptens

Camelids can produce antibodies devoid of light chains and CH1 domains (Hamers-Casterman, C. et al. (1993) Nature 363, 446-448). Camelid heavy-chain variable domains (VHH) have high affinities for protein antigens and the structures of two of these complexes have been determined (Desmyter, A. et al. (1996) Nature Struc. Biol. 3, 803-811; Decanniere, K. et al. (1999) Structure 7, 361-370). However, the small size of these VHHs and their monomeric nature bring into question their capacity to bind haptens. Here, we have successfully raised llama antibodies against the hapten azo-dye Reactive Red (RR6) and determined the crystal structure of the complex between a dimer of this hapten and a VHH fragment. The surface of interaction between the VHH and the dimeric hapten is large, with an area of ca. 300 A(2); this correlates well with the low-dissociation constant of 22 nM measured for the monomer. The VHH fragment provides an efficient combining site to the RR6, using its three CDR loops. In particular, CDR1 provides a strong interaction to the hapten through two histidine residues bound to its copper atoms. VHH fragments might, therefore, prove to be valuable tools for selecting, removing, or capturing haptens. They are likely to play a role in biotechnology extending beyond protein recognition alone.     

Modeling bispecific monoclonal antibody interaction with two cell membrane targets indicates the importance of surface diffusion

We have developed a mathematical framework for describing a bispecific monoclonal antibody interaction with two independent membrane-bound targets that are expressed on the same cell surface. The bispecific antibody in solution binds either of the two targets first, and then cross-links with the second one while on the cell surface, subject to rate-limiting lateral diffusion step within the lifetime of the monovalently engaged antibody-antigen complex. At experimental densities, only a small fraction of the free targets is expected to lie within the reach of the antibody binding sites at any time. Using ordinary differential equation and Monte Carlo simulation-based models, we validated this approach against an independently published anti-CD4/CD70 DuetMab experimental data set. As a result of dimensional reduction, the cell surface reaction is expected to be so rapid that, in agreement with the experimental data, no monovalently bound bispecific antibody binary complexes accumulate until cross-linking is complete. The dissociation of the bispecific antibody from the ternary cross-linked complex is expected to be significantly slower than that from either of the monovalently bound variants. We estimate that the effective affinity of the bivalently bound bispecific antibody is enhanced for about 4 orders of magnitude over that of the monovalently bound species. This avidity enhancement allows for the highly specific binding of anti-CD4/CD70 DuetMab to the cells that are positive for both target antigens over those that express only one or the other We suggest that the lateral diffusion of target antigens in the cell membrane also plays a key role in the avidity effect of natural antibodies and other bivalent ligands in their interactions with their respective cell surface receptors.     

Intrinsic binding effects in mixed counterionic polyelectrolyte systems: extension of condensation theory and comparison with voltammetric data

Voltammetric speciation data for the potassium/zinc/polymethacrylate system, recently obtained for various charge densities of the polyelectrolyte (Díaz-Cruz et al., Anal. Chim. Acta, 264 (1992) 163) and for different concentrations of monovalent counterions (van den Hoop and van Leeuwen, Anal. Chim. Acta, 273 (1993) 275), are compared with theoretical predictions computed according to a new thermodynamic model developed by Paoletti et al. (Biophys. Chem., 41 (1991) 73) and recently extended by Benegas and Paoletti (in preparation). The model allows: (i) the simultaneous condensation of both monovalent and divalent counterions and (ii) can account for a certain specific affinity of the polyelectrolyte for one type of the counterion over the other. For various charge densities of the polyelectrolyte, experimentally obtained speciation data for the K/Zn/PMA system agree well with theoretical predictions by considering an extra reduced molar affinity energy of -4RT for the Zn(2+) polyelectrolyte binding. The agreement between experimental and theoretical values for the distribution of Zn(2+) ions over the free and bound state becomes less perfect for relatively high concentrations of monovalent counterions.     

Binding and transepithelial transport of immunoglobulins by intestinal M cells: demonstration using monoclonal IgA antibodies against enteric viral proteins

M cells of intestinal epithelia overlying lymphoid follicles endocytose luminal macromolecules and microorganisms and deliver them to underlying lymphoid tissue. The effect of luminal secretory IgA antibodies on adherence and transepithelial transport of antigens and microorganisms by M cells is unknown. We have studied the interaction of monoclonal IgA antibodies directed against specific enteric viruses, or the hapten trinitrophenyl (TNP), with M cells. To produce monospecific IgA antibodies against mouse mammary tumor virus (MMTV) and reovirus type 1, Peyer's patch cells from mucosally immunized mice were fused with myeloma cells, generating hybridomas that secreted virus-specific IgA antibodies in monomeric and polymeric forms. One of two anti-MMTV IgA antibodies specifically bound the viral surface glycoprotein gp52, and 3 of 10 antireovirus IgA antibodies immunoprecipitated sigma 3 and mu lc surface proteins. 35S-labeled IgA antibodies injected intravenously into rats were recovered in bile as higher molecular weight species, suggesting that secretory component had been added on passage through the liver. Radiolabeled or colloidal gold-conjugated mouse IgA was injected into mouse, rat, and rabbit intestinal loops containing Peyer's patches. Light microscopic autoradiography and EM showed that all IgA antibodies (antivirus or anti-TNP) bound to M cell luminal membranes and were transported in vesicles across M cells. IgA-gold binding was inhibited by excess unlabeled IgA, indicating that binding was specific. IgG-gold also adhered to M cells and excess unlabeled IgG inhibited IgA-gold binding; thus binding was not isotype-specific. Immune complexes consisting of monoclonal anti-TNP IgA and TNP-ferritin adhered selectively to M cell membranes, while TNP-ferritin alone did not. These results suggest that selective adherence of luminal antibody to M cells may facilitate delivery of virus-antibody complexes to mucosal lymphoid tissue, enhancing subsequent secretory immune responses or facilitating viral invasion.     

Binding of a monoclonal antibody and its Fab fragment to supported phospholipid monolayers measured by total internal reflection fluorescence microscopy.

The association of an anti-dinitrophenyl monoclonal antibody and its Fab fragment with supported phospholipid monolayers composed of a mixture of dipalmitoylphosphatidylcholine and dinitrophenyl-conjugated dipalmitoylphosphatidylethanolamine has been characterized with total internal reflection fluorescence microscopy. The surface densities of bound antibodies were measured as a function of the antibody and Fab solution concentrations, and as a function of the solution concentration of dinitrophenylglycine. The apparent association constant of Fab fragments with surface-associated haptens was approximately 10-fold lower than the association constant for haptens in solution, and the apparent surface association constant for intact antibodies was only approximately 10-fold higher than the constant for Fab fragments. Data analysis with simple theoretical models indicated that, at most antibody surface densities, 50-90% of membrane-associated intact antibodies were attached to the surface by two antigen binding sites.     

Interaction of antibodies specific towards the 2,4-dinitrophenyl group and of their fragments with hapten

The interaction of hapten (ε-DNP lys) with native and S-sulfonated antibodies specific towards the 2,4-dinitrophenyl group, as well as the interaction with isolated chains and a complex obtained by mixing light, (L) and heavy (H) chains of these antibodies, were followed both by polarography and by equilibrium dialysis. With the S-sulfonated antibodies and with the mixture of H and L chains the binding heterogeneity observed in the original antibodies was much lowered or entirely removed. At the same time, the amount of active proteins in the sample decreased approximately by half. The association constants of modified antibodies were of the same order as the average association constants of the original antibodies. A slow increase of the amounts of hapten bound with proteins was observed on mixing the H and L chains and adding hapten. This slow reactivation was not obtained with the original or S-sulfonated antibodies and with isolated chains. It was shown that the reaction determining the kinetics of this reactivation (the slowest reaction) was not the association of H and L chains but the interaction of complexes of the H and L chains with hapten. It was reported previously that H chains were nonspecifically reactivated by binding L chains. The amount of hapten bound by the complex of H and L chains increased with increasing excess of L chains following a curve resembling the Langmuir isotherm. The limiting value of the amount of hapten bound when using antibody L chains was higher than in the case of nonspecific L chains.     

Interaction of antibodies with liposomes bearing fluorescent haptens

Kinetic and equilibrium aspects of the recognition of antigenic model membranes by antibodies have been studied. Monoclonal anti-fluorescein IgG and its monovalent Fab fragment were allowed to interact with a fluorescein-lipid hapten that was incorporated into phospholipid vesicles. The binding was assayed in the nanomolar hapten concentration range by monitoring the quenching of hapten fluorescence by antibody. The rate and strength of the binding depended on the lipid composition of the vesicles; cholesterol enhanced both. The biphasic binding kinetics observed at high antibody concentrations for some compositions, plus additional spectroscopic evidence, led us to hypothesize that the hapten existed in a composition-dependent equilibrium between at least two conformations: (1) extended away from the membrane surface, available for binding, and (2) sequestered at or in the surface, unavailable for binding. The rate and strength of IgG binding were always greater than those of Fab, indicating bivalent binding by the IgG. This binding was intra-vesicular, since no agglutination of the vesicles was detected.     

Occluded molecular surface: Analysis of protein packing

We describe a novel method to calculate the packing interactions in protein structural models. The method calculates the interatomic occluded surface areas for each atom in the protein model. The identification of, and degree of interaction with, neighboring atoms is accomplished by extending surface normal from a dot surface of each atom to the point of intersection with neighboring atoms. The combined occluded and non-occluded surface areas may be normalized for the amino acid composition of the protein providing a single parameter, the normalized protein surface ratio, which is diagnostic for native-like Structures. Individual residues in the model which are in infrequent occluded surface environments may be identified. The method provides a means to explicitly describe packing densities and packing environments of individual atoms in a protein model. Finally, the method allows estimation of the complementarity between any interacting molecules, for example a ligand binding to a receptor.     

Role of the non‐hypervariable FR3 D‐E loop in single‐domain antibody recognition of haptens and carbohydrates

Single‐domain antibodies (sdAbs), the variable domains of camelid heavy chain‐only antibodies, are generally thought to poorly recognize nonproteinaceous small molecules and carbohydrates in comparison with conventional antibodies. However, the structures of anti‐methotrexate, anti‐triclocarban and anti‐cortisol sdAbs revealed unexpected contributions of the non‐hypervariable “CDR4” loop, formed between β‐strands D and E of framework region 3, in binding. Here, we investigated the potential role of CDR4 in sdAb binding to a hapten, 15‐acetyl‐deoxynivalenol (15‐AcDON), and to carbohydrates. We constructed and panned a phage‐displayed library in which CDR4 of the 15‐AcDON‐specific sdAb, NAT‐267, was extended and randomized. From this library, we identified one sdAb, MA‐232, bearing a 14‐residue insertion in CDR4 and showing improved binding to 15‐AcDON by ELISA and surface plasmon resonance. On the basis of these results, we constructed a second set of phage‐displayed libraries in which the CDR4 and other regions of three hapten‐ or carbohydrate‐binding sdAbs were diversified. With the goal of identifying sdAbs with novel glycan‐binding specificities, we panned the library against four tumor‐associated carbohydrate antigens but were unable to enrich binding phages. Thus, we conclude that while CDR4 may play a role in binding of some rare hapten‐specific sdAbs, diversifying this region through molecular engineering is probably not a general solution to sdAb carbohydrate recognition in the absence of a paired V_L domain.     

Internalizing Cancer Antibodies from Phage Libraries Selected on Tumor Cells and Yeast-Displayed Tumor Antigens

A number of approaches have been utilized to generate antibodies to cancer cell surface receptors that can be used as potential therapeutics. A number of these therapeutic approaches, including antibody-drug conjugates, immunotoxins, and targeted nucleic acid delivery, require antibodies that not only bind receptor but also undergo internalization into the cell upon binding. We previously reported on the ability to generate cancer cell binding and internalizing antibodies directly from human phage antibody libraries selected for internalization into cancer cell lines. While a number of useful antibodies have been generated using this approach, limitations include the inability to direct the selections to specific antigens and to identify the antigen bound by the antibodies. Here we show that these limitations can be overcome by using yeast-displayed antigens known to be associated with a cell type to select the phage antibody output after several rounds of selection on a mammalian cell line. We used this approach to generate several human phage antibodies to yeast-displayed EphA2 and CD44. The antibodies bound both yeast-displayed and mammalian cell surface antigens, and were endocytosed upon binding to mammalian cells. This approach is generalizable to many mammalian cell surface proteins, results in the generation of functional internalizing antibodies, and does not require antigen expression and purification for antibody generation.     

Anti-hapten IgG antibodies bound to cell surface hapten: anti-IgG antibody prevents dissociation as measured with fluid phase hapten

We investigated the dissociation by fluid phase hapten of IgG antibodies bound to cell surface hapten in the presence and absence of anti-IgG antibodies. Dissociation was quantitated with fluid phase hapten, preventing reassociation of the anti-hapten antibodies. More than 90% of the anti-hapten IgG alone was prevented from reassociation by low concentrations of fluid phase hapten (nanogram to microgram range). In contrast, no dissociation of some IgG-anti-IgG complexes could be measured even at 24 hr incubation in the presence of very large excess of fluid phase hapten (100 mg/ml). We excluded aggregate formation between anti-hapten antibodies due to cross-linking by anti-antibodies as a cause for decreased dissociability by 1) performing the experiments in large excess of anti-antibody, 2) showing that the phenomenon was independent of anti-hapten antibody density, 3) showing that decreased dissociation also occurred at 4 degrees C, and 4) showing that aggregation by protein A did induce decreased dissociability, albeit three orders of magnitude lower than the anti-antibody. It was concluded that anti-antibody directly affected the "avidity" of cell hapten bound anti-hapten IgG in an unknown manner.     

Surface expression and partial characterization of an arsonate hapten-specific idiotype-bearing T-cell receptor

Anti-idiotype antibodies raised against the arsonate hapten idiotype have been used to detect arsonate-binding receptors on the surface of peripheral T cells of A/J mice and to isolate this material after biosynthetic labeling for partial chemical characterization. It was found that 2-3% of splenic T cells from arsonate-immune mice specifically bound the hapten using immunofluorescent keyhole limpet hemocyanin as a carrier. In double-immunofluorescence labeling experiments, a high proportion (approximately equal to 70%) of these cells also bound the (Fab')2 fragment of rabbit anti-idiotype antibody in exactly the same patches on the cell as the arsonate hemocyanin antigen. In addition, the anti-idiotype antibody inhibited the binding of the hapten-carrier complex to T cells by approximately equal to 70%. In parallel experiments, fowl antibodies against mouse (Fab')2 fragments bound to 100% of arsonate-binding T cells in the same cell-surface patches as the hapten, and were capable of inhibiting 100% of the hapten-binding cells. Capping, shedding, and resynthesis experiments indicated that the T cells synthesized their antigen-binding idiotype-bearing receptors. Immunoblots of unreduced detergent extracts of purified splenic T cells developed with anti-idiotype antibodies showed bands at 150,000 and 94,000 Da. Equal amounts of protein extracted from liver and analyzed in the same gels as the T-cell material failed to show any reactivity with anti-idiotype antibodies. To confirm the biosynthetic origin of the idiotype-positive materials, detergent extracts from 75Se-methionine- or [3H]leucine-labeled Con A-treated splenic T cells were reacted with anti-idiotype antibodies and the bound material was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of 2-mercaptoethanol the major band was at 68,000 Da, with variable minor levels of material at 45,000 Da, while when hapten was used to isolate the receptor a dominant 25,000- to 30,000-Da band was seen. We believe that the higher-molecular-weight materials are multimers of the 25,000-30,000 subunit.     

Kinetics of binding reactions of an antibody molecule with haptens on a membrane surface

Direct measurement has been made of the reaction rate of binding of a bivalent antibody and fluorescent haptens, which were covalently bound on a model membrane surface, by a method of stopped-flow fluorometry. The result was interpreted as indicating that the reaction takes place in two steps: (i) binding of a hapten with one of the two antigen-combining sites of an antibody molecule, and (ii) binding of another hapten with the other site of the antibody molecule in question. The rate of the second step was found to depend on the fluidity of the membrane.