A model of neutralization of Chlamydia trachomatis based on antibody and host cell aggregation on the elementary body surface

Humoral immunity is that aspect of specific immunity that is mediated by B lymphocytes and involves the neutralizing of pathogens by means of antibodies attaching to the pathogen's binding sites. Antibodies bind to and block ligand sites on the pathogen which prevents these sites from attaching to target cell receptors and so cell entry is inhibited. Many studies investigate the role of humoral immunity for protection against chlamydial challenge and they have shown that neutralization of the chlamydial body requires a large number of attached antibodies. Steric hindrance greatly influences the number of available sites that may be bound, reducing relative occupancy well below 100%. We model steric effects of antibody Fab fragment attachment indicating that they must be taken into consideration to accurately model valency, the number of available binding sites. We derive a partial differential equation for the number of antibody Fabs and host cell receptors that are aggregated to extracellular chlamydial elementary bodies. We consider steric effects in describing the size distribution of aggregates. Our theory is in good agreement with Monte Carlo simulations of binding. We use our theoretical prediction for the valency in a model for the in-host population dynamics of a chlamydial infection and we fit our model to experimental data.     

Molybdate-stabilized glucocorticoid receptor: evidence for a receptor heteromer

The composition of the molybdate-stabilized glucocorticoid receptor (GR) complex has been investigated with a monoclonal antibody against the steroid-binding Mr 94 000 (94K) GR protein. It was concluded that one antibody molecule binds one 94K GR molecule. This finding constituted the basis for calculating the number of antibodies bound to the molybdate-stabilized nonactivated GR complex, which has an Mr of 302 000 (302K). Gel filtration on Sephacryl S-400 and density gradient centrifugation showed that only one antibody molecule bound to the molybdate-stabilized GR complex (calculated relative molecular mass for the antibody--molybdate-stabilized GR complex, 456 000; relative molecular mass for one antibody molecule, 157 000). Furthermore, experiments performed with a second antibody immunoprecipitation assay in the presence of an excess of both antibody and GR confirmed the above results. The possibility of steric hindrance not allowing more than one antibody molecule to bind to the molybdate-stabilized GR complex could be excluded. These results suggest that the molybdate-stabilized GR complex with an Mr of 302K only contains one steroid-binding 94K GR molecule and therefore represents a heteromeric complex.     

Steric effects in antibody reactions with polyvalent antigen

Antigen valency has been defined (Singer, 1965) as the maximum number of epitopes per antigen which can be simultaneously occupied by antibody. If the epitopes are closely spaced, steric hindrance prevents the simultaneous occupancy of all epitopes. Current methods of estimating both the antigen valency and the association constant (Ka) from equilibrium binding data do not allow for the effects of steric hindrance. We have developed a theory which accounts rigorously for steric hindrance when monovalent ligands of quite general shape (antibodies) react reversibly with multivalent acceptor molecules (antigens). The surfaces of the acceptors are modelled by completely general two-dimensional lattices. Using this theory we demonstrate that curvature of Scatchard plots can arise from steric effects alone in the absence of other known causes such as cross-linking, cooperativity and heterogeneous epitope affinities. Our results generalize the conclusions of McGhee & von Hippel (1974) who dealt with one-dimension acceptor molecules such as DNA. We discuss inaccuracies in the estimation of both Ka and antigen valency using the traditional approach of fitting straight lines to Scatchard plots.     

A cellular receptor of human rhinovirus type 2, the very-low-density lipoprotein receptor,binds to two neighboring proteins of the viral capsid

The very-low-density lipoprotein receptor (VLDL-R) is a receptor for the minor-group human rhinoviruses (HRVs). Only two of the eight binding repeats of the VLDL-R bind to HRV2, and their footprints describe an annulus on the dome at each fivefold axis. By studying the complex formed between a selection of soluble fragments of the VLDL-R and HRV2, we demonstrate that it is the second and third repeats that bind. We also show that artificial concatemers of the same repeat can bind to HRV2 with the same footprint as that for the native receptor. In a 16-A-resolution cryoelectron microscopy map of HRV2 in complex with the VLDL-R, the individual repeats are defined. The third repeat is strongly bound to charged and polar residues of the HI and BC loops of viral protein 1 (VP1), while the second repeat is more weakly bound to the neighboring VP1. The footprint of the strongly bound third repeat extends down the north side of the canyon. Since the receptor molecule can bind to two adjacent copies of VP1, we suggest that the bound receptor "staples" the VP1s together and must be detached before release of the RNA can occur. When the receptor is bound to neighboring sites on HRV2, steric hindrance prevents binding of the second repeat.     

Integrating molecular detection and response to create self-signalling antibodies

Monoclonal antibodies are reproducible, specific, and cost-effective molecular probes; use outside the laboratory is, however, restricted by technical limitations. Addressing these constraints, the first self-signalling antibodies are now described, where specific antigen binding causes release of bound reporter from bispecific antibodies (BsAb) to generate a detectable signal. The report examines the concept that two different antibody binding sites in close proximity can promote interaction between molecules recognised by these sites, generating a signal by molecular crowding. Signal strength is found to increase with increasing homogeneity for a BsAb reactive with multimeric surfactant antigen; signal response is linear for a BsAb reactive with univalent small analyte deoxypyridinoline. Self-signalling is consistent with intramolecular steric hindrance. This is the first report detailing integration of two different functions, molecular detection and signal response, into BsAbs and with detection of large and small analytes, has generic application to antibody-based systems.     

Mitogen-like monoclonal anti-actin antibodies

Monoclonal antibodies (IgM kappa) have been produced to actin isolated electrophoretically from L cell extracts. These monoclonal anti-actin antibodies bind to intact L cells and modulate DNA synthesis and cell proliferation, much like affinity-purified polyclonal rabbit antibody to the same Mr 42,000 actin. In addition, monoclonal antibodies specific for actin from Entamoeba histolytica also bound to and modulated the growth of L cells. A monoclonal antibody directed against a neuroblastoma surface antigen did not produce stimulation of L cells, and the binding activity of anti-actin monoclonal antibody to L cells was removed by absorption with actin covalently coupled to Sepharose. These observations demonstrate the specificity of interaction between the anti-actin monoclonal antibodies and the surface of intact L cells. We conclude that a surface actin-like molecule on the L cell, when bound by specific monoclonal antibody, initiates a stimulatory signal which results in enhanced cellular metabolism.     

Monoclonal antibodies demonstrate limited structural homology between myosin isozymes from Acanthamoeba

We used a library of 31 monoclonal and six polyclonal antibodies to compare the structures of the two classes of cytoplasmic myosin isozymes isolated from Acanthamoeba: myosin-I, a 150,000-mol-wt, globular molecule; and myosin-II, a 400,000-mol-wt molecule with two heads and a 90-nm tail. This analysis confirms that myosin-I and -II are unique gene products and provides the first evidence that these isozymes have at least one structurally homologous region functionally important for myosin's role in contractility. Characterization of the 23 myosin-II monoclonal antibody binding sites by antibody staining of one-dimensional peptide maps and solid phase, competitive binding assays demonstrate that they bind to at least 15 unique sites on the myosin-II heavy chain. The antibodies can be grouped into six families, whose members bind close to one another. None of the monoclonal antibodies bind to myosin-II light chains and polyclonal antibodies against myosin-II light or heavy chain bind only to myosin-II light or heavy chains, respectively: no antibody binds both heavy and light chains. Six of eight monoclonal antibodies and one of two polyclonal sera that react with the myosin-I heavy chain also bind to determinants on the myosin-II heavy chain. The cross-reactive monoclonal antibodies bind to the region of myosin-II recognized by the largest family of myosin-II monoclonal antibodies. In the two papers that immediately follow, we show that this family of monoclonal antibodies to myosin-II binds to the myosin-II tail near the junction with the heads and inhibits both the actin-activated ATPase of myosin-II and contraction of gelled cytoplasmic extracts of Acanthamoeba cytoplasm. Further, this structurally homologous region may play a key role in energy transduction by cytoplasmic myosins.     

Study of the complex between porcine plasmin and alpha2-macroglobulin (author's transl)]

Porcine plasmin (EC 3.4.21.7) is obtained from plasminogen activated by human urokinase. This enzyme can bind, in an equimolecular ratio, to an alpha2-macroglobulin isolated from porcine serum. The number of active sites of plasmin has been determined through a burst titration of nitroaniline during the presteady-state hydrolysis of an amide substrate (N-alpha-carbobenzoxy-L-arginine-p-nitroanilide). The kinetic constants relative to a very sensitive ester substrate (N-alpha-carbobenzoxy-L-lysine nitrophenylester) hydrolysis have been measured. The binding of plasmin to alpha2-macroglobulin results in a complete inhibition of proteolytic activity, a reduction of active sites number and a decrease of esterolytic activity towards this substrate. In the complex, the residual activity (about 60%) is protected from protein inhibitors. Absorbance difference spectra show that 1 mol of alpha2-macroglobulin binds 1 mol of plasmin and 2 mol of trypsin. When plasmin is first bound to alpha2-macroglobulin, only 1 mol of trypsin can gain access tothe second site without removing the plasmin, showing that a steric hindrance is implicated in the inhibition performed by alpha2-macroglobulin binding.     

Antibody binding in proximity to the receptor/glycoprotein complex leads to a basal level of virus neutralization

Hypothetically, antibodies may neutralize enveloped viruses by diverse mechanisms, such as disruption of receptor binding, interference with conformational changes required for virus entry, steric hindrance, or virus aggregation. Here, we demonstrate that retroviral infection mediated by the avian sarcoma-leukosis virus (ASLV-A) envelope glycoproteins can be neutralized by an antibody directed against a functionally unimportant component of a chimeric receptor protein. Thus, the binding of an antibody in proximity to the retroviral envelope glycoprotein-receptor complex, without binding to the entry machinery itself, results in neutralization. This finding provides additional support for the hypothesis that steric hindrance is sufficient for antibody-mediated neutralization of retroviruses.     

Left-handed Z-DNA regions are present in negatively supercoiled bacteriophage PM2 DNA

Bacteriophage PM2 DNA is a 10-kb covalently closed circular (ccc) molecule with a reported superhelical density of sigma = -0.12. Here we describe the binding of anti-Z-DNA antibodies to PM2 form I DNA under high and low salt conditions. The binding to PM2 DNA has been demonstrated by competitive radioimmunoassay (RIA), retardation of the DNA:antibody complexes in agarose gels and visualization by electron microscopy. The antibody binding is dependent on the degree of negative supercoiling. Thus, PM2 form II and form III did not bind the antibody. The low salt RIA results indicated the presence of 200-400 bp of left-handed DNA per PM2 molecule. This could reduce the effective superhelical density to sigma = -0.04 to -0.08, a range comparable with those found for other ccc DNAs in vivo. Electron microscopy revealed that a maximum of 22 antibody molecules bind to PM2. Single-site restriction with HpaII of the fixed DNA:antibody complex showed a cluster of four to five antibody molecules bound near one end of the linear DNA molecule. The evidence presented indicates that PM2 DNA contains regions of left-handed conformation under physiological conditions (low salt concentration) as well as at high salt concentrations. In addition, electrophoretic analyses of PM2 topoisomers indicate the presence of left-handed regions at superhelical densities less than that of isolated PM2 DNA.     

Steric effects on multivalent ligand-receptor binding: exclusion of ligand sites by bound cell surface receptors.

Steric effects can influence the binding of a cell surface receptor to a multivalent ligand. To account for steric effects arising from the size of a receptor and from the spacing of binding sites on a ligand, we extend a standard mathematical model for ligand-receptor interactions by introducing a steric hindrance factor. This factor gives the fraction of unbound ligand sites that are accessible to receptors, and thus available for binding, as a function of ligand site occupancy. We derive expressions for the steric hindrance factor for various cases in which the receptor covers a compact region on the ligand surface and the ligand expresses sites that are distributed regularly or randomly in one or two dimensions. These expressions are relevant for ligands such as linear polymers, proteins, and viruses. We also present numerical algorithms that can be used to calculate steric hindrance factors for other cases. These theoretical results allow us to quantify the effects of steric hindrance on ligand-receptor kinetics and equilibria.     

Anti-idiotypic antibodies against an antibody to the platelet glycoprotein (GP) IIb-IIIa complex mimic GP IIb-IIIa by recognizing fibrinogen.

Binding of the adhesive ligand fibrinogen and the monoclonal antibody PAC1 to platelet glycoprotein (GP) IIb-IIIa is dependent on cell activation and inhibited by Arg-Gly-Asp (RGD)-containing peptides. Previously, we identified a sequence in a hypervariable region of PAC1 (mu-CDR3) that mimics the activity of the antibody. Here we examine whether monoclonal antibodies to this idiotypic determinant in PAC1 can mimic GP IIb-IIIa by binding to fibrinogen. Mice were immunized with a peptide derived from the mu-CDR3 of PAC1. Four antibodies were obtained that recognized fibrinogen as well as a recombinant form of the variable region of PAC1. However, they did not bind to other RGD-containing proteins, including von Willebrand factor, fibronectin, and vitronectin. Several studies suggested that these anti-PAC1 peptide antibodies were specific for GP IIb-IIIa recognition sites in fibrinogen. Three such sites have been proposed: two RGD-containing regions in the A alpha chain, and the COOH terminus of the gamma chain (gamma 400-411). Two of the antibodies inhibited fibrinogen binding to activated platelets, and all four antibodies bound to the fibrinogen A alpha chain on immunoblots. Antibody binding to immobilized fibrinogen was partially inhibited by monoclonal antibodies specific for the two A alpha chain RGD regions. However, the anti-PAC1 peptide antibodies also bound to plasmin-derived fibrinogen fragments X and D100, which contain gamma 400-411 but lack one or both A alpha RGD regions. This binding was inhibited by an antibody specific for gamma 400-411. When fragment D100 was converted to D80, which lacks gamma 400-411, antibody binding was reduced significantly (p less than 0.01). Electron microscopy of fibrinogen-antibody complexes confirmed that each antibody could bind to sites on the A alpha and gamma chains. These studies demonstrate that certain anti-PAC1 peptide antibodies mimic GP IIb-IIIa by binding to platelet recognition sites in fibrinogen. Furthermore, they suggest that the gamma 400-411 region of fibrinogen may exist in a conformation similar to that of an A alpha RGD region of the molecule.     

The Drosophila zeste protein binds cooperatively to sites in many gene regulatory regions: implications for transvection and gene regulation.

The Drosophila zeste protein binds in vitro to several sites in the white, Ultrabithorax, decapentaplegic, Antennapedia, and engrailed genes and to at least one site in the zeste gene itself. The distribution of these sites corresponds often with that of regulatory elements in these genes as defined by mutations or, in the case of white, by molecular analysis. A zeste binding site is frequently found in the immediate vicinity of the promoter. zeste binding sites are composed of two or more zeste recognition sequences T/CGAGT/CG. Isolated consensus sequences do not bind or footprint. Cooperative interactions are involved both in binding to a given site and between proteins bound at independent sites. zeste bound to one DNA molecule can in fact bind simultaneously to another DNA molecule. These results suggest a general role for zeste in bringing together distant regulatory elements controlling the activity of a target gene. In this model, transvection effects are a by-product of normal intragenic zeste action.     

Antibody recycling by engineered pH-dependent antigen binding improves the duration of antigen neutralization

For many antibodies, each antigen-binding site binds to only one antigen molecule during the antibody's lifetime in plasma. To increase the number of cycles of antigen binding and lysosomal degradation, we engineered tocilizumab (Actemra), an antibody against the IL-6 receptor (IL-6R), to rapidly dissociate from IL-6R within the acidic environment of the endosome (pH 6.0) while maintaining its binding affinity to IL-6R in plasma (pH 7.4). Studies using normal mice and mice expressing human IL-6R suggested that this pH-dependent IL-6R dissociation within the acidic environment of the endosome resulted in lysosomal degradation of the previously bound IL-6R while releasing the free antibody back to the plasma to bind another IL-6R molecule. In cynomolgus monkeys, an antibody with pH-dependent antigen binding, but not an affinity-matured variant, significantly improved the pharmacokinetics and duration of C-reactive protein inhibition. Engineering pH dependency into the interactions of therapeutic antibodies with their targets may enable them to be delivered less frequently or at lower doses.     

Hapten conformation in the combining site of antibodies that bind phenylphosphocholine.

We have shown previously that anti-phenylphosphocholine antibodies elicited by phosphocholine-keyhole limpet hemocyanin can be divided into two populations according to their ability to recognize the two hapten analogues p-nitrophenylphosphocholine (NPPC) and p-nitrophenyl 3,3-dimethylbutyl phosphate (NPDBP). These analogues differ from each other in that NPPC has a positively charged nitrogen in the choline moiety, whereas NPDBP lacks the positively charged nitrogen. Group II-A antibodies bind only NPPC, whereas group II-B antibodies bind both ligands. Here, by infrared and nuclear magnetic resonance spectroscopic investigations, we find that when free in solution NPPC has a predominantly fixed structure in which the termini approach each other, probably due to electrostatic interactions within the molecule; this "bent" structural feature is retained when the ligand is bound by antibody. In contrast, the structure of unbound NPDBP is less fixed, being characterized by rapidly interchanging conformations corresponding to an open chain structure with less overall proximity of the termini compared to NPPC. The overall shape of NPPC is essentially unaltered by binding, whereas in the case of NPDBP what was a minor conformation in the unbound state becomes the predominate conformation of the bound ligand. Thus, our results are consistent with these antibodies providing a molecular template for stabilizing the conformation of the bound ligand.     

Clearance kinetics and immunochemistry in rabbits of soluble antibody/DNA immune complexes. Effects of antibody class and DNA conformation

We examined the clearance kinetics in rabbits of soluble antibody/DNA immune complexes (IC) containing either IgG or IgM anti-DNA antibodies. Differences in the complement-mediated binding of these IC to rabbit blood cells (platelets) were also studied. Complexation of either double-stranded (ds) or single-stranded (ss) DNA with IgG anti-DNA tends to preclude in vivo DNA recognition mechanisms; the DNA is cleared as part of an IC at a rate slower than that of free DNA. Binding of ds- or ssDNA by IgM anti-DNA antibodies leads to formation of IC which are cleared more like free DNA, and this effect is most evident for ssDNA. However, although both IgG- and IgM-containing IC bound rapidly to blood cells in vivo, significant differences in their immunochemistry were apparent. For example, the DNA in IgM-containing IC was more susceptible to both in vivo and in vitro degradation. In addition, the binding of IgM-containing IC to rabbit platelets and human red blood cells was considerably more labile. Based on this systematic investigation of the soluble antibody/DNA IC that can potentially form in the circulation of a patient with systemic lupus erythematosus, it should be possible to formulate predictions regarding the relative pathogenic potential of these IC.     

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.     

Studies on the neutralization of herpes simplex virus. VIII. Significance of viral sensitization for inactivation by complement.

Early and late IgG of rabbits immunized with herpes virus showed, respectively, 8-fold and 2-fold enhancement of neutralization endpoint in the presence of complement (C). Kinetic curve experiments employing an appropriate amount of virus revealed that both neutralization and sensitization followed first-order reaction, and each IgG possessed a certain range of concentration where neutralization was negligible while sensitization was marked. Dose responses of neutralization and sensitization velocities demonstrated that the C enhancement of late IgG was about 7-fold and that of early IgG more than 20-fold. These facts suggested that the IgGs contained two different entities of complement-requiring (CRN) and non-requiring neutralizing (N) antibodies at different proportions, only the former being responsible for sensitization. The different CRN: N ratios obtained by the endpoint and kinetic methods may mean either that the two antibodies differ in avidity for the virus or that the number of critical sites per virion for CRN antibody is greater than that for N antibody. In this interpretation, sensitization by CRN antibody as well as neutralization by N antibody is thought to result from attachment of a single antibody molecule to the viral critical site. Alternative explanations, ascribing the mechanism of neutralization to steric hindrance of critical sites or to multiple hit of those sites by antibody, were denied by analyses of the present data.     

Structural similarities among enzyme pterin binding sites as demonstrated by a monoclonal anti-idiotypic antibody

BALB/c mice were immunized with a synthetic co-factor of the aromatic amino acid hydroxylases, 6,7-dimethyl-5,6,7,8-tetrahydropterin, conjugated to albumin. Hybridoma cell lines isolated from the immunized mice secreted monoclonal antibodies reacting specifically with the pterin molecule and monoclonal antibodies which were found to bind phenylalanine hydroxylase. Several lines of evidence were consistent with the anti-phenylalanine hydroxylase antibodies being anti-idiotype antibodies mimicking the pterin molecule and binding to the pterin binding site of phenylalanine hydroxylase. (a) An anti-idiotype monoclonal antibody, NS7, when reimmunized into mice produced anti-pterin antibodies consistent with NS7 being an internal image anti-idiotypic antibody. (b) NS7 antibody was prevented from binding to phenylalanine hydroxylase when a competitive inhibitor of phenylalanine hydroxylase enzyme activity, 6,7-dimethyl-7,8-dihydropterin, was bound to phenylalanine hydroxylase. (c) NS7 antibody was shown to bind to a wide range of pterin-requiring enzymes: phenylalanine, tyrosine and tryptophan hydroxylases, dihydropteridine reductase, dihydrofolate reductase, and sepiapterin reductase. Thus the NS7 antibody has successfully mimicked a common portion of the pterin cofactors utilized by these enzymes and demonstrated structure homology in their pterin binding sites despite their diverse function and little amino acid sequence homology except among the three aromatic amino acid hydroxylases.     

The carbohydrate at FcgammaRIIIa Asn-162. An element required for high affinity binding to non-fucosylated IgG glycoforms

FcgammaRIIIa plays a prominent role in the elimination of tumor cells by antibody-based cancer therapies. Non-fucosylated bisected IgGs bind this receptor with increased affinity and trigger FcgammaRIII-mediated effector functions more efficiently than native, fucosylated antibodies. In this study the contribution of the carbohydrates of both binding partners to the strength of the complex was analyzed. Glycoengineering of the antibody increased affinity for two polymorphic forms of soluble human FcgammaRIIIa (by up to 50-fold) but did not affect binding to the inhibitory FcgammaRIIb receptor. While the absence of carbohydrate at FcgammaRIIIa's Asn-162 increased affinity for native IgG, presumably due to the removal of steric hindrance caused by the bulky sugars, it unexpectedly reduced affinity for glycoengineered (GE) antibodies by over one order of magnitude, bringing the affinity down to the same level as for native IgG. We conclude that the high affinity between GE antibodies and FcgammaRIII is mediated by productive interactions formed between the receptor carbohydrate attached at Asn-162 and regions of the Fc that are only accessible when it is nonfucosylated. As FcgammaRIIIa and FcgammaRIIIb are the only human Fcgamma receptors glycosylated at this position, the proposed interactions explain the observed selective affinity increase of GE antibodies for only these receptors. Furthermore, we predict from our structural model that only one of the two Fc-fucose residues needs to be absent for increased binding affinity toward FcgammaRIII. This information can be exploited for the design of new antibodies with altered Fc receptor binding affinity and enhanced therapeutic potential.