Antibodies with infinite affinity

Here we report an approach to the design and production of antibody/ligand pairs, to achieve functional affinity far greater than avidin/biotin. Using fundamental chemical principles, we have developed antibody/ligand pairs that retain the binding specificity of the antibody, but do not dissociate. Choosing a structurally characterized antibody/ligand pair as an example, we engineered complementary reactive groups in the antibody binding pocket and the ligand, so that they would be in close proximity in the antibody/ligand complex. Cross-reactions with other molecules in the medium are averted because of the low reactivity of these groups; however, in the antibody/ligand complex the effective local concentrations of the complementary reactive groups are very large, allowing a covalent reaction to link the two together. By eliminating the dissociation of the ligand from the antibody, we have made the affinity functionally infinite. This chemical manipulation of affinity is applicable to other biological binding pairs.

In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei

Most eukaryotic telomeres contain a repeating motif with stretches of guanine residues that form a 3′-terminal overhang extending beyond the telomeric duplex region. The telomeric repeat of hypotrichous ciliates, d(T4G4), forms a 16-nucleotide 3′-overhang. Such sequences can adopt parallel-stranded as well as antiparallel-stranded quadruplex conformations in vitro. Although it has been proposed that guanine-quadruplex conformations may have important cellular roles including telomere function, recombination, and transcription, evidence for the existence of this DNA structure in vivo has been elusive to date. We have generated high-affinity single-chain antibody fragment (scFv) probes for the guanine-quadruplex formed by the Stylonychia telomeric repeat, by ribosome display from the Human Combinatorial Antibody Library. Of the scFvs selected, one (Sty3) had an affinity of Kd = 125 pM for the parallel-stranded guanine-quadruplex and could discriminate with at least 1,000-fold specificity between parallel or antiparallel quadruplex conformations formed by the same sequence motif. A second scFv (Sty49) bound both the parallel and antiparallel quadruplex with similar (Kd = 3–5 nM) affinity. Indirect immunofluorescence studies show that Sty49 reacts specifically with the macronucleus but not the micronucleus of Stylonychia lemnae. The replication band, the region where replication and telomere elongation take place, was also not stained, suggesting that the guanine-quadruplex is resolved during replication. Our results provide experimental evidence that the telomeres of Stylonychia macronuclei adopt in vivo a guanine-quadruplex structure, indicating that this structure may have an important role for telomere functioning.

Identification of an autoimmune serum containing antibodies against the Barr body

Transcriptional inactivation of one X chromosome in mammalian female somatic cells leads to condensation of the inactive X chromosome into the heterochromatic sex chromatin, or Barr body. Little is known about the molecular composition and structure of the Barr body or the mechanisms leading to its formation in female nuclei. Because human sera from patients with autoimmune diseases often contain antibodies against a variety of cellular components, we reasoned that some autoimmune sera may contain antibodies against proteins associated with the Barr body. Therefore, we screened autoimmune sera by immunofluorescence of human fibroblasts and identified one serum that immunostained a distinct nuclear structure with a size and nuclear localization consistent with the Barr body. The number of these structures was consistent with the number of Barr bodies expected in diploid female fibroblasts containing two to five X chromosomes. Immunostaining with the serum followed by fluorescence in situ hybridization with a probe against XIST RNA demonstrated that the major fluorescent signal from the autoantibody colocalized with XIST RNA. Further analysis of the serum showed that it stains human metaphase chromosomes and a nuclear structure consistent with the inactive X in female mouse fibroblasts. However, it does not exhibit localization to a Barr body-like structure in female mouse embryonic stem cells or in cells from female mouse E7.5 embryos. The lack of staining of the inactive X in cells from female E7.5 embryos suggests the antigen(s) may be involved in X inactivation at a stage subsequent to initiation of X inactivation. This demonstration of an autoantibody recognizing an antigen(s) associated with the Barr body presents a strategy for identifying molecular components of the Barr body and examining the molecular basis of X inactivation.

Patterning of functional antibodies and other proteins by photolithography of silane monolayers.

We have demonstrated the assembly of two-dimensional patterns of functional antibodies on a surface. In particular, we have selectively adsorbed micrometer-scale regions of biotinylated immunoglobulin that exhibit specific antigen binding after adsorption. The advantage of this technique is its potential adaptability to adsorbing arbitrary proteins in tightly packed monolayers while retaining functionality. The procedure begins with the formation of a self-assembled monolayer of n-octadecyltrimethoxysilane (OTMS) on a silicon dioxide surface. This monolayer can then be selectively removed by UV photolithography. Under appropriate solution conditions, the OTMS regions will adsorb a monolayer of bovine serum albumin (BSA), while the silicon dioxide regions where the OTMS has been removed by UV light will adsorb less than 2% of a monolayer, thus creating high contrast patterned adsorption of BSA. The attachment of the molecule biotin to the BSA allows the pattern to be replicated in a layer of streptavidin, which bonds to the biotinylated BSA and in turn will bond an additional layer of an arbitrary biotinylated protein. In our test case, functionality of the biotinylated goat antibodies raised against mouse immunoglobulin was demonstrated by the specific binding of fluorescently labeled mouse IgG.

Random phage mimotopes recognized by monoclonal antibodies against the pyruvate dehydrogenase complex-E2 (PDC-E2).

Dihydrolipoamide acetyltransferase, the E2 component of the pyruvate dehydrogenase complex (PDC-E2), is the autoantigen most commonly recognized by autoantibodies in primary biliary cirrhosis (PBC). We identified a peptide mimotope(s) of PDC-E2 by screening a phage-epitope library expressing random dodecapeptides in the pIII coat protein of fd phage using C355.1, a murine monoclonal antibody (mAb) that recognizes a conformation-dependent epitope in the inner lipoyl domain of PDC-E2 and uniquely stains the apical region of bile duct epithelium (BDE) only in patients with PBC. Eight different sequences were identified in 36 phage clones. WMSYPDRTLRTS was present in 29 clones; WESYPFRVGTSL, APKTYVSVSGMV, LTYVSLQGRQGH, LDYVPLKHRHRH, AALWGVKVRHVS, KVLNRIMAGVRH and GNVALVSSRVNA were singly represented. Three common amino acid motifs (W-SYP, TYVS, and VRH) were shared among all peptide sequences. Competitive inhibition of the immunohistochemical staining of PBC BDE was performed by incubating the peptides WMSYPDRTLRTS, WESYPDRTLRTS, APKTYVSVSGMV, and AALWGVKVRHVS with either C355.1 or a second PDC-E2-specific mAb, C150.1. Both mAbs were originally generated to PDC-E2 but map to distinct regions of PDC-E2. Two of the peptides, although selected by reaction with C355.1, strongly inhibited the staining of BDE by C150.1, whereas the peptide APKTYVSVSGMV consistently inhibited the staining of C355.1 on biliary duct epithelium more strongly than the typical mitochondrial staining of hepatocytes. Rabbit sera raised against the peptide WMSYPDRTLRTS stained BDE of livers and isolated bile duct epithelial cells of PBC patients more intensively than controls. The rabbit sera stained all size ducts in normals, but only small/medium-sized ductules in PBC livers. These studies provide evidence that the antigen present in BDE is a molecular mimic of PDC-E2, and not PDC-E2 itself.

Identification of peptides specific for cerebrospinal fluid antibodies in multiple sclerosis by using phage libraries.

The study of the origin and pathogenetic relevance of the oligoclonal antibodies present in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients has been hampered by a lack of specific ligands. We recently reported a general strategy, based on phage-displayed random peptide libraries, to identify ligands for disease-specific antibodies even in the absence of any information on the nature of the pathologic antigen. With this procedure, we identified several peptides specifically recognized by antibodies present in the CSF of MS patients. Using these peptides as reagents, we demonstrated that they mimic different natural epitopes and react with antibodies enriched in the CSF of MS patients. Antibodies recognizing the selected peptides are commonly found with equal frequency in the sera of MS patients and of normal individuals. In contrast, the repertoire of CSF antibodies appears to be individual-specific and is probably the result of a nonspecific immunodysregulation rather than a stereotyped response to a single antigen/agent.

Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.

Guanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.

A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities.

We report a novel approach to the generation of monoclonal antibodies based on the molecular cloning and expression of immunoglobulin variable region cDNAs generated from single rabbit or murine lymphocytes that were selected for the production of specific antibodies. Single cells secreting antibodies for a specific peptide either from gp116 of the human cytomegalovirus or from gp120 of HIV-1 or for sheep red blood cells were selected using antigen-specific hemolytic plaque assays. Sheep red blood cells were coated with specific peptides in a procedure applicable to any antigen that can be biotinylated. Heavy- and light-chain variable region cDNAs were rescued from single cells by reverse transcription-PCR and expressed in the context of human immunoglobulin constant regions. These chimeric murine and rabbit monoclonal antibodies replicated the target specificities of the original antibody-forming cells. The selected lymphocyte antibody method exploits the in vivo mechanisms that generate high-affinity antibodies. This method can use lymphocytes from peripheral blood, can exploit a variety of procedures that identify individual lymphocytes producing a particular antibody, and is applicable to the generation of monoclonal antibodies from many species, including humans.

Circumventing tolerance to generate autologous monoclonal antibodies to the prion protein.

Prion diseases are disorders of protein conformation and do not provoke an immune response. Raising antibodies to the prion protein (PrP) has been difficult due to conservation of the PrP sequence and to inhibitory activity of alpha-PrP antibodies toward lymphocytes. To circumvent these problems, we immunized mice in which the PrP gene was ablated (Prnp 0/0) and retrieved specific monoclonal antibodies (mAbs) through phage display libraries. This approach yielded alpha-PrP mAbs that recognize mouse PrP. Studies with these mAbs suggest that cellular PrP adopts an unusually open structure consistent with the conformational plasticity of this protein.

A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis. The virus does not replicate efficiently in cell cultures, and it is therefore difficult to assess infection-neutralizing antibodies and to evaluate protective immunity in vitro. To study the binding of the HCV envelope to cell-surface receptors, we developed an assay to assess specific binding of recombinant envelope proteins to human cells and neutralization thereof. HCV recombinant envelope proteins expressed in various systems were incubated with human cells, and binding was assessed by flow cytometry using anti-envelope antibodies. Envelope glycoprotein 2 (E2) expressed in mammalian cells, but not in yeast or insect cells, binds human cells with high affinity (Kd approximately 10(-8) M). We then assessed antibodies able to neutralize E2 binding in the sera of both vaccinated and carrier chimpanzees, as well as in the sera of humans infected with various HCV genotypes. Vaccination with recombinant envelope proteins expressed in mammalian cells elicited high titers of neutralizing antibodies that correlated with protection from HCV challenge. HCV infection does not elicit neutralizing antibodies in most chimpanzees and humans, although low titers of neutralizing antibodies were detectable in a minority of infections. The ability to neutralize binding of E2 derived from the HCV-1 genotype was equally distributed among sera from patients infected with HCV genotypes 1, 2, and 3, demonstrating that binding of E2 is partly independent of E2 hypervariable regions. However, a mouse monoclonal antibody raised against the E2 hypervariable region 1 can partially neutralize binding of E2, indicating that at least two neutralizing epitopes, one of which is hypervariable, should exist on the E2 protein. The neutralization-of-binding assay described will be useful to study protective immunity to HCV infection and for vaccine development.

Anti-idiotypic antibodies mimicking glycoprotein D of herpes simplex virus identify a cellular protein required for virus spread from cell to cell and virus-induced polykaryocytosis.

Glycoprotein D (gD) of herpes simplex virus 1 (HSV-1) is required for stable attachment and penetration of the virus into susceptible cells after initial binding. We derived anti-idiotypic antibodies to the neutralizing monoclonal antibody HD1 to gD of HSV-1. These antibodies have the properties expected of antibodies against a gD receptor. Specifically, they bind to the surface of HEp-2, Vero, and HeLa cells susceptible to HSV infection and specifically react with a Mr 62,000 protein in these and other (143TK- and BHK) cell lines. They neutralize virion infectivity, drastically decrease plaque formation by impairing cell-to-cell spread of virions, and reduce polykaryocytosis induced by strain HFEM, which carries a syncytial (syn-) mutation. They do not affect HSV growth in a single-step cycle and plaque formation by an unrelated virus, indicating that they specifically affect the interaction of HSV gD) with a cell surface receptor. We conclude that the Mr 62,000 cell surface protein interacts with gD to enable spread of HSV-1 from cell to cell and virus-induced polykaryocytosis.

Interactions of protein antigens with antibodies.

There are now several crystal structures of antibody Fab fragments complexed to their protein antigens. These include Fab complexes with lysozyme, two Fab complexes with influenza virus neuraminidase, and three Fab complexes with their anti-idiotype Fabs. The pattern of binding that emerges is similar to that found with other protein-protein interactions, with good shape complementarity between the interacting surfaces and reasonable juxtapositions of polar residues so as to permit hydrogen-bond formation. Water molecules have been observed in cavities within the interface and on the periphery, where they often form bridging hydrogen bonds between antibody and antigen. For the most part the antigen is bound in the middle of the antibody combining site with most of the six complementarity-determining residues involved in binding. For the most studied antigen, lysozyme, the epitopes for four antibodies occupy approximately 45% of the accessible surface area. Some conformational changes have been observed to accompany binding in both the antibody and the antigen, although most of the information on conformational change in the latter comes from studies of complexes with small antigens.

Antibodies against specific extracellular epitopes of the glucagon receptor block glucagon binding.

Polyclonal antibodies were prepared against synthetic peptides corresponding to four different extramembrane segments of the rat glucagon receptor. The antibodies bound specifically to native glucagon receptor as judged by immunofluorescence microscopy of cultured cells expressing a synthetic gene for the receptor. Antibodies to peptides designated PR-15 and DK-12 were directed against amino acid residues 103-117 and 126-137, respectively, of the extracellular N-terminal tail. Antibody to peptide KD-14 was directed against residues 206-219 of the first extracellular loop, and antibody to peptide ST-18, against the intracellular C-terminal tail, residues 468-485. The DK-12 and KD-14 antibodies, but not the PR-15 and ST-18 antibodies, could effectively block binding of 125I-labeled glucagon to its receptor in liver membranes. Incubation of these antibodies with rat liver membranes resulted in both a decrease in the maximal hormonal binding capacity and an apparent decrease in glucagon affinity for its receptor. These effects were abolished in the presence of excess specific peptide antigen. In addition, DK-12 and KD-14 antibodies, but not PR-15 and ST-18 antibodies, interfered with glucagon-induced adenylyl cyclase activation in rat liver membranes and behaved as functional glucagon antagonists. These results demonstrate that DK-12 and KD-14 antibodies are pharmacologically active glucagon antagonists and strongly suggest that residues 126-137 of the N-terminal tail and residues 206-219 of the first extracellular loop contain determinants of ligand binding and may comprise the primary ligand-binding site on the glucagon receptor.

Monoclonal antibodies inhibit in vitro fibrillar aggregation of the Alzheimer beta-amyloid peptide.

The beta-amyloid peptide, the hallmark of Alzheimer disease, forms fibrillar toxic aggregates in brain tissue that can be dissolved only by strong denaturing agents. To study beta-amyloid formation and its inhibition, we prepared immune complexes with two monoclonal antibodies (mAbs), AMY-33 and 6F/3D, raised against beta-amyloid fragments spanning amino acid residues 1-28 and 8-17 of the beta-amyloid peptide chain, respectively. In vitro aggregation of beta-amyloid peptide was induced by incubation for 3 h at 37 degrees C and monitored by ELISA, negative staining electron microscopy, and fluorimetric studies. We found that the mAs prevent the aggregation of beta-amyloid peptide and that the inhibitory effect appears to be related to the localization of the antibody-binding sites and the nature of the aggregating agents. Preparation of mAbs against "aggregating epitopes," defined as sequences related to the sites where protein aggregation is initiated, may lead to the understanding and prevention of protein aggregation. The results of this study may provide a foundation for using mAbs in vivo to prevent the beta-amyloid peptide aggregation that is associated with Alzheimer disease.

Crystal structure of the complex of a catalytic antibody Fab fragment with a transition state analog: structural similarities in esterase-like catalytic antibodies.

The x-ray structure of the complex of a catalytic antibody Fab fragment with a phosphonate transition-state analog has been determined. The antibody (CNJ206) catalyzes the hydrolysis of p-nitrophenyl esters with significant rate enhancement and substrate specificity. Comparison of this structure with that of the uncomplexed Fab fragment suggests hapten-induced conformational changes: the shape of the combining site changes from a shallow groove in the uncomplexed Fab to a deep pocket where the hapten is buried. Three hydrogen-bond donors appear to stabilize the charged phosphonate group of the hapten: two NH groups of the heavy (H) chain complementarity-determining region 3 (H3 CDR) polypeptide chain and the side-chain of histidine-H35 in the H chain (His-H35) in the H1 CDR. The combining site shows striking structural similarities to that of antibody 17E8, which also has esterase activity. Both catalytic antibody ("abzyme") structures suggest that oxyanion stabilization plays a significant role in their rate acceleration. Additional catalytic groups that improve efficiency are not necessarily induced by the eliciting hapten; these groups may occur because of the variability in the combining sites of different monoclonal antibodies that bind to the same hapten.