Haloperidol binding to monoclonal antibodies. Hypervariable region amino acid sequence determination

The primary sequences of five monoclonal antibodies (mAbs A-E) which bind with various affinities (Kd = 4-810 nM) to the D-2 dopaminergic antagonist, haloperidol, have been determined. Immunoglobulin light and heavy chain mRNA was isolated and gene sequence determined by primer extension in the presence of dideoxynucleotides. The pattern of insertions and deletions found within the hypervariable regions produce loops which differ in length from one antibody to another, and are directly responsible for establishing the gross architecture of the combining site. Two of the anti-haloperidol mAbs have long hypervariable loops which form a pocket-shaped combining site. Three other mAbs have deletions of 3 or 4 amino acids in the third heavy chain complementarity producing region which result in a groove-like combining site as determined by computer based molecular modeling. A discussion of the probable mechanism by which the given sequences were generated from various gene segments is also presented.     

Analysis of the binding site architecture of monoclonal antibodies to morphine by using competitive ligand binding and molecular modeling

The structural features of mAb directed against the opiate morphine were analyzed by using competitive ligand analog-binding studies, examination of the V region amino acid sequence, and computer-aided molecular modeling of the fragment V region. The antibody response in BALB/c mice to morphine is relatively restricted, in that all of the mAb examined in this study contained the same lambda L chain and very similar H chain V regions. A three-dimensional model of the antimorphine-binding site was constructed by using computational and graphic display techniques. Each of the six complementary-determining regions was constructed by using fragment replacement methods employing canonical loop conformations of known "parent" structures. Experimental competitive ligand-binding data and theoretical modeling suggest that a charged glutamate residue at position H:50 and aromatic side chains of residues H:33W, H:47W, H:58F, H:95W, H:101iY, and L:91W are key features in ionic and hydrophobic interactions with the ligand. This study represents the first use of theoretical and experimental modeling techniques to describe the Ag-binding site of a mouse fragment V region containing a lambda L chain.     

Active site structure and antigen binding properties of idiotypically cross-reactive anti-fluorescein monoclonal antibodies

This report includes complete VH and V kappa nucleotide and deduced amino acid sequences of idiotypically cross-reactive monoclonal anti-fluorescein antibodies that differed greater than 10(5)-fold in affinity. High affinity monoclonal antibody 4-4-20 and intermediate affinity antibodies 10-25, 5-14, 9-40, 12-40, and 3-24 utilized greater than or equal to 90% homologous VHIIIC germ-line genes. Extensive D segment length and sequence variability were observed; however, compensatory germ-line JH4 (4-4-20 and 3-24) or JH3 (10-25, 5-14, 9-40, and 12-40) sequence lengths resulted in H chain CDR3 + FR4 to be a constant 18 amino acids. In addition, each antibody and low affinity 3-13 rearranged greater than or equal to 96% homologous V kappa II genes to J kappa 1, except for 10-25 (J kappa 5) and 3-13 (J kappa 4). Resolved crystal structure of complexed fluorescein and 4-4-20 Fab fragments revealed residues HisL27d, TyrL32, ArgL34, SerL91, TrpL96, and TrpH33 acted as hapten contact residues. Antibodies 5-14, 9-40, 12-40, and 3-24 primary structures possessed identical contact residues as 4-4-20 except for the substitution of HisL34 for ArgL34. Thus, ArgL34 was implicated in the increased affinity of monoclonal antibody 4-4-20. Finally, it was difficult to correlate extensive H chain CDR3 residue heterogeneity directly with fluorescein binding and idiotypy.     

Biological activities of binding site specific monoclonal antibodies to prolactin receptors of rabbit mammary gland

The biological activity of three monoclonal antibodies (mAbs) against the rabbit mammary prolactin (PRL) receptor (M110, A82, and A917) were investigated using explants of rabbit mammary gland. The three mAbs which were all able to inhibit the binding of 125I-ovine prolactin to its receptor had different biological activities. Two mAbs (M110 and A82) were able to prevent the stimulating effect of PRL on casein synthesis when the molar ratio between the mAb and PRL was 100. At a lower concentration, M110 moved the PRL dose-response curve to the right by a factor of 2.4. This mAb was also effective in vivo, reducing milk production in a lactating rabbit, in a similar fashion to the prolactin lowering drug, CB-154. One mAb (A917) was able to mimic the action of PRL on both casein and DNA ([3H]thymidine incorporation) synthesis, whereas the other two mAbs were without any stimulatory effect. For this stimulatory effect to be observed, bivalency of the antibody was essential, since monovalent fragments, which were able to inhibit PRL binding, had no agonistic activity. The ability of the mAbs to induce a down-regulation of receptors was also studied. M110, which was equipotent to PRL in occupation of receptors, induced no down-regulation, while A917, which had full biological activity, induced only a small degree of down-regulation. These studies suggest that the binding domain of the receptor might be relatively complex, since only a part of this domain recognized by the antibody with PRL-like activity was able to induce hormonal action. Alternatively, only those antibodies able to microaggregate the receptors may possess PRL-like activity.     

Computer-aided molecular modeling of the binding site architecture for eight monoclonal antibodies that bind a high potency guanidinium sweetener

Computer-aided molecular modeling of the antibody binding site of eight different monoclonal antibodies (mAb) that bind the intense sweetener ligand (N-(p-cyanophenyl)-N'-diphenylmethyl) guanidine acetic acid was completed using canonical loop structures and framework regions from known immuno globulins as “parent structures” for the molecular scaffoldings. The models of the fragment variable (Fv) region of the mAb were analyzed/or the presence and location of residues predicted to be involved in ligand binding. Several binding site tryptophan residues in these models were located in positions that support previous flurospectroscopic observations of the mAb-ligand complexation. Computer-aided renderings of the electrostatic potential at the van der Waals surface of the Fv region were compared and found to be consistent with the ligand binding specificity profiles for the different mAb. The Fv model of mAb NC6.8 was consistent with the binding site features determined in the Fab structure recently solved by X-ray diffraction techniques. These Fv models should provide an adequate basis for site-directed mutagenesis experiments in order to characterize interactive motifs in the mAb binding site.     

Temperature-dependent binding of monoclonal antibodies to C hordein

The consensus octapeptide repeat motif of the barley seed storage protein C hordein, Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln, forms the epitope of two anti-prolamin monoclonal antibodies (Mabs), IFRN 0061 and 0614. The Mabs were found to exhibit unusual temperature-dependent binding characteristics, recognising C hordein and a peptide corresponding to the consensus repeat at 5°C but not at 37°C, as determined by enzyme-linked immunosorbent assay (ELISA). The K_d of IFRN 0614 for the consensus peptide was found to be 1.2×10¹² mol⁻¹ at 12°C, but no constant could be calculated at 37°C due to a lack of binding. Similar ELISA binding characteristics were observed with an anti-C hordein polyclonal antiserum and a Mab raised to the consensus peptide. Circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy showed that the protein and the consensus peptide exist in a temperature-dependent equilibrium of poly-L-proline II type structures and β-turn conformations. Whilst thermodynamic and kinetic effects may reduce antibody binding at higher temperatures, they cannot account for the complete loss of Mab recognition at higher temperatures. It seems likely that the Mabs preferentially recognise the Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln motif when presented in a conformation which may correspond to the poly-L-proline II type conformation which dominates the CD and FTIR spectra at 4-12°C.     

Recognition of the acetylcholine receptor binding site of a long-chain neurotoxin by toxin-specific monoclonal antibodies.

The present paper reports the preparation and characterization of two neutralizing monoclonal antibodies (Mabs), called MST1 and MST2, which bind at the central loop of a long-chain neurotoxin from cobra venom. The central loop is a critical region for the binding of the toxin to the nicotinic acetylcholine receptor. Some of the residues incorporated in the epitopes recognized by MST1 and MST2 have been identified on the basis of competition experiments using a set of 'chemical mutants' of the toxin. We show that MST1 and MST2 bind at the base and at the tip of the central loop of the toxin, respectively, however, only MST2 actually overlaps the acetylcholine receptor binding site. Accordingly, only MST2 is capable of recognizing all homologous toxins so far examined. MST2, therefore, mimicks, at least partially, the site by which the nicotinic acetylcholine receptor recognizes a long-chain neurotoxin.     

Characterization of monoclonal antibodies to the beta-adrenergic antagonist alprenolol as models of the receptor binding site

Antibodies to receptor ligands have been valuable in understanding the nature of receptor-ligand interactions. We have developed four monoclonal antibodies to the beta-adrenergic receptor antagonist alprenolol by immunizing A/J mice with (-)-alprenolol coupled to keyhole limpet hemocyanin. The antisera from these mice displayed specific [3H]dihydroalprenolol ([3H]DHA) binding that was inhibited by alprenolol, propranolol, and isoproterenol. Somatic cell fusion of spleen cells from the immunized mice to SP2/0 myeloma cells, followed by limited dilution subcloning, resulted in the isolation of four hybridomas (1B7, 5B7, 5D9, and 2G9) demonstrating three different classes of ligand binding characteristics. 1B7 had the highest binding affinity for antagonists based on Scatchard analysis (Kd [125I]- CYP = 1.4 X 10(-10) M; Kd [3H]DHA = 6.5 X 10(-9) M), and was the only antibody to demonstrate agonist-inhibition of [3H]DHA binding. Ki values computed from competitive inhibition curves of [3H]DHA binding to 1B7 resulted in a rank order of potency similar to that of beta-2-adrenergic receptors: (-)-propranolol greater than acebutolol amine greater than isoproterenol greater than (+)-propranolol greater than epinephrine greater than norepinephrine. 5B7 and 5D9 exemplified a second class of antibody. This pair had lower antagonist binding affinities (Kd [3H]DHA = 2 X 10(-8) M and 2.5 X 10(-7) M, respectively) and was stereoselective in binding receptor antagonists: (-)-propranolol greater than (+)-propranolol greater than acebutolol amine. Agonist inhibition of [3H]DHA binding to these antibodies could only be observed at very high concentrations (greater than 10(-4) M agonist), and was not dose-dependent. Finally, the class of anti-alprenolol monoclonal antibodies represented by 2G9 had the lowest antagonist binding affinity of all (IC50 alprenolol = 1 X 10(-5) M), did not demonstrate ligand stereoselectivity, and did not recognize agonists. We propose that antibodies raised against beta-adrenergic receptor ligands demonstrating stereoselective agonist binding will also demonstrate high affinity antagonist binding, and that they will closely parallel the binding characteristics of the receptor. According to this "agonist best-fit hypothesis," anti-idiotypic antibodies raised against the binding site of these idiotypes might contain true mirror images of the beta-adrenergic receptor binding site.     

Three-dimensional structure-activity relationship modeling of cocaine binding to two monoclonal antibodies by comparative molecular field analysis

Successful immunotherapy of cocaine addiction and overdoses requires cocaine-binding antibodies with specific properties, such as high affinity and selectivity for cocaine. We have determined the affinities of two cocaine-binding murine monoclonal antibodies (mAb: clones 3P1A6 and MM0240PA) for cocaine and its metabolites by [3H]-radioligand binding assays. mAb 3P1A6 (K(d) = 0.22 nM) displayed a 50-fold higher affinity for cocaine than mAb MM0240PA (K(d) = 11 nM) and also had a greater specificity for cocaine. For the systematic exploration of both antibodies' binding specificities, we used a set of approximately 35 cocaine analogues as structural probes by determining their relative binding affinities (RBAs) using an enzyme-linked immunosorbent competition assay. Three-dimensional quantitative structure-activity relationship (3D-QSAR) models on the basis of comparative molecular field analysis (CoMFA) techniques correlated the binding data with structural features of the ligands. The analysis indicated that despite the mAbs' differing specificities for cocaine, the relative contributions of the steric (approximately 80%) and electrostatic (approximately 20%) field interactions to ligand-binding were similar. Generated three-dimensional CoMFA contour plots then located the specific regions about cocaine where the ligand/receptor interactions occurred. While the overall binding patterns of the two mAbs had many features in common, distinct differences were observed about the phenyl ring and the methylester group of cocaine. Furthermore, using previously published data, a 3D-QSAR model was developed for cocaine binding to the dopamine reuptake transporter (DAT) that was compared to the mAb models. Although the relative steric and electrostatic field contributions were similar to those of the mAbs, the DAT cocaine-binding site showed a preference for negatively charged ligands. Besides establishing molecular level insight into the interactions that govern cocaine binding specificity by biopolymers, the three-dimensional images obtained reflect the properties of the mAbs binding pockets and provide the initial information needed for the possible design of novel antibodies with properties optimized for immunotherapy.     

Binding of synthetic peptides by a human monoclonal IgM with an unusual combining site structure

Using X-ray crystallography, a human monoclonal IgM cryoglobulin (Mez) was found to have an unusual combining site topography. Analysis of the unliganded Fv at 2.6 A resolution revealed that the HCDR3 had partitioned the active site into two compartments [Ramsland PA et al. 2000. Mol. Immunol. 37: 295-310]. The two cavities had dimensions and chemical properties that were compatible with the binding of peptides. In this study, libraries of peptides were prepared using solid-phase synthesis. Binding of the intact Mez IgM to these peptides was tested by enzyme-linked immunoassays. Screening of 400 dipeptides revealed that binding was markedly skewed toward amino acids with aromatic side-chains (Phe and Trp), especially when located in the second position. Preferential recognition of aromatic side-chains by Mez IgM was confirmed with larger peptides of three to five residues, but C-terminal positioning was not favored in these peptides. Mez IgM also showed binding propensities for acidic residues (Asp and Glu) as well as several other side-chains with different chemical properties, including His, Pro, Asn and Gln. Mez IgM recognized sets of overlapping octapeptides representing the sequences of the constant domains of human IgG1 heavy chains. These peptides represented similar stretches of polypeptide on the three-dimensional structures of all three constant domains (CH1, CH2 and CH3). Thus, Mez IgM may recognize structurally homologous regions of immunoglobulin domains, which were conserved during the evolution of the immune system.     

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.     

A very high affinity opioid binding site in rat brain: demonstration by computer modeling

We present substantial new evidence for at least four distinct types of opioid receptors in rat brain, using quantitative ligand binding studies and mathematical modeling. Three of these binding sites are consistent with the well established "mu", "delta" and "kappa" receptors. The fourth has two distinctive features: 1) extremely high affinity (dissociation constant less than 1 nM); 2) almost complete lack of specificity for the classical "delta" or "mu" selective ligands. These properties are consistent with the putative "mu1" receptor described by Pasternak and coworkers.     

Mapping the binding of monoclonal antibodies to histone H5

The binding sites of nine monoclonal antibodies along the polypeptide chain of histone H5 were mapped. Immunoblotting experiments with peptides generated from H5 by trypsin digestion, N-bromosuccinimide cleavage, and cyanogen bromide cleavage revealed that all of the monoclonal antibodies reacted with the globular region of H5 which is encompassed by amino acid residues 22-98. Within this globular segment, the epitopes could be subdivided into three regions. Monoclonals 1G11, 2E5, and 2H5 bind to residues 28-31. The close proximity of the epitopes was verified by a competitive enzyme-linked immunosorbent assay and by their binding pattern to a tryptic digest of H5. Monoclonals 4C6, 6E12, and 2E12 bind to a region encompassed by amino acids 28-53 while monoclonals 4H7, 1C3, and 3H9 bind to a region encompassed by residues 53-98. Precise localization of the epitopes in the primary sequence of H5 will allow detailed studies on the mode of binding of H5 to core particles in chromatin.     

Anti-idiotypic monoclonal antibodies to GM1 identify ganglioside binding proteins

Ganglioside stimulated neurite outgrowth may be due to gangliosidebinding to membrane proteins or to intercalation into the membrane.To test that ganglioside binding proteins could be found onneuronal surfaces, antiidiotypic ganglioside monoclonal antibodies(AIG mAbs) were generated to mimic the biological propertiesof the GM1 ganglioside. The AIG mAbs were identified by theirability to bind to a known GM1 binding protein, the ß-subunit of cholera toxin. For the two AIG mAbs studied, AIG5 andAIG20, binding to ß-CT was blocked most strongly byGM1. This data also suggests that AIG5 and AIG20 mimic differentbut overlapping epitopes of the ganglioside GM1. Western blottingand immunoprecipitation of mammalian tissues reveals four potentialganglioside binding proteins of molecular weight 93, 66, 57,and 45 kDa. Immunocytochemistry demonstrates neuronal surfacelabel with the AIG mAbs, which suggests that gangliosides, enrichedon the neuronal surface membrane, are co-localized with putativeganglioside binding proteins. In bioassays, the AIG mAbs promoteneuronal sprouting. This shows that these antibodies can beused to study the biological effects of ganglioside bindingto neuronal surface proteins, and the role of gangliosides inthe activation of neurite outgrowth. agonist antibody anti-idiotypic antibody gangliosides ganglioside binding proteins     

Structural predictions of the binding site architecture for monoclonal antibody NC6.8 using computer-aided molecular modeling, ligand binding, and spectroscopy.

Monoclonal antibody NC6.8 binds the superpotent sweetener ligand N-(p-cyanophenyl)-N'-(diphenylmethyl) guanidineacetic acid with high affinity (Kd = 53 nM). Using computer-aided molecular modeling and several experimental techniques, such as competitive ligand binding, absorbance spectroscopy, and fluorescence spectroscopy, we have predicted the structure of the variable domain fragment (Fv) and identified the key residues in the combining site of the antibody. We have identified nine specific amino acids as being involved in ligand recognition and complexation. Most notable are H:33W, which is responsible for ligand-induced tryptophan fluorescence quenching, H:56R, which forms a salt bridge with the carboxylate moiety of the ligand, and L:34H, which, deep in the binding site, interacts with the cyanophenyl portion of the ligand. Two residues located deep in the putative binding pocket, H:35E and H:50E, provide the negatively charged potential for interaction with the protonated aryl nitrogen and the positive guanidinium group. These modeling predictions were made before the solution of high-resolution structures of the native Fab (2.6 A) and the Fab-ligand complex (2.2 A). Comparisons between the theoretical model and experimental native and liganded Fab structures are made.     

Modes of binding of 2'-AMP to RNase T1. A computer modeling study.

The modes of binding of adenosine 2'-monophosphate (2'-AMP) to the enzyme ribonuclease (RNase) T1 were determined by computer modelling studies. The phosphate moiety of 2'-AMP binds at the primary phosphate binding site. However, adenine can occupy two distinct sites--(1) The primary base binding site where the guanine of 2'-GMP binds and (2) The subsite close to the N1 subsite for the base on the 3'-side of guanine in a guanyl dinucleotide. The minimum energy conformers corresponding to the two modes of binding of 2'-AMP to RNase T1 were found to be of nearly the same energy implying that in solution 2'-AMP binds to the enzyme in both modes. The conformation of the inhibitor and the predicted hydrogen bonding scheme for the RNase T1-2'-AMP complex in the second binding mode (S) agrees well with the reported x-ray crystallographic study. The existence of the first mode of binding explains the experimental observations that RNase T1 catalyses the hydrolysis of phosphodiester bonds adjacent to adenosine at high enzyme concentrations. A comparison of the interactions of 2'-AMP and 2'-GMP with RNase T1 reveals that Glu58 and Asn98 at the phosphate binding site and Glu46 at the base binding site preferentially stabilise the enzyme-2'-GMP complex.     

Stoichiometric binding of apolipoprotein B-specific monoclonal antibodies to low density lipoproteins

The structure of apolipoprotein B and its stoichiometry on plasma lipoproteins has been a major issue and one refractory to a variety of analyses. Immunochemical analyses represent an independent approach. Examinations of apolipoprotein B (apo-B) epitopes on human plasma low density lipoproteins (LDL) using monoclonal antibodies have consistently revealed the existence of extensive apo-B heterogeneity. In the present study, we have addressed the solution of the stoichiometry problem using quantitative analysis of the maximum number of identical antibodies that can be bound per LDL particle in which we take into account this ligand heterogeneity. We have estimated the molecular weight of apo-B by quantifying the number of times a given apo-B epitope is expressed on the surface of LDL. The quantitative binding of eight previously characterized monoclonal antibodies was measured in a fluid phase radioimmunoassay. The results were analyzed by Scatchard analysis and expressed on the basis of independent measurements of the maximum amount of LDL that could be bound by each antibody. Affinity constants for each of the eight antibodies varied between 8.5 X 10(7) and 80 X 10(7) M-1. For these same antibodies, the concentration of maximally bound antibody at a normalized LDL concentration of 1000 ng/ml was estimated to be 0.9-1.8 nM with a mean of 1.23 nM. Adopting a molecular mass from physicochemical analysis for LDL apo-B of 550,000 daltons, the molar ratio between bound antibody and LDL varied between 0.5 and 1.2 (mean 0.75 +/- 0.15). The results supported the hypothesis that apo-B is present as a single large molecular weight polypeptide in LDL.     

Isolation and functional effects of monoclonal antibodies binding to thymidylate synthase

Monoclonal antibodies against electrophoretically pure thymidylate synthase from HeLa cells have been produced. Antibodies (M-TS-4 and M-TS-9) from hybridoma clones were shown by enzyme-linked immunoassay to recognize thymidylate synthase from a variety of human cell lines, but they did not bind to thymidylate synthase from mouse cell lines. The strongest binding of antibodies was observed to enzyme from HeLa cells. These two monoclonal antibodies bind simultaneously to different antigenic sites on thymidylate synthase purified from HeLa cells, as reflected by a high additivity index and results of cross-linked radioimmunoassay. Both monoclonal antibodies inhibit the activity of thymidylate synthase from human cell lines. The strongest inhibition was observed with thymidylate synthase from HeLa cells. Monoclonal antibody M-TS-9 (IgM subclass) decreased the rate of binding of [3H]FdUMP to thymidylate synthase in the presence of 5,10-methylenetetrahydrofolate while M-TS-4 (IgG1) did not change the rate of ternary complex formation. These data indicate that the antibodies recognize different epitopes on the enzyme molecule.     

Homophilic binding of mouse monoclonal antibodies against GD3 ganglioside.

R24, a mouse IgG3 mAb against GD3 ganglioside, was shown to bind to itself in a homophilic manner. This was demonstrated by augmented binding of 125I-labeled R24 to the cell surface of GD3+ cells by unlabeled R24 and by direct binding of biotinylated R24 to R24 adsorbed on solid phase. Although homophilic binding was evident when R24 was bound to solid phase, R24-R24 aggregates could not be detected in solution under otherwise identical conditions. R24 bound to four other mAb (two IgG3, one IgG2a, one IgM) directed against GD3 but did not bind to a panel of 21 other mAb including other IgG3 mAb and mAb directed against non-GD3 ganglioside. Evidence implicating the GD3-binding site of R24 in homophilic binding included the following observations: 1) F(ab')2 fragments of R24 could bind to R24, 2) an antiidiotypic mAb against the GD3-binding site of R24 inhibited R24 homophilic binding, 3) an IgM anti-GD3 mAb also demonstrated homophilic binding to R24, and 4) homophilic binding was a function of immunoreactivity and avidity for GD3. R24 variants with 40-fold lower avidity for GD3 demonstrated a similar decrease in homophilic binding. Inasmuch as R24 bound to R24 F(ab')2 fragments and specifically to anti-GD3 mAb, it appeared that the target for homophilic binding was an epitope within the V region of anti-GD3 mAb. It is likely that homophilic interactions result in increased affinity of R24 for GD3 through increased effective valency of antibody-Ag complexes.