Modulation of the M2 muscarinic acetylcholine receptor activity with monoclonal anti-M2 receptor antibody fragments

Antibodies directed against the second extracellular loop of G protein-coupled receptors are known to have functional activities. From a partial agonist monoclonal antibody directed against the M2 muscarinic receptor, we constructed and produced a single chain variable fragment with high affinity for its target epitope. The fragment is able to recognize its receptor on Chinese hamster ovary cells transfected with the M2 muscarinic acetylcholine receptor to block the effect of carbachol on this receptor and to exert an inverse agonist activity on the basal activity of the receptor. The antibody fragment is also able to increase the basal rhythm of cultured neonatal rat cardiomyocytes and to inhibit in a non-competitive manner the negative chronotropic effect of carbachol. This antibody fragment is able to exert its inverse agonist activity in vivo on mouse heart activity. The immunological strategy presented here could be useful to develop specific allosteric inverse agonist reagents for G protein-coupled receptors.     

Muscarinic cholinergic receptor of rat cerebral cortex. Location and characterization of ligand binding site-carrying peptides in synaptosomal membranes and isolated neuronal perikarya.

A careful examination of the location and biochemical properties of the tryptic peptides identified by site-specific labelling of the muscarinic cholinergic receptor (mAChR) of rat cerebral cortex has been carried out. In brain synaptosomal membranes and isolated neuronal perikarya, mAChR labelled with [3H]propylbenzilylcholine mustard (PrBCM) was tryptically cleaved to peptides of Mr 50,000, 30,000. 18,000 and a limiting fragment of Mr 8000. All of these binding site-carrying fragments, characterized in terms of their content of carbohydrates and thiol groups, were quantitatively recovered as membrane-bound peptides. The delipidated [3H]PrBCM-labelled tryptic limiting fragment was found to be highly hydrophobic and insoluble in aqueous media. Experiments performed with proteinase on the tryptic limiting fragment suggest the existence of an ester linkage between the ligand and the peptide. The results strongly support the hydropathicity profile which predicts the location of the muscarinic receptor protein with respect to the membrane bilayer.     

乙酰胆碱对大鼠体外抗体生成的影响

目的：观察不同浓度乙酰胆碱（ＡＣｈ,１０－１０～１０－５ｍｏｌ／Ｌ）对大鼠体外抗体生成的影响,并初步探讨其作用机制,从细胞水平了解乙酰胆碱与免疫功能之间的关系。方法：用体外抗体生成的检测方法,用绵羊红细胞（ＳＲＢＣ）刺激大鼠肠系膜淋巴结Ｂ细胞转化成抗体形成细胞（ＡＦＣ）,然后检测其抗体生成量。结果：①１０－１０～１０－７ｍｏｌ／ＬＡＣｈ能显著抑制体外抗体生成,其中１０－８和１０－７ｍｏｌ／ＬＡＣｈ的作用较强,而１０－６和１０－５ｍｏｌ／ＬＡＣｈ无明显的抑制作用;②Ｍ型胆碱能受体激动剂毛果芸香碱（１０－８和１０－７ｍｏｌ／Ｌ）能明显减弱体外抗体生成,而Ｎ型受体激动剂烟碱（１０－８和１０－７ｍｏｌ／Ｌ）没有显著的减弱作用,Ｍ型受体拮抗剂阿托品（１０－７和１０－６ｍｏｌ／Ｌ）可完全阻断ＡＣｈ抑制体外抗体生成的作用;③ＡＣｈ分别在Ｂ细胞用ＳＲＢＣ刺激后３～４８ｈ中的６个不同时间与淋巴细胞作用,其抗体生成仍然是减少的。结论：ＡＣｈ可非浓度依赖性地抑制大鼠的体外抗体生成;此作用可能由Ｂ细胞上的Ｍ型胆碱能受体介导;且ＡＣｈ可能主要影响Ｂ细胞转化的后期过程。     

Electrophoretic characterization of muscarinic receptors under denaturating and nondenaturating conditions: computer-assisted Ferguson plot analysis

The physical properties of the covalently labeled [( 3H]propylbenzilycholine mustard) muscarinic acetylcholine receptor from rat brain were studied by sodium dodecyl sulfate-polyacrylamide electrophoresis and computer-assisted Ferguson plot analysis. No proteolytic degradation or dimerization of the ligand binding subunit was found. No clues for different molecular weight forms or anomalous migration of the muscarinic receptor were detected. The weighted regression analysis of Ferguson plots gave an apparent molecular mass of 64-65 kDa. A new method for the electrophoretic separation of native (quinuclidinyl[3H]benzilate labeled) muscarinic receptor-detergent complexes was used for the comparison of wheat germ agglutinin binding, and not lectin binding receptors which were obtained by selective solubilization from porcine striatum. For this purpose, the computer-assisted Ferguson plot analysis is particularly suitable, since it renders possible the statistical assessment of both size and charge differences. Both receptor-detergent complexes were found to differ; statistically significant in their net charge but not in their size. The data support the view that muscarinic receptors from different sources may differ considerably in their glycosylation and that the receptor from porcine striatum can reversibly associate with a low-molecular-mass component which contains sialic acid.     

Characterization of an antibody Fv fragment that binds to the human, but not to the rat neurotensin receptor NTS-1

The cDNAs coding for the heavy and light chain variable domains of an antibody, recognizing the human G-protein-coupled receptor for neurotensin, NTS-1, were obtained from a hybridoma cell line, B-N6. The Fv B-N6 fragment was expressed in Escherichia coli and purified. To characterize the properties of the antibody fragment, human and rat high-affinity neurotensin receptors were expressed in E. coli in functional form, linked at their N-termini to the maltose-binding protein. Fv B-N6 was found to compete for [3H]neurotensin binding to the human neurotensin receptor, but not to the rat neurotensin receptor, with IC50 values of 1.6 microM (membrane-bound receptor) and 1.9 microM (detergent-solubilized, purified receptor). The formation of a relatively stable complex of Fv B-N6 with purified human neurotensin receptor fusion protein was also demonstrated by gel filtration experiments. The Fv B-N6 fragment will be used to isolate a high-affinity binder to the human neurotensin receptor as a valuable tool for cocrystallization and receptor structure determination.     

Muscarinic receptors in the prenatal mouse embryo. Comparison of M35-immunohistochemistry with [3H]quinuclidinyl benzylate autoradiography

Muscarinic cholinergic receptors are widespread in nervous tissue and smooth muscsle or paracrine epithelial cells of various organs. In the embryo, muscarinic receptors are transitorily expressed in the early blastoderm and later on in blastemic tissues during morphogenesis. Recently, a monoclonal antibody (M35) against muscarinic receptor from calf brain became available. In the present study the use of M35-immunohistochemistry is compared to autoradiographic localization of muscarinic binding sites in the mouse embryo. The aim of the study is to test the suitability of the antibody for localization of muscrinic receptors in embryonic tissues. For autoradiography whole-body sagittal cryostat sections of the 17- and 18-day mouse embryo were covered with LKB-Ultrofilm after incubation with the radioactive ligand [³H] quinuclidinyl benzylate (QNB). For immunohistochemistry cryostat sections of formalin fixed tissues were used. In general, all tissues exhibiting ligand binding were also recognized by the antibody. M35-immunohistochemistry resulted in higher spatial resolution of receptor localization than [³H]QNB autoradiography. Definitive muscarinic receptors were observed in smooth muscle and the epithelial lining of the vascular, intestinal, respiratory and urinary system, in the brain, spinal cord and peripheral nerves. The embryonic type of the muscarinic receptor was detected in the mesothelium of lung and liver, in the nephrogenic blastema of the metanephros, and in lung mesenchyme. A large amount of embryonic muscarinic receptors was found in the remnants of the notochord and in the nucleus pulposus of the developing vertebral column. A function in morphogenesis is discussed of the embryonic muscarinic receptor.     

Antibody to Epitope Tag Induces Internalization of Human Muscarinic Subtype 1 Receptor

Abstract: We have studied the effect of an antibody against the epitope EYMPME on the internalization of the human muscarinic cholinergic receptor hm1 tagged with the epitope at the amino terminus. The antibody to the tag induces internalization of the hm1 receptor within minutes after exposure of human embryonic kidney 293 cells transfected with the tagged receptor. This antibody-induced internalization is reversible following removal of the antibody. In contrast to hm1 internalization induced by the agonist carbachol, internalization induced by antibody is not blocked by the muscarinic antagonist atropine. The mechanism of antibody-mediated internalization does not appear to involve receptor dimerization by the antibody, as Fab fragments derived from the antibody also induce internalization. The pathway of antibody-induced internalization, similar to the agonist-induced process, is mediated by clathrin-coated vesicles. Furthermore, antibody treatment does not result in any second messenger production, as measured by phosphoinositide accumulation. Our data show that internalization of a G protein-coupled receptor can be triggered by interaction of the amino terminus of the receptor with an exogenous ligand and can occur independently of second messenger production. This result suggests that the receptor can exist in multiple conformations, each mediating distinct downstream events.     

Generation by phage display and characterization of drug-target complex-specific antibodies for pharmacokinetic analysis of biotherapeutics

Anti-idiotypic antibodies play an important role in pre-clinical and clinical development of therapeutic antibodies, where they are used for pharmacokinetic studies and for the development of immunogenicity assays. By using an antibody phage display library in combination with guided in vitro selection against various marketed drugs, we generated antibodies that recognize the drug only when bound to its target. We have named such specificities Type 3, to distinguish them from the anti-idiotypic antibodies that specifically detect free antibody drug or total drug. We describe the generation and characterization of such reagents for the development of ligand binding assays for drug quantification. We also show how these Type 3 antibodies can be used to develop very specific and sensitive assays that avoid the bridging format.

Abbreviations: BAP: bacterial alkaline phosphatase; CDR: complementarity-determining regions in VH or VL; Fab: antigen-binding fragment of an antibody; HRP: horseradish peroxidase; HuCAL®: Human Combinatorial Antibody Libraries; IgG: immunoglobulin G; LBA: ligand binding assay; LOQ: limit of quantitation; NHS: normal human serum; PK: pharmacokinetics; VH: variable region of the heavy chain of an antibody; VL: variable region of the light chain of an antibody.     

Large scale preparation and characterization of membrane-bound and detergent-solubilized muscarinic acetylcholine receptor from pig atria

The muscarinic acetylcholine receptor (mAcChR) has been prepared from pig atrial membranes by new large scale procedures which result in 30-40 fold enrichment of the receptor in the membrane-bound state and a further three fold enrichment during solubilization. The membrane-bound receptor was prepared by differential and sucrose density gradient centrifugation in 25 mM imidazole, 1 mM EDTA, pH 7.4. A double extraction procedure using a mixed digitonin/cholate detergent was used to solubilize the receptor at a 60-70% yield. The membrane and solubilized preparations had specific activities of 3.5-5 and 8-12 pmol [3H]L-quinuclidinyl benzilate (QNB) binding sites per mg of protein, respectively. The presence of imidazole, which behaved as a weak muscarinic ligand, stabilized the receptor during solubilization and storage. Both the membrane-bound and detergent-solubilized mAcChR bound antagonists at a single class of sites and agonists at two subclasses of QNB sites. The proportion of high affinity agonist sites in the solubilized receptor was about 1/3 that in the membrane receptor. [3H]Propylbenzilylcholine mustard covalently labeled a single prominent atropine-sensitive component with an apparent molecular weight of 70-74,000 on SDS-polyacrylamide gels for both the membrane and solubilized receptor.     

Structural Microheterogeneity of the Muscarinic Cholinergic Receptor Is Not Related to Functional Diversity Identified by Differences in Affinity for Pirenzepine

The muscarinic receptor for acetylcholine shows a diversity in ligand binding properties and effector mechanisms which have suggested the existence of two subtypes (M1 and M2), to which the selective antagonist pirenzepine binds with markedly different affinities. The receptor from rat brain, covalently labelled with the alkylating antagonist tritiated propylbenzilylcholine mustard, displays a structural microheterogeneity on electrophoresis, covering the region of apparent molecular weight 66,000-76,000, with dominant components at 68,000 and 73,000. Selective inhibition by pirenzepine of labelling of the M1 receptor with tritiated mustard has been analysed on fluorographs of sodium dodecyl sulphate-polyacrylamide gels and shown to cause a uniform reduction in radioactive labelling of the broad receptor peak, rather than selectively inhibiting either the high- or low-molecular-weight regions of the band. It is further shown that although this receptor microheterogeneity is found for each of four brain regions studied, it is not found for the heart receptor, which gives a discrete labelled band of apparent molecular weight 72,000. It is therefore suggested that the structural microheterogeneity is the result of tissue-specific, posttranslational modification of the molecule, such as glycosylation, and is not directly related to the functional diversity of the receptor.     

Purification of the muscarinic acetylcholine receptor from porcine brain

The muscarinic acetylcholine receptor of porcine cerebrum has been purified to apparent homogeneity by affinity chromatography, with conjugated 3-(2'-aminobenzhydryloxy)tropane (ABT) as described previously (Haga, K., and Haga, T. (1983) J. Biol. Chem. 258, 13575-13579). In a single step purification using 900 ml of digitonin/cholate-solubilized preparations and 300 ml of the ABT-agarose gel, we obtained, in a yield of 10-15%, more than 250 pmol of muscarinic receptors which bind [3H]N-methylscopolamine with a specific activity of 1,000-5,000 pmol/mg of protein (1,000-5,000-fold purification). The muscarinic receptors eluted from the ABT-agarose gel with 0.1 mM atropine were adsorbed to hydroxylapatite and then recovered as a concentrated solution. Muscarinic receptors were further purified by rechromatography with the same gel or by gel permeation high pressure liquid chromatography. The amino acid composition of the purified receptor was determined, and the specific activity of the purified preparation was estimated to be 13,100 pmol/mg of protein on the basis of amino acid composition. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified receptors with or without radioiodination revealed a single, major band with an apparent Mr of 70,000 either by silver staining or radioautogram. The major band corresponded to the band which specifically bound [3H]propylbenzylcholine mustard (irreversible muscarinic ligand). The purified receptor showed essentially the same specificity for muscarinic ligands as unpurified receptors.     

A series of Fas receptor agonist antibodies that demonstrate an inverse correlation between affinity and potency

Receptor agonism remains poorly understood at the molecular and mechanistic level. In this study, we identified a fully human anti-Fas antibody that could efficiently trigger apoptosis and therefore function as a potent agonist. Protein engineering and crystallography were used to mechanistically understand the agonistic activity of the antibody. The crystal structure of the complex was determined at 1.9 Å resolution and provided insights into epitope recognition and comparisons with the natural ligand FasL (Fas ligand). When we affinity-matured the agonist antibody, we observed that, surprisingly, the higher-affinity antibodies demonstrated a significant reduction, rather than an increase, in agonist activity at the Fas receptor. We propose and experimentally demonstrate a model to explain this non-intuitive impact of affinity on agonist antibody signalling and explore the implications for the discovery of therapeutic agonists in general.     

Improving the developability of an anti-EphA2 single-chain variable fragment for nanoparticle targeting

Antibody-targeted nanoparticles have great promise as anti-cancer drugs; however, substantial developmental challenges of antibody modules prevent many candidates from reaching the clinic. Here, we describe a robust strategy for developing an EphA2-targeting antibody fragment for immunoliposomal drug delivery. A highly bioactive single-chain variable fragment (scFv) was engineered to overcome developmental liabilities, including low thermostability and weak binding to affinity purification resins. Improved thermostability was achieved by modifying the framework of the scFv, and complementarity-determining region (CDR)-H2 was modified to increase binding to protein A resins. The results of our engineering campaigns demonstrate that it is possible, using focused design strategies, to rapidly improve the stability and manufacturing characteristics of an antibody fragment for use as a component of a novel therapeutic construct.     

Eosinophils and airway nerves in asthma

In the lungs, neuronal M2 muscarinic receptors limit the release of acetylcholine from postganglionic cholinergic nerves. However, these receptors are not functional under certain circumstances in animal models of hyperreactivity such as occurs after exposure of sensitised animals to an allergen or during a respiratory tract virus infection. This loss of M2 receptor function leads to an increase in acetylcholine release from cholinergic nerves and thus is a mechanism for the vagally mediated hyperreactivity seen in these animals. Studies in animal models of hyperreactivity have shown that eosinophils localise to the airway nerves of sensitised animals after antigen challenge. Inhibiting this localisation of eosinophils either with an antibody to the eosinophil survival cytokine IL-5 or the eosinophil adhesion molecule VLA-4 prevents loss of M2 muscarinic receptor function. It is likely that eosinophil MBP is responsible for the loss of M2 receptor function, since inhibiting eosinophil MBP with an antibody or neutralising MBP with heparin prevents this loss of function. These data are also supported by ligand binding studies where it has been shown that eosinophil MBP is an allosteric antagonist at neuronal M2 muscarinic receptors. Loss of function of lung neuronal M2 muscarinic receptors may also occur under certain circumstances in patients with asthma, although the mechanisms are not yet established.     

Epiregulin Recognition Mechanisms by Anti-epiregulin Antibody 9E5: STRUCTURAL,FUNCTIONAL, AND MOLECULAR DYNAMICS SIMULATION ANALYSES*

Epiregulin (EPR) is a ligand of the epidermal growth factor (EGF) family that upon binding to its epidermal growth factor receptor (EGFR) stimulates proliferative signaling, especially in colon cancer cells. Here, we describe the three-dimensional structure of the EPR antibody (the 9E5(Fab) fragment) in the presence and absence of EPR. Among the six complementarity-determining regions (CDRs), CDR1–3 in the light chain and CDR2 in the heavy chain predominantly recognize EPR. In particular, CDR3 in the heavy chain dramatically moves with cis-trans isomerization of Pro¹⁰³. A molecular dynamics simulation and mutational analyses revealed that Arg⁴⁰ in EPR is a key residue for the specific binding of 9E5 IgG. From isothermal titration calorimetry analysis, the dissociation constant was determined to be 6.5 nm. Surface plasmon resonance analysis revealed that the dissociation rate of 9E5 IgG is extremely slow. The superimposed structure of 9E5(Fab)·EPR on the known complex structure of EGF·EGFR showed that the 9E5(Fab) paratope overlaps with Domains I and III on the EGFR, which reveals that the 9E5(Fab)·EPR complex could not bind to the EGFR. The 9E5 antibody will also be useful in medicine as a neutralizing antibody specific for colon cancer.     

Binding of an antagonistic monoclonal antibody to an intact and fragmented EGF-receptor polypeptide

A murine monoclonal antibody (No. 425) raised against human A431 carcinoma cells specifically immunoprecipitates the 170,000 molecular weight epidermal growth factor (EGF)-receptor from extracts of A431 cells as well as from extracts of human placenta and cultured fibroblasts, but does not recognize the murine receptor. Binding to the external domain of the human EGF-receptor was indicated by indirect immunofluorescent staining of fixed nonpermeable cells. The antibody binds to both glyco- and aglycoreceptor forms, indicating that the epitope is a part of the polypeptide chain. Binding of the antibody to the receptor is conformation dependent; i.e., denatured receptors lacking EGF-binding activity are not recognized by the antibody. The results of antibody binding studies indicate that the epitope is closely linked to the EGF binding active site, and is common to both high- and low-affinity EGF-receptors. Interaction of this epitope with the antibody inhibits EGF binding and bioactivity, and triggers receptor down-regulation, but does not generate EGFlike kinase-stimulatory or mitogenic responses either in vitro or in vivo. The antibody was tested for its ability to bind to domain-sized fragments of the 170-kDa EGF-receptor. It can recognize both the proteolytically generated 110-kDa EGF binding peptide, and a soluble 100-kDa EGF-receptor secreted by A431 cells. This indicates that the epitope recognized this antibody retains its conformation after proteolytic separation of the EGF binding domain from the rest of the receptor molecule.     

Molecular cloning and expression of a fifth muscarinic acetylcholine receptor

A cDNA of 2149 base pairs with an incomplete open reading frame (ORF) encoding amino acids 1-516 of a 531-amino acid protein highly homologous to muscarinic receptors was cloned from a rat brain cDNA library. The complete ORF was then deduced from a DNA fragment cloned from a rat genomic library. This ORF was subcloned into the eukaryotic expression vector p91023(B) under control of the adenovirus major late promoter and co-transfected with the thymidine kinase selection marker into muscarinic receptor-negative, thymidine kinase-negative murine L cells. Stable transformants were selected and tested for acquisition of muscarinic receptors by following appearance of specific binding sites for the muscarinic ligand [3H] N-methylscopolamine. Two cell lines, LM5.36 and LM5.40, were cloned and shown to express typical muscarinic receptor sites, thus confirming that the newly cloned ORF encodes a muscarinic receptor, the rat M5 muscarinic acetylcholine receptor. Tests for activities showed it to stimulate phosphoinositide hydrolysis in intact cells, without affecting positively or negatively adenylyl cyclase activity. The M5 receptor contains two putative glycosylation sites at its amino terminus and, based on hydropathicity analysis, is predicted to span the plasma membrane seven times. Like 17 other receptors of this class, the M5 receptor has 19 conserved amino acids, among which are 4 prolines located in the 4th, 5th, 6th, and 7th predicted transmembrane regions, conferring possible bends to these helices, and 2 cysteines, one in the 1st and the other in the 2nd extracellular loop, possibly providing for a disulfide bond. Similarity in amino acid composition and in patterns of antagonist binding and biologic effects suggest the M5 receptor to be M1-like.     

Selective inactivation of muscarinic receptor subtypes

Muscarinic receptors exist in multiple subtypes, denoted as M₁, M₂ M₃ and M₄, encoded by four distinct but related genes. A fifth gene product, m5, has also been predicted although this sequence awaits a pharmacological equivalent. Many tissues express more than one muscarinic receptor subtype, which may couple to different intracellular effectors and thus have different physiological roles. One way to characterize the role of each receptor is to selectively inactivate one receptor population, thus pharmacologically ‘isolating’ the muscarinic receptor subtype of interest. Selective receptor inactivation can be achieved using either a selective, irreversible antagonist, or protection using a selective, reversible antagonist against a non-selective irreversible antagonist. Therefore, combination of these two approaches may provide optimal selective inactivation. Several muscarinic alkylating agents have been identified, including phenoxybenzamine, EEDQ (N-Ethoxycarbonyl-1-ethoxy-1,2-dihydroquinoline) and propylbenzilylcholine mustard. These irreversible antagonists do not, in general, discriminate between muscarinic receptor subtypes and are frequently used to estimate the affinity and relative efficacy of muscarinic agonists. Consequently, use of these irreversible antagonists provides estimations of the ‘receptor reserve’ associated with a response mediated by muscarinic receptor activation. In contrast, 4-DAMP mustard (4-diphenylacetoxy-N-(2-chloroethyl)piperidine) selectively inactivates M₃ receptors, but will not discriminate between M₁ M ₂ or M₄ receptors. In the absence of highly selective alkylating agents, receptor protection by reversible antagonists may be used. Thus, reversible antagonists, such as pirenzepine, methoctramine or para-fluorohexahydrosiladifenidol, at appropriate fractional receptor occupancies, may protect M₁ M₂ or M₃ receptors against alkylation by phenoxybenzamine. Selective alkylation of M₃ receptors by 4-DAMP mustard is enhanced with concurrent M₂ protection. This approach has been applied to defining the role of these muscarinic receptor subtypes in the control of ileal smooth muscle tone. These data suggest that, in ileum, M₂ receptors may act to inhibit β-adrenoceptor activation, thereby offsetting relaxation, while M₃ receptors directly mediate contraction.     

The differential loss of [3H]pirenzepine vs [3H] (-) quinuclidinylbenzilate binding to soluble rat brain muscarinic receptors indicates that pirenzepine binds to an allosteric state of the muscarinic receptor

[3H]Pirenzepine [( 3H]PZ) and [3H] (-)Quinuclidinylbenzilate [( 3H] (-)QNB) specific binding to soluble rat brain muscarinic cholinergic receptors was assessed as a function of time subsequent to receptor solubilization. The soluble brain muscarinic receptor is stable at 4 degrees C when assayed by [3H] (-)QNB binding (t 1/2 = 80 hrs). In contrast the pirenzepine state of the receptor decays rapidly (t 1/2 = 3.0 hrs). Prior occupation of the receptor with [3H] (-)QNB or [3H]PZ increases the receptor stability by two to five fold (t 1/2 QNB greater than 1,000 hrs; t 1/2 PZ = 6.5 hrs). These data indicate that pirenzepine binds to an allosteric state of the muscarinic receptor and that caution should be employed in the assignment of receptor subtypes based solely upon the binding of ligands which recognize unique conformational states.