Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-03-19
2003-11-11
Spector, Lorraine (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S007210, C436S547000, C436S548000, C530S388100, C530S387100
Reexamination Certificate
active
06645723
ABSTRACT:
The present invention relates to a method for determining cytokine receptor, especially growth hormone receptor (GHR), activation, by the use of an antibody capable of discriminating between an activated and an inactive cytokine receptor conformation, especially GHR or growth hormone binding protein (GHBP) conformation, the method comprising: contacting the antibody and the cytokine receptor to form a complex, contacting a candidate ligand to the complex, measuring the antibody binding to inactive cytokine receptor and thereby discriminating between an activated and an inactive conformation.
It also relates to a method for screening for a functionally cytokine agonists and antagonists, especially functionally growth hormone (GH) agonists, among candidate ligands by the use of an antibody specific for activated cytokine receptor conformation. The invention also relates to an antibody directed to the hinge sparing subdomains I and II of the growth hormone receptor extracellular region and which is capable of discriminating between an activated and an inactive receptor conformation.
The invention also relates to the use of the antibody for e.g. characterization of growth hormone receptor interactions and for screening for compounds stabilizing the dimerization of GHR.
INTRODUCTION
The 22 kDa polypeptide human growth hormone (hGH), essential for normal growth and development, induces a variety or biological effects including linear growth, lactation, nitrogen retention, diabetogenic and insulin-like effects and macrophage activation (1-4). Each of these effects is initiated by hGH interaction with specific cell receptors. The hGH receptor (hGHR) belongs to a large cytokine receptor family (5) which includes receptors for prolactin, erythropoietin, interleukins (IL)-2, 4, and -6, granulocyte macrophage-colony-stimulating factor (GM-CSF) and granulocyte-colony-stimulating factor (G-CSF) (6-10). The three-domain organization of these receptors comprises a heavily N-glycosylated extracellular ligand binding domain, a single transmembrane segment and an intercellular domain, the last of which shares little sequence identity within the family (11). The hGHR extracellular region has two subdomains, one implicated mainly in hGH binding (subdomain I) and the other in hGHR dimerization (subdomain II) (7, 8).
The extracellular hGHR domain is found in serum in the form of a hormone binding protein (hGHBP) which binds hGH with approximately the same affinity and specificity as the intact receptor (12). Non-glycosylated recombinant bacterial hGHBP (13) has the same binding affinity (K
d
=0.4 nM) (14) and specificity for hGH as the mammalian binding protein. Crystallization studies of hGH and hGHBP show that a single hGH molecule binds two hGHBP molecules (15). Each hGH molecule is bivalent, containing two separate hGHBP binding sites; site I is a high affinity site and site II, a low affinity site. In contrast, the hGHBP is univalent, as it uses the same amino acids to bind either hGH site I or site II. An hGH excess dissociates the hGH-(hGHBP)
2
complex to form a monomeric complex. Biological response triggering requires sequential binding of the first receptor subunit to site I on a GH molecule, followed by binding of the second receptor to GH site II, forming the GH-(GHR)
2
complex (16). In addition to the hormone-receptor interactions, there is also substantial surface contact between the subdomain II subunits of the extracellular regions of the two receptor molecules. This sequential dimerization model has been confirmed using several approaches including crystallization, size exclusion chromatography, calorimetry and fluorescence quenching assays (14).
Although little is known of the hGHR signal transduction mechanism, several lines of evidence indicate that hGHR activation triggers tyrosine phosphorylation of JAK2 kinase (17) and some STAT transcription factors, mainly STAT2, STAT3 and STAT5 (18-21).
Earlier methods for determining whether a candidate ligand is an agonist or an antagonist to a growth hormone receptor, has been based on the theory that it must be determining whether the ligand forms a complex of one or two growth hormone receptors. See WO 92/21029.
However, Hardinger et al in J Biol Chem, 1996, Vol 271 (12) 6708-12, have found that both native hGH and hGH-G120R form complexes similar in size to that expected for hGH when bound to recombinant hGH binding protein. They further conclude. “our results indicates that the GH antagonist, hGH-G120R, is able to bind, dimerize, and internalize GHRs in a similar manner as hGH. However, the dimeric complex formed with hGH-G120R is not functional as in the hGH:GHR dimer.
We present here the novel finding that the measurement of a conformation change of the GH receptor is the relevant measurement for the determination whether a ligand is an agonist or an antagonist to the growth hormone receptor.
ABBREVIATIONS USED
EDC/NHS
N-ethyl-N′-(3-dimethylaminopropyl)
carbodiimidehydrochloride/ N-hydroxysuccinimide)
EIA
enzyme-linked immunoassay
FCS
fetal calf serum
GAM-PO
goat anti-mouse immunoglobulin antibody
G-CSF
granulocyte colony-stimulating factor
GM-CSF
granulocyte macrophage-colony-stimulating factor
hGH
human growth hormone
hGHBP
human growth hormone binding protein
hGHR
human growth hormone receptor
IL-2, IL-4, IL-6
interleukins 2, 4 and 6
PBS
phosphate-buffered saline
PO
peroxidase
OPD
o-phenylenediamine dihydrochloride
RAM
rabbit anti-mouse
sc
subcutaneous
SDS-PAGE
sodium dodecyl sulphate polyacrylamide gel
electrophoresis
THE INVENTION
We have developed a panel of human growth hormone receptor-specific monoclonal antibodies (mAb), used for the characterization of signals triggered by hGH binding. One of these was used here to characterize hormone/receptor interactions. This antibody, GHR05, is directed against the hinge spanning subdomains I and II of the receptor's extracellular region and recognizes uncomplexed hGHR on the cell surface. Binding assays indicate a higher affinity of GHR05 for the hGH-hGHR complex than for hGHR alone, indicating that the mAb better recognizes a receptor conformation induced following ligand binding. Antibody binding to the cell surface receptor increases upon receptor binding to growth hormone, but not when it binds a mutant form, hGHG120R, which does not trigger receptor activation. Growth hormone binding thus appears to lead to a conformational change in the receptor epitope recognized by GHR05, giving rise to the active dimer configuration, necessary for signal transduction. The antibody appears thus to activate the cells through conformational changes, which are different from those triggered by GH.
Binding assays indicate a higher affinity of GHR05 for the hGH-hGHR complex than for hGHR alone, indicating that the mAb better recognizes a receptor conformation induced following ligand binding.
Using a chimeric receptor-expressing, growth hormone-dependent murine cell line, we find that GHR05 binds to the receptor in the absence of hGH and delivers a signal leading to cell proliferation, i.e. activation. Finally, GHR05 treatment of IM-9 cells, a human cell line expressing a functional hGHR, leads to cell proliferation mediated by the generation of GH-specific signals, including phosphorylation of the JAK2 tyrosine kinase and activation of STAT5.
The invention is defined according to the attached claims.
The invention also includes screening against the monomer for prevention of the dissociation of the already-formed complex and blocking the activity of the preformed complexes.
The binding of the claimed antibody to GHR or to GHBP is not inhibited by soluble GH and the antibody has an ability to recognize the GHR and the GHBP in western blot
The antibody has also an ability to recognize in flow cytometry, all cells naturally expressing the GHR or cells transfected with a plasmid containing the gene that encodes the GHR. Another ability is to immunoprecipitate the BP-GH-1125-complex.
The screening of compounds with properties similar to GHR05 is possible by this invention.
GHR19
Alonso Carlos Martinez
Baron Leonor Kremer
Frade Jose Miguel Rodriguez
Garcia Jose Mario Mellado
Consejos Superior de Investigaciones Cientificas
Dinsmore & Shohl LLP
Spector Lorraine
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