WO1993020199A9 - Human gonadotropin receptor (fsh receptor) - Google Patents

Human gonadotropin receptor (fsh receptor)

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Publication number
WO1993020199A9
WO1993020199A9 PCT/EP1993/000780 EP9300780W WO9320199A9 WO 1993020199 A9 WO1993020199 A9 WO 1993020199A9 EP 9300780 W EP9300780 W EP 9300780W WO 9320199 A9 WO9320199 A9 WO 9320199A9
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Prior art keywords
leu
ser
fsh
asn
receptor
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PCT/EP1993/000780
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French (fr)
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WO1993020199A1 (en
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Definitions

  • the invention relates to the field of reproductive medicine, particularly human reproductive medicine.
  • Gonadotropins follicle stimulating hormone (FSH) , chorionic gonadotropin (CG) , luteinizing hormone (LH) and thyroid stimulating hormone (TSH)
  • FSH follicle stimulating hormone
  • CG chorionic gonadotropin
  • LH luteinizing hormone
  • TSH thyroid stimulating hormone
  • the present invention provides such a cell. It also provides a novel gonadotropin receptor, i.e. the human follicle stimulating hormone receptor.
  • the DNA sequence is useful for studying the characteristics of the receptor by site directed mutations, thereby enabling to elucidate the parts of the receptor involved in the various aspects of its functions.
  • the amino acid sequence can be used to produce synthetic peptides in order to identify the smallest peptide still having binding affinity for FSH.
  • polypeptides comprising the extracellular part of the FSH receptor can be constructed.
  • the peptides and polypeptides of the invention can also be used directly as competing compounds for the endogenous receptors or in diagnostic test kits testing for the ligand for the receptor.
  • Antibodies or antiserum directed against a polypeptide according to the invention have use in diagnostic immunoassay's and generation of anti- idiotype antibodies.
  • a more preferred use is the use as an antidote against overstimulation with FSH. This occurs regularly in IVF (in vitro fertilization) protocols resulting in ovarian hyperstimulation.
  • the antibodies may be formulated into pharmaceutical formulations by mixing with suitable pharmaceutical acceptable carriers in a manner known to those skilled in the art.
  • a specific polypeptide according to the invention in any of the embodiments described above can be used to produce antibodies, both polyclonal, monospecific and monoclonal. Such use of a polypeptide according to the invention and such an antibody fall within the scope of the invention.
  • polyclonal antibodies When polyclonal antibodies are desired, techniques for producing and processing polyclonal sera are known in the art (e.g. Mayer and Walter, eds, Immunochemical Methods in Cell and Molecular Biology, Academic Press, London, 1987) .
  • a selected mammal e.g. a rabbit is given (multiple) injections with one of the above-mentioned immunogens, e.g. corresponding to about 20 ⁇ g to about 80 ⁇ g of polypeptide per immunization. Immunization is carried out with an acceptable adjuvant, generally in equal volumes of immunogen and adjuvant.
  • Acceptable adjuvants include Freund's complete, Freund's incomplete, alum-precipitate or water-in-oil emulsions, with a preference for Freund's complete adjuvant for the initial immunization. For booster immunization Freund's incomplete adjuvant is preferred.
  • the initial immunization consists of the administration of approximately l ml emulsion at multiple subcutaneous sites on the backs of the rabbits. Booster immunizations utilizing an equal volume of immunogen are given at about one monthly intervals and are continued until adequate levels of antibodies are present in an individual rabbits serum. Blood is collected and serum isolated by methods known in the art.
  • Monospecific antibodies to each of the immunogens are affinity purified from polyspecific antisera by a modification of the method of Hall et al. (Nature 311, 379-387 1984) , prepared by immunizing rabbits as described above with the purified proteins.
  • Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogeneous binding characteristics for the relevant antigen.
  • Homogeneous binding as used herein refers to the ability of the antibody species to bind to a specific antigen or epitope.
  • Monoclonal antibody reactive against one of the above- mentioned immunogens can be prepared by immunizing inbred mice, preferably Balb/c with the appropriate protein by techniques known in the art (Kohler and Milstein, Nature 256; 495-497, 1975). Hybridoma cells are subsequently selected by growth in hypoxanthine, thymidine and aminopterin in an appropriate cell culture medium such as Dulbecco's modified Eagle's medium (DMEM) . Antibody producing hybridomas are cloned, preferably using the soft agar technique of MacPherson, (Soft Agar Techniques, Tissue Culture Methods and Applications, Kruse and Paterson, eds., Academic Press, 276, 1973) .
  • Discrete colonies are transferred into individual wells of culture plates for cultivation in an appropriate culture medium.
  • Antibody producing cells are identified by screening with the appropriate immunogen.
  • Immunogen positive hybridoma cells are maintained by techniques known in the art.
  • Specific anti-monoclonal antibodies are produced by cultivating the hybridomas in vitro or preparing ascites fluid in mice following hybridoma injection by procedures known in the art.
  • the polyclonal or monoclonal antibodies can show agonistic or antagonistic activity as compared to the activity of the natural ligand.
  • Specific antagonistic or agonistic activity may involve the interference of the antibodies with the signal transduction system in the cells expressing the FSH receptor, without competing for the binding of FSH to its receptor.
  • the DNA coding for the receptor or for an altered receptor can be inserted into a suitable vector for expression in either a prokaryotic or a eukaryotic host.
  • the hosts may be bacteria, phages, yeasts, funghi, animal cells or plant cells, preferred however are mammalian cells.
  • the vector into which the DNA is inserted may be any suitable vector. It may comprise suitable regulating elements such as promoters, enhancers, repressors, etc. If necessary, it may also comprise a signal sequence to transport the protein translated from the DNA to the surface or the outside of the host, even though the receptor does contain its own signal sequence.
  • a cell which preferably expresses no related receptors and more preferably no other receptors at all, and which cell expresses the receptor according to the invention in altered or unaltered form, in its (outer) membrane.
  • a signal producing system in the cell is needed.
  • the signal producing system may be the cell's own or may be cotransfected with the DNA coding for the receptor.
  • the signal will be provided by the so called second messenger system, which works through G- proteins which are associated with the receptor at its surface site. Any other signal producing system as well as second messenger initiated test system will be suitable, as long as the signal is somehow measurable.
  • Oligodeoxynucleotides were synthesized using the phosphoramidite method on an Applied Biosystems 381A DNA synthesizer. They correspond to nucleotide position 1237-1255 (transmembrane region II) and 1843-1861 (transmembrane region VII) of the hFSH-R (Minegish et al., Bioch.Bioph.Res.Comm. 175. 1125, 1991) . Both oligonucleotides were used as primers in a polymerase chain reaction (Maniatis et al.. Cold Spring Harbor Laboratory, "Molecular Cloning: A Laboratory Manual", 1989) to generate a 625bp DNA fragment amplified from human genomic DNA (Clonetech) . The resulting product from PCR was verified by DNA sequence analysis (Pharmacia,T7-sequencing kit) .
  • the partial hFSH-R amplification product obtained by PCR (section l.l.) was used as probe (Pharmacia, oligolabelling kit) to screen 3.6xl0 5 recombinant phages of the human testis cDNA library (section 1.2.).
  • Prehybridization of filters was carried out for 5 hours at 65 °C in a solution containing 6xSSC (0.9 mol/1 NaCl,0.09 mol/1 Na-citrate, pH 7.0), lOxDenhardt (lxDenhardt:200 ⁇ g/ml Ficoll-70 (Pharmacia), 200 ⁇ g/ml polyvinylpyrrolidone (Sigma) , 200 ⁇ g/ml bovine serum albumin (BSA, Sigma) , 50 ⁇ g/ml sheared and denatured herring sperm DNA (Sigma) , 9% dextran sulphate (Pharmacia), and 0.1% sodium dodecyl sulphate (SDS, Sigma) .
  • 6xSSC 0.9 mol/1 NaCl,0.09 mol/1 Na-citrate, pH 7.0
  • lOxDenhardt lxDenhardt:200 ⁇ g/ml Ficoll-70 (Pharmacia)
  • Hybridization was performed in the same solution by the addition of the 32 P-labelled DNA probe (2.3xl0 5 cpm/ml) . The mixture was incubated overnight at the same temperature. Filters were washed in solutions with decreasing salt concentrations (O.lxSSC, 65 °C) . Positive recombinant phages were purified by two successive rounds of phage titration and hybridization. Phage DNA inserts were isolated and subcloned in the endoR EcoRI site of pGEM3Z (Promega) and characterized by endoR mapping and DNA sequence analysis.
  • CHO cells (CHO Kl) were obtained from ATCC (CCL61) . They were cultured in M505 medium that consisted of a mixture (1:1) of Dulbecco's Modified Eagle's Medium (DMEM, Gibco 074-2100) and Nutrient mixture F12 (Ham's F12, Gibco 074-1700) supplemented with 2.5 mg/ml sodium bicarbonate (Baker), 55 ⁇ g/ml sodium pyruvate (Fluka) , 2.3 ⁇ g/ml 3-mercaptoethanol (Baker), 1.2 ⁇ g/ml ethanolamine (Baker), 360 ⁇ g/ml L- glutamine (Merck), 0.45 ⁇ g/ml sodiumselenite (Fluka), 62.5 ⁇ g/ml penicillin (Mycopharm) , 62.5 ⁇ g/ml streptomycin (Serva) , and 10% fetal calf serum (FCS, Bocknek) .
  • DMEM D
  • Recombinant constructs used for transformation of CHO cells consist of the expression vector pKCRhFSH-R (section 2.1.) and the selection vector pAG60MT2.
  • the latter vector was constructed by insertion of a 3kb human MTIl A -containing endoR Hindlll fragment (Karin and Richards, Nature 299, 797, 1982) into the endoR Hindlll site of pAG60 (Colbere-Garapin et al. , J.Mol.Biol 150, 1, 1981); the transcription of this gene was directed towards the tk-promoter that was located in front of the neomycin resistence gene.
  • CHOhFSH- RlCdlO refers to transformed CHO cells obtained after successive selection by neomycin and 10 ⁇ mol/1 CdCl 2 .
  • CHOhFSH-RlCdlO cells pellet were homogenised with a teflon glass homogeniser in ice-cold 10 mmol/1 Tris- HCl buffer, pH 7.4, supplemented with 0.25 mol/1 sucrose and 5 mmol/1 MgCl 2 « The homogenate was diluted to 2.5 x 10 6 cells (starting material)/ml with homogenisation buffer. For saturation experiments, aliquots homogenate (200 ⁇ l/tube) were incubated with increasing concentrations 125 I-hFSH (200 ⁇ l/tube; 4-150 pmol/1) with or without excess unlabeled recFSH (10 IU/100 ⁇ l/tube) in polypropylene microfuge tubes.
  • the assay buffer consisted of 10 mmol/1 Tris-HCl, pH 7.4, supplemented with 5 mmol/1 MgCl 2 and 1 g/1 bovine serum albumine (BSA; Sigma, St Louis, MO, USA) . After 24 h of incubation at room temperature (RT) , 500 ⁇ l ice-cold assay buffer was added, and bound and free hormone were separated by centrifugation (5 min at 15.000 x g) . The bound radioactivity was measured with a LKB gamma counter. The K-j (equilibrium dissociation constant) and B ⁇ ax (maximum binding capacity) were assessed by Scatchard plot analysis. Competition of antisera for 1 l ? ⁇ -FSH binding was assessed using the single cell clone mentioned previously expressing the human FSH-R, selected by subsequent exposure to G418 (800 ⁇ g/ml; Geneticin;
  • control serum that is included is a serum of saline-injected mice. The results are presented in
  • the CHOhFSH-RlCdlO cells were washed once, resuspended in M505, pH 7.4, supplemented with 10% FCS, and cultured in 24 well-plates (Nunclon; 0.1% gelatin coated) at a concentration of 2 x 10 5 cells/ml/well for 48 h at 37 °C in a humidified atmosphere of 5% C ⁇ 2/95% O 2 .
  • the remaining cells were treated with 0.5 ml 1-propanol which was followed by ultrasonification for 2 min. Subsequently, the content of each well was transfered to Eppendorf tubes and stored at -20°C.
  • a cAMP (RIANEN) kit NNN
  • the cellular samples were lyophilized using a Speed Vac and reconstituted in 0.5 ml kit buffer. The medium samples were measured directly using a callibration curve of standard cAMP in CHO cell culture medium.
  • GST-FSH-R 1 contains a 1000 basepair fragment coding for the complete extracellular N-terminus of the FSH- R.
  • Fusion proteins 2 and 3 contain only parts of this sequence: fusion protein 2 (GST-FSH-R 2) contains a 600 basepair fragment coding for the N-terminal part of the FSH-R present in GST-FSH-R 1; fusion protein 3 (GST-FSH-R 3) contains a 400 basepair fragment coding for the C-terminal part of the FSH-R present in GST- FSH-R 1) . These fragments were derived by polymerase chain reaction (PCR) on the FSH-R cDNA clones ( Figure 2A; seq. ID No. 1) . Via this procedure restriction sites were introduced, which facilitated subsequent cloning procedures.
  • PCR polymerase chain reaction
  • GST-FSH-R 1 we used the primers 5'-TGTCATCATCGGATC-3 ⁇ and 5'-TCTGAGGATGTTGTAC- 3' ; for GST-FSH-R 2 we used the primers 5'- TGTCATCATCGGATC-3 ' and 5'-AGGCAGGGAATGGATCC-3' ; for GST-FSH-R 3 we used the primers 5•-AGAACAAGGATCC-3' and 5'-TCTGAGGATGTTGTAC-3' .
  • Some primers were complementary to the FSH-R cDNA sequence (SEQ ID NO:l). Only the complementary sequence of the primers is shown.
  • the FSH-R fragments were cloned into one of the pGEX expression vectors (Smith and Johnson, Gene 67, 31, 1988) . Depending on the reading frame, either pGEX-1 (for the 1000 bp and 600 bp FSH-R fragments) or pGEX-3X (for the 400 bp FSH-R fragment) were used. In order to study expression of the fusion proteins, these constructs were transformed into E. coli MC1061.
  • IPTG isopropyl- ⁇ -D- thiogalactopyranoside
  • Triton-X-l00 was added to a final concentration of 1%, incubation was continued for 10 minutes on ice, and cells were sonified. The supernantant of the sonicate was incubated with gluthathione-agarose carrier (Pharmacia) for 30 minutes at 4°C. Carriers were washed several times with PBS, and finally with 50 mM Tris pH 8. The fusion proteins thus purified were used to generate antisera.
  • mice Six weeks old female BALB/c mice were injected intraperitoneally with 50 ⁇ g of bacterial fusion proteins in complete Freund's adjuvant. In addition, mice were immunized with membranes of 10' CHO cells transfected with the human FSH-R. For this purpose an FSH-R single cell clone was used that was obtained by subsequent G418 (800 ⁇ g/ml; Geneticin, Gibco) and cadmium (2.5 ⁇ M) selection. Two subsequent intramuscular injections were given at three weeks intervals with 50 ⁇ g fusion protein or membranes of 10 7' cells i.n i.ncomplete Freund's adjuvant. Three weeks after the third injection, mice were boosted intraperitoneally with 100 ⁇ g fusion protein or
  • Erythrocyte depleted spleen cells were prepared according to Steenbakkers et al. (J. Imm. Methods 152,
  • Denatured and reduced protein preparations were applied on sodium dodecylsulfate (SDS) polyacrylamide gels and blotted onto nitrocellulose filters.
  • SDS sodium dodecylsulfate
  • small (approximately 200 ng) and equivalent amounts of protein were applied to the gels.
  • Membranes were blocked with 20% FCS, and incubated for 2 hours at room temperature with different concentrations of antibody preparations. Blots were washed with Tris-buffered saline (TBS) Tween and incubated with Goat-anti-mouse alkaline phosphatase (AP) conjugate (Pro ega) for 45 minutes at room temperature.
  • TBS Tris-buffered saline
  • AP Goat-anti-mouse alkaline phosphatase conjugate
  • AP substrate nitrogen blue tetrazoline (NBT) / 5-bromo-4-chloro-3- indolyl phosphate (BCIP) ) reactions were done in 100 mM NaCl, 100 mM Tris pH 9.5, and 10 mM MgC12) .
  • CHO cells were attached to the bottom of 24 well plates (approximately 10" cells/well) for three days. Cells were washed in phosphate buffered saline (PBS) for 10 minutes. After fixation with 4% paraformaldehyde in PBS for one hour at room temperature, the cells were washed in PBS containing 0.05% normal swine serum (NSS) and 0.02% Triton-X-100 for 10 minutes. After blocking with NSS in PBS-Triton for 1 hour, cells were incubated with antisera for 16 hours at room temperature.
  • PBS phosphate buffered saline
  • NSS normal swine serum
  • Triton-X-100 Triton-X-100
  • Antibodies raised against the human FSH-R were characterized by Western immunoblotting, lmmunostai .m.ng, and by thei.r competi.ti.on for binding and cAMP generation.
  • Antisera raised against the bacterial GST-FSH-R fusions recognized these proteins on Western blots (Fig. 6C) . Often, these antisera also recognized GST alone (control lanes) . After preclearing with GST, antisera specifically reacted with the FSH-R part of the fusion proteins. A representative example is shown in Figure 6B.
  • the anti GST-FSH-Rl serum inhibited cAMP generation to the same degree at the anti GST-FSH-R3 serum (Fig. 8) .
  • the GST-FSH-Rl antiserum competed less than the anti GST-FSH-R3 serum for 125 I- FSH binding (Fig. 7) .
  • These antibodies are potentially interesting as antagonists since they do not have to compete with the high affinity FSH binding in order to block the biological effect of FSH.
  • TCT AAC AGG GTT TTT CTC TGC CAA GAG AGC AAG GTG ACA GAG ATT CCT 207 Ser Asn Arg Val Phe Leu Cys Gin Glu Ser Lys Val Thr Glu lie Pro
  • Cys lie Gly lie Tyr Leu Leu Leu lie Ala Ser Val Asp lie His Thr 410 415 420 425
  • CAA GCC CAA ATT TAT AGG ACA GAA ACT TCA TCC ACT GTC CAC AAC ACC 2079 Gin Ala Gin He Tyr Arg Thr Glu Thr Ser Ser Thr Val His Asn Thr 650 655 660 665
  • MOLECULE TYPE protein
  • Fig. 1 Physical maps of three hFSH-R cDNA clones and their position on their respective plasmids.
  • Fig. 2A Nucleotide sequence of the hFSH-R cDNA cloned in pGEM3Zcl (seq. ID No. 1).
  • Fig. 2B Amino acid sequence of the hFSH-R coded for by the hFSH-R cDNA in pGEM3Zcl (seq. ID No. 2).
  • Fig. 3 Binding of 125 I-hFSH to hFSH-R of a neomycin- CdCl2 (10 ⁇ mol/1) selected CHO pool. Cell membranes were incubated with increasing concentrations 125 ⁇ - hFSH in the absence or presence of excess unlabeled recFSH. The saturation curve of specifically bound 125 I-hFSH together with the derived Scatchard plot are shown. The values represent the mean of duplicate determinations. The calculated Kd and B ma ⁇ are presented in the Scatchard plot (insert) , where B/F is plotted against the specific bound.
  • Fig. 4 Dose dependent stimulation of intracellular cAMP by recFSH. The stimulations were repeated at different times of incubation. Values represent the mean of duplicate determinations.
  • Fig. 5 Dose dependent stimulation of extracellular (medium) cAMP by recFSH. The stimulations were repeated at different times of incubation. Values represent the mean of duplicate determinations.
  • Fig. 6A Western blot of GST-hFSH-R fusion proteins stained with Coomassie Blue.
  • SUBSTITUTESHEET Fig. 6B Western blot of GST-hFSH-R fusion proteins stained with antiserum raised against GST-hFSH-R3 fusion protein. The antiserum has been preclaered with GST proteins.
  • Fig. 6C Western blot of GST-hFSH-R fusion proteins stained with antiserum raised against GST-hFSH-Rl fusion protein.
  • Fig. 7 Interference of hFSH-R antisera with binding of 1 12255 II--FFSSHH ttoo CCHHOO ccee!lls expressing the human FSH receptor (hFSH-R) .
  • Fig. 8 Interference of hFSH-R antisera with FSH-induced cAMP generation.

Abstract

The present invention provides a new receptor for gonadotropins, amino acid sequences of said receptor, nucleic acid sequences coding for said receptor, recombinant hosts comprising such a nucleic acid as well as screening assays using said receptor and ligands such as antibodies for that receptor. In a more preferred embodiment the invention provides antibodies raised against the human FSH receptor. The compounds and methods of the invention will find their use in the field of reproductive medicine, particularly human reproductive medicines.

Description

HUMAN GONADOTROPIN RECEPTOR (FSH RECEPTOR)
The invention relates to the field of reproductive medicine, particularly human reproductive medicine.
In that field frequent use is made of gonadotropins or analogs thereof. Gonadotropins (follicle stimulating hormone (FSH) , chorionic gonadotropin (CG) , luteinizing hormone (LH) and thyroid stimulating hormone (TSH)) are a family of protein hormones with a common α-subunit and a hormone specific 3-subunit. All human gonadotropins have been studied intensively and much is known about them. Though there are clinical application's for the natural gonadotropins, either isolated from body fluids or produced by recombinant DNA technology, the natural gonadotropins often may not have the right combination of desired properties.
Therefore there is a need for altered gonadotropins, or molecules mimicking certain properties of gonadotropins. Especially interesting are derivatives with higher binding affinities for their respective receptors, either activating or blocking said receptors, derivatives with a longer residence time in the body or at the receptor and derivatives with a higher specificity for their receptors. This list of desirable alterations is of course not exhaustive.
However, in order to be able to study the effect of alterations made to the various gonadotropins, one must be able to study the interaction of the gonadotropin derivative with its receptor. The usual means to study this interaction is studying the in vitro binding of the altered gonadotropin to a tissue or a cell line known to express the receptor involved. Usually such cell lines or tissues express more than one receptor. Especially when testing altered gonadotropins, the presence of other receptors may interfere with the test for binding affinity for the gonadotropin receptor, because the binding affinity and specificity are altered. Therefore the need exists for cells which only express one receptor, namely the one tested for.
The present invention provides such a cell. It also provides a novel gonadotropin receptor, i.e. the human follicle stimulating hormone receptor.
Both its DNA sequence (SEQ ID NO: 1) as well as its amino acid sequence (SEQ ID NO: 2) are provided. The DNA sequence is useful for studying the characteristics of the receptor by site directed mutations, thereby enabling to elucidate the parts of the receptor involved in the various aspects of its functions. The amino acid sequence can be used to produce synthetic peptides in order to identify the smallest peptide still having binding affinity for FSH.
In this way polypeptides comprising the extracellular part of the FSH receptor can be constructed. The peptides and polypeptides of the invention can also be used directly as competing compounds for the endogenous receptors or in diagnostic test kits testing for the ligand for the receptor. Antibodies or antiserum directed against a polypeptide according to the invention have use in diagnostic immunoassay's and generation of anti- idiotype antibodies. A more preferred use is the use as an antidote against overstimulation with FSH. This occurs regularly in IVF (in vitro fertilization) protocols resulting in ovarian hyperstimulation. The antibodies may be formulated into pharmaceutical formulations by mixing with suitable pharmaceutical acceptable carriers in a manner known to those skilled in the art.
A specific polypeptide according to the invention in any of the embodiments described above can be used to produce antibodies, both polyclonal, monospecific and monoclonal. Such use of a polypeptide according to the invention and such an antibody fall within the scope of the invention.
When polyclonal antibodies are desired, techniques for producing and processing polyclonal sera are known in the art (e.g. Mayer and Walter, eds, Immunochemical Methods in Cell and Molecular Biology, Academic Press, London, 1987) . In short, a selected mammal, e.g. a rabbit is given (multiple) injections with one of the above-mentioned immunogens, e.g. corresponding to about 20 μg to about 80 μg of polypeptide per immunization. Immunization is carried out with an acceptable adjuvant, generally in equal volumes of immunogen and adjuvant. Acceptable adjuvants include Freund's complete, Freund's incomplete, alum-precipitate or water-in-oil emulsions, with a preference for Freund's complete adjuvant for the initial immunization. For booster immunization Freund's incomplete adjuvant is preferred. The initial immunization consists of the administration of approximately l ml emulsion at multiple subcutaneous sites on the backs of the rabbits. Booster immunizations utilizing an equal volume of immunogen are given at about one monthly intervals and are continued until adequate levels of antibodies are present in an individual rabbits serum. Blood is collected and serum isolated by methods known in the art.
Monospecific antibodies to each of the immunogens are affinity purified from polyspecific antisera by a modification of the method of Hall et al. (Nature 311, 379-387 1984) , prepared by immunizing rabbits as described above with the purified proteins. Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogeneous binding characteristics for the relevant antigen. Homogeneous binding as used herein refers to the ability of the antibody species to bind to a specific antigen or epitope.
Monoclonal antibody reactive against one of the above- mentioned immunogens can be prepared by immunizing inbred mice, preferably Balb/c with the appropriate protein by techniques known in the art (Kohler and Milstein, Nature 256; 495-497, 1975). Hybridoma cells are subsequently selected by growth in hypoxanthine, thymidine and aminopterin in an appropriate cell culture medium such as Dulbecco's modified Eagle's medium (DMEM) . Antibody producing hybridomas are cloned, preferably using the soft agar technique of MacPherson, (Soft Agar Techniques, Tissue Culture Methods and Applications, Kruse and Paterson, eds., Academic Press, 276, 1973) . Discrete colonies are transferred into individual wells of culture plates for cultivation in an appropriate culture medium. Antibody producing cells are identified by screening with the appropriate immunogen. Immunogen positive hybridoma cells are maintained by techniques known in the art. Specific anti-monoclonal antibodies are produced by cultivating the hybridomas in vitro or preparing ascites fluid in mice following hybridoma injection by procedures known in the art. The polyclonal or monoclonal antibodies can show agonistic or antagonistic activity as compared to the activity of the natural ligand. Specific antagonistic or agonistic activity may involve the interference of the antibodies with the signal transduction system in the cells expressing the FSH receptor, without competing for the binding of FSH to its receptor.
The DNA coding for the receptor or for an altered receptor can be inserted into a suitable vector for expression in either a prokaryotic or a eukaryotic host. The hosts may be bacteria, phages, yeasts, funghi, animal cells or plant cells, preferred however are mammalian cells.
The vector into which the DNA is inserted may be any suitable vector. It may comprise suitable regulating elements such as promoters, enhancers, repressors, etc. If necessary, it may also comprise a signal sequence to transport the protein translated from the DNA to the surface or the outside of the host, even though the receptor does contain its own signal sequence.
In order to make a test for binding activity of compounds for the receptors of the invention it will suffice to have a cell, which preferably expresses no related receptors and more preferably no other receptors at all, and which cell expresses the receptor according to the invention in altered or unaltered form, in its (outer) membrane. In order to test for activity in activating or . blocking the receptor a signal producing system in the cell is needed. The signal producing system may be the cell's own or may be cotransfected with the DNA coding for the receptor. Usually, the signal will be provided by the so called second messenger system, which works through G- proteins which are associated with the receptor at its surface site. Any other signal producing system as well as second messenger initiated test system will be suitable, as long as the signal is somehow measurable.
EXAMPLES
1. Molecular cloning of human follicle stimulating hormone receptor(hFSH-R)
1.1. Probe synthesis
Oligodeoxynucleotides were synthesized using the phosphoramidite method on an Applied Biosystems 381A DNA synthesizer. They correspond to nucleotide position 1237-1255 (transmembrane region II) and 1843-1861 (transmembrane region VII) of the hFSH-R (Minegish et al., Bioch.Bioph.Res.Comm. 175. 1125, 1991) . Both oligonucleotides were used as primers in a polymerase chain reaction (Maniatis et al.. Cold Spring Harbor Laboratory, "Molecular Cloning: A Laboratory Manual", 1989) to generate a 625bp DNA fragment amplified from human genomic DNA (Clonetech) . The resulting product from PCR was verified by DNA sequence analysis (Pharmacia,T7-sequencing kit) .
1.2. cDNA library
A human testis cDNA library in phage λgtll (Clonetech; lxlO6 independent clones) was titrated on the host E.coli Y1090" and preparation of library DNA onto nitrocellulose filters was as described (Huynh et al. , DNA Cloning Techniques, "A Practical Approach", 1984; Maniatis et al., ibid.). 1.3. Clone identification
The partial hFSH-R amplification product obtained by PCR (section l.l.) was used as probe (Pharmacia, oligolabelling kit) to screen 3.6xl05 recombinant phages of the human testis cDNA library (section 1.2.). Prehybridization of filters was carried out for 5 hours at 65 °C in a solution containing 6xSSC (0.9 mol/1 NaCl,0.09 mol/1 Na-citrate, pH 7.0), lOxDenhardt (lxDenhardt:200μg/ml Ficoll-70 (Pharmacia), 200 μg/ml polyvinylpyrrolidone (Sigma) , 200μg/ml bovine serum albumin (BSA, Sigma) , 50 μg/ml sheared and denatured herring sperm DNA (Sigma) , 9% dextran sulphate (Pharmacia), and 0.1% sodium dodecyl sulphate (SDS, Sigma) . Hybridization was performed in the same solution by the addition of the 32P-labelled DNA probe (2.3xl05cpm/ml) . The mixture was incubated overnight at the same temperature. Filters were washed in solutions with decreasing salt concentrations (O.lxSSC, 65 °C) . Positive recombinant phages were purified by two successive rounds of phage titration and hybridization. Phage DNA inserts were isolated and subcloned in the endoR EcoRI site of pGEM3Z (Promega) and characterized by endoR mapping and DNA sequence analysis.
2. Expression of hFSH-R in Chinese hamster ovary(CHO) cells
2.1. Construction of expression plasmids A complete hFSH-R encoding cDNA was reconstructed from two overlapping cDNA clones (pGEM3ZR13 and pGEM3ZR25) by use of the unique internal endoR BamHI site at position 686 (Minegish et al., ibid.). The complete hFSH-R cDNA (pGEM3Zcl) is located on a 2222bp endoR EcoRI fragment (see Fig. 1) . After isolation and filling in the endoR EcoRI sites with the Klenow fragment of DNA polymerase I (Pharmacia) the fragment was inserted into the unique endoR BamHI site (after filling in with Klenow fragment) of vector pKCR (O'Hara et al. ,Proc.Natl.Acad.Sci.USA 78., 1527,1981). The latter vector was modified in a way that the last exon region of the /3-globin gene was removed by digestion with endoR EcoRI and BamHI, filling in, and religation (position 1122-1196; van Ooyen et al. , Science 206, 337, 1979) and replacement of pBR322 for pBR327 sequences.
2.2. Growth,transformation,and selection of CHO cells
CHO cells (CHO Kl) were obtained from ATCC (CCL61) . They were cultured in M505 medium that consisted of a mixture (1:1) of Dulbecco's Modified Eagle's Medium (DMEM, Gibco 074-2100) and Nutrient mixture F12 (Ham's F12, Gibco 074-1700) supplemented with 2.5 mg/ml sodium bicarbonate (Baker), 55 μg/ml sodium pyruvate (Fluka) , 2.3 μg/ml 3-mercaptoethanol (Baker), 1.2 μg/ml ethanolamine (Baker), 360 μg/ml L- glutamine (Merck), 0.45 μg/ml sodiumselenite (Fluka), 62.5 μg/ml penicillin (Mycopharm) , 62.5 μg/ml streptomycin (Serva) , and 10% fetal calf serum (FCS, Bocknek) . Recombinant constructs used for transformation of CHO cells consist of the expression vector pKCRhFSH-R (section 2.1.) and the selection vector pAG60MT2. The latter vector was constructed by insertion of a 3kb human MTIlA-containing endoR Hindlll fragment (Karin and Richards, Nature 299, 797, 1982) into the endoR Hindlll site of pAG60 (Colbere-Garapin et al. , J.Mol.Biol 150, 1, 1981); the transcription of this gene was directed towards the tk-promoter that was located in front of the neomycin resistence gene.
For stable transformation the recombinant vectors pKCRhFSH-R and pAG60MT2 (molar ratio 10:1) were introduced in CHO cells by the calcium-phosphate precipitation method (Graham and van der Eb, Virology 52, 456, 1973) . To select CHO transformants for stable integration and expression of the neomycin gene the antibiotic G418 (Gibco) was added 24 hours post- transformation at a concentration of 0.8 mg/ml. After this primary selection phase a second selection was performed by subjecting the cells to increasing concentrations of CdCl2 as described (Greene et al., Mol.Endocrinology 4., 1465, 1990). In this way CHOhFSH- RlCdlO refers to transformed CHO cells obtained after successive selection by neomycin and 10 μmol/1 CdCl2.
3. Characterisation of hFSH binding and signal transduction
3.1. Hormones Highly purified (> 99%) lyophilized recombinant human FSH (recFSH batch 77; specific activity 10.661 (8.859 - 12.765) IU/mg in vitro bioactivity in terms of IS 70/45) was supplied by Diosynth (Oss, The Netherlands) . Purified iodinated pituitary human FSH (125I-hFSH; 3.3 - 7.4 MBq/μg) was obtained from New England Nuclear-Du Pont (NEN, Boston, MA, USA) .
3.2. Scatchard analysis of FSH binding
CHOhFSH-RlCdlO cells pellet were homogenised with a teflon glass homogeniser in ice-cold 10 mmol/1 Tris- HCl buffer, pH 7.4, supplemented with 0.25 mol/1 sucrose and 5 mmol/1 MgCl2« The homogenate was diluted to 2.5 x 106 cells (starting material)/ml with homogenisation buffer. For saturation experiments, aliquots homogenate (200 μl/tube) were incubated with increasing concentrations 125I-hFSH (200 μl/tube; 4-150 pmol/1) with or without excess unlabeled recFSH (10 IU/100 μl/tube) in polypropylene microfuge tubes. The assay buffer consisted of 10 mmol/1 Tris-HCl, pH 7.4, supplemented with 5 mmol/1 MgCl2 and 1 g/1 bovine serum albumine (BSA; Sigma, St Louis, MO, USA) . After 24 h of incubation at room temperature (RT) , 500 μl ice-cold assay buffer was added, and bound and free hormone were separated by centrifugation (5 min at 15.000 x g) . The bound radioactivity was measured with a LKB gamma counter. The K-j (equilibrium dissociation constant) and Bπιax (maximum binding capacity) were assessed by Scatchard plot analysis. Competition of antisera for 1l?~ -FSH binding was assessed using the single cell clone mentioned previously expressing the human FSH-R, selected by subsequent exposure to G418 (800 μg/ml; Geneticin;
Gibco) and Cadmium (2,5 μM) . -"^I-FSH binding was performed essentially as described above, but the ligand (50,000 counts per minute) was mixed with varying dilutions of antisera prior to addition to the cell pellets. Binding of radioactive label is expressed as a percentage of maximal binding (%B/B0) .
The control serum that is included is a serum of saline-injected mice. The results are presented in
Figure 7.
3.3. FSH-induced cAMP production
The CHOhFSH-RlCdlO cells were washed once, resuspended in M505, pH 7.4, supplemented with 10% FCS, and cultured in 24 well-plates (Nunclon; 0.1% gelatin coated) at a concentration of 2 x 105 cells/ml/well for 48 h at 37 °C in a humidified atmosphere of 5% Cθ2/95% O2. After this preincubation period, cells were washed with M505 supplemented with 5 μg/ml transferrin (Pentex) and 1 μg/ml insulin (Diosynth) and incubated under the same conditions in 1 ml M505 supplemented with 5 μg/ml transferrin, 1 μg/ml insulin, 1 mM 3-isobutyl-l-methylxanthine (Aldrich-Europe, Beerse, Belgium) and various concentrations recFSH. After 10 min, 1, 4 and 22 h of incubation the supernatant medium was removed and stored at -20°C until cAMP analysis. To determine the intracellular cAMP content the remaining cells were treated with 0.5 ml 1-propanol which was followed by ultrasonification for 2 min. Subsequently, the content of each well was transfered to Eppendorf tubes and stored at -20°C. Prior to cAMP analysis using a cAMP (RIANEN) kit (NEN) , the cellular samples were lyophilized using a Speed Vac and reconstituted in 0.5 ml kit buffer. The medium samples were measured directly using a callibration curve of standard cAMP in CHO cell culture medium.
Interference of antisera with FSH-induced cAMP generation was assessed using the single cell clone mentioned previously expressing the human FSH-R, selected by subsequent exposure to G418 (800 μg/ml; Geneticin; Gibco) and Cadmium (2.5 μM) . Second messenger generation experiments were performed essentially as described above, but the ligand (10 mU/ml recFSH) was mixed with varying dilutions of antisera prior to addition to the cells. The amount of extracellular cAMP generated was determined after a 24 hour incubation period. The control serum is one of saline-injected mice. The results are presented in Figure 8.
4. Raising of anti FSH-receptor antibodies.
4.1 Cloning of GST-FSH-receptor fusion protein constructs.
All recombinant DNA techniques were performed according to standard protocolls (Sambrook et al., in: Molecular Cloning, 2nd ed. , CSHL Press) . Three fusion proteins were generated, containing different parts of the extracellular region of the FSH-R, linked to glutathione-S-transferase (GST) . Fusion protein 1 (GST-FSH-R 1) contains a 1000 basepair fragment coding for the complete extracellular N-terminus of the FSH- R. Fusion proteins 2 and 3 contain only parts of this sequence: fusion protein 2 (GST-FSH-R 2) contains a 600 basepair fragment coding for the N-terminal part of the FSH-R present in GST-FSH-R 1; fusion protein 3 (GST-FSH-R 3) contains a 400 basepair fragment coding for the C-terminal part of the FSH-R present in GST- FSH-R 1) . These fragments were derived by polymerase chain reaction (PCR) on the FSH-R cDNA clones (Figure 2A; seq. ID No. 1) . Via this procedure restriction sites were introduced, which facilitated subsequent cloning procedures. For GST-FSH-R 1 we used the primers 5'-TGTCATCATCGGATC-3■ and 5'-TCTGAGGATGTTGTAC- 3' ; for GST-FSH-R 2 we used the primers 5'- TGTCATCATCGGATC-3 ' and 5'-AGGCAGGGAATGGATCC-3' ; for GST-FSH-R 3 we used the primers 5•-AGAACAAGGATCC-3' and 5'-TCTGAGGATGTTGTAC-3' . Some primers were complementary to the FSH-R cDNA sequence (SEQ ID NO:l). Only the complementary sequence of the primers is shown.
The FSH-R fragments were cloned into one of the pGEX expression vectors (Smith and Johnson, Gene 67, 31, 1988) . Depending on the reading frame, either pGEX-1 (for the 1000 bp and 600 bp FSH-R fragments) or pGEX-3X (for the 400 bp FSH-R fragment) were used. In order to study expression of the fusion proteins, these constructs were transformed into E. coli MC1061.
4.2 Expression of GST-FSH-R fusion proteins in E. coli
Bacteria containing the GST-FSH-R fusion protein constructs were grown at 37°C to ODg5o 1. To induce fusion protein expression isopropyl-β-D- thiogalactopyranoside (IPTG) was added at a concentration of 0.1 mM, and cells were grown at 22°C for 4 hours. Cells were collected by centrifugation and resuspended in phosphate buffered saline (PBS) containing 1 mg/ml lysozyme and 1 mM phenylmethylsulfonyl fluoride (PMSF) . After 20 minutes on ice, Triton-X-l00 was added to a final concentration of 1%, incubation was continued for 10 minutes on ice, and cells were sonified. The supernantant of the sonicate was incubated with gluthathione-agarose carrier (Pharmacia) for 30 minutes at 4°C. Carriers were washed several times with PBS, and finally with 50 mM Tris pH 8. The fusion proteins thus purified were used to generate antisera.
4.3 Immunisation of mice
Six weeks old female BALB/c mice were injected intraperitoneally with 50 μg of bacterial fusion proteins in complete Freund's adjuvant. In addition, mice were immunized with membranes of 10' CHO cells transfected with the human FSH-R. For this purpose an FSH-R single cell clone was used that was obtained by subsequent G418 (800 μg/ml; Geneticin, Gibco) and cadmium (2.5 μM) selection. Two subsequent intramuscular injections were given at three weeks intervals with 50 μg fusion protein or membranes of 10 7' cells i.n i.ncomplete Freund's adjuvant. Three weeks after the third injection, mice were boosted intraperitoneally with 100 μg fusion protein or
7 membranes of 10' cells in PBS. Four days after this final boost, sera and spleens were collected.
Erythrocyte depleted spleen cells were prepared according to Steenbakkers et al. (J. Imm. Methods 152,
69, 1992).
4.4 Western immunoblotting
Denatured and reduced protein preparations were applied on sodium dodecylsulfate (SDS) polyacrylamide gels and blotted onto nitrocellulose filters. In order to be able to compare reactivity with different protein preparations, small (approximately 200 ng) and equivalent amounts of protein were applied to the gels. Membranes were blocked with 20% FCS, and incubated for 2 hours at room temperature with different concentrations of antibody preparations. Blots were washed with Tris-buffered saline (TBS) Tween and incubated with Goat-anti-mouse alkaline phosphatase (AP) conjugate (Pro ega) for 45 minutes at room temperature. Subsequently, AP substrate (nitroblue tetrazoline (NBT) / 5-bromo-4-chloro-3- indolyl phosphate (BCIP) ) reactions were done in 100 mM NaCl, 100 mM Tris pH 9.5, and 10 mM MgC12) .
In order to show specific immunoreactivity with the FSH-R part of GST-FSH-R fusion proteins, antisera raised against bacterial fusion proteins were precleared with GST by pre-incubating sera several times with bacterially produced GST.
4.5 Immunostaining
CHO cells were attached to the bottom of 24 well plates (approximately 10" cells/well) for three days. Cells were washed in phosphate buffered saline (PBS) for 10 minutes. After fixation with 4% paraformaldehyde in PBS for one hour at room temperature, the cells were washed in PBS containing 0.05% normal swine serum (NSS) and 0.02% Triton-X-100 for 10 minutes. After blocking with NSS in PBS-Triton for 1 hour, cells were incubated with antisera for 16 hours at room temperature. After washing 3 times 10 minutes in PBS-NSS-Triton, cells were incubated with Swine-anti-mouse coupled to fluorescein isothiocyanate (FITC) for 1 hour at room temperature. Cells were washed in PBS and kept in mount solution to avoid fading of the fluorescent signal during examination with an inverted microscope. Results
hFSH-R cDNA
Screening of the human testis cDNA library with the hFSH-R specific DNA probe resulted in five recombinant phages positive in hybridization. Two of these were studied in more detail and their insert size and map location are shown in figure 1. In order to reconstruct a cDNA encoding the complete hFSH-R a combination was made between pGEM3ZR13 (endoR EcoRI-BamHI;position -84 to +686) and pGEM3ZR25(endoR BamHI-EcoRI; position +686 to +2138) giving pGEM3Zcl.
hFSH-R cDNA sequence
A complete DNA sequence analysis was performed of the 2222bp endoR EcoRI fragment of pGEM3Zcl (Fig. 2A; Seq. ID. No. 1)). Comparison of this sequence with the hFSH-R cDNA sequence of Minegish et al. (ibid.) gave rise to several modifications of the hFSH-R protein. Differences between both sequences are shown in Figure 2 together with their corresponding change in amino acid.
Scatchard analysis of FSH binding
Scatchard plot analysis of the saturation data gave a straight line (Fig. 3, inset), indicating the presence of a single class of high affinity binding sites with a K^ = 28.8 pmol/1 and max = 4.1 pmol/1, which equals approximately 2400 receptors per cell.
FSH-induced cAMP production Incubation of CHOhFSH-RlCdlO cells with increasing concentrations recFSH induced a dose- dependent increase in intracellular as well as extracellular (medium) cAMP at all incubation times. Intracellular cAMP reached its maximum at 1 h of incubation which was followed by a sharp decline reaching cAMP levels only slightly elevated compared to unstimulated conditions at 22 h of incubation (Fig. 4) . Concomitantly with the decline in intracellular cAMP extracellular cAMP increased reaching the highest levels at 22 h of incubation (Fig. 5) .
Characterization of antibodies against the human FSH-R
Antibodies raised against the human FSH-R were characterized by Western immunoblotting, lmmunostai .m.ng, and by thei.r competi.ti.on for
Figure imgf000019_0001
binding and cAMP generation.
Antisera raised against the bacterial GST-FSH-R fusions recognized these proteins on Western blots (Fig. 6C) . Often, these antisera also recognized GST alone (control lanes) . After preclearing with GST, antisera specifically reacted with the FSH-R part of the fusion proteins. A representative example is shown in Figure 6B.
Immunostaining of prefixed CHO cells expressing the transfected human FSH-R showed a positive reaction with the antisera raised against bacterial fusion proteins, whereas nontransfected CHO cells showed no immunostaining. In order to asses whether antisera would also detect the native and functional FSH-R, it was determined whether antisera would compete for binding of 12^I-FSH to CHO cells expressing the transfected human FSH-R. Indeed, a concentration-dependent competition for lώJI-FSH binding to transfected CHO cells was observed with anti-FSH-R antisera, whereas an irrelevant serum did not show this effect (Fig. 7) .
We also determined whether antisera would interfere with FSH-induced cAMP generation. As shown in Figure 8, antisera directed against the FSH-R inhibited the generation of cAMP in a concentration- dependent manner. Again, an irrelevant serum was inactive in this respect.
The anti GST-FSH-Rl serum inhibited cAMP generation to the same degree at the anti GST-FSH-R3 serum (Fig. 8) . However, the GST-FSH-Rl antiserum competed less than the anti GST-FSH-R3 serum for 125I- FSH binding (Fig. 7) . This suggests that in the anti GST-FSH-Rl serum antibodies are present that do not interfere with FSH binding but do block second messenger generation. These antibodies are potentially interesting as antagonists since they do not have to compete with the high affinity FSH binding in order to block the biological effect of FSH.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Akzo N.V.
(B) STREET: Velperweg 76
(C) CITY: Arnhem
(E) COUNTRY: The Netherlands
(F) POSTAL CODE (ZIP) : 6824 BM
(G) TELEPHONE: 04120 - 66379 (H) TELEFAX: 04120 - 50592
(ii) TITLE OF INVENTION: Human gonadotropin receptor
(iii) NUMBER OF SEQUENCES: 2
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO)
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: EP 92.200.886.7
(B) FILING DATE: 30-MAR-1992
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2222 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(ix) FEATURE:
(A) NAME/KEV: CDS
(B) LOCATION: 85..2169
(C) IDENTIFICATION METHOD: experimental
(D) OTHER INFORMATION: /product= "FSH receptor"
/evidence= EXPERIMENTAL (ix) FEATURE:
(A) NAME/KEY: primer_bind
(B) LOCATION: 136..151
(D) OTHER INFORMATION: /standard_name= "first primer for GST-FSH-Rl and for GST-FSH-R2"
(ix) FEATURE:
(A) NAME/KEY: primer_bind
(B) LOCATION: 1168..1183
(D) OTHER INFORMATION: /standard_name= "primer for GST-FSH-R3"
(ix) FEATURE:
(A) NAME/KEY: primer_bind
(B) LOCATION: 763..776
(D) OTHER INFORMATION: /standard_name-= "primer for GST-FSH-R3"
(ix) FEATURE:
(A) NAME/KEY: primer_bind
(B) LOCATION: complement (1167..1183)
(D) OTHER INFORMATION: /standard_name= "primer for GST-FSH-Rl and GST-FSH-R3"
(ix) FEATURE:
(A) NAME/KEY: primer_bind
(B) LOCATION: complement (770..787)
(D) OTHER INFORMATION: /standard_name= "primer for GST-FSH-R2"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GAATTCGGGG TGTGGAGCTT CTGAGATCTG TGGAGGTTTT TCTCTGCAAA TGCAGGAAGA 60
AATCAGGTGG ATGGATGCAT AATT ATG GCC CTG CTC CTG GTC TCT TTG CTG 111
Met Ala Leu Leu Leu Val Ser Leu Leu 1 5
GCA TTC CTG AGC TTG GGC TCA GGA TGT CAT CAT CGG ATC TGT CAC TGC 159 Ala Phe Leu Ser Leu Gly Ser Gly Cys His His Arg lie Cys His Cys 10 15 20 25
TCT AAC AGG GTT TTT CTC TGC CAA GAG AGC AAG GTG ACA GAG ATT CCT 207 Ser Asn Arg Val Phe Leu Cys Gin Glu Ser Lys Val Thr Glu lie Pro
30 35 40
TCT GAC CTC CCG AGG AAT GCC ATT GAA CTG AGG TTT GTC CTC ACC AAG 255 Ser Asp Leu Pro Arg Asn Ala lie Glu Leu Arg Phe Val Leu Thr Lys 45 50 55
CTT CGA GTC ATC CAA AAA GGT GCA TTT TCA GGA TTT GGG GAC CTG GAG 303 Leu Arg Val lie Gin Lys Gly Ala Phe Ser Gly Phe Gly Asp Leu Glu 60 65 70
TITU E HEET AAA ATA GAG ATC TCT CAG AAT GAT GTC TTG GAG GTG ATA GAG GCA GAT 351 Lys lie Glu lie Ser Gin Asn Asp Val Leu Glu Val lie Glu Ala Asp 75 80 85
GTG TTC TCC AAC CTT CCC AAA TTA CAT GAA ATT AGA ATT GAA AAG GCC 399 Val Phe Ser Asn Leu Pro Lys Leu His Glu lie Arg lie Glu Lys Ala 90 95 100 105
AAC AAC CTG CTC TAC ATC AAC CCT GAG GCC TTC CAG AAC CTT CCC AAC 447 Asn Asn Leu Leu Tyr lie Asn Pro Glu Ala Phe Gin Asn Leu Pro Asn 110 115 120
CTT CAA TAT CTG TTA ATA TCC AAC ACA GGT ATT AAG CAC CTT CCA GAT 495 Leu Gin Tyr Leu Leu lie Ser Asn Thr Gly lie Lys His Leu Pro Asp 125 130 135
GTT CAC AAG ATT CAT TCT CTC CAA AAA GTT TTA CTT GAC ATT CAA GAT 543 Val His Lys lie His Ser Leu Gin Lys Val Leu Leu Asp lie Gin Asp 140 145 150
AAC ATA AAC ATC CAC ACA ATT GAA AGA AAT TCT TTC GTG GGG CTG AGC 591 Asn lie Asn lie His Thr lie Glu Arg Asn Ser Phe Val Gly Leu Ser 155 160 165
TTT GAA AGT GTG ATT CTA TGG CTG AAT AAG AAT GGG ATT CAA GAA ATA 639 Phe Glu Ser Val lie Leu Trp Leu Asn Lys Asn Gly lie Gin Glu lie 170 175 180 185
CAC AAC TGT GCA TTC AAT GGA ACC CAA CTA GAT GAG CTG AAT CTA AGC 687 His Asn Cys Ala Phe Asn Gly Thr Gin Leu Asp Glu Leu Asn Leu Ser 190 195 200
GAT AAT AAT AAT TTA GAA GAA TTG CCT AAT GAT GTT TTC CAC GGA GCC 735 Asp Asn Asn Asn Leu Glu Glu Leu Pro Asn Asp Val Phe His Gly Ala 205 210 215
TCT GGA CCA GTC ATT CTA GAT ATT TCA AGA ACA AGG ATC CAT TCC CTG 783 Ser Gly Pro Val lie Leu Asp lie Ser Arg Thr Arg lie His Ser Leu 220 225 230^
CCT AGC TAT GGC TTA GAA AAT CTT AAG AAG CTG AGG GCC AGG TCG ACT 831 Pro Ser Tyr Gly Leu Glu Asn Leu Lys Lys Leu Arg Ala Arg Ser Thr 235 240 245
TAC AAC TTA AAA AAG CTG CCT ACT CTG GAA AAG CTT GTC GCC CTC ATG 879 Tyr Asn Leu Lys Lys Leu Pro Thr Leu Glu Lys Leu Val Ala Leu Met 250 255 260 265
GAA GCC AGC CTC ACC TAT CCC AGC CAT TGC TGT GCC TTT GCA AAC TGG 927 Glu Ala Ser Leu Thr Tyr Pro Ser His Cys Cys Ala Phe Ala Asn Trp 270 275 280
AGA CGG CAA ATC TCT GAG CTT CAT CCA ATT TGC AAC AAA TCT ATT TTA 975 Arg Arg Gin lie Ser Glu Leu His Pro lie Cys Asn Lys Ser lie Leu 285 290 295 AGG CAA GAA GTT GAT TAT ATG ACT CAG ACT AGG GGT CAG AGA TCC TCT 1023
Arg Gin Glu Val Asp Tyr Met Thr Gin Thr Arg Gly Gin Arg Ser Ser 300 305 310
CTG GCA GAA GAC AAT GAG TCC AGC TAC AGC AGA GGA TTT GAC ATG ACG 1071
Leu Ala Glu Asp Asn Glu Ser Ser Tyr Ser Arg Gly Phe Asp Met Thr
315 320 325
TAC ACT GAG TTT GAC TAT GAC TTA TGC AAT GAA GTG GTT GAC GTG ACC 1119
Tyr Thr Glu Phe Asp Tyr Asp Leu Cys Asn Glu Val Val Asp Val Thr 330 335 340 345
TGC TCC CCT AAG CCA GAT GCA TTC AAC CCA TGT GAA GAT ATC ATG GGG 1167
Cys Ser Pro Lys Pro Asp Ala Phe Asn Pro Cys Glu Asp lie Met Gly 350 355 360
TAC AAC ATC CTC AGA GTC CTG ATA TGG TTT ATC AGC ATC CTG GCC ATC 1215
Tyr Asn lie Leu Arg Val Leu lie Trp Phe lie Ser lie Leu Ala lie 365 370 375
ACT GGG AAC ATC ATA GTG CTA GTG ATC CTA ACT ACC AGC CAA TAT AAA 1263
Thr Gly Asn lie lie Val Leu Val lie Leu Thr Thr Ser Gin Tyr Lys 380 385 390
CTC ACA GTC CCC AGG TTC CTT ATG TGC AAC CTG GCC TTT GCT GAT CTC 1311
Leu Thr Val Pro Arg Phe Leu Met Cys Asn Leu Ala Phe Ala Asp Leu
395 400 405
TGC ATT GGA ATC TAC CTG CTG CTC ATT GCA TCA GTT GAT ATC CAT ACC 1359
Cys lie Gly lie Tyr Leu Leu Leu lie Ala Ser Val Asp lie His Thr 410 415 420 425
AAG AGC CAA TAT CAC AAC TAT GCC ATT GAC TGG CAA ACT GGG GCA GGC 1407
Lys Ser Gin Tyr His Asn Tyr Ala lie Asp Trp Gin Thr Gly Ala Gly 430 435 440
TGT GAT GCT GCT GGC TTT TTC ACT GTC TTT GCC AGT GAG CTG TCA GTC 1455
Cys Asp Ala Ala Gly Phe Phe Thr Val Phe Ala Ser Glu Leu Ser Val 445 450 455
TAC ACT CTG ACA GCT ATC ACC TTG GAA AGA TGG CAT ACC ATC ACG CAT 1503
Tyr Thr Leu Thr Ala lie Thr Leu Glu Arg Trp His Thr lie Thr His 460 465 470
GCC ATG CAG CTG GAC TGC AAG GTG CAG CTC CGC CAT GCT GCC AGT GTC 1551
Ala Met Gin Leu Asp Cys Lys Val Gin Leu Arg His Ala Ala Ser Val
475 480 485
ATG GTG ATG GGC TGG ATT TTT GCT TTT GCA GCT GCC CTC TTT CCC ATC 1599
Met Val Met Gly Trp He Phe Ala Phe Ala Ala Ala Leu Phe Pro He 490 495 500 505
TTT GGC ATC AGC AGC TAC ATG AAG GTG AGC ATC TGC CTG CCC ATG GAT 1647
Phe Gly He Ser Ser Tyr Met Lys Val Ser He Cys Leu Pro Met Asp 510 515 520 ATT GAC AGC CCT TTG TCA CAG CTG TAT GTC ATG TCC CTC CTT GTG CTC 1695 He Asp Ser Pro Leu Ser Gin Leu Tyr Val Met Ser Leu Leu Val Leu 525 530 535
AAT GTC CTG GCC TTT GTG GTC ATC TGT GGC TGC TAT ATC CAC ATC TAC 1743 Asn Val Leu Ala Phe Val Val He Cys Gly Cys Tyr He His He Tyr 540 545 550
CTC ACA GTG CGG AAC CCC AAC ATC GIG TCC TCC TCT AGT GAC ACC AGG 1791 Leu Thr Val Arg Asn Pro Asn He Val Ser Ser Ser Ser Asp Thr Arg 555 560 565
ATC GCC AAG CGC ATG GCC ATG CTC ATC TTC ACT GAC TTC CTC TGC ATG 1839 He Ala Lys Arg Met Ala Met Leu He Phe Thr Asp Phe Leu Cys Met 570 575 580 585
GCA CCC ATT TCT TTC TTT GCC ATT TCT GCC TCC CTC AAG GTG CCC CTC 1887 Ala Pro He Ser Phe Phe Ala He Ser Ala Ser Leu Lys Val Pro Leu 590 595 600
ATC ACT GTG TCC AAA GCA AAG ATT CTG CTG GTT CTG TTT CAC CCC ATC 1935 He Thr Val Ser Lys Ala Lys He Leu Leu Val Leu Phe His Pro He 605 610 615
AAC TCC TGT GCC AAC CCC TTC CTC TAT GCC ATC TTT ACC AAA AAC TTT 1983 Asn Ser Cys Ala Asn Pro Phe Leu Tyr Ala He Phe Thr Lys Asn Phe 620 625 630
CGC AGA GAT TTC TTC ATT CTG CTG AGC AAG TGT GGC TGC TAT GAA ATG 2031 Arg Arg Asp Phe Phe He Leu Leu Ser Lys Cys Gly Cys Tyr Glu Met 635 640 645
CAA GCC CAA ATT TAT AGG ACA GAA ACT TCA TCC ACT GTC CAC AAC ACC 2079 Gin Ala Gin He Tyr Arg Thr Glu Thr Ser Ser Thr Val His Asn Thr 650 655 660 665
CAT CCA AGG AAT GGC CAC TGC TCT TCA GCT CCC AGA GTC ACC AAT GGT 2127 His Pro Arg Asn Gly His Cys Ser Ser Ala Pro Arg Val Thr Asn Gly 670 675 680
TCC ACT TAC ATA CTT GTC CCT CTA AGT CAT TTA GCC CAA AAC 2170
Ser Thr Tyr He Leu Val Pro Leu Ser His Leu Ala Gin Asn 685 690 695
TAAAACACAAT GTGAAAATGT ATCTGAAAAA AAAAAAAAAA AAACCGGAAT TC 2222
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 695 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Ala Leu Leu Leu Val Ser Leu Leu Ala Phe Leu Ser Leu Gly Ser 1 5 10 15
Gly Cys His His Arg He Cys His Cys Ser Asn Arg Val Phe Leu Cys 20 25 30
Gin Glu Ser Lys Val Thr Glu He Pro Ser Asp Leu Pro Arg Asn Ala 35 40 45
He Glu Leu Arg Phe Val Leu Thr Lys Leu Arg Val He Gin Lys Gly 50 55 60
Ala Phe Ser Gly Phe Gly Asp Leu Glu Lys He Glu He Ser Gin Asn 65 70 75 80
Asp Val Leu Glu Val He Glu Ala Asp Val Phe Ser Asn Leu Pro Lys
85 90 95
Leu His Glu He Arg He Glu Lys Ala Asn Asn Leu Leu Tyr He Asn 100 105 110
Pro Glu Ala Phe Gin Asn Leu Pro Asn Leu Gin Tyr Leu Leu He Ser 115 120 125
Asn Thr Gly He Lys His Leu Pro Asp Val His Lys He His Ser Leu 130 135 140
Gin Lys Val Leu Leu Asp He Gin Asp Asn He Asn He His Thr He 145 150 155 160
Glu Arg Asn Ser Phe Val Gly Leu Ser Phe Glu Ser Val He Leu Trp 165 170 175
Leu Asn Lys Asn Gly He Gin Glu He His Asn Cys Ala Phe Asn Gly 180 185 190
Thr Gin Leu Asp Glu Leu Asn Leu Ser Asp Asn Asn Asn Leu Glu Glu 195 200 205 *
Leu Pro Asn Asp Val Phe His Gly Ala Ser Gly Pro Val He Leu Asp 210 215 220
He Ser Arg Thr Arg He His Ser Leu Pro Ser Tyr Gly Leu Glu Asn 225 230 235 240
Leu Lys Lys Leu Arg Ala Arg Ser Thr Tyr Asn Leu Lys Lys Leu Pro 245 250 255
Thr Leu Glu Lys Leu Val Ala Leu Met Glu Ala Ser Leu Thr Tyr Pro 260 265 270
Ser His Cys Cys Ala Phe Ala Asn Trp Arg Arg Gin He Ser Glu Leu 275 280 285
SUBSTITUTESHEET His Pro He Cys Asn Lys Ser He Leu Arg Gin Glu Val Asp Tyr Met 290 295 300
Thr Gin Thr Arg Gly Gin Arg Ser Ser Leu Ala Glu Asp Asn Glu Ser 305 310 315 320
Ser Tyr Ser Arg Gly Phe Asp Met Thr Tyr Thr Glu Phe Asp Tyr Asp 325 330 335
Leu Cys Asn Glu Val Val Asp Val Thr Cys Ser Pro Lys Pro Asp Ala 340 345 350
Phe Asn Pro Cys Glu Asp He Met Gly Tyr Asn He Leu Arg Val Leu 355 360 365
He Trp Phe He Ser He Leu Ala He Thr Gly Asn He He Val Leu 370 375 380
Val He Leu Thr Thr Ser Gin Tyr Lys Leu Thr Val Pro Arg Phe Leu 385 390 395 400
Met Cys Asn Leu Ala Phe Ala Asp Leu Cys He Gly He Tyr Leu Leu 405 410 415
Leu He Ala Ser Val Asp He His Thr Lys Ser Gin Tyr His Asn Tyr 420 425 430
Ala He Asp Trp Gin Thr Gly Ala Gly Cys Asp Ala Ala Gly Phe Phe 435 440 445
Thr Val Phe Ala Ser Glu Leu Ser Val Tyr Thr Leu Thr Ala He Thr 450 455 460
Leu Glu Arg Trp His Thr He Thr His Ala Met Gin Leu Asp Cys Lys 465 470 475 480
Val Gin Leu Arg His Ala Ala Ser Val Met Val Met Gly Trp He Phe 485 490 495
Ala Phe Ala Ala Ala Leu Phe Pro He Phe Gly He Ser Ser Tyr Met 500 505 510
Lys Val Ser He Cys Leu Pro Met Asp He Asp Ser Pro Leu Ser Gin 515 520 525
Leu Tyr Val Met Ser Leu Leu Val Leu Asn Val Leu Ala Phe Val Val 530 535 540
He Cys Gly Cys Tyr He His He Tyr Leu Thr Val Arg Asn Pro Asn 545 550 555 560
He Val Ser Ser Ser Ser Asp Thr Arg He Ala Lys Arg Met Ala Met 565 570 575
Leu He Phe Thr Asp Phe Leu Cys Met Ala Pro He Ser Phe Phe Ala 580 585 590 He Ser Ala Ser Leu Lys Val Pro Leu He Thr Val Ser Lys Ala Lys
595 600 605
He Leu Leu Val Leu Phe His Pro He Asn Ser Cys Ala Asn Pro Phe
610 615 620
Leu Tyr Ala He Phe Thr Lys Asn Phe Arg Arg Asp Phe Phe He Leu
625 630 635 640
Leu Ser Lys Cys Gly Cys Tyr Glu Met Gin Ala Gin He Tyr Arg Thr
645 650 655
Glu Thr Ser Ser Thr Val His Asn Thr His Pro Arg Asn Gly His Cys
660 665 670
Ser Ser Ala Pro Arg Val Thr Asn Gly Ser Thr Tyr He Leu Val Pro
675 680 685
Leu Ser His Leu Ala Gin Asn 690 695
SUBSTITUTESHEET LEGENDS
Fig. 1. Physical maps of three hFSH-R cDNA clones and their position on their respective plasmids.
Fig. 2A. Nucleotide sequence of the hFSH-R cDNA cloned in pGEM3Zcl (seq. ID No. 1).
Fig. 2B. Amino acid sequence of the hFSH-R coded for by the hFSH-R cDNA in pGEM3Zcl (seq. ID No. 2).
Fig. 3. Binding of 125I-hFSH to hFSH-R of a neomycin- CdCl2 (10 μmol/1) selected CHO pool. Cell membranes were incubated with increasing concentrations 125ι- hFSH in the absence or presence of excess unlabeled recFSH. The saturation curve of specifically bound 125I-hFSH together with the derived Scatchard plot are shown. The values represent the mean of duplicate determinations. The calculated Kd and Bmaχ are presented in the Scatchard plot (insert) , where B/F is plotted against the specific bound.
Fig. 4. Dose dependent stimulation of intracellular cAMP by recFSH. The stimulations were repeated at different times of incubation. Values represent the mean of duplicate determinations.
Fig. 5. Dose dependent stimulation of extracellular (medium) cAMP by recFSH. The stimulations were repeated at different times of incubation. Values represent the mean of duplicate determinations.
Fig. 6A. Western blot of GST-hFSH-R fusion proteins stained with Coomassie Blue.
SUBSTITUTESHEET Fig. 6B. Western blot of GST-hFSH-R fusion proteins stained with antiserum raised against GST-hFSH-R3 fusion protein. The antiserum has been preclaered with GST proteins.
Fig. 6C. Western blot of GST-hFSH-R fusion proteins stained with antiserum raised against GST-hFSH-Rl fusion protein.
Fig. 7. Interference of hFSH-R antisera with binding of 1 12255II--FFSSHH ttoo CCHHOO ccee!lls expressing the human FSH receptor (hFSH-R) .
Fig. 8. Interference of hFSH-R antisera with FSH-induced cAMP generation.

Claims

CLAIMS .
1) An antibody or an antigen-binding fragment thereof having specific binding activity for the human Follicle Stimulating Hormone receptor (FSH-R) .
2) An antibody or fragment thereof according to claim 1, characterised in that said antibody is a monoclonal antibody.
3) An antibody or fragment thereof according to claim 1 or 2, characterized in that said antibody or fragment has agonistic or antagonistic activity with respect to the activity of FSH.
4) An anti idiotypic antibody or a functional fragment thereof capable of binding to an antibody or a fragment according to claims 1 or 2.
5) Pharmaceutical formulation comprising a therapeutical amount of an antibody or antigen-binding fragment according to any of the claims l to 4 and a pharmaceutical acceptable carrier.
6) A proteinaceous substance having binding activity for at least human follicle stimulating hormone, characterized in that the proteinaceous substance is the human Follicle Stimulating Hormone (FSH) receptor or an analogon or a derivative or a fragment thereof.
7) A proteinaceous substance according to claim 6, characterized in that it comprises a sequence at least 70% homologous to the sequence of SEQ ID NO:2 or a part thereof. 8) A nucleic acid, characterized in that at least part of it codes for a proteinaceous substance according to claim 6 or 7.
9) A nucleic acid according to claim 8, characterized in that it is a DNA sequence which is at least 70% homologous to the sequence of SEQ ID NO:l or a part thereof.
10) An expression vector, characterized in that it comprises a DNA sequence according to claim 9 and suitable regulation sequences for expression.
11) A vector according to claim 10, characterized in that it comprises a signal sequence for transporting the expression product of a DNA sequence according to claim 9 to the surface site of a cell.
12) A transfected cell capable of expression, characterized in that it comprises a vector according to claim 10 or 11.
13) A transfected cell, characterized in that it expresses a proteinaceous substance according to claim 6 or 7.
14) A transfected cell according to claim 13, characterized in that said proteinaceous substance is expressed at the surface of the cell.
15) A transfected cell according to claim 13 or 14, characterized in that it is cotransfected with one or more elements of a signal producing system.
16) A transfected cell according to claim 14 or 15, characterized in that the proteinaceous substance induces a response in the signal producing system of the transfected cell.
17) A transfected cell according to claim 16, characterized in that the signal producing system gives a measurable signal.
18) A method for testing compounds for human gonadotropin like activity, characterized in that the compound is contacted with a transfected cell according to claim 17 and that the signal of the signal producing system is detected or measured.
19) A method for testing compounds for antagonistic activity of human gonadotropin like activity, characterized in that said compound is contacted with a transfected cell according to claim 17 together with a compound with known human gonadotropin like activity and that the presence or absence of a signal of the signal producing system is detected o,r measured.
20) A method according to claim 18 or 19, characterized in that the human gonadotropin like activity is human follicle stimulating hormone activity.
PCT/EP1993/000780 1992-03-30 1993-03-29 Human gonadotropin receptor (fsh receptor) WO1993020199A1 (en)

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EP92200886 1992-03-30

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WO1993020199A9 true WO1993020199A9 (en) 1994-01-06

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EP0950711A3 (en) * 1998-02-06 2003-09-17 Akzo Nobel N.V. Gonadotropin receptor
US7273932B1 (en) 1999-05-28 2007-09-25 The University Of Cincinnati Antisense oligonucleotides for fertility and menstrual cycle regulation and for chemopreventive and chemotherapeutic use
AU2001269014A1 (en) * 2000-05-18 2001-11-26 Bayer Aktiengesellschaft Regulation of human follicle stimulating hormone-like g protein-coupled receptor
US11865166B2 (en) 2016-08-29 2024-01-09 The Trustees Of The University Of Pennsylvania Nucleic acid encoding optimized immunogenic peptide that targets follicle stimulating hormone receptor
US20210340263A1 (en) * 2018-08-09 2021-11-04 The Wistar Institute Anti-Follicule Stimulating Hormone Receptor Antibodies
CN114605542B (en) * 2020-12-04 2024-03-01 康威(广州)生物科技有限公司 Preparation and application of anti-FSHR antibody and antibody-drug conjugate thereof

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US4652450A (en) * 1984-08-31 1987-03-24 Research Corporation Bacterial FSH binding inhibitor
US4921808A (en) * 1986-06-25 1990-05-01 The Albany Medical College Of Union University Method for determining follicle stimulating hormone
WO1988008719A1 (en) * 1987-05-12 1988-11-17 Aphton Corporation Reproductive hormone-specific immunocontraceptives and methods of using them
EP0471030B1 (en) * 1989-05-05 1994-12-14 Genentech, Inc. Glycoprotein hormone receptor molecules
CA2106061C (en) * 1991-03-15 2008-10-07 Christie A. Kelton Human follicle stimulating hormone receptor

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