LIGANDS POTENTIATING THE BIOACTIVITY OF GONADOTROPHINS

专利摘要:
The present invention relates to antibodies directed against follicle-stimulating hormone (FSH) capable of potentiating the bioactivity of gonadotropins.
公开号:FR3025518A1
申请号:FR1558078
申请日:2015-08-31
公开日:2016-03-11
发明作者:Elodie Kara；Jeremye Decourtye；Sophie Casteret；Marie-Christine Maurel
申请人:Repropharm SA；
IPC主号:

专利说明:

[0001] TECHNICAL FIELD The present invention relates to antibodies directed against follicle-stimulating hormone (FSH) capable of potentiating the bioactivity of gonadotropins.
[0002] The present invention finds its applications mainly in human and veterinary medicine, to induce ovulation in a female mammal. In the description below, references in brackets ([]) refer to the list of references at the end of the text.
[0003] State of the art Gonadotropins (or gonadotropins) are complex glycoprotein hormones that play a central role in the regulation of reproduction in vertebrates by acting on the functions of the gonads (ovaries and testes). Two of these hormones are secreted in all vertebrates: luteinizing hormone (LH) and follicle stimulating hormone (FSH). In two groups of mammals, equines and primates, there is also a chorionic gonadotropin (CG) secreted by the placenta: human choriogonadotropin (hCG) and equine choriogonadotropin (eCG), both of which act via LH receptors.
[0004] Luteinizing hormone (LH) is produced by gonadotropic cells of the anterior lobe of the pituitary gland under stimulation of GnRH, itself produced by the hypothalamus. LH stimulates testosterone production in males, while it is involved in ovarian cycle changes in females where it is responsible for terminal follicular growth and ovulation followed by transformation of the ruptured ovulatory follicle into a corpus luteum. . During the luteal phase of the menstrual cycle, LH stimulates the secretion of progesterone by the corpus luteum, essential for the early development and implantation of the embryo. LH consists of a subunit a common to all glycoprotein hormones of the same species (such as FSH, CG and thyroid stimulating hormone, TSH) and a subunit [3 responsible for The activity specificity of the hormone; activity that exists only if the two subunits are non-covalently associated in the form of a dimer. Follicle stimulating hormone (FSH) is produced by the ante-pituitary gland under stimulation of GnRH produced by the hypothalamus. In males, it stimulates Sertoli cells essential for spermatogenesis. In females, it is responsible for the recruitment of primordial, immature follicles, their growth and their differentiation into pre-ovulatory follicles by stimulating the FSH receptors of granulosa cells. FSH consists of two α and β subunits, and has a structure similar to that of LH. Only the dimer is able to stimulate FSH receptors. In females, the levels of LH and FSH are cyclic: very low during sexual rest or outside the ovulatory period, with a peak of secretion in the preovulatory period. Gonadotropins are used in veterinary and human medicine to induce ovulation in female mammals. Although effective, these treatments pose a health risk due to the use of hormones extracted from biological fluids (blood, urine) or tissue (pituitary), particularly in the veterinary field. This is the case of chorionic equine gonadotropin (eCG) extracted from pregnant mare blood, and porcine LH and FSH extracted from porcine pituitary glands. In the veterinary field, hCG extracted from urine of pregnant women, Chorulon® (MSD Laboratory) is also used. In the field of the human clinic, and particularly of the Medically Assisted Procreation (or PMA), hormones extracted from urine of menopausal women such as Fostimon® (Laboratoire Junévrier) which is a purified FSH and Menopur® are used. (Laboratoire Ferring Pharmaceuticals) which is a hMG (human menoposal gonadotropin), a mixture of FSH and LH and Endo5000 Chorionic Gonadotropin which is a purified hCG (Schering-Plow Laboratory). Recombinant human FSHs, such as Gonal-F® (Merck Serono Laboratory) and Puregon® (Merck Shering-Plow Laboratory) are also used; recombinant hCG and LH such as Ovidrel® and Luveris® (Merck Serono Laboratory). In addition, the repeated use of these hormones most often induces an immune reaction that neutralizes the effect of the hormones thus leading to a decrease in therapeutic efficacy. However, it has also been demonstrated in some cases that the immune reaction could produce antibodies capable of potentiating the activity of the hormone when it was co-administered (Patent EP 1518863) [1]. Since then, it has also been demonstrated three anti-LH monoclonal antibodies capable of potentiating its action as well as that of FSH for two of them (International Application WO 2012/066519) [2]. DESCRIPTION OF THE INVENTION The inventors have now obtained monoclonal antibodies produced against the FSH subunit [3], capable of potentiating its action as well as that of LH and hCG. These monoclonal antibodies are respectively called CA5 and CH10. The hybridoma that produced the CA5 antibody was deposited in accordance with the Budapest Treaty on 03/10/2013 at the CNCM (National Collection of Microorganisms Culture, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France), under number CNCM 1-4801. The hybridoma that produced the antibody CH10 was deposited in accordance with the Budapest Treaty on 03/10/2013 with the CNCM (National Collection of Culture of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris). Cedex 15, France), under number CNCM 1-4802. The nucleotide sequences of the variable regions of the heavy and light chains of the CA5 and CH10 antibodies were determined, the corresponding peptide sequences deduced. They are presented respectively in Tables 1 and 2 below.
[0005] 2 5 Table 1 monoclonal antibody CA5 heavy chain (VH) sequence GAGGTGAAGCTGGTGGAATCTGGAGGAGGCTTGGTACAGCCT GGGGGTTCTCTGAGACTCTCCTGTGCAACTTCTGGGTTCACCTT CAGTGATTTCTACATGGAGTGGGTCCGCCAGCCTCCAGGGAAG AGACTGGAGTGGATTGCTGCAAGTAGAAACAAAGCTAAGGATT ATACAACAGAGTACAGTGCATCTGTGAAGGGTCGGTTCATCGT CTCCAGAGACACTTCCCAAAGCATCCTCTACCTTCAGATGAATG CCCTGAGAGCTGAGGACACTGCCATTTATTTCTGTGCAAGAGAT nucleotide GCAAGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCT (SEQ ID NO: 1) 3025518-4 - CTGCA sequence EVKLVESGGGLVQPGGSLRLSCATSGFTFSDFYMEWVRQPPGKR LEVVIAASRNKAKDYTTEYSASVKGRFIVSRDTSQSILYLQMNALRA peptide EDTAIYFCARDARFAYWGQGTLVTVSA (SEQ ID NO: 2) light chain (VL ) Sequence GACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGT TGGAGAGAAGATTACTATGAGCTGCAAGTCCAGTCAGAGCCTTT TATATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCAGCAG AAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCAC TAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCT GGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAG ACCTGGCAGTTTATTACTGTCAGCAATATTATAGCTATCCTCGG ACGTTCGGTGGAGGCACCAAGCT nucleotide GGAAATCAAA (SEQ ID NO: 3) Sequence DIVMSQSPSSLAVSVGEKITMSCKSSQSLLYSSNQKNYLAWYQQK PGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAV peptide YYCQQYYSYPRTFGGGTKLEIK (SEQ ID NO: 4) Table 2 Monoclonal antibody CH10 heavy chain (VH) sequence GAGGTGCAGCTTGTTGAGTCTGGTGGAGGATTGGTGCAGCCTA AAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTC AATACCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGG GTTTGGAATGGGTTGCTCGCATAAGAAGTAAAAGTAATAATTAT GCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTC CAGAGATGATTCACAAAGCATGCTCTATCTGCAAATGAACAACT TGAAAACTGAGGACACAGCCATGTATTACTGTGTGAGACAGGAT TACTACGGTAGTAGCTACTTTGACTACTGGGGCCAAGGCACCA nucleotide CTCTCACAGTCTCCTCA (SEQ ID NO: 5) sequence EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNVVVRQAPGKG LEVVVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLK TEDTAMYYCVRQDYYGSSYFDYWGQGTTLTVSS peptide SEQ ID NO: 6) light chain (VL) sequence GACATTGTGATGACTCAGTCTCCAGCCACCCTGTCTGTGACTCC 3025518-5 - AGGAGATAGAGTCTCTCTTTCCTGCAGGGCCAGCCAGAGTATT nucleotide (SEQ ID NO: 7) AGCGA CTACTTACACTGGTATCAACAAAAATCACATGAGTCTCC AAGGCTTCTCATCAAATATGCTTCCCAATCCATCTCTGGGATCC CCTCCAGGTTCAGTGGCAGTGGATCAGGGTCAGATTTCACTCT CAGTATCAACAGTGTGGAACCTGAAGATGTTGGAGTGTATTACT GTCAAAATGGTCACAGCTTTCCGTACACGTTCGGAGGGGGGAC CAAGCTGGAAATAAAA sequence peptide DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPR LLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNG (SEQ ID NO: 8) HSFPYTFGGGTKLEIK The sequences encoding the CDRs (complementarity determining regions) were determined from the sequences of the variable regions of the heavy chains (VH-CDR) and light (VL-CDR) CA5 and CH10 antibodies above. The corresponding peptide sequences have been deduced and are presented respectively in Tables 3 and 4 below. Table 3 CA5 monoclonal antibody VH-CDR1 (SEQ ID NO: 9) GFTFSDFY VH-CDR2 (SEQ ID NO: 10) SRNKAKDYTT VH-CDR3 (SEQ ID NO: 11) ARDARFAY VL-CDR1 (SEQ ID NO: 12) QSLLYSSNQKNY VL VL-CDR3 (SEQ ID NO: 13) QQYYSYPRT Table 4 Monoclonal antibody CH10 VH-CDR1 (SEQ ID NO: 14) GFTFNTYA VH-CDR2 (SEQ ID NO: 15) IRSKSNNYAT VH-CDR3 (SEQ ID NO: 13) The present invention relates to a ligand of follicle-stimulating hormone (FSH) potentiating bioactivity. FSH, luteinizing hormone (LH) and chorionic gonadotropin (CG), characterized in that it comprises the paratope of an anti-subunit [3 FSH antibody. For the purposes of the present invention, the term "FSH anti-subunit antibody" is intended to mean any antibody obtained by immunizing an animal from the first 5 injections of FSH followed by several boosters with injection of the sub-unit. unit [3 of the FSH. Injections may be made from FSH of different mammals, for example ovine, human, bovine, caprine or porcine FSH, equine, canine, murine, etc., and [3 FSH subunits of homologous origin. or heterologous. Thus monoclonal antibodies CA5 and CH10 were obtained following immunization from sheep FSH and ovine FSH subunit [3]. In particular, the subject of the present invention is therefore a ligand according to the invention, characterized in that: the variable domain of the heavy chain contains the following CDRs: VH-CDR1, defined by the sequence GFTFSDFY (SEQ ID NO: 9); - VH-CDR2, defined by the sequence SRNKAKDYTT (SEQ ID NO: 10); - VH-CDR3, defined by the sequence ARDARFAY (SEQ ID NO: 11); and the variable domain of the light chain contains the following CDRs: VL-CDR1, defined by the sequence QSLLYSSNQKNY (SEQ ID NO: 12); VL-CDR2, defined by the sequence VVAS; - VL-CDR3, defined by the sequence QQYYSYPRT (SEQ ID NO: 13). In particular, the subject of the present invention is thus a ligand according to the invention, characterized in that: the variable domain of the heavy chain contains the following CDRs: VH-CDR1, defined by the sequence GFTFNTYA (SEQ ID NO: 14); - VH-CDR2, defined by the sequence IRSKSNNYAT (SEQ ID NO: 15); - VH-CDR3, defined by the sequence VRQDYYGSSYFDY (SEQ ID NO: 16); and the variable domain of the light chain contains the following CDRs: VL-CDR1, defined by the sequence QSISDY (SEQ ID NO: 17); VL-CDR2, defined by the sequence YAS; - VL-CDR3, defined by the sequence QNGHSFPYT (SEQ ID NO: 18). For the purpose of the present invention, the term "CDR" means the three hypervariable regions of the variable regions of the heavy and light chains of an antibody which constitute the elements of the paratope and make it possible to determine the complementarity of the antibody with the epitope. antigen. These three hypervariable regions are framed by four constant regions that constitute the FR or framework regions and give a stable configuration to the variable domain. A ligand according to the present invention is, for example: CA5 monoclonal antibody produced by hybridoma CNCM 1-4801; the monoclonal antibody CH10 produced by the hybridoma CNCM 1-4802; a Fab, Fab ', F (ab') 2, Fv, dsFv or scFv fragment, a nanobody of an antibody above. Preferably, it is a Fab fragment or an scFv fragment; a bi-, tri- or tetravalent form (diabodies, triabodies, tetrabodies) of two, three or four fragments of scFv, respectively; a recombinant antibody comprising the paratope of an antibody above and whose constant regions have been modified so as to minimize immunogenicity with respect to the animal or man for whom it is intended. For example, it is a chimeric antibody (humanized, ovine, caprinized, bovine, porcine, etc ...) or fully humanized, ovine, caprine, bovine, porcine As a non-limiting example, the nucleotide sequences scFv derived from the CA5 and CH10 antibodies were determined, the corresponding peptide sequences deduced, and are presented respectively in Tables 5 and 6 below. Table 5 scFv CA5 CAGGTGCAGCTGCAGCAGTCAGGCGGCGGCCTGGTACAACCT GGTGGCTCACTGCGCCTGAGCTGCGCAACCAGCGGTTTTACCT TTAGCGATTTCTACATGGAATGGGTTCGCCAACCGCCGGGTAA GCGTCTGGAATGGATCGCGGCGAGCCGTAACAAGGCGAAAGA TTATACCACTGAATATAGCGCGTCGGTGAAAGGTCGCTTCATTG Sequence TCTCGCGCGATACCAGCCAGTCGATTCTGTATCTGCAAATGAAT n cl u ic éotid GCCCTGCGTGCCGAAGACACGGCCATCTACTTCTGTGCGCGTG (SEQ ID NO: 19) ATGCACGCTTTGCCTATTGGGGCCAAGGCACCCTGGTGACCGT TAGCGCCGGTGGTGGCGGTTCAGGTGGTGGCGGTAGCGGTGG CGGTGGCTCAGATATTCAGATGACCCAGACCCCGTCAAGCCTG GCGGTGTCAGTCGGCGAAGAGATTACTATGAGCTGTAAAAGCT CGCAGAGCCTGCTGTACTCATCGAACCAGAAAAATTACCTGGC ATGGTATCAACAGAAGCCGGGTCAGTCGCCGAAACTGCTGATC 3025518-8 - TACTGGGCCTCAACCCGTGAGAGCGGCGTACCGGATCGCTTTA CTGGCAGCGGCAGCGGCACGGACTTTACGCTGACGATTAGCTC GGTGAAGGCCGAAGACCTGGCGGTTTATTATTGCCAACAGTAC TATAGCTACCCTCGTACCTTCGGCGGCGGCACGAAACTCGAGA TTAAACATCACCATCACCATCACTAACTCGAGATCAAGTAA QVQLQQSGGGLVQPGGSLRLSCATSGFTFSDFYMEWVRQPPGK RLEVVIAASRNKAKDYTTEYSASVKGRFIVSRDTSQSILYLQMNALR Peptide sequence AEDTAIYFCARDARFAYW GQGTLVTVSAGGGGSGGGGSGGGGS DIQMTQTPSSLAVSVGEEITMSCKSSQSLLYSSNQKNYLAWYQQK (SEQ ID NO 20) Table 6 PGQSPKWYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAV YYCQQYYSYPRTFGGGTKLEIKHHHHHH scFv CH10 CAGGTGCAGCTGCAGCAATCAGGCGGCGGCCTGGTCCAACCG AAAGGTAGCCTGAAACTGTCGTGCGCCGCCAGCGGCTTTACGT TCAACACTTACGCGATGAATTGGGTGCGTCAGGCGCCTGGTAA AGGCCTGGAATGGGTGGCACGCATCCGTTCAAAAAGCAACAAT TACGCGACGTATTATGCAGACAGCGTAAAAGATCGCTTTACCAT CAGCCGTGATGATTCACAGTCAATGCTGTACCTGCAAATGAATA ACCTGAAAACTGAAGACACTGCGATGTATTATTGTGTTCGCCAG GACTATTACGGTAGCTCGTATTTCGATTACTGGGGCCAAGGCA nucleotide sequence CCACCCTGACGGTGAGCTCGGGTGGCGGTGGCTCAGGTGGTG GTGGTAGCGGCGGTGGCGGTAGCGATATCCAGATGACCCAGA (SEQ ID NO: 21) CCCCGGCAACCCTGAGCGTTACCCCTGGTGACCGCGTTTCGCT GAGCTGCCGTGCCTCGCAGAGCATTTCGGACTATCTGCACTGG TATCAGCAAAAATCACACGAATCACCGCGTCTGCTGATTAAGTA CGCCAGCCAATCGATTAGCGGTATTCCGAGCCGCTTTTCGGGC TCGGGTTCGGGCTCGGATTTTACCCTGTCAATTAATAGCGTAGA GCCGGAAGATGTAGGCGTCTACTATTGTCAGAACGGCCATTCA TTCCCGTACACGTTTGGCGGCGGCACCAAGCTCGAGATTAAGC ATCACCATCATCACCATTAACTCGAGA TCAAGTAA QVQLQQSGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGK Peptide sequence GLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNL KTEDTAMYYCVRQDYYGSSYFDYVVGQGTTLTVSSGGGGSGGGG (SEQ ID NO 22) SGGGGSDIQMTQTPATLSVTPGDRVSLSCRASQSISDYLHWYQQ 3025518-9 - KSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGV YYCQNGHSFPYTFGGGTKLEIKHHHHHH The present invention also relates to a nucleotide sequence encoding a ligand according to the invention. The subject of the present invention is also a recombinant vector, in particular an expression vector, comprising a nucleotide sequence according to the invention. The subject of the present invention is also a host cell comprising a nucleotide sequence according to the invention or a recombinant vector according to the invention. For example, these are the CNCM 1-4801 and CNCM 1-4802 hybridomas or a cell transformed with a nucleotide sequence or a recombinant vector according to the invention. The present invention also relates to a process for producing a ligand according to the invention, characterized in that it comprises culturing in a suitable medium of host cells according to the invention, and the recovery of said ligand from of said culture.
[0006] The inventors have demonstrated that CA5 antibody strongly potentiates porcine, ovine, and bovine FSH and to a lesser extent than human FSH significantly. In addition, the inventors have demonstrated that the scFvs derived from the CA5 and CH10 antibodies have the same binding and potentiation properties as the antibodies from which they are derived.
[0007] The present invention also relates to a ligand according to the invention for use as a medicament, in particular for potentiating the bioactivity of FSH, LH, and chorionic gonadotropin (CG) for inducing ovulation in a mammal and to reduce the problems of hormone-dependent infertility or hypofertility in a male or female mammal. The present invention also relates to a complex formed of a ligand and a gonadotropin, or an active peptide thereof, capable of binding to said ligand and whose activity is potentiated by said ligand. For example, it is the complex of a ligand with LH, with the hormone chorionic gonadotropin (CG), or with FSH extracted from biological or recombinant tissues or fluids, or an active peptide of these capable of binding to said ligand and whose activity is potentiated by said ligand. The subject of the present invention is also a ligand or complex according to the invention for use as a medicament, in particular for potentiating the bioactivity of FSH, LH, and chorionic gonadotropin (CG) to induce ovulation. or even polyovulation in a female mammal or to reduce the problems of hormone-dependent infertility or hypofertility in a male or female mammal. The drug also increases the level of circulating endogenous progesterone secreted by one or more corpora lutea in a female mammal, thereby promoting early embryonic development and decreasing the risk of abortion.
[0008] The present invention also relates to a meat-producing method, wherein said method comprises administering ligand and / or complex of the invention to a non-human animal mammalian animal. The present invention also relates to a ligand and / or complex of the invention for use in the treatment of infertility or hormone dependent hypofertility in a mammal. In the case of a female mammal suffering from infertility or subfertility, the administration of the ligand or complex of the invention will allow to stimulate a natural procreation, medically assisted or artificial. It should be noted that the administration of the ligand or complex of the invention to a healthy female mammal will also make it possible to trigger ovulation in the context of natural or artificial procreation. For the purposes of the present invention, the term "steroid-dependent infertility / hypofertility" means infertility / hypofertility due to hormonal insufficiency, for example, low circulating concentrations of FSH and LH or an absence of these hormones resulting, for example, from an external cause. (For example pesticides) or internal (for example, pituitary or hypothalamic insufficiency or a problem of receptivity of the gonads to LH and / or FSH due to an abnormality of the LH, FSH, CG, or gonadotropin receptors, for example mutation or polymorphism of receptors). The ligands and complexes of the invention may be used in humans or animals, in particular sheep, cattle, goats, horses, pigs, murines, canines, camels, etc. The ligands, hormones or The complexes according to the invention can be administered either separately, sequentially or jointly by injection, for example intramuscularly, intravenously, intraperitoneally, subcutaneously, transcutaneously, intradermally, intraorbitally, intraocularly, ophthalmically, or transocularly, without alter their potentiating effect. The present invention also relates to a pharmaceutical composition comprising a ligand or complex of the invention and a pharmaceutically acceptable vehicle. The pharmaceutical composition may further comprise FSH and / or LH and / or chorionic gonadotropin hormone (CG). Other advantages may still be apparent to those skilled in the art upon reading the examples below, illustrated by the appended figures, given by way of illustration. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 illustrates the in vitro potentiating effect of monoclonal antibodies CA5 (A) and CH10 (B) on the bioactivity of ovine FSH (oFSH) on bovine granulosa cells. FIG. 2 represents the in vitro potentiating effect of monoclonal antibodies CA5 (A) and CH10 (B) on the ovine FSH bioactivity (oFSH) on a HEK 293 cell line stably transfected with the human FSH receptor. FIG. 3 represents the in vitro potentiating effect of monoclonal antibody CA5 on the ovine FSH bioactivity (oFSH) on a HEK 293 cell line stably transfected with the human FSH receptor and the Glosensor® vector. FIG. 4 represents the potentiating effect of monoclonal antibody CA5 on the bioactivity of porcine FSH (pFSH) on a HEK 293 cell line stably transfected with the human FSH receptor and the Glosensor® vector. FIG. 5 represents the in vitro potentiating effect of monoclonal antibody CA5 (A to E) and scFv CA5 (F) on the bioactivity of human FSH (hFSH) on a HEK 293 cell line stably transfected with the human FSH receptor and the Glosensor® vector. FIG. 6 represents the in vitro potentiating effect of the monoclonal antibody CH10 on the ovine FSH bioactivity (oFSH) on a HEK 293 cell line stably transfected with the human FSH receptor and the Glosensor® vector. FIG. 7 represents the potentiating effect of the monoclonal antibody CH10 on the bioactivity of human FSH (hFSH) in vitro on a H EK 293 cell line stably transfected with the human FSH receptor and the vector Glosensor®. FIG. 8 represents the in vivo potentiating effect of monoclonal antibody CA5 on the ovine FSH bioactivity (oFSH) (A) and on the bioactivity of human FSH (hFSH) Gonal-F®, Puregon® and Fostimon ® (B) in the female rat. FIG. 9 represents the in vivo potentiating effect of monoclonal antibody CA5 on the bioactivity of human FSH (hFSH) Gonal-F® (A) and the potentiating effect of scFv CA5 on the bioactivity of human FSHs (FIG. hFSH) Gonal-F® (A), Puregon® and Fostimon® (B) in female rats. FIG. 10 represents the in vivo potentiating effect of the monoclonal antibody CH10 on the ovine FSH bioactivity (oFSH) (A) and on the bioactivity of human FSH (hFSH) Gonal-F® (B) in the female rat. FIG. 11 represents the in vivo potentiating effect of the monoclonal antibody CH10 on the bioactivity of human FSH (hFSH) Puregon® and Fostimon® (A) and the potentiating effect of scFv CH10 on the bioactivity of human FSH (hFSH) Gonal-F® (B) in the female rat.
[0009] EXAMPLES EXAMPLE 1: OBTAINING THE LIGANDS OF THE INVENTION AND THEIR CHARACTERIZATION 1 / Immunization Strategy for the Mice The injections were all carried out intraperitoneally on mice (Balb / C). Five mice were used for each immunization strategy.
[0010] Mouse immunization strategy for CA5 antibody and CH10 antibody A first injection (JO) was performed with 100 μg of purified sheep FSH with complete Freund's adjuvant. Several booster injections were then performed according to the following sequence: J25 and J44: 100 μg of purified sheep FSH with incomplete Freund's adjuvant; J134 and J204: 50 μg of sheep FSH beta subunit with incomplete Freund's adjuvant; J217, J218 and J219: 30 μg of sheep FSH beta subunit without adjuvant; 5 - J220: fusion. 2 / Isotyping The isotyping of the CA5 and CH10 antibodies was carried out with the FastElysa isotyping kit marketed by RD Biotech (reference RDB 3255) according to the manufacturer's recommendations. The CA5 antibody is an immunoglobulin class IgG2a and isotype Kappa. The values of the optical densities (OD) obtained were 0.335 and 0.371 respectively.
[0011] The CH10 antibody is an immunoglobulin of IgM class and Kappa isotype. The values of the optical densities (OD) obtained were 0.2 and 0.124 respectively. 3 / Sequencing The nucleotide sequences of the variable part of the heavy (VH) and light (VL) chains of the CA5 and CH10 antibodies secreted by the CNCM1-4801 and CNCM1-4802 hybridomas respectively, were determined from their RNAs. messenger (mRNA) according to the protocol below.
[0012] The RNAs were extracted from the cells using the Nucleospin® RNA kit (Macherey Nagel, Germany) according to the manufacturer's recommendations. The purified RNA concentrations were estimated by measuring the absorbance (A) at 260 nm and their quality by the ratio A260nm / 280nm and visually after electrophoretic migration on agarose gel.
[0013] The complementary DNAs of the mRNAs were then synthesized using an oligo-dT18 as a primer by reverse transcription reaction with the enzyme M-MLV (Ref M1701, Promega, USA) according to the manufacturer's recommendations. The synthesis of the second strand of DNA was carried out by a polymerase chain reaction (PCR) according to the following protocol: at 4 μl of the retrotranscription reaction were added in a final volume of 50 μl; the reaction buffer (1X final), 200 μM each dNTPs, 300 nM sense and antisense primers, 1.25 U GoTaq polymerase (Ref M3175, Promega, USA). For amplification of the variable part of the light chains, 9 different primer pairs were used (MKRev2 to 8 + MKC5For) and 3 different couples for those of the heavy chains (CA5: VHRev1 + VHFor, CH10: VHRev1 + MpCFor ). Table 7: Nucleotide sequences of the primers used to sequence the heavy (VH) and light (VL) chains of the CA5 antibody. CA5 Antibody Heavy Chain (VH) Name Sequence 5'-3 'SEQ ID NO VHRev1 CGGGATCCTCTAGAGGTCCAACTGCAGGAGTCA GG SEQ ID NO: 23 VHFor CAGGGGCCAGTGGATAGAC SEQ ID NO: 24 Light Chain (VL) MKRev5 GACATTGTGATGACCCAGTCT SEQ ID NO: 25 MKC5For GGATACAGTTGGTGCAGCATC SEQ ID NO: Table 8: Nucleotide sequences of the primers used to sequence the 5 'portion of the heavy (VH) and light (VL) chains of the CA5 antibody. CA5 Antibody Heavy Chain (VH) Name Sequence 5'-3 'SEQ ID NO CA5VH_Fw CACTTTTACATGGTATCCAGTG SEQ ID NO: 27 CA5VH_Rev GTTTCTACTTGCAGCAATCCACT SEQ ID NO: 28 Light Chain (VL) CA5VL_Fw GAWTCACAGRCCCAGGTYC SEQ ID NO: 29 CA5VL_Rev CCCAGTAAATCAGCAGTTTAGGA SEQ ID NO: 30 Table 9: Nucleotide sequences of the primers used to sequence the heavy (VH) and light (VL) chains of the CH10 antibody. Antibody CH10 Heavy Chain (VH) Name Sequence 5'-3 'SEQ ID NO VHRev1 CGGGATCCTCTAGAGGTCCAACTGCAGGAGTCA GG SEQ ID NO: 23 MpCFor GGGGAAGACATTTGGGAAGG SEQ ID NO: 31 3025518 - 15 - Light Chain (VL) MKRev2 GATATTGTGATGACGCAGGCT SEQ ID NO: 32 MKRev3 GATATTGTGATAACCCAG SEQ ID NO: 33 MKRev4 GACATTGTGCTGACCCAATCT SEQ ID NO: 34 MKRev6 GATATTGTGCTAACTCAGTCT SEQ ID NO: 35 MKRev8 GACATCCAGCTGACTCAGTCT SEQ ID NO: 36 MKC5For GGATACAGTTGGTGCAGCATC SEQ ID NO: 37 Table 10: Nucleotide sequences of the primers used to sequence the 5 'part of the heavy chains (VH) and light (VL) of the CH10 antibody. Antibody CH10 Heavy chain (VH) Name Sequence 5'-3 'SEQ ID NO CH1 OVH_Fw ATGGTGTTGGGGCTGAAGTG SEQ ID NO: 38 CH1 OVH_Rev CAGTTCATGGCGTAGGTATTGA SEQ ID NO: 39 Light chain (VL) CH1 OVL_Fw TTCTGGAYTTCAGCCTCCAG SEQ ID NO: 40 CH1 OVL_Rev GATTGGGAAGCATATTTGATGAG SEQ ID NO: 41 The PCR program used consists of an initial denaturation of 2 min at 95 ° C followed by 30 denaturation cycles of 30 sec at 95 ° C, 30 sec hybridization at 47 ° C and amplification 1 min at 72 ° C. ° C and finally a final amplification of 5 min at 72 ° C. The PCR products obtained were desalted with the QlAquick® Gel extraction kit (Ref 28704, Qiagen GmbH, Germany) and then ligated with the plasmid pGEMT easy vector (Ref A1360, Promega, USA) to be transformed into bacteria. Plasmid DNA extracted from different bacterial clones was sent for assay by sequencing (Macrogen Europe, The Netherlands). The 5 'terminal nucleotide sequences of the VH and VL of the 2 antibodies were subsequently determined by the design of specific primers anchored in the leader sequences of the cDNAs (Fw primer). These primers were designed following the homology identification by alignment between the previously obtained VL and VH sequences and the IMGT / V-QUEST software database (Brochet et al., Nucl. Acids Res., 36: W503-508, 2008, Giudicelli et al., Cold Spring Harb Protoc., 2011 (6): 695-715, 2011) [3, 4] and the extraction of the IMGT / GENE- interest leader sequences. DB (Giudicelli et al., Nucl Acids Res., 33: D256-261, 2005) [5]. The antisense primers (Rev) were designed in the respective previously determined VH and VL sequences of each of the antibodies. The protocol used to obtain part 5 is the same as that described in the previous paragraph. The consensus nucleotide sequences were deduced from sequence alignment using MultAlin software (Corpet, Nucl Acids Res., 16 (22): 10881-10890, 1988) [6]. Polypeptide sequence transcription and annotation of CDRs were performed using IMGTN-QUEST software. The results are shown in Tables 11 to 14. Table 11: Nucleotide and peptide sequences of the heavy variable (VH) and light (VL) parts of the CA5 antibody. Antibody (CA5) Heavy chain (VH) sequence GAGGTGAAGCTGGTGGAATCTGGAGGAGGCTTGGTACAGCCTGG GGGTTCTCTGAGACTCTCCTGTGCAACTTCTGGGTTCACCTTCAG TGATTTCTACATGGAGTGGGTCCGCCAGCCTCCAGGGAAGAGAC TGGAGTGGATTGCTGCAAGTAGAAACAAAGCTAAGGATTATACAA CAGAGTACAGTGCATCTGTGAAGGGTCGGTTCATCGTCTCCAGAG ACACTTCCCAAAGCATCCTCTACCTTCAGATGAATGCCCTGAGAG CTGAGGACACTGCCATTTATTTCTGTGCAAGAGATGCAAGGTTTG nucleotide CTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA SEQ ID NO: 1 Sequence EVKLVESGGGLVQPGGSLRLSCATSGFTFSDFYMEWVRQPPGKRL EWIAASRNKAKDYTTEYSASVKGRFIVSRDTSQSILYLQMNALRAEDT AIYFCARDARFAYWGQGTLVTVSA peptide SEQ ID NO: 2 light chain (VL) sequence GACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTT GGAGAGAAGATTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTA TATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAA CCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGG GAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGAC AGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGC AGTTTATTACTGTCAGCAATATTATAGCTATCCTCGGACGTTCGGT GGAGGCACCAAGCTGGAAATCAAA nucleotide SEQ ID NO: 3 Sé SEQ ID NO: 4 3025518 DIVENCEQSPSSLAVSVGEKITMSCKSSQSLLYSSNQKNYLAWYQQPP GQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYY CQQYYSYPRTFGGTKLEIK TABLE 12: Nucleotide and Peptide Sequences of the Heavy (VH) and Light (VL) Variable Portions of the CH10 Antibody. CH10 antibody heavy chain (VH) sequence GAGGTGCAGCTTGTTGAGTCTGGTGGAGGATTGGTGCAGCCTAA AGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAA TACCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTT TGGAATGGGTTGCTCGCATAAGAAGTAAAAGTAATAATTATGCAAC ATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGA TGATTCACAAAGCATGCTCTATCTGCAAATGAACAACTTGAAAACT GAGGACACAGCCATGTATTACTGTGTGAGACAGGATTACTACGGT AGTAGCTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTC nucleotide TCCTCA SEQ ID NO: 5 Sequence EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGL EVVVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTE DTAMYYCVRQDYYGSSYFDYWGQGTTLTVSS peptide SEQ ID NO: 6 Light chain (VL) sequence GACATTGTGATGACTCAGTCTCCAGCCACCCTGTCTGTGACTCCA GGAGATAGAGTCTCTCTTTCCTGCAGGGCCAGCCAGAGTATTAGC GACTACTTACACTGGTATCAACAAAAATCACATGAGTCTCCAAGG CTTCTCATCAAATATGCTTCCCAATCCATCTCTGGGATCCCCTCCA GGTTCAGTGGCAGTGGATCAGGGTCAGATTTCACTCTCAGTATCA ACAGTGTGGAACCTGAAGATGTTGGAGTGTATTACTGTCAAAATG GTCACAGCTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAA nucleotide ATAAAA SEQ ID NO: 7 Sequence DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHVVYQQKSHESPRLL IKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFP YTFGGTKLEIK Peptide SEQ ID NO: 8 Table 13: CDR of heavy variable (VH) and light (VL) parts of CA5 VH-CDR1 antibody (SEQ ID NO: 9) GFTFSDFY VH-CDR2 (SEQ ID NO: 10) SRNKAKDYTT VH-CDR3 (SEQ ID NO: 11) ARDARFAY VL-CDR1 (SEQ ID NO: 12) QSLLYSSNQKNY VL-CDR2 WAS 3025518 - 18 - VL-CDR3 (SEQ ID NO: 13) QQYYSYPRT Table 14: CDR of the parts heavy (VH) and light (VL) variables of the antibody CH10 VH-CDR1 (SEQ ID NO: 14) GFTFNTYA VH-CDR2 (SEQ ID NO: 15) IRSKSNNYAT VH-CDR3 (SEQ ID NO: 16) VRQDYYGSSYFDY VL- CDR1 (SEQ ID NO: 17) QSISDY VL-CDR2 VL-CDR3 YAS (SEQ ID NO: 18) QNGHSFPYT 4 / Construction, production and characterization of scFv 5a / Construction of scFv antibody fragments Variable fragment synthesis genes single chain (scFv) derived from CA5 and CH10 antibodies were synthesized by ATG: Biosynthetics GmbH (Germany).
[0014] Each sequence was designed for the fusion of the heavy and light variable portions (SEQ ID NO: 1 / SEQ ID NO: 3 for CA5, SEQ ID NO: 5 / SEQ ID NO: 7 for CH10) linked by a coding sequence for the peptide (Gly4Ser) 3 ensuring the functionality of the protein and terminated by a sequence encoding the peptide His6 (peptide HIS-tag) which will allow the purification of scFvs. In order to allow their insertion into the expression plasmid, the sequences were flanked by the PstI and SalI restriction enzyme sites. Additional sequence was added between the 3 'end of the VL and the SalI site allowing deletion of the His6 peptide if desired. Codons were optimized for E. coli expression. A schematic representation of the construction of the scFvs synthesis genes is detailed below: VH Linker LQ I (G4S) 31 ...................... The antibody fragments were inserted between the PstI and XhoI enzyme sites of the expression plasmid pSVV1 (ATG: Biosynthetics GmbH, Germany) according to ES Ward et al. (VVard et al.). , Nature, 341: 544-546, 1989) [7] which contains under the control of a LacZ inducible promoter, a PeIB signal sequence which fuses in reading phase with the recombinant antibody fragment gene. , allows the addressing of the synthesized protein to the bacterial periplasm. In the periplasm, this signal sequence is removed by a peptidase. After control by sequencing the quality of the constructs, the plasmids pSW1-CA5 and pSVV1-CH10 were transformed by heat shock into HB2151 bacteria (T53040, Interchim, France) made competent (Li et al., J. Biotechnol. 9 (50): 8549-8554, 2010) [8].
[0015] Table 15: Nucleotide and oeotidia sequences of scFv CA5. CA5 scFv nucleotide sequence CAGGTGCAGCTGCAGCAGTCAGGCGGCGGCCTGGTACAACCT GGTGGCTCACTGCGCCTGAGCTGCGCAACCAGCGGTTTTACCT SEQ ID NO: 19 TTAGCGATTTCTACATGGAATGGGTTCGCCAACCGCCGGGTAAG CGTCTGGAATGGATCGCGGCGAGCCGTAACAAGGCGAAAGATT ATACCACTGAATATAGCGCGTCGGTGAAAGGTCGCTTCATTGTC TCGCGCGATACCAGCCAGTCGATTCTGTATCTGCAAATGAATGC CCTGCGTGCCGAAGACACGGCCATCTACTTCTGTGCGCGTGAT GCACGCTTTGCCTATTGGGGCCAAGGCACCCTGGTGACCGTTA GCGCCGGTGGTGGCGGTTCAGGTGGTGGCGGTAGCGGTGGCG GTGGCTCAGATATTCAGATGACCCAGACCCCGTCAAGCCTGGC GGTGTCAGTCGGCGAAGAGATTACTATGAGCTGTAAAAGCTCGC AGAGCCTGCTGTACTCATCGAACCAGAAAAATTACCTGGCATGG TATCAACAGAAGCCGGGTCAGTCGCCGAAACTGCTGATCTACTG GGCCTCAACCCGTGAGAGCGGCGTACCGGATCGCTTTACTGGC AGCGGCAGCGGCACGGACTTTACGCTGACGATTAGCTCGGTGA AGGCCGAAGACCTGGCGGTTTATTATTGCCAACAGTACTATAGC TACCCTCGTACCTTCGGCGGCGGCACGAAACTCGAGATTAAACA TCACCATCACCATCACTAACTCGAGATCAAGTAA Sequence QVQLQQSGGGLVQPGGSLRLSCATSGFTFSDFYMEVVVRQPPGKR LEWIAASRNKAKDYTTEYSASVKGRFIVSRDTSQSILYLQMNALRAE peptide SEQ ID NO: 20 DTAIYFCARDARFAYWGQGTLVTVSAGGGGSG GGGSGGGGSDIQ MTQTPSSLAVSVGEEITMSCKSSQSLLYSSNQKNYLAWYQQKPGQ SPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYC QQYYSYPRTFGGGTKLEIKHHHHHH 3025518 - 20 - Table 16: Nucleotide and nucleotide sequences of scFv CH10. CH10 scFv nucleotide sequence CAGGTGCAGCTGCAGCAATCAGGCGGCGGCCTGGTCCAACCG AAAGGTAGCCTGAAACTGTCGTGCGCCGCCAGCGGCTTTACGT SEQ ID NO: 21 TCAACACTTACGCGATGAATTGGGTGCGTCAGGCGCCTGGTAAA GGCCTGGAATGGGTGGCACGCATCCGTTCAAAAAGCAACAATTA CGCGACGTATTATGCAGACAGCGTAAAAGATCGCTTTACCATCA GCCGTGATGATTCACAGTCAATGCTGTACCTGCAAATGAATAAC CTGAAAACTGAAGACACTGCGATGTATTATTGTGTTCGCCAGGA CTATTACGGTAGCTCGTATTTCGATTACTGGGGCCAAGGCACCA CCCTGACGGTGAGCTCGGGTGGCGGTGGCTCAGGTGGTGGTG GTAGCGGCGGTGGCGGTAGCGATATCCAGATGACCCAGACCCC GGCAACCCTGAGCGTTACCCCTGGTGACCGCGTTTCGCTGAGC TGCCGTGCCTCGCAGAGCATTTCGGACTATCTGCACTGGTATCA GCAAAAATCACACGAATCACCGCGTCTGCTGATTAAGTACGCCA GCCAATCGATTAGCGGTATTCCGAGCCGCTTTTCGGGCTCGGG TTCGGGCTCGGATTTTACCCTGTCAATTAATAGCGTAGAGCCGG AAGATGTAGGCGTCTACTATTGTCAGAACGGCCATTCATTCCCG TACACGTTTGGCGGCGGCACCAAGCTCGAGATTAAGCATCACC ATCATCACCATTAACTCGAGATCAAGTAA Sequence QVQLQQSGGGLVQPKGSLKLSCAASGFTFNTYAMNVVVRQAPGKG LEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKT peptide SEQ ID NO: 22 EDTAMYYCVRQDYYGSSYFDYWGQGTTLTVSSGGG GSGGGGSG GGGSDIQMTQTPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSH ESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYC QNGHSFPYTFGGGTKLEIKHHHHHH b / Production of Recombinant Antibody Fragments - Bacterial Culture Pre-culture was performed in 5 ml of 2xYT medium containing 50 μg / ml ampicillin overnight at 37 ° C. The next day, 500 μl of this preculture was seeded in 500 ml of the same medium and grown at 37 ° C to 150 RPM until a OD600 'of 1.4 was obtained. The synthesis of scFv was induced by adding 0.1 mM IPTG 16 h at 16 ° C to 150 RPM. Extraction The culture medium was centrifuged for 30 minutes at 4500 g at 4 ° C. The rest of the preparation was carried out at 4 ° C. To extract the bacterial periplasm, the pellet was resuspended and incubated in 10 ml of TES (0.2 M Tris pH8, 0.5 M EDTA, 0.5 M sucrose) for 30 min to which 15 ml of TES diluted 1/4 to be incubated 30 min. The bacterial extract was centrifuged for 30 min at 10,000g. The supernatant was dialyzed against PBS overnight. The dialyzed supernatant was treated immediately to purify the scFv or stored at -20 ° C until use. The production of scFv in the periplasm was analyzed by Western blotting using an anti-His-Tag HRP antibody (Ref R93125 Life Technologies, France) according to the manufacturer's instructions for use. Purification The periplasm was centrifuged for 20 min at 5,000 g at 4 ° C. The supernatant was incubated with HIS-Select® Nickel Affinity Gel (Sigma-Aldrich, MO, USA) with stirring for 1h at 4 ° C. The gel was washed with 0.05 M sodium phosphate buffer, 0.3 M NaCl pH8 and then the same buffer supplemented with 20 mM imidazole to obtain a D0280 ', close to 0. The scFv was then eluted with 0.05 M sodium phosphate buffer, 0.3 M NaCl, 250 mM imidazole pH8. The eluate was dialyzed against PBS overnight. It is stored at -20 ° C. Quality control The purified scFv was analyzed by 15% polyacrylamide gel electrophoresis after staining with Coomassie blue and SephadexTM 75 10/300 GL column exclusion chromatography (Ref 17-5174-01 GE Healthcare, Germany). ). 5 / Specificity 3025518 - 22 - The specificity of the antibodies and their scFv was studied by ELISA technique. Each hormone tested was prepared at a concentration of 10 μg / ml in 0.1M sodium carbonate buffer pH 9.6 and dispensed at 100 μl per well on an ELISA plate. The adsorption time was 18 hours at + 4 ° C. After five washes, the wells were treated with 100 μl of PBS supplemented with 0.1% Tween and 1% BSA for 45 min at 37 ° C, then each antibody or scFv was dispensed at 100 μl / well and incubated for 1 hour at 37 ° C. On each hormone tested, the antibodies and scFvs were distributed at different concentrations in a range of 10 to 250 μg / ml for the antibodies and from 10 to 150 or 200 μg / ml for the scFvs. After five washes, a peroxidase-coupled secondary antibody (HRP) was dispensed at 100 μl / well and incubated for 1 hour at 37 ° C. According to the isotype of the monoclonal antibody studied, the secondary antibody was an anti-IgG1 HRP (Ref 115-035-205, Jackson ImmunoResearch Laboratories Inc.), an anti-IgG2a HRP (Ref 115-035- 206, Jackson Laboratories) or HRP anti-IgM (Ref 115-035-075, Jackson Laboratories). For scFv, an anti-His Tag HRP (R93125 Life Technologies, France) was used. After five washes, enzymatic activity was revealed with TMB dispensed at 100 μl / well. The time of revelation was 5 to 30 min at room temperature depending on the rate of reaction. After stopping the reaction with 1M H2504 (50 μl / well) the intensity of the color reaction (Optical Density) was measured using an ELISA plate spectrophotometer. For the CA5 and CH10 antibodies and their scFv, the percentage of cross-reactivity was calculated with respect to the values obtained with ovine FSH (oFSH) considered to be the 100% reference value. The percentage of cross-reaction was calculated conventionally by comparing the dose-response curves obtained with the concentration range of the antibody or scFv. From the curve obtained with the hormone reference: - either at the concentration giving 50% of the maximum optical density (ED 50). From the curve obtained with another hormone, B is the concentration corresponding to the same optical density value as that used to define A. The cross-reaction percentage is equal to A divided by B and multiplied by 100: (A / B) X 100]. Specificity of CA5 Antibody and its scFv Table 17 illustrates the percentages of cross-reactions of CA5 antibody with subunits (su) a and [3 of ovine FSH and the sub-units thereof. unit [3 of human FSH: 5 Table 17 CA5 oFSH su a oFSH s.u. 13 oFSH s.u. 13 hFSH Cross-reactivity 100% 6% 80% 50% The CA5 antibody recognizes very little the sheep subunit but strongly the ovine FSH [3 subunit (80%); it also crosses with the subunit [3 of the human FSH, less strongly (50%). Its specificity is anti-subunit [3 FSH. Table 18 illustrates the percentages of cross reactions of CA5 and scFv CA5 with porcine FSH (pFSH) and different human FSHs: Table 18 oFSH pFSH hFSH hFSH (Puregon) hFSH hMG (Gonal F) (Fostimon) (Menopur) CA5 100% 134% 128% 70% 76% 61% scFv CA5 100% 61% ND ND 10% 10% The CA5 antibody has a strong recognition of swine FSH and human FSH Gonal-F. It also crosses significantly with other human FSH between 61 and 76%. The CA5 antibody better recognizes the FSHs tested in their dimeric form which tends to indicate specificity against a conformational epitope. ScFv CA5 significantly recognizes pFSH (61%) and weakly hFSH (Fostimon) and hMG (Menopur). Cross-reaction on the other two human FSHs could not be measured (ND) due to too weak a link. The binding of the scFv CA5, as well as that of the whole antibody, thus seems to be dependent on the conformation of the hormone, probably impaired during the adsorption on the plastic of the ELISA plate. The specificity of scFv CA5 was assessed against porcine (pLH), ovine (oLH), bovine (bLH), eCG, and Chorulon and Endo 5000 hCGs. The results are shown in Table 19. Table 19 oLH pLH bLH eCG Chorulon Endo 5000 scFv CA5 25% 29% 33% ND 10% ND 3025518 - 24 - The binding of scFv CA5 is significant with respect to animal LH with a cross reaction between 35 and 40% . Conversely, only hCG Chorulon is poorly recognized (10%). The low binding on the other two adsorbed hCGs failed to quantify a cross-reaction. These results reinforce the hypothesis of conformational epitope specificity given the biological effects of CA5 and scFv obtained in vitro and in vivo on hCG activity (see results in Examples 2 and 3). This hypothesis is reinforced by the results obtained by western blotting by incubating the CA5 antibody on oFSH migrated on 5% polyacrilamide gel under denaturing or non-denaturing conditions. Only the [3 oFSH band was recognized under non-denaturing conditions and gave an important signal. No signal was observed on oFSH migrated under denaturing conditions. An estimate of the scFv dissociation constant Kd for the various FSHs, LHs and CGs studied was calculated on GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA, version 5) using the "One site - Specific binding" function in a saturation binding experiment mode (GraphPad PRISM software) The different values obtained are shown in tables 20 and 21. Table 20 scFv CA5 oFSH pFSH hFSH hFSH hFSH hMG Gonal-F Puregon Fostimon Menopur Kd (10-6M) 0.54 1.24 1.43 2.67 2.03 2.41 Table 21 scFv CA5 oLH pLH bLH eCG hCG hCG Chorulon Endo 5000 Kd (10-6M) ) 1.95 2.47 2.43 4.07 3.80 3.14 The comparison of dissociation constants Kd thus estimated indicates a higher affinity of scFv CA5 for ovine and porcine FSH with a value of 0.54 and 1.24 μM respectively With the exception of recombinant human FSH Gonal F (Kd 1.43 μM), scFv CA5 has a lower affinity for FS Human H (Kd from 2.03 to 2.67pM). Compared with oFSH and pFSH, scFv CA5 has a medium affinity for sheep and porcine LH (Kd of 1.95 and 2.47 pM, respectively). The Kd values estimated for hCG and eCG (Kd of 3.14 to 4.07 μM) indicate a lower affinity for scFv CA5 for these hormones. Specificity of the CH10 Antibody and its scFv Table 22 illustrates the percentages of cross-reactions of the CH10 antibody with the ovine (su) a and [3 subunits of the ovine FSH and the subunit [3 of the 5 human FSH: Table 22 CH10 oFSH su a oFSH s.u. 13 oFSH s.u. 13 hFSH Cross-reactivity 100% 43% 88% 40% The antibody CH10 preferentially recognizes the subunit [3 of ovine FSH (88%) and twice the subunit [3 of human FSH and the subunit of sheep unit (40% and 43%). According to these results, the specificity of CH10 is anti-subunit [3 FSH preferentially oFSH. It recognizes to a lesser degree, but in a non-negligible way, the sheep subunit unlike the CA5 antibody. All these results can lead to the hypothesis of an epitope involving primarily [3 but also a, on the association zone of the two subunits for example.
[0016] Table 23 illustrates the percent cross-reactions of CH10 and scFv CH10 obtained with porcine FSH (pFSH) and different human FSH: Table 23 oFSH pFSH hFSH hFSH (Puregon) hFSH hMG (Gonal F) (Fostimon) (Menopur) CH10 100% 24% 50% 71% 100% 48% scFv CH10 100% 175% 32% 67% 30% 61% The CH10 antibody and its scFv have a strong recognition of animal FSH and a cross reaction ranging from 30 to 100 % for human FSH.
[0017] The specificity of scFv CH10 was assessed for porcine (pLH), ovine (oLH), bovine (bLH), eCG, and Chorulon and Endo 5000 hCG. The results are shown in Table 24. Table 24 oLH pLH bLH eCG Chorulon Endo 5000 scFv CH10 68% 63% 52% ND ND ND The binding of scFv CH10 to animal LH is significant with a cross reaction between 52 and 68%. Conversely, the weak binding on the hCG and the adsorbed eCG did not make it possible to quantify a cross reaction. These results reinforce the hypothesis of specificity towards a conformational epitope, given the biological effects of CH10 and its scFv obtained in vitro and in vivo on the activity of hCG Chorulon and Endo 5000 (see results in Examples 2 and 3). An estimate of the dissociation constant Kd of the scFv CH10 against the various FSHs, LHs and CGs studied was calculated on GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA, version 5). using the "One site - Specific binding" function in a saturation binding experiment mode (GraphPad PRISM software) The values obtained are shown in Tables 25 and 26.
[0018] Table 25 scFv CH10 oFSH pFSH hFSH hFSH hFSH hMG Gonal-F Puregon Fostimon Menopur Kd (10-6M) 2.85 5.22 1.82 11.5 1.59 7.36 Table 26 scFv CH10 oLH pLH bLH eCG hCG hCG Chorulon Endo 5000 Kd (10-6M) 1.55 2.47 1.94 1.97 1.47 2.09 The dissociation constants Kd thus estimated indicate affinity of scFv CH10 for both ovine and porcine FSH (Kd 7.51 and 5.22 μM) than for the human FSH Gonal F, Fostimon and hMG Menopur (Kd of 1.82, 1.59 and 7.36 μM, respectively). Kd estimates for hCG and eCG (Kd of 1.47 to 2.09 μM) indicate good affinity for scFv CH10 for these hormones compared to FSH. EXAMPLE 2: IN VITRO MEASUREMENT OF THE POTENTIATING EFFECT OF THE LIGANDS OF THE INVENTION ON THE BIOACTIVITY OF FSH The demonstration of the potentiating effect of the ligands of the invention on the bioactivity of FSH was performed by comparing the biological response obtained with different types or cell lines stimulated either with FSH alone or with the FSH / monoclonal antibody (mAb) complex. In each case, the comparison of the dose-response curves obtained made it possible to quantify the in vitro potentiating effect of the MAb on the biological activity of the complexed FSH. The statistical analysis of the results was done by the Prism software (GraphPad Software Inc., San Diego, CA, USA, version 5). 1) On primary cultures of bovine granulosa cells The potentiating effect of CA5 and CH10 mAbs on the bioactivity of ovine FSH (oFSH) was firstly characterized on bovine granulosa cells expressing endogenous bovine FSH receptor. Hybridoma supernatants at the final concentration of 0.1 μg / ml of CA5 or CH10 antibody were incubated with a range of sheep or human FSH ranging from 3 ng / ml to 25 ng / ml, 30 min at 37 ° C. .
[0019] The bovine granulosa cells were punctured on cow ovaries from follicles of diameter ranging from 2 to 6 mm, according to the protocol described by Chopineau et al. (Mol Cell Endocrinol., 92 (2): 229-39, 1993) [8] and Wehbi et al. (Endocrinology, 151 (6): 2788-2799, 2010) [9]. Bovine granulosa cells suspended in McCoy's 5A medium (Lonza, Belgium, reference BE12-688F), prepared at 80,000 cells per 0.5 ml, were stimulated for 3 hours at 37 ° C with shaking. in the presence of IBMX 48 μg / ml (Sigma Aldrich, France, reference 15879), by a range of FSH ranging from 3 ng / ml to 25 ng / ml, alone or previously complexed with a monoclonal antibody according to the protocol below. above. The biological response measured was cAMP secretion. After centrifugation, cAMP produced was assayed in the culture supernatant using an ELISA kit (Biomedical Technologies Inc., MA, USA, BT-730). The results are shown in FIG. 1. The results show an amplification of the cAMP secretion by a factor of 1.3 times for CA5, by a factor of 5.5 times for CH10 on the activity of ovine FSH. . Statistical analysis by two-way ANOVA, GraphPad PRISM software shows a significant effect ranging from p <0.05 (*) for CA5 to p <0.01 (**) and p < 0.001 (***) for CH10. 2 / On the HEK293 cell line stably transfected with the human FSH receptor The potentiating effect of the MAbs on the FSH of different species was measured on HEK 293 cells stably expressing the human FSH 35 receptor. This system made it possible to measure the production of cAMP following the activation of the FSH receptor after stimulation with FSH alone or with the FSH / ACM complex for 1 hour at 37 ° C. For this, 60,000 cells were distributed in wells of 96-well plates (Becton Dickinson, NJ, USA, reference 353072) and cultivated 24h at 37 ° C., 5% CO 2 in a humid atmosphere, in 100 μl of MEM medium ( Ozyme, France, reference BE12-611F) containing 10% FCS (Lonza, Belgium, reference DE14-801F), penicillin / streptomycin 1% (Sigma Aldrich, France, reference P-4333) and G418 400 μg / ml (Sigma Aldrich). , France, reference A1720). After 2 hours of weaning in MEM medium, the cells were stimulated for 1 h at 37 ° C. The culture supernatant was recovered and assayed using an ELISA kit (Biomedical Technologies Inc., MA, USA, BT-730). The results express the amount of cAMP secreted at the endpoint. They were analyzed using Prism software (GraphPad Software Inc., San Diego, CA, USA, version 5). Figure 2 shows the potentiating effect of monoclonal antibodies CA5 and CH10 on the ovine FSH bioactivity (oFSH) in vitro on HEK 293 cells stably transfected with the human FSH receptor. For this, the cells were stimulated either with a range from 3 ng / ml to 32.5 ng / ml ovine FSH, or with the same pre-incubated FSH range points, 30 minutes at 37 ° C., with monoclonal antibody (0.1 μg / ml final concentration) prior to stimulation of the cells. A two-way analysis of variance (two-way ANOVA, GraphPad PRISM software) made it possible to compare the dose-response curves obtained with FSH alone or with the FSH / monoclonal antibody complex. The CA5 antibody showed a potentiating effect ranging from 160% to 200% on the activity of the oFSH; this effect is significant for the 32.5 ng / ml oFSH concentration (p <0.05). The CH10 antibody exerts a greater potentiating effect on the oFSH for all the tested concentrations ranging from 225% for the 3 ng / ml point (p <0.01) to 260% for the 10 ng / ml and 33 ng / ml points. ng / ml respectively (p <0.001). 3 / On the HEK293 cell line stably transfected with the human FSH receptor and with the Glosensor® system The potentiating effect of the mAbs on the FSHs of different species was measured in real time on HEK 293 cells stably expressing the Human FSH receptor and GloSensorTM vector (Promega, France). This cellular system made it possible to monitor cAMP production following stimulation of the FSH receptor by the agonist (FSH alone or FSH complex / monoclonal antibody) in real time. Following the binding of cAMP to the GloSensorTM protein, the GloSensorTM substrate (Promega, France, reference E1291) was hydrolysed and led to a measured luminescence emission using a PolarStar Optima reader (BMG Labtech, Germany) and expressed in RLU (Relative Luminescence Unit). This stable line was developed by the Biology and Biomedematics of Signaling Systems team at INRA Val de Loire, 37380 Nouzilly, France) and was made available free of charge for these 10 trials. For this, the EK 293 H cells were cultured at a rate of 80,000 cells per well of a 96-well white-bottom microplate (Dominique Dutscher, France, reference 655903) and cultured in 100 μl of MEM medium (Ozyme, France). , reference BE12-611F) supplemented with 10% FCS (Lonza, Belgium, reference DE14-801F), penicillin / streptomycin 1% (Sigma Aldrich, France, reference P-4333), Hygromycin B 200 μg / ml (Life TechnologiesTM, France, reference 10687010) and G418 400 μg / ml (Sigma Aldrich, France, reference A1720) overnight. After 2h of weaning in 100 μl of MEM medium supplemented with 1% BSA (PAA, France, reference K45012) and containing 4% of GloSensorTM substrate for 2 hours at room temperature, protected from light, the cell plate was put in the PolarStar Optima reader and a first reading was made for 5 minutes to measure the basal level of luminescence. The plate was then removed from the reader and 11 μl of ligand (FSH alone or FSH complex / monoclonal antibody) was added to obtain the indicated concentrations. The emitted luminescence was then measured for about 1h30. The results obtained were analyzed using Prism software (GraphPad Prism Software Inc., San Diego, CA, USA, version 5). The nonlinear function "log (agonist) versus response" was used to plot the response as a function of FSH concentration. This made it possible to characterize and compare the EC50 for FSH alone and the FSH complexed with the monoclonal antibody. For each example, the significant effect of the FSH / potentiating antibody complex was measured by bivariate variance analysis (two-way ANOVA, GraphPad PRISM software) by comparing the two curves in their entirety. CA5 Monoclonal Antibody FIG. 3 illustrates the production kinetics curves of cAMP expressed in relative units of luminescence as a function of time (in minutes) obtained at concentrations 0 - 0.1 - 0.3 - 1 and 3 nM ovine FSH alone or complexed with CA5 antibody. It is observed that the cells "stimulated" by the antibody alone, without FSH, show no response: the luminescent signal remains at its basal level (curve 3A). The CA5 antibody alone exerts no agonist or antagonist effect on the human FSH receptor expressed by the HEK 293 cells. In contrast, the monoclonal antibody CA5 (final concentration 6 nM), complexed with the oFSH, amplifies significantly. very significant and remarkable stimulating activity of the hormone. An increase in the maximal cellular response of 350% and 330% at the concentrations of 0.1 and 0.3 nM oFSH (curves B and C) and an increase of 230% and 140% respectively for the concentrations of 1 and 3 nM oFSH (curves D and E). The amplitude of the potentiating effect becomes less important with the highest concentrations of oFSH (1 and 3 nM) due to a saturation of the cellular response and the luminescent signal at 11000 RLU in this case. The EC50 value measured by GraphPad Prism is 2.36.10-9M for oFSH and 2.83.10-19M for the oFSH / CA5 complex resulting in an increase of 1 unit of LogEC50 (from 20 10-8'6 to 10- 954) when the FSH is complexed to the CA5 antibody. The difference between the two oFSH versus oFSH / CA5 curves is highly significant for all oFSH doses tested (p <0.001). The potentiating effect of the CA5 antibody measured on the porcine FSH is illustrated in FIG. 4. A 190% increase in the maximum response to 0.03 nM of pFSH is observed when this is complexed with CA5 antibody (final concentration 6 nM) (curve A), but this increase is not significant. The potentiating effect reaches 250% at the 0.1 nM concentration of pFSH (curve B) and is significant (p <0.01). The level of response obtained with 0.03 nM pFSH complexed with CA5 is equivalent to that obtained with pFSH 0.1 nM alone (500 RLU versus 585 RLU respectively); which means that the 0.03 nM / CA5 pFSH complex induces the same response amplitude as the pFSH alone at a concentration 3.3 times higher (0.1 nM, ie 0.5 Log). Finally, the potentiating effect of CA5 has been studied on the activity of recombinant human FSH (Gonal-F, SERONO Laboratory). Figure 5 illustrates the cAMP production kinetics curves expressed in relative units of luminescence versus time (minutes) obtained during stimulation with 0.03-0.1-0 concentrations. 2 - 0.3 nM FSH alone or complexed with CA5 antibody (6 nM). A potentiating effect of 235% was observed with the concentration 0.03 nM hFSH, 200% with 0.1 and 0.2 nM hFSH 5 then 170% with the highest concentration 0.3 nM hFSH due to a saturation of the cellular system (maximum 10530 RLU). The calculation of EC50 by GraphPad Prism indicated a value of 5.86.10-1 ° M for hFSH and 1.36.10-1 ° M for the hFSH / CA5 complex resulting in an increase of 0.63 in LogEC50 (from 10- 923 to 10-986) when the FSH is complexed with the CA5 antibody.
[0020] The potentiating effect of scFv CA5 was also studied on the bioactivity of hFSH at 0.01 nM. A significant increase of 160% (p <0.01) was obtained at the concentration of 36 nM scFv (Figure 5). This increase was similar to that of the whole antibody, bivalent CA5. This result means that the monovalent fragment has the same potentiating properties of FSH activity as the CA5 antibody. Although significant (p <001), the potentiating effect of CA5 antibody on the bioactivity of human FSH remains less important than on the bioactivity of ovine FSH for which an increase of one unit of Log was obtained between the EC50 of the oFSH and that of the oFSH / CA5 complex. Monoclonal Antibody CH10 The modulatory effect of the CH10 antibody was studied on ovine FSH (oFSH) and on human FSH (hFSH) (Gonal F, SERONO Laboratory). Figure 6 illustrates the enhancement effect of the CH10 antibody (10nM) on the bioactivity of the prepared oFSH at concentrations 0.01-0.03 and 0.1 nM. On the low concentrations (0.01 and 0.03 nM) we obtain an increase of 185% of the cellular response with the complex oFSH / CH10 (curves A and B). At the concentration of 0.1 nM oFSH, the increase is 312% with the complex oFSH / CH10 (curve C). These increases are very significant (p <0.001).
[0021] The potentiating effect of CH10 (1.3 nM) was measured on prepared human FSH from 0.1 nM to 3 nM (Figure 7A and B). A moderate but significant increase (p <0.001) ranging from 140% to 150% was observed. The EC50s measured by GraphPad Prism were 2.85 x 10-1 ° M for hFSH and 3.17 x 10 ° M for the hFSH / CH10 complex (Figure 7C). 3025518 - 32 - The potentiating effect of CH10 is more specifically on ovine, animal FSH. A weak effect of CH10 antibody was observed on human FSH.
[0022] EXAMPLE 3: IN VIVO MEASUREMENT OF THE POTENTIATING EFFECT OF THE LIGANDS OF THE INVENTION ON THE BIOACTIVITY OF FSH AND LH / CG IN THE RAT MODEL After being characterized in vitro, the potentiating effect of each monoclonal antibody was characterized in vivo, in the female rat for their effect on the bioactivity of FSH and in the male rat for their effect on the bioactivity of LH / CG, which they also recognize. To measure the FSH bioactivity, the protocol used was that of the biological assay described by Steelman and Pohley (Steelman SL, Pohley FM.
[0023] Endocrinology, 53: 604-616. 1953) [12]. In order to measure LH bioactivity, the protocol used was that of the assay described by Scobey et al (Scobey et al., Reprod Biol Endocr., 3: 61, 2005) [13]. The effect of antibodies on FSH activity was assessed using ovine and human FSH. The effect of antibodies on LH activity was evaluated on two hCG (human Chorionic Gonadotropin) preparations. Statistical analysis was performed with GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA, version 5). Results from experiments on batches of 5 animals, a non-parametric one-variable variance analysis (Kruskal Wallis test), followed by a Dunns correction, were applied or a non-parametric test was performed. parametric (Mann-Whitney test). For results on larger numbers (n> 30) from the compilation of several bioassays, a parametric test (unpaired Student t test) followed by a Bonferroni correction was applied. 1 / Potentiating effect of antibodies on the bioactivity of FSH in the Ratte The potentiating effect of CA5 and CH10 antibodies and their scFv was studied on ovine FSH and on various human FSH preparations used in human reproduction Gonal-F and Puregon (recombinant FSH from Merck Serono and Merck Schering-Plow laboratories respectively), Fostimon and Menopur (FSH extracts marketed by Genevrier and Merck Schering-Plow respectively). As described in the Steelman and Pohley protocol, 21-day immature rats received 2 morning and evening 5, 100 μl injections of a mixture of hCG and FSH with a constant amount of hCG (3.5 IU) supplemented with a variable amount of FSH ranging from 0.5 to 1.5 IU for human FSH (Gonal F, Puregon, Fostimon, Menopur) or from 0.5 to 2 μg for ovine FSH (extracted hormone) . Injections were performed subcutaneously at the nape of the neck. Each experiment comprised at least 4 batches: a lot treated with saline (cl serum), a lot treated with antibody or scFv alone, a lot treated with hCG + FSH, a batch treated with the mixture hCG / FSH supplemented with 2 μg antibody or purified scFv. In the case of treatment with the hormone / antibody or scFv complex, prior to injection, the FSH + antibody mixture was incubated for 20 min at 37 ° C or at room temperature, and then added to the hCG. The hCG can indifferently be mixed with the FSH during the incubation of the complex. On the fourth day, the rats were weighed, and their ovaries were removed, dissected and weighed. The results are expressed in milligrams of ovary / 100 grams of body weight. The increase in ovarian weight is proportional to the amount of bioactive FSH injected. This makes it possible to quantify and compare the bioactivity of the same amount of injected hormone alone or in complex with an antibody. Comparison of the bioactivity of the FSH injected alone or complexed with the antibody or scFv makes it possible to measure the differential of the response and thus to quantify the potentiating effect of the antibody or its scFv. Effect of CA5 antibody and its scFv Figure 8A illustrates a representative example of the effect of CA5 antibody on ovine FSH bioactivity. Each lot consisted of 5 females. The lot treated with the CA5 antibody injected alone had the same average ovarian weight as the control lot treated with saline (25.6 mg and 29.15 mg, respectively). The batch that received conventional hormone treatment (hCG 3.5 IU + oFSH 0.5 μg) gave a mean ovarian weight of 85.7 mg significantly higher than the control lot (p <0.05). The batch treated with ovine FSH previously complexed with the CA5 antibody had an average weight of 198 mg, a very significant increase in the FSH bioactivity of 231% compared to the batch having received conventional hormone treatment (p. <0.0001). The potentiating effect of CA5 on ovine FSH activity, in vivo, was analyzed on several experiments whose overall results are shown in Table 27. The increase in mean ovarian weight, recorded over three Experiments following stimulation with the hormone / CA5 complex is highly significant (p <0.0001). Table 27 lot Mean ± sem number effective statistics Serum (1) 25.6 ± 2.4 13 NS CA5 29.15 ± 4.6 15 hCG + oFSH 98.12 ± 6.37 13 *** hCG + oFSH + CA5 165 , 8 ± 11.54 12 p <0.0001 The effect of CA5 on human FSHs was also analyzed over a large population in several experiments. The results are shown in Table 28 below. Table 28 lot Mean ± SEM effective statistics hCG + hFSH 73.93 ± 1.525 77 *** p <0.0001 hCG + hFSH + CA5 128.3 ± 4.322 80 The increase in mean ovarian weight recorded in females treated with hFSH complex Gonal F / CA5 is 173%: the average ovarian weight goes from 73.93 mg in females treated with a standard 128.3 mg treatment in females treated with the hormone / CA5 complex. This difference is highly significant (p <0.0001, unpaired t-test). Finally, the potentiating effect was studied on two other preparations of human FSH (Puregon and Fostimon). The results shown in Figure 8B are a representative example. A significant increase of 153% was recorded on the activity of the human FSH Puregon (p <0.05) and 142% on the activity of the human FSH Fostimon (NS). In comparison, the increase in human FSH Gonal F activity was 179% in this experiment (p <0.05).
[0024] In conclusion, the CA5 antibody exerts a major potentiating effect on ovine FSH and an equally important potentiating effect on the activity of human FSH from different pharmaceutical preparations. The effect of scFv CA5 was studied in the same protocol as the whole antibody. Figure 9A illustrates the results obtained. The same potentiating effect on the activity of human FSH Gonal F was obtained with the whole antibody or with its scFv significantly (p <0.05).
[0025] Various modes of injection of the hormone / scFv mixtures were evaluated and compared with the conventional protocol (subcutaneous injection). Thus, a bioassay aimed to compare an intraperitoneal injection of the hormonal mixture with an injection of the hormonal mixture intraperitoneally followed by a second deferred injection of the scFv CA5 15 minutes later (FIG. 9A). In this case, a 146% increase was obtained in females treated with the hormone / scFv complex with an average ovarian weight ranging from 61 mg (hCG + hFSH Gonal F lot) to 89 mg (lot scFv then hCG + hFSH Gonal F). These results are important because they demonstrate that the potentiating effect of scFv CA5 can be established in vivo even if the scFv is injected alone, independently of the hormone and delayed. The hormone / scFv complex that can be formed in vivo in the treated animal. The potentiating effect of scFv CA5 was also studied on the activity of the human FSH Fostimon and Puregon (Figure 9B). A significant 140% increase was obtained on Puregon FSH activity (p <0.05) and a nonsignificant 126% increase on FSH Fostimon (NS) activity. Effect of CH10 antibody and its scFv The potentiating effect of CH10 on ovine FSH activity, in vivo, was analyzed in several experiments whose overall results are shown in Figure 10A. The comparison of three response experiments between the treated rats with the hCG + oFSH mixture and those treated with the hormones / CH10 complex shows a significant increase of 145% (p <0.01). Mean ovarian weights were 87.55 ± 3.724 mg in females treated with hCG + oFSH (n = 12) at 126.6 ± 9.6 mg / 100 g body weight in complex-treated rats. hormone / CH10 (n = 13). No effect of the CH10 antibody alone on the ovarian response was observed. The potentiating effect of CH10 on human FSH Gonal-F was also analyzed in large numbers in several experiments. The results are shown in Table 29 below and in Figure 10B. 3025518 - 36 - Table 29 lot Mean ± sem effective statistics hCG + hFSH GONAL F 73.93 ± 1.525 77 *** p <0.0001 hCG + hFSH Gonal F + CA5 128.3 ± 4.322 80 A 170% increase in the weight of ovaries was recorded in females treated with the Gonal F / CH10 complex. This difference is highly significant (p <0.0001, unpaired t-test).
[0026] The potentiating effect of CH10 has also been sought on Puregon and Fostimon human FSHs (FIG. 11A). A significant increase of 145% and 174% was obtained respectively (p <0.05). FIG. 11B illustrates the effect of the hFSH complex Gonal F / scFv CH10 in conventional injection subcutaneously and in time delayed intraperitoneal injection. A significant increase of 160% (p <0.05) was obtained by conventional injection and a 150% increase, although not significant, by independent injections of scFv CH10 and hFSH intraperitoneally. The potentiating effect of scFv CH10 can therefore be put in place in vivo, whether it is injected separately or preincubated with the hormone.
[0027] Potential Antibody Effect on LH / CG Bioactivity in the Rat Because of the very high cost of ovine LH, these bioassays were performed with readily available hCG in a very pure and inexpensive form. The effect of the antibodies was studied on two human extracted human hCG 20 (human Chorionic Gonadotropin) preparations, one used in human reproduction in the context of medically assisted procreation treatments: the ENDO 5000 (Schering-Plow laboratory) and the other used in veterinary medicine: the Chorulon (MSD laboratory). According to the protocol of Scobey et al. [13], the bioactivity of LH 25 or hCG has been quantified in relation to the increase in the weight of the seminal vesicles whose development is androgen-dependent. The weight varies proportionally to the activity of the hCG and thus makes it possible to quantify and compare the biological activity of the hormone injected alone or complexed with the antibody studied. The protocol was performed with 25-day-old pigs that were injected subcutaneously once daily for four days with 100 μl of 1.5 IU hCG or a 1.5 IU hCG + mixture. 2 μg of antibody incubated for 20 minutes at 37 ° C. On the fifth day, the rats were weighed and then sacrificed. Their seminal vesicles (VS) were removed, dissected and weighed. The weight of the seminal vesicles is expressed in mg / 100g of body weight in order to compare and combine the results obtained with different batches. In each experiment, each condition was tested on a batch of 5 rats. The same experiment was repeated several times. The effect of CA5 on both hCG preparations: Chorulon and Endo 5000 is shown in Figure 12. Histogram A is a representative example of a bioassay performed on 6 batches of 5 rats. A very significant potentiating effect (p <0.0001, Krustal and Wallis test) was obtained with the Chorulon / CA5 hCG complex with a 196% increase in seminal vesicle weight compared to the hCG alone lot. A significant effect was also obtained with hCG ENDO 5000 / CA5 complex with a weight increase of 193% (p <0.01). It was observed that the lot treated with the CA5 antibody alone showed no change in the weight of the seminal vesicles compared with the control animals treated with saline: the antibody alone had no effect on the target organ, unlike the complex . The sum of the results obtained during the repetitions of this bioassay with the two hCG confirms a highly significant potentiating effect (p <0.0001, unpaired t-test) of the hormone / CA5 complex: - on a strength of 53 and 56 animals respectively, the mean weight of SV was 28.8 mg / 100 g in rats treated with hCG Chorulon and 46.7 mg / 100 g in rats treated with the complex (162% increase) (Figure 12B) On a strength of 26 and 30 animals respectively, the average weight of the VS was 24.64 mg / 100 g in the hCG-treated rats ENDO 5000 and 48.13 mg / 100 g in the rats treated with the complex. (195% increase) (Figure 12C). The CH10 antibody also exhibited a significant potentiating effect on hCG Chorulon and hCG ENDO 5000, in vivo, in the rat. Figure 13A illustrates a representative case of bioassay performed on 6 batches of 5 rats. A very significant potentiating effect (p <0.0001, Krustal and Wallis test) was obtained with the Chorulon / CH10 hCG complex with a 193% increase in the weight of the seminal vesicles compared to the hCG alone lot.
[0028] A significant effect was also obtained on the batch treated with hCG 3025518 ENDO 5000 / CH10 complex with a weight increase of 199% (p <0.0001). It is observed that the batch treated with the CH10 antibody alone shows no change in the weight of the seminal vesicles compared to the control animals treated with physiological saline. CH10 not complexed with the hormone therefore has no specific effect on the target organ. The compilation of the results obtained during the repetitions of this bioassay with both hCG confirms a highly significant potentiating effect (p <0.0001, unpaired t-test) of the hormone / CH10 complex: - on a population of 34 and 35 animals respectively, the mean weight of the VS was 29.6 mg / 100 g in the Chorulon hCG treated rats versus 50.04 mg / 100 g in the complex treated rats (169% increase) (Figure 13B) on a strength of 13 and 15 animals respectively, the average weight of the VS was 24.64 mg / 100 g in the rats treated with hCG ENDO 5000 and 51.39 mg / 100 g in the rats treated with the complex (208% increase) (Figure 13C). EXAMPLE 4: IN VIVO MEASUREMENT OF THE POTENTIATING EFFECT OF THE LIGANDS OF THE INVENTION ON THE BIOACTIVITY OF ENDOGENOUS GONADOTROPINS IN EWES Having demonstrated and characterized the potentiating effect in vivo, monoclonal antibodies CA5 and CH10, in a rodent ( small animal), the aim was to study the effect of each antibody on the activity of FSH in a larger animal: the ewe.
[0029] For this, a study was carried out on sheep Ile de France, pubescent, all the same age, in order to assess the potentiating effect of antibodies on the own hormones of treated sheep (endogenous hormones). The study of specificity showed a strong binding of CA5 and CH10 antibodies for ovine FSH and a more variable binding for ovine LH. For this purpose, a treatment comprising only the injection of an antibody alone has been developed to evaluate its effectiveness. In the protocols implemented in sheep, each antibody was therefore injected alone and not previously incubated with exogenous FSH as was done in studies in rats. In addition, each antibody was injected into ewes free of any prior stimulation of the ovary: the animals did not receive any hormone treatment to stimulate ovulation with gonadotropin prior to injection of the antibody. The potentiating effect of the anti-FSH CA5 and CH10 antibodies was evaluated during protocols carried out during the sexual season (January) or at the end of the sexual season (end of March). The protocols were all performed on ewes whose ovulatory cycle was previously synchronized by placing a vaginal sponge impregnated with a progestagen (45 mg of fluorogestone acetate (FGA) - MSD) for 14 days. The potentiating effect was analyzed by comparing the ovulatory response (number of ovulations) and the establishment of one or more functional yellow bodies of good quality (amplitude of progesterone secretion) in control ewes (batch). physiological saline), ewes stimulated by porcine FSH treatment (FSH lot) and ewes stimulated by an antibody alone (antibody batch). In each protocol, a plasma LH assay was performed by ELISA to detect and date the pre-ovulatory peak of LH. To evaluate the ovulatory response, an endoscopic observation of the ovaries was performed by laparoscopy, under anesthesia, eight days after removal of the vaginal sponge, in order to count the number of yellow bodies and observe their appearance. To evaluate the functionality and quality of the yellow body (s), a quantitative ELISA of progesterone was performed from daily blood samples from the first to the 21st day after removal of the sponge. All statistical analyzes were done with GraphPad Prism Version 5.0 software (GraphPad, San Diego, CA, USA).
[0030] CA5 Antibody The potentiating effect of CA5 antibody (IgG) was evaluated in two protocols (1 and 2) during the sexual season and at the end of the sexual season. In protocol 1, carried out at the end of the sexual season: the batch "CA5 antibody" (n = 6) received three injections of CA5 purified intramuscularly: 2 mg 4 days before removal of the sponge, 1 mg before removal and 1mg when removing the sponge. the "control" lot (n = 5) was injected with physiological saline intramuscularly 24 hours before the sponge was removed; and the "FSH" lot (n = 5) was injected intramuscularly with 100 μg of porcine FSH (pFSH) 24 hours before the removal of the sponge and 90 μg 12h before removal of the sponge. Analysis of the ovulatory response gave the results shown in Table 30 below. Table 30 Lot serum Lot FSH Lot CA5 Statistics (1) Only Number of ewes per lot 5 5 6 2/5 Number of ewes ovulating per lot 2/5 (40%) 2/6 (35%) NS (40%) Number of corpus luteum per ovum ewe 1.5 ± 0.7 3 ± 2.8 1.5 ± 0.7 NS LH peak moment (hours after withdrawal) 72 ± 17 60 ± 0 78 ± 25 NS L Statistical analysis was done by Fisher's exact test. Compared to the control and FSH lots, the results obtained in lot CA5 do not show any significant effect on the ovulatory response. None of the 10 measured parameters show any trend. The progesterone secretion profile during the luteal phase obtained in the three batches is illustrated in FIG. 14. For each individual, the progesterone concentration values (ng / ml) were normalized by number of yellow bodies. Each curve represents the average of the progesterone values obtained in the females of each batch at a time t. A significant effect of the CA5 antibody was observed on the intensity of progesterone secretion per corpus luteum and on the onset of its placement (Figure 14A). Indeed, on the curve CA5, compared with the curves of the batches FSH and serum (1), there is a beginning of progesterone secretion as of D4 followed by a significant increase in the secretion which is maintained throughout the luteal phase. until the end of the cycle. The mean values of progesterone at Day 10 are 1.38 ng / ml - 0.92 and 0.52 ng / ml respectively for batches CA5, FSH and serum cl) and 2.58 - 1.7 and 1.18. ng / ml at D15. The comparison of the three curves was made by a paired nonparametric t test (Wilcoxon test). Thus, the curve of the CA5 lot is higher than the FSH lot curve and significantly different (p <0.01), similarly with the control lot curve (p <0.001). The curve of the FSH batch is higher and significantly different from that of the control lot (p <0.001). To quantify this significant and constant increase in the level of progesterone secretion to the end of the cycle in ewes under CA5 stimulation, an AUC calculation was performed with GraphPad Prism software version 5.0 . The results are shown in Figure 14B and show that the AUC of the CA5 curve (23.61 arbitrary units) tends to be higher than the AUC of the serum curve cl) (8 units) but this difference is not not significant. Likewise, the AUC of the FSH curve (12 units) is not significantly different from the control curve. In conclusion, the injection of CA5 into ewes gives the same results as a conventional treatment with FSH in terms of induction of ovulation but allows the faster establishment of the secretion of progesterone and the maintenance of a a more efficient functional corpus luteum with a higher circulating level of progesterone, guaranteeing a better success of early embryonic development and maintenance of pregnancy (decreased risk of abortion). In protocol 2, carried out during the sexual season: the batch "CA5 antibody" (n = 7) received a single intramuscular injection of 2 mg of CA5 antibody 24h before removal of the sponge. - the "control" lot (n = 9) was injected with saline intramuscularly 24 hours before sponge removal - the "FSH" lot (n = 11) received an injection intravenously 100 μg of 25 swine FSH (pFSH) muscle 24h prior to removal of the sponge and 90 μg 12h prior to removal of the sponge. The analysis of the ovulatory response gave the results presented in Table 31 below. The statistical analysis was done by Fisher's exact test.
[0031] Table 31 Serum lot Lot FSH Lot CA5 Statistics (1) Only Number of ewes per lot 9 11 7 Number of ewes ovulating by 7/7 ***, 4/9 (44%) 4/11 (36%) lot (100%) p <0.0001 3025518 - 42 - Number of corpus luteum per ewe lot 0.67 ± 0.3 0.9 ± 0.5 1.5 ± 0.4 NS, p = 0.06 Number of corpus luteum per number Ovulating ewes 1.5 ± 0.3 3 ± 1.4 1.5 ± 0.4 NS LH peak moment (hours after shrinkage) 64 ± 13 56 ± 7 57 ± 4.4 NS Compared to control and control lots FSH, the results obtained in lot CA5 show a very significant effect of the antibody injected alone on the ovulatory response. In fact, 100% of the females (7/7) who received an injection of 2 mg of antibody ovulated against 44% and 36% respectively for the lot serum c1) and the 5 lot FSH (p <0.0001, exact test of Fisher). The number of yellow bodies obtained per female on the total number of the lot is similarly higher in lot "CA5" and almost significantly (p = 0.06, Mann-Whitney t test): 1.5 yellow body versus 0 , 9 (FSH) and 0.67 (C1 serum) respectively. The average number of yellow bodies per ovumed female is not significantly different between the three batches. The mean time of appearance of the LH peak is not significantly different between the three lots. Nevertheless, there is a tendency for less variability in the arrival of the LH peak (hence the time of ovulation) in the CA5 lot compared to the FSH and especially serum O lot.
[0032] The secretion pattern of progesterone during the luteal phase following ovulation is illustrated in Figure 15A. For each individual, progesterone concentration values (ng / ml) were normalized by number of yellow bodies. Each curve in the figure represents the average of the progesterone values obtained in the females of each batch. A remarkable effect of CA5 antibody was observed on the intensity of progesterone secretion: a significant and very significant increase in progesterone secretion per corpus luteum was observed throughout the luteal phase compared to The average values at day 10 are 2.44 ng / ml, 1.3 and 0.62 ng / ml respectively for batches CA5, FSH and serum (1), 25 and 2.88 - 1.42 and 1.18 ng / ml at day 15. The comparison of the three curves was made by a paired nonparametric t test (VVilcoxon test). The CA5 lot curve is significantly higher and different from the FSH lot curve (p <0.01), similarly to the control lot curve (p <0.001). The curve of the FSH lot is significantly different from that of the control lot (p <0.001). To quantify this remarkable and constant increase in the level of progesterone secretion per corpus luteum throughout the luteal phase of the cycle in CA5-stimulated ewes, an area under the curve (AUC) calculation was performed. was made with GraphPad Prism software version 5.0. The results are shown in Fig. 15B and show that the AUC of the CA5 curve (24.20 units) is significantly higher by a factor of 3 of the AUC of the serum curve c1) (8.1) ( p <0.05, non-parametric Mann-Whitney t test). Conversely, the AUC of the FSH curve is not significantly different from the control curve.
[0033] In conclusion, the use of the CA5 antibody in the form of a single intramuscular injection of 2 mg gave very significantly better results than a conventional treatment with FSH by allowing: 1-induction of ovulation in 100% of the stimulated females 2-the development of yellow bodies of better quality with a secretion of progesterone much higher than that observed following FSH treatment and a very fast implementation as soon as D4. It should be emphasized that the impact of this additional property of CA5 compared to FSH treatment is very important. Indeed, the plasma concentration of progesterone is a key factor of embryonic development, particularly in its early stages.
[0034] The faster establishment of progesterone secretion and the maintenance of a more effective functional corpus luteum with a higher circulating level of progesterone is the guarantee of a better success of early embryonic development and maintenance of gestation with decreased risk of abortion.
[0035] All of the results indicate that the potentiating antibodies, particularly CA5 injected in vivo in the ewe, are capable of complexing the endogenous gonadotropic hormones of the animal and of potentiating the biological activity of the hormones proper to the animal.
[0036] The potentiating effect of CA5 antibody in ewes is capable of inducing ovarian stimulation stronger than conventional FSH hormone treatment: ovulation induction is 100% during sexual season and in all cases a significant increase in the circulating concentration of progesterone, 200 to 300% is maintained throughout the luteal phase. This additional effect is major in reducing the rates of failure of progestin-dependent embryonic development and the risks of abortion. Reference Lists 1- Patent EP 1518863 2- International Application WO 2012/066519 3-Pike et al., Nucl. Acids Res., 36: W503-508, 2008; 4- Giudicelli et al., Cold Spring Harb Protoc., 2011 (6): 695-715, 2011; 5- Giudicelli et al., Nucl. Acids Res., 33: D256-261, 2005 6- Corpet, Nucl. Acids Res., 16 (22): 10881-10890, 1988. Ward et al. Nature, 341: 544-546, 1989) Li et al., Afr. J. Biotechnol., 9 (50): 8549-8554, 2010 9- Chopineau et al., Mol. Cell Endocrinol., 92 (2): 229-239, 1993 Wehbi et al., Endocrinology, 151 (6): 2788-2799, 2010 11- Reverchon et al., Human Reprod., 27 (6): 1790 -1800, 2012 12- Steelman SL, Pohley FM., Endocrinology, 53: 604-616, 1953 13- Scobey et al., Reprod. Biol. Endocr. 3: 61, 2005

权利要求:
Claims (15)
[0001]
REVENDICATIONS1. Follicle-stimulating hormone (FSH) ligand potentiating the bioactivity of FSH, luteinizing hormone (LH) and chorionic gonadotropin (CG), characterized in that said ligand is an antibody or a fragment thereof ci and in that: the variable domain of the heavy chain contains the following CDRs: - VH-CDR1, defined by the sequence GFTFSDFY (SEQ ID NO: 9); - VH-CDR2, defined by the sequence SRNKAKDYTT (SEQ ID NO: 10); - VH-CDR3, defined by the sequence ARDARFAY (SEQ ID NO: 11); and the variable domain of the light chain contains the following CDRs: VL-CDR1, defined by the sequence QSLLYSSNQKNY (SEQ ID NO: 12); - VL-CDR2, defined by the WAS sequence; - VL-CDR3, defined by the sequence QQYYSYPRT (SEQ ID NO: 13).
[0002]
2. Ligand follicle-stimulating hormone (FSH) potentiating the bioactivity of FSH, luteinizing hormone (LH) and chorionic gonadotropin (CG), characterized in that said ligand is an antibody or a fragment of it and that: the variable domain of the heavy chain contains the following CDRs: - VH-CDR1, defined by the sequence GFTFNTYA (SEQ ID NO: 14); - VH-CDR2, defined by the sequence IRSKSNNYAT (SEQ ID NO: 15); - VH-CDR3, defined by the sequence VRQDYYGSSYFDY (SEQ ID NO: 16); and the variable domain of the light chain contains the following CDRs: VL-CDR1, defined by the sequence QSISDY (SEQ ID NO: 17); - VL-CDR2, defined by the sequence YAS; - VL-CDR3, defined by the sequence QNGHSFPYT (SEQ ID NO: 18).
[0003]
3. Ligand according to any one of claims 1 or 2, characterized in that the ligand is selected from the group consisting of Fab, Fab ', F (ab') 2, Fv, dsFv, scFv, diabodies, triabodies, tetrabodies and nanobodies.
[0004]
4. Ligand according to claim 1, characterized in that the ligand is monoclonal antibody CA5 produced by the hybridoma CNCM I-4801. 3025518 - 47 -
[0005]
5. Ligand according to claim 2, characterized in that the ligand is the monoclonal antibody CH10 produced by the hybridoma CNCM 1-4802. 5
[0006]
6. Ligand according to claim 3, characterized in that the peptide sequence of scFv is the sequence SEQ ID NO: 20 or SEQ ID NO: 22.
[0007]
Ligand according to any one of claims 1 to 6 for use as a medicament. 10
[0008]
A ligand-gonadotropin complex selected from: a complex of a ligand according to any one of claims 1 to 6 with FSH or an active peptide thereof; a complex of a ligand according to any one of claims 1 to 6 with LH or chorionic gonadotropin hormone (CG) or an active peptide thereof.
[0009]
9. Complex according to claim 8 for use as a medicament. 20
[0010]
The ligand for use according to claim 7 or complex for use according to claim 9, wherein said medicament is for inducing ovulation or polyovulation in a female mammal.
[0011]
The ligand for use according to claim 7 or complex for use according to claim 9, wherein said medicament is for increasing the endogenous circulating progesterone level in a female mammal.
[0012]
12. A pharmaceutical composition for use in the induction of ovulation or polyovulation in a female mammal, characterized in that it comprises a ligand according to any one of claims 1 to 6 and / or a The complex of claim 8 and a pharmaceutically acceptable carrier. 35
[0013]
13. Pharmaceutical composition according to claim 12, characterized in that it further comprises an FSH, an LH and / or a CG. 3025518 48 -
[0014]
A ligand according to any of claims 1 to 6 or a complex according to claim 8 for use in the treatment and / or prevention of infertility or subfertility in a mammal.
[0015]
A ligand according to any of claims 1 to 6 or a complex according to claim 8 for use in stimulating procreation in a healthy female mammal.

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法律状态:
2015-09-03| PLFP| Fee payment|Year of fee payment: 2 |

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2016-04-22| PLSC| Publication of the preliminary search report|Effective date: 20160422 |

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2016-07-29| PLFP| Fee payment|Year of fee payment: 3 |

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2017-08-30| PLFP| Fee payment|Year of fee payment: 4 |

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2018-01-19| TP| Transmission of property|Owner name: REPROPHARM VET, FR Effective date: 20171218 |

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2018-09-27| PLFP| Fee payment|Year of fee payment: 5 |

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2019-09-27| PLFP| Fee payment|Year of fee payment: 6 |

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2020-09-28| PLFP| Fee payment|Year of fee payment: 7 |

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2021-09-29| PLFP| Fee payment|Year of fee payment: 8 |

优先权:

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申请号 | 申请日 | 专利标题

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FR1458469|2014-09-10|

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FR1458469A|FR3025517B1|2014-09-10|2014-09-10|LIGANDS POTENTIATING THE BIOACTIVITY OF GONADOTROPHINS|

---

FR1558078|2015-08-31|

---

FR1558078A|FR3025518B1|2014-09-10|2015-08-31|LIGANDS POTENTIATING THE BIOACTIVITY OF GONADOTROPHINS|FR1558078A| FR3025518B1|2014-09-10|2015-08-31|LIGANDS POTENTIATING THE BIOACTIVITY OF GONADOTROPHINS|

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ES15770570T| ES2797752T3|2014-09-10|2015-09-10|Ligands that enhance the bioactivity of gonadotropins|

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JP2017534010A| JP6649385B2|2014-09-10|2015-09-10|Ligands that enhance the biological activity of gonadotropin|

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PT157705708T| PT3191514T|2014-09-10|2015-09-10|Ligands that potentiate the bioactivity of gonadotropins|

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US15/510,640| US10584166B2|2014-09-10|2015-09-10|Ligands that potentiate the bioactivity of gonadotropins|

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DK15770570.8T| DK3191514T3|2014-09-10|2015-09-10|Ligands that potentiate the bioactivity of gonadotrophins|

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EP15770570.8A| EP3191514B1|2014-09-10|2015-09-10|Ligands that potentiate the bioactivity of gonadotropins|

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RS20200702A| RS60501B1|2014-09-10|2015-09-10|Ligands that potentiate the bioactivity of gonadotropins|

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LTEP15770570.8T| LT3191514T|2014-09-10|2015-09-10|Ligands that potentiate the bioactivity of gonadotropins|

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PCT/FR2015/052414| WO2016038309A1|2014-09-10|2015-09-10|Ligands that potentiate the bioactivity of gonadotropins|

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HUE15770570A| HUE049357T2|2014-09-10|2015-09-10|Ligands that potentiate the bioactivity of gonadotropins|

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CN201580061187.9A| CN107108732B|2014-09-10|2015-09-10|Ligands that enhance the biological activity of gonadotropins|

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SI201531257T| SI3191514T1|2014-09-10|2015-09-10|Ligands that potentiate the bioactivity of gonadotropins|

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BR112017004739A| BR112017004739A2|2014-09-10|2015-09-10|follicle stimulating hormone ligands, ligand-gonadotropin complex, use of a binder, and pharmaceutical composition|

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PL15770570T| PL3191514T3|2014-09-10|2015-09-10|Ligands that potentiate the bioactivity of gonadotropins|

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CA2960132A| CA2960132A1|2014-09-10|2015-09-10|Ligands that potentiate the bioactivity of gonadotropins|

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HRP20200969TT| HRP20200969T1|2014-09-10|2020-06-18|Ligands that potentiate the bioactivity of gonadotropins|