Humanized variant antibodies or fragments thereof potentiating the bioactivity of gonadotropins

Abstract

The present invention relates to novel humanized variant antibodies or fragments thereof that are capable of potentiating the bioactivity of gonadotropins.

Description

[0001] HUMANIZED VARIANT ANTIBODIES OR FRAGMENTS THEREOF POTENTIATING GONADOTROPIN BIOACTY

[0002] Technical field

[0003] The present invention relates to novel humanized variant antibodies or fragments thereof, derived from the murine monoclonal antibody CF12 directed against human follicle-stimulating hormone (anti-FSH), and capable of potentiating the bioactivity of gonadotropins.

[0004] The present invention finds its applications mainly in reproductive medicine, in particular for inducing ovulation or polyovulation in a female mammal, restoring and / or stimulating spermatogenesis in a male mammal, or even for increasing steroidogenesis in a male or female mammal.

[0005] In the description below, the references in brackets ([ ]) refer to the list of references presented at the end of the text.

[0006] State of the art

[0007] Gonadotropins (or gonadotropins) are complex glycoprotein hormones that play a central role in regulating reproduction in vertebrates by affecting 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.

[0008] Luteinizing hormone (LH) is produced by the gonadotropic cells of the anterior lobe of the pituitary gland under stimulation of GnRH (Gonadotropin Releasing Hormone), itself produced by the hypothalamus. LH stimulates the production of testosterone in males, while it is involved in changes in the ovarian cycle in females where it is responsible for terminal follicular growth and ovulation and then the 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 early development and implantation of the embryo.

[0009] Follicle-stimulating hormone (or FSH) is produced by gonadotropin cells in the anterior lobe of the pituitary gland under the stimulation of GnRH produced by the hypothalamus. In males, it stimulates Sertoli cells, which are essential for spermatogenesis. In females, it is responsible for the recruitment of immature primordial follicles, their growth, and their differentiation into preovulatory follicles by stimulating FSH receptors on granulosa cells.

[0010] FSH, like LH, consists of an α-subunit common to all glycoprotein hormones of the same species (LH, FSH, Choriogonadotropin (CG) and thyroid-stimulating hormone (TSH)) and a specific β-subunit responsible for the specificity of the hormone's activity; activity that only exists if the two subunits are non-covalently associated in the form of a dimer. Only the dimeric form is capable of activating the receptor for which it is specific and of stimulating the target cells expressing this receptor. FSH has a structure similar to that of LH. In females, LH and FSH levels are cyclical: very low outside the ovulatory period, with a peak of secretion in the preovulatory period.

[0011] Gonadotropins are used in veterinary and human medicine to induce ovulation in female mammals. In the veterinary field, although effective, these treatments present a health risk due to the use of hormones extracted from biological fluids (blood, urine) or tissues (pituitary glands). This is the case for equine chorionic gonadotropin (eCG) extracted from the blood of pregnant mares, and porcine LH and FSH extracted from pig pituitary glands. In the veterinary field, an hCG extracted from the urine of pregnant women, Chorulon® (MSD Laboratory), is also used.

[0012] In the field of human clinical practice, and particularly in Assisted Reproduction (ART), hormones extracted from the urine of postmenopausal women are used, such as Fostimon® (Laboratoire Genévrier), which is a purified FSH, and Menopur® (Laboratoire Ferring Pharmaceuticals), which is an hMG (human menoposal gonadotropin), a mixture of FSH and LH. Recombinant human FSH are also used, such as Gonal-F® (Laboratoire Merck Serono), Puregon® (Laboratoire Merck Schering-Plough), Rekovelle® (Laboratoire Ferring Pharmaceuticals), and recombinant biosimilar FSH, Ovaleap® (Teva) and Bemfola® (Gedeon Richter); recombinant hCG and LH are also used, such as Ovitrelle® and Luveris® (Laboratoire Merck Serono). However, repeated use of these hormones can induce an immune reaction which can neutralize the effect of the hormones, thus leading to a reduction in therapeutic efficacy during subsequent treatments.

[0013] On the other hand, it has also been demonstrated in livestock treated with eCG also called PMSG (Pregnant Mare Serum Gonadotropin) that the immune reaction could, in certain cases, produce anti-eCG polyclonal antibodies capable of potentiating the FSH and LH bioactivities of the eCG injected during the treatments [1]. Studies have also shown that the purified fraction of these antibodies, co-administered with eCG, in vivo in immature female rats, led to significantly greater stimulation of the ovaries [2]. Since then, three anti-LH monoclonal antibodies have also been identified capable of potentiating its action as well as that of FSH for two of them [3], and, more recently, the murine anti-FSH monoclonal antibody CF12 capable of potentiating the bioactivity of FSH, LH and CG [4].

[0014] Description of the invention The inventors have now produced humanized variants derived from the murine anti-FSH monoclonal antibody CF12 [4] in which (i) the sequences of the CDRs Complementary Determining Region) (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 of the antigen) are identical to those of the monoclonal antibody CF12, and (ii) the sequences of the Framework (FRs) (regions surrounding the CDRs constituting the "framework" and giving a stable configuration to the variable domain) of the monoclonal antibody CF12 of which at least one FR carries at least one mutation, are inserted into a human immunoglobulin framework (e.g. Fc region of the gamma-4 immunoglobulin chain (IgG4)).These humanized variant antibodies unexpectedly exhibit the ability to potentiate FSH bioactivity in vitro and in vivo with an effect equal to or greater than that of the murine anti-FSH monoclonal antibody CF12. They can also potentiate LH and hCG bioactivity in vivo with a lower effect than the murine anti-FSH monoclonal antibody CF12; this presents the additional and novel advantage of favoring and better targeting FSH potentiation in a therapeutic indication.

[0015] The amino acid sequences of the variable regions of the heavy and light chains of the humanized variants of the murine anti-FSH monoclonal antibody CF12 have been synthesized and are presented in the table below.

[0016] Table 1

[0017] In Table 1, the sequences corresponding to the CDRs are underlined.

[0018] The present invention relates to novel humanized variant antibodies derived from a murine anti-human follicle-stimulating hormone (anti-FSH) monoclonal antibody which potentiates the bioactivity of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and human chorionic gonadotropin (hCG) (e.g. the murine anti-FSH antibody CF12 of sequence VH

[0019] “QGQMQQSGAELVKPGASVKLSCKTSGFTFSSSYISWLKQKPGQSLEWIAWIYAGTGGTS YNQKFTGKAQLTVDTSSSTAYMQFSSLTTEDSAIYYCARHGSYFDYWGQGTTLTVSS” (SEQ ID NO: 18) and VL sequence

[0020] "DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLES GIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIK" (SEQ ID NO: 19)) or fragments thereof, all binding FSH and potentiating the bioactivity of FSH, LH and hCG, and characterized in that: the variable domain of the heavy chain of said humanized variant antibody or fragment thereof contains the amino acid sequence QX2 QLVQSGAEVKKPGASVKVSCKX 24 SGFTFSSSYIX 35 WX 37 RQAPGQRLEWX 48 X 49 WIYAG TGGTSYX 61 Q KFX 65 GX 67 X 68 X 69 X 70 TX 72 DTSASTAYM EX 83 SSLRSEDTAVYYCARHGSYFDYW GQGTLVTVSS where 2 is V or G, X 24 is A or T, X 35 is H or S, X 37 is V or L, X 48 is I or M, X 49 is A or G, X 61 is S or N, X 65 is T or Q, X 67 is R or K, X 58 is A or V, X 69 is T or Q, X 70 is I or L, X 72 is V or R, X 83 is L or F (SEQ ID NO: 1), or the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAX 24 SGFTFSSSYISWLRQAPGKGLEWX 48 AWIYAGTGGT SYAQX 63 VKGRFX 69 X 70 SVDTSKNTAYLQMNSLRAEDTAVYYCARHGSYFDYWGQGTLVTVS S where 24 is A or T, X 48 is I or V, X 63 is S or K, X 69is Q or T, X 70 is L or I (SEQ ID NO: 2), or the amino acid sequence

[0021] QMQLVQSGPEVKKPGTSVKVSCKX 24 SGFTFSSSSYISWLRQARGQRLEWIAWIYAGTGGTS YAQKFQERVX 69 X 70 T DMSTSTAYMEFSSLRSEDTAVYYCAR HGSYFDYWGQGTLVTVSS where 24 is T or A, X 69 is Q or T, X 70 is L or I (SEQ ID NO: 3); and the variable domain of the light chain of said humanized variant antibody or fragment thereof contains the amino acid sequence DIX 3 X 4 TQSPX 9 SLX 12 X 13 SX 15 GX 17 RX 19 TIX 22 CX 24 X 25 SQSVDYDGDSYMX 38 WYQQKPGX 46 X 47 PKLLIYAASX 57 X 58 ESGVPX S4 RFSGSGSGTDFTLTISSLQX 84 EDX 87 AX 89 YYCQQSNEDPYTFG QGTKLEIK where X 3 is Q or V, X 4 is M or L, X 9 is D or SX 12 is A or S, X 13is A or V, X 15 is L or V, X 17 is E or D, X 19 is A or V, X 22 is N or T, X 24 is R or K, X 25 is A or S, X 38 is A or N, X 46 is Q or K, X 47 is P or A, X 57 is N or S, X 58 is R or L, X 64 is D or S, X 84 is A or P, X 87 is V or F, X 89 is V or T (SEQ ID NO: 4).

[0022] For the purposes of the present invention, the term "humanized variant antibody" means an antibody resulting from the insertion of the parental hypervariable regions (CDRs) of the murine anti-FSH monoclonal antibody CF12 into a human immunoglobulin framework, and of which at least one parental framework region (FR) has been mutated.

[0023] For the purposes of the present invention, the term "fragment thereof" means, for example, a fragment of a humanized variant antibody according to the invention: Fab, Fab', F(ab')2, Fv, dsFv, scFv, or scFv-Fc with a human Fc, nanobody. Preferably, it is a scFv fragment (single chain variable fragment). For example, a scFv fragment according to the present invention has the sequence SEQ ID NO: 17.

[0024] In particular, a humanized variant antibody or fragment thereof according to the present invention is characterized in that: the variable domain of the heavy chain of said humanized variant antibody or fragment thereof contains the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGFTFSSSYIHWVRQAPGQRLEWMGWIYAGTGGTS YSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARHGSYFDYWGQGTLVTVSS (SEQ ID NO: 5), or the amino acid sequence

[0025] <h2 style=";text-align:left;direction:ltr">QVQLVQSGAEVKKPGASVKVSCKASGFTFSSSYISWVRQAPGQRLEWMAWIYAGTGGTS YNQKFTGKVTITRDTSASTAYMELSSLRSEDTAVYYCARHGSYFDYWGQGTLVTVSS (SEQ ID NO: 6), or the sequence in amino acids<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0026] <h2 style=";text-align:left;direction:ltr"> QGQLVQSGAEVKKPGASVKVSCKASGFTFSSSYISWLRQAPGQRLEWIAWIYAGTGGTSY NQKFTGKVTITRDTSASTAYMELSSLRSEDTAVYYCARHGSYFDYWGQGTLVTVSS (SEQ ID NO: 7), or the sequence in amino acids<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0027] <h2 style=";text-align:left;direction:ltr"> QGQLVQSGAEVKKPGASVKVSCKASGFTFSSSYISWLRQAPGQRLEWIAWIYAGTGGTSY NQKFTGKATLTVDTSASTAYMELSSLRSEDTAVYYCARHGSYFDYWGQGTLVTVSS (SEQ ID NO: 8), or the sequence in amino acids<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0028] <h2 style=";text-align:left;direction:ltr"> QVQLVQSGAEVKKPGASVKVSCKTSGFTFSSSYISWLRQAPGQRLEWIAWIYAGTGGTSYS QKFQGRVQLTVDTSASTAYMEFSSLRSEDTAVYYCARHGSYFDYWGQGTLVTVSS (SEQ ID NO: 9), or the sequence of amino acids<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0029] <h2 style=";text-align:left;direction:ltr"> QVQLVQSGAEVKKPGASVKVSCKASGFTFSSSYISWLRQAPGQRLEWMAWIYAGTGGTSY SQKFQGRVTITVDTSASTAYMEFSSLRSEDTAVYYCARHGSYFDYWGQGTLVTVSS (SEQ ID NO: 10), or the sequence of amino acids<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0030] <h2 style=";text-align:left;direction:ltr">EVQLVESGGGLVQPGGSLRLSCATSGFTFSSSYISWLRQAPGKGLEWIAWIYAGTGGTSYA QKVKGRFQLSVDTSKNTAYLQMNSLRAEDTAVYYCARHGSYFDYWGQGTLVTVSS (SEQ ID NO: 11), or the sequence in amino acids<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0031] <h2 style=";text-align:left;direction:ltr"> EVQLVESGGGLVQPGGSLRLSCAASGFTFSSSYISWLRQAPGKGLEWVAWIYAGTGGTSY AQSVKGRFTISVDTSKNTAYLQMNSLRAEDTAVYYCARHGSYFDYWGQGTLVTVSS (SEQ ID NO: 12), or the sequence of amino acids<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0032] <h2 style=";text-align:left;direction:ltr"> QMQLVQSGPEVKKPGTSVKVSCKTSGFTFSSSYISWLRQARGQRLEWIAWIYAGTGGTSY AQKFQERVQLTVDMSTSTAYMEFSSLRSEDTAVYYCARHGSYFDYWGQGTLVTVSS (SEQ ID NO: 13), or the sequence of amino acids<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0033] (SEQ ID NO: 14); and the variable domain of the light chain of said humanized variant antibody or fragment thereof contains the amino acid sequence DIVMTQSPDSLAVSLGERATINCKSSQSVDYDGDSYMAWYQQKPGQPPKLLIYAASNRESG VPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGQGTKLEIK (SEQ ID NO: 15), or the amino acid sequence DIQLTQSPSSLSASVGDRVTITCRASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASSLESG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPYTFGQGTKLEIK (SEQ ID NO: 16).

[0034] For example, a humanized variant antibody or fragment thereof according to the present invention is characterized in that: the heavy chain variable domain of said humanized variant antibody or fragment thereof contains the sequence SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID NO: 7 or SEQ ID NO: 8, and the light chain variable domain of said humanized variant antibody or fragment thereof contains the sequence SEQ ID NO: 15; OR the heavy chain variable domain of said humanized variant antibody or fragment thereof contains the sequence SEQ ID NO: 9, or SEQ ID NO: 10, and the light chain variable domain of said humanized variant antibody or fragment thereof contains the sequence SEQ ID NO: 16;or the variable domain of the heavy chain of said humanized variant antibody or fragment thereof contains the sequence SEQ ID NO: 11 or SEQ ID NO: 12, and the variable domain of the light chain of said humanized variant antibody or fragment thereof contains the sequence SEQ ID NO: 16; or the variable domain of the heavy chain of said humanized variant antibody or fragment thereof contains the sequence SEQ ID NO: 13 or SEQ ID NO: 14, and the variable domain of the light chain of said humanized variant antibody or fragment thereof contains the sequence SEQ ID NO: 16.;

[0035] For example, a humanized variant antibody or fragment thereof according to the present invention is characterized in that it comprises the heavy chain variable domain and the light chain variable domain of the following respective sequences: SEQ ID NO: 9 and SEQ ID NO: 16 (CF12-13(HBV)-VL139); SEQ ID NO: 10 and SEQ ID NO: 16 (CF12-13(HCV)-VL139); SEQ ID NO: 11 and SEQ ID NO: 16 (CF12-366(HBV)-VL139); SEQ ID NO: 12 and SEQ ID NO: 16 (CF12-366(HCV)-VL139); SEQ ID NO: 5 and SEQ ID NO: 15 (CF12-13(H0)-VLO); SEQ ID NO: 6 and SEQ ID NO: 15 (CF12-13(H1)-VL0); SEQ ID NO: 7 and SEQ ID NO:

[0036] 15 (CF12-13(H2)-VL0); SEQ ID NO: 8 and SEQ ID NO: 15 (CF12-13(H3)-VL0); SEQ ID NO: 13 and SEQ ID NO: 16 (CF12-158(VHB)-VL139); SEQ ID NO: 14 and SEQ ID NO: 16 (CF12-158(VHC)-VL139).

[0037] For example, a humanized variant antibody or fragment thereof according to the present invention is characterized in that it comprises the heavy chain variable domain and the light chain variable domain of the following respective sequences: SEQ ID NO: 6 and SEQ ID NO: 15 (CF12-13(H1)-VL0); SEQ ID NO: 8 and SEQ ID NO: 15 (CF12-13(H3)-VL0); SEQ ID NO: 12 and SEQ ID NO: 16 (CF12-366(VHC)-VL139); SEQ ID NO: 9 and SEQ ID NO:

[0038] 16 (CF12-13(VHB)-VL139); SEQ ID NO: 11 and SEQ ID NO: 16 (CF12-366(VHB)-VL139); SEQ ID NO: 14 and SEQ ID NO: 16 (CF12-158VHC-VL139).

[0039] Preferably, a humanized variant antibody or fragment thereof according to the present invention is characterized in that it comprises the variable domain of the heavy chain containing the sequence SEQ ID NO: 8 or SEQ ID NO: 9, and the variable domain of the light chain containing the sequence SEQ ID NO: 15 or SEQ ID NO: 16, respectively. In particular, it is the humanized variant antibody or fragment thereof according to the present invention characterized in that it comprises the variable domain of the heavy chain containing the sequence SEQ ID NO: 8 and the variable domain of the light chain containing the sequence SEQ ID NO: 15; or the variable domain of the heavy chain containing the sequence SEQ ID NO: 9 and the variable domain of the light chain containing the sequence SEQ ID NO: 16.

[0040] Preferably, a humanized variant antibody or fragment thereof according to the present invention is characterized in that it comprises the variable domain of the heavy chain containing the sequence SEQ ID NO: 9, and the variable domain of the light chain containing the sequence SEQ ID NO: 16.

[0041] The present invention also relates to a pharmaceutical composition characterized in that it comprises a humanized variant antibody or fragment thereof according to the present invention, and a pharmaceutically acceptable vehicle.

[0042] “Pharmaceutically acceptable vehicle” means, for example, sterile isotonic solution, or sterile phosphate buffered saline (e.g. PBS), or a suitable formulation buffer.

[0043] According to a particular embodiment of the present invention, the pharmaceutical composition of the present invention may further comprise FSH, or LH, or a mixture of FSH and LH, or hMG, or a mixture of FSH and hMG, or a mixture of LH and hMG, or hCG, or a mixture of hCG and FSH, or a mixture of hCG and hMG. These hormones may be extracted or recombinant.

[0044] The present invention also relates to a humanized variant antibody or fragment thereof according to the present invention or a pharmaceutical composition according to the present invention, for use as a medicament, in particular for potentiating the bioactivity of FSH. For example, said medicament is intended to induce ovulation or polyovulation in a female mammal, and / or to stimulate steroidogenesis in a male or female mammal (for example by increasing the level of circulating endogenous estradiol and / or progesterone in a female mammal). For example, said medicament is intended to restore and / or stimulate spermatogenesis in a male mammal in order to increase the level of sperm production and their quality (mobility and morphology) and thus increase their number and concentration in the sperm.

[0045] The present invention also relates to a humanized variant antibody or fragment thereof according to the present invention or a pharmaceutical composition according to the present invention, for use in the prevention or treatment of infertility or subfertility in a male or female mammal. For example, it is the prevention or treatment of infertility in the case of hypogonadotropic hypogonadism, to restore and stimulate spermatogenesis in a male mammal or in the case of idiopathic or non-idiopathic non-obstructive azoospermia; or in the case of hypogonadotropic hypogonadism, to restore and stimulate oogenesis in a female mammal.For example, it is the prevention or treatment of subfertility in the case of oligozoospermia, oligoasthenospermia, oligoteratospermia, oligoasthenoteratospermia, asthenospermia, teratospermia or asthenoteratospermia in a male mammal; or in the case of anovulation, ovarian dysfunction or low ovarian reserve (low number of follicles in the antrum) in a female mammal.

[0046] In the case of a female mammal suffering from infertility or subfertility, the administration of the humanized variant antibody or fragment thereof according to the present invention or of the pharmaceutical composition according to the present invention, will allow ovarian stimulation with a view to natural procreation with or without artificial insemination, or medically assisted procreation (MAP) by Artificial Insemination (AI) or by In Vitro Fertilization (IVF) with or without ICSI (Intra Cytoplasmic Sperm Injection) or In Vitro Maturation (IVM) of the oocytes.

[0047] It should be noted that the administration of the humanized variant antibody or fragment thereof according to the present invention or of the pharmaceutical composition according to the present invention, to a healthy female mammal, will also make it possible to stimulate the ovary and trigger ovulation in the context of natural procreation with or without artificial insemination. The administration of the humanized variant antibody or fragment thereof according to the present invention or of the pharmaceutical composition according to the present invention, to a healthy female mammal, will make it possible to stimulate the recruitment and growth of ovarian follicles as well as the maturation of oocytes which will be punctured in the case of In Vitro Fertilization (IVF).

[0048] In the case of a male mammal suffering from infertility, such as hypogonadotropic hypogonadism, or subfertility, such as oligozoospermia, oligoasthenoteratospermia, oligoasthenospermia, oligoteratospermia, oligoasthenoteratospermia, asthenospermia, teratospermia or asthenoteratospermia, the administration of the humanized variant antibody or fragment thereof according to the present invention or of the pharmaceutical composition according to the present invention, will make it possible to stimulate or restore spermatogenesis, thus increasing the production and quality of spermatozoa to a level sufficient to allow natural fertilization with or without AI, or artificial fertilization in the case of conventional IVF or with ICSI.

[0049] For the purposes of the present invention, the term “hormone-dependent infertility / hypofertility” means infertility / hypofertility which may be due to hormonal insufficiency, for example low circulating concentrations of FSH or absence of this hormone resulting from an external cause (e.g. pesticides, endocrine disruptors) or internal cause (e.g. congenital or traumatic pituitary or hypothalamic insufficiency, cancer, surgical intervention, or problem of receptivity of the gonads to FSH due to an anomaly of the receptors or of the FSH, for example a mutation or a polymorphism of the FSH receptors).

[0050] For the purposes of the present invention, subfertility may also concern male or female mammals having normal circulating levels of FSH. In this case, the administration of the humanized variant antibody or fragment thereof according to the present invention or of the pharmaceutical composition according to the present invention, alone without exogenous hormones, will make it possible to potentiate the activity of endogenous FSH and to stimulate folliculogenesis and oocyte maturation in a female mammal and spermatogenesis in a male mammal.

[0051] For the purposes of the present invention, subfertility may also concern female mammals having a low ovarian reserve and described as "low responders" because they do not respond to ovarian stimulation hormonal treatments. In this case, the administration of the humanized variant antibody or fragment thereof according to the present invention, alone or in combination with an exogenous FSH and / or exogenous hMG treatment, will make it possible to amplify the response of the target cells to FSH and thus increase i) the number of stimulated ovarian follicles of sufficient size to perform a puncture and ii) the number of mature oocytes punctured, an essential condition for considering IVF.

[0052] The present invention also relates to a humanized variant antibody or fragment thereof according to the present invention or a pharmaceutical composition according to the present invention, for use in stimulating procreation in a female mammal.

[0053] The humanized variant antibody or fragment thereof according to the present invention or the pharmaceutical composition according to the present invention, can be used in humans or animals, in particular rodents, and non-human primates.

[0054] The humanized variant antibody or fragment thereof according to the present invention or the pharmaceutical composition according to the present invention, can be administered by injection, for example, intramuscularly, intravenously, intraperitoneally, subcutaneously, transcutaneously, intradermally, intraorbitally, intraocularly, ophthalmically, or transocularly, without altering their potentiating effect on the bioactivity of FSH, LH and / or hCG.Even if in the present description reference is made to a pharmaceutical composition, it is understood that each of the compounds of the composition can be administered concomitantly with the other compounds (for example in a single composition or in two compositions, each of these compositions comprising one or more of the aforementioned components, the mode of administration of each of the compounds or composition(s) being able to be identical or different), or independently of each other, for example successively, for example independent administrations of a humanized variant antibody or fragment thereof according to the present invention and of a hormone or mixture of hormones mentioned above, these administrations being carried out on the same mammal.These different administrations can be carried out, independently of each other or in a linked manner (composition or co-administration), by an identical or different mode of administration (injection, ingestion, topical application, etc.). The humanized variant antibody, or fragment thereof, can be administered once in the female and every two weeks for several months in the male. The humanized variant antibody, or fragment thereof, can be administered alone or in combination with an administration of a hormone or a mixture of hormones mentioned above, the latter being done once or several times per week, for two to several weeks.

[0055] BRIEF DESCRIPTION OF THE FIGURES

[0056] Figure 1 represents the binding of humanized variant antibodies according to the invention to FSH (A) and hCG (B).

[0057] Figure 2 represents the in vitro potentiating effect on human FSH of humanized variant antibodies according to the invention produced in transient CHO line: (A) HOLO, H1L0, H2L0 and H3L0 variants (B) 13VHB- and 13VHC-139VL variants (C) 158VHB-, 158VHC-, 366VHB- and 366VHC-139VL variants.

[0058] Figure 3 represents the in vitro potentiating effect on human FSH of humanized variant antibodies according to the invention produced in a stable CHO line: (A) H3L0, H1 L0 variants (B) 13VHB-, 158VHC-, 366VHB-, 366VHC-139VL variants (C) 13VHB-139VL variant.

[0059] Figure 4 represents the in vitro potentiating effect on different pharmaceutical preparations of human FSH and on hMG, of the humanized variant antibody 13VHB-139VL (SEQ ID NO: 9 and SEQ ID NO: 16): (A) on Fostimon® and Menopur® (B) on Bemfola® and Ovaleap® (C) on Puregon® and Rekovelle®.

[0060] Figure 5 represents the in vivo potentiating effect on human FSH and LH / hCG, in immature rats and rats, of humanized variant antibodies according to the invention produced in a transient line: (A) effect of the 366VHC-, 13VHB-, 158VHC-, 366VHB-, 13VHC- and 158VHB- 139VL variants on FSH in rats; (B) effect of the 366VHC-, 13VHB-, 158VHC-, 366VHB-, 13VHC- and 158VHB-139VL variants on LH / hCG in rats; (C) effect of the HOLO, H1L0, H2L0 and H3L0 variants on FSH in rats (D); effect of HOLO, H1 L0, H2L0 and H3L0 variants on hCG in rats; (E) ranking of the best variants according to their potentiating effect on FSH (F); ranking of the best variants according to their potentiating effect on hCG.

[0061] Figure 6 represents the in vivo potentiating effect on human FSH (A) and LH / CG (B) in immature rats and rats, respectively, of humanized variant antibodies (H1L0, H3L0, 13VHB-139VL, 366VHB-139VL, 366VHC-139VL, 158VHC-139VL) according to the invention, produced in a stable CHO line.

[0062] Figure 7 represents the in vivo potentiating effect on human FSH (A, C, D, E) and LH / CG (B) in immature male and female rats, respectively, of the humanized variant antibody 13VHB-139VL (SEQ ID NO: 9 and SEQ ID NO: 16).

[0063] Figure 8 represents the different stages of oocyte maturation (germinal vesicle (GV), metaphase 1 (ML) and metaphase 2 of meiosis (ML) stages) of female Cynomolgus monkeys, observed by optical and confocal microscopy.

[0064] Figure 9 represents the potentiating effect of the humanized H1L0 and 366VHC-139VL variants on estradiol (A) and progesterone (B) secretion in female cynomolgus monkeys given a single injection of the variant on the first day of treatment and a daily injection of 37.5 IU of human FSH for 12 days.

[0065] Figure 10 represents the potentiating effect of the humanized variants H3L0, 366VHB-139VL and 13VHB-139VL on the number and size of ovarian follicles observed on the thirteenth day of treatment in female cynomolgus monkeys having received a single injection of the variant on the first day of treatment and an injection of 37.5 IU of human FSH for 12 days.

[0066] Figure 11 represents the potentiating effect of the humanized H3L0 variants, 366VHB-139VL and 13VHB-139VL, on the secretion of estradiol (A) and progesterone (B) in female cynomolgus monkeys having received a single injection of the variant on the first day of treatment and a daily injection of 37.5 IU of human FSH for 12 days.

[0067] Figure 12 represents the potentiating effect of the humanized variant antibody 13VHB-139VL (SEQ ID NO: 9 and SEQ ID NO: 16) on the resumption and stimulation of spermatogenesis in adult azoospermic male rats treated with hCG + hMG in combination with 13VHB-139VL, by increasing testicular weight (A), testicular reserve (B) and epididymal sperm count (C and D).

[0068] Figure 13 represents the histological sections of azoospermic adult male rat testes, untreated (A), treated with hCG + hMG (B), and treated with hCG + hMG in combination with the humanized variant antibody 13VHB-139VL (SEQ ID NO: 9 and SEQ ID NO: 16) (C).

[0069] Figure 14 represents the potentiating effect of the humanized variant antibody 13VHB-139VL (SEQ ID NO: 9 and SEQ ID NO: 16) on the resumption and stimulation of spermatogenesis in adult male hypogonadal hpg - / - mice treated with FSH + hCG alone or in combination with 13VHB-139VL.

[0070] Figure 15 represents the potentiating effect of the humanized variant antibody 13VHB-139VL (SEQ ID NO: 9 and SEQ ID NO: 16) on the resumption and stimulation of spermatogenesis in adult male hypogonadal hpg - / - mice treated with hMG alone or in combination with 13VHB-139VL.

[0071] Figure 16 represents the potentiating effect of the humanized variant antibody 13VHB-139VL (SEQ ID NO: 9 and SEQ ID NO: 16) on the resumption and stimulation of spermatogenesis in adult male hpg - / - hypogonadal mice treated with FSH then with hMG alone or in combination with 13VHB-139VL: effect observed on testicular weight (A), testicular reserve (B) and epididymal sperm count (C).

[0072] Figure 17 represents the analysis of testicular cell populations by flow cytometry, in the untreated adult male hypogonadal hpg-l- mouse (A), treated with FSH then hMG (B), or treated with FSH then hMG in combination with the humanized variant antibody 13VHB-139VL (SEQ ID NO: 9 and SEQ ID NO: 16) (C).

[0073] Figure 18 represents the binding to human FSH, hMG and hCG of the scFv fragment whose peptide sequence (SEQ ID NO: 18) is derived from the humanized variant antibody 13VHB-139VL (SEQ ID NO: 9 and SEQ ID NO: 16) according to the invention.

[0074] Figure 19 represents the in vitro potentiating effect on human FSH (A) and hMG (B) of the humanized variant antibody 13VHB-139VL (SEQ ID NO: 9 and SEQ ID NO: 16) and its scFv (SEQ ID NO: 18) comparatively.

[0075] Figure 20 represents the in vitro potentiating effect of scFv 13VHB (SEQ ID NO: 18) on the LH bioactivity of hCG.

[0076] EXAMPLES

[0077] EXAMPLE 1: OBTAINING HUMANIZED VARIANTS OF THE CF12 ANTIBODY.

[0078] In these variants, the sequences of the ODRs (Complementary Determining Regions, hypervariable regions of the variable regions of the heavy and light chains of CF12 which constitute the elements of the paratope and make it possible to determine the complementarity of the antibody with the epitope of the antigen) are strictly identical to those of the CF12 monoclonal antibody. Conversely, the sequences of the Frameworks (FRs) (regions surrounding the CDRs constituting the "framework" and giving a stable configuration to the variable domain) carry at least one mutation on at least one FR of the CF12 monoclonal antibody. These structures were inserted into a human immunoglobulin framework (Fc region) of isotype lgG4.

[0079] Humanization of the murine monoclonal antibody CF12 was achieved by CDR-grafting. The three parental hypervariable regions CDRs of each CF12 heavy (VH) and light (VL) chain were grafted onto the framework of a human immunoglobulin acceptor variable domain FR (Framework) [5, 6]. The most homologous human framework sequences to the murine sequences were selected from human germline structure databases such as IgBIast (https: / / www.ncbi.nlm.nih.gov / igblast / ) or IMGT (http: / / www.imgt.org).The humanization method also considered the introduction of retro-mutations of certain human amino acids into murine frameworks to prevent loss of antibody affinity or stability due to structural incompatibilities between human frameworks and grafted murine CDRs [7, 8]. In order to identify the amino acids that would need to be retro-mutated, a 3-D model of the parental antibody was constructed in silico according to established protocols [9]. For this, the sequences of the variable heavy (VH) and light (VL) chains were numbered / annotated according to IMGT nomenclatures (Lefranc et al., 2015; Kabat et al., 1991; Chothia et al., 1987, 1989,1998) [10-14] in order to identify the frameworks and CDRs. The framework and CDR residues of VL and VH were then used to search for similar sequences of murine antibody structures resolved via protein BLAST.The structural models selected for the CDRs were grafted onto those selected for the frameworks. Finally, the reconstructed partial VL and VH models were manually subjected to mutagenesis and optimized (The PyMOL Molecular Graphics System, Version 1.8, Schrôdinger, LLC).

[0080] The best 3-D structure of the parental antibody was then constructed using software such as MOE and PAPS (Packing Angle Prediction Server: http: / / www.bioinf.org.uk / abs / paps / )

[0015] in order to find the best predicted tertiary arrangement and then optimized [16, 17]. Comparison between this model and those of humanized antibodies with selected germline sequences led to the construction by retro-mutations of the humanization variants of CF12.

[0081] The human germline sequences selected for humanization of the VH chain are: IGHV1-3*01, IGHV1-58*01, IGHV3-66*01.

[0082] The human germline sequences selected for VL chain humanization are: IGKV1-39*01 and IGKV4-V01.

[0083] The human germline sequences for VH and VL junctions are IGHJ4*01 and IGKJ2*01 respectively.

[0084] Ten humanized variants of CF12 were thus developed by inserting the VH and VL variable chains into a human immunoglobulin framework of isotype lgG4 carrying the N228P mutation and the R409K allotype.

[0085] The humanized variants are described in Table 1 below.

[0086] Table 1

[0087] These humanized variants were characterized with respect to their binding properties and with respect to their pharmacological effect in vitro and in vivo.

[0088] First, the variants were produced by transient expression in eukaryotic cells. Thus, the variants 13VHB-139VL, 13VHC-139VL, 366VHB-139VL, 366VHC-139VL, 158VHB-139VL, 158VHC-139VL were produced by transient chemical transfection of CHOEBNALT-85-1 E9 cells with pQMCF-1.2 plasmids containing the inserts of the DNA sequences to be expressed. The variants HOLO, H1 L0, H2L0 and H3L0 were produced by transient chemical transfection of HEK and CHO cells with Ml-mAbs' IgGP and IgK plasmids.

[0089] In a second step, the six best candidates 13VHB-139VL, 366VHB-139VL, 366VHC-139VL, 158VHC-139VL, HILO and H3L0 were produced in stable lines. The cell lines were generated by transfecting the gene of interest into the host cell line CHO-M (derived from CHO-K1) using microporation of SLXplasmid_220_Puro_BT+ plasmids, followed by one round of cloning and a second transfection with plasmids carrying another selection marker, SLXplasmid_221_Hygro_BT+ (SuperTransfection). Two additional rounds of single-cell dilution were performed to obtain highly productive super-transfected clonal cell lines.

[0090] In all cases, plasmid transfers into cells were performed by co-transfection of a vector carrying the VH gene of interest and a vector carrying the VL gene of interest at variable ratios between the two vectors. The variants produced were purified by protein A affinity chromatography.

[0091] EXAMPLE 2: MEASUREMENT OF THE BINDING ACTIVITY OF HUMANIZED VARIANTS ON FSH AND LH / hCG

[0092] The binding activity of humanized antibodies on recombinant human hormones FSH (GonalF® 1050 IU / 1.75 ml, Merck) and hCG (Ovitrelle® 250 micrograms, Merck) was studied by ELISA technique. Each hormone was prepared at a concentration of 10 pg / ml in 0.1 M sodium carbonate buffer pH 9.6 and distributed at 100 μl per well on an ELISA plate (MaxiSorp™, Nunc). The adsorption time of the hormones was 1 hour at 37°C followed by 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 1 hour at 37°C, then each antibody was distributed at 100 µl / well and incubated for 1 hour at 37°C. For each hormone evaluated, the antibodies were distributed at different concentrations within a range of 6.25 to 100 pg / ml.

[0093] After five washes, a secondary antibody coupled to peroxidase (H RP) (anti-human lgG4 Fc, Abeam 99817) diluted 1 / 2500 ewas distributed at a rate of 100 µl / well and incubated for 1 hour at 37°C. After five washes, the enzymatic activity was revealed with TM B distributed at a rate of 100 µl / well. The revelation time was 1 hour at room temperature. After stopping the reaction with 1 M H2SO4 (50 µl / well), the intensity of the colored reaction (Optical Density - OD) was measured using an ELISA plate spectrophotometer.

[0094] Table 2 below shows the optical density values ​​obtained with the ten variants at the concentration of 100pg / ml on FSH and hCG.

[0095] Table 2

[0096] The results obtained by ELISA technique demonstrate that the variants have a better binding to FSH than to hCG. They reveal a high variability in binding activity between the variants, the OD value ranging from 2.28 to 0.24.

[0097] The cross-linking obtained on hCG is weak and can be explained by the fact that human FSH and hCG have the same alpha subunit unlike the beta subunit which differs from one hormone to another.

[0098] The same analysis was conducted with the six selected variants, produced in a stable CHO line. The results obtained with a range of antibodies from 5 to 100 pg / ml on adsorbed FSH and hCG are illustrated in Figure 1. Each variant binds preferentially to human FSH (Figure 1A) and shows weaker binding to hCG (Figure 1B).

[0099] The binding levels obtained on FSH vary depending on the antibodies with an order of binding intensity that is generally identical between the two types of production (expression in transient lineage and in stable lineage).

[0100] EXAMPLE 3: IN VITRO MEASUREMENT OF THE POTENTIATING EFFECT OF HUMANIZED VARIANTS ON THE BIOACTIVITY OF FSH AND LH / hCG The potentiating effect of humanized variants on the bioactivity of human FSH was measured in vitro on HEK 293 cells expressing the human FSH receptor. The cells were stimulated with a range of human FSH (Gonal-f ®, Merck, France) alone or in combination with a humanized variant.

[0101] Comparison of the dose-response curve obtained with FSH or with the FSH / variant complex made it possible to quantify the potentiating effect of each of the ten variants on FSH activity and to select the best candidates based on this property.

[0102] On HEK 293 hFSHR GloSensor® cells

[0103] The potentiating effect of the humanized variants on FSH activity was evaluated in vitro on HEK 293 cells stably expressing on the one hand the human FSH receptor, and on the other hand a cyclic adenosine monophosphate (cAMP) sensor, GloSensor® (Promega reference E2401, Charbonnières-les-Bains, France). The cells were distributed at a rate of 80,000 cells per well, in 96-well dishes with white borders and backgrounds (Greiner Bio One reference 655083, Dominique Dutscher, Brumath, France), in EMEM medium (Lonza reference BE12-611 F, Ozyme, Saint Cyr l'Ecole, France) supplemented with 10% fetal calf serum (Eurobio CVFSVF00 01, Les Ulys, France) previously decomplemented, 1% penicillin / streptomycin (Lonza reference DE17-602E) and incubated overnight in a 37°C 5% CO2 incubator under a humid atmosphere.The next day, the cell medium was aspirated and replaced with EMEM culture medium containing 1% HEPES (Lonza reference BE17-737E, Ozyme, Saint Cyr l'Ecole, France) and GloSensor® substrate (Promega reference E1291, Charbonnières-les-Bains, France) at a rate of 4 μl of substrate in 100 μl of medium per well. After 2 hours of incubation at 21 °C in a humid atmosphere and protected from light, 10 μl of an FSH or FSH + variant preparation was added to the cells and the emitted luminescence was quantified using a LUMIstar Omega 96-well plate reader (BMG Labtech, Champigny sur Marne, France). Luminescence was measured for approximately 60 minutes.

[0104] The potentiating effect was quantified by comparing the dose-response curves obtained with a range of FSH concentrations (Gonal-f ®) from 10' 11 M to 10' 8M, prepared alone or in combination with a variant prepared at a fixed concentration of 10 pg / ml (M). The FSH+variant mixture was preincubated for 20 minutes at 37°C before deposition onto the cells. These dose-response curves represent the maximum luminescence signal obtained upon stimulation of cells at each concentration of FSH alone or with the variant.

[0105] The results obtained were analyzed with Prism software (GraphPad Prism Software Inc., San Diego, CA, USA, version 10.0.1). The nonlinear function “log (agonist) versus response” was used to plot the response as a function of FSH concentration and to characterize the EC50 of FSH alone, the EC50 of FSH complexed with the tested variant (potency) and the maximum signal of the response (efficacy) in each case. In order to be able to compare the curves with each other, the luminescent signal is expressed as a percentage relative to the maximum response obtained with FSH alone, which is set at 100%. The basal level of the response is set at 0%.

[0106] This study was first performed with the ten humanized variants produced in transiently transfected CHO cells in order to select them for their potentiating effect on FSH activity. The results obtained are illustrated in Figure 2A for the H0L0, H1L0, H2L0 and H3L0 variants, Figure 2B for the 13VHB- and 13VHC- 139VL variants and Figure 2C for the 158VHB-, 158VHC-, 366VHB- and 366VHC-139VL variants. The cellular response obtained with each variant was characterized by its maximum level (efficacy) and by its EC50 (potency). For all variants, it is observed that the FSH+variant complex induces an increase in the potency of the cAMP response, with an EC50 value 1.5 to 2 times higher than that obtained with FSH alone. For some variants, an increase in the maximum response with the FSH+variant complex of up to 120% compared to FSH alone is also observed (366 HBV and 366 HCV, H3L0, HOLO, 13HBV).This reflects a better efficiency of the cAMP response during stimulation with FSH+variant. The potentiation of FSH activity reaches a plateau in all cases, which suggests that the potentiating effect does not induce an uncontrolled phenomenon of hyperstimulation of the cells.

[0107] The six variants with the strongest potentiating effect on FSH activity were then sectioned and produced in a stable CHO cell line. These are the H3L0, H1L0 and 13VHB-, 158VHC-, 366VHB-, 366VHC-139VL variants.

[0108] Their potentiating effect on FSH activity was then characterized in vitro, according to the same protocol. The results obtained are illustrated in Figure 3A for the H3L0, H1 L0 variants and Figure 3B for the 13VHB-, 158VHC-, 366VHB- and 366VHC-139VL variants. Again, for all variants, the dose response curves obtained with the FSH+variant complexes have an EC50 value of 1.3 to 2.1 times higher than that obtained with FSH alone, reflecting an increase in the power of the cAMP response. With the exception of the 366VHC-139VL variant, an increase in the maximum response with the FSH+variant complex of up to 125% compared to FSH alone is also observed (in ascending order of efficacy: 366VHC-, 366VHB-, 13VHB-139VL, H1 L0, H3L0, 158VHC-139VL).

[0109] In conclusion, the potentiating effect on FSH is of variable amplitude depending on the variants. It results in an increase in the power of the cAMP response obtained with the variant+FSH complex and in an increase in the efficiency of the response with a higher maximum level of cAMP than that obtained with FSH alone.

[0110] Finally, the pharmacological characteristics of the 13VHB-139VL variant, chosen after numerous in vivo tests, and produced by its final clonal line, were measured and are illustrated in Figure 3C. The curves represent the average of three independent tests carried out with Gonal-f and show a doubled EC50 value compared to FSH alone (1.1.1 O' 10 M versus 2.08.10' 10 M respectively) and an increased efficacy of 130% in the presence of the variant, confirming its potentiating effect on FSH activity.

[0111] The potentiating properties of the 13VHB variant were also evaluated on other commercially available human FSH preparations and on hMG used in human reproductive medicine. These preparations are used in human medicine in women, for example, for ovarian stimulation treatments in the context of ART. The same experimental conditions were used as those described above for Gonal-f.

[0112] These are the purified extracted hormones:

[0113] Fostimon ® (FSH) (IBSA, Italy);

[0114] Menopur ® (purified hMG containing 50% FSH and 50% LH) (Ferring, France); and recombinant FSH:

[0115] Biosimilars of Follitropin alpha: Ovaleap ® (Teva, Germany) and Bemfola ® (Gedeon Richter, France);

[0116] Follitropin beta: Puregon ® (MSD, USA);

[0117] Follitropin delta: Rekovelle ® (Ferring, France).

[0118] The results are illustrated in Figure 4. For each hormone, three independent trials were performed each time and a representative example is shown. Figure 4A, illustrates the results obtained with the two extracted hormones Fostimon® and Menopur® and shows a doubled EC50 value in the presence of 13VHB and a maximum response of 120% compared to the hormone alone indicating better potency and efficacy of the response.

[0119] Figure 4B illustrates the potentiating effect of 13VHB on the activity of the two biosimilar recombinant FSHs, Bemfola® and Ovaleap®. The EC50 value was increased from 1.57 to 1.7 times and the maximum response was 130% compared to the hormone alone in the case of Bemfola® and lower in the case of Ovaleap® (110%).

[0120] The potentiating effect of 13VHB on the activity of follitropins beta (Puregon®) and delta (Rekovelle®) is illustrated in Figure 4C. In the presence of 13VHB, the efficacy was increased by 130% in the case of Rekovelle® and by 158% in the case of Puregon®. At the same time, the potency (EC50) was 2 to 2.3 times higher than that of the hormone alone in the case of Puregon and Rekovelle, respectively.

[0121] In conclusion, these results demonstrated that the potentiating effect of the 13VHB variant is exerted on all FSH and hMG preparations available on the market and used in human reproductive medicine. The combination of the 13VHB variant with these different types of FSH and hMG allows in all cases a better efficacy with an increase in the maximum level of the response of 20 to 60% compared to the hormone alone and a better potency with an increase in the EC50 by a factor of 1.6 to 2.3. EXAMPLE 4: IN VIVO MEASUREMENT OF THE POTENTIATING EFFECT OF HUMANIZED VARIANTS ON THE BIOACTIVITY OF FSH AND LH / hCG

[0122] The potentiating effect of the humanized variants on the bioactivity of FSH and hCG / LH was then studied and characterized in vivo in immature female and male rats, in order to evaluate the impact of the humanization of the sequence on the potentiating effect in vivo. For this, the effect of each variant injected in combination with hormonal treatment was systematically compared to that of the murine CF12 antibody and hormonal treatment alone.

[0123] Measurement of FSH activity in immature rats by Steelman and Pohlev's Bioassay

[0124] The potentiating effect of humanized variants on FSH activity was evaluated in vivo in 25-day-old immature Wistar Han rats (Charles River, L'ArbresIe, France). The protocol used was that of Steelman and Pohley

[0018] . The animals received a subcutaneous injection of 100 μl of [hCG 3.5 IU + hFSH 0.5 IU] or 100 μl of the mixture [hCG 3.5 IU + hFSH 0.5 IU + variant 2 pg] twice daily for 3 days. The dose of 2 pg corresponds to a concentration of about 0.3 pg / ml of blood if we assume a blood volume of 65 ml / kg in rats (University of Pittsburgh Policy for Regulating the Volume of Experimental Blood Sample Withdrawals in Laboratory Animals). The variants were formulated in sterile PBS and the hormones in the medium provided by the manufacturer for their preparation.

[0125] The 4th èmeOn day 1, the rats were euthanized, their ovaries were dissected and weighed in pairs. The increase in ovarian weight was proportional to the activity of the injected FSH. Each experimental group included five females.

[0126] Measurement of hCG / LH activity in immature rats using Scobev's Bioassav

[0127] The effect of the variants on LH / hCG activity was evaluated in vivo in immature male rats according to the protocol of Scobey et al.

[0019] . For this, 3-week-old Wistar Han rats received a subcutaneous injection of 100 μl of hCG 1.5 IU or 100 μl of the mixture (hCG 1.5 IU + a 2 pg variant), once a day for four days. The dose of 2 pg corresponds to a concentration of the order of 0.3 pg / ml of blood if we assume a blood volume of 65 ml / kg in rats (University of Pittsburgh Policy for Regulating the Volume of Experimental Blood Sample Withdrawals in Laboratory Animals). The variants were formulated in sterile PBS and the hormones in the medium provided by the manufacturer for their preparation.

[0128] The 5th èmeOn day 1, the animals were sacrificed, and the seminal vesicles were dissected and weighed. The weight of the seminal vesicles is proportional to the activity of the injected hCG. Each experimental group consisted of five females.

[0129] First, the ten variants expressed in the transient line were characterized with respect to their in vivo potentiating effect on FSH and LH / CG activity and compared to that of the murine antibody CF12. The objective was to select variants based on their FSH potentiating effect.

[0130] 1 / Effect of humanized variants including the 139VL light chain

[0131] A first series of six variants characterized by the sequence 139VL (SEQ ID NO: 16) was evaluated in both bioassays. These are the variants 13VHB, 13VHC, 158VHB, 158VHC, 366VHB and 366VHC.

[0132] Their potentiating effect on FSH activity was quantified by comparing the average ovarian weight of females in the variant+FSH group with that of females treated with hormones alone. The results are shown in Figure 5A and showed that the 366VHC, 158VHC and 13VHB variants have a potentiating effect close to that of the murine CF12 antibody. The average ovarian weight of females treated with the hCG / FSH+variant mixture is 1.91 to 1.82 times higher than with hormonal treatment alone, compared to 2 times in the group treated with hCG / FSH+CF12. The other three variants 366VHB, 13VHC and 158VHB have a potentiating effect that tends to be weaker with an increase in average ovarian weight ranging from 1.71 to 1.5 times greater compared to hormonal treatment alone.

[0133] The results obtained in the Scobey bioassay are illustrated in Figure 5B. They surprisingly and unexpectedly showed that the variants exhibit a more or less significant decrease in their potentiating effect on LH / CG activity compared to the murine CF12 antibody. Indeed, the average weight of the seminal vesicles of males treated with the hCG+variant mixture is 1.26 to 1.05 times higher than with hCG alone, compared to 1.71 times for animals treated with hCG+CF12. The decrease in the effect of the variants on LH / CG activity therefore represents a loss ranging from 45% to 67% compared to the murine CF12 antibody.

[0134] 21 Effect of humanized variants including the LO light chain

[0135] Similarly, the effect of the four variants comprising the VLO light chain (SEQ ID NO: 15) were characterized in both bioassays. These are the HOLO, H1L0, H2L0 and H3L0 variants.

[0136] Their potentiating effect on FSH activity is illustrated in Figure 5C. The H1 LO and H3L0 variants showed a potentiating effect equal to that of the murine CF12 antibody, with in all three cases a mean ovarian weight 1.67 times higher than in the group treated with hormones alone. The other two variants, H2L0 and particularly HOLD, had a weaker effect: the mean ovarian weight of females treated with hCG / FSH+variant was 1.5 and 1.4 times higher than in females receiving hormonal treatment alone, compared to 1.67 times in the group treated with hCG / FSH+CF12.

[0137] The results obtained in the Scobey bioassay are illustrated in Figure 5D and showed, surprisingly and unexpectedly, a potentiating effect on LH / hCG activity lower than that of the murine CF12 antibody. This decrease is variable according to the variants but in no case is the potentiating effect on LH cancelled. The average weight of the seminal vesicles of males treated with the hCG+variant mixture is 1.28 to 1.05 times higher than with hCG alone compared to 1.54 times for the batch treated with hCG+CF12. The decrease in the effect of the variants on LH therefore represents a loss ranging from 26% to 49% compared to the effect of the murine CF12 antibody.

[0138] 3 / Ranking of all humanized variants according to their potentiating effect on FSH and LH / hCG and selection of the six best

[0139] In order to integrate all the results obtained and compare the ten variants with each other, a calculation formula was used to quantify the % potentiating effect on FSH and LH of each variant compared to CF12.

[0140] In this calculation, the effect of CF12 represents 100% and the effect of hormone treatment alone is set to 0%.

[0141] The effect of each variant is expressed in % relative to CF12 according to the following formula: (AC) / (BC) *100, where

[0142] - A is the value obtained with the treatment (variant + hormone);

[0143] B is the value obtained with the treatment (CF12 + hormone);

[0144] This is the value obtained with hormonal treatment alone; the factor 100 allows the ratio to be expressed in %.

[0145] The ranking obtained in relation to the potentiating effect of FSH activity is illustrated in Figure 5E. Their potentiating effect varies from 95% to 50% compared to that of CF12 (100%). Five variants have a strong potentiating effect ranging from 100 to 80%: H3L0 (94%), H1 L0 (93.5%), 366VHC (91.5%), 13VHB (88%) and 158VHC (82.5%). The other five have a less significant effect ranging from 75 to 50% compared to CF12 (100%): H2L0 (74.5%), 366VHB (71%), HOLD (60%), 13VHC (59.5%) and 158VHB (50.5%).

[0146] The ranking obtained with respect to the potentiating effect on LH / hCG activity is illustrated in Figure 5F and highlighted a weaker potentiating effect of the variants, ranging from 52% to 5%, compared to that of CF12 (100%). Five variants have a potentiating effect ranging from 52% to 20%: HOLO (51.84%), H1 L0 (40.8%), 158VHB (36.6%), 13VHC (26.8%) and 13VHB (22.3%). The other five variants have a weaker effect ranging from 19% to 5%: 158VHC (17.9%), H2L0 (15%), 366VHB (7.4%), 366VHC (6.2%) and H3L0 (5%).

[0147] All these results indicate that humanization of the murine CF12 antibody sequence has allowed the potentiating effect of the variants on FSH activity to be preserved: 7 out of 10 variants have between 95 and 70% of the effect of the original murine antibody. Conversely, and unexpectedly and surprisingly, humanization has led to a more or less significant decrease in the potentiating effect of the variants on LH / hCG activity, with five of them having an effect below 20% of that of the original murine antibody. It is important to emphasize that in no case has this decrease led to a total disappearance of the potentiating effect on LH / hCG. All variants have retained a potentiating effect on LH / hCG to varying degrees.Surprisingly also, the impact of humanization on the FSH potentiating effect is not correlated with that observed on the LH potentiating effect: a variant showing a significant decrease in the effect on LH may have retained a strong potentiating effect on FSH activity, this is the case of H3L0. Conversely, the variant with the highest effect on LH activity (for example HOLO) has a reduced potentiating effect on FSH activity, less than 60%. Finally, some variants have a homogeneous ranking between the effect on FSH and on LH, this is the case of 158VHC for example.

[0148] A study of the impact of human germline sequences selected for humanization of the VH or VL chain on a possible modification of the potentiating effect showed no correlation between a negative impact on the FSH or LH effect and one or more particular germline sequences.

[0149] Potentiating effect on FSH and LH activity of the six humanized variants selected and produced in a stable line.

[0150] The ranking of the variants produced in the transient line with respect to their potentiating effect on FSH activity allowed the selection of the six best candidates. These variants were produced in a stable CHO line and their potentiating effect was controlled and measured in vivo with respect to the effect of the murine monoclonal antibody CF 12. The same calculation formula as previously was used to quantify in % the potentiating effect of the humanized variants on FSH and LH / CG activity with respect to CF12, whose effect is set at 100%. The effect of hormonal treatment alone is set at 0%.

[0151] The results shown in Figure 6A show that the potentiating effect on FSH activity remained very high and stable. The 366VHB, 13VHB and H3L0 variants are the most potentiating with an effect equal to 96.5, 94% and 81% respectively compared to that of CF12. The 366VHC and 158VHB variants have an effect equal to 74 and 71% compared to that of CF12 respectively. The H1 L0 variant is the only variant to show a decrease in its potentiating effect on FSH (54%).

[0152] The results in Figure 6B illustrate for all variants, a repeated decrease in the potentiating effect on LH / CG activity compared to the murine antibody CF12. Their ranking is globally identical to that of the ten variants produced in transient expression and marks a surprising drop in the amplitude of the potentiating effect on LH / CG representing 24% to 3% of the effect of CF12. The H1 L0 and 13VHB variants have a potentiating effect on LH / CG activity representing 24 and 20% respectively of the effect of CF12 and are the most efficient. The other four variants have a very reduced effect ranging from 6.35% to 4% of that of CF12.

[0153] Overall, the pharmacodynamic properties of the variants produced in stable lines were not modified with the exception of H1 L0, whose potentiating effect on FSH activity was reduced and, to a lesser extent, that on LH / CG activity.

[0154] In conclusion, the humanization of CF12 surprisingly led to variants that strongly potentiate FSH activity and weakly potentiate LH / CG activity, leading to a new molecule that is more specific in its potentiating action on FSH. These new properties will allow better targeting of cells expressing FSH receptors (e.g. granulosa cells in the ovary, Sertoli cells in the testis) and consequently better targeting and defining the potential therapeutic indications of the future drug.

[0155] Potentiating effect on FSH and LH activity of the 13VHB-139VL variant produced in its definitive stable lineage.

[0156] The potentiating effect of the variant selected as a first-rank candidate for drug development, 13VHB, produced in its final monoclonal CHO line, was verified and characterized on FSH (Figure 7A) and LH / CG (Figure 7B) activity. The results indicated that the final product of the 13VHB variant has a potentiating effect on FSH in the same order as CF12 with a mean ovarian weight increased by 1.7 times in rats treated with 13VHB + FSH / hCG compared to rats treated with FSH / hCG alone (Figure 7A). Comparatively, the potentiating effect on hCG activity remained low with a mean seminal vesicle weight increased by 1.3 times in rats treated with 13VHB + hCG compared to rats treated with hCG alone (Figure 7B). These results confirmed that 13VHB is a variant with a strong potentiating effect on FSH activity and a weaker potentiating effect on LH / CG activity.

[0157] In order to further characterize the potentiating effect on FSH, a dose-response study was performed with the same protocol of Steelman and Pohley

[0018] in immature female rats treated with increasing doses of the 13VHB variant, ranging from 0.005 pg to 8 pg / injection / 100 g body weight, combined with a fixed dose of FSH (Gonal-f) of 0.5 IU. The results are expressed as raw data (ovarian weight in mg per 100 g body weight) in Figure 7C and as a dose-response curve in Figure 7D. They clearly demonstrate a dose effect of the 13VHB variant in the FSH bioassay with an optimal dose of 2 pg / injection / 100 g body weight. The dose of 13VHB corresponding to 20% of the activity (EC20) is 0.486 pg / 100 g or 4.86 pg / injection / kg of body weight. That of the EC50 is 0.73 pg / 100 g or 7.3 pg / injection / kg of body weight.

[0158] The potentiating effect of 13VH was evaluated on other human FSH preparations used in human clinical practice in ART techniques, such as Gonal-f, to stimulate follicular growth and oocyte maturation. These are the recombinant follitropins alpha, Bemfola (Gedeon Richter, France) and Ovaleap (Theramex, France), Follitropin Beta Puregon and Follitropin Delta Rekovelle (Ferring, France). The extracted hMG Menopur (Ferring, France) composed of 50% hFSH and 50% hLH was also tested. Each hormonal preparation was injected at a single dose of 0.5 IU and 13VHB was injected at 2 pg / injection / 100 g of body weight. The results are shown in Figure 7E and represent the raw data obtained after the different treatments with or without 13VHB. They showed that the 13VHB variant exerts a potentiating effect on all FSH preparations tested with varying amplitude.An increase of 146% and 140% respectively in the mean ovarian weight was obtained in females treated with 13VHB in association with follitropins alpha, Benfola® and Ovaleap®, compared to females treated with the same hormone without 13VHB. The increase is 130% with follitropin delta and hMG (Menopur®) compared to females treated without 13VHB and 120% with follitropin beta, (Puregon®). In these last three cases, the effect is lower probably due to a difference in specific activity specific to each of these FSH preparations.

[0159] EXAMPLE 5: IN VIVO POTENTIALIZING EFFECT OF SELECTED HUMANIZED VARIANTS ON FOLLICULOGENESIS AND OOCYTE MATURATION IN FEMALE NON-HUMAN PRIMATES

[0160] The potentiating effect of the five best humanized variants selected in vitro, and in vivo in the immature female rat, was then evaluated in the female Cynomolgus monkey (Macaca fascicularis) in the context of ovarian stimulation treatments identical to those used in women for In Vitro Fertilization.

[0161] Three different protocols used in female reproductive medicine were thus adapted and developed for use in the monkey (Macaca fascicularis). A long agonist protocol, based on a two-week pretreatment with a GnRH agonist before ovarian stimulation treatment with FSH. In this case, the variant is used in combination with exogenous FSH treatment. A short antagonist protocol comprising ovarian stimulation either with a daily injection of FSH for 12 days or with a single injection of the variant on day 1 of the cycle. In this case, the variant was used alone without any injection of exogenous FSH and was compared to the classic treatment with FSH. In both cases, a daily injection of GnRH antagonist was performed when follicles of preovulatory size were observed during ultrasound monitoring.a short agonist protocol (or “flare-up” agonist protocol) comprising an injection of GnRH agonist on day 1 of the cycle and ovarian stimulation either with a daily injection of FSH for 9 days from day 6, or with a single injection of the variant on day 1 of the cycle. In this case, the variant was used alone without any injection of exogenous FSH and compared to the conventional treatment with exogenous FSH.

[0162] Potentiating Effect of Humanized Variants in Combination with Exogenous FSH in Long Agonist Treatment Mature females aged at least 36 months were treated with a long-acting GnRH agonist, Decapeptyl® LP (1.5 mg per female) on the first day of menstruation to inhibit the secretion of endogenous gonadotropins. A second injection of this agonist was administered 15 to 21 days after the first to cover the entire stimulation cycle. Ovarian stimulation treatment began 15 to 21 days after the first Decapeptyl LP injection. This treatment included:

[0163] - either 1 injection of human FSH (Gonal-f®, Merck KGaA, Germany) 37.5 IU per day for 12 days;

[0164] - either 1 injection of human FSH (Gonal-f®, Merck KGaA, Germany) 37.5 IU per day for 12 days and a single injection of potentiating antibody at a dose of 20 pg / kg, on the first day of menstruation, 20 minutes after the injection of Gonal-f. The dose of 20 pg / kg corresponds to a concentration of around 0.3 pg / ml of blood if we assume a blood volume of 65 ml / kg (University of Pittsburgh Policy for Regulating the Volume of Experimental Blood Sample Withdrawals in Laboratory Animals).

[0165] Transabdominal ultrasounds of the ovaries and blood samples were performed during ovarian stimulation treatment to monitor the growth of ovarian follicles and the secretion of steroidogenic hormones such as estradiol and progesterone, respectively.

[0166] Females were then injected with 2600 IU of hCG (Ovitrelle®, Merck KGaA, Germany) 36 hours after the last Gonal-f injection. Follicular puncture by laparotomy was performed 36 hours after the hCG injection.

[0167] After opening the abdomen, the ovaries were gently brought to the surface and the follicles were punctured using a 25 G needle and a 5 ml syringe, and G-MOPS PLUS culture medium (Vitrolife Reference 10130, Paris, France). The punctured follicular fluid was collected in 14 ml round-bottom Falcon tubes (Dutscher reference 352001, Brumath, France) containing 200 μl of heparin (Choay Heparin 25000 IU / 5 ml, Cheplapharm Arzneimittel GmbH, Greifswald, Germany).

[0168] The oocytes were then searched for in the follicular fluid under a binocular microscope and then decoronized by hyaluronidase treatment (BioCare Europe reference 90101-5X1ML, Rome, Italy). They were then observed under a microscope to determine their maturation stage evaluated according to the presence or absence of the first polar body. In order to confirm their maturation stage and control their quality, the oocytes were then fixed in a 4% paraformaldehyde (PAF) solution containing 0.5% Triton and 1% BSA for 30 min at 37°C, protected from light. The oocytes were then transferred to a 1% PBS / BSA solution and stored at 4°C until their analysis by confocal microscopy after labeling of DNA and tubulin by immunocytochemistry.

[0169] 1 / Effect of the 366VHC-139VL and H 1 LO variants A first series of nine females divided into 3 groups of 3 animals made it possible to evaluate the effect of the 366VHC-139VL and H1L0 variants in combination with FSH compared to treatment with FSH alone. These variants were produced in a transient CHO line and formulated in PBS.

[0170] To assess follicular growth during the three treatments, ultrasound monitoring of the ovaries was performed from the first day of treatment until follicular puncture (D15) to measure the size (in millimeters) and number of follicles. The results shown in Table 3 represent the number of total follicles and the number of large follicles larger than 3 mm observed on D13, the day of hCG injection. A size larger than 3 mm defines a preovulatory follicle. This size is sufficient to allow puncture.

[0171] Table 3: Number of follicles per ovary in monkeys treated with FSH, FSH+H1 LO and FSH+366VHC (mean ± SEM). * p<0.05 Greater effect of FSH+CF12-H1 L0 versus FSH (two-way A NOVA).

[0172] Treatments combining an antibody injection with FSH treatment resulted in the recruitment of a greater number of total follicles, detectable by ultrasound, with more large follicles. In the groups treated with FSH + H1 L0 or FSH + 366VHC, the number of total follicles was increased by 1.5 times. The number of follicles with a diameter greater than 3 mm was 2.64 times higher (*, p<0.05 versus FSH) with the FSH+H1 L0 treatment and 1.8 times higher with the FSH+366VHC treatment (non-significant). These results demonstrate that FSH treatment combining a humanized variant induced better ovarian stimulation, better recruitment and better follicular growth, particularly with H1 L0, whose effect was significantly greater than that of FSH alone (p<0.05).

[0173] Analysis of follicular punctures allowed to count the number of total oocytes, to sort immature oocytes at the Germinal Vesicle stage (GV stage) and Metaphase 1 (Ml stage) and mature oocytes at the Metaphase 2 stage of meiosis (Mil stage) characterized by the presence of a first polar body. The results presented in Table 4 illustrate the average number of total oocytes and mature oocytes (Mil) obtained with each of the three treatments. Table 4: Variation in the average number of total oocytes and mature oocytes punctured in monkeys treated with FSH, FSH+H1 L0 and FSH+366VHC- (mean ± SEM)

[0174] Monkeys treated with the variant + FSH combination yielded both more total oocytes and more mature oocytes than the group treated with FSH alone: ​​2.6 times more mature oocytes with the 366VHC variant and 3.15 times more with the H1 L0 variant. These results demonstrate that FSH treatment in combination with a potentiating variant induced better oocyte quality and better oocyte maturation, particularly in the case of the H1 L0 variant where 74% of the retrieved oocytes were mature oocytes compared to 54% with FSH alone.

[0175] This result is significant insofar as the success rate of In Vitro Fertilization is higher the greater the number of mature oocytes (Mil) collected.

[0176] Observation of oocytes by confocal microscopy after labeling of DNA and Tubulin, showed that the structure and quality of oocytes resulting from a treatment combining a humanized variant were completely normal and identical to oocytes resulting from a standard treatment with FSH alone. Figure 8 illustrates the different stages of oocyte maturation observed respectively by optical and confocal microscopy: immature oocytes at the Germinal Vesicle (GV) stage and at the Metaphase 1 (Ml) stage and mature oocyte at the Metaphase 2 stage of meiosis (Mil) characterized by the presence of the first Polar Globule (PG).

[0177] In order to characterize the effect of the variants on the steroidogenic response of the ovary, estradiol and progesterone secretion was monitored throughout the cycle from the beginning of stimulation (D1) to the end of the luteal phase (D30). The results are presented in Figure 9. The group of females treated with FSH+H1 L0 showed higher estradiol secretion compared to the other two groups throughout the follicular phase (Figure 9A). On D9, the estradiol concentration was significantly higher in the monkeys treated with FSH+H1 L0 compared to the females treated with FSH alone by a factor of 4 (p<0.001) or with FSH+366VHC by a factor of 3 (p<0.01) (Two way-ANOVA). This higher estradiol secretion is correlated with the better follicular growth and oocyte maturation observed in females treated with FSH+H1L0. There is no significant difference between the FSH and FSH+366VHC groups.

[0178] No difference in progesterone secretion was observed between the three treatments (Figure 9B). 21 Effect of H3L0, 366VHB-139VL and 13VHB-139VL variants

[0179] A second series of treatments was carried out in this same protocol on thirteen monkeys in order to evaluate the potentiating effect of three other humanized variants: H3VH-L0VL (H3L0), 366VHB-139VL (366VHB) and 13VHB-139VL (13VHB). These variants were produced in a stable CHO line. They were formulated and diluted in PBS.

[0180] The monkeys were divided into 4 groups: 3 groups of 3 monkeys treated with FSH alone, FSH+H3L0 or FSH+366VHB and one group of 4 monkeys treated with FSH+13VHB.

[0181] The potentiating effect of these 3 variants was evaluated in a superovulation treatment identical to the previous one using the same physiological parameters to monitor ovarian stimulation. The effect of the variants was compared to that of a standard treatment with FSH alone.

[0182] Follicular recruitment and growth was monitored by ultrasound of the ovaries from the first day of treatment until follicular puncture (day 15). Table 5 illustrates the size and number of follicles observed in the 4 groups.

[0183] Table 5: Size and number of follicles per ovary, in monkeys treated with FSH, FSH+H3L0, FSH+366VHB, FSH+13VHB (mean ± SEM).

[0184] The results indicate that ovarian stimulation was greater with the FSH+variant combination treatments compared to FSH alone, particularly with the FSH+366VHB and FSH+13VHB treatments. The number of follicles with a diameter greater than 4 mm was 7.2 times higher in the FSH+13VHB group compared to the FSH group and 4.2 times higher than in the FSH+366VHB group.

[0185] The number of follicles measured at day 13 and classified by size category is shown in Figure 10, for each batch of females. The batch treated with FSH+13VHB gave the greatest number of growing follicles and the largest follicle sizes. Thus, 18 follicles of 4-5 mm were observed on average in the FSH+13VHB group compared to 10 in the FSH+366VHB group and 3 in the FSH alone and FSH+H3L0 groups. This reflects better follicular growth and differentiation.

[0186] Follicle puncture was performed on day 15 of treatment and showed significant differences between treatments.

[0187] The number of total oocytes retrieved and among them the number of mature oocytes observed (Metaphase II Stage) for each treatment (FSH alone or in combination with an antibody injection on D1) are summarized in Table 6. Table 6: Number of total oocytes and mature oocytes (Mil) obtained in each experimental group. The results are expressed as mean ± SEM.

[0188] The results show that females treated with FSH in combination with a humanized variant had a higher total oocyte count and a higher number of mature oocytes. Females treated with the 13VHB variant gave the best results with 3 times more mature oocytes at the Mil stage than females treated with FSH alone: ​​25.5 MH versus 8.33 respectively. Treatment with the H3L0 variant gave 2.4 times more mature oocytes than with treatment with FSH alone: ​​20.33 Mil versus 8.33 respectively. Treatment with 13VHB therefore achieved the best oocyte maturation resulting in the highest rate of mature oocytes, developed at the Metaphase II stage of meiosis. This result is clinically very important because the success rate of IVF depends on the number of mature oocytes collected, only oocytes at the MH stage being suitable for fertilization.

[0189] An observation of the oocytes by confocal microscopy after labeling of DNA and Tubulin, showed again that the structure and quality of the oocytes resulting from a treatment combining the 366VHB, H3L0 or 13VHB variants are completely normal and identical to the oocytes resulting from a standard treatment with FSH alone.

[0190] In addition, the effect of the three variants in association with FSH treatment was also characterized on the steroidogenic response of the ovary during the treatment cycle. Monitoring of estradiol and progesterone secretion was measured and compared in the four groups of females treated with FSH, FSH+366VHB, FSH+H3L0, FSH+13VHB (Figure 11). The results illustrated in Figure 11A show very significantly that females treated with the 13VHB variant had a secretion of estradiol three times higher than with the other variants or FSH alone (p<0.001, Two-way ANOVA). This higher secretion of estradiol is correlated with the stronger follicular growth observed by ultrasound and the better maturation of oocytes leading to a number of mature oocytes 3 times higher with the 13VHB variant.These results clearly indicate that the 13VHB variant enabled better follicular growth and better oocyte maturation.

[0191] The results illustrated in Figure 11 B show very significantly that progesterone secretion is also higher in monkeys having received FSH treatment combining the 13VHB (p<0.01) and 366VHB (p<0.001, Two-way ANOVA) variants. This better secretion of progesterone reflects a better quality of the corpora lutea, a hormone essential for good early embryonic development and the prevention of early embryonic loss.

[0192] In conclusion, the overall results obtained with the five best humanized variants in this first protocol in non-human primates allowed the selection of the 13VHB variant as a first-rank candidate for drug development. The H3L0 variant was selected as a second-rank candidate given the higher number of mature oocytes obtained compared to FSH treatment alone or FSH in combination with the 366VHB variant.

[0193] Effect of the 13VHB variant administered alone, without exogenous FSH, as part of a Short Antagonist Treatment protocol.

[0194] The Short Antagonist protocol is widely used in women undergoing IVF. It has been adapted and used in cynomolgus monkeys to study the effect of the 13VHB variant injected alone on the activity of endogenous FSH.

[0195] In this case, the females received no human FSH injection and were treated only with the humanized antibody alone.

[0196] For this, females aged at least 36 months were treated:

[0197] - either with a single injection of the 13VHB variant at a dose of 20 pg / kg, on the first or second day of menstruation. This dose corresponds to a concentration of around 0.3 pg / ml of blood if we assume a blood volume of 65 ml / kg (University of Pittsburgh Policy for Regulating the Volume of Experimental Blood Sample Withdrawals in Laboratory Animals);

[0198] - or with 1 injection per day of 37.5 IU of human FSH (Gonal-f®, Merck Serono, France) for 12 days.

[0199] Each experimental group included two monkeys.

[0200] Transabdominal ultrasounds of the ovaries were performed during ovarian stimulation treatment to monitor the growth of ovarian follicles.

[0201] Natural ovulation was inhibited by injection of a GnRH antagonist (Cetrotide®, Merck Serono, France) when the growing follicles reached an average diameter of 3 mm, or at the latest at n ème day of the ovarian stimulation protocol. The females then received an injection of 2600 IU of hCG (Ovitrelle®, Merck Serono, France) 36 hours after the last Gonal-f injection.

[0202] Follicular puncture by laparotomy was performed 36 hours after hCG injection, according to the procedure described above in the Long Agonist protocol. The punctured follicular fluid was collected in 14 ml round-bottom Falcon tubes (Dutscher reference 352001, Brumath, France) containing 20 μl of heparin (Choay Heparin 25000 IU / 5 ml, Cheplapharm Arzneimittel GmbH, Greifswald, Germany). Oocytes were searched for in the follicular fluid under a binocular microscope and then decoronized by hyaluronidase treatment according to the same procedure as above and then observed under a microscope to determine their maturation stage. A portion of the oocytes was fixed in 4% paraformaldehyde (PAF) solution containing 0.5% Triton and 1% BSA for 30 min at 37°C, then transferred to 1% PBS / BSA solution and stored at 4°C until analysis. The other portion was used for parthenogenesis study.

[0203] The results obtained in each experimental group (n=2 monkeys) are presented in Table 7. They indicate the average number of total oocytes and mature oocytes (Mil) obtained per female during the puncture carried out on day 15 of treatment.

[0204] Table 7: Variation in the number of total oocytes and mature oocytes retrieved in monkeys treated either with FSH or with the 13VHB variant injected alone (mean ± SEM) in a short antagonist protocol.

[0205] The results demonstrate that the ovarian response obtained following treatment with 13VHB injected in a single dose at 20 pg / kg is very close to that obtained with a treatment comprising 12 injections of FSH. Indeed, 18.5 mature oocytes were obtained on average in the 13VHB batch compared to 20 in the FSH batch. The average number of total oocytes was 43.5 in the 13VHB batch compared to 46 in the FSH batch.

[0206] These results are important because they indicate that the 13VHB variant used as monotherapy, and administered in a single injection at the beginning of the cycle, is capable of potentiating the activity of endogenous FSH and of inducing sufficient stimulation of the ovary to result in the production of several mature oocytes in a manner identical and as effective as a treatment comprising 12 daily injections of exogenous FSH.

[0207] This allows us to consider the possibility of using a humanized variant according to the invention, alone, as monotherapy, in female or male patients suffering from infertility or subfertility and having a sufficient circulating level of FSH. In this case, treatment with a humanized variant according to the invention, e.g. 13VHB, would be used as a substitute for a treatment comprising multiple injections of exogenous FSH.

[0208] Effect of the 13VHB variant administered alone, without exogenous FSH, as part of a Short Agonist Treatment protocol

[0209] The effect of the 13VHB variant administered alone on endogenous FSH was also evaluated in another protocol used in women for IVF. This is a short agonist protocol (flare-up agonist protocol) which was adapted for use in cynomolgus monkeys. In this protocol, two monkeys aged at least 36 months received an injection of a long-acting GnRH agonist (Décapeptyl LP, Ipsen Pharma, France) on the first day of menstruation. Then,

[0210] - one received a single injection of the 13VHB variant at a dose of 20 pg / kg on the day of the GnRH agonist injection; - the other received a single injection of human FSH (Gonal-f®, Merck Serono, France) at 37.5 IU per day for 9 days. The first FSH injection was given 72 hours after the GnRH agonist injection.

[0211] Treatment with the variant alone has therefore been extremely simplified since it only includes two injections on the first day of menstruation (1 injection of antibody + 1 injection of the agonist) compared to 10 injections required in treatment with FSH (1 injection of agonist + 9 injections of FSH).

[0212] The females then received an injection of 2600 IU of hCG (Ovitrelle®, Merck Serono, France) on the thirteenth day of treatment. Follicular puncture by laparotomy was performed 36 hours after the hCG injection. After opening the abdomen, the ovaries were gently brought to the surface and the follicles were punctured using a 25G needle and a 5 ml syringe, and G-MOPS PLUS culture medium (Vitrolife Reference 10130, Paris, France). The punctured follicular fluid was collected in 14 ml round-bottom Falcon tubes (Dutscher reference 352001, Brumath, France) containing 200 μl of heparin (Choay Heparin 25000 IU / 5 ml, Cheplapharm Arzneimittel GmbH, Greifswald, Germany), and transported to the laboratory in a transport case at 37°C (Biotherm INC-RB1 CRYOLOGIC: transportable incubator for tubes, Laboratoires JCD, La Mulatière, France).

[0213] Throughout the ovarian stimulation treatment, transabdominal ultrasounds of the ovaries and blood tests were performed to monitor follicle growth and the secretion of hormones such as estradiol and progesterone, respectively.

[0214] Once in the laboratory, oocytes were searched for in the follicular fluid under a binocular microscope. For this, the 14 ml Falcon tubes containing the follicular fluid were emptied into Nunc cell culture petri dishes (reference 055061, Dominique Dutscher, Brumath, France). The oocytes thus identified were decoronized by treatment with hyaluronidase (BioCare Europe reference 90101-5X1ML, Rome, Italy), then observed under a microscope to determine their stage of maturation.

[0215] The results obtained in each experimental group (n=1 monkey) are summarized in Table 8. They indicate the number of total oocytes and mature oocytes (Mil) obtained per female during the follicular puncture carried out on D15.

[0216] Table 8: Number of total oocytes and mature oocytes retrieved from monkeys treated either with FSH or with the 13VHB variant alone in a "flare-up" agonist protocol (n=1 per group). Treatment with the 13VHB variant injected once at 20 pg / kg resulted in 15 mature oocytes, which is sufficient for IVF. This result is not very different from the 20 mature oocytes obtained in the monkey that received 9 FSH injections. Only the total number of oocytes is very different between the two females.

[0217] The results obtained with the 13VHB variant alone in this second type of protocol also indicate that it is capable of potentiating the activity of endogenous FSH and inducing stimulation of the ovary leading to a significant production of mature oocytes, sufficient to consider IVF.

[0218] They again confirm the possibility of considering the use of a humanized variant according to the invention, alone, in monotherapy, eg 13VHB, in women and in men with sufficient circulating levels of FSH.

[0219] All mature oocytes obtained in the two protocols testing the effect of the 13VHB antibody alone were then used in a parthenogenesis study.

[0220] Parthenogenesis study on mature oocytes punctured from monkeys treated with FSH alone or with the 13VHB variant alone.

[0221] The ability of mature oocytes to divide by parthenogenesis was assessed with stage Mil oocytes retrieved from monkeys treated either with FSH alone or with 13VHB alone as monotherapy, in the case of an antagonist protocol or a short agonist "flare-up" protocol.

[0222] For this, the oocytes were incubated for 2 minutes in a 5 μM ionomycin solution (Merck, France), followed by a 5-minute wash in G-MOPS medium (Vitrolife Reference 10130, Paris, France). The oocytes were then incubated for 4 hours in a 2 mM 6-Dimethylaminopurine (6-DMAP) solution (Merck, France), followed by a 5-minute wash in G-TL medium (Vitrolife Reference 10145, Paris, France). Finally, the oocytes were cultured for 5 days in a 50 μl drop of G-TL medium covered with 120 μl of mineral oil, in an incubator at 37°C 6% CO2 5% O2 89% N2. At the end of the 5 days of culture, the oocytes were analyzed by optical microscopy in order to observe the presence or absence of cell divisions.

[0223] The results obtained after 5 days of culture after parthenogenesis induction treatment are shown in Table 9.

[0224] Table 9: Percentage of mature oocytes divided by parthenogenesis after 5 days of culture, in the case of monkeys treated with FSH or with 13VHB alone. The results show that most of the treated oocytes exhibit parthenogenesis patterns: 89% divided in the case of monkeys treated with 13VHB and 90% in the case of monkeys treated with FSH. Only 11% and 10% of the oocytes in the two respective treatments did not divide.

[0225] There is therefore no difference between FSH and 13VHB treatment: in both cases, the mature oocytes collected have the same ability to divide by parthenogenesis, demonstrating the same oocyte quality with 13VHB treatment alone compared to standard treatment with FSH alone.

[0226] EXAMPLE 6: IN VIVO POTENTIALIZING EFFECT OF THE HUMANIZED ANTIBODY 13VHB-139VL (SEQ ID NO: 9 and SEQ ID NO: 16) ON SPERMATOGENESIS IN ADULT MALE RATS

[0227] The potentiating effect of the humanized antibody 13VHB-139VL (SEQ ID NO: 9 and SEQ ID NO: 16) was studied in males to assess its impact on spermatogenesis controlled by FSH and LH. FSH is involved in the early stages of spermatogenesis and in the stimulation of Sertoli cells, the nurse cells of germ cells essential for their maturation. LH is involved in the development of the later stages of spermatogenesis differentiation (spermatids and spermatozoa) and in the stimulation of Leydig cells secreting testosterone.

[0228] The potentiating effect of 13VHB antibody on spermatogenesis was evaluated in vivo in an experimental model of adult male rats pretreated with a GnRH inhibitor to induce hypogonadotropic hypogonadism causing total inhibition of spermatogenesis.

[0229] For this, 12-week-old Wistar Han rats (Charles River, L'ArbresIe, France) were pretreated with a GnRH antagonist (Firmagon® LP, Ferring, France), at a rate of one injection of 2 mg / kg on the first day of treatment and then a second injection 6 weeks later.

[0230] Hormonal treatment with or without 13VHB variant began one week after the second Firmagon injection, at which point spermatogenesis was completely inhibited. The animals were treated for eight weeks, a duration corresponding to a spermatogenesis cycle in rats (54 to 56 days). Animals treated with hormones alone received either a mixture of hCG (Ovitrelle®, Merck) at 2.5 IU / kg injected 5 times per week and hMG (Menopur®, Ferring) at a dose of 25 IU / kg injected 3 times per week (Hormone X1 batch), or the same mixture with a double dose of hormones (hCG 5 IU / kg injected 5 times per week and Menopur® 50 IU / kg injected 3 times per week) (Hormone X2 batch). Menopur® is an hMG (human Menoposal Gonadotropin) composed of an FSH+LH mixture in equivalent quantities (50% each).

[0231] In a third batch, the animals were treated with the mixture of hormone X1 and the 13VHB variant (batch HormoneX1+13VHB). The variant was injected at a dose of 20 pg / kg 3 times per week. This dose corresponds to a concentration of around 0.3 pg / ml of blood if we assume a blood volume of 65 ml / kg in rats (University of Pittsburgh Policy for Regulating the Volume of Experimental Blood Sample Withdrawals in Laboratory Animals). The 13VHB variant was formulated in sterile PBS.

[0232] A control group, treated only with the antagonist, received only physiological serum, without any hormonal treatment.

[0233] The study therefore involved six experimental groups, each comprising four animals: a control group (WT) not pretreated with Firmagon and not stimulated; an “inhibition control” group treated with Firmagon only; a group treated with Firmagon then physiological serum (saline); a group treated with Firmagon then with HormonesXI; a group treated with Firmagon then with HormonesXI + 13VHB; a group treated with Firmagon then with HormonesX2.

[0234] At the end of treatment, the animals were sacrificed and blood was collected intracardially into heparinized tubes and centrifuged. The testes and epididymides were removed and weighed.

[0235] The testes were then ground in Ham's F12 medium supplemented with 1% sodium pyruvate and 1% Hepes, and sonicated. The number of spermatid heads was then counted under a light microscope to assess sperm reserve. One testis from each experimental group was fixed in 10% formalin solution and embedded in paraffin for histological analysis. Thin sections of 5 μm thickness were cut along the transverse axis of the testis and stained with periodic acid-Schiff (PAS) and hematoxylin-eosin (H&E). Examination of the stained slides was performed using NDP Viewer software. Testicular sections were evaluated according to the OECD guidelines for histopathological evaluation (Part 2: Male Reproductive System).

[0236] The epididymides were cut into thin sections and incubated at room temperature for at least 4 hours in Ham's F12 medium supplemented with 1% sodium pyruvate and 1% Hepes. The preparations were then collected, filtered, and sperm were counted under light microscopy and by flow cytometry.

[0237] Potentiating effect of the 13VHB variant on the resumption of spermatopenesis.

[0238] The potentiating effect of the 13VHB variant was evaluated after 8 weeks of treatment, by comparing the responses of animals receiving hormonal treatment alone with those of males receiving hormonal treatment in combination with the antibody.

[0239] The testicular response was evaluated according to three criteria: the weight of the testes which is correlated with the intensity of spermatogenesis; the sperm reserve defined by the number of spermatids in the testes; the number of spermatozoa collected in the epididymides. The results obtained at the end of the 8 weeks of treatment are illustrated in Figure 12. They show first of all that the inhibition induced by the pretreatment with Firmagon was total with a residual testicular weight reduced by 8 times compared to the control group (WT) (Figure 12A), a total absence of spermatids (Figure 12B) and a total absence of epididymal spermatozoa (Figure 12C). The animals are totally azoospermic. The same is true for the group treated with Firmagon + saline whose spermatogenesis remained totally inhibited 8 weeks after the start of the stimulation treatments.

[0240] The effect of the different treatments on testicular weight is illustrated in Figure 12A. It is observed that animals receiving Hormones X1+13VHB variant treatment have a testicular weight 1.8 times higher than those receiving Hormones X1 treatment alone (3.13 mg / g body weight versus 1.83 respectively). Conversely, doubling the doses of hormones (Hormones X2) did not significantly improve the increase in testicular weight (2.19 mg / g body weight versus 1.83). The 13VHB antibody therefore allowed a much greater increase in testicular weight compared to hormonal treatment alone even with a double dose of hormones.

[0241] The effect on sperm reserve is illustrated in Figure 12B. The results show that the number of spermatids is 6, 3.10 7 with hormone treatment X1 and 3.4.10 7with Hormones X2. Again, doubling the dose of hormones did not improve the resumption of spermatogenesis compared to Hormones X1. Conversely, animals treated with the 13VHB variant in combination with Hormones X1 had a spermatid count 1.6 times higher than those in the Hormones X1 group alone (9.73.10 7 spermatids / g of testes versus 6.39.10 7 respectively) and 3 times higher compared to the Hormones X2 batch (9.73.10 7 spermatids / g of testes versus 3.35.10 7 ). Moreover, in a single spermatogenesis cycle, the effect of 13VHB allowed a complete restoration of spermatogenesis with a number of spermatids returned to a normal level, equal to that of the WT control batch without Firmagon treatment (9.7.10 7 and 8.1, 10 7spermatids / g of testes respectively). This demonstrates that the treatment combining the antibody was more effective and was the only one to allow a complete resumption of spermatogenesis in a single spermatogenesis cycle.

[0242] The results concerning the number of epididymal spermatozoa obtained at the end of the 8 weeks of the different treatments are illustrated in Figure 12C. It is observed that the number of epididymal spermatozoa is twice as high in the 13VHB+Hormones X1 batch compared to the Hormones X1 batch: 1.15.10 7 and 2.16.10 7 spermatozoa per 0.1 g of epididymis respectively. From 8 weeks (a single cycle of spermatogenesis) the treatment combining 13VHB + Hormones X1 therefore allowed a return to a normal level of epididymal spermatozoa identical to that of untreated control animals (WT): 2, 15.10 7 versus 1,8.10 7sperm per 0.1 g of epididymis respectively. Conversely, doubling the doses of hormones (HormonesX2) had no improving effect on the increase in sperm count compared to Hormones X1 treatment, demonstrating that treatment combining the 13VHB variant provides greater efficacy than standard hormonal treatment, even at double dose.

[0243] Figure 12D is a dot plot representation where each point represents the number of sperm counted per animal in each group. It is observed that all animals in the group associating the 13VHB variant responded to the treatment and all showed a complete recovery of spermatogenesis with a median value equal to that of the WT control group not pretreated with Firmagon. Conversely, groups treated with hormones X1 or X2 alone showed a high individual variability in response with a median value much lower than that of the group associating 13VHB.

[0244] In conclusion, the three physiological parameters analyzed demonstrate that the potentiating effect of the 13VHB variant associated with hormonal treatment (Hormone X1) allowed a much more intense resumption and stimulation of spermatogenesis than hormonal treatment alone, resulting in a return to a normal level of spermatogenesis after eight weeks of treatment, i.e., a single spermatogenesis cycle. In azoospermic animals, in one spermatogenesis cycle, it allowed the return to a normal level of spermatid and sperm count. This new treatment provides greater efficacy and would therefore allow a shorter treatment time in humans.

[0245] This study was supplemented by a histological analysis of testicular sections from each of the experimental batches. Three representative examples are illustrated in Figure 13.

[0246] The testis from the Firmagon control batch (Figure 13A) is markedly atrophic with small seminiferous tubules and reduced and disorganized interstitial tissue. The seminiferous tubules are poor in germ cells and devoid of any elongated spermatids, reflecting totally defective spermatogenesis.

[0247] The histology of the testes from the batch treated with hormones X1 (Figure 13B) and the batch treated with hormones X1 + 13VHB (Figure 13C) shows in both cases a denser structure. It is noted that the size of the seminiferous tubules is greater (diameter and surface area) in the testis of the batch hormones X1 + 13VHB attesting to a more intense spermatogenesis compared to the batch hormones alone X1. This is correlated with the obtaining of a higher average testes weight in the batch hormones X1 + 13VHB. Similarly, the presence of early and late stages of spermatogenesis in normal proportion was observed in both testes but the abundance of elongated spermatids is higher in the testis of the batch "hormones + 13VHB" reflecting again a stronger resumption of spermatogenesis.

[0248] The study of histological sections of the testes was completed by an image analysis in order to measure the average diameter of the seminiferous tubules (in pm) and the average area of ​​the seminiferous tubules (in pm 2 ) in each group. On each histological section, 100 circular sections of seminiferous tubules were thus analyzed. The results are illustrated in Table 10 below. Table 10: Mean values ​​of the diameters and areas of the seminiferous tubule sections according to the treatments (mean ± standard deviation). Comparison with

[0249] Hormones X1: ***p<0.001, ****p<0.0001 (Kruskal-Wallis test

[0250] A significantly higher mean diameter of the seminiferous tubules was observed in the [hormones X1 + 13VHB] group compared to the [hormones X1] group (***, p<0.001). Similarly, the mean area of ​​the tubules was significantly higher in the [hormones X1 + 13VHB] group compared to the [hormones X1] group (p<0.001). The control groups [treated with Firmagon] and [+saline] were very significantly lower than the groups treated with hormones alone or in association with 13VHB (****, p<0.0001).

[0251] All these results demonstrate that hormonal treatment in combination with 13VHB is significantly more effective than treatment with hormones alone, with significantly greater resumption and stimulation of spermatogenesis. After 8 weeks of treatment with X1 hormones + 13VHB (a single spermatogenesis cycle), the level of sperm production became identical to that of control WT animals not treated with Firmagon. In humans, this perspective would make it possible to significantly shorten the treatment times required to restart and stimulate spermatogenesis in an oligozoospermic individual, for example.

[0252] EXAMPLE 7: IN VIVO POTENTIALIZING EFFECT OF THE HUMANIZED ANTIBODY 13VHB-139VL (SEQ ID NO: 9 and SEQ ID NO: 16) ON SPERMATOGENESIS IN THE ADULT MALE hpg HYPOGONADICAL MOUSE

[0253] The effect of the 13VHB antibody on the stimulation of spermatogenesis was also evaluated in a model of congenital hypogonadotropic hypogonadism in adult male hpg mice. These mice carry a mutation (hpg) in the GnRH gene resulting in an absence of secretion of endogenous gonadotropic hormones (LH and FSH)

[0020] . In the homozygous state for the hpg mutation (hpg Males lack any spermatogenesis. They therefore constitute a "natural" model of hypogonadotropic hypogonadism and azoospermia in males.

[0254] These mice were obtained following the revitalization of embryos from the Jackson laboratory (The Jackson Laboratory, USA) and the line was maintained by Charles River laboratories (France). For each experiment, adult homozygous male mice, 8 weeks old, received hormonal treatment, alone or in combination with the 13VHB variant, for at least 7 weeks. This duration corresponds to a spermatogenesis cycle which is 35 days in mice. For the groups of animals treated with the 13VHB variant, the antibody was injected at a dose of 20 pg / kg two or three times per week. This dose corresponds to a concentration of around 0.26 pg of antibody per ml of blood if a blood volume of 75 ml / kg is assumed in mice (University of Pittsburgh Policy for Regulating the Volume of Experimental Blood Sample Withdrawals in Laboratory Animals). The 13VHB antibody was prepared in sterile PBS.

[0255] At the end of each treatment, the animals were sacrificed and blood was collected intracardially into heparinized tubes and centrifuged. The plasmas were then frozen at -20°C. The testes and epididymides were removed and weighed.

[0256] The testes were then ground in Ham's F12 medium supplemented with 1% sodium pyruvate and 1% Hepes, and sonicated. The number of spermatid heads was then counted under a light microscope to assess sperm reserve. One testis from each experimental group was fixed in 10% formalin solution and embedded in paraffin for histological analysis. Thin sections of 5 μm thickness were cut along the transverse axis of the testis and stained with periodic acid-Schiff (PAS) and hematoxylin-eosin (H&E). Examination of the stained slides was performed using NDP Viewer software. Testicular sections were evaluated according to the OECD guidelines for histopathological evaluation (Part 2: Male reproductive system).At the same time, an analysis of the testicular preparations by flow cytometry was carried out in order to measure the number of haploid (Spermatids), diploid (Spermatogonia, Spermatocytes II) and tetraploid (Spermatocytes I) cells and thus monitor the progress of the resumption of meiosis according to the treatments received by the animals.

[0257] The epididymides were cut into thin sections and incubated for at least 4 hours at room temperature in Ham's F12 medium supplemented with 1% sodium pyruvate and 1% Hepes. The preparations were then collected, filtered, and the spermatozoa were counted under a light microscope.

[0258] Three different hormonal treatment protocols were used in these trials. These protocols are used in human medicine for men suffering from hormonal infertility. This is the case, for example, for azoospermic, oligoasthenoteratospermic (OATS) or oligozoospermic patients.

[0259] Effect of the 13VHB variant in a stimulation protocol using FSH and hCG.

[0260] Two batches of male hpg mice were treated for seven weeks:

[0261] 6 mice received a mixture of FSH 1 Ul (Gonal®, Merck) + hCG 1 Ul (Ovitrelle®, Merck) injected 5 times per week; 7 mice received a mixture of FSH 1 Ul + hCG 1 Ul injected 5 times per week and the 13VHB variant injected at 20 pg / kg 3 times per week.

[0262] After 7 weeks of treatment, the animals were sacrificed, the epididymides were removed and the sperm preparations were analyzed. The results are expressed as the total number of sperm (millions) per animal. They are illustrated in Figure 14 and allow comparison of the resumption of spermatogenesis in the two treatments. In the untreated control homozygotes, no epididymal spermatozoa were observed, attesting that these males were totally azoospermic. In the homozygotes treated with the FSH + hCG mixture in association with the 13VHB variant, the average number of spermatozoa per animal was 1.4 times higher than that obtained in the males treated with the FSH + hCG mixture alone: ​​8.5.10 6 ± 0.5.10 6 spermatozoa against 6.2.10 6 ± 0.5.10 5 respectively. Although not significant, this trend reflects a greater recovery of spermatogenesis with treatment combining the 13VHB variant.

[0263] Effect of the 13VHB variant in a stimulation protocol using hMG.

[0264] The effect of the 13VHB variant was evaluated in combination with hormonal treatment using hMG (Menopur®, Ferring) at a dose of 25 IU / kg. hMG consists of an equimolar mixture of FSH and LH.

[0265] Two batches of male hpg mice were treated for seven weeks:

[0266] 8 mice received 25 IU / kg Menopur (hMG) injected 5 days per week;

[0267] 9 mice received 25 IU / kg Menopur (hMG) 5 days per week and the 13VHB variant injected at 20 pg / kg 2 days per week. The 13VHB variant was prepared in sterile PBS.

[0268] After 7 weeks of treatment, the animals were sacrificed, the epididymides were removed and the sperm preparations were analyzed. The results shown in Figure 15 show that the mean value of the sperm count in males treated with hMG in combination with the 13VHB variant was 3.5 times higher than that obtained in males treated with the hMG mixture alone: ​​0.35.10 6 ± 0.18.10 6 sperm per animal versus 0.1.10 6 ± 0.05.10 6 respectively. Although not significant, this trend again reflects a greater recovery of spermatogenesis with hMG treatment associating the 13VHB variant.

[0269] Effect of the 13VHB variant in a stimulation protocol combining treatment with FSH followed by treatment with hMG.

[0270] The effect of the 13VHB variant was evaluated in association with a third type of protocol comprising:

[0271] 7 weeks of treatment with 1 IU FSH (Gonal-f®, Merck) injected 5 days per week; followed by 7 weeks of treatment with 25 IU / kg hMG (Menopur®, Ferring) injected 5 days per week.

[0272] Two batches of male hpg mice were treated: one group of 8 mice received hormonal treatment alone; one group of 9 mice received hormonal treatment in association with the 13VHB variant injected at a dose of 20 pg / kg 2 days per week. The 13VHB variant was prepared in sterile PBS.

[0273] After 14 weeks of treatment, the animals were sacrificed, the testes and epididymides were removed, weighed, and the spermatid and spermatozoa preparations analyzed.

[0274] The results obtained are illustrated in Figure 16. They show that animals treated with the 13VHB variant associated with hormonal treatment have a higher testicular weight (Figure 16A), a number of spermatids in the testis (Figure 16B) and a number of epididymal spermatozoa (Figure 16C) than in animals treated with hormones alone.

[0275] Testicular weight was 1.45 times higher in animals receiving hormone therapy in combination with the 13VHB variant compared to animals receiving hormone therapy alone: ​​0.42 mg / g body weight versus 0.29 mg / g body weight, respectively. This difference was not significant (Figure 16A).

[0276] It was found that the batch that received hormonal treatment in association with the 13VH variant had an average number of spermatids 3.5 times higher than that of the batch that received hormonal treatment alone: ​​67.10 s ± 13.10 6spermatids / g of testicle versus 19.10 s ± 6, 1.10 6 spermatids / g of testicle respectively (Figure 16B). This difference is significant (*: p<0.05, Mann Whitney test).

[0277] Similarly, the batch that received hormonal treatment in association with the 13VH variant had an average number of epididymal spermatozoa 6 times higher than that of the batch that received hormonal treatment alone: ​​3, 1.10 s ± 1,14.10 s spermatozoa / 0.1 g of epididymis vs. 0.55.10 s ± 0.4.10 s respectively (Figure 16C). This difference is significant (*: p<0.05, Mann Whitney test).

[0278] Histological analysis of the testes

[0279] A histological analysis of testicular sections from each of the experimental batches was performed and showed a very atrophic structure of the testicle from a homozygous male with empty, small seminiferous tubules and reduced and destructured interstitial tissue. The histology of the testicles from the batch treated with hormones alone and hormones + 13VHB showed in both cases a denser structure with a larger size of the seminiferous tubules (diameter and surface area) in the testicle from the hormone + 13VHB batch.

[0280] Specifically, the mean diameter of the seminiferous tubules (pm) and the mean area of ​​the seminiferous tubules (pm 2 ) were measured by image analysis on 50 circular sections of seminiferous tubules from each histological section. The results are illustrated in Table 11a.

[0281] Table 11a: Mean values ​​of diameters and areas of seminiferous tubule sections according to treatments (mean ± standard deviation). Comparison with hormone treatment: **p<0.01, *p<0.05 (Kruskal-Wallis test).

[0282] A significantly higher mean diameter of the seminiferous tubules was observed in the [hormones + 13VHB] group by a factor of 1.2 compared to the [hormones] group (*, p<0.05). Similarly, the mean area of ​​the tubes was significantly higher in the [hormones + 13VHB] group by a factor of 1.4 compared to the [hormones] group (**, p<0.01). The treatment combining 13VHB therefore increased the development of the seminiferous tubules with a significantly higher mean diameter and mean area, attesting to a significantly greater recovery and stimulation of spermatogenesis compared to hormonal treatment alone.

[0283] Analysis of testicular cell populations by flow cytometry

[0284] An analysis of the different testicular cell populations was performed by flow cytometry. For this, a volume of 20 μl of cell suspension from the homogenate of each testicle was analyzed by flow cytometry after filtration on a 100 μm grid and labeling of the cell DNA with Hoechst 33342. The cells to be counted were selected by determining the area of ​​blue fluorescence emitted by Hoechst 33342 on the biparametric combining FSC and SSC. From this preliminary selection, a new biparametric histogram combining green autofluorescence and blue fluorescence of Hoescht 33342 is then used, on which are defined a cell population with 1C DNA (Spermatids, Spermatozoa), a cell population with 2C DNA (Spermatogonia, Spermatocytes II, somatic cells) and a cell population with 4C DNA (Spermatocytes I).

[0285] Representative individual results from each batch are shown in Figure 17. Figure 17A illustrates the case of a male that did not respond to treatment. Figure 17B illustrates the case of a male that responded to hormone treatment alone and Figure 17C illustrates the case of a male that responded to Hormones+13VHB treatment. It can be seen that the number of 4C and 2C events (cells with 4 and 2 chromatids) are close between the two treatments. Conversely, the number of haploid cells (1C events) in the animal treated with Hormones + 13VHB is 3.7 times higher than that treated with Hormones alone: ​​54,016 events versus 14,546 respectively. This result reflects the establishment of more intense spermatogenesis with a higher production of spermatids and spermatozoa in the animal treated with hormones + 13VHB.

[0286] The mean values ​​obtained with all the animals in the group treated with hormones alone and those in the group treated with hormones + 13VHB are illustrated in the following table 11b.

[0287] Table 11b

[0288] The results show that after hormonal treatment combining 13VHB, the average number of haploid cells (spermatids) increased by 12% compared to the group treated with hormones alone. Conversely, the average number of 4C cells (spermatocytes I) and 2C cells is numerically and proportionally slightly lower with hormonal treatment combining 13VHB: -9% and -3% respectively. This demonstrates that a more intense spermatogenesis was established in males having received 13VHB, resulting in a higher number of spermatids formed (1C), after the same duration of treatment. This greater resumption of spermatogenesis also results in a lower number of 4C cells, reflecting a more effective lifting of the blockage of spermatogenesis at the Spermatocytes 1 stage, characteristic of homozygous hpg (- / -) males.

[0289] The impact of these preclinical results is very important because they very clearly demonstrate a significant potentiating effect of a humanized variant antibody according to the invention, i.e. 13VHB, allowing a faster restoration, and a more intense and more effective stimulation of spermatogenesis in males with congenital azoospermia compared to standard hormonal treatment alone.

[0290] EXAMPLE 8 CONSTRUCTION, PRODUCTION AND FUNCTIONAL CHARACTERIZATION OF A MONOVALENT FRAGMENT OF THE 13VHB-139VL VARIANT (SEQ ID NO: 18)

[0291] A monovalent fragment was developed from the humanized 13VHB-139VL variant in scFv format. Its in vitro binding and potentiating properties of FSH and LH / CG were evaluated as described for variant selection, and compared to the full-length lgG4 variant.

[0292] Construction of the 13VHB variant scFv fragment The synthetic gene encoding the 13VHB scFv fragment derived from the 13VHB-139VL variant was synthesized by ATG:Biosynthetics GmbH (Germany). The 13VHB scFv was formed by fusing the CF12-13-VHB heavy chain sequence (SEQ ID NO: 9) and the CF12-VL139 light chain sequence (SEQ ID NO: 16) via a sequence encoding a linker [GGGGS^ (SEQ ID NO: 22) and the 3' addition of a sequence encoding 6x Histidine. The synthetic gene was designed by fusing the sequence starting at the HindIII enzyme site and the end of the sequence encoding the cytoplasmic targeting signal peptide PelB described in Ward et al.

[0021] with the 13VHB scFv gene terminated by an XhoI enzyme site sequence. The sequence described by Ward et al.

[0021] was modified by substituting thymine (T) at position 23 with a cytosine (C).The synthetic gene was inserted into the plasmid pUC19-PelB constructed as described by Ward et al.

[0021] between the HindIII and XhoI enzyme sites. After sequencing the quality of the construct, the plasmid pUC19-scFv 13VHB was transformed by heat shock into HB2151 bacteria (T53040, Interchim, France) made competent

[0022] .

[0293] Table 12: Peptide sequence of scFv 13VHB

[0294] Production of the recombinant antibody fragment

[0295] 1 / Bacterial culture

[0296] A pre-culture was carried out in 5 ml of 2xYT medium containing 100 pg / ml ampicillin overnight at 37°C 200 RPM. The next day, 500 μl of this pre-culture was inoculated into 500 ml of 2xYT medium containing 50 pg / ml ampicillin and grown at 37°C at 180 RPM until an OD600m of 1.5 was obtained. Synthesis of scFv was induced by the addition of 0.1 mM IPTG for 16 h at 16°C and 150 RPM.

[0297] 21 Extraction

[0298] The bacterial suspension was centrifuged for 30 min at 4000 g at °C. The rest of the preparation was carried out at °C. To extract the bacterial periplasm, the pellet was resuspended and incubated in 10 ml of TES buffer (0.2 M Tris pH 8, 0.5 mM EDTA, 0.5 M sucrose) for 30 min to which 15 ml of TES buffer diluted 14-fold was then added for a further 30 min. The bacterial extract was then centrifuged for 45 min at 10,000 g. The supernatant was dialyzed against 20 mM sodium phosphate buffer, 300 mM NaCl pH 7.4 overnight. The dialyzed supernatant was then processed immediately to purify the scFv or stored at -20 °C until use.

[0299] 3 / Purification

[0300] The periplasm was centrifuged for 20 min at 4000 g at 4°C. A concentrated imidazole solution was added to the supernatant to adjust the concentration to 10 mM. The supernatant was then incubated with Ni-NTA HisPur™ resin (88221, ThermoFisher Scientific, MA, USA) with shaking for 1 h at 4°C. The resin was then washed with 20 mM sodium phosphate buffer, 300 mM NaCl pH 7.4 supplemented with 20 mM imidazole until an OD2sonm was obtained close to 0. The 13VHB scFv was then eluted with 20 mM sodium phosphate buffer, 300 mM NaCl, 250 mM imidazole pH 7.4. The eluate was dialyzed against 10 mM potassium phosphate buffer, 150 mM NaCl pH 7.4 and stored at -80°C.

[0301] 4 / Quality control

[0302] To verify its purity, the purified fraction of the 13VHB scFv was analyzed by 15% polyacrylamide gel electrophoresis after Coomassie blue staining and by Western blotting with an anti-His tag antibody (R931-25, ThermoFisher Scientific, MA, USA). The results showed the presence of a single homogeneous band reflecting the presence of a single molecular form of the product, without any degraded form.

[0303] Binding activity of scFv 13VHB on h FSH and LH / hCG

[0304] The binding activity of scFv 13VHB on recombinant human hormones FSH (Gonal-f ®, Merck) and hCG (Ovitrelle ®, Merck) as well as on extracted hMG (Menopur ®, Ferring) was studied by ELISA technique. Each hormone evaluated was prepared at a concentration of 10 pg / ml in PBS buffer and distributed at 100 μl per well on an Immulon 2HB ELISA plate (Ref. 3455 Thermo Fisher, USA). The adsorption time was 18 hours at +4°C. After five washes, the wells were treated with 100 μl of PBS supplemented with 1% BSA for 1 hour at 37°C, then the scFv 13VHB prepared at different concentrations was distributed at 100 μl / well and incubated for 2 hours at 37°C. The concentration range of scFv prepared in PBS supplemented with 0.2% BSA was from 3.5 to 600 pg / ml.

[0305] After five washes, a secondary anti-His Tag antibody coupled to peroxidase (H RP) (Ref. R93125 Life technologies, France) diluted to 1 Z1000 èmein a 0.2% PBS-BSA buffer was distributed at a rate of 100 µl / well and incubated for 1 hour at 37°C. After five washes, the enzymatic activity was revealed with TMB (Ref 5120-0074 SeraCare Life Sciences, USA) distributed at a rate of 100 µl / well and incubated for 10 minutes at room temperature. After addition of 1 M H2SO4 (50 µl / well) the intensity of the colored reaction (Optical Density) was measured using an ELISA plate spectrophotometer. The results demonstrated a binding activity of the scFv on the three hormones. Figure 18 represents the binding curves expressed as a percentage compared to the 100% given by the signal obtained with the scFv at 600 pg / ml on FSH. The basal signal was set at 0%. Binding of scFV 13VHB was observed on all three hormones. Compared to FSH (100%), the maximum binding obtained on hMG was 47.7% and the maximum binding on hCG was 21.2%.The level of binding is different depending on the hormones, attesting to a binding specificity of the scFv similar to that of the 13VHB lgG4 variant, namely a better affinity on hFSH then in decreasing order on hMG and hCG.

[0306] Potentiating effect of scFv 13VHB in vitro on FSH bioactivity

[0307] The potentiating effect of scFv 13VHB on FSH activity was evaluated in vitro on HEK 293 cells (HiTSeeker HEK293 cells) expressing the human FSH receptor (Innovative Technologies in Biological Systems, reference P30117, Derio Bizkaia, Spain).

[0308] Cells were distributed at 80,000 cells per well in 96-well dishes with white borders and backgrounds (Greiner Bio One reference 655083, Dominique Dutscher, Brumath, France), in DMEM medium (Sigma-Aldrich reference D6429, Merck Millipore, Saint Quentin Fallavier, France) supplemented with 10% fetal bovine serum (GIBCO, reference A47668-01, Thermofisher, USA) previously decomplemented, 10 pg / ml puromycin (Invivogen reference ant-pr, Toulouse, France) and incubated overnight in a 37°C 5°C CO2 incubator under a humid atmosphere. The next day, the cell medium was aspirated and replaced with 30 μl of phosphate buffered saline (PBS) containing 1% hepes (Lonza reference BE17-737E). After 2 hours of incubation at 37°C, under a humid atmosphere and 5% CO2, the cells were stimulated for 1.5 hours with 15 μl of a range of concentrations of recombinant FSH (Gonal-f®, Merck) ranging from 10 -12 M to 5.10 -9M or extracted hMG (Menopur®, Ferring) ranging from 10 -11 M to 5.10 -8 M, prepared alone or in combination with the scFv or the full-length variant at a fixed concentration of 100 pg / ml (3.5 pM). The hormone + scFv or variant mixture was preincubated for 20 minutes at 37°C before deposition. After stimulation, cAMP was measured using a HitHunter® cAMP Assay for Biologicals kit (Eurofins Discoverx Products LLC reference 90-0075LM25, Fremont, USA). The emitted luminescence was quantified using a LUMIstar Omega 96-well plate reader (BMG Labtech, Champigny sur Marne, France). EC50 values ​​were calculated by Prism software (GraphPAd Prism Software Inc., San Diego, CA, USA, version 10.0.1).

[0309] The results obtained with Gonal-f are illustrated in Table 11. They show a potentiating effect of scFv 13VHB resulting in an increase in potency relative to the cAMP response compared to that obtained with FSH alone.

[0310] Table 11: EC50 values ​​of the dose response curves obtained after stimulation with Gonal-f, Gonal-f + scFv 13VHB and Gonal-f + whole 13VHB variant.

[0311] It is observed that TEC50 obtained with the FSH + scFv 13VHB complex is increased by 2.6 times compared to TEC50 of FSH alone. Comparatively, TEC50 is increased by a factor of 4 with the FSH + whole 13VHB variant complex.

[0312] The results obtained with Menopur® showed a potentiating effect of the scFv identical to that of the entire 13VHB variant as illustrated in Table 12.

[0313] Table 12: EC50 values ​​of the dose response curves obtained after stimulation with Menopur, Menopur + scFv 13VHB and Menopur + 13VHB variant.

[0314] The hMG + scFv 13VHB complex induces an increase in the power of the AM Pc response identical to that of the hMG + whole variant complex: the TEC50 value with the FSH + scFv mixture is increased by 2.7 times compared to the EC50 of hMG alone, and by 2.8 times with the whole 13VHB variant.

[0315] In conclusion, the monovalent scFv 13VHB fragment exerts a potentiating effect on FSH activity in vitro. This effect is very close to that of the whole 13VHB variant in the case of Gonal-f and identical to that of the whole 13VHB variant in the case of Menopur®.

[0316] To complete this study, a dose response was performed by stimulating the cells with an increasing range of the ligand (13VHB whole variant or its scFv) associated with a fixed dose of FSH hormone (Gonal-f, Merck) or hMG (Menopur®, Ferring).

[0317] For this, the same experimental protocol was used. The cells were stimulated for 1 h 30 min with 15 μl of a range of scFv concentrations ranging from 5.10' 9 M to 10' 5 M either with a range of concentrations of the entire 13VH variant ranging from 5.10' 10 M to 10' 6 M. In both cases, each concentration point of scFv or whole antibody was prepared in combination with FSH (Gonal-f) at a fixed concentration of 50 pM or with hMG (Menopur) at a fixed concentration of 1 nM. The scFv or whole antibody + hormone mixture was pre-incubated for 20 minutes at 37°C before deposition on the cells.

[0318] The scFv or whole antibody dose response (D / R) curves are shown in Figure 19. Figure 19A shows that the curves obtained with 50 pM FSH (Gonal-f) are relatively similar: in both cases, TEC50 is of the order of 10' 8 M with a factor of 1.6 in favor of the whole antibody. The Hill slope is 1.64 times steeper with the scFv than the whole antibody. Figure 19B also shows that the curves obtained with 1 nM hMG (Menopur) are relatively similar between the scFv and the whole antibody: TEC50 is of the order of 10' 8 M with a 1.7 times higher power in the case of 13VHB IgG. The Hill slope is also 1.57 times higher in the case of scFv. It should be noted that the TEC50 and Hill slope values ​​are very reproducible from one hormone to another: for example, TEC50 of scFv is 9.42.10' 8 M with FSH and 9.8.10' 8 M with hMG. Similarly the EC50 of the whole variant is 5.88.10' 8M with FSH and 5.77.10' 8 M with hMG. The Hill slope value obtained with scFv is 1.6 times higher on average in the case of both hormones.

[0319] These results demonstrate that the potentiating effect on FSH activity is not dependent on the bivalence of the 13VHB antibody, the monovalent scFV fragment also exerting the same effect.

[0320] Potentiating effect of scFv 13VHB in vitro on LH / CG bioactivity

[0321] The potentiating effect of scFv 13VHB on LH / CG activity was evaluated in vitro on HEK 293 cells expressing the human LH receptor (Innovative Technologies in Biological Systems, reference P30177, Derio Bizkaia, Spain).

[0322] Cells were distributed at 80,000 cells per well in 96-well dishes with white borders and backgrounds (Greiner Bio One reference 655083, Dominique Dutscher, Brumath, France), in DMEM medium (Sigma-Aldrich reference D6429, Merck Millipore, Saint Quentin Fallavier, France) supplemented with 10% fetal bovine serum (GIBCO, reference A47668-01, Thermofisher, USA) previously decomplemented, 10 pg / ml puromycin (Invivogen reference ant-pr, Toulouse, France) and incubated overnight in a 37°C 5% CO2 incubator under a humid atmosphere. The next day, the cell medium was aspirated and replaced with 30 μl of phosphate buffered saline (PBS) containing 1% hepes (Lonza reference BE17-737E). After 2 hours of incubation at 37°C, under a humid atmosphere and 5% CO2, the cells were stimulated for 1.5 hours with 15 μl of a range of hCG concentrations (Ovitrelle®, Merck) ranging from 10' 11 M at 5.10' 9M, prepared alone or in combination with scFv at a fixed concentration of 100 pg / ml (3.5 pM). The hCG + scFv mixture was preincubated for 20 minutes at 37°C before deposition.

[0323] After stimulation, cAMP was measured using a HitHunter® cAMP Assay for Biologicals kit (Eurofins Discoverx Products LLC reference 90-0075LM25, Fremont, USA). The emitted luminescence was quantified using a LUMIstar Omega 96-well plate reader (BMG Labtech, Champigny sur Marne, France).

[0324] The potentiating effect was evaluated by comparing the dose-response curves obtained with hCG alone and with the hCG + scFv 13VHB complex. The results are illustrated in Figure 20 and show a potentiating effect of scFv on hCG activity with a better power of the response in AM Pc. The EC50 value obtained with the hCG + scFv 13VHB complex is increased by a factor of 2 compared to that obtained with hCG alone: ​​it is 0.75.10' 10M with scFv 13VHB versus 1.5.10' 10 M with hCG alone. The value of the slope of the dose / response curve (Hill slope) is also increased in the case of stimulation with hCG + scFv 13VHB: it is 1.438 with scFv + hCG versus 1.312 with hCG alone.

[0325] In addition to its potentiating effect on FSH activity, these results demonstrate that the monovalent form of a humanized variant antibody, i.e. scFv 13VHB, is also capable of potentiating LH activity like the bivalent humanized variant antibody from which it is derived, i.e. the whole bivalent 13VHB variant.

[0326] EXAMPLE 9: IN VIVO POTENTIALIZING EFFECT OF THE HUMANIZED ANTIBODY 13VHB-139VL (SEQ ID NO: 9 and SEQ ID NO: 16) ON THE RESPONSE TO SUPEROVULATION TREATMENT IN A “POOR RESPONDER” FEMALE MOUSE MODEL

[0327] In women, a poor response to ovarian stimulation is often due to a reduced ovarian reserve (Antrum Follicular Count (AFC) less than 5). This parameter, associated with a low AMH (anti-Müllerian hormone) level, less than 1.2 ng / ml, is a poor prognosis in terms of the chance of success during IVF treatment.

[0328] To evaluate the potentiating effect of the humanized 13VHB-139VL variant antibody in such an animal model, adult MNRI mice were treated with chemotherapy and then received superovulation treatment.

[0329] Experimental protocol

[0330] In order to obtain such a female "Poor Responder" model, adult female NM RI mice, 8 weeks old, received a chemotherapy treatment consisting of a single intraperitoneal injection of 12 mg / kg busulfan and 120 mg / kg cyclophosphamide

[0023] . Sixty-one mice were thus treated. Six additional mice served as controls for histological studies of the ovaries: 2 "chemo" control mice, having received the chemotherapy treatment without superovulation treatment and 4 "wild type" mice having received no treatment.

[0331] Five to eight weeks after the injection of busulfan+cyclophosphamide, the mice were divided into two groups and subjected to superovulation treatment according to the following protocol: group 1 (n=30 mice) treated with a subcutaneous injection of 1.875 IU of

[0332] Menopur® (purified hMG containing 50% FSH and 50% LH) (Ferring, France) on day 1 and day 2 of the cycle. Group 2 (n=31 mice) treated with a subcutaneous injection of a mixture of 1.875 IU of Menopur® and the humanized 13VHB variant antibody at a dose of 20 pg / kg on day 1 and an injection of 1.875 IU of Menopur® on day 2 of the cycle.

[0333] In total, each mouse received 3.75 IU of Menopur®.

[0334] For both groups, ovulation was induced by an injection of 10 IU of hCG (Ovitrelle®, Merck Serono SA) followed by mating on day 3 of the cycle.

[0335] On day 4 of the cycle, the presence of vaginal plugs attesting to mating was verified.

[0336] On day 5 of the cycle, mice with a vaginal plug were sacrificed. After flushing the uterine horns, the embryos were harvested to determine the number of viable embryos. In addition, for 13 females from groups 1 and 2, the ovaries were removed for further histological analysis to count the number of corpora lutea and to study certain markers of folliculogenesis by immunocytochemistry.

[0337] For each group the following analyses were collected: the number of pregnant mice to calculate the pregnancy rate; the number of live embryos collected per pregnant female to calculate the fertilization rate.

[0338] From histological sections of the ovaries:

[0339] Measurement of total follicular density (N / mm 3) on 4 wild-type control mice with normal ovarian reserve and on 2 control mice treated with chemotherapy to confirm the decrease in ovarian reserve.

[0340] Counting the number of corpora lutea to assess ovulation rate.

[0341] Counting atretic follicles and calculating the percentage of atretic follicles to total follicles.

[0342] Evaluation of the apoptosis rate determined by the percentage of positive follicles after caspase-3 immunostaining.

[0343] Results

[0344] 1 / Control of chemotherapy treatment

[0345] To verify the impact of chemotherapy treatment on the ovarian reserve of females, a histological study of the ovaries of control "chemo" mice and untreated "wild type" mice was performed to quantify their follicular reserve. For this, 5 μm thick ovarian sections were stained with hematoxylin eosin (Merck, Germany) to count the number of total follicles present. The results are expressed as the number of follicles per cubic millimeter of ovary.

[0346] The results showed that the follicular density was 2,400 ± 1,070 follicles per cubic millimeter (mm 3 ) ovarian in control (untreated) mice and 665.2 ± 63.48 follicles per cubic millimeter (mm 3 ) of ovary in control “chemo” mice that received chemotherapy treatment but no superovulation treatment (Table 13).

[0347] Table 13: Evolution of follicular density in mice treated with chemotherapy compared to untreated control mice

[0348] ***, p<0.005; ANOVA-Newman-Keuls Chemotherapy treatment induced a significant decrease in follicular density, which represents a 72% drop in ovarian reserve compared to untreated "wild type" mice (***, p<0.005; ANOVA-Newman-Keuls).

[0349] The results significantly demonstrating the impact of chemotherapy treatment on the drop in the number of follicles, validate this experimental model of mice with low ovarian reserve.

[0350] 21 Results of superovulation treatment with hormone alone or hormone in combination with 13VHB antibody

[0351] Two days after mating, on day 5 of the cycle, the mice with a vaginal plug were sacrificed. The embryos were collected by flushing and observed under a binocular microscope to observe their stage of development and deduce the number of live embryos that had begun cell division.

[0352] The number of pregnant females and the average number of live embryos obtained per female are shown in Table 14.

[0353] Table 14: Number of pregnant mice and live embryos per treatment

[0354] The number of pregnant mice was significantly different between the group treated with hMG (Menopur®) alone and the group treated with hMG (Menopur®) in combination with the 13VHB antibody: 9 out of 30 mice were pregnant in the group treated with hMG alone and 17 out of 31 mice were pregnant in the group treated with hMG+13VHB (*: p <0.05; Fisher's exact test, one-tailed). Twice as many pregnant mice were therefore obtained in the group receiving 13VHB, which means that the number of mice responding to the superovulation treatment doubled thanks to the combination of the 13VHB antibody with hormonal treatment.

[0355] The mean number of live embryos per mouse was not significantly different between the two treatments: 23.67 ± 14.55 in the hMG group and 25.53 ± 20.54 in the hMG + 13VHB group.

[0356] A histological analysis of the ovaries was also carried out on 13 mice from each group to count the number of corpora lutea per ovary, deduce whether the female had ovulated and evaluate the average number of ovulations per female. The results are shown in Table 15. Table 15: Number of corpora lutea observed by ovarian histology and number of pregnant mice in each treatment group; calculation of ovulation rate and fertilization rate

[0357] *, p<0.05, One-tailed Fisher's exact test

[0358] Histological analysis of the ovaries showed that all 13 females in each treatment group (Menopur® or Menopur®+13VHB) had corpora lutea, indicating that all mice had ovulated. The average number of corpora lutea per ovary was not significantly different between the two groups: 23.3 ± 10.2 and 25.9 ± 5 in the Menopur® and Menopur®+13VHB groups, respectively. These results indicate that the ovulation rate (number of ovulated females / total number of females X100) was 100% in both treatments.

[0359] Although all mice ovulated, surprisingly, the number of mice that became pregnant after mating was significantly higher in the Menopur®+13VHB group (*, p<0.05; one-tailed Fisher's exact test). It was 6 out of 13 pregnant mice in the Menopur® group and 11 out of 13 pregnant mice in the Menopur®+13VHB group. The fertilization rate (number of pregnant mice / number of ovulating mice X100) increased from 46% in the Menopur® group to 85% in the Menopur®+13VHB group, this difference being significant (*, p<0.05; one-tailed Fisher's exact test). This 1.85-fold increase in fertilization rate surprisingly demonstrates that better oocyte maturation was achieved with 13VHB, which led to twice as many pregnant females. Combining 13VHB with hormonal treatment therefore doubled the number of "responding" females compared to conventional hormonal treatment.

[0360] Analysis of the degree of apoptosis in ovarian follicles was performed by classical histology and immunocytochemistry to verify the possible effect of 13VHB on apoptosis of ovarian follicles.

[0361] An initial assessment of follicle quality was performed on hematoxylin-eosin stained sections based on the following criteria: basement membrane integrity, cell density, presence or absence of pyknotic bodies, and oocyte integrity. On this basis, the number of atretic follicles and the number of total follicles were counted in both treatment groups. The results were expressed as the percentage of atretic follicles to total follicles per mouse (number of atretic follicles / number of total follicles X100). They are presented in Table 16.

[0362] Table 16: Study of follicle atresia in mice receiving superovulation treatment with Menopur® alone or Menopur® in combination with 13VHB by histological analysis

[0363] Statistical analysis was performed using a non-parametric Mann Whitney t-test. The results show that there is no significant difference in the percentage of atretic follicles in mice treated with hormone alone (19.94 ± 8.26) or with hormone+13VHB (16.78 ± 9.33). Therefore, treatment with 13VHB does not induce abnormal ovarian stimulation and does not increase the number of pyknotic figures and atresia in ovarian follicles.

[0364] This first classical histology study was completed by an immunocytochemical analysis using a caspase 3-specific antibody, as described by Amorim et al.

[0024] . Caspase 3 is one of the effector caspases, cysteine ​​proteases that play a key role in the execution of apoptosis by cleaving target proteins that maintain cell integrity. A positive labeling indicates the presence of active caspase 3 signifying the activation of apoptosis in the cell.

[0365] In this analysis, a follicle was defined as atretic when more than 50% of the granulosa cells and / or its oocyte showed positive caspase 3 staining. The results obtained on ovarian sections from mice in both treatment groups are shown in Table 17.

[0366] Table 17: Immunocytochemical analysis of ovaries by specific labeling of caspase 3 in mice having received superovulation treatment with Menopur® alone or Menopur® in combination with 13VHB Statistical analysis was performed using a non-parametric Mann Whitney t-test.

[0367] The results show that there is no significant difference in the percentage of caspase 3-positive follicles in the two treatment groups. The percentage of follicles showing caspase 3 activation is 7.66% in mice treated with hormone alone and 7.81% in mice treated with hormone+13VHB.

[0368] These results indicate that there is no difference in caspase 3 activation in females treated with 13VH B and that no adverse deleterious effects of the 13VH B antibody on ovarian function were demonstrated.

[0369] All the results obtained with this animal model of mice with low ovarian reserve significantly demonstrate an improving effect of the 13VHB antibody in the response to superovulation treatment, surprisingly leading to a doubling of the fertilization rate and the pregnancy rate in mice treated with hormone + 13VHB, compared to females having received conventional hormonal treatment. This potentiating effect of 13VHB in vivo in the unfavorable case of low responder mice, with a poor prognosis, surprisingly made it possible to double the number of females capable of responding to superovulation treatment. The scope of these results opens interesting perspectives in the development of an ovarian stimulation treatment in women with a poor prognosis, low responders for example.

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Claims

CLAIMS 1. Humanized variant antibody or fragment thereof potentiating the bioactivity of follicle-stimulating hormone (FSH), lutein-stimulating hormone (LH) and human Chorio-Gonadotropin (hCG), characterized in that: the variable domain of the heavy chain of said humanized variant antibody or fragment thereof contains the amino acid sequence QX 2 QLVQSGAEVKKPGASVKVSCKX 24 SGFTFSSSYIX 35 WX 37 RQAPGQRLEWX 48 X 49 WIYAG TGGTSYX 61 QKFX 65 GX 67 X 68 X 69 X 70 TX 72 DTSASTAYMEX 83 SSLRSEDTAVYYCARHGSYFDYW GQGTLVTVSS where 2 is V or G, X 24 is A or T, X 35 is H or S, X 37 is V or L, X 48 is I or M, X 49 is A or G, X 61 is S or N, X 65 is T or Q, X 67 is R or K, X 58 is A or V, X 69 is T or Q, X 70is I or L, X 72 is V or R, X 83 is L or F (SEQ ID NO: 1), or the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAX 24 SGFTFSSSYISWLRQAPGKGLEWX 48 AWIYAGTGGT SYAQX 63 VKGRFX 69 X 70 SVDTSKNTAYLQMNSLRAEDTAVYYCARHGSYFDYWGQGTLVTVS S where 24 is A or T, X 48 is I or V, X 63 is S or K, X 69 is Q or T, X 70 is L or I (SEQ ID NO: 2), or the amino acid sequence QMQLVQSGPEVKKPGTSVKVSCKX 24 SGFTFSSSYISWLRQARGQRLEWIAWIYAGTGGTS YAQKFQERVX 69 X 70 TVDMSTSTAYMEFSSLRSEDTAVYYCARHGSYFDYWGQGTLVTVSS where X 24 is T or A, X 69 is Q or T, X 70 is L or I (SEQ ID NO: 3); and the variable domain of the light chain of said humanized variant antibody or fragment thereof contains the amino acid sequence DIX 3 X 4 TQSPX 9 SLX 12 X 13 SX 15 GX 17 RX19 TIX 22 CX 24 X 25 SQSVDYDGDSYMX 38 WYQQKPGX 46 X 47 PKLLIYAASX 57 X 58 ESGVPX 64 RFSGSGSGTDFTLTISSLQX 84 EDX 87 AX 89 YYCQQSNEDPYTFG QGTKLEIK where X 3 is Q or V, X 4 is M or L, X 9 is D or S, X 12 is A or S, X 13 is A or V, X 15 is L or V, X 17 is E or D, X 19 is A or V, X 22 is N or T, X 24 is R or K, X 25 is A or S, X 38 is A or N, X 46 is Q or K, X 47 is P or A, X 57 is N or S, X 58 is R or L, X 64 is D or S, X 84 is A or P, X 87 is V or F, X 89 is V or T (SEQ ID NO: 4).

2. Humanized variant antibody or fragment thereof according to claim 1, characterized in that: the variable domain of the heavy chain of said humanized variant antibody or fragment thereof contains the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGFTFSSSYIHWVRQAPGQRLEWMGWIYAGTGGTS YSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARHGSYFDYWGQGTLVTVSS (SEQ ID NO: 5), or the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGFTFSSSYISWVRQAPGQRLEWMAWIYAGTGGTS YNQKFTGKVTITRDTSASTAYMELSSLRSEDTAVYYCARHGSYFDYWGQGTLVTVSS (SEQ ID NO: 6), or the amino acid sequence QGQLVQSGAEVKKPGASVKVSCKASGFTFSSSYISWLRQAPGQRLEWIAWIYAGTGGTSY NQKFTGKVTITRDTSASTAYMELSSLRSEDTAVYYCARHGSYFDYWGQGTLVTVSS (SEQ ID NO: 7), or the amino acid sequence QGQLVQSGAEVKKPGASVKVSCKASGFTFSSSYISWLRQAPGQRLEWIAWIYAGTGGTSY NQKFTGKATLTVDTSASTAYMELSSLRSEDTAVYYCARHGSYFDYWGQGTLVTVSS (SEQ ID NO: 8), or the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKTSGFTFSSSYISWLRQAPGQRLEWIAWIYAGTGGTSYS QKFQGRVQLTVDTSASTAYMEFSSLRSEDTAVYYCARHGSYFDYWGQGTLVTVSS (SEQ ID NO: 9), or the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGFTFSSSYISWLRQAPGQRLEWMAWIYAGTGGTSY SQKFQGRVTITVDTSASTAYMEFSSLRSEDTAVYYCARHGSYFDYWGQGTLVTVSS (SEQ ID NO: 10), or the amino acid sequence EVQLVESGGGLVQPGGSLRLSCATSGFTFSSSYISWLRQAPGKGLEWIAWIYAGTGGTSYA QKVKGRFQLSVDTSKNTAYLQMNSLRAEDTAVYYCARHGSYFDYWGQGTLVTVSS (SEQ ID NO: 11), or the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSSSYISWLRQAPGKGLEWVAWIYAGTGGTSY AQSVKGRFTISVDTSKNTAYLQMNSLRAEDTAVYYCARHGSYFDYWGQGTLVTVSS (SEQ ID NO: 12), or the amino acid sequence QMQLVQSGPEVKKPGTSVKVSCKTSGFTFSSSYISWLRQARGQRLEWIAWIYAGTGGTSY AQKFQERVQLTVDMSTSTAYMEFSSLRSEDTAVYYCARHGSYFDYWGQGTLVTVSS (SEQ ID NO: 13), or the amino acid sequence QMQLVQSGPEVKKPGTSVKVSCKASGFTFSSSYISWLRQARGQRLEWIAWIYAGTGGTSY AQKFQERVTITVDMSTSTAYMEFSSLRSEDTAVYYCARHGSYFDYWGQGTLVTVSS (SEQ ID NO: 14); and the variable domain of the light chain of said humanized variant antibody or fragment thereof contains the amino acid sequence DIVMTQSPDSLAVSLGERATINCKSSQSVDYDGDSYMAWYQQKPGQPPKLLIYAASNRESG VPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGQGTKLEIK (SEQ ID NO: 15), or the amino acid sequence DIQLTQSPSSLSASVGDRVTITCRASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASSLESG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPYTFGQGTKLEIK (SEQ ID NO: 16).

3. Humanized variant antibody or fragment thereof according to claim 2, characterized in that: the variable domain of the heavy chain of said humanized variant antibody or fragment thereof contains the sequence SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID NO: 7 or SEQ ID NO: 8, and the variable domain of the light chain of said humanized variant antibody or fragment thereof contains the sequence SEQ ID NO: 15; OR the variable domain of the heavy chain of said humanized variant antibody or fragment thereof contains the sequence SEQ ID NO: 9, or SEQ ID NO: 10, and the variable domain of the light chain of said humanized variant antibody or fragment thereof contains the sequence SEQ ID NO: 16; or the variable domain of the heavy chain of said humanized variant antibody or fragment thereof contains the sequence SEQ ID NO: 11 or SEQ ID NO: 12, and the variable domain of the light chain of said humanized variant antibody or fragment thereof contains the sequence SEQ ID NO: 16; or the variable domain of the heavy chain of said humanized variant antibody or fragment thereof contains the sequence SEQ ID NO: 13 or SEQ ID NO: 14, and the variable domain of the light chain of said humanized variant antibody or fragment thereof contains the sequence SEQ ID NO:

16.

4. Humanized variant antibody or fragment thereof according to claim 3, characterized in that it comprises the variable domain of the heavy chain and the variable domain of the light chain of the following respective sequences: SEQ ID NO: 9 and SEQ ID NO: 16; SEQ ID NO: 10 and SEQ ID NO: 16; SEQ ID NO: 11 and SEQ ID NO: 16; SEQ ID NO: 12 and SEQ ID NO: 16; SEQ ID NO: 5 and SEQ ID NO: 15; SEQ ID NO: 6 and SEQ ID NO: 15; SEQ ID NO: 7 and SEQ ID NO: 15; SEQ ID NO: 8 and SEQ ID NO: 15; SEQ ID NO: 13 and SEQ ID NO: 16; SEQ ID NO: 14 and SEQ ID NO:

16.

5. Humanized variant antibody or fragment thereof according to claim 4, characterized in that it comprises the variable domain of the heavy chain and the variable domain of the light chain of the following respective sequences: SEQ ID NO: 6 and SEQ ID NO: 15; SEQ ID NO: 8 and SEQ ID NO: 15; SEQ ID NO: 12 and SEQ ID NO: 16; SEQ ID NO: 9 and SEQ ID NO: 16; SEQ ID NO: 11 and SEQ ID NO: 16; SEQ ID NO: 14 and SEQ ID NO:

16.

6. Humanized variant antibody or fragment thereof according to claim 5, characterized in that it comprises the variable domain of the heavy chain containing the sequence SEQ ID NO: 8 or SEQ ID NO: 9, and the variable domain of the light chain containing the sequence SEQ ID NO: 15 or SEQ ID NO: 16, respectively.

7. Humanized variant antibody or fragment thereof according to claim 6, characterized in that it comprises the variable domain of the heavy chain containing the sequence SEQ ID NO: 9, and the variable domain of the light chain containing the sequence SEQ ID NO:

16.

8. Humanized variant antibody or fragment thereof according to claim 7, characterized in that it comprises the heavy chain of sequence SEQ ID NO: 20 and the light chain of sequence SEQ ID NO:

21.

9. Humanized variant antibody or fragment thereof according to any one of claims 1 to 8, wherein the fragment is a scFv, preferably a scFv of sequence SEQ ID NO:

17.

10. Pharmaceutical composition, characterized in that it comprises a humanized variant antibody or fragment thereof according to any one of claims 1 to 9, and a pharmaceutically acceptable vehicle.

11. Pharmaceutical composition according to claim 10, characterized in that it further comprises FSH, or LH, or a mixture of FSH and LH, or hMG (human Menopausal Gonadotropin), or a mixture of FSH and hMG, or a mixture of LH and hMG, or hCG, or a mixture of hCG and FSH, or a mixture of hCG and hMG.

12. A humanized variant antibody or fragment thereof according to any one of claims 1 to 9 or a pharmaceutical composition according to any one of claims 10 or 11, for use as a medicament.

13. A humanized variant antibody or fragment thereof according to any one of claims 1 to 9 or a pharmaceutical composition according to any one of claims 10 or 11, for use according to claim 12, wherein the medicament is for inducing ovulation or polyovulation in a female mammal.

14. A humanized variant antibody or fragment thereof according to any one of claims 1 to 9 or a pharmaceutical composition according to any one of claims 10 or 11, for use according to claim 12, wherein the medicament is for inducing or stimulating spermatogenesis in a male mammal.

15. A humanized variant antibody or fragment thereof according to any one of claims 1 to 9 or a pharmaceutical composition according to any one of claims 10 or 11, for use according to claim 12, wherein the medicament is for increasing steroidogenesis in a male or female mammal.

16. A humanized variant antibody or fragment thereof according to any one of claims 1 to 9 or a pharmaceutical composition according to any one of claims 10 or 11, for use in the prevention or treatment of infertility or subfertility in a male or female mammal.

17. A humanized variant antibody or fragment thereof according to any one of claims 1 to 9 or a pharmaceutical composition according to any one of claims 10 or 11, for use in stimulating procreation in a female mammal.

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