LONG ACTING LUTEINIZING HORMONE (LH) COMPOUND

Abstract

The present invention relates to a long acting biologically active luteinizing hormone (LH) compound comprising an LH agonist linked to a pharmaceutically acceptable molecule providing an in vivo plasma half-life of the LH agonist or LH compound which is increased substantially compared to the in vivo plasma half-life of an LH agonist administered in the same manner as the LH compound. The present invention relates to methods for controlled ovarian stimulation which can be used in conjunction with assisted reproduction technologies such as in vitro fertilisation, intra cytoplasmatic sperm injection, intra uterine insemination and in vitro maturation. In other aspects the invention relates to methods for inducing folliculogenesis and methods for providing luteal support for the corpora lutea.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to a long acting biologically active luteinizing hormone (LH) compound comprising an LH agonist linked to a pharmaceutically acceptable molecule, and to methods of preparing and using such LH compounds. These LH compounds have a protracted profile of action and are useful in assisted reproduction technology procedures, such as for promotion of fertility or treatment of infertility and for use in hypogonadotropic hypogonadal males and in boys with cryptorchidism. The modified LH have a protracted profile of action and are useful in combination with follicle stimulating hormone (FSH) for inducing follicular development in anovulatory women or for inducing controlled ovarian stimulation in the follicular phase of the menstrual cycle of a mammalian female subject. Furthermore, the present invention relates to methods for controlled ovarian stimulation, which can be used in conjunction with assisted reproduction technologies such as in vitro fertilisation (IVF), intra cytoplasmatic sperm injection (ICSI), intra uterine insemination (IUD, in vitro maturation (IVM), and induction of oculation. In other aspects the invention relates to methods for inducing folliculogenesis and methods for providing luteal and gestational support for corpora lutea.

BACKGROUND OF THE INVENTION

[0002] Assisted reproduction technology (ART) procedures typically require treatment with exogenous gonadotropins to stimulate growth and maturation of the ovarian follicles. When gonadotropins are used to treat anovulatory females, the goal is to replicate the normal menstrual cycle, when a single, dominant follicle matures prior to induction of ovulation. In contrast, for women undergoing in vitro fertilization (IVF), controlled ovarian stimulation (COS) is employed to stimulate the growth and maturation of several ovarian follicles, yielding multiple oocytes, which then are retrieved for use in the IVF procedure.

[0003] In connection with ART, COS, that secures development of multiple follicles it is essential to achieve the best possible chance for the patient to become pregnant. To obtain multiple follicle growth circulating levels of FSH need to surpass the physiological threshold level that triggers growth of responsive follicles for a longer period than the natural three to four day period. This is achieved by administration of exogenous FSH or by manipulating the pituitary gland to secrete enhanced amounts of FSH, and COS performed in the right way may easily result in the harvest of excess mature oocytes for in vitro fertilization (IVF).

[0004] In addition to stimulating follicular growth, an important function of FSH is to stimulate the development of LH-receptors on granulosa cells. LH-receptors are constitutively expressed on theca cells immediately surrounding the follicle and secure the production of--among other substances--androgens (i.e. androstenedione and testosterone) for conversion into oestrogens in the granulosa cell layer, but LH-receptors also have important functions in the granulosa cell layer of the follicle. Currently it is not known precisely when in follicular development LH-receptors become expressed on granulosa cells.

[0005] In the normal menstrual cycle, the LH-receptor is only activated by LH activity released from the pituitary, but hCG, which essentially is a pregnancy associated protein, may also bind and stimulate the LH-receptor. hCG has a longer half-life than LH and the accumulated in vivo activity of hCG (from equal doses of LH and hCG in an ampule) is usually considered to be 6 to 8 times higher than LH (Stockman P G W et al. Fertil Steril 1993;60:175, Giudice E et al. J Clin Res 2001;4:27).

[0006] Some preparations for COS only contain FSH while others contain a combination of FSH and LH-like activity (i.e. either LH or hCG alone or a mixture of LH and hCG). For instance, Menopur contains urine derived FSH and LH-like activity. In these preparations about 95 percent of the in vivo receptor mediated LH-like bioactivity derives from hCG due to its longer half-life (Van den Hooven H et al. RBM Online 2003; 7: 547). Recombinant LH is also available in a pure form as add on for COS (i.e. Luveris, Merck-Serono, Darmstadt, Germany). However, hCG for COS is only available in the presence of a product containing FSH and is not marketed in small doses to be used in connection with COS.

[0007] The clinical benefit of using LH-activity in connection with COS has been heavily debated during the past decade. Although a number of meta-analyses have suggested that the addition of LH-like activity, which in essence is provided via hCG, show an augmented baby-take-home rate as compared to pure FSH alone, this issue has not been clarified (Al-Inany H G et al., Reprod Biomed Online. 2008;16: 81-88, Westergaard L W, Cochrane Database Syst Rev 2003;1:CD003973., Al-Inany H G et al., Gynecol Endocrinol. 2009;25:372-8). Adding to the complexity are differences between the FSH isoform profile of the most frequently used FSH containing hCG (i.e. Menopur (highly purified hMG containing urine derived FSH, LH and hCG)) and the pure FSH (i.e. recombinant FSH, Puregon or Gonal F). However, there is no doubt that LH levels can be reduced below a threshold limit at which adding LH-like activity will be helpful and there is also an upper threshold limit above which negative effects on treatment outcome become apparent. Thus LH-like activity should ideally remain in a therapeutically narrow window.

[0008] LH and CG are very homologous. In comparison to LH, CG has a C-terminal glycosylated extension that has been shown to be important for the longer half-life of CG. Human LH and hCG are more than 80% identical in sequence. Although both LH and hCG binds to and activate the LH-receptor, both hormones exist as a family of iso hormones that differ in their oligosaccharide composition. Each of the different isoforms affects the receptor in a specific way and may elicit variable cellular responses (Burgon P G et al., Endocrinology, 1996;137:4827; Stanton P G et al., Mol Cell Endocrinol. 1996;125:133-141.), as have also been shown for the different FSH isoforms (Barrios-de-Tomasi J, et al. Mol Cell Endocrinol. 2002;186:189-98, Yding Andersen C & Ezcurra D, Reproductive Biology Insights 2011:4, 1-10). Thus the more subtle and fine-tuned effects of LH and hCG may actually differ. Recent studies presented at the ESHRE conference in Stockholm (July 2011) showed that LH acted much faster than HCG, but less efficient overall at the receptor level (L. Casarini et al., ESHRE Stockholm 2011-P312, Universita degli Studi di Modena, Italy). hCG is a pregnancy associated protein which is secreted following the implantation of the embryo starting around 8 days after ovulation. hCG is capable of stimulating the corpus luteum to remain active and continue its secretion of progesterone and other substances necessary for the pregnancy to become established. Despite the fact that levels of LH at that moment of the menstrual cycle are present in appreciable amounts, this level is insufficient to stimulate the corpus luteum further and unless the woman becomes pregnant the corpus luteum will regress, a menstrual bleeding will occur and a new menstrual cycle start. Although this difference between LH and hCG has puzzled science for some time, it has now been demonstrated that the LH-receptor (LH-R) changes during the luteal phase. The functional full-length receptor maintains its expression when hCG is present, whereas LH is unable to accomplish that (Dickinson R E et al., Endocrinology 150: 2873-2881, 2009). This demonstrates differences in the effect of LH and hCG during the luteal phase and this could suggest that LH and hCG also in the follicular phase of the menstrual cycle exert different effects at the receptor level.

[0009] It is now well recognised that LH-R expression on human granulosa cells is sufficient to drive follicular development from a diameter of around 10-12 millimetre and until ovulation with the presence of FSH in only small permissive amounts in connection with COS (Blockheel et al., 2009; Filicori et a., 1999). Thereby this stimulation resembles conditions of the natural menstrual cycle, in which levels of FSH is attenuated during the second half of the follicular phase, while levels of LH remain fairly constant and it has been shown that LH has a very strong stimulating effect on oestradiol production in granulosa cells from preovulatory follicles prior to the mid-cycle surge of gonadotropins. The ability to provide a more natural environment for the final maturation of the follicles is likely to provide oocytes that has an even better capacity to sustain fertilization, embryogenesis and implantation and subsequently result in a better reproductive outcome.

[0010] One of the most severe side-effects of COS is the occurrence of ovarian hyper stimulation syndrome (OHSS), which is a potential life threatening condition. Recent studies have shown that it is now possible to almost completely eliminate OHSS by the use of an agonist trigger for final follicular maturation (Humaidan P, Kol S, Papanikolaou E; Copenhagen GnRH Agonist Triggering Workshop Group. GnRH agonist for triggering of final oocyte maturation: time for a change of practice? Hum Reprod Update. 2011;17:510-24. PMID:21450755) without compromising the reproductive outcome. In combination with a GnRH antagonist down regulated pituitary function, a bolus of a GnRH agonist is capable of displacing the antagonist and cause a flare-up of gonadotropin release, which is then used as a signal for ovulation induction. However, subsequent to the flare up the agonist causes pituitary down regulation, which removes the stimulatory signals to the ovary. Removal of these stimuli also reduces the risk of OHSS. However, this down regulation also has a profound negative impact on the function of the corpus luteum and the reproductive outcome is unacceptably low. So in order to maintain a certain function of the corpus luteum it has successfully been attempted to add a bolus of hCG (1500 IU) at the time of oocyte retrieval and later on in the luteal phase. Alternatively daily injections of LH can rescue the luteal phase and provide a good reproductive outcome (A novel method of luteal supplementation with recombinant luteinizing hormone when a gonadotropin-releasing hormone agonist is used instead of human chorionic gonadotropin for ovulation triggering: a randomized prospective proof of concept study. Papanikolaou E G, Verpoest W, Fatemi H, Tarlatzis B, Devroey P, Tournaye H. Fertil Steril. 2011 Mar. 1;95(3):1174-7. Epub 2010 Oct. 27. PMID: 20979997).

[0011] Despite recent advances in ART, ovarian stimulation through exogenous gonadotropins is not uniformly successful due, in part, to varying individual responses to treatment with gonadotropins. This variability complicates patient management and can result in multiple births and potentially life-threatening complications.

[0012] The gonadotropins form a family of structurally related glycoprotein hormones. Typical members include chorionic gonadotropin (CG), follicle stimulating hormone (FSH; follitropin), luteinizing hormone (LH; lutropin) and thyroid stimulating hormone (TSH; thyrotropin). FSH, LH and TSH are present in most vertebrate species and are synthesized and secreted by the pituitary. CG has so far been found only in primates, including humans, and in horses and is synthesized by placental tissue. FSH and LH are the pituitary hormones essential for follicular maturation and luteinization in the female and for testis maturation and spermatogenesis in the male. Gonadotropins are secreted by the pituitary gland under the control of hypothalamic gonadotropin-releasing hormone (GnRH). Follicle stimulating hormone (FSH) and luteinizing hormone (LH) are the pituitary hormones essential for follicular maturation (follicular development) and luteinization. FSH is required for follicular recruitment (i.e., the early growth of ovarian follicles) at the beginning of the spontaneous menstrual cycle, and it also supports mid- and late-stage follicular development.

[0013] In recent years very pure preparations, of the gonadotropins have become available through the use of recombinant DNA technology (see for instance Boime et al., Seminars in Reproductive Endocrinology 10, 45-50, 1992: "Expression of recombinant human FSH, LH and CG in mammalian cells"). The recombinant gonadotropins are of constant quality i.e. have reproducible biochemical and biological properties. Genomic and cDNA clones have been prepared for all subunits and their primary structure has been resolved. Moreover, Chinese Hamster Ovary (CHO) cells have been transfected with human gonadotropin subunit genes and these cells are shown to be capable of secreting intact dimers (e.g. Keene et al (1989), J. Biol. Chem., 264, 4769-4775; Van Wezenbeek et al (1990), in From clone to Clinic (eds Crommelin D. J. A. and Schellekens H.), 245-251). It has been demonstrated that the biochemical and biological characteristics of e.g. recombinant FSH are almost identical to those of natural FSH (Mannaerts et al (1991), Endocrinology, 129, 2623-2630). Moreover, pregnancies were achieved after controlled ovarian superovulation using recombinant FSH (Germond et al (1992), Lancet, 339 ,1170; Devroey et al (1992), Lancet, 339, 1170-1171).

[0014] The gonadotropin may also be isolated from natural sources, e.g. from human urine, or the gonadotropin may be prepared in a (bio)synthetic way, c.f. by recombinant DNA techniques.

[0015] Gonadotrophins are widely used in clinical practice to treat women with WHO group II and WHO group I anovulation (World Health Organisation Technical Report 514, (1973)). Conventionally, follicular development is induced by administering hMG (human menopausal gonadotrophin) or u-hFSH (urinary human follicle stimulating hormone) at a dose of 75-150 IU/day. This dose is increased after a few days (usually five) by steps of 75 IU. It is rare to exceed 450 IU/day. When there is at least one follicle having a mean diameter of at least 18 mm and no more than two follicles having a mean diameter of at least 16 mm, a high dose (of 5000 IU for example) of hCG (human chorionic gonadotrophin) is administered to induce ovulation. This "conventional protocol" has been used successfully for more than 20 years. It carries some risks however, mainly in patients with polycystic ovaries or polysystic ovarian syndrome (PCOS).

[0016] These risks include the occurrence of OHSS, and a relatively high incidence of multiple pregnancies (Schenker et al, Fertil. Steril. 35: 105-123 (1981)). Although the majority of multiple pregnancies are twins, induction of ovulation contributes to one third of the high rank multiple births in the UK (Levene et al, Br. J Obstet. Gynacol. 99: 607-613 (1992)).

[0017] Careful monitoring during treatment by ultrasound (US) and assessment of serum oestradiol (E2) have reduced these risks but have not been able to prevent them in all patients. These problems are directly related to the difficulty of obtaining the growth of a single dominant follicle leading to non-physiological multifollicular development.

[0018] FSH is administered therapeutically to induce follicular development in anovulatory women and women undergoing COS. In traditional ovulatory stimulation methods, FSH is administered throughout treatment until shortly before the oocytes are retrieved. This continued stimulation by FSH usually causes multiple follicular development and can in combination with an exogenous bolus of hCG to induce ovulation lead to a potentially fatal condition, OHSS. It has now been estimated that COS is fatal to otherwise healthy patients in around 3 per 100,000 stimulation cycles. Decreasing the dosage of FSH can reduce the risk of OHSS, but low FSH dosages yield inadequate number of follicles and thus lower the chances of success in assisted reproduction.

[0019] LH functions during all stages of a normal menstrual cycle. LH stimulates the theca cells of the follicle to produce the androgen substrate which is converted into estrogen by the aromatase system in the granulosa cells. During the late stages of follicle maturation, approximately 5 to 7 days before ovulation, large ovarian follicles begin to express LH receptors in granulosa cells, which render those follicles responsive to LH for continued maturation and development. Hillier et al., Mol. Cell Endocrinol. 100:51 (1994), Campbell et al. J. Reprod. Fertil. 117:244 (1999). Next, a mid-cycle surge of LH triggers the final stage of follicular maturation and ovulation in a normal menstrual cycle. Ovulation follows the mid-cycle LH surge within 24 to 48 hours. Finally, in the second part of the menstrual cycle, the luteal phase, LH stimulates production of estrogen and progesterone in the corpus luteum of the ovary as it prepares the uterus for implantation and pregnancy.

[0020] In ovarian stimulation protocols, hCG can serve as a source of LH activity because hCG and LH act through the same receptor. Filicori et al. Human Reprod. 17:2009 (2002a); Martin et al., Fertil. Steril. 76: 0-49 (2002). Relative to LH, hCG has a longer half-life and, hence, is more potent in vivo than LH, although the literature tends to treat hCG and LH as fungible. Indeed, the scientific literature generally does not mention determining the source of LH activity in naturally-derived gonadotropin preparations.

[0021] The literature discloses using LH activity or low doses of hCG in combination with FSH throughout ovulatory stimulation, but guidance regarding effective amounts and timing of LH activity supplementation is lacking. For example, the abstract of Martin et al, Fertil. Steril. 76: O-49 (2002), discloses administering 2.5 .mu.g recombinant hCG daily (maintaining serum hCG levels from 1-3 mIU/mL) during ovulatory stimulation. Gordon et al. disclose administering 75 IU FSH with 0, 1, 25, and 75 IU LH activity. Human Reprod. 12 (Suppl. 1): 52 (1997a); ibid.: 53 (1997b).

[0022] Published studies disclose administering LH activity, throughout stimulation, at FSH to LH ratios of 150:0, 150:37.5, 150:75, and 150:150. Filicori et al. (2002a). Further, the literature documents supplementing FSH stimulation with 50 IU hCG/day (Filicori et al., J. Clin. Endocrinol. & Metabol. 84: 2659 (1999)), and protocols in which 150 IU FSH is administered for 7 days, followed by treatment with FSH-to-hCG ratios of 150:0, 50:50, 25:100, and 0:200 (ibid. 87:1156 (2002c) and US20080108571).

[0023] During the last 10 years, a new protocol has been designed (the "chronic low dose protocol") and tested in order to reduce further the incidence of the complications of gonadotrophin therapy (Seibel et al, Int. J Fertil., 29: 338-339 (1984); Buvat et al, Fertil. Steril., 52: 553-559 (1989); Hamilton-Fairley et al, Human Reprod. 6: 1095- 1099 (1991); Sagle et al, Fertil Steril., 55: 56-60 (1991); Shoham et al, Fertil. Steril., 55: 1051-1056 (1991); Meldrum, Fertil Steril., 55: 1039-1040 (1991)). This protocol starts with a low dose of FSH or hMG (75 IU/day) and no dose adjustment before seven or preferably 14 days of treatment. If a dose adjustment is required, this is made by incremental steps of only 37.5 IU. In addition, each subsequent increase may only be effected after seven days of treatment at a given dose. The concept of this chronic low dose protocol is to find the threshold amount of FSH necessary to promote unifolliculogenesis. Encouraging results have been published so far, showing that this approach reduces the mean number of preovulatory follicles, the average preovulatory E2 level and the size of the ovary at mid-luteal phase.

[0024] However, despite the use of the chronic low dose protocol, some treatment cycles still have to be cancelled due to an over-response (e. g. where there are more than 3 follicles with a mean diameter of 16 mm or more). In addition, the multiple pregnancy rate, although clearly improved when compared to the conventional protocol, is still higher than in spontaneous conception cycles i.e. 5-10% in induced ovulation as opposed to 1.5% in spontaneous cycles. This is due to the fact that development of a single pre-ovulatory follicle is obtained in only about two thirds to three quarters of the induced cycles and follicles having a mean diameter of 15 mm or less are usually not considered when assessing the number of pre-ovulatory follicles on the day of hCG administration (Buvat et al, FertiL Steril., 52: 553-559 (1989); Hamilton-Fairley et al, Human Reprod. 6: 1095-1099 (1991)). It is, however, not clear whether follicles with a mean diameter of 14 to 15 mm, or even less, on the day of hCG administration, will ovulate and lead to the release of a healthy fertilisable oocyte. Thus, it would be desirable to have improvements in FSH-induced follicular development treatment in which the rates of multiple pregnancy and cycle cancellation are reduced.

[0025] Antral follicle growth is induced by FSH. Continuously throughout life and up to the menopause, some follicles enter a growth phase which is interrupted by regression and atresia before reaching the full maturity stage of preovulatory status (Hillier, Hum. Reprod., 9: 181-191 (1994)). During the growth phase, most follicles could be rescued from atresia, provided that it is exposed to a sufficient concentration of FSH. The level of FSH required to prevent atresia and promote further growth of a follicle is called the "FSH threshold" level (Brown, Aus. NZJ Obstet. Gynecol., 18: 47-55 (1987). The FSH threshold level varies with time and, at a given time-point, the follicles which are currently in a growth phase have different FSH threshold levels. This is the rationale on which the "chronic low dose" protocol is based. A progressive and cautious increase in the dose of FSH is used for finding the threshold level of a minimal number of follicles, and hopefully achieving mono-ovulation.

[0026] It is known that LH also contributes to the phenomenon of follicle dominance and mono-ovulation. Indeed, although some LH is essential for E2 synthesis during follicular development, there is evidence that excessive exposure to LH will trigger follicular atresia and suppress granulosa cell proliferation. Developing follicles appear thus to have finite requirements for stimulation by LH, beyond which normal follicular development ceases. This is the "LH ceiling" concept (Hillier, Hum. Reprod., 9: 181-191 (1994)). It is believed that, at a given time-point, the follicles which are currently in a growth phase have different LH ceiling levels. It is suggested that the more mature follicles are more resistant to the atretic action of LH than less mature follicles.

[0027] Two cases of WHO group I anovulation treated by either FSH alone or hMG using a step-up protocol have been reported (Glasier et al, Journal of AEndocrinology, 119 A-159 (1988)). The "FSH alone" cycle had a much larger number of mature follicles than the hMG cycle, possibly supporting a role of LH in the atresia of secondary follicles. Afterwards two comparative studies were published. In a first cross-over study in 10 hypogonadotrophic hypogonadal women, a striking difference was recorded in terms of preovulatory E2 levels, but follicular count was not reported (Couzinet et al, J. Clin. Endocrinol. Metab. 66: 552-556 (1988)). A second cross-over study in 9 hypogonadotrophic hypogonadal women reported a mean number of follicles having a mean diameter of more than 16 mm on the day of hCG administration of 2.0 (0.7 in hMG-treated cycles and of 1.2 in FSH-treated cycles (Shoham et al, FertiL Steril., 55: 1051-1056 (1991)). No information is available on the number of smaller follicles.

[0028] More recently, the results of administering 150 IU hFSH (human FSH) and 75 IU r-hLH (recombinant human LH) to a single patient with unmeasurably low serum FSH, LH and E2 concentrations have been published (Hall et al, The Lancet, 344 (8918): 334-335 (1994)). Administration of r-hLH and r-hFSH caused E2 levels to be raised, and the total number of follicles of 10 mm or more in diameter to be reduced, as compared to administration of hFSH alone. However, the number of large follicles remained sufficiently high to suggest an unacceptably high multiple pregnancy rate.

[0029] A further study compared the effect of administering r-hLH (at a dose of either 300 IU/day or 750 IU/day) and r-hFSH to normal ovulatory women after treatment with FSH for stimulating multiple follicular development prior to intrauterine implantation (Sullivan et al, Journal of Clinical Endocrinology and Metabolism, 84,228-232, 1999)). The results indicate that serum E2 levels were raised in those women who received LH, although no measurements of the number and size of follicles were made and a multiple pregnancy occurred in the group receiving 750 IU/day of LH.

[0030] The literature documents other compositions that contain both FSH and LH activity, as well as use of FSH in combination with LH activity. For example, PCT application WO 00/67778, published Nov. 16, 2000, is directed to using LH or an equivalent amount of hCG in combination with FSH to induce follicular development in anovulatory women. More particularly, the '778 application discloses administering LH or "a biologically-active analogue thereof" in doses of 100 to 1500 IU per day (page 4, lines 26-29) and in FSH:LH ratios that range from 1:1.5 to 1:20 (id., lines 16-18).

[0031] U.S. Pat. No. 5,929,028 is directed to liquid formulations that contain one or more natural or recombinant gonadotropins, including FSH, LH, and hCG. The '028 patent discusses naturally derived compositions of human menopausal gonadotropin (hMG), which have FSH and LH activities in a ratio of approximately 1:1, but mentions no ratio of FSH to LH activity other than the 1:1 ratio of commercial hMG preparations.

[0032] Additionally, there are commercial formulations that contain both FSH and LH. Human-derived preparations are available containing 75 IU FSH with 75 IU LH activity (Pergonal, Humegon, Menogon, Repronex, and Menopur) and 75 IU FSH with 25 or 35 IU LH activity (Normegon and Pergogreen).

[0033] It is conventional wisdom, however, that "excessive" LH levels, albeit ill-defined, result in follicular atresia, suppression of granulosa cell proliferation, and premature luteinization. See, generally, Filicori, Fertil. Steril. 79: 253 (2003). Although recent work suggests otherwise, a notion persists in the field that LH activity levels must be within a certain range, and that levels below or above an "LH ceiling" impair normal follicle development. Shoham, Fertil. Steril. 70: 1170 (2002).

[0034] In summary, there is published evidence that supplementing FSH with LH activity during ovulation induction reduces the duration of treatment and the amount of gonadotropin used to achieve proper follicle development. Filicori et al. (1999), (2002b). On the other hand, the belief persists that "high" LH activity levels negatively impacts follicle development.

[0035] Despite the numerous advances in COS protocols there is a need for further improvement and to remove the occurrence of OHSS, to improve the subsequent implantation rates and to improve the convenience for the females undergoing assisted reproductive therapy as well as safety.

[0036] That belief has guided the conventional ovarian-stimulation paradigm, which involves administration of FSH throughout controlled ovarian stimulation. Exogenous LH activity is deemed unnecessary and even detrimental during the early to middle stages of follicular development. Accordingly, the traditional means of ovarian stimulation entail treatment with FSH alone, typically at 75-300 IU/day. In this traditional protocol, LH activity is administered to induce ovulation only after the follicle reaches a certain stage of development. Only recently has LH activity been administered throughout treatment, and the optimal amount and timing of LH activity that is effective in this context remains controversial.

[0037] In order for boys to develop normal fertility, both testicles need to be located outside of the body at a lower temperature in the scrotum. If one or both testicles remain at body temperature for prolonged periods of time, fertility may be compromised and the ability to produce functional sperm cells in adult life may be hampered. In order to reduce the negative impact on fertility by an undescended testicle, it is usually physically moved to the scrotum through an operation or by hormone treatment with hCG that cause the testicle(s) to move to the scrotum. hCG stimulates production of testicular steroid hormones by stimulating the Leydig cells to produce androgens. The exact mechanism of action of the increased levels of androgens in causing the testicule(s) to move to the scrotum is not known precisely.

[0038] The frequency of at least one undecended testicles among boys is about 3% of full-term and 30% of premature infant boys. However, during the first year of life the majority of testicles within the body arrive in the scrotum themselves (the majority within three months), making the true incidence of cryptorchidism around 1% overall. The effect of hCG is well documented but due to differences in patient age, treatment schedules, and possible inclusion of retractile testes, very divergent results have been reported and the true efficacy is not known. A number of different dosage schedules have been reported, ranging from 3-15 doses given twice a week (10 injections over 5 weeks is common). One of the most common schedules prescribes 250 IU/dose in young infants, 500 IU/dose in children 6 years or younger, and 1000 IU/dose in individuals older than 6 years.

[0039] Men with hypogonadotropic hypogonadism have an inability to carry out pituitary release of the gonadotropins LH and FSH. Various genetic defects may cause a defect in the hypothalamus resulting in a deficiency in the release of gonadotropin releasing hormone (GnRH), which in turn causes the pituitary to reduce release in FSH and LH. One such condition is the so-called Kallmann syndrome that affects approximately 1:10.000 males and 1:50.000 females. Apart from affecting the fertility, the main health problem to both men and women is oesteoporosis.

[0040] When levels of LH are low the androgen production in men is reduced and they are often infertile and show reduced male characteristics. Treatment is focused on restoring the deficient hormones. Males are administered hCG or testosterone. A number of different testosterone preparations are available; the more widely used ones only requires administering with monthly intervals. However, to induce sperm production and fertility in these men, it is required to with administration of hCG, because exogenously administered testosterone reaching the testicles via circulation seldom reaches intratesticular levels sufficient to cause sperm production. It appears more effective using hCG to stimulate the testicular androgen production sufficiently to provoke sperm production often in combination with FSH administration. Since sperm production from the spermatogonial stage to the fully mature spermatozoa takes 60 to 70 days it is often a lengthy process with multiple injections of hCG for initiation of sperm production.

SUMMARY OF THE INVENTION

[0041] The present inventors have realized that a modified mammalian CG or LH, e.g. human CG or human LH, that agonize and activate the LH receptor in a mammal and provides a biological body composition or concentration of the mammalian CG or LH, e.g. human CG or human LH,

[0042] a) sufficient to drive an antral follicle from about 5-6 mm, such as from 10 mm in diameter up to about 30 mm in diameter in which a maturing oocyte can finalize the maturation to be ready for resumption of the meiosis,

[0043] b) sufficient to drive androgen production in the early adolescent, about 1 year after birth of a male offspring or in puberty for both female and male subjects,

[0044] c) sufficient to support steroid production in hypogonadothrophic hypogonadal for both female and male subjects,

[0045] d) sufficient to sustain progesterone in the pen-, in the ovulatoric- and the post ovulatoric-phase of a mammalian subject with the object regulating the endometrium and womb for allowing implantation of a mammalian blastocyst,

[0046] e) sufficient to sustain a progesterone in the pen-, in the ovulatoric- and the post ovulatoric-phase of a mammalian subject with the object of preparing the endometrium and womb for implantation,

[0047] f) sufficient to drive androgen production in hypogonadotropic hypogonadism men with the object of provoking sperm production and augment androgen production.

[0048] g) sufficient to drive androgen production in boys with cryptorchidism with the object of provoking testis relocation to the scrotum.

[0049] h) sufficient to drive progesterone production in women with recurrent pregnancy with the object of reducing the risk of losing the pregnancy. is important for improving the present platform of the ARTs (except for b).

[0050] Accordingly, in a broad aspect the present invention relates to a long acting biologically active luteinizing hormone (LH) compound comprising an LH agonist linked to a pharmaceutically acceptable molecule providing an in vivo plasma half-life of the LH agonist or LH compound which is increased substantially compared to the in vivo plasma half-life of the LH agonist administered in the same manner as the LH compound.

[0051] In another aspect the present invention relates to a long acting biologically active luteinizing hormone (LH) compound comprising an LH agonist linked to a pharmaceutically acceptable molecule providing an in vivo plasma half-life of the LH agonist or LH compound which is increased substantially compared to in vivo plasma half-life of endogenous chorionic gonadotropin (CG).

[0052] In a further aspect the present invention relates to a long acting biologically active luteinizing hormone (LH) compound comprising a mammal CG or analog thereof or a mammal LH or analog thereof linked to a pharmaceutically acceptable molecule selected from a molecule having binding to a mammal neonatal Fc receptor, transferrin and a CH.sub.3(CH.sub.2).sub.nCO--, wherein n is 8 to 22 and a polymer.

[0053] The LH agonist may be of mammalian origin, and may be selected from a mammal CG, such as human CG, and equine CG, such as horse CG; or a mammal LH, such as human LH, cow LH, pig LH, horse LH, sheep LH, dog LH, cat LH, and goat LH. The LH agonist may also be an analog of a mammalian LH agonist, and typically the analog has at least 80% identity to the corresponding mammalian sequence of the LH agonist, such as chorionic gonadotropin or luteinizing hormone, such as at least 85% identity, 90% identity, 95% identity, 98% identity, or at least 99% identity.

[0054] The LH agonist may also be of non-mammalian origin, and may be selected from small organic molecules, peptides, polypeptides and proteins.

[0055] The LH agonist is linked to another molecule, preferably a pharmaceutically acceptable molecule, and it is this modified compound that herein is referred to as an LH compound. The LH agonist may be linked to the pharmaceutically acceptable molecule in various ways as described in the prior art literature, such as without limitation chemical coupling through a bifunctional linker, gene technologically by coupling the N-terminal or C-terminal of the LH agonist, such as hCG or hLH, to the pharmaceutically acceptable molecule, such as albumin. In particular, the N-terminal of albumin, e.g. human albumin, can be coupled to the C-terminal of the alfa-chain of hCG or hLH, or the C-terminal of the beta-chain of hCG or hLH or the C-terminal of albumin, e.g. human albumin, can be coupled to the N-terminal of the alfa-chain of hCG or hLH, or the N-terminal of the beta-chain of hCG or hLH. A linker sequence can be inserted between the albumin and the hCG or LH chain. The two chains in hCG and/or hLH, i.e. alfa and beta chains, can be coupled together through a linker peptide, thus producing one polypeptide sequence, which in turn can be linked, such as through chemical linking or genetically linking, to the pharmaceutically acceptable molecule.

[0056] The LH agonist may be linked to the pharmaceutically acceptable molecule through a stable linker or a more labile linker. Several linkers are known in the art, including bifunctional PEG molecules (e.g. see Paige et.al Pharmaceutical Research, vol. 12, no. 12, 1995), hydrolysable linkers (Shechter et al Bioconjugate Chem. 2005, 16, 913-920 and International Journal of Peptide Research and Therapeutics, Vol. 13, Nos. 1-2, June 2007 and WO2009095479), PDPH and EMCH see e.g. in WO2010092135. In the special case where chemical conjugation (linking of two or more molecules) of the LH agonist, such as hCG, to the pharmaceutically acceptable molecule, strongly reduce the functional LH activity it may be preferable to use a more labile linker that can release the functional LH agonist.

[0057] The LH agonist may be glycosylated in which case linking to the pharmaceutically acceptable molecule may be through such sugar moiety, or the sugar moiety may be inserted and used to create a link between the LH agonist and the pharmaceutically acceptable molecule.

[0058] The LH agonist may be linked to one or more pharmaceutically acceptable molecule(s) or one pharmaceutically acceptable molecule may be linked to one or more LH agonist(s), typically the LH agonist is linked to one or two pharmaceutically acceptable molecule(s), preferably one pharmaceutically acceptable molecule. For instance, one hCG is linked to one albumin, e.g. human albumin or modified albumin.

[0059] A further advantage of the LH compound of the present invention is that the pharmaceutically acceptable molecule provides a serum concentration of the LH agonist or LH compound sufficient to support the formation and maintenance of Corpus Luteum/corpora lutea (CL). Such advantage is obtained when an injection of the LH compound is given during the follicular phase of the menstrual cycle in connection with follicle stimulating hormone (FSH) treatment, preferably 5-10 days after initiation of FSH treatment.

[0060] A still further advantage of the LH compound of the present invention is that the pharmaceutically acceptable molecule provides a concentration of the LH agonist or LH compound to stimulate sufficient progesterone release from CL. Such advantage is obtained after an injection of the LH compound during the follicular phase of the menstrual cycle in connection with FSH treatment, preferably 5-10 days after initiation of FSH treatment or in connection with ovulation induction or in connection with embryo transfer or sometime in the luteal or gestational phases.

[0061] A further aspect of the present invention concerns a pharmaceutical composition comprising the LH compound, and optionally a pharmaceutically acceptable carrier or excipient. Such composition may comprise one or more LH compounds.

[0062] A still further aspect of the present invention relates to an LH compound of the present invention, for use in infertility treatment of a mammalian subject, such as assisted reproduction technologies treatment, e.g. IVF or ICSI treatment, or maldecensus of the testis or for the use of augmenting steroid production in hypoganadotropic hypogonadism. Typically, LH compound is for use in promoting fertility of a mammalian subject.

[0063] The present invention also concerns a method of infertility treatment of a mammalian subject comprising administering to a mammal in need thereof the LH compound of the present invention.

[0064] Moreover, the present invention concerns a method of promoting fertility of a mammalian subject comprising administering to a mammal in need thereof the LH compound of the present invention.

[0065] Furthermore, the present inventors have realized that a long-acting modified LH comprising a mammalian LH or analog thereof linked to a pharmaceutically acceptable molecule, e.g. human LH linked to e.g. fused to albumin, or fused to an Fc fragment of a mammalian antibody, or a variant of an Fc fragment of a mammalian antibody or conjugated to an acylation group or PEG, that agonize and activate the LH receptor in a mammal and provides an in vivo plasma half-life of the mammalian LH or analog thereof, or the modified LH which is from 2 to 48 hours, typically from 4 to 28 hours, such as 6-8 hours in a mammal. The modified LH either given in the follicular phase or as a luteal phase support is believed to improve safety, treatment outcome and patient convenience. This long-acting modified mammalian LH of the present invention with the specified in-vivo half-life is particularly useful in combination with FSH.

[0066] Accordingly, a further aspect of the present invention relates to a pharmaceutical composition comprising the modified LH of the present invention and an FSH or a molecule having FSH activity. The pharmaceutical composition may be one composition comprising both the modified LH and the FSH or the molecule having FSH activity, or may be a kit of parts comprising the modified LH and the FSH or the molecule having FSH activity in separate compositions, wherein such compositions may be administered simultaneously, sequentially, or separately.

[0067] The present invention also concerns a modified LH comprising a mammalian LH or analog thereof linked to a pharmaceutically acceptable molecule providing an in vivo plasma half-life of the mammalian LH or analog thereof, or the modified LH which is from 2 to 48 hours in a mammal, for use in combination with an FSH or a molecule having FSH activity for simultaneous, sequential or separate use to induce follicular development, such as paucifolliculogenesis or unifolliculogenesis, in anovulatory treatment of a mammalian female subject or induce COS in the follicular phase of the menstrual cycle of a mammalian female subject.

[0068] The present invention also concerns a method of inducing follicular development, such as paucifolliculogenesis or unifolliculogenesis, in anovulatory treatment of a mammalian female subject or induce COS in the follicular phase of the menstrual cycle of a mammalian female subject comprising administering to a mammal in need thereof an effective amount of the modified LH comprising a mammalian LH or analog thereof linked to a pharmaceutically acceptable molecule providing an in vivo plasma half-life of the mammalian LH or analog thereof, or the modified LH which is from 2 to 48 hours in a mammal, simultaneous, sequential or separate in combination with an FSH or a molecule having FSH activity.

[0069] In a further aspect the present invention relates to administering a LH compound during the first 12 weeks of gestation to women with recurrent pregnancy loss in order to enhance progesterone output by the CL and enhance the rate of deliveries of children.

[0070] Accordingly, in a further aspect the present invention relates to a modified LH comprising a mammalian LH or analog thereof linked to a pharmaceutically acceptable molecule providing an in vivo plasma half-life of the mammalian LH or analog thereof, or the modified LH which is from 2 to 48 hours in a mammal, typically from 4 to 28 hours, such as 6-8 hours. As stated above such modified LH is particularly useful in combination with FSH treatment.

[0071] The mammalian LH may be selected from a mammal LH, such as primate LH (e.g. abe or monkey LH), human LH, and horse LH. The LH may also be an analog of a mammalian LH, and typically the analog has at least 80% identity to the corresponding mammalian sequence of the LH, such as at least 85% identity, 90% identity, 95% identity, 98% identity, or at least 99% identity.

[0072] The mammalian LH is linked to another molecule, preferably a pharmaceutically acceptable molecule, and it is this modified LH that herein is referred to as a modified LH. The mammalian LH may be linked to the pharmaceutically acceptable molecule in various ways as described in the prior art literature, such as without limitation chemical coupling through a bifunctional linker, gene technologically by coupling the N-terminal or C-terminal of the LH, such as hLH, to the pharmaceutically acceptable molecule, such as albumin. In particular, the N-terminal of albumin, e.g. human albumin, can be coupled to the N-terminal of the alfa-chain of hLH, or the C-terminal of the beta-chain of hLH. The two chains in hLH, i.e. alfa and beta chains, can be coupled together through a linker peptide, thus producing one polypeptide sequence, which in turn can be linked, such as through chemical linking or genetically linking, to the pharmaceutically acceptable molecule.

[0073] The mammalian LH may be linked to the pharmaceutically acceptable molecule through a stable linker or a more labile linker. Several linkers are known in the art, including bifunctional PEG molecules (e.g. see Paige et.al Pharmaceutical Research, vol. 12, no. 12, 1995), hydrolysable linkers (Shechter et al Bioconjugate Chem. 2005, 16, 913-920 and International Journal of Peptide Research and Therapeutics, Vol. 13, Nos. 1-2, June 2007 and WO2009095479), PDPH and EMCH see e.g. in WO2010092135. In the special case where chemical conjugation (linking of two or more molecules) of the mammalian LH, such as hLH, to the pharmaceutically acceptable molecule, strongly reduce the functional LH activity it may be preferable to use a more labile linker that can release the mammalian LH.

[0074] The mammalian LH may be glycosylated in which case linking to the pharmaceutically acceptable molecule may be through such sugar moiety or the sugar moiety may be inserted and used to create a link between the LH agonist and the pharmaceutically acceptable molecule.

[0075] The mammalian LH may be linked to one or more pharmaceutically acceptable molecule(s) or one pharmaceutically acceptable molecule may be linked to one or more mammalian LH, typically the mammalian LH is linked to one to five, such as one or two pharmaceutically acceptable molecule(s). For instance, one hLH is linked to one albumin, e.g. human albumin or modified albumin.

[0076] A further aspect of the present invention concerns a pharmaceutical composition comprising the modified LH of the present invention, and optionally a pharmaceutically acceptable carrier or excipient. Such composition may comprise one or more modified LH.

[0077] A still further aspect of the present invention provides a method for assisted reproductive therapy in a female human, said method comprising

[0078] a. starting stimulation by administering FSH on cycle day 1-3 of a menstrual cycle,

[0079] b. administering a GnRH antagonist from day 4-7 of the stimulation until ovulation triggering,

[0080] c. providing an LH agonist to said female by administering at least one dosage of an LH agonist in the period from day 1-9 of the stimulation, said dosage being sufficient to stimulate follicle development until ovulation triggering,

[0081] d. discontinuing administration of FSH when at least one follicle has a diameter of 12-14 mm,

[0082] e. inducing ovulation with at least one dosage of a GnRH agonist when at least one follicle has a diameter of at least 15 mm.

[0083] The stimulation protocols of the present invention seek to improve known methods by discontinuing FSH administration when a suitable number of follicles have been recruited and by switching at this point to hCG (or LH) administration in order to ensure maturation of the largest follicles, while the more immature follicles do not develop further. It is known in the art that a high number of immature follicles at ovulation increase the risk of OHSS.

[0084] The risk of OHSS is further reduced by inducing ovulation by use of a GnRH agonist trigger shot thus obviating the need for administrating a high dosage of hCG to provoke ovulation.

[0085] In preferred embodiments of the invention, the hCG administered during the follicular stimulation is a long-acting hCG or long-acting LH. The use of long-acting hCG or LH has several advantages. Obviously there is increased patient compliance as the number of injections is reduced. It is also expected that this could reduce the risk of

[0086] OHSS even further, as there is no risk of accumulation of hCG or LH when the protein is administered only once or twice during the follicular phase.

[0087] It is also conceivable that the long-acting hCG or LH may be administered as a single dose during one of the very first days of the stimulation protocol. When making a bolus injection of a drug, such as a recombinant protein, there is almost always an initial surge in the serum level after which the serum level drops to a lower level which steadily decreases according to the serum half-life of the drug. During the initial days of the stimulation protocol the receptor for hCG or LH is not yet active on the granulosa cells, while they are constitutively expressed on the surrounding theca cells. The LH activity provided at this stage of follicular development is likely to enhance the androgen output by the theca cells. These androgens are likely to affect the granulosa cells to enhance their FSH receptor expression and thereby make them more sensitive to the exogenous administered FSH and thereby improve follicular health (Eilso Nielsen M, Rasmussen I A, Kristensen S G, Christensen S T, Mollg{dot over (a)}rd K, Wreford Andersen E, Byskov A G, Yding Andersen C: Expression of Androgen-receptor mRNA in granulosa cells from human small antral follicles and the corresponding follicular fluid concentrations of androgens are positively correlated to granulosa cell FSH receptor mRNA expression. Mol. Hum. Reprod. 2011;17:63-70. PMID: 20843821). This means that the surge resulting from a bolus hCG injection can improve granulosa cell responsiveness and be terminated before the receptor becomes active on the granulosa cells. Hereafter, a more suitable and stable serum level of hCG can be obtained for maturing and developing the right follicles.

[0088] In some embodiments of the invention, the long-acting hCG or LH administered during the stimulation phase is sufficient only to support the follicle development until ovulation. In other embodiments, the dosage is also sufficient to provide support for the luteal phase according to the invention.

[0089] In an alternative protocol, ovulation induction is performed by administering a relatively low dosage of hCG, the dosage being 2000 IU or less, such as 1500 IU or less, for example 1000 IU or less, such as 750 IU, 500 IU, or 200 IU. Preferably the dosage is between 1000 and 2000 IU. Ovulation induction can also involve co-administration of a GnRH agonist. This alternative protocol also carries an inherently low risk of OHSS, because the dosage of hCG for ovulation induction is significantly lower than the dosage used for ovulation induction in the prior art.

[0090] In another aspect the invention relates to a method for providing luteal support to a female undergoing assisted reproductive therapy, said method comprising administering an LH agonist during the luteal phase at least until 2 weeks after ovulation.

[0091] According to this aspect, the LH agonist is administered from around the time of ovulation or oocyte pick up and continued during the luteal phase. Preferably, the LH agonist administration continues until at least 28 days after ovulation.

[0092] Preferably the LH agonist is administered during the luteal phase is LH or an LH analogue or LH variant as described herein.

[0093] LH and hCG as well as long-acting versions of these hormones may be administered in dosages and at intervals as described in the present application. The female may be one who has undergone assisted reproductive therapy according to the invention. The female may also be a female who has undergone controlled ovarian stimulation according to the prior art, including females who have received a bolus injection of hCG for triggering of ovulation. The latter females will have sufficient serum levels of hCG to provide luteal support for approximately one week after ovulation triggering.

[0094] In preferred embodiments the LH agonist is administered until at least 6 weeks after ovulation, such as 7 weeks, for example 8 weeks, such as 9 weeks, for example 10 weeks after fertilisation.

[0095] The long-acting hCG or long-acting LH may be administered every 2nd day, such as every 3rd day, for example every 4th day, such as every 5th day, for example every 35 6th day, such as every 7th day, for example every 8th day, such as every 9th day, for example every 10th day during the ovulation induction phase and/or the luteal phase and/or gestational phase.

[0096] The long-acting hCG or long-acting LH may be also administered every 14th day, such as every 21st, for example every month or even less frequently during the ovulation induction phase and/or the subsequent luteal phase and/or the subsequent gestational phase.

[0097] The controlled ovarian stimulation methods of the present invention generally result in improvement involving one or more of the following parameters: biochemical pregnancies, live births, improved implantation rates, improved retention rates, reduced miscarriage rates, reduced ectopic miscarriage rates, reduced occurrence of OHSS, and improved convenience due to reduced or no need for progesterone administration in the luteal phase.

[0098] The follicular stimulation protocols of the present invention may be used in conjunction with in vitro fertilization (IVF), through intra cytoplasmatic sperm injection (ICSI), intra uterine insemination (IUD, in vitro maturation (IVM), or other forms derived thereof such as ovarian ovulation alone.

[0099] The methods for providing luteal support may be used in conjunction with any of the stimulation protocols mentioned above or in conjunction with any attempted pregnancy or actual pregnancy in which there is a need for stimulating the progesterone level.

[0100] In a further aspect the invention relates to a method for inducing folliculogenesis and supporting subsequent embryo implantation comprising:

[0101] a. starting stimulation by administering FSH on cycle day 1-3 of a menstrual cycle,

[0102] b. administering a GnRH antagonist from day 4-7 of the stimulation until ovulation,

[0103] c. administering an LH agonist by administering at least one dosage of hCG in the period from day 1-9, said at least one dosage of hCG being sufficient to stimulate follicle development until ovulation,

[0104] d. discontinuing administration of FSH when at least one follicle has a mean diameter of 12-14 mm, and

[0105] e. providing luteal support by administering one or more dosages of an LH agonist sufficient to provide a serum progesterone concentration of at least 20 nmol/L 7-10 days after ovulation. This method may result in monofolliculogenesis or paucifolliculogenesis. The method may also be used for stimulation of follicle development in anovulatory women. The method may also involve ovulation induction using hCG trigger short or GnRH trigger shot as part of a COS protocol.

[0106] A further aspect of the present invention is to provide a LH compound to such boys in order to provide a stable concentration of androgens and reduce the number of injection given to these young boys.

[0107] A still futher aspect of the present invention is to provide a LH compound to such men with hypogonadotropic hypogonadism in order to provide a stable concentration of hCG that can provoke testicular androgen production sufficiently to cause sperm production and to maintain androgens at an acceptable level.

BRIEF DESCRIPTION OF THE DRAWINGS

[0108] FIG. 1a. A schematic drawing of a typical short antagonist protocol with hCG/GnRHa triggering as known in the prior art. The drawing schematically shows daily administration of FSH from day 1 of the menstrual cycle and until ovulation triggering. A daily dose of GnRH antagonist is administered starting approximately on day 6 and until ovulation triggering. Ovulation is triggered by a bolus shot of hCG or GnRH agonist when follicles have reached a size of 16-18 mm diameter. 36 hrs later oocytes are harvested. Two days later, one or more fertilized embryos are transferred back to the uterus. In order to provide luteal support progesterone is administered e.g. vaginally or intramuscularly.

[0109] FIG. 1b. A schematic drawing of an alternative short antagonist protocol with hCG/GnRH agonist triggering. The protocol is based on administration of a long-acting FSH (Corifollitropin) on day one of the protocol. From day 6 or 7 and onwards daily dosages of recombinant or urinary FSH is administered to supplement the corifollitropin. GnRH antagonist, triggering and luteal support as for FIG. 1a.

[0110] FIG. 2a. An exemplary COS protocol of the invention, wherein the daily dosages of FSH are replaced by one bolus injection of a long-lasting FSH, such as Corifollitropin. Follicle development is supported by administration of long-acting hCG (S-hCG), which is administered at approximately day 6 depending on follicle diameter. A GnRH antagonist is administered from around day 6 of the protocol, and ovulation is induced with a GnRH agonist. Luteal support can be provided by progesterone or by using luteal methods of the invention (FIG. 4a or 4b).

[0111] FIG. 2b. A schematic illustration of one exemplary controlled ovarian stimulation (COS) protocol according to the invention. FSH is administered as daily dosages of urinary or recombinant FSH. FSH administration is discontinued from around day 6, corresponding to a follicle size of approximately 12-14 mm. A GnRH antagonist is administered as daily dosages starting day 6 and until ovulation triggering. On day 6 a bolus shot of long-acting (long-lasting) hCG or long-acting LH is administered. Ovulation is triggered by administration of a GnRH agonist. Luteal support is not shown and may be achieved by administration of progesterone as in FIG. 1a, or through the administration of one or more subcutaneous injections of hCG or long-acting hCG or LH or long-acting LH during the luteal phase.

[0112] FIG. 2c. An exemplary COS protocol of the invention, wherein follicle recruitment is supported by daily dosages of recombinant or urinary FSH. Follicle development is supported by daily dosages of hCG or LH from about day 6, corresponding to a follicle size of approximately 12-14 mm. A GnRH antagonist is administered as daily dosages starting day 6 and until ovulation triggering. Ovulation is triggered by administration of a GnRH agonist. Luteal support is not shown and may be achieved by administration of progesterone as in FIG. 1a, or through the administration of one or more subcutaneous injections of hCG or long-acting hCG or LH or long-acting LH during the luteal phase. In case of pregnancy, the luteal support may be continued until gestational week 5-10.

[0113] FIG. 3a. An exemplary COS protocol of the invention. Compared to the protocol in FIG. 2b, administration of long-acting (long lasting) hCG or LH is continued into the luteal phase to provide luteal support. In case of pregnancy, the luteal support may be continued until gestational week 5-10.

[0114] FIG. 3b. An exemplary COS protocol of the invention. Compared to the protocol in FIG. 3a, the hCG or LH is administered as daily injections of recombinant or urinary hCG or LH. The hCG may be replaced by equivalent LH dosages. In case of pregnancy, the luteal support may be continued until gestational week 5-10.

[0115] FIG. 3c. An exemplary COS protocol of the invention. Compared to the protocol in FIG. 3b, hCG/LH administration starts already from day 2 of the menstrual cycle as daily injections of hCG or LH. Alternatively the daily dosages may be replaced by equivalent dosages of one or more injections of long-acting hCG or long-acting LH. In case of pregnancy, the luteal support may be continued until gestational week 5-10.

[0116] FIG. 4a: An exemplary protocol of the invention illustrating luteal support. Irrespective of the stimulation protocol (not shown), daily dosages of urinary or recombinant LH or hCG provide luteal support during the luteal phase starting approximately day 2 after ovulation. The luteal support may be continued until gestational week 5 if the female is pregnant, for example until gestational week 10.

[0117] FIG. 4b. An exemplary protocol of the invention illustrating luteal support. Irrespective of the stimulation protocol (not shown), one or more doses of long-acting LH or long action hCG administered every 2 to 7 days provide luteal support during the luteal phase starting approximately day 2 after ovulation. The luteal support may continue until gestational week 5 if the female is pregnant, for example until gestational week 10.

[0118] FIG. 5: ClustalX2 alignment of gonadotropin alpha chains from different species. Sequences were retrieved from the Uniprot database. The Uniprot ID and accession number is given:

TABLE-US-00001 Human GLHA_HUMAN P01215 (SEQ ID NO: 1) Mouse GLHA_MOUSE P01216 (SEQ ID NO: 2) Rat GLHA_RAT P11962 (SEQ ID NO: 3)

[0119] Fully conserved residues are marked with black background and the corresponding amino acid residue shown in capital below the alignment. Semi-conserved residues are shown with grey background and are marked with a lower case below the alignment.

[0120] Alignment performed with ClustalX2 (Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., Higgins, D. G. (2007) Clustal W and Clustal X version 2.0. Bioinformatics, 23:2947-2948.)

[0121] FIG. 6: ClustalX2 alignment of Luteinizing hormone beta chains from various species:

Sequences were retrieved from the Uniprot database. The Uniprot ID and accession number is aiven

TABLE-US-00002 Human: LSHB_HUMAN P01229 (SEQ ID NO: 4) Mouse: LSHB_MOUSE O09108 (SEQ ID NO: 5) Rat: LSHB_RAT P01230 (SEQ ID NO: 6) Gorilla: LSHB_GORGO Q2Q1P1 (SEQ ID NO: 7) Chimpanzee: LSHB_PANTR Q2Q1P2 (SEQ ID NO: 8)

[0122] FIG. 7: ClustalX2 alignment of Follicle stimulating hormone beta chains from various species. Sequences were retrieved from the Uniprot database. The Uniprot ID and accession number is given:

TABLE-US-00003 Follitropin subunit beta Human: FSHB_HUMAN P01225 (SEQ ID NO: 10) Mouse: FSNB_MOUSE Q60687 (SEQ ID NO: 11) Rat: FSHB_RAT P18427 (SEQ ID NO: 12) Gorilla: FSHB_GORGO A1BN60 (SEQ ID NO: 13) Chimpanzee: FSHB_PANTR Q2PUH2 (SEQ ID NO: 14)

[0123] FIG. 8a: Non-reducing SDS PAGE of Conjugate) with rHA and hCG as controls.

[0124] FIG. 8b: Reducing SDS PAGE of Conjugate) with rHA and different concentrations of hCG as controls.

[0125] FIG. 8c: SEC-HPLC Analysis of purified Conjugate).

[0126] FIG. 9a: Non-reducing SDS PAGE of Conjugate3 with rHA and hCG as controls.

[0127] FIG. 9b: Reducing SDS PAGE of Conjugate3 with rHA and different concentrations of hCG as controls.

[0128] FIG. 9c: SEC-HPLC Analysis of purified Conjugate3.

[0129] FIG. 10a: Non-reducing SDS PAGE of Conjugate4 with rHA and hCG as controls.

[0130] FIG. 10b: Reducing SDS PAGE of Conjugate4 with rHA and different concentrations of hCG as controls.

[0131] FIG. 10c: SEC-HPLC Analysis of purified Conjugate4.

[0132] FIG. 11 a: Non-reducing SDS PAGE of Conjugate3V1 with K473P-rHA and hCG as controls.

[0133] FIG. 11b: Reducing SDS PAGE of Conjugate3V1 with K573P-rHA and different concentrations of hCG as controls.

[0134] FIG. 11c: SEC-HPLC Analysis of purified Conjugate3V1.

[0135] FIG. 12a: Non-reducing SDS PAGE of Conjugate4V1 with K573P-rHA and hCG as controls.

[0136] FIG. 12b: Reducing SDS PAGE of Conjugate4V1 with K573P-rHA and different concentrations of hCG as controls.

[0137] FIG. 12c: SEC-HPLC Analysis of purified Conjugate4V1.

[0138] FIG. 13: Confirmation of amplified single gene vectors prior to transfection s.

[0139] FIG. 14: Western Blot of Product 1-10 using anti-HSA (human serum albumin). under non-reducing (A) and reducing (B) conditions

[0140] FIG. 15: Western Blot of Product 1-10 using anti-gonadotropin common .alpha.-subunit under non-reducing (A) and reducing (B) conditions.

[0141] FIG. 16: Western Blot of Product 1-10 using anti-hCG .beta.-subunit under non-reducing (A) and reducing (B) conditions.

[0142] FIG. 17: SDS PAGE (non-reducing (A) and reducing (B)) analysis of products 1-10.

[0143] FIG. 18: SDS PAGE (non-reducing (A) and reducing (B)) analysis of products 11-12.

[0144] FIG. 19a: Measurement of in vitro activity of hCG, Conjugate3 and Conjugate4.

[0145] FIG. 19b: Measurement of in vitro activity of hCG, Conjugate3v1, Conjugate4V1, Product 11 and Product 12.

[0146] FIG. 19c: Measurement of in vitro activity of hCG, Product 2, Product 3, Product 4 and Product 5.

[0147] FIG. 19d: Measurement of in vitro activity of hCG, Product 7, Product 8, Product 9 and Product 10.

[0148] FIG. 20a: Measurement of in vivo activity of hCG after four daily doses.

[0149] FIG. 20b: Measurement of in vivo activity of hCG and Conjugate3 after four daily doses.

[0150] FIG. 20c: Measurement of in vivo activity of hCG and Conjugate3 after four daily doses.

[0151] FIG. 20d: Measurement of in vivo activity of hCG, Conjugate3 and Conjugate4 after four daily doses.

[0152] FIG. 20e: Measurement of in vivo activity of hCG, Conjugate3V1 and Conjugate4V1 after four daily doses.

[0153] FIG. 20f: Measurement of in vivo activity of hCG, Product 2, Product 3 and Product 8 after four daily doses.

[0154] FIG. 20g: Measurement of in vivo activity of hCG, Product 11 and Product 12 after four daily doses.

[0155] FIG. 20h: Measurement of in vivo activity of hCG, Product 4, Product 5 and Product 10 after four daily doses.

[0156] FIG. 20i: Measurement of in vivo activity of hCG and Product 7 after four daily doses.

[0157] FIG. 20j: Measurement of in vivo activity of hCG, Conjugate3, Product 11 and Product 12 after a single bolus injection on day 1.

[0158] FIG. 21a: Pharmacokinetic data of hCG, Conjugate3 and Conjugate3V1 in hypophysectomized male rats (linear scale).

[0159] FIG. 21b: Pharmacokinetic data of hCG, Conjugate3 and Conjugate3V1 in hypophysectomized male rats (logarithmic scale).

[0160] FIG. 21c: Standard curves of hCG, Conjugate3 and Conjugate3V1.

[0161] FIG. 21d: Calculation of terminal half-life for hCG, Conjugate3 and Conjugate3V1 in hypophysectomized male rats.

[0162] FIG. 22a: Pharmacokinetic data of hCG, Conjugate4V1, Product 11 and Product 12 in hypophysectomized male rats (linear scale).

[0163] FIG. 22b: Pharmacokinetic data of hCG, Conjugate4V1, Product 11 and Product 12 in hypophysectomized male rats (logarithmic scale).

[0164] FIG. 22c: Standard curves of hCG, Conjugate4V1, Product 11 and Product 12.

[0165] FIG. 22d: Calculation of terminal half-life for hCG, Conjugate4V1, Product 11 and Product 12 in hypophysectomized male rats.

[0166] FIG. 22e: Pharmacokinetic data of Product 7 and Product 10 in hypophysectomized male rats (linear scale).

[0167] FIG. 22f: Pharmacokinetic data of Product 7 and Product 10 in hypophysectomized male rats (logarithmic scale).

[0168] FIG. 22g: Standard curves of Product 7 and Product 10.

[0169] FIG. 22h: Calculation of terminal half-life for Product 7 and Product 10 in hypophysectomized male rats.

[0170] FIG. 23a: Pharmacokinetic data of hCG, LH,Conjugate3, Conjugate3V1, Product 2, Product 3 and Product 7 in normal adult male rats (linear scale).

[0171] FIG. 23b: Pharmacokinetic data of hCG, LH, Conjugate3, Conjugate3V1, Product 2, Product 3 and Product 7 in normal adult male rats (logarithmic scale).

[0172] FIG. 23c: Standard curves of LH and Product 7.

[0173] FIG. 23d: Calculation of terminal half-life for hCG, LH, Conjugate3, Conjugate3V1, Product 2, Product 3 and Product 7 in normal adult male rats.

DEFINITIONS

[0174] In the present context, the term "LH agonist" as used herein means a molecule of mammalian or non-mammalian origin that binds to and activates a luteinizing hormone receptor of a mammal, such as a human. The LH agonist may be a small organic molecule, a peptide, a polypeptide, a protein, and may be produced by synthetic methods, by recombinant means or be obtained from tissue or body fluids. The term "LH agonist" as used herein also includes pharmaceutically acceptable salts thereof. The term "LH agonist" includes hLH, hLH analogues and variants, and long-acting hLH. The term also includes hCG, hCG analogues and variants, and long-acting hCG.

[0175] As used herein, an "IU ratio" is the ratio of the number of IU of one component to the number of IU of another component. It is noteworthy that gonadotrophins may now be expressed in (mass/g) instead of biological IU. In this case, a conversion factor has to be used to translate the new value into IU. As used herein the in vivo plasma concentration in a mammal is measured by ELISA or another immunological method known to the person skilled in the art and expressed in IU per liter used interchangeable with IU/L.

[0176] In the present context, the term "a long acting biologically active luteinizing hormone (LH) compound" or "LH compound" (these terms are used interchangeable throughout the specification) as used herein means an LH agonist linked to a pharmaceutically acceptable molecule, such as an hCG or hLH with the pharmaceutically acceptable molecule bonded to it in order to modify the properties of said hCG or hLH. The term "LH compound" as used herein also includes pharmaceutically acceptable salts thereof.

[0177] In the present context, the term "a modified luteinizing hormone" or "modified LH" (these terms are used interchangeable throughout the specification) as used herein means a mammal LH linked to a pharmaceutically acceptable molecule, such as a hLH with the pharmaceutically acceptable molecule bonded to it in order to modify the properties of said hLH. The term "modified LH" as used herein also includes pharmaceutically acceptable salts thereof.

[0178] In the present context, the term "a molecule having binding to a mammal neonatal Fc receptor" as used herein means any pharmaceutically acceptable molecule having affinity to a mammal neonatal Fc receptor (FcRn), such as strong affinity, weak affinity or medium affinity. FcRn is active in adult epithelial tissue and expressed in the lumen of the intestines, pulmonary airways, nasal surfaces, vaginal surfaces, colon and rectal surfaces (U.S. Pat. No. 6,485,726). Fusion proteins comprised of FcRn binding partners (e.g., IgG, Fc fragments) can be effectively shuttled across epithelial barriers by FcRn, thus providing a non-invasive means to systemically administer a desired therapeutic molecule. Additionally, fusion proteins comprising an FcRn binding partner are endocytosed and protected by cells expressing the FcRn. Instead of being marked for degradation, these fusion proteins are recycled out into circulation again, thus increasing the in vivo half-life of these proteins. One approach to improve the efficacy of a therapeutic protein is to increase its serum persistence, thereby allowing higher circulating levels, less frequent administration and reduced doses. The half-life of an albumin fusion or conjugate or of an Fc fusion or conjugate depends in one instance on its pH-dependent binding to the FcRn. FcRn, which is expressed on the surface of endothelial cells, binds the albumin and/or the Fc in a pH-dependent manner and protects it from degradation. Some albumin variants or variants of Fc fragments that selectively bind stronger than the respective wild type to the FcRn at pH 6.0, but not pH 7.4, exhibit a longer terminal half-life in a variety of animal models. For the Fc containing molecules several mutations located at the interface between the CH2 and CH3 domains, such as T250Q/M428L (Hinton PR. et al., 2004. Engineered human IgG antibodies with longer serum half-lives in primates. J Biol Chem. 279(8):6213-6) and 252Y/S254T/T256E+H433K/N434F (Vaccaro C. et al., 2005. Engineering the Fc region of immunoglobulin G to modulate in vivo antibody levels. Nat Biotechnol. 23(10):1283-8), have been shown to increase the binding affinity to FcRn and the half-life of the Fc-variant containing molecule in vivo. However, there is not always a direct relationship between increased FcRn binding and improved half-life (Datta-Mannan A. et al., 2007. Humanized IgG1 Variants with Differential Binding Properties to the Neonatal Fc Receptor : Relationship to Pharmacokinetics in Mice and Primates. Drug Metab. Dispos. 35: 86-94). Variants of human albumin have in a similar way shown to increase the binding affinity to FcRn and the half-life of the albumin-variant containing molecule in vivo (cf. WO 2010/092135 and WO2011/051489).

[0179] In the present context, the term "an Fc fragment of a mammalian antibody" as used herein means a constant region, i.e. Fc fragment of a mammalian antibody or a fragment thereof wherein such mammalian antibody may be selected from IgM, IgG, IgA, IgD and IgE from a mammal, such as a primate, e.g. human, abe, or monkey; an equine, e.g. horse. A typical Fc fragment of a mammalian antibody is a recombinant Fc fragment of a human antibody, such as a recombinant Fc fragment of a human IgG antibody. The creation of fusion proteins comprised of immunoglobulin constant regions linked to a protein of interest, or fragment thereof, has been described (see, e.g., U.S. Pat. Nos. 5,155,027, 5,428, 130, 5,480,981, and 5,808,029). These molecules usually possess both the biological activity associated with the linked molecule of interest as well as the effector function, or some other desired characteristic, associated with the immunoglobulin constant region. Fusion proteins comprising an Fc portion of an immunoglobulin can bestow several desirable properties on a fusion protein including increased stability, increased serum half-life (see Capon et al. (1989) Nature 337:525) as well as binding to Fc receptors such as the neonatal Fc receptor (FcRn) (U.S. Pat. Nos. 6,086,875, 6,030,613, and 6,485,726).

[0180] In the present context, the term "a variant of an Fc fragment of a mammalian antibody" or "Fc variant" (used interchangeably throughout the present description) as used herein means the Fc fragment of a mammalian antibody, wherein one or more amino acid residues, such as 1-10 amino acid residues, of the Fc fragment have been substituted by other amino acid residues and/or wherein one or more amino acid residues, such as 1-10 amino acid residues, have been deleted from the Fc fragment and/or wherein one or more amino acid residues, such as 1-10 amino acid residues, have been added to the Fc fragment and/or wherein one or more amino acid residues, such as 1-10 amino acid residues, in the Fc fragment have been modified. Such addition or deletion of amino acid residues can take e.g. place at the N-terminal of the Fc fragment and/or at the C-terminal of the Fc fragment. Fc variant refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn (WO 97/34631). Native refers to an Fc that has not been modified by a human. WO 96/32478 describes exemplary Fc variants, as well as interaction with the salvage receptor. Thus, the term "Fc variant" in one embodiment comprises a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises sites that may be removed because they provide structural features or biological activity that are not required for the fusion molecules of the present invention. Thus, Fc variant comprises a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC). The Fc region of IgG can be modified according to well recognized procedures such as site directed mutagenesis and the like to yield modified IgG or Fc fragments or portions thereof that will be bound by FcRn. Such modifications include modifications remote from the FcRn contact sites as well as modifications within the contact sites that preserve or even enhance binding to the FcRn. For example the following single amino acid residues in human IgG1 Fc (Fcy1) can be substituted without significant loss of Fc binding affinity for FcRn: P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305A, T307A,L309A, Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, A330S, P331A, P331S, E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361 A, Q362A, Y373A, S375A D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A, and K447A, where for example P238A represents wild type proline substituted by alanine at position number 238. In addition to alanine, other amino acids may be substituted for the wild type amino acids at the positions specified above. Mutations may be introduced singly into Fc, giving rise to more than one hundred FcRn binding partners distinct from native Fc. Additionally, combinations of two, three, or more of these individual mutations may be introduced together, giving rise to hundreds more FcRn binding partners. Certain of the above mutations may confer new functionality upon the FcRn binding partner. For example, one embodiment incorporates N297 A, removing a highly conserved N-glycosylation site. The effect of this mutation is to reduce binding to immune effector cells and potentially decrease immunogenicity, thereby enhancing circulating half-life of the FcRn binding partner, and to render the FcRn binding partner incapable of binding to FcyRI, FcyRIIA, FcyRIIB, and FcyRIIIA, without compromising affinity for FcRn (Routledge et al. (1995) Transplantation 60:847; Friend et al. (1999) Transplantation 68:1632; Shields et al. (1995) J. Bioi. Chern. 276:6591). Additionally, at least three human Fc gamma receptors appear to recognize a binding site on IgG within the lower hinge region, generally amino acids 234-237. Therefore, another example of new functionality and potential decreased immunogenicity may arise from mutations of this region, as for example by replacing amino acids 233-236 of human IgG1 "ELLG" (SEQ ID NO:63) with the corresponding sequence from IgG2 "PVA" (with one amino acid deletion). It has been shown that FcyRI, FcyRII, and FcyRIII, which mediate various effector functions, will not bind to IgG1 when such mutations have been introduced (Ward and Ghetie (1995) Therapeutic Immunology 2:77 and Armour et al. (1999) Eur. J. Immunol. 29:2613). As a further example of new functionality arising from mutations described above, affinity for FcRn may be increased beyond that of wildtype in some instances. This increased affinity may reflect an increased "on" rate, a decreased "off" rate, or both an increased "on" rate and a decreased "off" rate. Mutations believed to impart an increased affinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al. (2001) J. Bioi. Chern. 276:6591). Furthermore, such variant(s) of the Fc fragment includes without limitation the "knob-into-hole" or "KnH" technology as described in for instance by Atwell et all J. Mol. Biol. (1997), 270, 26-35 and in Ridgway et al Protein Engineering, vol. 9, no. 7, pp 617-621, 1996. The term "knob-into-hole" or "KnH" technology as used herein refers to the technology directing the pairing of two polypeptides together in vitro or in vivo by introducing a protuberance (knob) into one polypeptide and a cavity (hole) into the other polypeptide at an interface in which they interact. For example, KnHs have been introduced in the Fc:Fc binding interfaces, CL:CHI interfaces or VH:VL interfaces of antibodies (e.g., US2007 /0178552, U.S. Ser. No. 12/811,207, US20100286374, WO96/027011, WO98/050431 and Zhu et al. (1997) Protein Science 6:781-788). This is especially useful in driving the pairing of two different heavy chains together during the manufacture of multispecific antibodies or heterodimeric Fc-fusion molecules. For example, multispecific antibodies having KnH in their Fc regions can further comprise single variable domains linked to each Fc region, or further comprise different heavy chain variable domains that pair with similar or different light chain variable domains. KnH technology can also be used to pair two different receptor extracellular domains together or any other polypeptide sequences that comprise different target recognition sequences (e.g., including affibodies, peptibodies and other Fc fusions). KnH technology can also be used to pair two different chains in e.g. gonadotropin molecules like FSH, LH, hCG or TSH in an Fc-fusion.

[0181] In the present context, the term "pharmaceutically acceptable salt" is intended to indicate salts which are not harmful to the mammalian subject to be treated. Such salts include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts. Acid addition salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric acids and the like. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids and the like. Further examples of pharmaceutically acceptable inorganic or organic acid addition salts include the pharmaceutically acceptable salts listed in J. Pharm. Sci. 66, 2, (1977) which is incorporated herein by reference. Examples of metal salts include lithium, sodium, potassium, magnesium salts and the like. Examples of ammonium and alkylated ammonium salts include ammonium, methylammonium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium, tetramethylammonium salts and the like.

[0182] In the present context, the term "chorionic gonadotropin" as used herein means chorionic gonadotropin of mammalian origin, e.g. primates such as human chorionic gonadotropin or equine chorionic gonadotropin such as horse chorionic gonadotropin, and recombinant chorionic gonadotropin, such as recombinant human chorionic gonadotropin, and analogues of such chorionic gonadotropins. As used herein "CG" and "chorionic gonadotropin" are interchangeable. When CG is an analogue of a chorionic gonadotropin of a mammal, such as hCG and recombinant hCG, said analogue is understood to be the compound obtained by substituting one or more amino acid residues in the CG, e.g. hCG, sequence with another natural or unnatural amino acid; and/or by adding one or more natural or unnatural amino acids to the CG, e.g. hCG, sequence; and/or by deleting one or more amino acid residue from the CG, e.g. hCG, sequence, wherein any of these steps may optionally be followed by further derivatization of one or more amino acid residues. In particular, such substitutions are conservative in the sense that one amino acid residue is substituted by another amino acid residue from the same group, i.e. by another amino acid residue with similar properties. Amino acids may conveniently be divided in the following groups based on their properties: Basic amino acids (such as arginine, lysine, histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine, cysteine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, proline, methionine and valine), aromatic amino acids (such as phenylalanine, tryptophan, tyrosine) and small amino acids (such as glycine, alanine, serine and threonine). Typically, the CG has at least 80% identity with hCG, and typically, has at least 20% of the CG in vivo activity of hCG.

[0183] In the present context, the term "luteinizing hormone" or "mammalian luteinizing hormone" as used herein means luteinizing hormone of mammalian origin, such as primates, e.g. human, or horse luteinizing hormone, and recombinant luteinizing hormone, such as recombinant human, horse, abe, or monkey luteinizing hormone, and analogues of such luteinizing hormones. As used herein "LH" and "luteinizing hormone" are interchangeable. When LH is an analogue of a luteinizing hormone of a mammal, such as hLH and recombinant hLH, said analogue is understood to be the compound obtained by substituting one or more amino acid residues in the LH, e.g. hLH, sequence with another natural or unnatural amino acid; and/or by adding one or more natural or unnatural amino acids to the LH, e.g. hLH, sequence; and/or by deleting one or more amino acid residue from the LH, e.g. hLH, sequence, wherein any of these steps may optionally be followed by further derivatization of one or more amino acid residues. In particular, such substitutions are conservative in the sense that one amino acid residue is substituted by another amino acid residue from the same group, i.e. by another amino acid residue with similar properties. Amino acids may conveniently be divided in the following groups based on their properties: Basic amino acids (such as arginine, lysine, histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine, cysteine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, proline, methionine and valine), aromatic amino acids (such as phenylalanine, tryptophan, tyrosine) and small amino acids (such as glycine, alanine, serine and threonine). Typically, the LH has at least 80% identity with hLH, and typically, has at least 20% of the LH in vivo activity of hLH. For purposes of the present invention, hLH, hLH analogues and variants, and long-acting hLH do not include hCG, hCG analogues and variants, and long-acting hCG.

[0184] In the present context, the term "follicular stimulating hormone" as used herein means follicular stimulating hormone of mammalian origin, such as human, equine, bovine, or porcine follicular stimulating hormone, and recombinant follicular stimulating hormone, such as recombinant human, equine, bovine, or porcine follicular stimulating hormone, and analogues of such follicular stimulating hormones. As used herein "FSH" and "follicular stimulating hormone" are interchangeable. For the stimulation of follicle growth the FSH may be derived exogenously or produced endogenously in the woman in amounts higher than normal, f.i. by providing clomiphene citrate that acts as an oestradiol antagonist on the pituitary.

[0185] When FSH is an analogue of a follicular stimulating hormone of a mammal, such as hFSH and recombinant hFSH, said analogue is understood to be the compound obtained by substituting one or more amino acid residues in the FSH, e.g. hFSH, sequence with another natural or unnatural amino acid; and/or by adding one or more natural or unnatural amino acids to the FSH, e.g. hFSH, sequence; and/or by deleting one or more amino acid residue from the FSH, e.g. hFSH, sequence, wherein any of these steps may optionally be followed by further derivatization of one or more amino acid residues. In particular, such substitutions are conservative in the sense that one amino acid residue is substituted by another amino acid residue from the same group, i.e. by another amino acid residue with similar properties. Amino acids may conveniently be divided in the following groups based on their properties: Basic amino acids (such as arginine, lysine, histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine, cysteine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, proline, methionine and valine), aromatic amino acids (such as phenylalanine, tryptophan, tyrosine) and small amino acids (such as glycine, alanine, serine and threonine). Recombinantly produced FSH or analog of FSH is typically derived from mammalian cell lines, such as a human or hamster cell lines, e.g. cell lines selected from CHO, BHK, and HEK. Typically, the FSH has at least 80% identity with hFSH, and typically, has at least 20% of the FSH in vivo activity of hFSH.

[0186] The term "a molecule having FSH activity" as used herein means a molecule that binds to and activates an FSH receptor when administered to a mammal e.g. such molecule may without limitation be selected from any one of small organic molecules.

[0187] In the present context, the term "a linker" as used herein means a valence bond or multifunctional moiety, such as a bifunctional moiety that separates the LH agonist, e.g. the mammalian LH, and the pharmaceutically acceptable molecule. The multifunctional moiety, such as bi- or trifunctional, is covalently linked to one or more LH agonist(s), such as one or more mammalian LH, and covalently linked to one or more pharmaceutically acceptable molecule(s) so as to create the LH compound, such as the modified LH. The linker may be stabile which means that no significant chemical reactions, e.g. hydrolysis, occurs at physiological conditions (e.g. temperature of 37.degree. Celcius and pH 7.4) over the time period of the treatment. This can be determined by stability studies known in the art. The linker may be labile which means that a chemical bond is broken, typically by hydrolysis, at physiologically relevant conditions (e.g. temperature of 37.degree. Celcius and pH 7.4). This can be determined by stability studies known in the art. The linker may be a chemical linker meaning that it is generated by organic chemistry outside a living cell. The linker may be a sugar moiety, such as a glycosylation on a protein, or may be chemically prepared and used to link the LH agonist, e.g. the mammalian LH, and the pharmaceutically acceptable molecule. The linker may be a disulphide bridge, such as a--S--S-- bond between two cysteine (Cys) amino acid residues in each of the LH agonist, e.g. the mammalian LH, and the pharmaceutically acceptable molecule. The linker may be a fused linker meaning that the LH compound, e.g. the modified LH, can be expressed in a living cell as one polypeptide or protein. The linker may be a hydrophilic linker that separates an LH agonist, e.g. the mammalian LH, and a pharmaceutically acceptable molecule with a chemical moiety, which comprises at least 5 non-hydrogen atoms where 30-50% of these are either N or 0. The linker may be hydrolysable as described in U.S. Pat. No. 6,515,100, U.S. Pat. No. 7,122,189, U.S. Pat. No. 7,700,551, WO2004089280, WO2006138572 and WO2009095479. Typical compounds useful as linkers in the present invention include those selected from the group having dicarboxylic acids, malemido hydrazides, PDPH, SPDP, LC-SPDP, GMBS, carboxylic acid hydrazides, and small peptides. More specific examples of compounds useful as linkers, according to the present invention, include: (a) dicarboxylic acids such as succinic acid, glutaric acid, and adipic acid; (b) maleimido hydrazides such as N-[maleimidocaproic acid]hydrazide (EMCH), N-[maleimidopropionic acid]hydrazide (MPH or BMPH), 4-[N-maleimidomethyl]cyclohexan-1-carboxylhydrazide, and N-[k-maleimidoundcanoic acid]hydrazide (KMUH), 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide (MPBH); (c) NHS-3-maleimidopropionate Succinimide ester (MPS-EDA); (d) PDPH linkers such as (3-[2-pyridyldithio] propionyl hydrazide) conjugated to sulfurhydryl reactive protein; (e) N-Succinimidyl 3-(2-pyridyldithio)-propionate (SPDP), (f) Succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate (LC-SPDP), (g) N-(.gamma.-Maleimidobutyryloxy)succinimide ester (GMBS), and (h) carboxylic acid hydrazides selected from 2-5 carbon atoms. Other non-peptide linkers are also possible. For example, alkyl linkers such as --NH--(CH2)m-C(O)--, wherein m is an integer selected from 2-20, could be used. These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl (e.g., C1 to C6) lower acyl, halogen (e.g., Cl, Br, I, F), CN, NH.sub.2, phenyl, etc. An exemplary non-peptide linker is a PEG linker. Additional linkers useful according to the present invention are described in U.S. Pat. No. 6,660,843. The LH compound of the present invention wherein the pharmaceutically acceptable molecule is fused to the LH agonist may optionally comprise at least one peptide linker. In one embodiment, the linker is comprised of amino acids linked together by peptide bonds, wherein the amino acids are selected from the twenty naturally occurring amino acids. In various embodiments the linker can comprise 1-5 amino acids, 1-10 amino acids, 1-20 amino acids, 10-50 amino acids, 50-100 amino acids, or 100-200 amino acids. In one embodiment the amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In one embodiment a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine. The linker in one embodiment can comprise the sequence Gn (equivalently, -(Gly)n-) (SEQ ID NO:64). The linker can in one embodiment comprise the sequence (GGS)n (SEQ ID NO:65) or (GGGGS)n (SEQ ID NO:66). In each instance, n is an integer, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples of linkers include, but are not limited to, GGG, SGGSGGS (SEQ ID NO:58), GGSGGSGGSGGSGGG (SEQ ID NO:59), GGSGGSGGSGGSGGSGGS (SEQ ID

[0188] NO:60), and GGGGSGGGGSGGGGS (SEQ ID NO:57). In one embodiment the linker is an 8-amino acid linker EFAGAAAV (SEQ ID NO:56).

[0189] Different techniques for linking two or more molecules together, such as the LH agonist, e.g. the mammalian LH, and the pharmaceutically acceptable molecule, and optionally via a multifunctional linker, such as bifunctional linker, are available in the prior art, and a suitable reference here is WO0158493, including all relevant documents listed and cited therein.

[0190] In the present context, the term "a pharmaceutically acceptable molecule" as used herein means a molecule selected from any one of small organic molecules, peptides, oligopeptides, polypeptides, proteins, receptors, glycosylations, sugars, polymers (e.g. polyethylene glycols, PEG), nucleic acids (e.g. DNA and RNA), hormones, which when linked to the LH agonist, e.g. the mammalian LH, increases the serum half-life of the LH agonist, e.g. the mammalian LH, or the LH compound, e.g. the modified LH. Typically, pharmaceutically acceptable molecules are without limitation albumin, such as human albumin, recombinant albumin, or polymer, such as PEG, e.g. PEG of a molecular weight of at least 10 kDa, such as from 10 kDa to 150 kDa. Furthermore, pharmaceutically acceptable molecules may be selected from a Fc fragment of a mammalian antibody, transferrin, albumin, such as human albumin, recombinant albumin, variants of albumin, CH.sub.3(CH.sub.2).sub.nCO--, wherein n is 8 to 22, or polymer, such as PEG, e.g. PEG of a molecular weight of at least 5 kDa, such as from 10 kDa to 150 kDa, typically 10 to 40 kDa.

[0191] In the present context, the term "in vivo plasma half-life" is used in its normal meaning, i.e., the time required for the amount of the LH agonist, e.g. mammalian LH, or LH compound, e.g. modified LH, in a biological system to be reduced to one half of its value by biological processes.

[0192] The term "serum half-life", which may be used interchangeably with "plasma half-life" or "half-life" is used in its normal meaning, i.e., the time required for the amount of the LH agonist, e.g. mammalian LH, or LH compound, e.g. modified LH, recombinant or urinary hCG or LH or FSH or long-acting hCG, long-acting LH or long-acting FSH in a biological system to be reduced to one half of its concentration. Thus as used herein, the "serum half-life" means the serum half-life in vivo. Determination of serum half-life is often more simple than determining functional half-life and the magnitude of serum half-life is usually a good indication of the magnitude of functional in vivo half-life. Preferably the serum half-life is measured in a mammal, more preferably in a species of Hominidae, such as Orang-utan, Chimpanzee or Gorillas, more preferably in humans. The serum half-lives mentioned in the present application are half-lives as determined in humans. An indication of the half-life or any change in half-life can also be obtained in rodents, such as mouse or rat or hamster. Furthermore half-life can be measured in larger mammals having a body weight in the same range as human beings or closer to human being body weight than rodents: preferably monkey, dog, pig, or cattle (calf). Gonadotropins which have a longer half life than recombinant or urinary gonadotropins (FSH, LH or hCG) are considered "long-acting" according to the present invention.

[0193] The term "increased" as used in connection with the plasma half-life is used to indicate that the relevant half-life of the LH compound, e.g. the modified LH, is statistically significantly increased relative to that of the LH agonist, e.g. the mammalian LH, as determined under comparable conditions. For instance the relevant half-life may be increased by at least about 25%, such as by at least about 50%, e.g., by at least about 100%, 150%, 200%, 250%, or 500%. Measurement of in vivo plasma half-life can be carried out in a number of ways as described in the literature. An increase in in-vivo plasma half-life may be quantified as a decrease in clearance or as an increase in mean residence time (MRT). LH compound, e.g. modified LH, of the present invention for which the clearance is decreased to less than 70%, such as less than 50%, such as less than 20%, such as less than 10% of the clearance of the LH agonist, e.g. mammalian LH, as determined in a suitable assay is said to have an increased in-vivo plasma half-life. LH compound, e.g. modified LH, of the present invention for which MRT is increased to more than 130%, such as more than 150%, such as more than 200%, such as more than 500% of the MRT of the LH agonist, e.g. the mammalian LH, in a suitable assay is said to have an increased in vivo plasma half-life. Clearance and mean residence time can be assessed in standard pharmacokinetic studies using suitable test animals. It is within the capabilities of a person skilled in the art to choose a suitable test animal for a given protein. Tests in human, of course, represent the ultimate test. Suitable test animals include normal, Sprague-Dawley male rats, mice and cynomolgus monkeys. Typically the mice and rats are injected in a single subcutaneous bolus, while monkeys may be injected in a single subcutaneous bolus or in a single iv dose. The amount injected depends on the test animal. Subsequently, blood samples are taken over a period of one to ten days as appropriate (depending on the sensitivity of the assay it may be as long as 30 days) for the assessment of clearance and MRT. The blood samples are conveniently analysed by ELISA techniques or other immunological techniques.

[0194] In the present context, the term "mammalian origin" as used herein means obtained from a mammal, thus an LH agonist of mammalian origin may for instance be a human CG or human LH obtained from tissue or blood of a mammal, or may be obtained by recombinant means, such as recombinant proteins, recombinant polypeptides, for instance an LH agonist of mammalian origin may be a recombinant mammalian CG or recombinant mammalian LH, for instance recombinant human CG or LH.

[0195] In the present context, the term "non-mammalian origin" as used herein means obtained from a source which is not a mammal, such as synthetic peptides, oligo peptides and polypeptides or small organic molecules, for instance an LH agonist of non-mammalian origin may be a small organic molecule or short peptide of 5 to 20 amino acids that binds and activates the LH receptor.

[0196] In the present context, the term "plasma concentration" as used herein means the concentration that can be measured in circulation at any given time after injection of the LH agonist.

[0197] In the present context, the term "an injection" as used herein means administration by the parenteral route such as by subcutaneous, intramuscular, intraperitoneal or intravenous injection by means of a syringe or other administration device.

[0198] In the present context, the term "FSH treatment" as used herein means standard follicular stimulating hormone treatment. FSH is required for follicular recruitment (i.e., the early growth of ovarian follicles) at the beginning of the spontaneous menstrual cycle, and it also supports mid- and late-stage folliculogenesis. FSH is administered therapeutically to induce folliculogenesis in anovulatory women and women undergoing COS. In traditional ovulatory stimulation methods, FSH is administered throughout treatment until the time that oocytes are retrieved.

[0199] In the present context, the term "analog" or "analogue" (used interchangeably throughout the present description) as used herein means a polypeptide or protein wherein one or more amino acid residues of the polypeptide or protein have been substituted by other amino acid residues and/or wherein one or more amino acid residues have been deleted from the peptide and/or wherein one or more amino acid residues have been added to the polypeptide or protein. Such addition or deletion of amino acid residues can take e.g. place at the N-terminal of the peptide and/or at the C-terminal of the peptide. A simple system may be used to describe analogues: for example, an hCG analogue comprising the mutation R133C designates an analogue wherein the naturally occurring R at position 133 of hCG has been substituted with C. Another example, a hLH analogue comprising the mutation L121C designates an analogue wherein the naturally occurring L at position 121 of hLH beta chain has been substituted with C Formulae of polypeptide or protein analogs are drawn using standard single letter abbreviation for amino acids used according to IUPAC-IUB nomenclature.

[0200] In the present context, the term "identity" as used herein refers to a relationship between the sequences of two or more proteins, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between proteins, as determined by the number of matches between strings of two or more amino acid residues. "Identity" measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms"). Identity of related proteins can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math., 48, 1073, (1988). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are described in publicly available computer programs. Preferred computer program methods to determine identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res., 12, 387, (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol., 215, 403-410, (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well known Smith Waterman algorithm may also be used to determine identity. For example, using the computer algorithm GAP (Genetics Computer Group, University of Wisconsin, Madison, Wis.), two proteins for which the percent sequence identity is to be determined are aligned for optimal matching of their respective amino acids (the "matched span", as determined by the algorithm). A gap opening penalty (which is calculated as 3 times the average diagonal; the "average diagonal" is the average of the diagonal of the comparison matrix being used; the "diagonal" is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1/10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. A standard comparison matrix (see Dayhoff et al., Atlas of Protein Sequence and Structure, vol. 5, supp.3 (1978) for the PAM 250 comparison matrix; Henikoff et al., Proc. Natl. Acad. Sci USA, 89, 10915-10919, (1992) for the BLOSUM 62 comparison matrix) is also used by the algorithm. Preferred parameters for a protein sequence comparison include the following: Algorithm: Needleman et al., J. Mol. Biol, 48, 443-453, (1970); Comparison matrix: BLOSUM 62 from Henikoff et al., Proc. Natl. Acad. Sci. USA, 89, 10915-10919, (1992); Gap Penalty: 12, Gap Length Penalty: 4, Threshold of Similarity: 0. The GAP program is useful with the above parameters. The aforementioned parameters are the default parameters for protein comparisons (along with no penalty for end gaps) using the GAP algorithm. Amino acid sequence homology/identity is conveniently determined from aligned sequences, using e.g. the ClustalW program, version 1.8, June 1999, using default parameters (Thompson et al., 1994, ClustalW: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Res., 22:4673-4680) and analyzed by use of GENEDOC version 2.5 (Nicholas et al., 1997 GeneDoc: Analysis and Visualization of Genetic Variation, EMBNEW.NEWS 4:14; Nicholas, K. B. and Nicholas H. B. Jr. 1997 GeneDoc: Analysis and Visualization of Genetic Variation).

[0201] The most abundant protein component in circulating blood of mammalian species is serum albumin, which is normally present at a concentration of approximately 3 to 4.5 grams per 100 millilitres of whole blood. Serum albumin is a blood protein of approximately 70,000 Dalton (Da) which has several important functions in the circulatory system. It functions as a transporter of a variety of organic molecules found in the blood, as the main transporter of various metabolites such as fatty acids and bilirubin through the blood, and, owing to its abundance, as an osmotic regulator of the circulating blood. In the present context, the term "an albumin" as used herein means albumin of mammalian origin or non-mammalian origin, such as human serum albumin that is described in Peters, T., Jr. (1996) All about Albumin: Biochemistry, Genetics and Medical, Applications pp 10, Academic Press, Inc., Orlando (ISBN 0-12-5521 10-3), or recombinant human albumin, or modified albumin, such as human albumin modified as described in WO2011051489 and WO2010092135.

[0202] WO2011051489 the specification relates to variants of a parent albumin having altered plasma half-life compared with the parent albumin. The present invention also relates to fusion polypeptides and conjugates comprising said variant albumin.

[0203] WO2010092135 based on the three-dimensional structure of albumin, the inventors have designed variant polypeptides (muteins) which have one or more cysteine residues with a free thiol group (hereinafter referred to as "thio-albumin"). The variant polypeptide may be conjugated through the sulphur atom of the cysteine residue to a conjugation partner such as a bioactive compound.

[0204] WO2005054286 the specification relates to proteins comprising Interleukin 11 (IL-11) (including, but not limited to, fragments and variants thereof), which exhibit thrombopoietic or antiinflammatory properties, fused to albumin (including, but not limited to fragments or variants of albumin).

[0205] WO2004083245 describes an agent having a greater half-life than naturally produced albumin in a patient with NS, the agent comprising an albumin-like first polypeptide bound to a second polypeptide.

[0206] WO03066681 describes a composition comprising a non-albumin protein stabilised by the addition of a highly purified recombinant human serum albumin. The non-albumin protein may be Factor VIII.

[0207] In the present context, the term "a polymer" as used herein means a molecule formed by covalent linkage of two or more monomers, wherein none of the monomers is an amino acid residue, except where the polymer is human albumin or another abundant plasma protein. The term "polymer" may be used interchangeably with the term "polymer molecule". The term is intended to cover carbohydrate molecules attached by in vitro glycosylation. Carbohydrate molecules attached by in vivo glycosylation, such as N- or O-glycosylation (as further described below) are referred to herein as "an oligosaccharide moiety". Except where the number of polymer molecules is expressly indicated, every reference to "a polymer", "a polymer molecule", "the polymer" or "the polymer molecule" as used in the present invention shall be a reference to one or more polymer molecule(s). The polymer may be a water soluble or water insoluble polymer, such as a PEG moiety. The PEG moiety may have an average size selected from the range of 500 Da to 200.000 Da, such as from 500 Da to 100.000 Da, such as from 2000 Da to 50.000 Da. Such PEG molecules may be retrieved from i.a. Shearwater Inc.

[0208] In the present context, the term "a pharmaceutical composition" as used herein means a composition containing an LH compound, e.g. a modified LH, of the present invention, and/or a modified LH of the present invention and an FSH, and optionally one or more pharmaceutically acceptable carriers or excipients, and may be prepared by conventional techniques, e.g. as described in Remington: The Science and Practice of Pharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19th edition, Easton, Pa. The compositions may appear in conventional forms, for example capsules, tablets, aerosols, solutions, suspensions or topical applications. Typically, the pharmaceutical compositions of the present invention may be formulated for parenteral administration e.g., by i.v. or subcutaneous injection, and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. Examples of oily or nonaqueous carriers, diluents, solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate), and may contain formulatory agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water. Oils useful in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils useful in such formulations include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. The parenteral formulations typically will contain from about 0.0001 to about 25%, such as from about 0.5 to about 25%, by weight of the active ingredient in solution. Preservatives and buffers may be used. In order to minimise or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 0.000001 to about 15% by weight, such as from about 0.000001 to about 5% by weight or from about 5 to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.

[0209] In the present context, the term "assisted reproduction technologies" as used herein means methods that intent to enhance the possibility of conceiving either naturally or by retrieving an oocyte and spermatozoa and perform in vitro fertilization, this may either be through in vitro fertilization (IVF) or by intra cytoplasmatic sperm injection (ICSI), intra uterine insemination (IUD, in vitro maturation (IVM), or other forms derived thereof.

[0210] The term "recurrent pregnancy loss" or "habitual abortion" as used herein (used interchangeably) happens in about 1% of fertile women, who unsuccessfully tried to conceive in three or more pregnancies and the pregnancy terminated before 12 weeks of gestation. Because embryo attachment and early implantation in the uterus are exquisitely controlled by the local hormonal milieu, endocrine disorders are frequently linked to failures in early gestation although a multitude of factors may result in a similar clinical picture. The uterus undergoes essential developmental changes during the preimplantation period, stimulated by estrogen and progesterone. Secreted by the CL, progesterone is important for the successful implantation and continuation of pregnancy. Therefore, conditions related to inadequate progesterone secretion by the CL are likely to negatively affect the outcome of the pregnancy.

[0211] The term "bolus" as used herein is the administration of a compound that is given to raise its concentration in blood, serum or plasma to an effective level. The administration can be given by any route of administration including oral, inhalation, intravenous administration, by intramuscular, intrathecal or subcutaneous injection.

[0212] The term "gonadotropin" as used herein is a naturally occurring hormone that belongs to a group of heterodimeric glycoproteins including follicle stimulating hormone (FSH), luteinising hormone (LH) and chorionic gonadotropin (CG), such as human chorionic gonadotropin. These hormones regulate gonadal function in the male and female. Each of these naturally occurring hormones is composed of two non-covalently linked subunits: an .alpha.-subunit, which is common to FSH, LH and hCG, and a .beta.-subunit, which is unique to each of them, and which confers biological specificity to each hormone. In all of the naturally occurring gonadotropins, each subunit has asparagine-linked (N-linked) oligosaccharide side chains. In the common .alpha.-subunit of the human hormones, these are attached at positions 52 and 78 (SEQ ID NO:1). In both human FSH and hCG, two N-linked oligosaccharide side chains are attached to the beta-subunit, at positions 7 and 24 in FSH (SEQ ID NO:10).

[0213] The alpha chain of preferred gonadotropins of the present invention is selected from the group consisting of sequences having at least 80% sequence identity to SEQ ID NOS:1, 2, or 3, more preferably 85%, more preferably 90%, more preferably 95%. Preferably, a variant comprises the conserved cysteine residues at the position and spacing of SEQ ID NO:1. In a particularly preferred embodiment, the gonadotropins comprise the human alpha-subunit having SEQ ID NO:1.

[0214] As with all glycoproteins, variations in oligosaccharide structure occur in the gonadotropins, resulting in an array of isoforms that are found within the pituitary gland and in circulation. Furthermore, there are differences in degree of terminal carbohydrate "capping" by sialic acid. The isoforms may be separated on the basis of their charge, which is largely determined by the number and distribution of sialylated N-linked oligosaccharides. Highly sialylated forms will have a more acidic pH and are termed "acidic". Less sialylated forms have comparatively higher pH's and are termed "basic".

[0215] As a consequence of their structural differences, gonadotropin isoforms differ in their capability to bind to target-cell receptors. The degree of sialylation affects their ability to survive in circulation. For example, in the case of FSH, highly acidic/sialylated isoforms have considerably longer plasma half-lives in animal models.

[0216] Days--the protocols of the present invention start at a certain point in the menstrual cycle. Day 1 of a menstrual cycle is the first day of menstruation. When reference is made to days of a stimulation protocol, day 1 is the day the first dosage of FSH is administered. Day 2 of a stimulation protocol is the day after etc. In the luteal phase, days are calculated either from the day of ovulation or from the day of oocyte pick up.

[0217] Follicle size--Ovarian function may be measured by gynecologic ultrasonography of follicular volume. Measurement of ovarian follicle diameter is routinely made using ultrasonography. Today, ovarian follicle volume can also be measured rapidly and automatically from three-dimensionally reconstructed ultrasound images (Salama S, Arbo E, Lamazou F, Levailllant J M, Frydman R, Fanchin R (April 2010). Reproducibility and reliability of automated volumetric measurement of single preovulatory follicles using SonoAVC". Fertil. Steril. 93 (6): 2069-73).

[0218] Urine-derived or urinary. The terms are used interchangably. The term refers to the origin of gonadotropins purified from urine.

[0219] In the present context, the term "infertility treatment" as used herein means methods that help the woman of an infertile couple or a single woman to conceive.

[0220] In the present context, the term "promoting fertility" as used herein means methods that will enhance the fertility of a couple, a woman or a man.

[0221] In the present context, the term "mammalian subject", "mammal" or "mammalian" (these terms are used interchangeable throughout the specification) as used herein means any mammal, such as a human, a cow, a pig, a horse, a sheep, a dog, a cat and a goat.

[0222] The terms "a" and "an" and "the" and similar referents as used in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0223] An "effective amount" of an LH compound as used herein means an amount of the LH compound to be administered sufficient to promote fertility in a mammal or to treat infertility in a mammal in need thereof. An amount adequate to accomplish this is defined as "an effective amount". Effective amounts for each purpose will depend on the severity of the condition as well as the weight and general state of the subject. It will be understood that determining an appropriate dosage may be achieved using routine experimentation, by constructing a matrix of values and testing different points in the matrix, which is all within the ordinary skills of a trained physician or veterinarian. Typical dosages of hCG administration will be from 50-500 IU daily or dosages of an LH compound that will provide a similar biological effect.

[0224] An "effective amount" of a modified LH as used herein means an amount of the modified LH to be administered sufficient to assist in inducing follicular development, such as paucifolliculogenesis or unifolliculogenesis, in anovulatory treatment of a mammalian female subject or inducing COS in the follicular phase of the menstrual cycle of a mammalian female subject in need thereof. As the modified LH is intended to be used in combination with FSH as described herein, the modified LH will "assist" in this treatment together with FSH. An amount adequate to accomplish this is defined as "an effective amount". Effective amounts for each purpose will depend on the severity of the condition as well as the weight and general state of the subject. It will be understood that determining an appropriate dosage may be achieved using routine experimentation, by constructing a matrix of values and testing different points in the matrix, which is all within the ordinary skills of a trained physician or veterinarian. Typical dosages of hFSH administration will be from 50-500 IU daily or dosages of a molecule having FSH activity that will provide a similar biological effect.

[0225] The term "acylation group" as used herein means an R--(C.dbd.O)-group, wherein R is selected from straight-chain or branched, saturated or unsaturated carbon chains, optionally comprising one or more O, N, S, or P, such as a straight-chain or branched alkane carboxylic acid. Various examples of suitable acylation groups are described in WO2006/037810, WO00/34331, WO2006/097537, WO2011/080103. In particular examples of suitable acylation groups have the structure CH.sub.3(CH.sub.2).sub.nCO--, wherein n is 4 to 40, e.g. 8 to 22, such as an acylation group selected from the group comprising CH.sub.3(CH.sub.2).sub.8CO--, CH.sub.3(CH.sub.2).sub.9CO--, CH.sub.3(CH.sub.2).sub.10CO--, CH.sub.3(CH.sub.2).sub.11CO--, CH.sub.3(CH.sub.2).sub.12CO--, CH.sub.3(CH.sub.2).sub.13CO--, CH.sub.3(CH.sub.2).sub.14CO--, CH.sub.3(CH.sub.2).sub.15CO--, CH.sub.3(CH.sub.2).sub.16CO--, CH.sub.3(CH.sub.2).sub.17CO--, CH.sub.3(CH.sub.2).sub.18CO--, CH.sub.3(CH.sub.2).sub.19CO--, CH.sub.3(CH.sub.2).sub.20CO--, CH.sub.3(CH.sub.2).sub.21CO--and CH.sub.3(CH.sub.2).sub.22CO--. Further examples of suitable acylation groups has the structure HOOC-(CH.sub.2).sub.nCO--, wherein n is 4 to 40, e.g. 12 to 20, typically, HOOC--(CH.sub.2).sub.14CO--, HOOC--(CH.sub.2).sub.15CO--, HOOC--(CH.sub.2).sub.16CO--, HOOC--(CH.sub.2).sub.17CO--and HOOC--(CH.sub.2).sub.18CO--. See also U.S. Pat. No. 5,905,140 for further examples of acylation groups.

[0226] The term "treatment" and "treating" as used herein in relation to a modified LH means the management and care of a patient for the purpose of inducing follicular development in anovulatory treatment of a mammalian female subject or induce COS in the follicular phase of the menstrual cycle of a mammalian female subject.

[0227] The term "treatment" and "treating" as used herein in relation to an LH compound means the management and care of a patient for the purpose of treating infertility or promoting fertility. The patient to be treated is preferably a mammal; in particular a human being, but it may also include animals, such as dogs, cats, horses, cows, sheep and pigs.

DESCRIPTION OF THE INVENTION

[0228] Long Acting Biologically Active Luteinizing Hormone Compound

[0229] The present invention relates to a long acting biologically active LH compound comprising an LH agonist linked to a pharmaceutically acceptable molecule, wherein the administration of the LH compound can be done once or twice in connection with ART procedures, especially in the follicular phase. This is a considerable advantage over the current ART procedures, and leads to improved infertility treatments.

[0230] Moreover, the present invention relates to a long acting biologically active LH compound comprising an LH agonist linked to a pharmaceutically acceptable molecule, wherein the administration of the LH compound can be done at regular intervals in connection with ART procedures to sustain luteal and gestational phase support. This is a considerable advantage over the current ART procedures, and leads to improved infertility treatments.

[0231] In a broad aspect the present invention relates to a long acting biologically active LH compound comprising an LH agonist linked to a pharmaceutically acceptable molecule providing an in vivo plasma half-life of the LH agonist or LH compound which is increased substantially compared to the in vivo plasma half-life of an LH agonist administered in the same manner as the LH compound.

[0232] In another broad aspect the present invention relates to a long acting biologically active LH compound comprising an LH agonist linked to a pharmaceutically acceptable molecule providing an in vivo plasma half-life of the LH agonist or LH compound which is increased substantially compared to in vivo plasma half-life of endogenous CG.

[0233] In a still further aspect the present invention relates to a long acting biologically active luteinizing hormone (LH) compound comprising a mammal CG or analog thereof or a mammal LH or analog thereof linked to a pharmaceutically acceptable molecule selected from a molecule having binding to a mammal neonatal Fc receptor, transferrin and a CH.sub.3(CH.sub.2).sub.nCO--, wherein n is 8 to 22 and a polymer.

[0234] In one embodiment the pharmaceutically acceptable molecule is selected from a molecule having binding to a mammal neonatal Fc receptor.

[0235] In a further embodiment the pharmaceutically acceptable molecule is selected from an albumin, such as human albumin, recombinant human albumin, a modified human albumin with increased binding to a mammal FcRn, a modified recombinant albumin with increased binding to a mammal FcRn. Typically, the pharmaceutically acceptable molecule is selected from recombinant human albumin (SEQ ID NO:20). Typically, the pharmaceutically acceptable molecule is selected from recombinant K573P human albumin (SEQ ID NO:21).

[0236] In a still further embodiment the pharmaceutically acceptable molecule is selected from an Fc fragment of a mammalian antibody, such as a recombinant Fc fragment of a mammalian antibody. Typically, the pharmaceutically acceptable molecule is selected from SEQ ID NO:22.

[0237] In a further embodiment the pharmaceutically acceptable molecule is selected from a variant of an Fc fragment of a mammalian antibody, such as a recombinant variant of an Fc fragment of a mammalian antibody. Typically, the pharmaceutically acceptable molecule is selected from a sequence having at least 80% identity, such as at least 90% identity, such as at least 95% identity to SEQ ID NO:22, disclaiming SEQ ID NO:22.

[0238] In a further embodiment the LH agonist may be selected from a small organic molecule, a peptide, a polypeptide, a protein, and may be produced by synthetic methods, recombinant means or be obtained from tissue or blood. In a particular embodiment the LH agonist is of non-mammalian origin. In another particular embodiment the LH agonist is of mammalian origin, such as a protein obtained by recombinant means.

[0239] In a still further embodiment the mammal CG or analog thereof or a mammal LH or analog thereof is selected from recombinant mammal CG or analog thereof or a recombinant mammal LH or analog thereof.

[0240] In a further embodiment the LH agonist is selected from a mammal CG or a mammal LH. When the LH agonist is a mammal CG it is typically a primate CG, e.g. a human CG or abe CG or monkey CG, but may also be selected from other mammalian species such as equine CG, e.g. horse CG. When the LH agonist is a mammal LH it is typically a primate LH, such as human LH, abe LH or monkey LH; the sequence of cow LH; the sequence pig LH; the sequence of equine LH, such as horse LH; the sequence of sheep LH; the sequence of dog LH; the sequence of cat LH; and the sequence of goat LH. Typically, the LH agonist is a human CG.

[0241] In a further embodiment, the LH agonist is selected from an analog of a mammal CG or an analog of a mammal LH. When the LH agonist is an analog of a mammal CG the analog has at least 80% identity to the corresponding mammalian sequence of chorionic gonadotropin, such as 85% identity, 90% identity, 95% identity, 98% identity. Typically, the LH agonist is an analog of a human CG having at least 80% identity to the corresponding human sequence of chorionic gonadotropin, such as 85% identity, 90% identity, 95% identity, 98% identity. When the LH agonist is an analog of a mammal LH the analog has at least 80% identity to the corresponding mammalian sequence of luteinizing hormone, such as 85% identity, 90% identity, 95% identity, 98% identity. Typically, the LH agonist is an analog of a human LH having at least 80% identity to the corresponding human sequence of luteinizing hormone, such as 85% identity, 90% identity, 95% identity, 98% identity.

[0242] When the LH agonist is selected from a polypeptide or protein, such as an analog of a mammal CG or an analog of a mammal LH, it may be glycosylated. Typically, the LH agonist is an hCG which is glycosylated.

[0243] It may also be that the LH compound as such is glycosylated, and the glycosylation may be on the LH agonist or on the pharmaceutically acceptable molecule, when said molecule is selected from a polypeptide or protein, such as human albumin.

[0244] The LH agonist may be linked to the pharmaceutically acceptable molecule in various ways, such as directly through a valence bond, or indirectly through a linker, which linker typically is a bifunctional linker, although it may also be a multifunctional linker. In further embodiments, the linker is selected from a chemical linker, a sugar moiety, a disulphide bridge, a fused linker, a hydrophilic linker, a hydrolysable linker. In a further embodiment the LH agonist is fused to the pharmaceutically acceptable molecule through a peptide linker. In a still further embodiment the LH agonist is fused directly to the pharmaceutically acceptable molecule, so as to create one polypeptide or protein, by expressing the LH compound from a host cell, such as a CHO cell or yeast cell. In a further embodiment the LH agonist is linked to the pharmaceutically acceptable molecule through a stable linker. In another embodiment the LH agonist is linked to the pharmaceutically acceptable molecule through a labile linker.

[0245] Accordingly, the LH agonist may be linked to the pharmaceutically acceptable molecule in various ways using techniques that are well-known in the prior art, and the present invention also comprises the situation where one or more LH agonist(s) is linked to one or more pharmaceutically acceptable molecule(s), such as two LH agonist linked to one pharmaceutically acceptable molecule, or one LH agonist linked to two pharmaceutically acceptable molecules. In a further embodiment the LH agonist is linked to one or two pharmaceutically acceptable molecule(s), preferably one pharmaceutically acceptable molecule.

[0246] In a further embodiment, the mammal CG or analog thereof or a mammal LH or analog thereof is linked to the pharmaceutically acceptable molecule and wherein the linker is selected from a chemical linker, optionally a bifunctional linker. Typically, the chemical linker is selected from a sugar moiety, a disulphide bridge, a hydrophilic linker, a hydrolysable linker, dicarboxylic acids, carboxylic acid hydrazides, maleimido hydrazides, PDPH, SPDP, LC-SPDP, GMBS, alkyl linkers, and PEG linkers. In a still further embodiment, the chemical linker is selected from succinic acid, glutaric acid, adipic acid, N-[maleimidocaproic acid]hydrazide (EMCH), N-[maleimidopropionic acid]hydrazide (MPH or BMPH), 4-[N-maleimidomethyl]cyclohexan-1-carboxylhydrazide, N-[k-maleimidoundcanoic acid]hydrazide (KMUH), 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide (MPBH), NHS-3-maleimidopropionate Succinimide ester (MPS-EDA), (3-[2-pyridyldithio]propionyl hydrazide) conjugated to sulfurhydryl reactive protein, N-Succinimidyl 3-(2-pyridyldithio)-propionate (SPDP), Succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate (LC-SPDP), N-(.gamma.-Maleimidobutyryloxy)succinimide ester (GMBS), carboxylic acid hydrazides having from 2-5 carbon atoms, --NH--(CH2).sub.m--C(O)--, wherein m is an integer from 2-20, optionally substituted with any non-sterically hindering group, such as C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 acyl, halogen (e.g., Cl, Br), CN, NH.sub.2, or phenyl.

[0247] In a further embodiment the mammal CG or analog thereof or a mammal LH or analog thereof is directly chemically linked to the pharmaceutically acceptable molecule. In a further embodiment the pharmaceutically acceptable molecule is linked to the alpha chain of the mammal CG or analog thereof or the mammal LH or analog thereof.

[0248] In a further embodiment the pharmaceutically acceptable molecule is linked to the beta chain of the mammal CG or analog thereof or the mammal LH or analog thereof.

[0249] In a still further embodiment the mammal CG or analog thereof or a mammal LH or analog thereof is fused to the pharmaceutically acceptable molecule selected from a molecule having binding to a mammal neonatal Fc receptor, such as an albumin, an Fc fragment of a mammalian antibody, or a variant of an Fc fragment of a mammalian antibody, optionally through a peptide linker.

[0250] In a further embodiment the peptide linker has at least 1 amino acid, such as from 1-200 amino acids, typically 1-50 amino acids wherein the amino acids are selected from the twenty naturally occurring amino acids. Typically, the peptide linker has from 1-40 amino acids, such as from 1-30, such as from 1-20, such as from 1-10 amino acids.

[0251] In a further embodiment the peptide linker is selected from a linker made up of amino acids selected from glycine, alanine, proline, asparagine, glutamine, and lysine. Typically, the peptide linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine. In particular, the peptide linker comprises a sequence selected from -(G)n- (SEQ ID NO:67), (GGS)n (SEQ ID NO:68) or (GGGGS)n (SEQ ID NO:69), wherein n is an integer of from 1-50. Typically n is an integer selected from 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0252] In another embodiment the mammal CG or analog thereof or a mammal LH or analog thereof is directly fused to the pharmaceutically acceptable molecule.

[0253] In a further embodiment the pharmaceutically acceptable molecule is fused to an N-terminal of the mammal CG or analog thereof.

[0254] In a still further embodiment the pharmaceutically acceptable molecule is fused to an N-terminal of the mammal LH or analog thereof.

[0255] In a further embodiment the pharmaceutically acceptable molecule is fused to the N-terminal of the alpha chain of the mammal CG or analog thereof.

[0256] In a still further embodiment the pharmaceutically acceptable molecule is fused to the N-terminal of the alpha chain of the mammal LH or analog thereof.

[0257] In a further embodiment the pharmaceutically acceptable molecule is fused to the N-terminal of the beta chain of the mammal CG or analog thereof.

[0258] In a still further embodiment the pharmaceutically acceptable molecule is fused to the N-terminal of the beta chain of the mammal LH or analog thereof.

[0259] In a further embodiment the pharmaceutically acceptable molecule is fused to a C-terminal of the mammal CG or analog thereof.

[0260] In a still further embodiment the pharmaceutically acceptable molecule is fused to a C-terminal of mammal LH or analog thereof.

[0261] In a further embodiment the pharmaceutically acceptable molecule is fused to the C-terminal of the alpha chain of the mammal CG or analog thereof.

[0262] In a still further embodiment the pharmaceutically acceptable molecule is fused to the C-terminal of the alpha chain of the mammal LH or analog thereof.

[0263] In a further embodiment the pharmaceutically acceptable molecule is fused to the C-terminal of the beta chain of the mammal CG or analog thereof.

[0264] In a still further embodiment the pharmaceutically acceptable molecule is fused to the C-terminal of the beta chain of the mammal LH or analog thereof.

[0265] In a further embodiment the mammal CG or analog thereof or a mammal LH or analog thereof is selected from one mammal CG or analog thereof. Typically, one hCG.

[0266] In a still further embodiment the mammal CG or analog thereof or a mammal LH or analog thereof is selected from one mammal LH or analog thereof. Typically, one hLH.

[0267] In a further embodiment the mammal CG or analog thereof or a mammal LH or analog thereof is selected from two mammal CG or analog thereof. Typically, two hCG.

[0268] In a still further embodiment the mammal CG or analog thereof or a mammal LH or analog thereof is selected from two mammal LH or analog thereof. Typically, two hLH.

[0269] In a further embodiment the pharmaceutically acceptable molecule is selected from one pharmaceutically acceptable molecule. Typically, one albumin or one Fc fragment or one variant of an Fc fragment.

[0270] In a still further embodiment the pharmaceutically acceptable molecule is selected from two pharmaceutically acceptable molecules. Typically, two albumins or two Fc fragments or two variants of an Fc fragment, or combinations thereof.

[0271] Preferred LH compounds of the present invention are selected from Conjugate1 (hCG-PDPH-rHA conjugate), Conjugate3 (hCG-SPDP-rHA conjugate), Conjugate4 (EDC activated hCG-PDPH-rHA conjugate), Conjugate3V1 (hCG-SPDP-rHA-K573P conjugate), and Conjugate4V1 (EDC activated hCG-PDPH-rHA-K573P conjugate).

[0272] Other preferred LH compounds of the present invention are selected from Product2 consisting of SEQ ID NO:9 and SEQ ID NO:26, Product3 consisting of SEQ ID NO:1 and SEQ ID NO:28, Product4 consisting of SEQ ID NO:9 and SEQ ID NO:27, Product5 consisting of SEQ ID NO:1 and SEQ ID NO:29 , Product7 consisting of SEQ ID NO:4 and SEQ ID NO:26, Product8 consisting of SEQ ID NO:1 and SEQ ID NO:30, Product9 consisting of SEQ ID NO:4 and SEQ ID NO:27, Product10 consisting of SEQ ID NO:1 and SEQ ID NO:31, Product11 consisting of SEQ ID NO:32 and SEQ ID NO:33, Product12 consisting of SEQ ID NO:32 and SEQ ID NO:34, Product13 consisting of SEQ ID NO:32 and SEQ ID NO:61, and Product14 consisting of SEQ ID NO:32 and SEQ ID NO:62.

[0273] In order to produce the LH compound which can be administered once or twice in connection with ART procedures, such LH compound when administered to a mammal should result in the LH agonist or LH compound having in vivo plasma half-life augmented at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, such as from 1.5 times to 25 times.

[0274] In a further embodiment the LH agonist has an in vivo plasma half-life of at least 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18, days, 19 days, such as from 2 to 20 days. In a further embodiment the LH compound has an in vivo plasma half-life of at least 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18, days, 19 days, such as from 2 to 20 days.

[0275] In a further embodiment the pharmaceutical acceptable molecule provides a biological body composition or concentration of the LH agonist or LH compound sufficient to drive an antral follicle from about 10 mm in diameter up to preovulatory stage at (i.e. about 15-30 mm in diameter) which a maturing oocyte can finalize the maturation to be ready for resumption of the meiosis.

[0276] In a still further embodiment the pharmaceutical acceptable molecule provides a biological body composition or concentration of the LH agonist or LH compound sufficient to drive androgen production in the early adolescent, about 1 year after birth of a male offspring or in puberty for both female and male subjects.

[0277] In a further embodiment the pharmaceutical acceptable molecule provides a biological body composition or concentration of the LH agonist or LH compound sufficient to support hypogonadothrophe hypogonade subjects.

[0278] In a still further embodiment the pharmaceutical acceptable molecule provides a biological body composition or concentration of the LH agonist or LH compound sufficient to sustain progesterone in the pen-, in the ovulatoric- and the post ovulatoric-phase of a mammalian subject with the object regulating the endometrium and womb for avoiding or allowing implantation of a mammalian blastocyst.

[0279] In a further embodiment the pharmaceutical acceptable molecule provides a biological body composition or concentration of the LH agonist or LH compound sufficient to sustain a progesterone in the pen, in the ovulatoric and the post ovulatoric phase of a mammalian subject with the object of preparing the endometrium and womb for implantation.

[0280] In a still further embodiment the pharmaceutically acceptable molecule provides a plasma concentration of the LH agonist or LH compound to support the formation and maintenance of CL, when an injection is given during the follicular phase of the menstrual cycle in connection with FSH treatment, preferably 5-10 days after initiation of FSH treatment.

[0281] In a further embodiment the pharmaceutically acceptable molecule provides a concentration of the LH agonist or LH compound to stimulate sufficient progesterone release from CL after an injection during the follicular phase of the menstrual cycle in connection with FSH treatment, preferably 5-10 days after initiation of FSH treatment.

[0282] In a still further embodiment the pharmaceutically acceptable molecule has binding to a neonatal Fc receptor (FcRn), such as a pH dependent binding allowing the LH compound to escape lysosomal degradation as described in Roopenian et.al. , "FcRn: the neonatal Fc receptor comes of age", Nature reviews, Immunology, vol. 7, p. 715.725, September 2007.

[0283] A typical pharmaceutically acceptable molecule which has binding to the FcRn is selected from an albumin, such as modified albumin with increased binding to FcRn, human albumin, or recombinant human albumin.

[0284] In a further embodiment the pharmaceutically acceptable molecule is selected from any one of small organic molecules, peptides, oligopeptides, polypeptides, proteins, receptors, glycosylations, acylation groups, sugars, polymers (e.g. polyethylene glycols, PEG), nucleic acids (e.g. DNA and RNA), and hormones. Typically, the pharmaceutically acceptable molecule is without limitation selected from an Fc fragment of mammalian antibody, transferrin, albumin, such as human albumin, recombinant albumin, variants of albumin; an acylation group, such as CH.sub.3(CH.sub.2).sub.nCO--, wherein n is 8 to 22; or polymer, such as PEG, e.g. PEG of a molecular weight of at least 5 kDa, such as from 10 kDa to 150 kDa, typically 10 to 40 kDa.

[0285] In a further embodiment the pharmaceutically acceptable molecule is selected from a polymer, such as PEG. Typically, the PEG moiety may have an average size selected from the range of 500 Da to 200.000 Da, such as from 500 Da to 100.000 Da, such as from 2000 Da to 50.000 Da.

[0286] A further aspect of the present invention relates to a pharmaceutical composition comprising the LH compound of the present invention, and optionally a pharmaceutically acceptable carrier or excipient. Typically, the pharmaceutical composition is for injection, such as subcutaneous injection.

[0287] In connection with ART procedures more than one medicament in the infertility treatment or in promoting fertility may be administered, either concomitantly or sequentially. It is therefore within the scope of the present invention to use an LH compound of the present invention in ART procedures, such as IVF or ICSI, for infertility treatment or in promoting fertility in combination with one or more other therapeutically active compound(s) normally used in the infertility treatment or in promoting fertility. By analogy, it is also within the scope of the present invention to use an LH compound of the present invention in combination with other therapeutically active compounds normally used in the infertility treatment or in promoting fertility in the manufacture of a medicament for said infertility treatment or in promoting fertility.

[0288] A further aspect of the present invention relates to the LH compound of the present invention for use in infertility treatment or promoting fertility of a mammalian subject, such as assisted reproduction technologies treatment, e.g. IVF or ICSI treatment, or maldecensus of the testes.

[0289] A further aspect of the present invention relates to the LH compound of the present invention for use in a method for assisted reproductive therapy in a female mammal wherein the LH compound is administered in a dosage one time, two times, three times or four times during the follicular phase, the dosage being sufficient to support the follicle development. The LH compound may be administered as single bolus injection(s). In one embodiment the dosage is also sufficient to provide luteal support.

[0290] A still further aspect of the present invention relates to the LH compound of the present invention for use in a method for assisted reproductive therapy in a female mammal wherein the LH compound is administered in a dosage one time, two times, three times or four times during the luteal phase at least until 2 weeks after ovulation. The LH compound may be administered as single bolus injection(s). In one embodiment the LH compound is administered for the first time after ovulation.

[0291] A further aspect of the present invention relates to the LH compound of the present invention for use in a method for assisted reproductive therapy in a female mammal wherein the LH compound is administered in a dosage one time, two times, three times or four times, during the gestational phase at least until 2 weeks after ovulation. The LH compound may be administered as single bolus injection(s). In one embodiment the LH compound is administered for the first time after ovulation.

[0292] A still further aspect of the present invention relates to the LH compound of the present invention for use in a method for treatment of recurrent pregnancy loss in a female mammal wherein the LH compound is administered in a dosage one time, two times, three times or four or more times, during the early gestational period until 12 weeks after conception. The LH compound may be administered as single bolus injection(s). In one embodiment the LH compound is administered for the first time after ovulation.

[0293] A further aspect of the present invention relates to the LH compound of the present invention for use in a method for enhancing progesterone production and optimizing chances for a successful pregnancy wherein the LH compound is administered in a dosage one time, two times, three times or four or more times, during the first 12 weeks of gestation. The LH compound may be administered as single bolus injection(s). In one embodiment the LH compound is administered for the first time after ovulation.

[0294] A further aspect of the present invention relates to the LH compound of the present invention for use in a method for assisted reproductive therapy in a female mammal wherein the LH compound is administered in a dosage once or twice, in connection with ovulation induction. The LH compound may be administered as single bolus injection(s).

[0295] In connection with ovulation triggering various treatment regimens may be used. In one embodiment a GnRH agonist is used for ovulation triggering. In another embodiment, an hCG is used for ovulation triggering.

[0296] A further aspect of the present invention relates to the LH compound of the present invention for use in promoting fertility or treatment of infertility of a hypogonadotropic hypogonadal male mammalian subject.

[0297] A still further aspect of the present invention relates to the LH compound of the present invention for use in promoting fertility or treatment of infertility of a young or adolescent male mammalian subject having cryptorchidism.

[0298] A still further aspect of the present invention relates to a method of infertility treatment of a mammalian subject comprising administering to a mammal in need thereof an effective amount of the LH compound of the present invention.

[0299] A further aspect of the present invention relates to a method of promoting fertility of a mammalian subject comprising administering to a mammal in need thereof an effective amount of the LH compound of the present invention.

[0300] In a further embodiment the mammalian subject is selected from a human, a cow, a pig, a horse, a sheep, a dog, a cat and a goat, typically a human subject.

[0301] In a further aspect the present invention relates to a method of preparing a long acting biologically active luteinizing hormone (LH) compound, such as any one of the herein disclosed conjugates of the present invention, comprising an LH agonist linked to a pharmaceutically acceptable molecule, the method comprising reacting an LH agonist with a linker attached to a pharmaceutically acceptable molecule, or reacting an LH agonist with a linker and then attaching said linker to a pharmaceutically acceptable molecule, or reacting a linker with a pharmaceutically acceptable molecule and then reacting an LH agonist with the linker attached to the pharmaceutically acceptable molecule, or by expressing the LH agonist and pharmaceutically acceptable molecule from a host cell.

[0302] Long-Acting Modified Mammalian LH

[0303] The present invention relates to a long-acting modified mammalian LH, e.g. human LH linked to e.g. fused to albumin, or conjugated to an acylation group or PEG, that agonize and activate the LH receptor in a mammal and provides an in vivo plasma half-life of the mammalian LH or analog thereof, or the modified LH which is from 2 to 48 hours in a mammal. The modified LH either given in the follicular phase or as a luteal phase support is believed to improve patient convenience and treatment outcome.

[0304] Furthermore, the use of a long acting modified mammalian LH will not interfere with the specific effects that the hyperglycosylated hCG secreted from the implanting embryo will exert and it will be possible for the patient at her earliest possible convenience to detect if she is pregnant by use of an ordinary pregnancy-test.

[0305] Collectively, these findings suggest that a long acting modified mammalian LH preparation, in which the specific effects of LH at the receptor level are maintained in combination with a constant presence in circulation will be able to optimise COS in the follicular phase of the menstrual cycle and thus final treatment outcome.

[0306] In a broad aspect the present invention relates to a modified LH comprising a mammalian LH or analog thereof linked to a pharmaceutically acceptable molecule providing an in vivo plasma half-life of the mammalian LH or analog thereof, or the modified LH which is from 2 to 48 hours in a mammal.

[0307] A further aspect of the present invention relates to a long acting biologically active luteinizing hormone (LH) compound comprising a mammal LH or analog thereof linked to a pharmaceutically acceptable molecule selected from a molecule having binding to a mammal neonatal Fc receptor, transferrin and a CH.sub.3(CH.sub.2).sub.nCO--, wherein n is 8 to 22 and a polymer for use in combination with an FSH or a molecule having FSH activity for simultaneous, sequential or separate use to induce follicular development, such as paucifolliculogenesis or unifolliculogenesis, in anovulatory treatment of a mammalian female subject or induce COS in the follicular phase of the menstrual cycle of a mammalian female subject.

[0308] In an embodiment the FSH is derived exogenously in the mammalian female subject. In another embodiment the FSH is produced endogenously in the mammalian female subject.

[0309] In a further embodiment the pharmaceutically acceptable molecule is selected from a molecule having binding to a mammal neonatal Fc receptor, such as an albumin, e.g. human albumin, recombinant human albumin, a modified human albumin with increased binding to a mammal FcRn, a modified recombinant albumin with increased binding to a mammal FcRn; an Fc fragment of a mammalian antibody, such as a recombinant Fc fragment of a mammalian antibody; and a variant of an Fc fragment of a mammalian antibody.

[0310] In a further embodiment the pharmaceutically acceptable molecule provides an in vivo plasma half-life of the mammal LH or analog thereof, or the modified LH which is from 2 to 48 hours in the mammalian female subject.

[0311] In a further embodiment the mammalian LH has an in vivo plasma half-life of at least 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, such as from 4 to 48 hours, 5 to 40 hours, 6 to 36 hours, 7 to 30 hours, 8 to 28 hours, 9 to 26 hours, or 10 to 24 hours, typically from 6 to 8 hours. In a further embodiment the modified LH has an in vivo plasma half-life of at least 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, such as from 4 to 48 hours, 5 to 40 hours, 6 to 36 hours, 7 to 30 hours, 8 to 28 hours, 9 to 26 hours, or 10 to 24 hours, typically from 6 to 8 hours.

[0312] Upon administration of the modified LH to a mammal it is important that a sufficient in vivo plasma concentration is reached and maintained for such time it takes to provide an effect in inducing follicular development in anovulatory treatment of a mammalian female subject or inducing COS in the follicular phase of the menstrual cycle of a mammalian female subject. In further embodiments the modified LH of the present invention provides an in vivo plasma concentration of the modified LH, the mammalian LH or a mixture thereof, in a mammal in the range of from 2 to 30 IU/L, such as 2 to 4 IU/L, 4 to 8 IU/L, 8 to 14 IU/L, 14 to 20 IU/L, or 20 to 30 IU/L.

[0313] The mammalian LH or analog thereof may be recombinant or synthetic, or a combination thereof and may be produced by synthetic methods, such as standard chemical methods, including synthesis by an automated procedure, or by recombinant means or be obtained from urine, tissue or blood. In a further embodiment the mammalian LH is a recombinant LH. In a still further embodiment the mammalian LH is recombinant human LH (rhLH).

[0314] In a further embodiment the mammalian LH may be selected from the sequence of human LH, the sequence of cow LH, the sequence of pig LH, the sequence of horse LH, the sequence of sheep LH, the sequence of dog LH, the sequence of cat LH, and the sequence of goat LH.

[0315] In a still further embodiment the mammalian LH analog is a recombinant LH. The mammalian LH analog which may be produced by recombinant means, is typically selected from an analog of the mammalian LH, wherein the analog has at least 80% identity to the corresponding mammalian sequence of LH, such as 85% identity, 90% identity, 95% identity, 98% identity. For instance, the mammalian LH analog is selected from an analog of the human LH, wherein the analog has at least 80% identity to the human sequence of LH, such as 85% identity, 90% identity, 95% identity, 98% identity. Or the mammalian LH analog is selected from an analog of the horse LH, wherein the analog has at least 80% identity to the horse sequence of LH, such as 85% identity, 90% identity, 95% identity, 98% identity.

[0316] The mammalian LH or analog of a mammalian LH, as used in accordance with the present invention may be glycosylated. Typically, the mammalian LH or analog is glycosylated, such as a hLH which is glycosylated.

[0317] It may also be that the modified LH as such is glycosylated, and the glycosylation may be on the mammalian LH or on the pharmaceutically acceptable molecule, when said molecule is selected from a polypeptide or protein, such as human albumin. The mammalian LH or analog thereof may be linked to the pharmaceutically acceptable molecule in various ways, such as directly through a valence bond, or indirectly through a linker, which linker typically is a bifunctional linker, although it may also be a multifunctional linker. In further embodiments, the linker is selected from a chemical linker, a sugar moiety, a disulphide bridge, a fused linker, a hydrophilic linker, a hydrolysable linker. In a further embodiment the mammalian LH or analog thereof is fused to the pharmaceutically acceptable molecule through a peptide linker. In a still further embodiment the mammalian LH or analog thereof is fused directly to the pharmaceutically acceptable molecule, so as to create one polypeptide or protein, by expressing the modified LH from a host cell, such as a CHO cell or yeast cell. In a further embodiment the mammalian LH or analog thereof is linked to the pharmaceutically acceptable molecule through a stable linker. In another embodiment the mammalian LH or analog thereof is linked to the pharmaceutically acceptable molecule through a labile linker.

[0318] Accordingly, the mammalian LH or analog thereof may be linked to the pharmaceutically acceptable molecule in various ways using techniques that are well-known in the prior art, and the present invention also comprises the situation where one or more mammalian LH or analog thereof is linked to one or more pharmaceutically acceptable molecule(s), such as two mammalian LH or analog thereof linked to one pharmaceutically acceptable molecule, or one mammalian LH or analog thereof linked to two pharmaceutically acceptable molecules. In a further embodiment the mammalian LH or analog thereof is linked to one or two pharmaceutically acceptable molecule(s), such as one pharmaceutically acceptable molecule.

[0319] In a still further embodiment the pharmaceutically acceptable molecule has binding to a neonatal Fc receptor (FcRn), such as a pH dependent binding allowing the modified LH to escape lysosomal degradation as described in Roopenian et.al. , "FcRn: the neonatal Fc receptor comes of age", Nature reviews, Immunology, vol. 7, p. 715.725, September 2007.

[0320] A typical pharmaceutically acceptable molecule which has binding to the FcRn is selected from an albumin, such as modified albumin with increased or reduced binding to FcRn, human albumin, or recombinant human albumin.

[0321] In a further embodiment the pharmaceutically acceptable molecule is selected from any one of small organic molecules, peptides, oligopeptides, polypeptides, proteins, receptors, glycosylations, acylation groups, sugars, polymers (e.g. polyethylene glycols, PEG), nucleic acids (e.g. DNA and RNA), and hormones. Typically, the pharmaceutically acceptable molecule is without limitation selected from a Fc fragment of mammalian antibody, transferrin, albumin, such as human albumin, recombinant albumin, variants of albumin; an acylation group, such as CH.sub.3(CH.sub.2).sub.nCO--, wherein n is 8 to 22; or polymer, such as PEG, e.g. PEG of a molecular weight of at least 5 kDa, such as from 10 kDa to 150 kDa, typically 10 to 40 kDa.

[0322] A further aspect of the present invention relates to a pharmaceutical composition comprising the modified LH of the present invention, and optionally a pharmaceutically acceptable carrier or excipient.

[0323] In connection with ART procedures, and in particular with inducing follicular development or COS as explained herein, more than one medicament may be administered, either concomitantly or sequentially. It is therefore within the scope of the present invention to use a modified LH of the present invention in ART procedures, such as inducing COS, for infertility treatment or in promoting fertility in combination with one or more other therapeutically active compound(s) normally used in the infertility treatment or in promoting fertility.

[0324] In a further aspect the present invention relates to a modified LH comprising a mammalian LH or analog thereof linked to a pharmaceutically acceptable molecule providing an in vivo plasma half-life of the mammalian LH or analog thereof, or the modified LH which is from 2 to 48 hours in a mammal for use in combination with an FSH or a molecule having FSH activity for simultaneous, sequential or separate use to induce follicular development in anovulatory treatment of a mammalian female subject or induce COS in the follicular phase of the menstrual cycle of a mammalian female subject. In one embodiment the FSH is selected from mammalian FSH, such as human FSH, in particular recombinant FSH, e.g. rhFSH. In a particular embodiment the FSH is selected from Puregon, Gonal F, Elonva, Fostinorm, Bravelle, Menopur.

[0325] In a further embodiment the combination of a modified LH of the present invention and an FSH or a molecule having FSH activity is to induce follicular development, such as paucifolliculogenesis or unifolliculogenesis, in anovulatory treatment of a mammalian female subject. In a still further embodiment the combination of a modified LH of the present invention and an FSH or a molecule having FSH activity is to induce COS in the follicular phase of the menstrual cycle of a mammalian female subject. Typically, in inducing follicular development or COS, the modified LH of the present invention and an FSH or a molecule having FSH activity are administered in a IU ratio range (FSH:modified LH) from 20:1 to 1:20. In further embodiments the FSH:modified LH is administered in the IU ratios range from 18:1 to 1:18, 15:1 to 1:15, 12:1 to 1:12, 9:1 to 1:9, 5:1 to 1:5, such as 4:1 to 1:4.

[0326] Although as stated above the FSH and modified LH may be administered simultaneously, sequentially or separately the combination is typically administered together either as a kit of parts comprising the modified LH and FSH in separate dosage forms that may be the same, e.g. two separate injections in a kit, such as subcutaneous injections, or as a pharmaceutical composition comprising the modified LH of the present invention and an FSH or a molecule having FSH activity, and optionally a pharmaceutically acceptable carrier or excipient. It is preferred that the modified LH and FSH be administered subcutaneously, preferably into the anterior abdominal wall. Formulations for parenteral administration will usually be sterile. Pharmaceutical formulations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended mammalian subject; aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents are also within the scope of the invention. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets. The formulations can be administered through a prefilled syringe, an auto-injector or a multidose auto-injector. Typically, LH compound and the FSH or the molecule having FSH activity are provided for simultaneous use in a pharmaceutical composition.

[0327] A still further aspect of the present invention relates to a method of inducing follicular development, such as paucifolliculogenesis or unifolliculogenesis, in anovulatory treatment of a mammalian female subject or induce COS in the follicular phase of the menstrual cycle of a mammalian female subject comprising administering to a mammal in need thereof an effective amount of the modified LH of the present invention simultaneous, sequential or separate in combination with an FSH or a molecule having FSH activity. In a further embodiment the FSH or the molecule having FSH activity is selected from mammalian FSH, such as human FSH, in particular recombinant FSH, e.g. rhFSH.

[0328] In a further embodiment the mammalian subject is selected from a human, a cow, a pig, a horse, a sheep, a dog, a cat and a goat, typically a human subject.

[0329] In a further aspect the present invention relates to a method of preparing a modified LH of the present invention, such as any one of the herein disclosed conjugates of the present invention, comprising a mammalian LH or analog thereof linked to a pharmaceutically acceptable molecule providing an in vivo plasma half-life of the mammalian LH or analog thereof, or the modified LH which is from 2 to 48 hours in a mammal, the method comprising reacting a mammalian LH or analog thereof with a linker attached to a pharmaceutically acceptable molecule, or reacting a mammalian LH or analog thereof with a linker and then attaching said linker to a pharmaceutically acceptable molecule, or reacting a linker with a pharmaceutically acceptable molecule and then reacting a mammalian LH or analog thereof with the linker attached to the pharmaceutically acceptable molecule, or by expressing the mammalian LH or analog thereof and pharmaceutically acceptable molecule from a host cell.

[0330] Methods of the Invention

[0331] FIG. 1a and 1b describe protocols for controlled ovarian stimulation as known in the prior art. The protocol in FIG. 1a starts with administration of FSH, recombinant or urinary, on day 1-3 of a menstrual cycle. FSH is administered in daily dosages until ovulation induction. From about day 6, a GnRH antagonist is administered to avoid a premature surge in LH prior to ovulation induction. Ovulation is induced by administering a trigger shot of 5,000 to 10,000 IU of recombinant hCG. Ovulation may alternatively be induced by GnRH agonist triggering. This is typically done when one to three follicles have a size of 17 mm. In order to provide luteal support, progesterone is administered vaginally or intramuscularly starting on the day of embryo transfer. The progesterone administration is continued at least until day 28 of the stimulation protocol. In many cases progesterone is administered until week or even until week 10 of the pregnancy.

[0332] In FIG. 1b, the daily dosages of FSH on days 1-6 are replaced by one dosage of long-acting FSH (corifollitropin). At around day 5-7 the serum level of corifollitropin is decreasing and daily dosages of recombinant or urinary FSH are given until ovulation triggering. The advantage of using corifollitropin is that the female does not need to visit the clinic and receive injections of FSH on days 2-5 or 6.

[0333] FIG. 2a is a schematic illustration of one embodiment of the stimulation protocol according to the present invention based on administration of long-acting gonadotropins.

[0334] In this case, long-acting FSH, such as corifollitropin alpha, is administered on day 1-3 of a menstrual cycle. In case of corifollitropin the dosage may be 100 and 150 .mu.g per female. The dosage and serum half-life is chosen so that the serum level of FSH decreases in the later stages of follicular phase so that further follicle recruitment is significantly reduced in the late phase of the follicular phase. This serves to reduce the number of follicles stimulated to develop and thereby reduce the risk of OHSS.

[0335] As in the known protocols, a GnRH antagonist is administered starting on day 4-7 of the stimulation protocol. Follicle development is stimulated by administering one dosage of a long-acting hCG on day 6 of the stimulation protocol or earlier. A long-acting LH can also be used. The dosage of long-acting hCG or LH is sufficient to stimulate follicle development and sufficiently low to reduce the risk of OHSS. The long acting hCG or LH is administered in a dosage giving a biological response similar to the response obtained with a serum level of 4-15 IU/litre. To further reduce the risk of OHSS, a GnRH agonist trigger shot is used to induce ovulation when at least one follicle has a diameter of at least 15 mm, preferably when 3 follicles have a diameter of 17 mm. Suitable dosages range between 0.1 and 1 mg when administered subcutaneously or intranasaly. Luteal phase support can be provided by administering either progesterone as in the known protocols (illustrated in FIG. 2a) or by administering an LH agonist as herein described and illustrated (FIGS. 4a and 4b).

[0336] FIG. 2b illustrates a protocol of the invention, wherein folliculogenesis is stimulated by administering recombinant or urine derived FSH as daily injections from day 1 until approximately day 6. Preferred daily dosages of FSH are 150-225 IU per day. The exact day of discontinuing FSH administration is determined by UV scanning and measurement of follicle diameter. When at least one follicle has reached a diameter of 12-14 mm, FSH administration is discontinued and a single dose of long-acting hCG or LH is administered to stimulate follicle development as described above. Oocyte induction and luteal support as described in FIG. 2a. In the regime of FIG. 2b, GnRH antagonist and agonist are administered as described for FIG. 2a.

[0337] FIG. 2c illustrates a protocol of the invention, wherein folliculogenesis and follicle development is stimulated by administering daily dosages of FSH as in FIG. 2b and daily dosages of hCG or LH starting from approximately day 6. Suitable dosages of hCG during the follicular phase include 25-400 IU per day, such as 50-300 IU per day, more preferably 100-300, such as 150-250, for example 175-225 IU per day. In the regime of FIG. 2c, the GnRH antagonist and agonist are administered as described for FIG. 2a. Luteal support is not illustrated here but may be either by progesterone administration or LH/hCG administration as illustrated in FIG. 4a or 4b.

[0338] In an alternative protocol based on administration of long-acting (long-lasting) hCG or LH illustrated in FIG. 3a, the FSH, GnRH antagonist and GnRH agonist are administered as described for the protocol in FIG. 2b. Follicle development and luteal support are provided by administering a long-acting hCG or LH at intervals of 2-7 days (illustrated by 5 days) starting e.g. from about day 6 in the follicular phase and continuing at least until pregnancy testing. In case of pregnancy, administration of long-acting LH or hCG can be continued until week 5, for example until week 10 of the pregnancy. A suitable dosage of long-acting hCG or LH during the follicular phase is a dosage giving a biological response similar to the response obtained with a serum level of 4-15 IU/litre.

[0339] In alternative protocols, recombinant or urinary hCG or LH is administered in daily dosages during the follicular phase and through the luteal phase (FIGS. 3b and 3c). The figures illustrate hCG administration, but it is likewise conceivable that LH is administered. Administration of hCG may commence on day 6 of the stimulation, but may likewise start earlier, such as on day 2 of the stimulation. Suitable dosages of urinary or recombinant hCG during the follicular phase include 25-400 IU per day, such as 50-300 IU per day, more preferably 100-300, such as 150-250, for example 175-225 IU per day. In the luteal phase it is preferred to use LH or an LH analogue or variant, as administration of recombinant or urinary hCG may result in a false detection of biochemical pregnancy. The majority of lateral flow device pregnancy tests rely on detection of urinary hCG. Suitable dosages of recombinant or urinary hCG in the luteal phase include 25-400 IU hCG per day, preferably 50-200 IU hCG per day, for example 75-200, such as 100-150 or 120-170 IU/day.

[0340] The protocol in FIG. 3c illustrates another protocol of the invention, in which hCG (or LH) is administered already from day 2 of the stimulation protocol. Otherwise the protocol is identical to the protocol of FIG. 3b. The follicle stimulation of the protocol in FIGS. 3b and 3c can also be performed with long-acting hCG or long-acting LH, which is administered as a single dosage on day 1, 2, or 3 of the stimulation protocol.

[0341] FIGS. 4a and 4b illustrate protocols for luteal support according to the invention. According to the illustrated embodiment (FIG. 4a), luteal support is provided by administering daily dosages of LH (or hCG) from around the time of oocyte harvest and continuing until day 28 of the protocol. A preferred dosage range of recombinant or urinary LH for luteal support includes daily dosages of 100-600 IU LH per day, preferably 150-450 IU LH per day, such as 200-400 IU LH per day, for example 250-350 IU/day. A preferred dosage range of recombinant or urinary hCG includes 25-400 IU hCG per day, preferably 50-200 IU hCG per day, for example 75-200, such as 100-150 or 120-170 IU/day.

[0342] Luteal support is continued until at least 2 weeks after ovulation, but may be continued until gestational week 5 or 10.

[0343] Similar results can be achieved by administering long-acting LH or long-acting hCG (FIG. 4b), one or more times during the luteal phase in a dosage giving a biological response similar to the response achieved by administering daily dosages of the preferred dosages of LH. The luteal support protocol can be used in conjunction with any type of ART.

[0344] The different protocols of the invention can be combined in many ways as long as they do not depart from the inventive concept of the invention as defined in the independent claims.

FSH

[0345] Follicle stimulating hormone (FSH) regulates the development, growth, pubertal maturation, and reproductive processes of the human body. In both males and females, FSH stimulates the maturation of germ cells. In males, FSH induces Sertoli cells to secrete inhibin and stimulates the formation of sertoli-sertoli tight junctions (zonula occludens). In females, FSH stimulates the growth and recruitment of immature Ovarian follicles in the ovary. In early (small) antral follicles, FSH is the major survival factor that rescues the follicles from apoptosis (programmed death of the somatic cells of the follicle and oocyte). In the luteal-follicle phase transition period the serum levels of progesterone and estrogen (primarily estradiol) decrease and no longer suppress the release of FSH, consequently FSH peaks at about day three (day one is the first day of menstrual cycle).

[0346] FSH is a heterodimeric glycoprotein. Each monomeric unit is a protein molecule with one or more oligosaccharide chains covalently linked to amino acid side chains; two of these monomeric units make the full, functional protein. The protein dimer contains 2 polypeptide units, labeled .alpha. and .beta. subunits. The a subunits of LH, FSH, TSH, and hCG are identical, and contain 92 amino acids (see FIG. 5). FSH has a .beta. subunit of 118 amino acids (FSHB), which confers its specific biologic action and is responsible for interaction with the FSH-receptor. The sugar part of the hormone is composed of fucose, galactose, mannose, galactosamine, glucosamine, and sialic acid, the latter being critical for its biologic half-life. The half-life of FSH is 3-4 hours.

[0347] The gene for the a subunit is located on chromosome 6p21.1-23. It is expressed in different cell types. The gene for the FSH .beta. subunit is located on chromosome 11p13, and is expressed in gonadotropes of the pituitary cells, controlled by GnRH, inhibited by inhibin, and enhanced by activin.

[0348] The beta-chain of preferred FSH of the present invention is selected from the group consisting of sequences having at least 80% sequence identity to SEQ ID NOS:10, 11, 12, 13, or 15, more preferably 85%, more preferably 90%, more preferably 95%. Preferably, a variant comprises the conserved cysteine residues at the position and spacing of SEQ ID NO:10. In a particularly preferred embodiment, the FSH comprises the human alpha-subunit having SEQ ID NO:10.

[0349] It will be understood by one of skill in the art that FSH may be substituted by a biologically active analogue, or by a compound that stimulates endogenous FSH secretion. In this latter class are included aromatase inhibitors, and anti-oestrogens such as tamoxifen and clomiphene citrate (CC). These compounds stimulate endogenous FSH secretion by removing the negative feedback exerted by oestrogen on the hypothalamus (either by antagonising oestrogen receptors, as is the case with CC and tamoxifen, or by greatly decreasing oestrogen concentrations, as is the case with aromatase inhibitors). Other types of FSH analogues include, for example single chain FSH analogues in which the [beta]-subunit is fused to the CTP of hCG, which in turn is fused to FSH [alpha]-subunit, as described in WO 96/05224 (single chain FSH-CTP).

[0350] In a further embodiment the FSH is selected from an analogue of a mammal FSH or an analogue of a mammal FSH. When the FSH is an analogue of a mammal FSH the analogue has at least 80% identity to the corresponding mammalian sequence of FSH, such as 85% identity, 90% identity, 95% identity, 98% identity. The sequence identity applies both to the alpha and beta chains of FSH.

[0351] The two most common forms used are urinary human menopausal gonadotropin (containing FSH and LH activity) and recombinant FSH (containing FSH without any LH activity). Owing to the relatively short half-life of all currently used FSH preparations, clinical protocols for induction of multi follicular development in women stimulated for IVF require daily injections. The use of long-acting versions of FSH, exhibiting prolonged half-lives, can be used to replace the daily injections of FSH.

[0352] FSH activity is normally given in IU following the pharmacopeia. Now pure preparations of FSH may also be manufactured and the activity given in mass (e.g. .mu.g per vial). In the present invention it is understood that activity given in IU correlates to FSH activity given with other units.

[0353] In one embodiment of the invention FSH is a long-acting FSH. By long-acting is intended a protein that has a serum half life which is at least 1.5 times the serum half-life of recombinant or urinary FSH, more preferably at least 2 times, more preferably at least 3 times, more preferably at least 4 times, more preferably at least 5 times, more preferably at least 7 times, more preferably at least 10 times, such as at least 15 times, for example at least 20 times, such as at least 25 times, for example at least 30 times, 40 times or 50 times or more.

[0354] Preferably the half-life of long-acting FSH is not longer than 72 hours, such as 48 hours. This makes it easier to control FSH administration in the later phases of folliculogenesis.

[0355] Long-acting FSH may for example be FSH-CTP, which is described in WO 93/06844, and has a wild type FSH [alpha]-subunit and a [beta]-subunit that consists of the wild type human FSH [beta]-subunit (SEQ ID NO:10) fused at its carboxyl terminal to the carboxy terminal peptide (CTP) of the [beta]-subunit of hCG (residues 118 to position 145 of the native hCG[beta]sequence, SEQ ID NO:9). The resulting beta-subunit has the sequence of SEQ ID NO:15.

[0356] Corifollitropin alfa is a glycoprotein produced in CHO cells by recombinant DNA technology. Corifollitropin alfa is designed as a sustained follicle stimulant with the same pharmacodynamic profile as (rec)FSH, but with a markedly prolonged duration of FSH activity. Due to its ability to initiate and sustain multiple follicular growth for up to a week, a single subcutaneous injection of the recommended dose of Corifollitropin (Elonva.RTM.) may replace some or all of the first seven injections of any daily (rec)FSH preparation in a COS treatment cycle. The long duration of FSH activity was achieved by adding the carboxyterminal peptide of the .beta.-subunit of human chorionic gonadotropin (hCG) to the .beta.-chain of human FSH (SEQ ID NO:10). Corifollitropin alfa does not display any intrinsic LH/hCG activity. Corifollitropin alfa has an average elimination half-life of 69 hours (59-79 hours).

[0357] Corifollitropin may preferably be administered as one bolus injection of 40-240 .mu.g per female mammal such as for example 60-220 .mu.g per female mammal, such as for example 80-200 .mu.g per female mammal, such as for example 100-180 .mu.g per female mammal or such as for example 100-150 .mu.g per female. Corifollitropin may in another embodiment be administered as one bolus injection of 40-120 .mu.g per female mammal, such as for example 60-100 .mu.g per female mammal, or such as for example 70-90 .mu.g per female mammal. In yet another embodiment corifollitropin is administered as one bolus injection of 120-240 .mu.g per female mammal, such as for example 140-220 .mu.g per female mammal, such as for example 160-200 .mu.g per female mammal or such as for example 170-190 .mu.g per female mammal. Corifollitropin is approved for dosages of 100 .mu.g for females under 60 kg and 150 .mu.g for females above 60 kg.

[0358] The administration of corifollitropin as one bolus injection is preferably on day 1-3 of the menstrual cycle, such as for example on day 1, day 2 or day 3 of the menstrual cycle.

[0359] Thus, in one preferred embodiment FSH is long-acting FSH, such as Corifollitropin, preferably administered as one bolus injection of 40-240 .mu.g per female mammal on day 1-3 of the menstrual cycle.

[0360] In aspects of the invention where FSH (or an analogue) is used in conjunction with COS techniques or regimens, appropriate doses and administration regimes will be apparent to a person skilled in the art and any appropriate dose and administration regime may be used.

[0361] For example FSH may be administered in a dosage giving a serum level of 1-50 IU FSH per litre, such as for example 2-40 IU FSH per litre, such as for example 3-35 IU FSH per litre, for example 4-30 IU FSH per litre, such as for example 5-25 IU FSH per litre, for example 7-20 IU FSH per, such as for example 10-15 IU FSH per litre during the follicular phase.

[0362] It is preferred that FSH is administered in a dosage giving a serum level of 5-25 IU FSH per litre, preferably 10-15 IU per litre during the follicular phase.

[0363] FSH may in one embodiment be administered at daily dosages of 50-600 IU FSH per day, preferably 100-300 IU FSH per day, such as 150-225 IU/day. In some patients showing a decreased response to FSH it may be desirable to use doses of up to 600 IU FSH per day. A typical regimen is as follows: the patient is started on 150 IU FSH per day. If follicular development is adequate the dose of 150 IU FSH/day may be maintained. If follicular development is inadequate the dose may be increased to 225, 300, 375, 450, 525 or 600 IU FSH/day. Ideally, the cumulative dose of FSH should not exceed 6000 IU/cycle.

[0364] FSH used in the methods of the invention can be from any source. Such sources will be well known to a person skilled in the field of ovulation induction and COS procedures. A urinary preparation of FSH may be used, e.g. hMG which contains FSH and LH activity at a 1:1 ratio.

[0365] Thus, in one embodiment of the present invention FSH is recombinant or urine-derived FSH administered at daily dosages of 50-600 IU FSH per day, preferably 100-300 IU FSH per day, such as 150-225 IU/day.

[0366] FSH may be administered starting on cycle day 1-3 of the menstrual cycle, such as for example on cycle day 1, for example on cycle day 2 or for example on cycle day 3 of the menstrual cycle.

[0367] Administration of FSH is discontinued when at least one follicle has a diameter of 12-14 mm, such as for example 12-13 mm or for example 13-14 mm. Administration of FSH may for example be discontinued on cycle day 4, 5, 7 or 8 of the menstrual cycle. In typical cases of the invention the administration of FSH is discontinued on cycle day 6 of the menstrual cycle. The purpose of discontinuing FSH administration is to stop further folliculogenesis and allow the largest follicles to mature. It is known in the art that there is a correlation between the number of follicles and the risk of OHSS.

[0368] When the administered FSH is recombinant or urine derived FSH, discontinuing FSH administration merely requires that no further FSH is administered. The serum level of FSH will then fall over the next couple of days to a level which will no longer stimulate folliculogenesis. When long-acting FSH is administered, e.g. corifollitropin alpha, discontinuing FSH administration means that the administration should be discontinued so that the level of serum FSH activity no longer stimulate folliculogenesis one or two days after the first follicles have reached the diameters described above. In the case of administration of e.g. corifollitropin alpha, it is preferred to administer just one dosage of corrifollitropin alpha on the first day of the stimulation protocol. If there is a need for further folliculogenesis, recombinant or urine derived FSH can be administered later in the stimulation cycle but before the follicles reach a size of 12-14 mm. In this way the serum level of FSH can be more easily controlled.

[0369] Generally speaking, the level of serum FSH activity, measured in IU or .mu.g per litre, should fall to a level which is below 50% of the serum level during the first 1-6 days of the stimulation protocol, more preferably below 25%, such as below 10%.

Luteinising Hormone (LH)

[0370] LH is a hormone produced by the anterior pituitary gland and is essential for reproduction both in males and females. In females, at the time of menstruation, FSH initiates follicular growth, specifically affecting granulosa cells. With the rise in oestrogens, LH receptors are also expressed on the maturing follicle that produces an increasing amount of estradiol. Eventually at the time of the maturation of the follicle, the oestrogen rise leads via the hypothalamic interface to the "positive feed-back" effect, a release of LH over a 24- to 48-hour period. This `LH surge` triggers ovulation, thereby not only releasing the egg but also initiating the conversion of the residual follicle into a corpus luteum that, in turn, produces progesterone to prepare the endometrium for a possible implantation. LH is necessary to maintain luteal function for the first two weeks. In case of a pregnancy, luteal function will be further maintained by the action of hCG (a hormone very similar to LH) from the newly established pregnancy. LH supports theca cells in the ovary that provide androgens and hormonal precursors for estradiol production. LH is a heterodimeric glycoprotein. Each monomeric unit is a protein molecule with one or more oligosaccharide chains covalently linked to amino acid side chains; two of these monomeric units make the full, functional protein. The protein dimer contains 2 polypeptide units, labeled alpha and beta subunits. The alpha subunits of LH, FSH, TSH, and hCG are identical, and contain 92 amino acids. LH has a beta subunit of 141 amino acids (LHB), which confers its specific biologic action and is responsible for interaction with the LH-receptor. This beta subunit contains an amino acid sequence that exhibits homologies with that of the beta subunit of hCG and both stimulate the same receptor. However, the hCG beta subunit contains an additional 24 amino acids, and the two hormones differ in the composition of their sugar moieties.

[0371] The beta chain of LH of the present invention is selected from the group consisting of sequences having at least 80% sequence identity to SEQ ID NOS:4, 5, 6, 7, and 8, more preferably 85%, more preferably 90%, more preferably 95%. Preferably, a variant comprises the conserved cysteine residues at the position and spacing of SEQ ID NO:4. In a particularly preferred embodiment, the LH comprises the human alpha-subunit having SEQ ID NO:4.

[0372] The different composition of these oligosaccharides affects bioactivity and speed of degradation and elimination. The biologic half-life of LH is 20 minutes, which is much shorter than that of FSH (3-4 hours) and hCG (24-30 hours).

[0373] It will be understood by one of skill in the art that LH may be substituted by a biologically active analogue, or by a compound that stimulates endogenous LH secretion. Analogues of LH include all molecules which exert the same physiological, biochemical or biological effects as LH, and/or bind to the same receptors as LH. Some analogues of LH may also include single chain LH. hCG is known to share some physiological actions with LH. Some examples of analogues of LH are as disclosed, for example in European patent no. EP 0 322 226 (Applied Research Systems), WO 92/22568 (University of Medicine & Dentistry of New Jersey), WO 96/05224 (Washington University), WO 90/09800 (Washington University), WO 93/06844 (Washington University), WO 98/43999 (Washington University), WO 99/25849 (Washington University), WO 00/61586 (Akzo Nobel).

[0374] LH and its analogues or the LH agonist may be selected from a small organic molecule, a peptide, a polypeptide, a protein, and may be produced by synthetic methods, recombinant means or be obtained from its natural sources, e.g. from urine, tissue or blood.

[0375] In one particular embodiment the LH agonist is recombinant or urine-derived LH. In another embodiment the LH agonist is of non-mammalian origin. In another particular embodiment the LH agonist is of mammalian origin, such as a protein obtained by recombinant means. In a further embodiment the LH agonist is selected from a mammal chorionic gonadotropin (CG) or a mammal LH. When the LH agonist is a mammal CG it is typically a primate CG, e.g. a human CG, but may also be selected from other mammalian species such as horse CG.

[0376] In a further embodiment the LH agonist is selected from an analogue of a mammal LH or an analogue of a mammal LH. When the LH agonist is an analogue of a mammal LH the analogue preferably has at least 80% identity to the corresponding mammalian sequence of luteinising hormone, such as 85% identity, 90% identity, 95% identity, 98% identity. The sequence variation can be in the alpha chain, in the beta chain or in both. The LH agonist may be an analogue of a human CG having at least 80% identity to the corresponding human sequence of chorionic gonadotropin, such as 85% identity, 90% identity, 95% identity, 98% identity. When the LH agonist is an analogue of a mammal LH the analogue has at least 80% identity to the corresponding mammalian sequence of luteinizing hormone, such as 85% identity, 90% identity, 95% identity, 98% identity. Typically, the LH agonist is an analogue of a human LH having at least 80% identity to the corresponding human sequence of luteinizing hormone, such as 85% identity, 90% identity, 95% identity, 98% identity.

[0377] When the LH agonist is selected from a polypeptide or protein, such as an analogue of a mammal CG or an analogue of a mammal LH, it may be glycosylated. Typically, the LH agonist is an hCG which is glycosylated. It may also be that the LH compound as such is glycosylated, and the glycosylation may be on the LH agonist or on the pharmaceutically acceptable molecule, when said molecule is selected from a polypeptide or protein, such as human albumin. The LH agonist may be linked to the pharmaceutically acceptable molecule in various ways, such as directly through a valence bond, or indirectly through a linker, which linker typically is a bifunctional linker, although it may also be a multifunctional linker. In further embodiments, the linker is selected from a chemical linker, a sugar moiety, a disulphide bridge, a fused linker, a hydrophilic linker, a hydrolysable linker. In a further embodiment the LH agonist is fused to the pharmaceutically acceptable molecule through a peptide linker. In a still further embodiment the LH agonist is fused directly to the pharmaceutically acceptable molecule, so as to create one polypeptide or protein, by expressing the LH compound from a host cell, such as a CHO cell or yeast cell. In a further embodiment the LH agonist is linked to the pharmaceutically acceptable molecule through a stable linker. In another embodiment the LH agonist is linked to the pharmaceutically acceptable molecule through a labile linker. Accordingly, the LH agonist may be linked to the pharmaceutically acceptable molecule in various ways using techniques that are well-known in the prior art, and the present invention also comprises the situation where one or more LH agonist(s) is linked to one or more pharmaceutically acceptable molecule(s), such as two LH agonist linked to one pharmaceutically acceptable molecule, or one LH agonist linked to two pharmaceutically acceptable molecules. In a further embodiment the LH agonist is linked to one or two pharmaceutically acceptable molecule(s), preferably one pharmaceutically acceptable molecule.

[0378] When the LH agonist is a mammal LH it is typically a human LH, but may also be selected from other mammalian species such as cow LH, pig LH, horse LH, sheep LH, dog LH, cat LH, and goat LH. Typically, the LH agonist is a human LH or human hCG. Normally, IVF involves daily luteal phase support treatment with progesterone during the first 2 weeks after embryo transfer. If gestation is confirmed on day 28 of the cycle daily luteal phase support treatment with progesterone may be extended for additional 5-10 weeks. In the present invention, administration of LH may however replace this daily treatment with progesterone. Providing luteal and gestational phase support by administering one or more dosages of an LH agonist may result in better development of oocytes, healthier oocytes, improved embryo implantation and retention, reduced biochemical pregnancy risk and may reduce the risk of OHSS.

[0379] In one embodiment the LH agonist used alone for luteal support is an LH analogue and not hCG or an hCG analogue. This is firstly because administration of hCG in the luteal phase may interfere with the biochemical pregnancy tests, which normally involve detection of urinary hCG secreted by the corpus luteum. Furthermore, administration of LH instead of hCG in the luteal phase is expected to reduce the risk of developing OHSS due to the different receptor affinities of the two proteins.

[0380] In another embodiment the LH agonist used alone for gestational support is an LH analogue and not hCG or an hCG analogue. This is firstly because administration of hCG in the gestational phase may interfere with the biochemical pregnancy tests, which normally involve detection of urinary hCG secreted by the corpus luteum. Furthermore, administration of LH instead of hCG in the gestational phase is expected to reduce the risk of developing OHSS due to the different receptor affinities of the two proteins.

[0381] Although both LH and hCG binds to and activate the LH-receptor, both hormones exist as a family of iso-hormones that differ in their oligosaccharide composition. Each of the different isoforms affects the receptor in a specific way and may elicit variable cellular responses (Burgon P G et al., Endocrinology, 1996;137:4827; Stanton PG et al., Mol Cell Endocrinol. 1996;125:133-141.), as have also been shown for the different FSH isoforms (Barrios-de-Tomasi J, et al. Mol Cell Endocrinol. 2002;186:189-98, Yding Andersen C & Ezcurra D, Reproductive Biology Insights 2011:4, 1-10). Thus the more subtle and fine-tuned effects of LH and hCG may actually differ. Recent studies presented at the ESHRE conference in Stockholm (July 2011) actually showed that LH acted much faster than hCG, but less efficient overall at the receptor level (L. Casarini et al., ESHRE Stockholm 2011-P312, Universita degli Studi di Modena, Italy). In a presentation by professor Peter Humaindan (ESHRE Stockholm 2011), it was further shown that addition of recombinant LH to recombinant FSH during COS significantly increased the oocyte yield as compared to equivalent doses of hCG added, suggesting specific LH effects at the receptor level. hCG is a pregnancy associated protein which is secreted following the implantation of the embryo around 8 days after conception. hCG is capable of stimulating the corpus luteum to remain active and continue its secretion of progesterone and other substances necessary for the pregnancy to become established. When hCG starts to be secreted from the implanting embryo, LH is present in appreciable amounts, but these levels are insufficient to stimulate the corpus luteum further and unless the woman becomes pregnant the corpus luteum will regress, a menstrual bleeding will occur and a new menstrual cycle start. So at this stage hCG preferentially stimulates the corpus luteum. Although this difference between LH and hCG has puzzled science for some time, it has now been demonstrated that the LH-receptor (LH-R) changes during the luteal phase. The functional full-length receptor maintains its expression when hCG is present, whereas LH is unable to accomplish that (Dickinson RE et al., Endocrinology 150: 2873-2881, 2009). This demonstrates differences in the effect of LH and hCG during the luteal phase.

[0382] The method for assisted reproductive therapy in a female mammal as described herein may in one embodiment further comprise providing luteal phase support by administering one or more dosages of an LH agonist replacing the current progesterone luteal phase support.

[0383] LH activity is normally given in IU following the pharmacopeia. Now pure preparations of LH may also be manufactured and the activity given in mass (e.g. .mu.g per vial). In the present invention it is understood that activity given in IU correlates to LH activity given with other units, such as molar units.

[0384] Urinary or recombinant or long-acting LH may be administered during the follicular phase instead of hCG (urinary or recombinant or long-acting) in dosages giving a response equivalent to the biological response provided by the dosages of urinary and recombinant hCG described herein.

[0385] The one or more dosages of LH agonist should be sufficient to provide a serum progesterone concentration of at least 5 nmol/L, such as at least 10 nmol/L, such as at least 15 nmol/L or such as at least 20 nmol/L at least until 5 days after ovulation or oocyte pick up, at least until 10 days after oocyte pick up, preferably at least until 14, more preferably at least until 21, more preferably at least until 28 days after ovulation or oocyte pick up. Preferably, the administration of an LH agonist may be continued beyond 28 days after ovulation or oocyte pickup such as up to 5 weeks, such as up to 6 weeks, for example up to 7 weeks, such as up to 8 weeks, for example up to 9 weeks, such as up to 10 weeks or more.

[0386] In one particular preferred embodiment, the method for assisted reproductive therapy further comprise providing luteal support by administering one or more dosages of an LH agonist sufficient to provide a serum progesterone concentration of at least 20 nmol/L at day 7 after oocyte pickup.

[0387] It is preferred that the one or more dosages in the luteal phase are sufficient to maintain a serum progesterone level of at least at 20 nmol/L at least until 10 days after oocyte pick up, preferably at least until 14, more preferably at least until 21, more preferably at least until 28 days after oocyte pick up.

[0388] It is preferred that the administered LH agonist is sufficient to provide a biological response similar to the response provided by a serum concentration of 4-12 IU recombinant or urinary LH per litre during the luteal phase, more preferably a serum concentration of 4-12 IU/L, such as 8-12 IU/L.

[0389] In a preferred embodiment recombinant or urine-derived LH is administered in the luteal phase at daily dosages of 100-600 IU LH per day, preferably 150-450 IU LH per day, such as 200-400 IU LH per day, for example 250-350 IU/day.

[0390] The LH agonist may in one embodiment be a long-acting LH exhibiting a prolonged serum half-life. The long-acting LH may comprise LH or an LH agonist linked to a chemical moiety such as a pharmaceutically acceptable molecule providing a serum half-life of the LH agonist or LH compound which is increased substantially when compared to the serum half-life of an LH agonist administered in the same manner as the LH compound or when compared to in vivo plasma half-life of endogenous chorionic gonadotropin (CG). The modified LH either given in the follicular phase or as a luteal phase support is believed to improve patient convenience and treatment outcome, when compared with conventional progesterone administration.

[0391] In one preferred embodiment the LH agonist is a long-acting LH comprising luteinizing hormone linked to a chemical moiety. In another preferred embodiment the LH agonist is a long-acting hCG comprising human chorionic gonadotropin linked to a chemical moiety.

[0392] In order to produce the long term LH compound which can be administered once or twice during the luteal phase in connection with assisted reproduction technology (ART) procedures, such long term LH compound when administered to a mammal should result in the LH agonist or LH compound having serum half-life of at least 1.5 times the half-life of LH, such as at least 2 times, at least 3 times, at least 4 times, preferably at least 5 times, more preferably at least 6 times or such as from 1.5 times to 25 times the half-life of LH.

[0393] In a preferred embodiment the LH agonist is a long-acting LH comprising luteinizing hormone linked to a chemical moiety, wherein the long-acting LH has a serum half-life of at least 6 times the half-life of LH.

[0394] When the long-acting LH comprises human chorionic gonadotropin linked to a chemical moiety, the serum half-life of the long term LH may for example be of at least 1.2 times the half-life of hCG, such as at least 1.3 times the half-life of hCG, at least 1.5 times the half-life of hCG, at least 2 times the half-life of hCG or such as at least 3 times the half-life of hCG.

[0395] The LH agonist may in one preferred embodiment be a long-acting hCG comprising human chorionic gonadotropin linked to a chemical moiety, wherein the long-acting hCG has a serum half-life of at least 1.5 times the half-life of hCG.

[0396] Luteal support is given to women before a biochemical pregnancy is detected, embryo implantation and pregnancy may be uncertain and the female might not get pregnant in a given cycle. Thus, the female mammal might have to undergo additional stimulation or ovulation cycles, and it is preferred that the half-life of the LH agonist administered during the luteal phase is no longer than 10 days, preferably no longer than 5 days, so that the level can fall below a level which can interfere with the subsequent ovulation or stimulation cycle in case the female mammal has to get a new treatment in the next cycle.

[0397] Examples of long-acting LH and hCG are found in the appended examples.

Human Chorionic Gonadotropin (hCG)

[0398] Human chorionic gonadotropin (hCG) is a glycoprotein hormone produced during pregnancy that is made by the developing embryo after conception and later by the syncytiotrophoblast (part of the placenta). Its role is to prevent the disintegration of the corpus luteum of the ovary and thereby maintain progesterone production that is critical during pregnancy in humans. hCG may have additional functions; for instance, it is thought that hCG affects the immune tolerance of the pregnancy. Early pregnancy testing, in general, is based on the detection or measurement of hCG.

[0399] Human chorionic gonadotropin interacts with the LHCG receptor and promotes the maintenance of the corpus luteum during the beginning of pregnancy, causing it to secrete the hormone progesterone. Progesterone enriches the uterus with a thick lining of blood vessels and capillaries so that it can sustain the growing fetus. Due to its highly-negative charge, hCG may repel the immune cells of the mother, protecting the fetus during the first trimester. It has also been hypothesized that hCG may be a placental link for the development of local maternal immunotolerance. For example, hCG-treated endometrial cells induce an increase in T cell apoptosis (dissolution of T-cells). These results suggest that hCG may be a link in the development of peritrophoblastic immune tolerance, and may facilitate the trophoblast invasion, which is known to expedite fetal development in the endometrium. It has also been suggested that hCG levels are linked to the severity of morning sickness in pregnant women.

[0400] Human chorionic gonadotropin is a glycoprotein composed of 244 amino acids with a molecular mass of 36.7 kDa. It is heterodimeric, with an alpha-subunit identical to that of luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and beta-subunit that is unique to hCG (SEQ ID NO:9).

[0401] The alpha-subunit is 92 amino acids long. The beta-subunit of hCG contains 145 amino acids encoded by six highly-homologous genes that are arranged in tandem and inverted pairs on chromosome 19q13.3-CGB (1, 2, 3, 5, 7, 8). The two subunits create a small hydrophobic core surrounded by a high surface area-to-volume ratio: 2.8 times that of a sphere. The vast majority of the outer amino acids are hydrophilic.

[0402] The beta chain of preferred hCG of the present invention is selected from the group consisting of sequences having at least 80% sequence identity to SEQ ID NO:9, more preferably 85%, more preferably 90%, more preferably 95%. Preferably, a variant comprises the conserved cysteine residues at the position and spacing of SEQ ID NO:9. In a particularly preferred embodiment, the hCG comprise the human alpha-subunit having SEQ ID NO:9.

[0403] hCG can be distinguished from LH by the presence in the beta-subunit of the C terminal peptide, CTP, consisting of amino acids 112-145 of SEQ ID NO:9. A distinction can be done immunologically and by sequencing.

[0404] The hCG that is used may be from any source, provided it is not contaminated with any materials (particularly other gonadotropins) which will substantially affect its action. Urinary hCG may be used, although it is preferred to use recombinant hCG (rhCG), because of its high purity. Similar conditions apply to the source of hCG for use in the present invention.

[0405] hCG activity is normally given in IU following the pharmacopeia. Now pure preparations of hCG may also be manufactured and the activity given in mass (e.g. .mu.g per vial). In the present invention it is understood that activity given in IU correlates to hCG activity given with other units, such as molar units.

[0406] Analogues of hCG include all molecules which exert the same physiological, biochemical or biological effects as hCG, and/or bind to the same receptors, as hCG. Luteinising hormone (LH) is known to share some physiological actions with hCG. Some analogues of hCG include single chain hCG, in which the C-terminus of the [bet]-subunit is fused to the N-terminus of the [alpha]-subunit (Sugahara et al., PNAS, 92, 1995, 2041-2045). Other examples of analogues are as is disclosed, for example in European patent no. EP 0 322 226 (Applied Research Systems), WO 92/22568 (University of Medicine & Dentistry of New Jersey), WO 96/05224 (Washington University), WO 90/09800 (Washington University), WO 93/06844 (Washington University), WO 98/43999 (Washington University), WO 99/25849 (Washington University).

[0407] In relation to fertility treatments, human chorionic gonadotropin is extensively used parentally as an ovulation inducer in lieu of luteinizing hormone. In the presence of one or more mature ovarian follicles, ovulation can be triggered by the administration of hCG. As ovulation will happen 24-36 hours after the injection of hCG, procedures can be scheduled to take advantage of this time sequence. Thus, patients that undergo IVF, in general, receive hCG to trigger the ovulation process, but have their eggs retrieved at about 36 hours after injection, a few hours before the eggs actually would be released from the ovary.

[0408] In the method of the present invention, at least one dosage of hCG is administered in the period from day 1-9 of the stimulation or of the menstrual cycle, the dosage being sufficient to stimulate follicle development until ovulation triggering. It is preferred that the administered hCG is sufficient to provide a biological response similar to the response provided by a serum concentration of 4-15 IU LH per litre during the luteal phase, more preferably a serum concentration of 4-12, or 5-12, such as 5-8 IU/L.

[0409] As described above, hCG may be recombinant or urine-derived. Thus, in one embodiment hCG is administered as daily dosages of recombinant or urine-derived hCG. The daily administration of hCG may for example be carried out until follicle maturation is triggered or ovulation is induced/triggered with a conventional bolus of hCG.

[0410] For daily administration of hCG during the follicular phase the dosage should be in the range of 25-4000 IU hCG/day, preferably 25-1000 IU hCG/day, more preferably 30-1000 or 30-500 IU hCG/day, most preferably 25-400 IU per day, such as 50-300 IU per day, more preferably 100-300, such as 150-250, for example 175-225 IU per day. It is also possible to administer hCG on a less frequent basis, for example every two, three, or four days, preferably every two days, until ovulation is triggered. In such a regimen, doses such as those outlined above may be used, although a dose of 50-400 IU hCG is preferred.

[0411] Thus, in one preferred embodiment the daily dosage of hCG when administered in the luteal phase is 25-400 IU hCG per day, preferably 50-200 IU hCG per day, for example 75-200, such as 100-150 or 120-170 IU/day.

[0412] In one embodiment of the present invention, hCG is a long-acting hCG exhibiting a prolonged serum half-life. The long-acting hCG may comprise hCG linked to a chemical moiety such as a pharmaceutically acceptable molecule providing a serum half-life of the hCG compound which is increased substantially when compared to the serum half-life of an hCG administered in the same manner as the long-acting hCG.

[0413] The half-life of the long-acting hCG may for example be of at least 1.2 times the half-life of hCG, such as at least 1.3 times the half-life of hCG, at least 1.5 times the half-life of hCG, at least 2 times the half-life of hCG or such as at least 3 times the half-life of hCG.

[0414] In one preferred embodiment hCG is a long-acting hCG with a serum half-life of at least 1.5 times the half-life of hCG.

[0415] In one embodiment the long-acting hCG is administered every 2.sup.nd day, such as every 3.sup.rd day, for example every 4.sup.th day, such as every 5.sup.th day, for example every 6.sup.th day, such as every 7.sup.th day, for example every 8.sup.th day, such as every 9.sup.th day, for example every 10.sup.th day during the ovulation induction phase and/or the subsequent luteal and/or gestational phases. In a further embodiment the long-acting hCG may be also administered every 14th day, such as every 21st, for example every month or even less frequently during the ovulation induction phase and/or the subsequent luteal phase and/or the subsequent gestational phase. As mentioned above, it is preferred to shift from hCG administration to LH administration in the luteal phase. The long-acting hCG is in one embodiment administered as a single bolus injection during the follicular phase, the dosage being sufficient to support the follicle development, and preferably also sufficient to provide luteal support.

[0416] In one embodiment of the present invention the long-acting hCG is administered as a single bolus injection when at least one follicle has a diameter of at least 8 mm, such as at least 9 mm, at least 10 mm, such as for example at least 11 mm, such as at least 13 mm, at least 14 mm or such as for example at least 15 mm.

[0417] In one preferred embodiment the long-acting hCG is administered as a single bolus injection when at least one follicle has a diameter of at least 12 mm.

[0418] The long-acting hCG is in one embodiment administered as a single bolus injection during FSH administration.

[0419] It is preferred that the hCG dosage in the follicular phase is sufficient to provide a biological response similar to the response provided by a serum concentration of 1-12 IU hCG per litre during the follicular phase.

[0420] It is further preferred that wherein hCG is recombinant or urine-derived hCG is administered at daily dosages of 25-300 IU hCG per day, preferably 50-200, such as 125-200 IU/day, for example 150-200 IU/day.

[0421] Preferably, hCG is administered daily from day 5 of the follicular phase and until ovulation triggering.

[0422] hCG may further be administered from day 2 of the follicular phase, so that hCG is administered from day 2 of the follicular phase and until ovulation.

Ovulation Triggering

[0423] The exact time of administration of the ovulation triggering treatment is determined by UV measurement of follicle diameter. Typically the triggering treatment is carried out when at least one follicle has a mean diameter of at least 15 mm. Preferably the triggering treatment is carried out when at least one follicle has a diameter of 16 mm, more preferably 17 mm. In many cases, the triggering treatment is carried out when at least 2 follicles have reached the indicated sizes, more preferably when at least 3 follicles have reached the indicated sizes. In a particularly preferred embodiment, ovulation triggering is induced when three or more follicles have a diameter of 17 mm.

[0424] In one aspect of the invention, the ovulation is triggered by administration of a therapeutically effective dosage of a GnRH agonist as herein described. In another aspect of the invention, ovulation is triggered by a relatively low dosage of hCG or an hCG analogue or variant or long-acting hCG. When recombinant or urinary hCG is used the triggering dosage is 2000 IU or less, such as 1500 IU or less, for example 1000 IU or less. A low dosage of hCG or analogue/variant/long-acting hCG can be supplemented with a dosage of a GnRH agonist.

Gonadotropin Releasing Hormone Agonist

[0425] A gonadotropin-releasing hormone agonist (GnRH agonist, GnRH-A) is a synthetic peptide modeled after the hypothalamic neuro hormone GnRH that interacts with the gonadotropin-releasing hormone receptor to elicit its biologic response, the release of the pituitary hormones FSH and LH. Agonists do not quickly dissociate from the GnRH receptor. As a result when administrating the GnRH agonist initially there is an increase in FSH and LH secretion (so-called "flare effect"). However after about ten days a profound hypogonadal effect (i.e. decrease in FSH and LH) is achieved through receptor down regulation by internalization of receptors. Generally this induced and reversible hypogonadism is the therapeutic goal.

[0426] In one aspect of the invention, a GnRH agonist is administered in order to trigger ovulation.

[0427] Examples of approved GnRH agonists include: leuprolide (Lupron, Eligard); buserelin (Suprefact, Suprecor); nafarelin (Synarel); histerelin (Supprelin); goserelin (Zoladex); deslorelin (Suprelorin, Ovuplant); Triptorelin.

[0428] Appropriate doses and administration regimes will be apparent to a person skilled in the art and any appropriate dose and administration regime may be used. The agonists can be administered subcutaneously or by initranasal spray.

[0429] By way of illustration, a commonly used and therapeutically effective dosage of Buserelin is 0.5 mg (subcutaneously) or 0.2 mg (intranasally). Triptorelin can be administered in a dosage of 0.2 mg subcutaneously and Leuprolide can be administered in a dosage of 1.0 mg subcutaneously.

Gonadotropin Releasing Hormone Antagonist

[0430] Gonadotropin-releasing hormone (GnRH) antagonists (receptor blockers) are a class of compounds that are similar in structure to natural GnRH (a hormone made by neurons in the hypothalamus) but that have an antagonistic effect. GnRH antagonists are peptide molecules that are made of multiple, often synthetically produced amino acids. GnRH antagonists compete with natural GnRH for binding to GnRH receptors, thus decreasing or blocking GnRH action in the body.

[0431] GnRH antagonists competitively and reversibly bind to GnRH receptors in the pituitary gland, blocking the release of luteinising hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary. In women, the reduction in LH subsequently leads to suppression of estrogen release from the ovaries.

[0432] Unlike the GnRH agonists, which cause an initial stimulation of the hypothalamic-pituitary-gonadal axis (HPGA), leading to a surge in oestrogen levels, GnRH antagonists have an immediate onset of action, rapidly reducing sex hormone levels without an initial surge.

[0433] Currently approved GnRH antagonists suitable for use in the methods of the present invention include the following: Cetrorelix; Ganirelix; Abarelix; Degarelix. Appropriate doses and administration regimes will be apparent to a person skilled in the art and any appropriate dose and administration regime may be used.

[0434] The above embodiments as well as the embodiments to be described hereunder should be seen as referring to any one of the aspects described herein as well as any one of the embodiments described herein unless it is specified that an embodiment relates to a certain aspect or aspects of the present invention.

[0435] All references, including publications, patent applications and patents, cited herein are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0436] All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

[0437] Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0438] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless other-wise indicated herein, and each separate value is incorporated into the specification as if it was individually recited herein. Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by "about," where appropriate).

[0439] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0440] The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise indicated. No language in the specification should be construed as indicating any element is essential to the practice of the invention unless as much is explicitly stated.

[0441] The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability and/or enforceability of such patent documents.

[0442] The description herein of any aspect or embodiment of the invention using terms such as "comprising", "having", "including" or "containing" with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that "consists of", "consists essentially of", or "substantially comprises" that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

[0443] This invention includes all modifications and equivalents of the subject matter recited in the aspects or claims presented herein to the maximum extent permitted by applicable law.

[0444] The present invention is further illustrated by the following examples which, however, are not to be construed as limiting the scope of protection. The features disclosed in the foregoing description and in the following examples may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

List of Embodiments

[0445] 1. A long acting biologically active luteinizing hormone (LH) compound comprising an LH agonist linked to a pharmaceutically acceptable molecule providing an in vivo plasma half-life of the LH agonist or LH compound which is increased substantially compared to the in vivo plasma half-life of an LH agonist administered in the same manner as the LH compound.

[0446] 2. The LH compound of embodiment 1 wherein the LH agonist is selected from a mammal CG or analog thereof or a mammal LH or analog thereof, such as recombinant hLH or hGH.

[0447] 3. The LH compound of any one of the preceding embodiments wherein the LH agonist is chemically linked to the pharmaceutically acceptable molecule, optionally through a bifunctional linker.

[0448] 4. The LH compound of any one of the preceding embodiments wherein the pharmaceutical acceptable molecule provides a biological body composition or concentration of the LH agonist or LH compound sufficient to drive an antral follicle from about 10 mm in diameter up to the preovulatory stage at about 15-30 mm in diameter in which a maturing oocyte can finalize the maturation to be ready for resumption of the meiosis.

[0449] 5. The LH compound of any one of the preceding embodiments wherein the pharmaceutical acceptable molecule provides a biological body composition or concentration of the LH agonist or LH compound sufficient to support hypogonadothrophic hypogonadal subjects.

[0450] 6. The LH compound of any one of the preceding embodiments wherein the pharmaceutical acceptable molecule provides a biological body composition or concentration of the LH agonist or LH compound sufficient to sustain progesterone in the pen-, in the ovulatoric- and the post ovulatoric- phase of a mammalian subject with the object regulating the endometrium and womb for avoiding or allowing implantation of a mammalian blastocyst.

[0451] 7. The LH compound of any one of the preceding embodiments wherein the pharmaceutically acceptable molecule provides a plasma concentration of the LH agonist or LH compound to support the formation and maintenance of Corpus Luteum/corpora lutea (CL), when an injection is given during the follicular phase of the menstrual cycle in connection with follicle stimulating hormone (FSH) treatment, preferably 5-10 days after initiation of FSH treatment.

[0452] 8. The LH compound of any one of the preceding embodiments wherein the LH agonist is selected from the sequence of human CG or human LH, the sequence of cow CG or cow LH, the sequence of pig CG or pig LH, the sequence of horse CG or horse LH, the sequence of sheep CG or sheep LH, the sequence of dog CG or dog LH, the sequence of cat CG or cat LH, and the sequence of goat CG or goat LH.

[0453] 9. The LH compound of any one of the preceding embodiments wherein the analog has at least 80% identity to the corresponding mammalian sequence of chorionic gonadotropin or luteinizing hormone, such as 85% identity, 90% identity, 95% identity, 98% identity.

[0454] 10. The LH compound of any one of the preceding embodiments wherein the LH agonist or LH compound having in vivo plasma half-life augmented at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, such as from 1.5 times to 25 times.

[0455] 11. The LH compound of any one of the preceding embodiments wherein the LH agonist or LH compound is glycosylated.

[0456] 12. The LH compound of any one of the preceding embodiments wherein the pharmaceutically acceptable molecule is selected from an albumin or a polymer, such as modified albumin with increased binding to FcRn, human albumin, recombinant human albumin or PEG.

[0457] 13. A pharmaceutical composition comprising the LH compound of any one of the preceding embodiments.

[0458] 14. The LH compound of any one of the preceding embodiments for use in promoting fertility of a mammalian subject, such as assisted reproduction technologies treatment.

[0459] 15. A modified luteinizing hormone comprising a mammalian LH or analog thereof linked to a pharmaceutically acceptable molecule providing an in vivo plasma half-life of the mammalian LH or analog thereof, or the modified LH which is from 2 to 48 hours in a mammal.

[0460] 16. The modified LH of embodiment 15 for providing an in vivo plasma concentration of the modified LH, the mammalian LH or a mixture thereof, in a mammal in the range of from 2 to 30 IU/L.

[0461] 17. The modified LH of any one of the preceding embodiments 15-16 wherein the mammalian LH or analog thereof is a recombinant LH, e.g. rhLH.

[0462] 18. The modified LH of any one of the preceding embodiments 15-17 wherein the modified LH comprises an analog of the mammalian LH, wherein the analog has at least 80% identity to the corresponding mammalian sequence of LH.

[0463] 19. The modified LH of any one of the preceding embodiments 15-18 wherein the mammalian LH is selected from the sequence of human LH, and the sequence of horse LH.

[0464] 20. The modified LH of any one of the preceding embodiments 15-19 wherein the pharmaceutically acceptable molecule is selected from any one of small organic molecules, peptides, oligopeptides, polypeptides, proteins, receptors, glycosylations, acylation groups, sugars, polymers (e.g. polyethylene glycols, PEG), nucleic acids (e.g. DNA and RNA), hormones, typically, pharmaceutically acceptable molecules are without limitation albumin, such as human albumin, recombinant albumin, variants of albumin, CH.sub.3(CH.sub.2).sub.nCO--, wherein n is 4 to 40, or polymer, such as PEG, e.g. PEG of a molecular weight of at least 5 kDa, such as from 10 kDa to 150 kDa, typically 10 to 40 kDa.

[0465] 21. The modified LH of any one of the preceding embodiments 15-20 wherein the mammalian LH or analog thereof is chemically linked to the pharmaceutically acceptable molecule, optionally through a bifunctional linker, or is fused to the pharmaceutically acceptable molecule, optionally through a peptide linker.

[0466] 22. The modified LH of any one of the preceding embodiments 15-21 wherein the mammalian LH or analog thereof, or modified LH is glycosylated.

[0467] 23. The modified LH of any one of the preceding embodiments 15-22 wherein the mammalian LH or analog thereof is linked to one or two pharmaceutically acceptable molecule(s), preferably one pharmaceutically acceptable molecule.

[0468] 24. A pharmaceutical composition comprising the modified LH of any one of the preceding embodiments 15-23.

[0469] 25. A pharmaceutical composition comprising the modified LH of any one of the preceding embodiments 15-23 and an FSH or a molecule having FSH activity.

[0470] 26. The modified LH of any one of the preceding embodiments 15-23 for use in combination with an FSH or a molecule having FSH activity for simultaneous, sequential or separate use to induce follicular development, such as paucifolliculogenesis or unifolliculogenesis, in anovulatory treatment of a mammalian female subject or induce COS in the follicular phase of the menstrual cycle of a mammalian female subject.

[0471] 27. The modified LH of embodiment 26 wherein administration of FSH:LH the IU ratios range from 20:1 to 1:20.

[0472] 28. The modified LH of any one of embodiments 25-27 wherein the FSH is selected from mammalian FSH, such as human FSH, in particular recombinant FSH, e.g. rhFSH.

EXAMPLES

Example 1

Method Producing Long Acting hCG

[0473] A long acting hCG is produced by chemical conjugation of hCG to human serum albumin or a variant of human serum albumin with selected improved or reduced affinity for the neonatal Fc receptor.

[0474] Chemical conjugation can be performed using a multitude of different chemistries and linkers known in the art, including linkers with a high covalent stability and linkers with lower covalent stability having the potential of releasing the active component from the albumin molecule typically by hydrolysation of a labile chemical bond.

[0475] Suitable attachment groups on the albumin molecule are apparent from the table below

TABLE-US-00004 Conjugation Attachment Examples of non- method/- group Amino acid peptide moiety Activated PEG Reference --NH2 N-terminal, Lys, Polymer, e.g. PEG, mPEG-SPA Shearwater His, Arg with amide or imine Tresylated Inc. Delgado group mPEG et al., critical reviews in Therapeutic Drug Carrier Systems 9(3, 4): 249- 304 (1992) --COOH C-term, Asp, Polymer, e.g, PEG, mPEG-Hz Shearwater Glu with ester or amide In vitro coupling Inc. group Oligosaccharide moiety --SH Cys Polymer, e.g. PEG, PEG- Shearwater with disulfide, Vinylsulphone Inc. Delgado critimaleimide PEG-maleimide et al., critical or vinyl sulfone In vitro coupling reviews in group Therapeutic Oligosaccharide Drug Carrier moiety Systems 9(3, 4): 249- 304 (1992) --OH Ser, Thr, --OH, Oligosaccharide In vivo O-linked Lys moiety glycosylation PEG with ester, ether, carbamate, carbonate --CONH2 Asn as part of an Oligosaccharide In vivo N-glycosylation moiety N-glycosylation site Polymer, e.g. PEG Aromatic Phe, Tyr, Trp Oligosaccharide In vitro coupling residue moiety --CONH2 Gln Oligosaccharide In vitro coupling Yan and moiety Wold, Biochemistry, 1984, Jul. 31; 23(16): 3759-65 Aldehyde Oxidized oligo- Polymer, e.g. PEG, PEGylation Andresz et Ketone saccharide PEG-hydrazide al., 1978, Makromol. Chem. 179: 301, WO92/16555, WO00/23114 Guanidino Oligosaccharide In vitro coupling Lundblad and moiety Noyes, Chemical Reagents for Protein Modification, CRC Press Inc., Florida, USA Imidazole His Oligosaccharide In vitro coupling As for ring moiety guanidine

[0476] Especially suitable is coupling to the free cysteine residue on the albumin molecule (Cys 34), e.g. by methods described in WO2010092135, especially the methods using PDPH (3-(2-pyridyldithio)propionyl hydrazide) to link albumin to hCG via a hydrazone link to hCG. In another aspect the method in WO2010092135 using EMCH ((3,3''-N-(s-maleimidocaproic acid) hydrazide) to link albumin to hCG via a hydrazone link to hCG is used.

[0477] Suitable attachment groups on the hCG molecule include those in the table above, and include chemistries for coupling to the glycosylation moieties of the hCG molecule. Coupling to the glycosylation moieties is preferred as these are expected not to have direct interaction with the hCG receptor and thereby the coupling will not interfere with the function.

[0478] Yet another coupling technology is described by Neose (see eg US2004/0126838) using enzymatic glycoconjugation. This technology can be used to link e.g. albumin to hCG using a suitable linker.

[0479] In the special case where chemical conjugation to the hCG molecule strongly reduce the functional activity it will be preferable to use a labile linker that can release a functional hCG. It is preferable to attach only one albumin molecule pr. hCG molecule.

[0480] In another instance the coupling of the hCG and the albumin molecule can be performed by genetic fusion of the two molecules. As the hCG molecule has two chains there are four different orientation possibilities:

[0481] Albumin-hCG(alpha chain)

[0482] Albumin-hCG(beta chain)

[0483] hCG(alpha chain)-albumin

[0484] hCG(beta chain)-albumin

[0485] Recombinant hCG packaged in a prefilled syringe in the product Ovitrelle.RTM. produced by Merck Serono are available containing 0.5 mL solution with 250 .mu.g recombinant hCG. The formulation excipients can be removed by dialysis and gel filtration. Albumin or albumin variants can be produced as described in WO2010092135. The hCG and the albumin can be conjugated using the PDPH or EMCH chemistry as described in WO2010092135.

Example 2

[0486] List of Sequences and Their UniProt (www.uniprot.ora) ID (name) and AC Accession

Glycoprotein Hormones Alpha Chain (see FIG. 5):

TABLE-US-00005

[0487] Human GLHA_HUMAN P01215 SEQ ID NO: 1 Mouse GLHA_MOUSE P01216 SEQ ID NO: 2 Rat GLHA_RAT P11962 SEQ ID NO: 3 >GLHA_HUMAN P01215_Mature 25-116 APDVQDCPECTLQENPFFSQPGAPILQCMGCCFSRAYPTPLRSKKTMLV QKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYHKS >GLHA_MOUSE P01216_Mature 25-120 LPDGDFIIQGCPECKLKENKYFSKLGAPIYQCMGCCFSRAYPTPARSKK TMLVPKNITSEATCCVAKAFTKATVMGNARVENHTECHCSTCYYHKS >GLHA_RAT P11962_Mature 25-120 LPDGDLIIQGCPECKLKENKYFSKLGAPIYQCMGCCFSRAYPTPARSKK TMLVPKNITSEATCCVAKSFTKATVMGNARVENHTDCHCSTCYYHKS

Luteinizing Hormone Beta Chain (see FIG. 6):

TABLE-US-00006

[0488] Human: LSHB_HUMAN P01229 SEQ ID NO: 4 Mouse: LSHB_MOUSE O09108 SEQ ID NO: 5 Rat: LSHB_RAT P01230 SEQ ID NO: 6 Gorilla: LSHB_GORGO Q2Q1P1 SEQ ID NO: 7 Chimpanzee: LSHB_PANTR Q2Q1P2 SEQ ID NO: 8 >LSHB_HUMAN P01229_Mature 21-141 SREPLRPWCHPINAILAVEKEGCPVCITVNTTICAGYCPTMMRVLQAVLP PLPQVVCTYRDVRFESIRLPGCPRGVDPVVSFPVALSCRCGPCRRSTSDC GGPKDHPLTCDHPQLSGLLFL >LSHB_PANTR Q2Q1P2_Mature 21-141 SREPLRPWCHPINATLAVEKEGCPVCITVNTTICAGYCPTMMRVLQAVLP PLPQVVCTYRDVRFESIRLPGCPRGVDPVVSFPVALSCRCGPCRRSTSDC GGPKDHPLTCDHPQLSGLLFL >LSHB_GORGO Q2Q1P1_Mature 21-141 SREPLRPRCRPINATLAVEKEGCPVCITVNTTICAGYCPTMMRVLQGVLP PLPQVVCTYRDVRFESIXLPGCPRGVDPMVSFPVALSCRCGPCHRSTSDC GGPNDHPLTCDHPQLSGLLFL >LSHB_MOUSE O09108_Mature 21-141 SRGPLRPLCRPVNATLAAENEFCPVCITFTTSICAGYCPSMVRVLPAALP PVPQPVCTYRELAFASVRLPGCPPGVDPIVSFPVALSCRCGPCRLSSSDC GGPRTQPMACDLPHLPGLLLL >LSHB_RAT P01230_Mature 21-141 SRGPLRPLCRPVNATLAAENEFCPVCITFTTSICAGYCPSMVRVLPAALP PVPQPVCTYRELRFASVRLPGCPPGVDPIVSFPVALSCRCGPCRLSSSDC GGPRTQPMTCDLPHLPGLLLF

Choriogonadotropin Beta (hCG-B):

TABLE-US-00007 Human: CGHB_HUMAN P01233 SEQ ID NO: 9 >CGHB_HUMAN P01233_Mature 21-165 SKEPLRPRCRPINATLAVEKEGCPVCITVNTTICAGYCPTMTRVLQGVLP ALPQVVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCALCRRSTTDC GGPKDHPLTCDDPREQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ

Follicle Stimulating Hormone (FIG. 7)

TABLE-US-00008

[0489] Follitropin subunit beta Human: FSHB_HUMAN P01225 SEQ ID NO: 10 Mouse: FSNB_MOUSE Q60687 SEQ ID NO: 11 Rat: FSHB_RAT P18427 SEQ ID NO: 12 Gorilla: FSHB_GORGO A1BN60 SEQ ID NO: 13 Chimpanzee: FSHB_PANTR Q2PUH2 SEQ ID NO: 14 >FSHB_HUMAN P01225_Mature 19-129 NSCELTNITIAIEKEECRFCISINTTWCAGYCYTRDLVYKDPARPKIQKT CTFKELVYETVRVPGCAHHADSLYTYPVATQCHCGKCDSDSTDCTVRGLG PSYCSFGEMKE >FSNB_MOUSE Q60687_Mature 21-130 SCELTNITISVEKEECRFCISINTTWCAGYCYTRDLVYKDPARPNTQKVC TFKELVYETVRLPGCARHSDSLYTYPVATECHCGKCDSDSTDCTVRGLGP SYCSFSEMKE >FSHB_RAT P18427_Mature 21-130 SCELTNITISVEKEECRFCISINTTWCEGYCYTRDLVYKDPARPNTQKVC TFKELVYETIRLPGCARHSDSLYTYPVATECHCGKCDSDSTDCTVRGLGP SYCSFGEMKE >FSHB_GORGO A1BN60_Mature 21-129 CELTNITIAIEKEECRFCISINTTWCAGYCYTRDLVYKDPARPNIQKTCT FKELVYETVRVPGCAHHADSLYTYPVATQCHCGKCDSDSTDCTVRGLGPS YCSFGEMKE >FSHB_PANTR Q2PUH2_Mature 21-129 CELTNITIAIEKEECRFCISINTTWCAGHCYTRDLVYKDPARPNIQKTCT FKELVYETVRVPGCAHHADSLYTYPVATQCHCGKCDSDSTDCTVRGLGPS YCSFGEMKE

Corifollitropin Alpha

[0490] Corifollitropin alpha consists of the gonadotropin alpha chain (SEQ ID NO:1) and the beta chain of FSH+the C-terminal 28 amino acids of hCG (marked in bold).

TABLE-US-00009 SEQ ID NO: 15 NSCELTNITIAIEKEECRFCISINTTWCAGYCYTRDLVYKDPARPKIQK TCTFKELVYETVRVPGCAHHADSLYTYPVATQCHCGKCDSDSTDCTVRG LGPSYCSFGEMKESSSSKAPPPSLPSPSRLPGPSDTPILPQ

Example 3

Method Producing Long Acting hLH

[0491] A long acting hLH is produced by chemical conjugation of hLH to human serum albumin or a variant of human serum albumin with selected improved or reduced affinity for the neonatal Fc receptor.

[0492] Chemical conjugation can be performed using a multitude of different chemistries and linkers known in the art, including linkers with a high covalent stability and linkers with lower covalent stability having the potential of releasing the active component from the albumin molecule typically by hydrolysation of a labile chemical bond.

[0493] Suitable attachment groups on the albumin molecule are apparent from the table below

TABLE-US-00010 Conjugation Attachment Examples of non- method/- group Amino acid peptide moiety Activated PEG Reference --NH2 N-terminal, Lys, Polymer, e.g. PEG, mPEG-SPA Shearwater His, Arg with amide or imine Tresylated Inc. Delgado group mPEG et al., critical reviews in Therapeutic Drug Carrier Systems 9(3, 4): 249- 304 (1992) --COOH C-term, Asp, Polymer, e.g, PEG, mPEG-Hz Shearwater Glu with ester or amide In vitro coupling Inc. group Oligosaccharide moiety --SH Cys Polymer, e.g. PEG, PEG- Shearwater with disulfide, Vinylsulphone Inc. Delgado critimaleimide PEG-maleimide et al., critical or vinyl sulfone In vitro coupling reviews in group Therapeutic Oligosaccharide Drug Carrier moiety Systems 9(3, 4): 249- 304 (1992) --OH Ser, Thr, --OH, Oligosaccharide In vivo O-linked Lys moiety glycosylation PEG with ester, ether, carbamate, carbonate --CONH2 Asn as part of an Oligosaccharide In vivo N-glycosylation moiety N-glycosylation site Polymer, e.g. PEG Aromatic Phe, Tyr, Trp Oligosaccharide In vitro coupling residue moiety --CONH2 Gln Oligosaccharide In vitro coupling Yan and moiety Wold, Biochemistry, 1984, Jul. 31; 23(16): 3759-65 Aldehyde Oxidized oligo- Polymer, e.g. PEG, PEGylation Andresz et Ketone saccharide PEG-hydrazide al., 1978, Makromol. Chem. 179: 301, WO92/16555, WO00/23114 Guanidino Oligosaccharide In vitro coupling Lundblad and moiety Noyes, Chemical Reagents for Protein Modification, CRC Press Inc., Florida, USA Imidazole His Oligosaccharide In vitro coupling As for ring moiety guanidine

[0494] Especially suitable is coupling to the free cysteine residue on the albumin molecule (Cys 34), e.g. by methods described in WO2010092135, especially the methods using PDPH (3-(2-pyridyldithio)propionyl hydrazide) to link albumin to hCG via a hydrazone link to hCG. In another aspect the method in WO2010092135 using EMCH ((3,3''-N-(.epsilon.-maleimidocaproic acid) hydrazide) to link albumin to hCG via a hydrazone link to hLH is used.

[0495] Suitable attachment groups on the hLH molecule include those in the table above, and include chemistries for coupling to the glycosylation moieties of the hLH molecule. Coupling to the glycosylation moieties is preferred as these are expected not to have direct interaction with the hLH receptor and thereby the coupling will not interfere with the function.

[0496] Yet another coupling technology is described by Neose (see e.g. US2004/0126838) using enzymatic glycoconjugation. This technology can be used to link e.g. albumin to hLH using a suitable linker.

[0497] In the special case where chemical conjugation to the hLH molecule strongly reduce the functional activity it will be preferable to use a labile linker that can release a functional hLHCG. It is preferable to attach only one albumin molecule pr. hLH molecule.

[0498] In another instance the coupling of the hLH and the albumin molecule can be performed by genetic fusion of the two molecules. As the hLH molecule has two chains there are four different orientation possibilities:

[0499] Albumin-hLH(alpha chain)

[0500] Albumin-hLH(beta chain)

[0501] hLH(alpha chain)-albumin

[0502] hLH(beta chain)-albumin

[0503] Recombinant hLH packaged as lyophilized powder in the product Luveris.RTM. produced by EMD Serono are available and can be reconstituted in 1.0 mL solution containing 82.5 IU recombinant hLH. The formulation excipients can be removed by dialysis and gel filtration. Albumin or albumin variants can be produced as described in WO2010092135. The recombinant hLH and the albumin can be conjugated using the PDPH or EMCH chemistry as described in WO2010092135.

Example 4

Covalent Attachment of SPA-PEG to hLH or Variants Thereof

[0504] Human LH and variants thereof are covalently linked to SPA-PEG 5000, SPA-PEG 12000 and SPA-PEG 20000 (NOF Corporation) as described below ("PEGylation of hLH and variants thereof in solution").

PEGylation of hLH and Variants Thereof in Solution

[0505] Human LH and variants thereof are PEGylated at a concentration of 250 .mu.g/ml in 50 mM sodium phosphate, 100 mM NaCl, pH 8.5. The molar surplus of PEG is 5-100 times with respect to PEGylation sites on the protein. The reaction mixture is placed in a thermo mixer for 30 minutes at 37.degree. C. 10 at 1200 rpm. After 30 minutes, quenching of the reaction is obtained by adding a molar excess of glycine.

[0506] Cation exchange chromatography is applied to remove excess PEG, glycine and other by-products from the reaction mixture. The PEGylation reaction mixture is diluted with 20 mM sodium citrate pH 2.5 until the ionic strength is less than 7 mS/cm. pH is adjusted to 2.5 using 5 N HCl. The mixture is applied to a SP-sepharose FF column equilibrated with 20 mM sodium citrate pH 2.5. Unbound material is washed off the column using 4 column volumes of equilibration buffer. PEGylated protein is eluted in three column volumes by adding 20 mM sodium citrate, 750 mM sodium chloride. Pure PEGylated hLH is concentrated and buffer exchange is performed using VivaSpin concentration devices, molecular weight cut-off (mwco): kDa.

Example 5

Production and Characterization of 1:1 Conjugates Between hCG and Recombinant Human Albumin or K573P Variant of Human Albumin

Materials

[0507] Recombinant Human Albumin (Recombumin, Novozymes Biopharma) was supplied as a 200 mg/mIsolution. The original vial was aliquoted into 50.times.1 ml aliquots in a laminar flow cabinet. Aliquots were stored refrigerated.

[0508] Recombinant Human Albumin variant K573P may be produced as described in WO2011051489. The compound (112 mg/ml) was stored refrigerated.

[0509] Recombinant hCG was produced from the product Ovitrelle (Merck Serono), and formulation excipients were removed by a buffer change using GE Healthcare, disposable PD-10 desalting columns as described for each conjugate. PDPH ((3-[2-pyridyldithio]propionyl hydrazide), EMCH (N-[maleimidocaproic acid]hydrazide)), SPDP (N -Succinimidyl 3-(2-pyridyldithio)-propionate) and EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) was purchased from Thermo Fisher Scientific Inc.

[0510] All other chemicals and materials was standard laboratory quality.

Methods

Size Exclusion HPLC (SEC-HPLC):

[0511] Analytical SEC-HPLC was undertaken using an Agilent HP1100 machine fitted with a multi-wavelength detector. Analytical columns used were: TSK g3000 SWXL (7.8 mm id.times.30 cm length) with TSK- SWXL guard column TSK g3000 SWXL (7.5 mm id.times.60 cm length) with TSK- SWXL guard column Prep-scale HPLC was undertaken using a Waters HPLC system. Prep columns used were:

[0512] Superdex 200 26/600 Hiload

[0513] Superdex 200 10/300 GL

All columns were run in 0.2 .mu.m filtered PBS at pH 7.4 unless described otherwise. Detection was at 280 nm. Typical run conditions/analysis times are shown in the Table below.

TABLE-US-00011 Flow rate Analysis time Column Method (ml/min) (min) Superdex 200 Manual 2.6 120 26/600 Hiload Superdex 200 SPRDX200.M 0.5 60 10/300 GL TSK g3000 30 cm hCG.M 1 15 TSK g3000 60 cm TSK60.M 1 30

SDS Non-Reducing Gels:

[0514] Optimal separation/resolution for SDS non-reducing gels was achieved using Novex 4-12% Tris-glycine gels. Running Buffer was Tris acetate SDS running buffer.

Sample preparation was as follows:

[0515] 5 .mu.l LDS sample buffer was added to 15 .mu.l sample. Incubate 5 minutes at room temperature

[0516] 10 .mu.l sample loaded onto gel

[0517] All gels were run at a constant voltage of 150V, run time .about.60 minutes.

[0518] Gels were washed for 5 minutes with deionised water, stained with Gel Code Blue Safe-Stain (ThermoFisher) and de-stained using deionised water.

[0519] SDS reducing Gels:

[0520] Optimal separation/resolution for SDS non-reducing gels was achieved using Novex 4-12% Tris-glycine gels. Running Buffer was Tris-glycine SDS running buffer.

Sample preparation was as follows:

[0521] 5 .mu.l LDS sample buffer

[0522] 2 .mu.l NuPAGE reducing agent

[0523] 15 .mu.l sample.

[0524] Samples were heated to 85 deg.C. for two minutes and 10 .mu.l sample was loaded onto the gel. All gels were run at a constant voltage of 150V, run time .about.60 minutes. Gels were washed and stained/de-stained as for non-reducing gels.

Spectrophotometric Methods (A280):

[0525] Measurement was performed using a Shimadzu UV-160 spectrophotometer with 1 cm quartz microcuvette over the range 220-320 nm, following baseline correction against the sample buffer.

Endotoxin Measurments:

[0526] Endotoxin measurements were taken using a Charles River Portable Test System (PTS) with a 1-0.01 EU/mL. Samples were analysed after dilution with 1/10 ratio of dispersal reagent (0.05 mL sample +0.1 mL dispersal agent +0.35 mL LAL water). A PTS endotoxin cartridge was used to analyse the samples post dilution.

A: Production of Conjugate1

[0527] Conjugate 1 is a 1:1:1 hCG-PDPH-Albumin conjugation produced by conjugation of PDPH linker to oxidized sialic acids on hCG and further coupling of hCG-PDPH to rHA via formation of a disulphide bond to the free cysteine at rHA as described for PDPH by the manufacturer.

Preparation of hCG:

[0528] Thirty syringes of Ovitrelle were pooled (totaling 15 ml, 7.5 mg hCG) and concentrated to .about.5 mg/ml using vivaspin 500 centrifugal concentrators in a bench top centrifuge at 13,000 rpm. The concentrated solution was buffer changed using three PD-desalting columns, equilibrated in phosphate buffered saline at pH 7.4. 500 .mu.l solution was loaded onto each column and washed in with 2.5 ml PBS. hCG was eluted in 1 ml PBS. The buffer changed hCG concentration was determined by A280 measurement using an extinction coefficient of 0.459 for a 1 mg/ml solution.

Periodate Oxidation of hCG:

[0529] A 1 mg/ml solution of sodium periodate in H2O was prepared. This was added to chilled hCG solution (44 .mu.l per mg hCG), wrapped in foil to protect from light, and placed on ice for 35 minutes. Unreacted periodate was removed from the reaction mixture using two PD-10 desalting columns, equilibrated in phosphate buffered saline at pH 7.4. The reaction mixture was divided into two equal aliquots, loaded onto columns, and washed in to a total volume of 2.5 ml with PBS. Oxidised hCG was eluted in 500 .mu.l aliquots of PBS. Protein containing peak fractions were identified by A280 measurement and pooled. The hCG concentration was determined by A280 measurement. The oxidised hCG solution was concentrated to .about.8 mg/ml hCG using vivaspin 500 centrifugal concentrator.

Reaction of Oxidised hCG with PDPH:

[0530] A 100 mg/ml solution of PDPH in dimethyl sulphoxide (DMSO) was prepared. This was added to the oxidised hCG solution to give a 32.5 fold molar excess of PDPH (3 .mu.l PDPH solution per mg hCG), mixed gently and placed in a water bath at 25 deg.C. for 3 hours. To remove unreacted crosslinker, the reaction mixture was loaded onto a PD-10 column equilibrated in PBS, and washed in to a total volume of 2.5 ml with PBS. The hCG-PDPH was eluted in 500 .mu.l aliquots of PBS. Peak fractions were identified by A280 measurement and pooled.

Preparation of rHA:

[0531] Recombumin rHA was purified to remove rHA dimer, prior to reaction with hCG-PDPH. The rHA (500 .mu.l) was loaded onto a Superdex 200 10/300 GL column and eluted in PBS as described in above. The monomer peak was collected. rHA concentration was determined by A280 measurement using a 1 mg/ml extinction coefficient of 0.51. The purified rHA was concentrated to .about.200 mg/ml using a vivaspin 500 centrifugal concentrator in a benchtop centrifuge at 13,000 rpm.

Reaction of hCG-PDPH with rHA:

[0532] Purified rHA was added to the hCG-PDPH solution to give a 5 fold molar excess of 10 rHA (12.8 mg rHA per mg hCG), mixed gently and placed in a waterbath at 25 deg.C. for 3 hours.

Purification of 1:1:1 Conjugate:

[0533] The reaction mixture was divided into two 500 .mu.l aliquots. The first aliquot was loaded onto a Superdex 200 10/300 GL column and eluted in PBS as described above. The conjugate peak was collected. This was repeated with the second aliquot of reaction mixture. Conjugate peaks were pooled and conjugate concentration was determined by A280 measurement using a 1 mg/ml extinction coefficient of 0.496.

[0534] The conjugate solution was re-concentrated tenfold, to a volume of 700 .mu.l, using vivaspin500 centrifugal concentrators. This concentrated conjugate solution was purified in 100 .mu.l aliquots using a Superdex 200 10/300 GL column as before.

[0535] The purified conjugate solution was filtered through a 0.2 .mu.m filter, pipetted into 50 .mu.l aliquots in sterile 500 .mu.l eppendorf tubes, and frozen.

[0536] The samples were analyzed by reducing and non-reducing SDS-PAGE (FIGS. 8a and 8b) and by SEC-HPLC (FIG. 8c).

[0537] Conjugate 1 stability was analyzed for 96 hours at four different conditions; 5 deg.C. (pH 7.4), 37 deg.C. (pH 7.4), 37 deg.C. (pH 6.4), and 37 deg.C. (pH 5.4). The samples were analyzed by SEC-HPLC.

TABLE-US-00012 Storage Conditions 5 deg. 37 deg. 37 deg. 37 deg. C. pH C. pH C. pH C. pH 7.4 7.4 6.4 5.4 t = 0 conjugate 89.9% -- -- -- monomer 7.5% -- -- -- t = 12 h conjugate .sup. 92% 70.4% 75.6% 69.9% monomer 8% 29.6% 24.4% 30.1% t = 24 conjugate .sup. 90% 53.2% 70.4% 65.6.1%.sup. monomer .sup. 10% 46.8% 29.6% 34.4% t = 48 conjugate 90.4% 32.5% 63.3% 57.0% monomer 9.6% 67.5% 36.7% 43.0% t = 96 conjugate 89.7% 20.4% 45.8% 46.6% monomer 10.3% 79.6% 54.2% 53.4%

The result of the stability analysis is that non-conjugated hCG is released at an initial speed of app. 40% per day.

B: Production of Coniuqate3

[0538] Conjugate3 is a 1:1:1 hCG-SPDH-Albumin conjugation produced by conjugation of SPDP linker to free amines (on N-termini or on lysine side chains) on hCG and further coupling of hCG-SPDP to rHA via formation of a disulphide bond to the free cysteine at rHA as described by the SPDP manufacturer.

Preparation of hCG:

[0539] Twenty syringes of Ovitrelle were pooled (10 ml, 5 mg hCG) and concentrated to app. 2 ml using 6 Vivaspin500 centrifugal concentrators in a benchtop centrifuge at 13,000 rpm. The concentrated solution was buffer changed using 2 PD-10 desalting columns, equilibrated in citrate buffer at pH 5.9. Half of the solution was loaded onto each column and washed in to a total volume of 2.5 ml with citrate buffer. The hCG was eluted in 500 .mu.l aliquots of citrate buffer. Peak fractions were identified by A280 measurement and pooled. The hCG concentration was determined by A280 measurement using an extinction coefficient of 0.459 and diluted to a concentration of 1 mg/ml with citrate buffer.

Reaction of hCG with SPDP:

[0540] A 2.5 mg/ml solution of SPDP in DMSO was prepared. This was added to the hCG solution to give a 10 fold molar excess of SPDP (50 .mu.l SPDP solution per mg hCG), mixed gently and placed in a water bath at 25 deg.C. for 1 hour. To remove unreacted crosslinker, half of the reaction mixture was loaded onto each of two PD10 columns equilibrated in phosphate buffered saline at pH 7.4, and washed in to a total volume of 2.5 ml with PBS. The hCG-SPDP conjugate was eluted in 500 .mu.l aliquots of PBS. Protein containing peak fractions were identified by A280 measurement and pooled.

Preparation of rHA:

[0541] The rHA was purified using a Superdex 200 26/600 Hiload column. The rHA (3 ml) was loaded onto the column and eluted in PBS as described above. The monomer peak was collected. The rHA concentration was determined by A280 measurement as before.

Reaction of hCG-SPDP with rHA:

[0542] Purified rHA was added to the hCG-SPDP solution to give a 5 fold molar excess of rHA (12.8 mg rHA per mg hCG), mixed gently and placed in a water bath at 25 deg.C. overnight. The reaction mixture (3 ml) was loaded onto a Superdex 200 26/600 Hiload column and eluted in PBS as described above. The conjugate peak was collected. The solution was sterile filtered, and placed in an incubator at 37 deg.C. for 48 hours, for hydrolysis of weakly bound crosslinker.

Final Purification of 1:1:1 Conjugate:

[0543] After hydrolysis the solution was concentrated to a volume of app. 3 ml, using vivaspin500 centrifugal concentrators. Concentrated solution was loaded onto a Superdex 200 26/600 Hiload column and eluted in PBS. The conjugate peak was collected. Conjugate concentration was determined by A280 measurement using a 1 mg/ml extinction coefficient of 0.496. The purified conjugate solution was concentrated to app. 1 mg/ml as before, filtered through 0.2 micom filter, pipetted into 50 .mu.l aliquots in sterile 500 .mu.l eppendorf tubes, and frozen.

[0544] The samples were analyzed by reducing and non-reducing SDS-PAGE (FIGS. 9a and 9b) and by SEC-HPLC (FIG. 9c).

[0545] Conjugate 3 stability was analyzed for 96 hours at 37 deg.C. (pH 7.4). The samples were analyzed by SEC-HPLC:

TABLE-US-00013 Time High MW (%) Conjugate (%) rHA + hCG monomer (%) 0 (start) 4.5 95.5 0 24 hours 2.5 95.2 2.3 48 hours 2.2 94.5 3.3 72 hours 4.7 91.0 4.3 96 hours 3.4 91.0 5.7

[0546] The result of the stability analysis was that conjugate 3 is reasonable stable and that non-conjugated hCG is released at an initial speed of app. 2% pr. day.

C: Production of Coniuqate4

[0547] Conjugate4 is a 1:1:1 hCG-PDPH-Albumin conjugation produced by conjugation of PDPH linker to hCG activated with EDC whereby carboxylic acid groups in hCG will react with PDPH. hCG-PDPH is further coupled to rHA via formation of a disulphide bond to the free cysteine at rHA as described by the PDPH and EDC manufacturer.

Preparation of hCG:

[0548] Twenty syringes of Ovitrelle were pooled (10 ml, 5 mg hCG) and concentrated to a volume of app. 1.5 ml using 6 Vivaspin500 centrifugal concentrators in a benchtop centrifuge at 13,000 rpm. The concentrated hCG solution was buffer changed using 3 PD-10 columns, equilibrated in MES buffer at pH 5.3. Approximately 500 .mu.l solution was loaded onto each column and washed into a total volume of 3 ml with MES buffer. hCG was eluted in 1.0 ml MES buffer. The hCG concentration was determined by A280 measurement using an extinction coefficient of 0.459.

Reaction of hCG with EDC and PDPH:

[0549] A 50 mg/ml solution of PDPH in DMSO was prepared. This was added to the hCG solution to give 5 mM PDPH in the reaction mixture (1.15 mg/ml PDPH) and mixed gently. A 50 mg/mIsolution of EDC in MES buffer was prepared and immediately added to the hCG/PDPH solution to give 2.5 mM EDC in the reaction mixture (0.48 mg/ml EDC). The reaction mixture was mixed gently and placed in a water bath at 25 deg.C. for 30 minutes. To remove unreacted crosslinker, half of the reaction mixture was loaded onto each of two PD-10 columns equilibrated in phosphate buffered saline at pH 7.4, and washed in to a total volume of 2.5 ml with PBS. The hCG-PDPH was eluted in 500 .mu.l aliquots of PBS. Protein containing peak fractions were identified by A280 measurement and pooled.

Preparation of rHA:

[0550] rHA was purified using a Superdex 200 26/600 Hiload column. The rHA (3 ml) was loaded onto the column and eluted in PBS. The monomer peak was collected. The rHA concentration was determined by A280 measurement as before.

Reaction of hCG-PDPH with rHA:

[0551] Purified rHA was added to the hCG-PDPH solution to give a 5 fold molar excess of rHA (12.8 mg rHA per mg hCG), mixed gently and placed in a water bath at 25 deg.C. for three hours. Half of the reaction mixture was loaded onto a Superdex 200 26/600 Hiload column after three hours. The second half was loaded two hours later, on completion of the first cycle. The conjugate solution was concentrated to a volume of app. 3 ml using vivaspin500 centrifugal concentrators. The solution was sterile filtered, and placed in an incubator at 37 deg.C, for 36 hours, for hydrolysis of weakly bound crosslinker.

Final Purification of 1:1 Conjugate:

[0552] The hydrolysed conjugate solution (3 ml) was loaded onto a Superdex 200 26/600 Hiload column and eluted in PBS as described above. The conjugate peak was collected. Conjugate concentration was determined by A280 measurement using a 1 mg/ml extinction coefficient of 0.496. The purified conjugate solution was concentrated to app. 1 mg/ml as before, filtered through 02 .mu.m filter, pipetted into 50 .mu.l aliquots in sterile 500 .mu.l eppendorf tubes, and frozen.

[0553] The samples were analyzed by reducing and non-reducing SDS-PAGE (FIGS. 10a and 10b) and by SEC-HPLC (FIG. 10c)

[0554] Conjugate 4 stability was analyzed for 96 hours at 37 deg.C. (pH 7.4). The samples were analyzed by SEC-HPLC:

TABLE-US-00014 Incubation time (h) % monomer 0 (start) 0 24 2.7 48 3.7 72 4.6 96 4.5

[0555] The result of the stability analysis was that conjugate 3 is reasonable stable and that non-conjugated hCG is released at an initial speed of app. 2% per day.

D: Production of Coniuqate3V1

[0556] Conjugate3V1 is rhCG-SPDP-rHA(K573P).

[0557] A conjugate of hCG and rHA variant (K573P) was prepared using SPDP crosslinker, using the method used to produce Conjugate3 as described above. The samples were analyzed by reducing and non-reducing SDS-PAGE (FIGS. 11a and 11b) and by SEC-HPLC (FIG. 11c).

[0558] Conjugate 3V1 stability was analyzed for 96 hours at 37 deg.C. (pH 7.4). The samples were analyzed by SEC-HPLC:

TABLE-US-00015 Incubation time (h) % monomer 0 (start) 0 24 3.7 48 4.8 72 5.5 96 6.2

[0559] The result of the stability analysis was that conjugate 3V1 is reasonable stable and that non-conjugated hCG is released at an initial speed of app. 2% per day.

E: Production of Coniuqate4V1

[0560] Conjugate4V1 is rhCG-PDPH-rHA(K573P).

[0561] A conjugate of hCG and rHA variant (K573P) was prepared using EDC and PDPH crosslinkers, using the method used to produce Conjugate4 as described above. The samples were analyzed by reducing and non-reducing SDS-PAGE (FIGS. 12a and 12b) and by SEC-HPLC (FIG. 12c).

TABLE-US-00016 Incubation time (h) % monomer 0 (start) 0 24 2.8 48 3.3 72 4.3 96 5.7

[0562] The result of the stability analysis was that conjugate 4V1 is reasonable stable and that non-conjugated hCG is released at an initial speed of app. 2% pr day

Example 6

[0563] Production of hCG-Albumin Fusions and hLH-Albumin Fusions.

Construction of Expression Plasmids

[0564] Genes encoding the gonadotropin common .alpha.-subunit, the hCG .beta.-subunit and the hLH .beta.-subunit and fusions of these three with the gene of human serum albumin at either the 3'-end or at the 5'-end was constructed by assembly of synthetic oligonucleotides using polymerase chain reaction (PCR). The sequence encoding for the natural human signal sequences of the relevant genes was included, and for human serum albumin the gene encoding for the natural pro-peptide was included. The codon usage of the genes was optimized for high expression in mammalian cells. The relevant genes are:

TABLE-US-00017 Gene# Sequence ID Gene encoding Gene1 SEQ ID NO: 44 .alpha.-chain (348 bases) Gene2 SEQ ID NO: 45 hCG .beta.-chain (495 bases) Gene3 SEQ ID NO: 46 LH .beta.-chain (423 bases) Gene4 SEQ ID NO: 47 Wt Albumin + .alpha.-chain (2103 bases) Gene5 SEQ ID NO: 48 .alpha.-chain + wt Albumin (2103 bases) Gene6 SEQ ID NO: 49 Wt Albumin + hCG .beta.-chain (2262 bases) Gene7 SEQ ID NO: 50 hCG .beta.-chain + wt Albumin (2250 bases) Gene8 SEQ ID NO: 51 Wt Albumin + LH .beta.-chain (2190 bases) Gene9 SEQ ID NO: 52 LH .beta.-chain + wt Albumin (2178 bases)

[0565] The gene sequences were synthesised by GeneArt AG and sub-cloned into pEE12.4 and pEE6.4 vectors respectively as shown in Tables below

TABLE-US-00018 Product Number Product name First Gene Second Gene 1 hCG Gene1 Gene2 2 hCG-wtA-.alpha.-N Gene4 Gene2 3 hCG-wtA-.beta.-N Gene1 Gene6 4 hCG-wtA-.alpha.-C Gene5 Gene2 5 hCG-wtA-.beta.-C Gene1 Gene7 6 LH Gene1 Gene3 7 LH-wtA-.alpha.-N Gene4 Gene3 8 LH-wtA-.beta.-N Gene1 Gene8 9 LH-wtA-.alpha.-C Gene5 Gene3 10 LH-wtA-.beta.-C Gene1 Gene9

[0566] N-terminal restriction site Hind III and the C-terminal restriction site EcoRI were used. In short, 5 .mu.g of lyophilised shuttle vector as produced by GeneArt was resuspended in 50 .mu.l endotoxin free, sterile water. 10 .mu.l of the generated 100 ng/ml DNA solution was mixed with 2.5 .mu.l each of EcoRI and HindIII high-fidelity restriction enzymes, 5 .mu.l of 10.times. NEB buffer and 30 .mu.l endotoxin free, sterile water on ice. Samples were then incubated at 37.degree. C. for 2 hours. 8.3 .mu.l of 6.times. DNA loading buffer was added and samples electrophoresed at 120 V for 40-60 min on a 1% w/v agarose gel stained with ethidium bromide. 10 .mu.l Lonza SimplyLoad Tandem DNA ladder was used as reference ladder. The agarose gel was imaged using a BioSpectrum Imaging System (UVP).

[0567] The relevant fragments were gel-extracted using a QIAquick gel extraction kit according to manufacturer's instructions. Ligations were set-up using a 1:6 and a 1:12 ratio of vector backbone to insert DNA, 1 .mu.l T4 quick ligase, 20 .mu.l of 2.times. T4 quick ligation buffer, reaction volume adjusted to 20 .mu.l with endotoxin-free, sterile water when necessary and samples incubated at room temperature for 15 minutes. 10 .mu.l aliquots of the ligation reaction were used to transform One Shot Top 10 Chemically Competent Escherichia coli cells using the heat-shock method according to manufacturer's instructions. Cells were spread onto ampicillin-containing (50 .mu.g/ml) Luria Bertani agar plates and incubated overnight at 37.degree. C. until bacterial colonies were evident. To screen for recombinants, single bacterial colonies were picked into 15 ml Luria Bertani (LB) medium containing 50 .mu.g/ml ampicillin and incubated at 37.degree. C. for 6 hours with shaking. Vector DNA was isolated from 10 ml of these growth cultures using the QIAGEN miniprep system and eluted in 30 .mu.l EB buffer. Positive recombinants were identified by digestion with HindIII and EcoRI. Aliquots of generated vectors were sent for gene sequencing by 3rd party using vector specific forward (GCTGACAGACTAACAGACTGTTCC (SEQ ID NO:70)) and reverse (CAAATGTGGTATGGCTGA (SEQ ID NO:71)) primers. The Table below shows the GS vectors used for each gene.

[0568] The relevant fragments were gel-extracted using a QIAquick gel extraction kit according to manufacturer's instructions. Ligations were set-up using a 1:6 and a 1:12 ratio of vector backbone to insert DNA, 1 .mu.l T4 quick ligase, 20 .mu.l of 2.times. T4 quick ligation buffer, reaction volume adjusted to 20 .mu.l with endotoxin-free, sterile water when necessary and samples incubated at room temperature for 15 minutes. 10 .mu.l aliquots of the ligation reaction were used to transform One Shot Top 10 Chemically Competent Escherichia coli cells using the heat-shock method according to manufacturer's instructions. Cells were spread onto ampicillin-containing (50 .mu.g/ml) Luria Bertani agar plates and incubated overnight at 37.degree. C. until bacterial colonies were evident. To screen for recombinants, single bacterial colonies were picked into 15 ml Luria Bertani (LB) medium containing 50 .mu.g/ml ampicillin and incubated at 37.degree. C. for 6 hours with shaking. Vector DNA was isolated from 10 ml of these growth cultures using the QIAGEN miniprep system and eluted in 30 .mu.l EB buffer. Positive recombinants were identified by digestion with HindIII and EcoRI. Aliquots of generated vectors were sent for gene sequencing by 3rd party using vector specific forward (GCTGACAGACTAACAGACTGTTCC (SEQ ID NO:70)) and reverse (CAAATGTGGTATGGCTGA (SEQ ID NO:71)) primers. The Table below shows the GS vectors used for each gene.

TABLE-US-00019 Gene Sub-cloned into 1 pEE12.4 2 pEE6.4 3 pEE6.4 4 pEE12.4 5 pEE12.4 6 pEE6.4 7 pEE6.4 8 pEE6.4 9 pEE6.4

DNA Amplification:

[0569] For DNA amplification, 5 ml of the growth cultures produced during the colony screening were used to inoculate 1.5 L Luria Bertani (LB) medium containing 50 .mu.g/ml ampicillin, and incubated 37.degree. C. overnight with shaking at 220 rpm. Vector DNA was isolated using the QIAGEN Plasmid Plus Gigaprep system. In all instances, DNA concentration was measured using a Nanodrop 1000 spectrophotometer (Thermo-Scientific) and adjusted to 1 mg/ml with endotoxin-free, sterile water. FIG. 13 show confirmation of the gene sizes.

Routine Culture of CHOK1SV Cells:

[0570] CHOK1SV cells were cultured in CD-CHO media supplemented with 6 mM glutamine. Cells were incubated in a shaking incubator at 36.5.degree. C., 10% CO2, 85% humidity, 140 rpm. Cells were routinely sub-cultured every 3-4 days, seeding at 2.times.105 cells/ml and were propagated in order to have sufficient cells available for transfection. Cells were discarded by passage 20.

Transient Transfections of CHOK1SV Cells:

[0571] Transient transfections were performed using CHOK1SV cells which had been in culture a minimum two weeks. Cells were sub-cultured 24 h prior to transfection and cell viability was >99% at the time of transfection. All transfections were carried out via electroporation using the Gene Pulse MXCeII (Bio-Rad), a plate based system for electroporation. For each transfection, viable cells were resuspended in pre-warmed media to 2.86.times.107 cells/ml. 80 .mu.g DNA (40 .mu.g per single gene vector) was aliquotted into each well and 700 .mu.l cell suspension added. Cells were electroporated at 300 V, 1300 .mu.F. Transfected cells were transferred to pre-warmed media in Erlenmeyer flasks and the wells rinsed twice with pre-warmed media which was also transferred to the flasks. Transfected cell cultures were incubated in a shaking incubator at 36.5.degree. C., 10% CO2, 85% humidity, 140 rpm for 6 days. Cell viability was measured at the time of harvest using a Cedex HiRes automated cell counter (Roche).

Purification of Albumin Linked Products:

[0572] For all purifications, culture supernatant was harvested and clarified by centrifugation at 2000 rpm, 10 mins. Clarified supernatant was concentrated approximately 10 times to approximately 100-150 ml using Tangential-Flow-Filtration (TFF) with a 30 kDa MWCO filter. The concentrated supernatant was purified using 5 ml of CaptureSelect HSA resin (BAC, 191.2970.05) which was packed into a 10/50 Tricorn column (GE Healthcare, 28-4064-14) at a flow rate of 2 ml/min. The column was equilibrated and washed with 50 mM sodium phosphate, 125 mM sodium chloride (PBS buffer), pH 7.4 after loading of cell culture supernatant. Elution was initiated with 20 mM Tris, 2 M magnesium chloride, pH 7.4. After each run the column was cleaned in place with PBS buffer, pH 2.0.

Purification of Wild Type hCG and LH:

[0573] Clarified supernatant was concentrated approximately 10 times to approximately 100-150 ml using Tangential-Flow-Filtration (TFF) with a 10 kDa MWCO filter. The concentrated supernatant was purified using a HiTrap Capto Q column (5 ml, GE Healthcare, 11-0013-03) at a flow of 5 ml/min. The column was equilibrated and washed with 20 mM Tris, pH 8.0 after loading of cell culture supernatant. Elution was initiated by applying a linear elution gradient to 20 mM Tris, 1 M sodium chloride, pH 8.0 over 20 column volumes. After each run the column was cleaned in place with 0.5 M NaOH.

Analysis of Products 1-10 by SDS PAGE

[0574] Reduced samples were prepared for analysis by mixing with NuPage 4.times. LDS sample buffer (Invitrogen, NP0007) and NuPage 10.times. sample reducing agent (Invitrogen, NP0009), and incubated at 70.degree. C., 10 min. For non-reduced samples, the reducing agent and heat incubation were omitted. Samples were electrophoresed on 1.5 mm NuPage 4-12% Bis-Tris Novex pre-cast gels (Invitrogen, NP0315) with NuPage MES SDS running buffer under denaturing conditions. 10 .mu.l aliquots of SeeBlue Plus 2 pre-stained molecular weight standard (Invitrogen, LC5925) and of a control antibody or hCG protein at 1 mg/ml were included on the gel. 10 .mu.l of each sample at 1 mg/ml were loaded onto the gel. Once electrophoresed, gels were stained with InstantBlue (TripleRed, ISB01L) for 30 min at room temperature. Images of the stained gels were analysed on a BioSpectrum Imaging System (UVP) (see FIG. 17).

Analysis of Products 1-10 by Western Blot Methods

[0575] Gels, prepared as described for SDS PAGE with the inclusion of an appropriate control (Human Serum Albumin (Abcam, ab7473) or hCG (Ovitrelle, Serono), were transferred onto nitrocellulose membrane (0.2 .mu.m pore size) using X-Cell II Blot module (Invitrogen) in NuPAGE transfer buffer (Invitrogen) over 1.5 hours at 25 V, 100-125 mA. The Western Blot was performed using Western Breeze Chromogenic Western Blot Immunodetection kits (Invitrogen), according to manufacturers instructions. Briefly, membranes were blocked for 30 min, room temperature and washed 2.times.20 ml H2O. Membranes were incubated with primary antibody solution, for 1 hour at room temperature. The membrane was washed with 4.times.20 ml wash solution and incubated with secondary antibody solution for 30 min at room temperature. The membrane was once again washed with 4.times.20 ml of wash solution followed by 2.times.20 ml H2O, incubated in 5 ml Chromogenic substrate until bands developed, then rinsed 2.times.20 ml H2O and dried. Images of the dried membranes were analysed on a BioSpectrum Imaging System (UVP).

Anti-HSA Western Blot:

[0576] This Western Blot used a goat anti-Human Serum Albumin antibody (Abcam, ab19180) as primary antibody. It was used at 1:2000 dilution (0.5 .mu.g/ml final concentration). A Goat Western Breeze Chromogenic Western Blot Immunodetection kit was used (Invitrogen, WB7107) See FIG. 14.

Anti-hCG Alpha Chain Western Blot:

[0577] This Western Blot used a polyclonal goat anti-hCG alpha chain (Abcam, ab20712) as primary antibody. It was used at 1:10000 dilution (0.6 .mu.g/ml final concentration). A Goat Western Breeze Chromogenic Western Blot Immunodetection kit was used (Invitrogen, WB7107), see FIG. 15.

Anti-hCG Beta Chain Western Blot:

[0578] This Western Blot used a monoclonal mouse anti-hCG beta chain antibody (Abcam, ab9582) as primary antibody. It was used at 1:333.33 dilution (0.6 .mu.g/ml final concentration). A Mouse Western Breeze Chromogenic Western Blot Immunodetection kit was used (Invitrogen, WB7103), see FIG. 16.

[0579] The Western blot analysis identified and confirmed successfully the individual building blocks of the products.

Analysis by SEC

[0580] Duplicate samples were analysed by SE-HPLC on an Agilent 1200 series HPLC system, using a Zorbax GF-250 4 .mu.m 4.6 mm ID.times.25 cm column (Agilent) or a Zorbax GF-250 4 .mu.m 9.2 mm ID.times.25 cm column (Agilent). Aliquots of sample at a concentration of 1 mg/ml were filtered through a 0.2 .mu.m filter prior to injection. 20 or 100 .mu.l aliquots were injected respectively and run at 1 ml/min for 5 to 15 minutes. Soluble aggregate levels were analysed using Chemstation software.

Example

[0581] Production of hCG-Fc Fusion and LH-Fc Fusion

Construction of Expression Plasmids

[0582] Genes encoding fusion of the gonadotropin common .alpha.-subunit and a linker (GGGGSGGGGSGGGGS (SEQ ID NO:57)) with the Fc of a human IgG1, fusion of the hCG .beta.-subunit and a linker (GGGGSGGGGSGGGGS (SEQ ID NO: 57)) with the Fc of a human IgG1 and fusion of the hLH .beta.-subunit and a linker (GGGGSGGGGSGGGGS (SEQ ID NO: 57)) with the Fc of a human IgG1 was constructed by assembly of synthetic oligonucleotides using polymerase chain reaction (PCR). The sequence encoding for the natural human signal sequences of the relevant genes was included. The codon usage of the genes was optimized for high expression in mammalian cells. The relevant genes are:

TABLE-US-00020 Gene # Sequence ID Protein chain Gene10 SEQ ID NO: 53 .alpha.-chain + link + Fc (1074 bases) Gene11 SEQ ID NO: 54 hCG .beta.-chain + link + Fc + His-tag (1239 bases) Gene12 SEQ ID NO: 55 LH .beta.-chain + link + Fc + His-tag (1167 bases)

The gene sequences were synthesised by GeneArt AG and sub-cloned into pEE12.4 and pEE6.4 vectors respectively as shown in Tables below. All work was performed as described in Example 6.

TABLE-US-00021 Product Number Product name First Gene Second Gene 11 hCG-Fc Gene10 Gene11 12 LH-Fc Gene10 Gene12

TABLE-US-00022 Gene Sub-cloned into 10 pEE12.4 11 pEE6.4 12 pEE6.4

FIG. 13 show confirmation of the gene sizes.

[0583] DNA amplification, culture and transfection of CHOK1SV cells were performed as described in Example 6.

Purification:

[0584] Protein A purification was used to purify the Fc-fusion products. Clarified supernatant was purified using a pre-packed 5 ml HiTrap MabSelect SuRE column (GE Healthcare, 11-0034-94) on an AKTA purifier (10 ml/min). The column was equilibrated with 50 mM sodium phosphate, 125 mM sodium chloride, pH 7.3, washed with 50 mM sodium phosphate and 1 M sodium chloride pH 7.3 and eluted with 10 mM sodium formate, pH 3.5. Eluted fractions were immediately pH adjusted to pH 7.3.

Analysis of Products 11 and12 by SDS PAGE

[0585] The purified materials were analyzed by reduced and non-reduced SDS PAGE as described in Example 6 (see FIG. 18).

Analysis by SEC-HPLC

[0586] The purified materials were further analyzed by Size Exclusion Chromatography as described in Example 6 confirming purity and identity.

Example 8

Measurement of In Vitro Activity of hCG and LH Variants:

[0587] The MLTC-1 line (murine leydig tumor cell line MLTC-1 (ATCC-CRL-2065)), expressing the LH/hCG receptors was seeded at an appropriate cell density and challenged with LH/ hCG variants on day 1 of culture. In response to this challenge the steroidogenic pathway was activated in the cells and progesterone and testosterone produced and secreted.

[0588] Cells were propagated in RPMI medium and for the assay seeded at a concentration of 80000 c/ml or 8000 c/well.

Cell Culture:

[0589] Day 0: seeding of the MLTC cells

[0590] Day 1-T=0 h:

[0591] Exposure according to experimental setup

[0592] Day 1-T=4 h:

[0593] Collection of the spent medium for progesterone and/or testosterone quantification.

[0594] Spent medium of the duplo wells is pooled and stored at -20.degree. C.

[0595] Day 2- T=24 h:

[0596] Collection of the spent medium for progesterone and/or testosterone quantification.

[0597] Spent medium of the duplo wells is pooled and stored at -20.degree. C.

Steroid Quantification in Spent Medium:

[0598] The levels of steroid hormone was measured using the Meso Scale Discovery Multi-Spot Assay System:

[0599] The spent medium are added to a MULTI-SPOT 96-well Human Progesterone or Testosterone Plate.

[0600] 1. Add 25 .mu.L/well of Detection Reagent (solution of Diluent 22 containing diluted SULFO-TAG progesterone).

[0601] 2. Add 25 .mu.L/well Calibrator or sample and incubate at room temperature with shaking for 1 hour.

[0602] 3. Prepare SECTOR.RTM. instrument such that the plate can be read immediately following Read Buffer addition.

[0603] 4. Wash plates 3 times with PBS.

[0604] 5. Add 150 .mu.L/well 1.times. Read Buffer T. Avoid bubbles. The use of an electronic multi-pipettor at moderate speed setting is recommended.

[0605] 6. Read the plate on the SECTOR instrument. All buffers and reference standards provided by the vendor.

[0606] EC-50 is calculated for all compounds using Prism software from Graphpad Software, Inc. Graphs are fitted using non-linear regression with or without fixed slopes and/or max and min values.

[0607] The method was applied to the compounds from example 5 and the products from Example 6 and Example 7. The results are shown in FIGS. 19a-d.

Example 9

In Vivo Potency of LH/hCG Variants

[0608] The objective of the study was to determine the potency of hCG/LH test material with respect to its HCG stimulating activities in the LH assay. The effect on growth stimulation of the seminal vesicles in immature male rats was assessed. Different dosing regimens, e.g. daily dosing, dosing every other day or dosing at day one, two, three or four were employed. The LH compounds produced in example 5 and the products produced in example 6 and example 7 were compared to the reference material Ovitrelle. The results were presented as weights of seminal vesicles after dosing of Ovitrelle, and the LH compounds at varying levels over four days.

[0609] Both reference and test material were reconstituted daily in PBS-albumin buffer (0.1% albumin) and concentrations adjusted prior to administration. Administration was subcutaneously at the neck at 0.2 ml/rat.

[0610] Male SPF Wistar rats at 21 to 23 days of age at arrival were used. Rats within a weight range of no more than 10 g on the first day of dose administration were used in the study. On day 5, 24 hours after last dosing, the rats were euthanised by an overdose of CO2/O2 anaesthesia. Seminal vesicles were removed and trimmed and blot-dried. The weight of the seminal vesicles were determined and recorded.

[0611] The method was applied to the compounds from Example 5 and the products from Example 6 and Example 7. The results are shown in FIGS. 20a-j.

Example 10

Measurement of hCG Content in Rat Serum.

[0612] Serum collected from rats exposed to LH containing products were sent to Bioscientia GmbH, Institut fur Medizinische Diagnostik GmbH, Konrad-Adenauer-Str. 17, 55218 Ingelheim, Germany for analysis, using the ADVIA Centaur Total hCG (ThCG) assay.

[0613] The ADVIA Centaur Total hCG (ThCG) assay is a two-site sandwich immunoassay using direct chemiluminometric technology, which uses constant amounts of two antibodies. The first antibody, in the Lite Reagent, is a polyclonal goat anti-hCG antibody that has been affinity purified and labeled with acridinium ester. The second antibody, in the Solid Phase, is a purified monoclonal mouse anti-hCG antibody, which is covalently coupled to paramagnetic particles. These two antibodies are specific for different epitopes that are present on both the free .beta. subunit and the .beta. subunit of intact hCG.

The system automatically performs the following actions:

[0614] dispenses 50 .mu.L of sample into a cuvette

[0615] dispenses 100 .mu.L of Lite Reagent and 450 .mu.L of Solid Phase and incubates for 7.5 minutes at 37.degree. C.

[0616] separates, aspirates, and washes the cuvettes with reagent water3

[0617] dispenses 300 .mu.L each of Acid Reagent and Base Reagent to initiate the chemiluminescent reaction

[0618] reports results according to the selected option, as described in the system operating instructions or in the online help system

[0619] A direct relationship exists between the amount of hCG present in the serum sample and the amount of relative light units (RLUs) detected by the system. Dilution curves of the respective hCG containing products were used for calibration of the data obtained.

Example 11

Measurement of Content of LH Like Immunoreactivity in Rat Serum.

[0620] Serum collected from rats exposed to LH containing products were sent to Bioscientia GmbH, Institut fur Medizinische Diagnostik GmbH, Konrad-Adenauer-Str. 17, 55218 Ingelheim, Germany for analysis, using the Cobas.RTM. Luteinizing Hormone ECLIA (Elecsys) assay.

[0621] The Elecsys LH assay employs two monoclonal antibodies specifically directed against human LH. The two specific antibodies used recognize particular conformations, with the biotinylated antibodies detecting an epitope constructed from both subunits whereas the antibody with the ruthenium complexa label detects an epitope from the .beta.-subunit. As a result, the Elecsys LH assay shows negligible cross-reactivity with FSH, TSH, hCG, hGH, and hPL.

Test Principle

[0622] 1st incubation: 20 .mu.L of sample, a biotinylated monoclonal LH-specific antibody, and a monoclonal LH-specific antibody labeled with a ruthenium complex form a sandwich complex.

[0623] 2nd incubation: After addition of streptavidin-coated microparticles, the complex becomes bound to the solid phase via interaction of biotin and streptavidin.

[0624] The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed with ProCell/ProCell M. Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier.

[0625] Results are determined via a calibration curve which is instrument-specifically generated by 2-point calibration and a master curve provided via the reagent barcode. Dilution curves of the respective LH containing products were used for calibration of the data obtained.

Example 12

PK Data in Hypophysectomized Male Wistar Rats

[0626] The pharmacokinetic profiles of the LH compounds were measured in hypophysectomized male Wistar rats.

[0627] The LH compounds were administered subcutaneously at varying dosages at time 0 hours. Blood was sampled from the rats at varying time points--the first 4 blood samples from each rat was collected by sublingual bleeding using 19G single use needles. The 5th and terminal blood sample was collected under anaesthesia. Serum was prepared according to following instruction:

[0628] The blood samples were collected in SST tubes with clotting activator, Sarstedt RefNo. 41.1500.005. The samples were inverted 5.times. and then allowed to clot for 30 minutes at ambient temperature. The samples were centrifuged immediately after clotting for 10 minutes at 2500G, 20.degree. C. The serum was split into two storage vials -50 .mu.l in each vial. 300 .mu.l PBS/BSA is added to one of the vials.

The Serum Samples were Frozen at -15.degree. C. within 60 Minutes after Centrifugation.

[0629] The serum labels of the LH compounds were measured as described in example for hCG containing compounds and as described in example 11 for LH containing compounds. The method was applied to the compounds from example 5 and to the products from example 6 and example 7. The results are shown in FIG. 21a-d and FIG. 22a-h.

Example 13

PK Data in Normal Adult Male Rats

[0630] The pharmacokinetic profiles of the LH compounds were measured in normal Sprague Dawley male rats. The LH compounds were administered subcutaneously at varying dosages at time 0 hours. Blood was sampled from the rats at varying time points--the first 4 blood samples from each rat was collected by sublingual bleeding using 19 G single use needles. The 5th and terminal blood sample was collected under anaesthesia. Serum was prepared according to following instruction:

[0631] The blood samples were collected in SST tubes with clotting activator, Sarstedt RefNo. 41.1500.005. The samples were inverted 5.times. and then allowed to clot for 30 minutes at ambient temperature. The samples were centrifuged immediately after clotting for 10 minutes at 2500 G, 20.degree. C. The serum was split into two storage vials -100 .mu.l in each vial. 250 .mu.l PBS/BSA was added to one of the vials. The serum samples were frozen at .ltoreq.-15.degree. C. within 60 minutes after centrifugation. The serum levels of the LH compounds were measured as described in example 10 for hCG containing compounds and as described in example 11 for LH containing compounds. The method was applied to the compounds from Example 5 and to the products from Example 6. The results are shown in FIG. 23a-d.

Example 14

[0632] Comparison of hCG given in the late follicular phase as a substitute for FSH and luteal phase support given as daily injections of low dose r-hCG or r-LH as compared to a standard GnRH antagonist protocol supplemented with luteal phase progesterone administration.

Background

[0633] It is becoming increasingly clear that the current method of supporting the luteal phase for optimizing chances of implantation and establishment of a pregnancy is poorly defined. Further the current regimes of administering luteal phase support do not appear to provide sufficient progesterone concentrations in all patients to secure optimal results. In addition, the mode of administration of the most commonly used luteal phase support products have a number of side effects that reduce patients' compliance and acceptance.

[0634] The aim of the present study is to determine whether it is possible to develop new stimulation protocols in which no luteal phase progesterone administration is required by combining follicular phase administration of low-dose hCG (i.e. 150-200 IU per day) as a substitute for FSH stimulation in the late follicular phase, while using a GnRH agonist injection for ovulation induction. The use of a GnRH agonist for ovulation induction is known to reduce pituitary output of gonadotropins resulting in an insufficient corpus luteum function. However, the risk of ovarian hyper stimulation syndrome (OHSS) is simultaneously reduced to a near negligible level. In order to secure a proper luteal phase sustaining the establishment of a pregnancy, as well as maintaining the risk of OHSS at low levels, the present study will either administer daily injections of r-hCG (125 IU per day) or r-LH (i.e. 300 IU per day) from the day of oocyte pickup to stimulate the corpus luteum function and augment the endogenous production of progesterone without administration of exogenous progesterone in connection with a GnRH agonist ovulation trigger.

Material and Methods

[0635] It is planned to perform two studies, one on each of two clinics, including a total of 90 women per clinic in a randomized clinical evaluation.

Inclusion criteria:

[0636] 1. Female age between 25 and 40 years

[0637] 2. Baseline FSH and LH <12 IU/l

[0638] 3. Menstrual cycle length between 25-34 days

[0639] 4. Body Mass Index (BMI) between 18 and 30

[0640] 5. Both ovaries present and absence of uterine abnormalities

Exclusion Criteria

[0640]

[0641] 1. The presence of only one ovary.

[0642] 2. Uterine abnormalities

[0643] 3. Polycystic ovarian syndrome

[0644] 4. Diabetes, epilepsy, lever, kidney, heart disease including metabolic diseases as judged by the treating doctor

[0645] 5. Allergy towards any substance present in the drugs used for administration.

[0646] 6. Earlier participation in the study

Hormonal Treatment

[0647] Treatment group-I: From cycle day two recombinant FSH (r-hFSH; Gonal-F, Merck-Serono, Hellerup, Denmark) is administered in a fixed dose for the first 4 days. The dose is either 150 or 225 IU per day depending on age, BMI, basal FSH, antral follicle count and the ovarian volume. After 4 days, doses can be adjusted depending on the ovarian response.

[0648] When at least four follicles reach a diameter of 12 mm, the daily FSH dose (irrespective of which specific dose was used initially) is exchanged with 200 IU hCG daily (r-hCG, Ovitrelle, Merck-Serono, Hellerup, Denmark)--see instructions for dilution below. To prevent a premature LH rise, a fixed GnRH antagonist protocol is used commencing on stimulation day 5 in the morning. On this day, 0.25 mg/day GnRH antagonist (Cetrotide, Merck-Serono, Hellerup, Denmark) is given s.c. daily and will be continued until and including the day of ovulation induction. When three or more follicles reach a diameter of 17 mm ovulation is induced in all patients by the administration of a single bolus of GnRH agonist, such as 0.5 mg buserelin s.c. (Suprefact; Sanofi-Aventis, Horsholm, Denmark) followed by oocyte pick up (OPU) 34 hours later. In connection with the OPU, at around 35 hours after the buserelin injection, administration of 125 IU IU r-hCG (r-hCG, Ovitrelle, Merck-Serono, Hellerup, Denmark)(see instructions for dilution below) daily will be initiated for luteal phase support and stimulation of endogenous progesterone production. Administration of r-hCG will be continued until the pregnancy test is performed. No exogenous progesterone is administered.

[0649] Treatment group-II: From cycle day two recombinant FSH (r-hFSH; Gonal-F, Merck-Serono, Hellerup, Denmark) is administered in a fixed dose of 225 IU daily. Also from cycle day two a fixed dose of 150 IU of hCG daily (r-hCG, Ovitrelle, Merck-Serono, Hellerup, Denmark) will be administered--see instructions for dilution below. To prevent a premature LH rise, a fixed GnRH antagonist protocol is used commencing on stimulation day 5 in the morning. On this day, 0.25 mg/day GnRH antagonist (Cetrotide, Merck-Serono, Hellerup, Denmark) is given s.c. daily and will be continued until and including the day of ovulation induction.

[0650] When at least four follicles reach a diameter of 12-13 mm, the daily FSH dose is discontinued while the dose of hCG will continue with 150 IU daily until ovulation induction.

[0651] When three or more follicles reach a diameter of 17 mm ovulation is induced in all patients by the administration of a single bolus GnRH antagonist of 0.5 mg buserelin s.c. (Suprefact; Sanofi-Aventis, Horsholm, Denmark) followed by oocyte pick up (OPU) 34 hours later. In connection with the OPU, at around 35 hours after the buserelin injection, administration of 125 IU IU r-hCG (r-hCG, Ovitrelle, Merck-Serono, Hellerup, Denmark)(see instructions for dilution below) daily will be initiated for luteal phase support and stimulation of endogenous progesterone production. Administration of r-hCG will be continued until the pregnancy test is performed. No exogenous progesterone is administered.

[0652] Treatment group-III: From cycle day two recombinant FSH (r-hFSH; Gonal-F, Merck-Serono, Hellerup, Denmark) is administered in a fixed dose for the first 4 days. The dose is either 150 or 225 IU per day depending on age, BMI, basal FSH, antral follicle count and the ovarian volume. After 4 days, doses can be adjusted depending on the ovarian response. To prevent a premature LH rise, a fixed GnRH antagonist protocol is used commencing on stimulation day 5 in the morning. On this day, 0.25 mg/day GnRH antagonist (Cetrotide, Merck-Serono, Hellerup, Denmark) is given s.c. daily and will be continued until and including the day of ovulation induction. When three or more follicles reach a diameter of 17 mm ovulation is induced in all patients by administration of a single bolus of 0.5 mg buserelin s.c. (Suprefact; Sanofi-Aventis, Horsholm, Denmark) followed by oocyte pick up (OPU) 34 hours later. In connection with the OPU, at around 35 hours after the buserelin injection, administration of 300 IU r-LH (r-LH, Luveris, Merck-Serono, Hellerup, Denmark) daily will be initiated for luteal phase support and stimulation of endogenous progesterone production. Administration of r-LH will be continuned until the pregnancy test is performed. No exogenous progesterone is administered.

[0653] Control group: From cycle day two recombinant FSH (r-hFSH; Gonal-F, Merck-Serono, Hellerup, Denmark) is administered in a fixed dose for the first 4 days. The dose is either 150 or 225 IU per day depending of age, BMI, basal FSH, antral follicle count and the ovarian volume. After 4 days, doses can be adjusted depending on the ovarian response. To prevent a premature LH rise, a fixed GnRH antagonist protocol is used commencing on stimulation day 5 in the morning. On this day, 0.25 mg/day GnRH antagonist (Cetrotide, Merck-Serono, Hellerup, Denmark) is given s.c. daily and will be continued until and including the day of ovulation induction. When three or more follicles reach a diameter of 17 mm ovulation will be induced in all patients by administration of a single bolus of 250 .mu.g r-hCG (r-hCG, Ovitrelle, Merck-Serono, Hellerup, Denmark) followed by oocyte pick up (OPU) 34-35 hours later. For luteal phase support daily micronized progesterone vaginally, 90 mg per day (Crinone; Merck-Serono, Hellerup, Denmark) and oestradiol 4 mg per day orally (Estrofem; Novo Nordisk, Copenhagen, Denmark) will be administered, commencing the day after OPU and continuing until the day of the pregnancy test.

[0654] For all for groups: Laboratory procedures will follow the participating clinics normal procedures and will be independent of the randomization. A maximum of two embryos will be transferred on day 2 after retrieval. All laboratory parameters including fertilization rate, cleavage rate will be monitored. A biochemical pregnancy is defined by a plasma .beta.-hCG concentration .gtoreq.10 IU/ion day 12 after ET. Clinical pregnancy is defined as an intrauterine gestational sac with a heartbeat 3 weeks after a positive hCG-test.

Randomization

[0655] Participating patients will be randomized to one of four groups on stimulation day 1.

Blood Samples and Hormone Assays

[0656] Blood samples will be collected on 1) the day of ovulation induction, 2) the day of OPU 3) the day of OPU plus seven and 4) on day 14 after OPU. Serum aliquots (the sample is divided into two equal ampoules) are kept frozen at -20 .degree. C. for subsequent analysis of LH, progesterone and hCG. The hormones will be measured using each participating laboratory's in house assay.

Outcome Measures

[0657] The primary outcome is the mid-luteal phase progesterone level. Secondary outcome measures include ongoing pregnancy rate, the rate of early pregnancy loss and the OHSS rate.

Dilution of r-hCG for Stimulation

[0658] One ampoule of r-hCG (r-hCG, Ovitrelle, Merck-Serono, Hellerup, Denmark) contains 250 .mu.g r-hCG corresponding to approximately 6.500 IU. Using a sterile 2 ml syringe with an injection needle 1 ml of liquid should be drawn from a bottle with 10 ml sterile physiological saline by penetrating the rubber stopper. The content of the ampoule should subsequently be injected into the remaining 9 ml saline in the bottle. The concentration of hCG will now constitute 650 IU/ml in the bottle. In order to provide the patient stimulation with 200 IU hCG 0.3 ml (or precisely 195 IU) from the bottle should be redrawn and injected via a sterile 1 ml syringe.

[0659] In order to retrieve a total of 125 IU hCG, 0.19 ml from the bottle should be redrawn and injected via a sterile 1 ml syringe.

[0660] In order to retrieve a total of 150 IU hCG, 0.23 ml from the bottle should be redrawn and injected via a sterile 1 ml syringe.

[0661] The r-hCG for stimulation should be prepared fresh every day.

Participants and Clinical Activity

[0662] Two fertility clinics participate in the study. One fertility clinic undertakes a trial comprising treatment group I and treatment group II and a control group including a total of 90 patients (3 groups of 30 patients). The other fertility clinic undertakes a trial comprising treatment group I, treatment group III and a control group including a total of 90 patients (3 groups of 30 patients).

Example 15

[0663] hCG administered in the late follicular phase as a substitute for FSH and luteal phase support administered as daily injections of low dose r-hCG or r-LH were compared to a standard GnRH antagonist protocol supplemented with luteal phase progesterone administration. Thus wild-type hCG was administered as daily small doses to illustrate the effect of S-hCG as described in this patent application.

Background

[0664] It has become increasingly clear that the current method of supporting the luteal phase to optimize chances of implantation and establishment of pregnancy is poorly defined. Further the current regimes of administering luteal phase support do not appear to provide sufficient progesterone concentrations in all patients to secure optimal results. In addition, the mode of administration of the most commonly used luteal phase support products have a number of side effects that reduce patients' compliance and acceptance.

[0665] The aim of the present study was to determine whether it would be possible to develop a new stimulation protocol in which no luteal phase progesterone administration was required by combining follicular phase administration of low-dose hCG (i.e. 150-200 IU per day) as a substitute for FSH stimulation in the late follicular phase, while using a GnRH agonist injection for ovulation induction. The use of a GnRH agonist for ovulation induction is known to reduce pituitary output of gonadotropins resulting in an insufficient corpus luteum function. However, the risk of ovarian hyper stimulation syndrome (OHSS) is simultaneously reduced to a near negligible level. In order to secure a proper luteal phase sustaining the establishment of a pregnancy, as well as maintaining the risk of OHSS at low levels, patients in the present study were either administered daily injections of r-hCG (125 IU per day) or r-LH (i.e. 300 IU per day) from the day of oocyte pickup to stimulate the corpus luteum function and augment the endogenous production of progesterone without administration of exogenous progesterone in connection with a GnRH agonist ovulation trigger.

Material and Methods

[0666] A total of 32 women were included in this prospective randomized trial, which is detailed below:

Inclusion Criteria:



[0667] 1. Female age between and 40 years

[0668] 2. Baseline FSH and LH <12 IU/l

[0669] 3. Menstrual cycle length between 25-34 days

[0670] 4. Body Mass Index (BMI) between 18 and 30

[0671] 5. Both ovaries present and absence of uterine abnormalities

Exclusion Criteria

[0671]

[0672] 1. The presence of only one ovary.

[0673] 2. Uterine abnormalities

[0674] 3. Polycystic ovarian syndrome

[0675] 4. Diabetes, epilepsy, liver, kidney, heart disease including metabolic diseases as judged by the treating doctor

[0676] 5. Allergy towards any substance present in the drugs used for administration.

[0677] 6. Earlier participation in the study

Hormonal Treatment

[0678] Treatment group-I: From cycle day two recombinant FSH (r-hFSH; Gonal-F, Merck-Serono, Hellerup, Denmark) was administered in a fixed dose for the first 4 days. The dose was either 150 or 225 IU per day depending on age, BMI, basal FSH, antral follicle count and the ovarian volume. After 4 days, doses were adjusted depending on the ovarian response.

[0679] When at least four follicles had reached a diameter of 12 mm, the daily FSH dose (irrespective of which specific dose was used initially) was exchanged with 200 IU hCG daily (r-hCG, Ovitrelle, Merck-Serono, Hellerup, Denmark)--see instructions for dilution below. To prevent a premature LH rise, a fixed GnRH antagonist protocol was used commencing on stimulation day 5 in the morning. On this day, 0.25 mg GnRH antagonist (Cetrotide, Merck-Serono, Hellerup, Denmark) was adminstered s.c. daily and continued until and including the day of ovulation induction. When three or more follicles had reached a diameter of 17 mm ovulation was induced in all patients by the administration of a single bolus of a GnRH agonist, such as 0.5 mg buserelin s.c. (Suprefact; Sanofi-Aventis, Horsholm, Denmark) followed by oocyte pick up (OPU) 34 hours later. In connection with the OPU, at around 35 hours after the buserelin injection, administration of 125 IU r-hCG (r-hCG, Ovitrelle, Merck-Serono, Hellerup, Denmark)(see instructions for dilution below) daily was initiated for luteal phase support and stimulation of endogenous progesterone production. Administration of r-hCG was continued until the pregnancy test was performed. No exogenous progesterone was administered.

[0680] Treatment group-II: From cycle day two recombinant FSH (r-hFSH; Gonal-F, Merck-Serono, Hellerup, Denmark) was administered in a fixed dose of 225 IU daily. Also from cycle day two a fixed dose of 150 IU of hCG daily (r-hCG, Ovitrelle, Merck-Serono, Hellerup, Denmark) was administered--see instructions for dilution below. To prevent a premature LH rise, a fixed GnRH antagonist protocol was used commencing on stimulation day 5 in the morning. On this day, 0.25 mg GnRH antagonist (Cetrotide, Merck-Serono, Hellerup, Denmark) was given s.c. daily and continued until and including the day of ovulation induction.

[0681] When at least four follicles had reached a diameter of 12-13 mm, the daily FSH dose was discontinued while the dose of hCG was continued with 150 IU daily until ovulation induction.

[0682] When three or more follicles had reached a diameter of 17 mm ovulation was induced in all patients by the administration of a single bolus GnRH antagonist of 0.5 mg buserelin s.c. (Suprefact; Sanofi-Aventis, Horsholm, Denmark) followed by oocyte pick up (OPU) 34 hours later. In connection with the OPU, at around 35 hours after the buserelin injection, administration of 125 IU r-hCG (r-hCG, Ovitrelle, Merck-Serono, Hellerup, Denmark)(see instructions for dilution below) daily was initiated for luteal phase support and stimulation of endogenous progesterone production. Administration of r-hCG was continued until the pregnancy test was performed. No exogenous progesterone was administered.

[0683] Treatment group-III: From cycle day two recombinant FSH (r-hFSH; Gonal-F, Merck-Serono, Hellerup, Denmark) was administered in a fixed dose for the first 4 days. The dose was either 150 or 225 IU per day depending on age, BMI, basal FSH, antral follicle count and the ovarian volume. After 4 days, doses were adjusted depending on the ovarian response. To prevent a premature LH rise, a fixed GnRH antagonist protocol was used commencing on stimulation day 5 in the morning. On this day, 0.25 mg GnRH antagonist (Cetrotide, Merck-Serono, Hellerup, Denmark) was given s.c. daily and continued until and including the day of ovulation induction. When three or more follicles had reached a diameter of 17 mm ovulation was induced in all patients by administration of a single bolus of 0.5 mg buserelin s.c. (Suprefact; Sanofi-Aventis, Horsholm, Denmark) followed by oocyte pick up (OPU) 34 hours later. In connection with the OPU, at around 35 hours after the buserelin injection, administration of 300 IU r-LH (r-LH, Luveris, Merck-Serono, Hellerup, Denmark) daily was initiated for luteal phase support and stimulation of endogenous progesterone production. Administration of r-LH was continuned until the pregnancy test was performed. No exogenous progesterone was administered.

[0684] Control group (treatment group 4): From cycle day two recombinant FSH (r-hFSH; Gonal-F, Merck-Serono, Hellerup, Denmark) was administered in a fixed dose for the first 4 days. The dose was either 150 or 225 IU per day depending of age, BMI, basal FSH, antral follicle count and the ovarian volume. After 4 days, doses were adjusted depending on the ovarian response. To prevent a premature LH rise, a fixed GnRH antagonist protocol was used commencing on stimulation day 5 in the morning. On this day, 0.25 mg GnRH antagonist (Cetrotide, Merck-Serono, Hellerup, Denmark) was given s.c. daily and continued until and including the day of ovulation induction. When three or more follicles had reached a diameter of 17 mm ovulation was induced in all patients by administration of a single bolus of 250 .mu.g r-hCG (r-hCG, Ovitrelle, Merck-Serono, Hellerup, Denmark) followed by oocyte pick up (OPU) 34-35 hours later. For luteal phase support daily micronized progesterone vaginally, 90 mg per day (Crinone; Merck-Serono, Hellerup, Denmark) and estradiol 4 mg per day orally (Estrofem; Novo Nordisk, Copenhagen, Denmark) was administered, commencing the day after OPU and continued until the day of the pregnancy test.

[0685] For all for groups: Laboratory procedures followed the participating clinics normal procedures and were independent of the randomization. A maximum of two embryos were transferred on day 2 after retrieval. All laboratory parameters including fertilization rate and cleavage rate were monitored. A biochemical pregnancy was defined by a plasma .beta.-hCG concentration .gtoreq.10 IU/l on day 12 after ET. Clinical pregnancy was defined as an intrauterine gestational sac with a heartbeat 3 weeks after a positive hCG-test.

Randomization

[0686] Participating patients were randomized to one of four groups on stimulation day 1.

Blood Samples and Hormone Assays

[0687] Blood samples were collected on 1) the day of ovulation induction, 2) the day of OPU 3) the day of OPU plus seven and 4) on day 14 after OPU. Serum aliquots (the sample was divided into two equal ampoules) were kept frozen at -20.degree. C. for subsequent analysis of LH, progesterone and hCG. The hormones were measured using each participating laboratory's in house assay.

Outcome Measures

[0688] The primary outcome was the mid-luteal phase progesterone level.

Dilution of r-hCG for Stimulation

[0689] One ampoule of r-hCG (r-hCG, Ovitrelle, Merck-Serono, Hellerup, Denmark) contains 250 .mu.g r-hCG corresponding to approximately 6.500 IU. Using a sterile 2 ml syringe with an injection needle 1 ml of liquid was drawn from a bottle with 10 ml sterile physiological saline by penetrating the rubber stopper. The content of the ampoule was subsequently injected into the remaining 9 ml saline in the bottle. The concentration of hCG did now constitute 650 IU/ml in the bottle. In order to provide the patient stimulation with 200 IU hCG 0.3 ml (or precisely 195 IU) from the bottle was redrawn and injected via a sterile 1 ml syringe.

[0690] In order to retrieve a total of 125 IU hCG, 0.19 ml from the bottle was redrawn and injected via a sterile 1 ml syringe.

[0691] In order to retrieve a total of 150 IU hCG, 0.23 ml from the bottle was redrawn and injected via a sterile 1 ml syringe.

[0692] The r-hCG for stimulation was prepared fresh every day.

Results

TABLE-US-00023

[0693] Progesterone concentration (nmol/l) (mean .+-. SEM) Treatment Treatment Treatment Treatment group 1 group 2 group 3 group 4 No. of patients 11 6 4 11 Day of 5.3 .+-. 1.8 6.5 .+-. 3.1 2.7 .+-. 1.6 4.4 .+-. 1.9 ovulation induction Day of oocyte 10 .+-. 3 18 .+-. 11 37 .+-. 24 19 .+-. 6 pickup (OPU) OPU + 7 351 .+-. 118 448 .+-. 91 448 .+-. 294 211 .+-. 52 Day of hCG 88 .+-. 33 168 .+-. 72 126 .+-. 124 49 .+-. 23 testing

[0694] The data clearly demonstrate that the proposed dosing regiments of rhCG in the follicular phase and in the luteal phase and the proposed dosing regimen for stimulation of progesterone production by rhLH in the luteal phase show a pronounced positive effect on the mid-luteal phase progesterone production.

Pharmacological Methods

Example 16

[0695] How to Determine the Biopotency of a Long Acting LH Compound, such as hCG Linked to Human Albumin (SUS-hCG)

[0696] The biopotency of SUS-hCG will be determined using one of two established in vivo assays. The pharmacopaeia and authorities ask for the Van Hell bioassay. (Van Hell etal., Acta Endocrin. 47: 409 (1964) which determine the LH biological activity of LH-containing gonadotropin products measuring the seminal vesicle weight gain. The ovarian ascorbic acid depletion assay, which measures the decrease in ovarian ascorbic acid in response to exogenous LH treatments ministered to pseudo-pregnant rats (Parlow AF: Bioassay of pituitary luteinizing hormone by depletion of ovarian ascorbic acid. In Human Pituitary Gonadotropins Edited by: Albert A. Springfield; C C Thomas; 1961:300-320). This latter assay shows greater sensitivity for detecting LH bioactivity compared to the first mentioned pharmacopaeia described assay being almost one order of magnitude more sensitive.

[0697] Further the in vitro bioactivity of SUS-hCG will be determined using standard cell assays such as the MA10 Leydig cell bioassay disclosed Ascoli, Endocrinology 108: 88 (1981) or the mouse Leydig cell assay in which LH induced increase in testosterone in vitro by mouse Leydig cells is measured by standard immunological techniques such as RIA assay (Van Damme et al., Acta Endocrinol. (Copenh.) 1974:77;655).

[0698] For all assays the bioactivity of SUS-hCG will be compared to recombinant hCG and human urine derived hCG and by using The National Institute of Biological Standards and Controls (NIBSC Herts, UK) appropriate standards.

[0699] The amount hCG protein in a given composition will be determined using standard immunological techniques such as ELISA assay or RIA assay and characterized by Western blotting and measurement of total protein content using Bradford and/or Lowry assays.

Example 17

[0700] How to Determine the Biopotency of a Long Acting Modified LH (S-LH), such as hLH Linked to an Acylation Group, PEG or Human Albumin in Combination with FSH

[0701] The biopotency of S-LH will be determined using one of two established in vivo assays. The pharmacopaeia and authorities ask for the Van Hell bioassay. (Van Hell 30 etal., Acta Endocrin. 47: 409 (1964) which determine the LH biological activity of LH-containing gonadotropin products measuring the seminal vesicle weight gain. The ovarian ascorbic acid depletion assay, which measures the decrease in ovarian ascorbic acid in response to exogenous LH treatments ministered to pseudo-pregnant rats (Parlow 32 AF: Bioassay of pituitary luteinizing hormone by depletion of ovarian ascorbic acid. In Human Pituitary Gonadotropins Edited by: Albert A. Springfield; C C Thomas; 1961:300-320). This latter assay shows greater sensitivity for detecting LH bioactivity compared to the first mentioned pharmacopaeia described assay being almost one order of magnitude more sensitive.

[0702] Further the in vitro bioactivity of S-LH will be determined using standard cell assays such as the MA10 Leydig cell bioassay disclosed Ascoli, Endocrinology 108: 88 (1981) or the mouse Leydig cell assay in which LH induced increase in testosterone in vitro by mouse Leydig cells is measured by standard immunological techniques such as RIA assay (Van Damme et al., Acta Endocrinol. (Copenh.) 1974:77;655).

[0703] For all assays the bioactivity of S-LH will be compared to recombinant hCG, recombinant LH and human urine derived hCG and by using The National Institute of Biological Standards and Controls (NIBSC Herts, UK) appropriate standards.

[0704] The amount LH protein in a given composition will be determined using standard immunological techniques such as ELISA assay or RIA assay and characterized by Western blotting and measurement of total protein content using Bradford and/or Lowry assays.

[0705] The effect of S-LH in combination with FSH to sustain multiple follicular development and embryo development in vivo will be performed in mice as described by Yding Andersen C et al., Requirements for human chorionic gonadotropin and recombinant human luteinizing hormone for follicular development and maturation. J. Assist. Reprod. Gen., 1999, 16, 536-541, in relation to the native LH and hCG hormones. Mice will be stimulated with a fixed dose of FSH to induce multiple follicular development and in combination with varying amounts of LH/hCG activity. The mice will be induced to ovulate and be mated to a male. Later the mice will be killed and the oviduct will be recovered and flushed to determine the number of blastocysts present. The number of blastocysts will in a semi quantitative way express the potency of the LH component.

Example 18

[0706] How to Determine the Bio Potency of a Long-Acting Modified LH, such as hLH Linked to an Acylation Group, PEG or human Albumin in Combination with FSH

[0707] The bio potency of long-acting LH may be determined using one of two established in vivo assays. The pharmacopaeia and authorities ask for the Van Hell bioassay. (Van Hell et al., Acta Endocrin. 47: 409 (1964) which determine the LH biological activity of LH containing gonadotropin products measuring the seminal vesicle weight gain. The ovarian ascorbic acid depletion assay, which measures the decrease in ovarian ascorbic acid in response to exogenous LH treatments ministered to pseudo-pregnant rats (Parlow A F: Bioassay of pituitary luteinizing hormone by depletion of ovarian ascorbic acid. In Human Pituitary Gonadotropins Edited by: Albert A. Springfield; C C Thomas; 1961:300-320). This latter assay shows greater sensitivity for detecting LH bioactivity compared to the first mentioned pharmacopaeia described assay being almost one order of magnitude more sensitive.

[0708] Further the in vitro bioactivity of long-acting LH will be determined using standard cell assays such as the MA10 Leydig cell bioassay disclosed Ascoli, Endocrinology 108: 88 (1981) or the mouse Leydig cell assay in which LH induced increase in testosterone in vitro by mouse Leydig cells is measured by standard immunological techniques such as RIA assay (Van Damme et al., Acta Endocrinol. (Copenh.) 1974:77;655).

[0709] For all assays the bioactivity of long-acting LH will be compared to recombinant hCG, recombinant LH and human urine derived hCG and by using The National Institute of Biological Standards and Controls (NIBSC Herts, UK) appropriate standards. The amount of LH protein in a given composition will be determined using standard immunological techniques such as ELISA assay or RIA assay and characterized by Western blotting and measurement of total protein content using Bradford and/or Lowry assays.

Example 19

Combined PK/PD Study Data in Normal and Hypophysectomized Male Rats.

[0710] The LH compounds are administered subcutaneously at varying dosages at time 0 hours. Blood is sampled from the rats at varying time points but at least daily for up to four week. The first blood samples from each rat are collected by sublingual bleeding using 19 G single use needles. The terminal blood sample is collected under anaesthesia. Both reference and test material are reconstituted daily in PBS-albumin buffer (0.1% albumin) and concentrations adjusted prior to administration. Administration was subcutaneously at the neck at 0.2 ml/rat.

[0711] For the study in hypophysectimised rats, male Wistar rats, 95-110 g, are purchased from Taconic-M&B and hypophysectomised using a trans auricular procedure. For the study in normal rats, male Sprague Dawley rats weighing approximately 250 g are used.

Serum is prepared according to following instruction:

[0712] The blood samples are collecrted in SST tubes with clotting activator, Sarstedt RefNo. 41.1500.005. The samples are inverted 5.times. and then allowed to clot for 30 minutes at ambient temperature. The samples are centrifuged immediately after clotting for 10 minutes at 2500 G, 20.degree. C. The serum is split into two storage vials -50 .mu.l in each vial. 300 .mu.l PBS/BSA is added to one of the vials.

[0713] The serum samples are frozen at .ltoreq.-15.degree. C. within 60 minutes after centrifugation. The serum levels of the LH compounds are measured as described in example X and Z. The serum levels of testosterone are measured as described in example Y On the last day the rats are euthanised by an overdose of CO2/O2 anaesthesia. Seminal vesicles are removed and trimmed and blot-dried. The weight of the seminal vesicles are determined and recorded.

Sequence CWU 1

1

71192PRTHomo sapiens 1Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro 1 5 10 15 Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys 20 25 30 Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu 35 40 45 Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser 50 55 60 Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr 65 70 75 80 Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser 85 90 296PRTMus musculus 2Leu Pro Asp Gly Asp Phe Ile Ile Gln Gly Cys Pro Glu Cys Lys Leu 1 5 10 15 Lys Glu Asn Lys Tyr Phe Ser Lys Leu Gly Ala Pro Ile Tyr Gln Cys 20 25 30 Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro Ala Arg Ser Lys 35 40 45 Lys Thr Met Leu Val Pro Lys Asn Ile Thr Ser Glu Ala Thr Cys Cys 50 55 60 Val Ala Lys Ala Phe Thr Lys Ala Thr Val Met Gly Asn Ala Arg Val 65 70 75 80 Glu Asn His Thr Glu Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser 85 90 95 396PRTRattus norvegicus 3Leu Pro Asp Gly Asp Leu Ile Ile Gln Gly Cys Pro Glu Cys Lys Leu 1 5 10 15 Lys Glu Asn Lys Tyr Phe Ser Lys Leu Gly Ala Pro Ile Tyr Gln Cys 20 25 30 Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro Ala Arg Ser Lys 35 40 45 Lys Thr Met Leu Val Pro Lys Asn Ile Thr Ser Glu Ala Thr Cys Cys 50 55 60 Val Ala Lys Ser Phe Thr Lys Ala Thr Val Met Gly Asn Ala Arg Val 65 70 75 80 Glu Asn His Thr Asp Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser 85 90 95 4121PRTHomo sapiens 4Ser Arg Glu Pro Leu Arg Pro Trp Cys His Pro Ile Asn Ala Ile Leu 1 5 10 15 Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr 20 25 30 Ile Cys Ala Gly Tyr Cys Pro Thr Met Met Arg Val Leu Gln Ala Val 35 40 45 Leu Pro Pro Leu Pro Gln Val Val Cys Thr Tyr Arg Asp Val Arg Phe 50 55 60 Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asp Pro Val Val 65 70 75 80 Ser Phe Pro Val Ala Leu Ser Cys Arg Cys Gly Pro Cys Arg Arg Ser 85 90 95 Thr Ser Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp His 100 105 110 Pro Gln Leu Ser Gly Leu Leu Phe Leu 115 120 5121PRTMus musculus 5Ser Arg Gly Pro Leu Arg Pro Leu Cys Arg Pro Val Asn Ala Thr Leu 1 5 10 15 Ala Ala Glu Asn Glu Phe Cys Pro Val Cys Ile Thr Phe Thr Thr Ser 20 25 30 Ile Cys Ala Gly Tyr Cys Pro Ser Met Val Arg Val Leu Pro Ala Ala 35 40 45 Leu Pro Pro Val Pro Gln Pro Val Cys Thr Tyr Arg Glu Leu Ala Phe 50 55 60 Ala Ser Val Arg Leu Pro Gly Cys Pro Pro Gly Val Asp Pro Ile Val 65 70 75 80 Ser Phe Pro Val Ala Leu Ser Cys Arg Cys Gly Pro Cys Arg Leu Ser 85 90 95 Ser Ser Asp Cys Gly Gly Pro Arg Thr Gln Pro Met Ala Cys Asp Leu 100 105 110 Pro His Leu Pro Gly Leu Leu Leu Leu 115 120 6121PRTRattus norvegicus 6Ser Arg Gly Pro Leu Arg Pro Leu Cys Arg Pro Val Asn Ala Thr Leu 1 5 10 15 Ala Ala Glu Asn Glu Phe Cys Pro Val Cys Ile Thr Phe Thr Thr Ser 20 25 30 Ile Cys Ala Gly Tyr Cys Pro Ser Met Val Arg Val Leu Pro Ala Ala 35 40 45 Leu Pro Pro Val Pro Gln Pro Val Cys Thr Tyr Arg Glu Leu Arg Phe 50 55 60 Ala Ser Val Arg Leu Pro Gly Cys Pro Pro Gly Val Asp Pro Ile Val 65 70 75 80 Ser Phe Pro Val Ala Leu Ser Cys Arg Cys Gly Pro Cys Arg Leu Ser 85 90 95 Ser Ser Asp Cys Gly Gly Pro Arg Thr Gln Pro Met Thr Cys Asp Leu 100 105 110 Pro His Leu Pro Gly Leu Leu Leu Phe 115 120 7121PRTGorilla gorillaMOD_RES(68)..(68)Any amino acid 7Ser Arg Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu 1 5 10 15 Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr 20 25 30 Ile Cys Ala Gly Tyr Cys Pro Thr Met Met Arg Val Leu Gln Gly Val 35 40 45 Leu Pro Pro Leu Pro Gln Val Val Cys Thr Tyr Arg Asp Val Arg Phe 50 55 60 Glu Ser Ile Xaa Leu Pro Gly Cys Pro Arg Gly Val Asp Pro Met Val 65 70 75 80 Ser Phe Pro Val Ala Leu Ser Cys Arg Cys Gly Pro Cys His Arg Ser 85 90 95 Thr Ser Asp Cys Gly Gly Pro Asn Asp His Pro Leu Thr Cys Asp His 100 105 110 Pro Gln Leu Ser Gly Leu Leu Phe Leu 115 120 8121PRTPan paniscus 8Ser Arg Glu Pro Leu Arg Pro Trp Cys His Pro Ile Asn Ala Thr Leu 1 5 10 15 Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr 20 25 30 Ile Cys Ala Gly Tyr Cys Pro Thr Met Met Arg Val Leu Gln Ala Val 35 40 45 Leu Pro Pro Leu Pro Gln Val Val Cys Thr Tyr Arg Asp Val Arg Phe 50 55 60 Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asp Pro Val Val 65 70 75 80 Ser Phe Pro Val Ala Leu Ser Cys Arg Cys Gly Pro Cys Arg Arg Ser 85 90 95 Thr Ser Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp His 100 105 110 Pro Gln Leu Ser Gly Leu Leu Phe Leu 115 120 9145PRTHomo sapiens 9Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu 1 5 10 15 Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr 20 25 30 Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val 35 40 45 Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asp Val Arg Phe 50 55 60 Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asn Pro Val Val 65 70 75 80 Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg Ser 85 90 95 Thr Thr Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp 100 105 110 Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu 115 120 125 Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro 130 135 140 Gln 145 10111PRTHomo sapiens 10Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Ala Ile Glu Lys Glu Glu 1 5 10 15 Cys Arg Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala Gly Tyr Cys 20 25 30 Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Lys Ile Gln 35 40 45 Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val Arg Val Pro 50 55 60 Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro Val Ala Thr 65 70 75 80 Gln Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys Thr Val 85 90 95 Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Gly Glu Met Lys Glu 100 105 110 11110PRTMus musculus 11Ser Cys Glu Leu Thr Asn Ile Thr Ile Ser Val Glu Lys Glu Glu Cys 1 5 10 15 Arg Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala Gly Tyr Cys Tyr 20 25 30 Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Asn Thr Gln Lys 35 40 45 Val Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val Arg Leu Pro Gly 50 55 60 Cys Ala Arg His Ser Asp Ser Leu Tyr Thr Tyr Pro Val Ala Thr Glu 65 70 75 80 Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys Thr Val Arg 85 90 95 Gly Leu Gly Pro Ser Tyr Cys Ser Phe Ser Glu Met Lys Glu 100 105 110 12110PRTRattus norvegicus 12Ser Cys Glu Leu Thr Asn Ile Thr Ile Ser Val Glu Lys Glu Glu Cys 1 5 10 15 Arg Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Glu Gly Tyr Cys Tyr 20 25 30 Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Asn Thr Gln Lys 35 40 45 Val Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Ile Arg Leu Pro Gly 50 55 60 Cys Ala Arg His Ser Asp Ser Leu Tyr Thr Tyr Pro Val Ala Thr Glu 65 70 75 80 Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys Thr Val Arg 85 90 95 Gly Leu Gly Pro Ser Tyr Cys Ser Phe Gly Glu Met Lys Glu 100 105 110 13109PRTGorilla gorilla 13Cys Glu Leu Thr Asn Ile Thr Ile Ala Ile Glu Lys Glu Glu Cys Arg 1 5 10 15 Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala Gly Tyr Cys Tyr Thr 20 25 30 Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Asn Ile Gln Lys Thr 35 40 45 Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val Arg Val Pro Gly Cys 50 55 60 Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro Val Ala Thr Gln Cys 65 70 75 80 His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys Thr Val Arg Gly 85 90 95 Leu Gly Pro Ser Tyr Cys Ser Phe Gly Glu Met Lys Glu 100 105 14109PRTPan paniscus 14Cys Glu Leu Thr Asn Ile Thr Ile Ala Ile Glu Lys Glu Glu Cys Arg 1 5 10 15 Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala Gly His Cys Tyr Thr 20 25 30 Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Asn Ile Gln Lys Thr 35 40 45 Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val Arg Val Pro Gly Cys 50 55 60 Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro Val Ala Thr Gln Cys 65 70 75 80 His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys Thr Val Arg Gly 85 90 95 Leu Gly Pro Ser Tyr Cys Ser Phe Gly Glu Met Lys Glu 100 105 15139PRTArtificial SequenceDescription of Artificial Sequence Synthetic Fusion between human FSHB and the C-terminal 28 amino acids of hCG sequence 15Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Ala Ile Glu Lys Glu Glu 1 5 10 15 Cys Arg Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala Gly Tyr Cys 20 25 30 Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Lys Ile Gln 35 40 45 Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val Arg Val Pro 50 55 60 Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro Val Ala Thr 65 70 75 80 Gln Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys Thr Val 85 90 95 Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Gly Glu Met Lys Glu Ser 100 105 110 Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu 115 120 125 Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln 130 135 16116PRTHomo sapiens 16Met Asp Tyr Tyr Arg Lys Tyr Ala Ala Ile Phe Leu Val Thr Leu Ser 1 5 10 15 Val Phe Leu His Val Leu His Ser Ala Pro Asp Val Gln Asp Cys Pro 20 25 30 Glu Cys Thr Leu Gln Glu Asn Pro Phe Phe Ser Gln Pro Gly Ala Pro 35 40 45 Ile Leu Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro 50 55 60 Leu Arg Ser Lys Lys Thr Met Leu Val Gln Lys Asn Val Thr Ser Glu 65 70 75 80 Ser Thr Cys Cys Val Ala Lys Ser Tyr Asn Arg Val Thr Val Met Gly 85 90 95 Gly Phe Lys Val Glu Asn His Thr Ala Cys His Cys Ser Thr Cys Tyr 100 105 110 Tyr His Lys Ser 115 17165PRTHomo sapiens 17Met Glu Met Phe Gln Gly Leu Leu Leu Leu Leu Leu Leu Ser Met Gly 1 5 10 15 Gly Thr Trp Ala Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile 20 25 30 Asn Ala Thr Leu Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr 35 40 45 Val Asn Thr Thr Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val 50 55 60 Leu Gln Gly Val Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg 65 70 75 80 Asp Val Arg Phe Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val 85 90 95 Asn Pro Val Val Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu 100 105 110 Cys Arg Arg Ser Thr Thr Asp Cys Gly Gly Pro Lys Asp His Pro Leu 115 120 125 Thr Cys Asp Asp Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro 130 135 140 Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr 145 150 155 160 Pro Ile Leu Pro Gln 165 18141PRTHomo sapiens 18Met Glu Met Leu Gln Gly Leu Leu Leu Leu Leu Leu Leu Ser Met Gly 1 5 10 15 Gly Ala Trp Ala Ser Arg Glu Pro Leu Arg Pro Trp Cys His Pro Ile 20 25 30 Asn Ala Ile Leu Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr 35 40 45 Val Asn Thr Thr Ile Cys Ala Gly Tyr Cys Pro Thr Met Met Arg Val 50 55 60 Leu Gln Ala Val Leu Pro Pro Leu Pro Gln Val Val Cys Thr Tyr Arg 65 70 75 80 Asp Val Arg Phe Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val 85 90 95 Asp Pro Val Val Ser Phe Pro Val Ala Leu Ser Cys Arg Cys Gly Pro 100 105 110 Cys Arg Arg Ser Thr Ser Asp Cys Gly Gly Pro Lys Asp His Pro Leu 115 120 125 Thr Cys Asp His Pro Gln Leu Ser Gly Leu Leu Phe Leu 130 135 140 19609PRTHomo sapiens 19Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 35 40 45 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65

70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys 180 185 190 Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205 Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys 210 215 220 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265 270 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 275 280 285 Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu 290 295 300 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 305 310 315 320 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330 335 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys 370 375 380 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 405 410 415 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 450 455 460 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 530 535 540 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 565 570 575 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585 590 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 595 600 605 Leu 20585PRTHomo sapiens 20Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 21585PRTArtificial SequenceDescription of Artificial Sequence Synthetic Human serum albumin K573P variant sequence 21Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Pro Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 22227PRTHomo sapiens 22Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 2315PRTArtificial SequenceDescription of Artificial Sequence Synthetic linker sequence 23Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 24242PRTArtificial SequenceDescription of Artificial Sequence Synthetic linker + human Fc sequence 24Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 1 5 10 15 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 20 25 30 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 35 40 45 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 50 55 60 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 65 70 75 80 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 85 90 95 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 100 105 110 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 115 120 125 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 130 135 140 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 145 150 155 160 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 165 170 175 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 180 185 190 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 195 200 205 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 210 215 220 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 225 230 235 240 Gly Lys 25248PRTArtificial SequenceDescription of Artificial Sequence Synthetic Linker + Fc + His-tag sequence 25Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 1 5 10 15 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 20 25 30 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 35 40 45 Ser Arg Thr Pro Glu Val Thr Cys Val

Val Val Asp Val Ser His Glu 50 55 60 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 65 70 75 80 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 85 90 95 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 100 105 110 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 115 120 125 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 130 135 140 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 145 150 155 160 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 165 170 175 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 180 185 190 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 195 200 205 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 210 215 220 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 225 230 235 240 Gly Lys His His His His His His 245 26677PRTArtificial SequenceDescription of Artificial Sequence Synthetic Mature wt Albumin + alpha-chain sequence 26Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu Ala Pro Asp Val Gln Asp Cys 580 585 590 Pro Glu Cys Thr Leu Gln Glu Asn Pro Phe Phe Ser Gln Pro Gly Ala 595 600 605 Pro Ile Leu Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr 610 615 620 Pro Leu Arg Ser Lys Lys Thr Met Leu Val Gln Lys Asn Val Thr Ser 625 630 635 640 Glu Ser Thr Cys Cys Val Ala Lys Ser Tyr Asn Arg Val Thr Val Met 645 650 655 Gly Gly Phe Lys Val Glu Asn His Thr Ala Cys His Cys Ser Thr Cys 660 665 670 Tyr Tyr His Lys Ser 675 27677PRTArtificial SequenceDescription of Artificial Sequence Synthetic Mature alpha-chain + wt Albumin sequence 27Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro 1 5 10 15 Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys 20 25 30 Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu 35 40 45 Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser 50 55 60 Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr 65 70 75 80 Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser Asp Ala His Lys 85 90 95 Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys 100 105 110 Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe 115 120 125 Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr 130 135 140 Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr 145 150 155 160 Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr 165 170 175 Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu 180 185 190 Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val 195 200 205 Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu 210 215 220 Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr 225 230 235 240 Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala 245 250 255 Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro 260 265 270 Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln 275 280 285 Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys 290 295 300 Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe 305 310 315 320 Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu 325 330 335 Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu 340 345 350 Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys 355 360 365 Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu 370 375 380 Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp 385 390 395 400 Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp 405 410 415 Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp 420 425 430 Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr 435 440 445 Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys 450 455 460 Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile 465 470 475 480 Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln 485 490 495 Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr 500 505 510 Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys 515 520 525 Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr 530 535 540 Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro 545 550 555 560 Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg 565 570 575 Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys 580 585 590 Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu 595 600 605 Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu 610 615 620 Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met 625 630 635 640 Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys 645 650 655 Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln 660 665 670 Ala Ala Leu Gly Leu 675 28730PRTArtificial SequenceDescription of Artificial Sequence Synthetic Mature human Albumin + hCG beta-chain sequence 28Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu Ser Lys Glu Pro Leu Arg Pro 580 585 590 Arg Cys Arg Pro Ile Asn Ala Thr Leu Ala Val Glu Lys Glu Gly Cys 595 600 605 Pro Val Cys Ile Thr Val Asn Thr Thr Ile Cys Ala Gly Tyr Cys Pro 610 615 620 Thr Met Thr Arg Val Leu Gln Gly Val Leu Pro Ala Leu Pro Gln Val 625 630 635 640 Val Cys Asn Tyr Arg Asp Val Arg Phe Glu Ser Ile Arg Leu Pro Gly 645 650 655 Cys Pro Arg Gly Val Asn Pro Val Val Ser Tyr Ala Val Ala Leu Ser 660 665 670 Cys Gln Cys Ala Leu Cys Arg Arg Ser Thr Thr Asp Cys Gly Gly Pro 675 680 685 Lys Asp His Pro Leu Thr Cys Asp Asp Pro Arg Phe Gln Asp Ser Ser 690 695 700 Ser Ser Lys Ala Pro Pro Pro Ser

Leu Pro Ser Pro Ser Arg Leu Pro 705 710 715 720 Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln 725 730 29730PRTArtificial SequenceDescription of Artificial Sequence Synthetic Mature human hCG beta-chain + wt human Albumin sequence 29Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu 1 5 10 15 Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr 20 25 30 Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val 35 40 45 Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asp Val Arg Phe 50 55 60 Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asn Pro Val Val 65 70 75 80 Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg Ser 85 90 95 Thr Thr Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp 100 105 110 Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu 115 120 125 Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro 130 135 140 Gln Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly 145 150 155 160 Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu 165 170 175 Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr 180 185 190 Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp 195 200 205 Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr 210 215 220 Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu 225 230 235 240 Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn 245 250 255 Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe 260 265 270 His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala 275 280 285 Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys 290 295 300 Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala 305 310 315 320 Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala 325 330 335 Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly 340 345 350 Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe 355 360 365 Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr 370 375 380 Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp 385 390 395 400 Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile 405 410 415 Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser 420 425 430 His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro 435 440 445 Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr 450 455 460 Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala 465 470 475 480 Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys 485 490 495 Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His 500 505 510 Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu 515 520 525 Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly 530 535 540 Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val 545 550 555 560 Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly 565 570 575 Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro 580 585 590 Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu 595 600 605 His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu 610 615 620 Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu 625 630 635 640 Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala 645 650 655 Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr 660 665 670 Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln 675 680 685 Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys 690 695 700 Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu 705 710 715 720 Val Ala Ala Ser Gln Ala Ala Leu Gly Leu 725 730 30706PRTArtificial SequenceDescription of Artificial Sequence Synthetic Mature human wt Albumin + human LH beta-chain sequence 30Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu Ser Arg Glu Pro Leu Arg Pro 580 585 590 Trp Cys His Pro Ile Asn Ala Ile Leu Ala Val Glu Lys Glu Gly Cys 595 600 605 Pro Val Cys Ile Thr Val Asn Thr Thr Ile Cys Ala Gly Tyr Cys Pro 610 615 620 Thr Met Met Arg Val Leu Gln Ala Val Leu Pro Pro Leu Pro Gln Val 625 630 635 640 Val Cys Thr Tyr Arg Asp Val Arg Phe Glu Ser Ile Arg Leu Pro Gly 645 650 655 Cys Pro Arg Gly Val Asp Pro Val Val Ser Phe Pro Val Ala Leu Ser 660 665 670 Cys Arg Cys Gly Pro Cys Arg Arg Ser Thr Ser Asp Cys Gly Gly Pro 675 680 685 Lys Asp His Pro Leu Thr Cys Asp His Pro Gln Leu Ser Gly Leu Leu 690 695 700 Phe Leu 705 31706PRTArtificial SequenceDescription of Artificial Sequence Synthetic Mature human LH beta-chain + human wt Albumin sequence 31Ser Arg Glu Pro Leu Arg Pro Trp Cys His Pro Ile Asn Ala Ile Leu 1 5 10 15 Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr 20 25 30 Ile Cys Ala Gly Tyr Cys Pro Thr Met Met Arg Val Leu Gln Ala Val 35 40 45 Leu Pro Pro Leu Pro Gln Val Val Cys Thr Tyr Arg Asp Val Arg Phe 50 55 60 Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asp Pro Val Val 65 70 75 80 Ser Phe Pro Val Ala Leu Ser Cys Arg Cys Gly Pro Cys Arg Arg Ser 85 90 95 Thr Ser Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp His 100 105 110 Pro Gln Leu Ser Gly Leu Leu Phe Leu Asp Ala His Lys Ser Glu Val 115 120 125 Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val 130 135 140 Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His 145 150 155 160 Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala 165 170 175 Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly 180 185 190 Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met 195 200 205 Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu 210 215 220 Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu 225 230 235 240 Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu 245 250 255 Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala 260 265 270 Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu 275 280 285 Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp 290 295 300 Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys 305 310 315 320 Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala 325 330 335 Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val 340 345 350 Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His 355 360 365 Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr 370 375 380 Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys 385 390 395 400 Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn 405 410 415 Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu 420 425 430 Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu 435 440 445 Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val 450 455 460 Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys 465 470 475 480 Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp 485 490 495 Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn 500 505 510 Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu 515 520 525 Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu 530 535 540 Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys 545 550 555 560 His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val 565 570 575 Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp 580 585 590 Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys 595 600 605 Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn 610 615 620 Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys 625 630 635 640 Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His 645 650 655 Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe 660 665 670 Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys 675 680 685 Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu 690 695 700 Gly Leu 705 32334PRTArtificial SequenceDescription of Artificial Sequence Synthetic Mature human alpha-chain + linker + human Fc sequence 32Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro 1 5 10 15 Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys 20 25 30 Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu 35 40 45 Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser 50 55 60 Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr 65 70

75 80 Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser Gly Gly Gly Gly 85 90 95 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr 100 105 110 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 115 120 125 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 130 135 140 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 145 150 155 160 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 165 170 175 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 180 185 190 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 195 200 205 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 210 215 220 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 225 230 235 240 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 245 250 255 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 260 265 270 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 275 280 285 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 290 295 300 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 305 310 315 320 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 33393PRTArtificial SequenceDescription of Artificial Sequence Synthetic Mature human hCG beta-chain + linker + human Fc + Histag sequence 33Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu 1 5 10 15 Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr 20 25 30 Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val 35 40 45 Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asp Val Arg Phe 50 55 60 Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asn Pro Val Val 65 70 75 80 Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg Ser 85 90 95 Thr Thr Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp 100 105 110 Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu 115 120 125 Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro 130 135 140 Gln Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 145 150 155 160 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 165 170 175 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 180 185 190 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 195 200 205 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 210 215 220 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 225 230 235 240 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 245 250 255 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 260 265 270 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 275 280 285 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 290 295 300 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 305 310 315 320 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 325 330 335 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 340 345 350 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 355 360 365 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 370 375 380 Pro Gly Lys His His His His His His 385 390 34369PRTArtificial SequenceDescription of Artificial Sequence Synthetic Mature human LH beta-chain + linker + human Fc + Histag sequence 34Ser Arg Glu Pro Leu Arg Pro Trp Cys His Pro Ile Asn Ala Ile Leu 1 5 10 15 Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr 20 25 30 Ile Cys Ala Gly Tyr Cys Pro Thr Met Met Arg Val Leu Gln Ala Val 35 40 45 Leu Pro Pro Leu Pro Gln Val Val Cys Thr Tyr Arg Asp Val Arg Phe 50 55 60 Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asp Pro Val Val 65 70 75 80 Ser Phe Pro Val Ala Leu Ser Cys Arg Cys Gly Pro Cys Arg Arg Ser 85 90 95 Thr Ser Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp His 100 105 110 Pro Gln Leu Ser Gly Leu Leu Phe Leu Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro 130 135 140 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 145 150 155 160 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 165 170 175 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 180 185 190 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 195 200 205 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 210 215 220 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 225 230 235 240 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 245 250 255 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 260 265 270 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 275 280 285 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 290 295 300 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 305 310 315 320 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 325 330 335 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 340 345 350 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys His His His His His 355 360 365 His 35701PRTArtificial SequenceDescription of Artificial Sequence Synthetic wt human Albumin with signal peptide and propeptide + human alpha-chain sequence 35Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 35 40 45 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys 180 185 190 Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205 Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys 210 215 220 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265 270 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 275 280 285 Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu 290 295 300 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 305 310 315 320 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330 335 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys 370 375 380 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 405 410 415 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 450 455 460 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 530 535 540 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 565 570 575 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585 590 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 595 600 605 Leu Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn 610 615 620 Pro Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys 625 630 635 640 Cys Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met 645 650 655 Leu Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys 660 665 670 Ser Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His 675 680 685 Thr Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser 690 695 700 36701PRTArtificial SequenceDescription of Artificial Sequence Synthetic Human alpha-chain with signal peptide + wt human Albumin sequence 36Met Asp Tyr Tyr Arg Lys Tyr Ala Ala Ile Phe Leu Val Thr Leu Ser 1 5 10 15 Val Phe Leu His Val Leu His Ser Ala Pro Asp Val Gln Asp Cys Pro 20 25 30 Glu Cys Thr Leu Gln Glu Asn Pro Phe Phe Ser Gln Pro Gly Ala Pro 35 40 45 Ile Leu Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro 50 55 60 Leu Arg Ser Lys Lys Thr Met Leu Val Gln Lys Asn Val Thr Ser Glu 65 70 75 80 Ser Thr Cys Cys Val Ala Lys Ser Tyr Asn Arg Val Thr Val Met Gly 85 90 95 Gly Phe Lys Val Glu Asn His Thr Ala Cys His Cys Ser Thr Cys Tyr 100 105 110 Tyr His Lys Ser Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys 115 120 125 Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala 130 135 140 Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn 145 150 155 160 Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu 165 170 175 Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr 180 185 190 Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala 195 200 205 Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp 210 215 220 Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys 225 230 235 240 Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr 245 250 255 Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe 260 265 270 Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala 275 280 285 Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu 290 295 300 Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln 305 310 315 320 Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser 325 330 335 Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr 340 345 350 Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu 355 360 365 Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln 370 375 380 Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu 385 390 395 400 Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala 405 410 415 Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys 420 425 430 Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr 435 440 445 Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg 450 455 460 Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala 465 470 475 480 Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu 485 490 495 Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu 500

505 510 Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr 515 520 525 Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg 530 535 540 Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys 545 550 555 560 Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu 565 570 575 Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys 580 585 590 Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu 595 600 605 Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr 610 615 620 Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys 625 630 635 640 Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr 645 650 655 Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu 660 665 670 Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly 675 680 685 Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu 690 695 700 37754PRTArtificial SequenceDescription of Artificial Sequence Synthetic Human wt Albumin with signal peptide and propeptide + human hCG beta-chain sequence 37Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 35 40 45 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys 180 185 190 Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205 Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys 210 215 220 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265 270 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 275 280 285 Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu 290 295 300 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 305 310 315 320 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330 335 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys 370 375 380 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 405 410 415 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 450 455 460 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 530 535 540 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 565 570 575 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585 590 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 595 600 605 Leu Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr 610 615 620 Leu Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr 625 630 635 640 Thr Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly 645 650 655 Val Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asp Val Arg 660 665 670 Phe Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asn Pro Val 675 680 685 Val Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg 690 695 700 Ser Thr Thr Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp 705 710 715 720 Asp Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser 725 730 735 Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu 740 745 750 Pro Gln 38750PRTArtificial SequenceDescription of Artificial Sequence Synthetic Human hCG beta-chain with signal peptide + human wt Albumin sequence 38Met Glu Met Phe Gln Gly Leu Leu Leu Leu Leu Leu Leu Ser Met Gly 1 5 10 15 Gly Thr Trp Ala Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile 20 25 30 Asn Ala Thr Leu Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr 35 40 45 Val Asn Thr Thr Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val 50 55 60 Leu Gln Gly Val Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg 65 70 75 80 Asp Val Arg Phe Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val 85 90 95 Asn Pro Val Val Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu 100 105 110 Cys Arg Arg Ser Thr Thr Asp Cys Gly Gly Pro Lys Asp His Pro Leu 115 120 125 Thr Cys Asp Asp Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro 130 135 140 Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr 145 150 155 160 Pro Ile Leu Pro Gln Asp Ala His Lys Ser Glu Val Ala His Arg Phe 165 170 175 Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe 180 185 190 Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val 195 200 205 Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala 210 215 220 Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys 225 230 235 240 Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys 245 250 255 Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp 260 265 270 Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met 275 280 285 Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu 290 295 300 Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu 305 310 315 320 Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala 325 330 335 Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp 340 345 350 Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu 355 360 365 Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu 370 375 380 Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val 385 390 395 400 Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu 405 410 415 Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn 420 425 430 Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu 435 440 445 Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro 450 455 460 Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val 465 470 475 480 Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu 485 490 495 Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu 500 505 510 Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala 515 520 525 Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro 530 535 540 Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe 545 550 555 560 Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr 565 570 575 Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser 580 585 590 Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala 595 600 605 Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln 610 615 620 Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys 625 630 635 640 Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu 645 650 655 Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe 660 665 670 Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile 675 680 685 Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala 690 695 700 Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val 705 710 715 720 Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu 725 730 735 Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu 740 745 750 39730PRTArtificial SequenceDescription of Artificial Sequence Synthetic Human wt Albumin with signal peptide and propeptide + human LH beta-chain sequence 39Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 35 40 45 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys 180 185 190 Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205 Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys 210 215 220 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265 270 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 275 280 285 Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu 290 295 300 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 305 310 315 320 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330 335 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys 370 375 380 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 405 410 415 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 450 455 460 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 530 535

540 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 565 570 575 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585 590 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 595 600 605 Leu Ser Arg Glu Pro Leu Arg Pro Trp Cys His Pro Ile Asn Ala Ile 610 615 620 Leu Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr 625 630 635 640 Thr Ile Cys Ala Gly Tyr Cys Pro Thr Met Met Arg Val Leu Gln Ala 645 650 655 Val Leu Pro Pro Leu Pro Gln Val Val Cys Thr Tyr Arg Asp Val Arg 660 665 670 Phe Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asp Pro Val 675 680 685 Val Ser Phe Pro Val Ala Leu Ser Cys Arg Cys Gly Pro Cys Arg Arg 690 695 700 Ser Thr Ser Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp 705 710 715 720 His Pro Gln Leu Ser Gly Leu Leu Phe Leu 725 730 40726PRTArtificial SequenceDescription of Artificial Sequence Synthetic Human LH beta-chain with signal peptide + human wt Albumin sequence 40Met Glu Met Leu Gln Gly Leu Leu Leu Leu Leu Leu Leu Ser Met Gly 1 5 10 15 Gly Ala Trp Ala Ser Arg Glu Pro Leu Arg Pro Trp Cys His Pro Ile 20 25 30 Asn Ala Ile Leu Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr 35 40 45 Val Asn Thr Thr Ile Cys Ala Gly Tyr Cys Pro Thr Met Met Arg Val 50 55 60 Leu Gln Ala Val Leu Pro Pro Leu Pro Gln Val Val Cys Thr Tyr Arg 65 70 75 80 Asp Val Arg Phe Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val 85 90 95 Asp Pro Val Val Ser Phe Pro Val Ala Leu Ser Cys Arg Cys Gly Pro 100 105 110 Cys Arg Arg Ser Thr Ser Asp Cys Gly Gly Pro Lys Asp His Pro Leu 115 120 125 Thr Cys Asp His Pro Gln Leu Ser Gly Leu Leu Phe Leu Asp Ala His 130 135 140 Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe 145 150 155 160 Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro 165 170 175 Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys 180 185 190 Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His 195 200 205 Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr 210 215 220 Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn 225 230 235 240 Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu 245 250 255 Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu 260 265 270 Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro 275 280 285 Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala 290 295 300 Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu 305 310 315 320 Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys 325 330 335 Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe 340 345 350 Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu 355 360 365 Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr 370 375 380 Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp 385 390 395 400 Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu 405 410 415 Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala 420 425 430 Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala 435 440 445 Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys 450 455 460 Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro 465 470 475 480 Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr 485 490 495 Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala 500 505 510 Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu 515 520 525 Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe 530 535 540 Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser 545 550 555 560 Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser 565 570 575 Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp 580 585 590 Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr 595 600 605 Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn 610 615 620 Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro 625 630 635 640 Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr 645 650 655 Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu 660 665 670 Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val 675 680 685 Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp 690 695 700 Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser 705 710 715 720 Gln Ala Ala Leu Gly Leu 725 41358PRTArtificial SequenceDescription of Artificial Sequence Synthetic Human alpha-chain with signal peptide + linker + human Fc sequence 41Met Asp Tyr Tyr Arg Lys Tyr Ala Ala Ile Phe Leu Val Thr Leu Ser 1 5 10 15 Val Phe Leu His Val Leu His Ser Ala Pro Asp Val Gln Asp Cys Pro 20 25 30 Glu Cys Thr Leu Gln Glu Asn Pro Phe Phe Ser Gln Pro Gly Ala Pro 35 40 45 Ile Leu Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro 50 55 60 Leu Arg Ser Lys Lys Thr Met Leu Val Gln Lys Asn Val Thr Ser Glu 65 70 75 80 Ser Thr Cys Cys Val Ala Lys Ser Tyr Asn Arg Val Thr Val Met Gly 85 90 95 Gly Phe Lys Val Glu Asn His Thr Ala Cys His Cys Ser Thr Cys Tyr 100 105 110 Tyr His Lys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 130 135 140 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 145 150 155 160 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 165 170 175 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 180 185 190 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 195 200 205 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 210 215 220 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 225 230 235 240 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 245 250 255 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 260 265 270 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 275 280 285 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 290 295 300 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 305 310 315 320 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 325 330 335 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 340 345 350 Ser Leu Ser Pro Gly Lys 355 42413PRTArtificial SequenceDescription of Artificial Sequence Synthetic Human hCG beta-chain with signal peptide + linker + human Fc + Histag sequence 42Met Glu Met Phe Gln Gly Leu Leu Leu Leu Leu Leu Leu Ser Met Gly 1 5 10 15 Gly Thr Trp Ala Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile 20 25 30 Asn Ala Thr Leu Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr 35 40 45 Val Asn Thr Thr Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val 50 55 60 Leu Gln Gly Val Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg 65 70 75 80 Asp Val Arg Phe Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val 85 90 95 Asn Pro Val Val Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu 100 105 110 Cys Arg Arg Ser Thr Thr Asp Cys Gly Gly Pro Lys Asp His Pro Leu 115 120 125 Thr Cys Asp Asp Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro 130 135 140 Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr 145 150 155 160 Pro Ile Leu Pro Gln Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 165 170 175 Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 180 185 190 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 195 200 205 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 210 215 220 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 225 230 235 240 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 245 250 255 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 260 265 270 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 275 280 285 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 290 295 300 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 305 310 315 320 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 325 330 335 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 340 345 350 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 355 360 365 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 370 375 380 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 385 390 395 400 Leu Ser Leu Ser Pro Gly Lys His His His His His His 405 410 43389PRTArtificial SequenceDescription of Artificial Sequence Synthetic Human LH beta-chain with signal peptide + linker + human Fc + Histag sequence 43Met Glu Met Leu Gln Gly Leu Leu Leu Leu Leu Leu Leu Ser Met Gly 1 5 10 15 Gly Ala Trp Ala Ser Arg Glu Pro Leu Arg Pro Trp Cys His Pro Ile 20 25 30 Asn Ala Ile Leu Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr 35 40 45 Val Asn Thr Thr Ile Cys Ala Gly Tyr Cys Pro Thr Met Met Arg Val 50 55 60 Leu Gln Ala Val Leu Pro Pro Leu Pro Gln Val Val Cys Thr Tyr Arg 65 70 75 80 Asp Val Arg Phe Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val 85 90 95 Asp Pro Val Val Ser Phe Pro Val Ala Leu Ser Cys Arg Cys Gly Pro 100 105 110 Cys Arg Arg Ser Thr Ser Asp Cys Gly Gly Pro Lys Asp His Pro Leu 115 120 125 Thr Cys Asp His Pro Gln Leu Ser Gly Leu Leu Phe Leu Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His 145 150 155 160 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 165 170 175 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 180 185 190 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 195 200 205 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 210 215 220 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 225 230 235 240 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 245 250 255 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 260 265 270 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 275 280 285 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 290 295 300 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 305 310 315 320 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 325 330 335 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 340 345 350 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 355 360 365 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys His 370 375 380 His His His His His 385 44348DNAArtificial SequenceDescription of Artificial Sequence Synthetic Human alpha-chain with signal peptide optimized (348 bases) sequence 44atggactact accggaagta cgccgccatc ttcctcgtga ccctgtccgt gttcctgcac 60gtgctgcact ctgcccccga tgtgcaggac tgccctgagt gcaccctgca ggaaaaccca 120ttcttcagcc agcctggcgc ccctatcctg cagtgcatgg gctgctgctt ctcccgggct 180taccccaccc ctctgcggtc caagaaaacc atgctggtgc agaaaaacgt gacctccgag 240tctacctgct gcgtggccaa gtcctacaac agagtgaccg tgatgggcgg cttcaaggtg 300gaaaaccaca ccgcctgcca ctgctccacc tgttactacc acaagtcc 34845495DNAArtificial SequenceDescription of Artificial Sequence Synthetic Human hCG beta-chain with signal peptide, opdimized (495 bases) sequence 45atggaaatgt tccagggcct ccttctcctg ctgctgctgt ctatgggcgg cacctgggcc 60tccaaagagc ctctgaggcc ccggtgcaga cccatcaatg ctaccctggc cgtggaaaaa 120gagggctgcc ccgtgtgcat caccgtgaac accaccatct gcgccggcta ctgccctacc 180atgaccagag tgctgcaggg cgtgctgcct gctctgcctc aggtcgtgtg caactaccgg 240gacgtgcgct tcgagtccat cagactgcct ggctgcccca gaggcgtgaa ccctgtggtg 300tcttacgccg tggccctgtc

ttgccagtgc gccctgtgca gaagatccac caccgattgt 360ggcggcccta aggaccaccc tctgacctgc gacgaccctc ggttccagga ctcctccagc 420tctaaggccc ctccaccttc cctgcctagc ccttctagac tgccaggccc ttccgacacc 480cccatcctgc ctcag 49546423DNAArtificial SequenceDescription of Artificial Sequence Synthetic Human LH beta-chain with signal peptide, optimized (423 bases) sequence 46atggaaatgc tgcagggcct ccttctcctg ctgctgctgt ctatgggcgg agcctgggcc 60tctagagagc cactgaggcc ttggtgccac cccatcaatg ccatcctggc cgtggaaaaa 120gagggctgcc ccgtgtgcat caccgtgaac accaccatct gcgccggcta ctgccccacc 180atgatgagag tgctgcaggc cgtgctgccc cctctgcctc aggtcgtgtg cacctacaga 240gatgtgcgct tcgagtccat ccggctgcct ggctgtccta gaggcgtgga ccctgtggtg 300tctttccctg tggccctgtc ctgcagatgc ggcccttgca gaagatccac ctccgactgt 360ggcggcccta aggaccaccc tctgacctgc gatcaccctc agctgtccgg cctgctgttc 420ctg 423472103DNAArtificial SequenceDescription of Artificial Sequence Synthetic Human wt Albumin with signal peptide and propeptide + human alpha-chain (2103 bases) sequence 47atgaaatggg tcacctttat ctccctgctg ttcctgttct cctccgccta ctctcggggc 60gtgttcagaa gggacgccca caagtctgag gtggcccacc ggttcaagga cctgggcgag 120gaaaacttca aggccctggt gctgatcgcc ttcgcccagt acctgcagca gtgccccttc 180gaggaccacg tgaagctcgt gaacgaagtg accgagttcg ccaagacctg cgtggccgat 240gagtccgccg agaactgcga caagtccctg cacaccctgt tcggcgacaa gctgtgtacc 300gtggccaccc tgagagaaac ctacggcgag atggccgact gctgcgccaa gcaggaacct 360gagcggaacg agtgcttcct gcagcacaag gacgacaacc ccaacctgcc cagactcgtg 420cggcctgagg tggacgtgat gtgcaccgcc ttccacgaca acgaggaaac cttcctgaag 480aagtacctgt acgagatcgc cagacggcac ccctacttct acgcccccga gctgctgttc 540ttcgccaagc ggtacaaggc cgccttcacc gagtgttgcc aggccgccga taaggccgct 600tgcctgctgc ctaagctgga cgagctgagg gatgagggca aggcctcctc tgccaagcag 660agactgaagt gcgcctccct gcagaagttc ggcgagcggg cctttaaggc ctgggccgtg 720gctagactgt cccagagatt ccccaaggcc gagtttgccg aggtgtccaa gctcgtgacc 780gacctgacca aggtgcacac cgagtgctgt cacggcgacc tgctggaatg cgccgacgac 840agagccgatc tggccaagta catctgcgag aaccaggact ccatctcctc caagctgaaa 900gagtgctgcg agaagcctct gctggaaaag tcccactgta tcgccgaggt ggaaaacgac 960gagatgcccg ccgacctgcc ttctctggcc gccgacttcg tggaatccaa ggacgtgtgc 1020aagaactacg ccgaggccaa ggatgtgttc ctgggcatgt ttctgtacga gtacgctcgg 1080cggcaccccg actactctgt ggtgctgctg ctgagactgg ctaagaccta cgagacaacc 1140ctggaaaaat gctgcgccgc tgccgacccc cacgagtgtt acgccaaggt gttcgacgag 1200ttcaagccac tggtggaaga accccagaac ctgatcaagc agaattgcga gctgttcgag 1260cagctgggcg agtacaagtt ccagaacgcc ctgctcgtgc ggtacaccaa gaaagtgccc 1320caggtgtcca cccccaccct ggtggaagtg tcccggaacc tgggcaaagt gggctccaag 1380tgctgcaagc accctgaggc caagcggatg ccttgcgccg aggactacct gtccgtggtg 1440ctgaaccagc tgtgcgtgct gcacgaaaag acccccgtgt ccgacagagt gaccaagtgt 1500tgcaccgagt ccctcgtgaa cagacggccc tgcttctccg ccctggaagt ggacgagaca 1560tacgtgccca aagagttcaa cgccgagaca ttcaccttcc acgccgacat ctgcaccctg 1620tccgagaaag agcggcagat caagaaacag accgctctgg tggaactcgt gaagcacaag 1680cccaaggcca ccaaagaaca gctgaaggcc gtgatggacg acttcgccgc ctttgtggaa 1740aagtgctgta aagccgatga caaagagaca tgcttcgccg aagagggcaa gaaactggtg 1800gccgcctctc aggctgcact gggactggct ccagacgtgc aggactgccc tgagtgcacc 1860ctgcaggaaa acccattctt cagccagcct ggcgccccta tcctgcagtg catgggctgc 1920tgcttcagcc gggcttaccc cacccctctg cggtccaaga aaaccatgct ggtgcagaaa 1980aacgtgacct ccgagtctac ctgctgtgtg gccaagtcct acaatagagt gaccgtgatg 2040ggcggcttca aagtggaaaa ccacaccgcc tgccactgct ccacctgtta ctaccacaag 2100tcc 2103482103DNAArtificial SequenceDescription of Artificial Sequence Synthetic Human alpha-chain with signal peptide + human wt Albumin (2103 bases) sequence 48atggactact accggaagta cgccgccatc ttcctcgtga ccctgtccgt gttcctgcac 60gtgctgcact ctgcccccga tgtgcaggac tgccctgagt gcaccctgca ggaaaaccca 120ttcttcagcc agcctggcgc ccctatcctg cagtgcatgg gctgctgctt ctcccgggct 180taccccaccc ctctgcggtc caagaaaacc atgctggtgc agaaaaacgt gacctccgag 240tctacctgct gcgtggccaa gtcctacaac agagtgaccg tgatgggcgg cttcaaggtg 300gaaaaccaca ccgcctgcca ctgctccacc tgttactacc acaagtccga cgcccacaag 360agcgaggtgg cccacagatt caaggacctg ggcgaggaaa acttcaaggc cctggtgctg 420atcgccttcg cccagtacct gcagcagtgc cccttcgagg accacgtgaa gctcgtgaac 480gaagtgaccg agttcgccaa gacctgtgtg gccgacgagt ccgccgagaa ctgcgacaag 540tctctgcaca ccctgttcgg cgacaagctg tgcaccgtgg ccaccctgag agaaacctac 600ggcgagatgg ccgactgctg cgccaagcag gaacctgagc ggaacgagtg cttcctgcag 660cacaaggacg acaaccccaa cctgcccaga ctcgtgcggc ctgaggtgga cgtgatgtgc 720accgccttcc acgacaacga ggaaaccttc ctgaagaagt acctgtacga gatcgccaga 780cggcacccct acttctacgc ccccgagctg ctgttcttcg ccaagcggta caaggccgcc 840ttcaccgagt gttgccaggc cgccgataag gccgcttgcc tgctgcctaa gctggacgag 900ctgagggatg agggcaaggc ctcctctgcc aagcagagac tgaagtgcgc ctccctgcag 960aagttcggcg agcgggcctt taaggcctgg gccgtggcta gactgtccca gagattcccc 1020aaggccgagt ttgccgaggt gtccaagctc gtgaccgacc tgaccaaggt gcacaccgag 1080tgctgtcacg gcgacctgct ggaatgcgcc gacgacagag ccgatctggc caagtacatc 1140tgcgagaacc aggactccat ctcctccaag ctgaaagagt gctgcgagaa gcctctgctg 1200gaaaagtccc actgtatcgc cgaagtggaa aacgacgaga tgcccgccga cctgccttct 1260ctggccgccg acttcgtgga atccaaggac gtgtgcaaga actacgccga ggccaaggat 1320gtgttcctgg gcatgtttct gtacgagtac gctcggcggc accccgacta ctctgtggtg 1380ctgctgctga gactggctaa gacctacgag acaaccctgg aaaaatgctg cgccgctgcc 1440gacccccacg agtgttacgc caaggtgttc gacgagttca agccactggt ggaagaaccc 1500cagaacctga tcaagcagaa ttgcgagctg ttcgagcagc tgggcgagta caagttccag 1560aacgccctgc tcgtgcggta caccaagaaa gtgccccagg tgtccacccc caccctggtg 1620gaagtgtccc ggaacctggg caaagtgggc tccaagtgct gcaagcaccc tgaggccaag 1680cggatgcctt gcgccgagga ctacctgagc gtggtgctga accagctgtg tgtgctgcac 1740gaaaagaccc ccgtgtccga tagagtgacc aagtgttgca ccgagtccct cgtgaacaga 1800cggccttgct tctccgccct ggaagtggac gagacatacg tgcccaaaga gttcaacgcc 1860gagacattca ccttccacgc cgacatctgc accctgtctg agaaagagcg gcagatcaag 1920aaacagaccg ctctggtgga actcgtgaag cacaagccca aggccaccaa agaacagctg 1980aaggccgtga tggacgactt cgccgccttt gtggaaaagt gctgtaaagc cgatgacaaa 2040gagacatgct tcgccgaaga gggcaagaaa ctggtggccg cctctcaggc tgctctggga 2100ctg 2103492262DNAArtificial SequenceDescription of Artificial Sequence Synthetic Human wt Albumin with signal peptide and propeptide + human hCG beta-chain, optimized (2262 bases) sequence 49atgaaatggg tcacctttat ctccctgctg ttcctgttct cctccgccta ctctcggggc 60gtgttcagaa gggacgccca caagtctgag gtggcccacc ggttcaagga cctgggcgag 120gaaaacttca aggccctggt gctgatcgcc ttcgcccagt acctgcagca gtgccccttc 180gaggaccacg tgaagctcgt gaacgaagtg accgagttcg ccaagacctg cgtggccgat 240gagtccgccg agaactgcga caagtccctg cacaccctgt tcggcgacaa gctgtgtacc 300gtggccaccc tgagagaaac ctacggcgag atggccgact gctgcgccaa gcaggaacct 360gagcggaacg agtgcttcct gcagcacaag gacgacaacc ccaacctgcc cagactcgtg 420cggcctgagg tggacgtgat gtgcaccgcc ttccacgaca acgaggaaac cttcctgaag 480aagtacctgt acgagatcgc cagacggcac ccctacttct acgcccccga gctgctgttc 540ttcgccaagc ggtacaaggc cgccttcacc gagtgttgcc aggccgccga taaggccgct 600tgcctgctgc ctaagctgga cgagctgagg gatgagggca aggcctcctc tgccaagcag 660agactgaagt gcgcctccct gcagaagttc ggcgagcggg cctttaaggc ctgggccgtg 720gctagactgt cccagagatt ccccaaggcc gagtttgccg aggtgtccaa gctcgtgacc 780gacctgacca aggtgcacac cgagtgctgt cacggcgacc tgctggaatg cgccgacgac 840agagccgatc tggccaagta catctgcgag aaccaggact ccatctcctc caagctgaaa 900gagtgctgcg agaagcctct gctggaaaag tcccactgta tcgccgaggt ggaaaacgac 960gagatgcccg ccgacctgcc ttctctggcc gccgacttcg tggaatccaa ggacgtgtgc 1020aagaactacg ccgaggccaa ggatgtgttc ctgggcatgt ttctgtacga gtacgctcgg 1080cggcaccccg actactctgt ggtgctgctg ctgagactgg ctaagaccta cgagacaacc 1140ctggaaaaat gctgcgccgc tgccgacccc cacgagtgtt acgccaaggt gttcgacgag 1200ttcaagccac tggtggaaga accccagaac ctgatcaagc agaattgcga gctgttcgag 1260cagctgggcg agtacaagtt ccagaacgcc ctgctcgtgc ggtacaccaa gaaagtgccc 1320caggtgtcca cccccaccct ggtggaagtg tcccggaacc tgggcaaagt gggctccaag 1380tgctgcaagc accctgaggc caagcggatg ccttgcgccg aggactacct gtccgtggtg 1440ctgaaccagc tgtgcgtgct gcacgaaaag acccccgtgt ccgacagagt gaccaagtgt 1500tgcaccgagt ccctcgtgaa cagacggccc tgcttctccg ccctggaagt ggacgagaca 1560tacgtgccca aagagttcaa cgccgagaca ttcaccttcc acgccgacat ctgcaccctg 1620tccgagaaag agcggcagat caagaaacag accgctctgg tggaactcgt gaagcacaag 1680cccaaggcca ccaaagaaca gctgaaggcc gtgatggacg acttcgccgc ctttgtggaa 1740aagtgctgta aagccgatga caaagagaca tgcttcgccg aagagggcaa gaaactggtg 1800gccgcctctc aggctgctct gggcctgtct aaagagcctc tgcggcctcg gtgccggcct 1860atcaatgcta ccctggccgt ggaaaaagag ggctgccccg tgtgcatcac cgtgaacacc 1920accatctgcg ccggctactg ccctaccatg acaagggtgc tgcagggcgt gctgcctgct 1980ctgcctcagg tcgtgtgcaa ctaccgggac gtgcgcttcg agtccatcag actgcctggc 2040tgccccagag gcgtgaaccc tgtggtgtct tacgccgtgg ccctgtcttg ccagtgcgcc 2100ctgtgcagaa gatccaccac cgattgtggc ggccctaagg accaccctct gacctgcgac 2160gaccctcggt tccaggacag ctccagctct aaggcccctc caccttccct gcctagccct 2220tctagactgc caggcccttc cgacacccct atcctgcctc ag 2262502250DNAArtificial SequenceDescription of Artificial Sequence Synthetic Human hCG beta-chain with signal peptide + human wt Albumin, optimized (2250 bases) sequence 50atggaaatgt tccagggcct ccttctcctg ctgctgctgt ctatgggcgg cacctgggcc 60tccaaagagc ctctgaggcc ccggtgcaga cccatcaatg ctaccctggc cgtggaaaaa 120gagggctgcc ccgtgtgcat caccgtgaac accaccatct gcgccggcta ctgccctacc 180atgaccagag tgctgcaggg cgtgctgcct gctctgcctc aggtcgtgtg caactaccgg 240gacgtgcgct tcgagtccat cagactgcct ggctgcccca gaggcgtgaa ccctgtggtg 300tcttacgccg tggccctgtc ttgccagtgc gccctgtgca gaagatccac caccgattgt 360ggcggcccta aggaccaccc tctgacctgc gacgaccctc ggttccagga ctcctccagc 420tctaaggccc ctccaccttc cctgcctagc ccttctagac tgccaggccc ttccgacacc 480cccatcctgc cacaggatgc ccacaagtct gaggtggccc accggttcaa ggacctgggc 540gaggaaaact tcaaggccct ggtgctgatc gccttcgccc agtacctgca gcagtgcccc 600ttcgaggacc acgtgaagct cgtgaacgaa gtgaccgagt tcgccaagac ctgcgtggcc 660gatgagtccg ccgagaactg cgacaagtcc ctgcacaccc tgttcggcga caagctgtgt 720accgtggcca ccctgagaga aacctacggc gagatggccg actgctgcgc caagcaggaa 780cctgagcgga acgagtgctt tctgcagcac aaggacgaca accctaacct gcctcggctc 840gtgcggcctg aggtggacgt gatgtgtacc gccttccacg acaacgagga aaccttcctg 900aagaagtacc tgtacgagat cgccagacgg cacccctact tctacgcccc cgagctgctg 960ttcttcgcca agcggtacaa ggccgccttc accgagtgtt gccaggccgc cgataaggcc 1020gcttgcctgc tgcctaagct ggacgagctg agggatgagg gcaaggcctc ctctgccaag 1080cagagactga agtgcgcctc cctgcagaag ttcggcgagc gggcctttaa ggcctgggcc 1140gtggctagac tgtcccagag attccccaag gccgagtttg ccgaggtgtc caagctcgtg 1200accgacctga ccaaggtgca caccgagtgc tgtcacggcg acctgctgga atgcgccgac 1260gacagagccg atctggccaa gtacatctgc gagaaccagg acagcatctc ctccaagctg 1320aaagagtgtt gcgagaagcc tctgctggaa aagtcccact gtatcgccga ggtggaaaac 1380gacgagatgc ccgccgacct gccttctctg gccgccgact tcgtggaatc caaggacgtg 1440tgcaagaact acgccgaggc caaggatgtg ttcctgggca tgtttctgta cgagtacgct 1500cggcggcacc ccgactactc tgtggtgctg ctgctgagac tggccaagac ctacgagaca 1560accctggaaa agtgctgcgc cgctgccgac cctcacgagt gttacgccaa ggtgttcgac 1620gagttcaagc cactggtgga agaaccccag aacctgatca agcagaattg cgagctgttc 1680gagcagctgg gcgagtacaa gttccagaac gccctgctcg tgcgctacac caagaaagtg 1740ccccaggtgt ccacccccac cctggtggaa gtgtcccgga acctgggcaa agtgggctcc 1800aagtgctgca agcaccctga ggccaagcgg atgccttgcg ccgaggacta cctgtccgtg 1860gtgctgaacc agctgtgcgt gctgcacgaa aagacccccg tgtccgacag agtgaccaag 1920tgttgcaccg agtccctcgt gaacagacgg ccctgcttct ccgccctgga agtggacgag 1980acatacgtgc ccaaagagtt caacgccgag acattcacct tccacgccga catctgcacc 2040ctgtccgaga aagagcggca gatcaagaaa cagaccgctc tggtggaact cgtgaagcac 2100aagcccaagg ccaccaaaga acagctgaag gccgtgatgg acgacttcgc cgcctttgtg 2160gaaaaatgtt gcaaggccga tgacaaagag acatgcttcg ccgaagaggg caagaaactg 2220gtggccgcct ctcaggctgc tctgggactg 2250512190DNAArtificial SequenceDescription of Artificial Sequence Synthetic Human wt Albumin with signal peptide and propeptide + human LH beta-chain, optimized (2190 bases) sequence 51atgaaatggg tcacctttat ctccctgctg ttcctgttct cctccgccta ctctcggggc 60gtgttcagaa gggacgccca caagtctgag gtggcccacc ggttcaagga cctgggcgag 120gaaaacttca aggccctggt gctgatcgcc ttcgcccagt acctgcagca gtgccccttc 180gaggaccacg tgaagctcgt gaacgaagtg accgagttcg ccaagacctg cgtggccgat 240gagtccgccg agaactgcga caagtccctg cacaccctgt tcggcgacaa gctgtgtacc 300gtggccaccc tgagagaaac ctacggcgag atggccgact gctgcgccaa gcaggaacct 360gagcggaacg agtgcttcct gcagcacaag gacgacaacc ccaacctgcc cagactcgtg 420cggcctgagg tggacgtgat gtgcaccgcc ttccacgaca acgaggaaac cttcctgaag 480aagtacctgt acgagatcgc cagacggcac ccctacttct acgcccccga gctgctgttc 540ttcgccaagc ggtacaaggc cgccttcacc gagtgttgcc aggccgccga taaggccgct 600tgcctgctgc ctaagctgga cgagctgagg gatgagggca aggcctcctc tgccaagcag 660agactgaagt gcgcctccct gcagaagttc ggcgagcggg cctttaaggc ctgggccgtg 720gctagactgt cccagagatt ccccaaggcc gagtttgccg aggtgtccaa gctcgtgacc 780gacctgacca aggtgcacac cgagtgctgt cacggcgacc tgctggaatg cgccgacgac 840agagccgatc tggccaagta catctgcgag aaccaggact ccatctcctc caagctgaaa 900gagtgctgcg agaagcctct gctggaaaag tcccactgta tcgccgaggt ggaaaacgac 960gagatgcccg ccgacctgcc ttctctggcc gccgacttcg tggaatccaa ggacgtgtgc 1020aagaactacg ccgaggccaa ggatgtgttc ctgggcatgt ttctgtacga gtacgctcgg 1080cggcaccccg actactctgt ggtgctgctg ctgagactgg ctaagaccta cgagacaacc 1140ctggaaaaat gctgcgccgc tgccgacccc cacgagtgtt acgccaaggt gttcgacgag 1200ttcaagccac tggtggaaga accccagaac ctgatcaagc agaattgcga gctgttcgag 1260cagctgggcg agtacaagtt ccagaacgcc ctgctcgtgc ggtacaccaa gaaagtgccc 1320caggtgtcca cccccaccct ggtggaagtg tcccggaacc tgggcaaagt gggctccaag 1380tgctgcaagc accctgaggc caagcggatg ccttgcgccg aggactacct gtccgtggtg 1440ctgaaccagc tgtgcgtgct gcacgaaaag acccccgtgt ccgacagagt gaccaagtgt 1500tgcaccgagt ccctcgtgaa cagacggccc tgcttctccg ccctggaagt ggacgagaca 1560tacgtgccca aagagttcaa cgccgagaca ttcaccttcc acgccgacat ctgcaccctg 1620tccgagaaag agcggcagat caagaaacag accgctctgg tggaactcgt gaagcacaag 1680cccaaggcca ccaaagaaca gctgaaggcc gtgatggacg acttcgccgc ctttgtggaa 1740aagtgctgta aagccgatga caaagagaca tgcttcgccg aagagggcaa gaaactggtg 1800gccgcctctc aggctgctct gggcctgtct agagagcctc tgaggccttg gtgccacccc 1860atcaatgcca tcctggccgt ggaaaaagag ggctgccccg tgtgcatcac cgtgaacacc 1920accatctgcg ccggctactg ccccaccatg atgagggtgc tgcaggccgt gctgcctcca 1980ctgcctcagg tcgtgtgtac ctaccgggac gtgcgcttcg agtccatcag actgcctggc 2040tgccctagag gcgtggaccc tgtggtgtct ttccctgtgg ccctgtcctg cagatgcggc 2100ccttgcagaa gatccacctc cgactgtggc ggccctaagg accaccctct gacctgcgat 2160cacccccagc tgtccggcct gctgtttctg 2190522178DNAArtificial SequenceDescription of Artificial Sequence Synthetic Human LH beta-chain with signal peptide + human wt Albumin (2178 bases) sequence 52atggaaatgc tgcagggcct ccttctcctg ctgctgctgt ctatgggcgg agcctgggcc 60tctagagagc cactgaggcc ttggtgccac cccatcaatg ccatcctggc cgtggaaaaa 120gagggctgcc ccgtgtgcat caccgtgaac accaccatct gcgccggcta ctgccccacc 180atgatgagag tgctgcaggc cgtgctgccc cctctgcctc aggtcgtgtg cacctacaga 240gatgtgcgct tcgagtccat ccggctgcct ggctgtccta gaggcgtgga ccctgtggtg 300tctttccctg tggccctgtc ctgcagatgc ggcccttgca gaagatccac ctccgactgt 360ggcggcccta aggaccaccc tctgacctgc gatcaccctc agctgtccgg cctgctgttc 420ctggacgccc acaagtctga ggtggcccac cggttcaagg acctgggcga ggaaaacttc 480aaggccctgg tgctgatcgc cttcgcccag tacctgcagc agtgcccctt cgaggaccac 540gtgaagctcg tgaacgaagt gaccgagttc gccaagacct gcgtggccga tgagtccgcc 600gagaactgcg acaagtccct gcacaccctg ttcggcgaca agctgtgtac cgtggccacc 660ctgagagaaa cctacggcga gatggccgac tgctgcgcca agcaggaacc tgagcggaac 720gagtgctttc tgcagcacaa ggacgacaac cccaacctgc ccagactcgt gcggcctgag 780gtggacgtga tgtgcaccgc cttccacgac aacgaggaaa ccttcctgaa gaagtacctg 840tacgagatcg ccagacggca cccctacttc tacgcccccg agctgctgtt cttcgccaag 900cggtacaagg ccgccttcac cgagtgttgc caggccgccg ataaggccgc ttgcctgctg 960cctaagctgg acgagctgcg ggatgagggc aaggcctctt ctgccaagca gcggctgaag 1020tgcgcctccc tgcagaagtt tggcgagcgg gcctttaagg cctgggccgt ggctagactg 1080tcccagagat tccccaaggc cgagtttgcc gaggtgtcca agctcgtgac cgacctgacc 1140aaagtgcaca ccgagtgctg tcacggcgac ctgctggaat gcgccgacga cagagccgac 1200ctggccaagt acatctgcga gaaccaggac tccatctcct ccaagctgaa agagtgttgc 1260gagaagcctc tgctggaaaa gtcccactgt atcgccgagg tggaaaacga cgagatgccc 1320gccgacctgc cttctctggc cgccgacttc gtggaatcca aggacgtgtg caagaactac 1380gccgaggcca aggatgtgtt cctgggcatg tttctgtacg agtacgctcg gcggcacccc 1440gactactctg tggtgctgct gctgagactg gccaagacct acgagacaac cctggaaaag 1500tgctgcgccg ctgccgaccc tcacgagtgt tacgccaagg tgttcgacga gttcaagcca 1560ctggtggaag aaccccagaa cctgatcaag cagaattgcg agctgttcga gcagctgggc 1620gagtacaagt tccagaacgc cctgctcgtg cggtacacca agaaagtgcc ccaggtgtcc 1680acccccaccc tggtggaagt gtcccggaac ctgggcaaag tgggctccaa gtgctgcaag 1740caccctgagg ccaagcggat gccttgcgcc gaggactacc tgtccgtggt gctgaaccag 1800ctgtgcgtgc tgcacgaaaa gacccccgtg tccgacagag tgaccaagtg ttgcaccgag 1860tccctcgtga acagacggcc ctgcttctcc gccctggaag tggacgagac atacgtgccc 1920aaagagttca acgccgagac attcaccttc cacgccgaca tctgcaccct gtccgagaaa 1980gagcggcaga tcaagaaaca gaccgctctg gtggaactcg tgaagcacaa gcccaaggcc 2040accaaagaac agctgaaggc cgtgatggac gacttcgccg cctttgtgga aaaatgttgc 2100aaggccgatg acaaagagac atgcttcgcc gaagagggca agaaactggt

ggccgcctct 2160caggctgctc tgggactg 2178531074DNAArtificial SequenceDescription of Artificial Sequence Synthetic Human alpha-chain with signal peptide + linker + human Fc, optimized (1074 bases) sequence 53atggactact accggaagta cgccgccatc ttcctcgtga ccctgtccgt gttcctgcac 60gtgctgcact ctgcccccga tgtgcaggac tgccctgagt gcaccctgca ggaaaaccca 120ttcttcagcc agcctggcgc ccctatcctg cagtgcatgg gctgctgctt ctcccgggct 180taccccaccc ctctgcggtc caagaaaacc atgctggtgc agaaaaacgt gacctccgag 240tctacctgct gcgtggccaa gtcctacaac agagtgaccg tgatgggcgg cttcaaggtg 300gaaaaccaca ccgcctgcca ctgctccacc tgttactacc acaagtccgg cggaggcgga 360tctggcggcg gaggttctgg tggtggtggc tccgataaga cccacacctg tcccccttgt 420cccgcccctg aactgctggg aggcccttct gtgttcctgt tccccccaaa gcccaaggac 480accctgatga tctcccggac ccccgaagtg acctgcgtgg tggtggatgt gtcccacgag 540gaccctgaag tgaagttcaa ttggtacgtg gacggcgtgg aagtgcacaa cgccaagacc 600aagcccagag aggaacagta caactccacc taccgggtgg tgtccgtgct gaccgtgctg 660catcaggatt ggctgaacgg caaagagtac aagtgcaagg tgtccaacaa ggccctgcct 720gcccccatcg aaaagaccat ctccaaggcc aagggccagc cccgggaacc ccaggtgtac 780acactgcccc ctagcaggga cgagctgacc aagaaccagg tgtccctgac ctgtctcgtg 840aagggcttct acccctccga tatcgccgtg gaatgggagt ccaacggcca gcctgagaac 900aactacaaga ccaccccccc tgtgctggac tccgacggct cattcttcct gtactccaag 960ctgacagtgg acaagtcccg gtggcagcag ggcaacgtgt tctcctgctc cgtgatgcac 1020gaggccctgc acaaccacta cacccagaag tccctgtccc tgagccccgg caaa 1074541239DNAArtificial SequenceDescription of Artificial Sequence Synthetic Human hCG beta-chain + linker + human Fc + Histag, optimized (1239 bases) sequence 54atggaaatgt tccagggcct ccttctcctg ctgctgctgt ctatgggcgg cacctgggcc 60tccaaagagc ctctgaggcc ccggtgcaga cccatcaatg ctaccctggc cgtggaaaaa 120gagggctgcc ccgtgtgcat caccgtgaac accaccatct gcgccggcta ctgccctacc 180atgaccagag tgctgcaggg cgtgctgcct gctctgcctc aggtcgtgtg caactaccgg 240gacgtgcgct tcgagtccat cagactgcct ggctgcccca gaggcgtgaa ccctgtggtg 300tcttacgccg tggccctgtc ttgccagtgc gccctgtgca gaagatccac caccgattgt 360ggcggcccta aggaccaccc tctgacctgc gacgaccctc ggttccagga ctcctccagc 420tctaaggccc ctccaccttc cctgcctagc ccttctagac tgccaggccc ttccgacacc 480cccatcctgc cacagggtgg cggaggatct ggcggaggcg gttctggcgg cggtggctct 540gataagaccc acacctgtcc cccttgccct gcccctgaac tgctgggagg ccctagcgtg 600ttcctgttcc ccccaaagcc caaggacacc ctgatgatct cccggacccc cgaagtgacc 660tgcgtggtgg tggacgtgtc ccacgaggac cctgaagtga agttcaattg gtacgtggac 720ggcgtggaag tgcacaacgc caagaccaag cccagagagg aacagtacaa ctccacctac 780cgggtggtgt ccgtgctgac cgtgctgcac caggattggc tgaacggcaa agagtacaag 840tgcaaggtgt ccaacaaggc cctgcctgcc cccatcgaaa agaccatctc caaggccaag 900ggccagcccc gggaacccca ggtgtacaca ctgcccccta gcagggacga gctgaccaag 960aaccaggtgt ccctgacctg tctcgtgaag ggcttctacc cctccgatat cgccgtggaa 1020tgggagtcca acggccagcc tgagaacaac tacaagacca ccccccctgt gctggactcc 1080gacggctcat tcttcctgta ctccaagctg acagtggaca agtcccggtg gcagcagggc 1140aacgtgttct cctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagtcc 1200ctgtccctga gccccggcaa gcaccatcac caccaccat 1239551167DNAArtificial SequenceDescription of Artificial Sequence Synthetic Human LH beta-chain with signal peptide + linker + human Fc + Histag, optimized (1167 bases) sequence 55atggaaatgc tgcagggcct ccttctcctg ctgctgctgt ctatgggcgg agcctgggcc 60tctagagagc cactgaggcc ttggtgccac cccatcaatg ccatcctggc cgtggaaaaa 120gagggctgcc ccgtgtgcat caccgtgaac accaccatct gcgccggcta ctgccccacc 180atgatgagag tgctgcaggc cgtgctgccc cctctgcctc aggtcgtgtg cacctacaga 240gatgtgcgct tcgagtccat ccggctgcct ggctgtccta gaggcgtgga ccctgtggtg 300tctttccctg tggccctgtc ctgcagatgc ggcccttgca gaagatccac ctccgactgt 360ggcggcccta aggaccaccc tctgacctgc gatcaccctc agctgtccgg cctgctgttt 420ctgggaggcg gaggatctgg cggaggcggt tctggtggtg gcggctctga taagacccac 480acctgtcccc cttgccctgc ccctgaactg ctgggaggcc cttccgtgtt cctgttcccc 540ccaaagccca aggacaccct gatgatctcc cggacccccg aagtgacctg cgtggtggtg 600gatgtgtccc acgaggaccc tgaagtgaag ttcaattggt acgtggacgg cgtggaagtg 660cacaacgcca agaccaagcc cagagaggaa cagtacaact ccacctaccg ggtggtgtcc 720gtgctgaccg tgctgcacca ggattggctg aacggcaaag agtacaagtg caaggtgtcc 780aacaaggccc tgcctgcccc catcgaaaag accatctcca aggccaaggg ccagccccgg 840gaaccccagg tgtacacact gccccctagc agggacgagc tgaccaagaa ccaggtgtcc 900ctgacctgtc tcgtgaaggg cttctacccc tccgatatcg ccgtggaatg ggagtccaac 960ggccagcctg agaacaacta caagaccacc ccccctgtgc tggactccga cggctcattc 1020ttcctgtact ccaagctgac agtggacaag tcccggtggc agcagggcaa cgtgttctcc 1080tgcagcgtga tgcacgaggc tctgcacaac cactacaccc agaagtccct gtccctgagc 1140cccggcaagc accatcacca ccaccat 1167568PRTArtificial SequenceDescription of Artificial Sequence Synthetic linker sequence 56Glu Phe Ala Gly Ala Ala Ala Val 1 5 5715PRTArtificial SequenceDescription of Artificial Sequence Synthetic linker sequence 57Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 587PRTArtificial SequenceDescription of Artificial Sequence Synthetic linker sequence 58Ser Gly Gly Ser Gly Gly Ser 1 5 5915PRTArtificial SequenceDescription of Artificial Sequence Synthetic linker sequence 59Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gly 1 5 10 15 6018PRTArtificial SequenceDescription of Artificial Sequence Synthetic linker sequence 60Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 1 5 10 15 Gly Ser 61387PRTArtificial SequenceDescription of Artificial Sequence Synthetic hCG beta chain + linker + Fc sequence 61Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu 1 5 10 15 Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr 20 25 30 Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val 35 40 45 Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asp Val Arg Phe 50 55 60 Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asn Pro Val Val 65 70 75 80 Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg Ser 85 90 95 Thr Thr Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp 100 105 110 Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu 115 120 125 Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro 130 135 140 Gln Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 145 150 155 160 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 165 170 175 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 180 185 190 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 195 200 205 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 210 215 220 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 225 230 235 240 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 245 250 255 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 260 265 270 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 275 280 285 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 290 295 300 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 305 310 315 320 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 325 330 335 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 340 345 350 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 355 360 365 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 370 375 380 Pro Gly Lys 385 62363PRTArtificial SequenceDescription of Artificial Sequence Synthetic LH Beta chain + linker + Fc sequence 62Ser Arg Glu Pro Leu Arg Pro Trp Cys His Pro Ile Asn Ala Ile Leu 1 5 10 15 Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr 20 25 30 Ile Cys Ala Gly Tyr Cys Pro Thr Met Met Arg Val Leu Gln Ala Val 35 40 45 Leu Pro Pro Leu Pro Gln Val Val Cys Thr Tyr Arg Asp Val Arg Phe 50 55 60 Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asp Pro Val Val 65 70 75 80 Ser Phe Pro Val Ala Leu Ser Cys Arg Cys Gly Pro Cys Arg Arg Ser 85 90 95 Thr Ser Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp His 100 105 110 Pro Gln Leu Ser Gly Leu Leu Phe Leu Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro 130 135 140 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 145 150 155 160 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 165 170 175 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 180 185 190 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 195 200 205 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 210 215 220 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 225 230 235 240 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 245 250 255 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 260 265 270 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 275 280 285 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 290 295 300 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 305 310 315 320 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 325 330 335 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 340 345 350 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 355 360 634PRTHomo sapiens 63Glu Leu Leu Gly 1 6410PRTArtificial SequenceDescription of Artificial Sequence Synthetic linker sequence 64Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 6530PRTArtificial SequenceDescription of Artificial Sequence Synthetic linker sequence 65Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 1 5 10 15 Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser 20 25 30 6650PRTArtificial SequenceDescription of Artificial Sequence Synthetic linker sequence 66Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 35 40 45 Gly Ser 50 6750PRTArtificial SequenceDescription of Artificial Sequence Synthetic linker sequence 67Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 15 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 20 25 30 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 35 40 45 Gly Gly 50 68150PRTArtificial SequenceDescription of Artificial Sequence Synthetic linker sequence 68Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 1 5 10 15 Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25 30 Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser 35 40 45 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 50 55 60 Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 65 70 75 80 Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser 85 90 95 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 100 105 110 Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 115 120 125 Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser 130 135 140 Gly Gly Ser Gly Gly Ser 145 150 69250PRTArtificial SequenceDescription of Artificial Sequence Synthetic linker sequence 69Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 35 40 45 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 50 55 60 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 65 70 75 80 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 85 90 95 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 100 105 110 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 145 150 155 160 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 165 170 175 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 180 185 190 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 195 200 205 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 225 230 235 240 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 245 250 7024DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 70gctgacagac taacagactg ttcc 247118DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 71caaatgtggt atggctga 18

Claims

1. A long acting biologically active luteinizing hormone (LH) compound comprising a mammal CG or analog thereof or a mammal LH or analog thereof linked to a pharmaceutically acceptable molecule selected from a molecule having binding to a mammal neonatal Fc receptor, transferrin and a CH.sub.3(CH.sub.2).sub.nCO--, wherein n is 8 to 22 and a polymer.

2. The LH compound of claim 1 wherein the pharmaceutically acceptable molecule is selected from a molecule having binding to a mammal neonatal Fc receptor.

3. The LH compound of claim 1 wherein the pharmaceutically acceptable molecule is selected from an albumin, an Fc fragment of a mammalian antibody, and a variant of an Fc fragment of a mammalian antibody.

4. The LH compound of claim 1 wherein the pharmaceutically acceptable molecule is selected from an albumin such as human albumin, recombinant human albumin, a modified human albumin with increased binding to a mammal FcRn, a modified recombinant albumin with increased binding to a mammal FcRn.

5. The LH compound of claim 1 wherein the mammal CG or analog thereof or a mammal LH or analog thereof is selected from recombinant mammal CG or analog thereof or mammal LH or analog thereof.

6. The LH compound of claim 5 wherein the recombinant mammal CG or analog thereof is selected from the sequence of primate CG, such as human CG, abe CG or monkey CG; and the sequence of equine CG, such as horse CG or wherein the recombinant mammal LH or analog thereof is selected from the sequence of primate LH, such as human LH, abe LH or monkey LH; the sequence of cow LH; the sequence pig LH; the sequence of equine LH, such as horse LH; the sequence of sheep LH; the sequence of dog LH; the sequence of cat LH; and the sequence of goat LH.

7. The LH compound of claim 1 wherein the mammal CG or analog thereof or a mammal LH or analog thereof is selected from an analog that has at least 80% identity to the corresponding mammalian sequence of chorionic gonadotropin or luteinizing hormone, respectively, such as 85% identity, 90% identity, 95% identity, 98% identity.

8. The LH compound of claim 1 wherein the pharmaceutically acceptable molecule is selected from PEG, e.g. PEG of a molecular weight of at least 5 kDa, such as from 10 kDa to 150 kDa, typically 10 to 40 kDa.

9. The LH compound of claim 1 wherein the chemical linker is selected from a sugar moiety, a disulphide bridge, a hydrophilic linker, a hydrolysable linker, dicarboxylic acids, carboxylic acid hydrazides, maleimido hydrazides, PDPH, SPDP, LC-SPDP, GMBS, alkyl linkers, and PEG linkers.

10. The LH compound of claim 1 wherein the mammal CG or analog thereof or a mammal LH or analog thereof is directly chemically linked to the pharmaceutically acceptable molecule, wherein the pharmaceutically acceptable molecule is linked to the alfa chain of the mammal CG or analog thereof or the mammal LH or analog thereof or wherein the pharmaceutically acceptable molecule is linked to the beta chain of the mammal CG or analog thereof or the mammal LH or analog thereof.

11. The LH compound of claim 1 wherein the mammal CG or analog thereof or a mammal LH or analog thereof is fused to the pharmaceutically acceptable molecule selected from a molecule having binding to a mammal neonatal Fc receptor, such as an albumin, an Fc fragment of a mammalian antibody, or a variant of an Fc fragment of a mammalian antibody, optionally through a peptide linker.

12. The LH compound of claim 11 wherein the peptide linker has at least 1 amino acid, such as from 1-200 amino acids, typically 1-50 amino acids wherein the amino acids are selected from the twenty naturally occurring amino acids.

13. The LH compound of claim 11 wherein the mammal CG or analog thereof or a mammal LH or analog thereof is directly fused to the pharmaceutically acceptable molecule.

14. The LH compound of claim 11 wherein the pharmaceutically acceptable molecule is fused to an N-terminal of the mammal CG or analog thereof or an N-terminal of the mammal LH or analog thereof, wherein the pharmaceutically acceptable molecule is fused to the N-terminal of the alfa chain of the mammal CG or analog thereof or the N-terminal of the alfa chain of the mammal LH or analog thereof or wherein the pharmaceutically acceptable molecule is fused to the N-terminal of the beta chain of the mammal CG or analog thereof or the N-terminal of the beta chain of the mammal LH or analog thereof.

15. The LH compound of claim 11 wherein the pharmaceutically acceptable molecule is fused to a C-terminal of the mammal CG or analog thereof or a C-terminal of mammal LH or analog thereof, wherein the pharmaceutically acceptable molecule is fused to the C-terminal of the alfa chain of the mammal CG or analog thereof or the C-terminal of the alfa chain of the mammal LH or analog thereof or wherein the pharmaceutically acceptable molecule is fused to the C-terminal of the beta chain of the mammal CG or analog thereof or the C-terminal of the beta chain of the mammal LH or analog thereof.

16. The LH compound of claim 1 wherein the mammalian CG or analog thereof or the mammalian LH or analog thereof is glycosylated.

17. The LH compound of claim 1 wherein mammal CG or analog thereof or a mammal LH or analog thereof is selected from one mammal CG or analog thereof or one mammal LH or analog thereof.

18. The LH compound of claim 1 selected from Conjugate) (hCG-PDPH-rHA conjugate), Conjugate3 (hCG-SPDP-rHA conjugate), Conjugate4 (EDC activated hCG-PDPH-rHA conjugate) Conjugate3V1 (hCG-SPDP-rHA-K573P conjugate), Conjugate4V1 (EDC activated hCG-PDPH-rHA-K573P conjugate) Product2 consisting of SEQ ID NO:9 and SEQ ID NO:26, Product3 consisting of SEQ ID NO:1 and SEQ ID NO:28, Product4 consisting of SEQ ID NO:9 and SEQ ID NO:27, Product5 consisting of SEQ ID NO:1 and SEQ ID NO:29, Product7 consisting of SEQ ID NO:4 and SEQ ID NO:26, Product8 consisting of SEQ ID NO:1 and SEQ ID NO:30, Product9 consisting of SEQ ID NO:4 and SEQ ID NO:27, Product10 consisting of SEQ ID NO:1 and SEQ ID NO: 31, Product11 consisting of SEQ ID NO:32 and SEQ ID NO: 33, Product12 consisting of SEQ ID NO:32 and SEQ ID NO:34, Product13 consisting of SEQ ID NO:32 and SEQ ID NO:61, Product14 consisting of SEQ ID NO:32 and SEQ ID NO:62.

19. A composition comprising the compound of claim 1.

20. A method for assisted reproductive therapy in a female human, said method comprising a. starting stimulation by administering FSH on cycle day 1-3 of a menstrual cycle, b. administering a GnRH antagonist from day 4-7 of the stimulation until ovulation triggering, c. providing an LH compound of claim 1 to said female by administering at least one dosage of an LH agonist in the period from day 1-9 of the stimulation, said dosage being sufficient to stimulate follicle development until ovulation triggering, d. discontinuing administration of FSH when at least one follicle has a diameter of 12-14 mm, e. inducing ovulation with at least one dosage of a GnRH agonist when at least one follicle has a diameter of at least 15 mm.

Images