Fusion proteins comprising follicle-stimulating hormone and antigen-binding fragments to serum albumin, and compositions and uses thereof

Abstract

The present application relates to a fusion protein comprising a follicle-stimulating hormone and an antigen-binding fragment for serum albumin, and uses thereof. Accordingly, the present application provides a recombinant fusion protein comprising an antigen-binding fragment that binds to serum albumin and human follicle-stimulating hormone linked to the antigen-binding fragment, as well as a long-acting human follicle-stimulating hormone formulation comprising the recombinant fusion protein as an active ingredient. TIFF2025533090000088.tif76170

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119 to Korean Application No. 10-2022-0126516, filed October 4, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Reference to an electronically submitted sequence listing This application contains a Sequence Listing, which has been submitted via EFS-Web in XML format and is incorporated herein by reference in its entirety. This XML copy, created on October 4, 2023, is titled 2662-0007WO01_SEQL_ST26 and is 130,077 bytes in size.

[0003] 1. Field The present disclosure relates to fusion proteins comprising a follicle-stimulating hormone and an antigen-binding fragment to serum albumin and uses thereof. [Background technology]

[0004] 2. Description of Related Technology Infertility refers to the inability to conceive even after more than one year of normal marital relations. The causes of infertility are estimated to be male (40%), female (40%), mixed (10%), and unknown (10%). In the case of female infertility, the main causes include ovulation disorders, hormonal abnormalities, and uterine abnormalities. In the case of male infertility, the main causes include hormonal abnormalities, testicular abnormalities due to harmful substances or trauma, and sperm abnormalities. Assisted reproductive technologies (ARTs) for the treatment of female infertility include in vitro fertilization and embryo transfer (IVF-ET), gamete intrafallopian tube transfer (GIFT), zygote intrafallopian tube transfer (ZIFT), and intracytoplasmic sperm injection (ICSI). These ARTs essentially involve the ovulation induction process using follicle-stimulating hormone (FSH). Furthermore, FSH is used for therapeutic purposes in women suffering from anovulation. Furthermore, FSH is also used to treat male infertility caused by sperm abnormalities.

[0005] Human follicle-stimulating hormone (hFSH) is a glycoprotein hormone produced by the pituitary gland and secreted into the endocrine system. It has a dimeric structure consisting of an α subunit and a β subunit. The α subunit is a 92-amino acid glycoprotein common to glycoproteins such as luteinizing hormone (LH), human chorionic gonadotropin (hCG), and thyroid-stimulating hormone (TSH). It has two N-glycosylation sites at asparagine positions 52 and 78. The β subunit is a 111-amino acid glycoprotein specific to hFSH. It has two N-glycosylation sites at asparagine positions 7 and 24. hFSH is a glycoprotein in which glycans account for approximately 40% of its total molecular weight. It is known that such glycosylation plays an important role in hFSH's receptor binding ability and signal transduction activity.

[0006] hFSH is known to play an important role in the differentiation and growth of male and female germ cells. Therefore, it is used to mature multiple follicles in women undergoing assisted reproductive technology for infertility treatment, or to treat anovulation. hFSH is also used to increase sperm production and sperm count in men. Urinary hFSH purified from female urine has low efficiency and unstable supply, posing many risks, especially in terms of safety. In this regard, genetically engineered preparations prepared using genetic engineering technology are commonly used, but have the limitation that their short in vivo half-life requires frequent administration.

[0007] Given this technological background, it is necessary to develop a long-acting hormone preparation as a technology to improve the therapeutic effect of hFSH and increase convenience for patients.

[0008] Antigen-binding fragments that bind to serum albumin are provided in U.S. Patent Nos. 9,879,077 and 11,773,176, each of which is incorporated herein by reference in its entirety. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 9,879,077 [Patent Document 2] U.S. Patent No. 11,773,176 Summary of the Invention

[0010] overview A recombinant fusion protein is provided that includes an antigen-binding fragment that binds to serum albumin and human follicle-stimulating hormone (hFSH).

[0011] Nucleic acids encoding the recombinant fusion proteins, expression vectors containing the nucleic acids, and cells transformed with the expression vectors are provided.

[0012] Provided are long-acting hFSH formulations or pharmaceutical compositions for infertility treatment, each of which comprises a recombinant fusion protein as an active ingredient.

[0013] Additional aspects will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of presented aspects of the present disclosure.

[0014] Disclosed herein is a recombinant fusion protein comprising (a) human follicle-stimulating hormone (hFSH) comprising an hFSH α subunit and an hFSH β subunit, and (b) an antigen-binding fragment (Fab) that binds to serum albumin. In some embodiments, the hFSH α subunit and the hFSH β subunit are linked to the terminal region of the Fab. In some embodiments, the hFSH α subunit and the hFSH β subunit are independently linked to the end of the heavy chain constant 1 domain and the end of the light chain constant domain of the Fab. In some embodiments, the hFSH α subunit and the hFSH β subunit are independently linked to the end of the heavy chain constant 1 domain and the end of the light chain constant domain of the Fab by a linker. In other embodiments, the hFSH α subunit is linked to the end of the light chain constant domain, and the hFSH β subunit is linked to the end of the heavy chain constant 1 domain of the Fab.

[0015] In some embodiments, the linker comprises 1 to 50 amino acids. In some embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOs: 16, 70-85, and 88.

[0016] In some embodiments, the hFSH alpha subunit comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 1. In some embodiments, the hFSH alpha subunit comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the hFSH beta subunit comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 4. In some embodiments, the hFSH beta subunit comprises the amino acid sequence of SEQ ID NO: 4.

[0017] In some embodiments, in the fusion proteins disclosed herein, the heavy and light chains of the Fab are non-covalently linked.

[0018] Fab is (a) a heavy chain complementarity-determining domain 1 (CDR1) comprising the amino acid sequence of SYGIS (SEQ ID NO:22); a heavy chain complementarity-determining domain 2 (CDR2) comprising the amino acid sequence of TIFF2025533090000002.tif4128; and heavy chain complementarity-determining domain 3 (CDR3) containing the amino acid sequence of TIFF2025533090000003.tif4128; (b) a heavy chain CDR1 comprising the amino acid sequence of SYGIS (SEQ ID NO:22); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025533090000004.tif4128; and heavy chain CDR3 containing the amino acid sequence of TIFF2025533090000005.tif4128; (c) a heavy chain CDR1 comprising the amino acid sequence of NYGIH (SEQ ID NO:26); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025533090000006.tif4128; and Heavy chain CDR3 containing the amino acid sequence of TIFF2025533090000007.tif4128; (d) a heavy chain CDR1 comprising the amino acid sequence of SYAMS (SEQ ID NO:29); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025533090000008.tif4128; and heavy chain CDR3 containing the amino acid sequence of TIFF2025533090000009.tif4128; (e) a heavy chain CDR1 comprising the amino acid sequence of AYWIA (SEQ ID NO:32); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025533090000010.tif4128; and a heavy chain CDR3 comprising the amino acid sequence of LYSGSYSP (SEQ ID NO:34); or (f) a heavy chain CDR1 comprising the amino acid sequence of AYSMN (SEQ ID NO:35); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025533090000011.tif4128; and Heavy chain CDR3 containing the amino acid sequence of TIFF2025533090000012.tif4128 a heavy chain comprising a heavy chain variable domain comprising (g) light chain CDR1 containing the amino acid sequence of TIFF2025533090000013.tif4128; a light chain CDR2 comprising the amino acid sequence of GASRLES (SEQ ID NO:39); and a light chain CDR3 comprising the amino acid sequence of QQSDSVPVT (SEQ ID NO:40); (h) light chain CDR1 containing the amino acid sequence of TIFF2025533090000014.tif4128; a light chain CDR2 comprising the amino acid sequence of AASSLQS (SEQ ID NO:42), and Light chain CDR3 containing the amino acid sequence of TIFF2025533090000015.tif4128; (i) light chain CDR1 containing the amino acid sequence of TIFF2025533090000016.tif4128; a light chain CDR2 comprising the amino acid sequence of DASNRAT (SEQ ID NO:45); and Light chain CDR3 containing the amino acid sequence of TIFF2025533090000017.tif4128; (j) light chain CDR1 containing the amino acid sequence of TIFF2025533090000018.tif4128; a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:48); and a light chain CDR3 comprising the amino acid sequence of QQYGSSPRT (SEQ ID NO:49); (k) light chain CDR1 containing the amino acid sequence of TIFF2025533090000019.tif4128; a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:48); and a light chain CDR3 comprising the amino acid sequence of QKYSSYPLT (SEQ ID NO:51); or (l) light chain CDR1 containing the amino acid sequence of TIFF2025533090000020.tif4128; a light chain CDR2 comprising the amino acid sequence of GASTGAT (SEQ ID NO:53), and Light chain CDR3 containing the amino acid sequence of TIFF2025533090000021.tif4128 a light chain comprising a light chain variable domain comprising may include:

[0019] In some embodiments, in the fusion proteins disclosed herein, the heavy chain variable domain comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:35, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:36, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:37, and the light chain variable domain comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:52, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:53, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:54.

[0020] In some embodiments, the heavy chain variable domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:55, 56, 57, 58, 59, or 60. In some embodiments, the light chain variable domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:61, 62, 63, 64, 65, 66, or 67. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:55, 56, 57, 58, 59, or 60, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:61, 62, 63, 64, 65, 66, or 67. In some embodiments, the heavy chain constant domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:68. In some embodiments, the light chain constant domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:69 or 91.

[0021] In some embodiments, the heavy chain variable domain comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:35, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:36, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:37, and the light chain variable domain comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:52, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:53, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:54.

[0022] In some embodiments, the heavy chain variable domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:55, 56, 57, 58, 59, or 60. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:60.

[0023] In some embodiments, the light chain variable domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:61, 62, 63, 64, 65, 66, or 67. In some embodiments, the light chain variable domain comprises the amino acid sequence of SEQ ID NO:67.

[0024] In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:55, 56, 57, 58, 59, or 60, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:61, 62, 63, 64, 65, 66, or 67. In some embodiments, the heavy chain constant domain comprises the amino acid sequence.

[0025] In some embodiments, the light chain constant domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:69 or 91.

[0026] In some embodiments, the Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:6 and a light chain comprising the amino acid sequence of SEQ ID NO:14. In some embodiments, the Fab comprises a heavy chain constant 1 domain comprising the amino acid sequence of SEQ ID NO:68 linked to a heavy chain variable domain; and a light chain constant domain comprising the amino acid sequence of SEQ ID NO:69 or 91 linked to a light chain variable domain. In some embodiments, the recombinant fusion protein comprises (a) an antigen-binding fragment that binds to serum albumin, comprising a heavy chain variable domain comprising a heavy chain complementarity determining region (HCDR) comprising the amino acid sequence of SEQ ID NOs: 35, 36, and 37, a light chain variable domain comprising a light chain complementarity determining region (LCDR) comprising the amino acid sequence of SEQ ID NOs: 52, 53, and 54, a heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 68 linked to the heavy chain variable domain, and a light chain constant domain comprising the amino acid sequence of SEQ ID NO: 69 or 91 linked to the light chain variable domain, and (b) hFSH comprising an hFSH alpha subunit and an hFSH beta subunit, wherein the hFSH alpha subunit and the hFSH beta subunit are linked to the terminal regions of the antigen-binding fragment that binds to serum albumin.

[0027] Also disclosed herein are nucleic acid molecules encoding any of the recombinant fusion proteins disclosed herein.

[0028] Further disclosed herein are expression vectors comprising any of the nucleic acid molecules disclosed herein.

[0029] Disclosed herein are cells transformed with any of the expression vectors disclosed herein.

[0030] Disclosed herein are compositions comprising any of the recombinant fusion proteins disclosed herein. Also disclosed herein are pharmaceutical compositions comprising any of the compositions disclosed herein and a pharmaceutically acceptable carrier. Also disclosed are kits comprising any of the compositions disclosed herein and a label containing instructions for use.

[0031] Disclosed herein is a method of inducing superovulation in a subject in need thereof, comprising applying a pharmaceutical composition disclosed herein to in vitro fertilization-embryo transfer (IVF-ET), gamete intrafallopian tube transfer (GIFT), zygote intrafallopian tube transfer (ZIFT), intracytoplasmic sperm injection (ICSI), or in vitro fertilization. Disclosed herein is a method of treating anovulation, hypogonadism, or polycystic ovary syndrome in a subject in need thereof, comprising administering to the subject a pharmaceutical composition disclosed herein.

[0032] Further disclosed herein are long-acting human follicle-stimulating hormone (hFSH) compositions comprising the recombinant fusion protein disclosed herein as an active ingredient. In some embodiments, the long-acting hFSH compositions are applied in in vitro fertilization-embryo transfer (IVF-ET), gamete intrafallopian tube transfer (GIFT), zygote intrafallopian tube transfer (ZIFT), intracytoplasmic sperm injection (ICSI), or in vitro fertilization to induce superovulation in a subject. In some embodiments, the long-acting hFSH compositions are used to treat anovulation, hypogonadism, or polycystic ovary syndrome.

[0033] In some embodiments, the composition is administered to the subject subcutaneously or intramuscularly. [Brief explanation of the drawings]

[0034] These and other aspects, features, and advantages of particular embodiments of the present disclosure will become more apparent from the following description taken in conjunction with the accompanying drawings. [Figure 1] 1A and 1B are diagrams schematically showing the structures of SAFA-FSH proteins, with FIG. 1A showing the structure of SAFA-FSH-A protein and FIG. 1B showing the structure of SAFA-FSH-B. [Figure 2] 2A and 2B are diagrams showing cleavage maps of expression vectors for preparing SAFA-FSH protein, where FIG. 2A shows the expression vector for the heavy chain of the SAFA-FSH protein and FIG. 2B shows the expression vector for the light chain of the SAFA-FSH protein. [Figure 3] 1 shows the size analysis of SAFA-FSH protein by SDS-PAGE at 1.175 μg / well and 2.35 μg / well under reducing conditions (R), non-reducing conditions with heat (NR(B)), and non-reducing conditions without heat (NR(NB)). [Figure 4] 1 shows the results of analyzing the purity of SAFA-FSH protein by SE-HPLC. [Figure 5] 5A and 5B show the results of isoelectric point analysis of SAFA-FSH protein, with FIG. 5A showing the chromatographic peak of SAFA-FSH protein and FIG. 5B showing the band of SAFA-FSH protein. [Figure 6] 1 shows the molecular weights of the light and heavy chains in the SAFA-FSH protein as determined by capillary electrophoresis. [Figure 7] 1 shows the molecular weights of the light and heavy chains in the SAFA-FSH protein by intact mass analysis. [Figure 8] 1 shows the results of N-terminal sequencing of the light and heavy chains in the SAFA-FSH protein. [Figure 9]9A and 9B show the binding strength between SAFA-FSH protein and human serum albumin, with FIG. 9A showing the binding strength under pH 7.4 conditions and FIG. 9B showing the binding strength under pH 6.0 conditions. [Figure 10] 1 shows the expression of human follicle-stimulating hormone receptor on the surface of a human FSHR-stabilized cell line. [Figure 11] 1 shows the binding between the SAFA-FSH protein and the human follicle-stimulating hormone receptor. [Figure 12] Figures 12A and 12B show the level of follicle-stimulating hormone's cAMP-regulating ability upon addition of SAFA-FSH protein, with Figure 12A showing the results obtained by adding the protein at a concentration of 10 ng / ml and Figure 12B showing the results obtained by adding the protein at a concentration of 303 pM. [Figure 13] 1 shows the change in blood concentration after intravenous administration of Gonal-F protein at a concentration of 88 μg / kg using an animal model. [Figure 14] 1 shows changes in blood concentration after intravenous administration of SAFA-FSH protein at a concentration of 200 μg / kg using an animal model. [Figure 15] 1 shows changes in blood concentration after intravenous administration of SAFA-FSH protein at a concentration of 600 μg / kg using an animal model. [Figure 16] Figures 16A and 16B show the ovarian growth-promoting effect using an animal model after administration of SAFA-FSH protein, where Figure 16A shows the results of a quantitative comparison of changes in ovarian mass over time, and Figure 16B shows the results of visual confirmation of ovarian changes with the naked eye. [Figure 17] Figures 17A-17C show the efficacy of SAFA-FSH protein using an animal model of hypogonadism, with Figure 17A comparing the mean serum testosterone levels from weeks 1 to 4 from the start of the experiment, Figure 17B comparing the mean serum testosterone levels from weeks 5 to 8 from the start of the experiment, and Figure 17C comparing the mean serum testosterone levels from weeks 9 to 12 from the start of the experiment. [Figure 18]Figures 18A to 18D show the effectiveness of SAFA-FSH protein using an animal model 9 weeks after the start of the experiment, with Figure 18A showing the results comparing testis weight, Figure 18B showing the results comparing testis index values ​​(i.e., testis / body weight ratio), Figure 18C showing the results comparing total sperm count, and Figure 18D showing the results comparing sperm count recovery levels. DETAILED DESCRIPTION OF THE INVENTION

[0035] Detailed Description As used herein, the term "recombinant fusion protein" refers to a protein in which two or more proteins are artificially linked. In some embodiments, this term refers to a protein in which the alpha (α) and beta (β) subunits of FSH are linked to an antigen-binding fragment of serum albumin, i.e., an anti-serum albumin Fab antibody fragment. In some embodiments, a recombinant fusion protein can be obtained by expressing a fusion gene (expression vector) in a cell expression system in which a gene sequence encoding FSH is linked to a gene sequence encoding an antigen-binding fragment of anti-serum albumin. In such a recombinant fusion protein, FSH and the anti-serum albumin Fab antibody fragment can be linked to each other directly or via a connector such as a linker. In some embodiments, the recombinant fusion protein can be one in which FSH, a linker, a heavy chain comprising the heavy chain region of the antigen-binding fragment of serum albumin, and a light chain comprising the light chain region of the antigen-binding fragment of serum albumin are linked via a non-covalent bond.

[0036] As used herein, the term "serum albumin" refers to a protein that constitutes the basic substance of cells and plays an important role in maintaining the osmotic pressure between blood vessels and tissues by keeping bodily fluids within the blood vessels. In addition, the term "antigen-binding fragment against serum albumin" can refer to an anti-serum albumin antibody that specifically binds to an epitope of serum albumin or an antigen-binding fragment of the antibody molecule.

[0037] As used herein, the term "antigen-binding fragment that binds to serum albumin" refers to a functional unit that, as a fusion partner of hFSH, the active component of a recombinant fusion protein, can contribute to significantly improving the pharmacokinetic properties of the recombinant fusion protein while maintaining the inherent biological activity or therapeutic effect of hFSH. The antigen-binding fragment can be, for example, a human anti-serum albumin (SA) Fab, the details of which are as disclosed in Korean Patent Registration No. 10-1576561 and U.S. Patent Nos. 9,879,077 and 11,773,176, each of which is incorporated herein by reference in its entirety.

[0038] The term "hFSH" is a glycoprotein hormone produced by the pituitary gland and secreted into the endocrine system. It has a dimeric structure containing α and β subunits. This term may be used interchangeably with the term "human ovarian stimulating hormone." hFSH and its α and β subunits can be obtained from previously published databases, such as https: / / www.ncbi.nlm.nih.gov / , and may optionally include variants of amino acid / nucleotide sequences with 80% or greater sequence identity / homology to the aforementioned amino acid / nucleotide sequences. Meanwhile, in women, FSH can stimulate follicles in the ovaries during the follicular phase of the menstrual cycle, while in men, FSH helps stimulate sperm production in the testes. Therefore, hFSH is widely used as a sex hormone to improve or treat infertility caused by reproductive cycle or reproductive organ dysfunction. However, despite its therapeutic effects, low production efficiency and short administration intervals due to its short in vivo half-life pose a burden to patients seeking infertility treatment.

[0039] Under these technical circumstances, we have made great efforts to develop a long-acting hFSH formulation, and as a result, we have prepared a recombinant fusion protein in which the antigen-binding fragment SL335, which binds to serum albumin, is fused to hFSH having a dimeric structure containing FSH α subunit and FSH β subunit. The recombinant fusion protein has been confirmed to have improved pharmacokinetic properties and a therapeutic effect on promoting ovarian growth, thereby completing the present disclosure.

[0040] Disclosed herein is a recombinant fusion protein comprising (a) human follicle-stimulating hormone (hFSH) comprising an hFSH α subunit and an hFSH β subunit, and (b) an antigen-binding fragment (Fab) that binds to serum albumin. In some embodiments, the hFSH α subunit and the hFSH β subunit are linked to the terminal region of the Fab. In some embodiments, the hFSH α subunit and the hFSH β subunit are independently linked to the end of the heavy chain constant 1 domain and the end of the light chain constant domain of the Fab. In some embodiments, the hFSH α subunit and the hFSH β subunit are independently linked to the end of the heavy chain constant 1 domain and the end of the light chain constant domain of the Fab by a linker. In other embodiments, the hFSH α subunit is linked to the end of the light chain constant domain, and the hFSH β subunit is linked to the end of the heavy chain constant 1 domain of the Fab.

[0041] Fab is (a) a heavy chain complementarity-determining domain 1 (CDR1) comprising the amino acid sequence of SYGIS (SEQ ID NO:22); a heavy chain complementarity-determining domain 2 (CDR2) comprising the amino acid sequence of TIFF2025533090000022.tif4128; and heavy chain complementarity-determining domain 3 (CDR3) containing the amino acid sequence of TIFF2025533090000023.tif4128; (b) a heavy chain CDR1 comprising the amino acid sequence of SYGIS (SEQ ID NO:22); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025533090000024.tif4128; and Heavy chain CDR3 containing the amino acid sequence of TIFF2025533090000025.tif4128; (c) a heavy chain CDR1 comprising the amino acid sequence of NYGIH (SEQ ID NO:26); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025533090000026.tif4128; and Heavy chain CDR3 containing the amino acid sequence of TIFF2025533090000027.tif4128; (d) a heavy chain CDR1 comprising the amino acid sequence of SYAMS (SEQ ID NO:29); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025533090000028.tif4128; and heavy chain CDR3 containing the amino acid sequence of TIFF2025533090000029.tif4128; (e) a heavy chain CDR1 comprising the amino acid sequence of AYWIA (SEQ ID NO:32); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025533090000030.tif4128; and a heavy chain CDR3 comprising the amino acid sequence of LYSGSYSP (SEQ ID NO:34); or (f) a heavy chain CDR1 comprising the amino acid sequence of AYSMN (SEQ ID NO:35); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025533090000031.tif4128; and Heavy chain CDR3 containing the amino acid sequence of TIFF2025533090000032.tif4128 a heavy chain comprising a heavy chain variable domain comprising (g) light chain CDR1 containing the amino acid sequence of TIFF2025533090000033.tif4128; a light chain CDR2 comprising the amino acid sequence of GASRLES (SEQ ID NO:39); and a light chain CDR3 comprising the amino acid sequence of QQSDSVPVT (SEQ ID NO:40); (h) light chain CDR1 containing the amino acid sequence of TIFF2025533090000034.tif4128; a light chain CDR2 comprising the amino acid sequence of AASSLQS (SEQ ID NO:42), and Light chain CDR3 containing the amino acid sequence of TIFF2025533090000035.tif4128; (i) light chain CDR1 containing the amino acid sequence of TIFF2025533090000036.tif4128; a light chain CDR2 comprising the amino acid sequence of DASNRAT (SEQ ID NO:45); and Light chain CDR3 containing the amino acid sequence of TIFF2025533090000037.tif4128; (j) light chain CDR1 containing the amino acid sequence of TIFF2025533090000038.tif4128; a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:48); and a light chain CDR3 comprising the amino acid sequence of QQYGSSPRT (SEQ ID NO:49); (k) light chain CDR1 containing the amino acid sequence of TIFF2025533090000039.tif4128; a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:48); and a light chain CDR3 comprising the amino acid sequence of QKYSSYPLT (SEQ ID NO:51); or (l) light chain CDR1 containing the amino acid sequence of TIFF2025533090000040.tif4128; a light chain CDR2 comprising the amino acid sequence of GASTGAT (SEQ ID NO:53), and Light chain CDR3 containing the amino acid sequence of TIFF2025533090000041.tif4128 a light chain comprising a light chain variable domain comprising may include:

[0042] In some embodiments, the heavy chain variable domain comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:35, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:36, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:37, and the light chain variable domain comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:52, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:53, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:54.

[0043] In some embodiments, the heavy chain variable domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:55, 56, 57, 58, 59, or 60. In some embodiments, the light chain variable domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:61, 62, 63, 64, 65, 66, or 67. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:55, 56, 57, 58, 59, or 60, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:61, 62, 63, 64, 65, 66, or 67. In some embodiments, the heavy chain constant domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:68. In some embodiments, the light chain constant domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:69 or 91.

[0044] In some embodiments, the heavy chain of the Fab comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 13 and the amino acid sequence of SEQ ID NO: 6.

[0045] In some embodiments of the recombinant proteins disclosed herein, the heavy chain variable domain comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:35, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:36, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:37, and the light chain variable domain comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:52, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:53, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:54.

[0046] In some embodiments, the heavy chain variable domain comprises an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:55, 56, 57, 58, 59, or 60.

[0047] In some embodiments, the light chain variable domain comprises an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:61, 62, 63, 64, 65, 66, or 67.

[0048] In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:55, 56, 57, 58, 59, or 60, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:61, 62, 63, 64, 65, 66, or 67.

[0049] In some embodiments, the Fab comprises a heavy chain variable domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:55, 56, 57, 58, 59, or 60, and a light chain variable domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:61, 62, 63, 64, 65, or 66 or 67, respectively, or any combination of heavy chain and light chain variable domains disclosed herein. For example, a Fab may comprise a heavy chain variable domain comprising an amino acid sequence at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:60, and a light chain variable domain comprising an amino acid sequence at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:67.

[0050] In some embodiments, the heavy chain constant domain comprises an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:68.

[0051] In some embodiments, the light chain constant domain comprises an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:69 or 91.

[0052] In some embodiments, the recombinant fusion protein may comprise a heavy chain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 19. In some embodiments, the Fab comprises a heavy chain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 19. H -C H1 In some embodiments, the Fab comprises a heavy chain domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:13 (V domain). L -Cκ domain).

[0053] In some embodiments, the recombinant fusion protein may comprise a heavy chain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 19; and a light chain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 13. The recombinant protein may have significantly improved pharmacokinetic properties while maintaining the inherent biological activity of FSH.

[0054] In some embodiments, the Fab is selected from the group consisting of SEQ ID NO:6 (V H -C H1 a heavy chain domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:13 (V domain),L -Cκ domain).

[0055] In some embodiments, the heavy chain variable domain (VH) may comprise a heavy chain complementarity determining region (HCDR) comprising the amino acid sequence of SEQ ID NO:35, 36, or 37. For example, the heavy chain variable domain (VH) may comprise the amino acid sequence of SEQ ID NO:60, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or more sequence identity to the amino acid sequence of SEQ ID NO:60. The C-terminus of the heavy chain variable domain (VH) may be linked to the heavy chain constant 1 domain (CH1). Here, the heavy chain constant 1 domain (CH1) may comprise the amino acid sequence of SEQ ID NO:68, or a sequence having at least 80%, 85%, 90%, 95%, 99% or more sequence identity to the amino acid sequence of SEQ ID NO:68, although the embodiment is not particularly limited thereto.

[0056] In some embodiments, the light chain variable domain (VL) may comprise a light chain complementarity determining region (LCDR) comprising the amino acid sequence of SEQ ID NO:52, 53, and 54. For example, the light chain variable domain (VL) may comprise the amino acid sequence of SEQ ID NO:67, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or more sequence identity to the amino acid sequence of SEQ ID NO:67. The C-terminus of the light chain variable domain (VL) may be linked to a light chain constant domain (CL), wherein the light chain constant domain (CL) may comprise the amino acid sequence of SEQ ID NO:69 or 91, for example, a sequence having at least 80%, 85%, 90%, and 95% or more sequence identity to the amino acid sequence of SEQ ID NO:69 or 91, although the embodiment is not particularly limited thereto.

[0057] As used herein, the term "identity" refers to the overall relatedness between polymer molecules, for example, between nucleic acids (e.g., DNA molecules and / or RNA molecules) and / or between polypeptides. For example, a polypeptide is considered "substantially identical" if its amino acid sequence has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity. The percent identity of two nucleic acid or polypeptide sequences can be calculated, for example, by aligning the two sequences for optimal comparison (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences can be ignored for comparison). For example, the length of the sequence aligned for comparison is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleic acid or polypeptide sequences at corresponding positions are then compared. The determination of percent identity between two sequences and the comparison of these sequences can be achieved using mathematical algorithms. As is well known to those skilled in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms available in commercial computer programs, including, in particular, BLASTN for nucleotide sequences, and BLASTP, gapped BLAST, and PSIBLAS for amino acid sequences.

[0058] In some embodiments, the hFSH α subunit and the hFSH β subunit can be linked to the terminal region of the antigen-binding fragment that binds to serum albumin. For example, the hFSH α subunit and the hFSH β subunit can be linked to the C-terminus or N-terminus of the antigen-binding fragment that binds to serum albumin. The hFSH α subunit and the hFSH β subunit can be independently linked to the end of the constant 1 domain of the heavy chain (CH1) and the end of the constant domain of the light chain (CL), and more specifically, the N-terminus of the hFSH α subunit and the hFSH β subunit can be independently linked to the C-terminus of the constant 1 domain of the heavy chain (CH1) and the C-terminus of the constant domain of the light chain (LC). More specifically, (1) the hFSH α subunit can be linked to the end, e.g., the C-terminus, of the constant domain (CL) of the light chain, and the hFSH β subunit can be linked to the end, e.g., the C-terminus, of the constant 1 domain (CH1) of the heavy chain, or (2) the hFSH α subunit can be linked to the end, e.g., the C-terminus, of the constant 1 domain (CH1) of the heavy chain, and the hFSH β subunit can be linked to the end, e.g., the C-terminus, of the constant domain (CL) of the light chain.

[0059] In some embodiments, FSHα comprises an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1. FSHα can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 1. In some embodiments, FSHα comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encoding FSHα comprises the nucleotide sequence of SEQ ID NO: 2 or 3.

[0060] In some embodiments, FSHβ comprises an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:4. FSHβ may comprise an amino acid sequence having at least 90% identity to SEQ ID NO:4. In some embodiments, FSHβ comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, the nucleic acid molecule encoding FSHβ comprises the nucleotide sequence of SEQ ID NO:5 or 9.

[0061] In some embodiments, in the fusion proteins disclosed herein, the heavy and light chains of the Fab are non-covalently linked.

[0062] In some embodiments, the antigen-binding fragment that binds to serum albumin can be linked to the aforementioned hFSH, for example, hFSH α and β subunits, via a linker. For example, the linker can link the hFSH subunit to any one region selected from the C-terminus of the constant 1 domain of the heavy chain (CH1), the N-terminus of the variable domain of the heavy chain (VH), the C-terminus of the constant domain of the light chain (CL), and the N-terminus of the variable domain of the light chain (VL). Furthermore, the linker can be modified for use as needed.

[0063] For example, the linker can be a polypeptide containing 5 to 400 amino acids, 5 to 200 amino acids, or 5 to 200 amino acids. Such peptide linkers can contain Gly, Asn, and Ser residues, and can also contain neutral amino acids such as Thr and Ala. Amino acid sequences suitable for peptide linkers are known in the art. Furthermore, the copy number "n" can be adjusted to optimize the linker to achieve appropriate separation between functional moieties or to maintain necessary interactions between moieties. Other linkers are known in the art, such as a GS linker, which not only adds polar amino acid residues to improve water solubility but also adds amino acid residues such as T and A to maintain flexibility.

[0064] Thus, the linker can be a flexible linker containing G, S, and / or T, A residues. The linker can be (GpSs) n or (SpGs) n where, independently, p is an integer from 1 to 10, s is 0 or an integer from 0 to 10, p + s is an integer less than or equal to 20, and n is an integer from 1 to 20. More specifically, examples of linkers include: TIFF2025533090000042.tif41166, where n can be an integer from 1 to 20, or from 1 to 10.

[0065] Furthermore, the linker may comprise the amino acid sequence of SEQ ID NO: 85 or 88, although the embodiment is not particularly limited thereto.

[0066] In some embodiments, the linker comprises 1 to 50 amino acids. In some embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOs:16, 70-85, and 88. In some embodiments, the recombinant fusion protein may comprise (i) a heavy chain comprising the heavy chain fragment SL335 of SEQ ID NO:6, the linker of SEQ ID NO:88, and the FSH alpha subunit of SEQ ID NO:1; and a light chain comprising the light chain fragment SL335 of SEQ ID NO:91, the linker of SEQ ID NO:85, and the FSH beta subunit of SEQ ID NO:4, or (ii) a heavy chain comprising the heavy chain fragment SL335 of SEQ ID NO:6, the linker of SEQ ID NO:85, and the FSH beta subunit of SEQ ID NO:4; and a light chain comprising the light chain fragment SL335 of SEQ ID NO:91, the linker of SEQ ID NO:88, and the FSH alpha subunit of SEQ ID NO:1. The recombinant fusion protein can include, for example, the amino acid sequence of SEQ ID NOs: 19 and 21, or the amino acid sequence of SEQ ID NOs: 95 and 97. The recombinant fusion protein can have significantly improved pharmacokinetic properties while maintaining the inherent biological activity of hFSH.

[0067] In some embodiments, recombinant fusion proteins (SAFA-FSH A and SAFA-FSH B) are prepared, comprising: an antigen-binding fragment that binds to serum albumin; and an hFSH subunit fused to the C-terminus of the constant 1 domain of the heavy chain (CH1) and the constant domain of the light chain (CL), respectively. The recombinant fusion proteins retain the biological activity of each factor, specifically, their ability to bind to human serum albumin and the FSH receptor, respectively, and therefore may have extended in vivo half-lives and improved therapeutic effects, such as ovarian growth promotion.

[0068] In some embodiments, the heavy chain variable domain comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 35, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and The light chain variable domain comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:52, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:53, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:54.

[0069] In some embodiments, the heavy chain variable domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:55, 56, 57, 58, 59, or 60. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:60.

[0070] In some embodiments, the light chain variable domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:61, 62, 63, 64, 65, 66, or 67. In some embodiments, the light chain variable domain comprises the amino acid sequence of SEQ ID NO:67.

[0071] In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:55, 56, 57, 58, 59, or 60, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:61, 62, 63, 64, 65, 66, or 67. In some embodiments, the heavy chain constant domain comprises the amino acid sequence.

[0072] In some embodiments, the light chain constant domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:69 or 91.

[0073] In some embodiments, the Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:6 and a light chain comprising the amino acid sequence of SEQ ID NO:13. In some embodiments, the Fab comprises a heavy chain constant 1 domain comprising the amino acid sequence of SEQ ID NO:68 linked to a heavy chain variable domain; and a light chain constant domain comprising the amino acid sequence of SEQ ID NO:69 or 91 linked to a light chain variable domain.

[0074] Antigen-binding fragments of antibodies, or antibody fragments, refer to fragments that retain antigen-binding function and include Fab, F(ab'), F(ab')2, Fv, etc. The Fab antibody fragment has a structure containing light and heavy chain variable regions, a light chain constant region, and a heavy chain constant region (CH) that has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH domain. F(ab')2 antibodies are generated when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds. Recombinant techniques for generating Fv fragments, the minimum antibody fragment containing only the heavy and light chain variable regions, are described in PCT International Publication Nos. WO88 / 10649, WO88 / 106630, WO88 / 07085, WO88 / 07086, and WO88 / 09344. In two-chain Fvs, the heavy chain variable region and the light chain variable region are linked via a non-covalent bond. In single-chain Fvs (scFvs), the heavy chain variable region and the light chain variable region are generally linked at the C-terminus directly or via a covalent peptide linker. Therefore, single-chain Fvs (scFvs) can have a dimer-like structure, similar to two-chain Fvs. Such antibody fragments can be obtained using protease hydrolases (for example, Fab fragments can be obtained by cleaving whole antibodies with papain, and F(ab')2 fragments can be obtained by cleaving whole antibodies with pepsin), and they can also be produced by recombinant gene technology.

[0075] In some embodiments, an antigen-binding fragment against serum albumin may comprise a heavy chain region comprising the amino acid sequence of SEQ ID NO:6; and a light chain region comprising the amino acid sequence of SEQ ID NO:13. In some embodiments, a nucleic acid molecule encoding a heavy chain region comprising the amino acid sequence of SEQ ID NO:6 may have the nucleotide sequence of SEQ ID NO:11 or 12. In some embodiments, a nucleic acid molecule encoding a light chain region comprising the amino acid sequence of SEQ ID NO:13 may have the nucleotide sequence of SEQ ID NO:14 or 15.

[0076] As used herein, the term "antibody" (singular or plural) is a term of the art and can be used interchangeably herein to refer to a molecule having an antigen-binding site that specifically binds to an antigen. Antibodies include, for example, monoclonal antibodies, recombinantly produced antibodies, human antibodies, resurfaced antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFvs), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), bispecific antibodies, and multispecific antibodies.

[0077] An antibody can be any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG 2a or IgG 2b ) can be.

[0078] As used herein, the terms "bioeffector moiety," "antigen-binding domain," "antigen-binding region," "antigen-binding site," and similar terms refer to the portion of a recombinant protein (e.g., the complementarity-determining region (CDR)) that contains the amino acid residues that confer the recombinant protein its specificity for an antigen. The antigen-binding region can be derived from any animal species, including cats, rodents (e.g., mice, rats, or hamsters), and humans.

[0079] As used herein, the terms "variable region" and "variable domain" are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a portion of either the light or heavy chain, typically the amino-terminal 110-120 amino acids in the mature heavy chain and approximately 90-115 amino acids in the mature light chain, which vary extensively in sequence among antibodies and are used to determine the binding and specificity of a particular antibody for its specific antigen. Sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while more highly conserved regions within the variable domain are called framework regions (FRs). While not wishing to be bound by a particular mechanism or theory, the CDRs of the light and heavy chains are believed to be primarily responsible for the interaction and specificity of the antibody with the antigen. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region comprises rodent or mouse CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In some embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FR).

[0080] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody. The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0081] As used herein, the term "heavy chain (HC or CH)" refers to both a full-length heavy chain and a fragment thereof, the full-length heavy chain comprising a variable region domain VH containing an amino acid sequence with sufficient variable region (VR) sequence to confer specificity for an antigen, and three constant region domains CH1, CH2, and CH3. As used herein, the term "light chain (LC or CL)" refers to both a full-length light chain and a fragment thereof, the full-length light chain comprising a variable region domain VL containing an amino acid sequence with sufficient VR sequence to confer specificity for an antigen, and a constant region domain CL.

[0082] The heavy and light chain constant domains can be derived from IgG1 antibody constant domains, and in any one or more thereof, the cysteine, an amino acid used in the disulfide bond between the light and heavy chain domains, can be conserved, deleted, or substituted with an amino acid residue other than cysteine. For example, the heavy chain constant domain can comprise an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:68, and the light chain constant domain can comprise an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:69 or 91. Deletion or substitution of cysteines within the domains can contribute to improved expression levels of recombinant proteins in transformed cells during the process of producing the above-mentioned recombinant proteins. In some embodiments, (i) one or more cysteines in the heavy chain constant domain and / or (ii) one or more cysteines in the light chain constant domain located at the interchain disulfide bond between the light chain and the heavy chain are conserved, deleted, and / or substituted with an amino acid residue other than cysteine.

[0083] As used herein, the terms "constant region" and "constant domain" are interchangeable and have the meanings generally used in the art. The constant region is an antibody portion, for example, the carboxyl-terminal portion of the light chain and / or heavy chain, which is not directly involved in binding the antibody to an antigen, but can exhibit various effector functions, such as interacting with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence than the immunoglobulin variable domain.

[0084] As used herein, the term "heavy chain" when used in reference to an antibody can refer to any distinct type, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain, which give rise to antibodies of the IgA, IgD, IgE, IgG, and IgM classes, respectively, and include subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0085] As used herein, the term "light chain" when used in reference to an antibody can refer to any distinct type, for example, kappa (Cκ) or lambda (Cλ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In a specific embodiment, the light chain is a human light chain.

[0086] "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D Affinity can be expressed as the equilibrium dissociation constant (K D ), and the equilibrium association constant (K A ) can be measured and / or expressed in a number of ways known in the art, including, but not limited to, K D is k off / k on is calculated from the quotient of A is k on / k off It is calculated from the quotient of k on refers to the association rate constant of, for example, an antibody to an antigen, and k off refers to, for example, the dissociation of an antibody against an antigen. on and k offcan be determined by techniques known to those skilled in the art, such as BIAcore® or KinExA.

[0087] In some embodiments, the binding affinity of the recombinant fusion proteins disclosed herein is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold higher than that of human FSH, or any range therein, e.g., 2-fold to 10-fold higher.

[0088] As used herein, "epitope" is a term used in the art and refers to the localized region of an antigen that an antibody can specifically bind to.Epitope can be, for example, a continuous amino acid sequence of a polypeptide (linear or continuous epitope), or epitope can be, for example, a sequence of two or more non-contiguous regions of a polypeptide (single or multiple polypeptides) (stereoscopic, non-linear, discontinuous, or non-contiguous epitope).In some embodiments, the epitope that an antibody binds to can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA assay, hydrogen / deuterium exchange combined with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assay, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). In the case of X-ray crystallography, crystallization can be achieved using any method known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson, A. (1976) J. Biol. Chem. 251:6300-6303).Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques and refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds. Wyckoff HW et al.,; US 2004 / 0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49 (Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed. Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt 10):1316-1323). Mutagenesis mapping studies can be accomplished using any method known to those skilled in the art. For a description of mutagenesis techniques, including alanine scanning mutagenesis techniques, see, for example, Champe M et al., (1995) J Biol Chem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244:1081-1085. In some embodiments, the epitope of the antibody is determined using alanine scanning mutagenesis studies.

[0089] As used herein, the terms "immunospecifically bind," "immunospecifically recognize," "specifically bind," and "specifically recognize" are similar terms in the context of antibodies, and such binding refers to a molecule that binds to an antigen (e.g., an epitope, immune complex, or binding partner of an antigen-binding site) as understood by one of skill in the art. For example, a molecule that specifically binds to an antigen can generally bind to other peptides or polypeptides with lower affinity, as determined by, for example, immunoassays, BIAcore®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In some embodiments, a molecule that immunospecifically binds to an antigen has a K A At least 2 logs, 2.5 logs, 3 logs, 4 logs or more greater than K A It binds to the antigen.

[0090] In some embodiments, molecules that immunospecifically bind to an antigen do not cross-react with other proteins under similar binding conditions. In some embodiments, molecules that immunospecifically bind to an antigen do not cross-react with other proteins. In some embodiments, provided herein are recombinant proteins that bind to a specified antigen with higher affinity than to another unrelated antigen. In certain embodiments, provided herein are recombinant proteins that bind to a specified antigen (e.g., human serum albumin) with 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more affinity than to another unrelated antigen, as measured, for example, by radioimmunoassay, surface plasmon resonance, or equilibrium binding exclusion. In some embodiments, the degree of binding of a recombinant protein described herein to an unrelated protein is less than 10%, 15%, or 20% of the binding of an antibody to the specified antigen, as measured, for example, by radioimmunoassay.

[0091] In some embodiments, provided herein are recombinant proteins that bind to antigens of various species, such as cats, rodents (e.g., mice, rats, or hamsters), and humans. In some embodiments, provided herein are recombinant proteins that bind to human antigens with higher affinity than to antigens of other species. In certain embodiments, provided herein are recombinant proteins that bind to human antigens with 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more higher affinity than to other species, as measured, for example, by radioimmunoassay, surface plasmon resonance, or equilibrium binding exclusion. In some embodiments, the recombinant proteins described herein that bind to human antigens will bind to antigen proteins of other species with less than 10%, 15%, or 20% of the binding of an antibody to human antigen proteins, as measured, for example, by radioimmunoassay, surface plasmon resonance, or equilibrium binding exclusion.

[0092] As used herein, the term "host cell" can be any type of cell, for example, a primary cell, a cell in culture, or a cell derived from a cell line. In some embodiments, the term "host cell" refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to, for example, possible mutations or environmental influences that may occur later, or the integration of the nucleic acid molecule into the host cell genome.

[0093] In some aspects, the recombinant proteins described herein can be described by their VL domain alone, their VH domain alone, their three VL CDRs alone, or their three VH CDRs alone. See, for example, Rader C et al., (1998) PNAS 95: 8910-8915, which is incorporated herein by reference in its entirety, describing the humanization of a murine anti-αvβ3 antibody by identifying complementary light or heavy chains from a human light or heavy chain library, respectively, to produce humanized antibody variants with similar or higher affinity than that of the original antibody. See also Clackson T et al., (1991) Nature 352:624-628, which is incorporated herein by reference in its entirety, describing a method for generating antibodies that bind to a specific antigen by using a specific VL domain (or VH domain) to screen a library for complementary variable domains. The screen yielded 14 new partners for the specific VH domain and 13 new partners for the specific VL domain that were strong binders, as determined by ELISA. See also Kim SJ & Hong HJ, (2007) J Microbiol 45:572-577, which is incorporated herein by reference in its entirety, describing a method for generating antibodies that bind to a specific antigen by using a specific VH domain to screen a library (e.g., a human VL library) for complementary VL domains; the selected VL domains could then be used to guide the selection of additional complementary (e.g., human) VH domains.

[0094] In one aspect, provided herein is a recombinant protein that specifically binds to serum albumin (e.g., human serum albumin) and comprises a Chothia VL CDR in the VL. In one aspect, provided herein is an antibody that specifically binds to serum albumin (e.g., human serum albumin) and comprises a Chothia VH CDR in the VH. In one aspect, provided herein is an antibody that specifically binds to serum albumin (e.g., human serum albumin) and comprises a Chothia VL CDR in the VL and a Chothia VH CDR in the VH. In one embodiment, an antibody that specifically binds to serum albumin (e.g., human serum albumin) comprises one or more CDRs, wherein the Chothia and Kabat CDRs have the same amino acid sequence. In one embodiment, provided herein is an antibody that specifically binds to serum albumin and comprises a combination of Kabat and Chothia CDRs.

[0095] In certain aspects, the CDRs of an antibody can be determined according to the AbM numbering scheme, which refers to the AbM hypervariable regions, which represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.).

[0096] In some embodiments, the position of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of an antibody described herein may be varied by 1, 2, 3, 4, 5, or 6 amino acid positions, so long as immunospecific binding to an antigen is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). For example, the positions defining the CDRs of an antibody described herein may be varied by shifting the N-terminal and / or C-terminal boundaries of the CDRs by 1, 2, 3, 4, 5, or 6 amino acids relative to the CDR positions of an antibody described herein, so long as immunospecific binding to an antigen(s) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the length of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of an antibody described herein may be varied (e.g., made shorter or longer) by 1, 2, 3, 4, 5, or more amino acids, so long as immunospecific binding to the antigen(s) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).

[0097] In some embodiments, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein can be 1, 2, 3, 4, 5 or more amino acids shorter than one or more of the CDRs described herein, so long as immunospecific binding to an antigen(s) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein can be 1, 2, 3, 4, 5 or more amino acids longer than one or more of the CDRs described herein, so long as immunospecific binding to an antigen(s) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the amino terminus of a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein may be extended by 1, 2, 3, 4, 5 or more amino acids compared to one or more of the CDRs described herein, so long as immunospecific binding to an antigen(s) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the carboxy terminus of a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein may be extended by 1, 2, 3, 4, 5 or more amino acids compared to one or more of the CDRs described herein, so long as immunospecific binding to an antigen(s) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).In other embodiments, the amino terminus of a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein may be shortened by 1, 2, 3, 4, 5 or more amino acids compared to one or more of the CDRs described herein, so long as immunospecific binding to an antigen(s) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In some embodiments, the carboxy terminus of a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein can be shortened by 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs described herein, so long as immunospecific binding to an antigen(s) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). Any method known in the art, for example, the binding assays and conditions described in the "Examples" section herein, can be used to determine whether immunospecific binding to an antigen(s) is maintained.

[0098] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0099] The recombinant proteins disclosed herein can be fused or conjugated (e.g., covalently or non-covalently linked) to a detectable label or substance. Examples of detectable labels or substances include enzyme labels, such as glucose oxidase; iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121 In), and technetium (99 These include radioisotopes such as Tc; luminescent labels such as luminol; and fluorescent labels such as fluorescein and rhodamine, as well as biotin. Such labeled antibodies can be used to detect antigen proteins.

[0100] Polynucleotides, vectors, and cells According to some aspects of the present disclosure, there are provided nucleic acids encoding the recombinant fusion proteins, expression vectors comprising the nucleic acids, and cells transformed with the expression vectors.

[0101] The nucleic acids, expression vectors, and transformed cells either directly contain the recombinant fusion proteins or nucleic acids encoding the recombinant fusion proteins, or use these, and therefore a description of the common features among them will be omitted.

[0102] For example, recombinant fusion proteins can be produced by isolating nucleic acids encoding the recombinant fusion proteins. After isolating the nucleic acids, the resulting nucleic acids can be inserted into replicable vectors for further cloning (i.e., DNA amplification) or further expression. In this regard, some aspects provide vectors containing nucleic acids.

[0103] As used herein, the term "nucleic acid" comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and the term "nucleotide," which is the basic structural unit in nucleic acids, includes not only natural nucleotides but also analogs in which the sugar or base moiety is modified.

[0104] Nucleic acid is also understood to include a nucleotide sequence that shows substantial identity to the nucleotide sequence. Substantial identity refers to a nucleotide sequence that shows at least 80% homology, more preferably at least 90% homology, and most preferably at least 95% homology when the nucleotide sequence of the present disclosure and another sequence are aligned to correspond as closely as possible and the aligned sequences are analyzed using an algorithm commonly used in the art.

[0105] DNA encoding a recombinant fusion protein can be readily isolated or synthesized using conventional procedures (e.g., by using an oligonucleotide probe capable of specifically binding to DNA encoding the recombinant fusion protein). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0106] As used herein, the term "vector" refers to a plasmid vector as a means for expressing a target gene in a host cell, examples of which include plasmid vectors; cosmid vectors; viral vectors, such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors. In the vector, the nucleic acid encoding the recombinant fusion protein can be operably linked to a promoter.

[0107] As used herein, the phrase "operably linked" refers to a functional linkage between a control sequence for nucleic acid expression (e.g., a promoter, a signal sequence, or an array of binding sites for transcriptional regulators) and another nucleic acid sequence, whereby the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0108] In some embodiments, when prokaryotic cells are used as hosts, a strong promoter capable of initiating transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for translation initiation, and a transcription / translation termination sequence are generally included. In one or more embodiments, for example, when eukaryotic cells are used as hosts, promoters derived from the genomes of mammalian cells (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) can be used, and these promoters generally have polyadenylation sequences as transcription termination sequences. In one or more embodiments, the vector can be fused to other sequences to facilitate purification of recombinant fusion proteins expressed from the vector. Examples of fused sequences include glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), 6xHis (hexahistidine; Qiagen, USA), etc. The vector may contain, as a selectable marker, an antibiotic resistance gene commonly used in the art, such as genes resistant to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0109] Some aspects of the present disclosure provide cells transformed with the aforementioned vectors. Cells used to produce the recombinant fusion proteins of the present disclosure can be prokaryotic cells, yeast cells, or higher eukaryotic cells, but the embodiments are not particularly limited thereto. Prokaryotic host cells include Escherichia coli, Bacillus strains such as B. subtilis and B. thuringiensis, Streptomyces, Pseudomonas (e.g., P. putida), Proteus mirabilis, and Staphylococcus (e.g., S. carnosus). However, animal cells are of greatest interest, and examples of useful host cell lines include, but are not limited to, COS-7, BHK, CHO (GS null CHO-K1), CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, WI-38, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0110] As used herein, the term "transformation" refers to a molecular biological technique in which a DNA fragment or a plasmid carrying a foreign gene of a different type from that of the original cell enters a cell and combines with the DNA present in the original cell, thereby changing the genetic traits of the original cell. Transformation can also refer to the insertion of an expression vector containing a recombinant fusion protein gene into a host cell.

[0111] Disclosed herein are nucleic acid molecules that encode the recombinant proteins disclosed herein.

[0112] Disclosed herein are expression vectors that include the nucleic acid molecules disclosed herein.

[0113] Disclosed herein are cells transformed with the expression vectors disclosed herein.

[0114] The nucleic acids, expression vectors, and transformed cells either directly contain or use the above-mentioned recombinant proteins or nucleic acids encoding the recombinant proteins, and therefore a description common to them will be omitted.

[0115] For example, in some aspects, recombinant proteins can be produced by isolating nucleic acids encoding the recombinant proteins. The nucleic acids are isolated and inserted into replicable vectors for further cloning (DNA amplification) or further expression. Based on this, other aspects relate to vectors containing the nucleic acids.

[0116] As used herein, the term "nucleic acid" or "nucleic acid molecule" comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides as the basic units of nucleic acids include not only naturally occurring nucleotides but also analogs having modified sugar or base moieties.

[0117] Nucleic acid is understood to include a nucleotide sequence that shows substantial identity to the nucleotide sequence. Substantial identity means a nucleotide sequence that shows at least 80% homology, more specifically at least 90% homology, and most specifically at least 95% homology when the nucleotide sequence of the present disclosure and another optional sequence are aligned to correspond to each other as closely as possible and the aligned sequences are analyzed using an algorithm commonly used in the art.

[0118] DNA encoding a recombinant protein is readily isolated or synthesized using conventional processes (e.g., by using an oligonucleotide probe capable of specifically binding to DNA encoding the recombinant protein). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0119] As used herein, the term "vector" includes, as a means for expressing a target gene in a host cell, a plasmid vector; a cosmid vector; a viral vector, such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. In a vector, a nucleic acid encoding a recombinant protein is operably linked to a promoter.

[0120] "Operably linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., promoter, signal sequence, array of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the control sequence directs the transcription and / or translation of the other nucleic acid sequence.

[0121] When prokaryotic cells are used as hosts, a strong promoter capable of directing transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter), a ribosome binding site for translation initiation, and transcription / translation termination sequences are generally included. For example, when eukaryotic cells are used as hosts, promoters derived from mammalian cell genomes (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or mammalian virus promoters (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) can be used, and polyadenylation sequences are generally used as transcription termination sequences. In some cases, vectors can be fused with additional sequences to facilitate purification of recombinant proteins expressed therefrom. Examples of fused sequences include glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), 6X His (hexahistidine; Quiagen, USA), etc. The vector contains, as a selectable marker, an antibiotic resistance gene commonly used in the art, such as genes resistant to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0122] In yet another aspect, the present disclosure provides a cell transformed with the above-described vector. The cells used to produce the recombinant proteins of the present disclosure can be, but are not limited to, prokaryotic cells, yeast cells, or higher eukaryotic cells. Prokaryotic host cells can be used, for example, Escherichia coli, Bacillus strains such as Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, and Staphylococcus (e.g., Staphylococcus carnosus). However, animal cells are of greatest interest, and examples of useful host cell lines include, but are not limited to, COS-7, BHK, CHO (GS null CHO-K1), CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0123] As used herein, the term "transformation" refers to a molecular biological technique in which a DNA fragment or plasmid carrying a foreign gene of a different type from that of the original cell changes the genetic make-up of a cell by invading the cell and combining with the DNA in the original cell. Transformation refers to the insertion of an expression vector containing a gene for a recombinant protein into a host cell.

[0124] Provided herein are nucleic acid molecules comprising nucleotide sequences encoding the recombinant proteins described herein (e.g., variable light chain regions and / or variable heavy chain regions) that immunospecifically bind to an antigen, as well as vectors, e.g., vectors comprising such polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells). Provided herein are polynucleotides comprising nucleotide sequences encoding any of the antibodies provided herein, as well as vectors comprising such polynucleotide sequences, e.g., expression vectors for their efficient expression in host cells, e.g., mammalian cells.

[0125] As used herein, an "isolated" polynucleotide or nucleic acid molecule is one that is separated from other nucleic acid molecules present in the nucleic acid molecule's natural source (e.g., in a mouse or human). Furthermore, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium if produced by recombinant technology, or substantially free of chemical precursors or other chemicals if chemically synthesized. For example, the term "substantially free" includes preparations of polynucleotides or nucleic acid molecules that have less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (particularly less than about 10%) of other materials, such as cellular material, culture medium, other nucleic acid molecules, chemical precursors, and / or other chemicals. In some embodiments, the nucleic acid molecule(s) encoding the antibodies described herein are isolated or purified.

[0126] Provided herein are polynucleotides comprising nucleotide sequences encoding antibodies that immunospecifically bind to an antigenic polypeptide (e.g., human serum albumin) and that comprise an amino acid sequence described herein, as well as antibodies that compete with such antibodies (e.g., in a dose-dependent manner) for binding to the antigenic polypeptide or that bind to the same epitope as such antibodies.

[0127] Provided herein is a polynucleotide comprising a nucleotide sequence encoding the light chain or heavy chain of the antibody described herein.The polynucleotide can comprise a nucleotide sequence encoding the light chain comprising the VL FR and CDR of the antibody described herein.The polynucleotide can comprise a nucleotide sequence encoding the heavy chain comprising the VH FR and CDR of the antibody described herein.

[0128] Provided herein are polynucleotides comprising nucleotide sequences encoding recombinant proteins comprising three VH chain CDRs (e.g., comprising VL CDR1, VL CDR2, and VL CDR3 of an antibody against human serum albumin described herein) and a Fab comprising three VH chain CDRs (e.g., comprising VH CDR1, VH CDR2, and VH CDR3 of an antibody against human serum albumin described herein).

[0129] Provided herein is a polynucleotide comprising a nucleotide sequence encoding a recombinant protein comprising a VL domain.

[0130] In certain embodiments, the polynucleotide described herein comprises a nucleotide sequence encoding a recombinant protein provided herein comprising a light chain variable region comprising an amino acid sequence described herein (e.g., SEQ ID NO:61, 62, 63, 64, 65, 66, or 67), wherein the antibody immunospecifically binds to serum albumin.

[0131] In certain embodiments, the polynucleotides described herein comprise a nucleotide sequence encoding an antibody provided herein comprising a heavy chain variable region comprising an amino acid sequence described herein (e.g., SEQ ID NO:55, 56, 57, 58, 59, or 60), wherein the antibody immunospecifically binds to serum albumin.

[0132] In specific aspects, provided herein are polynucleotides comprising nucleotide sequences encoding antibodies comprising a light chain and a heavy chain (e.g., separate light and heavy chains). With respect to the light chain, in some embodiments, the polynucleotides provided herein comprise a nucleotide sequence encoding a κ light chain. In other embodiments, the polynucleotides provided herein comprise a nucleotide sequence encoding a λ light chain. In still other embodiments, the polynucleotides provided herein comprise a nucleotide sequence encoding an antibody described herein comprising a human κ light chain or a human λ light chain. In some embodiments, the polynucleotides provided herein comprise a nucleotide sequence encoding an antibody that immunospecifically binds to serum albumin, wherein the antibody comprises a light chain, and wherein the amino acid sequence of the VL domain can comprise the amino acid sequence set forth in SEQ ID NO:61, 62, 63, 64, 65, 66, or 67, and wherein the constant region of the light chain comprises the amino acid sequence of a κ light chain constant region.

[0133] Also provided herein are polynucleotides encoding antibodies or fragments thereof that are optimized, for example, by codon / RNA optimization, substitution with heterologous signal sequences, and elimination of mRNA instability elements. Methods for generating optimized nucleic acids encoding antibodies or fragments thereof (e.g., light chains, heavy chains, VH domains, or VL domains) for recombinant expression by introducing codon changes and / or eliminating inhibitory regions in mRNA can be performed accordingly by adapting the optimization methods described in, for example, U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498. For example, potential splice sites and instability elements (e.g., A / T or A / U-rich elements) within RNA can be mutated without changing the amino acids encoded by the nucleic acid sequence to increase the stability of RNA for recombinant expression. This modification can take advantage of the degeneracy of the genetic code, for example, by using alternative codons for the same amino acid. In some embodiments, conservative variations, e.g., altering one or more codons to encode a similar amino acid having a similar chemical structure and properties and / or function as the original amino acid, may be desirable.

[0134] In certain embodiments, optimized polynucleotide sequences encoding the antibodies or fragments thereof (e.g., VL or VH domains) described herein can hybridize to antisense (e.g., complementary) polynucleotides of non-optimized polynucleotide sequences encoding the antibodies or fragments thereof (e.g., VL or VH domains) described herein. In specific embodiments, optimized nucleotide sequences encoding the antibodies or fragments described herein hybridize to antisense polynucleotides of non-optimized polynucleotide sequences encoding the antibodies or fragments thereof described herein under high stringency conditions. In some embodiments, optimized nucleotide sequences encoding the antibodies or fragments thereof described herein hybridize to antisense polynucleotides of non-optimized nucleotide sequences encoding the antibodies or fragments thereof described herein under high stringency, intermediate, or low stringency hybridization conditions. Information regarding hybridization conditions has been described, see, e.g., US 2005 / 0048549 (e.g., paragraphs 72-73), which is incorporated herein by reference.

[0135] Polynucleotides can be obtained by any method known in the art, and the nucleotide sequence of polynucleotides can be determined.The nucleotide sequences encoding the antibodies and modified versions of these antibodies described herein can be determined using methods well known in the art, that is, nucleotide codons known to encode specific amino acids are assembled in such a way as to generate nucleic acids encoding antibodies.Such polynucleotides encoding antibodies can be assembled from chemically synthesized oligonucleotides (for example, as described in Kutmeier G et al., (1994), BioTechniques 17: 242-246), which simply involves synthesizing overlapping oligonucleotides containing portions of the sequence encoding antibodies, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0136] Alternatively, polynucleotides encoding the antibodies or fragments thereof described herein can be generated from nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Such PCR amplification methods can be used to obtain nucleic acid containing sequences encoding the light and / or heavy chains of the antibody. Such PCR amplification methods can be used to obtain nucleic acid containing sequences encoding the variable light and / or variable heavy chain regions of the antibody. The amplified nucleic acid can be cloned into a vector for expression in a host cell and for further cloning, e.g., to generate chimeric and humanized antibodies.

[0137] If a clone containing a nucleic acid encoding a particular antibody or fragment thereof is not available but the sequence of the antibody molecule or fragment thereof is known, nucleic acid encoding the immunoglobulin or fragment can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library or a cDNA library generated from any tissue or cell that expresses the antibody, such as hybridoma cells selected to express an antibody described herein, or nucleic acid isolated therefrom, e.g., polyA+ RNA) by PCR amplification using synthetic primers capable of hybridizing to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for the particular gene sequence to identify cDNA clones from the cDNA library that encode the antibody, for example. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

[0138] DNA encoding the recombinant proteins described herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the recombinant protein). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from the CHO GS System™ (Lonza)), or myeloma cells that do not otherwise produce immunoglobulin proteins, to obtain synthesis of the recombinant protein in the recombinant host cells.

[0139] To generate antibodies, PCR primers containing the VH or VL nucleotide sequence, restriction sites, and flanking sequences for protecting the restriction sites can be used to amplify the VH or VL sequence in the scFv clone. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing a heavy chain constant region, such as the human gamma 4 constant region, and the PCR-amplified VL domain can be cloned into a vector expressing a light chain constant region, such as the human kappa or lambda constant region. In some embodiments, the vector for expressing the VH or VL domain contains an EF-1α promoter, a secretion signal, a cloning site for the variable domain, a constant domain, and a selection marker such as neomycin. The VH and VL domains can also be cloned into a single vector expressing the necessary constant regions. Then, using techniques known to those skilled in the art, the heavy chain conversion vector and the light chain conversion vector are co-transfected into a cell line to generate a stable or transient cell line expressing a full-length antibody, such as an IgG.

[0140] The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the murine sequences, or by covalently joining all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0141] Also provided are polynucleotides that hybridize to the polynucleotides encoding the antibodies described herein under high, intermediate, or low stringency hybridization conditions. In specific embodiments, the polynucleotides described herein hybridize to the polynucleotides encoding the VH domains and / or VL domains provided herein under high, intermediate, or low stringency hybridization conditions.

[0142] Hybridization conditions are described in the art and are known to those skilled in the art. For example, hybridization under stringent conditions may involve hybridization to filter-bound DNA in 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2xSSC / 0.1% SDS at about 50-65°C; hybridization under highly stringent conditions may involve hybridization to filter-bound nucleic acid in 6xSSC at about 45°C, followed by one or more washes in 0.1xSSC / 0.2% SDS at about 68°C. Hybridization under other stringent hybridization conditions is known to those skilled in the art and has been described, for example, see Ausubel FM et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, pp. 6.3.1-6.3.6 and 2.10.3.

[0143] Another aspect provides a recombinant vector comprising a gene encoding FSH and a nucleic acid encoding an antigen-binding fragment of serum albumin. Yet another aspect provides a cell transformed with the vector.

[0144] Disclosed herein are nucleic acid molecules encoding a heavy chain region comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 6. Disclosed herein are nucleic acid molecules encoding a light chain region comprising a nucleotide sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 14 or 15.

[0145] Disclosed herein are nucleic acid molecules encoding a light chain region comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 13. Disclosed herein are nucleic acid molecules encoding a light chain region comprising a nucleotide sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 14 or 15.

[0146] In some embodiments, disclosed herein are nucleic acids that each encode the heavy chain region of SEQ ID NO:6 and the light chain region of SEQ ID NO:13. In some embodiments, the nucleic acid encoding the heavy chain region of SEQ ID NO:6 may be represented by SEQ ID NO:7 or SEQ ID NO:8, and the nucleic acid encoding the light chain region of SEQ ID NO:13 may be represented by SEQ ID NO:14 or SEQ ID NO:15.

[0147] (a) the promoter; (b) a first nucleic acid molecule encoding a light chain that binds to serum albumin, and (c) a second nucleic acid molecule encoding the heavy chain and a biologically active effector moiety (e.g., FSH) and a linker. An expression vector comprising: Further disclosed herein is an expression vector wherein the promoter, first nucleic acid sequence, and second nucleic acid molecule are operably linked, and the second nucleic acid molecule can encode one, two, three, four, five, six, or more biologically active effector moieties and a linker.

[0148] Also disclosed herein is an expression vector comprising: (a) the promoter; and (b) a nucleic acid molecule encoding a heavy chain variable domain as disclosed herein and a heavy chain constant domain as disclosed herein.

[0149] Also disclosed herein is an expression vector comprising: (a) the promoter; and (b) a nucleic acid molecule encoding an IL18BP as disclosed herein, a heavy chain variable domain as disclosed herein, and a heavy chain constant domain as disclosed herein.

[0150] Also disclosed herein is an expression vector comprising: (a) the promoter; and (b) a nucleic acid molecule encoding a light chain variable domain as disclosed herein and a light chain constant domain as disclosed herein.

[0151] Also disclosed herein is an expression vector comprising: (a) the promoter; and (b) a nucleic acid molecule encoding FSH as disclosed herein, a light chain variable domain as disclosed herein, and a light chain constant domain as disclosed herein. One, two, three, or more expression vectors or nucleic acid molecules can be expressed to produce a desired recombinant protein.

[0152] In some embodiments, the first nucleic acid molecule or vector comprises a nucleic acid sequence encoding a recombinant protein comprising an antigen-binding fragment comprising a heavy chain, wherein the heavy chain comprises a heavy chain variable domain and a heavy chain constant domain, wherein the heavy chain variable domain comprises: (1) a heavy chain complementarity-determining domain 1 (CDR1) comprising the amino acid sequence of SYGIS (SEQ ID NO: 22); a heavy chain complementarity-determining domain 2 (CDR2) comprising the amino acid sequence of TIFF2025533090000043.tif4128; and heavy chain complementarity-determining domain 3 (CDR3) containing the amino acid sequence of TIFF2025533090000044.tif4128; (2) a heavy chain CDR1 comprising the amino acid sequence of SYGIS (SEQ ID NO:22); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025533090000045.tif4128; and Heavy chain CDR3 containing the amino acid sequence of TIFF2025533090000046.tif4128; (3) a heavy chain CDR1 comprising the amino acid sequence of NYGIH (SEQ ID NO: 26); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025533090000047.tif4128; and Heavy chain CDR3 containing the amino acid sequence of TIFF2025533090000048.tif4128; (4) a heavy chain CDR1 comprising the amino acid sequence of SYAMS (SEQ ID NO: 29); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025533090000049.tif4128; and Heavy chain CDR3 containing the amino acid sequence of TIFF2025533090000050.tif4128; (5) a heavy chain CDR1 comprising the amino acid sequence of AYWIA (SEQ ID NO: 32); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025533090000051.tif4128; and a heavy chain CDR3 comprising the amino acid sequence of LYSGSYSP (SEQ ID NO:34); or (6) a heavy chain CDR1 comprising the amino acid sequence of AYSMN (SEQ ID NO: 35); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025533090000052.tif4128; and Heavy chain CDR3 containing the amino acid sequence of TIFF2025533090000053.tif4128.

[0153] A second nucleic acid molecule or vector is disclosed herein, which comprises a nucleic acid sequence encoding a recombinant protein comprising an antigen-binding fragment comprising a light chain, wherein the light chain comprises a light chain variable domain and a light chain constant domain, wherein the light chain variable domain comprises: (7) light chain CDR1 containing the amino acid sequence of TIFF2025533090000054.tif4128; a light chain CDR2 comprising the amino acid sequence of GASRLES (SEQ ID NO:39); and a light chain CDR3 comprising the amino acid sequence of QQSDSVPVT (SEQ ID NO:40); (8) light chain CDR1 containing the amino acid sequence of TIFF2025533090000055.tif4128; a light chain CDR2 comprising the amino acid sequence of AASSLQS (SEQ ID NO:42), and Light chain CDR3 containing the amino acid sequence of TIFF2025533090000056.tif4128; (9) light chain CDR1 containing the amino acid sequence of TIFF2025533090000057.tif4128; a light chain CDR2 comprising the amino acid sequence of DASNRAT (SEQ ID NO:45); and Light chain CDR3 containing the amino acid sequence of TIFF2025533090000058.tif4128; (10) light chain CDR1 containing the amino acid sequence of TIFF2025533090000059.tif4128; a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:48); and a light chain CDR3 comprising the amino acid sequence of QQYGSSPRT (SEQ ID NO:49); (11) light chain CDR1 containing the amino acid sequence of TIFF2025533090000060.tif4128; a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:48); and a light chain CDR3 comprising the amino acid sequence of QKYSSYPLT (SEQ ID NO:51); or (12) light chain CDR1 containing the amino acid sequence of TIFF2025533090000061.tif4128; a light chain CDR2 comprising the amino acid sequence of GASTGAT (SEQ ID NO:53), and Light chain CDR3 containing the amino acid sequence of TIFF2025533090000062.tif4128.

[0154] For example, a nucleic acid molecule encoding FSH can be ligated to the first or second nucleic acid molecule or vector described above.

[0155] In other embodiments, the first nucleic acid molecule can comprise a nucleic acid sequence encoding a Fab comprising: a heavy chain variable domain comprising (1) above and a light chain variable domain comprising (7) above; a heavy chain variable domain comprising (2) above and a light chain variable domain comprising (8) above; a heavy chain variable domain comprising (3) above and a light chain variable domain comprising (9) above; a heavy chain variable domain comprising (4) above and a light chain variable domain comprising (10) above; a heavy chain variable domain comprising (5) above and a light chain variable domain comprising (11) above; a heavy chain variable domain comprising (6) above and a light chain variable domain comprising (12) above; or any and all combinations of the heavy chain variable domains and light chain variable domains described above. In some embodiments, the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab(SL335) comprising: the heavy chain variable domain comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:35, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:36, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:37, and the light chain variable domain comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:52, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:53, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:54. The first or second nucleic acid molecule can encode FSH alpha and beta.

[0156] In other embodiments, the first nucleic acid molecule or vector comprises a nucleic acid sequence encoding a Fab comprising a heavy chain variable domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:55, 56, 57, 58, 59, or 60. In some embodiments, the second nucleic acid molecule or vector comprises a nucleic acid sequence encoding a Fab comprising a light chain variable domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:61, 62, 63, 64, 65, 66, or 67. A nucleic acid molecule encoding FSH can be ligated to the first or second nucleic acid molecule or vector.

[0157] In some embodiments, the first nucleic acid molecule or vector comprises a nucleic acid sequence encoding a Fab comprising a heavy chain variable domain comprising an amino acid sequence at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:55, 56, 57, 58, 59, or 60, and a light chain variable domain comprising an amino acid sequence at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:61, 62, 63, 64, 65, or 66 or 67.

[0158] In some embodiments, the first nucleic acid molecule is SEQ ID NO:6 (V H -C H1 a heavy chain domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:13 (V domain); L-Cκ domain) and a light chain domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity thereto.

[0159] In some embodiments, the bioactive effector moiety is FSH. In some embodiments, the nucleic acid molecule encodes an FSH alpha protein comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:1. In some embodiments, the nucleic acid molecule encoding FSH alpha comprises a nucleotide sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:2 or 3. For example, the first nucleic acid molecule can comprise a nucleotide sequence encoding an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to one or more of SEQ ID NO:1, e.g., SEQ ID NO:2 or 3.

[0160] In some embodiments, the nucleic acid molecule encodes an FSHβ protein comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:4. In some embodiments, the nucleic acid molecule encoding FSHβ comprises a nucleotide sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:5. For example, a first nucleic acid molecule can comprise a nucleotide sequence encoding an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to one or more of SEQ ID NO:4, e.g., SEQ ID NO:5.

[0161] Recombinant expression of an antibody or fragment thereof described herein that specifically binds (e.g., the heavy or light chain of an antibody described herein) involves construction of an expression vector containing a polynucleotide encoding the antibody or fragment. Once a polynucleotide encoding an antibody or fragment thereof described herein (e.g., a heavy or light chain variable domain) is obtained, vectors for the production of the antibody molecule can be generated by recombinant DNA technology using techniques well known in the art. Accordingly, methods for preparing a protein by expressing a polynucleotide containing a nucleotide sequence encoding an antibody or antibody fragment (e.g., a light or heavy chain) are described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing antibody or antibody fragment (e.g., a light or heavy chain) coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors containing a nucleotide sequence encoding an antibody molecule described herein, an antibody heavy or light chain, a heavy or light chain variable domain of an antibody or fragment thereof, or a heavy or light chain CDR, operably linked to a promoter. Such vectors can contain, for example, nucleotide sequences encoding the constant region of an antibody molecule (see, e.g., WO86 / 05807 and WO89 / 01036; and U.S. Patent No. 5,122,464), and the variable domain of an antibody can be cloned into such a vector for expression of an entire heavy chain, an entire light chain, or both the entire heavy and light chains.

[0162] The expression vector can be transferred to a cell (e.g., a host cell) by conventional techniques and the resulting cells can then be cultured by conventional techniques to produce an antibody described herein.

[0163] A variety of host-expression vector systems can be utilized to express the described antibody molecules. Such host-expression systems represent vehicles in which a coding sequence of interest may be produced and subsequently purified, but also represent cells which, when transformed or transfected with the appropriate nucleotide coding sequence, are capable of expressing the antibody molecules described herein in situ. These include microorganisms such as bacteria (e.g., Escherichia coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing the antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody coding sequences; plant cell systems (e.g., green algae such as Chlamydomonas reinhardtii) infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody coding sequences; or mammalian cell systems (e.g., COS typhimurium) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells. In some embodiments, cells for expressing an antibody described herein (e.g., an antibody comprising any one of the CDRs of antibody pab1949 or pab2044) are CHO cells, e.g., CHO cells from CHO GS System™ (Lonza).In some embodiments, cells for expressing the antibodies described herein are human cells, e.g., human cell lines. In some embodiments, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In some embodiments, bacterial cells such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells), particularly for expression of whole recombinant antibody molecules, are used for expression of recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary (CHO) cells in combination with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus are effective expression systems for antibodies (Foecking MK & Hofstetter H (1986) Gene 45: 101-105; and Cockett MI et al., (1990) Biotechnology 8: 662-667). In certain embodiments, the antibodies described herein are produced by CHO cells or NS0 cells. In some embodiments, expression of the nucleotide sequence encoding the antibodies described herein is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0164] In bacterial systems, numerous expression vectors can be advantageously selected depending on the intended use of the expressed antibody molecule. For example, when large quantities of such antibodies are produced for the preparation of pharmaceutical compositions of antibody molecules, a vector directing the expression of high-level fusion protein products that are easily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2: 1791-1794), in which the antibody coding sequence can be individually ligated into the vector in frame with the lac Z coding region to produce a fusion protein; pIN vector (Inouye S & Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G & Schuster SM (1989) J Biol Chem 24: 5503-5509). For example, pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0165] In mammalian host cells, a number of viral-based expression systems can be utilized. When adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated into an adenovirus transcription / translation control complex, such as the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in an infected host (see, e.g., Logan J & Shenk T (1984) PNAS 81: 3655-3659). Specific initiation signals may also be required for efficient translation of the inserted antibody coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, eg, Bitter G et al., (1987) Methods Enzymol 153:516-544).

[0166] In addition, a host cell strain can be selected that modulates the expression of the inserted sequence or modifies and processes the gene product in the specific manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. An appropriate cell line or host system can be selected to ensure the correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10 and HsS78Bst cells. In some embodiments, the recombinant proteins (e.g., antibodies comprising CDRs) described herein are produced in mammalian cells, such as CHO cells.

[0167] In some aspects, rather than using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.) and a selectable marker. After introduction of the foreign DNA / polynucleotide, engineered cells can be grown in an enriched medium for 1-2 days and then switched to a selective medium. The selectable marker on the recombinant plasmid confers resistance to the selection, allowing cells to stably integrate the plasmid into their chromosomes and grow to form foci that can then be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines expressing the antibodies or fragments thereof described herein. Such engineered cell lines can be particularly useful in screening and evaluating compositions that interact directly or indirectly with antibody molecules.

[0168] A number of selection systems can be used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell 11(1): 223-232), hypoxanthine guanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS 48(12): 2026-2034), and adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3): 817-823) genes, which can be used in tk-, hgprt-, or aprt- cells, respectively. Antimetabolite resistance can also be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler M et al., (1980) PNAS 77(6): 3567-3570; O'Hare K et al., (1981) PNAS 78: 1527-1531); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-2076); neo, which confers resistance to the aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3: 87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan RC (1993) Science 260: 926-932; and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62: 191-217; Nabel GJ & Felgner PL (1993) Trends Biotechnol 11(5): 211-215; and hygro, which confers resistance to hygromycin (Santerre RF et al., (1984) Gene 30(1-3): 147-156).

[0169] Once an antibody molecule described herein is produced by recombinant expression, it can be purified by any method known in the art for the purification of immunoglobulin molecules, such as, for example, chromatography (e.g., ion exchange, affinity, particularly by affinity for a specific antigen following Protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for the purification of proteins. Additionally, the antibodies described herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0170] In specific embodiments, the antibodies described herein are isolated or purified. Generally, an isolated antibody is substantially free of other antibodies having antigen specificities different from the isolated antibody. For example, in some embodiments, preparations of the antibodies described herein are substantially free of cellular material and / or chemical precursors. The term "substantially free of cellular material" includes preparations of antibodies in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, antibodies that are substantially free of cellular material include preparations of antibodies that have less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins") and / or antibody variants, e.g., antibody preparations with different post-translationally modified forms of the antibody. When an antibody or fragment is recombinantly produced, it is also generally substantially free of culture medium, i.e., culture medium accounts for less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation.When an antibody or fragment is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals involved in the synthesis of the protein.Thus, such preparations of antibodies or fragments have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody or fragment of interest.In some embodiments, the antibodies described herein are isolated or purified.

[0171] antibody production Yet another aspect provides a method for preparing a recombinant protein, comprising: (a) culturing cells; and (b) recovering the recombinant protein from the cultured cells. The cells can be cultured in a variety of media. Commercially available culture media can be used without limitation. Any other necessary supplements known to those skilled in the art can also be included at appropriate concentrations. Culture conditions, such as temperature and pH, will be those previously used with the host cells selected for expression and will be apparent to those skilled in the art. The recombinant protein can be recovered by removing impurities, for example, by centrifugation or ultrafiltration, and purifying the resulting product, for example, by affinity chromatography. Other additional purification techniques, such as anion exchange or cation exchange chromatography, hydrophobic interaction chromatography, or hydroxylapatite chromatography, can also be used.

[0172] The recombinant proteins disclosed herein can be produced by any method known in the art for the synthesis of antibodies, for example, by chemical synthesis or recombinant expression technology. The methods described herein, unless otherwise indicated, use conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields, within the skill of the art. These techniques are described, for example, in the references cited herein and are fully explained in the literature. For example, Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al. ,See Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annually updated); Current Protocols in Immunology, John Wiley & Sons (1987 and annually updated); Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0173] In some embodiments, a recombinant protein described herein is an antibody (e.g., a recombinant antibody) that is prepared, expressed, produced, or isolated by any means involving, for example, synthesis of a DNA sequence or production via genetic engineering. In certain embodiments, such an antibody comprises a sequence (e.g., a DNA sequence or an amino acid sequence) that does not naturally occur within the antibody germline repertoire of an animal or mammal (e.g., a human) in vivo.

[0174] In some aspects, provided herein are methods for producing a recombinant protein disclosed herein, comprising culturing a cell or host cell as described herein. In some aspects, provided herein are methods for producing a recombinant protein, comprising expressing (e.g., recombinantly expressing) an antibody using a cell or host cell described herein (e.g., a cell or host cell comprising a polynucleotide encoding an antibody described herein). In some embodiments, the cell is an isolated cell. In some embodiments, an exogenous polynucleotide has been introduced into the cell. In some embodiments, the method further comprises purifying the antibody obtained from the cell or host cell.

[0175] Antibodies can be prepared using a variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma technology, including those known in the art and taught, for example, in Harlow E & Lane D, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling GJ et al.,: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981). The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. For example, monoclonal antibodies can be recombinantly produced from host cells exogenously expressing the antibodies described herein.

[0176] A "monoclonal antibody," as used herein, is an antibody produced by a single cell (e.g., a hybridoma or host cell producing a recombinant antibody), wherein the antibody immunospecifically binds to an antigen (e.g., human serum albumin), e.g., as determined by ELISA or other antigen-binding or competitive binding assays known in the art or in the Examples provided herein. In certain embodiments, a monoclonal antibody may be a chimeric or humanized antibody. In certain embodiments, a monoclonal antibody is a monovalent or polyvalent (e.g., bivalent) antibody. In certain embodiments, a monoclonal antibody may be a Fab fragment or a F(ab')2 fragment. The monoclonal antibodies described herein can be made by hybridoma methods, e.g., as described in Kohler G & Milstein C (1975) Nature 256: 495, or can be isolated, e.g., from phage libraries, using techniques such as those described herein. Other methods for the preparation of clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art (see, e.g., Chapter 11 in Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., supra).

[0177] Methods for producing and screening specific antibodies using hybridoma technology are routine and well-known in the art. For example, in the hybridoma method, mice or other suitable host animals, such as sheep, goats, rabbits, rats, hamsters, or macaques, are immunized to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the antigen used for immunization (e.g., human serum albumin). Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells. Some aspects of the present disclosure provide methods for preparing recombinant fusion proteins, including (a) culturing cells; and (b) recovering the recombinant fusion protein from the cultured cells. Cells can be cultured in various media. Any commercially available medium can be used as a culture medium without limitation. All other necessary supplements known to those skilled in the art can also be included at appropriate concentrations. Culture conditions, such as temperature, pH, etc., will be readily applied to the host cells selected for expression and will be apparent to those skilled in the art. The recombinant fusion protein can be purified, for example, by centrifugation or ultrafiltration to remove impurities, and the resulting product can be purified, for example, using affinity chromatography, etc. Other purification techniques can be used, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, etc.

[0178] Compositions and Uses Thereof Some aspects of the present disclosure provide long-acting hFSH formulations that include a recombinant fusion protein as an active ingredient.

[0179] Since long-acting hFSH preparations use the aforementioned recombinant fusion protein as the active ingredient, a description of the content common to both will be omitted.

[0180] The recombinant fusion protein, which is the active ingredient of the preparation, can be fused to an antigen-binding fragment that binds to serum albumin, thereby extending the in vivo half-life of the recombinant protein and thus improving the therapeutic effect of hFSH.

[0181] In some embodiments, follitropin α (trade name: Gonal-F), commonly used in infertility treatment, has a half-life of approximately 24 hours in the human body, thus necessitating frequent administration to patients. However, the recombinant fusion product according to some embodiments of the present disclosure not only exhibits an approximately three-fold longer half-life in rats compared to follitropin α, but also exhibits an approximately three-fold higher ovarian growth-promoting effect than Gonal-F, as determined by measuring and comparing ovarian volume under similar experimental conditions. Therefore, long-acting hFSH preparations can be used in assisted reproductive technologies for infertility treatment, such as in vitro fertilization-embryo transfer (IVF-ET), gamete intrafallopian tube injection (GIFT), zygote intrafallopian tube injection (ZIFT), and intracytoplasmic sperm injection (ICSI). Long-acting hFSH preparations can also be applied to IVF techniques to induce superovulation in subjects, or can be used to treat anovulation, hypogonadism, and polycystic ovary syndrome.

[0182] In some embodiments, the long-acting hFSH preparation can be provided in the following forms: a pharmaceutical composition for treating infertility, comprising (a) a pharmaceutically effective amount of a recombinant fusion protein and (b) a pharmaceutically acceptable carrier; a method for treating infertility by administering a pharmaceutical composition comprising the recombinant fusion protein; and a pharmaceutical use of the recombinant fusion protein for treating infertility.

[0183] As used herein, the term "treatment" refers to any action that improves or beneficially alters the symptoms of infertility by administering a pharmaceutical composition.

[0184] The term "infertility", which is the disease treated by the pharmaceutical composition, refers to a condition in which pregnancy does not occur even after one year or more of normal marital relations, and is treated or expected to be treated through the administration of FSH. Infertility can include both female infertility, including anovulation, and male infertility caused by sperm abnormalities.

[0185] The pharmaceutically acceptable carrier contained in the pharmaceutical composition may be one that is conventionally used in formulation, and examples thereof include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc., but the embodiment is not particularly limited thereto. The pharmaceutical composition of the present disclosure may further include, in addition to the above-mentioned components, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc.

[0186] The pharmaceutical composition can be administered orally or parenterally. Preferably, the administration can be parenteral, and in the case of parenteral administration, the pharmaceutical composition can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc. In some embodiments, the pharmaceutical composition can be administered via subcutaneous or intramuscular injection.

[0187] The pharmaceutical composition is administered in a pharmaceutically effective amount. As used herein, "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level can be determined based on factors such as the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, co-administered drugs, and other factors well known in the medical field. The pharmaceutical composition can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered simultaneously, separately, or sequentially with conventional therapeutic agents, and can be administered in a single dose or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and such an amount can be easily determined by those skilled in the art. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a target disease or condition, and the amount can vary depending on the subject's characteristics, estradiol concentration, cause of infertility, and purpose of administration.

[0188] The pharmaceutical compositions can be prepared in unit dosage form or in multi-dose containers by formulating them with pharmaceutically acceptable carriers and / or excipients according to methods readily practiced by those skilled in the art to which this disclosure pertains, where the dosage form can be a solution, suspension, emulsion, extract, powder, suppository, powdered drug, granules, tablet, or capsule in an oily or aqueous medium, and can further include dispersing agents or stabilizers.

[0189] For example, a pharmaceutical composition can include: (a) a pharmaceutically effective amount of the recombinant protein; and (b) a pharmaceutically acceptable carrier.

[0190] In some embodiments, the in vivo half-life of the pharmaceutical composition may exhibit a 2- to 20-fold increase when compared to that of human FSH. The in vivo half-life, when compared to that of human FSH, can be, for example, about 2.5-fold to about 3.5-fold, about 3.5-fold to about 6-fold increase, about 4-fold to about 6-fold increase, about 4.5-fold to about 6-fold increase, about 5-fold to about 6-fold increase, about 5.5-fold to about 6-fold increase, about 3-fold to about 5.5-fold increase, about 3.5-fold to about 5.5-fold increase, about 4-fold to about 5.5-fold increase, about 4.5-fold to about 5.5-fold increase, about 5-fold to about 5.5-fold increase, about 3-fold to about 5-fold increase, about 3.5-fold to about 5-fold increase, about 4-fold to about 5-fold increase, about 4.5-fold to about 5-fold increase, about 3-fold to about 4.5-fold increase, about 3.5-fold to about 4.5-fold increase, about 4-fold to about 4.5-fold increase, or any factor or factor range derived therefrom. In some embodiments, the in vivo half-life of human FSH can be assessed following subcutaneous injection of human FSH.

[0191] In some embodiments, pharmaceutical compositions can reduce the white blood cell level in blood.White blood cells can be, for example, neutrophils, monocytes, basophils, or a combination thereof.In some embodiments, the reduced white blood cell level can be sustained and maintained for up to 20 days after administration, up to 15 days after administration, up to 12 days after administration, up to 10 days after administration, up to 8 days after administration, up to 7 days after administration, or any range derived therefrom.

[0192] Pharmaceutical compositions can be prepared in unit dosage form or in multi-dose containers by formulating them with pharmaceutically acceptable carriers and / or excipients according to methods readily practiced by those skilled in the art to which this disclosure pertains, and in this case, the formulations can be in the form of solutions, suspensions, or emulsions in oily or aqueous vehicles, or in the form of extracts, suppositories, powders, granules, tablets, or capsules, and the formulations can further include dispersing agents or stabilizers.

[0193] Provided herein are compositions comprising the recombinant proteins described herein having a desired purity in a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Also disclosed herein are pharmaceutical compositions comprising the recombinant proteins described herein and a pharmaceutically acceptable excipient. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed.

[0194] The pharmaceutical compositions according to some aspects can be used after being formulated in the form of oral preparations, for example, powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., topical preparations, suppositories, or sterile injectable preparations, according to common methods, and for formulation, the pharmaceutical compositions can include suitable carriers, excipients, or diluents commonly used in the preparation of pharmaceutical compositions.

[0195] Carriers, excipients, or diluents can include various compounds or mixtures, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

[0196] When formulated, it can be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like.

[0197] Solid formulations for oral administration can be prepared by mixing the recombinant fusion protein with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate or talc can also be used.

[0198] Oral liquid preparations may include suspensions, oral solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. may be included.

[0199] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may contain propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. Suppository bases may include witepsol, macrogol, Tween 61, cocoa butter, laurin butter, glycerin gelatin, etc.

[0200] Disclosed herein is a method of inducing superovulation in a subject in need thereof, comprising applying a pharmaceutical composition disclosed herein to in vitro fertilization-embryo transfer (IVF-ET), gamete intrafallopian tube transfer (GIFT), zygote intrafallopian tube transfer (ZIFT), intracytoplasmic sperm injection (ICSI), or in vitro fertilization. Disclosed herein is a method of treating anovulation, hypogonadism, or polycystic ovary syndrome in a subject in need thereof, comprising administering to the subject a pharmaceutical composition disclosed herein.

[0201] Further disclosed herein are long-acting human follicle-stimulating hormone (hFSH) compositions comprising the recombinant fusion protein disclosed herein as an active ingredient. In some embodiments, the long-acting hFSH compositions are applied in in vitro fertilization-embryo transfer (IVF-ET), gamete intrafallopian tube transfer (GIFT), zygote intrafallopian tube transfer (ZIFT), intracytoplasmic sperm injection (ICSI), or in vitro fertilization to induce superovulation in a subject. In some embodiments, the long-acting hFSH compositions are used to treat anovulation, hypogonadism, or polycystic ovary syndrome.

[0202] As used herein, the term "treat" or "treatment" refers to any effect whereby the symptoms of a disease or condition are improved, eliminated, or otherwise favorably altered by administering a composition disclosed herein.

[0203] Also disclosed herein is the use of the compositions disclosed herein for inducing superovulation in a subject in need thereof. Also disclosed herein is the use of the compositions disclosed herein for the manufacture of a medicament for inducing superovulation in a subject in need thereof. Also disclosed herein is the use of the compositions disclosed herein for the manufacture of a medicament for inducing superovulation in a subject in need thereof.

[0204] The pharmaceutical compositions described herein may be useful for enhancing, inducing, or activating the activity of the recombinant proteins disclosed herein to treat diseases or conditions.

[0205] Compositions to be used for in vivo administration can be sterile, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0206] The long-acting hFSH composition can be applied in in vitro fertilization-embryo transfer (IVF-ET), gamete intrafallopian tube transfer (GIFT), zygote intrafallopian tube transfer (ZIFT), intracytoplasmic sperm injection (ICSI), or in vitro fertilization to induce superovulation in a subject. In some embodiments, the long-acting hFSH composition is used to treat anovulation, hypogonadism, or polycystic ovary syndrome.

[0207] In some embodiments, the pharmaceutical composition may be for preventing or treating adult-onset Still's disease, comprising a recombinant fusion protein as an active ingredient. Adult-onset Still's disease is a multifactorial systemic autoinflammatory disease with symptoms similar to those of systemic juvenile idiopathic arthritis. It is an inflammatory disease that occurs in adults, but the exact mechanism of the disease remains unknown. Adult-onset Still's disease is characterized by elevated serum FSH levels and downregulation of the in vivo FSH antagonist. Meanwhile, it has been reported that more than 50% of patients with adult-onset Still's disease who received recombinant FSH drugs at doses of 80 mg / head and 160 mg / head, respectively, responded to the drug. Clinical trials have also reported that the drug has a half-life of approximately 30 to 40 hours in humans and is effective when administered three times a week. Therefore, the drug presents a problem in that it must be administered frequently to patients as a subcutaneous injection. In one exemplary embodiment, recombinant fusion protein has been confirmed to show about 3.5 times longer half-life in rats than recombinant FSH, and has the same and similar activity even when administered in small doses.Therefore, recombinant fusion protein can be effectively used to treat adult-onset Still's disease.

[0208] Disclosed herein are compositions comprising the recombinant fusion proteins disclosed herein and a carrier. Disclosed herein are pharmaceutical compositions comprising the recombinant fusion proteins disclosed herein and a pharmaceutically acceptable carrier. Disclosed herein are kits comprising the compositions disclosed herein and a label containing instructions for use.

[0209] Disclosed herein is a method of inducing superovulation in a subject in need thereof, comprising applying a pharmaceutical composition disclosed herein to in vitro fertilization-embryo transfer (IVF-ET), gamete intrafallopian tube transfer (GIFT), zygote intrafallopian tube transfer (ZIFT), intracytoplasmic sperm injection (ICSI), or in vitro fertilization. Disclosed herein is a method of treating anovulation, hypogonadism, or polycystic ovary syndrome in a subject in need thereof, comprising administering to the subject a pharmaceutical composition disclosed herein.

[0210] Further disclosed herein are long-acting human follicle-stimulating hormone (hFSH) compositions comprising the recombinant fusion protein disclosed herein as an active ingredient. In some embodiments, the long-acting hFSH compositions are applied in in vitro fertilization-embryo transfer (IVF-ET), gamete intrafallopian tube transfer (GIFT), zygote intrafallopian tube transfer (ZIFT), intracytoplasmic sperm injection (ICSI), or in vitro fertilization to induce superovulation in a subject. In some embodiments, the long-acting hFSH compositions are used to treat anovulation, hypogonadism, or polycystic ovary syndrome.

[0211] In some embodiments, the composition is administered to the subject subcutaneously or intramuscularly.

[0212] Yet another aspect provides a pharmaceutical composition comprising the recombinant fusion protein as an active ingredient, specific details of which are provided above.

[0213] Route of administration and dosage The pharmaceutical compositions of the present disclosure can be administered to a subject through a variety of routes of administration, including oral, transdermal, subcutaneous, intravenous, and intramuscular routes of administration.

[0214] The amount of the recombinant protein or composition disclosed herein that will be effective in the treatment and / or prevention of a condition will depend on the nature of the disease, and can be determined by standard clinical techniques.

[0215] In the present disclosure, the amount of the recombinant protein disclosed herein to be actually administered is determined in light of various relevant factors, including the disease to be treated, the selected administration route, the age, sex, and weight of the patient, and the severity of the disease, as well as the type of biologically active polypeptide as the active ingredient. Because the recombinant protein of the present disclosure has excellent sustainability in the blood, the number and frequency of administration of peptide preparations containing the recombinant protein of the present disclosure can be significantly reduced.

[0216] The pharmaceutical composition is administered in a pharmaceutically effective amount. As used herein, "pharmacologically effective amount" or "effective amount" in the context of administering a therapy to a subject refers to the amount of therapy that achieves the desired preventive or therapeutic effect. The effective dose level can be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, and co-administered drugs, as well as other factors well known in the medical field. The pharmaceutical composition can be administered as a single therapeutic agent or in combination with other therapeutic agents, and can be administered simultaneously with existing therapeutic agents, separately, or sequentially, in one dose or in a few divided doses. Taking all factors into consideration, it is important to administer the composition in the minimum amount sufficient to achieve maximum effect without any side effects, and this amount can be easily determined by those skilled in the art. As used herein, the term "pharmacologically effective amount" refers to an amount sufficient to treat or induce the disease or condition described herein, for example, to induce superovulation or treat anovulation, hypogonadism, or polycystic ovary syndrome.

[0217] The appropriate dosage of the pharmaceutical composition varies depending on the patient's condition, body weight, disease severity, drug formulation, administration route and duration, but can be appropriately selected by those skilled in the art. However, to achieve the desired effect, the pharmaceutical composition can be administered at a daily dose of 0.0001 mg / kg to 2,000 mg / kg, specifically 0.001 mg / kg to 2,000 mg / kg. Administration can be performed once a day or in several divided doses.

[0218] The exact dosage employed in the composition also depends on the route of administration and the severity of the disease, and should be determined according to the judgment of the doctor and the individual patient's circumstances.For example, the effective dosage can also vary depending on the administration means, target site, patient's physiological condition (including age, weight and health condition), other drugs administered, or whether treatment is preventive or therapeutic.Usually, the patient is human, but can also be non-human, such as pets, for example, dogs and cats.The treatment dosage is optimally titrated to optimize safety and effectiveness.

[0219] In certain embodiments, in vitro assays are employed to help identify optimal dosage ranges. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0220] In some embodiments, the recombinant fusion protein may be administered at a dose of 0.001 mg / kg to 2,000 mg / kg, for example, 0.001 mg / kg to 0.01 mg / kg, 0.1 mg / kg to 1 mg / kg, 1.5 mg / kg to 2 mg / kg, 4 mg / kg to 10 mg / kg, 15 mg / kg to 20 mg / kg, 30 mg / kg to 40 mg / kg, 60 mg / kg to 80 mg / kg, 100 mg / kg to 200 mg / kg, or any dose or dose range derived therefrom.

[0221] Pharmaceutical compositions for treating diseases or inducing conditions can be administered via various routes to mammals, e.g., rats, mice, livestock, humans, etc. For example, oral, rectal or intravenous, intramuscular, subcutaneous, or intrathecal administration, or by intracerebroventricular injection, all modes of administration may be contemplated.

[0222] Pharmaceutical compositions can be administered orally or parenterally.Specifically, pharmaceutical compositions can be administered parenterally, and in this case, can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration.In some embodiments, it can be administered in the form of subcutaneous injection.When administered orally, proteins or peptides are digested, and therefore it is necessary to formulate oral compositions by coating the active ingredient or protecting it from degradation in the stomach.In addition, pharmaceutical compositions can be administered by any device that can deliver active substances to target cells.

[0223] Yet another aspect provides a health functional food composition for preventing or ameliorating a condition or disease, the health functional food composition comprising a recombinant fusion protein as an active ingredient. Yet another aspect provides a health functional food composition for preventing or ameliorating a condition or disease, the health functional food composition comprising a recombinant fusion protein as an active ingredient.

[0224] Regarding the health functional food composition, when the recombinant fusion protein is used as an additive for the health functional food and can be used appropriately according to a general method, the recombinant fusion protein can be added as it is, or can be used together with other foods or food ingredients. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use, such as prevention, health, treatment, etc.

[0225] The formulation of the health functional food can be in the form of powder, granules, pills, tablets, and capsules, as well as in the form of a general food or drink.

[0226] The type of food is not particularly limited, and examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, etc., and may include all foods within common sense.

[0227] Generally, in the preparation of food or beverages, the recombinant fusion protein can be added in an amount of 15 parts by weight or less, specifically 10 parts by weight or less, per 100 parts by weight of the raw material. However, for long-term intake for health and hygiene purposes or health management purposes, the amount can be adjusted to be below the above range. Furthermore, since the present disclosure uses fractions derived from natural products, there are no safety issues. Therefore, the amount can exceed the above range.

[0228] Among health functional foods, beverages can contain various flavorings or natural carbohydrates as additional ingredients, as in common beverages. The natural carbohydrates can include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Sweeteners that can be used include natural sweeteners such as thaumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame. The proportion of natural carbohydrates can be about 0.01 g to 0.04 g, specifically about 0.02 g to 0.03 g, per 100 mL of the beverage according to the present disclosure.

[0229] In addition, health functional food compositions according to some aspects can contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonation agents used in carbonated beverages. Additionally, the compositions of the present disclosure can contain fruit pulp for preparing natural fruit juices, fruit juice drinks, and vegetable drinks. These ingredients can be used alone or in mixtures. The proportion of these additives is not limited, but is generally selected from the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health functional food composition.

[0230] As described above, the recombinant fusion protein can exhibit an approximately 3.5-fold increased half-life in rats, for example, when compared to human recombinant FSH, and exhibits biological activity at a level similar to that of FSH not fused to SL335. Thus, the recombinant fusion protein can exhibit similar efficacy with less frequent administration, allowing patients to administer the drug at more convenient intervals.

[0231] kit Provided herein are kits containing one or more recombinant proteins or conjugates thereof described herein. Provided herein are kits containing a composition disclosed herein and a label containing instructions for its use. Provided herein are pharmaceutical packs or kits containing one or more containers filled with one or more of the components of the pharmaceutical compositions described herein, such as one or more recombinant proteins provided herein. In some embodiments, the kits contain the pharmaceutical compositions described herein and any prophylactic or therapeutic agents, such as those described herein. Optionally associated with such container(s) may be a notice in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, reflecting agency approval of the manufacture, use, or sale for human administration. Also provided herein are kits that can be used in the above-described methods. In some embodiments, the kits contain a recombinant protein described herein, e.g., a purified recombinant protein, in one or more containers. In some embodiments, the kits described herein contain a substantially isolated antigen(s) (e.g., human serum albumin) that can be used as a control. In other embodiments, the kits described herein further include a control antibody that does not react with the serum albumin antigen. In other embodiments, the kits described herein include one or more elements for detecting binding of the recombinant protein to the serum albumin antigen (e.g., the recombinant protein can be conjugated to a detectable substrate such as a fluorescent compound, an enzyme substrate, a radioactive compound, or a luminescent compound, or a second antibody that recognizes the first antibody can be conjugated to a detectable substrate). In specific embodiments, the kits provided herein can include a recombinantly produced or chemically synthesized serum albumin antigen. The serum albumin antigen provided in the kit can also be bound to a solid support. In some embodiments, the detection means of the above-mentioned kits includes a solid support to which the serum albumin antigen is bound.Such kits can also include an unconjugated reporter-labeled anti-human antibody or anti-mouse / rat antibody. Upon binding of the antibody to serum albumin, the antigen can be detected by binding of the reporter-labeled antibody.

[0232] Reference will now be made in detail to the embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the embodiments may have different forms and should not be construed as limited to the description set forth herein. Accordingly, the embodiments will be described below merely by reference to the figures to illustrate aspects of the present specification. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. Phrases such as "at least one," when preceding a list of elements, modify the entire list of elements and not individual elements of the list.

[0233] The present disclosure will now be described in detail with reference to the following examples, which are for illustrative purposes only and are not intended to limit the scope of the present disclosure. [Example]

[0234] Example 1. Preparation of a recombinant fusion protein containing follicle-stimulating hormone and an antigen-binding fragment against serum albumin In this example, recombinant fusion proteins containing follicle-stimulating hormone (FSH) and an antigen-binding fragment for serum albumin were prepared. In the recombinant fusion proteins shown in Figure 1, the human FSH β subunit and the human FSH α subunit were each linked via a peptide linker to the C-terminal region of the human anti-serum albumin Fab antibody fragment "SL335." Recombinant fusion proteins, namely, SAFA-FSH-A and SAFA-FSH-B proteins, were prepared by varying the combination of heavy chain fragments or light chain fragments linked to the human FSH α / β subunits. Specifically, in this example, (i) SAFA-FSH-A protein was prepared, which contained a heavy chain composed of the SL335 heavy chain fragment of SEQ ID NO:6, a linker of SEQ ID NO:88, and the FSH α subunit of SEQ ID NO:1; and a light chain composed of the SL335 light chain fragment of SEQ ID NO:14, a linker of SEQ ID NO:85, and the FSH β subunit of SEQ ID NO:4; and (ii) SAFA-FSH-B protein was prepared, which contained a heavy chain composed of the SL335 heavy chain fragment of SEQ ID NO:6, a linker of SEQ ID NO:85, and the FSH β subunit of SEQ ID NO:4; and a light chain composed of the SL335 light chain fragment of SEQ ID NO:14, a linker of SEQ ID NO:88, and the FSH α subunit of SEQ ID NO:1.

[0235] 1-1. Preparation of expression vectors for recombinant fusion proteins The genes used in the expression vectors according to some embodiments were subjected to codon optimization by Cosmogenetech Co., Ltd. (South Korea), and the genetic information of the light chain fragment and / or heavy chain fragment of the serum albumin-binding Fab antibody (SL335) was the same as SEQ ID NOs: 7, 8, 15, and 16, the genetic information of the peptide linker was the same as SEQ ID NOs: 86, 87, 89, and 90, and the genetic information of the FSH α subunit and FSH β subunit genes was the same as the FSH α subunit (according to UniProKB P01215) and FSH β subunit (according to UniProKB P01225) (SEQ ID NOs: 2, 3, 5, and 9).

[0236] The recombinant genes for SAFA-FSH heavy chain, i.e., SL335H-linker-FSHα and SL335H-linker-FSHβ (SEQ ID NOs: 20 and 96), prepared by gene synthesis and the pD2535NT(ATUM) expression vector, were treated with BbsI restriction enzyme (Takara, Japan) to cleave the BbsI site, and then treated with T4 DNA ligase (Takara, Japan) to ligate them into the expression vector. Furthermore, the recombinant genes of SAFA-FSH light chains, i.e., SL335L-linker-FSHβ and SL335L-linker-FSHα (SEQ ID NOs:94 and 98), prepared by gene synthesis and the pD2359 (ATUM) expression vector were treated with BsrGI (NEB, USA) and BbsI (Takara, Japan) restriction enzymes, respectively, to cleave the BsrGI and BbsI sites, and then treated with T4 DNA ligase (Takara, Japan), respectively, to ligate them into the expression vectors. Specifically, the expression vectors for preparing SAFA-FSH-A and SAFA-FSH-B proteins according to the embodiment are as shown in FIG. 2.

[0237] 1-2. Preparation of transient expression cells ExpiCHO-S™ cells (ThermoFisher Scientific) were added to a 125 ml culture flask containing expression medium (ExpiCHO Expression Medium, Thermo Fisher Scientific), and then cultured in a shaking incubator at 37°C, 140 rpm, 5% CO2, and 80% humidity. Then, for the preparation of transient expression cells, the cultured cells were diluted to 6.0 × 10 6 The cells were seeded in a culture medium at a concentration of 1000 cells / ml and then transfected with the plasmid vectors (pD2535NT and pD2539) containing the heavy and light chain genes prepared in Example 1-1. The transfected cells were then cultured in a shaking incubator under the same conditions as above for 16 hours, after which they were treated with ExpiCHO feed and enhancer. On the third day of culture, ExpiCHO feed was added, the incubator temperature was set to 37°C, and the cells were cultured for 8 days. After completion of the culture, the resulting culture medium was centrifuged at 4,000 rpm for 15 minutes at 4°C to separate the cells from the culture medium. The separated culture medium was then passed through a 0.2 μm filter paper to remove impurities.

[0238] On the other hand, the expression patterns and productivity of recombinant fusion proteins containing FSH and an antigen-binding fragment of serum albumin, i.e., SAFA-FSH-A and SAFA-FSH-B proteins, were compared and confirmed, and it was confirmed that the SAFA-FSH-B protein has a relatively stable structure and exhibits excellent productivity (not shown). Therefore, in the following experiments, the SAFA-FSH-B protein was selected as the recombinant fusion protein according to the embodiment.

[0239] 1-3. Preparation of stabilized cell lines A stabilized cell line was prepared using HD-BIOP3 GS null CHO-K1 cells (Horizon Discovery). Specifically, 3.0 × 10 cells were used. 5The cells were seeded at a concentration of 1.0 × 10 cells / ml in CD FortiCHO (Thermo Fisher Scientific) medium supplemented with 4 mM L-glutamine and cultured in a shaking incubator at 37°C, 5% CO2, and humidity of 80% or higher. 6 The cells were seeded at a concentration of 1000 cells / ml, and the SAFA-FSH-B plasmid vectors (pD2535NT and pD2539) prepared in Example 1-1 were added to OptiPRO SFM medium along with Freestyle Max Reagent (Invitrogen, Carlsbad, California). After transfection with the vectors, the transfected cells were cultured for 2 days at 37°C, 5% CO2, and 80% or higher humidity. To select stable pools, the medium was replaced with L-glutamine-free CD FortiCHO medium by centrifugation, and the cells were treated with 50 μM methionine sulfoximine (MSX) (Sigma-Aldrich, St. St. Louis, Missouri) and 10 μg / ml puromycin (Thermo Fisher Scientific) every 2 days to remove cells not injected with the vector. Thereafter, the medium was replaced with CD FortiCHO medium containing both MSX and puromycin at intervals of 7 to 10 days by centrifugation, and the cell count was increased to 5.0 × 10 5 The cells were cultured for 21 days at a constant concentration of 1.0 × 10 cells / ml. When the viability recovered to 90% or more, the cells were cultured at 1.0 × 10 cells / ml. 7 Stocks were prepared at a concentration of cells / ml.

[0240] 1-4. Isolation and purification of SAFA-FSH protein Protein samples present in the culture medium of Examples 1-3 were purified by sequential affinity chromatography (AC), multimodal anion exchange chromatography (MAEX), and cation exchange chromatography (CEX). Specifically, AC treatment was performed by binding the culture medium to the resin using CaptureSelect CH1-XL resin (Thermo Fisher, USA) at a flow rate of 10 ml / min, and then the recombinant fusion protein was recovered by pH elution. The AC-purified protein was subjected to MAEX using Capo Adhere ImpRes resin (Cytiva, Sweden) at a flow rate of 5 ml / min, and then the recombinant fusion protein was recovered by FT mode. Finally, CEX was performed using CM Sepharose® Fast Flow resin at a flow rate of 5 ml / min. These separation and purification methods yielded SAFA-FSH-B protein.

[0241] Example 2. Confirmation of the physical properties of SAFA-FSH protein 2-1. Size analysis To confirm the size of the recombinant fusion protein prepared in Example 1, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed. Specifically, protein samples were prepared under reducing and non-reducing conditions using non-reducing (NR) 4x SDS sample buffer (Thermo Fisher Scientific). For reducing conditions, samples were heated at 100°C for 5 minutes, and an unheated sample was prepared alongside to compare the heat-dependent protein shape and size. Protein size markers (SMOBio, Taiwan) were prepared to compare the protein size under each condition. The prepared protein samples were quantified onto a 4-15% Mini-protein TGX precast gel (15 wells, Bio-Rad) to contain 1.175 μg or 2.35 μg per well, respectively, and then subjected to electrophoresis in tris-glycine SDS running buffer at 150 V for 50 minutes. After electrophoresis was completed, the SDS-PAGE gel was stained with EZ-Gel staining solution (DoGenBio, South Korea) for 1 h and then destained with distilled water for 1 day.

[0242] As shown in Figure 3, under reducing (R) and non-reducing (NR(B)) conditions with 5 minutes of heating, the heavy and light chain proteins underwent additional N-linked glycosylation, resulting in a heavy chain band at approximately 40 kDa, higher than the theoretical size of 34.109 kDa, and a light chain band at 40-45 kDa, higher than the theoretical size of 37.081 kDa. On the other hand, under non-reducing (NR(NB)) conditions without heating, a protein band corresponding to the intact form of SAFA-FSH, consisting of the heavy and light chains, was observed at approximately 80 kDa, higher than the theoretical size of 71.19 kDa (heavy chain: 34.109 kDa, light chain: 37.081 kDa).

[0243] 2-2. Purity assay To measure the purity of the recombinant fusion protein according to the embodiment prepared in Example 1, SE-HPLC was performed on it. First, an HPLC apparatus equipped with a TSKgel G3000SWXL 7.8 × 300 mm column (Tosoh Bioscience, Japan) and an Alliance HPLC system (Waters, Milford, MA) was equilibrated with 200 mM sodium phosphate pH 7.0 buffer containing 100 mM NaCl. The sample to be analyzed was prepared by dilution with 200 mM sodium phosphate, 100 mM NaCl, and pH 7.0 buffer, and 50 μg of the prepared sample was loaded onto the column. SE-HPLC analysis was performed for 40 minutes under conditions of a flow rate of 0.5 ml / min and a maximum pressure of 1,000 psi, and the purity was measured at a wavelength of A280 nm.

[0244] As a result, as shown in FIG. 4, it was confirmed that the SAFA-FSH protein had a purity of 99% or more.

[0245] 2-3.Isoelectric point analysis To measure the isoelectric point of the recombinant fusion protein according to the embodiment prepared in Example 1, isoelectric focusing (IEF) analysis was performed. Specifically, analysis was performed using a pH 3-10 IEF gel (Invitrogen™ Novex™ pH 3-10 IEF Protein Gel, ThermoFisher Scientific, USA) and capillary isoelectric focusing (cIEF) analysis was performed using a PA800 (SCIEX, USA) device. Five μg of sample was loaded onto the pH 3-10 IEF gel and run under the following conditions: 100 V for 1 hour, 200 V for 1 hour, and 500 V for 1 hour. The sample was fixed with 12% trichloroacetic acid (TCA) and stained with Coomassie Brilliant Blue (CBB). The protein pI was then analyzed using ChemiDoc MP Imagers (Bio-Rad). Furthermore, for cIEF analysis by using a neutral capillary, 50 μm id × 45 cm, and cIEF gel (polymer solution) (Beckman Coulter, Inc.), 10 μL of 10 mg / ml SAFA-FSH and cIEF peptide markers (pI 10, 7.0, 5.5, 4.1, Beckman Coulter, Inc.) were mixed and subsequently titrated against the neutral capillary to analyze the isoelectric point of SAFA-FSH protein using 32 Karat software.

[0246] As a result, as shown in Figure 5, a band and a peak were observed between pI 6.5 and pI 9.2, and these bands and peaks appeared due to N-linked glycans between the FSH α subunit and the FSH β subunit.

[0247] 2-4. Analysis of the molecular weight of light and heavy chains To measure the molecular weight of the recombinant fusion protein according to the embodiment prepared in Example 1, capillary electrophoresis and intact mass spectrometry were performed. To confirm the change in protein size caused by the N-linked glycans contained in the FSH α subunit and FSH β subunit, the protein was treated under reducing conditions with PNGase F (NEB, USA), which cleaves N-linked glycans, and the change in molecular weight was then confirmed using SDS-PAGE and capillary electrophoresis.

[0248] For SDS-PAGE, the PNGase F-treated SAFA-FSH sample was aliquoted onto a 4-15% Mini-protein TGX precast gel (15 wells, Bio-Rad) so that 2 μg was contained per well, and then electrophoresed in tris-glycine SDS running buffer at 150 V for 50 minutes. After electrophoresis, the SDS-PAGE gel was stained with EZ-Gel staining solution (DoGenBio, South Korea) for 1 hour and then destained with distilled water for 1 day to confirm the cleavage of N-linked glycans by PNGase F treatment.

[0249] Samples not treated with PNGase F and samples in which N-linked glycans were cleaved by PNGase F enzyme treatment were subjected to a bare fused silica capillary (50 μm ID × 57 cm, Beckman Coulter) and SDS-MW size standards (10 kDa–225 kDa, Beckman Coulter) using a PA800 instrument, and the protein size and molecular weight were confirmed using 32 Karat software.

[0250] As a result, as shown in Table 1 and Figure 6, it was confirmed that the mass of the light chain of the SAFA-FSH protein (SL335L + FSHα) was approximately 39.22 kDa, and the mass of the heavy chain of the protein (SL335H - FSHβ) was approximately 40.45 kDa.

[0251] [Table 1]

[0252] Furthermore, intact mass spectrometry was performed under reducing conditions (20 mM DTT, 37 °C) using an ACQUTY I-Class UPLC (Waters) and a SYNAPT G2-SI MS / MS system (Waters). Results were analyzed using Flexanalysis Flexcontrol software version 3.0.

[0253] As a result, as shown in Table 2 and Figure 7, it was confirmed that the mass of the light chain of the SAFA-FSH protein (SL335L + FSHα) was approximately 37.94 kDa, and the mass of the heavy chain of the protein (SL335H - FSHβ) was approximately 41.74 kDa.

[0254] [Table 2]

[0255] 2-5. Sequencing To determine the sequence of the recombinant fusion protein according to the embodiment prepared in Example 1, N-terminal sequencing and peptide mapping analysis of the heavy and light chains of the protein were performed. Specifically, N-terminal sequencing and peptide mapping analysis of the heavy and light chains of the SAFA-FSH protein were performed using a Dionex UHPLC (Thermo Fisher Scientific) and a Q-TOF 5600+ MS / MS system (AB SCIEX, CA, USA). An Acquity UPLC® BEH130 C4, 1.7 μm column was used for the measurement using acetonitrile (ACN; JT Baker) as the mobile phase at a flow rate of 0.3 ml / min.

[0256] As a result, as shown in FIG. 8, the N-terminal sequences of the heavy and light chains of the SAFA-FSH protein were confirmed to be 100%.

[0257] Example 3. Confirmation of the biological properties of SAFA-FSH protein 3-1. Analysis of binding ability to human serum albumin The binding ability of SAFA-FSH protein to human serum albumin was analyzed using Bio-Layer Interferometry (BLI) and the Octet® BLI system (Octet® Red, Sartorius, Germany). Human serum albumin (Sigma) was immobilized on an AR2G biosensor chip (Sartorius, Germany) at a concentration of 30 μg / mL in 10 mM acetate buffer, pH 5.0. SAFA-FSH protein was added at concentrations of 6.25 nM, 3.125 nM, 1.56 nM, and 0.78 nM (association: 600 s, dissociation: 900 s) and the binding ability of both proteins was confirmed under conditions of pH 7.4 or pH 6.0.

[0258] [Table 3]

[0259] As a result, as shown in Table 3 and FIG. 9, it was confirmed that SAFA-FSH protein and human serum albumin can form an effective bond at pH 7.4 or pH 6.0.

[0260] 3-2. Confirmation of binding to FSH receptor Using a human FSHR stable cell line (HEK293, Creative Biogene, USA) expressing the human FSH receptor on its surface, the binding between SAFA-FSH protein and the FSH receptor was analyzed using a CytoFLEX flow cytometer (Beckman Coulter, USA).

[0261] First, human FSHR stable cells were cultured in MACS buffer (PBS containing 0.5% BSA and 2 mM EDTA) at 1 × 10 6 The cells were lysed at a concentration of 1.0 × 10 cells / ml and then added to 100 μl (1.0 × 10 5The cells were added to a 1.7 ml tube in a volume of 100 μl (cells / test). The cells were then centrifuged at 500 × g for 5 minutes at 4 °C. After removing the supernatant from the 1.7 ml tube, 100 μl each of human FSHR APC-labeled antibody (R&D systems, USA) and APC mouse IgG2a isotype control (R&D systems, USA), diluted 1:25 and 1:100 with MACS buffer, were added to the same tube and allowed to react for 30 minutes at 4 °C. 500 μl of MACS buffer was added to the tube, and the cells were centrifuged at 500 × g for 5 minutes at 4 °C. The supernatant was then removed to remove unbound antibody. 200 μl of 0.4% PFA was added to the tube, and the cells were then loosened and fixed using a pipette. Human FSHR expression was confirmed by measuring APC sensitivity using a CytoFLEX device. As shown in Figure 10, high levels of human FSH receptor expression were confirmed on the surface of the human FSHR stable cells.

[0262] Subsequently, the binding between SAFA-FSH protein and human FSHR was confirmed using a human FSHR stable cell line in which the expression of human FSHR was confirmed. Specifically, human FSHR stable cells were placed in MACS buffer (PBS containing 0.5% BSA and 2 mM EDTA) at 1 × 10 6 The cells were lysed at a concentration of 1.0 × 10 cells / ml and then added to 100 μl (1.0 × 10 5A 1.7 ml tube was added to a 1.7 ml tube in a volume of 1000 cells / test. The cells were then centrifuged at 500 × g for 5 minutes at 4 °C. After removing the supernatant from the 1.7 ml tube, 100 μl of each of SAFA-FSH protein and SL335 fragment diluted in MACS buffer to concentrations ranging from 1,000 nM to 0.064 nM was added to the same tube and incubated for 30 minutes at 4 °C. 500 μl of MACS buffer was added to the tube, and the cells were centrifuged at 500 × g for 5 minutes at 4 °C. The supernatant was then removed to remove unbound protein. 100 μl of goat anti-human IgG Fd-FITC antibody (Southern Biotech, USA) diluted 1:500 in MACS buffer was added to the tube, and the incubation was incubated for 30 minutes at 4 °C. 500 μl of MACS buffer was added to the tube, and the cells were centrifuged at 500 × g for 5 minutes at 4 °C. The supernatant was then removed to remove unbound antibody. 200 μl of 0.4% PFA was added to the tube, and then the cells were loosened and fixed using a pipette. The binding between SAFA-FSH protein and human FSHR was confirmed by measuring FITC sensitivity with a CytoFLEX device.

[0263] As a result, as shown in Figure 11, the SL335 fragment did not form an effective bond with the human FSH receptor present on the surface of FSHR stable cells, but the SAFA-FSH protein was able to form an effective bond with the human FSH receptor.

[0264] 3-3. Confirmation of cAMP regulatory ability The ability of SAFA-FSH protein to regulate cAMP was confirmed using the KGN cell line, a human ovarian granulosa cell line. Specifically, 1 mL of KGN cells was cultured at 3.0 × 10 in MEM + 10 FBS medium in a 12-well culture plate. 5The cells were cultured overnight at a concentration of 1000 viable cells / well / mL. After incubating the cells with 0.5 mM 3-isobutyl-1-methylxanthine for 15 minutes, 0.5 mM IBMX was added and the cells were incubated for 1 hour with SAFA-FSH protein, Gonal-F, or FSH IS at a concentration of 10 ng / ml or 303 pM in MEM medium supplemented with 1% FBS (NIBSC 08 / 282). cAMP levels were measured in the supernatant obtained after centrifugation of the cells at 500 × g for 5 minutes at 4°C using a cAMP enzyme immunoassay kit (R&D, USA).

[0265] [Table 4]

[0266] As a result, as shown in Table 4 and FIG. 12, it was confirmed that the SAFA-FSH protein can contribute to the regulation of cAMP levels by acting on the human ovarian granulosa cell line.

[0267] Example 4. Pharmacokinetic evaluation of SAFA-FSH protein The absorption, distribution, biotransformation, and excretion of the recombinant fusion protein according to the embodiment prepared in Example 1 were confirmed by pharmacokinetic evaluation. Specifically, 17 healthy 7-week-old male rats (specific pathogen-free (SPF) rats, Hsd: Sprague Dawley® SDR®) were purchased from Koatech (South Korea) and acclimated for 7 days. Healthy individuals among these rats were used in this experiment. In each experimental group, five rats were intravenously administered a single dose of the SAFA-FSH protein and Gonal-F protein (follitropin alpha, rhFSH) of Example 1. The SAFA-FSH protein was administered at a dose of 200 μg / kg or 600 μg / kg, and the Gonal-F protein was administered at a dose of 88 μg / kg.

[0268] Then, 0.5 ml of whole blood was collected from the jugular vein according to a predetermined blood collection schedule (single intravenous administration of SAFA-FSH at doses of 200 μg / kg and 600 μg / kg, before administration of the test substance (0), and 5, 15, 30, 1, 1.5, 3, 5, 10, 24, 48, 72, 120, 144, and 168 hours after administration of the test substance (15 time points in total); single intravenous administration of Gonal-F at a dose of 88 μg / kg, before administration of the test substance (0), and 5, 15, 30, 1, 1.5, 3, 5, 10, 24, 48, and 72 hours after administration of the test substance (12 time points in total)). Serum was separated by centrifugation at 13,000 rpm for 2 minutes and stored in a deep freezer (-70°C). Next, the protein concentration in the plasma was quantitatively analyzed by ELISA.

[0269] As a result, as shown in Figures 13 to 15, it was confirmed that the maximum serum concentration (Cmax) of SAFA-FSH protein was equivalent to or approximately 1.2 times higher than that of Gonal-F protein. Furthermore, the elimination half-life of SAFA-FSH protein was 27.1 hours or 10.4 hours, which was 2.6 times longer than that of Gonal-F protein. Based on these experimental results, it can be seen that the recombinant fusion protein increases the elimination half-life in the body and provides convenience to patients by allowing for longer drug administration intervals.

[0270] Example 5. Pharmacodynamic evaluation of SAFA-FSH protein The pharmacological effect of the recombinant fusion protein according to the embodiment prepared in Example 1 on the treatment of female infertility was confirmed by pharmacodynamic evaluation. Specifically, using 20 two-week-old immature female Sprague Dawley rats (30 g to 40 g), the SAFA-FSH protein and Gonal-F protein of Example 1 were subcutaneously administered to the rats under the experimental conditions shown in Table 5, and follicle-stimulating ovarian growth was confirmed. From the time of administration of the test substance until day 7, the ovaries were removed from the rats and weighed to confirm ovarian growth due to the SAFA-FSH protein.

[0271] [Table 5]

[0272] As a result, as shown in Figure 16, among the Gonal-F protein administration groups, the hFSH 240 / rat ED group induced effective ovarian growth compared to the control group, while the hFSH 240 / rat E3D group, which had a longer administration interval, had no effect on ovarian growth. On the other hand, the SAFA-FSH protein administration group according to this example induced effective ovarian growth despite the long administration interval of 3 days. In particular, ovarian growth was induced at a level equivalent to or approximately three times higher than that of the hFSH 240 / rat ED group.

[0273] To further confirm the efficacy of SAFA-FSH protein, a pharmacodynamic study was conducted using an experimental animal model of hypogonadism induced by administering Diphereline PR injection to male Sprague-Dawley rats. Specifically, 5-week-old male rats were divided into seven groups of five or six rats per group. Body weight gain was measured by recording the weight of the experimental animals at 7-day intervals. Diphereline PR injection (Ipsen, Paris, France) was administered subcutaneously at a dose of 5 mg / kg to all experimental groups except Group 1 at the start of the experiment and three weeks after the experiment. All experimental groups except for Groups 1 and 2 received subcutaneous administration of human chorionic gonadotropin (hCG) protein (LG Chem, South Korea) at a dose of 20 IU / kg three times a week for 9 weeks. Recombinant FSH protein was administered subcutaneously at a dose of 1.85 μg / kg (25 IU / kg) three times a week (Group 4) or once every 5 days (Group 5). SAFA-FSH protein was administered subcutaneously at a dose of 9.87 μg / kg once every 5 days (Group 6) or once every 10 days (Group 7). To measure testosterone levels in each experimental group, rats were anesthetized and blood samples were collected weekly. The collected blood was centrifuged at 3,000 rpm for 10 minutes, and serum was separated. Testosterone levels were measured using electrochemiluminescence at Seoul Clinical Laboratories, South Korea. Nine weeks after administration of the test substances, the rats were sacrificed. To measure sperm counts from sacrificed rats, the cauda epididymis was placed on a 6 cm plate containing 10 mL DPBS, minced with a scalpel, and incubated in saline for 15 minutes at 37°C and 5% CO2. It was then heat-treated at 60°C for 1 minute, followed by immediate cooling at 24°C. Appropriately diluted samples were used to count sperm using a Neubauer hemocytometer.

[0274] As a result, no differences in body weight were observed between the groups treated with the test substance throughout the experimental period. Furthermore, as shown in Figure 17, mean serum testosterone levels from 1 to 4 weeks after administration of the test substance were unchanged among all test groups (Figure 17A). Meanwhile, mean serum testosterone levels increased in Group 1, which did not receive diferrelin injections, from 5 to 8 weeks after administration of the test substance, whereas mean serum testosterone levels were lower in Group 2, which received diferrelin injections. Groups 3, 4, 5, 6, and 7, which were treated with hCG, showed increased mean serum testosterone levels compared to Group 2, which did not receive hCG (Figure 17B). Furthermore, mean serum testosterone levels in Group 2 remained lower than those in Group 1 from 9 to 12 weeks after administration of the test substance. Groups 3, 4, 5, 6, and 7 showed increased mean serum testosterone levels compared to Group 2. Notably, the mean serum testosterone levels in Groups 5 and 6 were significantly higher than those in Group 1 (Figure 17C).

[0275] Furthermore, at 9 weeks after the administration of hCG and recombinant FSH (rFSH), rats were sacrificed and their testes were weighed. Testis weights in Group 2 were lower than those in Group 1, whereas those in Groups 3, 4, 5, 6, and 7 were higher than those in Group 2 (Figure 18A). Furthermore, the testis index (i.e., testis / body weight ratio) showed similar results to those for testis weight (Figure 18B). The total sperm count in Group 2 was reduced by 68% compared to Group 1, and the sperm count in Group 3 did not recover compared to Group 2. Furthermore, sperm counts were restored in Group 4, which received rFSH three times every 7 days, and Group 5, which received rFSH once every 5 days. Sperm counts were also restored in Group 6, which received SAFA-FSH once every 5 days, and Group 7, which received SAFA-FSH once every 10 days (Figures 18C and 18D).

[0276] In view of the above results, it can be seen that SAFA-FSH (recombinant fusion protein) can lengthen the drug administration interval and achieve a high therapeutic effect.

[0277] According to one or more embodiments, a recombinant fusion protein comprising an antigen-binding fragment that binds to serum albumin and human follicle-stimulating hormone has improved pharmacokinetic properties, including increased in vivo half-life, and can exhibit continuous therapeutic effects, such as ovarian growth promotion.

[0278] Therefore, the recombinant fusion protein can be used as the active ingredient in human follicle-stimulating hormone preparations for the treatment of infertility.

[0279] It should be understood that the embodiments described herein should be considered for illustrative purposes only, and not for purposes of limitation. The description of a feature or aspect within each embodiment should typically be considered applicable to other similar features or aspects in other embodiments. Although one or more embodiments have been described with reference to the drawings, it will be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the present disclosure, as defined by the following claims.

[0280] All of the various aspects, embodiments, and options described herein can be combined in any and all variations. All publications, patents, and patent applications mentioned in this specification are incorporated by reference herein to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A recombinant fusion protein comprising (a) human follicle-stimulating hormone (hFSH) comprising an hFSH α subunit and an hFSH β subunit, and (b) an antigen-binding fragment (Fab) that binds to serum albumin.

2. 2. The recombinant fusion protein of claim 1, wherein the hFSH alpha subunit and the hFSH beta subunit are linked to the terminal regions of the Fab.

3. 3. The recombinant fusion protein of claim 1 or 2, wherein the hFSH alpha subunit and the hFSH beta subunit are independently linked to the ends of the heavy chain constant 1 domain and the light chain constant domain of the Fab.

4. The recombinant fusion protein of any one of claims 1 to 3, wherein the hFSH alpha subunit and the hFSH beta subunit are independently linked by linkers to the ends of the heavy chain constant 1 domain and the light chain constant domain of the Fab.

5. 5. The recombinant fusion protein of any one of claims 1 to 4, wherein the hFSH alpha subunit is linked to the end of the light chain constant domain and the hFSH beta subunit is linked to the end of the heavy chain constant 1 domain of the Fab.

6. 6. The recombinant fusion protein of claim 4 or 5, wherein the linker comprises 1 to 50 amino acids.

7. 7. The recombinant fusion protein of claim 6, wherein the linker comprises an amino acid sequence of any one of SEQ ID NOs: 16, 70-85, and 88.

8. 8. The recombinant fusion protein of any one of claims 1 to 7, wherein the hFSH alpha subunit comprises an amino acid sequence having at least 90% identity to SEQ ID NO:

1.

9. 9. The recombinant fusion protein of any one of claims 1 to 8, wherein the hFSH alpha subunit comprises the amino acid sequence of SEQ ID NO:

1.

10. 10. The recombinant fusion protein of any one of claims 1 to 9, wherein the hFSH beta subunit comprises an amino acid sequence having at least 90% identity to SEQ ID NO:

4.

11. 11. The recombinant fusion protein of any one of claims 1 to 10, wherein the hFSH beta subunit comprises the amino acid sequence of SEQ ID NO:

4.

12. Fab, (a) a heavy chain complementarity-determining domain 1 (CDR1) comprising the amino acid sequence of SYGIS (SEQ ID NO:22); a heavy chain complementarity-determining domain 2 (CDR2) comprising the amino acid sequence a heavy chain complementarity-determining domain 3 (CDR3) comprising the amino acid sequence of (b) a heavy chain CDR1 comprising the amino acid sequence of SYGIS (SEQ ID NO:22); a heavy chain CDR2 comprising the amino acid sequence of a heavy chain CDR3 comprising the amino acid sequence of: (c) a heavy chain CDR1 comprising the amino acid sequence of NYGIH (SEQ ID NO:26); a heavy chain CDR2 comprising the amino acid sequence of a heavy chain CDR3 comprising the amino acid sequence of: (d) a heavy chain CDR1 comprising the amino acid sequence of SYAMS (SEQ ID NO:29); a heavy chain CDR2 comprising the amino acid sequence of a heavy chain CDR3 comprising the amino acid sequence of: (e) a heavy chain CDR1 comprising the amino acid sequence of AYWIA (SEQ ID NO:32); a heavy chain CDR2 comprising the amino acid sequence of a heavy chain CDR3 comprising the amino acid sequence of LYSGSYSP (SEQ ID NO:34); or (f) a heavy chain CDR1 comprising the amino acid sequence of AYSMN (SEQ ID NO:35); a heavy chain CDR2 comprising the amino acid sequence of heavy chain CDR3 comprising the amino acid sequence a heavy chain comprising a heavy chain variable domain comprising (g) a light chain CDR1 comprising the amino acid sequence a light chain CDR2 comprising the amino acid sequence of GASRLES (SEQ ID NO:39); and a light chain CDR3 comprising the amino acid sequence of QQSDSVPVT (SEQ ID NO:40); (h) a light chain CDR1 comprising the amino acid sequence a light chain CDR2 comprising the amino acid sequence of AASSLQS (SEQ ID NO:42), and a light chain CDR3 comprising the amino acid sequence of: (i) a light chain CDR1 comprising the amino acid sequence a light chain CDR2 comprising the amino acid sequence of DASNRAT (SEQ ID NO:45); and a light chain CDR3 comprising the amino acid sequence of: (j) a light chain CDR1 comprising the amino acid sequence a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:48); and a light chain CDR3 comprising the amino acid sequence of QQYGSSPRT (SEQ ID NO:49); (k) a light chain CDR1 comprising the amino acid sequence a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:48); and a light chain CDR3 comprising the amino acid sequence of QKYSSYPLT (SEQ ID NO:51); or (l) a light chain CDR1 comprising the amino acid sequence a light chain CDR2 comprising the amino acid sequence of GASTGAT (SEQ ID NO:53), and a light chain CDR3 comprising the amino acid sequence a light chain comprising a light chain variable domain comprising 12. The recombinant fusion protein of any one of claims 1 to 11, comprising:

13. the heavy chain variable domain comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 35, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and 13. The recombinant fusion protein of any one of claims 1 to 12, wherein the light chain variable domain comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

54.

14. 14. The recombinant fusion protein of any one of claims 1 to 13, wherein the heavy chain variable domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 55, 56, 57, 58, 59, or 60.

15. 15. The recombinant fusion protein of any one of claims 1 to 14, wherein said heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:

60.

16. 16. The recombinant fusion protein of any one of claims 1 to 15, wherein the light chain variable domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 61, 62, 63, 64, 65, 66, or 67.

17. 17. The recombinant fusion protein of any one of claims 1 to 16, wherein said light chain variable domain comprises the amino acid sequence of SEQ ID NO:

67.

18. 18. The recombinant fusion protein of any one of claims 1 to 17, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 55, 56, 57, 58, 59, or 60, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 61, 62, 63, 64, 65, 66, or 67.

19. 19. The recombinant fusion protein of any one of claims 1 to 18, wherein said heavy chain constant domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:

68.

20. 20. The recombinant fusion protein of any one of claims 1 to 19, wherein said light chain constant domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 69 or 91.

21. 21. The recombinant fusion protein of any one of claims 1 to 20, wherein said Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:6 and a light chain comprising the amino acid sequence of SEQ ID NO:

91.

22. 22. The recombinant fusion protein of any one of claims 1 to 21, wherein said Fab comprises a heavy chain constant 1 domain comprising the amino acid sequence of SEQ ID NO: 68 linked to said heavy chain variable domain; and a light chain constant domain comprising the amino acid sequence of SEQ ID NO: 69 or 91 linked to said light chain variable domain.

23. A nucleic acid encoding the recombinant fusion protein of any one of claims 1 to 22.

24. 24. An expression vector comprising the nucleic acid of claim 23.

25. A cell transformed with the expression vector of claim 24.

26. 23. A composition comprising the recombinant fusion protein of any one of claims 1 to 22 and a carrier.

27. 23. A pharmaceutical composition comprising the recombinant fusion protein of any one of claims 1 to 22 and a pharmaceutically acceptable carrier.

28. 28. A kit comprising the composition of claim 26 or 27 and a label containing instructions for use.

29. 28. A method of inducing superovulation in a subject in need thereof, comprising applying the pharmaceutical composition of claim 27 to in vitro fertilization-embryo transfer (IVF-ET), gamete intrafallopian tube transfer (GIFT), zygote intrafallopian tube transfer (ZIFT), intracytoplasmic sperm injection (ICSI), or in vitro fertilization.

30. 30. A method of treating anovulation, hypogonadism, or polycystic ovary syndrome in a subject in need thereof, comprising administering to said subject the pharmaceutical composition of claim 27.

31. 31. The method of claim 29 or 30, wherein the composition is administered to the subject subcutaneously or intramuscularly.

32. A long-acting human follicle-stimulating hormone (hFSH) composition comprising as an active ingredient a recombinant fusion protein according to any one of claims 1 to 22.

33. 33. The long-acting hFSH formulation of claim 32, applied in in vitro fertilization-embryo transfer (IVF-ET), gamete intrafallopian tube transfer (GIFT), zygote intrafallopian tube transfer (ZIFT), intracytoplasmic sperm injection (ICSI), or in vitro fertilization to induce superovulation in a subject.

34. 34. The long-acting hFSH formulation of claim 33, used for the treatment of anovulation, hypogonadism, or polycystic ovary syndrome.

35. 34. The long-acting hFSH formulation of claim 33, which is administered subcutaneously or intramuscularly.

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