CN106496331B - FSH-Fc fusion protein and preparation method and application thereof - Google Patents
FSH-Fc fusion protein and preparation method and application thereof Download PDFInfo
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- CN106496331B CN106496331B CN201610982738.1A CN201610982738A CN106496331B CN 106496331 B CN106496331 B CN 106496331B CN 201610982738 A CN201610982738 A CN 201610982738A CN 106496331 B CN106496331 B CN 106496331B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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- Gastroenterology & Hepatology (AREA)
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Abstract
The invention belongs to the field of human reproductive disease treatment and assisted reproduction, and relates to a novel long-acting gonadotropin (FSH) and a preparation method thereof, and application thereof in assisted reproduction, wherein the FSH fusion protein is folded into a stable heterodimer form under the double actions of non-covalent combination between an FSH α subunit and an FSH β subunit and a locking and buckling Fc chain locking structure.
Description
Technical Field
The invention belongs to the field of human reproductive disease treatment and assisted reproduction. The present invention relates to gonadotropin growth hormone. More particularly, the present invention relates to a novel long-acting gonadotropin, a process for its preparation, and its use in assisted reproduction.
Background
FSH is a glycosylated protein synthesized and secreted by anterior pituitary basophilic cells, natural FSH consists of α and β subunits, of which the α subunit is identical to the α subunit of chorionic Hormone (HCG) secreted by the pituitary gland, and the β subunit has its specificity, which is the determining factor for the bioactivity of FSH.it is known that the α subunit of FSH consists of 92-96 amino acids, the β subunit consists of 109-115 amino acids, the correct higher structure between the two subunits is maintained by internal disulfide bonds, and in addition N-glycosylation has a greater influence on the formation of the correct higher structure, FSLH is also strongly associated with the formation of the correct higher structure, FSH is mainly associated with FSLH receptor through the C-terminal of β subunit, FSLH produces two signaling effects, one is an activating aromatase, the other effect is to induce the production of the receptor for luteinizing Hormone, and the cell receptor for the ovarian Follicle Stimulating Hormone (FSLH) and the ovarian Follicle Stimulating Hormone secretion, thus the ovarian Follicle Stimulating Hormone and ovarian Follicle Stimulating Hormone, which are the combined with the ovarian growth promoting action of ovarian Follicle Stimulating Hormone in vivo, Stimulating Hormone, thus the ovarian Follicle Stimulating Hormone, Stimulating Hormone, Stimulating Hormone, Stimulating Hormone, Stimulating Hormone.
FSH can be isolated from pituitary or urine (EP322, 438) or prepared recombinantly (US5,639, 640, US5,156, 957, US4,923, 805, US4,840, 896, EP211,894). The purity of the recombinant human FSH is better than that of the extracted FSH, the curative effect of the recombinant human FSH is not greatly different from that of the extracted FSH, but both types of FSH need to be continuously administrated for 8-10 days to stimulate the follicular development of women, and the FSH is subcutaneously injected for at least 1 time every day to bring much inconvenience and pain to patients. Long acting recombinant FSH is one direction to solve these problems.
The current design of a corresponding product (elonva) developed and marketed in 2010 by merck, germany, which has the bioactivity of natural FSH and a half-life of 69h in humans, but due to the problems of immunogenicity, etc., the clinical administration scheme is changed to one week for the first administration and then to 3 days for continuous administration, i.e., the design of long-acting FSH still faces great difficulties and needs to be improved.
Fusion of FSH with albumin or Fc fragment of IgG is a common technique for increasing the in vivo half-life of active proteinsThe patent publication US0, 186,662 discloses that the half-life in the rat body is prolonged to 60 hours by constructing homo-and heterodimers of FSH-Fc fusion protein, while the specific activity is also improved, but it is difficult to completely separate the homo-and heterodimers of the by-products FSH α or FSH β from the target product FSH αβ heterodimer when recombinant FSH is prepared in this design, unless a purification tag such as HIS is additionally introduced, and the introduction of the purification tag limits the application of clinical treatment2The homodimer of (a) and thus the co-product interference facing homodimer is also required in constructing recombinant expression cell lines and purifying products, making the quality control of drugs more challenging.
The invention aims to provide an improved FSH-Fc fusion protein and a preparation method thereof, so as to obtain a long-acting heterodimer FSH-Fc fusion protein with the activity and uniform quality.
Disclosure of Invention
The inventive idea and principle of the present invention is that referring to the characteristic that the natural FSH structure is fused between the α and β subunits via non-covalent tight linkage to form heterodimers, the different single-chain Fc fragments are generated by different site-directed mutagenesis of certain amino acids on the binding surface of the natural Fc duplex, such that the amino acids in the CH3domain on one Fc chain are mutated to amino acids with small side chains, forming a "padlock" structure, and the corresponding amino acids on the other Fc chain are mutated to amino acids with large side chains, forming a "padlock" structure, which results in dimerization of two heteromonohol fragments, which is more prone to form heterodimers, thus the stability of the fusion between the heteromono Fc fragments is achieved by direct Fc-subunit fusion of the FSH α and β subunits via the non-covalent tight linkage, or by direct Fc-linker fusion of the natural FSH subunits with the N-Fc-linker "(i-Fc-linker) is directly fused to the N-Fc-subunit of the FSH subunit, i chain-Fc-linker-Fc-linker-Fc-linker-Fc-linker-Fc-linker-molecule, which is directly fused to form a loop-Fc-loop-Fc-loop-Fc-dimerization-protein under the same-dimerization-induced by a direct-dimerization process, which is directly-dimerization-induced by direct-dimerization-linking process, which is directly under the same-induced by direct.
The greatest difference between the present invention and the FSH-Fc fusion proteins of the prior art (US20050186662 and CN201210476665) is that FSH α and β subunits are fused to the N-terminus of the "padlock" and "padlock" Fc chains, respectively, or vice versa.in the prior art, the non-covalent binding dimerization between the α and β subunits of FSH is not combined with the Fc "locker" dimerization.the principle of the present invention is based on, on one hand, the CH3 structural design concept of "knob-int-holes" for the heavy chain ligation.Ridgway JB, Presta LG, Carp.Protein Eng.1996 Jul; 9 latch 7: 21. the knob-interlocking chains are incorporated herein by reference. the design of knob-button antibodies in particular in the context of heavy chains which, specifically, the heavy chain of antibodies, which form a heavy chain heterodimers with a smaller than the CH3, or which forms a heavy chain heterodimers with a side chains which are formed by the reverse side chain binding of the amino acid sequence of the CH3, or by the reverse side chain of the amino acid sequence of the CH-int-linker peptide.
In general, when bispecific antibodies are prepared using recombinant expression systems, by direct co-transfection of antibody A and antibody B genes, the proportion of spontaneously formed heterodimers AB in the expression product is less than 25%, whereas the proportion of AB heterodimers produced by the CH3 mutation is increased to more than 50%. To further increase the heterodimer ratio, the heterodimer ratio obtained by transiently transfected cells can be increased to 90% by optimizing the transfection-encoding DNA ratio, but in stably transfected cells, it is difficult to obtain a heterodimeric bispecific antibody of more than 50%. In industrial production, it is often necessary to use a stably expressing cell line to produce a recombinant antibody. Therefore, in the preparation of bispecific antibodies, additional mutations need to be introduced in the Fab fragment to increase heterodimer stability (progression in overlapping the chain association assay in biphenically modified IgG antibodies. Klein C et al, MAbs.2012; 4(6): 653-63.).
In the present invention, since there is a natural non-covalent tight association between the FSH- α subunit and the FSH- β subunit, and specific interaction between them promotes β 1 β heterodimerization, the α subunit of FSH is fused directly or indirectly via a linking element to the N-terminus of the "padlock Fc" chain, while the β subunit of FSH is fused directly or indirectly via a linking element to the N-terminus of the "padlock Fc" chain, or vice versa, the α subunit of FSH is fused directly or indirectly via a linking element to the N-terminus of the "padlock Fc" chain, while the β subunit of FSH is fused directly or indirectly via a linking element to the N-terminus of the "padlock Fc" chain, so that molecular interactions exist that stabilize the heterodimer structure between the N-terminal N- α subunit of the fusion protein and the FSH- β subunit, between the C-terminal "padlock" Fc chain and the "Fc chain fragment of the" Fc chain, under both interactions, the structural stability of FSH α -84-FSH β -Fck is improved, the stability of the FSH- β -Fck dimer is improved when the FSH-8225-25-b-t-.
In one aspect, the present invention provides a long-acting FSH fusion protein, said FSH fusion protein comprising two different peptide chains, one of which is FSH α subunit directly or indirectly linked to peptide chain FSH α -Fch formed at the N-terminus of the padlock Fc chain (Fch) via a linking element, the other of which is FSH β subunit directly or indirectly linked to FSH β -fck formed at the N-terminus of the padlock Fc chain (Fck) via a linking element, the two peptide chains, i.e. FSH α -Fch and FSH β -Fck, fold into a stable heterodimer in the form of FSH α -Fch-FSH β -Fck under the dual action between the non-covalent binding between the FSH α subunit and the FSH β subunit and the latch structure on the padlock Fc chain and the padlock Fc chain CH 3.
Or vice versa, in the long-acting FSH fusion protein of the present invention, one of the FSH α subunits is directly or indirectly linked to the peptide chain FSH α -Fck formed at the N-terminus of the buckled Fc chain (Fck) via a linking element, and the other is the peptide chain FSH β -fch formed at the N-terminus of the padlock Fc (fch) fragment via a FSH β subunit which is also linked to the N-terminus of the padlock Fc (fch) fragment via a linking element, so as to form another stable form of heterodimer FSH α -Fck-FSH β -Fch under the dual actions of the above-mentioned non-covalent binding action and padlock structure.
The FSH in the present invention is mammalian FSH, preferably human FSH.
The padlock Fc chain has an amino acid sequence shown in SEQ ID NO. 1. Or the padlock Fc chain comprises an amino acid sequence obtained by deleting, adding or substituting 1, 2 or 3 residues on SEQ ID NO. 1.
The button Fc chain has an amino acid sequence shown in SEQ ID NO. 2. Or the buckled Fc chain comprises an amino acid sequence obtained by deleting, adding or substituting 1, 2 or 3 residues on SEQ ID NO. 2.
The peptide chain FSH α -Fch has an amino acid sequence shown in SEQ ID NO.3, or an amino acid sequence obtained by deleting, adding or substituting 1, 2 or 3 residues on the SEQ ID NO. 3.
The peptide chain FSH β -Fck has an amino acid sequence shown in SEQ ID NO.4 or an amino acid sequence obtained by deleting, adding or substituting 1, 2 or 3 residues on the SEQ ID NO. 4.
In a specific embodiment of the invention, the long-acting FSH fusion protein comprises two different peptide chains FSH α -Fch and FSH β -Fck, wherein said FSH α -Fch has the amino acid sequence shown in SEQ ID No.3, and said FSH β -Fck has the amino acid sequence shown in SEQ ID No. 4.
The present invention also provides a vector comprising a cDNA encoding the peptide chain FSH α -Fch. in a specific embodiment of the present invention, the peptide chain FSH α -Fch has the amino acid sequence shown in SEQ ID NO. 3.
The present invention also provides a vector comprising a cDNA encoding the peptide chain FSH α -Fck in a specific embodiment of the present invention, the peptide chain FSH β -Fck has the amino acid sequence shown in SEQ ID NO. 4.
The invention also provides a vector which contains the cDNA coding SEQ ID NO.3 and the cDNA coding SEQ ID NO. 4. By expressing the FSH fusion protein in mammalian cells such as NS0 cells, CHO cells, HEK293 cells and the like, the invention realizes the aim of prolonging the in vivo half-life of FSH on the premise of keeping the bioactivity of FSH, has higher purity of an expression product, is easy to prepare, has more feasibility in a production process, and further provides a method which can reduce the administration frequency and still can play the therapeutic role of FSH in reproduction-related diseases.
In another aspect, the invention provides the use of the long-acting FSH fusion protein for the preparation of a medicament for the treatment of polycystic ovary syndrome or other anovulatory disease or disorder.
In another aspect, the invention provides the use of the long-acting FSH fusion protein for the preparation of a pro-ovarian hyperstimulation for use in assisted reproductive techniques.
In another aspect, the present invention provides a method for preparing a long-acting FSH fusion protein, comprising (1) synthesizing two different peptide chain-encoding cDNA sequences, one of which is peptide chain FSH α -Fch in which FSH α subunit is directly or indirectly linked to the N-terminus of a padlock Fc chain (Fch) through a linking element, and the other of which is peptide chain FSH β -Fck in which FSH β subunit is directly or indirectly linked to the N-terminus of a padlock Fc chain (Fck) through a linking element, (2) introducing the cDNA sequences encoding peptide chain FSH α -Fch and FSH β -Fck into a mammalian expression vector in an enzyme-cleaved linkage manner to obtain a recombinant double expression vector, and (3) transforming mammalian cells with the recombinant double expression vector obtained in step (2), and recovering and purifying the obtained FSH fusion protein.
The FSH-Fc fusion protein of the present invention can be purified from host cells by standard experimental means. Including, but not limited to, affinity purification with protein a.
Fc in the context of the present invention refers to the CH2 and CH3 fragments of the constant regions of human immunoglobulin chains, particularly the constant regions of immunoglobulin heavy chains. The Fc region of a natural human immunoglobulin varies from individual to individual with respect to amino acid differences. The human immunoglobulin Fc of the present invention also includes all changes to the amino acids of the native human immunoglobulin Fc sequence, including, but not limited to, changes to certain amino acids local to the Fc region, such as mutations including certain amino acid positions in the hinge region, certain amino acid mutations that reduce immune function, or other nonsense mutations.
In a particular embodiment, the human immunoglobulin Fc used in the present invention comprises at least one immunoglobulin hinge region, a CH2 domain and a CH3domain, in particular a mutant of human IgG4 Fc.
Mammalian host cells to which the present invention relates include, but are not limited to, CHO, HEK 293. The host cell may also be a yeast or prokaryotic cell such as e.coli.
Therefore, the present invention provides a method for increasing the half-life of FSH in vivo and facilitating the preparation of FSH-FC fusion proteins, while maintaining the bioactivity of FSH.
Drawings
FIG. 1 is a schematic diagram of a heterodimer of FSH α -Fch-FSH β -Fck fusion protein, subunit 1.α, subunit 2.β, fragment 3.IgGCH2, variant fragment 4. padlock IgG CH3, and variant fragment 5. padlock IgG CH 3.
FIG. 2 is a graph showing the results of the determination of the biological activity of FSH α -Fch-FSH β -Fck.
Examples
Example 1 construction of FSH α -Fch-FSH β -Fck heterodimer expression plasmid
Coding DNA sequences of FSH α -Fch (SEQ ID NO:3) and FSH β -Fck (SEQ ID NO:4) were synthesized based on the amino acid sequence of FSH α subunit (Gene bank No. NP-000726.1), human FSH β subunit amino acid sequence (Gene bank No. NP-000501.1), and the amino acid sequence of the latticed Fc chain shown in SEQ ID NO:1 (containing T148S, L150A, Y189V mutations, forming a smaller side chain structure) and the amino acid sequence of the latticed Fc chain shown in SEQ ID NO:2 (containing T148W mutations, forming a larger side chain structure), respectively, and the coding DNA sequences of FSH α -Fch (SEQ ID NO:3) and FSH β -Fck (SEQ ID NO:4) were introduced with restriction endonuclease SapI at the upstream and downstream of the coding sequences, respectively.
After the coding DNA is synthesized, FSH α -Fch is inserted into an expression vector pQKPX1 by utilizing a SapI restriction enzyme cutting site to generate a pQKPX1- α plasmid, FSH β -Fck is inserted into a pQKPX2 vector, an FSH β -Fck expression element in the pQKPX2 vector is cut off by utilizing NotI and PvuI restriction enzyme cutting sites and is inserted into a pQKPX1- α plasmid to generate a double expression plasmid pQKPX- αβ.
The successfully constructed double expression plasmid pQKPX- αβ was taken and digested with PvuI restriction enzyme, the digested product was separated and purified by 0.8% agarose electrophoresis, and a linearized DNA fragment of about 11000bp in size was recovered and prepared for transfection of CHO-K1 cells.
Example 2 expression and purification of FSH α -Fch-FSH β -Fck heterodimer protein
2.1 Stable expression of FSH α -Fch-FSH β -Fck heterodimer
CHO-K1(ATCC, CCL-61) cells acclimated to serum-free medium at 5X 106Inoculating cells/ml into 30ml CD-CHO culture medium, and culturing for 24hr until cell density reaches 1-2 × 106cells/ml. The cell suspension was centrifuged at 800rpm for 5min, the supernatant was discarded, the cells were washed 2 times with 50ml of CD CHO medium, the supernatant was discarded, and the cell suspension was resuspended in 1ml of CD CHO medium. Cells and plasmids were arranged at 1X 107cell: 40. mu.g DNA, setting electroporation transfection parameters: the voltage is 300V; capacitor 900 muAnd F, performing electric pulse, transferring the transfected cells into 100ml of CD-CHO culture medium, and subpackaging the cells into 10 96-well plates with each plate hole being 100 mu L. After 24 hours, 100 mul of MSX working solution is respectively added for pressure screening. After the cell plate was cultured for 3 weeks, cell clones were formed in the plate wells. Selecting the monoclonal cells with good separation degree, transferring the cells to a 24-pore plate and a 6-pore plate in sequence for amplification culture, quantitatively detecting culture supernatant by using an HPLC-ProteinA chromatographic column, selecting 10 cell clones with higher expression level for further culture in a shake flask, and carrying out non-reduction SDS-PAGE electrophoresis detection on the culture supernatant.
2.2 purification of the FSH α -Fch-FSH β -Fck heterodimer protein
SDS-PAGE electrophoresis and western-blotting detection show that the main components in the culture supernatant are heterodimers and partial degradation products, and homodimers can hardly be detected or a very small amount of homodimers can be contained. Purification can therefore be carried out by proteinA affinity chromatography in combination with ion exchange. The cell supernatant was centrifuged, and the supernatant was collected and filtered through a 0.22 μm filter to prepare a sample. The Protein A affinity chromatography column was equilibrated with 20mM PB buffer containing 50mM NaCl at pH7.0, and the filtered sample was applied, rinsed with the equilibration buffer after the application, and then eluted with 0.1M citric acid at pH3.0 to remove the target Protein. After one-step purification, HPLC results show that the purity reaches more than 95%. Further purifying by anion exchange, using Hitrap Q HP ion exchange column, loading after balancing with 20mM PB buffer solution, eluting with 20mM PB buffer solution containing 1M NaCl, and detecting the purity of the eluted target protein by HPLC to reach 99%.
Example 3 detection of the biological Activity of FSH α -Fch-FSH β -Fck heterodimers
The biological activity of FSH recombinant protein is determined by adopting a rat ovary weight increasing method according to the pharmacopoeia of China, the HCG is used for assisting a tested FSH medicament, immature female rats of 21 days old are stimulated, ovarian follicle growth and egg cell generation are triggered, and the biological activity of the tested medicament can be clearly evaluated by measuring the weight of ovaries at the end of administration.
Example 4 determination of half-life of FSH α -Fch-FSH β -Fck heterodimers
According to 0.1mg/kg body weight, a single administration of FSH α -Fch-FSH β -Fck heterodimer fusion protein is performed subcutaneously to macaques, 0.6mL of venous blood is collected at 2, 4,8 and 12hr, 2, 3, 4,8, 10 and 12day after the administration, FSH content in samples is determined by sandwich ELISA, PK parameters of FSH in macaques blood serum are calculated by WinNonlin software, and the results are shown in the following table 1. pharmacokinetic parameters show that the FSH α -Fch-FSH β -Fck heterodimer fusion protein of the invention has higher area under the drug time curve, and the half-life is calculated to be about 248 hours, while the half-life of simultaneously determined fruinafine is 17.3 hours, and the half-life of the FSH α -Fch-FSH β -Fck heterodimer is 14 times that of the latter (table 1).
TABLE 1 pharmacokinetic parameters of FSH α -Fch-FSH β -Fck heterodimer fusion proteins
Sequence listing
<110> Beijing Qikang Xingheng biological medicine science and technology Limited
<120> novel FSH-Fc fusion protein, preparation method and application thereof
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Claims (12)
1. A long-acting FSH fusion protein comprises two different peptide chains, wherein one peptide chain is FSH α -Fch formed by connecting an FSH α subunit to a locked Fc chain FchN end directly or indirectly through a linking element, the other peptide chain is FSH β -Fck formed by connecting an FSH β subunit to a locked Fc chain FckN end directly or indirectly through a linking element, and the peptide chains FSH α -Fch and FSH β -Fck are folded into a stable heterodimer in the form of FSH α -Fch-FSH β -Fck under the double actions of non-covalent combination between the FSH α subunit and the FSH β subunit and a locking structure on the locked Fc chain CH 3.
2. A long-acting FSH fusion protein comprises two different peptide chains, wherein one peptide chain is FSH α -Fck formed by connecting an FSH α subunit to an FckN end of a button Fc chain directly or indirectly through a linking element, the other peptide chain is FSH α -Fch formed by connecting an FSH β subunit directly or indirectly through a linking element to an N end of a padlock Fcfch fragment, and the peptide chains FSH α -Fck and FSH α -Fch are folded into a stable heterodimer in the form of FSH α -Fck-FSH β -Fch under the double effects of non-covalent combination between the FSH α subunit and the FSH β subunit and a lock structure on the padlock and the button Fc chain CH 3.
3. The long-acting FSH fusion protein of claim 1 or 2, wherein said FSH is human FSH.
4. The long-acting FSH fusion protein of any of claims 1 to 3, wherein said Fc is IgG4 Fc.
5. The long-acting FSH fusion protein of claim 4, wherein said padlock Fc chain Fch has T148S, L150A, Y189V mutations compared to wild-type IgG4 Fc; the buckled Fc chain Fck has a T148W mutation compared to wild-type IgG4 Fc.
6. The long-acting FSH fusion protein of claim 5 wherein the padlock Fc chain has an amino acid sequence as set forth in SEQ ID NO. 1 or by deletion, addition or substitution of 1, 2 or 3 residues in SEQ ID NO. 1; the button Fc chain has an amino acid sequence shown in SEQ ID NO. 2 or obtained by deleting, adding or substituting 1, 2 or 3 residues on the SEQ ID NO. 2.
7. The long-acting FSH fusion protein of claim 1, wherein said peptide chain FSH α -Fch has the amino acid sequence shown in SEQ ID No.3 or 1, 2 or 3 residue amino acid sequence deleted, added or substituted on SEQ ID No. 3.
8. The long-acting FSH fusion protein of claim 1, wherein said peptide chain FSH α -Fck has the amino acid sequence shown in SEQ ID No.4 or 1, 2 or 3 residue amino acid sequence deleted, added or substituted on SEQ ID No. 4.
9. The long-acting FSH fusion protein of claim 7 or 8 wherein the peptide chain FSH α -Fch has an amino acid sequence shown in SEQ ID NO.3 or 1, 2 or 3 residues deleted, added or substituted on SEQ ID NO.3 and the peptide chain FSH α -Fck has an amino acid sequence shown in SEQ ID NO.4 or 1, 2 or 3 residues deleted, added or substituted on SEQ ID NO. 4.
10. Use of a long-acting FSH fusion protein as claimed in any one of claims 1 to 9 in the manufacture of a medicament for the treatment of polycystic ovary syndrome or other anovulatory disease or disorder.
11. Use of a long acting FSH fusion protein as claimed in any one of claims 1 to 9 for the preparation of a pro-ovarian hyperstimulation for use in assisted reproductive techniques.
12. A method of preparing a long-acting FSH fusion protein according to any of claims 1 to 9.
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| CN1993048A (en) * | 2002-02-21 | 2007-07-04 | 惠氏公司 | Follistatin domain containing proteins |
| CN103429619A (en) * | 2011-03-17 | 2013-12-04 | 雷蒙特亚特特拉维夫大学有限公司 | Bi- and monospecific, asymmetric antibodies and method of generating same |
| CN103539861A (en) * | 2013-11-01 | 2014-01-29 | 广州诺新生物技术有限公司 | Long-acting recombinant human follicle-stimulating hormone-Fc fusion protein (hFSH-Fc) |
| WO2015103928A1 (en) * | 2014-01-08 | 2015-07-16 | 上海恒瑞医药有限公司 | Il-15 heterogeneous dimer protein and uses thereof |
| WO2016001810A1 (en) * | 2014-07-01 | 2016-01-07 | Pfizer Inc. | Bispecific heterodimeric diabodies and uses thereof |
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| CN1993048A (en) * | 2002-02-21 | 2007-07-04 | 惠氏公司 | Follistatin domain containing proteins |
| CN103429619A (en) * | 2011-03-17 | 2013-12-04 | 雷蒙特亚特特拉维夫大学有限公司 | Bi- and monospecific, asymmetric antibodies and method of generating same |
| CN103539861A (en) * | 2013-11-01 | 2014-01-29 | 广州诺新生物技术有限公司 | Long-acting recombinant human follicle-stimulating hormone-Fc fusion protein (hFSH-Fc) |
| WO2015103928A1 (en) * | 2014-01-08 | 2015-07-16 | 上海恒瑞医药有限公司 | Il-15 heterogeneous dimer protein and uses thereof |
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