WO2018032787A1

WO2018032787A1 - Highly glycosylated human growth hormone fusion protein, and manufacturing method and application of same

Info

Publication number: WO2018032787A1
Application number: PCT/CN2017/079873
Authority: WO
Inventors: 李强; 陈思; 郑云程; 李子瑞; 马心鲁
Original assignee: 安源医药科技（上海）有限公司
Priority date: 2016-08-19
Filing date: 2017-04-10
Publication date: 2018-02-22
Also published as: CN106256835A; CN107286248A; CN107286248B

Abstract

A highly glycosylated human growth hormone fusion protein, comprising, from the N-terminus to the C-terminus, of a human growth hormone (hGH), a flexible peptide linker (L), and a human chorionic gonadotropin β-subunit carboxy-terminal rigid peptide (CTP), and an Fc fragment of a human immunoglobulin. Further provided is a highly efficient manufacturing method of the fusion protein. The fusion protein has improved in vivo pharmaceutical efficacy and extended circulation half-life in vivo when compared to a recombinant hGH, greatly reducing administering frequency and improving bioavailability of a drug. The fusion protein also has a simple and highly efficient manufacturing process.

Description

High glycosylated human growth hormone fusion protein, preparation method and use thereof

Technical field

The present invention relates to the field of fusion proteins, and more particularly to a highly glycosylated human growth hormone fusion protein, a preparation method thereof and use thereof.

Background technique

Human growth hormone (hGH) is a protein hormone secreted by eosinophils in the anterior pituitary gland. It is composed of 191 amino acids. Its physiological function is mainly to promote substance metabolism and growth. hGH mediates action on cartilage tissue by growth-promoting factors or insulin-like growth factors, increasing bone length, thereby promoting linear growth of the human body. More functions of hGH have been discovered in recent years, including promoting skeletal muscle and cardiomyocyte growth, promoting protein synthesis, regulating immune system function, and enhancing immune defense capabilities. The large-scale clinical application of hGH has also expanded from the prevention and treatment of childhood growth hormone deficiency in children with short stature to burns, acute pancreatitis, and aging in the elderly. Its clinical indications are still expanding.

Children and adults can be treated with exogenous growth hormone supplementation in the absence of growth hormone. The in vivo elimination half-life of natural hGH is about 0.3 hours. The commercial half-life of recombinant human growth hormone is about 2-3 hours. It is cleared by the liver and kidneys soon after injection, so it needs to be administered subcutaneously every day. Bring a lot of pain to the patient. In addition, long-term injection of recombinant human growth hormone produces antibodies in a small number of patients and affects efficacy. Therefore, there is a need for a technique that can maintain a high activity while prolonging the circulating half-life of hGH. The construction of recombinant long-acting, high activity, reduced immunogenicity and high bioavailability of human growth hormone has become the primary goal of next-generation drug development.

In order to stabilize proteins and prevent enzymatic and renal clearance, such as polyethylene glycol (PEG) modification (Sada et al., J. Ferment Bioeng 71: 137-139, 1991), glycosylation modification (USA) Patent No. US 7,217,689) and methods of fusion with other proteins (WO 93/15199) to promote absorption in the body, prevention of degradation and renal clearance. The PEG-modified protein can prevent hydrolysis without causing serious side effects. However, the coupling of PEG to the target protein belongs to non-specific covalent binding. This random coupling hinders the interaction between the target protein and its receptor to varying degrees. In vivo activity is reduced. Increased glycosylation can increase the molecular weight of the target protein, especially for proteins that are at the renal filtration threshold (~30KD), which can reduce the sensitivity of proteins to protease hydrolysis. Korean Patent Application Publication No. KR10-2013-0029713 discloses a method of attempting to increase the half-life in vivo by introducing at least one mutation into the α-1 antitrypsin to add an N-glycosyl group. There are two types of glycosylation modifications of proteins, including O-glycans and N-glycans. However, the attachment and addition of a sugar chain can cause the physiologically active protein to be inactivated, and the selection site of the site of the physiologically active protein additionally attached to the sugar chain is also very narrow, thereby introducing an additional glycosylation site to the protein for glycosylation. Engineering technology has not been widely used.

Another way to improve protein pharmacokinetics and in vivo stability is to link the gene of a physiologically active protein to an expression gene of a protein with high stability and to generate a fusion protein by genetic recombination techniques. An Fc fusion protein is a novel recombinant protein produced by fusion of a biologically active functional protein molecule (a soluble ligand, a receptor or other biologically active substance requiring an extended half-life) with an Fc fragment by techniques such as genetic engineering. The fusion protein not only retains the biological activity of the functional protein molecule, but also prolongs the half-life of the fusion protein and enhances its stability. The interchain disulfide bond of the Fc fragment facilitates the formation of a dimer of the fusion molecule, thereby enhancing ligand binding. The ability to enhance biological activity; the introduction of an Fc fragment also facilitates increased expression levels of the fusion molecule in mammalian cells. Thus the manufacture of a fusion protein containing an Fc region linked to a human IgG protein will help to prolong the circulating half-life of the drug and/or increase its biological activity. Patent CN102875683 discloses a long-acting recombinant human growth hormone Fc fusion protein, which adds a flexible peptide linker to hGH and human IgG Fc variants to reduce steric hindrance effects, with the expectation of prolonging serum half-life, biological activity Increased, thereby improving pharmacokinetics and efficacy. However, since the active site of hGH is mainly at the C-terminus, other proteins fused at the C-terminus greatly affect the activity and function of hGH. The CN102875683 patent shows that the dimerized hGH-L-vFc has a molar activity of 1.2 nM. The active phase is still less than ideal for recombinant hGH. According to the present invention, it is found that simply extending the flexible peptide linker and increasing the spatial distance between the Fc and the hGH end of the hGH cannot solve the problem, and other methods are needed to solve the problem that the fusion ligand affects the activity of hGH. The present invention aims to find a new fusion method to further increase the serum half-life and in vivo activity of the fusion protein.

CTP is a short peptide derived from the carboxy terminus of the β-subunit of human chorionic gonadotropin (hCG). Four reproductive-related peptide hormones, follicle stimulating hormone (FSH), luteinizing hormone (LH), thyrotropin (TSH), and chorionic gonadotropin (hCG) contain the same alpha-subunits and their respective specific Beta-subunit. Compared with the other three hormones, the half-life of hCG is significantly prolonged, mainly due to the unique carboxy terminal peptide (CTP) on its β-subunit (Fares FA et al. Proc Natl Acad Sci USA. 89: 4304–4308, 1992). CTP contains 37 amino acid residues with four O-glycosylation sites. The terminal is a sialic acid residue. CTP can increase the glycosylation level of the protein and increase the activity of the target protein, while the negatively charged, highly sialylated CTP can resist the clearance of the kidney, thereby prolonging the half-life of the protein in vivo. U.S. Patent No. 13,195,931 discloses a method for ligating the growth hormone half-life by only about 6-fold by attaching the chorionic gonadotropin carboxy-terminal peptide (CTP) to hGH, but does not mention several hGH/CTP chimeras. The in vitro activity of the protein; the in vivo efficacy showed that the chimeric protein fused with one or two CTP molecules was inferior to recombinant hGH, and only CTP-hGH-CTP-CTP was a chimeric protein with three CTP molecules. The efficacy is slightly higher than recombinant hGH. The present inventors have creatively used a CTP polypeptide having a plurality of O-glycosyl sites as part of a linker peptide for ligation of hGH and Fc fragments, rather than acting as a fusion ligand due to its natural glycosylation site In addition, the half-life of the fusion protein can be further prolonged, the bioavailability is improved, and the steric hindrance effect of the fusion ligand Fc on hGH is greatly reduced, thereby maintaining high biological activity.

In summary, many protein fusion methods have been constructed for the development of long-acting protein drugs, but to date there have been no long-acting hGH preparations with very high activity and half-life. Therefore, there is an urgent need in the art to develop long-acting hGH which is long-acting, highly active, simple in purification steps, and convenient for industrial production.

Summary of the invention

In order to solve the problem of short half-life and poor biological activity of recombinant hGH, a high-glycosylated long-acting hGH fusion protein was designed and prepared. After long-term and in-depth research, the inventors have designed a unique peptide linker for the first time to reduce the steric hindrance between fusion molecules. The fusion protein consisting of the C-terminus of hGH and Fc is linked by a flexible peptide and a rigid peptide. Unexpectedly, this fusion protein was approximately one-fold more potent than the in vitro activity of hGH-L-vFc developed by the inventors of the present invention (see, Chinese Patent No. CN102875683). In addition, an unexpected technical effect has been obtained, that is, the bioavailability of the fusion protein is also greatly improved.

In a first aspect of the invention, a recombinant hGH fusion protein comprising human growth hormone (hGH), a flexible peptide linker (L), at least one human chorionic gonadotropin from N-terminus to C-terminus is provided Hormone β-subunit carboxy terminal rigid peptide (CTP) and human immunoglobulin Fc fragment; wherein the Fc fragment is preferably a human IgG Fc variant (denoted as vFc).

Wherein the flexible peptide linker is preferably non-immunogenic and produces a sufficient distance between hGH and Fc to minimize steric effects between each other. Preferably, a flexible peptide linker comprising two or more of the following amino acids: Gly (G), Ser (S), Ala (A) and Thr (T) is used. Preferably, the flexible peptide linker comprises G and S residues. The length of the linker peptide is very important for the activity of the fusion protein. For the purposes of the present invention, preferably, the flexible peptide linker amino acid composition has the structural formula of (GS) _a (GGS) _b (GGGS) _c (GGGGS) _d , wherein a, b, c and d are greater than or equal to 0 An integer, and a+b+c+d≥1.

In some embodiments of the invention, the peptide linker is selected from the group consisting of:

(a) L1: GSGGGSGGGGSGGGGS;

(b) L2: GSGGGGSGGGGSGGGGSGGGGSGGGGS;

(c) L3: GSGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS;

(d) L4: GGGGSGGGGSGGGGSGGGGS.

Wherein the human chorionic gonadotropin β-subunit carboxy terminal peptide rigid peptide (CTP) is selected from the full length sequence consisting of amino acids 113 to 145 of the carboxy terminus of human chorionic gonadotropin β subunit or a fragment thereof (hereinafter referred to as CTP), specifically, the CTP rigid peptide comprises SEQ ID NO: 1 or a truncated sequence thereof.

Preferably, the CTP rigid peptide comprises at least 2 glycosylation sites; for example, in a preferred embodiment of the invention, the CTP comprises 2 glycosylation sites, exemplarily, the CTP comprises SEQ ID NO: 10 amino acids at the 1N terminus, ie SSSS*KAPPPS* (* represents a glycosylation site); or the CTP comprises 14 amino acids at the C-terminus of SEQ ID NO: 1 , ie S*RLPGPS*DTPILPQ; In one embodiment, the CTP comprises three glycosylation sites, exemplarily, the CTP comprises 16 amino acids of the N-terminus of SEQ ID NO: 1 , ie SSSS*KAPPPS*LPSPS*R; In one embodiment, the CTP comprises four glycosylation sites, exemplarily, the CTP sequence comprises 28, 29, 30, 31, 32 or 33 amino acids and begins with human chorionic gonadotropin beta subunit The 113th, 114th, 115th, 116th, 117th or 118th place ends at 145th place. Specifically, the CTP comprises 28 amino acids of the N-terminus of SEQ ID NO: 1, namely SSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ. Each possibility represents a separate embodiment of the invention.

In other embodiments, the CTP rigid units provided herein are at least 70% homologous to the native CTP amino acid sequence; in other embodiments, the CTP rigid units provided herein are at least 80% homologous to the native CTP amino acid sequence; In other embodiments, the CTP rigid units provided herein are at least 90% homologous to the native CTP amino acid sequence; in other embodiments, the CTP rigid sheets provided by the present invention The element is at least 95% homologous to the native CTP amino acid sequence.

Preferably, in an embodiment of the invention, the CTP rigid peptide may preferably comprise the following sequence elements:

(i) CTP ¹ : SSSSKAPPPSLPSPSRLPGPSDTPILPQ;

(ii) CTP ² : PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ;

(iii) CTP ³ : SSSSKAPPPS;

(iv) CTP ^4: SRLPGPSDTPILPQ.

The fusion protein of the present invention may further comprise two or more of the above CTP rigid peptide sequence units. As an embodiment of the present invention, the CTP comprises two CTP ³ units: SSSSKAPPPSSSSSKAPPPS (CTP ^3- CTP ³ , Or expressed as (CTP ³ ) ₂ ).

Wherein the extended half-life portion is preferably a self-immunoglobulin IgG, IgM, IgA Fc fragment; more preferably an Fc fragment from human IgG1, IgG2, IgG3 or IgG4 and variants thereof; further, the human IgG Fc variant comprises in the wild At least one amino acid modification in a human IgG Fc, and the variant has reduced effector function (ADCC and/or CDC effect) and/or enhanced binding affinity to the neonatal receptor FcRn. Further, the human IgG Fc variant may be selected from the group consisting of:

(a) vFcγ ₁ : human IgG1 hinge region, CH2 and CH3 region containing the Leu234Val, Leu235Ala and Pro331Ser mutations (such as the amino acid sequence shown in SEQ ID NO: 4);

(b) vFcγ _2-1 : human IgG2 hinge region, CH2 and CH3 region containing the Pro331Ser mutation (such as the amino acid sequence shown in SEQ ID NO: 5);

(c) vFcγ _2-2 : human IgG2 hinge region, CH2 and CH3 regions containing the Pro331Ser, Thr250Gln and Met428Leu mutations (such as the amino acid sequence shown in SEQ ID NO: 6);

(d) vFcγ ₄ : human IgG4 hinge region, CH2 and CH3 region containing the Ser228Pro and Leu235Ala mutations (such as the amino acid sequence shown in SEQ ID NO: 7).

Preferably, the amino acid sequence of the hGH-L-CTP-vFc fusion protein is set forth in SEQ ID NO: 2.

In a second aspect of the invention, a DNA molecule encoding the recombinant hGH-L-CTP-vFc fusion protein of the first aspect of the invention is provided, preferably wherein the DNA sequence has the sequence shown in SEQ ID NO: Nucleotide sequence.

According to a third aspect of the invention, a vector comprising the DNA sequence of the second aspect of the invention is provided.

According to a fourth aspect of the present invention, a host cell comprising a third party of the present invention is provided The vector is surfaced or transfected with the above vector.

In a specific embodiment of the invention, the host cell is a CHO derived cell line DXB-11.

Preferably, the host cell produces a recombinant hGH-L-CTP-vFc fusion protein according to the first aspect of the invention in more than 30 μg/million cells per 24 hours in its growth medium.

In a fifth aspect of the invention, a method for the preparation of the recombinant hGH-L-CTP-vFc fusion protein of the first aspect of the invention, the method comprising:

(a) introducing a DNA encoding a fusion protein into a CHO cell to generate a CHO-derived cell line;

(b) a high-yield cell line expressing more than 30 μg/10 ⁶ (million) cells per 24 hours during the screening step (a);

(c) culturing step (b) the selected cell strain to express the fusion protein;

(d) Harvesting the fermentation broth obtained in the step (c) to purify the fusion protein.

Preferably, the CHO-derived cell line in step (a) is DXB-11.

In a sixth aspect of the invention, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient or diluent, and an effective amount of said hGH-L-CTP- vFc fusion protein.

In a further aspect, the present invention provides the hGH-L-CTP-vFc fusion protein for treating growth and development disorders caused by insufficient secretion of endogenous growth hormone, and the short and chronic renal function caused by Turner syndrome. Depletion, Prader-Willi syndrome or idiopathic short stature.

In still another aspect, the present invention provides the use of the fusion protein for the preparation of a medicament for the treatment of osteoporosis, in particular for slowing bone loss due to aging.

In summary, the fusion protein structure of the present invention has the following characteristics:

1. The fusion ligand human IgG Fc variant used in the fusion protein is non-lytic, which reduces the effector function triggered by binding to FcγRs and Clq;

2. The fusion protein prepared by the invention has good stability in fermentation, purification process and storage process;

3. By connecting hGH and Fc variants by L-CTP, CTP rigid peptide can not only further prolong its half-life in vivo, but also increase the spatial distance between fusion molecules by blocking the glycosylation side chain, and promote hGH and Fc segments. Each folds to form the correct three-dimensional conformation without affecting each other's biological activity, and the biological activity of hGH-L-CTP-vFc is greatly improved compared with hGH-L-vFc;

4. The fusion protein has reduced immunogenicity, which reduces the risk of inducing neutralizing antibody production;

Compared with PEG-rhGH, hGH-L-CTP-vFc has improved bioavailability, low fluctuation of blood concentration, faster onset, and extended half-life of circulating in vivo, which can reduce the frequency of injection and improve patient compliance. Sex

Compared with recombinant hGH, hGH-L-CTP-vFc has a simple purification step and high purification efficiency.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

Detailed description of the invention:

IgG Fc variant

Non-lytic Fc variant

The Fc element is derived from the constant region Fc fragment of immunoglobulin IgG, which plays an important role in eradicating the immune defense of pathogens. The effector function of Fc-mediated IgG is exerted through two mechanisms: (1) binding to cell surface Fc receptors (FcγRs), digestion of pathogens by phagocytosis or cleavage or killer cells via antibody-dependent cellular cytotoxicity (ADCC) pathway , or (2) binding to C1q of the first complement component C1, eliciting a complement-dependent cytotoxicity (CDC) pathway, thereby lysing the pathogen. For the treatment of human application, when the hGH-L-CTP-vFc fusion protein binds to the hGH receptor on the surface of the target cell, the Fc region of the fusion protein preferably does not have adverse effector functions and thus does not dissolve. Or remove these target cells. Therefore, the Fc region of hGH-Fc must be insoluble, and the Fc region is preferably inactive in binding to FcγRs and Clq to trigger effector functions. Among the four human IgG subtypes, IgG1 and IgG3 efficiently bind to FcγRs, and the binding affinity of IgG4 to FcγRs is low, and the binding of IgG2 to FcγRs is too low to be determined, so human IgG2 has almost no ADCC effect. In addition, human IgG1 and IgG3 can also efficiently bind to C1q to activate the complement cascade. Human IgG2 binds relatively weakly to C1q, whereas IgG4 does not bind to C1q (Jefferis R et al, Immunol Rev, 1998, 163: 59-76), so the human IgG2 CDC effect is also weak. Clearly, none of the native IgG subtypes are well suited for the production of hGH fusion proteins. In order to obtain non-lytic Fc without effector function, the most effective method is to mutate the complement and receptor binding domain of the Fc fragment, modulate the binding affinity of Fc to related receptors, reduce or eliminate ADCC and CDC effects, and retain only Fc. Long cycle half-life characteristics without cytotoxicity. More non-lytic Fc variants For the mutation-containing site, see Shields RL et al, J Biol Chem, 2001, 276(9): 6591-604 or Chinese invention patent CN 201280031137.2.

Fc variants with improved pharmacokinetics

The plasma half-life of IgG depends on its binding to FcRn, which typically binds at pH 6.0 and dissociates at pH 7.4 (plasma pH). By studying the binding sites of the two, the site of binding to FcRn on IgG was engineered to increase the binding ability at pH 6.0. Mutations in some residues of the human Fcγ domain important for binding to FcRn have been shown to increase serum half-life. In particular, in human Fcγ1, when three residues were replaced by the other 19 conventional amino acids, some point mutations showed increased FcRn binding affinity (Hinton et al, J Biol Chem, 279(8): 6213-6216, 2004). Hinton et al. found that two mutants, T250Q and M428L, increased the binding to FcRn by 3 and 7 fold, respectively. At the same time, two sites were mutated, and the binding was increased by 28 times. In rhesus monkeys, the M428L or T250QM428L mutant showed a 2-fold increase in plasma half-life (Paul R. Hinton et al, J Immunol, 2006, 176: 346-356). In addition, studies on the T250Q/M428L mutation in the Fc segment of five humanized antibodies not only improved the interaction of Fc with FcRn, but in subsequent in vivo pharmacokinetic tests, it was found to be administered by subcutaneous injection, Fc mutation. The antibody has improved pharmacokinetic parameters compared to wild-type antibodies, increased in vivo exposure, reduced clearance, and improved subcutaneous bioavailability (Datta-Mannan A et al. MAbs. Taylor & Francis, 2012, 4(2): 267- 273.).

Carboxyl terminal peptide (CTP)

CTP is a short peptide derived from the carboxy terminus of the β-subunit of human chorionic gonadotropin (hCG). Four reproductive-related peptide hormones, follicle stimulating hormone (FSH), luteinizing hormone (LH), thyrotropin (TSH), and chorionic gonadotropin (hCG) contain the same alpha-subunit and their respective specific beta - Yaki. Compared with the other three hormones, the half-life of hCG is significantly prolonged, mainly due to the unique carboxy terminal peptide (CTP) on its β-subunit (Fares FA et al, Proc Natl Acad Sci USA, 1992, 89: 4304-4308). ). CTP contains 37 amino acid residues with four O-glycosylation sites and a terminal sialic acid residue. Negatively charged, highly sialylated CTP is resistant to the clearance of the kidneys, thereby prolonging the half-life of the protein in the body. Thus, the inventors creatively add at least one CTP polypeptide after a suitable length of flexible linker peptide such that the half-life of the fusion protein is further extended and the bioavailability is increased.

Furthermore, by adding a CTP peptide between hGH and an Fc variant, it corresponds to the addition of a rigid linker peptide. This aspect ensures that the N-terminally fused hGH does not affect the binding site of the Fc variant and FcRn, thereby affecting the half-life; in addition, the Fc Protein A binding site is important for the purification step in the preparation process, and the CTP is guaranteed to N- The end-fused hGH also does not "cover" its binding site to Protein A. On the other hand, the addition of CTP also allows the Fc fragment of about 25 kD size to not interfere with the correct folding of the N-terminally fused hGH, thereby causing a decrease or loss of its biological activity/function. A rigid CTP polypeptide having a plurality of glycosyl side chains, which forms a stable stereoconfiguration relative to the random coiling of a flexible linker peptide such as (GGGGS)n, which causes the hGH and Fc segments to fold independently. The correct three-dimensional conformation does not affect each other's biological activity.

Fusion protein and production method thereof

The fusion proteins of the invention are typically prepared by biosynthetic methods. According to the nucleotide sequence of the present invention, one skilled in the art can conveniently prepare the nucleic acid of the present invention by various known methods. These methods are, for example but not limited to, PCR, DNA synthesis, etc. For specific methods, see J. Sambrook, Molecular Cloning Experiment Guide. As an embodiment of the present invention, the nucleic acid sequence of the present invention can be constructed by a method of segmentally synthesizing a nucleotide sequence and performing overlap extension PCR.

The invention also provides an expression vector comprising a sequence encoding a fusion protein of the invention and an expression control sequence operably linked thereto. By "operably linked" or "operably linked" is meant a condition in which portions of a linear DNA sequence are capable of modulating or controlling the activity of other portions of the same linear DNA sequence. For example, if a promoter controls the transcription of a sequence, then it is operably linked to the coding sequence.

The expression vector may be a commercially available vector such as, but not limited to, pcDNA3, pIRES, pDR, pUC18 or the like which can be used for expression of a eukaryotic cell system. One skilled in the art can select a suitable expression vector based on the host cell.

According to the restriction enzyme map of the known empty-load expression vector, the skilled person can prepare the present invention by inserting the coding sequence of the fusion protein of the present invention into a suitable restriction site by restriction enzyme cleavage and splicing according to a conventional method. Recombinant expression vector.

The invention also provides a host cell expressing a fusion protein of the invention comprising a coding sequence for a fusion protein of the invention. The host cell is preferably a eukaryotic cell such as, but not limited to, CHO, COS Cells, 293 cells, RSF cells, etc. In a preferred embodiment of the present invention, the cell is a CHO cell which can express the fusion protein of the present invention well, and a fusion protein having good binding activity and good stability can be obtained.

The present invention also provides a method for producing a fusion protein of the present invention using recombinant DNA, the steps of which include:

1) providing a nucleic acid sequence encoding a fusion protein (such as the sequence of SEQ ID NO: 3);

2) inserting the nucleic acid sequence of 1) into a suitable expression vector to obtain a recombinant expression vector;

3) introducing the recombinant expression vector of 2) into a suitable host cell;

4) culturing the transformed host cell under conditions suitable for expression;

5) Collect the supernatant and purify the fusion protein product.

Introduction of the coding sequence into a host cell can employ a variety of known techniques in the art such as, but not limited to, calcium phosphate precipitation, protoplast fusion, lipofection, electroporation, microinjection, reverse transcription, phage Transduction method, alkali metal ion method.

For the culture and expression of host cells, see Olander RM Dev Biol Stand 1996; 86:338. The cells and debris in the suspension can be removed by centrifugation and the clear solution collected. It can be identified by agarose gel electrophoresis.

The fusion protein obtained as described above can be purified to a substantially uniform property, such as a single band on SDS-PAGE electrophoresis. For example, when the recombinant protein is secreted, a commercially available ultrafiltration membrane can be used to separate the protein, for example, from Millipore, Pellicon, etc., and the expression supernatant is first concentrated. The concentrate may be further purified by gel chromatography or by ion exchange chromatography. For example, anion exchange chromatography (DEAE, etc.) or cation exchange chromatography. The gel matrix may be a matrix commonly used for protein purification such as agarose, dextran, polyamide, and the like. The Q- or SP- group is a preferred ion exchange group. Finally, the purified product may be further purified by hydroxyapatite adsorption chromatography, metal chelate chromatography, hydrophobic interaction chromatography and reversed-phase high performance liquid chromatography (RP-HPLC). All of the above purification steps can utilize different combinations to ultimately achieve a substantially uniform protein purity.

The expressed fusion protein can be purified using an affinity chromatography column containing a specific antibody, receptor or ligand of the fusion protein. Depending on the nature of the affinity column used, the fusion polypeptide bound to the affinity column can be eluted using conventional methods such as high salt buffer, pH change, and the like. Alternatively, the amino terminus or carboxy terminus of the fusion protein may also contain one or more polypeptide fragments as a protein tag. Any suitable label can be used in the present invention. For example, the tags may be FLAG, HA, HAl, c-Myc, 6-His or 8-His, etc. These tags can be used to purify the fusion protein.

Pharmaceutical composition

The present invention also provides a pharmaceutical composition comprising an effective amount (e.g., 0.000001 to 90% by weight; preferably 0.1 to 50% by weight; more preferably 5 to 40% by weight) of the fusion protein of the present invention, and pharmaceutically acceptable Accepted carrier. Generally, an effective amount of a fusion protein of the invention can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably, the pH is from about 6 to about 8. The term "effective amount" or "effective amount" refers to an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals. A "pharmaceutically acceptable" ingredient is one which is suitable for use in humans and/or mammals without excessive adverse side effects (such as toxicity, irritation, and allergies), i.e., materials having a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.

Pharmaceutically acceptable carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. Usually, the pharmaceutical preparation should be matched to the mode of administration, and the pharmaceutical composition of the present invention can be prepared into an injection form, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparation of the present invention can also be formulated into a sustained release preparation.

The effective amount of the fusion protein of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, pharmacokinetic parameters of the fusion protein such as bioavailability, metabolism, half-life, etc.; severity of the disease to be treated by the patient, body weight of the patient, immune status of the patient, administration Ways, etc. In general, when the fusion protein of the present invention is administered at a dose of about 0.00001 mg to 50 mg/kg of animal body weight per day (preferably 0.0001 mg to 10 mg/kg of animal body weight), a satisfactory effect can be obtained. For example, several separate doses may be administered per day, or the dose may be proportionally reduced, as is critical to the condition of the treatment.

DRAWINGS

Figure 1 shows the nucleotide sequence and deduced amino acid sequence of the fusion protein of the Spe I-EcoR I fragment in the pAPG-3 expression vector. The position of the peptide linker in the mature protein is indicated by a dotted line. In the Fc region, the bold nucleotides and corresponding amino acid variants are underlined.

Figure 2 shows the ability of hGH fusion protein to stimulate proliferation of Nb2-11 cells.

Figure 3 shows the growth curves of each group after administration of the hGH fusion protein.

Figure 4 shows the histological observation of the tibia of rats after administration of hGH fusion protein. Note: A: model group; B: TL group; C: TM group; D: TH group; E: SL group; F: SM group; G: SH group.

detailed description

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions.

Example 1. Construction of an expression plasmid encoding a hGH fusion protein

The gene sequences encoding the hGH leader peptide and mature hGH as well as the different lengths of flexible peptide linkers, different lengths of CTP rigid peptides and different IgG Fc variants are artificially optimized CHO cell-preferred codons obtained by chemical synthesis. In order to facilitate insertion of the fragment of interest into a specific site of the expression vector, there is a restriction endonuclease site at the 5' and 3' ends of the synthesized fragment, respectively SpeI and EcoRI. The fusion gene after sequencing verification was digested with SpeI and EcoRI, and then inserted into the corresponding restriction sites of the expression plasmid PXY1A1 which was modified with PCDNA3.1 as a template to obtain a fusion gene expression plasmid pAPG. The PXY1A1 plasmid includes, but is not limited to, the following important expression components: 1) human cytomegalovirus early promoter and mammalian cells with high exogenous expression of the desired enhancer; 2) dual screening marker with kanamycin resistance in bacteria Sexuality, G418 resistance in mammalian cells; 3) Murine dihydrofolate reductase (DHFR) gene expression cassette, when the host cell is DHFR gene-deficient, methotrexate (MTX) can amplify the fusion gene And the DHFR gene (see U.S. Patent 4,399,216).

As shown in Table 1, the present invention constructs a series of hGH-Fc fusion proteins having flexible peptide linkers of different lengths, CTP compositions, and also different positions, as well as IgG Fc variant (vFc) elements of several different subtypes. . The nucleotide sequence of APG-3 and the translated amino acid sequence are shown in Figure 1.

Table 1. Composition of several hGH fusion proteins constructed

Example 2. Transient expression of different fusion proteins and in vitro activity assay

The series of expression plasmids obtained in Example 1 were transfected into 3×10 ⁷ CHO cells in a 30 ml shake flask using DNAFect LT reagent (ATGCell), and the transfected cells were grown in serum-free growth medium for 5 days, according to The concentration of the fusion protein in the supernatant was determined by the corresponding ELISA method, and its in vitro biological activity was determined by the method described in Example 5. The ELISA results showed that the transient expression levels of several plasmids under these conditions were similar, but their activities showed large differences (Table 2).

Table 2. Comparison of in vitro activity EC ₅₀ values of various fusion proteins transiently expressed

Among them, we defined the molar activity of APG-1 as 100%. The activity of APG-2 was 80.9% of APG-1, indicating that the elongation of the peptide linker alone did not effectively improve the activity of the fusion protein, i.e., the steric hindrance effect of each other could not be attenuated with the extension of the peptide linker. Even an excessively long peptide linker not only does not increase the activity of the fusion protein, but instead causes the protein to fold in error and is secreted as an inactive multimer. We speculate that the long flexible peptide linker makes the fusion protein more flexible, allowing it to rotate freely, which may make the steric structure of hGH closer to the Fc region, and the addition of CTP between the two increases considerably. Up A rigid peptide linker at one end keeps them away from each other. More importantly, CTP contains multiple glycosyl side chains. CTP can form a fixed spatial conformation with respect to the random coiled form of flexible peptide linkers, which can effectively separate the different functions of the fusion protein. Zone, which is more conducive to the two parts are independently folded into the correct three-dimensional conformation, maintaining a high activity. The correctness of this speculation was verified by comparison of the activities of APG-1, APG-2 and APG-3. The activity of APG-4 in which CTP was placed at the Fc C-terminus was only 61.3% of APG-3 in which CTP was placed at the Fc N-terminus, and the activity of APG-4 was similar to that of APG-1 without CTP. The activity of APG-3 is higher than that of APG-4, and the activities of APG-5, APG-6 and APG-7 are much higher than other fusion proteins. The above results confirmed that CTP is critical for the activity of the fusion protein, and that CTP is placed at the N-terminus of the Fc to increase the activity of the fusion protein.

Example 3, Expression of fusion protein in transfected cell lines

The recombinant expression plasmid is transfected into a mammalian host cell line to express the hGH-L-CTP-vFc fusion protein. In order to stabilize high levels of expression, a preferred host cell line is a DHFR enzyme deficient CHO-cell (see U.S. Patent 4,818,679). A preferred method of transfection is electroporation, and other methods can be used, including calcium phosphate co-sedimentation, lipofection, and protoplast fusion. In electroporation, 5 × 10 ⁷ cells were placed in a cuvette with a Gene Pulser Electroporator (Bio-Rad Laboratories, Hercules, CA) set to an electric field of 300 V and a capacitance of 1050 μFd, followed by electroporation by adding 50 μg of the plasmid. Two days after transfection, the medium was changed to a growth medium containing 0.6 mg/mL G418. Transfectants resistant to G418 drugs can grow into cell populations visible to the naked eye after about 2 weeks of transfection. Transfectants resistant to the selected drug were screened by an ELISA assay for anti-human IgG Fc. Quantitative analysis of the amount of fusion protein expression can also be performed using an anti-hGH ELISA. The wells that produced high levels of Fc fusion protein were subcloned by limiting dilution of 96 well plates.

In order to achieve a higher level of expression of the fusion protein, it is preferred to co-amplify with the DHFR gene which is inhibited by the MTX drug. The transfected fusion protein gene was co-amplified with the DHFR gene in growth medium containing increasing concentrations of MTX. Subclones with limiting dilution were able to grow transfectants in up to 6 [mu]M MTX medium. The secreted rate was determined for further analysis of the subcloned cell lines. A cell line having a secretion rate level of more than about 30 (preferably about 50) μg/10 ⁶ (i.e., millions) of cells per 24 hours is adapted to suspension culture using serum-free growth medium. The fusion protein is then purified using conditioned medium.

Example 4: Purification and Characterization of Fusion Protein

The conditioned medium containing the fusion protein was titrated to pH 7-8 with 1 N NaOH and then filtered through a 0.45 micron nitrocellulose filter. The filtrate was loaded onto a phosphate buffered saline (PBS) equilibrated Protein A column. After the fusion protein is bound to the Protein A column, the effluent components are discarded. The column was washed with PBS until the OD value at 280 nm was less than 0.01. The bound fusion protein was then eluted with 0.1 M citrate buffer pH 3.75. The eluate was neutralized with 0.4 volume of 1 M K ₂ HPO ₄ and the fractions containing the purified protein were combined and dialyzed against PBS. It was then filtered through a 0.22 micron nitrocellulose filter and stored at 4 °C. Protein products were identified and analyzed by SDS-PAGE under non-reducing conditions. The fusion protein was quantified by BCA protein analysis using BSA as a standard.

Example 5: In vitro biological activity analysis of fusion protein

The in vitro biological activity of the recombinant hGH-L-CTP-Fc fusion protein was examined using Nb2-11 cell proliferation. Mouse lymphoma cell Nb2-11 (US ATCC cell bank) was cultured normally in Fischer's medium containing 10% fetal bovine serum. Using a serum-free medium, the fusion protein was diluted 3-fold at 1000 ng/ml, and 8 samples of different concentrations were obtained, 100 μl per well, added to a 96-well plate, and the medium was a negative control. The cells in the log phase growth phase were taken, and the cells were washed once with serum-free medium to adjust the density to 3 × 10 ⁶ cells per ml, and 100 μl per well was added to the above 96-well plate. The cells were cultured for 48 hours at 37 ° C in a 5% CO ₂ incubator, and cell proliferation was measured using a CCK-8 kit (Cell Counting Kit, purchased from Shanghai Shengsheng Biotechnology Co., Ltd.). The absorbance at 450 nm was measured with a microplate reader, and the OD reading was plotted against the concentration of the fusion protein, and the resulting dose response curve was used to calculate the biological activity of the fusion protein.

Figure 2 shows the ability of hGH fusion protein to stimulate proliferation of Nb2 cells. Table 3 EC50 values _{(half maximal effective concentration, EC 50} ) different fusion proteins. Since the amino acid at the C-terminus of growth hormone is closely related to its function, Fc directly linked to the C segment of hGH affects its biological activity. After the addition of the linker peptide to hGH and Fc, the activity of the hGH fusion protein is increased. As can be seen from the results, the activity of APG-3 was nearly doubled compared with APG-1. This may be due to the fact that CTP exerts its own function on the one hand, and on the other hand, as a rigid linker peptide between Fc and hGH, CTP is coupled with a flexible peptide, which allows the fusion protein to fold into a more favorable three-dimensional structure, ensuring hGH Biological activity.

Table 3. EC ₅₀ values of hGH fusion proteins

Example 6. Determination of in vivo biological activity of fusion protein

Pharmacodynamic studies of long-acting in vivo growth of fusion proteins. Four-week-old SPF male SD rats weighing 60-80 g were selected and provided by the Experimental Animal Center of China National Food and Drug Administration. Two weeks before the test, the pituitary was removed under the condition of cleansing. The recovery period was 2 weeks after the pituitary surgery. Before the administration, the qualified healthy animals whose body weight change was less than ±10% before the operation were selected. The pituitary rats were randomly divided according to their body weight. There are 7 groups of 7 each. The mode of administration was subcutaneous injection in the neck. The medium dose was converted to 1.26 IU/kg/d in clinical dose, and the setting dose was 1:3. PEG-rhGH (Changchun Jinsai Pharmaceutical Co., Ltd.) high, medium and low doses. The groups were 27 IU/kg/7d (SH), 9 IU/kg/7d (SM) and 3 IU/kg/7d (SL), respectively. The high, medium and low doses of APG-3 were 27 IU/kg/7d (TH). , 9 IU / kg / 7d (TM), 3 IU / kg / 7d (TL). Different doses of APG-3 fusion protein and PEG-rhGH were administered once a week, ie, d1, d7, d14, d21, and d28, for 5 weeks, for a total of 5 times, and the model group was given a vehicle.

First, the study of rat body weight changes

Each rat was weighed at the same time every day after administration, and the weight gain of the rats was calculated every day, and the experiment was terminated on the 35th day (d35), and the rats were weighed. The difference between the body weight (bw _i ) and the pre-dose weight (bw ₁ ) of each animal on the day after administration, that is, the weight gain Δbw (g) that day (if necessary, an autopsy can be performed after the end of the experiment, and the saddle is cut. In the area, visually check for the presence or absence of pituitary remains, and remove animals with pituitary remains. Weight gain calculation formula: Δbw = bw _{i -} bw ₀ , where: Δbw: weight gain; bw ₀ : pre-dose (d0) body weight, bw _i : body weight one day after administration. Measurement data by mean ± standard deviation

It is indicated that the difference of the mean of the multiple sets of calculated data is compared by the homogeneity of the variance of the F test, and the paired t test is used for statistical analysis of each group. Figure 3 shows the growth curves of each group after administration.

The pituitary rats were 7 weeks old when they started to be administered. They were adult rats once a week. After 5 weeks, the rats were 12 weeks old and were adult rats. After the growth period, the growth was about to stagnate. . As can be seen from Table 4, the body weight of the model group (only the vehicle was given) did not change, and there was no increase, while the body weight of APG-3 and the positive control drug PEG-rhGH increased significantly (P<0.01). The effect relationship indicates that APG-3, like PEG-rhGH, increases body growth and increases protein synthesis. After the third dose, the Δbw of each dose group showed a significant difference; on the 35th day after the administration, APG-3 had a very significant effect on the weight gain of the rat, and the high dose APG-3 induced the pituitary. The weight gain (Δbw value) of the resected rats was about 1.5 times higher than that of the high dose PEG-rhGH group; while the high dose PEG-rhGH induced weight gain was slightly better than the medium dose APG-3, and the growth trend of the growth curve from d26 Smooth. This may Because APG-3 should have a longer in vivo half-life than PEG-rhGH, this in vivo accumulation effect makes the APG-3 group have a steeper growth curve after the third administration.

Table 4. Changes in body weight of rats before and after administration

Note: Compared with the model group, ^## P<0.01.

Second, rat cognitive function research

After the termination of the experiment (d35), the rats were subjected to a dark avoidance test. The rat escaping instrument uses a plastic box that is divided into two chambers, light and dark. There is an arched small door between the light and dark boxes. The bottom of the two boxes are copper grids. The bottom of the chamber has a copper grid and can be energized. The voltage is 36V. One day before the experiment, the rats were first placed in a bright room illuminated by strong light, and they were found to find the passage between the light and dark chambers and enter the dark room. The rats were repeatedly trained until they could be found within 60s. And enter the dark room, after the rat enters the dark room, close the channel between the light and dark room, the dark room is energized to give 36V, 50Hz AC electrical stimulation once, lasting about 5s, let the rats rest for 30s and then repeat the above process until In the Ming room, he did not dare to enter the darkroom for more than 300s. The rats were repeatedly trained in the darkness reaction box, and the rats were shocked and fled to the bright room. On the day of the experiment, the rats were first placed in a clear box, adapted to 5 min, and the small door was opened. The time when the rat entered the dark box from the bright box was the darkness incubation period. The rats entered the dark box, and the bottom of the dark box was stimulated by alternating current. Rats Then escaped into the dark room, within 5 minutes, the number of times the rats were shocked by entering the black box was the number of shuttles. The darkness meter automatically records the number of times the rat enters the darkroom within 5 minutes, which is the number of shuttles, and the time of entering the darkroom for the first time, which is the darkness incubation period, and statistical analysis (measured by the pharmacological laboratory, the instrument used is the pharmacology room of Beijing Drug Testing Institute). ).

Avoiding darkness avoidance experiments is a common method for screening nootropics. It is established by using the animal's good darkness (light and dark) and fear and memory of aversive stimuli (such as electric shock). One of the methods of memory performance in rats. Judging criteria are that the longer the animal avoids dark latency, the less the number of shuttles, and the stronger the animal's ability to learn and remember. It can be seen from Table 5 that the model group has the shortest latency, the most frequent shuttle times, and the worst learning and memory ability. Compared with the model group, the APG-3 dose group has a long time to avoid darkness (prolonged by 290%~ 1220%), the APG-3 high- and medium-dose group and the PEG-rhGH middle-dose group and the model group were significantly different (P<0.05) (the SH group has a large gap within the group, there is no statistical significance) At the same time, the number of shuttles in each dose group of APG-3 was significantly reduced, and the difference was significant compared with the model group (P<0.05). The results showed that APG-3, like the positive drug PEG-rhGH, had a certain effect on improving the cognitive function and puzzle of pituitary rats.

Table 5, rat darkness avoidance experiment statistics

Note: Compared with the model group, ^# P<0.05, ^## P<0.01.

3. Study on the content of insulin-like factor-1 in rats

IGF-1 is an important growth factor on the growth axis. It has a variety of physiological functions. In addition to regulating the growth of the body, it has a mitotic effect on many cell lines, including promoting osteoblast proliferation and differentiation, and increasing osteoblast activity. And the amount that prevents osteoblast apoptosis. Growth hormone is regulated by growth of IGF-1. At present, more studies suggest that changes in serum IGF-1 levels are relatively reliable and sensitive indicators for determining the efficacy of GH therapy.

At the end of the above experiment, blood was taken from the venous plexus of the rat, about 0.5 mL each, after standing for 30 minutes, centrifuged at 6000 rpm for 10 min at 4 ° C, and the rat serum was separated by about 0.2 mL/room, and stored at -20 ° C for use. At the time of testing, the insulin-like factor (IGY-1) was determined by the method of radioimmunoassay kit (HY-082, RIA KIT, Beijing Huaying Biotechnology Research Institute). Measurement data by mean ± standard deviation

It is indicated that the difference of the mean of the multiple sets of calculated data is compared by the homogeneity of the variance of the F test, and the paired t test is used for statistical analysis of each group. The results are shown in Table 6.

After 5 weeks of administration, serum IGF-1 was significantly increased in the high-dose, medium-dose, and APG-3 groups of PEG-rhGH compared with the model group (P<0.05 or P<0.01), and PEG-rhGH was low. There was no significant change in IGF-1 content between the dose group and the model group. The IGF-1 content of each APG-3 dose group was generally higher than that of PEG-rhGH. It can be seen from the results that the growth promoting effect of APG-3 and the ability to stimulate IGF-1 synthesis is superior to PEG-rhGH.

Table 6. Statistics of serum IGF-1 after administration

Note: Compared to the model group, ^## P<0.01.

Fourth, rat tail length, liver weight and bone plate width study

After the rats were sacrificed before dosing d0 and the test end d35 carbon dioxide asphyxiation, the length of the tail root to the tip of each rat, ie the length of the tail (cm), was measured by a measuring tape. The formula for increasing the length of the tail was: ΔL=L ₃₅ –L ₀ , where : ΔL: tail length increased; L ₀ : d0 tail length before administration; L ₃₅ : d35 tail length after administration. The sacrificed rats were harvested from the liver, the attached tissues were removed, washed with physiological saline, and the filter paper was blotted to dry the surrounding blood (g).

The sacrificed rats were harvested and the left humerus was removed. The attached muscles and connective tissues were removed. Neutral 10% formaldehyde was stored. The tissue samples were excised from the median sagittal plane at the top of the proximal humerus and harvested to a thickness of 3 mm. The paraffin was embedded and sectioned. After staining with hematoxylin eosin (HE), the neutral resin was sealed, observed by light microscopy and photomicrographed. Quantitatively measure the width of the humerus epiphyseal plate (growth plate cartilage band), one slice per mouse, and measure each slice in a non-overlapping field of view. Using a uniform standard, select the width of 20 bone plate on the slice to calculate each one quantitatively. The average of the width of the rat bone plate, and finally the average of the entire group. Measurement data by mean ± standard deviation

It is indicated that the difference of the mean of the multiple sets of calculated data is compared by the homogeneity of the variance of the F test, and the paired t test is used for statistical analysis of each group. The results are shown in Table 7.

It can be seen from Table 7 that since the model group rats did not grow, the results showed that the tail length of the model group was almost unchanged before and after administration, and the liver weight was also the lightest, and the width of the humerus epiphyseal plate was also the smallest; Compared with APG-3 and PEG-rhGH, the tail length and liver weight of the rats in each dose group were significantly increased (P<0.01), and the width of the humerus epiphyseal plate was significantly widened (P<0.01 or P<0.05). The results indicate that both APG-3 and PEG-rhGH have pharmacological effects that promote the growth of the body.

Table 7. Statistics of tail length, liver weight, and epiphyseal plate width after administration

Note: Compared to the model group, ^## P<0.01.

V. Study on bone mineral density in rats

After each rat was sacrificed, the right femur was removed, the attached muscles and connective tissue were removed, and the femoral head was removed. Each femur was stored in a test tube containing saline at 4 ° C, and the test was taken out and dried. HOLOGIC dual energy X The line bone densitometer (Hologic, USA) was used to determine the bone mineral density (BMD) and bone mineral content (BMC) of the rat femur using special software for small animals. At the time of measurement, 7 rats in each group were measured together, and the BMD and BMC values of the central and distal ends of the femur were separately measured and measured. Measurement data by mean ± standard deviation

It is indicated that the difference of the mean of the multiple sets of calculated data is compared by the homogeneity of the variance of the F test, and the paired t test is used for statistical analysis of each group. The results are shown in Table 8.

As can be seen from Table 8, the BMD and BMC values of the central and distal femurs of the model group were the smallest. Compared with the model group, the BMD and BMC at the central and distal ends of each dose group of APG-3 and PEG-rhGH were compared. Significantly elevated (P < 0.01 or P < 0.05). Bone mineral density is the mineral mass contained in the unit bone area in the bone tissue of the body, and is one of the important factors affecting bone strength. It is a commonly used index for evaluating bone strength. The results of this experimental study show that APG-3 and PEG-rhGH have the same effect of increasing bone mineral content, can promote bone calcification, increase bone density, promote bone cell proliferation, increase its activity, and strengthen bone mineralization process. The bone mass is increased; therefore, it is clinically expected to be used for the treatment of osteoporosis, especially for relieving bone loss due to aging.

Table 8. Statistics of femur bone mineral density and bone mineral content in rats after administration

Note: Compared with the model group, ^# P<0.05, ^## P<0.01.

Sixth, pathological histological examination

After the rats were sacrificed, the left lower extremity humerus was taken, and the muscles and connective tissues were removed and stored in neutral formalin. The tibia specimen was subjected to conventional decalcification and embedding, and a 4 μm thick tissue section was prepared, and the longitudinal section was taken for HE staining. The sections were subjected to pathological observation and microphotographing (×2), and the image pro insight 8.0 software was used for bone morphology measurement. Sections of 7 rats in each group, each section was taken for central field of view measurement, calculation of bone area (Tissear Area, T.Ar); Trabecular Bone Area (Tb. Ar), trabecular circumference ( Trabecular Perimeter, Tb. Pm), Trabecular thickness (Tb. Th), Trabecular number (Tb. N), Trabecular separation (Tb. Sp). Measurement data by mean ± standard deviation

It is indicated that the difference of the mean of the multiple sets of calculated data is compared by the homogeneity of the variance of the F test, and the paired t test is used for statistical analysis of each group. The results are shown in Table 9.

Table 9. Statistical data of tibia trabecular measurement results of rat rats after administration

Note: Compared with the model group, ^## P<0.01, ^# P<0.05.

Continued Table 9. Statistical data of tibia trabecular measurement results of rat rats after administration

Note: Compared with the model group, ^## P<0.01, ^# P<0.05.

The pathological sections of the left tibia of the rats were observed by morphological observation, micrograph and data analysis. The conclusions were as follows: except for the largest separation of the trabecular bone in the model group, the other parameters were the lowest; the pathological observation was The trabecular bone of the model group is slender and pale, with poor structural integrity, with distortion and fracture. The trabecular bone is short and the growth is not good. The size of the medullary cavity is different, and the hematopoietic cells in the cavity are reduced. The trabecular thickness of each dose group increased, and the APG-3 medium dose group increased (P<0.01). The other parameters of APG-3 increased significantly (P<0.01 or P<0.05). As shown in Fig. 4, the pathological morphology was observed as TH group, TM group and SH group. The trabecular bone was relatively thick, full and dark, and the trabecular bone structure was intact, without distortion or fracture, and the growth was good. Smaller, the number of red blood cells in the lumen is larger, and the trabecular bone structure of the TL group is better than the model group. The greater the separation of the trabecular bone, the greater the distance between the trabecular bone, the bone The poorer the structure, the increased bone resorption, and osteoporosis may occur. Compared with the model group, the trabecular bone separation of the APG-3 and PEG-rhGH groups was significantly reduced, and the trabecular bone separation of the APG-3 high- and medium-dose groups and the PEG-rhGH high-dose group was reduced. Statistical significance (P<0.01 or P<0.05), only from trabecular bone separation: model group>SL>TL>SM>TM>SH>TH, suggesting that APG-3 and PEG-rhGH can improve bone quality loose.

Example 7, Pharmacokinetic Determination of Fusion Protein

SPF male Sprague-Dawley rats (provided by the Experimental Animal Center of China Food and Drug Administration) at 3-4 weeks of age were randomly divided into 3 groups, 3 in each group, and a single subcutaneous administration of 27 IU/kg PEG- The APG-3 fusion protein of rhGH (Changchun Jinsai Pharmaceutical Co., Ltd.), 27 IU/kg (high dose group) and 9 IU/kg (low dose group) was used to investigate the changes of plasma concentration and time. Blood was collected from the eyelids at 0, 1, 2, 4, 8, 24, 48, 96 and 168 hours after administration. The blood was placed at room temperature for 30 min and then centrifuged at 5000 r/min for 10 min. The serum was separated and stored at -20 °C. Serum hGH levels were determined at various time points using an ELISA specific for hGH. The main pharmacokinetic parameters of each group were calculated by the software PKSOLVER. The results of the pharmacokinetic parameters of each group are shown in Table 10.

Table 10. Pharmacokinetic parameters of hGH fusion protein in pituitary rats

As can be seen from the results, the elimination phase half-life of PEG-rhGH drug on SD rats excised from the pituitary gland was 9.89 h, and the peak time was about 24 h. The T _1/2 of the high-dose and medium-dose groups of the fusion protein were 11 h and 12 h, respectively, and did not change substantially with the change in dose. Compared with PEGylated rhGH, the APG-3 fusion protein has a longer half-life. On the one hand, the combined Fc variant effectively prolongs the half-life, and on the other hand introduces a CTP rigid structure. The negatively charged, highly sialylated CTP is resistant to the kidney. Its scavenging effect further extends the half-life of the fusion protein. The peak time of the fusion protein drug was about 8 hours, and the APG-3 fusion protein was more effective than the 24-hour peak of PEG-rhGH. Thus, APG-3 exhibits superior performance in terms of biological activity, bioavailability, and pharmacokinetics compared to PEG-rhGH.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

A recombinant human growth hormone fusion protein, characterized in that the fusion protein comprises human growth hormone, a flexible peptide linker, at least one carboxy terminal rigid peptide of human chorionic gonadotropin β subunit, and N-terminal to C-terminal Human immunoglobulin Fc fragment.
The fusion protein according to claim 1, wherein the human immunoglobulin Fc fragment is a human IgG Fc variant.
The fusion protein of claim 2, wherein the human IgG Fc variant comprises at least one amino acid modification in a wild-type human immunoglobulin Fc, and the variant has reduced effector function and/or The binding affinity to the neonatal receptor FcRn is enhanced.
The fusion protein of claim 3, wherein the IgG Fc variant is selected from the group consisting of:

(a) human IgG1 hinge region, CH2 and CH3 regions containing the Leu234Val, Leu235Ala and Pro331Ser mutations;

(b) human IgG2 hinge region, CH2 and CH3 regions containing the Pro331Ser mutation;

(c) human IgG2 hinge region, CH2 and CH3 regions containing the Pro331Ser, Thr250Gln and Met428Leu mutations;

(d) Human IgG4 hinge region, CH2 and CH3 regions containing the Ser228Pro and Leu235Ala mutations.
The fusion protein according to claim 1, wherein the flexible peptide linker comprises two or more amino acids selected from the group consisting of glycine, serine, alanine and threonine.
The fusion protein according to claim 5, wherein said flexible peptide linker has an amino acid composition of the formula: (GS)a(GGS)b(GGGS)c(GGGGS)d, wherein a, b, c and d is an integer greater than or equal to 0, and a+b+c+d≥1.
The fusion protein according to claim 6, wherein the amino acid of the flexible peptide linker is selected from the group consisting of:

(a) GSGGGSGGGGSGGGGS;

(b) GSGGGGSGGGGSGGGGSGGGGSGGGGS;

(c) GSGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS;

(d) GGGGSGGGGSGGGGSGGGGS.
The fusion protein according to claim 1, wherein the carboxy terminal rigid peptide of the human chorionic gonadotropin beta subunit comprises SEQ ID NO: 1 or a truncated sequence thereof, and comprises at least two glycosyl groups. Chemical site.
The fusion protein according to claim 8, wherein the carboxy terminal rigid peptide of the human chorionic gonadotropin beta subunit is selected from the group consisting of the following sequences:

(i) SSSSKAPPPSLPSPSRLPGPSDTPILPQ;

(ii) PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ;

(iii) SSSSKAPPPS;

(iv) SRLPGPSDTPILPQ.
The fusion protein according to claim 1, wherein the carboxy terminal rigid peptide of the human chorionic gonadotropin beta subunit has at least 70%, 80%, 90 of the rigid peptide sequence of claim 8 or 9. % or 95% identity.
The fusion protein according to claim 1, wherein the fusion protein comprises 1, 2, 3, 4 or more human chorionic gonadotropin beta subunits according to claim 8, 9 or 10. The carboxy terminal rigid peptide.
The fusion protein according to claim 1, wherein the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 2.
A DNA molecule encoding the fusion protein of any of claims 1-12.
The DNA molecule according to claim 13, wherein the DNA molecule comprises the nucleotide sequence shown in SEQ ID NO: 3.
A vector comprising the DNA molecule of claim 13 or 14.
A host cell comprising the vector of claim 15 or transfected with the vector of claim 15.
A method of preparing a recombinant fusion protein according to any one of claims 1 to 12, comprising the steps of:

(a) introducing a DNA encoding a fusion protein into a CHO cell to generate a CHO-derived cell line;

(b) a high-yield cell line expressing more than 30 μg/10 6 (million) cells per 24 hours during the screening step (a);

(c) culturing step (b) the selected cell strain to express the fusion protein;

(d) harvesting the fermentation broth obtained in step (c), purifying the fusion protein;
The method of recombinant fusion protein according to claim 17, wherein the CHO-derived cell line in step (a) is DXB-11.
A pharmaceutical composition, characterized in that the composition comprises a fusion protein according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier.
Use of the fusion protein according to any one of claims 1 to 12 for the preparation of a medicament for the treatment of a disease caused by insufficient secretion of endogenous growth hormone, including growth and development disorders, and shortness caused by Turner syndrome. Chronic renal failure, Prader-Willi syndrome or idiopathic short stature.
Use of a fusion protein according to any of claims 1-12 for the preparation of a medicament for the treatment of osteoporosis.