A recombinant cyclic human growth hormone-fc fusion protein and application thereof

Abstract

The application discloses a kind of recombinant cyclic human growth hormone-Fc fusion protein and application thereof.The fusion protein is sequentially connected by Fc fragment, human growth hormone, Fc fragment from N-terminal to C-terminal using two connection peptides to form a molecule, and forms a pair or more pairs of disulfide bond or does not form disulfide bond after the folding of two Fc fragments, to constitute cyclic Fc-hGH-Fc.The refolding ratio of the cyclic Fc-hGH-Fc fusion protein is higher than the yield of monomer Fc / Fc-hGH expression refolding, and the form of Fc / Fc homodimer and double body hGH-Fc / hGH-Fc is less, which is convenient for later purification.The fermentation preparation process of the fusion protein of the application is simple, convenient for purification, high in yield and low in cost.The fusion protein of the application weakens the protein clearance effect caused by growth hormone receptor by using the steric hindrance effect of Fc, further prolongs the half-life of drug and increases the efficacy.

Description

Technical Field

[0001] This invention relates to the field of genetically engineered drugs, specifically to a recombinant cyclic human growth hormone-Fc fusion protein and its applications. Background Technology

[0002] Human growth hormone (hGH) is a polypeptide hormone (SEQ ID NO:1) synthesized, stored, and secreted by growth hormone cells in the pituitary gland. The hGH molecule consists of 191 amino acid residues with a relative molecular mass of 22,124 Daltons. Under physiological conditions, growth hormone binds to its primary receptor (hGHR) on the surface of target cells, activating corresponding downstream pathways. Growth hormone promotes development and cell proliferation in animals and humans. Its main physiological function is to promote the synthesis of various tissues, especially proteins, and to stimulate the growth of articular cartilage and epiphyseal cartilage. Growth hormone promotes height increase; a deficiency leads to growth arrest.

[0003] Clinical studies have shown that wild-type growth hormone has a very short half-life in the human body (approximately 2 hours). Therefore, traditional recombinant human growth hormone therapy requires daily subcutaneous injections over a long period (up to 6 months or more). Frequent injections are inconvenient for patients, and the psychological stress associated with injections reduces their quality of life. Furthermore, patient adherence to dosing frequency is a significant factor hindering treatment effectiveness, and the cost is also relatively high. To address these issues, the development of long-acting recombinant human growth hormone is essential. Currently, several recombinant human growth hormone products are available on the market both domestically and internationally. These include Pfizer's Genotropin, Eli Lilly's Humatrope, Genentech's Nutropin, Novo Nordisk's Norditropin, Genotropin, Cyproheptadine, and Ansumeng. However, these are all short-acting products requiring daily injections. Therefore, the market urgently needs a new, long-acting, and safe growth hormone drug.

[0004] Fc fusion proteins are a safe and effective technology for prolonging the plasma half-life of protein drugs. Currently, the FDA has approved as many as nine Fc fusion proteins. Because Fc proteins originate from conserved regions of human immunoglobulins, they possess advantages such as high safety and low immunogenicity. Most fusion protein drugs exist primarily in homodimer form, while hGH-Fc / hGH-Fc dimeric fusion proteins, compared to monomeric forms (i.e., Fc / hGH-Fc), shorten the drug's half-life and increase dosing frequency due to increased growth hormone receptor-mediated protein clearance. Compared to homodimers, monomeric Fc fusion proteins (Fc / hGH-Fc) containing protein drugs linked to only one Fc domain have improved half-life or biological activity. In 2017, Shanghai Xingdijin Biotechnology Co., Ltd. disclosed an Fc fusion protein (Fc / hGH-Fc) fused with only one hGH domain in a patent application, which demonstrated superior efficacy compared to the hGH-Fc / hGH-Fc dimeric form in animal experiments. However, because Fc / hGH-Fc is a heterodimer expressed using mammalian cells, homodimer impurities inevitably arise during the production process. These impurities include Fc / Fc fusion proteins without growth hormone and hGH-Fc / hGH-Fc fusion proteins containing two growth hormone molecules. Consequently, in the later stages of cell saturation, hGH-Fc / hGH-Fc and Fc / Fc, due to their similar properties, are difficult to remove during purification, and their proportions are relatively high. Therefore, Fc / hGH-Fc suffers from process instability and difficulty in scaling up production, leading to decreased drug-like properties. Furthermore, although Fc / hGH-Fc shows significant improvements in efficacy and pharmacokinetics compared to the hGH-Fc / hGH-Fc homodimer protein, its half-life remains insufficient due to factors such as growth hormone receptor-mediated protein degradation.

[0005] To develop longer-acting hGH, research institutions and pharmaceutical companies have attempted to develop modified or engineered hGH with different structures. For example, PEGylated hGH fusion proteins: CN 108794634A and CN 108136043B are formed by two coupling processes using PEG with aldehyde groups at both ends to create a monomeric Fc / hGH-PEG-Fc protein, or by expressing Fc and hGH-Fc in a dual-plasmid configuration to form a monomeric Fc / hGH-Fc fusion protein; or CN 108136043A is formed by subsequent chemical coupling to create an hGH-fatty acid modified protein. However, some of these modified or engineered hGHs still have unsatisfactory performance and significant drawbacks. For instance, PEGylated growth hormone, while significantly increasing its half-life and prolonging its effect, often suffers from activity loss during the PEGylation process, resulting in reduced specific activity. Alternatively, the subsequent chemical modification methods are complex and require sophisticated manufacturing processes. Meanwhile, literature reports (J Clin Endocrinol Metab, June 2020, 105(6):1–13) that preclinical studies in primates have shown that cadaveric autopsies reveal PEG accumulation in the ependymal cells of the choroid plexus, leading to toxicity. This is a significant reason limiting the widespread application of PEGylated hGH. Furthermore, literature reports that long-term use of PEG can cause the development of drug-resistant antibodies and fat collapse at the injection site, further limiting the widespread use of PEGylated hGH.

[0006] In conclusion, in order to facilitate long-term clinical use and improve efficacy, it is necessary to develop safe, effective, simple, low-cost, and high-yield novel long-acting growth hormone products. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the primary objective of this invention is to provide a recombinant cyclic human growth hormone-Fc fusion protein. The cyclic fusion protein of this invention exhibits a higher refolding rate than monomeric expression and does not form Fc / Fc homodimers or dimers (hGH-Fc / hGH-Fc), facilitating subsequent purification. Furthermore, compared to the traditional Fc / hGH-Fc heterodimer form, the cyclic molecule in this invention contains only one polypeptide chain, which improves production efficiency and reduces production costs. The fermentation preparation process of the fusion protein of this invention is simple, easy to purify, yields high output, and is low in cost. The fusion protein of this invention utilizes the steric hindrance effect of Fc to weaken the protein clearance effect caused by the growth hormone receptor, thereby further extending the drug's half-life. Further animal experiments show that the cyclic growth hormone Fc-fusion protein of this invention exhibits a longer half-life and superior efficacy in rat experiments compared to the traditional Fc / hGH-Fc heterodimer form.

[0008] Another object of the present invention is to provide the encoding gene of the above-mentioned recombinant cyclic human growth hormone-Fc fusion protein.

[0009] Another object of the present invention is to provide the application of the above-mentioned recombinant cyclic human growth hormone-Fc fusion protein.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] In a first aspect of the invention, a recombinant cyclic human growth hormone-Fc fusion protein is provided, wherein the molecular linkage is Fc-hGH-Fc. The fusion protein comprises human growth hormone, two linking peptides, and two identical or different Fc fragments. Specifically, two linking peptides are used to sequentially link a first Fc monomer fragment, human growth hormone, and a second Fc monomer fragment from the N-terminus to the C-terminus to form a molecule. One linking peptide is located between the first Fc fragment and the human growth hormone, and the other linking peptide is located between the human growth hormone and the other Fc fragment. The amino acid sequences of the two linking peptides are identical or different. After folding, the two Fc fragments form one or more pairs of disulfide bonds or no disulfide bonds. Because the two Fc fragments can spontaneously form a dimer, the entire molecule is thus linked end-to-end to form the cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc (e.g., ...). Figure 1 (As shown).

[0012] The amino acid sequence of the growth hormone is shown in SEQ ID NO:1.

[0013] In some embodiments, the Fc fragment is selected from the Fc fragment of human IgG, preferably the Fc fragment of human IgG4 or IgG1; more preferably the Fc fragment of human IgG4.

[0014] In some embodiments, the sum of the amino acid numbers of the two linker peptides is ≥5. The linker peptides are preferably sequences with low immunogenicity and flexible structure, and more preferably GS repeats, GGGGS repeats, or random sequences containing only the seven amino acids Q, S, E, P, T, G, and A.

[0015] In another preferred embodiment, the amino acid sequence of the recombinant cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc is shown in SEQ ID NO:2.

[0016] In a second aspect of the invention, a DNA molecule encoding the recombinant cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc described in the first aspect of the invention is provided.

[0017] In a third aspect of the invention, an expression vector comprising the DNA molecule described in the second aspect of the invention is provided, preferably a eukaryotic or prokaryotic cell expression vector.

[0018] In a fourth aspect of the invention, a host cell comprising the expression vector described in the third aspect of the invention is provided, wherein the cell is selected from eukaryotic cells or prokaryotic cells; preferably Escherichia coli.

[0019] In a fifth aspect of the invention, a method for producing the recombinant cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc described in the first aspect of the invention is provided, comprising the following steps:

[0020] (a) Under suitable expression conditions, the host cells described in the fourth aspect of the present invention are cultured to obtain a culture medium containing the expression of the cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc;

[0021] (b) The cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc was isolated from the culture medium.

[0022] In another preferred embodiment, in step (b), the separation includes: high-pressure disruption of bacterial cells, washing twice with PBS buffer, and recovery of inclusion bodies; dissolution of inclusion bodies; refolding of the fusion protein Fc-hGH-Fc; and purification of the fusion protein Fc-hGH-Fc.

[0023] In a sixth aspect of the invention, a pharmaceutical composition is provided comprising a therapeutically effective amount of the recombinant cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc described in the first aspect of the invention and a pharmaceutically acceptable carrier.

[0024] In a seventh aspect of the invention, the use of the recombinant cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc described in the first aspect of the invention or the pharmaceutical composition described in the sixth aspect of the invention in the preparation of a medicament for treating GH deficiency-related diseases is provided.

[0025] The beneficial effects of the above technical solutions are as follows:

[0026] (1) The fusion protein fermentation preparation process of the present invention is simple, easy to purify, and has a high yield. 7g of target protein can be obtained from one liter of fermentation culture, and the cost is low.

[0027] (2) The refolding rate of the fusion protein of the present invention is higher than that of the monomer expression refolding. The refolding rate of the cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc of the present invention is 65%, while the refolding rate of the monomer Fc / hGH-Fc is 25%. It does not form Fc / Fc homodimers or hGH-Fc / hGH-Fc homodimers, which facilitates the subsequent purification.

[0028] (3) The fusion protein of the present invention weakens the protein clearance effect caused by growth hormone receptor by using the steric hindrance effect of Fc, thereby further prolonging the half-life of the drug.

[0029] (4) Compared with currently available PEGylated long-acting GH formulations, this invention does not introduce the risks of hepatotoxicity and nephrotoxicity associated with PEGylation. Compared with eukaryotic cell expression, it also reduces the risk of immunogenicity caused by glycosylation.

[0030] (5) The molecular structure in this invention has better efficacy than Fc / hGH-Fc in animal experiments. Attached Figure Description

[0031] Figure 1 Schematic diagram of the structure of the cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc;

[0032] Figure 2 SDS-PAGE gel images of Fc and hGH-Fc fusion protein expression and cyclic Fc-hGH-Fc fusion protein expression during the preparation of monomeric Fc / hGH-Fc fusion protein;

[0033] A is a 10% SDS-PAGE gel image of the fermentation of only Fc during the preparation of the monomeric Fc / Fc-hGH fusion protein; B is a 10% SDS-PAGE gel image of the fermentation of hGH-Fc during the preparation of the monomeric Fc / Fc-hGH fusion protein; C is a 10% SDS-PAGE gel image of the fermentation and harvesting of the cyclic Fc-hGH-Fc fusion protein; D is the protein molecular weight marker.

[0034] Figure 3 Quantitative detection of Fc-hGH-Fc fusion protein after bacterial lysis;

[0035] Figure 4Denaturation and renaturation SDS-PAGE gel images of inclusion bodies of cyclic Fc-hGH-Fc and monomeric Fc / hGH-Fc fusion proteins;

[0036] A is a 4%-12% SDS-PAGE gradient gel image of the cyclic Fc-hGH-Fc fusion protein after refolding. Lane 1 shows the reduced state of the protein after refolding, lane 2 shows the protein marker, and lane 3 shows the non-reduced state after refolding. B is an SDS-PAGE gel image of the monomeric Fc / hGH-Fc fusion protein after refolding. Lane 1 shows the non-reduced state of the protein after refolding, lane 2 shows the protein marker, and lane 3 shows the reduced state after refolding. C is the protein molecular weight marker.

[0037] Figure 5 Graphs showing the detection of various indicators of the purified cyclic Fc-hGH-Fc fusion protein;

[0038] A shows the SEC-HPLC result of the purified cyclic Fc-hGH-Fc fusion protein, with a purity of 97.28%.

[0039] B shows the UPLC result of the purified cyclic Fc-hGH-Fc fusion protein, with a purity of 95%.

[0040] C represents the LC-Mass assay result of the purified cyclic Fc-hGH-Fc fusion protein, which is 77654.5 Da. Its theoretical molecular weight is 77653.8658 Da.

[0041] D represents the 4%-12% Nu-PAGE gel image detection result of the purified cyclic Fc-hGH-Fc fusion protein, with a loading amount of 5 μg / well;

[0042] Figure 6 Graphs showing the detection of various indicators of the purified Fc / hGH-Fc fusion protein;

[0043] A represents the SEC-HPLC result of the purified Fc / hGH-Fc fusion protein, with a purity of 97.89%.

[0044] B represents the UPLC result of the purified monomeric Fc / hGH-Fc fusion protein, with a purity of 94.5%.

[0045] C represents the LC-Mass assay result of 73252.00 Da for the purified monomeric Fc / hGH-Fc fusion protein, with a theoretical molecular weight of 73251.67 Da.

[0046] D represents the 8%-15% SDS-PAGE gel image detection result of the purified monomeric Fc / hGH-Fc fusion protein, with a loading amount of 5 μg / well;

[0047] Figure 7 Growth-promoting efficacy test of monomeric Fc / hGH-Fc and cyclic Fc-hGH-Fc in a pituitary-removed rat model: comparison of AUC of weight gain among different groups of rats.

[0048] A represents the comparison of AUC (Amount of Weight Gain) among rat groups, and B is a bar chart of weight gain in A.

[0049] Figure 8 Growth-promoting efficacy test of monomeric Fc / hGH-Fc and cyclic Fc-hGH-Fc in a pituitary-removed rat model: comparison of AUC of body length increase among different groups of rats;

[0050] A represents the comparison of AUC (Amount of Count) in rat body length growth among groups, and B is a bar chart of body length growth in A.

[0051] Figure 9 Growth-promoting efficacy of monomeric Fc / hGH-Fc and cyclic Fc-hGH-Fc in a pituitary-removed rat model: comparison of tail length growth AUC among different groups.

[0052] A represents the intergroup comparison of AUC for tail length growth in rats, and B is a bar chart of tail length growth in A.

[0053] Figure 10 Growth-promoting efficacy of monomeric hGH-Fc / Fc and cyclic Fc-hGH-Fc in a pituitary-removed rat model: Comparison of serum IGF-1 levels among the high-dose rat groups.

[0054] A shows the comparison of serum IGF-1 levels among groups in rats, and B is a line graph showing the changes in serum IGF-1 levels in A. Detailed Implementation

[0055] Terminology Definition

[0056] As used herein, the terms "fusion protein of the present invention," "cyclic Fc-hGH-Fc fusion protein," "recombinant cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc," "cyclic Fc-hGH-Fc," and "Fc-hGH-Fc" are used interchangeably and refer to the fusion protein described in the first aspect of the present invention, which is composed of human growth hormone, two linking peptides, and two identical or different Fc fragments. That is, two linking peptides are used to sequentially link the Fc fragment, human growth hormone, and Fc fragment from the N-terminus to the C-terminus to form a molecule. One linking peptide is present between the Fc fragment and human growth hormone, and the other linking peptide is present between human growth hormone and another Fc fragment. The amino acid sequences of the two linking peptides are identical or different, and the two Fc fragments form one or two pairs of disulfide bonds after folding.

[0057] As used herein, the terms "Fc / hGH-Fc", "Fc / Fc-hGH", "hGH-Fc / Fc", "monomer Fc / hGH-Fc", "monomer hGH-Fc / Fc", "Fc / hGH-Fc heterodimeric protein", and "hGH-Fc / Fc heterodimeric protein" are used interchangeably (the amino acid sequence of Fc in Fc / hGH-Fc is shown in SEQ ID NO:3 and the amino acid sequence of hGH-Fc in Fc / hGH-Fc is shown in SEQ ID NO:4). This protein refers to the heterodimeric protein Fc / hGH-Fc formed by the fusion of monomeric Fc and hGH-Fc, where there are one or more pairs of disulfide bonds between the two Fc molecules. The two monomeric Fc molecules in this heterodimeric protein may be identical or different.

[0058] In this invention, there are no particular limitations on the applicable immunoglobulins, which can be derived from humans or other mammals.

[0059] Immunoglobulin elements, or mutants and derivatives thereof. Preferably, they are human immunoglobulins, such as IgG, IgE, IgA, IgM, or combinations thereof.

[0060] Human immunoglobulin G comprises four subclasses: IgG1, IgG2, IgG3, and IgG4. These four subclasses share significant structural similarities, each containing four regions: one variable region (VH) and three constant regions (CH1, CH2, and CH3). The Fc fragment consists of two constant regions (CH2-CH3), with one or more disulfide bonds in the CH2 region, allowing two Fc fragment monomers to covalently bind as a homodimer. Under normal physiological conditions, the concentration of IgG in human plasma is highest for IgG1, followed by IgG2, with lower concentrations of IgG3 and IgG4.

[0061] A preferred Fc element is a human IgG4-Fc fragment, or a mutant or derivative thereof.

[0062] In this invention, the Fc fragment linked to GH (e.g., hGH) via a linker peptide originates from the constant region of immunoglobulin IgG. Among the four human IgG subtypes, IgG1, IgG2, IgG3, and IgG4 can all bind to hGH or its variants via linker peptides.

[0063] Starting with wild-type IgG4-Fc (whose amino acid sequence is shown in SEQ ID NO: 5), various mutations were performed to obtain IgG4-Fc containing FL234-235AK, N297E, N315Q, N384Q mutations and the removal of the K at the C-terminus.

[0064] This invention provides a fusion protein that optionally contains a linker peptide (also called a adaptor protein or linker peptide). The size and complexity of the linker peptide can affect the activity of the protein. Generally, the linker peptide should have sufficient length and flexibility to ensure that the two linked proteins have sufficient spatial freedom to perform their functions. At the same time, the formation of α-helices or β-sheets in the linker peptide should be avoided to prevent its impact on the stability of the fusion protein. Furthermore, the linker peptide should preferably contain amino acids with low immunogenicity.

[0065] The length of the linker peptide is generally 1-80 amino acids, preferably 2-50 amino acids, and even more preferably 5-40 amino acids.

[0066] In this invention, the preferred length of the linker peptide is 30-40 amino acids.

[0067] The preferred flexible linker peptide is SEPATSGSETPGTSESATPESGPGTSTEPSEG, which contains 32 amino acids.

[0068] Preparation of fusion proteins

[0069] As used in this article, "separated" refers to a substance that has been separated from its original environment (if it is a natural substance, the original environment is considered to be natural).

[0070] The initial environment is the natural environment. For example, polynucleotides and polypeptides in their natural state within living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if separated from other substances in their natural state.

[0071] As used in this article, "isolated recombinant fusion protein" refers to a recombinant fusion protein that is essentially free of its natural counterparts.

[0072] Other proteins, lipids, carbohydrates, or other substances. Those skilled in the art can purify recombinant fusion proteins using standard protein purification techniques. Essentially pure proteins produce a single master band on a non-reducing polyacrylamide gel.

[0073] The full-length nucleotide sequence or fragment thereof of the fusion protein or its elements (such as GH, Fc) of this invention can generally be obtained by PCR.

[0074] It can be obtained by amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on publicly available nucleotide sequences, especially open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequences are long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0075] Once the relevant sequence is obtained, recombination can be used to obtain large quantities of the same sequence. This is typically done by...

[0076] The clone is inserted into a vector, then transferred into cells, and the relevant sequence is then isolated from the proliferated host cells using conventional methods.

[0077] In addition, artificial synthesis methods can be used to synthesize relevant sequences, especially when the fragment length is short. Typically, ...

[0078] By first synthesizing multiple small fragments and then connecting them, a very long sequence can be obtained.

[0079] The method of amplifying DNA / RNA using PCR technology is preferred for obtaining the gene of the present invention. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0080] This invention also relates to vectors comprising the polynucleotides of this invention, and to the encoding of proteins using the vectors or fusion proteins of this invention.

[0081] The host cell generated by genetic engineering of the coding sequence, and the method for generating the protein described in this invention through recombination technology.

[0082] Using conventional recombinant DNA technology, the polynucleotide sequence of this invention can be used to express or produce recombinant DNA.

[0083] Protein. Generally, the following steps are involved:

[0084] (1) Transform or transduce suitable host cells using the polynucleotide (or variant) encoding the protein of the present invention, or using a recombinant expression vector containing the polynucleotide;

[0085] (2) Host cells cultured in a suitable culture medium;

[0086] (3) Isolate and purify proteins from culture media or cells.

[0087] Methods well known to those skilled in the art can be used to construct the coding DNA sequence containing the protein of the present invention and suitable...

[0088] Expression vectors for transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology. The DNA sequence can be efficiently linked to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0089] Furthermore, the expression vector preferably contains one or more selective marker genes to provide for selecting transformations.

[0090] Phenotypic traits of host cells, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0091] Vectors containing the appropriate DNA sequence and a suitable promoter or control sequence can be used for transformation of suitable...

[0092] When the host cell is in a suitable environment, it enables the expression of proteins.

[0093] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell.

[0094] Eukaryotic cells, such as mammalian cells. Representative examples include: bacterial cells of Escherichia coli and Streptomyces; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, NSO, COS7, or 293.

[0095] Transforming host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is the original...

[0096] In the case of nucleobiota such as *E. coli*, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be performed by electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0097] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the method used...

[0098] The host cells are used, and the culture medium can be selected from various conventional culture media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature change or chemical induction), and the cells are cultured for a further period.

[0099] As used in this article, "inclusion bodies" refer to high-density, insoluble protein particles that form when exogenous genes are expressed in prokaryotic cells, especially when they are expressed efficiently in Escherichia coli.

[0100] As used in this article, "inclusion body denaturation" refers to the process of redissolving inclusion bodies in an aqueous solution using detergents such as high-concentration urea or guanidine hydrochloride, or other conditions.

[0101] As used in this article, "inclusion body refolding" refers to the process by which, under suitable conditions, the re-dissolved inclusion bodies fold back into functional proteins.

[0102] The proteins used in the above methods can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If

[0103] Proteins can be separated and purified by various separation methods, taking advantage of their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0104] Pharmaceutical Composition and Administration

[0105] The present invention also provides a composition containing an effective amount of the fusion protein of the present invention, and pharmaceutically feasible...

[0106] Acceptable carrier. Typically, the fusion protein of the present invention can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8.

[0107] As used herein, the terms "effective amount" or "effective dose" refer to a dose that produces a functional or active effect in humans and / or animals.

[0108] The amount that is acceptable to humans and / or animals.

[0109] As used in this article, "pharmaceutically acceptable" ingredients are those suitable for use in humans and / or mammals without adverse effects.

[0110] Side effects (such as toxicity, irritation, and allergic reactions) are associated with substances that have a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents.

[0111] The pharmaceutical compositions of the present invention contain a safe and effective amount of the fusion protein of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. The pharmaceutical compositions are preferably manufactured under aseptic conditions. The dosage of the active ingredient is a therapeutically effective amount. The pharmaceutical formulations of the present invention can also be formulated into sustained-release formulations.

[0112] The effective amount of the fusion protein of this invention can vary depending on the administration method and the severity of the disease to be treated.

[0113] The preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials).

[0114] (Test). The factors mentioned include, but are not limited to: the pharmacokinetic parameters of the fusion protein of the present invention, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the patient's weight, the patient's immune status, the route of administration, etc.

[0115] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0116] Unless otherwise stated, all raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Experimental methods in the following examples that do not specify specific conditions are generally performed under standard conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 4th edition), or as recommended by the manufacturer.

[0117] In the following examples, the Fc in the cyclic Fc-hGH-Fc fusion protein and the monomeric Fc / hGH-Fc were mutated from the wild-type Fc segment of IgG4 (amino acid sequence as shown in SEQ ID NO:5), and the hGH was a wild-type sequence from a gene library (amino acid sequence as shown in SEQ ID NO:1). The amino acid sequence of the cyclic Fc-hGH-Fc fusion protein is shown in SEQ ID NO:2; the amino acid sequence of the Fc in the monomeric Fc / hGH-Fc is shown in SEQ ID NO:3; and the amino acid sequence of the hGH-Fc in Fc / hGH-Fc is shown in SEQ ID NO:4.

[0118] Example 1: Design, synthesis and plasmid construction of a circular Fc-hGH-Fc fusion protein

[0119] 1. Expression plasmid construction

[0120] Primers were designed, a new plasmid pET41a-Fc-hGh-Fc was constructed, and it was transformed into BL21(DE3) strain for expression testing. The amino acid sequence of the expressed fusion protein is shown in SEQ ID NO.2, and the structural schematic diagram of the circular Fc-hGH-Fc fusion protein is shown in [Figure number missing]. Figure 1 As shown, the basic process is as follows: primer design, PCR amplification, vector enzyme digestion, recombination, sequencing, plasmid extraction, transformation of BL21 (DE3), low-level expression, and high-level expression.

[0121] Primers were synthesized by Beijing Qingke Xinyue Biotechnology Co., Ltd. Following the procedures described in *Molecular Cloning*, overlap PCR was performed to obtain the target fragment. Then, recombination ligation of the fragment and a universal vector was carried out, followed by transformation, sequencing, and bacterial preservation. 2. Plasmid Extraction

[0122] Extraction was performed according to the procedures outlined in the Qiagen Mini-prep Kit and Qiagen Endofree Maxi-prep Kit. Sequencing was then used to confirm the correct target gene sequence.

[0123] Example 2. Expression and inclusion body preparation of cyclic Fc-hGH-Fc and monomeric Fc / hGH-Fc fusion proteins

[0124] The cyclic Fc-hGH-Fc fusion protein of this invention can be effectively expressed in prokaryotic systems, with advantages such as high expression levels, short expression time, and low cost. The entire fermentation expression process takes only three days. The implementation scheme is as follows:

[0125] 1. Preparation of Escherichia coli expressing circulated Fc-hGH-Fc

[0126] Following molecular biology methods, the plasmid obtained in Example 1 was transformed into BL21(DE3) competent cells. The bacterial culture containing the target protein was added to a 250 mL Erlenmeyer flask containing Kan broth and cultured overnight at 30 °C with shaking at 220 rpm. The next day, the overnight culture was transferred at a ratio of 1:50 to a 500 mL TB Erlenmeyer flask (2 L capacity) containing Kan resistance. Initial OD... 600 Incubate at approximately 0.1, 37°C, 220 rpm until OD reaches [value missing]. 600 =1.5 (approximately 2.5 h); cool to 30°C; add 0.5 mM IPTG to a final concentration; incubate overnight at 30°C and 220 rpm; then discard the supernatant and retain the precipitate.

[0127] 2. Expression of the cyclic Fc-hGH-Fc fusion protein

[0128] The bacterial strain obtained in step 1 was inoculated at a 1% inoculum in 50 mL of LB medium and cultured for 4-5 h as seed culture. This seed culture was then added to a 2.8 L fermenter containing FDMO medium to begin fermentation. The fermentation conditions were set as follows: stirring speed 200 rpm, culture temperature 37 ℃, aeration rate 3 L / min, pH 7.0, pressure 0 Bar, dissolved oxygen 100%. During the culture, the temperature was maintained at 37 ℃, pH 7.0, and the pressure at 0 Bar. The stirring speed and aeration rate were gradually increased to maintain a certain dissolved oxygen concentration. When the dissolved oxygen level rose, fed-batch feeding was initiated. When the cell density reached an OD600 value of 53, a final concentration of 0.5 mM IPTG was added, and the temperature was set to 37 ℃ for induction. Samples were taken every hour from 0 h of induction until the end of fermentation. These samples were used for SDS-PAGE analysis. Fermentation was terminated after 6 hours of induction, followed by centrifugation, discarding the supernatant, and retaining the bacterial cells. The expression of the cyclic Fc-hGH-Fc fusion protein produced insoluble inclusion bodies. The cells were then subjected to high-pressure disruption and washed twice with PBS buffer at pH 7.4 to recover the inclusion bodies. In this process, the expression and disruption of the fusion protein only required one complete operation to obtain a high yield of inclusion bodies. 467g of wet cells and 215g of inclusion bodies were obtained from 2.8L of fermentation broth.

[0129] The entire process of expressing the cyclic Fc-hGH-Fc fusion protein and cleaving and washing the inclusion bodies took 3 days.

[0130] 3. Expression of monomeric Fc / Fc-hGH fusion protein and fragmentation and washing of inclusion bodies

[0131] The same plasmid was constructed for the monomeric Fc / hGH-Fc. The monomeric Fc / hGH-Fc expressed in the prokaryotic system was expressed using the same method as the circular Fc-hGH-Fc fusion protein. However, the expression process was carried out in two separate containers: one containing only Fc and the other containing hGH-Fc. After the two batches of expression, the cells were lysed and washed separately using the same buffer conditions as those used for lysing and washing the circular Fc-hGH-Fc, resulting in inclusion bodies containing only Fc or Fc-hGH.

[0132] 4. Protein expression and detection of inclusion body fragmentation and washing.

[0133] The results are as follows Figure 2 As shown, the cyclic Fc-hGH-Fc fusion protein and the monomeric Fc / hGH-Fc fusion protein were mainly expressed in inclusion bodies during fermentation.

[0134] 5. Detection of concentration of cyclic Fc-hGH-Fc fusion protein

[0135] The expression level of the cyclic Fc-hGH-Fc fusion protein was detected by UPLC quantification. The UPLC quantification method was performed at a flow rate of 0.4 ml / min at 50℃ for a total of 16 min, with a detection UV of 215 nm. Mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B was an acetonitrile solution containing 0.085% trifluoroacetic acid. The column was first equilibrated at a ratio of 95% A / 5% B for 1 minute. After 1.5 minutes, the ratio of A to B was increased to the initial protein elution ratio of 70% / 30%, followed by a 10-minute linear gradient elution, gradually increasing the proportion of B to 60%. The column was then rinsed with 90% B for 1.5 minutes, and finally reequilibrated to the initial equilibration ratio. Inclusion body standards were selected from previously purified proteins, and a standard curve was constructed using a multi-point method for quantification. The concentration of the analyte was determined based on the peak area (the concentration of the analyte was controlled within the concentration range detected by the standard curve). The results are as follows: Figure 3 As shown: After lysis, the cyclic Fc-hGH-Fc was detected. Approximately 7g of the target protein can be obtained from 1 liter of fermentation culture.

[0136] 6. Conclusion

[0137] The expression and inclusion body preparation of cyclic Fc-hGH-Fc fusion proteins are simpler and easier to operate than those of monomeric Fc / Fc-hGH fusion proteins, requiring only one fermentation and one lysis and washing process, saving both time and cost. Simultaneously, most of the protein precipitates, resulting in a high recovery rate within the inclusion bodies, which lays the foundation for subsequent refolding and increased yield.

[0138] Example 3. Refolding of cyclic Fc-hGH-Fc fusion protein with control monomeric Fc / Fc-hGH fusion protein

[0139] 1. The refolding process of the cyclic Fc-hGH-Fc fusion protein

[0140] After lysis of the cyclic Fc-hGH-Fc fusion protein, refolding can be performed. 50g of inclusion bodies (approximately 0.65L of fermentation broth) are dissolved in 15 times their volume (750ml) of dissolving buffer, containing 4-8M urea and 1-10mM DTT (final concentration) at pH 8.5. Quantification is performed after 2-3 hours at room temperature, yielding approximately 4400mg of the target protein. This determines the concentration of the cyclic Fc-hGH-Fc fusion protein in the buffer, preparing for subsequent refolding. After 2-3 hours of dissolution, the buffer is diluted to a concentration of 2-4mg / ml of the cyclic Fc-hGH-Fc fusion protein, with 1.5-3M urea, pH 8, and 2-5mM cysteine ​​added. The mixture is stirred overnight at room temperature. The refolding rate is assessed by UPLC the next day. 2800mg of well-refolded target protein was obtained, representing a refolding rate of 65%.

[0141] 2. The refolding process of monomeric Fc / hGH-Fc fusion protein

[0142] The refolding process for monomeric Fc / hGH-Fc is essentially the same as that for cyclic Fc-hGH-Fc fusion protein. Since expression and inclusion body preparation are performed separately, the inclusion bodies also need to be dissolved separately at a ratio of 15 w / v for quantification. Based on the quantification results, they are mixed at a 1:1 molar ratio (10g Fc and 20g hGH-Fc), resulting in approximately 6.2g of protein in the refolding solution. Subsequent operations are the same as for cyclic Fc-hGH-Fc fusion protein. After overnight refolding, 1.54g of monomeric Fc / hGH-Fc protein is obtained, with a refolding rate of 25%. This process requires two quantifications, and errors in the quantification process can easily lead to uneven proportions during later mixing and refolding, potentially resulting in Fc, hGH-Fc, Fc / Fc, or hGH-Fc / hGH-Fc combinations. These components are very similar to the monomeric Fc / hGH-Fc fusion protein in terms of properties and structural characteristics, making them difficult to distinguish during the later purification process and increasing the difficulty of purification.

[0143] 3. Detection of refolding results of cyclic Fc-hGH-Fc and monomeric Fc / hGH-Fc proteins

[0144] Figure 3 This is a graph showing the quantitative detection of the cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc fusion protein after bacterial rupture. Figure 4These are SDS-PAGE gel images of the cyclic Fc-hGH-Fc fusion protein and the monomeric Fc / hGH-Fc fusion protein before and after renaturation. The SDS-PAGE images show that after renaturation, the desired proteins are formed: the cyclic Fc-hGH-Fc fusion protein and the monomeric Fc / Fc-hGH.

[0145] 4. Conclusion

[0146] Throughout the refolding process, the cyclic Fc-hGH-Fc fusion protein is simple to process, has high refolding efficiency, and few impurities. 1L of E. coli fermentation broth can express 7g of the target protein, and from expression to lysis to final refolding, 4.3g of the target protein can be obtained. This is significantly higher than the expression and refolding yield of monomeric Fc / hGH-Fc, and the refolding solution contains fewer impurities in Fc / Fc and hGH-Fc / hGH-Fc, making subsequent purification easier. Similarly, compared to the monomeric Fc / hGH-Fc fusion protein (CN 108794634A) GH-Linker-Fc / Fc expressed in the eukaryotic system, its expression level is only 3.2 g / L. After purification with GE's Mab-selectsure affinity medium, there are still 6% hGH-Fc / hGH-Fc and Fc / Fc and 1.2% other impurities. Therefore, the cyclic Fc-hGH-Fc fusion protein implemented in this scheme has obvious industrial value advantages.

[0147] Example 4. Purification process of cyclic human growth hormone-Fc fusion protein (Fc-hGH-Fc) and control group monomeric Fc / hGH-Fc fusion protein.

[0148] 1. Purification process of cyclic Fc-hGH-Fc fusion protein

[0149] Purification of fusion proteins primarily employs chromatographic methods, such as anion exchange chromatography, cation exchange chromatography, gel filtration chromatography, hydrophobic chromatography, and reversed-phase chromatography. Different combinations of these purification steps can be used to achieve essentially uniform protein purity. Affinity chromatography columns containing specific antibodies, receptors, or ligands of the fusion protein, such as recombinant rProtein A and rProtein G, can also be used to purify the expressed fusion protein. Since no homodimers are generated during refolding, subsequent purification is easier. After purification, the yield of the cyclic Fc-hGH-Fc fusion protein from the refolding buffer to the final product was 14%: the final product had a UPLC purity of 94.2% and a SEC-HPLC purity of 97.28%. Figure 5 As shown.

[0150] In the UPLC quantitative analysis, the flow rate was 50℃; 0.4 ml / min; for a total of 16 min; and the detection UV was 215 nm. Mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B was an acetonitrile solution containing 0.085% trifluoroacetic acid. The column was first equilibrated at a ratio of 95% A / 5% B for 1 min. After 1.5 min, the proportion of B was increased to 30% of the initial protein elution concentration, followed by a 10-minute linear gradient elution, gradually increasing the proportion of B to 60%. The column was then rinsed with 90% B for 1.5 min, and finally reequilibrated to the initial equilibration ratio. Inclusion body standards were selected from previously purified proteins, and a standard curve was constructed using a multi-point method for quantification. The concentration of the analyte was determined based on the peak area.

[0151] SEC-HPLC can monitor protein aggregates, concentration, and purity. The instrument used was an Agilent 1100. Detection was performed using a TSK gel G2000SWxl column, 7.8 mm * 300 mm. Method: Flow rate 0.4 ml / min, column temperature room temperature, UV 280. Mobile phase: 2xPBS, 5% IPA / water solution, pH 3.0. Run time: 45 minutes. The BIO-RAD protein marker (Gel Filtration Standard, CAT#: 151-1901) was used as a molecular weight reference.

[0152] The purified sample was analyzed by LC-Mass. A Waters ACQUITY UPLC Protein BEH C4 300A column (P / N: 186004496) was used for HPLC analysis. The flow rate was 0.4 mL / min, the column temperature was 70 °C, and the UV detection was 215 / 280 nm. Mobile phase A was an aqueous solution containing 0.1% FA, and mobile phase B was an acetonitrile solution containing 0.1% FA. The column was first equilibrated at a ratio of 95% A / 5% B for 1 minute. After 1.5 minutes, the proportion of B was increased to 20% of the initial protein elution ratio, followed by a 10-minute linear gradient elution, gradually increasing the proportion of B to 60%. The column was then flushed with 95% B for 1 minute, and finally reequilibrated to the initial ratio. The mass spectrometry was set to Full scan mode, cation polarity, Cone voltage of 70V, Mass range of High (400-7000 m / z), Scan rate of 1Hz, Capillary voltage of 1.5KV, and Desolvation temperature of 550℃.

[0153] 2. Purification process of human growth hormone-Fc fusion protein Fc / hGH-Fc

[0154] Because the Fc / Fc-hGH fusion protein produces a mixture of various fusion products in the refolding solution, including (hGH-Fc / hGH-Fc), homodimeric Fc fusion protein (Fc / Fc), and monomeric human growth hormone-Fc protein (hGH-Fc), the generation of numerous impurities leads to low yield, low purity, and instability. Furthermore, since these impurities have extremely similar physical and biochemical properties to the target protein, they are difficult to remove completely during purification using affinity columns, ion exchange columns, hydrophobic columns, and mixed packing materials. Ultimately, SEC columns are used to remove residual hGH-Fc / hGH-Fc, Fc / Fc, Fc, and hGH-Fc to achieve high-purity monomeric Fc / hGH-Fc fusion protein. After four rounds of purification, including the final SEC step, the yield of the monomeric Fc / hGH-Fc fusion protein from the refolding buffer to the final product was 14%. The final product had a UPLC purity of 94.5% and a SEC-HPLC purity of 97.89%, with an endotoxin level of 0.17 EU / mg. A total of 222 mg of target protein was obtained (10 g of monomeric Fc inclusion bodies and 20 g of monomeric human growth hormone-Fc protein (hGH-Fc) inclusion bodies; approximately 6.2 g of protein was dissolved in the refolding buffer after mixing; subsequent refolding procedures were the same as those for cyclic Fc-hGH-Fc fusion protein). After overnight refolding, 1.54 g of monomeric Fc / hGH-Fc fusion protein was obtained, with a refolding rate of 25%. After purification, 222 mg of monomeric Fc / hGH-Fc fusion protein was obtained).

[0155] After purification, the results of various protein parameters of the monomeric Fc / hGH-Fc fusion protein are shown in Table 1 and 2. Figure 6 As shown.

[0156] Table 1. Preparation process of cyclic Fc-hGH-Fc fusion protein and control group monomeric Fc / hGH-Fc fusion protein, and detection results of various protein indicators during the process.

[0157]

[0158] 3. Conclusion:

[0159] By comparing the fermentation to production process of cyclic Fc-hGH-Fc fusion protein and monomeric Fc / hGH-Fc fusion protein (Table 1), it can be seen that due to the simple molecular structure and high stability of cyclic Fc-hGH-Fc fusion protein, the refolding process in the early stage avoids the generation of a mixture of various similar properties. Therefore, the use of gel filtration chromatography to remove residual hGH-Fc / hGH-Fc, Fc / Fc, monomeric human growth hormone-Fc protein (hGH-Fc), and monomeric Fc protein can be avoided during the purification process, thus obtaining high-purity cyclic Fc-hGH-Fc fusion protein. Compared to the monomeric Fc / hGH-Fc fusion protein (CN 108794634A) GH-Linker-Fc / Fc expressed in the eukaryotic system, due to the presence of a mixture of similar components in the cell lysate, even after purification using GE's Mabselectsure affinity medium, 6% hGH-Fc / hGH-Fc, Fc / Fc, and 1.2% impurities remained. Therefore, the cyclic Fc-hGH-Fc fusion protein produced by this method demonstrates a greater industrial value advantage in purification.

[0160] Example 5. Detection of the activity of cyclic Fc-hGH-Fc fusion protein and control monomeric Fc / hGH-Fc fusion protein using Biacore SPR-8K.

[0161] The specific steps are as follows:

[0162] 1) Sample dilution:

[0163] The running buffer was 20 mM Hepes buffer, pH 7.4, 0.05% Tween-20.

[0164] The extracellular soluble domain of the hGH Receptor ligand was constructed and expressed by our company (GHR, GenBank: AAA52555.1) at a concentration of 10 μg / ml. It was immobilized on a CM5 chip in acetate buffer at pH 4.0, with an immobilization volume of 1000 RU.

[0165] The concentration gradients of the analytes cyclic Fc-hGH-Fc and monomeric Fc / hGH-Fc were: 0 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM.

[0166] 2) The SPR running procedure was as follows: flow rate 30 μl / min, binding time 200 seconds, and dissociation time 1000 seconds. The final results were fitted using SPR data analysis software to obtain the dissociation constant K. D The values ​​are shown in Table 2 below.

[0167] Table 2. Dissociation constant K of the extracellular soluble region of the cyclic Fc-hGH-Fc fusion protein and the control group monomeric Fc / hGH-Fc fusion protein and its ligand hGHReceptor. D

[0168]

[0169] SPR test data result analysis:

[0170] The results of the protein binding experiment with hGH-Receptor are shown in Table 2. In this test, the cyclic Fc-hGH-Fc fusion protein (KD=18.4nM) and the control monomeric Fc / hGH-Fc fusion protein (KD=7.2nM) showed that the affinity of the cyclic Fc-hGH-Fc for the receptor GHR was about 2 to 3 times weaker than that of the monomeric Fc / hGH-Fc. This indicates that in the Fc-hGH-Fc molecule, because both the N-terminus and C-terminus of hGH are connected to the Fc protein, the Fc creates a certain steric hindrance to the binding of hGH to the receptor GHR, weakening its binding to the receptor GHR, thereby weakening receptor-mediated hGH degradation and prolonging the drug's half-life in vivo.

[0171] Example 6. Pharmacodynamic experiment of cyclic human growth hormone-Fc fusion protein (Fc-hGH-Fc) in rats.

[0172] 1. Experimental Design and Methods

[0173] The pituitary gland was surgically removed from young female SD rats via the ear canal. All rats underwent pituitary removal surgery via ear canal puncture after induction anesthesia with isoflurane. A stereotactic pituitary resection instrument was used to aspirate and examine the pink pituitary tissue. Rats were weighed at 2 and 3 weeks post-surgery. Rats with a weight gain within ±10% and a healthy appearance were selected as successful model rats. Forty-two model rats were selected based on their weight gain rate, with a weight gain rate (2–3 weeks post-surgery) of 1.8 ± 1.6%. Animals were randomly and evenly divided into seven groups based on their body weight and weight gain rate: a model control group (0 nmol / kg), low (2.6 nmol / kg) and high (24.0 nmol / kg) dose groups of monomeric Fc / hGH-Fc, low (2.6 nmol / kg) and high (24.0 nmol / kg) dose groups of cyclic Fc-hGH-Fc, and low (2.6 nmol / kg) and high (24.0 nmol / kg) dose groups of cytosine (a PEG-modified hGH), with six animals in each group. Cytosine was used as a positive control in this experiment.

[0174] After grouping, the drug was administered subcutaneously once a week for 5 consecutive weeks, for a total of 5 administrations, with a dosage of 2.0 mL / kg. Body weight was measured daily after administration, and body length and tail length were measured weekly. The area under the curve (AUC) of weight gain was calculated. （体重增长克数） Area under the body length growth curve (AUC) （体长增长量） and the area under the tail length growth curve AUC （尾长增长量） Blood samples were collected from the orbital venous plexus of rats in the high-dose group before the first administration and on days 2, 4, 6, and 8 to separate serum, and the insulin-like growth factor 1 (IGF-1) content was measured by ELISA.

[0175] 2. Data Processing and Statistical Analysis

[0176] Excel 2013 and SPSS 22.0 were used for data entry and statistical analysis. The area under the curve (AUC) for weight gain (grams), body length gain (body length gain), and tail length gain (tail length gain) were calculated using the trapezoidal area method. Levene's test was used to test for homogeneity of variance in the quantitative data. If the variances among multiple groups were homogeneous (p>0.01), the results of the one-way ANOVA LSD test were directly used for pairwise comparisons between groups. If the variances among multiple groups were unequal (p<0.01), the Mann-Whitney U nonparametric test or the two independent samples t-test was used for pairwise comparisons between groups. All data are presented as follows: x ± s (standard deviation) represents the mean. p < 0.05 indicates that the difference is statistically significant.

[0177] 3. Experimental data on the growth-promoting effects of protein on a pituitary-removed rat model.

[0178] Comparison of AUC values ​​for weight gain in rats between groups is shown in the figure. Figure 7 Compared with the model control group, the AUC of the test sample monomeric Fc / hGH-Fc and cyclic Fc-hGH-Fc dose groups and the Jinsaizen dose groups after administration were significantly higher. （体重增长克数） All were significantly greater than the model control group ( p <0.05). Comparison between different samples at the same dose: AUC of monomeric Fc / hGH-Fc and low-dose cyclic Fc-hGH-Fc group. （体重增长克数） All were lower than the low-dose group of Kinsey ( p <0.05, p = 0.05).

[0179] Comparison of AUC for rat body length growth between groups is shown in the figure. Figure 8 Compared with the model control group, the AUC of the test sample monomeric Fc / hGH-Fc and cyclic Fc-hGH-Fc dose groups and the Jinsaizen dose groups after administration were significantly higher. （体长增长量）All were significantly greater than the model control group ( p <0.05). Comparison between different samples at the same dose, the AUC of the low-dose monomeric Fc / hGH-Fc group was [data missing]. （体长增长量） Significantly less than the low-dose group of Kinsey ( p <0.05). The AUC of the low-dose cyclic Fc-hGH-Fc group was... （体长增长量） Significantly greater than the monomeric Fc / hGH-Fc low-dose group ( p <0.05).

[0180] Comparison of AUC values ​​for rat tail length growth between groups is shown in the figure. Figure 9 Compared with the model control group, the AUC of the test sample monomeric Fc / hGH-Fc and cyclic Fc-hGH-Fc dose groups and the Jinsai Zeng dose groups were significantly lower. （尾长增长量） All were significantly greater than the model control group ( p <0.05). Comparing different samples at the same dose, cyclic Fc-hGH-Fc showed a greater increase than monomeric Fc / hGH-Fc, and both were higher than the low-dose group of Jinsai. AUC of monomeric Fc / hGH-Fc, cyclic Fc-hGH-Fc, and the high-dose group of Jinsai. （尾长增长量） There were no significant differences between the pairs.

[0181] Changes in serum IGF-1 levels in rats in each high-dose group are shown below. Figure 10 Before administration, there were no significant differences in serum IGF-1 levels among the monomeric Fc / hGH-Fc, cyclic Fc-hGH-Fc, and high-dose Jinsai groups. On day 2, there was no significant difference in serum IGF-1 levels between the high-dose monomeric Fc / hGH-Fc group and the high-dose Jinsai group, while the high-dose cyclic Fc-hGH-Fc group had lower levels than the high-dose Jinsai group. However, from day 4 until the end of the experiment, the serum IGF-1 levels in the high-dose cyclic Fc-hGH-Fc Jinsai group were significantly higher than those in the monomeric Fc / hGH-Fc group. There were no significant differences between the cyclic Fc-hGH-Fc and high-dose Jinsai groups. The serum IGF-1 levels in the monomeric Fc / hGH-Fc and cyclic Fc-hGH-Fc high-dose groups were slightly lower than those in the high-dose Jinsai group.

[0182] 4. Bioactivity test results

[0183] from Figures 7-10As can be seen, the cyclic Fc-hGH-Fc fusion protein modified by this invention exhibits significantly better performance than the monomeric Fc / hGH-Fc in terms of both in vitro binding to GH-receptor and in vivo growth in body weight, tail length, and IGF-1 levels in blood. Compared to long-acting growth promoters like Jinsaizeng on the Chinese market, its activity is equal to or even higher than Jinsaizeng's efficacy. Furthermore, the cyclic Fc-hGH-Fc fusion protein also demonstrates excellent long-lasting growth-promoting effects.

[0184] Summary and Discussion

[0185] In summary, the modified cyclic Fc-hGH-Fc structure of this invention enables high-level expression in prokaryotes during production, ensuring no dimer form of impurity Fc fusion protein is generated during prokaryotic expression. Therefore, its refolding and purification processes are simpler and yield higher outputs compared to monomeric Fc / hGH-Fc expressed separately in eukaryotic cells or prokaryotes, significantly reducing the production cost and time of long-acting growth hormone. The growth hormone produced using this design and process maintains high activity and excellent long-lasting effects. Furthermore, it avoids the risk of hepatotoxicity and nephrotoxicity associated with the introduction of foreign substances (such as PEGylation), and reduces the risk of immunogenicity from glycosylation compared to eukaryotic cell expression.

[0186] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. sequence list <110> Beijing Zhidao Biotechnology Co., Ltd. <120> A recombinant cyclic human growth hormone-Fc fusion protein and its application <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 191 <212> PRT <213> Human (Homo sapiens) <400> 1 Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg 1 5 10 15 Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu 20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro 35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg 50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu 65 70 75 80 Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val 85 90 95 Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp 100 105 110 Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser 130 135 140 Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160 Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe 165 170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe 180 185 190 <210> 2 <211> 697 <212> PRT <213> Artificial Sequence <400> 2 Ala Gly Pro Cys Pro Ala Pro Glu Ala Lys Gly Gly Pro Ser Val Phe 1 5 10 15 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 20 25 30 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 35 40 45 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 50 55 60 Lys Pro Arg Glu Glu Gln Phe Glu Ser Thr Tyr Arg Val Val Ser Val 65 70 75 80 Leu Thr Val Leu His Gln Asp Trp Leu Gln Gly Lys Glu Tyr Lys Cys 85 90 95 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 100 105 110 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 115 120 125 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 130 135 140 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Gln Gly 145 150 155 160 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 165 170 175 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 180 185 190 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 195 200 205 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Ser Glu Pro 210 215 220 Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro 225 230 235 240 Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Phe Pro Thr 245 250 255 Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg 260 265 270 Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr 275 280 285 Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser 290 295 300 Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr 305 310 315 320 Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile 325 330 335 Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn 340 345 350 Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys 355 360 365 Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly 370 375 380 Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp 385 390 395 400 Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu 405 410 415 Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile 420 425 430 Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Ser Glu Pro Ala 435 440 445 Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu 450 455 460 Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ala Gly Pro Cys 465 470 475 480 Pro Ala Pro Glu Ala Lys Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 485 490 495 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 500 505 510 Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp 515 520 525 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 530 535 540 Glu Gln Phe Glu Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 545 550 555 560 His Gln Asp Trp Leu Gln Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 565 570 575 Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 580 585 590 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu 595 600 605 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 610 615 620 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Gln Gly Gln Pro Glu Asn 625 630 635 640 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 645 650 655 Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn 660 665 670 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 675 680 685 Gln Lys Ser Leu Ser Leu Ser Leu Gly 690 695 <210> 3 <211> 219 <212> PRT <213> Artificial Sequence <400> 3 Ser Cys Ala Pro Glu Ala Lys Gly Gly Pro Ser Val Phe Leu Phe Pro 1 5 10 15 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 20 25 30 Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn 35 40 45 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 50 55 60 Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 65 70 75 80 Leu His Gln Asp Trp Leu Gln Gly Lys Glu Tyr Lys Cys Lys Val Ser 85 90 95 Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 110 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 115 120 125 Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe 130 135 140 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Gln Gly Gln Pro Glu 145 150 155 160 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 165 170 175 Phe Leu Val Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 180 185 190 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 195 200 205 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 4 <211> 432 <212> PRT <213> Artificial Sequence <400> 4 Met Ser Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met 1 5 10 15 Leu Arg Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu 20 25 30 Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln 35 40 45 Asn Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser 50 55 60 Asn Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile 65 70 75 80 Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg 85 90 95 Ser Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val 100 105 110 Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly 115 120 125 Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr 130 135 140 Tyr Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys 145 150 155 160 Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu 165 170 175 Thr Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly 180 185 190 Phe Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro 195 200 205 Gln Gly Ala Pro Gln Ser Cys Ala Pro Lys Ala Glu Gly Gly Pro Ser 210 215 220 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 225 230 235 240 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 245 250 255 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 260 265 270 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 275 280 285 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 290 295 300 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 305 310 315 320 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 325 330 335 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys 340 345 350 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 355 360 365 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 370 375 380 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 385 390 395 400 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 405 410 415 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 420 425 430 <210> 5 <211> 219 <212> PRT <213> Artificial Sequence <400> 5 Ser Cys Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 1 5 10 15 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 20 25 30 Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn 35 40 45 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 50 55 60 Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 65 70 75 80 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 85 90 95 Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 110 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 115 120 125 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 130 135 140 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 145 150 155 160 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 165 170 175 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 180 185 190 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 195 200 205 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215

Claims

1. A recombinant cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc, characterized in that, The amino acid sequence of the recombinant cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc is shown in SEQ ID NO:

2.

2. A DNA molecule encoding the recombinant cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc as described in claim 1.

3. An expression vector comprising the DNA molecule of claim 2, characterized in that, The vector is a eukaryotic or prokaryotic cell expression vector.

4. A host cell comprising the expression vector of claim 3, characterized in that, The cells are selected from eukaryotic or prokaryotic cells.

5. The host cell according to claim 4, characterized in that, The cells in question are Escherichia coli.

6. A method for producing the recombinant cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc according to claim 1, comprising the following steps: (a) Culturing the host cells of claim 5 under suitable expression conditions; (b) The human growth hormone-Fc fusion protein Fc-hGH-Fc was isolated from the host cells.

7. The method according to claim 6, characterized in that, In step (b), the separation includes: high-pressure disruption of bacterial cells, washing twice with PBS buffer, and recovery of inclusion bodies; disruption and elution of inclusion bodies to ensure that the target protein is not present in the supernatant; refolding of the fusion protein Fc-hGH-Fc; and purification of the fusion protein Fc-hGH-Fc.

8. A pharmaceutical composition, characterized in that, The recombinant cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc of claim 1 comprises a therapeutically effective amount and a pharmaceutically acceptable carrier.

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