Fc-hGH fusion protein, coding nucleic acid, vector, engineering cell and construction and preparation method and application of Fc-hGH fusion protein

Abstract

The invention relates to the technical field of biological medicines, in particular to an Fc-hGH fusion protein, coding nucleic acid, a carrier, an engineering cell as well as a construction and preparation method and application of the Fc-hGH fusion protein. From an N terminal to a C terminal, the Fc-hGH fusion protein comprises hGH and an IgG4 Fc mutant which are connected in sequence; compared with a wild type IgG4 Fc, the IgG4 Fc mutant has the following mutations: F234A, L235A, M428L and N434S. The invention also discloses a preparation method of the IgG4 Fc mutant. The Fc fragment variant of the IgG4 and the growth hormone form the fusion protein, and through the design of the Fc fragment variant of the IgG4, the ADCC effect, the ADCP effect and the CDC effect caused by the combination of an Fc region and Fc gamma R are eliminated, the in-vivo distribution of the drug is changed, the in-vivo half-life period of the drug is prolonged, and the molecular stability can be improved.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to Fc-hGH fusion proteins, encoding nucleic acids, vectors, engineered cells, their construction and preparation methods, and applications. Background Technology

[0002] Recombinant human growth hormone has the same amino acid sequence and spatial conformation as human growth hormone, and has the same biological effects as endogenous human growth hormone. It can promote the growth of bones, internal organs and the whole body, promote protein synthesis, and affect fat and mineral metabolism, playing a key role in human growth and development.

[0003] Traditional long-acting human growth hormones include:

[0004] CN110256575A discloses a long-acting recombinant human growth hormone (rhGH)-Fc fusion protein. The fusion protein consists of human growth hormone, a linker peptide, and an Fc fragment. From the N-terminus to the C-terminus, the fusion protein comprises human growth hormone, a linker peptide, and an Fc fragment, with a total amino acid sequence of 431 amino acids. The first 191 amino acids are the human growth hormone sequence, positions 192 to 206 are the linker peptide sequence, and positions 207 to 431 are the Fc fragment of human IgG. The Fc fragment is selected from the Fc fragment of human IgG, preferably the Fc fragment of human IgG4. The Fc fragment of the fusion protein includes four amino acid mutations: S228P, F234A, L235A, and V308P. The residues in the IgG Fc region are numbered according to the Kabat Eu Index.

[0005] CN101970492B discloses an Fc variant with altered binding to FcRn, the Fc variant having one to five amino acid modifications compared to IgG1, IgG2, IgG3, or IgG4 of the parental Fc polypeptide, wherein the Fc variant comprises amino acid modifications V259I and V308F, wherein the Fc variant exhibits increased binding to FcRn compared to the IgG Fc polypeptide, and wherein the variant is numbered according to the EU index of Kabat et al.

[0006] However, the stability of recombinant human growth hormone fusion proteins constructed using traditional Fc fragments needs to be improved. Summary of the Invention

[0007] Based on this, one or more embodiments of this application provide Fc-hGH fusion protein, encoding nucleic acid, vector, engineered cells, methods for their construction and preparation, and applications. These include the following technical solutions:

[0008] One or more embodiments of this application provide an Fc-hGH fusion protein, from the N-terminus to the C-terminus, the Fc-hGH fusion protein comprising hGH and an IgG4 Fc mutant connected in sequence;

[0009] Compared to the wild-type IgG4 Fc, the IgG4 Fc mutant has the following mutations: F234A, L235A, M428L, and N434S.

[0010] In some embodiments of this application, the Fc-hGH fusion protein satisfies one or more of the conditions shown in (1) and (2) below:

[0011] (1) The IgG4 Fc mutant also has the mutation S228P;

[0012] (2) The N-terminus of the hGH and the IgG4 Fc mutant are linked by or without a linker peptide;

[0013] Optionally, the linker peptide contains 4 to 27 amino acids;

[0014] Optionally, the structural formula of the linker peptide is (GGGGS)n, where n is 2 to 4.

[0015] In some embodiments of this application, the Fc-hGH fusion protein satisfies one of the conditions shown in (1) and (2):

[0016] (1) The hinge region of the IgG4 Fc mutant has the same length as the hinge region of the wild-type IgG4 Fc;

[0017] Optionally, the amino acid sequence of the IgG4 Fc mutant is shown as positions 207-435 in SEQ ID NO.3;

[0018] Optionally, the amino acid sequence of the Fc-hGH fusion protein is shown in SEQ ID NO.1 or SEQ ID NO.3;

[0019] (2) The hinge region of the IgG4 Fc mutant is shorter than that of the wild-type IgG4 Fc;

[0020] Optionally, the hinge region of the IgG4 Fc mutant contains 3 to 5 amino acids;

[0021] Optionally, the amino acid sequence of the IgG4 Fc mutant is shown as positions 192-412 in SEQ ID NO.2;

[0022] Optionally, the amino acid sequence of the Fc-hGH fusion protein is shown in SEQ ID NO.2 or SEQ ID NO.4.

[0023] One or more embodiments of this application provide a nucleic acid molecule that encodes the Fc-hGH fusion protein.

[0024] One or more embodiments of this application provide a vector comprising the nucleic acid molecule described above.

[0025] One or more embodiments of this application provide a cell that expresses the Fc-hGH fusion protein, the nucleic acid molecule, or the vector.

[0026] One or more embodiments of this application provide a method for constructing the cell described above, the method comprising the step of introducing the nucleic acid molecule or the vector into a target cell.

[0027] One or more embodiments of this application provide a method for preparing the Fc-hGH fusion protein, the method comprising the steps of culturing the cells and isolating the Fc-hGH fusion protein from the resulting culture.

[0028] One or more embodiments of this application provide the use of the Fc-hGH fusion protein, the nucleic acid molecule, the vector, or the cell in the preparation of a medicament for human growth hormone deficiency and diseases caused by human growth hormone deficiency.

[0029] One or more embodiments of this application provide a drug comprising the Fc-hGH fusion protein.

[0030] Compared with traditional technologies, this application has the following advantages:

[0031] This application proposes to form a fusion protein by combining a variant of the Fc fragment of IgG4 with growth hormone (hGH). By designing the variant of the Fc fragment of IgG4, not only are the ADCC, ADCP, and CDC effects caused by the binding of the Fc region to FcγR eliminated, but the high activity of growth hormone is maintained, the distribution of the drug in vivo is altered, the half-life of the drug in vivo is prolonged, and the molecular stability is improved. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1This is a schematic diagram of the Fc-hGH fusion protein structure.

[0034] Figure 2 Sensor diagram for detecting the affinity of Fc-hGH fusion protein for human GHR.

[0035] Figure 3 Sensor maps (multiple concentrations) for detecting the affinity of Fc-hGH fusion protein for human GHR.

[0036] Figure 4 Sensor diagram for detecting the affinity of Fc-hGH fusion protein for human CD32a (R167 / H167).

[0037] Figure 5 This is a sensor image for detecting the affinity of the Fc-hGH fusion protein for human CD32b.

[0038] Figure 6 Sensor diagram for detecting the affinity of Fc-hGH fusion protein for human CD16a (F176V / 176F).

[0039] Figure 7 This is a sensor image for detecting the affinity of the Fc-hGH fusion protein for human CD16b.

[0040] Figure 8 Sensor diagram for detecting the affinity of Fc-hGH fusion protein for human C1q.

[0041] Figure 9 A trend graph showing the weight gain in grams of rats in each group after drug administration. Detailed Implementation

[0042] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0044] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0045] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0046] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0047] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0048] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0049] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0050] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0051] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0052] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0053] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0054] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0055] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0056] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0057] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0058] Fc fusion proteins are formed by binding the Fc fragment of immunoglobulins to other biologically active functional protein molecules using genetic engineering techniques.

[0059] Hinge region: It is an elastic structural domain located between the CH1 and CH2 functional regions of the immunoglobulin heavy chain. It is mainly found in IgG, IgA and IgD, and connects the Fab segment (antigen-binding fragment) and the Fc segment (crystallizable fragment).

[0060] Fcγ receptors (FcγRs) are a class of cell surface receptors that can bind to the Fc terminus of antibodies and mainly generate signals within the cell through their ITAM activation sequence. Based on receptor affinity, Fcγ receptors are divided into three main classes: high-affinity receptors FcγRI (CD64), low-affinity receptors FcγRII (CD32), and FcγRIII (CD16).

[0061] ADCC (antibody-dependent cell-mediated cytotoxicity) refers to the action of antibody-dependent cell-mediated cytotoxicity. This occurs when the Fab fragment of an antibody binds to antigenic epitopes on virus-infected or tumor cells, while its Fc fragment binds to the FcR on the surface of killer cells (NK cells, macrophages, neutrophils, etc.). This mediates the direct killing of target cells by killer cells, and is an important mechanism of action for therapeutic antibody drugs against tumors.

[0062] ADCP (antibody-dependent cellular phagocytosis): This refers to antibody-dependent cell-mediated phagocytosis, in which monocytes, macrophages, neutrophils, and dendritic cells recognize and engulf disease cells by expressing FcγRIIa (CD32a), FcγRI (CD64), and FcγRIIIa (CD16a).

[0063] CDC (complement-dependent cytotoxicity) is an antibody-mediated immune response mechanism, the core of which lies in the formation of the membrane attack complex through the classical complement activation pathway.

[0064] L234A: This indicates that the leucine (L) at position 234 has been mutated to alanine (A).

[0065] Differential Scanning Fluorescence (DSF) is a method for evaluating protein thermal stability by slowly heating a sample on a quantitative PCR instrument and detecting the amount of fluorescent dye binding to proteins whose structure has changed during the heating process.

[0066] Melting temperature (Tm): refers to the temperature at which the absorbance increases to half of its maximum value, and is called the melting point or melting point of DNA.

[0067] Currently, the growth hormone products on the market are mainly short-acting. Human growth hormone has a plasma half-life of only 0.5 to 2.0 hours in the human body. Short-acting growth hormone requires daily injections to achieve maximum effect, and the treatment cycle is relatively long (3 months to 3 years). Frequent injections cause inconvenience to patients, reduce patient compliance, and are prone to missed injections, thus reducing treatment effectiveness. Therefore, the development of long-acting growth hormone is essential.

[0068] Clinical needs have driven long-acting growth hormone to become a hot research topic for major pharmaceutical companies, with a very broad market prospect. Domestically marketed long-acting growth hormones mainly extend the half-life of growth hormone through PEGylation to achieve a long-lasting effect. The technologies for long-acting growth hormones already on the market and under development both domestically and internationally primarily involve five methods: PEGylation, fusion proteins (including Fc, CTP (carboxy-terminal peptide), and HSA (human serum albumin)), non-covalently bound proteins, microsphere sustained-release, and prodrugs (proprietary linkers and inert carriers methoxy polyethylene glycol (PEG)).

[0069] Currently, there are three dosage forms of human growth hormone products on the market: short-acting powder for injection, short-acting aqueous injection, and long-acting. Powder for injection requires dissolution, which is cumbersome and poses a risk of contamination; its market share has been declining year by year. Aqueous injections are simple and convenient to administer, reducing the risk of environmental pollution during injection, and are currently the mainstream product. However, human growth hormone has a plasma half-life of only 0.5-2.0 hours, requiring daily injections to achieve maximum effect, and the treatment cycle is long (3 months to 3 years), causing significant patient discomfort, reducing medication adherence, and increasing the risk of missed injections, thus reducing treatment efficacy. Long-acting growth hormone can be injected once a week, reducing the number of injections by 313 per year, decreasing the frequency of administration by 86%, improving patient adherence while maintaining efficacy. Existing long-acting growth hormone fusion proteins have a certain binding affinity to FcγR, which can induce ADCC, ADCP, and CDC effects.

[0070] A first aspect of this application provides an Fc-hGH fusion protein, from the N-terminus to the C-terminus, wherein the Fc-hGH fusion protein comprises hGH and an IgG4 Fc mutant connected sequentially;

[0071] Compared to the wild-type IgG4 Fc, the IgG4 Fc mutant has the following mutations: F234A, L235A, M428L, and N434S.

[0072] This application proposes to form a fusion protein by combining a variant of the Fc fragment of IgG4 with growth hormone (hGH). By designing the Fc fragment variant of IgG4, not only are the ADCC, ADCP, and CDC effects caused by the binding of the Fc region to FcγR eliminated, the drug distribution in vivo is altered, and the half-life of the drug in vivo is prolonged, but the molecular stability is also improved.

[0073] In some embodiments of this application, the Fc-hGH fusion protein satisfies one or more of the conditions shown in (1) and (2) below:

[0074] (1) The IgG4 Fc mutant also has the mutation S228P;

[0075] (2) The N-terminus of the hGH and the IgG4 Fc mutant are linked by or without a linker peptide;

[0076] Optionally, the linker peptide contains 4 to 27 amino acids, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acids;

[0077] Optionally, the structural formula of the linker peptide is (GGGGS)n, where n is 2 to 4, for example, 2, 3, or 4.

[0078] In some embodiments of this application, the Fc-hGH fusion protein satisfies one of the conditions shown in (1) and (2):

[0079] (1) The hinge region of the IgG4 Fc mutant has the same length as the hinge region of the wild-type IgG4 Fc;

[0080] Optionally, the amino acid sequence of the IgG4 Fc mutant is as shown in positions 207-435 of SEQ ID NO.3, or has a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence shown.

[0081] Optionally, the amino acid sequence of the Fc-hGH fusion protein is as shown in SEQ ID NO.1 or SEQ ID NO.3, or has a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the shown sequence;

[0082] (2) The hinge region of the IgG4 Fc mutant is shorter than that of the wild-type IgG4 Fc;

[0083] Optionally, the hinge region of the IgG4 Fc mutant contains 3 to 5 amino acids;

[0084] Optionally, the amino acid sequence of the IgG4 Fc mutant is as shown in positions 192-412 of SEQ ID NO.2, or has a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence shown.

[0085] Optionally, the amino acid sequence of the Fc-hGH fusion protein is as shown in SEQ ID NO.2 or SEQ ID NO.4, or has a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the shown sequence.

[0086] "Identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if every position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared multiplied by 100. For example, in optimal sequence alignment, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in two sequences match or are homologous, then the two sequences are 95% homologous. Typically, comparisons are made when aligning two sequences to give the maximum percentage of identity. For example, comparisons can be performed using the BLAST algorithm, where the algorithm's parameters are chosen to give the maximum match between the sequences over the entire length of each reference sequence. The following references relate to the BLAST algorithm commonly used in sequence analysis: BLAST ALGORITHMS: Altschul, S.F. et al., (1990) J. Mol. Biol. 215:403-410; Gish, W. et al., (1993) Nature Genet. 3:266-272; Madden, T.L. et al., (1996) Meth. Enzymol. 266:131-141; Altschul, S.F. et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J. et al., (1997) Genome Res. 7:649-656. Other common BLAST algorithms, such as those provided by NCBI BLAST, are also well-known to those skilled in the art.

[0087] A second aspect of this application provides a nucleic acid molecule that encodes the Fc-hGH fusion protein.

[0088] As used herein, the term "nucleic acid molecule" refers to DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0089] The nucleic acid molecules in this application primarily refer to isolated nucleic acid molecules. "Isolated" means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth media. Generally, the term "isolated" is not intended to mean the complete absence of these materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compounds described herein.

[0090] A third aspect of this application provides a carrier comprising the aforementioned nucleic acid molecule.

[0091] The term "vector," also known as "expression vector," refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop to which an additional DNA segment can be linked. In another embodiment, the vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. The vectors disclosed herein are capable of autonomous replication in host cells that have been introduced into them (e.g., bacterial vectors with bacterial origins of replication and augmented mammalian vectors) or can be integrated into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome (e.g., non-augmented mammalian vectors). In general, vectors may be selected from, but are not limited to, mammalian cell viruses, bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, or combinations thereof.

[0092] A fourth aspect of this application provides a cell that expresses the Fc-hGH fusion protein, including the nucleic acid molecule or the vector.

[0093] The term "cell," also known as "host cell," refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of *Escherichia coli* or *Salmonella*; members of the Bacillaceae family, such as *Bacillus subtilis*; *Pneumococcus*; *Streptococcus*; and *Haemophilus influenzae*. Suitable microorganisms include *Saccharomyces cerevisiae* and *Pichia pastoris*. Suitable animal host cell lines include CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0094] The terms “cell,” “cell line,” and “cell culture” used herein are used interchangeably, and all such names include progeny. Therefore, “transformant” and “transformed cell” include primary test cells and cultures derived from them, regardless of passage number. It should also be understood that, due to intentional or unintentional mutations, all progeny cannot be exactly identical in DNA content. This includes mutant progeny with the same function or biological activity as those screened from the original transformed cells. Where different names are intended, the context will be clear.

[0095] A fifth aspect of this application provides a method for constructing the cell described above, the method comprising the step of introducing the nucleic acid molecule or the vector into a target cell.

[0096] A sixth aspect of this application provides a method for preparing the Fc-hGH fusion protein, the method comprising the steps of culturing the cells and isolating the Fc-hGH fusion protein from the resulting culture.

[0097] A seventh aspect of this application provides the use of the Fc-hGH fusion protein, the nucleic acid molecule, the carrier, or the cell in the preparation of a medicament for human growth hormone deficiency and diseases caused by human growth hormone deficiency.

[0098] An eighth aspect of this application provides a medicament comprising the aforementioned Fc-hGH fusion protein.

[0099] "Drug" or "pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0100] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivering antibody or antigen-binding fragments. Carriers can be anti-adhesion agents, adhesives, coatings, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterial agents, or antifungal agents), sweeteners, absorption delay agents, wetting agents, emulsifiers, buffers, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (such as olive oil), saline, buffer solutions, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

[0101] A ninth aspect of this application provides a treatment method for human growth hormone deficiency and diseases caused by human growth hormone deficiency, the treatment method comprising administering to a subject a therapeutically effective amount of the above-described Fc-hGH fusion protein or the above-described drug.

[0102] "Administration," "giving," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "giving," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Administration," "giving," and "treatment" also mean the treatment of, for example, cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0103] "Treatment" means administering an oral or topical therapeutic agent, such as a composition containing any of the antibodies or antigen-binding fragments of this application, to a patient who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves symptoms of one or more diseases to induce the regression of such symptoms or inhibit their progression to any clinically measured extent. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical test that a physician or other healthcare professional typically uses to assess the severity or progression of the symptoms. Although the embodiments of this application (e.g., treatment methods or products) may be ineffective in alleviating symptoms of each target disease, they should reduce symptoms of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0104] "Effective amount" or "effective dose" means the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic outcome. For prophylactic use, beneficial or desired outcomes include eliminating or reducing risk, mitigating severity, or delaying the onset of a condition, including the condition itself, its complications, and the biochemical, histological, and / or behavioral symptoms of intermediate pathological phenotypes present during the development of the condition. For therapeutic use, beneficial or desired outcomes include clinical outcomes such as reducing the incidence of various target antigen-related conditions of this application or improving one or more symptoms of said conditions, reducing the dosage of other agents required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of the target antigen-related condition in a patient.

[0105] In this document, the term "treatment" and its various parts of speech, "treatment and / or treating and / or treat," refer to all processes that may involve slowing, interrupting, blocking, controlling, preventing, or reversing the progression of the disease described herein, but do not necessarily indicate the complete elimination of all symptoms. Treatment may also include pain prevention. Treatment includes administering the antibodies of the present invention for treating human diseases or conditions that would benefit from reduced NGF activity, and includes: (a) inhibiting further progression of the disease, i.e., preventing its development; (b) alleviating the disease, i.e. causing the remission of the disease or condition or relieving its symptoms or complications; and (c) preventing the onset of symptomatic diseases.

[0106] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0107] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0108] This application describes a fusion protein formed by combining a variant of the Fc fragment of IgG4 with growth hormone (hGH). Compared to the wild-type IgG4 Fc fragment, the IgG4 Fc fragment variant exhibits the following mutations in the CH2 region: F234A, L235A, M428L, and N434S. Mutating specific sites in the wild-type IgG4 Fc fragment helps prevent Fab arm exchange, improves molecular stability, eliminates ADCC, ADCP, and CDC effects caused by Fc region binding to FcγR, alters drug distribution in vivo, and prolongs the drug's half-life in vivo.

[0109] Example 1. Construction and expression of human growth hormone Fc fusion protein (Fc-hGH fusion protein)

[0110] A DNA sequence encoding the Fc-hGH fusion protein was synthesized and inserted into the pcDNA3.4 expression vector to construct a full-length expression plasmid. Expression was performed in ExpiCHO-S cell cultures, and the supernatant was purified using a protein A affinity column to obtain GH21–GH24, respectively. The molecular design is shown in Table 1.

[0111] Table 1. Design of Fc-hGH fusion proteins

[0112]

[0113] The structure of the Fc-hGH fusion protein is shown in [see details]. Figure 1 .

[0114] Protein concentration was determined by SDS-PAGE. Under non-reducing conditions, the purified fusion protein migrated as a ~100 kDa band in SDS-PAGE, while under reducing conditions it migrated as a ~50 kDa band. The purity of the fusion protein was assessed by SEC-HPLC, indicating it was ≥90%.

[0115] The amino acid sequences of GH21~GH24 are as follows:

[0116] 1. GH21 (IgG4-S228P-FALA-LS-Full-length Hinge)

[0117] >GH21(SEQ ID NO.1)

[0118] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLGK*

[0119] 2. GH22 (IgG4-FALA-LS-truncated Hinge)

[0120] >GH22(SEQ ID NO.2)

[0121] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFPSCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLGK*

[0122] 3. GH23 (3*G4S-IgG4-S228P-FALA-LS-Full-length Hinge)

[0123] >GH23(SEQ ID NO.3)

[0124] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFGGGGSGGGGSGGGGSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLGK*

[0125] 4. GH24 (3*G4S-IgG4-FALA-LS-Truncated Hinge)

[0126] > GH24 (SEQ ID NO.4)

[0127] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDS NVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFGGGGSGGGGSGGGGSPSCPAPEAAG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLGK(447)*

[0128] 5. Fc of wild-type IgG4

[0129] > SEQ ID NO.5:

[0130]

[0131] The residues in the IgG Fc region are numbered according to the Kabat Eu Index.

[0132] Example 2. Assay of the binding activity of Fc-hGH fusion protein to human growth hormone receptor (GHR)

[0133] A 96-well ELISA microtiter plate was coated with 1 μg / mL human growth hormone receptor protein (Acro, GHR-H5222) using CBS (carboxylated polystyrene) at 4°C. The plate was washed twice with 0.05% PBST (phosphate-buffered saline containing Tween-20) and blocked at 37°C with 3% MPBS (phosphate-buffered saline containing skim milk powder) for 1 h, followed by a single wash with 0.05% PBST. Then, the Fc-hGH fusion protein was added to the blocking wells at an initial concentration of 300 nM, serially diluted 3-fold (100 μL / well), and incubated at room temperature for 2 h. After washing three times with 0.05% PBST, 0.1 μg / mL mouse anti-human IgG Fc [HRP] (horseradish peroxidase) was added to the plate (100 μL / well) and incubated at room temperature for 45 min. Wash six times with 0.05% PBST, and add TMB (3,3',5,5'-tetramethylbenzidine) chromogenic solution to the wells and react for 10 min. Use a microplate reader to measure the absorbance at 450 nm and calculate the EC50 (half-maximal effective concentration) of each fusion protein for binding to human GHR.

[0134] The EC50 values ​​of the binding activity of each fusion protein to human GHR are shown in Table 2. The results indicate that all Fc-hGH fusion proteins can bind to human GHR, exhibiting a concentration gradient-dependent trend. In terms of binding activity, GH21 (using a full-length hinge) showed better binding activity than GH22 (using a truncated hinge) as the linker peptide; the GH23Fc-hGH fusion protein using a 3*G4S-full-length hinge exhibited the best binding activity.

[0135] Table 2 Results of the assay for the binding activity of Fc-hGH fusion protein to human GHR

[0136]

[0137] Note: EC50 is the concentration of the fusion protein at which 50% of the maximum binding activity is achieved; the smaller the EC50, the greater the binding activity.

[0138] Example 3. Affinity detection of Fc-hGH fusion protein with human GHR

[0139] The Fc-hGH fusion protein was injected onto a Protein A chip as a stationary phase, and human GHR (same as in Example 2) was used as the analyte for detection. Affinity was determined using a Biacore 8K and a Biacore T200. The dissociation rate (kd) and binding rate (ka) constants were obtained using the corresponding evaluation software. The equilibrium dissociation constant (KD) was calculated based on the ratio of kd to ka.

[0140] Note: Biacore 8K and Biacore T200 are biomolecular interaction analysis instruments based on surface plasmon resonance (SPR) technology; Biacore instruments monitor molecular binding processes in real time using SPR technology and can directly calculate KD without labeling.

[0141] 3.1 Single concentration detection of Fc-hGH fusion protein SPR

[0142] All experimental data were analyzed using Biacore 8K and Biacore T200 evaluation software. The key parameters for detecting the affinity of Fc-hGH fusion proteins GH21–GH24 with human GHR were: capture time 12 s, binding time 60 s, dissociation time 360 ​​s, flow rate 30 μL / min, and fusion protein concentration 400 nM; chip surface regeneration time 30 s, flow rate 30 μL / min, and regeneration buffer 10 mM glycine hydrochloride, pH 1.5.

[0143] SPR-based signal sensing diagrams (see the SPR-based signal sensing diagram for the affinity detection of the Fc-hGH fusion protein with human GHR). Figure 2 The binding rate ka of human GHR to the fusion protein molecule on the chip surface and the dissociation rate kd of the human GHR-fusion protein complex after binding were tracked. The affinity constant KD was obtained by calculating the ratio of kd to ka, and the results are shown in Table 3. The results show that there is no significant difference in KD of the fusion protein as a whole.

[0144] Table 3. Affinity data of Fc-hGH fusion protein with human GHR

[0145]

[0146] The smaller the KD value, the higher the affinity and the stronger the binding ability.

[0147] 3.2 Detection of multiple concentrations of Fc-hGH fusion protein SPR

[0148] All experimental data were analyzed using Biacore 8K evaluation software version 4.0. The key parameters for detecting the affinity of the Fc-hGH fusion protein GH23 for human GHR were: capture time 30 s, binding time 60 s, dissociation time 360 ​​s, flow rate 30 μL / min, and fusion protein concentration gradients of 400 nM, 200 nM, 100 nM, 50 nM, and 25 nM; chip surface regeneration time 30 s, flow rate 30 μL / min, and regeneration buffer of 10 mM glycine hydrochloride at pH 1.5. The dissociation constant KD was obtained by fitting the final results using SPR data analysis software.

[0149] The results are as follows Figure 3As shown in Table 4, the results indicate that, in terms of receptor affinity, the fusion protein has a low dissociation constant KD value for human GHR, suggesting that the fusion protein has a high affinity for and strong binding ability to human GHR.

[0150] Table 4. Affinity data of Fc-hGH fusion protein with human GHR (multiple concentrations)

[0151]

[0152] Example 4. Evaluation of the in vitro biological activity of Fc-hGH fusion protein based on Nb2-11 cell proliferation.

[0153] Target cells were collected by centrifugation, and the supernatant was discarded. Cells were resuspended in assay buffer (Fischer's medium + 1% horse serum + 0.075% sodium bicarbonate + 0.05 mmol / L β-mercaptoethanol). Working solutions for GH21–GH24 were prepared using assay buffer (starting at 20 nmol / L, with 4-fold serial dilutions, for a total of 8 concentration gradients). Target cell density was adjusted, and the cell suspension was transferred to 96-well plates. System control (Human growth hormone (Sansure)) / positive control TJ101 working solutions were transferred to the corresponding wells of the 96-well plate. The 96-well plate was incubated at 37°C / 5% CO2 for 30 h. After incubation, the plate was removed, CellCounting-Lite working solution was added, and the plate was incubated at room temperature for 5 min. Chemiluminescence values ​​were read using a microplate reader.

[0154] Based on the correlation between relative chemiluminescence signal values ​​and sample concentrations, a dose-response curve is established. The EC20 can be obtained using the following four-parameter equation. 50 The relative value is: Y = Bottom + (Top - Bottom) / (1 + 10^((LogEC50 - X) * HillSlope)). Where X = Log sample concentration, and Y = Relative Luminescence Unit (RLU).

[0155] The biological activity of the Fc-hGH fusion protein was determined by the obtained dose-response curves, and the results are shown in Table 5. As can be seen from the table, the EC50 values ​​of GH23 and GH24 are significantly lower than those of GH21 and GH22. This indicates that, with the same functional protein (human growth hormone) and the same CH2 and CH3 sequences, different hinge region designs result in significant differences in the biological activity of Fc-hGH fusion protein molecules. The biological activities of GH23 and GH24 are significantly better than those of GH21 and GH22.

[0156] Table 5. Fitting parameters for dose-response curves in cell proliferation experiments of control and test samples

[0157]

[0158] Example 5. Affinity detection of Fc-hGH fusion protein with neonatal Fc receptor (FcRn)

[0159] The affinity of the Fc-hGH fusion protein for human FcRn (Acro, FCN-H52W7, pH 6.0 and pH 7.4) was measured. Human FcRn was injected onto a CM5 chip coupled with a histidine-tagged antibody as the stationary phase, and the Fc-hGH fusion protein was used as the analyte. Affinity was measured using a Biacore 8K and a Biacore T200 at pH 6.0 and pH 7.4, respectively. The dissociation rate (kd) and binding rate (ka) constants were obtained using the corresponding evaluation software. The equilibrium dissociation constant (KD) was calculated based on the ratio of kd to ka.

[0160] Human FcRn was used as a ligand, and the results were analyzed using Biacore 8K evaluation software version 4.0 at pH 6.0 and pH 7.4. The key parameters for detecting the affinity of Fc-hGH fusion proteins GH23 and TJ101 (fusion protein control) with FcRn were: capture time 30 s, binding time 60 s, dissociation time 60 s, flow rate 30 μL / min, and fusion protein concentration gradients of 400 nM, 200 nM, 100 nM, 50 nM, and 25 nM; chip surface regeneration time 30 s, flow rate 30 μL / min, and regeneration buffer of 10 mM glycine hydrochloride at pH 1.5. The dissociation constant KD was obtained by fitting the final results with SPR data analysis software.

[0161] The results are shown in Table 6. The results indicate that at pH 6.0, GH23 has a better affinity for human FcRn than TJ101; at pH 7.4, neither GH23 nor TJ101 binds to human FcRn. This suggests that when the fusion protein is in the acidic environment of the endosome, GH23 binds to human FcRn more effectively than TJ101, and the two proteins can be normally separated at physiological pH, ensuring the successful release of Fc-hGH and its biological function.

[0162] Table 6. Affinity data between Fc-hGH fusion protein and human FcRn

[0163]

[0164] Example 6. Affinity detection of Fc-hGH fusion protein with FcγR and C1q

[0165] Affinity assays were performed on the Fc-hGH fusion protein with human CD32a (R167 and H167) (Sino biological, 10374-H08H, 10374-H08H1), human CD32b (Sino biological, 10259-H08H), human CD16a (F176V and F176) (Sino biological, 10389-H08H1, 10389-H08H), human CD16b (Sino biological, 11046-H08H), and human C1q (Sigma, C1740). The binding affinity of the Fc-hGH fusion protein with FcγR for inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and C1q for complement-dependent cytotoxicity (CDC) was measured. For human C1q detection, the Fc-hGH fusion protein was injected onto a Protein A chip as the stationary phase, and human C1q was used as the analyte. For human CD64 detection, human CD64 was injected onto a CM5 chip coupled with a histidine-capturing antibody as the stationary phase, and the Fc-hGH fusion protein was used as the analyte. Human CD32a (R167 and H167), human CD32b, human CD16a (F176V and F176), and human CD16b were immobilized on a CM5 chip via amino-coupling reaction as the stationary phase, and the Fc-hGH fusion protein was used as the analyte. Affinity was determined using Biacore 8K and Biacore T200. The dissociation rate (kd) and binding rate (ka) constants were obtained using the corresponding evaluation software. The equilibrium dissociation constant (KD) was calculated based on the ratio of kd to ka.

[0166] 6.1 Human CD32a(R167) / human CD32a(H167), human CD32b, human CD16a (F176V / 176F), or human CD16b was used as the ligand, and the Fc-hGH fusion protein was used as the analyte. SPR operating parameters were: coupling level (RU) of 1500–2000, binding time of 60 s, dissociation time of 60 s, flow rate of 30 μL / min, and fusion protein concentration gradients of 1000 nM, 500 nM, 250 nM, 125 nM, and 62.5 nM; chip surface regeneration time of 30 s, flow rate of 30 μL / min, and regeneration buffer of 3 M magnesium chloride. The dissociation constant KD was obtained by fitting the final results using SPR data analysis software.

[0167] The results are as follows Figure 4 , Figure 5 , Figure 6 and Figure 7As shown in the results, no binding of GH23 to human CD32a (R167 / H167), human CD32b, human CD16a (F176V / 176F), or human CD16b was detected, indicating that GH23 has no ADCC effect.

[0168] 6.2 GH23 was used as the ligand, and human C1q was used as the analyte. SPR operating parameters were: capture time 30 s, binding time 60 s, dissociation time 360 ​​s, flow rate 30 μL / min, and fusion protein concentration gradients of 40 nM, 20 nM, 10 nM, 5 nM, and 2.5 nM; chip surface regeneration time 30 s, flow rate 30 μL / min, and regeneration buffer of 10 mM glycine hydrochloride at pH 1.5. The dissociation constant KD was obtained by fitting the final results using SPR data analysis software.

[0169] The results are as follows Figure 8 As shown in the figure. The results did not detect the binding of GH23 to human C1q, indicating that GH23 has no CDC effect.

[0170] Example 7. Pharmacokinetic study of Fc-hGH fusion protein after a single subcutaneous administration in rats.

[0171] In vivo pharmacokinetic experiments were conducted on rats using the Fc-hGH fusion protein GH23, with TJ101 as the control group. The specific experimental procedures are as follows:

[0172] SPF-grade SD rats aged 6-8 weeks were selected, with an equal number of males and females. The rats were randomly divided into four groups: a low-dose TJ101 group, a high-dose TJ101 group, a low-dose GH23 group, and a high-dose GH23 group; six rats were in each group. Administration was a single subcutaneous injection. The human growth hormone groups received short-acting human growth hormone (human growth hormone standard) at a dose of 2 mg / kg; the low-dose TJ101 and low-dose GH23 groups received it at a dose of 1 mg / kg; and the high-dose TJ101 and high-dose GH23 groups received it at a dose of 2 mg / kg.

[0173] The test substance was diluted to the given concentration using 1×PBS according to the above dosing regimen. Rat body weight was measured twice weekly after administration. Blood and serum samples were collected from each group of animals (at least 50 µL per animal) before administration and at 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 72 h, 96 h, 120 h, 168 h, 216 h, 264 h, and 336 h after administration. The rat serum samples were stored at -80°C. Serum samples were analyzed using a bioanalytical method specific to human growth hormone to obtain blood drug concentrations at each time point, and pharmacokinetic (PK) parameters were calculated.

[0174] Serum drug concentrations of SD rats were measured at various time points. Prism8 was used to plot the drug concentration versus time curves for each test substance. The area under the drug concentration-time curve (AUC) of the Fc-hGH fusion protein was greater than that of hGH, indicating that the Fc-hGH fusion protein remained in the rat body for a significantly longer time than hGH. The main pharmacokinetic parameters for each group were calculated, and the results are shown in Tables 7 and 8. Comprehensive analysis of the PK parameters of the test substances under different dosages and sexes showed that GH23 was superior to TJ101.

[0175] A comprehensive analysis of the blood drug concentration during the terminal elimination phase in each animal showed relatively small inter-individual differences. Therefore, the final T1 / 2 value should be representative and reflect the half-life of the test substance in the relevant species.

[0176] Table 7. PK parameters of Fc-hGH fusion protein in SD rats - 1 mg / kg groups (ng / mL, Mean±SD)

[0177]

[0178] Table 8. PK parameters of Fc-hGH fusion protein in SD rats - 2 mg / kg groups (ng / mL, Mean±SD)

[0179]

[0180] Elimination half-life (t1 / 2): The time required for the drug concentration to decrease by half, reflecting the rate at which the drug is eliminated from the body (the longer the half-life, the longer the drug remains in the body).

[0181] Time to peak concentration (Tmax): The time it takes to reach peak concentration, reflecting the rate of drug absorption.

[0182] Peak concentration (Cmax): The highest concentration of a drug reached in the body, reflecting the extent of drug absorption or the effectiveness of the administered dose.

[0183] Area under the drug-time curve (AUC): The area enclosed by the drug concentration-time curve and the horizontal axis, reflecting the total exposure of the drug in the body (the larger the AUC, the higher the exposure).

[0184] Vz_obs: Reflects the extent to which a drug is distributed in the body (theoretical "volume"). The larger the value, the easier it is for the drug to be distributed from the blood to the tissues.

[0185] Cl_obs reflects the plasma volume of drugs cleared by the body per unit time and is a core parameter for assessing drug elimination capacity. It is mainly related to clearance pathways such as hepatic metabolism and renal excretion. The higher the clearance rate, the faster the drug is eliminated from the body.

[0186] Example 8. Determination of the biological activity of Fc-hGH fusion protein in pituitary deprivation rats

[0187] The biological activity of Fc-hGH fusion protein was determined according to the pituitary rat body weight method in General Chapter 1219 of Part IV of the 2020 edition of the Chinese Pharmacopoeia.

[0188] Male SD rats aged 3-4 weeks were selected and subjected to pituitary resection via ear canal puncture after anesthesia with isoflurane. The surgery was performed using a stereotactic pituitary resection instrument. Rats were weighed at 2 and 3 weeks post-modeling. At least 56 rats with a body weight change rate ≤ 7% (3-week weight - 2-week weight) / pre-modeling weight × 100% were selected and randomly divided into 7 groups according to their body weight change rate from smallest to largest: model control group, low- and high-dose short-acting rhGH (human growth hormone standard) control groups, low- and high-dose TJ101 control groups, and low- and high-dose GH23 groups, with 8 rats in each group. Subcutaneous injection of 0.5 mL per rat was initiated after grouping. Groups 1 and 4-7 received a single dose, while groups 2 and 3 received a daily dose for 6 consecutive days. The first day of administration was defined as D1, the following day as D2, and so on. The short-acting rhGH group was administered once daily for 6 consecutive days; the model control group, TJ101 control group, and GH23 group received a single dose. From day 1 to day 7, rats were weighed at approximately the same time each day, and the weight gain (in grams) was calculated as: weight (Dn) – weight (D1). On day 7, all rats were euthanized by isoflurane-induced anesthesia and exsanguination. Dissection was performed to check for pituitary remnants, and photographs were taken. Bilateral tibias were harvested, and tibial length was measured using calipers. Tibias were fixed in formalin, decalcified, and paraffin-embedded. After toluidine blue staining, the entire section was digitally scanned to measure the epiphyseal plate width. Four values ​​were recorded for each tibia, and the average of the eight values ​​from both sides was taken. Grouping and administration regimens are shown in Table 9 below, and experimental results are shown in Tables 10 and 11 below. The trend chart of weight gain (in grams) after administration in each group is shown below. Figure 9 .

[0189] Table 9. Grouping and Dosing Regimens

[0190]

[0191] Table 10. Tibial length and epiphyseal plate width in rats of different groups (Mean±SD, n=8)

[0192]

[0193] Table 11. Weight gain (g) in rats of different groups (Mean ± SD, n = 8)

[0194]

[0195] The results showed that, during the 6-day dosing period, with the same total molar dose, the effect of GH23 in promoting animal weight gain and tibial epiphyseal plate growth was similar to that of TJ101 (control), and its effect in promoting animal weight gain was superior to that of short-acting rhGH administered once daily with the same total molar dose.

[0196] Example 9. Stability Study of Fc-hGH Fusion Protein

[0197] The Fc-hGH fusion protein GH23 and the control drug TJ101 were measured according to the conditions and sampling time points in Table 12 to study the stability of the fusion protein under the set conditions. The buffer system for the test drugs was PBS, pH 7.2.

[0198] Table 12. Conditions for studying the stability of Fc-hGH fusion protein

[0199]

[0200] 9.1 Thermal stability (DSF-Tm)

[0201] The onset temperature (Tonset) and melting temperature (Tm) of the Fc-hGH fusion proteins GH23 and TJ101 (control) at time 0 are shown in Table 13. From the Tm values, the Tm1 of both molecules is above 60℃, indicating good thermal stability, and there is no significant difference between molecules.

[0202] Table 13. DSF-Tm Detection Results

[0203]

[0204] 9.2 Stability at 25℃

[0205] The Fc-hGH fusion protein GH23 molecule was tested at 25℃ for 0 to 28 days, and the results are shown in Table 14. SEC-HPLC and CE-SDS-NR results were stable with no significant fluctuations. The SEC-HPLC results for the TJ101 molecule were stable with no significant fluctuations, but the CE-SDS-NR results showed a clear trend: the percentage of pre-peaks gradually increased from 7.82% at T0 to 12.52% at D28, while the percentage of the main peak gradually decreased from 89.89% at T0 to 84.48% at D28, indicating an increase in fragment peaks and suggesting a possible degradation of the molecule.

[0206] Table 14. Stability test results at 25℃

[0207]

[0208] 9.3 Freeze-thaw stability

[0209] Fc-hGH fusion proteins GH23 and TJ101 (control) were frozen at -70℃ for more than 2 hours, then thawed at 25℃ for at least 2 hours as one freeze-thaw cycle. Purity was tested after 3 and 5 freeze-thaw cycles, respectively.

[0210] The results are shown in Table 15. No significant changes were observed in any component of the Fc-hGH fusion protein GH23 in SEC-HPLC or CE-SDS-NR analysis. TJ101 showed a 2.92% decrease in its main peak in SEC-HPLC, but no significant changes in any component in CE-SDS-NR analysis. In summary, repeated freeze-thaw cycles had minimal impact on all fusion protein molecules. The GH23 molecule exhibited better freeze-thaw tolerance than TJ101.

[0211] Table 15. Results of Freeze-Thaw Stability Test

[0212]

[0213] Comprehensive analysis shows that the Fc-hGH fusion protein GH23 possesses excellent receptor affinity, in vitro activity, pharmacokinetic properties (longer half-life), and in vivo activity. From the drug stability test results, GH23 exhibits superior stability compared to the existing fusion protein TJ101. Therefore, modifying growth hormone with the IgG4Fc fragment variant containing F234A, L235A, M428L, and N434S mutations in the CH2 region, as described in this invention, can improve the performance of growth hormone.

[0214] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0215] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. An Fc-hGH fusion protein, characterized in that, From the N-terminus to the C-terminus, the Fc-hGH fusion protein comprises hGH and an IgG4 Fc mutant connected in sequence; Compared to the wild-type IgG4 Fc, the IgG4 Fc mutant has the following mutations: F234A, L235A, M428L, and N434S.

2. The Fc-hGH fusion protein according to claim 1, characterized in that, The Fc-hGH fusion protein satisfies one or more of the following conditions (1) and (2): (1) The IgG4 Fc mutant also has the mutation S228P; (2) The N-terminus of the hGH and the IgG4 Fc mutant are linked by or without a linker peptide; Optionally, the linker peptide contains 4 to 27 amino acids; Optionally, the structural formula of the linker peptide is (GGGGS)n, where n is 2 to 4.

3. The Fc-hGH fusion protein according to any one of claims 1 to 2, characterized in that, The Fc-hGH fusion protein satisfies one of the conditions shown in (1) and (2): (1) The hinge region of the IgG4 Fc mutant has the same length as the hinge region of the wild-type IgG4 Fc; Optionally, the amino acid sequence of the IgG4 Fc mutant is shown as positions 207-435 in SEQ ID NO.3; Optionally, the amino acid sequence of the Fc-hGH fusion protein is shown in SEQ ID NO.1 or SEQ ID NO.3; (2) The hinge region of the IgG4 Fc mutant is shorter than that of the wild-type IgG4 Fc; Optionally, the hinge region of the IgG4 Fc mutant contains 3 to 5 amino acids; Optionally, the amino acid sequence of the IgG4 Fc mutant is shown as positions 192-412 in SEQ ID NO.2; Optionally, the amino acid sequence of the Fc-hGH fusion protein is shown in SEQ ID NO.2 or SEQ ID NO.

4.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the Fc-hGH fusion protein according to any one of claims 1 to 3.

5. A carrier, characterized in that, The carrier includes the nucleic acid molecule as described in claim 4.

6. A cell, characterized in that, The cells express the Fc-hGH fusion protein of any one of claims 1 to 3, including the nucleic acid molecule of claim 4 or the vector of claim 5.

7. The method for constructing cells according to claim 6, characterized in that, The construction method includes the step of introducing the nucleic acid molecule of claim 4 or the vector of claim 5 into target cells.

8. The method for preparing the Fc-hGH fusion protein according to any one of claims 1 to 3, characterized in that, The preparation method includes the steps of culturing the cells of claim 6 and isolating the Fc-hGH fusion protein from the resulting culture.

9. The use of the Fc-hGH fusion protein of any one of claims 1 to 3, the nucleic acid molecule of claim 4, the vector of claim 5, or the cell of claim 6 in the preparation of medicaments for human growth hormone deficiency and diseases caused by human growth hormone deficiency.

10. A drug, characterized in that, The drug comprises the Fc-hGH fusion protein according to any one of claims 1 to 3.

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