Growth hormone fusion protein and application thereof

Abstract

The invention provides a growth hormone fusion protein and application thereof. The fusion protein comprises an Fc mutant, the Fc mutant comprises an amino acid sequence as shown in SEQ ID NO: 49, or the Fc mutant comprises a first peptide chain and a second peptide chain, and the C end of the second peptide chain is connected with the N end of the first peptide chain; wherein a, compared with a wild type IgG4Fc fragment, the first peptide chain has the following mutations: F234V and L235E; or S298N, T299A, and Y300S, or a combination thereof; compared with a hinge region of wild type IgG4, the second peptide chain has S28P mutation; or b, compared with a wild type IgG4Fc fragment, the first peptide chain 1) has 231st-239th amino acid deletion; or (2) compared with a wild type IgG1Fc fragment, the IgG1Fc fragment has the following mutations: the 228th to 239th amino acid deletion, H268Q, Y296F, A330S and P331S. The fusion protein has high binding activity with a human growth hormone receptor, ADCC and CDC effects can be eliminated, Nb2-11 cell proliferation is promoted, and therefore human growth hormone abnormality related diseases are effectively treated or prevented.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, specifically, the present invention relates to a growth hormone fusion protein and its application, more specifically, the present invention relates to an Fc mutant, a fusion protein, a nucleic acid molecule, an expression vector, a recombinant cell, a composition, a pharmaceutical use and a drug. Background Art

[0002] Growth hormone deficiency is a well-recognized clinical syndrome associated with numerous metabolic abnormalities, including abnormal body composition, decreased physical fitness, altered lipid metabolism, decreased bone mass, increased insulin resistance, and reduced quality of life. Most metabolic abnormalities associated with growth hormone deficiency can be reversed by recombinant human growth hormone (rhGH) replacement. Traditional treatment for growth hormone deficiency involves daily subcutaneous injections of rhGH. However, the injection schedule is too frequent and inconvenient for many patients, raising concerns about poor treatment compliance, which may lead to reduced efficacy. Long-acting rhGH preparations can not only reduce the number of injections, thereby improving compliance, but also help improve the efficacy of GH therapy.

[0003] Studies have been conducted to prepare fusion proteins using immunoglobulin Fc to increase the half-life of fusion proteins (CheolRyong Ku et al., Eur J Endocrinol. 2018 Sep; 179(3): 169-179; Wolfgang Glaesner et al., Diabetes Metab Res Rev. 2010 May; 26(4): 287-96.). Since the biological activity of growth hormone requires a certain spatial effect, improper connection of Fc in the fusion protein will cause a steric hindrance effect. However, studies have found that the use of GGGGSGGGSGGGGS or partial fragments of Genexine GX-H9 to connect with Fc all have problems such as low binding affinity to human growth hormone receptors, low activity in promoting cell proliferation, and low in vitro activity.

[0004] Therefore, there is still an urgent need for a fusion protein that has high binding activity to human growth hormone receptor and can promote T cell proliferation. Summary of the invention

[0005] This application is filed based on the inventor's discovery of the following issues and facts:

[0006] In the first aspect of the present invention, the present invention proposes an Fc mutant. According to an embodiment of the present invention, it comprises: a first peptide chain, wherein the first peptide chain 1) has a mutation at the following sites compared to the wild-type IgG4 Fc fragment: at least one of the 234th, 235th, 298th, 299th, and 300th positions; or 231-239th positions; or 2) has a mutation at the following sites compared to the wild-type IgG1 Fc fragment: at least one of the 228th-239th positions, and / or, at least one of the 268th, 296th, 330th, and 331st positions. The inventors performed the above mutations on the Fc fragment of IgG4 or IgG1 antibodies respectively. Mutations at different sites have different effects. The Fc mutant according to the embodiment of the present invention can effectively reduce the fragment content in the preparation process of the Fc fragment, avoid the formation of half antibodies, and eliminate ADCC and CDC effects. The fusion protein prepared using the Fc mutant described in the present application has a strong binding activity with the human growth hormone receptor, can eliminate ADCC and CDC effects, promote Nb2-11 cell proliferation, and can effectively treat or prevent diseases related to abnormal human growth hormone.

[0007] According to an embodiment of the present invention, the above-mentioned Fc mutant may further include at least one of the following additional technical features:

[0008] According to an embodiment of the present invention, the Fc mutant cannot mutate the amino acid at position 235 to alanine.

[0009] According to an embodiment of the present invention, the sequence of the Fc mutant is not SEQ ID NO:69.

[0010] Amino acid sequence of the Fc mutant exclusion sequence (first peptide chain 6) (having F234V and L235A mutations compared to the Fc fragment of the wild-type IgG4 antibody):

[0011] APEVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQID NO:69).

[0012] According to an embodiment of the present invention, the Fc mutant cannot have, and only has, amino acid mutations at positions 234 and 235.

[0013] According to an embodiment of the present invention, the Fc mutant further includes a second peptide chain, the C-terminus of the second peptide chain is connected to the N-terminus of the first peptide chain, and the second peptide chain has the following mutation sites: 1) compared with the hinge region of wild-type IgG4, it has a mutation at the following site: position 228; or 2) has a wild-type IgD fragment 1 or an IgD fragment 1 mutant.

[0014] The hinge region of wild-type IgG4 includes:

[0015] ESKYGPPCPSCP (SEQ ID NO: 72).

[0016] According to an embodiment of the present invention, the wild-type IgD fragment 1 includes amino acids 287-298 of wild-type IgD.

[0017] According to an embodiment of the present invention, the IgD fragment 1 mutant has mutations at the following sites compared to the wild-type IgD fragment 1: at least one of positions 291-296.

[0018] According to an embodiment of the present invention, the Fc mutant further includes a third peptide chain, the C-terminus of the third peptide chain is connected to the N-terminus of the second peptide chain, and the third peptide chain has at least one of the following mutation sites compared to the wild-type IgD fragment 2: position 263, position 264, position 266, position 267, position 268, position 269, position 270, position 271, position 272, position 273, position 274, position 275, position 276, position 277, position 278, position 280, position 281, position 283, position 284, position 285, position 286, position 287, position 288, position 289, position 290, and position 291.

[0019] According to an embodiment of the present invention, the wild-type IgD fragment 2 includes amino acids 262-291 of wild-type IgD.

[0020] According to an embodiment of the present invention, the first peptide chain 1) has mutations at the following sites compared to the wild-type IgG4 Fc fragment: positions 234 and 235; or positions 298, 299, 300; or positions 231-239; or 2) has mutations at the following sites compared to the wild-type IgG1 Fc fragment: positions 228-239, 268, 296, 330 and 331.

[0021] According to an embodiment of the present invention, the second peptide chain 1) has a mutation at the following position compared to the hinge region of wild-type IgG4: position 228; or 2) has a wild-type IgD fragment 1 or an IgD fragment 1 mutant.

[0022] According to an embodiment of the present invention, the IgD fragment 1 mutant has the following mutations compared to the wild-type IgD fragment 1: positions 291-296.

[0023] According to an embodiment of the present invention, the third peptide chain has the following mutation sites compared to the wild-type IgD fragment 2: 1) positions 266, 267, 268, 277, 278, 280, 283, 285, 286 and 287; or 2) positions 266, 277, 278, 280, 283, 285, 286 and 287. and 287th; or 3) 266th, 269th, 270th, 271st, 273rd, 274th, 275th, 277th, 278th, 281st, and 283rd; or 4) 263rd, 264th, 266th, 269th, 270th, 272nd, 274th, 276th, 284th, 287-291st.

[0024] According to an embodiment of the present invention, the first peptide chain 1) has the following mutations compared to the wild-type IgG4 Fc fragment: F234A and L235E; or F234V and L235E; or S298N, T299A and Y300S; or amino acid deletion at positions 231-239; or 2) has the following mutations compared to the wild-type IgG1 Fc fragment: amino acid deletion at positions 228-239, H268Q, Y296F, A330S and P331S. According to a specific embodiment of the present invention, mutations at different sites have different effects, and therefore, the Fc mutants obtained by combining the various mutation sites have different effects, such as, F234V&L235E can eliminate the ADCC effect, and S298N&T299A&Y300S can eliminate the ADCC and CDC effects.

[0025] According to an embodiment of the present invention, the second peptide chain has the following mutation compared to the hinge region of wild-type IgG4: S228P; or 2) has wild-type IgD fragment 1 or IgD fragment 1 mutant. As mentioned above, mutations at different sites have different effects, so the Fc mutants obtained by combining various mutation sites have different effects, among which S228P can prevent the IgG4 constant region from forming a half antibody.

[0026] According to an embodiment of the present invention, the IgD fragment 1 mutant has the following mutations compared to the wild-type IgD fragment 1: P291C, S292H, H293P, T294R, Q295L, P296S.

[0027] According to an embodiment of the present invention, the third peptide chain has mutations such as: 1) R266E, G267R, G268E, K277E, E278K, Q280E, R283E, T285E, K286G and T287E compared to the wild-type IgD fragment 2; or 2) R266K, K277E, E278K, Q280N, R283E, T285E, K286R and T287E; or 3) R266K, E269D, E270D, K271E, K273E, E274D, K275E, K277E, E278K, E281Q and R283G; or 4) N263Q, T264S, R266K, E269D, E270D, K272R, E274D, E276D, E284D and 287-291 deletions.

[0028] Wild-type IgD fragment 2 includes the following amino acid sequence:

[0029] RNTGRGGEEKKKEKEKEEQEERETKTPECP (SEQ ID NO: 44).

[0030] According to an embodiment of the present invention, the first peptide chain has an amino acid sequence shown in any one of SEQ ID NOs: 55, 56, 57, 58, 68, and 69.

[0031] According to an embodiment of the present invention, the second peptide chain has an amino acid sequence shown in any one of SEQ ID NOs: 59, 60, and 61.

[0032] According to an embodiment of the present invention, the third peptide chain has an amino acid sequence shown in any one of SEQ ID NOs: 62, 63, 64, and 65.

[0033] According to an embodiment of the present invention, the wild-type IgG4, wild-type IgG1, and wild-type IgD are human antibodies.

[0034] According to an embodiment of the present invention, the Fc mutant includes: 1) an amino acid sequence shown in any one of SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, and 54; or 2) an amino acid sequence that has at least 90% identity with the amino acid sequence shown in any one of SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, and 54.

[0035] In the second aspect of the present invention, the present invention proposes a fusion protein. According to an embodiment of the present invention, it includes a first peptide segment, the first peptide segment includes a biologically active molecule functional region; a second peptide segment, the second peptide segment includes an Fc mutant, and the Fc mutant is as defined in the first aspect, wherein the N-terminus of the second peptide segment is connected to the C-terminus of the first peptide segment. The fusion protein according to the embodiment of the present invention has a strong binding activity with the human growth hormone receptor, can effectively eliminate ADCC and CDC effects, promote Nb2-11 cell proliferation, and effectively treat or prevent diseases related to abnormal human growth hormone.

[0036] According to an embodiment of the present invention, the above fusion protein may further include at least one of the following additional technical features:

[0037] According to an embodiment of the present invention, the first peptide segment comprises growth hormone or a growth hormone analog, or a growth hormone functional region or a growth hormone analog functional region.

[0038] According to an embodiment of the present invention, the first peptide segment comprises human growth hormone or a functional region of human growth hormone.

[0039] According to an embodiment of the present invention, the human growth hormone has an amino acid sequence as shown in SEQ ID NO: 1 or a sequence having at least 90% identity thereto.

[0040] According to an embodiment of the present invention, the fusion protein further includes a connecting peptide.

[0041] According to an embodiment of the present invention, the N-terminus of the connecting peptide is connected to the C-terminus of the first peptide segment, and the C-terminus of the connecting peptide is connected to the N-terminus of the second peptide segment.

[0042] According to an embodiment of the present invention, the connecting peptide has the amino acid sequence described in SEQ ID NO:46.

[0043] According to an embodiment of the present invention, compared with the amino acid sequence shown in SEQ ID NO:46, at least one GGGGS of the connecting peptide has at least one of S>T, a G mutation added at the nitrogen terminal, and an L mutation added at the carbon terminal.

[0044] (G4S)n (SEQ ID NO: 46), wherein n is an integer greater than 0, preferably n is 2-6.

[0045] According to an embodiment of the present invention, the connecting peptide has an amino acid sequence shown in SEQ ID NO:11.

[0046] According to an embodiment of the present invention, the fusion protein comprises: 1) an amino acid sequence shown in any one of SEQ ID NO: 21, 22, 23, 24, 25, 26, 27, 28; or 2) an amino acid sequence having at least 90% or at least 95% identity with the amino acid sequence shown in any one of SEQ ID NO: 21, 22, 23, 24, 25, 26, 27, 28. In the present application, the inventor unexpectedly found that the fusion protein obtained by combining the second peptide segment with the connecting peptide has significantly better binding activity to human growth hormone receptor and Nb2-11 cell proliferation promoting effect.

[0047] In the third aspect of the present invention, the present invention proposes a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the optimized Fc mutant described above or the fusion protein described above. The Fc mutant obtained by the nucleic acid molecule according to the embodiment of the present invention can effectively avoid the formation of half antibodies, and eliminate ADCC and CDC effects. The obtained fusion protein has a strong binding activity with the human growth hormone receptor, can effectively eliminate ADCC and CDC effects, promote Nb2-11 cell proliferation, and effectively treat or prevent diseases related to abnormal human growth hormone.

[0048] According to an embodiment of the present invention, the above nucleic acid molecule may further include at least one of the following additional technical features:

[0049] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0050] According to an embodiment of the present invention, the nucleic acid molecule has a nucleotide sequence as shown in any one of SEQ ID NO:32, 33, 34, 35, 36, 37, 38, 39 or SEQ ID NO:13, 14, 15, 16, 17, 18, 19, 20.

[0051] It should be noted that, for the nucleic acids mentioned in the specification and claims of the present invention, those skilled in the art should understand that they actually include any one or both of the complementary double strands. For convenience, in the specification and claims, although only one strand is given in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid sequence in the present application includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.

[0052] In the fourth aspect of the present invention, the present invention proposes an expression vector. According to an embodiment of the present invention, the aforementioned nucleic acid molecule is carried. When the aforementioned nucleic acid molecule is connected to the vector, the nucleic acid molecule can be directly or indirectly connected to the control element on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, they are not from the vector itself. Of course, the nucleic acid molecule can be operably connected to the control element. Herein, "operably connected" means connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, can play their expected functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, plasmids, bacteriophages, etc. After the expression vector according to some specific embodiments of the present invention is introduced into a suitable receptor cell, the expression of the aforementioned Fc mutant or fusion protein can be effectively achieved under the mediation of the regulatory system, thereby achieving the in vitro acquisition of the Fc mutant or fusion protein in large quantities.

[0053] According to an embodiment of the present invention, the above expression vector may further include at least one of the following additional technical features:

[0054] According to an embodiment of the present invention, the expression vector is a eukaryotic expression vector, a prokaryotic expression vector or a viral vector.

[0055] According to an embodiment of the present invention, the virus comprises a lentivirus.

[0056] In a fifth aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell carries the aforementioned nucleic acid molecule, expression vector, or expresses the aforementioned Fc mutant or fusion protein. The recombinant cell according to an embodiment of the present invention can be used for in vitro expression and mass production of the aforementioned Fc mutant or fusion protein.

[0057] According to an embodiment of the present invention, the above-mentioned recombinant cell may further include at least one of the following additional technical features:

[0058] According to an embodiment of the present invention, the recombinant cell is obtained by introducing the aforementioned expression vector into a host cell.

[0059] According to an embodiment of the present invention, the expression vector is introduced into the host cell by electrotransduction.

[0060] According to an embodiment of the present invention, the recombinant cell is a eukaryotic cell.

[0061] According to an embodiment of the present invention, the recombinant cell is a mammalian cell.

[0062] According to some specific embodiments of the present invention, the eukaryotic cells do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0063] It should be noted that the recombinant cells of the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells or bacteriophages. The prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic cells may be fungi including Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as armyworms, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc. In some embodiments, the recombinant cells of the present invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells or COS cells, and do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0064] It should be noted that the "suitable conditions" described in the specification of this application refer to conditions suitable for the expression of the Fc mutant or fusion protein described in this application. It is easy for those skilled in the art to understand that the conditions suitable for the expression of the Fc mutant or fusion protein include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the Fc mutant or fusion protein according to the specific environment of the laboratory.

[0065] In the sixth aspect of the present invention, the present invention proposes a composition. According to an embodiment of the present invention, it includes: the fusion protein, nucleic acid molecule, expression vector or recombinant cell described above. As mentioned above, the fusion protein, and the fusion protein obtained after the expression of the nucleic acid molecule, expression vector or recombinant cell can not only effectively bind to the human growth hormone receptor, but also effectively promote the proliferation of Nb2-11 cells. Therefore, the fusion protein contained in the composition according to the embodiment of the present invention or the expressed fusion protein can bind to the human growth hormone receptor, promote the proliferation of Nb2-11 cells, and effectively treat or prevent diseases related to abnormal growth hormone.

[0066] Those skilled in the art will appreciate that the composition includes food compositions, pharmaceutical compositions, etc. The composition includes combinations separated in time and / or space, as long as they can work together to achieve the purpose of the present invention. For example, the ingredients contained in the composition can be applied to the subject as a whole, or separately. When the ingredients contained in the composition are applied to the subject separately, each ingredient can be applied to the subject simultaneously or sequentially.

[0067] In the seventh aspect of the present invention, the present invention provides the use of the above-mentioned fusion protein, nucleic acid molecule, expression vector, recombinant cell or composition in the preparation of a drug. According to an embodiment of the present invention, the drug is used to treat or prevent diseases related to abnormal growth hormone.

[0068] According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:

[0069] According to an embodiment of the present invention, the abnormal growth hormone-related diseases include at least one selected from the following: childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner syndrome, Prader-Willi syndrome, renal failure, diseases caused by the alienation state during chemotherapy and AIDS treatment, and intrauterine growth retardation.

[0070] In the eighth aspect of the present invention, the present invention proposes a drug. According to an embodiment of the present invention, it includes the fusion protein, nucleic acid molecule, expression vector, recombinant cell or composition described above. As mentioned above, the fusion protein, and the fusion protein obtained after the expression of the nucleic acid molecule, expression vector, or recombinant cell can not only effectively bind to the human growth hormone receptor, but also effectively promote the proliferation of Nb2-11 cells. Therefore, the fusion protein contained in the drug according to the embodiment of the present invention or the expressed fusion protein can bind to the human growth hormone receptor, promote the proliferation of Nb2-11 cells, and effectively treat or prevent diseases related to abnormal growth hormone.

[0071] According to an embodiment of the present invention, the above-mentioned medicine may further include at least one of the following additional technical features:

[0072] According to an embodiment of the present invention, the medicine further includes a pharmaceutically acceptable carrier, including any solvent, solid excipient, diluent, adhesive, disintegrant, or other liquid excipient, dispersant, flavoring agent or suspending agent, surfactant, isotonic agent, thickener, emulsifier, preservative, solid adhesive, glidant or lubricant, etc., suitable for a specific target dosage form. Except for the scope that any conventional excipient is incompatible with the compound of the present invention, such as any adverse biological effect produced or interaction with any other component of the pharmaceutically acceptable composition in a harmful manner, their use is also the scope considered by the present invention.

[0073] For example, the fusion protein of the present invention can be incorporated into a drug suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These drugs can be prepared in various forms. For example, liquid, semisolid and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions), dispersants or suspending agents, tablets, pills, powders, liposomes and suppositories. Typical drugs are in the form of injection solutions or infusion solutions. The fusion protein can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.

[0074] In the ninth aspect of the present invention, the present invention proposes a method for preventing or treating diseases related to abnormal growth hormone. According to an embodiment of the present invention, it includes administering to a subject at least one of the following: 1) the fusion protein described above; 2) the nucleic acid molecule described above; 3) the expression vector described above; 4) the recombinant cell described above; 5) the composition described above; and 6) the drug described above. As described above, the fusion protein, and the fusion protein obtained after the expression of the nucleic acid molecule, expression vector, or recombinant cell, can not only effectively bind to the human growth hormone receptor, but also effectively promote the proliferation of Nb2-11 cells. Therefore, the above-mentioned substances and the compositions and drugs containing the above-mentioned substances can effectively treat or prevent diseases related to abnormal growth hormone.

[0075] According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

[0076] According to an embodiment of the present invention, the abnormal growth hormone-related diseases include at least one selected from the following: childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner syndrome, Prader-Willi syndrome, renal failure, diseases caused by the alienation state during chemotherapy and AIDS treatment, and intrauterine growth retardation. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 is a schematic diagram of the structure of a fusion protein according to an embodiment of the present invention;

[0078] Figure 2 This is a result diagram of the ADCC effect induced by fusion protein 9# according to an example of the present invention. DETAILED DESCRIPTION

[0079] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0080] In the process of describing the present invention, the relevant terms in this document are explained and illustrated. These explanations and illustrations are only for the convenience of understanding of the scheme and cannot be regarded as limitations on the protection scheme of the present invention.

[0081] The fusion protein of the present invention is usually prepared by a biosynthetic method. According to the nucleotide sequence of the present invention, a person skilled in the art can easily prepare the encoding nucleic acid of the present invention by various known methods. These methods include, but are not limited to, PCR, artificial DNA synthesis, etc. For specific methods, see J. Sambrook, "Molecular Cloning Laboratory Guide". As an embodiment of the present invention, the encoding nucleic acid sequence of the present invention can be constructed by synthesizing the nucleotide sequence in segments and then performing overlap extension PCR.

[0082] In the present application, the "fusion protein" refers to a product obtained by fusing an antibody fragment with other biologically active proteins using genetic engineering technology.

[0083] In this application, the term "mutant" may refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.

[0084] In the present application, the antibody "Fc fragment" includes the CH2 and CH3 regions of the antibody, such as a wild-type IgG4 Fc fragment includes the CH2 and CH3 regions of a wild-type IgG4 antibody, and a wild-type IgG1 Fc fragment includes the CH2 and CH3 regions of a wild-type IgG1 antibody.

[0085] In the present application, "half-antibody" refers to two antibody fragments formed after the disulfide bond of the antibody Fc fragment is broken. For example, the half-antibody formed by the Fc fragment of the wild-type human IgG4 antibody is a fragment containing the CH2 and CH3 regions of the wild-type human IgG4 antibody, and the half-antibody formed by the Fc fragment of the wild-type human IgG1 antibody is a fragment containing the CH2 and CH3 regions of the wild-type human IgG1 antibody.

[0086] In this application, the term "nucleotide" refers to ribonucleotides, deoxynucleotides or modified forms of either type of nucleotide, and combinations thereof.

[0087] The term "host cell" used herein refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The term "transformed" or "transfected" used herein refers to the introduction of nucleic acid (eg, vector) into a cell by various techniques known in the art.

[0088] In this application, the terms "identity", "homology" or "similarity" are used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 48:443); the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:24 ... .70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0089] In the present application, the amino acid numbering of the Fc fragment of IgG4 or IgG1 is based on the EU numbering system. For example, the "S228P" means that the serine at position 228 according to the EU numbering system is replaced by proline.

[0090] In the present application, the amino acid numbering method of the IgD fragment refers to the literature: Takahashi, Nobuhiro, et al. "Complete Amino Acid Sequence of the δ Heavy Chain of Human Immunoglobulin D." Proceedings of the National Academy of Sciences of the United States of America, vol. 79, no. 9, 1982, pp. 2850-54, http: / / www.jstor.org / stable / 11720. Accessed 12Apr. 2022.

[0091] In some embodiments, the present invention provides a fusion protein, whose general structural formula is represented by XLY, wherein X is a first biologically active molecule; L is absent or a connecting peptide; Y is a second biologically active molecule; - is a peptide bond; and the active molecule X or Y is selected from a protein or protein domain, a polypeptide, an antibody or an antibody fragment.

[0092] According to some specific embodiments of the present invention, the above fusion protein may further include at least one of the following additional technical features:

[0093] According to some specific embodiments of the present invention, X is human growth hormone.

[0094] According to some specific embodiments of the present invention, the amino acid sequence of human growth hormone includes SEQ ID NO: 1 or an amino acid sequence having at least 80-99% identity thereto or at least a portion of SEQ ID NO: 1.

[0095] Human growth hormone (hGH) amino acid sequence:

[0096] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQI FKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF (SEQ ID NO: 1).

[0097] According to some specific embodiments of the present invention, Y is an Fc fragment or a variant thereof.

[0098] According to some specific embodiments of the present invention, the amino acid sequence of the Fc fragment includes SEQ ID NO:29, SEQ ID NO:30, or an amino acid sequence that is at least 80-99% identical to any one of them, or at least a portion of any one of them; or the amino acid sequence of the variant of the Fc fragment includes an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or an amino acid sequence that is at least 80-99% identical to any one of them, or at least a portion of any one of them.

[0099] The amino acid sequence of Fc fragment variant 1 includes:

[0100] ESKYGPPCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:2).

[0101] The amino acid sequence of Fc fragment variant 2 includes:

[0102] ESKYGPPCPPCPAPEVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:3).

[0103] The amino acid sequence of Fc fragment variant 3 includes:

[0104] ESKYGPPCPPCPAPEVEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:4).

[0105] The amino acid sequence of Fc fragment variant 4 includes:

[0106] ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNNASRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:5).

[0107] The amino acid sequence of Fc fragment variant 5 includes:

[0108] TPECCHPRLSLGVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:6).

[0109] The amino acid sequence of Fc fragment variant 6 includes:

[0110] TPECPSHTQPLGVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVKFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:7).

[0111] The amino acid sequence of the wild-type IgG4 fragment includes:

[0112] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:29).

[0113] The amino acid sequence of the wild-type IgG1 Fc fragment includes:

[0114] PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:30).

[0115] According to some specific embodiments of the present invention, the amino acid sequence of the fusion protein includes an amino acid sequence selected from: SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or an amino acid sequence that has at least 80-99% identity with any one of them, or at least a portion of any one of them.

[0116] Amino acid sequence of fusion protein 8# (bioactive molecule functional region-Fc mutant 1):

[0117] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDL LKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFRNTGEREEEKKKEKEEKEEEEEEEGEPECPESKYGP PCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 21).

[0118] Amino acid sequence of fusion protein 9# (bioactive molecule functional region-Fc mutant 2):

[0119] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFRNTGKGGEEKKKEKEEKENEEEEEREPECPESKYGPPCPPCPAPEVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 22).

[0120] Amino acid sequence of fusion protein 10# (bioactive molecule functional region - Fc mutant 4):

[0121] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFRNTGKGGDDEKEDEEEKEQQEGETKTPECPESKYGPPCPPCPAPEVEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 23).

[0122] Amino acid sequence of fusion protein 11# (bioactive molecule functional region-(G4S)3-Fc mutant 5):

[0123] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNNASRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 24).

[0124] Amino acid sequence of fusion protein 12# (bioactive molecule functional domain - GS linker peptide mutant - Fc mutant 3):

[0125] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFGGGGGSGGGGTGGGGSLESKYGPPCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 25).

[0126] Amino acid sequence of fusion protein 13# (biologically active molecule functional region-Fc mutant 6):

[0127] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYD LLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFRQSGKGGDDKRKDKDKEEQEERDTKESKYGPPCP PCPAPEFLGGGSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNNASRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQID NO: 26).

[0128] Amino acid sequence of fusion protein 14# (biologically active molecule functional region-(G4S)3-Fc mutant 7):

[0129] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFGGGGSGGGGSGGGGSTPECCHPRLSLGVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:27).

[0130] Amino acid sequence of fusion protein 15# (bioactive molecule functional region-(G4S)3-Fc mutant 8):

[0131] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFGGGGSGGGGSGGGGSTPECPSHTQPLGVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVKFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:28).

[0132] According to some specific embodiments of the present invention, the connecting peptide contains one or more amino acids selected from glycine, serine, alanine and threonine.

[0133] According to some specific embodiments of the present invention, the amino acid sequence of the connecting peptide includes an amino acid sequence selected from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or an amino acid sequence having at least 80-99% identity with any one of them, or at least a portion of any one of them.

[0134] Linker 1 (third peptide chain 1) amino acid sequence:

[0135] RNTGEREEEKKKEKEEKEEEEEEEGEPECP (SEQ ID NO: 8).

[0136] Linker 2 (third peptide chain 2) amino acid sequence:

[0137] RNTGKGGEEKKKEKEEKENEEEEEREPECP (SEQ ID NO: 9).

[0138] Linker 3 (third peptide chain 3) amino acid sequence:

[0139] RNTGKGGDDEKEDEEEKEQQEGETKTPECP (SEQ ID NO: 10).

[0140] Linker 4 amino acid sequence:

[0141] GGGGGSGGGGTGGGGSL (SEQ ID NO: 11).

[0142] Linker 5 (third peptide chain 4) amino acid sequence:

[0143] RQSGKGGDDKRKDKDKEEQEERDTK (SEQ ID NO: 12).

[0144] In some embodiments, the present invention provides a nucleotide sequence encoding the aforementioned connecting peptide.

[0145] In some embodiments, the present invention provides a nucleotide sequence encoding the above-mentioned fusion protein.

[0146] In some embodiments, the present invention provides a vector comprising the nucleotide sequence described above.

[0147] According to some specific embodiments of the present invention, the above-mentioned carrier may further include at least one of the following additional technical features:

[0148] According to some specific embodiments of the present invention, the vector proposed by the present invention is an expression vector.

[0149] In the present application, the term "vector" generally refers to a nucleic acid molecule that can self-replicate in a suitable host, which transfers the inserted nucleic acid molecule into and / or between host cells. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used to express the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell that contains the vector.

[0150] In some embodiments, the present invention provides an engineered cell, characterized in that the engineered cell comprises the fusion protein described above, or the engineered cell comprises the nucleotide sequence or expression vector described above.

[0151] In the present application, the term "engineered cell" refers to a cell having unique stable genetic traits obtained by modifying or recombining the genetic material of a host cell using genetic engineering technology or cell fusion technology.

[0152] In some embodiments, the present invention provides a pharmaceutical composition comprising the above-mentioned fusion protein.

[0153] According to some specific embodiments of the present invention, the above-mentioned pharmaceutical composition may further include at least one of the following additional technical features:

[0154] According to some specific embodiments of the present invention, the pharmaceutical composition is a pharmaceutical composition for oral, intravenous administration, such as bolus injection or by continuous infusion over a period of time, by subcutaneous, intramuscular, intraarterial, intraperitoneal, intrapulmonary, intracerebrospinal, intraarticular, intrasynovial, intrathecal, intralesional, or inhalation route such as intranasal, usually by intravenous or subcutaneous administration.

[0155] According to some specific embodiments of the present invention, the dosage form of the pharmaceutical composition is tablet, capsule, spray, injection, lyophilized powder injection or prefilled injection.

[0156] In some embodiments, the present invention provides a method for preparing the aforementioned fusion protein, which comprises: (a) culturing the aforementioned engineered cells to obtain a culture medium containing the fusion protein; and (b) isolating the fusion protein from the culture medium.

[0157] In some embodiments, the present invention provides the use of the aforementioned fusion protein or pharmaceutical composition in the preparation of a drug for treating or preventing a disease caused by lack of human growth hormone.

[0158] In the present application, the term "treatment" is used to refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, especially humans, including: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit an individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0159] The names and numbers of the sequences in this application are shown in Table 1:

[0160] Table 1:

[0161]

[0162]

[0163] Compared with the prior art, the present invention has at least one of the following beneficial technical effects:

[0164] (1) Long-acting human growth hormone fusion protein E of the present invention C50 The value is low, and it has strong binding activity with human GHR, especially the binding activity of human growth hormone fusion protein 8#, 9#, 10#, and 15# is significantly better than Genexine GX-H9 and GH+G / Slinker+IgG4-1 protein.

[0165] (2) The long-acting human growth hormone fusion protein of the present invention has a good proliferation effect on Nb2-11 cells; the proliferation activity of human growth hormone fusion proteins 8#, 9#, 10#, and 12# is significantly better than that of Genexine GX-H9 and GH+G / S linker+IgG4-1 protein.

[0166] (3) The long-acting human growth hormone fusion protein of the present invention has a good effect on the activity of luciferase expressed by reporter gene cells. Among them, human growth hormone fusion proteins 8#, 9#, 10#, and 12# are better than Genexine GX-H9 and GH+G / Slinker+IgG4-1 proteins in promoting the activity of luciferase expressed by reporter gene cells.

[0167] (4) The third peptide chain provided by the present invention can enhance the binding activity of the human growth hormone fusion protein comprising the third peptide chain with human GHR, the proliferation activity of Nb2-11 cells and the activity of reporter gene cells expressing luciferase.

[0168] (5) The SEQ ID NO: 54 provided by the present invention is conducive to improving the binding activity between the human growth hormone fusion protein of the second biologically active molecule comprising the amino acid sequence of SEQ ID NO: 54 and human GHR.

[0169] (6) The third peptide chain of SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 provided by the present invention is beneficial to improving the proliferation activity of the human growth hormone fusion protein containing the third peptide chain on Nb2-11 cells.

[0170] (7) The second biologically active molecule shown in SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:50 provided by the present invention is beneficial for improving the proliferation activity of human growth hormone fusion protein containing the second biologically active molecule on Nb2-11 cells.

[0171] (8) The third peptide chain of SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 provided by the present invention is beneficial for improving the activity of luciferase expressed by reporter gene cells of human growth hormone fusion protein containing the third peptide chain.

[0172] (9) The second biologically active molecule shown in SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:50 provided by the present invention is beneficial for increasing the activity of luciferase expressed by reporter gene cells containing the human growth hormone fusion protein containing the second biologically active molecule.

[0173] (10) The fusion protein provided by the present invention has a longer in vivo half-life.

[0174] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0175] Example 1 Preparation of human growth hormone fusion protein expression vector

[0176] In the present application, human growth hormone (SEQ ID NO: 1) is connected with various antibody Fc mutants (SEQ ID NO: 47-54, the bold font in the sequence indicates the second peptide chain) to construct fusion proteins, wherein some fusion proteins have connecting peptides. The specific structure of the fusion protein with connecting peptides is as follows: Figure 1 As shown, the connecting peptide is GGGGSGGGGSGGGGS (SEQ ID NO: 45) or is obtained by mutating it, and the antibody Fc mutant is obtained by mutating the wild-type IgG4 Fc fragment (SEQ ID NO: 71) or the IgG1 Fc fragment (SEQ ID NO: 30), and the mutation method includes point mutation, adding wild-type IgG4 and / or IgD antibody fragments or their corresponding mutants, etc. The specific experimental operation is as follows:

[0177] The nucleotide sequence encoding human growth hormone (SEQ ID NO:31), the nucleotide sequence encoding the connecting peptide and the nucleotide sequences encoding various antibody Fc mutants (SEQ ID NO:32-39) were combined, and the nucleotide sequences SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20 encoding the fusion proteins were cloned into the expression vector pCDNA3.4 (purchased from Thermo Fisher) by total gene synthesis and molecular cloning technology, respectively, to obtain the fusion protein expression vectors of each nucleotide sequence.

[0178] Human growth hormone (hGH) amino acid sequence:

[0179] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQI FKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF (SEQ ID NO: 1).

[0180] Amino acid sequence of the first peptide chain 1 (having F234V and L235E mutations compared to the wild-type IgG4 Fc fragment):

[0181] APEVEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQID NO:55).

[0182] Amino acid sequence of the first peptide chain 2 (having S298N, T299A and Y300S mutations compared to the wild-type IgG4 Fc fragment):

[0183] APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNNASRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQID NO:56).

[0184] Amino acid sequence of the first peptide chain 3 (compared to the wild-type IgG4 Fc fragment, amino acids 231-239 are missing):

[0185] VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:57).

[0186] Amino acid sequence of the first peptide chain 4 (compared to the wild-type IgG1 Fc fragment, with amino acid deletions at positions 228-239, H268Q, Y296F, A330S and P331S mutations):

[0187] VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVKFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:58).

[0188] Amino acid sequence of the first peptide chain 5 (having F234A and L235E mutations compared to the Fc fragment of the wild-type IgG4 antibody):

[0189] APEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQID NO:68).

[0190] Amino acid sequence of the second peptide chain 1:

[0191] ESKYGPPCPPCP (SEQ ID NO: 59).

[0192] Amino acid sequence of the second peptide chain 2 (IgD fragment 1 mutant):

[0193] TPECCHPRLSLG (SEQ ID NO: 60).

[0194] Amino acid sequence of the second peptide chain 3 (wild-type IgD fragment 1):

[0195] TPECPSHTQPLG (SEQ ID NO: 61).

[0196] Amino acid sequence of the third peptide chain 1:

[0197] RNTGEREEEKKKEKEEKEEEEEEEGEPECP (SEQ ID NO: 62).

[0198] Amino acid sequence of the third peptide chain 2:

[0199] RNTGKGGEEKKKEKEEKENEEEEEREPECP (SEQ ID NO: 63).

[0200] Amino acid sequence of the third peptide chain 3:

[0201] RNTGKGGDDEKEDEEEKEQQEGETKTPECP (SEQ ID NO: 64).

[0202] Amino acid sequence of the third peptide chain 4:

[0203] RQSGKGGDDKRKDKDKEEQEERDTK (SEQ ID NO: 65).

[0204] Fc mutant 1 (first peptide chain 5-second peptide chain 1-third peptide chain 1) amino acid sequence:

[0205] RNTGEREEEKKKEKEEKEEEEEGEPECPESKYGPPCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:47).

[0206] Amino acid sequence of Fc mutant 2 (first peptide chain 6-second peptide chain 1-third peptide chain 2):

[0207] RNTGKGGEEKKKEKEEKENEEEEEREPECPESKYGPPCPPCPAPEVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:48).

[0208] Amino acid sequence of Fc mutant 3 (first peptide chain 5-second peptide chain 1):

[0209] ESKYGPPCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:49)

[0210] Amino acid sequence of Fc mutant 4 (first peptide chain 1-second peptide chain 1-third peptide chain 3):

[0211] RNTGKGGDDEKEDEEEKEQQEGETKTPECPESKYGPPCPPCPAPEVEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:50).

[0212] Amino acid sequence of Fc mutant 5 (first peptide chain 2-second peptide chain 1):

[0213] ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNNASRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:51).

[0214] Fc mutant 6 (first peptide chain 2-second peptide chain 1-third peptide chain 4) amino acid sequence:

[0215] RQSGKGGDDKRKDKDKEEQEERDTKESKYGPPCPPCPAPEFLGGPSSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNNASRVVSVLTVLHQDWLNGKEYKCK VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:52).

[0216] Fc mutant 7 (first peptide chain 3-second peptide chain 2) amino acid sequence:

[0217] TPECCHPRLSLGVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:53).

[0218] Fc mutant 8 (first peptide chain 4-second peptide chain 3) amino acid sequence:

[0219] TPECPSHTQPLGVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVKFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:54).

[0220] Linker amino acid sequence:

[0221] GGGGGSGGGGTGGGGSL (SEQ ID NO: 11).

[0222] Nucleotide sequence encoding human growth hormone (hGH):

[0223] TTTCCCACCATTCCTCTGAGCAGACTGTTCGACAACGCCATGCTGAGAGCCCACAGACTGCACCAGCTGGCCTTTGACACATACCAGGAGTTCGAGGAGGCCTACATCCCCAAGGAGCAGAAGTACAGCTTCCTGCAGAACCCCCAGACCAGCCTGTGCTTCAGCGAGAGCATCCCCACCCCCAGCAATAGAGAGGAGACACAGCAGAAGAGCAACCTGGAGCTGCTGAGAATCAGCCTGCTGCTGATCCAGAGCTGGCTGGAGCCCGTTCAATTTCTGAGAAGCGTGTTCGCCAACAGCCTGGTGTACGGCGCCAGCGATTCTAATGTGTACGACCTGCTGAAGGACCTGGAGGAGGGCATCCAGACCCTGATGGGCAGACTGGAGGACGGCTCTCCTAGAACCGGACAAATTTTCAAGCAGACCTACAGCAAGTTCGACACCAACAGCCACAACGACGACGCCCTGCTGAAGAACTACGGCCTGCTGTACTGCTTCAGAAAGGACATGGACAAGGTGGAGACATTCCTGAGAATCGTGCAGTGCAGAAGCGTGGAGGGCAGCTGCGGATTC(SEQ ID NO:31).

[0224] Nucleotide sequence encoding Fc mutant 1:

[0225] AGAAACACCGGCGAGAGAGAGGAAGAGAAGAAGAAAGAGAAAGAGGAAAAAGAAGAGGAAGAGGAAGAAGAGGGCGAGCCTGAGTGCCCCGAGTCTAAGTATGGACCTCCTTGTCCTCCATGTCCAGCTCCAGAAGCTGAAGGCGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCAAGAGGATCCTGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACAGCACCTATAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCAAGGGAACCCCAGGTTTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCAGATGGCAAGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCTCTGAGCCTGAGCCTGGGCAAA(SEQ ID NO:32).

[0226] Nucleotide sequence encoding Fc mutant 2:

[0227] AGAAACACCGGCAAGGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGGAGAAGGAGAACGAGGAGGAGGAGGAGAGAGAGCCCGAGTGCCCTGAAAGCAAATACGGACCCCCTTGCCCTCCCTGCCCTGCTCCTGAAGTTGCTGGAGGACCTAGCGTGTTTCTGTTTCCCCCTAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGAGCCAAGAGGACCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAAACCAAGCCCAGAGAGGAGCAGTTCAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGGCCTGCCCAGCAGCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTATACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGCCAACCCGAAAATAATTACAAGACCACACCTCCCGTGCTGGACAGCGATGGATCATTTTTCCTGTACAGCAGACTGACCGTGGACAAGAGCAGATGGCAGGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGAGCCTGGGCAAA(SEQ ID NO:33).

[0228] Nucleotide sequence encoding Fc mutant 3:

[0229] GAATCTAAGTACGGCCCTCCTTGTCCTCCATGTCCAGCTCCAGAAGCTGAAGGCGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCAAGAGGATCCTGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACAGCACCTATAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCAAGGGAACCCCAGGTTTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCAGATGGCAAGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCTCTGAGCCTGAGCCTGGGCAAA(SEQ ID NO:34).

[0230] Nucleotide sequence encoding Fc mutant 4:

[0231] AGAAACACTGGAAAAGGCGGCGACGACGAGAAAGAGGACGAGGAAGAGAAAGAGCAGCAAGAGGGCGAGACAAAGACCCCTGAGTGCCCCGAGTCTAAGTACGGCCCTCCTTGTCCTCCATGTCCAGCTCCAGAAGTGGAAGGCGGCCCATCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCAAGAGGATCCTGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACAGCACCTATAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCAAGGGAACCCCAGGTTTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCCGGTGGCAAGAGGGAAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCTCTGAGCCTGAGCCTGGGCAAA(SEQ ID NO:35).

[0232] Nucleotide sequence encoding Fc mutant 5:

[0233] GAGTCTAAGTACGGCCCTCCTTGTCCTCCATGTCCAGCTCCAGAGTTTCTCGGCGGACCCTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCAAGAGGATCCTGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTTAACAACGCCAGCAGAGTGGTGTCCGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCAAGGGAACCCCAGGTTTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCAGATGGCAAGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCTCTGAGCCTGAGCCTGGGCAAA(SEQ ID NO:36).

[0234] Nucleotide sequence encoding Fc mutant 6:

[0235] AGACAGTCTGGAAAAGGCGGCGACGACAAGCGCAAGGATAAGGACAAAGAGGAACAAGAAGAACGCGACACCAAAGAGTCTAAGTACGGCCCTCCTTGTCCTCCATGTCCAGCTCCAGAGTTTCTCGGCGGACCCTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCAAGAGGATCCTGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTTAACAACGCCAGCAGAGTGGTGTCCGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCAAGGGAACCCCAGGTTTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCAGATGGCAAGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCTCTGAGCCTGAGCCTGGGCAAA(SEQ ID NO:37).

[0236] Nucleotide sequence encoding Fc mutant 7:

[0237] ACACCTGAGTGCTGTCACCCTAGACTGAGCCTGGGAGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCAAGAGGATCCTGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACAGCACCTATAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCAAGGGAACCCCAGGTTTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCAGATGGCAAGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCTCTGAGCCTGTCTCTGGGCAAG(SEQ ID NO:38).

[0238] Nucleotide sequence encoding Fc mutant 8:

[0239] ACACCTGAGTGTCCTAGCCACACACAGCCCCTGGGAGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCAAGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACAGCACCTATAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACCAAGAATCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGAGCCGGTGGCAGCAGGGCAATGTGTTCAGCTGTAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCAAA(SEQ ID NO:39).

[0240] Nucleotide sequence encoding the third peptide chain 1:

[0241] AGAAACACCGGCGAGAGAGAGGAAGAGAAGAAGAAAGAGAAAGAGGAAAAAGAAGAGGAAGAGGAAGAAGAGGGCGAGCCTGAGTGCCCC(SEQ ID NO:40).

[0242] Nucleotide sequence encoding the third peptide chain 2:

[0243] AGAAACACCGGCAAGGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGGAGAAGGAGAACGAGGAGGAGGAGGAGAGAGCCCGAGTGCCCT (SEQ ID NO: 41).

[0244] The nucleotide sequence encoding the third peptide chain 3:

[0245] AGAAACACTGGAAAAGGCGGCGACGACGAGAAAGAGGACGAGGAAGAGAAAGAGCAGCAAGAGGGCGAGACAAAGACCCCTGAGTGCCCC (SEQ ID NO: 42).

[0246] The nucleotide sequence encoding the third peptide chain 4 is:

[0247] AGACAGTTCTGGAAAAGGCGGCGACGACAAGCGCAAGGATAAGGACAAAGAGGAACAAGAAGAACGCGACACCAAA (SEQ ID NO: 66).

[0248] Nucleotide sequence encoding the linker peptide (Linker4):

[0249] ggcggaggcggaggatctggtggcggaggaacaggcggtggtggaagcctg (SEQ ID NO: 67).

[0250] Nucleotide sequence encoding fusion protein 8#:

[0251]

[0252] Nucleotide sequence encoding fusion protein 9#:

[0253]

[0254] Nucleotide sequence encoding fusion protein 10#:

[0255]

[0256] Nucleotide sequence encoding fusion protein 11#:

[0257]

[0258] Nucleotide sequence encoding fusion protein 12#:

[0259]

[0260] Nucleotide sequence encoding fusion protein 13#:

[0261]

[0262] Nucleotide sequence encoding fusion protein 14#:

[0263]

[0264] Nucleotide sequence encoding fusion protein 15#:

[0265]

[0266] The Fc fragment of wild-type IgG4 includes:

[0267] APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQID NO:71).

[0268] Example 2 Expression of human growth hormone fusion protein

[0269] In this example, multiple plasmids obtained in Example 1 were transfected into Expi CHO-S (Gibco, A29133) host cells, and the chemical transfection reagent Polyplus-FectoPRO (polyplus, 116-010) was used to transiently express the long-acting human growth hormone fusion protein, the corresponding amino acid sequence of which is SEQ ID NO: 21-28. The specific experimental operation is as follows:

[0270] (1) Expi CHO-S cells were passaged one day before transient infection and the cell density was adjusted to 3×10 6 Then, the cell culture flask was returned to the shaker (37°C, 8% CO2) for further culture.

[0271] (2) On the day of transient infection (1 L per molecule): Take Expi CHO-S cell fluid for cell counting and adjust the cell density to 6×10 6 cells / mL.

[0272] (3) Preparation of transfection complex: 16 sterile cell culture flasks were taken, 8 of which were marked "DNA" and 8 were marked "FectoPRO". The transfection reagent Polyplus-FectoPRO was added to the "FectoPRO" flask; 60 mL of Opti-MEM solution was added to all 8 "DNA" flasks and 500 μg of the fusion protein expression vectors including each nucleotide sequence obtained in Example 1 was added to each "DNA" flask; the mixture was mixed to obtain a fusion protein expression vector dilution solution, and the fusion protein expression vector dilution solutions including each nucleotide sequence were added to the "FectoPRO" flasks respectively and mixed. After incubation at room temperature for 10 min, the mixture was added to the cell solution and shaken. The cell culture flasks were returned to the shaker for further culture and transfection.

[0273] (4) 18-22 hours after transfection, add appropriate amount of OPM-CHO ProFeed and test the biochemical indexes of the cell fluid. According to the test results of biochemical indexes, supplement glucose to 6g / L. Start testing Titer 4 days after transfection, and feed and sugar every other day. When the cell viability is <80%, CHO cell fermentation broth is obtained, and the supernatant can be harvested for purification.

[0274] Example 3 Purification of human growth hormone fusion protein

[0275] In this example, the CHO cell fermentation broth obtained in Example 2 was subjected to secondary centrifugation (primary centrifugation conditions: 3,000×g, 30 min; secondary centrifugation conditions: 12,000×g, 20 min), and the supernatant was collected and filtered through a 0.2 μm filter for later use.

[0276] Protein A affinity chromatography: Use an aqueous solution containing 20 mM phosphate and 150 mM sodium chloride at pH 7.2 to equilibrate the chromatography column for at least 3 column volumes, load the clarified filtrate after filtration, and retain the feed on the chromatography column for 5 minutes. After loading the sample, use an aqueous solution containing 20 mM phosphate and 150 mM sodium chloride at pH 7.2 to equilibrate the chromatography column for at least 1 column volume, and use a buffer containing 50 mM HAc at pH 4.5, a buffer containing 50 mM HAc at pH 4.0, and a buffer containing 50 mM HAc at pH 3.5 in sequence to elute the target protein. When an absorption peak appears, the peak range is 50 mAU-peak-50 mAU, and a fusion protein with SEC purity that meets the requirements (>98.0) is obtained. ExpiCHO-S cells expressing SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28 were fermented and chromatographed to obtain fusion protein 8#, fusion protein 9#, fusion protein 10#, fusion protein 11#, fusion protein 12#, fusion protein 13#, fusion protein 14# and fusion protein 15#, respectively.

[0277] Example 4 In vitro receptor binding activity assay of human growth hormone fusion protein

[0278] In this example, the purified fusion protein obtained in Example 3 was subjected to in vitro receptor (human growth hormone receptor, Human GHR) binding activity detection, and Genexine GX-H9 (SEQ ID NO: 70) and GH+G / S linker+IgG4-1 (SEQ ID NO: 43) were used as control groups. The specific experimental operation was as follows:

[0279] Human GHR was diluted to prepare 0.5μg / mL coating solution, and 100μL / well was added to the ELISA plate, and the coating was carried out at 2-8℃ for more than 12 hours. The residual solution of the coating plate was discarded, and 1% BSA-PBST was added to each well, 300μL, and blocked at 37℃ for 1 hour. 300μL of PBST was added to each well and washed 3 times. The recombinant long-acting human growth hormone fusion protein, Genexine GX-H9, and GH+G / S linker+IgG4-1 were diluted to 5μg / mL, and then diluted 5 times to 8 gradient concentrations, 100μL / well was added to the ELISA plate, and incubated at 37℃ for 1 hour. After the incubation, 300μL of PBST was added to each well, washed 3 times, and then Goat antihuman IgG Fc-HRP diluted 10000 times with 1% BSA-PBST was added, and 100μL / well was added. After incubation at 37℃ for 1 hour, 300μL of PBST was added to each well, washed 3 times, and patted dry. Add TMB colorimetric solution, 100 μL per well. After 5 minutes of reaction at room temperature, add 2M H2SO4 aqueous solution to terminate the reaction, 100 μL / well. Place the terminated ELISA plate on an ELISA reader and read the absorbance OD at a wavelength of 450 nm. 450 The EC50 of each fusion protein's binding activity to human GHR was calculated.

[0280] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNV YDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFGSGGGSGGGGSGGGGSESKYGPPCPPCPA PEFAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:43).

[0281] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNV YDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFRNTGRGGEEKKKEKEKEEQEERETKTPEC PSHTQPLGVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:70).

[0282] The specific experimental results are shown in Table 2. (1) The long-acting human growth hormone fusion protein of the present invention has a low EC50 value and has a strong binding activity with Human GHR. In particular, the human growth hormone fusion proteins 8#, 9#, 10#, and 15# encoded by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 20 have significantly better binding activity to Human GHR than Genexine GX-H9 and GH+G / Slinker+IgG4-1; among them, the human growth hormone fusion protein 15# has the highest binding activity to human GHR, followed by the human growth hormone fusion protein 9#.

[0283] (2) Human growth hormone fusion protein 15# contains the second biologically active molecule encoded by SEQ ID NO:54, indicating that SEQ ID NO:54 is beneficial to improving the binding activity of human growth hormone fusion protein with human GHR.

[0284] Table 2:

[0285]

[0286] Example 5 Effect of human growth hormone fusion protein on Nb2-11 cell proliferation

[0287] This example uses the purified fusion protein obtained in Example 3 for experiment, and uses the above-mentioned Genexine GX-H9 (commercially available product) and GH+G / S linker+IgG4-1 as control groups. The specific experimental operation is as follows:

[0288] The cells were collected and cultured at 1 × 10 5 The concentration of cells was suspended in culture medium (PRMI 1640 culture medium, containing 1% FBS and 50 μM β-mercaptoethanol). Each 50 μL cell sample was added to each well of a 96-well cell culture plate. The above cells were cultured separately with 50 μL of analysis culture medium containing 0.051 ng / mL-3000 ng / mL concentration gradients of long-acting human growth hormone fusion protein, Genexine GX-H9, and GH+G / S linker+IgG4-1. The above cell plate was cultured in a 37°C, 5% CO2 humidified incubator for 96 hours, and then 50 μL ellTiter-Glo Luminescent Cell ViabilityAssay (Promega, G7571) was added to each well. After 10 minutes, the chemiluminescent signal was detected by an enzyme reader. The biological activity of the recombinant long-acting human growth hormone fusion protein was determined by the obtained dose-response curve.

[0289] The specific experimental results are shown in Table 3, among which, (1) the long-acting human growth hormone fusion protein of the present invention has a good proliferation effect on Nb2-11 cells; the proliferation activity of human growth hormone fusion proteins 8#, 9#, 10#, and 12# encoded by SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:17 is significantly better than that of Genexine GX-H9 and GH+G / Slinker+IgG4-1; among them, the human growth hormone fusion protein 10# has the highest proliferation activity, followed by human growth hormone fusion protein 8#, and then human growth hormone fusion protein 9#.

[0290] (2) The third peptide chain encoded by SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 is beneficial to improving the proliferation activity of human growth hormone fusion protein on Nb2-11 cells.

[0291] (3) The second biologically active molecule encoded by SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:50 is beneficial to improving the proliferation activity of the human growth hormone fusion protein on Nb2-11 cells.

[0292] Table 3:

[0293]

[0294]

[0295] Example 6 Experiment on biological activity of human growth hormone fusion protein on reporter gene cells

[0296] This example uses the purified human growth hormone fusion protein obtained in Example 3 for experiment, and uses the above-mentioned Genexine GX-H9 (commercially available product) and GH+G / S linker+IgG4-1 as control groups. The specific experimental operation is as follows:

[0297] Reporter gene cells (293-GHR / STAT5 cell line) in logarithmic growth phase were obtained, digested with trypsin and plated (Costar, 3917), 4×10 4 cells / well, 50μL / well, and incubate the 96-well white plate in a 37°C, 5% CO2 incubator overnight. Add gradient dilutions of recombinant long-acting human growth hormone fusion protein to the cells, with a starting concentration of 100nM, 5-fold gradient dilution, 10 gradient concentrations, and add the dilutions to the cells at a volume of 50μL / well. Incubate the 96-well white plate in a 37°C, 5% CO2 incubator for 6 hours. After the incubation, take out the 96-well white plate and Nano-Glo Luciferase Assay kit (promega, N112B), equilibrate to room temperature, add reaction substrate to the sample wells at a volume of 50μL / well, and place at room temperature for 10 minutes. Use an enzyme reader (Promega, GM2000) to record the luminescence signal value, with protein concentration as the X-axis and luminescence signal value as the Y-axis. Use GraphPad Prisim 5 for four-parameter fitting to calculate EC 50 value.

[0298] The specific experimental results are shown in Table 4, where:

[0299] (1) The recombinant long-acting human growth hormone fusion protein of the present invention has a good effect on the activity of luciferase expressed by reporter gene cells. Among them, the human growth hormone fusion proteins 8#, 9#, 10#, and 12# encoded by SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:17 have better activities in promoting the expression of luciferase by reporter gene cells than Genexine GX-H9 and GH+G / S linker+IgG4-1; among them, the human growth hormone fusion protein 9# has the highest activity in promoting the expression of luciferase by reporter gene cells, followed by human growth hormone fusion protein 8#, and then human growth hormone fusion protein 10#.

[0300] (2) The third peptide chain encoded by SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 is beneficial to improving the activity of luciferase expressed by the reporter gene cells of the human growth hormone fusion protein.

[0301] (3) The second biologically active molecule encoded by SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:50 is beneficial for increasing the activity of luciferase expressed by reporter gene cells containing human growth hormone fusion protein containing the second biologically active molecule.

[0302] Table 4:

[0303]

[0304]

[0305] Example 7 Detection of ADCC effect of human growth hormone fusion protein-induced effector cells Jurkat-CD16a-luc on target cells CHO-K1-PD-L1 / GHR

[0306] 7.1 Methods

[0307] The sample information is shown in Table 5:

[0308] Table 5:

[0309]

[0310] 3.2 Cell treatment

[0311] Treatment of target cells CHO-K1-PD-L1 / GHR: Perform subculture treatment 2 days before the experiment, discard the supernatant, wash the cells with 5 mL PBS, add 1 mL of 0.25% Trypsin-EDTA, digest in a 37°C incubator for 1 min, add 6 mL of culture medium 1 (450 mL F12 medium + 50 mL FBS) to terminate digestion, resuspend to a single cell suspension, discard 5 mL of cell suspension, add 8 mL of culture medium 1 (450 mL F12 medium + 50 mL FBS), and add 120 μL of G418, the concentration of G418 is 600 μg / mL, mix well, and place in the incubator for continued culture.

[0312] Effector cell Jurkat-CD16a-luc treatment: Perform subculture treatment 2 days before the experiment, mix the cell suspension by pipetting, take 5 mL of cell suspension into a new T75 culture flask, then add 15 mL of culture medium 2 (450 mL RPMI Medium 1640 + 50 mL FBS), 320 μL of G418, the concentration of G418 is 800 μg / mL, 60 μL of Hygromycin B, the concentration of Hygromycin B is 150 μg / mL, mix well, and continue culturing in the incubator.

[0313] 3.3 Experimental process

[0314] On the first day, the target cell CHO-K1-PD-L1 / GHR density was adjusted to 1.83×10 5 cells / mL, 50μL / well plated overnight; the next day, the SBT123 test molecules (molecule 9# and Genexine GX-H9), positive control M7824 and negative control Acterma were diluted to 0.1mg / mL in assay medium 2 (RPMI Medium 1640), diluted to 24μg / mL in the second stage, and then diluted 3-fold in 9 gradients, 25μL / well added to the cell plate, mixed, placed in a 37°C incubator for 45min, and then the cell plate was taken out; the density of the effector cells Jurkat-CD16a-luc was adjusted to 1.47×10 with assay medium 2 (RPMI Medium 1640). 6 cells / mL, 25μL / well was added to the cell plate, mixed, placed in a 37℃ incubator for 5.5h, and then the cell plate was taken out to equilibrate at room temperature, and One-GloLuciferase Assay System was added to the cell plate at 100μL / well, and pipetted 10 times to mix; after lysing at room temperature in the dark for 10min, the chemiluminescent signal value was detected by an enzyme reader. The results are as follows Figure 2 As shown, after adding the positive control PD-L1 antibody M7824, the negative control IL-6R antibody Acterma and the SBT123 test molecule to the ADCC experiment, the positive control M7824 induced effector cells Jurkat-CD16a-luc to have a significant ADCC effect on the target cells CHO-K1-PD-L1 / GHR, the negative control Acterma had no ADCC effect, and the SBT123 test molecule had no ADCC effect.

[0315] Example 8 Human growth hormone fusion protein induces CDC effect of human serum complement on target cells CHO-K1-PD-L1 / GHR

[0316] 8.1 Methods

[0317] The sample information is shown in Table 6:

[0318] Table 6:

[0319]

[0320] (2) Cell treatment

[0321] Treatment of target cells CHO-K1-PD-L1 / GHR: Perform subculture treatment 2 days before the experiment, discard the supernatant, wash the cells with 5 mL PBS, add 1 mL of 0.25% Trypsin-EDTA, digest in a 37°C incubator for 1 min, add 6 mL of culture medium 1 (450 mL F12 medium + 50 mL FBS) to terminate digestion, resuspend to a single cell suspension, discard 5 mL of cell suspension, add 8 mL of culture medium 1 (450 mL F12 medium + 50 mL FBS), and add 120 μL of G418, the concentration of G418 is 600 μg / mL, mix well, and place in the incubator for continued culture.

[0322] (3) Experimental process

[0323] On the first day, the target cell density of CHO-K1-PD-L1 / GHR was adjusted to 2.0×10 5cells / mL, 50 μL / well was plated overnight; on the next day, the SBT123 test molecules (molecule 9# and Genexine GX-H9), positive control Avelumab and negative control Acterma were first diluted to 0.1 mg / mL with differentiation medium (99% F12+1% FBS), second diluted to 15 μg / mL, and then 3-fold diluted 7 gradients, 50 μL / well were added to the cell plate, mixed, placed in a 37°C incubator for co-incubation for 30 minutes, and then the cell plate was taken out; 30% complement (containing 70% differentiation medium + 30% complement) was added, 50 μL / well, mixed, placed in a 37°C incubator for co-incubation for 6 hours, the cell plate was taken out and equilibrated at room temperature, and CellTiter-Glo Luminescent Cell Viability Assay was added, 50 μL / well was added to the cell plate, and the pipette was pipetted 10 times to mix; after lysing at room temperature in the dark for 10 minutes, the chemiluminescent signal value was detected by an enzyme reader. The results showed that after adding the positive control PD-L1 antibody Avelumab, the negative control IL-6R antibody Acterma and the SBT123 test molecule to the CDC experiment, the positive control Avelumab induced human serum complement and had a significant CDC effect on the target cells CHO-K1-PD-L1 / GHR, the negative control Acterma had no CDC effect, and the SBT123 test molecule had no CDC effect.

[0324] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0325] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An Fc mutant, characterized in that: The Fc mutant comprises the amino acid sequence shown in SEQ ID NO: 49, or The Fc mutant comprises a first peptide chain and a second peptide chain, wherein the C-terminus of the second peptide chain is connected to the N-terminus of the first peptide chain, wherein a. The first peptide chain has the following mutations compared to the wild-type IgG4 Fc fragment: F234V and L235E; or S298N, T299A and Y300S; the second peptide chain has a mutation of S228P compared to the hinge region of the wild-type IgG4; or b. The first peptide chain 1) has a deletion of amino acids 231-239 compared to the wild-type IgG4 Fc fragment; or 2) has the following mutations compared to the wild-type IgG1 Fc fragment: deletion of amino acids 228-239, H268Q, Y296F, A330S and P331S; the second peptide chain has a wild-type IgD fragment 1 or an IgD fragment 1 mutant; The wild-type IgD fragment 1 includes amino acids 287-298 of wild-type IgD; Compared with the wild-type IgD fragment 1, the IgD fragment 1 mutant has mutations at the following sites: P291C, S292H, H293P, T294R, Q295L, and P296S.

2. The Fc mutant according to claim 1, characterized in that The Fc mutant further includes a third peptide chain, the C-terminus of the third peptide chain is connected to the N-terminus of the second peptide chain, and the third peptide chain has at least one of the following mutation sites compared to the wild-type IgD fragment 2: position 263, position 264, position 266, position 267, position 268, position 269, position 270, position 271, position 272, position 273, position 274, position 275, position 276, position 277, position 278, position 280, position 281, position 283, position 284, position 285, position 286, position 287, position 288, position 289, position 290, position 291; Optionally, the wild-type IgD fragment 2 includes amino acids 262-291 of wild-type IgD; Optionally, the third peptide chain has the following mutation sites compared to the wild-type IgD fragment 2: 1) positions 266, 267, 268, 277, 278, 280, 283, 285, 286 and 287; or 2) positions 266, 277, 278, 280, 283, 285, 286 and 287. 287; or 3) 266, 269, 270, 271, 273, 274, 275, 277, 278, 281 and 283; or 4) 263, 264, 266, 269, 270, 272, 274, 276, 284, 287-291; Optionally, the third peptide chain has mutations such as: 1) R266E, G267R, G268E, K277E, E278K, Q280E, R283E, T285E, K286G and T287E compared to the wild-type IgD fragment 2; or 2) R266K, K277E, E278K, Q280N, R283E, T285E, K286R and T28 7E; or 3) R266K, E269D, E270D, K271E, K273E, E274D, K275E, K277E, E278K, E281Q, and R283G; or 4) N263Q, T264S, R266K, E269D, E270D, K272R, E274D, E276D, E284D, and 287-291 deletions.

3. The Fc mutant according to any one of claims 1 to 2, characterized in that: The first peptide chain has an amino acid sequence shown in any one of SEQ ID NOs: 55, 56, 57, 58, 68, and 69; Optionally, the second peptide chain has an amino acid sequence shown in any one of SEQ ID NOs: 59, 60, and 61; Optionally, the third peptide chain has an amino acid sequence shown in any one of SEQ ID NO: 62, 63, 64, and 65; Optionally, the wild-type IgG4, wild-type IgG1, and wild-type IgD are human antibodies; Optionally, the Fc mutant comprises: 1) the amino acid sequence shown in any one of SEQ ID NO: 48, 49, 50, 51, 52, 53, 54; or 2) An amino acid sequence that is at least 90% identical to the amino acid sequence shown in any one of SEQ ID NOs: 48, 49, 50, 51, 52, 53, and 54.

4. A fusion protein, characterized in that include: A first peptide segment, wherein the first peptide segment includes a biologically active molecule functional region; A second peptide segment, wherein the second peptide segment comprises an Fc mutant, and the Fc mutant is as defined in any one of claims 1 to 3, wherein the N-terminus of the second peptide segment is connected to the C-terminus of the first peptide segment.

5. The fusion protein according to claim 4, characterized in that The first peptide segment comprises growth hormone or a growth hormone analog, or a growth hormone functional region or a growth hormone analog functional region, preferably human growth hormone or a human growth hormone functional region; Optionally, the human growth hormone has an amino acid sequence as shown in SEQ ID NO: 1 or at least 90% identical thereto; Optionally, the fusion protein further comprises a connecting peptide; Optionally, the N-terminus of the connecting peptide is connected to the C-terminus of the first peptide segment, and the C-terminus of the connecting peptide is connected to the N-terminus of the second peptide segment; Optionally, the connecting peptide has the amino acid sequence described in SEQ ID NO:46; Preferably, compared with the amino acid sequence shown in SEQ ID NO: 46, at least one GGGGS of the connecting peptide has at least one of S>T, a G mutation added to the nitrogen terminal, and an L mutation added to the carbon terminal; Optionally, the connecting peptide has the amino acid sequence shown in SEQ ID NO:

11.

6. The fusion protein according to any one of claims 4 to 5, characterized in that include: 1) the amino acid sequence shown in any one of SEQ ID NO: 22, 23, 24, 25, 26, 27, 28; or 2) An amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence shown in any one of SEQ ID NOs: 22, 23, 24, 25, 26, 27, and 28.

7. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the Fc mutant according to any one of claims 1 to 3 or the fusion protein according to any one of claims 4 to 6; Optionally, the nucleic acid molecule is DNA; Optionally, the nucleic acid molecule has a nucleotide sequence as shown in any one of SEQ ID NO:33, 34, 35, 36, 37, 38, 39 or SEQ ID NO:14, 15, 16, 17, 18, 19, 20.

8. An expression vector, characterized in that Carrying the nucleic acid molecule according to claim 7; Optionally, the expression vector is a eukaryotic expression vector, a prokaryotic expression vector or a viral vector; Optionally, the virus comprises a lentivirus.

9. A recombinant cell, characterized in that Carrying the nucleic acid molecule of claim 7, the expression vector of claim 8, or expressing the Fc mutant of any one of claims 1 to 3 or the fusion protein of any one of claims 4 to 6; Optionally, the recombinant cell is obtained by introducing the expression vector of claim 8 into a host cell; Optionally, the expression vector is introduced into the host cell by electrotransduction; Optionally, the recombinant cell is a eukaryotic cell; Optionally, the recombinant cell is a mammalian cell.

10. A composition, characterized in that include: The fusion protein according to any one of claims 4 to 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8 or the recombinant cell according to claim 9.

11. Use of the fusion protein according to any one of claims 4 to 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8, the recombinant cell according to claim 9, or the composition according to claim 10 in the preparation of a drug for treating or preventing a disease related to abnormal growth hormone; Optionally, the abnormal growth hormone-related diseases include at least one selected from the following: childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner syndrome, Prader-Willi syndrome, renal failure, diseases caused by alienation during chemotherapy and AIDS treatment, and intrauterine growth retardation.

12. A drug, characterized in that include: The fusion protein according to any one of claims 4 to 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8, the recombinant cell according to claim 9, or the composition according to claim 10.

Images