Polyethylene glycol site-directed modified heteropolymer protein or polypeptide as well as preparation method and application thereof

Abstract

The invention discloses a polyethylene glycol site-directed modified heteropolymer protein or polypeptide as well as a preparation method and application thereof. Firstly, a method for performing site-specific polyethylene glycol modification on protein or polypeptide is provided, and comprises the following steps: providing protein or polypeptide to be modified, the protein or polypeptide to be modified comprises a target N-terminal alpha-amino group and a non-target N-terminal alpha-amino group, and performing mutation blocking on the non-target N-terminal alpha-amino group; and carrying out site-specific modification reaction on the target N-terminal alpha-amino group and the aldehyde group or ketone group derivative of polyethylene glycol to generate the protein or polypeptide subjected to site-specific modification by polyethylene glycol. The polyethylene glycol fixed-point single-modified heteropolymer protein or polypeptide product modified by the method has a definite and controllable modification position, can bring more definite half-life period prolonging and biased composition activity, and can be better applied to drug treatment.

Description

Polyethylene glycol site-directed modification of heteromeric proteins or polypeptides and preparation method and application thereof Technical Field

[0001] The present invention belongs to the field of biopharmaceutical technology, and specifically relates to a polyethylene glycol site-specifically modified heteromeric protein or polypeptide and a preparation method and application thereof. Background Art

[0002] Since 1977, when a research team led by Professor Frank F. Davis at Rutgers University discovered that covalently coupling proteins to long-chain polyethylene glycol could prolong their half-life and reduce immunogenicity (Abuchowski et al. 1977a, 1977b), chemical modification with polyethylene glycol has been widely applied to a wide range of bioactive drug molecules and has gradually become one of the most important delivery methods for long-acting protein and peptide drugs. After years of development, the technology for PEG-modified drugs entered the industrialization and practical stage in the 1980s. In 1990, the FDA approved the world's first PEG-modified drug (PEG-modified adenosine deaminase for the treatment of severe combined immunodeficiency), developed by Enzon Corporation in the United States. This drug not only prolongs its half-life but also significantly reduces its toxicity, improving patients' quality of life. In the following decades, polyethylene glycol modification technology has developed rapidly, and scientists have successfully developed polyethylene glycol-modified adenylate deaminase (PEG-ADA), asparaginase (PEG-L-asparaginase), interferon α-2b ( Protein drugs such as interferon α-2b), interferon α-2a (peginterferon α-2a), recombinant human granulocyte colony-stimulating factor (pegfil grastim), as well as polyethylene glycol-modified camptothecin (PEG-CPT), doxorubicin liposomes, etc.

[0003] However, due to technical limitations, many early PEG-modified drugs used random and multiple modifications. This type of modification often resulted in a mixture of drugs with non-uniform structures, making it difficult to control the batch-to-batch variability in drug activity and raising significant uncertainty about safety. With the development of PEG-modified drug technology, there has been a trend toward obtaining drugs with more defined structures through site-specific modification of a single PEG. Currently, commercially available PEG-modified drugs primarily use PEG aldehyde derivatives to site-specifically modify the N-terminal α-amino group of a protein, or PEG maleimide derivatives or vinyl sulfones to site-specifically modify the thiol groups of the cysteine ​​side chains of a protein. However, this site-specific modification method only has a single-site modification effect on single-chain proteins or proteins containing a single free cysteine ​​thiol group. When used to modify dimeric proteins or proteins containing multiple free cysteine ​​thiol groups, it is no longer a single-site modification.

[0004] Summary of the Invention

[0005] One object of the present invention is to provide a method for preparing a polyethylene glycol site-specifically modified heteromeric protein or polypeptide.

[0006] Another object of the present invention is to provide a polyethylene glycol site-specific modification of heteromeric proteins or polypeptides.

[0007] Another object of the present invention is to provide related applications of the polyethylene glycol site-specific modification of heteromeric proteins or polypeptides.

[0008] The present invention provides a novel method for site-specific modification of heteromeric proteins or polypeptides with polyethylene glycol. The method can achieve site-specific modification of the heteromeric protein or polypeptide composition by mutating the heteromeric protein or polypeptide composition, blocking the N-terminal α-amino group that is not to be modified with polyethylene glycol, and retaining only the N-terminal α-amino group that is to be modified with polyethylene glycol, and then reacting the retained N-terminal α-amino group with an aldehyde derivative or a keto derivative of polyethylene glycol.

[0009] Specifically, in one aspect, the present invention provides a method for site-specific polyethylene glycol modification of a protein or polypeptide, comprising:

[0010] a) providing a protein or polypeptide to be modified, wherein the protein or polypeptide to be modified comprises a target N-terminal α-amino group and a non-target N-terminal α-amino group, and performing mutation blocking on the non-target N-terminal α-amino group;

[0011] b) subjecting the target N-terminal α-amino group to a site-directed modification reaction with an aldehyde or ketone derivative of polyethylene glycol to generate a polyethylene glycol site-directed modified protein or polypeptide.

[0012] In the present invention, the "target N-terminal α-amino group" refers to the N-terminal α-amino group to be modified with polyethylene glycol, and the "non-target N-terminal α-amino group" refers to the N-terminal α-amino group not to be modified with polyethylene glycol.

[0013] The method of the present invention is used to carry out site-specific modification of heteromeric proteins or polypeptides with polyethylene glycol, which has a clear and controllable modification position, can bring more clear half-life extension and biased composition activity to the modified product, and is better applied to drug treatment purposes.

[0014] According to some specific embodiments of the present invention, in the method of the present invention for site-specific polyethylene glycol modification of a protein or polypeptide, the protein or polypeptide is a heteromeric protein or polypeptide.

[0015] According to some specific embodiments of the present invention, in the method of the present invention for site-directed polyethylene glycol modification of a protein or polypeptide, the process of mutating and blocking the non-target N-terminal α-amino group comprises:

[0016] constructing a gene encoding the amino acid sequence of the mutation-blocked protein or polypeptide into a vector;

[0017] Transform the vector into host bacteria and extract the endotoxin-removing plasmid;

[0018] transfecting the plasmid into host cells to express the target protein;

[0019] The target product is harvested, i.e., the protein or peptide after the non-target N-terminal α-amino group is mutated and blocked.

[0020] According to some specific embodiments of the present invention, in the method of the present invention for site-specific polyethylene glycol modification of a protein or polypeptide, the host cell is preferably a 293F cell.

[0021] According to some specific embodiments of the present invention, in the method of the present invention for site-specific polyethylene glycol modification of a protein or polypeptide, the protein or polypeptide is a heteromeric protein or polypeptide comprising 2-5 N-terminal α-amino groups.

[0022] According to some specific embodiments of the present invention, in the method of the present invention for site-specific polyethylene glycol modification of a protein or polypeptide, the heteromeric protein or polypeptide comprises one target N-terminal α-amino group and 1-2 non-target N-terminal α-amino groups.

[0023] According to some specific embodiments of the present invention, in the method of the present invention for site-specific polyethylene glycol modification of a protein or polypeptide, the heteromeric protein or polypeptide is a short half-life protein.

[0024] According to some specific embodiments of the present invention, in the method of the present invention for site-specific polyethylene glycol modification of a protein or polypeptide, the short half-life protein is a di-subunit heteromeric protein or polypeptide.

[0025] According to some specific embodiments of the present invention, in the method of the present invention for site-specific polyethylene glycol modification of a protein or polypeptide, the di-subunit heteromeric protein or polypeptide is selected from one or more of antibodies, cytokines, cytokine receptors, cytokine fusion proteins, hormones, hemoglobin, and G protein-coupled receptors.

[0026] According to some specific embodiments of the present invention, in the method of the present invention for site-specific polyethylene glycol modification of a protein or polypeptide, the cytokine is selected from one or more of interleukins, interferons, lymphotoxins, growth factors, and chemokines.

[0027] According to some specific embodiments of the present invention, in the method of the present invention for site-directed polyethylene glycol modification of a protein or polypeptide, the antibody is selected from a monoclonal antibody, a polyclonal antibody, and an antibody fragment. More specifically, the antibody fragment preferably comprises an antibody Fab fragment.

[0028] According to some specific embodiments of the present invention, in the method of the present invention for site-specific polyethylene glycol modification of a protein or polypeptide, the hormone is selected from one or more of follicle-stimulating hormone, luteinizing hormone, luteinizing hormone-releasing hormone, thyroid-stimulating hormone, and chorionic gonadotropin.

[0029] According to some specific embodiments of the present invention, in the method of the present invention for site-specific polyethylene glycol modification of a protein or polypeptide, the interleukin is one or more of interleukin 6, interleukin 12, interleukin 23, interleukin 27, and interleukin 35.

[0030] According to some specific embodiments of the present invention, in the method of the present invention for site-specific polyethylene glycol modification of a protein or polypeptide, the interleukin 12 includes human interleukin-12 and mouse interleukin 12.

[0031] According to some specific embodiments of the present invention, in the method of the present invention for site-specific polyethylene glycol modification of a protein or polypeptide, the interleukin 12 includes two subunits, p35 and p40.

[0032] According to some specific embodiments of the present invention, in the method of the present invention for site-directed polyethylene glycol modification of a protein or polypeptide, the process of mutating and blocking the non-target N-terminal α-amino group comprises:

[0033] By adding, reducing or replacing one or several amino acid residues, the non-target N-terminal first amino acid of the protein or polypeptide to be modified is mutated to one or more of proline, glutamine, glutamic acid, asparagine, aspartic acid or non-natural amino acids.

[0034] According to some specific embodiments of the present invention, in the method of the present invention for site-directed polyethylene glycol modification of a protein or polypeptide, the mutated amino acid may be proline or glutamine.

[0035] According to some specific embodiments of the present invention, in the method for site-specific polyethylene glycol modification of a protein or polypeptide of the present invention, the aldehyde or keto derivative of polyethylene glycol is selected from one or more of polyethylene glycol acetaldehyde derivatives, polyethylene glycol propionaldehyde derivatives, and polyethylene glycol butyraldehyde derivatives. Furthermore, the polyethylene glycol propionaldehyde derivative is preferably monomethoxy polyethylene glycol propionaldehyde and / or two-arm polyethylene glycol propionaldehyde.

[0036] On the other hand, the present invention also provides a polyethylene glycol site-specific modified protein or polypeptide, wherein the non-polyethylene glycol-modified N-terminus in the amino acid sequence of the protein or polypeptide is blocked by mutation compared to the wild-type protein or polypeptide.

[0037] According to some specific embodiments of the present invention, the polyethylene glycol site-directed modified protein or polypeptide of the present invention is prepared according to the method for site-directed polyethylene glycol modification of a protein or polypeptide according to the present invention.

[0038] According to some specific embodiments of the present invention, the polyethylene glycol site-directed modification protein or polypeptide of the present invention has a molecular weight of 5 to 40 KD, preferably 10 to 30 KD, and more preferably about 20 KD.

[0039] According to some specific embodiments of the present invention, in the polyethylene glycol site-directed modification protein or polypeptide of the present invention, one end of the polyethylene glycol structure is connected to the target N-terminus of the protein or polypeptide for modification, and the other end of the polyethylene glycol structure is modified with an immunoconjugate or a targeting fragment.

[0040] According to some specific embodiments of the present invention, in the PEGylated site-modified proteins or polypeptides of the present invention, the immunoconjugate or targeting fragment is intended to further extend the half-life of the PEGylated site-modified protein or enhance its targeting properties. Immunoconjugates or targeting fragments with such functions can be selected with reference to existing technologies in the relevant field. For example, the immunoconjugate or targeting fragment can be selected from albumin, albumin fusion proteins, antibodies, cytokines, cytokine receptors, cytokine fusion proteins, hormones, hemoglobin, G protein-coupled receptors, PAS, HES, CTP of the β subunit of human chorionic gonadotropin, PEG, and XTEN. Preferably, the cytokine is selected from interleukins, interferons, lymphotoxins, growth factors, and chemokines. Preferably, the antibody is selected from monoclonal antibodies, polyclonal antibodies, and antibody fragments. Preferably, the antibody fragment is the Fc region of an IgG2 or IgG4 antibody. Preferably, the antibody fragment is a tumor-targeting antibody fragment. Preferably, the hormone is selected from follicle-stimulating hormone, luteinizing hormone, luteinizing hormone-releasing hormone, thyroid-stimulating hormone, and chorionic gonadotropin. Preferably, the interleukin is interleukin-12, interleukin-2, interleukin-15, interleukin-23, interleukin-27, interleukin-35, etc. Preferably, the immunoconjugate or targeting fragment has a different protein or polypeptide structure from the other end of the polyethylene glycol structure.

[0041] According to some specific embodiments of the present invention, the polyethylene glycol site-directed modification protein or polypeptide of the present invention, wherein the amino acid sequence of the protein or polypeptide includes one or more of the following amino acid sequences: SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5; SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8; SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13; SEQ ID NO.14, 15, SEQ ID NO.16; SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28; SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35.

[0042] According to some specific embodiments of the present invention, the polyethylene glycol site-directed modification protein or polypeptide of the present invention, wherein the structure of the protein or polypeptide is selected from one or more of the following proteins or polypeptides:

[0043] A mutant human interleukin-12, wherein the amino acid sequence of the p35 subunit is shown in any one of SEQ ID NOs. 3-5;

[0044] A mutant human interleukin-12, wherein the amino acid sequence of the p40 subunit is shown in any one of SEQ ID NOs. 6-8;

[0045] A mutant murine interleukin-12, the amino acid sequence of its p35 subunit is shown in any one of SEQ ID NOs. 11-13;

[0046] A mutant murine interleukin-12, the p40 subunit amino acid sequence of which is shown in any one of SEQ ID NOs. 14-16;

[0047] A mutant human luteinizing hormone, wherein the amino acid sequence of its α subunit is shown in any one of SEQ ID NOs. 26-28, and the amino acid sequence of its β subunit is shown in SEQ ID NO. 18;

[0048] A mutant mouse luteinizing hormone, wherein the amino acid sequence of its α subunit is shown in any one of SEQ ID NOs. 33-35, and the amino acid sequence of its β subunit is shown in SEQ ID NO. 30;

[0049] A mutant human thyroid-stimulating hormone, wherein the amino acid sequence of its α subunit is shown in any one of SEQ ID NOs. 26-28, and the amino acid sequence of its β subunit is shown in SEQ ID NO. 19;

[0050] A mutant mouse thyroid-stimulating hormone, wherein the amino acid sequence of its α subunit is shown in any one of SEQ ID NOs. 33-35, and the amino acid sequence of its β subunit is shown in SEQ ID NO. 31;

[0051] A mutant human follicle-stimulating hormone, wherein the amino acid sequence of its α subunit is shown in any one of SEQ ID NOs. 26-28, and the amino acid sequence of its β subunit is shown in SEQ ID NO. 20;

[0052] A mutant mouse follicle-stimulating hormone, wherein the amino acid sequence of its α subunit is shown in any one of SEQ ID NOs. 33-35, and the amino acid sequence of its β subunit is shown in SEQ ID NO. 32;

[0053] The mutated human chorionic gonadotropin, whose α subunit amino acid sequence is shown in any one of SEQ ID NOs. 26-28, and whose β subunit amino acid sequence is shown in any one of SEQ ID NOs. 21-25.

[0054] On the other hand, the present invention also provides a drug, which includes the polyethylene glycol site-directed modification protein or polypeptide of the present invention and a pharmaceutically acceptable excipient.

[0055] According to some specific embodiments of the present invention, the dosage form of the drug of the present invention includes a liquid preparation and / or a lyophilized preparation;

[0056] According to some specific embodiments of the present invention, the drug of the present invention has administration routes including injection, oral administration, external application or a combination of multiple routes.

[0057] On the other hand, the present invention also provides the use of the polyethylene glycol site-specific modified protein or polypeptide or the drug in the preparation of anti-tumor drugs.

[0058] According to some specific embodiments of the present invention, the anti-tumor drug of the present invention can be administered alone or in combination with radiotherapy, chemotherapy, myeloablative therapy, CAR-T therapy, CAR-NK therapy, antibody targeted therapy, oncolytic virus therapy and / or gene vaccine therapy to patients.

[0059] In another aspect, the present invention further provides a protein or polypeptide that can be used as a precursor or intermediate for preparing the polyethylene glycol site-directed modification protein or polypeptide of the present invention. Specifically, the amino acid sequence of the protein or polypeptide includes one or more of the following:

[0060] SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5; SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8; SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13; SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16; SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28; SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35.

[0061] According to some specific embodiments of the present invention, the protein or polypeptide of the present invention that can be used as a precursor or intermediate for preparing the polyethylene glycol site-directed modification protein or polypeptide of the invention is selected from one or more of the following proteins or polypeptides:

[0062] A mutant human interleukin-12, wherein the amino acid sequence of the p35 subunit is shown in any one of SEQ ID NOs. 3-5;

[0063] A mutant human interleukin-12, wherein the amino acid sequence of the p40 subunit is shown in any one of SEQ ID NOs. 6-8;

[0064] A mutant murine interleukin-12, the amino acid sequence of its p35 subunit is shown in any one of SEQ ID NOs. 11-13;

[0065] A mutant murine interleukin-12, the p40 subunit amino acid sequence of which is shown in any one of SEQ ID NOs. 14-16;

[0066] A mutant human luteinizing hormone, wherein the amino acid sequence of its α subunit is shown in any one of SEQ ID NOs. 26-28, and the amino acid sequence of its β subunit is shown in SEQ ID NO. 18;

[0067] A mutant mouse luteinizing hormone, wherein the amino acid sequence of its α subunit is shown in any one of SEQ ID NOs. 33-35, and the amino acid sequence of its β subunit is shown in SEQ ID NO. 30;

[0068] A mutant human thyroid-stimulating hormone, wherein the amino acid sequence of its α subunit is shown in any one of SEQ ID NOs. 26-28, and the amino acid sequence of its β subunit is shown in SEQ ID NO. 19;

[0069] A mutant mouse thyroid-stimulating hormone, wherein the amino acid sequence of its α subunit is shown in any one of SEQ ID NOs. 33-35, and the amino acid sequence of its β subunit is shown in SEQ ID NO. 31;

[0070] A mutant human follicle-stimulating hormone, wherein the amino acid sequence of its α subunit is shown in any one of SEQ ID NOs. 26-28, and the amino acid sequence of its β subunit is shown in SEQ ID NO. 20;

[0071] A mutant mouse follicle-stimulating hormone, wherein the amino acid sequence of its α subunit is shown in any one of SEQ ID NOs. 33-35, and the amino acid sequence of its β subunit is shown in SEQ ID NO. 32;

[0072] The mutated human chorionic gonadotropin, whose α subunit amino acid sequence is shown in any one of SEQ ID NOs. 26-28, and whose β subunit amino acid sequence is shown in any one of SEQ ID NOs. 21-25.

[0073] On the other hand, the present invention also provides a nucleic acid molecule encoding the protein or polypeptide of the present invention.

[0074] According to some specific embodiments of the present invention, the nucleic acid molecule of the present invention comprises one or more of the following nucleotide sequences:

[0075] SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.40; SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.43; SEQ ID NO.46, SEQ ID NO.47, SEQ ID NO.48; SEQ ID NO.49, SEQ ID NO.50, SEQ ID NO.51; SEQ ID NO.61, SEQ ID NO.62, SEQ ID NO.63; SEQ ID NO.68, SEQ ID NO.69, SEQ ID NO.70.

[0076] In some specific embodiments of the present invention, the N-terminal α-amino group of other subunits of the heteromeric protein or polypeptide composition is blocked by genetic mutation, including by adding, deleting, or replacing the N-terminal amino acid of the blocking subunit to proline (Pro, P), which forms a ring on its own and blocks the N-terminal α-amino group; or by mutating the N-terminal amino acid of the blocking subunit to glutamine, glutamic acid, asparagine, or aspartic acid, and reacting the side chain carboxyl group with the N-terminal α-amino group to form a ring using glutamine cyclase or other means; or by inserting non-natural amino acids to block the N-terminal α-amino group or reduce the reaction probability of the N-terminal α-amino group with an aldehyde or keto derivative of polyethylene glycol. Furthermore, by reacting the aldehyde or keto derivative of polyethylene glycol with the heteromeric protein or polypeptide composition and controlling the reaction conditions to maintain an acidic pH, the aldehyde or keto derivative of polyethylene glycol reacts only with the retained N-terminal α-amino group and not with the ε-amino group of the amino acid side chain.

[0077] In some specific embodiments of the present invention, the N-terminal α-amino group of the redundant subunit is blocked by gene mutation technology, so that the aldehyde derivative or ketone derivative of polyethylene glycol can be used to modify the N-terminal α-amino group of the target subunit of the heteropolymer protein or polypeptide composition. The obtained product has a clear and consistent structure, and therefore has clear long-term, safety and effectiveness when used in drug therapy. At the same time, by blocking different subunits, the position of polyethylene glycol modification can be changed, and by changing the steric hindrance brought about by the modification position of polyethylene glycol, the binding ability of the protein or polypeptide composition to the receptor protein is changed, affecting the downstream signaling pathway, bringing about changes in the drug treatment effect, and achieving better drug treatment purposes. When using double-end modified polyethylene glycol derivatives, antibody fragments can be introduced to enhance the drug's transport and targeting capabilities, or other drugs can be introduced to achieve the effect of combined therapy.

[0078] The amino acid and nucleotide sequences mentioned in the present invention are as follows:

[0079] SEQ ID NO.1 Amino acid sequence of wild-type human IL-12P35 subunit:

[0080] SEQ ID NO.2 Amino acid sequence of wild-type human IL-12P40 subunit:

[0081] SEQ ID NO.3 amino acid sequence of human IL-12P35 subunit mutation:

[0082] SEQ ID NO.4 amino acid sequence of human IL-12P35 subunit mutation:

[0083] SEQ ID NO.5 Amino acid sequence of human IL-12P35 subunit mutation:

[0084] SEQ ID NO.6 Amino acid sequence of human IL-12P40 subunit mutation:

[0085] SEQ ID NO.7 amino acid sequence of human IL-12P40 subunit mutation:

[0086] SEQ ID NO.8 amino acid sequence of human IL-12P40 subunit mutation:

[0087] SEQ ID NO.9 amino acid sequence of the wild-type murine IL-12P35 subunit:

[0088] SEQ ID NO.10 Amino acid sequence of the wild-type mouse IL-12P40 subunit:

[0089] SEQ ID NO.11 amino acid sequence of mouse IL-12P35 subunit mutation:

[0090] SEQ ID NO.12 amino acid sequence of the mutant murine IL-12P35 subunit:

[0091] SEQ ID NO.13 amino acid sequence of mouse IL-12 P35 subunit mutation:

[0092] SEQ ID NO.14 amino acid sequence of mouse IL-12P40 subunit mutation:

[0093] SEQ ID NO.15 amino acid sequence of mouse IL-12P40 subunit mutation:

[0094] SEQ ID NO.16 amino acid sequence of mouse IL-12P40 subunit mutation:

[0095] SEQ ID NO.17 Amino acid sequence of wild-type human glycoprotein hormone α subunit:

[0096] SEQ ID NO.18 amino acid sequence of wild-type human luteinizing hormone (hLH) β subunit:

[0097] SEQ ID NO.19: Amino acid sequence of wild-type human thyroid stimulating hormone (hTSH) β subunit:

[0098] SEQ ID NO.20 Amino acid sequence of wild-type human follicle-stimulating hormone (hFSH) β subunit:

[0099] SEQ ID NO.21 Amino acid sequence of wild-type human chorionic gonadotropin (hCG) β1 subunit:

[0100] SEQ ID NO.22 Amino acid sequence of wild-type human chorionic gonadotropin (hCG) β2 subunit:

[0101] SEQ ID NO.23 Amino acid sequence of wild-type human chorionic gonadotropin (hCG) β3 subunit:

[0102] SEQ ID NO.24 amino acid sequence of wild-type human chorionic gonadotropin (hCG) β5 subunit:

[0103] SEQ ID NO.25 Amino acid sequence of wild-type human chorionic gonadotropin (hCG) β7 subunit:

[0104] SEQ ID NO.26 Amino acid sequence of mutant human glycoprotein hormone α subunit:

[0105] SEQ ID NO.27 amino acid sequence of mutant human glycoprotein hormone α subunit:

[0106] SEQ ID NO.28 amino acid sequence of mutant human glycoprotein hormone α subunit:

[0107] SEQ ID NO.29 amino acid sequence of the wild type of mouse glycoprotein hormone α subunit:

[0108] SEQ ID NO.30 Amino acid sequence of the wild type of mouse luteinizing hormone (mLH) β subunit:

[0109] SEQ ID NO.31 amino acid sequence of the wild-type murine thyroid-stimulating hormone (mTSH) β subunit:

[0110] SEQ ID NO.32 Amino acid sequence of the wild-type murine follicle-stimulating hormone (mFSH) β subunit:

[0111] SEQ ID NO.33 amino acid sequence of the mutant α subunit of mouse glycoprotein hormone:

[0112] SEQ ID NO.34 amino acid sequence of the mutant α subunit of mouse glycoprotein hormone:

[0113] SEQ ID NO.35 amino acid sequence of the mutant α subunit of mouse glycoprotein hormone:

[0114] SEQ ID NO.36 Nucleotide sequence of wild-type human IL-12P35 subunit:

[0115] SEQ ID NO.37 Nucleotide sequence of wild-type human IL-12P40 subunit:

[0116] SEQ ID NO.38 Nucleotide sequence of human IL-12P35 subunit mutation:

[0117] SEQ ID NO.39 Nucleotide sequence of human IL-12P35 subunit mutation:

[0118] SEQ ID NO.40 Nucleotide sequence of human IL-12P35 subunit mutation:

[0119] SEQ ID NO.41 Nucleotide sequence of human IL-12P40 subunit mutation:

[0120] SEQ ID NO.42 Nucleotide sequence of human IL-12P40 subunit mutation:

[0121] SEQ ID NO.43 Nucleotide sequence of human IL-12P40 subunit mutation:

[0122] SEQ ID NO.44: nucleotide sequence of the wild-type murine IL-12 P35 subunit:

[0123] SEQ ID NO.45: nucleotide sequence of the wild-type murine IL-12P40 subunit:

[0124] SEQ ID NO.46 Nucleotide sequence of mouse IL-12 P35 subunit mutation:

[0125] SEQ ID NO.47 Nucleotide sequence of mouse IL-12 P35 subunit mutation:

[0126] SEQ ID NO.48 Nucleotide sequence of mouse IL-12 P35 subunit mutation:

[0127] SEQ ID NO.49 Nucleotide sequence of mouse IL-12P40 subunit mutation:

[0128] SEQ ID NO.50 Nucleotide sequence of mouse IL-12P40 subunit mutation:

[0129] SEQ ID NO.51 Nucleotide sequence of mouse IL-12P40 subunit mutation:

[0130] SEQ ID NO.52 nucleotide sequence of wild-type human glycoprotein hormone α subunit:

[0131] SEQ ID NO.53 Nucleotide sequence of wild-type human luteinizing hormone (hLH) β subunit:

[0132] SEQ ID NO.54 nucleotide sequence of wild-type human thyroid-stimulating hormone (hTSH) β subunit:

[0133] SEQ ID NO.55 Nucleotide sequence of wild-type human follicle-stimulating hormone (hFSH) β subunit:

[0134] SEQ ID NO.56: nucleotide sequence of wild-type human chorionic gonadotropin (hCG) β1 subunit:

[0135] SEQ ID NO.57 nucleotide sequence of wild-type human chorionic gonadotropin (hCG) β2 subunit:

[0136] SEQ ID NO.58 nucleotide sequence of wild-type human chorionic gonadotropin (hCG) β3 subunit:

[0137] SEQ ID NO.59: nucleotide sequence of wild-type human chorionic gonadotropin (hCG) β5 subunit:

[0138] SEQ ID NO.60 Nucleotide sequence of wild-type human chorionic gonadotropin (hCG) β7 subunit:

[0139] SEQ ID NO.61 Nucleotide sequence of human glycoprotein hormone α subunit mutant:

[0140] SEQ ID NO.62 Nucleotide sequence of human glycoprotein hormone α subunit mutant:

[0141] SEQ ID NO.63 Nucleotide sequence of human glycoprotein hormone α subunit mutant:

[0142] SEQ ID NO.64 nucleotide sequence of the wild type of mouse glycoprotein hormone α subunit:

[0143] SEQ ID NO.65 Nucleotide sequence of the wild type of mouse luteinizing hormone (mLH) β subunit:

[0144] SEQ ID NO.66: nucleotide sequence of the wild-type murine thyroid-stimulating hormone (mTSH) β subunit:

[0145] SEQ ID NO.67 Nucleotide sequence of the wild type of murine follicle-stimulating hormone (mFSH) β subunit:

[0146] SEQ ID NO.68 Nucleotide sequence of the mutant type of mouse glycoprotein hormone α subunit:

[0147] SEQ ID NO.69 Nucleotide sequence of the mutant type of mouse glycoprotein hormone α subunit:

[0148] SEQ ID NO.70 Nucleotide sequence of the mutant type of mouse glycoprotein hormone α subunit:

[0149] definition

[0150] In order to provide a clear description of the present invention, a number of terms are defined below.

[0151] As used herein, "heteromeric composition", "heteromeric protein", "heterodimer protein or polypeptide", and "heteromultimeric protein or polypeptide" refer to a protein substance or polypeptide substance, which is a protein or polypeptide composition composed of at least two different amino acid sequences, and may be a composition of one or more proteins or polypeptides that are natural, expressed by recombinant cells, synthesized by cell-free technology, or synthesized by chemical synthesis technology.

[0152] "Interleukin-binding protein" refers to a receptor fragment or antibody fragment that can specifically bind to a certain type of interleukin.

[0153] "Cytokine binding protein" refers to a receptor fragment or antibody fragment that can specifically bind to a certain type of cytokine.

[0154] “Antibody,” “monoclonal antibody,” and “polyclonal antibody” refer to immunoglobulin molecules, which are heterotetrameric proteins produced by combining two identical heavy chains and two identical light chains, and which specifically bind to an antigen through an antigen-binding site composed of a light chain variable region (VL) and a heavy chain variable region (VH), thereby eliciting an antigen-specific humoral immune response.

[0155] “Antibody fragment” and “antigen-binding peptide” refer to antibody fragments having antigen-binding ability, and include fragments produced by cleaving antibodies with protein-cleaving enzymes and recombinantly produced single-chain fragments, and examples thereof include Fab, F(ab')2, scFv, diabodies, triabodies, sdAbs, and VHHs.

[0156] "Fc region of an immunoglobulin or a portion thereof" and "Fc segment" refer to a protein comprising the heavy chain constant region 2 (CH2) and the heavy chain constant region 3 (CH3) of an immunoglobulin, but excluding its heavy chain variable region and light chain and light chain constant region (CL1), and may further comprise the hinge region of the heavy chain constant region, as well as Fc fragments improved thereon.

[0157] "Nucleic acid" or "nucleic acid sequence" refers to a deoxyribonucleotide or ribonucleotide polymer in single- or double-stranded form and, unless otherwise limited, includes known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. Unless otherwise stated, a particular nucleic acid sequence includes its complementary sequence.

[0158] "Mutation" refers to the addition, deletion or substitution of a nucleic acid sequence, which results in a change in the amino acid sequence encoded by the nucleic acid sequence.

[0159] The term "encoding" in relation to a specific nucleic acid includes reference to a nucleic acid that contains the information for translation into a specific protein. This information is specified through the use of codons.

[0160] "Blocking" refers to changing the type of amino acid at the first position of the N-terminus by mutation, enzyme catalysis, chemical synthesis, etc., resulting in the loss or loss of reactivity of the α-amino group that should exist at the N-terminus, making it unable to react with polyethylene glycol aldehyde derivatives or keto derivatives.

[0161] “Polyethylene glycol aldehyde derivative” refers to a compound formed by modifying polyethylene glycol by introducing at least one aldehyde group at any position of the polyethylene glycol.

[0162] “Polyethylene glycol keto derivative” refers to a compound formed by modifying polyethylene glycol by introducing at least one keto group at any position of the polyethylene glycol.

[0163] "Site-directed single modification" refers to the introduction of a polyethylene glycol molecule at a specific position of the protein

[0164] "Double-end modified derivatives" refer to the introduction of active groups at both ends of polyethylene glycol, including at least one aldehyde group or one ketone group, and one other active group. This ensures that one polyethylene glycol molecule can undergo coupling reactions with two different biological macromolecules or chemical small molecules.

[0165] "Coupling" and "coupling reaction" refer to the process of obtaining a molecule by a certain chemical reaction between two molecules of a substance.

[0166] "Modification" refers to connecting one substance to another substance by coupling or physical means, and this connection can be a chemical bond connection or a physical connection.

[0167] A "host cell" is a cell that can support the replication or expression of an expression vector. The host cell can be a prokaryotic cell, such as E. coli, or a eukaryotic cell, such as a yeast cell, an insect cell, an amphibian cell, or a mammalian cell.

[0168] As used herein, "polypeptide," "peptide," and "protein" are used interchangeably and include polymers of amino acid residues.

[0169] The term "residue" or "amino acid residue" or "amino acid" includes reference to an amino acid incorporated into a protein, polypeptide, or peptide (collectively, "peptide"). The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may include known analogs of a natural amino acid that may function in a similar manner to the naturally occurring amino acids.

[0170] "Transfection," "stable transfection," and "transient transfection" refer to the uptake of an expression vector into a host cell, regardless of whether any coding sequence is actually expressed. A variety of transfection methods are known to those skilled in the art. For example, transfection can be accomplished by electroporation in the presence of an expression vector and a high concentration of calcium phosphate, by using a bacteriophage or viral expression vector for insertion into a host cell, by mechanical insertion of nucleic acid, or even by culturing host cells in the presence of unpackaged nucleic acid fragments. Successful transfection is generally confirmed when any indication of manipulation of the vector of interest appears in the host cell.

[0171] An "expression system" or "expression vector" refers to a nucleic acid sequence containing a desired coding sequence and control sequences in operable association so that a host transformed with these sequences can produce the encoded protein. To achieve transformation, the expression system may be contained on a vector; however, the relevant nucleic acid molecule may also subsequently be integrated into the host chromosome. BRIEF DESCRIPTION OF THE DRAWINGS

[0172] FIG1 is a DOT detection diagram of some products of human modified IL-12.

[0173] FIG2 is a diagram showing the DOT detection of some products of mouse-derived modified IL-12.

[0174] FIG3 is an SDS-PAGE image of the purification results of mP40Q.

[0175] FIG4 is a bar graph showing the expression levels of some mutant human IL-12.

[0176] FIG5 shows the non-reducing gel and reducing gel images of SDS-PAGE of purified mPEG-pALD-20KD-mP40Q and mPEG-pALD-20KD-mIL-12.

[0177] FIG6 shows the mIL-12 content in the serum of experimental animals.

[0178] FIG7 shows the IFN-γ content in the serum of experimental animals.

[0179] FIG8 shows the killing ratio of NK92 cells to Jurkat cells stimulated by PEG-IL-12-1 and PEG-IL-12-5.

[0180] FIG9 shows the killing ratio of NK92 cells to K562 cells stimulated by PEG-IL-12-1 and PEG-IL-12-5.

[0181] FIG10 shows the killing ratio of NK92 cells to SK-BR3 cells stimulated by PEG-IL-12-1 and PEG-IL-12-5.

[0182] FIG11 shows the killing ratio of NK92 cells to NCI-N87 cells stimulated by PEG-IL-12-1 and PEG-IL-12-5.

[0183] FIG12 is a photograph of non-reducing and reducing SDS-PAGE of purified hCG.

[0184] FIG13 is a diagram showing the purification results of PEG-modified native hCG and hCG-A1P detected by SDS-PAGE. DETAILED DESCRIPTION

[0185] In order to have a clearer understanding of the technical characteristics, purposes and beneficial effects of the present invention, now in conjunction with specific embodiments and the technical scheme of the present invention is described in detail below, it should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. For those skilled in the art, various changes and / or modifications easily thought of within the spirit of the present invention are all identified as being encompassed within the protection scope of the present invention. In the embodiments, each raw reagent material is commercially available, and the experimental method without specifying specific conditions is conventional method and normal condition known in the art, or according to the conditions recommended by the instrument manufacturer.

[0186] Example 1. Mutated IL-12

[0187] IL-12 is a heterodimeric cytokine having two subunits, P35 and P40. The present invention blocks the N-terminal α-amino group of one of the subunits through gene mutation.

[0188] Blocking the p35 subunit of human IL-12 involves mutating the first arginine (Arg1) at the N-terminus of the p35 subunit to proline (Arg1Pro), glutamine (Arg1Gln), or asparagine (Arg1Asn). In this example, the specific amino acid sequences of the mutated human IL-12 p35 subunit can be found in SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5.

[0189] Blocking the p40 subunit of human IL-12 involves mutating the first N-terminal isoleucine (Ile1) of the p40 subunit to proline (Ile1Pro), glutamine (Ile1Gln), or asparagine (Ile1Asn). In this example, the specific amino acid sequences of the mutated p40 subunit of human IL-12 can be found in SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8.

[0190] Blocking the p35 subunit of murine IL-12 involves mutating the first N-terminal arginine (Arg1) of the p35 subunit to proline (Arg1Pro), glutamine (Arg1Gln), or asparagine (Arg1Asn). In this example, the specific amino acid sequences of the murine IL-12 p35 subunit mutations can be found in SEQ ID NOs. 11-13.

[0191] Blocking the P40 subunit of murine IL-12 involves mutating the first N-terminal methionine (Met1) of the P40 subunit to proline (Met1Pro), glutamine (Met1Gln), or asparagine (Met1Asn). In this example, the specific amino acid sequences of the mutant murine IL-12 P40 subunits can be found in SEQ ID NOs. 14-16.

[0192] Additionally, IL-12 proteins or fragments thereof may include various modified proteins or peptides, i.e., variants. Such modifications may be performed by replacing, deleting, or adding at least one amino acid residue of wild-type IL-12 without altering the function of IL-12. These variant proteins or peptides may be 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the wild-type protein.

[0193] In some embodiments, the variant is obtained by substituting at least one amino acid residue of the wild-type protein without changing the protein's function. Traditionally, alanine can be substituted for the wild-type amino acid residue. This substitution can be a conservative amino acid substitution that has no or minimal effect on the overall protein charge, i.e., polarity or hydrophobicity. For conservative amino acid substitutions, see Table 1.

[0194] Table 1

[0195] For each amino acid, additional conservative substitutions include "homologs" of the amino acid. Specifically, a "homolog" refers to an amino acid obtained by inserting a methylene group (CH2) into the β-side chain of the amino acid side chain. Examples of "homologs" may include homophenylalanine, homoarginine, homoserine, etc., but are not limited thereto. As used herein, the term "IL-12 protein" may be used as a concept including "IL-12 protein and fragments thereof." Unless otherwise specified, the terms "protein," "polypeptide," and "peptide" may be used as interchangeable concepts.

[0196] The nucleic acid sequence corresponding to the amino acid sequence was synthesized using whole-genome sequence synthesis technology. For example, the amino acid sequence of the P35 subunit was constructed into the vector pxc17.4, with restriction enzyme cleavage sites of HindIII and EcoRI; the amino acid sequence of the P40 subunit was constructed into the vector pCDNA3.1+, with restriction enzyme cleavage sites of HindIII and EcoRI. The synthesized vector sequence was optimized based on 293F cells, avoiding restriction enzyme cleavage sites such as HindIII, EcoRI, SalI, NotI, PvuI, BamHI, and ScaI. After all nucleic acid sequences were optimized, a Kozak sequence (GCCACC) and a signal peptide were introduced at the N-terminus, and a TAA termination codon was added to the C-terminus. Of note, six His residues were added to the C-terminus of the weakly expressed P35 subunit as a purification tag to reduce the number of purification steps and the difficulty.

[0197] The synthesized vector containing the target gene was transformed and shaken, and then the endotoxin-removing plasmid was extracted and sequenced. Then, wild-type and modified IL-12 (each modified IL-12 is composed of a P35 and P40 subunit) were prepared according to the combination in Table 2. The expression vectors with P35 nucleic acid sequence and P40 nucleic acid sequence were co-transfected into 293F cells for transient expression. The expression vectors containing P35 and P40 were used in a 1:1 ratio, the concentration of the expression vector was 0.8ug / ml, the transfection reagent was 1.2ul / ml, the transfection system was opti-MEM, and the transfection reagent was One day before transfection, 1x 10 6 Prepare 500ml cell suspension at 100 cells / ml. Culture cells overnight at 37°C, 125rpm and 5% CO2. For transfection, take the corresponding volume of Add to a 50ml empty tube; In a second 50ml test tube, dilute the DNA plasmid to the desired concentration in serum-free medium to a final volume of 10% of the total volume. Gently mix evenly; Pour the diluted DNA into the pure Reagent, immediately mix the solution and incubate at room temperature for 10 minutes; Pour the transfection mixture into the cells and mix it thoroughly with the cell suspension. If necessary, add the transfection mixture at a final concentration of 0.5 μl / ml 2 hours after transfection. After 6 hours, cells were replaced with serum-free medium from Opim and cultured for 48 hours. Cell counts were monitored and, when cell viability was above 80%, cells were fed at a rate of 5% and culture continued. Otherwise, cells were harvested and tested for protein expression using DOT. The test results are shown in Figures 1 and 2. Figure 1 shows the expression of some products of human-derived recombinant IL-12. The darker the black, the more protein is expressed. It can be seen that combinations 1, 4, 6, and 7 have higher expression levels. Figure 2 shows the expression of some mouse-derived IL-12s, from which it can be seen that combinations 8, 10, and 13 all have higher expression levels. The expressed protein was purified by His affinity chromatography column, and the purity and molecular weight were confirmed by SDS-PAGE. Figure 3 shows the purification results of mP40Q, where lane 1 S is the cell supernatant stock solution expressing the protein, lane 2 FT is the flow-through after purification, in which most of the target protein has been captured, lane 3 is the collection liquid after column flushing, which contains only impurities but no target protein, lanes 4-8 are samples collected by volume of column eluate, most of which contain target protein, among which 2B3 contains the most target protein, lane 9 is a standard used as a control, and lane 10 is a marker lane used as a reference for molecular weight.

[0198] Table 2

[0199] The nucleic acid molecule may further include a signal sequence or a leader sequence. As used herein, the term "signal sequence" refers to a fragment that guides the secretion of bioactive molecule drugs and fusion proteins and is cut off after translation in the host cell. The signal sequence of the present invention is a polynucleotide that encodes an amino acid sequence that initiates protein movement through the endoplasmic reticulum (ER) membrane. Useful signal sequences in the present invention include: a signal peptide of a human interleukin 12 P35 subunit, a signal peptide of a human interleukin 12P40 subunit, a signal peptide of a mouse interleukin 12P35 subunit, and / or a signal peptide of a mouse interleukin 12 P40 subunit.

[0200] The characteristics of signal peptides are well known in the art, and signal peptides generally have 16 to 30 amino acids, but they can contain more or less number of amino acid residues. Conventional signal peptides are composed of three regions: an alkaline N-terminal region, a central hydrophobic region, and a more polar C-terminal region. The central hydrophobic region includes 4 to 12 hydrophobic residues, which fix the signal sequence by the membrane lipid bilayer during the translocation of the immature polypeptide. After activation, the signal sequence is usually cut off in the lumen of the ER by a cellular enzyme called a signal peptidase. Specifically, the signal sequence can be a secretory signal sequence for tissue plasminogen activator (tPa), a signal sequence for herpes simplex virus glycoprotein D (HSV gDs), or growth hormone. Preferably, the secretory signal sequence used in higher eukaryotic cells including mammals can be used. In addition, as a secretory signal sequence, the signal sequence contained in wild-type IL-7 can be used, or it can be used after replacing a codon with a high expression frequency in the host cell.

[0201] The expression levels of some mutant human IL-12 in this example are shown in Figure 4. It can be seen that the expression levels of different mutations vary, with the expression level of the P35 mutation being significantly lower than that of the P40 mutation. The expression levels of different mutations in the N-terminal amino acid of P40 also vary, with hP40P having the highest expression level, followed by hP40Q.

[0202] The obtained hP40Q, hP35Q, mP40Q, and mP35Q proteins were diluted to 2 mg / mL with ultrapure water at pH 6.0. 28 μL of 1 ng / mL glutamine cyclase was added and incubated at 37°C for 5 hours to convert the N-terminal glutamine of the IL-12 protein to pyroglutamate. After the incubation, the proteins were purified again using a His affinity column. The N-terminal α-amino-blocked proteins were obtained for subsequent coupling reactions with polyethylene glycol aldehyde derivatives.

[0203] Example 2

[0204] This example provides a method for synthesizing monodisperse 20KD monomethoxypolyethylene glycol propionaldehyde (mPEG-pALD-20K) site-specifically modified murine interleukin-12mP40Q and mP35Q. The specific preparation method is as follows:

[0205] (1) 5 mg of mouse interleukin-12 mP40Q or mP35Q were dissolved in 0.05 M acetic acid / sodium acetate buffer at pH 5.0, and stirred at 20°C to prepare an interleukin-12 solution with a final concentration of 1.0 mg / mL.

[0206] (2) 14.3 mg of monomethoxy polyethylene glycol propionaldehyde with a molecular weight of 20 KD (purchased from Xiamen Sinobond Biotechnology Co., Ltd., model: 06020101412) was dissolved in pure water to prepare a monomethoxy polyethylene glycol propionaldehyde aqueous solution; then the solution was added dropwise to the interleukin 12 solution, wherein the molar ratio of the monomethoxy polyethylene glycol propionaldehyde to the interleukin 12 was 10:1.

[0207] (3) 0.135 mg of sodium cyanoborohydride (the molar ratio of sodium cyanoborohydride to interleukin 12 was 30:1) was dissolved in water to prepare a sodium cyanoborohydride aqueous solution, which was then added to the interleukin 12 solution. The mixture was stirred at 20° C. for 24 h and the reaction was monitored by UPLC, as shown in Table 1 and Figure 1 below.

[0208] (4) After the reaction is completed, the product is purified by first filtering it through an ultrafiltration tube with a cut-off flow rate of 30KD, and then purifying it through ion exchange chromatography to obtain purified monodisperse 20KD monomethoxypolyethylene glycol propionaldehyde-modified interleukin-12.

[0209] The reaction process of the preparation method of this embodiment is shown as follows:

[0210] The value of n is approximately 454.

[0211] In this example, by controlling the above reaction conditions, mPEG-pALD-20KD was selectively site-specifically coupled to the α-amino group at the N-terminus of the P35 subunit of mouse interleukin 12mP40Q or site-specifically coupled to the α-amino group at the N-terminus of the P40 subunit of mouse interleukin 12mP35Q.

[0212] The purity of the purified mPEG-pALD-20KD-mP40Q and mPEG-pALD-20KD-mIL-12 was detected by SDS-PAGE, and the electrophoresis patterns are shown in FIG5 . In Figure 5, lanes 1 and 2 show the non-reducing gel and reducing gel results of mPEG-pALD-20KD-mP40Q, respectively, and lanes 3 and 4 show the non-reducing gel and reducing gel results of mPEG-pALD-20KD-mIL-12, respectively. The product purities are all above 95%. Comparison of the reducing and non-reducing SDS-PAGE gel images reveals that PEG modification is primarily on the P35 subunit of mPEG-pALD-20KD-mP40Q, while modifications are found on both the P40 and P35 subunits of mPEG-pALD-20KD-mIL-12. This demonstrates that this method can achieve site-specific single modification of the IL-12 heterodimer by blocking the N-terminal α-amino group of one subunit of the heterodimer, allowing the polyethylene glycol aldehyde derivative to react only with the N-terminal α-amino group of the other subunit.

[0213] Those skilled in the art can simply extend this technology and use this method for site-specific polyethylene glycol modification of other heteromeric proteins or polypeptides.

[0214] Example 3

[0215] An IFN-γ detection ELISA kit was used to detect the IFN-γ production of NK-92 cells stimulated by mPEG-pALD-20KD-mP40Q and mPEG-pALD-20KD-mP35Q (Example 2) and mIL-12, and the difference in activity between mPEG-pALD-20KD-mP40Q and mPEG-pALD-20KD-mP35Q and mIL-12, as well as the degree of toxicity reduction, were evaluated. The specific method is as follows:

[0216] (1) Take the cultured NK-92 cells and record the passage number. Observe the cell status under a microscope. If the cells are in good condition, collect the cells in a 15ml or 50ml sterile centrifuge tube, centrifuge at 600rpm for 5 minutes, discard the supernatant, and then resuspend the cells in 5ml of fresh complete culture medium and count them.

[0217] (2) Based on the cell count results, dilute the cell suspension to 2×105 cells / ml with fresh complete culture medium, and then add the cell suspension to a 96-well plate, 100 μl per well.

[0218] (3) Filter sterilize the sample and dilute it in a gradient with NK-92 complete medium to a final concentration of 1000 to 0.46 U / ml. Add the diluted sample to a 96-well plate containing 100 μl of cell suspension. The final culture volume is 200 μl and the final cell density is 1 × 105 cells / ml. Stimulate the cells for 24 ± 2 hours.

[0219] (4) After stimulation, centrifuge the 96-well plate at 1000 rpm for 5 minutes and dilute 60-fold with the standard diluent (1X Dilution buffer R) in the ELISA kit. Use a 10 μl 8-channel pipette to draw 5 μl of the supernatant and add it to 295 μl of the standard diluent. Use a plate washer to shake at high speed for 5 minutes. Then take 100 μl of the diluted supernatant and add it to the ELISA plate wells of the IFN-γ detection kit. Add 50 μl of biotinylated antibody, mix well, cover with a sealing film, and incubate at room temperature (18-25°C) for 120 minutes. After incubation, wash the plate four times with washing solution and pat dry.

[0220] (5) Then add 100 μl of streptavidin-HRP and incubate at room temperature (18-25°C) for 20 minutes. After incubation, wash the plate four times with washing solution and pat dry.

[0221] (6) Add 100 μl of TMB to each well and incubate at room temperature (18-25°C) in the dark for 5-30 minutes. The liquid in the well gradually turns blue. After incubation at room temperature, add 100 μl of Stop solution to each well to terminate the reaction. Detect the OD value at 450 nm using a SpectraMax M2 microplate reader.

[0222] Result processing: GraphPad Prism 6 software was used to process the data. OD value was used as the vertical axis and dilution factor as the horizontal axis. A four-parameter fitting was used to obtain the activity curve, top and bottom values, and R2 value. The test standard requires an R2 value of ≥ 0.95. The activity of the test sample was calculated according to the following formula:

[0223] Where Pr is the biological activity of the standard, that is, the biological activity of the WHO IL-12 standard = 1×10 4 U / ml; Ds is the predilution factor of the test sample; Dr is the predilution factor of the standard; Es is the dilution factor of the test sample equivalent to half the effective dose of the standard; Er is the dilution factor of the standard equivalent to half the effective dose. Specific activity of the test sample (U / mg) = test sample potency (U / ml) / test sample concentration (mg / ml).

[0224] The experimental results are shown in Table 3 below.

[0225] Table 3

[0226] As shown in the experimental results in Table 3, compared to the IL-12 standard, the activity of mPEG-pALD-20KD-mP40Q, the product obtained in Example 2, decreased by 13-fold, and the activity of mPEG-pALD-20KD-mP35Q decreased by 6.2-fold. This decrease in activity means that to produce the same amount of IFN-γ, PEG-modified IL-12 requires a larger dose than IL-12. Conversely, at the same dose, PEG-modified IL-12 has fewer side effects, facilitating the administration of a larger single dose and maintaining stable blood drug concentrations for a longer period without significant side effects. The doses of PEG-modified IL-12 used in subsequent pharmacodynamic studies were determined based on the experimental results obtained in Example 3.

[0227] Example 4

[0228] This example provides an analytical experiment on the effect of the duration of drug action of mPEG-pALD-20KD-mP40Q prepared in Example 2 in normal mice. By comparing the duration of action of mPEG-pALD-20KD-mP40Q prepared in Example 2 with that of wild-type mIL-12 in normal mice, it is confirmed that PEG modification of long-acting interleukin-12 prolongs the duration of drug action.

[0229] Four-week-old C57BL / 6 normal mice were randomly divided into five groups based on body weight. Two mice in each group were subcutaneously administered the test substance once, according to the dosing schedule shown in Table 4. Whole blood samples (100 μL each) were collected via the tail vein at 6, 12, 24, 72, 120, 192, and 264 hours. Blood was collected into sterile EP tubes, allowed to stand at room temperature for 30 minutes, and then centrifuged at 3000 g for 10 minutes to obtain serum. Interleukin-12 and IFN-γ levels in the serum of each group were quantified by ELISA.

[0230] Table 4

[0231] The results are shown in Figures 6 and 7. The results show that interleukin-12 in the serum of the PEG-IL-12 group was still detectable after 120 hours, while it was detectable after 24 hours in the high-dose native mIL-12 group and only after 12 hours in the medium- and low-dose groups, becoming undetectable thereafter. This suggests that PEG modification can prolong the half-life of interleukin-12. IFN-γ assays revealed that the PEG-modified interleukin-12 group produced significantly more IFN-γ than the native mIL-12 group. The native mIL-12 group reached a peak at 24 hours before declining to normal levels, indicating that the IFN-γ response lags behind that of interleukin-12. Among the PEG-modified interleukin-12 groups, levels decreased after 24 hours, while those in the medium- and low-dose groups continued to rise after 264 hours, with a more pronounced lag. Overall, PEG modification can prolong the half-life of interleukin-12, providing a sustained therapeutic effect.

[0232] Example 5

[0233] In this example, the same method as in Example 2 was used to prepare mPEG-bALD-20KD-modified human interleukin 12hP40Q (PEG-IL-12-5) and hP35Q (PEG-IL-12-1), and used them for in vitro stimulation of NK92 cells for tumor killing experiments.

[0234] The density of starved NK92 cells was adjusted to 1×10 5 / mL, divided into eight portions, plated into 6-well plates, 1.5mL per well, and mPEG-bALD-20K-hP40Q was prepared at 0.088nmol / L, 0.88nmol / L, and 8.8nmol / L, mPEG-bALD-20K-hP35Q was prepared at 0.132nmol / L, 1.32nmol / L, and 13.2nmol / L, and hIL-12 was prepared at 0.142nmol / L. They were added to the above 6-well plates and stimulated at 37°C, 5% CO2 for 48 hours, and a group without drug addition was set as a control group. The stimulated NK92 cells were then harvested, centrifuged at 1300r / min for 5min, and resuspended in culture medium to 1×10 6 / mL. Tumor cells Jurkat, K562, SK-BR3, NCI-N87 were adjusted to a cell density of 2×10 5 / mL and plated into 96-well plates, 50 μL per well. Stimulated NK92 cells were then added for co-culture at a ratio of 5:1 for NK92:Jurkat, 30:1 for NK92:K562, 40:1 for NK92:SK-BR3, and 30:1 for NK92:NCI-N87. A control group consisting of NK92 cells with natural death and a medium-free control group were established. Tumor-killing ability was analyzed using a CCK8 assay 24 hours after co-culture.

[0235] The tumor killing ability of NK92 cells stimulated by PEG-IL-12-1 and PEG-IL-12-5 is shown in Figures 8 to 11. Figure 8 shows the killing ratio of NK92 cells to kill Jurkat cells stimulated by PEG-IL-12-1 and PEG-IL-12-5. The tumor killing ratios of the low-dose concentration groups of PEG-IL-12-1 and PEG-IL-12-5 were lower than those of the IL-12 group, and the killing ratios of the medium-dose concentration group and the high-dose concentration group were higher than those of IL-12, but only PEG-IL-12-1 was significant. Figure 9 shows the killing ratio of NK92 cells to kill K562 cells stimulated by PEG-IL-12-1 and PEG-IL-12-5. The results are similar to those of Jurkat. The killing ratios of the medium-dose concentration group and the high-dose concentration group were higher than those of IL-12, and there were significant differences between PEG-IL-12-1 and PEG-IL-12-5. Figures 10 and 11 show the killing ratios of SK-BR3 and NCI-N87 cells stimulated by PEG-IL-12-1 and PEG-IL-12-5, respectively. In solid tumor killing experiments, a higher effector-target ratio is required, and the killing effect of the medium-dose group has become less significant. Only the high-dose group showed significantly better killing ability than the IL-12 group.

[0236] Example 6: Gene mutations of follicle-stimulating hormone, luteinizing hormone, and chorionic gonadotropin

[0237] Follicle-stimulating hormone, luteinizing hormone, and chorionic gonadotropin are a class of heterodimeric proteins with the same α-subunit-glycoprotein hormone α subunit and different β-subunits. The present invention blocks the N-terminal α-amino group of the α subunit through gene mutation, which can be used for site-specific modification of the three proteins.

[0238] Blocking the α-subunit of the human glycoprotein hormone includes mutating the first N-terminal alanine (Ala1) to proline (Ala1Pro), glutamine (Ala1Gln), or asparagine (Ala1Asn). In this example, the specific amino acid sequences of the mutated α-subunit of the human glycoprotein hormone are shown in: SEQ ID NO. 26, SEQ ID NO. 27, and SEQ ID NO. 28.

[0239] Blocking the α-subunit of the mouse glycoprotein hormone involves mutating the first N-terminal leucine (Leu1) to proline (Leu1Pro), glutamine (Leu1Gln), or asparagine (Leu1Asn). In this example, the specific amino acid sequences of the mutated α-subunit of the human glycoprotein hormone are shown in SEQ ID NO. 33, SEQ ID NO. 34, and SEQ ID NO. 35.

[0240] Additionally, the follicle-stimulating hormone, luteinizing hormone, or chorionic gonadotropin proteins or fragments thereof may include various modified proteins or peptides, i.e., variants. Such modifications may be performed by replacing, deleting, or adding at least one protein of the wild-type follicle-stimulating hormone, luteinizing hormone, or chorionic gonadotropin without altering the function of the follicle-stimulating hormone, luteinizing hormone, or chorionic gonadotropin. These different proteins or peptides may be 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the wild-type protein.

[0241] Traditionally, wild-type amino acid residues are substituted with alanine, but this substitution can be a conservative amino acid substitution that has no or weak effect on the overall protein charge, i.e., polarity or hydrophobicity. For conservative amino acid substitutions, reference can be made to Table 1.

[0242] For each amino acid, additional conservative substitutions include "homologs" of the amino acid. Specifically, "homologs" refer to amino acids obtained by inserting a methylene group (CH2) into the β-side chain of the amino acid side chain. Examples of "homologs" may include homophenylalanine, homoarginine, homoserine, etc., but are not limited thereto. As used herein, the term "follicle-stimulating hormone, luteinizing hormone, chorionic gonadotropin protein" may be used as a concept including "follicle-stimulating hormone, luteinizing hormone, chorionic gonadotropin protein and fragments thereof". Unless otherwise specified, the terms "protein", "polypeptide" and "peptide" may be used as interchangeable concepts.

[0243] The nucleic acid sequence corresponding to the amino acid sequence was synthesized using whole-genome sequence synthesis technology. For example, the amino acid sequence of the P35 subunit was constructed into the vector pxc17.4, with restriction enzyme cleavage sites of HindIII and EcoRI; the amino acid sequence of the P40 subunit was constructed into the vector pCDNA3.1+, with restriction enzyme cleavage sites of HindIII and EcoRI. The synthesized vector sequence was optimized based on 293F cells, avoiding restriction enzyme cleavage sites such as HindIII, EcoRI, SalI, NotI, PvuI, BamHI, and ScaI. After all nucleic acid sequences were optimized, a kozak sequence (GCCACC) and a signal peptide were introduced into the N-terminus, and a TAA termination codon was added to the C-terminus. It is worth noting that six His residues were added to the C-terminus of the α subunit as purification tags to reduce the number of purification steps and the difficulty.

[0244] The synthesized vector containing the target gene is transformed into bacteria, and then the endotoxin-removing plasmid is extracted and sequenced.

[0245] Subsequently, the α subunit nucleic acid sequence and the β subunit nucleic acid sequence were combined according to Table 5 to prepare wild-type and mutant follicle-stimulating hormone, luteinizing hormone or chorionic gonadotropin (each mutant hormone is composed of an α subunit and a β subunit), and the expression vectors carrying the α subunit nucleic acid sequence and the β subunit nucleic acid sequence were co-transfected into 293F cells for transient expression. The expression vectors containing the α subunit and the β subunit were used in a 1:1 ratio, the concentration of the expression vector was 0.8ug / ml, the transfection reagent was 1.2ul / ml, the transfection system was Opti-MEM, and the transfection reagent was One day before transfection, 1x 10 6 Prepare 500ml cell suspension at 100 cells / ml. Culture cells overnight at 37°C, 125rpm and 5% CO2. For transfection, take the corresponding volume of Add to a 50ml empty tube; In a second 50ml test tube, dilute the DNA plasmid to the desired concentration in serum-free medium to a final volume of 10% of the total volume. Gently mix evenly; Pour the diluted DNA into the pure Reagent, immediately mix the solution and incubate at room temperature for 10 minutes; Pour the transfection mixture into the cells and mix it thoroughly with the cell suspension. If necessary, add the transfection mixture at a final concentration of 0.5 μl / ml 2 hours after transfection. After 6 hours, the cells were replaced with serum-free medium and cultured. The cells were counted and monitored for 48 hours. When the cell viability was higher than 80%, the cells were fed at a rate of 5% and cultured. Otherwise, the cells were harvested and tested and purified.

[0246] Table 5

[0247] The nucleic acid molecule may further include a signal sequence or a leader sequence. As used herein, the term "signal sequence" refers to a fragment that guides the secretion of bioactive molecule drugs and fusion proteins and is cut off after translation in the host cell. The signal sequence of the present invention is a polynucleotide that encodes an amino acid sequence that initiates protein movement through the endoplasmic reticulum (ER) membrane. Useful signal sequences in the present invention include: a signal peptide of a human interleukin 12P35 subunit, a signal peptide of a human interleukin 12P40 subunit, a signal peptide of a mouse interleukin 12P35 subunit, and a signal peptide of a mouse interleukin 12P40 subunit.

[0248] The feature of signal peptide is well known in the art, and signal peptide generally has 16 to 30 amino acids, but they can comprise more or less number of amino acid residues. Conventional signal peptide is made up of three regions: alkaline N-terminal region, central hydrophobic region and more polar C-terminal region. Central hydrophobic region comprises 4 to 12 hydrophobic residues, which fix the signal sequence by membrane lipid bilayer during immature polypeptide translocation. After startup, the signal sequence is usually referred to as the cellular enzyme of signal peptidase in the lumen of ER and cuts off. Specifically, the signal sequence can be the secretion signal sequence of tissue plasminogen activator (tPa), the signal sequence of herpes simplex virus glycoprotein D (HSV gDs), or growth hormone. Preferably, the secretion signal sequence used in the higher eukaryotic cells including mammals etc. can be used. In addition, as secretion signal sequence, the signal sequence included in wild type hFSH, hLH, hTSH, hCG, mFSH, mLH, mTSH can be used, or it can be used after replacing the codon with high expression frequency in host cell.

[0249] Transiently expressed hCG protein was purified using a His affinity chromatography column, and purity and molecular weight were confirmed by SDS-PAGE. Figure 12 shows non-reduced and reduced SDS-PAGE images of purified hCG. Lanes 1 and 6 show the non-reduced and reduced results for native hCG, lanes 2 and 7 show the non-reduced and reduced results for hCG-A1P, lanes 3 and 8 show the non-reduced and reduced results for hCG-A1Q, and lanes 4 and 9 show the non-reduced and reduced results for hCG-A1N. All of these proteins are of high purity and suitable for PEG modification.

[0250] Example 7

[0251] This example provides the synthesis of a monodisperse 20KD monomethoxypolyethylene glycol propionaldehyde (mPEG-pALD-20K) site-specifically modified mutant human chorionic gonadotropin (hCG-A1P). The site-specific modification of polyethylene glycol to proline (P) is simpler than that to glutamine (Q) because proline can form a ring in its natural state without the need for enzyme catalysis. The purity of the site-specific modified product is also relatively high. Unlike glutamine, there is an incomplete cyclization catalyzed by glutamine cyclase, which results in some subunits that should be closed still being reacted. The specific preparation method is as follows:

[0252] (1) 5 mg of mutant human chorionic gonadotropin (hCG-A1P) was dissolved in 0.05 M acetic acid / sodium acetate buffer at pH 5.0 and stirred at 20°C to prepare an interleukin-12 solution with a final concentration of 1.0 mg / mL.

[0253] (2) 14.3 mg of monomethoxy polyethylene glycol propionaldehyde with a molecular weight of 20KD (purchased from Xiamen Sinobond Biotechnology Co., Ltd., model: 06020101412) was dissolved in pure water to prepare a monomethoxy polyethylene glycol propionaldehyde aqueous solution; then the solution was added dropwise to the interleukin 12 solution, wherein the molar ratio of the monomethoxy polyethylene glycol propionaldehyde to the mutant human chorionic gonadotropin (hCG-A1P) was 10:1.

[0254] (3) 0.135 mg of sodium cyanoborohydride (the molar ratio of sodium cyanoborohydride to interleukin-12 is 30:1) was dissolved in water to prepare a sodium cyanoborohydride aqueous solution, which was then added to the mutant human chorionic gonadotropin (hCG-A1P) solution and stirred at 20°C for 24 hours.

[0255] The reaction process of the preparation method of this embodiment is shown as follows:

[0256] The value of n is approximately 454.

[0257] In this example, by controlling the above reaction conditions, the selective site-specific coupling modification of mPEG-pALD-20KD on the α-amino group at the N-terminus of the β subunit of human chorionic gonadotropin hCG was achieved.

[0258] The purity of the purified mPEG-pALD-20KD-hCG-A1P was assessed by SDS-PAGE, and the electrophoretic pattern is shown in FIG13 . In FIG13 , the product purity was above 90%. Lanes 1 and 2 represent PEG-modified native hCG, and lanes 3 and 4 represent PEG-modified hCG-A1P. Comparison of the reducing and non-reducing SDS-PAGE gel images reveals that PEG can randomly modify both subunits of native hCG, but selectively modifies the β subunit of mPEG-pALD-20KD-hCG-A1P. This demonstrates that this method can achieve site-specific single modification of the hCG heterodimer by blocking the N-terminal α-amino group of the α subunit of the heterodimer, allowing the polyethylene glycol aldehyde derivative to react only with the N-terminal α-amino group of the β subunit.

[0259] Researchers in this field can simply extend this technology and use this method for site-specific polyethylene glycol modification of other heteromeric proteins or polypeptides.

Claims

1. A method for site-specific polyethylene glycol modification of a protein or polypeptide, comprising: a) providing a protein or polypeptide to be modified, wherein the protein or polypeptide to be modified comprises a target N-terminal α-amino group and a non-target N-terminal α-amino group, and performing mutation blocking on the non-target N-terminal α-amino group; b) subjecting the target N-terminal α-amino group to a site-directed modification reaction with an aldehyde or ketone derivative of polyethylene glycol to generate a polyethylene glycol site-directed modified protein or polypeptide.

2. The method according to claim 1, wherein The protein or polypeptide is a heteromeric protein or polypeptide; Preferably, the process of blocking the non-target N-terminal α-amino group by mutation includes: constructing a gene encoding the amino acid sequence of the mutation-blocked protein or polypeptide into a vector; Transform the vector into a host bacterium and extract the endotoxin-removing plasmid; Transfecting the plasmid into a host cell to express the target protein; wherein the host cell is preferably a 293F cell; The target product is harvested, i.e., the protein or peptide after the non-target N-terminal α-amino group is mutated and blocked.

3. The method according to claim 1 or 2, wherein: The protein or polypeptide is a heteromeric protein or polypeptide comprising 2-5 N-terminal α-amino groups; Preferably, the heteromeric protein or polypeptide comprises one target N-terminal α-amino group and 1-2 non-target N-terminal α-amino groups; Preferably, the two-subunit heteromeric protein or polypeptide is selected from one or more of antibodies, cytokines, cytokine receptors, cytokine fusion proteins, hormones, hemoglobin, and G protein-coupled receptors; Preferably, the cytokine is selected from one or more of interleukins, interferons, lymphotoxins, growth factors, and chemokines; Preferably, the antibody is selected from a monoclonal antibody, a polyclonal antibody, and an antibody fragment; the antibody fragment preferably includes the Fab segment of an antibody; Preferably, the hormone is selected from one or more of follicle-stimulating hormone, luteinizing hormone, luteinizing hormone-releasing hormone, thyroid-stimulating hormone, and chorionic gonadotropin; Preferably, the interleukin is one or more of interleukin 6, interleukin 12, interleukin 23, interleukin 27, and interleukin 35.

4. The method according to any one of claims 1 to 3, wherein: The process of blocking the non-target N-terminal α-amino group by mutation includes: By adding, reducing or replacing one or several amino acid residues, the non-target N-terminal first amino acid of the protein or polypeptide to be modified is mutated to one or more of proline, glutamine, glutamic acid, asparagine, aspartic acid or non-natural amino acids.

5. The method according to any one of claims 1 to 4, wherein: The aldehyde or ketone derivative of polyethylene glycol is selected from one or more of polyethylene glycol acetaldehyde derivatives, polyethylene glycol propionaldehyde derivatives and polyethylene glycol butyraldehyde derivatives; Furthermore, the polyethylene glycol propionaldehyde derivative is monomethoxy polyethylene glycol propionaldehyde and / or two-arm polyethylene glycol propionaldehyde.

6. A polyethylene glycol site-directed modification protein or polypeptide, wherein the non-PEG-modified N-terminus in the amino acid sequence of the protein or polypeptide is blocked by mutation compared to the wild-type protein or polypeptide; Preferably, the polyethylene glycol site-directed modification protein or polypeptide is prepared according to the method according to any one of claims 1-5.

7. The polyethylene glycol site-directed modification protein or polypeptide according to claim 6, wherein One end of the polyethylene glycol structure is connected to the target N-terminus of the protein or polypeptide for modification, and the other end of the polyethylene glycol structure is modified with an immunoconjugate or a targeting fragment.

8. The polyethylene glycol site-directed modification protein or polypeptide according to claim 6 or 7, having a molecular weight of 5 to 40 KD, preferably 10 to 30 KD.

9. A drug comprising the polyethylene glycol site-directed modification protein or polypeptide according to any one of claims 6 to 8, and further comprising a pharmaceutically acceptable excipient; Furthermore, the dosage form of the drug includes a liquid preparation and / or a lyophilized preparation; Furthermore, the administration routes of the drug include injection, oral administration, external application or a combination of multiple routes.

10. Use of the polyethylene glycol site-directed modification protein or polypeptide according to any one of claims 6 to 8 or the drug according to claim 9 in the preparation of anti-tumor drugs.

11. The use according to claim 10, wherein: The anti-tumor drug is administered to the patient alone or in combination with radiotherapy, chemotherapy, myeloablative therapy, CAR-T therapy, CAR-NK therapy, antibody targeted therapy, oncolytic virus therapy and / or gene vaccine therapy.

12. A protein or polypeptide comprising one or more of the following amino acid sequences: SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5; SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8; SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13; SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16; SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28; SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.

35.

13. The protein or polypeptide according to claim 12, which is selected from one or more of the following proteins or polypeptides: A mutant human interleukin-12, wherein the amino acid sequence of the p35 subunit is shown in any one of SEQ ID NOs. 3-5; A mutant human interleukin-12, wherein the amino acid sequence of the p40 subunit is shown in any one of SEQ ID NOs. 6-8; A mutant murine interleukin-12, the amino acid sequence of its p35 subunit is shown in any one of SEQ ID NOs. 11-13; A mutant murine interleukin-12, the p40 subunit amino acid sequence of which is shown in any one of SEQ ID NOs. 14-16; A mutant human luteinizing hormone, wherein the amino acid sequence of its α subunit is shown in any one of SEQ ID NOs. 26-28, and the amino acid sequence of its β subunit is shown in SEQ ID NO. 18; A mutant mouse luteinizing hormone, wherein the amino acid sequence of its α subunit is shown in any one of SEQ ID NOs. 33-35, and the amino acid sequence of its β subunit is shown in SEQ ID NO. 30; A mutant human thyroid-stimulating hormone, wherein the amino acid sequence of its α subunit is shown in any one of SEQ ID NOs. 26-28, and the amino acid sequence of its β subunit is shown in SEQ ID NO. 19; A mutant mouse thyroid-stimulating hormone, wherein the amino acid sequence of its α subunit is shown in any one of SEQ ID NOs. 33-35, and the amino acid sequence of its β subunit is shown in SEQ ID NO. 31; A mutant human follicle-stimulating hormone, wherein the amino acid sequence of its α subunit is shown in any one of SEQ ID NOs. 26-28, and the amino acid sequence of its β subunit is shown in SEQ ID NO. 20; A mutant mouse follicle-stimulating hormone, wherein the amino acid sequence of its α subunit is shown in any one of SEQ ID NOs. 33-35, and the amino acid sequence of its β subunit is shown in SEQ ID NO. 32; The mutated human chorionic gonadotropin, whose α subunit amino acid sequence is shown in any one of SEQ ID NOs. 26-28, and whose β subunit amino acid sequence is shown in any one of SEQ ID NOs. 21-25.

14. A nucleic acid molecule encoding the protein or polypeptide according to any one of claims 12-13.

15. The nucleic acid molecule according to claim 14, comprising one or more of the following nucleotide sequences: SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.40; SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.43; SEQ ID NO.46, SEQ ID NO.47, SEQ ID NO.48; SEQ ID NO.49, SEQ ID NO.50, SEQ ID NO.51; SEQ ID NO.61, SEQ ID NO.62, SEQ ID NO.63; SEQ ID NO.68, SEQ ID NO.69, SEQ ID NO.70.

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