CN111944008A

CN111944008A - Method for mutating protein and obtained mutant protein

Info

Publication number: CN111944008A
Application number: CN201910399622.9A
Authority: CN
Inventors: 胡辉
Original assignee: Shanghai Gaipu Biotechnology Co Ltd
Current assignee: Shanghai Gaipu Biotechnology Co Ltd
Priority date: 2019-05-14
Filing date: 2019-05-14
Publication date: 2020-11-17

Abstract

The present invention provides a method of altering the binding capacity of a protein to its binding partner and mutant proteins obtained by such a method. The method comprises the following steps: 1) increasing glycosylation sites in the protein; and 2) obtaining a mutant protein comprising a glycosylation modification at said increased glycosylation site. The mutant protein obtained by the method of the invention has the required biological activity while reducing the binding capacity with the binding partner. The mutant protein obtained by the method is structurally close to the natural protein, so that the immunogenicity is extremely low. The method of the invention has simple operation, easy expansion and convenient quality control.

Description

Method for mutating protein and obtained mutant protein

Technical Field

The present invention relates to the field of protein engineering. In particular, the present invention relates to novel methods of site-directed mutagenesis of proteins and the resulting mutant proteins that have altered binding capacity to a binding partner as compared to the original protein.

Background

Site-directed mutagenesis techniques are the substitution, insertion or deletion of specific nucleotides in a known DNA series to alter individual amino acid residues in the structure of an enzyme. Site-directed mutagenesis is the alteration of a particular nucleotide, and random mutagenesis is performed on a stretch of gene sequence most likely to affect the function and properties of the enzyme, thereby producing a series of mutated enzyme molecules. There are several methods for site-directed mutagenesis, such as oligonucleotide primer-mediated recombinant PCR site-directed mutagenesis, restriction endonuclease fragment substitution, cassette mutagenesis, and chemical total synthesis. Site-directed mutagenesis is an enzyme which purposefully changes a certain active group or module of the enzyme on the basis of the structure and function of the known enzyme so as to generate new properties, and is also called rational molecular design.

Protein Engineering (Protein Engineering) has been over 20 years old since the first time that humans have adapted site-directed mutagenesis to an active site of an enzyme of known structure and mechanism. This technology now enables the desired engineering of protein viability, specificity, stability and folding. Engineered proteins have been successfully used in the pharmaceutical industry and in the treatment of many significant diseases. The molecular design of protein engineering medicine is the characteristic of the third generation protein engineering medicine, the mutual relation between protein structure-function-activity is researched, the spatial structure of protein is designed, the structural rule of protein molecule and the relation with biological function are taken as the basis, the existing protein is directionally modified, designed and constructed through controlled gene modification and gene synthesis, and then the biomolecule with new medicine development value can be obtained through the application of high-flux screening technology, the development of processing technology and other strategies. On the basis of the design of novel proteins, polypeptides or other metabolic molecules, including vaccines, enzymes, antibodies, therapeutic peptides and other biomolecules, and finally the production of novel proteins which have better performance than the proteins existing in the nature and meet the requirements of human society. Essentially all modifications of the protein sequence and structure can be made using these methods to achieve the desired functional objectives.

It is well known in the art that the functions performed by proteins in vivo are often very complex. Therefore, in practical applications, it is more desirable to adjust the functions of a certain protein in different directions. That is, some functions or activities are desired to be down-regulated, but some functions or activities need to be maintained, even up-regulated.

In addition, current site-directed mutagenesis approaches can increase immunogenicity or adversely affect protein structural function. For example, IL-2 has a dual function in the immune response in that it not only mediates the expansion and activity of effector cells, but is also critically involved in the maintenance of peripheral immune tolerance. Thus, patients receiving high dose IL-2 treatment often experience severe cardiovascular, pulmonary, renal, hepatic, gastrointestinal, neurological, dermal, blood and systemic adverse events. Most of these side effects can be explained by the formation of the so-called vascular (or capillary) leak syndrome (VLS). Low dose IL-2 regimens have been tested in patients to avoid VLS, however, at the cost of reduced therapeutic outcome.

Several approaches have been taken to overcome the problems associated with IL-2 immunotherapy. For example, IL-2 is mutated to reduce its toxicity and/or to increase its efficacy. However, none of the known IL-2 mutants have been shown to overcome the above-mentioned problems associated with IL-2 immunotherapy, including toxicity caused by induced VLS, tumor tolerance caused by induced AICD and immunosuppression caused by activation of Timf cells. For example, Rochellicate corporation (CN103492411A) mutated amino acids in IL-2 at three positions F42A, Y45A, and L72G, reduced the affinity of the IL-2 protein for high affinity IL-2 receptors and retained the affinity of the mutant IL-2 protein for medium affinity IL-2 receptors, but also reduced the biological activity of IL-2. In addition, large-scale amino acid residue substitutions can also readily enhance the immunogenicity of the resulting protein mutants.

Thus, there is an urgent need in the art for new methods to alter the binding ability of a protein to its binding partner, while still maintaining the desired biological activity of the protein and without significantly enhancing the immunogenicity of the resulting protein mutants.

Disclosure of Invention

The object of the present invention is to provide a novel method of mutating a protein such that the binding capacity of the protein to its binding partner can be reduced, while at the same time the desired biological activity of the protein can be retained and the immunogenicity of the resulting protein mutant is not significantly enhanced.

Further, the invention provides a protein mutant obtained by the protein mutation method, glycosylation modification is added at the binding site, the binding site of the mutant protein and immune cells is shielded, and the functional structure of the mutant protein is hardly influenced. The mutant has altered binding ability to a binding partner compared to the wild-type protein, but retains the desired biological activity of the original protein, and has not significantly enhanced immunogenicity and an extended half-life.

In a first aspect, the present invention provides a method of altering the binding capacity of a protein to its binding partner, the method comprising the steps of:

1) increasing glycosylation sites in the protein; and

2) obtaining a mutant protein comprising a glycosylation modification at said increased glycosylation site.

In particular embodiments, the altering the binding capacity of a protein to its binding partner is decreasing the binding capacity of a protein to its binding partner.

In preferred embodiments, the protein comprises two or more binding sites for its ligand or receptor, and the binding at one site is eliminated or reduced, while the protein or polypeptide retains its particular biological activity.

In a preferred embodiment, said increasing of glycosylation sites of step 1) is the creation of glycosylation sites in the protein by site-directed mutagenesis of amino acid residues.

In preferred embodiments, the glycosylation site is an N-sugar site or an O-sugar site; n sugar sites are preferred.

In a preferred embodiment, the number of amino acid residues of the site-directed mutation is less than 10, preferably 1-8, 1-4, 1-2 amino acid residues; or the number of amino acid residues of the site-directed mutation is less than 10%, preferably less than 5%, preferably less than 3%, more preferably less than 2% compared to the number of amino acid residues of the protein before mutation. Most preferably 1 amino acid residue is mutated.

In a preferred embodiment, the obtaining of the mutant protein comprising a glycosylation modification at said increased glycosylation site of step 2) is by expressing a gene encoding the mutant protein in a host cell; or obtaining the amino acid sequence of the mutant protein through in vitro synthesis or prokaryotic host cell expression, and then carrying out glycosylation modification on glycosylation sites added in the mutant protein through in vitro chemical or biochemical enzyme catalysis.

In a preferred embodiment, the method further comprises the step of testing the resulting mutant protein for its binding ability to its binding partner and for the desired biological activity of the mutant protein.

In a preferred embodiment, the protein is an interleukin, an interferon, a tumor necrosis factor superfamily, a colony stimulating factor, a chemokine, a growth factor, or the like, preferably IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, IL-17, IL-23, a growth hormone; more preferably IL-2, IL-15, human growth hormone.

In a preferred embodiment, the method results in a mutant protein having 50% less, more preferably 60% less, more preferably 70% less, more preferably 80% less, more preferably 90% less, more preferably 95% less binding capacity to its binding partner than the wild-type protein, and most preferably the method results in a mutant protein that is not bound at all to its binding partner.

In a preferred embodiment, the method results in a mutant protein that is less immunogenic than mutant proteins obtained by traditional amino acid point mutation.

In a second aspect, the present invention provides a mutant protein produced by the method of the first aspect.

In a preferred embodiment, the method results in a mutant protein having a 50% reduced binding capacity to its binding partner, more preferably a 60% reduced binding capacity, more preferably a 70% reduced binding capacity, more preferably a 80% reduced binding capacity, more preferably a 90% reduced binding capacity, more preferably a 95% reduced binding capacity, compared to the wild-type protein, and most preferably the method results in a mutant protein that is not bound at all to its binding partner.

In a preferred embodiment, the in vivo half-life of the mutant protein is 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, 4 times or more, 4.5 times or more, 5 times or more, 5.5 times or more, 6 times or more, 6.5 times or more, 7 times or more, 7.5 times or more, 8 times or more, 8.5 times or more, 9 times or more, 9.5 times or more, 10 times or more, 11 times or more, 12 times or more, 13 times or more, 14 times or more, 15 times or more, 16 times or more, 17 times or more, 18 times or more, 19 times or more, 20 times or more of the corresponding unmutated protein.

In a third aspect, the present invention provides a polynucleotide encoding the mutant protein of the second aspect.

In a fourth aspect, the present invention provides an expression vector comprising the polynucleotide of the third aspect.

In a fifth aspect, the present invention provides a host cell comprising the expression vector of the fourth aspect or having integrated in its genome the polynucleotide of the third aspect.

In a preferred embodiment, the host cell is a eukaryotic cell; preferably yeast, insect cells, animal cells; more preferably animal cells; most preferably mammalian cells, including but not limited to CHO cells, 293 cells, SP/20 cells, NS0 cells; among them, CHO cells are most preferable.

In a sixth aspect, the present invention provides a pharmaceutical composition comprising the mutant protein of the second aspect and optionally a pharmaceutically acceptable excipient.

In a seventh aspect, the present invention provides a method of producing the mutant protein of the second aspect, the method comprising the steps of:

1) culturing the host cell of the fifth aspect under conditions suitable for expression of the mutant protein; and

2) optionally isolating and purifying the mutant protein obtained in step 1);

or

1) Obtaining the amino acid sequence of the mutant protein;

2) (ii) glycosylation modification by in vitro chemical or biochemical enzyme catalysis of increased glycosylation sites in the mutant protein; and

3) optionally isolating and purifying the mutant protein obtained in step 2).

In a preferred embodiment, the amino acid sequence of the mutant protein is obtained by in vitro synthesis or expression in a prokaryotic host cell.

In an eighth aspect, the present invention provides the use of a mutant protein according to the second aspect in the manufacture of a medicament.

In a preferred embodiment, the drug may be one that is used for the same purpose as the unmutated protoprotein; or a drug for a different purpose from the original protein.

In a ninth aspect, the present invention provides a method of treatment comprising the step of administering to a patient in need of treatment of a disease a mutant protein according to the second aspect or a pharmaceutical composition according to the sixth aspect.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the binding of IL-2 mutants to CD25 using an enzyme linked immunosorbent assay;

FIG. 2 shows the cell proliferation of CTLL-2 in response to rhIL-2 and mutant interleukin-2;

FIG. 3 shows NK92 cell proliferation in response to rhIL-2 and mutant interleukin-2;

FIGS. 4a-j show the sequences SEQ ID NO 1-10, respectively.

Detailed Description

The inventors have made extensive and intensive studies and have unexpectedly found that the binding ability between a protein or polypeptide and its binding partner can be changed by performing glycosylation modification in the protein or polypeptide, and that the desired biological activity of the resulting mutant protein or polypeptide can be retained. The present invention has been completed based on this finding.

Site-directed mutagenesis techniques

Site-directed mutagenesis is a protein engineering technique that substitutes, inserts or deletes specific nucleotides in a known DNA sequence based on the structure and function of a known protein, thereby generating a mutein molecule with novel properties.

The positioning mutation technology can change the physicochemical property of the protein, for example, improve the stability of protein drugs; enhancing the solubility of the protein drug; improving biological properties, including but not limited to altering the specificity of an enzyme for a substrate, increasing the activity of an enzyme and improving affinity, specificity, and the like.

Site-directed mutagenesis techniques may be used to enhance or eliminate the binding activity of a ligand to a receptor, enzyme, and substrate by mutating amino acids in the binding domain, which results in a change in properties such as secondary structure and charge. If the mutated amino acid is located at the critical antigen-antibody site, the amino acid change will probably cause the change of the charge and secondary structure of the site, and the ligand and receptor, enzyme and substrate can not be combined to achieve the mutation. At the same time, the original antibody can not recognize the site, and a new antigen is formed. This is a possible occurrence of such conventional mutagenesis methods.

Glycosylation modification of proteins

Protein glycosylation is a complex post-translational modification process, and glycosylation modifications are performed at specific sites of a protein, usually at asparagine residues (N-link) or serine/threonine residues (O-link), N-linked glycosylation modifications usually occur at Asn-X-Ser/Thr (X is a non-proline amino acid), O-linked glycosylation modifications usually occur at serine (Ser) or threonine (Thr) residues, and O-glycosidic bonds are formed by the hydroxyl groups of N-acetylgalactosamine (Gal-NAc) and Ser/Thr.

In addition to the classical N-glycosylation sequence Asn-X-Ser/Thr, non-classical N-glycosylation sequences also exist. For example, the Asn-X-Cys sequence is the first recognized non-classical glycosylation sequence. In addition, in addition to the classical tripartite sequence and the Asn-X-Cys sequence described above, several new glycosylation sites have been increasingly reported.

Thus, based on the teachings of the present invention and the common general knowledge in the art and subsequent discovery of glycosylation sequences, one skilled in the art can generate glycosylation sites in the protein or polypeptide to be engineered by site-directed mutagenesis to modify glycosylation in the protein or polypeptide. These mutant proteins obtained using the newly discovered glycosylation sites should also fall within the scope of the present invention.

Methods of the invention and resulting mutant proteins

The method of the invention obtains mutant protein containing glycosylation modification at the increased glycosylation sites by increasing the glycosylation sites in the protein or polypeptide, thereby changing the binding capacity of the protein and the binding partner thereof. In a particular embodiment, the mutant protein obtained by the method of the invention has reduced binding capacity to its binding partner.

Herein, "protein and its binding partner" refers to a protein and a substance capable of binding thereto, such as, but not limited to, receptors and ligands, enzymes and substrates thereof.

In the method of the present invention, the binding ability between the protein or peptide chain and the substrate or to other proteins or polypeptides is altered by generating N-glycosylation or O-glycosylation by amino acid mutation in the protein or peptide chain. The invention may be used to reduce the binding activity between ligand and receptor, enzyme and substrate by at least 10%, preferably by at least 30%, 50%, 70% to 100% compared to unmutated protein or polypeptide; most preferably 70% to 100%.

One skilled in the art can determine the newly added N-glycosylation site or O-glycosylation site required by the present invention according to the protein to be modified; preferably the N-sugar site, single site mutation to Asparagine (ASN). In a specific embodiment, the number of mutated amino acid residues is less than 10, preferably 1-8, 1-6, 1-4, 1-2 amino acid residues, most preferably 1 amino acid residue is mutated; alternatively, the number of amino acid residues of the site-directed mutation is less than 10%, preferably less than 5%, preferably less than 3%, more preferably less than 2% compared to the number of amino acid residues of the protein prior to mutation.

The mutant protein or polypeptide of the present invention is preferably expressed in eukaryotic cells and obtained by cell culture. Yeast, insect cells, animal cells can be selected, and transgenic animals can also be selected. In particular embodiments, the host cell is a eukaryotic cell; preferably yeast, insect cells, animal cells; more preferably animal cells; mammalian cells are most preferred, including but not limited to CHO cells, 293 cells, SP/20 cells, NS0 cells, with CHO cells being most preferred.

In other embodiments, proteins may also be obtained using in vitro synthesis or fermentation of prokaryotic bacterial expression, followed by in vitro enzymatic catalysis, etc. to achieve similar results. In vivo and in vitro modification protocols can achieve the same goal, i.e., site-directed protein glycosylation.

The method of the invention introduces a glycosylation mutation site to form a section of sugar chain, and utilizes the steric hindrance of the sugar chain to cause the abnormal combination between the ligand and the receptor and between the enzyme and the substrate, thereby reducing or eliminating the related biological activity. In the traditional site-directed mutagenesis technology, amino acids with larger differences with the original amino acid characteristics are generally selected, so that the secondary structure is changed maximally by utilizing the least mutations, and the aim of reducing or eliminating the related biological activity is fulfilled. However, as described above, such conventional methods inevitably result in an increase in the immunogenicity of the resulting mutant protein. In the method of the present invention, the mutation for newly adding a sugar chain can select and utilize the original amino acid sequence without requiring a large number of site mutations.

In light of the teachings of the present invention, one skilled in the art will appreciate that mutant proteins of the present invention have glycosylation modifications not possessed by the wild-type protein, i.e., the glycosylation pattern of the mutant protein is different from that of the wild-type protein. The glycosylation modification can be realized by expressing the coding sequence of the mutant protein with the glycosylation sites increased in a host cell, or the mutant protein can be obtained by in vitro synthesis, so as to realize glycosylation in vitro. It will also be understood by those skilled in the art that the host cell may be a eukaryotic cell, including but not limited to yeast cells, insect cells, mammalian cells, and the like. The glycosylation pattern of mutant proteins obtained from different host cells may differ. For example, when yeast cells or insect cells are used as host cells, the glycoform of the resulting mutant protein may be of non-human origin. Those skilled in the art will recognize that non-human glycoforms can be further modified into human glycoforms.

Based on the teachings of the present invention and prior art approaches to glycosylation modification, one skilled in the art can design glycosylation sites in proteins having binding partners to obtain glycosylation modified mutant proteins and test their binding activity to the original binding partner as well as other biological activities.

After obtaining the mutant proteins of the present invention, one skilled in the art can also examine the binding ability of the resulting mutant proteins to their binding partners and the desired biological activity of the mutant proteins.

The methods of the invention may be applied to a variety of proteins having a binding partner, including but not limited to interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemokines, growth factors, and the like, preferably IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, IL-17, IL-23, growth hormone; more preferably IL-2, IL-15, human growth hormone.

The mutant proteins obtained by the method of the invention have significantly reduced binding capacity to their binding partners compared to the wild-type protein, most preferably the mutant proteins obtained by the method do not bind to their binding partners at all; in addition to a significant reduction in binding capacity to its binding partner, the mutant proteins of the invention can also possess a desired biological activity, for example, from 50% to 130% of the desired biological activity; the mutant proteins of the invention also have significantly increased in vivo half-lives, e.g., 1.5-fold or more to 20-fold or more, over the unmutated original protein.

Pharmaceutical compositions of the invention and their administration

On the basis of the mutant protein, the invention also provides a pharmaceutical composition. In a specific embodiment, the pharmaceutical composition of the invention comprises the mutant protein of the invention and optionally a pharmaceutically acceptable excipient.

The person skilled in the art can decide on the particular adjuvants to be used in a particular situation.

Optionally, the composition of the present invention further comprises a pharmaceutically acceptable excipient. If desired, pharmaceutically acceptable excipients may be added to the mutant polypeptides, fusion proteins or conjugates of the invention to form a composition.

Exemplary excipients include, but are not limited to, those selected from the group consisting of: sugars, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, amino acids, and combinations thereof. Sugars, such as sugars, derivatised sugars, may be present as excipients. Specific sugar excipients include, for example: monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, etc.; disaccharides such as lactose, sucrose, trehalose, and the like; polysaccharides such as dextran, starch, and the like; and sugar alcohols such as mannitol, xylitol, maltitol, sorbitol, inositol, cyclodextrin and the like.

Excipients may also include inorganic salts or buffers such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and combinations thereof.

The composition may also include an antimicrobial agent for preventing or suppressing the growth of microorganisms. Non-limiting examples of antimicrobial agents suitable for one or more embodiments of the present invention include benzalkonium chloride, benzyl alcohol, phenol, phenethyl alcohol, and combinations thereof.

Antioxidants may also be present in the composition. Antioxidants are used to prevent oxidation and thus prevent deterioration of the protein, conjugate or other components of the formulation. Antioxidants suitable for use in one or more embodiments of the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, and combinations thereof.

The surfactant may be present as an excipient. Exemplary surfactants include: polysorbates, such as "tween 20" and "tween 80", and pluronics, such as F68 and F88; sorbitan esters; lipids, such as phospholipids (e.g., lecithin and other phosphatidylcholines), fatty acids, and fatty esters; steroids, such as cholesterol.

The acid or base may be present in the composition as an excipient. Non-limiting examples of acids that may be used include those selected from the group consisting of: hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, phosphoric acid, and combinations thereof. Examples of suitable bases include, but are not limited to, bases selected from the group consisting of: sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, and combinations thereof.

One or more amino acids that may be present in the composition as excipients are described herein. In this regard, exemplary amino acids include arginine, lysine, and glycine.

The amount of conjugate (i.e., the conjugate formed between the active agent and the polymeric agent) in the composition will vary depending on a number of factors, but the optimal amount of the composition when stored in a unit dose container (e.g., a vial) will be a therapeutically effective dose. In addition, the pharmaceutical preparation may be housed in one syringe. A therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the drug in order to determine which amount produces the clinically desirable endpoint.

The amount of any individual excipient in the composition will vary depending on the activity of the excipient and the particular needs of the composition. Typically, the optimum amount of any individual excipient is determined by routine experimentation, i.e., by preparing compositions containing varying amounts of excipient (ranging from low to high), examining stability and other parameters, and then determining the range at which optimum performance is obtained without significant adverse effects.

Generally, however, the excipient will be present in the composition in an amount of from about 1% to about 99%, preferably from about 5% to about 98%, more preferably from about 15 to about 95%, by weight of the excipient, and most preferably at a concentration of less than 30% by weight.

These compositions encompass all types of formulations and those particularly suitable for injection, such as powders or lyophilizates and liquid preparations which can be reconstituted. Examples of diluents suitable for reconstituting the solid composition prior to injection include bacteriostatic water for injection, 5% dextrose in water, phosphate buffered saline, sterile water, deionized water, and combinations thereof. In the case of liquid pharmaceutical compositions, solutions and suspensions are envisaged.

The compositions of one or more embodiments of the present invention are typically (though not necessarily) administered by injection, and thus are generally liquid solutions or suspensions immediately prior to administration. The pharmaceutical preparations may also take other forms such as syrups, creams, ointments, tablets, powders, and the like. Other modes of administration are also included, such as pulmonary, rectal, transdermal, transmucosal, oral, intrathecal, intratumoral, peritumoral, intraperitoneal, subcutaneous, intraarterial, and the like.

The invention also provides a method for administering a therapeutically effective dose to a patient for treating a responsive condition. The substance may be injected (e.g., intramuscularly, subcutaneously, and parenterally). Types of formulations suitable for parenteral administration include, inter alia, ready-to-inject solutions, dry powders combined with a solvent prior to use, ready-to-inject suspensions, dry insoluble compositions combined with a vehicle prior to use, and emulsions and liquid concentrates diluted prior to administration.

Uses and methods of use of the mutant proteins of the invention

As described above, the mutant proteins of the present invention have reduced binding ability to their binding partners, do not have significantly increased immunogenicity, and are capable of possessing desirable biological activities. Therefore, the mutant protein of the invention can be used for preparing corresponding medicaments. It will be appreciated by those skilled in the art that the agent may be one for the same use as the unmutated native protein or may be one for a different use than the native protein.

Administration of the mutant protein or pharmaceutical composition or medicament of the invention to a patient may treat the corresponding disease.

The invention has the advantages that:

1. the invention opens up a new technical means for the mutant protein to change the binding capacity of the protein and the binding partner thereof;

2. the mutant protein or polypeptide obtained by the invention comprises two or more binding sites with the ligand or the receptor thereof, the binding effect of one site is eliminated or weakened, and the protein or polypeptide can still retain the specific biological activity;

3. the mutant protein obtained by the invention shields the binding site of the mutant protein and immune cells through glycosylation modification at the binding site, and hardly influences the functional structure of the mutant protein, thereby having extremely low immunogenicity;

4. because the mutant protein obtained by the invention can not completely eliminate the protein in vivo and only generates competitive inhibition, the therapeutic application of the mutant protein obtained by the invention is better than that of an antibody;

5. the molecular design method of the invention is simple and is convenient for application on different molecules;

6. the invention has simple operation, easy expansion and convenient quality control; and

7. the half-life period of the mutant protein obtained by the invention in vivo is obviously prolonged.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.

Examples

Example 1 Synthesis of mutant Interleukin-2 (IL-2) proteins

1. Gene synthesis

The nucleotide sequence encoding the amino acid sequence of a mutant of interleukin-2 (IL-2) protein was obtained by an automated gene synthesis method. In some embodiments, the HIS tag is added at the end of the gene fragment to facilitate purification; in some examples, IgG1-Fc was added to the ends of the gene fragments to facilitate purification. The gene fragment is flanked by single restriction enzyme cleavage sites. All gene synthesis sequences are designed with a 5' DNA sequence encoding a leader peptide that targets secretion of the protein in eukaryotic cells.

Number of mutations	Mutation site	Example mutant names	Protein tag	Sequence of
					2	3、39	IL-2gm1(T3A,M39N)	HIS	SEQ ID NO:1
2	3、49	IL-2gm2(T3A,K49N)	HIS	SEQ ID NO:2
					2	3、73	IL-2gm3(T3A,A73T)	HIS	SEQ ID NO:3
2	3、73	IL-2gm3(T3A,A73S)	HIS	SEQ ID NO:4
					2	3、109	IL-2gm7(T3A,D109N)	HIS	SEQ ID NO:5

2. Plasmid construction

The synthesized gene was subcloned into pcDNA3.4 plasmid using molecular biology reagents according to the manufacturer's instructions.

3. Expression of mutant Interleukin-2 (IL-2) proteins

Transfection of plasmids was performed using Expi293F cells (Thermo Fisher Scientific) at 37 ℃ with 8% CO₂The cultivation is carried out on a shaker (VWR Scientific) and the day before transfection is inoculated in shake Flasks (Corning Erlenmeyer flashes) according to the manufacturer's instructions.

Cell supernatant suspensions from day two, day four and day five were collected for western blot experiments.

Example 2 expression of CD25 protein

Gene synthesis

The nucleotide sequence encoding the amino acid sequence of the CD25 protein (SEQ ID NO:8) was obtained by an automated gene synthesis method. SEQ ID NO 9 shows the amino acid sequence of the linker (GGGSGGGSGGGSGGGS). In some examples, the gene fragment was co-expressed with IgG1-Fc via a linker to facilitate purification. The gene fragment is flanked by single restriction enzyme cleavage sites. All gene synthesis sequences are designed with a 5' DNA sequence encoding a leader peptide that targets secretion of the protein in eukaryotic cells. Exemplary leader peptide sequences are given in SEQ ID NO 10. The synthesized gene was subcloned into pcDNA3.4 plasmid using molecular biology reagents according to the manufacturer's instructions.

The plasmid transfection was carried out using Expi293F cells (Thermo Fisher Scientific), cultured at 37 ℃ in a shaker (VWR Scientific) at 8% CO2, inoculated in shake Flasks (Corning Erlenmeyer flashes) the day before transfection and the transfection procedure was carried out according to the manufacturer's instructions.

Example 3 affinity assays for the detection of binding to CD25 Using ELISA, Fortebio or biacore

The inventor utilizes an enzyme-linked immunosorbent assay to detect the binding force of the IL-2 mutant and the CD 25.

CD25 (from example 2) was coated into 96-well high adsorption microplates (3590, Costar), washed and blocked. The sample to be tested is diluted to the appropriate concentration and added to the well. TMB was developed and the signal was read in each well using a microplate reader (M5, Molerlder Devices) at a wavelength of 450/650 nm. The rhIL-2 is recombinant human interleukin-2 (Quanqi) for injection.

TABLE 1 summary of binding activity of rhIL-2 to IL-2 mutants and CD25

Concentration (ug/ml)	1	0.2	0.04	0
					IL-2gm1	0.013	0.011	0.011	0.011
IL-2gm2	0.459	0.317	0.11	0.012
					IL-2gm3	0.153	0.057	0.015	0.01
IL-2gm7	0.12	0.03	0.012	0.01
					rhIL-2	1.481	1.014	0.661	0.01

Note: IL-2gm concentration point binding activity was compared to rhIL-2 (binding activity 100%).

The results are shown in FIG. 1. As can be seen from FIG. 1, at the experimental concentrations, it is evident that rhIL-2 is bound and that the binding activity is compared with the magnitude of the dose-dependent binding: rhIL-2> IL-2gm2> IL-2gm3> IL-2gm7> IL-2gm 1. At these concentrations, there was no dose-dependence and no binding could be judged, including IL-2gm 1. It was also demonstrated that IL-2gm2, IL-2gm3 and IL-2gm7 lost some of their ability to bind CD 25.

Example 4 cell proliferation assay Using CTLL2 cells

In this example, the inventors evaluated the activity of rhIL-2 and the mutant interleukin-2 of example 1 in a cell proliferation assay using CTLL2 cells.

The same number of CTLL-2 cells (mouse cytotoxic T lymphocyte cell line, IL-2 dependent, surface highly expressed CD 25.) were inoculated into the assay plates, then rhIL-2 and IL-2 mutants were added according to concentration gradients, and after 48 hours of incubation, cell Titer Glo Luminescent buffer was added. The effect of different concentrations of rhIL-2 and IL-2 mutants on cell proliferation was determined by measuring the amount of ATP in the cells by chemiluminescence (SpectraMaxM5) and the number of cells in each well. Data were analyzed using GraphPad Prism7 software and curves were fitted with Nonlinear regression. EC for cell proliferation derived from non-linear regression analysis of dose-response curves₅₀Value (concentration of test compound required to exhibit 50% of maximum response).

The results are shown in FIG. 2. Table 2 below summarizes the proliferation of CTLL-2 cells in response to rhIL-2 and mutant interleukin-2.

TABLE 2 summary of CTLL-2 cell proliferation in response to rhIL-2 and mutant interleukin-2

Test sample	EC₅₀(nM)	Potency (%). relative to rhIL-2
			rhIL-2	2.90E-03	100％
IL-2gm1-His	8.34E+01	0.003％
			IL-2gm2-His	6.38E-02	4.547％
IL-2gm3-His	1.65E-01	1.763％
			IL-2gm7-His	3.65E-01	0.794％

The inventors measured the activity of rhIL-2 and mutant interleukin-2 using a cell proliferation assay, and a summary of the results is shown in Table 1. All tested items induced CTLL-2 cell growth in a dose-dependent manner. In the case of comparable cell proliferation fold, EC₅₀The greater the activity of CTLL2, the less stimulating it to grow. This change is due to CD25 binding affected by its mutant protein, which retains the activation of IL-2R signaling by IL-2R β γ heterodimer, and thus the cells are efficiently expanded after increasing concentrations. All IL-2 mutants had a potency of no more than 4.547% relative to rhIL-2. It was demonstrated that IL-2gm (1, 2, 3, 7) can abolish binding to CD25 on the cell surface of CTLL2, withoutThe formation of IL-2R α β γ heterotrimers results in a reduction in stimulatory effects. Preferably, IL-2gm1, IL-2gm3, IL-2gm7 are reduced by more than 50-fold relative to rhIL-2. Of these, IL-2gm1 and IL-2gm7 are most pronounced.

Example 5 cell proliferation assay Using NK92 cells

The present inventors evaluated the activity of rhIL-2 and the mutant interleukin-2 of example 1 in a cell proliferation assay using NK92 cells.

The same amount of NK92 cells (NK-92 cells are an IL-2 dependent NK cell line derived from peripheral blood mononuclear cells of a 50 year old white male with aggressive non-Hodgkin lymphoma, the surface part of which expresses CD 25.) were inoculated into the plates, and then rhIL-2 and IL-2 mutants were added according to a concentration gradient, and after incubation for 72 hours, cell Titer Glo luminescence buffer was added. The effect of different concentrations of rhIL-2 and IL-2 mutants on cell proliferation was determined by measuring the amount of ATP in the cells by chemiluminescence (SpectraMaxM5) and the number of cells in each well. Data were analyzed using GraphPad Prism7 software and curves were fitted with Nonlinear regression. EC for cell proliferation derived from non-linear regression analysis of dose-response curves₅₀Value (concentration of test compound required to exhibit 50% of maximum response).

The results are shown in FIG. 3. Table 3 below summarizes the proliferation of NK92 cells in response to rhIL-2 and mutant interleukin-2.

TABLE 3 overview of NK92 cell proliferation in response to rhIL-2 and mutant interleukin-2

Test sample	EC₅₀(nM)	Potency (%). relative to rhIL-2
			rhIL-2	1.93E-03	100
IL-2gm1-His	3.85E+01	0.005％
			IL-2gm2-His	1.12E-01	1.721％
IL-2gm3-His	2.21E+00	0.088％
			IL-2gm7-His	5.57E-01	0.347％

The activity of rhIL-2 and mutant interleukin-2 was measured using a cell proliferation assay, and a summary of the results is shown in Table 2. All tested items induced NK92 cell growth in a dose-dependent manner. In the case of comparable cell proliferation fold, EC₅₀The larger, the less activity was demonstrated to stimulate the growth of NK 92. All IL-2 mutants had a potency of no more than 1.721% relative to rhIL-2. IL-2gm (1, 2, 3, 7) was shown to abolish binding to CD25 on the cell surface of CTLL2, and no IL-2R α β γ heterotrimer was formed resulting in reduced stimulatory effects.

Example 6 Synthesis and assay of mutants of human growth hormone protein

The nucleotide sequence of the amino acid sequence of the mutant (called HGHgm1 and HGHgm2 for short) of the recombinant human growth hormone protein is obtained by an automatic gene synthesis method. In some examples, the purification is facilitated by adding HIS tags to the ends of the gene fragments. The gene fragment is flanked by single restriction enzyme cleavage sites. All gene synthesis sequences are designed with a 5' DNA sequence encoding a leader peptide that targets secretion of the protein in eukaryotic cells.

Transfection of plasmids was performed using Expi293F cells (Thermo Fisher Scientific) at 37 ℃ with 8% CO₂The cultivation is carried out on a shaker (VWR Scientific) and the day before transfection is inoculated in shake Flasks (Corning Erlenmeyer flashes) according to the manufacturer's instructions. After 5 days of culture, cells were harvested and culture supernatants were purified for detection.

And detecting the binding force of the HGH mutant and the GHR by using an enzyme-linked immunosorbent assay.

GHR (mouse Growth Hormone Receptor, 50043-M08H-50, Sinobiological) was coated in 96-well high adsorption microwell plates (3590, Costar), washed and blocked. The sample to be tested is diluted to the appropriate concentration and added to the well. TMB was developed and the microplate reader (M5, Molerlder Devices) wavelength was 450/650nm and the signal value was read for each well. HGH is GH, Human (Z03012-50, Gen).

TABLE 4 summary of HGH mutants and GHR binding Activity

Concentration (ug/ml)	1	0.2	0.04	0
					HGHgm1	0.549	0.217	0.11	0.012
HGHgm2	0.011	0.01	0.01	0.014
					HGH	1.881	1.214	0.661	0.01

Note: the binding activity at each concentration point of HGH was controlled by HGH (binding activity 100%).

As can be seen from the table, it is evident that HGH is bound at the experimental concentrations and that the binding activity of hghghghghghghm 1 is weakly dose-dependent. At these concentrations, hghghghghghghghgm 2 had no dose-dependence and we could be judged as no binding. It can be judged that hghghghghghghm 2 has lost its ability to bind GHR. The HGH mutant obtained by this method may have reduced or eliminated binding capacity to its binding partner GHR.

Example 7 half-life Studies of mutant proteins of the invention

The present inventors further studied the in vivo half-life of the mutant proteins obtained by the method of the present invention, and found that the in vivo half-life of the mutant proteins obtained by the method of the present invention was increased to various degrees.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shanghai Gepu Biotechnology Ltd

<120> method for mutating protein and resulting mutant protein

<130> P2019-0279

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Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

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Asn Pro Lys Leu Thr Arg Asn Leu Thr Phe Lys Phe Tyr Met Pro Lys

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Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

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Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

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Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

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Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

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Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile

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Ile Ser Thr Leu Thr His His His His His His

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Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

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Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

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Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

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Asn Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

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Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

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Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

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Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

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Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile

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Ile Ser Thr Leu Thr His His His His His His

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Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

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Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

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Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

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Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

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Pro Leu Glu Glu Val Leu Asn Leu Thr Gln Ser Lys Asn Phe His Leu

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Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

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Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

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Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile

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Ile Ser Thr Leu Thr His His His His His His

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Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

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Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

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Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

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Pro Leu Glu Glu Val Leu Asn Leu Ser Gln Ser Lys Asn Phe His Leu

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Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

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Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

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Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr His His His His His His

130 135

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Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

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Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asn Glu Thr Ala

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Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile

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Ile Ser Thr Leu Thr His His His His His His

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Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg

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Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu

20 25 30

Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro

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Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg

50 55 60

Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu

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Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val

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Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp

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Leu Leu Lys Asp Leu Glu Glu Gly Asn Gln Thr Leu Met Gly Arg Leu

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Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser

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Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr

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Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe

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Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe His

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His His His His His

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Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg

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Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu

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Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro

35 40 45

Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg

50 55 60

Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu

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Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val

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Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp

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Leu Leu Lys Asp Leu Glu Asn Gly Thr Gln Thr Leu Met Gly Arg Leu

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Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser

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Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr

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Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe

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Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe His

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His His His His His

195

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Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys

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Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg

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Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly

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Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser

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Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln

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Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp

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Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn

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Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr

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Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu

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Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln

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Leu Ile Cys Thr Gly Glu Met Glu Thr Ser Gln Phe Pro Gly Glu Glu

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Lys Pro Gln Ala Ser Pro Glu Gly Arg Pro Glu Ser Glu Thr Ser Cys

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Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

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Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

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Claims

1. A method of altering the binding ability of a protein to its binding partner, the method comprising the steps of:

1) increasing glycosylation sites in the protein; and

2. The method of claim 1, wherein the altering the binding capacity of the protein to its binding partner is decreasing the binding capacity of the protein to its binding partner.

3. A mutant protein produced by the method of claim 1.

4. A polynucleotide encoding the mutant protein of claim 2.

5. An expression vector comprising the polynucleotide of claim 2.

6. A host cell comprising the expression vector of claim 5 or having integrated in its genome the polynucleotide of claim 4.

7. A pharmaceutical composition comprising the mutant protein of claim 2 and optionally a pharmaceutically acceptable excipient.

8. A method of making the mutant protein of claim 3, the method comprising the steps of:

1) culturing the host cell of claim 6 under conditions suitable for expression of the mutant protein; and

2) optionally isolating and purifying the mutant protein obtained in step 1);

or

1) Obtaining the amino acid sequence of the mutant protein;

3) optionally isolating and purifying the mutant protein obtained in step 2).

9. Use of the mutant protein of claim 3 in the manufacture of a medicament.

10. A method of treatment comprising the step of administering the mutant protein of claim 2 or the pharmaceutical composition of claim 7 to a patient in need of treatment for a disease.