WO2021228253A1 - Mutant protein for proliferating regulatory t cell - Google Patents

Abstract

Disclosed are an IL-2 mutant, a fusion protein or conjugate comprising the IL-2 mutant, and a pharmaceutical composition comprising the IL-2 mutant, fusion protein or conjugate. Compared with wild-type IL-2, the IL-2 mutant has reduced binding to IL-2Rβγ dimer, retains the biological activity thereof, and preferentially stimulates the proliferation of regulatory T cells. The IL-2 mutant can be used for treating autoimmune diseases, and does not have various side effects caused by immunotherapy by means of using natural IL-2.

Description

A mutant protein of proliferation regulatory T cells Technical field

The present invention relates to the field of protein engineering. Specifically, the present invention relates to a novel interleukin-2 (IL-2) mutant and its preparation method. Compared with wild-type IL-2, the interleukin-2 (IL-2) mutant and its binding partner, IL The binding ability of -2 receptor β subunit and IL-2 receptor γ subunit is decreased, and the corresponding biological activity is maintained, which can better stimulate the proliferation of regulatory T cells.

Background technique

IL-2 is a member of the interleukin family produced by activated T cells. It can stimulate the proliferation, development and differentiation of T cells by binding to interleukin 2 receptors on the cell surface. IL-2 receptor IL-2R is composed of three subunits: α, β and γ chains. According to its affinity for IL-2, IL-2R can be divided into high affinity receptors (αβγ chain complex), medium affinity receptors (βγ chain complex), low affinity receptors (only α chain or αγ chain complex) ) And pseudo-high affinity receptors (αβ chain complex) 4 types. IL-2 can trigger intracellular signal transduction only after binding to high-affinity receptors or medium-affinity receptors.

Different types of T cells have different sensitivity to IL-2, and regulatory T cells (Treg cells) are more sensitive to IL-2 than other cells. Because IL-2Rα can be continuously expressed on the surface of Treg cells for a long time, Treg cells are more sensitive to IL-2 than NK, Teff and other cells when the body is not stimulated by foreign antigens. Using the dose difference of IL-2 to selectively affect Treg and Teff, can regulate the immune system.

Currently, low-dose IL-2 therapy has been applied to type 1 diabetes (T1D), systemic lupus erythematosus (SLE), chronic graft-versus-host disease (GVHD) And other autoimmune diseases. However, IL-2 has short in vivo half-life, poor stability, short injection treatment window period, and difficult to grasp the safe dose and treatment course for immunosuppression and inflammation at the administration site.

Regulatory T cells (Treg) can express the transcription factor FOXP3 intracellularly, so they can be distinguished from effector T cells by CD4+CD25+FOXP3+. Defects and mutations in the FOXP3 gene lead to the destruction of self-tolerance and the formation of autoimmune diseases due to the defect or lack of function of Treg. IL-2 can stimulate T cell proliferation by binding to medium-affinity βγ dimers, and it can also stimulate T cell proliferation by binding to high-affinity αβγ trimers. When the affinity of βγ dimer and IL-2 is weakened, IL-2 mutants will preferentially stimulate Treg cells with αβγ receptors than wild-type IL-2, while avoiding stimulation of other types of T cells that mainly express βγ receptors Of proliferation. Therefore, reducing the ability of IL-2 to bind to the intermediate affinity receptor (βγ chain complex) can reduce the stimulating effect of IL-2 on effector T cells, thereby increasing the ratio of Treg cells to effector T cells, and improving the effect of Treg or Treg. Autoimmune response caused by functional impairment.

Therefore, a new type of interleukin-2 (IL-2) mutant that better stimulates the proliferation of regulatory T cells is required for the treatment of different autoimmune diseases.

Summary of the invention

The purpose of the present invention is to provide a novel IL-2 mutant. Compared with wild-type IL-2, the IL-2 mutant of the present invention can reduce the binding ability of its binding partner IL-2 receptor β subunit and IL-2 receptor γ subunit, and maintain the corresponding biological activity , Can better stimulate the proliferation of regulatory T cells.

In the first aspect, the present invention provides an IL-2 mutant. Compared with wild-type IL-2, the amino acid residues of the IL-2 mutant are mutated, which reduces the protein pair of the mutant IL-2. Affinity The affinity of the IL-2 receptor.

In a preferred embodiment, the medium-affinity IL-2 receptor contains only the IL-2 receptor β subunit and the IL-2 receptor γ subunit without the IL-2 receptor α subunit.

In a preferred embodiment, the IL-2 mutant can increase the ratio of CD3+CD4+FoxP3+ cells to CD3+CD4+FoxP3-.

In a preferred embodiment, the IL-2 mutant can increase the ratio of CD3+FoxP3+ cells to CD3+FoxP3-.

In a specific embodiment, the IL-2 mutant is mutated at the amino acid residue at position 90 corresponding to wild-type IL-2.

In a specific embodiment, compared to wild-type IL-2, the amino acid residues of the IL-2 mutant are mutated, thereby increasing artificial glycosylation sites.

In a preferred embodiment, the glycosylation site is an N sugar site or an O sugar site; preferably an N sugar site.

In a specific embodiment, the amino acid residues of the IL-2 mutant are mutated compared to wild-type IL-2.

In a preferred embodiment, the IL-2 mutant has the following amino acid residue mutations at position 90 corresponding to the wild-type IL-2 protein: N90T, N90S, N90V, N90I, N90M, N90L, N90Y, N90W, N90R , N90A, N90G, N90F, N90H, N90K, N90Q, N90D, N90E, N90P, N90C;

Preferably, the IL-2 mutant has the following amino acid residue mutations at position 90 corresponding to wild-type IL-2: N90T, N90S, N90V, N90I, N90M, N90L;

More preferably, the IL-2 mutant has the following amino acid residue mutations at position 90 corresponding to wild-type IL-2: N90T, N90S;

Most preferably, the IL-2 mutant has the following amino acid residue mutation at position 90 corresponding to wild-type IL-2: N90T.

In a preferred embodiment, the IL-2 mutant has the following amino acid residue mutations at position 3 corresponding to the wild-type IL-2 protein: T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P ; Preferred T3A.

In a preferred embodiment, the IL-2 mutant is mutated at position 125 cys: C125L, C125S, C125A; preferably C125S.

In the second aspect, the present invention provides a fusion protein or conjugate comprising the IL-2 mutant described in the first aspect and a non-IL-2 functional part.

In a preferred embodiment, the non-IL-2 functional part is selected from the following group:

Fc fragments, including but not limited to: Fc fragments of human IgG1, IgG2, IgG3, IgG4, and Fc fragment mutants with a homology of more than 90%;

Human serum albumin (HSA);

Anti-HSA antibodies and fragments

Anti-albumin polypeptide or antibody;

Transferrin;

Human chorionic gonadotropin β subunit carboxy terminal peptide (CTP);

Elastin-like peptide (ELP);

Antigen binding part.

In a preferred embodiment, the antigen binding portion is:

Antibodies or active antibody fragments;

Fab molecules, scFv molecules and VHH molecules; or

Cell receptor or ligand.

In a preferred embodiment, the IL-2 mutant and the non-IL-2 functional part in the fusion protein can be directly connected or connected via a linker; the linker can be a repeating sequence of AAA or GS, including But it is not limited to the repetitive sequence of G3S or the repetitive sequence of G4S; for example, (G3S)4.

In a preferred embodiment, the IL-2 mutant or fusion protein can be further modified as follows to form a conjugate:

Polyethylene glycol modification (PEGylation);

Polysialylation modification (PSAization);

Saturated fatty acid modification;

Hyaluronic acid modification (Hyaluronic acid, HA);

Polyamino acid modification (proline-alamine-serine polymer, PAS).

In a third aspect, the present invention provides a polynucleotide encoding the IL-2 mutant described in the first aspect or the fusion protein or conjugate described in the second aspect.

In a specific embodiment, the polynucleotide is DNA or RNA.

In the 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, which contains the expression vector of the fourth aspect, or the genome of the host cell integrates the polynucleotide of the third aspect.

In a preferred embodiment, the host cell is a eukaryotic cell; preferably yeast, insect cell, animal cell; more preferably animal cell; most preferably mammalian cell, such as Chinese hamster ovary cell.

In the sixth aspect, the present invention provides a pharmaceutical composition comprising the IL-2 mutant protein of the first aspect or the fusion protein or conjugate of the second aspect and a pharmaceutically acceptable Of accessories.

In the seventh aspect, the present invention provides the use of the IL-2 mutant described in the first aspect or the fusion protein described in the second aspect in the preparation of drugs for autoimmune diseases.

In a preferred embodiment, the disease is a disease in which IL-2 is used for immunotherapy.

In a preferred embodiment, the disease is immune disease, human immunodeficiency virus HIV infection, hepatitis C virus HCV infection, rheumatoid arthritis, atopic dermatitis and the like.

In a preferred embodiment, the immune disease, human immunodeficiency virus HIV infection, hepatitis C virus HCV infection, rheumatoid arthritis, atopic dermatitis, etc. are treated by stimulating the immune system or by proliferating immune cells.

In the eighth aspect, the present invention provides an IL-2 mutant that has a mutation at the 90th amino acid residue and the 39th amino acid residue corresponding to wild-type IL-2.

In a specific embodiment, the IL-2 mutant has the following amino acid residue mutations at position 90 corresponding to wild-type IL-2: N90T, N90S, N90V, N90I, N90M, N90L, N90Y, N90W, N90R, N90A, N90G, N90F, N90H, N90K, N90Q, N90D, N90E, N90P, N90C;

Preferably, the IL-2 mutant has the following amino acid residue mutations at position 90 corresponding to wild-type IL-2: N90T, N90S, N90V, N90I, N90M, N90L;

More preferably, the IL-2 mutant has the following amino acid residue mutations at position 90 corresponding to wild-type IL-2: N90T, N90S;

Most preferably, the IL-2 mutant has the following amino acid residue mutation at position 90 corresponding to wild-type IL-2: N90T.

In a specific embodiment, the IL-2 mutant has the following amino acid residue mutations at position 39 corresponding to wild-type IL-2: M39I, M39L, M39Q, M39A, M39V;

Preferably, the IL-2 mutant has the following amino acid residue mutations at position 39 corresponding to wild-type IL-2: M39I, M39L.

In a specific embodiment, the IL-2 mutant preferentially stimulates T regulatory cells compared to wild-type IL-2.

In a specific embodiment, the T regulatory cell is a CD25 ⁺ T regulatory cell or a T regulatory cell that highly expresses CD25.

In the ninth aspect, the present invention provides a fusion protein or conjugate comprising the IL-2 mutant described in the eighth aspect and a non-IL-2 functional part.

In a specific embodiment, the non-IL-2 functional part is an Fc fragment.

In the tenth aspect, the present invention provides a polynucleotide encoding the IL-2 mutant of the seventh aspect or the fusion protein or conjugate of the eighth aspect; preferably, the The polynucleotide is DNA or RNA.

In the eleventh aspect, the present invention provides an expression vector comprising the polynucleotide of the tenth aspect.

In a twelfth aspect, the present invention provides a host cell comprising the expression vector of the eleventh aspect, or the genome of the host cell integrates the polynucleotide of the tenth aspect.

In the thirteenth aspect, the present invention provides a pharmaceutical composition comprising the IL-2 mutant of the seventh aspect or the fusion protein or conjugate of the eighth aspect, and a pharmaceutical composition Accepted excipients.

In a thirteenth aspect, the present invention provides the use of the IL-2 mutant described in the seventh aspect or the fusion protein or conjugate described in the eighth aspect in the preparation of a medicament for the treatment of a disease in an individual.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them one by one here.

Description of the drawings

Figure 1 shows the binding capacity of IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc to IL-2Rα detected by Biacore;

Figure 2 shows the binding capacity of IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc to IL-2Rβγ dimer detected by Biacore;

Figure 3 shows how the interleukin-2 mutant and wild-type IL-2 of the present invention stimulate the proliferation of NK92 cells;

Figure 4 shows the sequence SEQ ID NO: 1-5 used in the present invention;

Figure 5 shows the proliferation of NK92 cells in response to IL-2-HSA and mutant IL-2;

Figure 6 shows the phosphorylation level of p-STAT5 in different cell populations;

Figure 7 shows the ratio of p-STAT5 levels in the FOXP3+/FOXP3-cell population;

Figure 8 shows the expansion of induced Treg cells (limited to CD3+CD4+CD25+Foxp3+) in mice;

Figure 9 shows the levels of induced Treg cells (limited to CD3+CD4+CD25+Foxp3+) in mice;

Figure 10 shows the expansion of induced Treg cells (limited to CD3+CD4+Foxp3+) in mice;

Figure 11 shows the levels of induced Treg cells (limited to CD3+CD4+Foxp3+) in mice;

Figure 12 shows the amplification of induced CD8+Tregs (limited to CD3+CD8+Foxp3+) in mice; and

Figure 13 shows the sequence SEQ ID NO: 6-18 used in the present invention.

Detailed ways

After extensive and in-depth research, the inventors unexpectedly discovered that a new type of IL-2 mutant after site-directed mutation of IL-2 polypeptide can reduce the binding to IL-2Rβγ dimer, retain its biological activity, and preferentially Stimulates the proliferation of regulatory T cells. Therefore, the IL-2 mutant of the present invention can be used for the treatment of autoimmune diseases, but it does not have various side effects caused by immunotherapy with natural IL-2. The present invention has been completed on this basis.

IL-2 mutant of the present invention

In the present invention, site-directed mutagenesis changes the amino acid residues of the IL-2 polypeptide, thereby changing the binding ability or affinity of IL-2 to its receptor, but can retain biological activity. The IL-2 mutant of the present invention can stimulate the proliferation of regulatory T cells (Treg), and compared with wild-type IL-2, its side effects are also significantly reduced, thereby achieving better therapeutic purposes.

The IL-2 mutant of the present invention is preferably expressed in eukaryotic cells and obtained by cell culture. You can choose yeast, insect cells, animal cells, or transgenic animals. In a specific embodiment, the host cells are eukaryotic cells; preferably yeast, insect cells, animal cells; animal cells are preferably mammalian cells, including but not limited to CHO cells, 293 cells, SP/20 cells, and NS0 cells. Optionally, the IL-2 mutant of the present invention can be obtained by cell-free expression, in vitro synthesis and other technical means.

When yeast cells or insect cells are used as host cells, the glycoform of the IL-2 mutant that may be obtained is non-human. Those skilled in the art know that non-human glycoforms can be further transformed into adult glycoforms.

In other embodiments, prokaryotic expression fermentation or in vitro cell-free synthesis can also be used to obtain IL-2 mutants.

The IL-2 mutant of the present invention is mutated at position 90 corresponding to wild-type IL-2. Therefore, in a specific embodiment, the IL-2 mutant of the present invention has the following amino acid residue mutations at position 90 corresponding to the wild-type IL-2 protein: N90T, N90S, N90V, N90I, N90M, N90L, N90Y, N90W, N90R, N90A, N90G, N90F, N90H, N90K, N90Q, N90D, N90E, N90P, N90C; preferably N90T, N90S, N90V, N90I, N90M, N90L; more preferably N90T, N90S; most preferably N90T.

Based on conventional practices in the field, the original O-sugar sites in IL-2 polypeptides can also be eliminated. The removal of O-sugar does not affect the biological activity of IL-2. The structure of O-sugar is complex and analysis is difficult. In order to reduce the complexity of production quality control For sex, genetic engineering mutation technology can usually be used to eliminate the glycosylation site. Therefore, the IL-2 mutant of the present invention may have the following amino acid residue mutations at position 3 corresponding to the wild-type IL-2 protein: T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K and T3P; preferably T3A . During the purification and renaturation of IL-2 gene products, mismatching disulfide bonds or the formation of disulfide bonds between molecules will reduce the activity of IL-2. Point mutations have been used to mutate cysteine at position 125 to leucine or serine, so that only one disulfide bond can be formed, which ensures the activity in the refolding process of IL-2. There are also reports that protein engineering technology is used to produce a new type of rIL-2, where the 125th cysteine of the IL-2 molecule is changed to alanine, and the specific activity of IL-2 is significantly higher than that of natural IL-2 after the modification. Therefore, the IL-2 mutant of the present invention can have the following amino acid residue mutations at position 125 corresponding to the wild-type IL-2 protein: C125L, C125A, C125S; preferably C125S.

Based on the IL-2 mutant corresponding to wild-type IL-2 at position 90 mutated, the present inventors further found that the amino acid residue at position 90 and amino acid 39 corresponding to wild-type IL-2 The residues are mutated. Compared with wild-type IL-2, the thus obtained IL-2 mutant preferentially stimulates T regulatory cells; especially CD25 ⁺ T regulatory cells or T regulatory cells with high CD25 expression.

In a specific embodiment, the IL-2 mutant has the following amino acid residue mutations at position 90 corresponding to wild-type IL-2: N90T, N90S, N90V, N90I, N90M, N90L, N90Y, N90W, N90R, N90A, N90G, N90F, N90H, N90K, N90Q, N90D, N90E, N90P, N90C; preferably N90T, N90S, N90V, N90I, N90M, N90L; more preferably N90T, N90S; most preferably N90T; at the same time, corresponding to the wild type The following amino acid residue mutations occurred at position 39 of IL-2: M39I, M39L, M39Q, M39A, M39V; preferably M39I, M39L.

"Corresponds to"

The term "corresponding to" used herein has the meaning commonly understood by those of ordinary skill in the art. Specifically, "corresponding to" means that after two sequences are aligned for homology or sequence identity, one sequence corresponds to a designated position in the other sequence. Therefore, for example, "corresponding to wild-type IL-2" means to align a certain amino acid sequence with the amino acid sequence of wild-type IL-2, and find a site on the amino acid sequence that corresponds to wild-type IL-2.

Fusion protein or conjugate of the present invention

Based on the IL-2 mutants of the present invention, those skilled in the art know that the IL-2 mutants of the present invention and other functional parts other than IL-2 can be made into fusion proteins or conjugates. In this context, conjugate refers to a water-soluble polymer covalently linked to the residues of the mutant IL-2 polypeptide. In a specific embodiment, the non-IL-2 functional part includes but is not limited to: Fc fragment, human serum albumin (HSA), anti-HSA antibodies and fragments, transferrin, human chorionic gonadotropin β subunit C-terminal peptide (CTP), elastin-like peptide (ELP) and antigen-binding portion, including cytokine or cytokine antibody, specifically interleukin, interferon, tumor necrosis factor superfamily, colony stimulation Factors, chemokines, growth factors, etc.

Based on conventional operations in the art, those skilled in the art know how to obtain a fusion protein or conjugate containing the IL-2 mutant of the present invention. For example, the IL-2 mutant of the present invention can be directly connected to other non-IL-2 functional parts, or can be connected through a linker. The linker may be a repetitive sequence of AAA or GS, including but not limited to _{a repetitive sequence of G 3} S or a repetitive sequence of G4S; for example, (G ₃ S) ₄ .

Further, the IL-2 mutant or fusion protein conjugate can also be modified with polyethylene glycol (PEGylation), polysialylated (PSA), saturated fatty acid, and hyaluronic acid (Hyaluronic acid, HA) or polyamino acid modification (proline-alamine-serine polymer, PAS) to form conjugates.

Bispecific antibody or trispecific antibody of the present invention

Diseases are usually caused by a variety of pathogenic factors. Blocking multiple targets at the same time may achieve better therapeutic effects. Therefore, bispecific antibodies (BsAbs) have emerged. Tumor immunotherapy is currently a new direction in the treatment of tumors. Bispecific antibodies can bind two different antigens, so the development prospects in the field of tumor therapy are very broad. Bispecific antibodies were originally prepared by chemical coupling or hybridoma hybridization. Nowadays, the rapid development of DNA recombination technology has revolutionized the structure of bispecific antibodies, which are mainly divided into two categories: IgG type containing Fc region and non-IgG type without Fc region. The structure of IgG-type bispecific antibodies is similar to that of monoclonal antibodies. The relative molecular weight of the protein is relatively large, and the plasma half-life is long. The structure of non-IgG bispecific antibodies is more diverse, the relative molecular weight of the protein is smaller, the tissue permeability is stronger, but the plasma half-life is shorter.

Based on the IL-2 mutant of the present invention, those skilled in the art know that the IL-2 mutant of the present invention can be covalently linked to the antibody domain. In a specific embodiment, the antibody domain includes, but is not limited to: IgG type antibodies and non-IgG type antibodies. In a preferred embodiment, the antibody domain may be an antibody or active antibody fragments thereof, Fab molecules, scFv molecules and VHH molecules, immunoglobulin molecules, receptor protein molecules or ligand protein molecules; the immunoglobulin The molecule can be an IgG molecule.

Based on conventional operations in the art, those skilled in the art know how to obtain the bispecific antibody comprising the IL-2 mutant of the present invention. For example, the IL-2 mutant of the present invention can be directly connected to other non-IL-2 functional parts, or can be connected through a linker. The linker may be a repetitive sequence of AAA or GS, including but not limited to _{a repetitive sequence of G 3} S or a repetitive sequence of G ₄ S; for example, (G ₃ S) ₄ .

Optionally, the mutants of the present invention can be coupled with T cell surface antigen antibodies, and can also be coupled with tumor cell surface antigen antibodies. Preferably, the mutants of the present invention can be coupled with T cell surface antigen antibodies.

Optionally, the mutants of the present invention can be coupled with T cell surface antigen antibodies to form bispecific antibodies, and can also be coupled with tumor cell surface antigen antibodies to form bispecific antibodies. Optionally, the mutants of the present invention can be coupled with T cell surface antigen antibodies to form trispecific antibodies, and can also be coupled with tumor cell surface antigen antibodies to form trispecific antibodies. Optionally, the mutants of the present invention can be coupled with T cell surface antigen antibodies or tumor cell surface antigen antibodies to form trispecific antibodies.

The pharmaceutical composition of the present invention and its administration method

On the basis of the IL-2 mutant of the present invention, the present invention also provides a pharmaceutical composition. In a specific embodiment, the pharmaceutical composition of the present invention comprises the IL-2 mutant of the present invention or the fusion protein or conjugate of claim 5 or the bispecific antibody or trispecific antibody of claim 7 Antibody and optional pharmaceutically acceptable excipients.

Optionally, the composition of the present invention further comprises a pharmaceutically acceptable excipient. If desired, pharmaceutically acceptable excipients can be added to the IL-2 mutant polypeptide, fusion protein or conjugate, bispecific antibody or trispecific antibody of the present invention to form a composition.

Use and method of use of the IL-2 mutant of the present invention

As described above, the IL-2 mutant of the present invention can reduce the affinity of the mutant IL-2 protein to the medium-affinity IL-2 receptor, while retaining the biological activity of IL-2, thereby better stimulating regulatory T Cells (Treg) proliferate. Therefore, the IL-2 mutant, fusion protein, conjugate, bispecific antibody or trispecific antibody, and pharmaceutical composition of the present invention can be prepared into corresponding drugs. The drug can be used to expand regulatory T cells (Treg) in vitro or treat diseases that use IL-2 for immunotherapy. In a specific embodiment, the disease is systemic lupus erythematosus (SLE), autoimmune disease, diabetes, human immunodeficiency virus HIV infection, hepatitis C virus HCV infection, rheumatoid arthritis, atopic dermatitis, etc. .

Advantages of the present invention:

1. The IL-2 mutant protein of the present invention reduces the binding to IL-2Rβγ dimer.

2. The structure of the IL-2 mutant of the present invention is closer to that of natural IL-2, avoiding the influence of mutation on other structural sites of the protein, and retaining biological activity;

3. The interleukin 2 mutant protein of the present invention can be used for the treatment of autoimmune diseases, but it does not have various side effects caused by immunotherapy with natural IL-2.

4. Compared with other IL-2 mutants in the prior art, the IL-2 mutant of the present invention has lower immunogenicity;

5. The IL-2 mutant of the present invention is convenient for production and quality control, and generally does not require an in vitro re-modification process, reducing steps to improve production efficiency;

6. The IL-2 mutant of the present invention is convenient to form a bifunctional or multifunctional fusion protein or immune composition with other molecules;

7. The IL-2 mutant of the present invention can be used for immunotherapy, but will not cause vascular (or capillary) leakage syndrome (VLS) caused by natural IL-2; and

8. The in vivo half-life of the IL-2 mutant of the present invention is significantly prolonged.

9. Compared with wild-type IL-2, the IL-2 mutants of the present invention (mutations 39 and 90) preferentially stimulate T regulatory cells; especially CD25 ⁺ T regulatory cells or T regulatory cells with high CD25 expression cell.

The present invention will be further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples usually follow the conventional conditions as described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions described by the manufacturer The suggested conditions.

Example

Example 1. Synthesis of mutant interleukin-2 (IL-2) protein

The coding sequences of IL-2 mutant IL-2-gmB2-hIgG4Fc (SEQ ID NO: 1) and wild-type IL2-N-hIgG4Fc (SEQ ID NO: 2) including the coding sequence of human IgG4Fc were constructed by molecular cloning. Into the eukaryotic expression vector to prepare IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc expression vectors. 293E cells cultured in Freestyle medium were used for transient transfection and expression of IL-2 mutant molecules. ^{Twenty-four hours before transfection, 150 ml of 0.5×10 6} cells/ml 293E cells were inoculated in a 1L cell culture flask and cultured in a 37°C 5% CO2 incubator with a shaker at 120 rpm. During transfection, 150μl of 293fectin was added to 2.85ml of OptiMEM, mixed thoroughly, and incubated for 2 minutes at room temperature; meanwhile, 150μg of plasmids used to express IL-2 molecules were diluted to 3ml with OptiMEM. The above-mentioned diluted transfection reagent and plasmid were mixed thoroughly, incubated at room temperature for 15 minutes, then all the mixture was added to the cells, mixed, and incubated in a 37°C 5% CO ₂ incubator with a shaker at 120 rpm for 7 days. Collect the cell culture supernatant, filter the supernatant with a 0.22 micron filter membrane, and then purify it with a Q-HP ion exchange chromatography column (GE), using 20mM Tris 0-500mM NaCl, pH 8.0 linear elution, continuous by volume Collect samples. Use 4-20% gradient gel (GenScript) to detect the collected components by SDS-PAGE, and combine the samples according to the electrophoresis purity.

Number of mutations	Example Mutant Name	Protein tag		sequence
3	IL-2gmB2 (T3A, N90T, C125A)	IgG4Fc	SEQ ID NO:1

Example 2. Preparation of receptor protein

To study the binding ability of IL-2 mutant molecules with IL-2Rα receptor and IL-2Rβγ heterodimerization receptor, human IL-2Rα receptor and IL-2Rβγ heterodimerization receptor were prepared protein.

The design of the human IL-2Rα receptor is to link the coding sequence of the extracellular domain of IL-2Rα to the 6×His Tag coding sequence (SEQ ID NO: 3) and clone it into a eukaryotic expression vector. 293E cells cultured in Freestyle medium were used for transient transfection and expression of IL-2Rα receptor. ^{Twenty-four hours before transfection, 150 ml of 0.5×10 6} cells/ml 293E cells were inoculated in a 1L cell culture flask and cultured in a 37°C 5% CO2 incubator with a shaker at 120 rpm. During transfection, 150μl of 293fectin was added to 2.85ml of OptiMEM, mixed well, and incubated for 2 minutes at room temperature; meanwhile, 150μg of the plasmid used to express IL-2Rα receptor was diluted to 3ml with OptiMEM. The above-mentioned diluted transfection reagent and plasmid are mixed thoroughly, incubated at room temperature for 15 minutes, then all the mixture is added to the cells, mixed, and incubated in a 37°C 5% CO2 incubator with a shaker at 120 rpm for 7 days. The cell culture supernatant was collected, and the supernatant was filtered with a 0.22 micron filter membrane, and then purified using a Ni-NTA affinity chromatography column (GE), and eluted under the condition of 20mM PB-0.5M NaCl-100mM imidazole. The purified protein was detected by SDS-PAGE using 4-20% gradient gel (GenScript).

The design of human IL-2Rβγ heterodimerization receptor utilizes the pairing characteristics of CH1 and CL to complete the heterologous pairing. hIL2Rβ (SEQ ID NO: 4) and hIL2Rγ (SEQ ID NO: 5) were cloned into eukaryotic expression vectors, respectively. 293E cells cultured in Freestyle medium were used for transient transfection and expression of IL-2Rβγ heterodimerization receptor. ^{Twenty-four hours before transfection, 150 ml of 0.5×10 6} cells/ml 293E cells were inoculated in a 1L cell culture flask and cultured in a 37°C 5% CO2 incubator with a shaker at 120 rpm. During transfection, first take 150μl of 293fectin and add it to 2.85ml OptiMEM, mix well, incubate at room temperature for 2 minutes; meanwhile, it will be used to express IL-2Rβγ heterodimerization receptor (named: hIL2Rβ,γECD-His) Each 75μg of plasmid was diluted to 3ml with OptiMEM. The above-mentioned diluted transfection reagent and plasmid are mixed thoroughly, incubated at room temperature for 15 minutes, then all the mixture is added to the cells, mixed, and incubated in a 37°C 5% CO2 incubator with a shaker at 120 rpm for 7 days. Collect the cell culture supernatant, filter the supernatant with a 0.22 micron filter membrane, and then purify it with a MabSelect SuRe affinity chromatography column (GE), eluting at 20 mM citrate-sodium citrate, pH 3.0 , Adjust the pH to neutral with 1M Tris base. The purified protein was detected by SDS-PAGE using 4-20% gradient gel (GenScript).

Example 3. Using biacore to detect binding affinity experiment

In order to study the affinity of the IL-2 mutant relative to the wild-type receptor, the recombinant monomer IL-2Rα subunit was used to determine the IL-2 mutant molecule IL-2-gmB2-hIgG4Fc by Biacore 8K (GE) under the following conditions And the affinity of wild-type IL2-N-hIgG4Fc to human IL-2Rα subunit: Immobilize human IL-2Rα subunit on CM5 chip (190RU). IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc were used as analytes in HBS-EP buffer at 25°C. For IL-2Rα, the analyte concentration was reduced from 200 nM to 1.526 nM (1:2 dilution), and the flow was 30 μl/min (binding time 180 seconds, dissociation time 300 seconds). For IL-2Rα, regeneration is performed with 20mM NaOH, 30ul/min for 10 seconds. For IL-2Rα, 1:1 binding is used, RI≠0, Rmax=global fit data.

The results are shown in Figure 1; the maximum R value of IL-2-gmB2-hIgG4Fc is 25, and the maximum R value of IL2-N-hIgG4Fc is 25. Therefore, IL-2-gmB2-hIgG4Fc retains the affinity for IL-2Rα dimer relative to wild-type IL2-N-hIgG4Fc.

Under the following conditions, recombinant hIL2Rβ, γECD-His heterodimer was used to determine the heterodimerization of IL-2 mutant molecules IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc to human IL-2Rβγ by Biacore 8K (GE) Affinity of the aggregate: IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc were immobilized on the Protein A chip (100RU). Recombinant hIL2Rβ,γECD-His heterodimer was used as analyte in HBS-EP buffer at 25°C. For IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc, the analyte concentration is reduced from 100nM to 0.78nM (1:2 dilution), and the flow rate is 30μl/min (binding time 180 seconds, dissociation time 300 seconds) . For IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc, 10mM Glycine (pH 1.5) for regeneration, 30ul/min, 30 seconds. For IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc, 1:1 binding was used, RI≠0, Rmax=local fitting data.

The results are shown in Figure 2; the maximum R value of IL-2-gmB2-hIgG4Fc is 1.7, and the maximum R value of IL2-N-hIgG4Fc is 4.0. As shown in Figure 2, IL-2-gmB2-hIgG4Fc has a reduced affinity for IL-2Rβγ dimer compared to wild-type IL2-N-hIgG4Fc. Therefore, IL-2-gmB2-hIgG4Fc retains its affinity with IL-2Rα and decreases its affinity with IL-2Rβγ dimer relative to wild-type IL2-N-hIgG4Fc.

Example 4. Using NK92 cells for cell proliferation analysis

NK92 cells are an IL-2-dependent NK cell line derived from peripheral blood mononuclear cells of a 50-year-old white male with rapidly progressive non-Hodgkin's lymphoma. The cell surface expresses IL-2 receptor α, β and γ chains, and the cell surface markers are the same as those of regulatory T cells. Therefore, the present inventors used NK92 cells to evaluate the activities of IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc in cell proliferation analysis.

Harvest NK92 cells in the logarithmic growth phase, wash them with basal medium MEM-α, and culture them (5000 cells/well) with different concentrations of IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc in the experiment Base (from Gibco (Cat. No. 32561-037) MEM-α medium, supplemented with 12.5% fetal bovine serum and 12.5% horse serum), incubate at 37°C in a 5% carbon dioxide incubator for 48 hours. Add 100μl of ATP detection substrate CellTiter-Glo (from Promega (Cat. No. G7571)) to each well, and detect the full-wavelength fluorescence value by the end-point method in a microplate reader (purchased from Molecular Devices (Model I3x)).

The results are shown in Figure 3; cell proliferation analysis was used to measure the activity of IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc, and it was found that all test products induced the growth of NK92 cells in a dose-dependent manner. Therefore, IL-2-gmB2-hIgG4Fc retains biological activity compared to wild-type IL2-N-hIgG4Fc.

Example 5. Synthesis of mutant interleukin-2 (IL-2) protein

The coding sequences of IL-2 mutant IL-2-gmB5-hIgG4Fc (SEQ ID NO: 6) and IL-2-gmB6-hIgG4Fc (SEQ ID NO: 7) including the human IgG4Fc coding sequence were constructed by molecular cloning methods. Into the eukaryotic expression vector to prepare the expression vector of IL-2 mutant. Similarly, the IL-2 mutants IL-2-gmB2-HSA (SEQ ID NO: 8), IL-2-gmB5-HSA (SEQ ID NO: 9), IL-2 including the human HSA coding sequence were separately obtained by molecular cloning. -2-gmB6-HSA (SEQ ID NO: 10), IL-2-gmB7-HSA (SEQ ID NO: 11), IL-2-gmB8-HSA (SEQ ID NO: 12), IL-2-gmB9- HSA (SEQ ID NO: 13), IL-2-gmB10-HSA (SEQ ID NO: 14), IL-2-HSA wild type (SEQ ID NO: 15), IL-2-gmB11-HSA (SEQ ID NO :16), IL-2-gmB12-HSA (SEQ ID NO: 17), IL-2-gmB13-HSA (SEQ ID NO: 18) coding sequence was constructed into eukaryotic expression vector to prepare IL-2 mutant Expression vector. 293E cells cultured in Freestyle medium were used for transient transfection and expression of IL-2 mutant molecules. ^{Twenty-four hours before transfection, 150 ml of 0.5×10 6} cells/ml 293E cells were inoculated in a 1L cell culture flask, and cultured on a shaker at 120 rpm in a 37°C 5% CO _{2 incubator.} During transfection, 150μl of 293fectin was added to 2.85ml of OptiMEM, mixed thoroughly, and incubated for 2 minutes at room temperature; meanwhile, 150μg of plasmids used to express IL-2 molecules were diluted to 3ml with OptiMEM. The above-mentioned diluted transfection reagent and plasmid were mixed thoroughly, incubated at room temperature for 15 minutes, then all the mixture was added to the cells, mixed, and incubated in a 37°C 5% CO ₂ incubator with a shaker at 120 rpm for 7 days. Collect the cell culture supernatant, filter the supernatant with a 0.22 micron filter membrane, and then purify it with a Q-HP ion exchange chromatography column (GE), using 20mM Tris 0-500mM NaCl, pH 8.0 linear elution, continuous by volume Collect samples. Use 4-20% gradient gel (GenScript) to detect the collected components by SDS-PAGE, and combine the samples according to the electrophoresis purity.

Number of mutations	Example Mutant Name	Protein tag		sequence
4	IL-2gmB5 (T3A, M39L, N90T, C125A)	IgG4Fc	SEQ ID NO: 6
4	IL-2gmB6(T3A, M39I, N90T, C125A)	IgG4Fc	SEQ ID NO: 7
3	IL-2gmB2(T3A, N90T, C125A)	HSA	SEQ ID NO: 8
4	IL-2gmB5 (T3A, M39L, N90T, C125A)	HSA	SEQ ID NO: 9
4	IL-2gmB6(T3A, M39I, N90T, C125A)	HSA	SEQ ID NO: 10
4	IL-2gmB7(T3A, M39I, N90V, C125A)	HSA	SEQ ID NO: 11
4	IL-2gmB8 (T3A, M39I, N90I, C125A)	HSA	SEQ ID NO: 12
4	IL-2gmB9(T3A, M39I, N90M, C125A)	HSA	SEQ ID NO: 13
4	IL-2gmB10 (T3A, M39I, N90L, C125A)	HSA	SEQ ID NO: 14
2	IL-2 (T3A, C125A)	HSA	SEQ ID NO: 15
3	IL-2gmB11(T3A, N90R, C125A)	HSA	SEQ ID NO: 16
3	IL-2gmB12(T3A, N90W, C125A)	HSA	SEQ ID NO: 17
3	IL-2gmB13(T3A, N90Y, C125A)	HSA	SEQ ID NO: 18

Example 6. Using NK92 cells for cell proliferation analysis

NK92 cells are an IL-2-dependent NK cell line derived from peripheral blood mononuclear cells of a 50-year-old white male with rapidly progressive non-Hodgkin's lymphoma. The cell surface expresses IL-2 receptor α, β and γ chains, and the cell surface markers are the same as those of regulatory T cells. Therefore, the inventors used NK92 cells to evaluate the activity of IL-2 mutants and wild-type IL2-HSA in cell proliferation analysis.

The method is the same as in Example 4. The results are shown in Figure 5; cell proliferation analysis was used to measure the activity of IL-2 mutant and wild-type IL2-HSA, and it was found that all test products induced the growth of NK92 cells in a dose-dependent manner. Therefore, compared to wild-type IL2-HSA, IL-2gmB2-HSA, IL-2gmB5-HSA, IL-2gmB6-HSA, IL-2gmB7-HSA, IL-2gmB8-HSA, IL-2gmB9-HSA, IL-2gmB10- HSA retains biological activity.

Example 7. Human PBMC activation method (p-STAT5)

Collect blood from healthy people into blood collection tubes containing heparin and separate PBMCs. Resuspend PBMCs in complete medium (RPMI-1640 containing 10% FBS) and inoculate them in 96 wells ^{with a cell number of 1*10 6 /50μl/well} In the board. Dilute wild-type IL2-HSA, IL-2gmB2-HSA, IL-2gmB5-HSA, IL-2gmB6-HSA with complete medium, starting at a final concentration of 2000nM, 10 times dilution, 6 gradients, 50μl/well to stimulate PBMC. After incubating in a 37°C incubator for 20 minutes, centrifuge to discard the supernatant, add CD3 (Sino Biological)&CD4&CD8&CD56(BioLegend)&CD25(BD) flow cytometry antibody for cell expression staining, wash and centrifuge to discard the supernatant; then use preheated PBMC Fix with Cytofix buffer (BD) at 37°C for 10 minutes, wash and centrifuge to discard the supernatant; then use Phosflow Permeabilization Buffer III (BD) at 4°C for 30 minutes, wash and centrifuge to discard the supernatant; use FOXP3 (BioLegend) & p-STAT5 (BD) flow cytometry antibody and isotype control antibody for intranuclear molecular staining, washing, centrifugation, discarding the supernatant, resuspending PBMC in Staining buffer (PBS containing 1% BSA), and finally using flow cytometry to detect and analyze different cell populations STAT5 Phosphorylation.

Treg cells are defined as CD3 ⁺ CD4 ⁺ CD25 ⁺ Foxp3 ⁺ , CD4 ⁺ T cells are defined as CD3 ⁺ CD4 ⁺ , CD8 ⁺ T cells are defined as CD3 ⁺ CD8 ⁺ , and NK cells are defined as CD3 ⁻ /CD56 ⁺ .

As shown in Figure 6, in the p-STAT5 stimulation assay, IL-2gmB2-HSA, IL-2gmB5-HSA and IL-2gmB6-HSA compared to the wild-type IL-2-HSA protein on CD4 ⁺ T, CD8 ⁺ T and The signal strength of NK is weakened. This is because the three mutant proteins in this experiment reduced their affinity with IL-2Rβγ dimer. The signal intensity of IL-2gmB2-HSA, IL-2gmB5-HSA and IL-2gmB6-HSA relative to the wild-type IL-2-HSA protein to Treg is equivalent at 2-200nM. The signal intensity of the three mutants at 2000 nM was significantly higher than that of wild-type IL-2-HSA. This indicates that the mutant IL-2 has a stronger ability to stimulate Treg proliferation than the wild-type IL-2-HSA.

As shown in Figure 7, in the p-STAT5 stimulation assay, ^{the MFI of CD3 +} CD4 ⁺ Foxp3 ^{+ in CD4 +} T cells was compared with the MFI of CD3 ⁺ CD4 ⁺ Foxp3 ^- . In addition, IL-2gmB2-HSA, IL-2gmB5-HSA and IL-2gmB6-HSA stimulate ^{the FoxP3 activation signal in CD4 +} T cells to be stronger. ^{Furthermore, comparing the MFI of CD3 +} Foxp3 ⁺ with the MFI of CD3 ⁺ Foxp3 ^- in T cells (limited to CD3 ⁺ ), it is found that IL-2gmB2-HSA and IL-2gmB5 are higher than 0.2nM. -HSA and IL-2gmB6-HSA stimulate the FoxP3 activation signal in T cells to be stronger.

It shows that the mutant IL-2-HSA is more inclined to stimulate the activation of ^{Foxp3 + cell population than the wild-type IL-2-HSA.}

Example 8. In vivo evaluation

In order to determine whether IL-2 mutants affect T cell responses in vivo, the IL-2 mutant fusion protein was administered to mice and different T cell subpopulations were monitored. C57BL/6 mice aged 6-8 weeks were assigned to 4 groups (n=6), G1 group was normal saline group, G2 group IL-2-N-hIgG4Fc wild type group (0.05mg/kg), G3 Group IL-2gmB2-N-hIgG4Fc (0.05mg/kg), G4 group IL-2gmB2-N-hIgG4Fc (0.015mg/kg). The mice were administered by subcutaneous injection on day 0 and day 7. On the 10th day, the T cell subsets in the blood were determined by flow cytometry. Add CD45(Biolegend)&CD3&CD4&CD8&CD25&NK1.1(BD) flow cytometry antibody to the mouse blood for cell expression staining. After surface staining, it is treated with 1X Lysing Buffer (BD555899) and the supernatant is centrifuged to discard the supernatant. PBS) resuspend the cells and count them. The cells were then fixed with 1×Foxp3 Fixation/Permeabilization Solution (invitrogen) at room temperature for 30 minutes, and then washed with 1×Permeabilization Buffer (invitrogen) for permeabilization, and centrifuged to discard the supernatant; use FOXP3 (BD) flow cytometry antibody and isotype control antibody (Invitrogen) Perform nuclear molecular staining, wash and centrifuge, discard the supernatant, resuspend the cells in Staining buffer, and finally use flow cytometry to detect and analyze different T cell subpopulations.

As shown in Figure 8, in a method of defining Treg, in the 4 groups of animals, compared with the G1 group, the Treg cells of the G2 group and the G3 group (defined as CD3 ⁺ CD4 ⁺ CD25 ⁺ Foxp3 ⁺ ) accounted for the proportion of CD3 ⁺ CD4 ^{The proportion of +} is higher, and the proportion of G3 is higher than that of G2. However, the ratio of Treg cells (limited to CD3 ⁺ CD4 ⁺ CD25 ⁺ Foxp3 ⁺ ) in the G4 group to CD3 + CD4 + was slightly lower than that in the G1 group.

As shown in Figure 9, in the four groups of animals, compared with the G1 group, ^{the MFI fluorescence value of Treg cells in the G3 and G4 groups (limited to CD3 +} CD4 ⁺ CD25 ⁺ Foxp3 ⁺ ) was higher, indicating that CD3 ⁺ CD4 ⁺ CD25 ⁺ Foxp3 ⁺ is more strongly expressed in the PBMCs of these two groups of animals. The G2 group is the opposite. CD3 ⁺ CD4 ⁺ CD25 ⁺ Foxp3 ⁺ can inhibit the function and migration of DCs, effector T cells (Th1, Th2, Th17 cells) in allergic reactions, and can inhibit CD8 ⁺ T cells that mediate inflammation.

As shown in Figure 10, in another Treg definition method, among the 4 groups of animals, compared with the G1 group, the Treg cells (defined as CD3 ⁺ CD4 ⁺ Foxp3 ⁺ ) in the G2 group, G3 group and G4 group accounted for the proportion of CD3 ⁺ CD4 ⁺ has a higher proportion, and the proportion of G3 is higher than that of G2, and the proportion of G2 is higher than that of G4.

As shown in Figure 11, in another Treg definition method, in the 4 groups of animals, compared with the G1 group, the Treg cells of the G2 group, G3 group and G4 group (limited to CD3 ⁺ CD4 ⁺ Foxp3 ⁺ ) The fluorescence value of MFI is higher, and G4>G3>G2>G1. It shows ^{that the expression of CD3 +} CD4 ⁺ Foxp3 ⁺ in the PBMC of animals also becomes stronger as the value of MFI increases. Thereby, CD8+ T cells that mediate inflammation can be better inhibited.

As shown in Figure 12, we also monitored CD8 ⁺ Tregs (limited to CD3 ⁺ CD8 ⁺ Foxp3 ⁺ ). This type of T cell is produced by naive CD8 ⁺ T cells in the presence of IL-4 and IL-12. The main role of the reaction is to inhibit the activation of naive and effector T cells, inhibit the IgG/IgE antibody response, and inhibit the expression of IL-4. In the 4 groups of animals, relative to the G1 group, the ^{proportion of CD8 +} Tregs (limited to CD3 ⁺ CD8 ⁺ Foxp3 ⁺ ) in G2, G3 and G4 groups to CD8 + T cells (limited to CD3 ⁺ CD8 ⁺ ) Higher, and G4>G3>G2>G1. It shows that the ^{higher the proportion of CD3 +} CD8 ⁺ Foxp3 ⁺ /CD3 ⁺ CD8 ⁺ , the stronger the ability to inhibit the activation of naive and effector T cells.

In vivo evaluation in mice showed that the mutant IL-2gmB2 group preferentially amplifies Treg proliferation, which is better than the IL-2 wild-type group, showing a good ability to directionally amplify Treg.

All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (22)

1. An IL-2 mutant which has a mutation at the 90th amino acid residue corresponding to wild-type IL-2.
2. The IL-2 mutant of claim 1, wherein the IL-2 mutant has the following amino acid residue mutations at position 90 corresponding to wild-type IL-2: N90T, N90S, N90V, N90I, N90M, N90L, N90Y, N90W, N90R, N90A, N90G, N90F, N90H, N90K, N90Q, N90D, N90E, N90P, N90C;

   Preferably, the IL-2 mutant has the following amino acid residue mutations at position 90 corresponding to wild-type IL-2: N90T, N90S, N90V, N90I, N90M, N90L;

   More preferably, the IL-2 mutant has the following amino acid residue mutations at position 90 corresponding to wild-type IL-2: N90T, N90S;

   Most preferably, the IL-2 mutant has the following amino acid residue mutation at position 90 corresponding to wild-type IL-2: N90T.
3. The IL-2 mutant of claim 1 or 2, wherein the IL-2 mutant preferentially stimulates T regulatory cells compared with wild-type IL-2.
4. A fusion protein or conjugate comprising the IL-2 mutant of any one of claims 1-3 and a non-IL-2 functional part.
5. The fusion protein or conjugate according to claim 4, wherein the non-IL-2 functional part is an Fc fragment.
6. A polynucleotide encoding the IL-2 mutant of any one of claims 1-3 or the fusion protein or conjugate of claim 4 or 5; preferably, the The polynucleotide is DNA or RNA.
7. An expression vector comprising the polynucleotide of claim 6.
8. A host cell comprising the expression vector according to claim 7, or the genome of the host cell is integrated with the polynucleotide according to claim 6.
9. A pharmaceutical composition comprising the IL-2 mutant according to any one of claims 1-3 or the fusion protein or conjugate according to claim 4 or 5, and a pharmaceutically acceptable Of accessories.
10. Use of the IL-2 mutant according to any one of claims 1 to 3 or the fusion protein or conjugate according to claim 4 or 5 in the preparation of a medicament for the treatment of a disease in an individual.
11. An IL-2 mutant having mutations at the 90th amino acid residue and 39th amino acid residue corresponding to wild-type IL-2.
12. The IL-2 mutant of claim 11, wherein the IL-2 mutant has the following amino acid residue mutations at position 90 corresponding to wild-type IL-2: N90T, N90S, N90V, N90I, N90M, N90L, N90Y, N90W, N90R, N90A, N90G, N90F, N90H, N90K, N90Q, N90D, N90E, N90P, N90C;

    Preferably, the IL-2 mutant has the following amino acid residue mutations at position 90 corresponding to wild-type IL-2: N90T, N90S, N90V, N90I, N90M, N90L;

    More preferably, the IL-2 mutant has the following amino acid residue mutations at position 90 corresponding to wild-type IL-2: N90T, N90S;

    Most preferably, the IL-2 mutant has the following amino acid residue mutation at position 90 corresponding to wild-type IL-2: N90T.
13. The IL-2 mutant of claim 11, wherein the IL-2 mutant has the following amino acid residue mutations at position 39 corresponding to wild-type IL-2: M39I, M39L, M39Q, M39A, M39V;

    Preferably, the IL-2 mutant has the following amino acid residue mutations at position 39 corresponding to wild-type IL-2: M39I, M39L.
14. The IL-2 mutant of any one of claims 11-13, wherein the IL-2 mutant preferentially stimulates T regulatory cells compared to wild-type IL-2.
15. The IL-2 mutant of claim 14, wherein the T regulatory cell is a CD25 ⁺ T regulatory cell or a T regulatory cell that highly expresses CD25.
16. A fusion protein or conjugate comprising the IL-2 mutant of any one of claims 11-15 and a non-IL-2 functional part.
17. The fusion protein or conjugate according to claim 16, wherein the non-IL-2 functional part is an Fc fragment.
18. A polynucleotide encoding the IL-2 mutant of any one of claims 11-15 or the fusion protein or conjugate of claim 16 or 17; preferably, the The polynucleotide is DNA or RNA.
19. An expression vector comprising the polynucleotide of claim 18.
20. A host cell comprising the expression vector according to claim 19, or the genome of the host cell is integrated with the polynucleotide according to claim 18.
21. A pharmaceutical composition comprising the IL-2 mutant according to any one of claims 11-15 or the fusion protein or conjugate according to claim 16 or 17, and pharmaceutically acceptable Of accessories.
22. Use of the IL-2 mutant of any one of claims 11-15 or the fusion protein or conjugate of claim 16 or 17 in the preparation of a medicament for the treatment of a disease in an individual.

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