A mutant protein that proliferates regulatory T cells

Abstract

The present invention provides an IL-2 mutant, a fusion protein, an antibody or a conjugate comprising the IL-2 mutant, and a pharmaceutical composition comprising the IL-2 mutant, fusion protein, antibody or conjugate. Compared with wild-type IL-2, the IL-2 mutant has reduced binding to the IL-2Rβγ dimer while retaining its biological activity, preferentially stimulating the proliferation of regulatory T cells. The IL-2 mutant of the present invention can be used to treat autoimmune diseases without the various side effects of immunotherapy using native IL-2.

Description

Technical Field

[0001] The present invention relates to the field of protein engineering. Specifically, the present invention relates to novel interleukin-2 (IL-2) mutants and methods for preparing the same. Compared to wild-type IL-2, the interleukin-2 (IL-2) mutants have reduced binding ability to their binding partners, the IL-2 receptor β subunit and the IL-2 receptor γ subunit, while maintaining corresponding biological activities, thereby better stimulating the proliferation of regulatory T cells. Background Art

[0002] IL-2 is a member of the interleukin family produced by activated T cells. It stimulates T cell proliferation, development, and differentiation by binding to the interleukin-2 receptor (IL-2R) on the cell surface. The IL-2 receptor, IL-2R, is composed of three subunits: α, β, and γ chains. Based on their affinity for IL-2, IL-2R can be divided into four types: high-affinity receptors (αβγ chain complex), intermediate-affinity receptors (βγ chain complex), low-affinity receptors (complexes containing only α or αγ chains), and pseudo-high-affinity receptors (αβ chain complex). IL-2 can only trigger intracellular signaling after binding to high-affinity or intermediate-affinity receptors.

[0003] Different types of T cells have varying sensitivities to IL-2, with regulatory T cells (Treg cells) being more sensitive to IL-2 than other cells. Because IL-2Rα is persistently expressed on the surface of Treg cells, Treg cells are more sensitive to IL-2 than NK cells, Teff cells, and other cells in the absence of external antigen stimulation. Utilizing differential IL-2 doses to selectively affect Treg and Teff cells can modulate the immune system.

[0004] Currently, low-dose IL-2 therapy has been used to treat autoimmune diseases such as type 1 diabetes (T1D), systemic lupus erythematosus (SLE), and chronic graft-versus-host disease (GVHD). However, IL-2 suffers from issues such as a short half-life in vivo, poor stability, a short therapeutic window for injection, and difficulty determining a safe dose and duration of treatment to balance immunosuppression and inflammation at the injection site.

[0005] Regulatory T cells (Tregs) express the transcription factor FOXP3 intracellularly and can be distinguished from effector T cells by their CD4+CD25+FOXP3+ identity. FOXP3 gene defects and mutations lead to a breakdown of self-tolerance and the development of autoimmune diseases due to Treg deficiency or dysfunction. IL-2 can stimulate T cell proliferation by binding to intermediate-affinity βγ dimers or high-affinity αβγ trimers. When the affinity of βγ dimers for IL-2 is reduced, mutant IL-2 preferentially stimulates Treg cells expressing αβγ receptors compared to wild-type IL-2, while avoiding stimulation of other T cell types that primarily express βγ receptors. Therefore, reducing IL-2's ability to bind to intermediate-affinity receptors (βγ chain complexes) can reduce IL-2's stimulatory effect on effector T cells, thereby increasing the ratio of Treg cells to effector T cells and ameliorating autoimmune responses caused by Treg or Treg dysfunction.

[0006] Therefore, novel interleukin-2 (IL-2) mutants that better stimulate the proliferation of regulatory T cells are needed for the treatment of various autoimmune diseases. Summary of the Invention

[0007] The present invention aims to provide a novel IL-2 mutant. Compared to wild-type IL-2, the IL-2 mutant of the present invention has reduced binding to its binding partners, the IL-2 receptor β subunit and the IL-2 receptor γ subunit, while maintaining corresponding biological activities, and can better stimulate the proliferation of regulatory T cells.

[0008] In a first aspect, the present invention provides an IL-2 mutant, wherein compared with wild-type IL-2, the amino acid residues of the IL-2 mutant are mutated, thereby reducing the affinity of the mutant interleukin-2 protein for the medium-affinity IL-2 receptor.

[0009] In a preferred embodiment, the intermediate affinity IL-2 receptor comprises only IL-2 receptor β subunit and IL-2 receptor γ subunit without IL-2 receptor α subunit.

[0010] In a preferred embodiment, the IL-2 mutant can increase the ratio of CD3+FoxP3+ cells to CD3+FoxP3- cells.

[0011] In a specific embodiment, the IL-2 mutant is mutated at the amino acid residue corresponding to position 90 of wild-type IL-2.

[0012] In a specific embodiment, compared to wild-type IL-2, the amino acid residues of the IL-2 mutant are mutated, thereby adding artificial glycosylation sites.

[0013] In a preferred embodiment, the glycosylation site is an N-glycan site or an O-glycan site; preferably an N-glycan site.

[0014] In a specific embodiment, the IL-2 mutant has an amino acid residue mutated compared to wild-type IL-2.

[0015] 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: N90A, N90G, N90V, N90I, N90M, N90L, N90F, N90Y, N90W, N90H, N90R, N90K, N90Q, N90D, N90E, N90P, N90T, N90B, N90C, N90S, N90Z, N90I;

[0016] 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;

[0017] More preferably, the IL-2 mutant has the following amino acid residue mutations at position 90 corresponding to wild-type IL-2: N90T, N90S;

[0018] Most preferably, the IL-2 mutant has the following amino acid residue mutation at position 90 corresponding to wild-type IL-2: N90T.

[0019] In a preferred embodiment, the IL-2 mutant has the following amino acid residue mutations at positions 3 corresponding to the wild-type IL-2 protein: T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K and T3P; preferably T3A.

[0020] In a preferred embodiment, the IL-2 mutant has a mutation in the cys site 125: C125L, C125S, C125A; preferably C125S.

[0021] In a second aspect, the present invention provides a fusion protein or conjugate, wherein the fusion protein or conjugate comprises the IL-2 mutant according to the first aspect and a non-IL-2 functional part.

[0022] In a preferred embodiment, the non-IL-2 functional part is selected from the group consisting of:

[0023] Fc fragments, including but not limited to: Fc fragments of human IgG1, IgG2, IgG3, IgG4, and Fc fragment mutants thereof with homology of more than 90%;

[0024] Human serum albumin (HSA);

[0025] Anti-HSA antibodies and fragments

[0026] anti-albumin peptides or antibodies;

[0027] transferrin;

[0028] human chorionic gonadotropin β-subunit carboxyl-terminal peptide (CTP);

[0029] elastin-like peptide (ELP);

[0030] Antigen binding portion.

[0031] In a preferred embodiment, the antigen binding portion is:

[0032] Antibodies or active antibody fragments thereof;

[0033] Fab molecules, scFv molecules and VHH molecules; or

[0034] Cell receptor or ligand.

[0035] 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 through a linker; the linker can be a repeating sequence of AAA or GS, including but not limited to a repeating sequence of G3S or a repeating sequence of G4S; for example (G3S)4.

[0036] In a preferred embodiment, the IL-2 mutant or fusion protein can be further modified as follows to form a conjugate:

[0037] polyethylene glycol modification (PEGylation);

[0038] Polysialylation modification (PSA);

[0039] saturated fatty acid modification;

[0040] Hyaluronic acid modification (HA);

[0041] Polyamino acid modification (proline-alamine-serine polymer, PAS-ylation).

[0042] In a third aspect, the present invention provides a polynucleotide encoding the IL-2 mutant according to the first aspect or the fusion protein or conjugate according to the second aspect.

[0043] In a fourth aspect, the present invention provides an expression vector comprising the polynucleotide described in the third aspect.

[0044] In a fifth aspect, the present invention provides a host cell, wherein the host cell comprises the expression vector described in the fourth aspect, or the genome of the host cell is integrated with the polynucleotide described in the third aspect.

[0045] 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.

[0046] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the IL-2 mutant protein described in the first aspect or the fusion protein or conjugate described in the second aspect and a pharmaceutically acceptable excipient.

[0047] In a seventh aspect, the present invention provides use of the IL-2 mutant described in the first aspect or the fusion protein described in the second aspect in the preparation of a medicament for autoimmune diseases.

[0048] In a preferred embodiment, the disease is a disease for which IL-2 is used for immunotherapy.

[0049] In a preferred embodiment, the disease is cancer, immune disease, human immunodeficiency virus (HIV) infection, hepatitis C virus (HCV) infection, rheumatoid arthritis, atopic dermatitis, etc.

[0050] In a preferred embodiment, the cancer, 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.

[0051] 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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The binding ability of IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc to IL-2Rα detected by Biacore is shown;

[0053] Figure 2 The binding affinity of IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc to IL-2Rβγ dimer detected by Biacore is shown;

[0054] Figure 3shows that the interleukin-2 mutant of the present invention and wild-type IL-2 stimulate the proliferation of NK92 cells; and

[0055] FIG4 shows the sequences SEQ ID NO: 1-5 used in the present invention. DETAILED DESCRIPTION

[0056] After extensive and in-depth research, the inventors unexpectedly discovered that novel IL-2 mutants, resulting from site-directed mutagenesis of the IL-2 polypeptide, can reduce binding to the IL-2Rβγ dimer while retaining its biological activity and preferentially stimulating the proliferation of regulatory T cells. Therefore, the IL-2 mutants of the present invention can be used to treat autoimmune diseases without the various side effects associated with immunotherapy using native IL-2. This foundation led to the completion of the present invention.

[0057] IL-2 mutants of the present invention

[0058] In the present invention, amino acid residues in the IL-2 polypeptide are altered through site-directed mutagenesis, thereby modifying the binding ability or affinity of IL-2 to its receptor while retaining biological activity. The IL-2 mutants of the present invention can stimulate the proliferation of regulatory T cells (Tregs) and have significantly reduced side effects compared to wild-type IL-2, thereby achieving better therapeutic goals.

[0059] The IL-2 mutants of the present invention are preferably expressed in eukaryotic cells and obtained through cell culture. Yeast, insect cells, animal cells, or even transgenic animals can be used as host cells. In specific embodiments, the host cells are eukaryotic cells; preferably yeast, insect cells, or animal cells; preferably mammalian cells, including but not limited to CHO cells, 293 cells, SP / 20 cells, and NS0 cells. Optionally, the IL-2 mutants of the present invention can be obtained by cell-free expression, in vitro synthesis, or other techniques.

[0060] When yeast cells or insect cells are used as host cells, the glycoforms of the IL-2 mutants that may be obtained are non-human. Those skilled in the art will appreciate that the non-human glycoforms can be further transformed into human glycoforms.

[0061] In other embodiments, IL-2 mutants can also be obtained using prokaryotic bacterial expression fermentation or in vitro cell-free synthesis.

[0062] The IL-2 mutants of the present invention are mutated at position 90 corresponding to wild-type IL-2. Therefore, in a specific embodiment, the IL-2 mutants of the present invention have the following amino acid residue mutations at position 90 corresponding to the wild-type IL-2 protein: N90A, N90G, N90V, N90I, N90M, N90L, N90F, N90Y, N90W, N90H, N90R, N90K, N90Q, N90D, N90E, N90P, N90T, N90B, N90C, N90S, N90Z and N90I; preferably N90T, N90S, N90V, N90I, N90M, N90L; more preferably N90T, N90S; most preferably N90T.

[0063] Based on conventional practices in the art, it is also possible to eliminate the existing O-glycan site in the IL-2 polypeptide. While removal of the O-glycan does not affect the biological activity of IL-2, the O-glycan structure is complex and difficult to analyze. To reduce the complexity of production quality control, genetic engineering mutagenesis techniques are often used to eliminate this glycosylation site. Therefore, the IL-2 mutants of the present invention can include the following amino acid residue mutations at positions corresponding to wild-type IL-2 protein: T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P; T3A is preferred. During the purification and renaturation of the IL-2 gene product, mispairing of disulfide bonds or the formation of intermolecular disulfide bonds can reduce IL-2 activity. Point mutations have been used to mutate the cysteine ​​at position 125 to leucine or seryl, limiting the formation of only one disulfide bond and ensuring activity during IL-2 renaturation. There are also reports of using protein engineering techniques to produce novel rIL-2, replacing the cysteine ​​residue at position 125 of the IL-2 molecule with an alanine residue. The modified IL-2 exhibits significantly increased specific activity compared to native IL-2. Therefore, the IL-2 mutants of the present invention may include the following amino acid residue mutations at position 125 corresponding to the wild-type IL-2 protein: C125L, C125A, or C125S; C125S is preferred.

[0064] "Corresponding to"

[0065] As used herein, the term "corresponding to" has the meaning commonly understood by those skilled in the art. Specifically, "corresponding to" refers to a position in one sequence that corresponds to a specified position in another sequence after alignment for homology or sequence identity. Thus, for example, "corresponding to wild-type IL-2" means aligning an amino acid sequence with the amino acid sequence of wild-type IL-2 to identify the position in the amino acid sequence that corresponds to wild-type IL-2.

[0066] Fusion proteins or conjugates of the present invention

[0067] Based on the IL-2 mutants of the present invention, those skilled in the art will appreciate that the IL-2 mutants of the present invention can be prepared into fusion proteins or conjugates with other functional parts other than IL-2. In this context, a conjugate refers to a water-soluble polymer covalently linked to the residues of a mutant IL-2 polypeptide. In a specific embodiment, the non-IL-2 functional part includes, but is not limited to, an Fc fragment, human serum albumin (HSA), anti-HSA antibodies and fragments, transferrin, human chorionic gonadotropin β subunit carboxyl-terminal peptide (CTP), elastin-like peptide (ELP), and an antigen-binding portion, and also includes cytokines, specifically interleukins, interferons, tumor necrosis factor superfamily, colony-stimulating factors, chemokines, growth factors, and the like.

[0068] Based on routine procedures in the art, those skilled in the art will know how to obtain fusion proteins or conjugates comprising the IL-2 mutants of the present invention. For example, the IL-2 mutants of the present invention can be directly linked to other non-IL-2 functional moieties or connected via a linker. The linker can be a repeating sequence of AAA or GS, including but not limited to a repeating sequence of G3S or a repeating sequence of G4S; for example, (G3S)4.

[0069] Furthermore, the IL-2 mutant or fusion protein conjugate can be modified with polyethylene glycol (PEGylation), polysialylation (PSA), saturated fatty acid, hyaluronic acid (HA) or polyamino acid (PAS) to form a conjugate.

[0070] Bispecific antibodies or trispecific antibodies of the present invention

[0071] Diseases are often caused by multiple pathogenic factors. Simultaneously blocking multiple targets may achieve better therapeutic effects, leading to the emergence of bispecific antibodies (BsAbs). Tumor immunotherapy is a new approach in tumor treatment. Bispecific antibodies can bind to two different antigens, and therefore have promising development prospects in the field of tumor treatment. Bispecific antibodies were initially prepared using chemical coupling or hybridoma hybridization methods. Today, the rapid development of recombinant DNA technology has revolutionized the structure of bispecific antibodies, which are now classified into two main categories: IgG-type bispecific antibodies containing an Fc region and non-IgG-type bispecific antibodies without an Fc region. IgG-type bispecific antibodies have a structure similar to monoclonal antibodies, with a larger relative molecular weight and a long plasma half-life. Non-IgG-type bispecific antibodies have a more diverse structure, a smaller relative molecular weight, and enhanced tissue penetration, but a shorter plasma half-life.

[0072] Based on the IL-2 mutants of the present invention, those skilled in the art will appreciate that the IL-2 mutants of the present invention can be covalently linked to antibody domains. In specific embodiments, the antibody domains include, but are not limited to, IgG antibodies and non-IgG antibodies. In preferred embodiments, the antibody domains can be antibodies or active antibody fragments thereof, Fab molecules, scFv molecules, VHH molecules, immunoglobulin molecules, receptor protein molecules, or ligand protein molecules; the immunoglobulin molecules can be IgG molecules.

[0073] Based on routine procedures in the art, those skilled in the art will understand how to obtain bispecific antibodies comprising the IL-2 mutants of the present invention. For example, the IL-2 mutants of the present invention can be linked directly to other non-IL-2 functional moieties or via a linker. The linker can be a repeating sequence of AAA or GS, including but not limited to a repeating sequence of G3S or a repeating sequence of G4S; for example, (G3S)4.

[0074] Optionally, the mutant of the present invention can be coupled with an antibody against a T cell surface antigen, or can be coupled with an antibody against a tumor cell surface antigen. Preferably, the mutant of the present invention can be coupled with an antibody against a T cell surface antigen.

[0075] The optional mutants of the present invention can be coupled with antibodies against T cell surface antigens to form bispecific antibodies, and can also be coupled with antibodies against tumor cell surface antigens to form bispecific antibodies. The optional mutants of the present invention can be coupled with antibodies against T cell surface antigens to form trispecific antibodies, and can also be coupled with antibodies against tumor cell surface antigens to form trispecific antibodies. The optional mutants of the present invention can be coupled with antibodies against T cell surface antigens or antibodies against tumor cell surface antigens to form trispecific antibodies.

[0076] Pharmaceutical composition of the present invention and its administration method

[0077] Based on the IL-2 mutants of the present invention, the present invention also provides pharmaceutical compositions. In specific embodiments, the pharmaceutical compositions comprise the IL-2 mutants of the present invention, the fusion protein or conjugate of claim 5, or the bispecific antibody or trispecific antibody of claim 7, and optionally, pharmaceutically acceptable excipients.

[0078] Optionally, the composition of the present invention further comprises a pharmaceutically acceptable excipient. If desired, a pharmaceutically acceptable excipient 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.

[0079] Uses and methods of use of the IL-2 mutants of the present invention

[0080] As described above, the IL-2 mutants of the present invention can reduce the affinity of the mutant IL-2 protein for the medium-affinity IL-2 receptor while retaining the biological activity of IL-2, thereby better stimulating the proliferation of regulatory T cells (Treg). Therefore, the IL-2 mutants, fusion proteins, conjugates, bispecific antibodies or trispecific antibodies, and pharmaceutical compositions of the present invention can be prepared into corresponding drugs. The drugs can be used to expand regulatory T cells (Treg) in vitro or treat diseases that use IL-2 for immunotherapy. In specific embodiments, the diseases are systemic lupus erythematosus (SLE), autoimmune diseases, diabetes, human immunodeficiency virus (HIV) infection, hepatitis C virus (HCV) infection, rheumatoid arthritis, atopic dermatitis, etc.

[0081] Advantages of the present invention:

[0082] 1. The IL-2 mutant protein of the present invention has reduced binding to IL-2Rβγ dimer.

[0083] 2. The structure of the IL-2 mutant of the present invention is closer to that of native IL-2, avoiding the effect of mutation on other structural sites of the protein and retaining biological activity;

[0084] 3. The interleukin-2 mutant protein of the present invention can be used to treat autoimmune diseases, but does not have the various side effects caused by immunotherapy using natural IL-2.

[0085] 4. Compared with other IL-2 mutants in the prior art, the IL-2 mutants of the present invention have lower immunogenicity;

[0086] 5. The IL-2 mutant of the present invention is easy to produce and control quality, generally does not require in vitro modification, and thus reduces steps and improves production efficiency;

[0087] 6. The IL-2 mutants of the present invention are convenient for forming bifunctional or multifunctional fusion proteins or immune compositions with other molecules;

[0088] 7. The IL-2 mutants of the present invention can be used for immunotherapy, but will not cause vascular (or capillary) leak syndrome (VLS) caused by native IL-2; and

[0089] 8. The in vivo half-life of the IL-2 mutant of the present invention is significantly prolonged.

[0090] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental procedures in the following examples, for which specific conditions are not specified, were generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer.

[0091] Example

[0092] Example 1. Synthesis of mutant interleukin-2 (IL-2) protein

[0093] The coding sequences of the IL-2 mutant IL-2-gmB2-hIgG4Fc (SEQ ID NO: 1) and wild-type IL2-N-hIgG4Fc (SEQ ID NO: 2) including the human IgG4Fc coding sequence were constructed into eukaryotic expression vectors by molecular cloning to prepare IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc expression vectors. Transient transfection expression of IL-2 mutant molecules was performed using 293E cells cultured in Freestyle medium. 24 hours before transfection, 0.5×10 6 150 ml of 293E cells at a concentration of 5 cells / ml were cultured in a 37°C, 5% CO2 incubator with a shaker at 120 rpm. For transfection, 150 μl of 293fectin was added to 2.85 ml of OptiMEM, mixed thoroughly, and incubated at room temperature for 2 minutes. Simultaneously, 150 μg of the plasmid for expressing IL-2 was diluted to 3 ml using OptiMEM. The diluted transfection reagent and plasmid were thoroughly mixed and incubated at room temperature for 15 minutes. The entire mixture was then added to the cells, mixed thoroughly, and cultured in a 37°C, 5% CO2 incubator with a shaker at 120 rpm for 7 days. The cell culture supernatant was collected, filtered through a 0.22 μm filter, and purified using a Q-HP ion exchange chromatography column (GE) using a linear elution using 20 mM Tris 0-500 mM NaCl, pH 8.0, with samples collected continuously by volume. The collected fractions were detected by SDS-PAGE using 4-20% gradient gel (GenScript), and the samples were combined according to the electrophoretic purity.

[0094] Number of mutations mutation site Example Mutant Name Protein Tagging sequence 2 90 IL-2gmB2(T3A,N90T) IgG4Fc SEQ ID NO: 1

[0095] Example 2. Preparation of receptor protein

[0096] In order to study the binding ability of IL-2 mutant molecules to IL-2Rα receptor and IL-2Rβγ heterodimeric receptor, human IL-2Rα receptor and IL-2Rβγ heterodimeric receptor proteins were prepared.

[0097] The human IL-2Rα receptor was designed by linking the IL-2Rα extracellular domain coding sequence with the 6×His Tag coding sequence (SEQ ID NO: 3) and cloning it into a eukaryotic expression vector. Transient transfection of IL-2Rα receptor was performed using 293E cells cultured in Freestyle medium. 24 hours before transfection, 0.5×10 6 150 ml of 293E cells at a concentration of 5 cells / ml were cultured in a 37°C 5% CO2 incubator on a shaker at 120 rpm. During transfection, 150 μl of 293fectin was added to 2.85 ml of OptiMEM, mixed thoroughly, and incubated at room temperature for 2 minutes. At the same time, 150 μg of the plasmid used to express the IL-2Rα receptor was diluted to 3 ml using OptiMEM. The diluted transfection reagent and plasmid were thoroughly mixed and incubated at room temperature for 15 minutes. Then, the entire mixture was added to the cells, mixed thoroughly, and cultured in a 37°C 5% CO2 incubator on a shaker at 120 rpm for 7 days. The cell culture supernatant was collected, filtered with a 0.22 μm filter membrane, and then purified using a Ni-NTA affinity chromatography column (GE), eluted under the conditions of 20 mM Pb-0.5 M NaCl-100 mM imidazole. The purified protein was detected by SDS-PAGE using a 4-20% gradient gel (GenScript).

[0098] The design of the human IL-2Rβγ heterodimeric receptor utilizes the characteristics of CH1 and CL pairing to complete heterologous pairing. hIL2Rβ (SEQ ID NO: 4) and hIL2Rγ (SEQ ID NO: 5) were cloned into eukaryotic expression vectors respectively. Transient transfection expression of the IL-2Rβγ heterodimeric receptor was performed in 293E cells cultured in Freestyle medium. 24 hours before transfection, 0.5×10 6150 ml of 293E cells at a concentration of 1 cell / ml were cultured in a 37°C, 5% CO2 incubator with a shaker at 120 rpm. For transfection, 150 μl of 293fectin was added to 2.85 ml of OptiMEM, mixed thoroughly, and incubated at room temperature for 2 minutes. Simultaneously, 75 μg of each plasmid expressing the IL-2Rβγ heterodimeric receptor (hIL2Rβ,γECD-His) was diluted to 3 ml in OptiMEM. The diluted transfection reagent and plasmids were thoroughly mixed and incubated at room temperature for 15 minutes. The entire mixture was then added to the cells, mixed thoroughly, and cultured in a 37°C, 5% CO2 incubator with a shaker at 120 rpm for 7 days. The cell culture supernatant was collected and filtered through a 0.22-μm filter. The supernatant was then purified using a MabSelect SuRe affinity chromatography column (GE). Elution was performed in 20 mM citric acid-sodium citrate, pH 3.0, and the pH was adjusted to neutral with 1 M Tris base. The purified protein was analyzed by SDS-PAGE using a 4-20% gradient gel (GenScript).

[0099] Example 3. Affinity test of binding receptor using biacore

[0100] To investigate the affinity of IL-2 mutants relative to wild-type receptors, the affinity of IL-2 mutant molecules IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc for the human IL-2Rα subunit was determined using Biacore 8K (GE) using recombinant monomeric IL-2Rα subunits under the following conditions: The human IL-2Rα subunit was immobilized on a CM5 chip (190 RU). 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) at a flow rate of 30 μl / min (association time 180 seconds, dissociation time 300 seconds). Regeneration of IL-2Rα was performed with 20 mM NaOH at 30 μl / min for 10 seconds. For IL-2Rα, data were fitted globally using 1:1 binding, RI≠0, and Rmax = .

[0101] The results are as follows Figure 1 wherein the Rmax value of IL-2-gmB2-hIgG4Fc is 25, and the Rmax value of IL2-N-hIgG4Fc is 25. Therefore, relative to wild-type IL2-N-hIgG4Fc, IL-2-gmB2-hIgG4Fc retains affinity for IL-2Rα dimer.

[0102] The affinity of the IL-2 mutant molecules IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc for the human IL-2Rβγ heterodimer was determined using Biacore 8K (GE) using recombinant hIL2Rβ,γ ECD-His heterodimer under the following conditions: IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc were immobilized on a Protein A chip (100 RU). Recombinant hIL2Rβ,γ ECD-His heterodimer was used as the analyte in HBS-EP buffer at 25°C. For IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc, the analyte concentration was reduced from 100 nM to 0.78 nM (1:2 dilution) at a flow rate of 30 μl / min (association time 180 seconds, dissociation time 300 seconds). For IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc, regeneration was performed with 10 mM Glycine (pH 1.5) at 30 μl / min for 30 seconds. For IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc, a 1:1 binding ratio was used, RI ≠ 0, and Rmax = local fit data.

[0103] The results are as follows Figure 2 As shown in FIG; wherein the maximum value of R of IL-2-gmB2-hIgG4Fc is 1.7, and the maximum value of R of IL2-N-hIgG4Fc is 4.0. Figure 2 As shown, IL-2-gmB2-hIgG4Fc has reduced affinity for IL-2Rβγ dimers relative to wild-type IL2-N-hIgG4Fc. Therefore, IL-2-gmB2-hIgG4Fc retains affinity for IL-2Rα and has reduced affinity for IL-2Rβγ dimers relative to wild-type IL2-N-hIgG4Fc.

[0104] Example 4. Cell proliferation analysis using NK92 cells

[0105] NK92 cells are an IL-2-dependent NK cell line derived from peripheral blood mononuclear cells of a 50-year-old Caucasian male with rapidly progressive non-Hodgkin's lymphoma. They express the IL-2 receptor α, β, and γ chains on their cell surface, a cell surface marker shared with regulatory T cells. Therefore, the present inventors evaluated the activity of IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc in a cell proliferation assay using NK92 cells.

[0106] NK92 cells in the logarithmic growth phase were harvested, washed once with basal medium MEM-α, and incubated (5000 cells / well) with various concentrations of IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc in experimental medium (MEM-α medium from Gibco (Cat. No. 32561-037) supplemented with 12.5% ​​fetal bovine serum and 12.5% ​​horse serum) in a 37°C, 5% CO2 incubator for 48 hours. 100 μl of the ATP detection substrate CellTiter-Glo (from Promega (Cat. No. G7571)) was added to each well, and full-wavelength fluorescence was measured using an end-point microplate reader (Molecular Devices (Model I3x)).

[0107] The results are as follows Figure 3 As shown in the figure, the activity of IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc was measured using a cell proliferation assay, and it was found that all tested articles induced NK92 cell growth in a dose-dependent manner. Therefore, IL-2-gmB2-hIgG4Fc retains biological activity relative to wild-type IL2-N-hIgG4Fc.

[0108] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto. Sequence Listing <110> Shanghai Gaipu Biotechnology Co., Ltd. <120> A mutant protein that proliferates regulatory T cells <130> P2020-0861 <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 377 <212> PRT <213> Artificial Sequence <400> 1 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val 1 5 10 15 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 225 230 235 240 Gly Gly Gly Ser Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu 245 250 255 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 260 265 270 Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 275 280 285 Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 290 295 300 Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 305 310 315 320 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Thr Val Ile 325 330 335 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 340 345 350 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 355 360 365 Ala Gln Ser Ile Ile Ser Thr Leu Thr 370 375 <210> 2 <211> 377 <212> PRT <213> Artificial Sequence <400> 2 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val 1 5 10 15 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 225 230 235 240 Gly Gly Gly Ser Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu 245 250 255 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 260 265 270 Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 275 280 285 Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 290 295 300 Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 305 310 315 320 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 325 330 335 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 340 345 350 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 355 360 365 Ala Gln Ser Ile Ile Ser Thr Leu Thr 370 375 <210> 3 <211> 257 <212> PRT <213> Artificial Sequence <400> 3 Met Asp Ser Tyr Leu Leu Met Trp Gly Leu Leu Thr Phe Ile Met Val 1 5 10 15 Pro Gly Cys Gln Ala Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro 20 25 30 His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn 35 40 45 Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr 50 55 60 Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys 65 70 75 80 Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro 85 90 95 Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro 100 105 110 Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro 115 120 125 Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val 130 135 140 Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His 145 150 155 160 Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His Gly Lys Thr Arg 165 170 175 Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly Glu Met Glu Thr Ser Gln 180 185 190 Phe Pro Gly Glu Glu Lys Pro Gln Ala Ser Pro Glu Gly Arg Pro Glu 195 200 205 Ser Glu Thr Ser Cys Leu Val Thr Thr Thr Asp Phe Gln Ile Gln Thr 210 215 220 Glu Met Ala Ala Thr Met Glu Thr Ser Ile Phe Thr Thr Glu Tyr Gln 225 230 235 240 Asp Asp Asp Asp Lys Ser Gly Gly Gly Gly Ser His His His His His 245 250 255 His <210> 4 <211> 356 <212> PRT <213> Artificial Sequence <400> 4 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro 20 25 30 Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu 35 40 45 Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp 50 55 60 Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln 65 70 75 80 Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu 85 90 95 Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly 100 105 110 Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser Ser Gly Gly 115 120 125 Gly Gly Ser His His His His His His Asp Asp Asp Asp Lys Ala Val 130 135 140 Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser Arg Ala Asn Ile 145 150 155 160 Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp Thr Ser Cys Gln 165 170 175 Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln Thr Cys Glu Leu 180 185 190 Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu Gly Ala 195 200 205 Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val Thr Leu Arg Val 210 215 220 Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala Ile Gln Asp Phe 225 230 235 240 Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile Ser Leu Gln Val 245 250 255 Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp Glu Ile Ser Gln 260 265 270 Ala Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu Ala Arg Thr Leu 275 280 285 Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu Thr Leu Lys Gln 290 295 300 Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro Asp Thr Gln Tyr 305 310 315 320 Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu Phe Thr Thr Trp 325 330 335 Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys Pro Ala Ala Leu 340 345 350 Gly Lys Asp Thr 355 <210> 5 <211> 381 <212> PRT <213> Artificial Sequence <400> 5 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 20 25 30 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 35 40 45 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 50 55 60 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 65 70 75 80 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 85 90 95 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 100 105 110 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Ser Gly Gly Gly 115 120 125 Gly Ser His His His His His His Asp Asp Asp Asp Lys Leu Asn Thr 130 135 140 Thr Ile Leu Thr Pro Asn Gly Asn Glu Asp Thr Thr Ala Asp Phe Phe 145 150 155 160 Leu Thr Thr Met Pro Thr Asp Ser Leu Ser Val Ser Thr Leu Pro Leu 165 170 175 Pro Glu Val Gln Cys Phe Val Phe Asn Val Glu Tyr Met Asn Cys Thr 180 185 190 Trp Asn Ser Ser Ser Glu Pro Gln Pro Thr Asn Leu Thr Leu His Tyr 195 200 205 Trp Tyr Lys Asn Ser Asp Asn Asp Lys Val Gln Lys Cys Ser His Tyr 210 215 220 Leu Phe Ser Glu Glu Ile Thr Ser Gly Cys Gln Leu Gln Lys Lys Glu 225 230 235 240 Ile His Leu Tyr Gln Thr Phe Val Val Gln Leu Gln Asp Pro Arg Glu 245 250 255 Pro Arg Arg Gln Ala Thr Gln Met Leu Lys Leu Gln Asn Leu Val Ile 260 265 270 Pro Trp Ala Pro Glu Asn Leu Thr Leu His Lys Leu Ser Glu Ser Gln 275 280 285 Leu Glu Leu Asn Trp Asn Asn Arg Phe Leu Asn His Cys Leu Glu His 290 295 300 Leu Val Gln Tyr Arg Thr Asp Trp Asp His Ser Trp Thr Glu Gln Ser 305 310 315 320 Val Asp Tyr Arg His Lys Phe Ser Leu Pro Ser Val Asp Gly Gln Lys 325 330 335 Arg Tyr Thr Phe Arg Val Arg Ser Arg Phe Asn Pro Leu Cys Gly Ser 340 345 350 Ala Gln His Trp Ser Glu Trp Ser His Pro Ile His Trp Gly Ser Asn 355 360 365 Thr Ser Lys Glu Asn Pro Phe Leu Phe Ala Leu Glu Ala 370 375 380

Claims

1. A fusion protein comprising an IL-2 mutant and a non-IL-2 functional portion, wherein the non-IL-2 functional portion is an Fc fragment, and the amino acid sequence of the fusion protein is shown in SEQ ID NO:

1.

2. A polynucleotide encoding the fusion protein according to claim 1.

3. An expression vector comprising the polynucleotide according to claim 2. 4 . A host cell, comprising the expression vector according to claim 3 , or the genome of the host cell is integrated with the polynucleotide according to claim 2 .

5. The host cell according to claim 4, wherein The host cell is a eukaryotic cell.

6. The host cell according to claim 5, wherein The host cell is a yeast or animal cell.

7. The host cell according to claim 6, wherein The host cell is an animal cell.

8. The host cell according to claim 7, wherein The host cell is a mammalian cell.

9. The host cell according to claim 8, wherein The host cell is a Chinese hamster ovary cell.

10. The host cell according to claim 7, wherein The host cell is an insect cell. Use of the fusion protein according to claim 1 for in vitro proliferation of regulatory T cells.

Images