Site-specifically conjugated PEGylated interleukin-2 variants with receptor affinity preferences and their uses

Abstract

Site-specifically PEG-modified interleukin-2 (IL-2) variants are provided. These variants have improved preference for Treg cells and extended half-life compared to parent IL-2. Also provided are compositions containing the IL-2 variants, nucleic acids encoding the IL-2 variants, vectors containing the nucleic acids, and host cells. Furthermore, therapeutic uses of the IL-2 variants and pharmaceutical compositions containing them are also provided.

Description

[Technical Field]

[0001] The present invention relates to PEGylated interleukin-2 (IL-2) variants and uses thereof. In particular, the present invention relates to PEGylated IL-2 variants with improved affinity for Treg cells and extended half-life compared to parent IL-2. The present invention also provides compositions comprising the IL-2 variants, nucleic acids encoding the IL-2 variants, vectors containing the nucleic acids, and host cells. The present invention also provides therapeutic uses of the IL-2 variants and pharmaceutical compositions containing them. [Background technology]

[0002] Regulatory T cells (Tregs) are a type of CD4+CD25+ T cell discovered in the 1970s. They are negative immunoregulatory cells whose primary function is to regulate immune responses and maintain immune homeostasis. Treg cells are central to immune homeostasis, maintaining tolerance to self-antigens and regulating immune responses to foreign antigens. Abnormalities in the number and function of Treg cells have been observed in various autoimmune diseases, including rheumatoid arthritis, type 1 diabetes, nephritis, multiple sclerosis, and systemic lupus erythematosus. Therefore, the development of methods to restore the number and function of Tregs for the treatment of various autoimmune diseases has attracted attention in recent years.

[0003] Interleukin-2 (IL-2) is a multifunctional cytokine expressed in various immune cells. It is primarily synthesized by activated T cells, especially helper T cells, and exerts biological functions such as regulating the body's immune response and enhancing the body's immune surveillance. In eukaryotic cells, human IL-2 (uniprot: P60568) is synthesized as a 153-amino acid precosed polypeptide, which is converted to the mature secreted IL-2 (containing 133 amino acids) after the removal of the N-terminal 20 amino acids.

[0004] In most cases, IL-2 exerts its effects via the IL-2 receptor (IL-2R). IL-2R is composed of three subunits: α (CD25), β (CD122), and γ (CD132). Different chains and their combinations result in receptor forms with different affinities for IL-2: (1) low-affinity receptor (IL-2Rα; CD25), (2) intermediate-affinity receptor (IL-2Rβγ), and (3) high-affinity receptor (IL-2Rαβγ). Cytotoxic CD8 + T cells and natural killer (NK) cells primarily express the intermediate-affinity receptor (IL-2Rβγ), whereas Treg cells, due to their high constitutive expression of CD25, primarily express the high-affinity receptor for IL-2 (IL-2Rαβγ) on their cell surface.

[0005] IL-2 binds to different IL-2 receptors and has two functions in the immune response: mediating the proliferation and activation of effector cells and maintaining peripheral immune tolerance. Specifically, as an immune system stimulator, it binds to the β and γ subunits to activate T cell proliferation and differentiation, induce the production of cytotoxic T lymphocytes (CTLs), stimulate the production, proliferation, and activation of natural killer (NK) cells, and exhibit antitumor activity. Meanwhile, an imbalance between Treg cells and effector T cells (Teff) is known to be the underlying cause of various autoimmune diseases. The IL-2Rα subunit on Treg cells significantly enhances the binding ability of IL-2 to the IL-2 receptor β and γ subunits. Therefore, IL-2 is thought to play an effective role in the treatment of autoimmune diseases.

[0006] To improve the efficacy of IL-2 in autoimmune diseases, existing ideas for engineering IL-2 in the prior art include: 1) extending the half-life of native IL-2 using PEG, Fc, etc. (e.g., CN201980034052). However, due to the bidirectional regulatory mechanism of native IL-2, PEGylated native IL-2 cannot completely eliminate the proliferative effects on Teff cells and NK cells, thereby affecting its therapeutic efficacy. 2) preparing IL-2 mutants that alter the receptor preference of IL-2. Because IL-2 itself is poorly stable, its mutants are usually less stable. Therefore, conventional random mutation methods for finding molecules with suitable properties cannot be applied to IL-2 protein. Even if IL-2 mutants (such as those disclosed in CN100366742C) are obtained, their short half-lives limit the feasibility of developing IL-2. 3) preparing IL-2 mutants to extend their half-life and fuse them to Fc or PEG. For example, CN201880081506 discloses a method for fusing an IL2 variant with Fc, and CN201580053284 discloses a method for fusing an IL2 variant containing a D109C substitution with PEG. These schemes address these issues to some extent. However, the IL-2 variants disclosed in CN201880081506, which have a combination of mutations at multiple sites, pose the risk of immunogenicity and half-life due to significant differences from the native protein sequence. The PEGylated IL2 variant containing N88R+D109C disclosed in CN201580053284 only extends half-life compared to the unmodified IL2 (N88R) variant, but its selectivity is not significantly improved, and the molecule still has significant NK cell activation activity.

[0007] Therefore, there remains a need to develop new IL-2 mutant molecules that have a high preference for Treg cells and a long half-life, particularly IL-2 mutant molecules that have advantageous properties for production and purification and improved pharmacodynamics. Summary of the Invention

[0008] The present invention meets these needs in part by providing novel PEG site-specifically conjugated IL-2 mutant molecules that have improved IL-2 receptor selectivity / preference, extended half-life, and / or improved developability compared to the parent IL-2.

[0009] In this application, we have obtained polyethylene glycol site-specifically conjugated interleukin-2 variants with improved selectivity, enhanced Treg activation, and enhanced in vivo efficacy through mutational modification of different sites in the parent IL-2, followed by site-specific PEGylation (referred to as PEGylated IL-2 variants or IL-2 variants; specific IL-2 variant molecules may also be referred to as "PEG-IL-2" molecules, usually followed by a description of the specific amino acid mutations found in the IL-2 molecule).

[0010] Thus, a first aspect of the present application provides novel PEGylated IL-2 variants that have one or more of the following advantages: (i) Enhanced binding preference for the receptor IL-2Rαβγ; (ii) reduced or abolished binding to IL-2Rβγ; (iii) It effectively activates STAT5 phosphorylation in Treg cells in vitro, but has no activating effect on STAT5 phosphorylation in Teff cells and NK cells. (iv) It activates Treg cells to a higher degree in vivo, but has no activating effect on Teff cells and NK cells. (v) Prolonged half-life, i.e., reduced dosing frequency. (vi) Improving the safety of IL-2 mutants by optimizing the PEG modification site. (vii) The number of mutations in the IL-2 protein is very low, reducing the immunogenic risk of the mutant protein in the body. (viii) The side effects of IL-2 are mainly caused by the activation of NK cells. However, the IL-2 variant of the present application has reduced or eliminated affinity for IL-2Rβγ, and therefore does not substantially activate NK cells. As a result, the toxic side effects of the IL-2 variant are significantly reduced or minimized.

[0011] In one aspect, the present application provides IL-2 variants comprising modifications of specific amino acid residues relative to the parent IL-2 with natural or unnatural amino acids, such modifications preferably being amino acid substitutions.

[0012] According to certain aspects, the IL-2 variants provided herein comprise amino acid substitutions for site-specific conjugation to a PEG molecule, whereby the PEG-conjugated IL-2 variants disclosed herein may also be referred to as PEGylated variants.

[0013] According to one aspect, the PEGylated IL-2 variants provided herein comprise one or more amino acid mutations at amino acid positions 33, 75, 77, 84, 88, 91, and 109, where the numbering of these amino acid positions is with reference to the parent sequence set forth in SEQ ID NO: 1. A PEG molecule is conjugated to any of the amino acids at positions 33, 75, 77, 84, 91, and 109.

[0014] In certain embodiments, the PEG molecule comprises a chemically reactive group for binding to a protein, such as a maleimide group, an o-dithiopyridyl group, an azide group, a p-toluenesulfonic acid group or a methanesulfonic acid group, or a hydroxylamine group (oxyamine). In certain embodiments, the PEG molecule is conjugated to a cysteine ​​residue, an unnatural amino acid, or any other method disclosed in the prior art. In still other embodiments, the PEG molecule comprises a chemically reactive group attached to an amino acid residue, such as an amino group, an alkynyl group, or a free thiol group. In certain embodiments, the chemically reactive group is a maleimide or iodoacetamide group and is conjugated to the free thiol group of a cysteine ​​residue. In certain embodiments, the chemically reactive group is a hydroxylamine group and is conjugated to a p-acetylphenylalanine residue. In certain embodiments, the chemically reactive group is an azide group and is conjugated to NE-propargyloxycarbonyl-L-lysine.

[0015] According to a particular embodiment, the PEG molecule may be a linear or branched molecule.

[0016] According to a particular embodiment, the average molecular weight of the PEG molecule is 5-100 KDa, preferably 20 K-100 K, more preferably 40 K-80 K.

[0017] According to certain aspects, the PEGylated IL-2 variants provided herein comprise one or more amino acid substitutions selected from N33C, S75C, N77C, D84C, N88C, N88R, D109C, and V91C, where the numbering of these amino acid positions is with reference to the parent sequence set forth in SEQ ID NO: 1.

[0018] According to certain aspects, the PEGylated IL-2 variants provided herein comprise one or more amino acid substitutions selected from N33Prk, N77Prk, or N88R, where the numbering of these amino acid positions is with reference to the parent sequence set forth in SEQ ID NO: 1.

[0019] According to certain aspects, the PEGylated IL-2 variants provided herein comprise one or more amino acid substitutions selected from N33pAcF, N77pAcF, or N88R, where the numbering of these amino acid positions is with reference to the parent sequence set forth in SEQ ID NO: 1.

[0020] According to a specific embodiment, in the PEGylated IL-2 variant provided herein, the PEG molecule is conjugated to a cysteine ​​at a position selected from positions 33, 75, 77, 84, 91, and 109 of the IL-2 molecule. Preferably, the PEG molecule is conjugated to one of these cysteines. More preferably, the PEG molecule is conjugated to the sulfhydryl group of the cysteine ​​at the corresponding position via a maleimide group contained therein. Most preferably, the PEG molecule is conjugated to the cysteine ​​at position 77 of the IL-2 molecule. According to a specific embodiment, each IL-2 molecule is conjugated to one PEG molecule. According to a specific embodiment, in the PEGylated IL-2 variant provided herein, the PEG molecule is conjugated to an unnatural amino acid (e.g., p-acetylphenylalanine) at a position selected from positions 33, 75, 77, 84, 91, and 109 of the IL-2 molecule. Preferably, the PEG molecule is conjugated to one of the unnatural amino acids (e.g., p-acetylphenylalanine). More preferably, the PEG molecule is conjugated to the unnatural amino acid at the corresponding position (e.g., p-acetylphenylalanine) via a hydroxylamine group contained therein. Most preferably, the PEG molecule is conjugated to the unnatural amino acid at position 77 of the IL-2 molecule (e.g., p-acetylphenylalanine). According to a specific embodiment, each IL-2 molecule is conjugated to one PEG molecule. According to a specific embodiment, in the PEGylated IL-2 variants provided herein, the PEG molecule is conjugated to an unnatural amino acid at a position selected from positions 33, 75, 77, 84, 91, and 109 of the IL-2 molecule (e.g., NE-propargyloxycarbonyl-L-lysine). Preferably, the PEG molecule is conjugated to one of the unnatural amino acids (e.g., NE-propargyloxycarbonyl-L-lysine). More preferably, the PEG molecule is conjugated to the unnatural amino acid at the corresponding position (eg, NE-propargyloxycarbonyl-L-lysine) via an azide group contained therein.Most preferably, the PEG molecule is conjugated to the unnatural amino acid at position 77 of the IL-2 molecule (e.g., NE-propargyloxycarbonyl-L-lysine). According to a particular embodiment, each IL-2 molecule is conjugated to one molecule of PEG.

[0021] According to certain aspects, the PEGylated IL-2 variants provided herein comprise the mutation N88R, and the PEG molecule is conjugated to a cysteine ​​residue or an unnatural amino acid (e.g., p-acetylphenylalanine, NE-propargyloxycarbonyl-L-lysine) at a position selected from positions 33, 75, 77, and 109 of the IL-2 molecule.

[0022] According to a specific embodiment, the PEGylated IL-2 variant provided herein comprises the mutation N88R, and the PEG molecule is conjugated to the cysteine ​​residue at position 77 of the IL-2 molecule or to an unnatural amino acid (e.g., p-acetylphenylalanine, NE-propargyloxycarbonyl-L-lysine). According to a preferred embodiment, the PEG molecule is a linear molecule. According to a preferred embodiment, the average molecular weight of the PEG molecule is 5-100 KDa, preferably 20 KDa-100 KDa, and more preferably 40 KDa-80 KDa.

[0023] According to one embodiment, the extended half-life of the PEGylated IL-2 variants provided herein is at least 3 hours, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 9 days. Such extended half-lives allow the IL-2 variants of the present application to be administered less frequently.

[0024] According to one embodiment, the PEGylated IL-2 variants provided herein have reduced binding affinity to the IL-2Rβγ receptor compared to the parent IL-2, e.g., the signal level of the STAT5 reporter gene at high concentrations is reduced by 5%-50%, preferably 15%-30%, of the IL-2Rβγ receptor.

[0025] According to one aspect, the PEGylated IL-2 variants provided herein increase IL-2-mediated CD25 cell activation and / or proliferation compared to the parent IL-2. + The cells are Treg cells. In one embodiment, the ability of an IL-2 variant to activate Treg cells is determined by detecting a change in the number of Treg cells expanded by the IL-2 variant in a pharmacodynamic (PD) study in wild-type mice. In one embodiment, the IL-2 variant of the invention increases the number of expanded Treg cells by at least 1-fold, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or 15-fold, compared to the parent IL-2 protein, as measured in a pharmacodynamic (PD) study in wild-type mice. In another embodiment, the ability and preference of an IL-2 variant to activate Treg cells is determined by detecting a change in the expansion of Treg, NK, and CD8+ T cells by the IL-2 variant in a pharmacodynamic and pharmacokinetic study in cynomolgus monkeys. The IL-2 variants of the invention increase the ratio of expanded Treg cells to CD3+ T cells in cynomolgus monkeys by at least 1-fold, for example 2-fold, 10-fold, 20-fold, 30-fold, 50-fold, 75-fold, or 90-fold. The ratio of expanded NK cells to PBMCs in cynomolgus monkeys is no more than 2-fold, and the ratio of expanded CD8+ T cells to CD3+ T cells in cynomolgus monkeys is no more than 2-fold.

[0026] According to one aspect, the IL-2 variants of the invention have a reduced IL-2-induced CD8 + Eliminates or reduces activation of T cells and NK cells.

[0027] In another embodiment, the IL-2 variants of the present invention may be conjugated to other non-immunogenic polymers. Examples of such polymers include recombinant non-immunogenic amino acid polymers, such as XTEN polymers, PAS polymers, and elastin-like polypeptides (ELPs). XTEN polymers contain a chain of A, E, G, P, S, and T amino acids expressed in Escherichia coli (Schellenberger, V., et al., 2009, Nat Biotechnol. 27:1186-90). PAS polymers are unstructured peptide polymers composed of repeating sequences of the amino acid residues P, A, and S (Schlapschy, M., et al., 2007, Protein Eng Des Sel. 20:273-84). Elastin-like polypeptides (ELPs) are random sequences consisting of repeating VPGxG sequences, primarily found in elastin. Here, x represents any protein other than proline (Doreen M. Floss et al., 2010, Trends Biotechnol. 28:37-45).

[0028] According to one embodiment, such non-immunogenic polymers may serve as an alternative to PEG molecules for conjugation to the aforementioned IL-2 variants.

[0029] In a second aspect, the present application provides pharmaceutical compositions and combination products comprising the IL-2 variants.

[0030] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0031] In a third aspect, the present application provides nucleic acids encoding the IL-2 variants of the invention, as well as vectors and host cells comprising said nucleic acids.

[0032] According to a fourth aspect, the present application provides a method for preparing the PEGylated IL-2 variant, the method comprising culturing the host cell of the third aspect under conditions suitable for expressing the IL-2 variant having the specific amino acid mutation, recovering the expressed variant, and subjecting the IL-2 variant to site-specific PEGylation.

[0033] According to a fifth aspect, the present application also provides methods of treating disease or reducing organ transplant rejection using the IL-2 variants and pharmaceutical compositions of the present invention.

[0034] According to a sixth aspect, the present application provides the use of the IL-2 variants and pharmaceutical compositions of the present invention in the manufacture of a medicament for the treatment of disease or the reduction of organ transplant rejection.

[0035] In one aspect, the present application provides IL-2 variants and pharmaceutical compositions of the present invention for use in the treatment of disease.

[0036] In one embodiment of the fifth and sixth aspects, the disease is an inflammatory disease (e.g., autoimmune disease, ankylosing spondylitis, amyotrophic lateral sclerosis, hepatitis C associated vasculitis, sclerosing cholangitis, inflammatory myopathy, relapsing polychondritis, Behcet's disease), coronary artery disease, macrophage activation syndrome, acute lung injury, hypertension, Takayasu's disease, Duchenne muscular dystrophy, transient ischemic attack, ischemic heart disease, Wiskott-Aldrich syndrome, bone marrow transplantation, or a transplant indication (e.g., corneal transplantation, pancreatic islet transplantation, or skin transplantation). According to certain embodiments, the autoimmune disease is rheumatoid arthritis, autoimmune encephalitis, type 1 diabetes, nephritis, multiple sclerosis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, Sjogren's syndrome, psoriasis, plaque psoriasis, alopecia areata, dermatomyositis, scleroderma, myasthenia gravis, demyelinating disease, inflammatory bowel disease, ulcerative colitis, Crohn's disease, autoimmune glomerulonephritis, pulmonary-renal hemorrhagic syndrome, graft-versus-host disease, organ transplant rejection, autoimmune hepatitis, autoimmune vasculitis, atopic dermatitis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, or idiopathic leukopenia. In particular embodiments, the autoimmune disease is rheumatoid arthritis, type 1 diabetes, nephritis, multiple sclerosis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, scleroderma, myasthenia gravis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, or atopic dermatitis.

[0037] According to a seventh aspect, the present application provides a method for improving the activation preference of the IL-2 receptor and significantly increasing the half-life of an IL-2 variant, the method comprising conjugating PEG to the cysteine ​​residue at position 77.

[0038] According to an eighth aspect, the present application provides a method for selectively stimulating regulatory T cells, the method comprising contacting a T cell population or peripheral blood with a PEGylated IL-2 variant as described above, thereby selectively stimulating STAT5 phosphorylation in regulatory T cells compared to non-regulatory T cells or NK cells.

[0039] According to one aspect, the present application provides a method for selectively stimulating regulatory T cells in a subject, the method comprising administering to the subject (e.g., a rodent or non-human primate) a PEGylated IL-2 variant as described above to selectively stimulate the proliferation of regulatory T cells relative to non-regulatory T cells or NK cells. According to one aspect, the method results in an increase in the ratio of regulatory T cells to non-regulatory T cells or the ratio of regulatory T cells to NK cells compared to before administration.

[0040] The present invention will be further described with reference to the following drawings and specific embodiments. However, these drawings and specific embodiments should not be construed as limiting the scope of the present invention. Modifications that can be easily conceived by those skilled in the art are also intended to be included within the spirit of the present invention and the scope of the appended claims. [Brief explanation of the drawings]

[0041] [Figure 1] FIG. 1 shows the signal values ​​of the IL-2βγ dual receptor activated with each PEG-IL-2 mutant at a concentration of 65.2 nM. [Figure 2] FIG. 2 shows the activity of each PEG-IL-2 variant at different concentrations when activating the IL-2βγ dual receptor. [Figure 3] FIG. 3 shows the activity of each PEG-IL-2 variant when activated against the IL-2αβγ triple receptor at different concentrations. [Figure 4] FIG. 4 shows the activity of each PEG-IL-2 variant when used at different concentrations to activate the IL-2βγ dual receptor. [Figure 5] FIG. 5 shows normalized signal values ​​when βγ dual receptors were activated with each PEG-IL-2 variant at a concentration of 250 nM. [Figure 6] FIG. 6 shows the activity of each PEG-IL-2 variant when used at different concentrations to activate the IL-2αβγ triple receptor. [Figure 7]FIG. 7 shows the effect of 80K-1PEG-IL2-N88R-N77C and 80K-1PEG-IL2-N88R-D109C on the body weight of wild-type mice after administration. [Figure 8] Figure 8 shows SDS-PAGE profiles of IL-2-N88R-N77C modified with PEGs of different molecular weights. Left: Coomassie brilliant blue staining, right: iodine staining. Lane 1: marker, lane 2: 80K-1PEG-IL2-N88R-N77C, lane 3: 60K-1PEG-IL2-N88R-N77C, lane 4: 40K-1PEG-IL2-N88R-N77C, lane 5: 20K-1PEG-IL2-N88R-N77C. [Figure 9] FIG. 9 shows the activation of human Treg cells by IL2-N88R-N77C mutants modified with PEG of different molecular weights. [Figure 10] FIG. 10 shows the activation of human NK cells by the IL2-N88R-N77C mutant modified with PEG of different molecular weights. [Figure 11] FIG. 11 shows the activation of human CD8+ T cells by IL2-N88R-N77C mutants modified with PEG of different molecular weights. [Figure 12] FIG. 12 shows an increase in peripheral Treg cells in mice after a single administration of IL2-N88R-N77C mutants modified with PEG of different molecular weights. [Figure 13] FIG. 13 shows the effect of IL2-N88R-N77C mutant modified with PEG of different molecular weights on peripheral CD8+ T cells in mice after a single administration. [Figure 14] FIG. 14 shows the effect of IL2-N88R-N77C mutant modified with PEG of different molecular weights on peripheral NK cells in mice after a single administration. [Figure 15] FIG. 15 shows PK concentration-time curves in mice after a single administration of IL2-N88R-N77C mutants modified with PEG of different molecular weights. [Figure 16]FIG. 16 shows the effect of IL2-N88R-N77C mutant modified with PEG of different molecular weights on ear thickness in a DTH mouse model. [Figure 17] FIG. 17 shows concentration-time curves in cynomolgus monkeys after administration of IL2-N88R-N77C mutants modified with PEG of different molecular weights. [Figure 18] FIG. 18 shows the effect of the IL2-N88R-N77C mutant modified with PEG of different molecular weights on peripheral NK cells in cynomolgus monkeys after administration. [Figure 19] FIG. 19 shows an increase in peripheral Treg cells in cynomolgus monkeys after administration of IL2-N88R-N77C mutants modified with PEG of different molecular weights. [Figure 20] FIG. 20 shows the effect of the IL2-N88R-N77C mutant modified with PEG of different molecular weights on peripheral CD8+ T cells in cynomolgus monkeys after administration. [Figure 21] FIG. 21 shows an increase in the intensity of CD25 protein expression in peripheral Treg cells of cynomolgus monkeys after administration of the IL2-N88R-N77C mutant modified with PEG of different molecular weights. [Figure 22] FIG. 22 shows an increase in the expression intensity of Foxp3 protein in peripheral Treg cells in cynomolgus monkeys after administration of the IL2-N88R-N77C mutant modified with PEG of different molecular weights. [Figure 23A-B] Figures 23A-E show the effects of the IL2-N88R-N77C mutant modified with PEG of different molecular weights on hematological indices (Na+, Cl-, EOS, ALB, CRP) in cynomolgus monkeys after administration. [Figure 23C-D] Same as above. [Figure 23E] Same as above. [Figure 24A]Figures 24A-C show the binding preference of each PEG-IL-2 variant with phenylalanine analog mutations to the IL-2 receptor. Specifically, Figure 24A shows the activity of each PEG-IL-2 variant when activating the IL-2βγ dual receptor at different concentrations. Figure 24B shows the normalized signal value of each PEG-IL-2 variant when activating the IL-2βγ dual receptor at a concentration of 250 nM. Figure 24C shows the activity of each PEG-IL-2 variant when activating the IL-2αβγ triple receptor at different concentrations. [Figure 24B-C] Same as above. [Figure 25] Figures 25A-C show the binding preference of each PEG-IL-2 variant with lysine analog mutations to the IL-2 receptor. Specifically, Figure 25A shows the activity when activating the IL-2βγ dual receptor at different concentrations. Figure 25B shows the normalized signal value of each PEG-IL-2 variant when activating the IL-2βγ dual receptor at a concentration of 250 nM. Figure 25C shows the activity of each PEG-IL-2 variant when activating the IL-2αβγ triple receptor at different concentrations. DETAILED DESCRIPTION OF THE INVENTION

[0042] Unless otherwise defined, all technical and scientific terms used in this disclosure have the meanings commonly understood by one of ordinary skill in the art. For purposes of the present invention, the following terms are defined below.

[0043] The term "about," when used in connection with a numerical value, is meant to include a range of numerical values ​​from a lower limit of 5% below that numerical value to an upper limit of 5% above that numerical value.

[0044] The term "and / or" should be understood to mean either any one of several alternatives or a combination of any two or more alternatives.

[0045] As used herein, the terms "comprising" or "including" mean the inclusion of the recited elements, integers, or steps, but do not exclude other elements, integers, or steps. When the terms "comprising" or "including" are used in this disclosure, unless otherwise specified, they also encompass situations consisting only of the recited elements, integers, or steps. For example, reference to an IL-2 variant "comprising" or "including" a particular mutation or combination of mutations is intended to encompass IL-2 variants having only that particular mutation or combination of mutations.

[0046] The term "parent interleukin-2" is used interchangeably with "parent IL-2" and "parent IL2" and refers to a parent IL-2 protein used as a template for introducing a mutation or combination of mutations of the present invention, preferably a native IL-2 protein, such as a native IL-2 protein from human, mouse, rat, or non-human primate. It refers to native IL-2 proteins from mouse, rat, or non-human primate, preferably human IL-2, in both unprocessed (e.g., signal peptide included) and processed (e.g., signal peptide removed) forms, preferably mature IL-2 proteins, such as the mature human IL-2 protein (uniprot:P60568). Parent "interleukin-2" also includes naturally occurring allelic and splice variants, isoforms, homologs, and species homologs of IL-2. In some embodiments, the parent IL-2 may contain amino acid mutations, compared to the native IL-2 protein, that do not affect binding to the IL-2 receptor. For example, human IL-2 proteins with substitutions at position 125, such as C125S and C125A, are included within the parent IL-2 of the present invention. This substitution removes the unpaired cysteine ​​residue at position 125 of IL-2, improving the stability of IL-2 and preventing IL-2 dimer formation. Examples include human IL-2 proteins that do not have an alanine at position 1, and combinations of the above mutations, such as IL-2 that do not have an alanine at position 1 and also have a C125S substitution.

[0047] Depending on the system for expressing a protein having IL-2 activity, the IL-2 protein may be either non-glycosylated or glycosylated. That is, the IL-2 protein may be non-glycosylated, or the IL-2 protein may be glycosylated. In one or more preferred embodiments, the IL-2 protein is non-glycosylated. The IL-2 protein may also be modified to contain and / or substitute one or more amino acid residues, e.g., lysine, cysteine, or arginine, to provide a site for polymer attachment via an atom in the side chain of the modified amino acid. Examples of substitutions in the IL-2 protein are described in U.S. Pat. No. 5,206,344. Furthermore, the IL-2 protein can be modified to contain a non-natural amino acid residue. Amino acid residues and techniques for adding non-natural amino acid residues are well known to those skilled in the art.

[0048] The amino acid mutation may be any of an amino acid substitution, deletion, insertion, or addition. In one embodiment, the amino acid mutation is one or more amino acid substitutions, for example, a single amino acid substitution or a combination of multiple amino acid substitutions. Amino acid deletions and insertions may occur at the amino and / or carboxy termini of the polypeptide sequence, or in internal regions of the polypeptide sequence. The amino acid substitutions of the present invention optionally include conservative amino acid substitutions.

[0049] Amino acid substitutions referred to in the present disclosure are described as single amino acid substitutions: (original amino acid residue / position / substituted amino acid residue). For example, a substitution of asparagine at position 88 with arginine is represented as N88R. When a combination of substitutions at multiple specific positions is represented, the individual amino acid substitutions are connected by the symbol "-". For example, the combination of substitutions N88R and D109C is represented as N88R-D109C.

[0050] References to amino acid positions in an IL-2 protein or IL-2 sequence refer to the amino acid sequence of the parent mature human IL-2 protein as set forth in SEQ ID NO: 1. Corresponding amino acid positions in other IL-2 proteins or polypeptides (full-length sequences or truncated fragments) are identified by aligning such amino acid sequences with SEQ ID NO: 1. Accordingly, in the present invention, amino acid positions in IL-2 proteins or polypeptides are numbered according to SEQ ID NO: 1, unless otherwise indicated. For example, reference to "N88" refers to the asparagine residue at position 88 of SEQ ID NO: 1, or the amino acid residue at the corresponding position in other IL-2 polypeptide sequences after alignment.

[0051] "Percent sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window. Preferably, sequence identity can be determined over the entire length of a reference sequence (e.g., SEQ ID NO: 1). Alignment methods for comparing multiple sequences are well known in the art. Suitable algorithms for determining percent sequence identity include, for example, the BLAST and BLAST 2.0 algorithms (see Altschul et al., Nuc. Acids Res. 25: 3389-402, 1977 and Altschul et al. J. Mol. Biol. 215: 403-10, 1990). Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information. For purposes of this application, identity will be determined using the Basic Local Alignment Search Tool, available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi, with default parameters.

[0052] "Affinity" or "binding affinity" refers to the intrinsic binding capacity that reflects the interaction between members of a binding pair. The affinity of a molecule X for a binding partner Y is determined by the dissociation and association rate constants (k dis and k on ) and the equilibrium dissociation constant (K D ) The binding affinity can be measured by common methods known in the art.

[0053] Polyethylene glycol (PEG) is a water-soluble polymer of ethylene glycol that offers advantages such as low toxicity, excellent amphiphilicity, low immunogenicity, and biocompatibility. PEG is available as linear or branched molecules of various sizes and can be modified with chemically reactive groups for conjugation to proteins. Various forms of PEG molecules have been developed as commercial products. Protein modification with PEG can be performed by random modification (e.g., because proteins typically contain multiple lysines, the ε-NH2 or α-NH2 of lysines is often used as the modification site, resulting in a mixture of randomly modified polyethylene glycols) or site-specific modification. Site-specific modification typically involves modifying cysteines in proteins. Most cysteine ​​residues in proteins form disulfide bonds to stabilize the protein's three-dimensional structure, but a small number of free (unpaired) cysteine ​​residues are usually present buried within the protein. Conjugation of PEG to free cysteines in proteins typically requires either a protein with a naturally occurring free cysteine ​​residue or the introduction of a new free cysteine ​​residue. Introducing a free cysteine ​​into a protein typically faces the following risks: On the one hand, the newly introduced cysteine ​​may form an incorrect intrachain disulfide bond with other cysteines in the protein, causing the protein to misfold; on the other hand, it may form an interchain disulfide bond with other molecules, causing the protein to aggregate. For example, Wang et al. found that a mutated cysteine ​​residue in IL-2 reduced the activity of IL-2 due to an incorrect disulfide bond (Wang, A., et al., 1984 Science. 224: 1431-3).

[0054] The term "PEGylated IL-2 mutant" refers to an IL-2 protein to which PEG is covalently attached. The IL-2 protein may be wild-type IL-2 (e.g., human IL-2 molecule) or a mutant IL-2 molecule (e.g., C125S-substituted human IL-2, N88R-substituted human IL-2). Because wild-type IL-2 has a short half-life in vivo, high doses or frequent repeated administration are usually required to maintain a necessary amount of IL-2 in the circulation. Therefore, wild-type IL-2 must be modified to extend its serum half-life. The PEGylated IL-2 mutant of the present application extends the half-life of IL-2, allowing Treg cells to be effectively stimulated even with lower doses or less frequent administration of the IL-2 protein. As a result, IL-2 toxicity to the subject is reduced, the subject's tolerance to IL-2 is increased, and compliance with IL-2 therapy is improved.

[0055] Polynucleotides, Vectors, and Hosts The present invention provides nucleic acids encoding the aforementioned IL-2 variants or PEGylated conjugates. Polynucleotide sequences encoding the muteins of the present invention can be generated by de novo solid-phase DNA synthesis using methods well known in the art or by PCR mutagenesis of an existing sequence encoding the parent IL-2. Furthermore, the polynucleotides and nucleic acids of the present invention can comprise a segment encoding a secretory signal peptide, operably linked to the segment encoding the mutein of the present invention, thereby directing the secretory expression of the mutein of the present invention.

[0056] The present invention also provides a vector comprising the nucleic acid of the present invention. In one embodiment, the vector is an expression vector, such as a eukaryotic or prokaryotic expression vector. In a preferred embodiment, the expression vector of the present invention is, for example, a pcDNA3.1 expression vector, a pBV220 expression vector, a p1128 expression vector, or a pET30a(+) expression vector.

[0057] The present invention also provides prokaryotic and eukaryotic host cells containing the nucleic acid or vector. Host cells suitable for replicating and supporting the expression of the mutant IL-2 protein or PEGylated conjugate are well known in the art. By transfecting or transducing such cells with a specific phenotype vector and culturing the vector-containing cells in large quantities to inoculate large-scale fermenters, sufficient quantities of the IL-2 mutant or PEGylated conjugate can be obtained for clinical use. In one embodiment, the host cell is a prokaryotic cell, such as Escherichia coli (see Fischer et al. (1995) Biotechnol. Appl. BioIL-2m. 21(3):295-311). In another embodiment, the host cell is a eukaryotic cell. In another embodiment, the host cell is selected from yeast cells or mammalian cells (e.g., CHO cells or 293 cells). For example, polypeptides can be produced in bacteria, particularly when glycosylation is not desired. After expression, the polypeptide can be isolated from the bacterial cell paste into a soluble fraction, which can then be further purified. In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi and yeast, are suitable cloning or expression hosts for polypeptide-encoding vectors. This includes fungal and yeast strains that have been "humanized" in their glycosylation pathways to produce polypeptides with partially or fully human glycosylation patterns. See Gerngross, Nat Biotech 22, 1409-1414 (2004) and Li et al., Nat Biotech 24, 210-215 (2006).

[0058] Pharmaceutical compositions and pharmaceutical preparations The present invention encompasses compositions (e.g., pharmaceutical compositions or pharmaceutical formulations) comprising the IL-2 variants or PEGylated conjugates thereof, and compositions comprising polynucleotides encoding the IL-2 variants or PEGylated conjugates thereof. These compositions may optionally contain suitable pharmaceutical adjuvants known in the art, such as pharmaceutical carriers, pharmaceutical excipients, e.g., buffers, etc.

[0059] Pharmaceutical carriers suitable for use in the present invention include sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. For injectable solutions, saline, aqueous dextrose, and glycerol solutions can also be used as liquid carriers. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, and the like. For information on the use and application of excipients, see also "Handbook of Pharmaceutical Excipients," Fifth Edition, R.C. Rowe, P.J. Seskey, and S.C. Owen, Pharmaceutical Press, London, Chicago. If desired, the composition may also contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, capsules, powders, sustained-release formulations, etc. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin, etc.

[0060] The formulations of the present invention can be prepared by mixing the IL-2 variant or PEGylated conjugate of the present invention having the desired purity with one or more optional formulation additives (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of a lyophilized formulation or aqueous solution. Examples of lyophilized antibody formulations include those described in U.S. Pat. No. 6,267,958. Examples of aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO 2006 / 044908. The latter formulations include histidine acetate buffer. Furthermore, sustained-release formulations can also be prepared. Preferred examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the protein. The matrices are in the form of shaped articles, such as films or microcapsules.

[0061] According to one embodiment, the pharmaceutical composition of the present invention comprises a buffer solution having a pH of 6 to 8, such as a PBS buffer or a histidine buffer. According to one embodiment, the PBS buffer solution is, for example, a PBS buffer solution having a pH of about 7.4. According to one embodiment, the histidine buffer solution is, for example, a histidine buffer solution having a pH of about 6.5, containing 10 mM histidine, 5% sorbitol, and 0.02% polysorbate 80. The pharmaceutical composition of the present invention is preferably storage stable in such a buffer solution.

[0062] The pharmaceutical compositions or formulations of the present invention may optionally contain one or more other active ingredients, preferably active ingredients whose complementary activities do not interfere with each other, depending on the particular indication being treated. For example, it may be desirable to provide other active ingredients for the treatment of autoimmune diseases in an amount effective for that purpose, appropriately combined.

[0063] Treatment methods and uses As used herein, the terms "individual" and "subject" are used interchangeably to refer to mammals, including, but not limited to, domestic animals (e.g., cows, sheep, cats, dogs, horses, etc.), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the subject is a human.

[0064] As used herein, the term "treatment" refers to a clinical intervention aimed at altering the natural course of disease in the individual being treated. Desirable therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of direct or indirect pathological consequences of disease, prevention of metastasis, slowing of disease progression, amelioration or palliation of the disease state, and alleviation or improvement of prognosis.

[0065] In one aspect, the present invention provides a method for suppressing the immune system of a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising an IL-2 variant or PEGylated conjugate of the present invention. The IL-2 variant of the present invention has high activity and selectivity for Treg cells and inhibits cytotoxic CD8 + The IL-2 variants of the present invention have a reduced stimulatory effect on T cells and NK cells and can therefore be used in lower doses to suppress the immune system of a subject.

[0066] Thus, in one aspect, the present invention provides a method for suppressing an immune response in a subject, comprising administering to the subject an effective amount of an IL-2 variant or a PEGylated conjugate thereof of the present invention. In one aspect, the IL-2 variant or a PEGylated conjugate thereof of the present invention is administered to a subject suffering from an autoimmune disease to suppress the immune response.

[0067] In another aspect, the present invention relates to a method for treating a disease, such as an autoimmune disease, in a subject, comprising administering to the subject an effective amount of an IL-2 variant or a PEGylated conjugate thereof, or a pharmaceutical composition as described herein.

[0068] The muteins of the present invention (and pharmaceutical compositions comprising the muteins or PEGylated conjugates thereof and, optionally, additional therapeutic agents) can be administered in any suitable manner. Such administration methods include parenteral, intrapulmonary, intranasal, and, optionally, intralesional administration for localized treatment. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any suitable route, for example, injections, such as intravenous or subcutaneous injections, depending on whether the administration is short-term or long-term. Various administration schedules are contemplated by the present disclosure. Examples include, but are not limited to, single doses, multiple doses, bolus administration, pulse infusion, etc.

[0069] The dosage of the mutant protein of the present invention (when used alone or in combination with one or more other therapeutic agents) suitable for the prevention or treatment of disease will vary depending on the type of disease being treated, the severity and course of the disease, whether the administration is for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history, and the judgment of the treating physician.

[0070] According to another aspect, the present invention also provides the use of an IL-2 variant, composition or PEGylated conjugate of the invention in the preparation of a medicament for use in the aforementioned methods (e.g. treatment). According to one aspect, the present invention also provides the IL-2 variant, composition and PEGylated conjugate of the invention for use in the treatment of disease. [Example]

[0071] To facilitate understanding of the present invention, the following examples are provided, however, these examples do not limit the scope of protection of the present invention and should not be construed as such.

[0072] Unless otherwise specified, the experimental methods described in the following examples were all carried out using methods known in the art using default parameters, steps, etc., and all experimental materials used were commercially available unless otherwise specified. Unless specific techniques or conditions are described, they were carried out according to methods described in literature in the art or product instructions. Reagents and equipment without a specified manufacturer were all standard commercially available products.

[0073] Example 1: Construction, expression and purification of interleukin-2 mutants Based on the protein sequence of human mature IL-2 (C125S) (SEQ ID NO: 1), cDNAs encoding IL-2 mutants with each amino acid mutation listed in Table 1 were designed according to the mutation sites indicated. An initiation codon and an EcoRI restriction enzyme site were added to the 5' end of each cDNA, and a termination codon and a BamHI restriction enzyme site were added to the 3' end. The cDNAs were prepared by DNA synthesis and cloned into the temperature-inducible expression vector p1128. The expression vector p1128 containing the corresponding cDNA sequence was transformed into DH5α-sensitive cells using the calcium chloride method. Positive clones were isolated from kanamycin-resistant plates, and the sequences were confirmed before use as expression strains for each mutant protein.

[0074] SEQ ID NO: 1 (IL-2, containing C125S) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT

[0075] [Table 1]

[0076] The strains expressing each IL-2 mutant protein were inoculated into Terrific Broth (TB broth) and cultured at 35°C and 250 rpm until logarithmic growth phase. The temperature was then raised to 42°C and cultured for 4 hours to induce expression of each mutant protein. The bacterial solution was then centrifuged at 13,900 g to recover the bacterial suspension. The bacteria were washed, resuspended in a buffer containing 50 mM Tris-HCl and 5 mM EDTA, pH 8.0, disrupted at 700-1,000 atmospheres, and centrifuged at 13,900 g to recover the inclusion bodies.

[0077] Next, the IL-2 mutein present as inclusion bodies was reconstituted and purified. Specifically, the inclusion bodies were washed with a wash buffer containing 2% Triton X-100 and centrifuged to collect the precipitate. The washed inclusion bodies were dissolved in 7 M guanidine hydrochloride solution, the guanidine hydrochloride concentration was reduced to 2 M, and the supernatant was collected by centrifugation. The supernatant was dialyzed overnight against 10 mM NaAc (pH 4.5) containing 5% trehalose, with one buffer exchange during the dialyzed period. After reconstitution, the solution was filtered to remove the precipitate and purified using CM Sepharose FF ion exchange chromatography with 0-500 mM NaCl solution. The buffers used for CM Sepharose FF ion exchange chromatography were: Buffer A: 30 mM PB phosphate buffer, pH 6.5; Buffer B: 30 mM PB phosphate buffer, 1 M NaCl, pH 6.5. The chromatography column was equilibrated with 3-5 column volumes of buffer A, and the reconstituted solution was then loaded onto the chromatography column. After loading, the chromatography column was washed with buffer A, and then gradient elution was performed with 0 to 50% buffer B in a volume 10 times the column volume, and the purified sample was collected.

[0078] Thus, various IL-2 mutants were obtained with specific mutation sites as listed in Table 1. Each IL-2 mutant was named based on its mutation site, as shown in Table 1. For example, mutant IL2-N88C is an IL-2 mutant in which the asparagine at amino acid position 88 of the human IL-2 protein sequence is substituted with cysteine, and mutant IL2-N88R-N33C is an IL-2 mutant in which the asparagine at amino acid position 88 of the human IL-2 protein sequence is substituted with arginine and the asparagine at amino acid position 33 of the human IL-2 protein sequence is substituted with cysteine.

[0079] Example 2: Polyethylene glycol modification and purification of IL-2 mutants Each IL-2 mutant obtained in Example 1 was modified with polyethylene glycol. The PEG used in this example contains a maleimide group that specifically reacts with the cysteine ​​residue at the specific mutation site of the mutant, allowing the corresponding IL-2 mutant to be site-specifically modified with PEG. Specifically, purified IL-2 mutant protein was isolated and adjusted to pH 6.5 and a protein concentration of 3 mg / ml. PEG-maleimide (e.g., 80 kDa PEG, Xiamen Sinopeg) was added at a molar ratio of IL-2 mutant:PEG of 1:5, and the mixture was allowed to react overnight at 2-8°C.

[0080] The reaction solution was purified using a GE MacroCapSP ion exchange column. The reaction solution was diluted 10-fold with ultrapure water, the pH was adjusted to 5.2, and the sample was injected onto the chromatography column. The buffers used for the MacroCapSP ion exchange column were as follows: Buffer A: 40 mM sodium acetate, pH 5.2; Buffer B: 40 mM sodium acetate, 1 M NaCl, pH 5.2. The chromatography column was first equilibrated with 3-5 volumes of Buffer A, and then the resulting PEGylated IL-2 mutant solution was injected onto the column. After injection, the chromatography column was washed with Buffer A and then gradient eluted with 10 volumes of Buffer B from 0 to 50% to recover and characterize the PEG-modified IL-2 mutant protein. The modified mutant protein was named according to the following convention: PEG MW-nPEG-IL-2-amino acid substitution, where n represents the number of PEGs attached to one IL-2 molecule. For example, the IL-2 variant 80k-1PEG-IL-2-V91C refers to an IL-2 variant in which one molecule of PEG with a molecular weight of 80k is attached to a substituted cysteine ​​residue at amino acid position 91 of the IL-2 molecule.

[0081] Example 3: Binding preferences of different PEG-mutated IL-2 variants to the IL-2 receptor In this example, the IL-2 mutants obtained in Example 2 that were PEG-modified at different sites were tested for binding to various IL-2 receptors, and the biological activities of the corresponding mutants were also tested.

[0082] Binding of IL-2 to the β and γ subunits of its receptor induces phosphorylation of STAT5, followed by dimerization and nuclear translocation to promote target gene transcription. Thus, in this example, the ability of mutant IL-2 polypeptides to induce signal transduction through the IL-2 receptor is assessed by measuring the phosphorylation of STAT5.

[0083] 1. Construction of 293EC18 / βγ / JAK3 / STAT5 and 293EC18 / αβγ / JAK3 / STAT5 cells Adherent 293EC18 cells were cultured in a conventional manner until they reached approximately 90% confluence. After trypsin digestion, the cells were cultured in cell culture medium at 4 × 10 5The cells were resuspended at 2000 cells / ml and 50 μl / well was added to two 96-well cell culture plates. After incubation at 37° C. for 48 hours, the supernatant was aspirated and fresh culture medium was added. Four plasmids, pcDNA3.1(+) / CD122 (constructed by Nanjing Novoacine Biotechnology Co., Ltd., Uniprot P14784, lMet-551Val integrated into the pcDNA3.1(+) plasmid), pcDNA3.1 / puro / CD132 (constructed by Nanjing Novoacine Biotechnology Co., Ltd., Uniprot P31785, lMet-551Thr integrated into the pcDNA3.1 / zeo plasmid), pCMV / JAK3 (Sino Biological), and pGL4.52 / STAT5RE (Promega), were transiently transfected into 293EC18 cells using a cationic transfection reagent (ExFect Transfection Reagent, Vazyme) according to the manufacturer's protocol. The resulting cells were designated 293EC18 / βγ / JAK3 / STAT5 cells. Five plasmids were selected: pcDNA3.1 / zeo / CD25 (constructed by Nanjing Novoacine Biotechnology Co., Ltd., Uniprot P015891Met-551Ile, constructed in the plasmid pcDNA3.1 / zeo), pcDNA3.1(+) / CD122 (constructed by Nanjing Novoacine Biotechnology Co., Ltd., Uniprot P14784, 1Met-551Val integrated into the plasmid pcDNA3.1(+)), pcDNA3.1 / puro / CD132 (constructed by Nanjing Novoacine Biotechnology Co., Ltd., Uniprot P31785, 1Met-551Thr integrated into the plasmid pcDNA3.1 / zeo), and pCMV / JAK3 (constructed by Sino 293EC18 cells were transiently transfected with PGL4.52 / STAT5RE (Promega) and 293EC18 / αβγ / JAK3 / STAT5 cells. The transfected cells were incubated at 37°C and 5% CO for an additional 6 hours.

[0084] 2. Testing the activation effect of each IL2 mutant on βγ and αβγ Each IL2 variant was serially diluted 4-fold with PBS buffer to create a total of 11 dilutions. Each IL2 variant sample was diluted 2-fold and added to the cell culture plate at 50 μl / well. The cell culture plate was then placed in a 37°C, 5% CO2 incubator and incubated overnight. The cell culture plate was then kept at room temperature for 20 ± 5 minutes to allow the plate to equilibrate to room temperature. The Bio-Lite Luciferase Assay System reagent was mixed and equilibrated at room temperature and added to the cell culture plate at 100 μl / well. The plate was mixed on a microplate shaker at 450 rpm for 2 minutes, then left to stand at room temperature for 3 minutes. Chemiluminescence readings were measured using an enzyme plate reader.

[0085] The logarithm of the final concentration of each IL-2 mutant sample was plotted on the X-axis and the chemiluminescence detection results on the Y-axis. Data were analyzed using a four-parameter logistic regression model in GraphPad Prism 9. The results are shown in Figures 1-3 and Table 2.

[0086] [Table 2]

[0087] The N88R single point mutation disclosed in the prior art significantly reduces the ability to activate Teff cells, but still retains a relatively strong ability to activate NK cells, posing a potential safety risk. This is also confirmed in the present application. See, for example, the results in Figure 10.

[0088] As shown in Figure 1, of all the tested compounds, 80k-1PEG-IL2-N88R-N33C, 80k-1PEG-IL2-N88R-S75C, and 80k-1PEG-IL2-N88R-N77C all had lower IL-2Rβγ dual receptor activation effects at a concentration of 65.2 nM than the unPEG-modified control IL-2N88R. In particular, 80k-PEG-IL2-N88R-N77C showed the most pronounced decrease in activation ability. As shown in Figure 2, the IL-2Rβγ dual receptor activation effects of each mutant were significantly reduced at all concentrations compared to the unPEG-modified control IL-2N88R. This suggests that the IL-2N88R-based mutants 80kJ-1PEG-IL2-N88R-N33C, 80kJ-1PEG-IL2-N88R-S75C, and 80kJ-1PEG-IL2-N88R-N77C further reduce the affinity for IL-2Rβγ, with 80kJ-1PEG-IL2-N88R-N77C showing the most significant reduction in activation.

[0089] The results in Table 2 and Figure 3 demonstrate that all of the PEG-modified IL-2 variants obtained in this study maintain excellent IL-2Rαβγ triple receptor activation ability compared to the unPEG-modified control IL2-N88R. In particular, 80kJ-1PEG-IL2-N88R-N33C and 80kJ-1PEG-IL2-D84C showed even stronger IL-2Rαβγ triple receptor activation than IL-2-N88R.

[0090] Analysis indicated that the PEGylated IL-2 variants (80kJ-1PEG-IL2-N88R-N33C, 80kJ-1PEG-IL2-N88R-S75C, and 80kJ-1PEG-IL2-N88R-N77C) better retained affinity for the IL-2Rαβγ triple receptor while minimizing affinity for the IL-2Rβγ dual receptor, suggesting a high T cell preference, i.e., the ability to stimulate Treg cells in vivo while largely avoiding activation of effector T cells (Teff).

[0091] Example 4: Effect of modification with polyethylene glycol of different molecular weights on the receptor binding preference of IL-2 mutants Following the method of Example 2, IL2 variants modified with PEG of different molecular weights were prepared and purified as shown in Table 3. See Example 2 for naming conventions.

[0092] [Table 3]

[0093] The binding preference of each polyethylene glycol-modified mutant obtained in this example for the IL2 receptor was evaluated by reporter assay according to the method of Example 3. The results are shown in FIGS.

[0094] [Table 4]

[0095] The results in Figures 4 and 5 indicate that PEG-IL2-N88R-N77C had the lowest binding affinity to the IL-2Rβγ dual receptor and the lowest activation effect. PEG-IL2-N88R-D109C exhibited activation effects comparable to those of the control IL2-N88R, and both were higher than those of PEG-IL2-N88R-N77C. This suggests that the additional substitution of a cysteine ​​at amino acid residue 77 of IL-2 followed by PEG modification further reduces its affinity for βγ compared to the control IL2-N88R.

[0096] As shown in Figure 6, compared with the control IL-2N88R, the IL-2 variants with the additional mutations N77C or D109C and modified with PEG of different molecular weights at the corresponding sites substantially retained their affinity for the IL-2Rαβγ triple receptor and their activation effects were comparable to those of the control. Furthermore, the 80k PEG-modified variant 80K-1PEG-IL2-N88R-N77C had slightly higher activation activity and retained stronger Treg cell preference than 80K-1PEG-IL2-N88R-D109C.

[0097] These studies demonstrated that PEG-IL2-N88R-N77C has the significant advantage of further reducing the affinity of IL-2 for the IL-2Rβγ dual receptor while better maintaining its affinity for the IL-2Rαβγ triple receptor. Therefore, the following experiments were carried out using PEG-IL2-N88R-N77C as a candidate.

[0098] Example 5: Effects of 80K-1PEG-IL2-N88R-D109C and 80K-1PEG-IL2-N88R-N77C on mice The effects of 80K-1PEG-IL2-N88R-D109C and 80K-1PEG-IL2-N88R-N77C on mouse body weight and physiological characteristics were investigated using Balb / c mice. SPF-grade Balb / c mice were purchased and divided into groups of three. Treatment began on the day of group assignment. The test substance or PBS (negative control group) was administered subcutaneously at a dose of 1 mg / kg once every three days for a total of three doses. Mice were weighed before each administration and three days after the third administration. The results are shown in Figure 7. Body weight in the 80K-1PEG-IL2-N88R-D109C-treated group began to decrease after the second administration and was significantly lower than the negative control group three days after the third administration (p = 0.009). In contrast, body weight in the 80K-1PEG-IL2-N88R-N77C group was not significantly different from the negative control group. Furthermore, visual observation revealed that mice treated with 80K-1PEG-IL2-N88R-D109C showed symptoms such as lethargy, decreased mobility, frequent curling up, and shaggy coats, whereas mice treated with 80K-1PEG-IL2-N88R-N77C showed normal mobility and smooth, shiny coats in good condition, similar to those of negative control mice. The experimental results showed that 80K-1PEG-IL2-N88R-N77C had minimal toxic side effects, while 80K-1PEG-IL2-N88R-D109C showed relatively obvious toxic side effects in mice.

[0099] Example 6: Construction of PEG-IL2-N88R-N77C mutants modified with PEG of different molecular weights Following the methods of Examples 1 and 2, PEGs of different molecular weights (20 kDa, 40 kDa, 60 kDa, and 80 kDa) were prepared to modify the IL2-N88R-N77C mutant. That is, PEGs of different molecular weights were attached to the cysteine ​​at position 77 of the mutant. The results are shown in Figure 8. The results demonstrate that the 80K-1PEG-IL2-N88R-N77C, 60K-1PEG-IL2-N88R-N77C, 40K-1PEG-IL2-N88R-N77C, and 20K-1PEG-IL2-N88R-N77C molecules were successfully prepared.

[0100] The IL2-N88R-N77C mutation site was identified using LC-MS as follows: IL-2 and IL2-N88R-N77C were digested with trypsin, and the resulting peptide fragments were analyzed by LC-MS. The mutation site was determined by comparing changes in characteristic peptide fragments. The results are shown in Table 5. In IL2-N88R-N77C, characteristic peptide fragments containing the mutated amino acid were detected, whereas those containing the original amino acid were not.

[0101] [Table 5]

[0102] The PEG conjugation site of 60K-1PEG-N88R-N77C and 80K-1PEG-N88R-N77C was analyzed using LC-MS as follows. IL2-N88R-N77C and PEG-IL2-N88R-N77C were digested with trypsin, and the resulting peptide fragments were analyzed by LC-MS. The PEG conjugation site was identified by comparing the reduction in specific peptide fragments before and after PEGylation. The results are shown in Table 6. The C77FHLRPR peptide fragment in the PEG conjugate was reduced to less than 5% of that in IL2-N88R-N77C, indicating that the PEG conjugation site was the cysteine ​​at position 77 of the IL-2 mutant, with a site-specific modification rate of over 95%.

[0103] [Table 6]

[0104] Example 7: Activation of P-STAT5 in different cell subsets of human PBMCs by IL-2 (N88R-N77C) mutants modified with PEG of different molecular weights In this example, the activating effect of each mutant prepared in Example 6 on P-STAT5 in different cell subsets (Treg, CD8+ T cells, NK cells) derived from human PBMC (Miaoshun Biotechnology) was detected.

[0105] Tregs and CD8 + T cells Human PBMC cells were thawed and cultured at 1.5 × 10 cells / ml in RPMI 1640 medium containing 10% FBS. 6 The IL-2 mutants were resuspended at 1000 nM / ml. 100 μl of each was added to each well of a V-bottom 96-well culture plate. Each IL-2 variant prepared in Example 6 was serially diluted 10-fold, with 1000 nM used as the working concentration in the first well. 100 μl of each serial dilution was added to each well of the cultured cells. The cells were stimulated at 37°C for 30 minutes and then stopped on ice. The cells were treated with commercially available fixatives and permeabilizers (BD Biosciences 558049 and 558050) and stained with antibodies against CD3, CD4, CD25, and CD8 according to conventional methods. P-STAT5 antibody staining was also performed (Table 7), and the mean fluorescence intensity (MFI) of P-STAT5 in CD3+ / CD4+ / CD25+ (Treg cells) and CD8+ / CD3+ (CD8+ T cells) was measured by flow cytometry.

[0106] NK cells Human PBMCs were thawed and NK cells were isolated using a commercially available kit (Miltenyi Biotec, 130-092-657) and an LS separation column (Miltenyi Biotec, 130-042-401). The cells were diluted to 2.5 × 10 in RPMI 1640 complete medium. 5 The cells were resuspended at 1000 / ml and added to a 96-well plate at 25,000 cells / well. - CD56 +The percentage of cells was detected by flow cytometry to determine the cell purification efficiency. Ten-fold gradient dilutions (1000 nM-1 nM) of each IL-2 variant prepared in Example 5 were prepared using RPMI 1640 complete medium, and 100 μl of each serial dilution was added to each well of cells cultured twice. After stimulation at 37°C for 30 minutes, the cells were treated with commercially available fixatives and permeabilization agents (BD Biosciences 558049 and 558050) and stained with P-STAT5 antibody to detect the mean fluorescence intensity (MFI) of cellular P-STAT5.

[0107] The experimental results are shown in Figures 9 to 11. IL-2-N88R-N77C modified with PEG of different molecular weights exhibited similar behavior in activating Tregs in PBMCs, and activated NK and CD8 + Table 8 shows that the IL-2-N88R-N77C mutant modified with 20 kDa PEG had no activating effect on T cells. 50 EC of P-STAT5 activation in Treg cells compared with values 50 Furthermore, the IL-2-N88R-N77C mutant modified with 40-80k PEG induced a similar level of Treg activation.

[0108] [Table 7]

[0109] [Table 8]

[0110] Example 8: Pharmacodynamics (PD) study of PEG-IL2-N88R-N77C in mice In this example, the pharmacodynamics (PD) of each IL-2 variant obtained in Example 6 was investigated after a single administration to mice. SPF-grade Balb / c mice were purchased and randomly assigned to groups according to body weight, with three mice in each group. Drug administration began on the day of group assignment. Information on the dosage and group assignment is shown in Table 9. G1 to G5 were administered once, and G6 and G7 were administered once daily, for a total of three doses. Thirty microliters of blood was collected from each group. For the PEGylated IL2 variants and negative control, blood was collected on days 0 (before administration), 2, 3, 4, 5, 6, and 7 (G3, G4, and G5 only). For IL2 and IL2-N88R, blood was collected on days 0 (before administration), 1, 2, and 3. The number of each cell subpopulation was measured by flow cytometry. Table 10 lists the antibodies against the staining markers used in flow cytometry, and Table 11 lists the gating settings.

[0111] [Table 9]

[0112] [Table 10]

[0113] [Table 11]

[0114] The experimental results are shown in Figures 12 to 14. All PEGylated IL-2 variants (Groups G1, G2, G3, and G4) significantly activated Treg cells at a dose of 1 mg / kg, as indicated by an increase in the percentage of Treg cells. As the molecular weight of PEG increased, both the timing and magnitude of the peak Treg cell percentage increased. No significant difference was observed between 60K-1PEG-IL2-N88R-N77C (Group G3) and 80K-1PEG-IL2-N88R-N77C (Group G4). Both treatments reached peak activation on day 6, resulting in a maximum increase of approximately 15-fold in the percentage of Treg cells, significantly higher than those observed in the IL-2 group (Group G6) and the IL-2-N88R-treated group (Group G7) (Figure 12). In contrast, the PEGylated IL-2 variants tested did not significantly activate CD8+ T cells or NK cells (Figures 13 and 14).

[0115] Example 9: Pharmacokinetic study of PEG-IL2-N88R-N77C in mice In this example, various IL-2 variants modified with PEG of different molecular weights obtained in Example 5 were administered to mice in a single dose, and the pharmacokinetics (PK) of the mice was then measured. Balb / c mice were purchased, with four mice per group. Each IL-2 variant to be tested was administered subcutaneously once at a dose of 1 mg / kg. Blood was collected via the orbit at different time points after administration, and plasma was separated. Plasma drug concentrations were measured by ELISA. Main pharmacokinetic parameters were calculated, and CT curves were plotted.

[0116] The results are shown in Figure 15 and Table 12. Increasing the molecular weight of the PEG modifying the IL-2 variants gradually decreased the drug metabolism rate of the corresponding variants. The half-lives of the 60K and 80K modified variants were significantly longer than those of the 20K and 40K modified variants, resulting in increased drug exposure. Modification with polyethylene glycols with molecular weights of 40K or higher significantly reduced the clearance (CL) of IL-2 in mice. The mean half-lives of the 60K and 80K modified variants were 20.78 and 25.23 hours, respectively, approximately 2.7-3.3 times longer than those of the 20K and 40K modified variants. The mean residence times (MRT) of the 60K and 80K modified variants were 39.45 and 40.08 hours, respectively, showing similar values. Pharmacokinetic data indicated that the 60K and 80K variants had more favorable in vivo metabolic profiles, supporting the extension of dosing intervals and the reduction of dosing frequency.

[0117] The main pharmacokinetic parameters are summarized below. [Table 12]

[0118] Example 10: KLH-induced delayed-type hypersensitivity (DTH) model in BALB / C mice The anti-inflammatory effects of 60K-1PEG-IL2-N88R-N77C and 80K-1PEG-IL2-N88R-N77C were evaluated in a keyhole limpet hemocyanin (KLH)-induced delayed-type hypersensitivity ear swelling model in Balb / c mice. SPF-grade Balb / c mice were randomly assigned to groups (see Table 13 for group assignment and dosing schedule). On the day of group assignment, each group, except for group G1, was sensitized by subcutaneous injection of KLH / CFA emulsion at four sites in the abdominal region. The KLH / CFA emulsion was prepared by emulsifying KLH and complete frenzy adjuvant (CFA) at a 1:1 ratio. Each mouse was injected with 100 μg of KLH / CFA emulsion (25 μg / injection). Drug administration began on the day of sensitization. Five days later, 5 μL of KLH diluted with saline at 1 μg / μL was injected intradermally into both ears of each mouse using a microsyringe. From day 5 to day 9, ear thickness was measured daily using a micrometer screw.

[0119] [Table 13]

[0120] After KLH stimulation, no significant increase in ear thickness was observed in unsensitized mice in group G1, but ear swelling was observed in the other groups, with ear thickness peaking at 24 hours in the negative control group (G2). The PEGylated IL-2 mutant drug-treated groups (G4, G5, G6, and G7) significantly suppressed the increase in ear thickness in a dose-dependent manner, demonstrating that the test drug suppressed the inflammatory response in the DTH mouse model (Figure 16).

[0121] Example 11: Pharmacodynamic and pharmacokinetic studies of PEG-IL2-N88R-N77C in cynomolgus monkeys In this study, 60K-1PEG-IL2-N88R-N77C and 80K-1PEG-IL2-N88R-N77C were subcutaneously administered to cynomolgus monkeys (two monkeys per group, one male and one female). Blood samples were collected before and at different time points after administration, and the effects on the following immune cells were evaluated by flow cytometry. See Table 14 for the administration method and grouping. Blood samples were collected before and after the first cycle (4 hours, 8 hours, 24 hours, 48 ​​hours, 72 hours, 120 hours, 168 hours, 240 hours, and 336 hours) and after the second cycle (8 hours, 24 hours, 48 ​​hours, 72 hours, 120 hours, 168 hours, and 240 hours), and plasma drug concentrations were measured. The gating settings used for flow cytometry are shown in Table 15. Pharmacokinetic parameters were analyzed using software. The results are shown in Tables 16 and 17.

[0122] [Table 14]

[0123] [Table 15]

[0124] [Table 16]

[0125] [Table 17]

[0126] The PK study results (Figure 17) showed that after two consecutive subcutaneous administrations (Q2W), the two test substances exhibited similar half-lives and MRTs in cynomolgus monkeys. The half-lives of 60K-1PEG-IL2-N88R-N77C and 80K-1PEG-IL2-N88R-N77C were 129.3-167.0 hours and 133.6-216.2 hours, respectively, which are significantly longer than the half-lives of IL-2 or IL-2-Fc fusion proteins reported in the literature. This is primarily due to the average molecular weight of the test substances exceeding 70 kD, which is greater than the glomerular filtration limit, resulting in reduced renal clearance and significantly prolonged half-lives.

[0127] The PD study showed that the PEG-modified IL-2 variant prepared in this application significantly stimulated an increase in peripheral Treg cell counts in cynomolgus monkeys at a dose of 400 μg / kg, with a maximum increase of more than 90-fold compared to the baseline before administration, with an average peak of 75-fold (Figure 19). Three weeks after administration, the average increase in Treg cells remained approximately 14-fold, demonstrating sustained and potent Treg cell-stimulating activity in monkeys. Detection of NK cells and CD8+ T cells showed that despite significant Treg cell proliferation, the PEG-modified IL-2 variant prepared in this application did not induce significant changes in the numbers of NK cells and CD8+ T cells (Figures 18 and 20). This reflects the optimized preference for Treg cell activation.

[0128] In the detection of NK cells and CD8+ T cells, despite the significant proliferation of Treg cells, the PEG-modified IL-2 variant prepared in the present application did not induce significant changes in the numbers of NK cells and CD8+ T cells (Figures 18 and 20). This reflects the optimized preference for Treg cell activation. The increase in CD25 after the second administration was significantly higher than after the first administration, peaking on day 7 after administration. The peak of Foxp3 appeared around day 4 after the first and second administrations. These data indicate that peripheral Treg cells expanded after administration, and both CD25 and Foxp3 expression increased.

[0129] Example 12: Preliminary safety study of PEG-IL2-N88R-N77C in cynomolgus monkeys In this study, 60K-1PEG-IL2-N88R-N77C and 80K-1PEG-IL2-N88R-N77C were subcutaneously administered to rhesus monkeys (2 monkeys per group, 1 male and 1 female). The doses and groupings are shown in Table 18. A 3-week recovery period was allowed after the experiment.

[0130] [Table 18]

[0131] All animals in each group survived at the doses and for the administration period described above, and no abnormalities were observed in body weight, body temperature, respiration, electrocardiogram, or blood pressure. At doses of 50 μg / kg and 400 μg / kg, no obvious abnormalities were observed macroscopically during the administration period. At the high dose of 1200 μg / kg, abnormal feces were observed, and male animals in each group showed reddened skin (whole body, groin, and hind legs) or peeling. Cytokines IFN-γ, TNF-α, IL-5, IL-6, and IL-10 were measured before the third administration and 24, 48, 72, and 120 hours after the third administration, but no increases in cytokines were observed. Necropsies were performed after the recovery period, and no drug-related organ abnormalities were observed.

[0132] After the second and third administrations, the serum of all test animals was tested for anti-drug antibodies (ADA), but all samples were negative.

[0133] Hematology and blood chemistry showed a decrease in Na+ and Cl-, an increase in eosinophils (EOS), a slight decrease in albumin (ALB), and a significant increase in C-reactive protein (CRP) in some animals after the final administration (Figure 23).

[0134] These results indicate that 60k-1PEG-IL2-N88R-N77C and 80k-1PEG-IL2-N88R-N77C induced significant activation and proliferation of peripheral blood Treg cells at doses of 400 μg / kg or less, without significant toxic reactions, suggesting reliable safety within this effective dose range. At a high dose of 1200 μg / kg, side effects known to be associated with IL-2 administration, such as diarrhea, increased white blood cell count, decreased albumin, and increased CRP, were observed.

[0135] These data predict that the therapeutic window of treatment with 60k-1PEG-IL2-N88R-N77C and 80k-1PEG-IL2-N88R-N77C is significantly broader than that of IL-2.

[0136] Example 13: Construction, expression, and purification of interleukin-2 mutants with unnatural amino acid (phenylalanine analog) mutations Interleukin-2 mutants with unnatural amino acids (phenylalanine analogs) were obtained using a standard method (An enhanced system for unnatural amino acid mutagenesis in E. coli. J Mol Biol. 2010 Jan 15;395(2):361-74). Based on the protein sequence of human mature IL-2 (SEQ ID NO: 1), the parent amino acid codon at each site was replaced with the amber codon TAG according to the mutation sites shown in Table 19. cDNAs encoding the corresponding IL-2 mutants with each codon mutation shown in Table 19 were then designed. An initiation codon and an NdeI restriction enzyme site were added to the 5' end of each cDNA, and a stop codon and an EcoRI restriction enzyme site were added to the 3' end. The cDNAs were synthesized by DNA synthesis and cloned into the expression vector pET30a(+). BL21(DE3) sensitive cells were cotransformed with the expression vector pET30a(+) containing the corresponding cDNA sequence and the auxiliary plasmid pEVOL-pAcF, which specifically transfects p-acetyl-L-phenylalanine (pAcF). Positive clones were isolated from kanamycin and chloramphenicol double-resistant plates, sequenced, and used as expression strains for each mutant protein.

[0137] [Table 19]

[0138] The IL2-pAcF mutant protein expression strains were inoculated into GMML medium and cultured at 37°C and 250 rpm in a shaker until the bacterial concentration reached an OD600 of 0.6-0.8. Expression of each mutant protein was induced for 4 hours by adding 1 mM p-acetylphenylalanine, 0.2% arabinose, and 1 mM IPTG, and the bacteria were then collected by centrifugation at 13,900 g. The bacteria were washed, resuspended in 50 mM Tris-HCl, 5 mM EDTA, pH 8.0 buffer, disrupted at 700-1,000 atmospheres, and centrifuged at 13,900 g to collect inclusion bodies.

[0139] Next, purification was carried out with reference to Example 1 to obtain purified interleukin-2 unnatural amino acid variants.

[0140] Example 14: Polyethylene glycol modification and purification of IL-2 variants with unnatural amino acid mutations The IL-2 unnatural amino acid variants obtained in Example 13 were modified and purified according to the references (CN Patent Publication No. 110078814 and CN Patent No. 104163864). The PEG used in this example contained a hydroxyamine group (oxyamine), which specifically reacts with p-acetylphenylalanine residues at specific mutation sites in the variants, allowing PEG to site-specifically modify the corresponding IL-2 variants. Specifically, the purified IL-2 variant protein was collected and the solution was changed to 10 mM sodium acetate, 5% trehalose, pH 4.5 buffer, the protein concentration was adjusted to 3 mg / ml, and PEG-hydroxylamine (20-80 kJ, e.g., 20 kJ, 40 kJ, 60 kJ, 80 kJ, Xiamen Sinopeg) was added at a molar ratio of IL-2 variant:PEG-hydroxylamine:NH4Cl of 1:5. NH4Cl and acetylhydrazine were added as catalysts, and the reaction was allowed to proceed at 2-8°C for at least 48 hours.

[0141] Next, purification was carried out with reference to Example 2 to obtain purified PEG-modified IL-2 non-natural amino acid mutant protein.

[0142] Example 15: IL-2 receptor binding preference of PEG-modified IL-2 variants with different unnatural amino acids (phenylalanine analogs) Referring to Example 3, the phosphorylation of STAT5 was used to evaluate the ability of the PEG-modified IL-2 mutants with unnatural amino acid mutations prepared in Example 14 to induce signaling through the mIL-2 receptor.

[0143] The results are shown in Figure 24 and Table 20. The PEGylated IL-2 mutants containing unnatural amino acid (phenylalanine analog) mutations obtained in Example 14 were found to retain their binding affinity to the IL-2Rαβγ triple receptor while exhibiting reduced affinity for the IL-2Rβγ dual receptor. In particular, when the PEG molecular weight exceeded 40 kJ, the STAT5 reporter gene signal mediated by the IL-2Rβγ dual receptor at high concentrations was reduced to 15% to 30% of that induced by IL-2. This suggests an increased preference for Treg cells, i.e., a preference for in vivo activation of Treg cells, resulting in greater avoidance of effector T cell (Teff) activation.

[0144] [Table 20]

[0145] Example 16: Construction, expression, and purification of interleukin-2 mutants with unnatural amino acid (lysine analog) mutations According to the reference method (Swiderska KW, Szlachcic A, Czyrek A, Zakrzewska M, Otlewski J. Site-specific conjugation of fibroblast growth factor 2 (FGF2) based on incorporation of alkyne-reactive unnatural amino acid. Bioorg Med Chem. 2017 Jul 15;25(14):3685-3693) and the basic construction method of Example 13, the parent amino acid at each mutation site was replaced with N-ε-propargyloxycarbonyl-L-lysine (Prk) according to the mutation sites shown in Table 20, and the corresponding interleukin-2 mutants with unnatural amino acid (lysine analog) mutations were obtained by corresponding verification, expression, and purification.

[0146] [Table 21]

[0147] Example 17: Polyethylene glycol modification and purification of IL-2 variants with unnatural amino acid (lysine analog) mutations The IL-2 mutants obtained in Example 16 were PEG-modified and purified according to the reference (Chinese Patent Application Publication No. 101265298). The PEG used in this example (20-80k, e.g., 20k, 40k, 60k, 80k, Xiamen Sinopeg) contains an azide group that reacts specifically with the alkyne group in the Prk structure of the mutants at specific mutation sites, allowing the PEG to site-specifically modify the corresponding IL-2 mutants. Specifically, the IL2-Prk mutant proteins were ultrafiltered into 25 mM PB at a concentration of 2.5 mg / ml or higher and pH 6.5. 2.5 mg of the liquid-exchanged IL2-Prk mutant protein was taken and a 10x molar ratio of PEG starting material was added. THPTA was added to the reaction solution to a final concentration of 1 mM, followed by CuSO₄·5H₂O to a final concentration of 0.2 mM, sodium ascorbate to a final concentration of 10 mM, and aminoguanidine to a final concentration of 5 mM. The reaction was carried out at room temperature for approximately 3 hours, and the PEG-modified protein was purified by MacroCap SP chromatography. After the polyethylene glycol modification reaction, purification was carried out as described in Example 2 to obtain purified PEG-modified IL-2 mutant protein bearing lysine analogs.

[0148] Example 18: Binding preference of PEG-modified IL-2 variants with different unnatural amino acid (lysine analog) mutations to the IL-2 receptor Referring to Example 3, the ability of the PEG-modified IL-2 mutants having lysine analogue mutations prepared in Example 17 to induce signal transduction through the IL-2 receptor was evaluated using STAT5 phosphorylation.

[0149] The results are shown in Figure 25 and Table 22. The results demonstrate that PEGylated IL-2 with unnatural amino acid (lysine analog) mutations obtained in Example 17 retains its binding affinity to the IL-2Rαβγ triple receptor while reducing its affinity to the IL-2Rβγ dual receptor. In particular, when the PEG molecular weight exceeded 40 kJ, the STAT5 reporter gene signal mediated by the IL-2Rβγ dual receptor at high concentrations was reduced to 15% to 30% of the signal induced by IL-2. This suggests an increased preference for Treg cells, i.e., an increased preference for Treg cell activation in vivo, leading to a greater suppression of effector T cell (Teff) activation.

[0150] [Table 22]

Claims

1. An IL-2 variant having at least 95% sequence identity with the parent sequence described in SEQ ID NO: 1, wherein the substitution includes one or more positions selected from N88, N77, N33, S75, D84, V91, and D109 in the sequence described in SEQ ID NO: 1, and the said positions are substituted with natural or non-natural amino acids.

2. The IL-2 variant according to claim 1, wherein the non-natural amino acid is selected from a lysine analog, a cysteine ​​analog or histidine analog, a phenylalanine analog, a non-natural amino acid containing an aromatic side chain, a non-natural amino acid containing an azide group, a non-natural amino acid containing an alkyne group, or a non-natural amino acid containing an aldehyde or ketone group.

3. The IL-2 variant according to claim 1, wherein the non-natural amino acid is selected from phenylalanine analogs or lysine analogs, the phenylalanine analog is p-acetylphenylalanine, and the lysine analog is N-E-propargyloxycarbonyl-L-lysine.

4. The IL-2 variant according to claim 1, wherein the substitution is one or more selected from N88R, N77C, N33C, S75C, D84C, N88C, V91C, and D109C; or one or more selected from N88R, N77 pAcF, or N33 pAcF; or one or more selected from N88R, N77Prk, or N33Prk.

5. The IL-2 variant according to claim 1, wherein the substitution includes a combination of the substitution of N88 and a substitution selected from any one of N77, N33, S75, D84, V91, and D109.

6. The IL-2 variant according to claim 1, wherein the substitution includes a combination of the substitution of N88R and a substitution selected from any one of N77, N33, S75, D84, V91 and D109, and the amino acid substitution other than N88R is modified by a water-soluble polymer, the water-soluble polymer being a PEG molecule.

7. The IL-2 variant according to claim 6, wherein the PEG molecule is a linear molecule or a branched molecule, and the average molecular weight of the PEG molecule is 5 to 100 kDa, or 20 to 100 kDa, or 40 to 80 kDa.

8. The IL-2 variant according to claim 7, wherein the PEG molecule comprises a chemically reactive group for conjugation with a protein, and the chemically reactive group is selected from a maleimide group, a hydroxylamine group, or an azide group.

9. The IL-2 mutant according to claim 1, wherein the IL-2 mutant includes an N88R substitution, and a PEG molecule is conjugated to the substituted residue at position 77, the substituted residue at position 77 is selected from cysteine, p-acetylphenylalanine, or N-E-propargyloxycarbonyl-L-lysine.

10. A pharmaceutical composition comprising the IL-2 variant described in any one of claims 1 to 9.

11. A nucleic acid molecule encoding the IL-2 variant according to any one of claims 1 to 9.

12. A vector comprising the nucleic acid molecule described in Claim 11.

13. A host cell comprising the nucleic acid molecule described in Claim 11, or a vector comprising the nucleic acid molecule.

14. A method for preparing an IL-2 mutant according to any one of claims 1 to 9, 1) A host cell containing a nucleic acid molecule encoding the IL-2 mutant is cultured under conditions suitable for expressing the IL-2 mutant. 2) The expressed mutant is collected, 3) Modify the IL-2 variant at a specific site with a PEG molecule, which is a water-soluble polymer. A method that includes the act of doing so.

15. A formulation for treating a target disease or reducing organ transplant rejection, comprising an effective amount of the IL-2 variant described in any one of claims 1 to 9.

16. Use of an IL-2 variant according to any one of claims 1 to 9, or a pharmaceutical composition containing the IL-2 variant, in the preparation of a drug for treating a disease or reducing organ transplant rejection.

17. The disease is an inflammatory disease (e.g., autoimmune disease, ankylosing spondylitis, amyotrophic lateral sclerosis, hepatitis C-associated vasculitis, sclerosing cholangitis, inflammatory muscle disease, relapsing polychondritis, Behçet's disease), coronary artery disease, macrophage activation syndrome, acute lung injury, hypertension, Takayasu's arteriovenous arthritis, Duchenne muscular dystrophy, transient ischemic attack, ischemic heart disease, Wiscott-Aldrich syndrome, bone marrow transplant, or a condition for transplantation (e.g., corneal transplant, islet transplant, skin transplant), preferably the autoimmune disease is rheumatoid arthritis, autoimmune encephalitis The preparation according to claim 15, which is a type 1 diabetes mellitus, nephritis, multiple sclerosis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, Sjögren's syndrome, psoriasis, psoriasis vulgaris, alopecia areata, dermatomyositis, scleroderma, myasthenia gravis, demyelination disease, inflammatory bowel disease, ulcerative colitis, Crohn's disease, autoimmune glomerulonephritis, pulmonary and renal hemorrhagic syndrome, graft-versus-host disease, organ transplant rejection, autoimmune hepatitis, autoimmune vasculitis, atopic dermatitis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, or idiopathic leukopenia.

18. The aforementioned diseases include inflammatory diseases (e.g., autoimmune diseases, ankylosing spondylitis, amyotrophic lateral sclerosis, hepatitis C-associated vasculitis, sclerosing cholangitis, inflammatory muscle diseases, relapsing polychondritis, Behçet's disease), coronary artery disease, macrophage activation syndrome, acute lung injury, hypertension, Takayasu's arteriovenous syndrome, Duchenne muscular dystrophy, transient ischemic attack, ischemic heart disease, Wiscott-Aldrich syndrome, bone marrow transplantation, or indications for transplantation (e.g., corneal transplantation, islet transplantation, skin transplantation), preferably, the aforementioned autoimmune diseases include rheumatoid arthritis and autoimmune encephalitis. The use according to claim 16, which is type 1 diabetes, nephritis, multiple sclerosis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, Sjögren's syndrome, psoriasis, psoriasis vulgaris, alopecia areata, dermatomyositis, scleroderma, myasthenia gravis, demyelination disease, inflammatory bowel disease, ulcerative colitis, Crohn's disease, autoimmune glomerulonephritis, pulmonary and renal hemorrhagic syndrome, graft-versus-host disease, organ transplant rejection, autoimmune hepatitis, autoimmune vasculitis, atopic dermatitis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, or idiopathic leukopenia.

19. A method for improving the activation preference of the IL-2 receptor and extending the half-life of an IL-2 mutant, 1) A step of substituting asparagine at position 77 of IL-2 with a natural amino acid or a non-natural amino acid, wherein the natural amino acid or non-natural amino acid is selected from cysteine, p-acetylphenylalanine, or N-E-propargyloxycarbonyl-L-lysine, and 2) A step of obtaining an IL-2 variant by site-specifically modifying the amino acid with the substituted position at position 77 using a water-soluble polymer, and 3) Optionally, a step of recovering the IL-2 mutant. A method that includes this.

20. The method according to claim 19, wherein the water-soluble polymer is PEG, and the PEG molecule is a linear molecule or a branched molecule.

21. The method according to claim 20, wherein the average molecular weight of the PEG molecules is 5 to 100 kDa, or 20 kDa to 100 kDa, or 40 kDa to 80 kDa.

22. The method according to claim 20 or 21, wherein the PEG molecule comprises a chemically reactive group for conjugation to a protein, and the chemically reactive group is selected from a maleimide group, a hydroxylamine group, or an azide group.

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