An interleukin 2 mutant

Abstract

The present application provides a kind of interleukin 2 mutant, fusion protein, antibody or conjugate comprising the mutant and pharmaceutical composition comprising the mutant, fusion protein, antibody or conjugate. Compared with wild type interleukin 2, the mutant eliminates the binding with IL-2R alpha, reduces the binding with IL-2R beta gamma dimer, but maintains the stimulation of tumor immune cells, including but not limited to T cells and NK cells proliferation. The IL-2 mutant of the present application eliminates the affinity for high affinity IL-2 receptor, while reducing the affinity for medium affinity IL-2 receptor. The interleukin 2 mutant of the present application is more suitable for fusion protein, antibody conjugate. The mutation site of the interleukin 2 mutant of the present application is very few relative to wild type interleukin 2, so the potential immunogenicity is very low. At the same time, there is no various side effects produced by immunotherapy with natural IL-2.

Description

Technical Field

[0001] This invention relates to the field of protein engineering. Specifically, it relates to novel interleukin-2 (IL-2) mutants and their preparation methods. Compared to wild-type IL-2, the IL-2 mutants eliminate binding to IL-2Rα and reduce binding to IL-2Rβγ dimers, while retaining the ability to stimulate the proliferation of tumor immune cells, including but not limited to T cells and NK cells. The IL-2 mutants of this invention are more suitable for fusion proteins and antibody conjugates. They can be used for immunotherapy without the various side effects associated with immunotherapy using natural IL-2. Background Technology

[0002] Interleukin-2 (IL-2) is a type I cytokine produced by activated CD4+ and CD8+ T cells, also known as a T cell growth factor. Activated dendritic cells (DCs), natural killer (NK) cells, and NKT cells can also produce IL-2. IL-2 is a multifunctional cytokine that plays a crucial role in lymphocyte homeostasis.

[0003] IL-2 can increase NK cell activity and mediate activation-induced cell death. IL-2 can induce T cell expansion to enhance adoptive immunotherapy. IL-2 can promote the proliferation and development of regulatory T cells (Tregs) and maintain Treg homeostasis and function. It was approved by the FDA in the 1980s for the treatment of melanoma and renal cell carcinoma. However, due to the short half-life of IL-2 in vivo (approximately 15 minutes), approximately 60,000-720,000 IU / kg needs to be injected every 8 hours during treatment, leading to some adverse reactions. Furthermore, high doses of IL-2 can cause vascular (or capillary) leakage syndrome (VLS) in patients. Low-dose IL-2 regimens have been tested in patients to avoid VLS; however, this comes at the cost of reduced treatment outcomes.

[0004] IL-2 has three receptor subunits: IL-2Rα, IL-2Rβ, and IL-2Rγ. Based on the different binding subunits, they can be classified into receptors with different affinities. IL-2Rαβγ has high affinity (Kd 10). -11 IL-2Rβγ has moderate affinity (Kd 10) -9 ), IL-2Rα has low affinity (Kd 10), -8IL-2 receptors. Different types of receptors are expressed on the surface of different cells. Regulatory T cells (Treg) express high-affinity receptors (IL-2Rαβγ) and are sensitive to low doses of IL-2; while effector T cells (Teff) express intermediate-affinity receptors (IL-2Rβγ) and are sensitive to high doses of IL-2.

[0005] IL-2 is a multifunctional cytokine. Low doses of IL-2 can selectively stimulate the proliferation of Treg cells, and its therapeutic mechanism is that Treg cells express a high-affinity IL-2 receptor (IL-2Rαβγ) on their surface. In clinical trials, low doses of IL-2 have shown good efficacy in treating autoimmune diseases such as systemic lupus erythematosus (SLE) and type 1 diabetes (T1D), and it is expected to become a potential drug for treating autoimmune diseases. Teff cells express a moderate-affinity receptor on their surface and are sensitive to high doses of IL-2. High doses of IL-2 can induce the proliferation of Teff cells and are used to treat cancer and AIDS, but high doses of IL-2 produce a series of toxicities during treatment, such as arrhythmias, dyspnea and hypoxia, and pulmonary edema. IL-2 is a known effective growth factor for both T and NK cells, but its application is limited by the aforementioned side effects.

[0006] Tumor immunotherapy has become an effective treatment for cancer in recent years. It utilizes PD-1 or PD-L1 inhibitors, as well as other similar immunosuppressants such as CTLA-4 and CD-47 antibodies, to induce T cells and NK cells to kill tumor cells. In recent years, bispecific antibodies have gained increasing attention in tumor immunotherapy. For example, Roche has used a PD-1 monoclonal antibody coupled with an IL-2 tetrap mutant (US20180326010) for tumor immunotherapy. This IL-2 tetrap mutant (CN103492411A) mutates amino acids at three sites: F44A, Y45A, and L72G in IL-2, reducing the affinity of the IL-2 protein for high-affinity IL-2 receptors while retaining the affinity of the mutant IL-2 protein for medium-affinity IL-2 receptors. However, the resulting IL-2 mutant also exhibits reduced biological activity.

[0007] Therefore, there is a need in this field to utilize IL-2 for tumor immunization and to stably produce IL-2 mutants that can effectively treat tumors. Summary of the Invention

[0008] This invention provides an interleukin-2 mutant protein, a fusion protein comprising the interleukin-2 mutant protein, an antibody or conjugate thereof, and a pharmaceutical composition comprising the interleukin-2 mutant protein, the fusion protein, the antibody or conjugate thereof. Compared to wild-type interleukin-2, the interleukin-2 mutant of this invention eliminates binding to IL-2Rα and reduces binding to IL-2Rβγ dimers, while still retaining the desired biological activity to stimulate the proliferation of tumor immune cells, including but not limited to T cells and NK cells. The interleukin-2 mutant protein of this invention is more suitable for fusion protein and antibody conjugates. The interleukin-2 mutant protein of this invention has extremely low potential immunogenicity. The interleukin-2 mutant protein of this invention can overcome the problems associated with IL-2 immunotherapy.

[0009] In a first aspect, the present invention provides an IL-2 mutant, wherein the amino acid residues of the IL-2 mutant are mutated compared with wild-type IL-2, thereby altering the binding ability of IL-2 to its receptor; the IL-2 mutant eliminates the affinity for high-affinity IL-2 receptors and reduces the affinity for medium-affinity IL-2 receptors.

[0010] In a preferred embodiment, the high-affinity IL-2 receptor is a heterotrimeric form of the IL-2 receptor, which is composed of a receptor α subunit, a receptor β subunit, and a receptor γ subunit; the intermediate-affinity IL-2 receptor contains only the IL-2 receptor β subunit and the IL-2 receptor γ subunit, but lacks the IL-2 receptor α subunit.

[0011] In a preferred embodiment, compared with wild-type IL-2, the binding affinity of the IL-2 mutant to the high-affinity IL-2 receptor is reduced by more than 55%, more preferably by more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, and most preferably the IL-2 mutant does not bind to the high-affinity IL-2 receptor;

[0012] The binding affinity of the IL-2 mutant to the intermediate-affinity IL-2 receptor is reduced by more than 10%, more preferably by more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99%; most preferably, the binding affinity of the IL-2 mutant to the intermediate-affinity IL-2 receptor is reduced compared to the binding affinity of wild-type IL-2 to the intermediate-affinity IL-2 receptor.

[0013] In a preferred embodiment, the IL-2 mutant retains the ability to activate tumor immune cells, including but not limited to the proliferation of T cells and NK cells.

[0014] In a specific embodiment, the IL-2 mutant has a mutation at amino acid residues at positions 42 and 44 corresponding to wild-type IL-2.

[0015] In a specific embodiment, the IL-2 mutant has an amino acid residue mutation at one or two sites corresponding to wild-type IL-2: 42 and 44.

[0016] In a preferred embodiment, the IL-2 mutant has a mutation at position 42 corresponding to wild-type IL-2, selecting from the group consisting of: F42N, F42A, F42G, F42P, F42S, F42T, F42E, F42D; or

[0017] The IL-2 mutant has an amino acid residue mutation selected from the group consisting of F44A, F44G, F44P, F44S, F44T, F44E, and F44D at position 44, corresponding to wild-type IL-2.

[0018] In a preferred embodiment, the IL-2 mutant undergoes the following mutation at one or two amino acid residues corresponding to wild-type IL-2: F42N, F44T.

[0019] In a preferred embodiment, the IL-2 mutant eliminates the O sugar site.

[0020] In a preferred embodiment, the IL-2 mutant undergoes a mutation at position 3 corresponding to wild-type IL-2, thereby eliminating the O sugar site.

[0021] In a preferred embodiment, the IL-2 mutant has the following amino acid residue mutations at position 3 of the wild-type IL-2 protein: T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P; T3A is preferred.

[0022] In a preferred embodiment, the IL-2 mutant is mutated at position 125 of the cys site: C125L, C125S, C125A; preferably C125S.

[0023] In a second aspect, the present invention provides an IL-2 mutant in which the amino acid residues are mutated compared to wild-type IL-2; the IL-2 mutant eliminates affinity for high-affinity IL-2 receptors while reducing affinity for medium-affinity IL-2 receptors.

[0024] In a preferred embodiment, the high-affinity IL-2 receptor is a heterotrimeric form of the IL-2 receptor, which is composed of a receptor α subunit, a receptor β subunit, and a receptor γ subunit; the intermediate-affinity IL-2 receptor contains only the IL-2 receptor β subunit and the IL-2 receptor γ subunit, but lacks the IL-2 receptor α subunit.

[0025] In a preferred embodiment, compared with wild-type IL-2, the binding affinity of the IL-2 mutant to the high-affinity IL-2 receptor is reduced by more than 55%, more preferably by more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, and most preferably the IL-2 mutant does not bind to the high-affinity IL-2 receptor;

[0026] The binding affinity of the IL-2 mutant to the intermediate-affinity IL-2 receptor is reduced by more than 10%, more preferably by more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99%; most preferably, the binding affinity of the IL-2 mutant to the intermediate-affinity IL-2 receptor is reduced compared to the binding affinity of wild-type IL-2 to the intermediate-affinity IL-2 receptor.

[0027] In a preferred embodiment, the IL-2 mutant retains the ability to activate tumor immune cells, including but not limited to the proliferation of T cells and NK cells.

[0028] In a specific implementation, the IL-2 mutant has mutated amino acid residues compared to wild-type IL-2, thereby affecting its binding to the IL-2 receptor.

[0029] In a specific embodiment, the IL-2 mutant has a mutation at amino acid residues at positions 42 and 44 corresponding to wild-type IL-2.

[0030] In a specific embodiment, the IL-2 mutant has an amino acid residue mutation at one or more sites corresponding to wild-type IL-2: 42 and 44.

[0031] In a preferred embodiment, the IL-2 mutant has a mutation at position 42 corresponding to wild-type IL-2: F42N, F42A, F42G, F42Q, F42E, F42D, F42P, F42S, F42T, F42K, F42R, F42V.

[0032] In a preferred embodiment, the IL-2 mutant has a mutation at position 44 corresponding to wild-type IL-2: F44T, F44A, F44G, F44Q, F44E, F44D, F44P, F44S, F44N, F44K, F44R, F44V.

[0033] In a preferred embodiment, the IL-2 mutant eliminates the O sugar site.

[0034] In a preferred embodiment, the IL-2 mutant undergoes a mutation at position 3 corresponding to wild-type IL-2, thereby eliminating the O sugar site.

[0035] In a preferred embodiment, the IL-2 mutant has the following amino acid residue mutations at position 3 of the wild-type IL-2 protein: T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P; T3A is preferred.

[0036] In a preferred embodiment, the IL-2 mutant mutates the cys site at position 125: C125L, C125S, or C125A; preferably C125S. In a preferred embodiment, the non-IL-2 functional portion is selected from the group consisting of:

[0037] In a third aspect, the present invention provides a fusion protein or conjugate comprising the IL-2 mutant and non-IL-2 functional portion described in the first or second aspect.

[0038] In a preferred embodiment, the non-IL-2 functional portion is selected from the group consisting of:

[0039] Fc fragments, including but not limited to: Fc fragments of human IgG1, IgG2, IgG3, and IgG4, and Fc fragment mutants with more than 90% homology;

[0040] Human serum albumin (HSA);

[0041] Anti-HSA antibodies and their fragments;

[0042] Anti-albumin peptides or antibodies;

[0043] Transferrin;

[0044] Human chorionic gonadotropin β-subunit carboxyl-terminal peptide (CTP);

[0045] Elastin-like peptide (ELP);

[0046] Antigen-binding portion.

[0047] Cell receptors or ligands.

[0048] In a preferred embodiment, the IL-2 mutant in the fusion protein can be directly linked to the non-IL-2 functional part, or they can be linked by a linker; the linker can be a repeating sequence of AAA or GS, including but not limited to repeating sequences of G3S or G4S; for example, (G3S)4.

[0049] In a preferred embodiment, the IL-2 mutant or fusion protein may be further modified to form a conjugate:

[0050] Polyethylene glycol modification (PEGylation);

[0051] Polysialylation modification (PSA);

[0052] Saturated fatty acid modification;

[0053] Hyaluronic acid modification (HA);

[0054] Polyamino acid modification (proline-alamine-serine polymer, PAS modification).

[0055] In a fourth aspect, the present invention provides a bispecific antibody or a trispecific antibody, the bispecific antibody or trispecific antibody comprising the IL-2 mutant and antibody portion described in the first or second aspect.

[0056] In a preferred embodiment, the antibody portion is selected from the group consisting of:

[0057] Fc fragments, including but not limited to: Fc fragments of human IgG1, IgG2, IgG3, and IgG4, and Fc fragment mutants with more than 90% homology;

[0058] In a preferred embodiment, the antigen-binding portion is:

[0059] Antibody or its active antibody fragment;

[0060] Fab molecules, scFv molecules, and VHH molecules; or

[0061] In a preferred embodiment, the IL-2 mutant in the bispecific or trispecific antibody can be directly linked to the non-IL-2 functional part, or they can be linked by a linker; the linker can be a repeating sequence of AAA or GS, including but not limited to repeating sequences of G3S or G4S; for example, (G3S)4.

[0062] In a preferred embodiment, the non-IL-2 functional portion of the IL-2 mutant can be linked with other cytokines or tumor markers to form a trispecific antibody.

[0063] In a fifth aspect, the present invention provides a polynucleotide encoding the IL-2 mutant described in the first or second aspect, or the fusion protein or conjugate described in the third aspect, or the bispecific antibody or trispecific antibody described in the fourth aspect.

[0064] In a sixth aspect, the present invention provides an expression vector comprising the polynucleotide described in the fifth aspect.

[0065] In a seventh aspect, the present invention provides a host cell comprising the expression vector described in the sixth aspect, or the genome of the host cell integrating the polynucleotides described in the fifth aspect.

[0066] In a preferred embodiment, the host cell is a eukaryotic cell; yeast, insect cells, or animal cells are preferred; animal cells are more preferred; and mammalian cells, such as Chinese hamster ovary cells, are most preferred.

[0067] In an eighth aspect, the present invention provides a pharmaceutical composition comprising the IL-2 mutant described in the first or second aspect, or the fusion protein or conjugate described in the third aspect, or the bispecific or trispecific antibody described in the fourth aspect, and pharmaceutically acceptable excipients.

[0068] In a ninth aspect, the present invention provides the use of the IL-2 mutant described in the first or second aspect, or the fusion protein or conjugate described in the third aspect, or the bispecific or trispecific antibody described in the fourth aspect, in the preparation of a medicament for the in vitro expansion of T lymphocytes, natural killer NK cells, or for the treatment of an individual’s disease.

[0069] In a preferred embodiment, the disease is one for which IL-2 is used for immunotherapy.

[0070] In a preferred embodiment, the disease is cancer, immune disease, human immunodeficiency virus (HIV) infection, hepatitis C virus (HCV) infection, rheumatoid arthritis, atopic dermatitis, etc.

[0071] In a preferred embodiment, the cancer, systemic lupus erythematosus, immune diseases, diabetes, HIV infection, HCV infection, rheumatoid arthritis, atopic dermatitis, etc., are treated by stimulating the immune system or by proliferating immune cells.

[0072] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0073] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0074] Figure 1 SDS-PAGE analysis of purified products of IL-2 mutant and wild-type IL-2 fusion protein; In the figure, M: Prestained Protein Ladder; 1: IL-2-HSA; 7: IL-2gm17-HSA;

[0075] Figure 2 The binding affinity of IL-2 mutant, IL-2 wild-type and IL-2Rα was shown using Biacore, with the maximum R value of IL-2gm17-HSA being 0 and the maximum R value of IL-2-HSA being 140.

[0076] Figure 3 The binding affinity of IL-2 mutant, IL-2 wild-type, and IL-2Rβγ dimer was shown using Biacore assays; the maximum R-value for IL-2gm17-HSA was 2.5, and the maximum R-value for IL-2-HSA was 17. Figure 3 As shown, IL-2gm17-HSA has a lower affinity for the IL-2Rβγ dimer compared to IL-2-HSA;

[0077] Figure 4 The study showed the proliferation of NK92 cells stimulated by interleukin-2 mutant and wild-type IL-2.

[0078] Figure 5 The effects of IL-2gm17-HSA and wild-type IL-2-HSA on NK cell proliferation were shown.

[0079] Figure 6 The effects of IL-2gm17-HSA and wild-type IL-2-HSA on the proliferation of Treg cells were shown; and

[0080] Figure 7a -f indicates the sequence SEQ ID NO:1-6 used in this invention. Detailed Implementation

[0081] Through extensive and in-depth research, the inventors unexpectedly discovered that novel IL-2 mutants, resulting from site-directed mutagenesis leading to glycosylation modification of the IL-2 peptide, or novel IL-2 mutant peptides without glycosylation modification, can eliminate the affinity of the IL-2 protein for high-affinity IL-2 receptors and reduce the affinity of the mutant IL-2 protein for medium-affinity IL-2 receptors, while retaining the biological activity of IL-2. This allows them to stimulate the proliferation of tumor immune cells, including but not limited to T cells and NK cells, thereby achieving therapeutic effects. This mutant is particularly suitable for fusion protein / antibody linkage compositions, achieving tumor inhibition or therapeutic effects by stimulating the proliferation of immune cells. Based on this, the present invention was completed.

[0082] The IL-2 mutant of the present invention

[0083] In this invention, site-directed mutagenesis alters the amino acid residues of the IL-2 peptide, thereby changing the binding affinity or affinity of IL-2 to its receptor while preserving its biological activity. The IL-2 mutant of this invention not only better stimulates the proliferation of tumor immune cells, including but not limited to T cells and NK cells, but also significantly reduces side effects compared to wild-type IL-2, thus achieving better therapeutic results.

[0084] The IL-2 mutant of the present invention is preferably expressed in eukaryotic cells and obtained through cell culture. Yeast, insect cells, animal cells, or transgenic animals can be selected. In a specific embodiment, the host cell is a eukaryotic cell; yeast, insect cells, or animal cells are preferred; the animal cell can be a mammalian cell, including but not limited to CHO cells, 293 cells, SP / 20 cells, and NSO cells.

[0085] When yeast cells or insect cells are used as host cells, the resulting IL-2 mutant may have a non-human glycoform. Those skilled in the art will know that the non-human glycoform can be further modified to a human glycoform.

[0086] In other embodiments, IL-2 mutants can also be obtained by prokaryotic bacterial expression fermentation or in vitro cell-free synthesis.

[0087] Regarding the number of introduced mutation sites, amino acid residue mutations can occur at either of the two sites, positions 42 and 44, in wild-type IL-2. Therefore, the IL-2 mutant of the present invention can have the following amino acid residue mutations at position 42 corresponding to the wild-type IL-2 protein: F42N, F42A, F42G, F42Q, F42E, F42D, F42P, F42S, F42T, F42K, F42R, F42V; and / or at position 44 corresponding to the wild-type IL-2 protein: F44T, F44A, F44G, F44Q, F44E, F44D, F44P, F44S, F44N, F44K, F44R, F44V;

[0088] Preferably, the IL-2 mutant has a mutation at position 42 corresponding to wild-type IL-2, consisting of an amino acid residue selected from the group consisting of: F42N, F42A, F42G, F42P, F42S, F42T, F42E, F42D; or

[0089] The IL-2 mutant has an amino acid residue mutation selected from the group consisting of F44A, F44G, F44P, F44S, F44T, F44E, and F44D at position 44, corresponding to wild-type IL-2.

[0090] Based on conventional practices in the field, the original O-glycan site in the IL-2 polypeptide can also be eliminated. Removing the O-glycan does not affect the biological activity of IL-2. However, the O-glycan structure is complex and difficult to analyze. To reduce the complexity of production quality control, genetic engineering mutation technology can usually be used to eliminate this glycosylation site. Therefore, the IL-2 mutant of the present invention can have the following amino acid residue mutations at position 3 corresponding to the wild-type IL-2 protein: T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P; preferably T3A. During the purification and refolding process of the IL-2 gene product, mismatched disulfide bonds or intermolecular disulfide bond formation will reduce the activity of IL-2. Point mutations have been used to mutate cysteine ​​at position 125 to leucine or serine, so that only one type of disulfide bond can be formed, thus ensuring the activity during the IL-2 refolding process. There are also reports of producing novel rIL-2 using protein engineering techniques, by replacing cysteine ​​at position 125 of the IL-2 molecule with alanine. This modified IL-2 exhibits a significantly increased specific activity compared to natural IL-2. Therefore, the IL-2 mutant of this invention can undergo the following amino acid residue mutations at position 125 corresponding to the wild-type IL-2 protein: C125L, C125A, C125S; preferably C125S.

[0091] "corresponds to"

[0092] As used herein, the term "corresponds to" has the meaning commonly understood by one of ordinary skill in the art. Specifically, "corresponds to" means that, after homology or sequence identity alignment, one sequence corresponds to a specific position in another sequence. Thus, for example, "corresponds to wild-type IL-2" means that an amino acid sequence is aligned with the amino acid sequence of wild-type IL-2, and a site on that amino acid sequence that corresponds to wild-type IL-2 is found.

[0093] The fusion protein or conjugate of the present invention

[0094] Based on the IL-2 mutant of the present invention, those skilled in the art will understand that the IL-2 mutant of the present invention can be combined with other non-IL-2 functional parts to form fusion proteins or conjugates. In this document, a conjugate refers to a water-soluble polymer covalently linking residues of the mutant IL-2 polypeptide. In specific embodiments, the non-IL-2 functional parts include, but are not limited to: Fc fragments, human serum albumin (HSA), anti-HSA antibodies or antibody fragments, transferrin, human chorionic gonadotropin β-subunit carboxyl-terminal peptide (CTP), elastin-like peptide (ELP), and antigen-binding moieties, and also include cytokines, specifically interleukins, interferons, tumor necrosis factor superfamily, colony-stimulating factors, chemokines, growth factors, etc.

[0095] Based on conventional practices in the art, those skilled in the art will know how to obtain fusion proteins or conjugates containing the IL-2 mutant of the present invention. For example, the IL-2 mutant of the present invention can be directly linked to other non-IL-2 functional parts, or it can be linked using a linker. The linker can be a repeating sequence of AAA or GS, including but not limited to repeating sequences of G3S or G4S; for example, (G3S)4.

[0096] Furthermore, the IL-2 mutant or fusion protein conjugate can also be modified with polyethylene glycol (PEGylation), polysialylated (PSA), saturated fatty acid, hyaluronic acid (HA), or proline-alamine-serine polymer (PAS) to form conjugates.

[0097] The bispecific or trispecific antibodies of the present invention

[0098] Diseases are often caused by multiple pathogenic factors, and simultaneously blocking multiple targets may achieve better therapeutic effects. Therefore, bispecific antibodies (BsAbs) have emerged. Tumor immunotherapy is a new direction in cancer treatment. Bispecific antibodies can bind to two different antigens, thus showing great promise in the field of cancer treatment. Initially, bispecific antibodies were prepared using chemical conjugation or hybridoma hybridization methods. Today, the rapid development of DNA recombination technology has revolutionized the structure of bispecific antibodies, mainly dividing them into two categories: IgG types containing an Fc region and non-IgG types lacking an Fc region. IgG type bispecific antibodies have a structure similar to monoclonal antibodies, with a relatively large molecular weight and a long plasma half-life. Non-IgG type bispecific antibodies have more diverse structural forms, a relatively small molecular weight, and stronger tissue penetration, but a shorter plasma half-life.

[0099] Based on the IL-2 mutant of the present invention, those skilled in the art will understand that the IL-2 mutant of the present invention can be covalently linked to an antibody domain. In specific embodiments, the antibody domain includes, but is not limited to, IgG type antibodies and non-IgG type antibodies. In a preferred embodiment, the antibody domain may be an antibody or its active antibody fragment, a Fab molecule, a scFv molecule and a VHH molecule, an immunoglobulin molecule, a receptor protein molecule or a ligand protein molecule; the immunoglobulin molecule may be an IgG molecule.

[0100] Based on conventional practices in the art, those skilled in the art know how to obtain bispecific antibodies containing the IL-2 mutant of the present invention. For example, the IL-2 mutant of the present invention can be directly linked to other non-IL-2 functional parts, or it can be linked using a linker. The linker can be a repeating sequence of AAA or GS, including but not limited to repeating sequences of G3S or G4S; for example, (G3S)4.

[0101] Optionally, the mutants of the present invention can be coupled with T cell surface antigen antibodies or with tumor cell surface antigen antibodies. Preferably, the mutants of the present invention can be coupled with T cell surface antigen antibodies.

[0102] Optionally, the mutants of the present invention can be coupled with T cell surface antigens / antibodies to form bispecific antibodies, and can also be coupled with tumor cell surface antigens / antibodies to form bispecific antibodies. Optionally, the mutants of the present invention can be coupled with T cell surface antigens / antibodies to form trispecific antibodies, and can also be coupled with tumor cell surface antigens / antibodies to form trispecific antibodies. Optionally, the mutants of the present invention can be coupled with either T cell surface antigens / antibodies or tumor cell surface antigens / antibodies to form trispecific antibodies.

[0103] The pharmaceutical composition and administration method of the present invention

[0104] Based on the IL-2 mutant of the present invention, the present invention also provides pharmaceutical compositions. In specific embodiments, the pharmaceutical compositions of the present invention comprise the IL-2 mutant of the present invention, or the fusion protein or conjugate described herein, or the bispecific antibody or trispecific antibody described herein, and optionally pharmaceutically acceptable excipients.

[0105] Optionally, the compositions of the present invention further comprise a pharmaceutically acceptable excipient. If desired, a pharmaceutically acceptable excipient may be added to the IL-2 mutant peptide, fusion protein or conjugate, bispecific antibody or trispecific antibody of the present invention to form a composition.

[0106] Uses and methods of using the IL-2 mutant of the present invention

[0107] As described above, the IL-2 mutant of the present invention can eliminate the affinity of the IL-2 protein for high-affinity IL-2 receptors and reduce the affinity of the mutant IL-2 protein for medium-affinity IL-2 receptors, while retaining the biological activity of IL-2, thereby better stimulating the proliferation of tumor immune cells, including but not limited to T cells and NK cells. Therefore, the IL-2 mutant, fusion protein, conjugate, bispecific antibody or trispecific antibody, and pharmaceutical composition of the present invention can be prepared into corresponding drugs. These drugs can be used to expand T lymphocytes, natural killer NK cells, or to treat diseases treated with IL-2 immunotherapy. In a specific embodiment, the disease is cancer; for example, cancer that requires treatment by stimulating the immune system or by proliferating immune cells. In a specific embodiment, the disease can be systemic lupus erythematosus, immune diseases, diabetes, HIV infection, HCV infection, rheumatoid arthritis, atopic dermatitis, etc.

[0108] This invention can also be used as a substitute for wild-type IL-2 in in vitro expanded cells during cell therapy processes such as CAT-T and CAR-NK.

[0109] Advantages of this invention:

[0110] 1. The IL-2 mutant protein of the present invention eliminates the affinity for high-affinity IL-2 receptors and reduces the affinity for medium-affinity IL-2 receptors, while retaining the biological activity of IL-2;

[0111] 2. Compared with other IL-2 mutants in the prior art, the IL-2 mutant of the present invention has very few mutation sites. Only two mutation sites are needed to achieve the effect of reducing affinity while retaining biological activity.

[0112] 3. The IL-2 mutant protein of the present invention avoids the reduction of specific activity by superimposed mutations, and can affect the binding of two different receptor regions by mutations at only two sites, thereby reducing potential immunogenicity;

[0113] 4. The IL-2 mutant of the present invention is easy to produce and control in terms of quality, and generally does not require an in vitro re-modification process, thus reducing steps and improving production efficiency;

[0114] 5. The IL-2 mutant of the present invention is readily compatible with other molecules to form bifunctional or multifunctional fusion proteins or immune compositions;

[0115] 6. The IL-2 mutant of the present invention can be used for immunotherapy but does not cause vascular (or capillary) leakage syndrome (VLS) caused by natural IL-2; and

[0116] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0117] Example

[0118] Example 1. Synthesis of mutant interleukin-2 (IL-2) protein

[0119] 1. Gene synthesis

[0120] The nucleotide sequence encoding the mutant amino acid sequence of interleukin-2 (IL-2) protein was obtained using an automated gene synthesis method. In the examples, an HSA tag was added to the end of the gene fragment to facilitate purification; this tag is also a common method for extending the half-life of protein drugs. The gene fragment was flanked by a single restriction endonuclease cleavage site. All synthesized gene sequences were designed to have a 5' DNA sequence encoding a leader peptide that enables targeted protein secretion in eukaryotic cells.

[0121] Number of mutations mutation site Example Mutant Name protein tags 3 3, 42 and 44 IL-2gm17(T3A,F42N,F44T) HSA

[0122] 2. Plasmid construction

[0123] The synthesized gene was subcloned into the pTT5 plasmid using molecular biology reagents according to the manufacturer's instructions.

[0124] 3. Expression of mutant interleukin-2 (IL-2) protein

[0125] The IL-2 mutant molecule was named IL-2gm17-HSA (SEQ ID NO:1) and wild-type IL-2-HSA (SEQ ID NO:2). These were constructed into eukaryotic expression vectors using molecular cloning techniques to prepare IL-2gm17-HSA and IL-2-HSA expression vectors, respectively. Transient transfection expression of the IL-2 mutant molecule was performed on 293E cells cultured in Freestyle medium. 24 hours before transfection, 0.5 × 10⁶ cells were seeded in 1L cell culture flasks. 6 150 ml of 293E cells / ml were cultured in a 37°C, 5% CO2 incubator at 120 rpm on a shaker. For transfection, 150 μl of 293fectin was added to 2.85 ml of OptiMEM, mixed thoroughly, and incubated at room temperature for 2 minutes. Simultaneously, 150 μg of the plasmid for expression was diluted to 3 ml using OptiMEM. The diluted transfection reagent and plasmid were thoroughly mixed, incubated at room temperature for 15 minutes, and then the entire mixture was added to the cells and mixed thoroughly. Cells were cultured at 37°C, 5% CO2 incubator at 120 rpm for 7 days. The cell culture supernatant was collected, filtered through a 0.22 μm filter, and then purified using a Q-HP ion exchange chromatography column (GE) with linear elution of 20 mM Tris and 0-500 mM NaCl at pH 8.0. Samples were collected continuously by volume. Each collected fraction was analyzed by SDS-PAGE using a 4–20% gradient gel (GenScript), and the samples were then combined according to their electrophoretic purity. The purity of the combined protein was then determined by SDS-PAGE, and the results are as follows: Figure 1 As shown. Lane M is the protein marker, lane 1 is IL-2-HSA, and lane 7 is IL-2gm17-HSA.

[0126] Example 2. Preparation of receptor protein

[0127] To investigate the binding ability of IL-2 mutant molecules to IL-2Rα receptor and IL-2Rβγ heterodimerized receptor, human IL-2Rα receptor and IL-2Rβγ heterodimerized receptor proteins were prepared.

[0128] The human IL-2Rα receptor was designed by linking the coding sequence of the extracellular domain of IL-2Rα with the coding sequence of the 6×His Tag (SEQ ID NO:3) and cloning it into a eukaryotic expression vector. Transient transfection expression of the IL-2Rα receptor was performed using 293E cells cultured in Freestyle medium. 24 hours before transfection, 0.5 × 10⁶ cells were seeded in 1L cell culture flasks. 6150 ml of 293E cells / ml were cultured in a 37°C, 5% CO2 incubator at 120 rpm on a shaker. For transfection, 150 μl of 293fectin was added to 2.85 ml of OptiMEM, mixed thoroughly, and incubated at room temperature for 2 minutes. Simultaneously, 150 μg of the plasmid expressing the IL-2Rα receptor was diluted to 3 ml using OptiMEM. The diluted transfection reagent and plasmid were thoroughly mixed and incubated at room temperature for 15 minutes. The mixture was then added to the cells, mixed thoroughly, and cultured for 7 days at 37°C, 5% CO2 on a shaker at 120 rpm. The cell culture supernatant was collected, filtered through a 0.22 μm filter, and then purified using a Ni-NTA affinity chromatography column (GE) under the conditions of 20 mMPB-0.5 M NaCl-100 mM imidazole. The purified protein was detected by SDS-PAGE using a 4–20% gradient gel (GenScript).

[0129] The human IL-2Rβγ heterodimerizing receptor was designed using the "Knobs into Holes" technique. The coding sequence for the extracellular domain of IL-2Rβ was ligated with the Fc fragment encoding "Knobs" (SEQ ID NO:4), and cloned into a eukaryotic expression vector. Similarly, the coding sequence for the extracellular domain of IL-2Rγ was ligated with the Fc fragment encoding "Holes" (SEQ ID NO:5), and cloned into a eukaryotic expression vector. Transient transfection expression of the IL-2Rβγ heterodimerizing receptor was performed using 293E cells cultured in Freestyle medium. 24 hours before transfection, 0.5 × 10⁶ cells were seeded in 1L cell culture flasks. 6 150 ml of 293E cells / ml were cultured in a 37°C, 5% CO2 incubator at 120 rpm on a shaker. For transfection, 150 μl of 293fectin was added to 2.85 ml of OptiMEM, mixed thoroughly, and incubated at room temperature for 2 minutes. Simultaneously, 75 μg of plasmids expressing the IL-2Rβγ heterodimer receptor were diluted to 3 ml using OptiMEM. The diluted transfection reagents and plasmids were thoroughly mixed and incubated at room temperature for 15 minutes. The mixture was then added to the cells, mixed thoroughly, and cultured at 37°C, 5% CO2 in a 120 rpm shaker for 7 days. The cell culture supernatant was collected, filtered through a 0.22 μm filter, and purified using a MabSelect SuRe affinity chromatography column (GE) at 20 mM citrate-sodium citrate, pH 3.0. The pH was adjusted to neutral with 1 M Tris base. Proteins were purified by SDS-PAGE analysis using a 4–20% gradient gel (GenScript).

[0130] Example 3. Affinity test for receptor binding using biacore

[0131] To investigate the affinity of IL-2 mutants relative to wild-type and receptors, the affinity of the recombinant monomeric IL-2Rα subunit for the human IL-2Rα subunit was determined using a Biacore 8K (GE) spectrometer under the following conditions: the human IL-2Rα subunit was immobilized on a CM5 chip (190 RU). IL-2gm17-HSA and IL-2-HSA were used as analytes in HBS-EP buffer at 25 °C. For IL-2Rα, the analyte concentration was reduced from 200 nM to 1.526 nM (1:2 dilution), and the flow rate was 30 μl / min (binding time 180 s, dissociation time 300 s). For IL-2Rα, regeneration was performed with 20 mM NaOH at 30 μl / min for 10 s. For IL-2Rα, a 1:1 binding ratio was used, RI ≠ 0, and the maximum R value was equal to the global fit data.

[0132] The results are as follows Figure 2 As shown, the maximum R value for IL-2gm17-HSA is 0, while the maximum R value for IL-2-HSA is 140. IL-2gm17-HSA eliminates its affinity for IL-2Rα compared to IL-2-HSA.

[0133] The affinity of recombinant IL-2Rβγ heterodimer to human IL-2Rβγ heterodimer was determined using a Biacore 8K (GE) instrument under the following conditions: human hIL-2Rβ,γECD-N-hIgG1Fc was immobilized on a Protein A chip (400 RU). IL-2gm17-HSA and IL-2-HSA were used as analytes in HBS-EP buffer at 25 °C. For IL-2Rβγ, the analyte concentration was reduced from 200 nM to 1.5625 nM (1:2 dilution), and the flow rate was 30 μl / min (binding time 180 s, dissociation time 300 s). For IL-2Rβγ, regeneration was performed using 10 mM Glycine (pH 1.5), 30 μl / min, for 30 s. For IL-2Rβγ, a 1:1 binding is used, RI≠0, and the maximum value of R = the locally fitted data.

[0134] The results are as follows Figure 3 As shown, the maximum R-value for IL-2gm17-HSA is 2.5, and the maximum R-value for IL-2-HSA is 17. (As...) Figure 3As shown, IL-2gm17-HSA reduces the affinity for the IL-2Rβγ dimer compared to IL-2-HSA. Therefore, IL-2gm17-HSA eliminates the affinity for IL-2Rα and reduces the affinity for the IL-2Rβγ dimer compared to IL-2-HSA.

[0135] Example 4. Cell proliferation analysis using NK92 cells

[0136] NK92 cells are an IL-2-dependent NK cell line derived from peripheral blood mononuclear cells of a 50-year-old Caucasian male with rapidly progressive non-Hodgkin's lymphoma. A portion of its cell surface expresses CD25. IL-2gm1-HSA (SEQ ID NO:6), an IL-2 mutant that eliminates the affinity of interleukin-2 protein for high-affinity IL-2 receptors while reducing its affinity for intermediate-affinity IL-2 receptors. The inventors used NK92 cells to evaluate the activities of IL-2gm17-HSA and IL-2-HSA in cell proliferation assays.

[0137] NK-92 cells in the logarithmic growth phase were harvested, washed once with MEM-α basal medium, and then (5000 cells / well) were incubated with different concentrations of IL-2gm17-HSA, IL-2gm1-HSA, and IL-2-HSA in experimental medium (MEM-α medium from Gibco (catalog number 32561-037), supplemented with 12.5% ​​fetal bovine serum and 12.5% ​​horse serum) at 37°C and 5% CO2 for 48 h. 100 μl of CellTiter-Glo (from Promega (catalog number G7571)) was added to each well, and the fluorescence values ​​were detected at all wavelengths using an endpoint method on a microplate reader (Molecular Devices (model I3x)).

[0138] The activities of IL-2gm17-HSA and IL-2-HSA were measured using cell proliferation analysis, and... Figure 4 An overview of the results is shown below. All test items induced NK92 cell growth in a dose-dependent manner. At comparable cell proliferation rates, EC... 50The larger the concentration, the weaker the activity of IL-2gm1-HSA in stimulating NK92 cell growth. This change is due to the influence of the mutant protein on CD25 binding, while the IL-2gm1-HSA mutant protein retains the activation of IL-2R signaling through IL-2Rβγ heterodimers, so cells still proliferate effectively even with increased concentrations. The specific activity of IL-2gm1-HSA in stimulating NK92 cell proliferation relative to IL-2-HSA was 1.07%, indicating that IL-2gm1-HSA eliminates the binding of CD25 to some NK92 cells expressing CD25, resulting in a weakened stimulatory effect due to the lack of IL-2Rαβγ heterotrimer formation. The IL-2gm1-HSA effect on NK92 cell proliferation was approximately 100-fold lower than that of IL-2-HSA. The IL-2gm17-HSA mutant protein not only affects binding to CD25 but also weakens the activation of IL-2R signaling via the IL-2Rβγ heterodimer. Therefore, even at increased concentrations, cells still achieved effective proliferation. The specific activity of IL-2gm17-HSA compared to IL-2-HSA in stimulating NK92 cell proliferation was 0.0055%, demonstrating that IL-2gm17-HSA eliminates the binding of CD25 to some NK92 cells expressing CD25, and the absence of IL-2Rαβγ heterotrimer formation also reduces the stimulatory effect. The proliferation effect of IL-2gm17-HSA on NK92 cells was reduced by approximately 10,000-fold compared to IL-2-HSA. This indicates that the IL-2gm17-HSA mutant protein weakens the activation of IL-2R signaling via the IL-2Rβγ heterodimer, thus allowing cells to proliferate effectively and retain their biological activity even at increased concentrations.

[0139] Example 5. Measurement of IL-2 mutant-induced PBMC proliferation.

[0140] Fresh blood samples were collected from healthy Chinese individuals (n=2) in heparin sodium tubes, and PBMCs were isolated. The PBMCs were resuspended in RPMI-1640 medium (containing 10% FBS) and inoculated into 48-well plates (1*10⁻⁶). 6 PBMCs (cells / well) were stimulated with different concentrations of IL-2 gm17-HSA and wild-type IL-2-HSA and co-cultured at 37°C and 5% CO2 for 6 days. FACS staining was performed using surface and intracellular marker antibodies to detect different cell populations. All samples were obtained using an LSR Tortessa™ cell analyzer.

[0141] NK cells are limited to CD3- / CD56+, while Treg cells are limited to CD3+CD4+CD25+Foxp3+.

[0142] The proliferation of NK cells after incubation for 6 days with different concentrations of IL-2gm17-HSA and wild-type IL-2-HSA is as follows: Figure 5 As shown.

[0143] At concentrations of 0-20 nM, IL-2gm17-HSA had a slightly weaker effect on stimulating NK cell proliferation compared to wild-type IL-2-HSA; at concentrations of 20-1000 nM, IL-2gm17-HSA had a significantly stronger effect on stimulating NK cell proliferation compared to wild-type IL-2-HSA.

[0144] The proliferation of Treg cells after incubation for 6 days with different concentrations of IL-2gm17-HSA and wild-type IL-2-HSA is as follows: Figure 6 As shown.

[0145] At concentrations of 0-20 nM, IL-2gm17-HSA slightly reduced the proliferative effect of IL-2-HSA on Treg cells compared to wild-type IL-2-HSA; at concentrations of 20 nM and 1000 nM, the proliferative effect of IL-2gm17-HSA on Treg cells was significantly weakened compared to wild-type IL-2-HSA.

[0146] In this experiment, IL-2gm17-HSA significantly stimulated the proliferation of NK cells and significantly inhibited the proliferation of Treg cells.

[0147] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> Shanghai Gaipu Biotechnology Co., Ltd. <120> An interleukin-2 mutant <130> P2020-0860 <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 721 <212> PRT <213> Artificial Sequence <400> 1 Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Asn Lys Thr Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Ala Ala Ala Asp Ala His Lys Ser Glu Val Ala 130 135 140 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 145 150 155 160 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 165 170 175 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 180 185 190 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 195 200 205 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 210 215 220 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 225 230 235 240 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 245 250 255 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 260 265 270 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 275 280 285 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys 290 295 300 Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 305 310 315 320 Leu Arg Asp Glu Gly Lys Ser Ser Ala Lys Gln Arg Leu Lys Cys 325 330 335 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 340 345 350 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 355 360 365 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 370 375 380 Asp Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 385 390 395 400 Cys Glu Asn Gln Asp Ser Ile Ser Ser Leu Lys Glu Cys Cys Glu 405 410 415 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 420 425 430 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 435 440 445 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 450 455 460 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 465 470 475 480 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys 485 490 495 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 500 505 510 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 515 520 525 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 530 535 540 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 545 550 555 560 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 565 570 575 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 580 585 590 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 595 600 605 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 610 615 620 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 625 630 635 640 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 645 650 655 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 660 665 670 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 675 680 685 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 690 695 700 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 705 710 715 720 Leu <210> 2 <211> 721 <212> PRT <213> Artificial Sequence <400> 2 Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys ​​​​​​Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Ala Ala Ala Asp Ala His Lys Ser Glu Val Ala 130 135 140 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 145 150 155 160 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 165 170 175 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 180 185 190 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 195 200 205 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 210 215 220 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 225 230 235 240 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 245 250 255 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 260 265 270 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 275 280 285 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Phe Thr Glu Cys 290,295,300 Cys Gln Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 305 310 315 320 Leu Arg Asp Glu Gly Lys Ser Ser Ala Lys Gln Arg Leu Lys Cys 325 330 335 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 340 345 350 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 355 360 365 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 370 375 380 Asp Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 385 390 395 400 Cys Glu Asn Gln Asp Ser Ile Ser Ser Leu Lys Glu Cys Cys Glu 405 410 415 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 420 425 430 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 435 440 445 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 450 455 460 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 465 470 475 480 Thr Thr Thr Thr Thr Glu Lys Cys 485,490,495 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 500 505 510 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu And Lys Gln Asn Cys 515 520 525 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 530 535 540 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 545 550 555 560 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 565 570 575 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 580 585 590 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 595 600 605 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 610 615 620 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 625 630 635 640 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 645 650 655 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 660 665 670 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 675 680 685 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 690 695 700 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 705 710 715 720 Leu <210> 3 <211> 257 <212> PRT <213> Artificial Sequence <400> 3 Met Asp Ser Tyr Leu Leu Met Trp Gly Leu Leu Thr Phe Ile Met Val 1 5 10 15 Pro Gly Cys Gln Ala Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro 20 25 30 His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn 35 40 45 Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr 50 55 60 Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys 65 70 75 80 Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro 85 90 95 Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro 100 105 110 Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro 115 120 125 Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val 130 135 140 Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His 145 150 155 160 Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His Gly Lys Thr Arg 165 170 175 Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly Glu Met Glu Thr Ser Gln 180 185 190 Phe Pro Gly Glu Glu Lys Pro Gln Ala Ser Pro Glu Gly Arg Pro Glu 195 200 205 Ser Glu Thr Ser Cys Leu Val Thr Thr Thr Asp Phe Gln Ile Gln Thr 210 215 220 Glu Met Ala Ala Thr Met Glu Thr Ser Ile Phe Thr Thr Glu Tyr Gln 225 230 235 240 Asp Asp Asp Asp Lys Ser Gly Gly Gly Gly Ser His His His His His 245 250 255 His <210> 4 <211> 477 <212> PRT <213> Artificial Sequence <400> 4 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Ala Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 20 25 30 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 35 40 45 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 50 55 60 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 65 70 75 80 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 85 90 95 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 100 105 110 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 115 120 125 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 130 135 140 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 145 150 155 160 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys 165 170 175 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 180 185 190 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 195 200 205 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 210 215 220 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 225 230 235 240 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly 245 250 255 Gly Ser Asp Asp Asp Asp Lys Ala Val Asn Gly Thr Ser Gln Phe Thr 260 265 270 Cys Phe Tyr Asn Ser Arg Ala Asn Ile Ser Cys Val Trp Ser Gln Asp 275 280 285 Gly Ala Leu Gln Asp Thr Ser Cys Gln Val His Ala Trp Pro Asp Arg 290 295 300 Arg Arg Trp Asn Gln Thr Cys Glu Leu Leu Pro Val Ser Gln Ala Ser 305 310 315 320 Trp Ala Cys Asn Leu Ile Leu Gly Ala Pro Asp Ser Gln Lys Leu Thr 325 330 335 Thr Val Asp Ile Val Thr Leu Arg Val Leu Cys Arg Glu Gly Val Arg 340 345 350 Trp Arg Val Met Ala Ile Gln Asp Phe Lys Pro Phe Glu Asn Leu Arg 355 360 365 Leu Met Ala Pro Ile Ser Leu Gln Val Val His Val Glu Thr His Arg 370 375 380 Cys Asn Ile Ser Trp Glu Ile Ser Gln Ala Ser His Tyr Phe Glu Arg 385 390 395 400 His Leu Glu Phe Glu Ala Arg Thr Leu Ser Pro Gly His Thr Trp Glu 405 410 415 Glu Ala Pro Leu Leu Thr Leu Lys Gln Lys Gln Glu Trp Ile Cys Leu 420 425 430 Glu Thr Leu Thr Pro Asp Thr Gln Tyr Glu Phe Gln Val Arg Val Lys 435 440 445 Pro Leu Gln Gly Glu Phe Thr Thr Trp Ser Pro Trp Ser Gln Pro Leu 450 455 460 Ala Phe Arg Thr Lys Pro Ala Ala Leu Gly Lys Asp Thr 465 470 475 <210> 5 <211> 503 <212> PRT <213> Artificial Sequence <400> 5 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Ala Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 20 25 30 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 35 40 45 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 50 55 60 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 65 70 75 80 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 85 90 95 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 100 105 110 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 115 120 125 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 130 135 140 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 145 150 155 160 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys 165 170 175 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 180 185 190 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 195 200 205 Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 210 215 220 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 225 230 235 240 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly 245 250 255 Gly Ser Asp Asp Asp Asp Lys Leu Asn Thr Thr Ile Leu Thr Pro Asn 260 265 270 Gly Asn Glu Asp Thr Thr Ala Asp Phe Phe Leu Thr Thr Met Pro Thr 275 280 285 Asp Ser Leu Ser Val Ser Thr Leu Pro Leu Pro Glu Val Gln Cys Phe 290 295 300 Val Phe Asn Val Glu Tyr Met Asn Cys Thr Trp Asn Ser Ser Ser Glu 305 310 315 320 Pro Gln Pro Thr Asn Leu Thr Leu His Tyr Trp Tyr Lys Asn Ser Asp 325 330 335 Asn Asp Lys Val Gln Lys Cys Ser His Tyr Leu Phe Ser Glu Glu Ile 340 345 350 Thr Ser Gly Cys Gln Leu Gln Lys Lys Glu Ile His Leu Tyr Gln Thr 355 360 365 Phe Val Val Gln Leu Gln Asp Pro Arg Glu Pro Arg Arg Gln Ala Thr 370 375 380 Gln Met Leu Lys Leu Gln Asn Leu Val Ile Pro Trp Ala Pro Glu Asn 385 390 395 400 Leu Thr Leu His Lys Leu Ser Glu Ser Gln Leu Glu Leu Asn Trp Asn 405 410 415 Asn Arg Phe Leu Asn His Cys Leu Glu His Leu Val Gln Tyr Arg Thr 420 425 430 Asp Trp Asp His Ser Trp Thr Glu Gln Ser Val Asp Tyr Arg His Lys 435 440 445 Phe Ser Leu Pro Ser Val Asp Gly Gln Lys Arg Tyr Thr Phe Arg Val 450 455 460 Arg Ser Arg Phe Asn Pro Leu Cys Gly Ser Ala Gln His Trp Ser Glu 465 470 475 480 Trp Ser His Pro Ile His Trp Gly Ser Asn Thr Ser Lys Glu Asn Pro 485 490 495 Phe Leu Phe Ala Leu Glu Ala 500 <210> 6 <211> 721 <212> PRT <213> Artificial Sequence <(400)> 6 Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Asn Leu Thr Phe Lys Phe Tyr Met Pro Lys 35(40) 45 Note: There seems to be a minor formatting issue in the original where '35(40)' is written in a non-standard way. I've tried to represent it as '35(40)' in the translation as best as possible while maintaining the overall structure. If this is incorrect, more context would be needed to accurately translate it.Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Ala Ala Ala Asp Ala His Lys Ser Glu Val Ala 130 135 140 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 145 150 155 160 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 165 170 175 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 180 185 190 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 195 200 205 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 210 215 220 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 225 230 235 240 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 245 250 255 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 260 265 270 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 275 280 285 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Phe Thr Glu Cys 290,295,300 Cys Gln Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 305 310 315 320 Leu Arg Asp Glu Gly Lys Ser Ser Ala Lys Gln Arg Leu Lys Cys 325 330 335 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 340 345 350 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 355 360 365 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 370 375 380 Asp Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 385 390 395 400 Cys Glu Asn Gln Asp Ser Ile Ser Ser Leu Lys Glu Cys Cys Glu 405 410 415 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 420 425 430 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 435 440 445 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 450 455 460 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 465 470 475 480 Thr Thr Thr Thr Thr Glu Lys Cys 485,490,495 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 500 505 510 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu And Lys Gln Asn Cys 515 520 525 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 530 535 540 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 545 550 555 560 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 565 570 575 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 580 585 590 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 595 600 605 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 610 615 620 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 625 630 635 640 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 645 650 655 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 660 665 670 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 675,680,685 Ala Phe Val Glu Lys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 690,695,700 Glu Glu Gly Lys Lys Leu Val Val Ser Ser Gln Ala Leu Gly 705 710 715 720 Leu

Claims

1. An interleukin-2 mutant, characterized in that, The amino acid sequence of the IL-2 mutant is shown in SEQ ID NO:

1.

2. A polynucleotide encoding the IL-2 mutant of claim 1.

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

4. A host cell comprising the expression vector of claim 3, or the genome of the host cell having the polynucleotide of claim 2 integrated therein.

5. The host cell as described in claim 4, characterized in that, The host cell is a eukaryotic cell.

6. The host cell as described in claim 5, characterized in that, The host cell is yeast, insect cell, or animal cell.

7. The host cell as described in claim 6, characterized in that, The host cell is an animal cell.

8. The host cell as described in claim 7, characterized in that, The host cell is a mammalian cell.

9. The host cell as described in claim 8, characterized in that, The host cells are Chinese hamster ovary cells.

10. A pharmaceutical composition comprising the IL-2 mutant of claim 1, with pharmaceutically acceptable excipients.

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