IL-2 mutant protein that proliferates immune cells

Abstract

The present invention provides IL-2 mutant proteins, fusion proteins or conjugates comprising the IL-2 mutant proteins, and pharmaceutical compositions comprising the IL-2 mutant proteins, fusion proteins or conjugates. Compared to wild-type IL-2 protein, the IL-2 mutant proteins of the present invention eliminate or reduce affinity for the high-affinity IL-2 receptor and essentially maintain affinity for the intermediate-affinity IL-2 receptor.

Description

Technical Field

[0001] The present invention relates to the field of protein engineering. Specifically, the present invention relates to a novel interleukin-2 (IL-2) mutant and a method for preparing the same. The interleukin-2 (IL-2) mutant has a reduced binding ability compared to the original protein of wild-type IL-2 and its binding partner, the IL-2 receptor α subunit, but maintains the binding ability to the IL-2 receptor β subunit and the IL-2 receptor γ subunit, as well as the corresponding biological activity, and can better stimulate tumor immune cells, including but not limited to the proliferation of T effector cells and NK cells.

Background Art

[0002] Interleukin-2 (IL-2) is a type of immune system cell growth factor that regulates the cell activity of white blood cells in the immune system, promotes the proliferation of Th0 and CTL, and also participates in antibody responses, hematopoiesis, and tumor monitoring. IL-2 mediates its effects by binding to the IL-2 receptor (IL-2R). The IL-2R is composed of three chains, α, β, and γ. Different combinations of them can generate receptor forms with different affinities for IL-2. The IL-2Rγ chain alone cannot bind to IL-2. The βγ chain is a medium-affinity IL-2R, and the αβγ chain is a high-affinity IL-2R.

[0003] IL-2 is mainly synthesized by activated T cells, especially helper T cells. It stimulates the proliferation and differentiation of T cells, induces the generation of cytotoxic T lymphocytes (CTL) and the differentiation of peripheral blood lymphocytes into cytotoxic cells and lymphokine-activated killer (LAK) cells, promotes the expression of cytokines and cytolytic molecules by T cells, promotes the proliferation and differentiation of B cells and the synthesis of immunoglobulins through B cells, and stimulates the generation, proliferation, and activation of natural killer (NK) cells.

[0004] The ability of IL-2 to expand lymphocyte populations in vivo and enhance the effector functions of these cells confers an anti-tumor effect of IL-2 and makes IL-2 immunotherapy an attractive treatment option for certain metastatic cancers. Accordingly, high-dose IL-2 therapy is approved for patients with metastatic renal cell carcinoma and malignant melanoma. However, IL-2 has a dual function in the immune response, as it not only mediates the proliferation and activation of effector cells but is also critically involved in maintaining peripheral immune tolerance.

[0005] Associated with IL-2 immunotherapy are the side effects caused by recombinant human IL-2 treatment. Patients receiving high-dose IL-2 therapy often experience severe cardiovascular, pulmonary, renal, hepatic, gastrointestinal, neurological, dermatological, hematological, and systemic adverse events, which require intensive monitoring and in-patient management. Most of these side effects can be explained by the formation of the so-called vascular (or capillary) leak syndrome (VLS), which is a pathological increase in vascular permeability leading to fluid overflow in multiple organs (e.g., edema in the lungs and skin and damage to hepatocytes) and loss of intravascular fluid (decrease in blood pressure and compensatory increase in heart rate). Solutions with low-dose IL-2 have been tested to avoid VLS; however, this comes at the cost of reducing the treatment outcome.

[0006] Tumor immunotherapy is a method for effectively treating tumors in recent years. Through PD-1 or PD-L1 inhibitors and other similar immune inhibitors such as CTLA-4 and CD-47 antibodies, the killing of tumor cells by T cells and NK cells is achieved in vivo. IL-2 is a known effective growth promoter for T cells and NK cells, but its application is limited due to the above side effects.

[0007] Cell immunotherapy has also been a method for effectively treating tumors or autoimmune diseases in recent years. Autologous immune cells are collected from the human body, cultured in vitro, expanded thousands of times in number, and after enhancing their targeted killing function, they are returned to the human body to kill pathogens, cancer cells, and mutant cells in the blood and tissues, disrupt immune tolerance, activate and enhance the body's immune capacity, and it is believed that a dual effect of treatment and healthcare can be obtained. These include cytokine-induced killer (CIK) cell therapy, dendritic cell (DC) therapy, DC + CIK cell therapy, natural killer (NK) cell therapy, DC-T, CART, CAR-NK cell therapy, etc. IL-2 is often used in its in vitro proliferation process, and wild-type IL-2 needs to more effectively proliferate IL-2 of immune cells such as CD8 because it simultaneously proliferates immune cells that do not kill tumors.

[0008] To overcome these problems associated with IL-2 immunotherapy, several approaches have been used in this field. For example, IL-2 can be combined with specific anti-IL-2 monoclonal antibodies to enhance the IL-2 therapeutic effect in vivo (Kamimura et al., J Immunol 177, 306-14 (2006), Boyman et al., Science 311, 1924-27 (2006)). Mutant IL-2 can be mutated in various ways to reduce its toxicity and / or improve its effect. For example, Hu et al. (Blood 101, 4853-4861 (2003), US Patent Publication No. 2003 / 0124678) replace the arginine residue at position 38 of IL-2 with tryptophan to eliminate the vascular permeability activity of IL-2. Shanafelt et al. (Nature Biotechnol 18, 1197-1202 (2000)) mutate asparagine 88 to arginine to enhance the selective proliferation of T cells and reduce the proliferation of NK cells with toxic side effects. Heaton et al. (Cancer Res 53, 2597-602 (1993), US Patent No. 5229109) introduce two mutations, Arg38Ala and Phe42Lys, to reduce the secretion of inflammatory cytokines by NK cells. Gillies et al. (US Patent Publication No. 2007 / 0036752) replace three residues of IL-2 (Asp20Thr, Asn88Arg, and Gln126Asp) to reduce VLS. Gillies et al. (W02008 / 0034473) enhance the effect by reducing the interaction with CD25 and the activation of Timf cells by substituting amino acids at the interface of the Arg38Trp and Phe42Lys mutant IL-2 and CD25. For the same purpose, Wittrup et al. (W02009 / 061853) obtain an IL-2 mutant that has increased affinity for CD25 but does not activate the receptor and thus acts as an antagonist. The purpose of introducing the mutation is to disrupt the interaction with the receptor β subunit and / or γ subunit.However, none of these known IL-2 mutants overcomes the above problems associated with IL-2 immunotherapy, namely, the induction of toxicity caused by VLS, the induction of tumor tolerance caused by AICD, and the immunosuppression caused by the activation of Timf cells. Rocheglicat (CN103492411A) mutated the amino acids at three sites of F42A, Y45A, and L72G of IL-2 to reduce the affinity of the IL-2 protein for the high-affinity IL-2 receptor and maintain the affinity of the mutant IL-2 protein for the medium-affinity IL-2 receptor. At the same time, the biological activity of the obtained IL-2 mutant also decreases. For example, the University of Miami (CN107074967A) constructed a fusion protein, which contains a first polypeptide of interleukin-2 or its functional variant or fragment and a second polypeptide fused to the open reading frame of the first polypeptide. Here, the second polypeptide contains the extracellular domain of interleukin-2 receptor α (IL-2Rα) or its functional variant or fragment. Here, the fusion protein has IL-2 activity. The constructed fusion protein eliminates the high-affinity IL-2Rα binding effect of IL-2 on the cell surface by self-binding. However, the fusion protein has self- or intermolecular interactions and is prone to generate multimers, which is disadvantageous for production and quality control. The fusion protein has a balance of binding and dissociation in vivo and cannot completely eliminate the high-affinity IL-2Rα binding effect on the cell surface and can still bind to cell surface IL-2Rα.

[0009] Therefore, in this field, it is necessary to improve the effectiveness of IL-2 and stably and conveniently produce enhanced IL-2 polypeptides. SUMMARY OF THE INVENTION

[0010] An object of the present invention is to provide a novel IL-2 mutant. Compared with wild-type IL-2, the IL-2 mutant of the present invention can overcome problems related to IL-2 immunotherapy while still maintaining the biological activities required.

[0011] In a first aspect, the present invention provides an IL-2 mutant. Compared with wild-type IL-2, the amino acid residues of the IL-2 mutant are mutated, so that the binding ability between IL-2 and its receptor is modified. The affinity of the IL-2 mutant for the high-affinity IL-2 receptor is eliminated or decreased, and basically maintains the affinity for the intermediate-affinity IL-2 receptor.

[0012] In a preferred embodiment, the high-affinity IL-2 receptor is the 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 only contains the IL-2 receptor β subunit and the IL-2 receptor γ subunit, and does not contain the IL-2 receptor α subunit.

[0013] In a preferred embodiment, compared with wild-type IL-2, the binding affinity of the IL-2 mutant for the high-affinity IL-2 receptor is decreased by 55% or more, more preferably 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, and most preferably the IL-2 mutant does not bind to the high-affinity IL-2 receptor.

[0014] The binding affinity between the IL-2 mutant and the intermediate-affinity IL-2 receptor is 50% or more, more preferably 60% or more, 70% or more, 80% or more, 90% or more, 95% or more of the binding affinity between wild-type IL-2 and the intermediate-affinity IL-2 receptor. Most preferably, the binding affinity between the IL-2 mutant and the intermediate-affinity IL-2 receptor corresponds to the binding affinity between wild-type IL-2 and the intermediate-affinity IL-2 receptor.

[0015] In a preferred embodiment, the IL-2 mutant maintains the growth effect of activated tumor immune cells including, but not limited to, T effector cells and NK cells.

[0016] In a specific embodiment, amino acid residue mutations occur at one or more sites among positions 39, 49, 73, and 109 corresponding to wild-type IL-2 in the IL-2 mutant.

[0017] In a preferred embodiment, amino acid residue mutations occur at any one of positions 39, 49, 73, and 109 corresponding to wild-type IL-2 in the IL-2 mutant.

[0018] In a preferred embodiment, amino acid residue mutations occur only at position 39 corresponding to wild-type IL-2 in the IL-2 mutant.

[0019] In a preferred embodiment, amino acid residue mutations occur at position 39 corresponding to wild-type IL-2 and optionally at one or more sites among positions 49, 73, and 109 in the IL-2 mutant, amino acid residue mutations occur at position 49 corresponding to wild-type IL-2 and at one or more sites among positions 39, 73, and 109 in the IL-2 mutant, amino acid residue mutations occur at position 73 corresponding to wild-type IL-2 and at one or more sites among positions 39, 49, and 109 in the IL-2 mutant, amino acid residue mutations occur at position 109 corresponding to wild-type IL-2 and at one or more sites among positions 39, 49, and 73 in the IL-2 mutant.

[0020] In a preferred embodiment, mutations occur at one, two, three, or four sites among positions 39, 49, 73, and 109 corresponding to wild-type IL-2 in the IL-2 mutant.

[0021] In a preferred embodiment, in the wild-type IL-2, one or more amino acid residue mutations occur among M39D, M39E, M39Q, M39N, M39A, K49N, A73T, A73S, and D109N to form the IL-2 mutant.

[0022] In a preferred embodiment, in the wild-type IL-2, amino acid residue point mutations occur at M39D, M39E, M39Q, M39N or M39A, preferably at M39D, M39E, M39Q or M39N, more preferably at M39D, M39E or M39Q, still more preferably at M39D or M39E, and most preferably at M39D to form the IL-2 mutant.

[0023] In a preferred embodiment, the IL-2 mutant eliminates the O-glycosylation site.

[0024] In a preferred embodiment, a mutation occurs at position 3 corresponding to wild-type IL-2 in the IL-2 mutant, thereby eliminating the O-glycosylation site.

[0025] In a preferred embodiment, amino acid residue mutations occur at position 3 corresponding to the wild-type IL-2 protein, such as T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P, preferably at T3A, to form the IL-2 mutant.

[0026] In a preferred embodiment, the IL-2 mutant mutates at the cys site at position 125, such as C125L, C125S, C125A, preferably at C125S.

[0027] In a second aspect, the present invention provides a fusion protein or conjugate, which comprises the IL-2 mutant according to the first aspect and a non-IL-2 functional part. In a preferred embodiment, the non-IL-2 functional part is Fc fragments including the Fc fragments of human IgG1, IgG2, IgG3, IgG4, and Fc fragments of mutants with a homology of 90% or more thereof, human serum albumin (HSA), anti-HSA antibody or antibody fragment, transferrin, human chorionic gonadotropin β subunit carboxyl-terminal peptide (CTP), elastin-like peptide (ELP), and selected from the group consisting of antigen-binding moieties.

[0028] In a preferred embodiment, the antigen-binding moiety is an antibody or an active antibody fragment thereof, a Fab molecule, an scFv molecule, and a VHH molecule, or a cell receptor or a ligand.

[0029] In a preferred embodiment, the IL-2 mutant and the non-IL-2 functional moiety of the fusion protein can be directly connected or connected via an adapter, and the adapter can be, for example, a G 3 S repeat sequence such as (G3S)4 or a G 4 S repeat sequence, and can be an AAA or GS repeat sequence including but not limited to these.

[0030] In a preferred embodiment, the IL-2 mutant or fusion protein can be further modified as follows to form a conjugate: polyethylene glycol modification (PEGylation), polysialylation modification (PSAylation), saturated fatty acid modification, hyaluronic acid modification (Hyaluronic acid, HA), polyamino acid modification (proline-alamine-serine polymer, PASylation).

[0031] In a third aspect, the present invention provides a polynucleotide, which encodes the IL-2 mutant according to the first aspect, the fusion protein or conjugate according to the second aspect.

[0032] In a fourth aspect, the present invention provides an expression vector, which contains the polynucleotide according to the third aspect.

[0033] In a fifth aspect, the present invention provides a host cell, which contains the expression vector according to the fourth aspect, or the polynucleotide according to the third aspect is integrated into the genome of the host cell.

[0034] In a preferred embodiment, the host cell is a eukaryotic cell, preferably a yeast, insect cell, animal cell, and may be a mammalian cell.

[0035] In a sixth aspect, the present invention provides a cell-free expression mode, and the expression system contains the expression vector according to the fourth aspect.

[0036] In a seventh aspect, the present invention provides a pharmaceutical composition, which contains the IL-2 mutant according to the first aspect, the fusion protein or conjugate according to the second aspect, and a pharmaceutically acceptable adjuvant.

[0037] In an eighth aspect, the present invention provides the use of the IL-2 mutant according to the first aspect or the fusion protein according to the second aspect in the in vitro proliferation of T lymphocytes and natural killer NK cells or in the preparation of drugs for the treatment of diseases in an individual.

[0038] In a preferred embodiment, the disease is a disease that is immunotherapeutically treated by applying IL-2.

[0039] 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.

[0040] In a preferred embodiment, the cancer, immune disease, human immunodeficiency virus (HIV) infection, hepatitis C virus (HCV) infection, rheumatoid arthritis, atopic dermatitis, etc. are treated by stimulating the immune system or proliferating immune cells.

[0041] In a ninth aspect, the present invention provides the IL-2 mutant according to the first aspect or the fusion protein according to the second aspect, which is used for in vitro proliferation of T lymphocytes and natural killer (NK) cells or treatment of individual diseases.

[0042] In a preferred embodiment, the disease is a disease that is immunotherapeutically treated by applying IL-2.

[0043] 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.

[0044] In a preferred embodiment, the cancer is a cancer that is treated by stimulating the immune system or proliferating immune cells.

[0045] In a tenth aspect, the present invention provides a treatment method including administering the IL-2 mutant according to the first aspect, the fusion protein or conjugate according to the second aspect, or the pharmaceutical composition according to the sixth aspect to a patient in need of IL-2 immunotherapy.

[0046] In a preferred embodiment, the treatment method treats cancer or immune diseases, human immunodeficiency virus (HIV) infection, hepatitis C virus (HCV) infection, rheumatoid arthritis, atopic dermatitis, etc. by stimulating the immune system or proliferating immune cells.

[0047] It should be understood that within the scope of the present invention, new or preferred technical solutions can be formed by combining each of the above technical features of the present invention with the technical features specifically described below (for example, in the embodiments). Due to space limitations, it will not be repeated here.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0049] After extensive and detailed research, the inventors of the present invention unexpectedly discovered that a novel IL-2 mutant polypeptide with glycosylation modification after site-specific mutation of the IL-2 polypeptide can eliminate or reduce the affinity of the IL-2 protein for the high-affinity IL-2 receptor, maintain the affinity of the mutant IL-2 protein for the medium-affinity IL-2 receptor, maintain the biological activity of IL-2, and better stimulate the proliferation of tumor immune cells including but not limited to T effector cells and NK cells, thereby achieving the purpose of treatment. Based on this, the present invention was completed.

[0050] Site-directed mutagenesis technology Site-directed mutagenesis is a protein engineering technology that generates mutant protein molecules with new traits by substituting, inserting, or deleting specific nucleotides in a known DNA sequence based on the structure and function of a known protein.

[0051] Site-directed mutagenesis technology can change the physical and chemical properties of proteins. For example, it can improve the stability of protein drugs, enhance the solubility performance of protein drugs, change the specificity of enzymes for substrates, improve enzyme activity, and improve affinity and specificity, thereby improving biological properties.

[0052] Site-directed mutagenesis technology can mutate the amino acids in the binding domain to increase or eliminate the binding activity between the ligand and the receptor, and between the enzyme and the substrate. Such mutations can modify the properties of the protein, resulting in changes in characteristics such as the secondary structure or high-level structure and the charge it possesses. When the mutated amino acid is finally in an important antigen-antibody interaction site, the change in this amino acid will result in changes in the charge and secondary or high-level structure of this site, making it unable to bind between the ligand and the receptor, and the purpose of the mutation is achieved. At the same time, the original antibody can no longer recognize this site, that is, a new antigen is formed. This inevitably occurs in such existing mutagenesis methods.

[0053] The IL-2 mutant of the present invention In the present invention, by introducing amino acid residues into the IL-2 polypeptide via site-directed mutagenesis, the binding mode between the IL-2 polypeptide and the IL-2R receptor is modified. The affinity between the modified IL-2 mutant of the present invention and the IL-2 receptor is modified. In a specific embodiment, the IL-2 mutant of the present invention eliminates or reduces the affinity for the high-affinity IL-2 receptor, while maintaining the affinity for the medium-affinity IL-2 receptor and maintaining the biological activity of IL-2. Therefore, the IL-2 mutant of the present invention can not only better stimulate the proliferation of tumor immune cells including but not limited to T effector cells and NK cells, but also significantly reduce its side effects compared with wild-type IL-2, so that better therapeutic purposes can be achieved.

[0054] When the IL-2 mutant or fusion protein of the present invention has glycosylation modification, preferably eukaryotic cell expression is used and obtained by cell culture. Yeast, insect cells, and animal cells can be selected, and genetically modified animals can be selected. In a specific embodiment, the host cell is a eukaryotic cell, preferably yeast, insect cells, or animal cells. When the IL-2 mutant or fusion protein of the present invention does not have glycosylation modification, cell-free expression, expression in E. coli, yeast, etc. can be used, and cell-free expression and yeast cells are preferred.

[0055] When yeast cells or insect cells are used as host cells, the glycoform of the obtained IL-2 mutant may be non-human. Those skilled in the art know that non-human glycoforms can be further converted into adult glycoforms.

[0056] In other embodiments, the IL-2 mutant can be obtained using prokaryotic expression fermentation or in vitro cell-free synthesis, and then the IL-2 mutant correctly glycosylated by methods such as in vitro enzyme catalysis can be obtained. In vivo and in vitro modification solutions can achieve the same purpose, namely, site-specific glycosylation of IL-2.

[0057] Glycosylation modification and mutation of proteins Protein glycosylation is a complex post-translational modification process. Glycosylation modification is performed at specific sites of the protein. Usually, the modification sites are asparagine residues (N-linked) or serine / threonine residues (O-linked). N-linked glycosylation modification generally occurs at Asn-X-Ser / Thr (X is a non-proline amino acid, called the N-glycosylation site), and O-linked glycosylation modification generally occurs at serine (Ser) or threonine (Thr) residues (called the O-glycosylation site), forming an O-glycoside bond by N-acetylgalactosamine (Gal-NAc) and the hydroxyl group of Ser / Thr. Correspondingly, the glycosylation site introduced into wild-type IL-2 by site-specific mutation is an artificial glycosylation site.

[0058] The inventors increase the three-dimensional spatial structure on the binding surface between the receptor protein and the ligand protein by artificially introducing glycosylation mutations so that the receptor and the ligand cannot come into direct contact. At the same time, it can be seen that when mutations occur at specific sites, glycosylation modification may significantly change the binding between the receptor and the ligand. Different from multiple previous amino acid site-specific mutations, the present invention greatly reduces the number of amino acid mutations. Conventional site-specific mutations change the hydrophilic or hydrophobic regions of the binding surface of the protein after multiple amino acids, or change its high-level structure so that two proteins cannot form a stable binding state after contact. In the present invention, after glycosylation mutation, the binding surface is cleaved by sugar chains, so the two proteins cannot be brought close to each other. Under the conditions of appropriate site selection, its ability to eliminate binding is much higher than that of the conventional mutation method, or by changing only one amino acid such as methionine at position 39 of the amino acid, the purpose of reducing or eliminating the binding between the ligand and the receptor can be achieved.

[0059] Specifically, the inventors selected the possible binding regions of interleukin-2 and interleukin-2 receptor and the mutation points in the amino acid sequences near them, used the original amino acid sequence containing the ASN, Thr or Ser sites as much as possible, and preferentially selected the first two amino acid mutations at the Thr or Ser sites. After glycosylation is completed, the amino acids at the mutation points are masked by sugar chains, and the immunogenicity is minimized. Mutations of Thr or Ser after the Asn site are also feasible solutions.

[0060] The point mutation at position 39 may change the ability to bind to the receptor regardless of glycosylation. Since there is only a single point mutation, the potential immunogenicity generated is very small.

[0061] Traditionally, it is considered that amino acids containing polymer amino acids and complex aromatic rings have greater immunogenicity, and multiple-point mutations produce more obvious immunogenicity. Due to the large molecular weight of sugar chains, especially N-glycans, which have complex 2-antennal, 3-antennal to 4-antennal structures (Jonathan J. Lyons et al., Glycans instructing immunity: the emerging role of altered glycosylation in clinical immunology, Front. Pediatr., June 11, 2015), any mutation near the binding site affects the binding between two proteins.

[0062] According to the teachings of the present invention, those skilled in the art know how to introduce fewer mutation sites into wild-type IL-2 by site-directed mutagenesis. In order to reduce the immunogenicity of the obtained IL-2 mutant, the IL-2 mutant of the present invention needs to reduce the number of mutation sites as much as possible and use the existing amino acid residues of natural IL-2 to generate new glycosylation sites. Therefore, the structure of the obtained IL-2 mutant is close to the structure of natural IL-2, avoiding the influence of mutations on other structural sites of the protein and maintaining biological activity. In a specific embodiment, mutations of amino acid residues occur at one or more sites of 39, 49, 73 and 109 of wild-type IL-2 (that is, one, two, three or four sites). In a specific embodiment, in wild-type IL-2, mutations of one or more amino acid residues among M39D, M39E, M39Q, M39N, M39A, K49N, A73T, A73S and D109N occur, preferably point mutations of M39D, M39E, M39Q, M39N or M39A, more preferably point mutations of M39D, M39E, M39Q or M39N, more preferably point mutations of M39D, M39E or M39Q, more preferably point mutations of M39D or M39E, and most preferably point mutation of M39D occurs.

[0063] Based on the conventional practices in this field, the original O-glycosylation site of the IL-2 polypeptide can also be eliminated. The removal of O-glycosylation does not affect the biological activity of IL-2. O-glycosylation has a complex structure and is difficult to analyze. In order to reduce the complexity of production quality control, genetic engineering mutation techniques are usually used to eliminate the glycosylation site. Therefore, in the IL-2 mutant of the present invention, mutations of amino acid residues of T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K and T3P can occur at position 3 corresponding to the wild-type IL-2 protein, and preferably a mutation of the amino acid residue of T3A can occur. During the purification and refolding of the IL-2 gene product, for example, mismatching of disulfide bonds or the formation of intermolecular disulfide bonds may both reduce the activity of IL-2. By using current site-directed mutagenesis to mutate cysteine at position 125 to leucine or serine to form only one disulfide bond, the activity during the IL-2 refolding process is guaranteed. Furthermore, there are also reports on using protein engineering techniques to produce novel rIL-2, changing cysteine at position 125 of the IL-2 molecule to alanine so that the activity of the modified IL-2 is significantly increased compared to natural IL-2. Therefore, in the IL-2 mutant of the present invention, mutations of amino acid residues of C125L, C125A, C125S can occur at position 125 corresponding to the wild-type IL-2 protein, and preferably a mutation of the amino acid residue of C125S can occur.

[0064] The IL-2 mutant protein of the present invention eliminates or reduces the affinity for the high-affinity IL-2 receptor, but can basically maintain the affinity of the IL-2 mutant for the intermediate-affinity IL-2 receptor. As used herein, "eliminates or reduces" refers to a situation where the change exceeds 50% compared to the original level (for example, compared to the affinity between wild-type IL-2 and the high-affinity IL-2 receptor). Therefore, "eliminating or reducing the affinity of the IL-2 mutant for the high-affinity IL-2 receptor" as described herein means that the binding affinity between the IL-2 mutant of the present invention and the high-affinity IL-2 receptor is reduced by 50% or more, more preferably 60% or more, 70% or more, 80% or more, 90% or more, 95% or more compared to the wild-type IL-2 protein, and most preferably the IL-2 mutant protein does not bind to the high-affinity IL-2 receptor. Similarly, "basically maintaining the affinity of the IL-2 mutant for the intermediate-affinity IL-2 receptor" as described herein means that the binding affinity between the IL-2 mutant and the intermediate-affinity IL-2 receptor is 50% or more, more preferably 60% or more, 70% or more, 80% or more, 90% or more, 95% or more of the binding affinity between wild-type IL-2 and the intermediate-affinity IL-2 receptor. In a preferred embodiment, the binding affinity between the IL-2 mutant and the intermediate-affinity IL-2 receptor corresponds to the binding affinity between wild-type IL-2 and the intermediate-affinity IL-2 receptor.

[0065] Furthermore, due to the very large individual differences among subjects, the inventors concluded that some IL-2 mutant proteins of the prior art showed relatively large differences in different molecular structures or between different experimental batches. For example, they showed relatively large differences at low and high concentrations. However, the IL-2 mutant of the present invention can exhibit excellent technical effects under both low and high concentrations.

[0066] "corresponds to" The term "corresponding to" described in this specification has the meaning generally understood by those skilled in the art. Specifically, "corresponding to" indicates the position where one array corresponds to the specified position of the other array after comparing the homology or identity of the two arrays. Thus, for example, "corresponding to wild-type IL-2" means comparing the amino acid sequences of a specific amino acid sequence and wild-type IL-2 and finding the site on the amino acid sequence that corresponds to wild-type IL-2.

[0067] Methods for measuring sequence homology or identity known to those skilled in the art include, but are not limited to, Computational Molecular Biology, Lesk, A.M. (ed.), Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W. (ed.), Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M. and Griffin, H.G. (eds.), Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J. (eds.), M Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). A preferred method for measuring identity is to obtain the maximum matching between the sequences being tested. Methods for measuring identity are compiled into computer programs available to the public. Preferred computer program methods for measuring identity between two sequences include, but are not limited to, the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S.F. et al., 1990). The public can obtain the BLASTX program (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S. et al., 1990) from NCBI and other sources. The well-known Smith Waterman algorithm can also be used for identity measurement.

[0068] The fusion protein or conjugate of the present invention Based on the IL-2 mutants of the present invention, those skilled in the art will understand that the IL-2 mutants of the present invention and other non-IL-2 functional moieties can be made into fusion proteins or conjugates. As used herein, conjugate means a water-soluble polymer that covalently binds to the residues of the mutant IL-2 polypeptide. In a specific embodiment, the non-IL-2 functional moiety includes, but is not limited to, an Fc fragment, human serum albumin (HSA), an anti-HSA antibody or antibody fragment, transferrin, the carboxy-terminal peptide (CTP) of the human chorionic gonadotropin β subunit, an elastin-like peptide (ELP), and an antigen-binding moiety. In a preferred embodiment, the antigen-binding moiety can be an antibody or an active antibody fragment thereof, a Fab molecule, a scFv molecule, a VHH molecule, an immunoglobulin molecule, a receptor protein molecule, or a ligand protein molecule, and the immunoglobulin molecule can be an IgG molecule.

[0069] Based on the general operations in this field, those skilled in the art can know the method for obtaining a fusion protein or conjugate containing the IL-2 mutant of the present invention. For example, the IL-2 mutant of the present invention can be directly linked to other non-IL-2 functional moieties or can be linked via an adapter. The adapter can be a repetitive sequence of G 4 such as (G3S) 3 and can be a repetitive sequence of AAA or GS including, but not limited to, repetitive sequences of G4S.

[0070] Furthermore, polyethylene glycol modification (PEGylation), polysialylation modification (PSAylation), saturated fatty acid modification, hyaluronic acid modification (Hyaluronic acid, HA), or polyamino acid modification (proline-alamine-serine polymer, PASylation) can also be performed on the IL-2 mutant or fusion protein conjugate to form a conjugate.

[0071] The pharmaceutical composition of the present invention and its administration method Based on the IL-2 mutant of the present invention, the present invention further provides a pharmaceutical composition. In a specific embodiment, the pharmaceutical composition of the present invention comprises the IL-2 mutant or fusion protein or conjugate of the present invention, and optionally a pharmaceutically acceptable adjuvant.

[0072] Optionally, the composition of the present invention further comprises a pharmaceutically acceptable excipient. If desired, a pharmaceutically acceptable excipient can be added to the IL-2 mutant polypeptide, fusion protein or conjugate of the present invention to form a composition.

[0073] Exemplary excipients include, but are not limited to, those excipients selected from the group consisting of sugars, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, amino acids and combinations thereof. Sugars such as sugars, derivatized sugars (e.g., sugar alcohols, aldonic acids, esterified sugars and / or sugar polymers) can be present as excipients. Specific sugar excipients include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc., disaccharides such as lactose, sucrose, trehalose, cellobiose, etc., polysaccharides such as raffinose, maltodextrin, dextran, starch, etc., and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, sorbitol (glucitol), inositol, cyclodextrin, etc.

[0074] The excipient can further comprise an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and combinations thereof.

[0075] The composition may further comprise an antimicrobial agent that prevents or inhibits the growth of microorganisms. Non-limiting examples of antimicrobial agents suitable for one or more embodiments of the present invention are benzalkonium chloride, benzethonium chloride, benzyl alcohol, phenol, phenethyl alcohol, and combinations thereof.

[0076] An oxidizing agent may also be present in the composition. By preventing oxidation, the antioxidant prevents the alteration of proteins, conjugates or other components of the formulation. Antioxidants suitable for one or more embodiments of the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol and combinations thereof.

[0077] Surfactants can be present as excipients. Exemplary surfactants include polysorbates such as "Tween 20" and "Tween 80" and pluronics such as F68 and F88, sorbitan esters, phospholipids (e.g., lecithin and other phosphatidylcholines), lipids such as fatty acids and fatty esters, and steroids such as cholesterol.

[0078] An acid or base can be present in the composition as an excipient. Non-limiting examples of acids that can be used include acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, sulfuric acid, fumaric acid and combinations thereof. Suitable examples of bases include bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumarate and combinations thereof (but not limited thereto).

[0079] Here, one or more amino acids that may be present as excipients in the composition are restricted. In this aspect, exemplary amino acids include arginine, lysine and glycine.

[0080] The amount of conjugate in the composition (i.e., the conjugate formed between the active agent and the polymeric reagent) depends on several factors, but will be the most preferred therapeutically effective amount when stored in a unit dose container of the composition (e.g., one vial). Further, the pharmaceutical formulation can be housed in one syringe. The therapeutically effective amount can be determined experimentally to produce the desired endpoint pharmacologically by repeatedly administering increasing amounts of the drug.

[0081] The amount of any individual excipient in the composition will vary depending on the activity of the excipient and the specific needs of the composition. Typically, the most preferred amount of any individual excipient is determined by common experimentation, i.e., preparing compositions containing different amounts of the excipient (ranging from low to high), examining stability and other parameters, and then determining the range that gives the most desirable refinement without significant adverse effects.

[0082] However, overall, the excipient is present in an amount of about 1 wt% to about 99 wt%, preferably about 5 wt% to about 98 wt%, more preferably about 15 wt% to about 95 wt% of the excipient in the composition, and most preferably the concentration is less than 30 wt%.

[0083] These compositions include all kinds of formulations, such as reconstitutable formulations, lyophilized agents and liquid agents, and formulations particularly suitable for injection. Examples of diluents suitable for reconstituting the solid composition before injection include bacteriostatic water for injection, 5% dextrose in water, phosphate buffered saline, sterile water, deionized water, and combinations thereof. In the case of liquid pharmaceutical compositions, solutions and suspensions are contemplated.

[0084] The composition of one or more embodiments of the present invention is typically (but not necessarily) a liquid solution or suspension immediately before administration because it is administered by injection. The pharmaceutical formulation can also use other forms such as syrups, emulsions, ointments, tablets, powders, etc. Other modes of administration are further included, such as transpulmonary, rectal, transdermal, transmucosal, oral, intrathecal, intratumoral, peritumoral, intraperitoneal, subcutaneous, intraarterial, etc.

[0085] The present invention further provides a method of administering, in a therapeutically effective amount, to a patient having a reactive symptom. It can be injected (e.g., intramuscularly, subcutaneously, and parenterally). Types of formulations suitable for parenteral administration include solutions that can be used immediately for injection, dry agents combined with a solvent before use, suspensions that can be used immediately for injection, dry insoluble compositions combined with a solvent before use, and emulsions and liquid concentrates that are diluted before administration, in particular.

[0086] The method of administration can be used to treat any symptom that can be treated or prevented by administering the mutant protein, fusion protein, or conjugate. Those skilled in the art know which symptoms a particular composition can effectively treat. For example, it is used to treat patients having a disease selected from the group consisting of renal cell carcinoma, metastatic melanoma, hepatitis C virus (HCV), human immunodeficiency virus (HIV), acute myeloid leukemia, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, juvenile rheumatoid arthritis, atopic dermatitis, breast cancer, and bladder cancer.

[0087] The actual dose administered varies depending on the age, weight, and general condition of the subject, as well as the severity of the symptom to be treated and the judgment of the healthcare professional. A therapeutically effective amount is known to those skilled in the art and / or is described in relevant reference documents and literature. Generally, the range of therapeutically effective amounts is a dose of about 0.001 mg to 1000 mg, preferably 0.01 mg / day to 100 mg / day, and more preferably within a dose of 0.10 mg / day to 50 mg / day. A predetermined dose can be administered periodically until symptoms such as organophosphorus poisoning are alleviated and / or completely resolved.

[0088] The unit dosage can be administered according to various dosing schedules depending on the judgment of the clinician, the needs of the patient, etc. Specific dosing schedules are known to those skilled in the art or can be determined experimentally using conventional methods. Exemplary dosing schedules include, but are not limited to, twice daily, once daily, three times a week, twice a week, once a week, twice a month, once a month dosing, and any combination thereof. When the clinical evaluation items are achieved, the administration of the composition is interrupted.

[0089] The present invention will be described in connection with preferred specific embodiments of the present invention, but it should be understood that the foregoing description and the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Other aspects, advantages, and variations within the scope of the present invention will be apparent to those skilled in the art.

[0090] Use and method of use of the IL-2 mutant of the present invention As described above, the IL-2 mutant of the present invention eliminates or reduces the affinity of the IL-2 protein for the high-affinity IL-2 receptor, maintains the affinity of the mutant IL-2 protein for the intermediate-affinity IL-2 receptor, and maintains the biological activity of IL-2, thereby better stimulating the proliferation of tumor immune cells including, but not limited to, T effector cells and NK cells. Therefore, the IL-2 mutant, fusion protein, conjugate or pharmaceutical composition of the present invention can be prepared into corresponding drugs. The drugs can be used to proliferate T lymphocytes and natural killer NK cells in vitro or to treat diseases in which IL-2 is used in immunotherapy. In a specific embodiment, the disease is cancer, for example, cancer that needs to be treated by stimulating the immune system or proliferating immune cells. In a specific embodiment, the disease can be an immune disease, human immunodeficiency virus HIV infection, hepatitis C virus HCV infection, rheumatoid arthritis, atopic dermatitis, etc.

[0091] The present invention can be used as a substitute for wild-type IL-2 when proliferating cells in vitro during cell therapies such as CAT-T, CAR-NK, etc.

[0092] The advantages of the present invention are as follows. 1. The IL-2 mutant protein of the present invention reduces or eliminates the affinity with the high-affinity IL-2 receptor and basically maintains the affinity for the medium-affinity IL-2 receptor. 2. The structure of the IL-2 mutant of the present invention is close to the structure of natural IL-2, avoiding the influence of mutations on other structural sites of the protein and maintaining biological activity. 3. Compared with other IL-2 mutants of the prior art, the IL-2 mutant of the present invention has lower immunogenicity. 4. The IL-2 mutant of the present invention has a simple molecular design and is convenient for application to different molecules. 5. The IL-2 mutant of the present invention is convenient for production and quality control, generally does not require an in vitro re-modification process, and reduces steps for improving production efficiency. 6. The IL-2 mutant of the present invention is convenient for forming bifunctional or multifunctional fusion proteins or immune compositions with other molecules. 7. The IL-2 mutant of the present invention can be used in immunotherapy and will not cause the vascular (or capillary) leak syndrome (VLS) caused by natural IL-2.

[0093] Hereinafter, the present invention will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and do not limit the scope of the present invention. In the following examples, experimental methods without indicating specific conditions usually follow conventional conditions such as those described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions proposed by the manufacturer.

Examples

[0094] Example 1. Synthesis of Mutant Interleukin-2 (IL-2) Protein 1. Gene Synthesis The nucleotide sequence encoding the amino acid sequence of the mutant of interleukin-2 (IL-2) protein is obtained by an automated gene synthesis method. In some examples, adding a HIS tag to the end of the gene fragment is advantageous for purification. In some examples, adding IgG1-Fc to the end of the gene fragment is advantageous for purification. At the same time, the Fc tag is also a common means to extend the half-life of protein pharmaceuticals. There is a single restriction endonuclease cleavage site on the side of the gene fragment. All gene synthesis sequences are designed to have a 5' end DNA sequence encoding a leading peptide that targets the secretion of the protein in eukaryotic cells.

[0095] [Table 1]

[0096] 2. Plasmid Construction The synthesized gene is cloned into the pcDNA3.4 plasmid, and the method uses molecular biology reagents according to the manufacturer's instructions.

[0097] 3. Expression of Mutant Interleukin-2 (IL-2) Protein Expi293F cells (Thermo Fisher Scientific) are used to transfect the plasmid, and the cells are cultured in a shaker (VWR Scientific) under the conditions of 37°C and 8% CO 2 and inoculated into shaking flasks (Corning Erlenmeyer Flasks) the day before transfection, and the transfection method is carried out according to the manufacturer's instructions. On the 2nd, 4th, and 5th days, the cell supernatant suspensions are collected respectively, and Western blotting is carried out to confirm the protein expression.

[0098] Example 2. Expression of CD25 protein Gene synthesis The nucleotide sequence encoding the amino acid sequence of CD25 protein (SEQ ID NO: 23) is obtained by an automated gene synthesis method. SEQ ID NO: 24 (GGGSGGGSGGGSGGGS) is the amino acid sequence of the adapter. In some examples, the gene fragment is co-expressed with IgG1-Fc via the adapter, which is advantageous for purification. There is a single restriction endonuclease cleavage site on the sides of the gene fragment. All gene synthesis sequences are designed to have a 5' end DNA sequence encoding a leading peptide that targets the secretion of the protein in eukaryotic cells. SEQ ID NO: 25 shows an exemplary leading peptide sequence. The synthesized gene is cloned into the pcDNA3.4 plasmid, and the method uses molecular biology reagents according to the manufacturer's instructions.

[0099] Expi293F cells (Thermo Fisher Scientific) are used to transfect the plasmid, and the cells are cultured in a shaker (VWR Scientific) at 37 °C and 8% CO 2 under the conditions, inoculated into shaking flasks (Corning Erlenmeyer Flasks) the day before transfection, and the transfection method is carried out according to the manufacturer's instructions. On the 2nd, 4th, and 5th days, the cell supernatant suspensions are collected respectively, and Western blotting is performed to confirm the expression of the protein.

[0100] Example 3. Detection experiment of binding affinity with CD25 using ELISA, Fortebio or biacore The inventors detect the binding ability between the IL-2 mutant and CD25 using enzyme-linked immunosorbent assay.

[0101] CD25 (derived from Example 2) was coated onto a 96-well high-adsorption microplate (3590, Costar), washed, and blocked. The samples to be tested were diluted to an appropriate concentration and added to the wells. Color development was carried out with TMB, and the signal values of each well were read at a wavelength of 450 / 650 nm using an ELISA reader (M5, Molerlder Devies). rhIL-2 is recombinant human interleukin-2 for injection (Quanqi).

[0102]

Table 2

[0103] The results are shown in Figure 1. As can be seen from the figure, it is clearly evident that rhIL-2 is bound under the experimental concentrations. When comparing the dose-dependent binding sizes of the binding activities, rhIL-2 > IL-2gm2 > IL-2gm4 > IL-2gm11 > IL-2gm3 > IL-2gm7 > IL-2gm12 > IL-2gm13. Under these concentrations, those without dose-dependence can be judged not to bind, and these include IL-2gm1, IL-2gm5, IL-2gm6, IL-2gm8, IL-2gm9, IL-2gm10, IL-2gm14, IL-2gm15, IL-2qm (CN103492411A). IL-2gm1, IL-2gm5, IL-2gm6, IL-2gm8, IL-2gm9, IL-2gm10, IL-2gm14, IL-2gm15 completely lost their binding ability to CD25, and IL-2gm2, IL-2gm4, IL-2gm11, IL-2gm3, IL-2gm7, IL-2gm12, IL-2gm13 proved to have partially lost their binding ability to CD25.

[0104] Example 4. Cell proliferation analysis using CTLL2 cells In this example, the inventors evaluated the activities of rhIL-2 and the mutant interleukin-2 of Example 1 in cell proliferation analysis using CTLL2 cells.

[0105] The same number of CTLL-2 cells (a mouse cytotoxic T lymphocyte cell line belonging to IL-2 dependence and highly expressing CD25 on the cell surface) were inoculated onto the experimental plates, and then rhIL-2 and IL-2 mutants were added according to the concentration gradient and incubated for 48 hours, and then put into cell Titer Glo Luminescent buffer. By detecting the intracellular ATP content (SpectraMax M5) by chemiluminescence and detecting the number of cells in each well, the effects of different concentrations of rhIL-2 and IL-2 mutants on cell proliferation were detected. The data were analyzed with GraphPad Prism7 software and the curves were fitted by nonlinear regression analysis. From the nonlinear regression analysis of the dose-response curve, the EC 50 value (the concentration of the test compound required to show 50% of the maximum response) was obtained.

[0106] Cell proliferation analysis was used to measure the activities of rhIL-2 and mutant interleukin-2 (His-tag), and the summary of the results is shown in Table 2 and Figure 2. All test parts induced the cell growth of CTLL-2 in a dose-dependent manner. When the cell proliferation multiples corresponded, the EC 50The larger it is, the more likely it is proven that its activity to stimulate the growth of CTLL2 is weaker. Such a change is due to the effect of its mutant protein on the binding to CD25. The mutant protein maintains IL-2R signaling through the activation of the IL-2Rβγ heterodimer. After the concentration increases, the cells are still effectively amplified. The effectiveness of all IL-2 mutants (His-tag) against rhIL-2 does not exceed 8.314%. IL-2gm(1-15) can remove the binding to CD25 and does not form the IL-2Rαβγ heterotrimer, proving that the stimulating effect decreases. Preferably, IL-2gm1, IL-2gm3, IL-2gm5, IL-2gm6, IL-2gm7, IL-2gm8, IL-2gm9, IL-2gm10, IL-2gm12, IL-2gm14 and IL-2gm15 had a proliferation effector exceeding 50-fold compared to the control sample rhIL-2. Here, IL-2gm1, IL-2gm6, and IL-2gm14 exceeded 100-fold.

[0107]

Table 3

[0108] Cell proliferation analysis was used to measure the activities of rhIL-2 and mutant interleukin-2 (Fc-tag), and the summary of the results is shown in Table 3 and Figure 3. All test articles induced CTLL-2 growth in a dose-dependent manner. When the cell proliferation fold corresponded, EC 50The larger it is, the weaker its activity to stimulate CTLL2 growth is proven. Such a change is due to the effect of its mutant protein on the binding to CD25. The mutant protein maintains IL-2R signaling by IL-2Rβγ heterodimer activation. After the concentration increases, the cells are still effectively amplified. The effectiveness of all IL-2 mutants (Fc-tag) against rhIL-2 does not exceed 31.546%. Similarly, the dimeric form of IL-2gm(4 - 6) can remove the binding to CD25 and does not form IL-2Rαβγ heterotrimer, proving that the stimulating effect decreases. Preferably, IL-2qm-Fc, IL-2gm5, and IL-2gm6 have a growth effect on CTLL2 more than 50 times that of the control sample rhIL-2. Here, no significant cell growth is observed for IL-2qm-Fc and IL-2gm6-Fc in the test concentration range.

[0109]

Table 4

[0110] Example 5. Cell proliferation analysis using NK92 cells The inventors evaluate the activities of rhIL-2 and the mutant interleukin-2 of Example 1 in cell proliferation analysis using NK92 cells. The same number of NK92 cells (NK-92 cells are an IL-2-dependent NK cell line derived from peripheral blood mononuclear cells of a 50-year-old white male suffering from aggressive non-Hodgkin lymphoma. Some of its cell surfaces express CD25.) were inoculated onto the experimental plates, and then rhIL-2 and IL-2 mutants were added according to the concentration gradient. After incubating for 72 hours, cell Titer Glo Luminescent buffer was added. By detecting the intracellular ATP content (SpectraMaxM5) by chemiluminescence and detecting the number of cells in each well, the effects of different concentrations of rhIL-2 and IL-2 mutants on cell proliferation were detected. The data were analyzed with GraphPad Prism7 software, and the curves were fitted by nonlinear regression analysis. From the nonlinear regression analysis of the dose-response curve, the EC 50 value (the concentration of the test compound required to show 50% of the maximum response) was obtained.

[0111] Cell proliferation analysis was used to measure the activities of rhIL-2 and mutant interleukin-2 (His-tag), and the summary of the results is shown in Table 4 and Figure 4. All test components induced the cell growth of NK92 in a dose-dependent manner. When the cell proliferation fold is corresponding, it is considered that the larger the EC 50 is, the weaker its activity to stimulate the growth of NK92 is proved. Such a change is due to the effect of its mutant protein on the binding to CD25. Since the mutant protein maintains the IL-2R signal transduction by the activation of the IL-2Rβγ heterodimer, after the concentration increases, the cells are still effectively amplified. The effectiveness of all IL-2 mutants (His-tag) against rhIL-2 does not exceed 1.721%. In some NK92 cells expressing CD25 on the surface of IL-2gm(1~15), the binding to CD25 was removed and the IL-2Rαβγ heterotrimer was not formed, which proved that the stimulating effect decreased. Here, IL-2gm1, IL-2gm5 and IL-2gm14 are the most obvious, and the proliferation effect decreases by more than 100.

[0112]

Table 5

[0113] The activities of rhIL-2 and mutant interleukin-2 (Fc-tag) were measured using cell proliferation assays, and the summary of the results is shown in Table 5 and Figure 5. All test articles induced NK92 cell growth in a dose-dependent manner. When the cell growth fold is corresponding, the larger the EC 50 is, the weaker its activity to stimulate NK92 growth is proved. Such changes are due to the influence of the mutant protein on its binding to CD25. The mutant protein maintains IL-2R signaling through IL-2Rβγ heterodimer activation. After the concentration increases, the cells are still effectively amplified. The efficacy of all IL-2 mutants (Fc-tag) against rhIL-2 does not exceed 34.783%. Similarly, the dimers of IL-2qm-Fc and IL-2gm(4-6) can reduce the binding of some NK92 cells expressing CD25 to CD25, and do not form IL-2Rαβγ heterotrimers, proving that the stimulating effect is reduced. Preferably, IL-2qm-Fc and IL-2gm6-Fc are reduced by more than 100-fold compared with rhIL-2. However, the growth fold of IL-2qm against NK92 cells is significantly smaller than that of other mutant proteins.

[0114]

Table 6

[0115] Example 6. Half-life study of the mutant protein of the present invention The inventors further studied the in vivo half-life of the mutant protein obtained by the method of the present invention, and found that the in vivo half-lives of the mutant proteins obtained by the method of the present invention all increased to varying degrees.

[0116] Example 7. Synthesis of IL-2 mutant protein (HSA-tag) The inventors synthesized the following IL-2 mutants.

[0117] [Table 7]

[0118] The coding sequences of the IL-2 mutant molecules IL-2gm1, IL-2gm4, IL-2gm6, and IL-2gm7 and the HSA coding sequence were ligated and constructed into a eukaryotic expression vector via molecular cloning means to prepare an expression vector for the IL-2 mutant molecule. Using 293E cells cultured in Freestyle medium, transient transfection expression of the IL-2 mutant molecule was performed. Twenty-four hours before transfection, 150 ml of 293E cells at 0.5×10 6 cells / ml were inoculated into a 1 L cell culture flask and cultured with shaking at 120 rpm in an incubator at 37°C and 5% CO 2 . At the time of transfection, first, 150 μl of 293fectin was taken, placed in 2.85 ml of OptiMEM, mixed well and uniformly, and incubated at room temperature for 2 minutes. At the same time, 150 μg of the plasmid for expressing the IL-2 molecule was diluted to 3 ml with OptiMEM respectively. The diluted transfection reagent and plasmid were mixed well and incubated at room temperature for 15 minutes. Next, all the mixtures were added to the cells and mixed uniformly, and cultured with shaking at 120 rpm in an incubator at 37°C and 5% CO 2 for 7 days. The cell culture supernatant was collected, filtered through a 0.22 μm filter membrane, and then purified using a Q-HP ion exchange chromatography column (GE), eluted linearly under pH 8.0 conditions using 20 mM Tris - 500 mM NaCl, and samples were continuously collected according to the dosage. SDS-PAGE detection was performed on each collected component using a 4 - 20% gradient gel (Genscript), and the samples were combined according to the electrophoresis purity.

[0119] Example 8. Preparation of Receptor Protein To study the binding ability of the IL-2 mutant molecule to the IL-2Rα receptor and the IL-2Rβγ heterodimerization receptor, human-derived IL-2Rα receptor and IL-2Rβγ heterodimerization receptor proteins are prepared and used for Biacore affinity detection. The design of the human-derived IL-2Rα receptor is to ligate the IL-2Rα extracellular domain coding sequence to the 6×His Tag coding sequence (SEQ ID NO: 31) and clone it into a eukaryotic expression vector. Transient transfection expression of the IL-2Rα receptor is performed using 293E cells cultured in Freestyle medium. 24 hours before transfection, 150 ml of 293E cells at 0.5×10 6 cells / ml are inoculated into a 1 L cell culture flask and cultured with shaking at 120 rpm in a 37°C, 5% CO 2 incubator. At the time of transfection, first take 150 μl of 293Fectin, put it into 2.85 ml of OptiMEM, mix well, incubate at room temperature for 2 minutes, and at the same time, dilute 150 μg of the plasmid for expressing the IL-2Rα receptor to 3 ml with OptiMEM. Mix the diluted transfection reagent and plasmid well, incubate at room temperature for 15 minutes, then put all the mixture into the cells and mix evenly, and culture with shaking at 120 rpm in a 37°C, 5% CO 2 incubator for 7 days. Collect the cell culture supernatant, filter the supernatant with a 0.22 μm filter membrane, and then purify it using a Ni-NTA affinity chromatography column (GE) and elute it under the condition of 20 mM PB - 0.5 M NaCl - 100 mM imidazole. Perform SDS-PAGE detection on the purified protein using a 4 - 20% gradient gel (Genscript).

[0120] The design of the human-derived IL-2Rβγ heterodimerization receptor utilizes the "Knobs into Holes" technology. The IL-2Rβ extracellular domain-encoding sequence is ligated to the "Knobs" Fc fragment (SEQ ID NO: 32), cloned into a eukaryotic expression vector. The IL-2Rγ extracellular domain-encoding sequence is ligated to the "Holes" Fc fragment (SEQ ID NO: 33) and cloned into a eukaryotic expression vector. Using 293E cells cultured in Freestyle medium, transient transfection expression of the IL-2Rβγ heterodimerization receptor is performed. 24 hours before transfection, inoculate 150 ml of 293E cells at 0.5×10 6 cells / ml into a 1 L cell culture flask and shake-culture at 37°C and 5% CO 2 in an incubator at 120 rpm. At the time of transfection, first take 150 μl of 293fectin, put it into 2.85 ml of OptiMEM, mix well, incubate at room temperature for 2 minutes. At the same time, dilute 75 μg of the plasmid for expressing the IL-2Rβγ heterodimerization receptor to 3 ml each with OptiMEM. Mix the diluted transfection reagent and plasmid well, incubate at room temperature for 15 minutes, then add all the mixtures to the cells and mix uniformly, and shake-culture at 37°C and 5% CO 2 in an incubator at 120 rpm for 7 days. Collect the cell culture supernatant, filter the supernatant with a 0.22 μm filter membrane, then purify it using a MabSelect SuRe affinity chromatography column (GE), elute under the conditions of 20 mM citric acid-sodium citrate, pH 3.0, and adjust the pH to neutral with 1 M Tris base. Perform SDS-PAGE detection on the purified protein using a 4-20% gradient gel (Genscript).

[0121] Example 9. Detection experiment of binding affinity with the receptor using Biacore To study the affinity of the IL-2 mutants for the receptor compared to the wild type, the affinity of the IL-2 mutants and wild-type IL-2-HSA for the human IL-2Rα subunit was measured via Biacore 8K (GE) using the recombinant monomeric IL-2Rα subunit under the following conditions, and the human IL-2Rα subunit was immobilized on a CM5 chip (190 RU). The IL-2 mutants and IL-2-HSA in HBS-EP buffer were used as analytes at 25°C. For IL-2Rα, the analyte concentration decreased from 200 nM to 1.526 nM (1:2 dilution) and flowed at 30 μl / min (the binding time was 180 s and the dissociation time was 300 s). For IL-2Rα, it was regenerated with 20 mM NaOH at 30 ul / min for 10 s. For IL-2Rα, 1:1 binding, RI≠0, and R maximum value = global fitting data were used.

[0122] Under the following conditions, the affinity of the IL-2 mutants and wild-type IL-2-HSA for the human IL-2Rβγ heterodimer was measured via Biacore 8K (GE) using the recombinant IL-2Rβγ heterodimer, and the human hIL-2Rβ and γ ECD-N-hIgG1Fc were immobilized on a Protein A chip (400 RU). The IL-2 mutants and IL-2-HSA in HBS-EP buffer were used as analytes at 25°C. For IL-2Rβγ, the analyte concentration decreased from 200 nM to 1.5625 nM (1:2 dilution) and flowed at 30 μl / min (the binding time was 180 s and the dissociation time was 300 s). For IL-2Rβγ, it was regenerated with 10 mM Glycine (pH 1.5) at 30 ul / min for 30 s. For IL-2Rβγ, 1:1 binding, RI≠0, and R maximum value = local fitting data were used. The results are as follows.

[0123]

Table 8

[0124] Affinity with the human IL-2Rα subunit compared with wild-type IL-2-HSA: IL-2gm1-HSA, IL-2gm6-HSA, IL-2gm1a-HSA, IL-2gm1b-HSA, and IL-2gm1c-HSA show no binding, while IL-2gm4-HSA and IL-2gm1d-HSA show binding.

[0125] Affinity with the human recombinant IL-2Rβγ heterodimer compared with wild-type IL-2-HSA: IL-2gm1-HSA, IL-2gm4-HSA, IL-2gm6-HSA, IL-2gm1a-HSA, IL-2gm1b-HSA, IL-2gm1c-HSA, and IL-2gm1d-HSA correspond to the wild type.

[0126] Therefore, IL-2gm1-HSA, IL-2gm1a-HSA, IL-2gm1b-HSA, and IL-2gm1c-HSA are preferred target molecules.

[0127] Figure 8A shows the affinity of IL-2gm1-HSA, IL-2gm4-HSA, IL-2gm6-HSA, IL-2gm1a-HSA, IL-2gm1b-HSA, IL-2gm1c-HSA, IL-2gm1d-HSA, and wild-type IL-2-HSA with the human IL-2Rα subunit.

[0128] Figure 8B shows the affinity of IL-2gm1-HSA, IL-2gm4-HSA, IL-2gm6-HSA, IL-2gm1a-HSA, IL-2gm1b-HSA, IL-2gm1c-HSA, IL-2gm1d-HSA, and wild-type IL-2-HSA with the human recombinant IL-2Rβγ heterodimer.

[0129] Example 10. Cell proliferation assay using NK92 cells The NK-92 cell is an IL-2-dependent NK cell line derived from peripheral blood mononuclear cells of a 50-year-old white male suffering from aggressive non-Hodgkin lymphoma. It expresses CD25 on some of its cell surfaces. The inventors of the present invention evaluate the activities of IL-2gm1-HSA, IL-2gm4-HSA, IL-2gm6-HSA, and wild-type IL-2-HSA in cell proliferation assays using NK92 cells.

[0130] NK92 cells in the logarithmic growth phase were collected, washed once with the basal medium MEM-α, and then co-cultured with different concentrations of IL-2gm1-HSA, IL-2gm4-HSA, IL-2gm6-HSA, and wild-type IL-2-HSA in an experimental medium (MEM-α medium purchased from Gibco (product number 32561-037) supplemented with 12.5% fetal bovine serum and 12.5% horse serum) in a 37 °C, 5% carbon dioxide incubator for 48 hours. 100 μl of the ATP detection substrate CellTiter-Glo (purchased from Promega (product number G7571)) was added to each well, and the full wavelength fluorescence value was detected by the defect method using an ELISA reader (purchased from Molecular Devices (model I3x)).

[0131] The activities of IL-2gm1-HSA, IL-2gm4-HSA, IL-2gm6-HSA, and the wild-type IL-2 molecule IL-2-HSA were measured using cell proliferation assays, and the results are shown in Figure 9. All test articles induced the growth of NK92 cells in a dose-dependent manner. When the cell growth multiples corresponded, EC 50The larger it is, the weaker its activity to stimulate the growth of NK92 is proven. Such a change is due to the effect of its mutant protein on the binding to CD25. The IL-2gm1-HSA mutant protein maintains IL-2R signaling by IL-2Rβγ heterodimer activation. After the concentration increases, the cells can still be effectively proliferated. Compared with IL-2-HSA, the specific activity of IL-2gm1-HSA to stimulate NK92 cell proliferation is 1.07%. IL-2gm1-HSA removes the binding to CD25 in some NK92 cells expressing CD25 on the surface and does not form the IL-2Rαβγ heterotrimer, proving that the stimulating effect decreases. Compared with IL-2-HSA, the effect of IL-2gm1-HSA to stimulate NK92 cell proliferation decreases by about 100 times. Compared with IL-2-HSA, the specific activity of IL-2gm4-HSA to stimulate NK92 cell proliferation is 1.78%. Compared with IL-2-HSA, the specific activity of IL-2gm6-HSA to stimulate NK92 cell proliferation is 0.087%. After the concentration increases, the cells can still be effectively proliferated and maintain biological activity.

[0132] Example 11. Measurement of IL-2 Mutant-Induced IFN-γ Release Collect NK92 cells in the logarithmic growth phase, wash them once with the basal medium MEM-α, and then co-culture them with different concentrations of IL-2gm1-HSA, IL-2gm4-HSA, IL-2gm6-HSA and wild-type IL-2-HSA in the experimental medium (MEM-α medium purchased from Gibco (product number 32561-037) supplemented with 12.5% fetal bovine serum and 12.5% horse serum) in a 37 °C, 5% carbon dioxide incubator for 24 hours. Collect the supernatant and analyze the release of IFN-γ using an anti-human IFN-γ ELISA kit (#SIF50) from R&D.

[0133] As shown in Figure 10A, the results show that IL-2gm1-HSA, IL-2gm4-HSA and wild-type IL-2-HSA all have a stimulating effect on interferon γ.

[0134] As shown in Figure 10B, the results show that both IL-2gm6-HSA and wild-type IL-2-HSA have a stimulatory effect on interferon γ.

[0135] Example 12. Measurement of the stimulation of IL-2 mutants on PBMC proliferation The inventors collected fresh blood samples from healthy Chinese people (n = 2) in heparin sodium tubes, separated PBMCs, and then resuspended the PBMCs in RPMI-1640 medium (containing 10% FBS). After that, the cells were inoculated into 48-well plates (1 * 10 6 cells / well), stimulated with different concentrations of IL-2gm6-HSA and wild-type IL-2-HSA, and co-cultured in a 37 °C, 5% carbon dioxide incubator for 6 days. FACS staining was performed with cell surface and intracellular marker antibodies to detect different cell populations, and all samples were obtained through a cell analyzer using LSR Fortessa (trademark).

[0136] NK cells are limited to CD3- / CD56+, and Treg cells are limited to CD3+CD4+CD25+Foxp3+.

[0137] The results are as shown in Figures 11A - B. Here, Figure 11A shows that at a concentration of 4 nM, compared with wild-type IL-2-HSA, the stimulatory effect of IL-2gm6-HSA on the proliferation of NK cells is slightly worse, and at a concentration of 100 nM, compared with wild-type IL-2-HSA, the stimulatory effect of IL-2gm6-HSA on the proliferation of NK cells increases significantly. Figure 11B shows that at concentrations of 4 nM and 100 nM, compared with wild-type IL-2-HSA, the stimulatory effect of IL-2gm6-HSA on the proliferation of Treg cells all decreases significantly.

[0138] Therefore, in this experiment, IL-2gm6-HSA has a significant proliferative stimulatory effect on NK cells and a significant proliferative inhibitory effect on Treg cells.

[0139] Next, the inventors measure the effect of IL-2gm1-HSA and wild-type IL-2-HSA on stimulating PBMC proliferation.

[0140] NK cells are limited to CD3- / CD56+, and CD4+ cells are limited to CD3+ / CD4+.

[0141] The results are as shown in FIGS. 12A - B. Here, FIG. 12A shows that under the stimulation of 0 - 500 nM sample concentration, IL-2gm1-HSA significantly increases the stimulating effect on the proliferation of NK cells compared with wild-type IL-2-HSA, and FIG. 12B shows that under the concentration stimulation conditions of 0 - 500 nM, IL-2gm1-HSA significantly decreases the stimulating effect on the proliferation of CD4+ T cells compared with wild-type IL-2-HSA.

[0142] Therefore, in this experiment, IL-2gm1-HSA has a significant proliferation-stimulating effect on NK cells and a significant proliferation-inhibiting effect on CD4+ T cells.

[0143] All documents mentioned in the present invention are cited as references in this application as if each document was individually cited as a reference. Further, after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms are also included in the scope defined by the appended claims of this application.

Sequence Listing Free-Text

[0144] SEQ ID NO: 1: IL-2gm1 SEQ ID NO: 2: IL-2gm1-HSA SEQ ID NO: 3: IL-2gm2 SEQ ID NO: 4: IL-2gm3 SEQ ID NO: 5: IL-2gm3 SEQ ID NO: 6: IL-2gm7 SEQ ID NO: 7: IL-2gm8 SEQ ID NO: 8: IL-2gm4 SEQ ID NO: 9: IL-2gm4-Fc Accession No. 10: IL-2gm9 Accession No. 11: IL-2gm10 Accession No. 12: IL-2gm11 Accession No. 13: IL-2gm12 Accession No. 14: IL-2gm5 Accession No. 15: IL-2gm5-Fc Accession No. 16: IL-2gm13 Accession No. 17: IL-2gm14 Accession No. 18: IL-2gm15 Accession No. 19: IL-2gm6 Accession No. 20: IL-2gm6-Fc Accession No. 21: IL-2gm6-HSA Accession No. 22: IL-2qm-Fc Accession No. 23: CD25 protein Accession No. 24: Linker Accession No. 25: Leader peptide Accession No. 26: IL-2-gm1-HSA Accession No. 27: IL-2-gm4-HSA Accession No. 28: IL-2-gm6-HSA Accession No. 29: IL-2-gm7-HSA Accession No. 30: Wild-type IL-2-HSA Accession No. 31: IL-2R alpha Accession No. 32: IL-2R beta Accession No. 33: IL-2R gamma Accession No. 34: IL-2-gm1a-HSA Accession No. 35: IL-2-gm1b-HSA Accession No. 36: IL-2-gm1c-HSA Accession No. 37: IL-2-gm1d-HSA

Claims

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

34.

2. A polynucleotide encoding the IL-2 mutant according to Claim 1.

3. An expression vector, characterized in that it contains the polynucleotide according to Claim 2.

4. A host cell, characterized in that it contains the expression vector according to Claim 3, or the polynucleotide according to Claim 2 is integrated into the genome of the host cell.

5. A pharmaceutical composition, characterized in that it contains the IL-2 mutant according to Claim 1 and a pharmaceutically acceptable adjuvant.

6. A drug containing the IL-2 mutant according to Claim 1 for the treatment of a disease in an individual, characterized in that the disease is a disease in which IL-2 is used in its immunotherapy.

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