An IL-2 derivative with improved water solubility

Abstract

This invention provides an IL-2 derivative with improved water solubility. While retaining its original good activity, the hydrophobicity is reduced and the hydrophilic properties are enhanced by introducing mutations at specific sites and fusing a short hydrophilic peptide at the end. This allows for the use of aqueous purification throughout the IL-2 production process, reducing environmental pollution and lowering costs. Furthermore, the modified, highly water-soluble IL-2 facilitates the adjustment of protein concentration and formulation requirements after drug development.

Description

Technical Field

[0001] This invention relates to the field of protein engineering, and more specifically to an IL-2 derivative with improved water solubility. Background Technology

[0002] Interleukin-2 (IL-2), discovered in 1976 and initially called T-cell growth factor (TCGF), is a globular glycoprotein that plays a crucial role in maintaining the normal function of T lymphocytes and NK cells. Natural IL-2 is a polypeptide (SEQ ID NO: 1) consisting of 133 amino acid residues with a molecular weight of approximately 15 kDa. IL-2 has three cysteine ​​residues located at positions 58, 105, and 125, with the cysteine ​​at position 125 not forming a disulfide bond with the other cysteine ​​residues. Post-translational modifications include threonine glycosylation at position 3, and the formation of disulfide bonds between the cysteine ​​residues at positions 58 and 105, resulting in its essential higher-order structure, primarily composed of four α-helices and several loops (Bazan et al., Science 257, 410-413 (1992)). IL-2 is mainly produced by activated T cells. It can promote the proliferation and differentiation of T cells, maintain T cell activity, stimulate the generation, proliferation and activation of natural killer (NK) cells, induce the generation of cytotoxic T lymphocytes (CTLs), and induce and activate lymphokine-activated killer (LAK) cells and tumor-infiltrating lymphocytes. It can also promote the expression of cytokines and cell lysis molecules by T cells and promote the proliferation of B cells (Waldmann et al., Nat Rev Immunol, 595-601 (2009)). These cells have the effect of killing cells infected by exogenous microorganisms and cancerous cells, either directly or indirectly. Therefore, IL-2 has good antiviral and anticancer effects and broad clinical application potential. IL-2 mediates its action by binding to the IL-2 receptor (IL-2R), which consists of three subunits: α (CD25), β (CD122), and γ (CD132). The α receptor is highly expressed on the surface of suppressor T cells (Tregs) and some endothelial cells, while the β and γ receptor subunits are highly expressed on effector T cells (Teffs) and NK cells. The α (CD25), β (CD122), and γ (CD132) receptor subunits can form αβγ heterotrimers or βγ heterodimers. IL-2 has different affinities for complex forms of different receptor subunits. IL-2 has the highest affinity for the αβγ trimeric receptor and a moderate affinity for the dimer receptor formed by β and γ (about 100-fold lower). IL-2 can transmit intracellular signals and exert cellular immune functions after binding to both types of receptor subunits (Minami et al., Annu Rev Immunol 11, 245-268 (1993)).Currently, in clinical applications, IL-2 primarily utilizes its property of preferentially binding to high-affinity receptors on the surface of Treg cells at low doses to suppress the body's immune response. High doses of IL-2 can effectively activate effector T cells and NK cells, thereby killing tumors. Human ALDESLEUKIN, a recombinant human IL-2, was approved by the FDA in 1992 for the treatment of metastatic renal cell carcinoma, and in 1998, it was also approved for the treatment of metastatic melanoma. Aldelikun was the first human IL-2 drug approved by the FDA for cancer treatment. Although it has shown good clinical efficacy, its poor water solubility and tendency to aggregate make it prone to producing anti-drug antibodies (ADA) in the body. This necessitates the addition of toxic SDS (sodium dodecyl sulfate) to enhance its water solubility, but long-term use of SDS can cause harm to the human body.

[0003] Furthermore, during the purification of IL-2, the high hydrophobicity of wild-type IL-2 and its near-neutral isoelectric point result in poor water solubility. Therefore, during the refolding and purification processes, organic phases are needed to improve purity and yield. For example, reverse-phase chromatography is used to purify the target protein IL-2 in literature (MP Weir et al. Biochem. 1987 Jul 1; 245(1):85-91. Purification and renaturation of recombinant human interleukin-2) and patents EP0268110B1 and US4992271A1. Reverse-phase chromatography typically requires high concentrations of organic solvents, such as acetonitrile, methanol, ethanol, or isopropanol. Currently, both developed countries in Europe and America, as well as China, have strict restrictions on the discharge of highly polluting organic solvents. In addition, the large-scale use of organic solvents requires specialized equipment and workshops, leading to increased production costs. Therefore, the introduction of reverse-phase chromatography has become a significant factor limiting IL-2 production.

[0004] Simultaneously, due to the poor water solubility of IL-2, additives such as nonionic polymeric detergents are required in formulation preparation to improve IL-2 solubility, while the concentration must be controlled at a low level. For example, patent US20120264817A1 mentions using docetaxel to prepare an aqueous solution; EP0268110B1 uses Tween 80, dextrose, SDS, PEG4000, Triton 305, and monostearate, with an optimal concentration of 0.1-2 mg / ml and pH 6-7; patent US4992271A1 uses 2.5% HSA or PPF to prepare a powder. The addition of additives increases the cost of IL-2 production. Furthermore, some additives, such as SDS and Triton 305, have certain cytotoxic properties, and long-term use can cause harm to the human body. Therefore, the instability and high cost of IL-2 have become bottlenecks in its production. Summary of the Invention

[0005] To overcome the problems in existing technologies, the present invention aims to provide a novel IL-2 derivative. While retaining its original good activity, this derivative reduces hydrophobicity and enhances hydrophilicity by introducing a mutation at a specific site and fusing a short hydrophilic peptide at the terminal. This allows for the entire IL-2 production process to utilize aqueous purification, reducing environmental pollution and lowering costs. Furthermore, since the cysteine ​​residue at position 125 of IL-2 does not form disulfide bonds with other cysteine ​​residues within the molecule, it could potentially form a dimer impurity with another wild-type IL-2 molecule through intermolecular disulfide bonds. Therefore, a mutation is introduced at position 125 of IL-2 to increase protein stability. The modified, highly water-soluble IL-2 facilitates the concentration and formulation requirements of the drug-grade protein. Its structural schematic diagram is shown below. Figure 1 .

[0006] In a first aspect, the present invention provides an IL-2 derivative, wherein, compared with wild-type IL-2 SEQ ID NO: 1, the IL-2 derivative undergoes a first amino acid mutation at position 19 (Leu) of the amino acid sequence shown in SEQ ID NO: 1, and introduces a hydrophilic polypeptide at the C-terminus.

[0007] In a preferred embodiment, the first amino acid mutation is an amino acid substitution selected from the group consisting of: L19R, L19Q, L19E, L19A, and L19H.

[0008] In a preferred embodiment, the amino acid sequence of the hydrophilic polypeptide is shown in SEQ ID NO:2, specifically SEPATSGSETPGSEPATSGSETPG.

[0009] In a preferred embodiment, the above-mentioned IL-2 derivative undergoes a second amino acid mutation at position 125 (Cys) of the amino acid sequence shown in SEQ ID NO: 1; the second amino acid mutation is preferably C125A or C125S.

[0010] In a preferred embodiment, the amino acid sequence of the above-mentioned IL-2 derivative is shown in any one of SEQ ID NO: 3-7.

[0011] In a second aspect, the present invention provides a polynucleotide encoding any of the IL-2 derivatives described in the first aspect.

[0012] In a third aspect, the present invention provides an expression vector comprising the polynucleotides described in the second aspect.

[0013] In a fourth aspect, the present invention provides a host cell comprising the expression vector described in the third aspect, or the genome of the host cell having the polynucleotides described in the second aspect integrated therein.

[0014] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the IL-2 derivative described in the first aspect and pharmaceutically acceptable excipients.

[0015] In a sixth aspect, the present invention provides the use of the IL-2 derivative described in the first aspect or the pharmaceutical composition described in the fifth aspect in the preparation of a disease medicament for immunotherapy using IL-2.

[0016] 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.

[0017] In a preferred embodiment, the cancer is treated by stimulating the immune system or by proliferating immune cells.

[0018] The beneficial effects of this invention are:

[0019] 1. This invention significantly improves the hydrophilicity of IL-2 derivatives without affecting molecular activity by introducing a specific point mutation, Leu19, and adding a hydrophilic polypeptide at the end. The solubility in neutral aqueous solution is more than 20 times higher than that of wild-type IL-2. The modified IL-2 does not require reverse-phase chromatography during the entire purification process, greatly saving production costs and protecting the environment.

[0020] 2. The modified IL-2 molecule, due to its increased hydrophilicity, has significantly improved solubility in neutral water environments, which is beneficial for drug formulation.

[0021] 3. Aldesleukin is an FDA-approved IL-2 molecule used to treat metastatic renal cell carcinoma. Because proteins are highly hydrophobic and prone to aggregation, aldesleukin often fails to develop into a drug-resistant antibody due to the formation of aggregates. To address this issue, pharmaceutical companies add 0.02% SDS to the formulation of aldesleukin. However, SDS is a toxic and potent protein denaturant that can cause harm to the human body with long-term use. Because the modified IL-2 molecule in this invention does not require the addition of other substances and is less prone to aggregate formation, it is also less likely to induce antibody production. Attached Figure Description

[0022] Figure 1 Schematic diagram of the structure of IL-2 derivatives;

[0023] Figure 2 Protein expression gel;

[0024] Figure 3 : Gel image of protein after refolding;

[0025] Figure 4 : Gel image of the final protein sample;

[0026] Figure 5 Cell proliferation experiments of IL-2 derivatives and wild-type IL-2 on CTLL2 cells; Detailed Implementation

[0027] Terminology Definition

[0028] As used herein, the terms "IL-2 mutant," "IL-2 derivative," "IL-2 derivative protein," and "modified IL-2 molecule" are used interchangeably to refer to an IL-2 derivative as described in the first aspect of this invention, which, compared to wild-type IL-2 SEQ ID NO: 1, may have a hydrophilic polypeptide introduced at the C-terminus, or the IL-2 derivative may or may not have a first amino acid mutation at position 19 (Leu) and position 125 (C125A) of the amino acid sequence shown in SEQ ID NO: 1.

[0029] As used herein, the term "wild-type IL-2" refers to wild-type IL-2, whose amino acid sequence is shown in SEQ ID NO: 1.

[0030] As used herein, the term “IL-2wt(C125A)” refers to IL-2 containing the mutant C125A, the amino acid sequence of which is shown in SEQ ID NO: 8.

[0031] As used herein, the term "amino acid mutation" encompasses amino acid substitution, deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be performed to achieve the final construct, provided the final construct possesses the desired properties. Preferred amino acid mutations are amino acid substitutions, where one amino acid is replaced with another amino acid having a different structure and / or chemical properties. Preferred amino acid substitutions include hydrophilic charged or uncharged amino acids such as Ser, Thr, Ala, Gly, Glu, Arg, His, and Lys. Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc.

[0032] When referring to variants derived from wild-type proteins, references to amino acid substitutions such as "L19H" refer to the position number (19) of the original residue leucine (L), followed by the substituted residue histidine (H).

[0033] As used herein, the term "fusion protein" refers to a protein obtained by fusing the mutant protein with a hydrophilic polypeptide. The hydrophilic polypeptide is located at the C-terminus or N-terminus of the mutant protein.

[0034] As used herein, the term "hydrophilic polypeptide" refers to a random polypeptide sequence composed of a number of hydrophilic amino acids, which may be randomly combined from the following amino acids: Arg, His, Lys, Asp, Glu, Gly, Pro, Ala, Ser, Thr, Asn, Gln, with preferred amino acids including Asp, Glu, Gly, Pro, Ala, Ser, Thr, Asn, Gln.

[0035] General methods

[0036] Typically, the preparation of the IL-2 derivatives of this invention can be performed using the procedures disclosed herein and by well-known recombinant DNA techniques, including, for example, polymerase chain reaction (PCR), preparation of plasmid DNA, cleavage of DNA with restriction enzymes, preparation of oligonucleotides, ligation of DNA, isolation of mRNA, introduction of DNA into suitable cells, transformation or transfection of the host, and culture of the host. Additionally, fusion molecules can be separated and purified using centrifugal agents and well-known electrophoresis, centrifugation, and chromatography methods. For general disclosures of these methods, see Sam brook et al., Molecular Cloning: A Laboratory Manual (2nd edition (1989)); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York (1989).

[0037] The gene encoding the variant protein of the present invention includes restriction enzyme digestion and ligation as basic steps for generating DNA encoding the desired fusion. The ends of the DNA fragment may need to be modified before ligation, and this can be achieved by filling protrusions, removing the end portions of nuclease (e.g., ExoIII) deletion fragments, site-directed mutagenesis, or by adding new base pairs via PCR.

[0038] Multiple linkers and adaptors can be used to facilitate the ligation of selected fragments. Expression constructs are typically assembled in stages using several rounds of restriction enzyme digestion, ligation, and E. coli transformation. Many cloning vectors suitable for constructing expression constructs are known in the art (lambda.ZAP, Agilent; pET, EMD Millipore), and specific choices are not critical to this invention.

[0039] The choice of cloning vector will be influenced by the gene transfer system selected to introduce the expression construct into the host cell. At the end of each stage, the resulting construct can be analyzed by restriction enzyme digestion, DNA sequencing, hybridization, and PCR.

[0040] Site-directed mutagenesis is commonly used to introduce specific mutations into genes encoding the IL-2 derivatives of the present invention using methods known in the art. See, for example, U.S. Patent Application Publication 2004 / 0171154; Storici et al., 2001, Nature Biotechnology 19:773-776; Kren et al., 1998, Nat. Med. 4:285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:15-16. Any site-directed mutagenesis procedure can be used in this invention. Many commercial kits are available for preparing variants of the present invention.

[0041] Various promoters (transcription initiation regulatory regions) can be used according to the present invention. The choice of a suitable promoter depends on the proposed expression host. Promoters from heterologous sources can be used, provided they are functional in the chosen host.

[0042] The IL-2 derivative can be expressed in E. coli without the signal sequence, and the protein is recovered from the inclusion bodies and refolded into its active form.

[0043] The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule used to introduce a specific gene operatively associated with it and to direct its expression in target cells. This term includes vectors as autonomously replicating nucleic acid structures as well as vectors incorporated into the genome of the host cell to which they are introduced. The expression vector of the present invention comprises an expression cassette. The expression cassette allows transcription of large amounts of stable mRNA. Once the expression vector is in the target cell, a gene-encoded ribonucleic acid molecule or protein is generated through a cellular transcription and / or translation system. In one embodiment, the expression vector of the present invention comprises an expression cassette containing a polynucleotide sequence encoding the IL-2 derivative of the present invention.

[0044] The term "transformation" as used herein has the meaning commonly understood by those skilled in the art, referring to the process of introducing exogenous DNA into a host. Methods of transformation include any method of introducing nucleic acids into cells, including but not limited to electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0045] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably to refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include the initially transformed cells and their derived progeny (regardless of passage number). Progeny may not be identical to parental cells in terms of nucleic acid contents, but may contain mutations. This document includes mutant progeny with the same function or biological activity as those screened or selected from the original transformed cells.

[0046] The host cell culture described in this article can be carried out according to conventional methods in the field, including but not limited to plate culture, shake flask culture, batch culture, continuous culture and fed-batch culture, and various culture conditions such as temperature, time and pH of the culture medium can be adjusted appropriately according to actual conditions.

[0047] As used herein, the term "high-affinity IL-2 receptor" refers to the heterotrimeric form of the IL-2 receptor, which consists of a receptor γ subunit (also known as the universal cytokine receptor γ subunit, γc, or CD132), a receptor β subunit (also known as CD122 or p70), and a receptor α subunit (also known as CD25 or p55). In contrast, the term "intermediate-affinity IL-2 receptor" refers to an IL-2 receptor that contains only the γ and β subunits and lacks the α subunit (see, for example, Olejniczak and Kasprzak, Med Sci Monit 14, RA179-189 (2008)).

[0048] “Affinity” refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless otherwise indicated, as used herein, “binding affinity” refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., receptor and ligand). The affinity of molecule X for its partner Y is typically expressed as a dissociation constant (KD), which is the ratio of dissociation to binding rate constants (Kdissociation and Kbinding, respectively). Thus, equal affinities may contain different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by established methods known in the art, including those described herein.

[0049] The affinity of mutant or wild-type IL-2 peptides for various forms of the IL-2 receptor can be determined by surface plasmon resonance (SPR) using standard instruments such as a BIAcore instrument (GE Healthcare) and receptor subunits (e.g., those obtainable through recombinant expression) as described in the examples (see, for example, Shanafelt et al., Nature Biotechnol 18, 1197-1202 (2000)). Alternatively, the affinity of IL-2 mutants for different forms of the IL-2 receptor can be assessed using cell lines known to express one or another of these receptor forms. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0050] The "effective amount" of a drug refers to the amount necessary to induce physiological changes in the cells or tissues to which it is administered. The "therapeutic effective amount" of a drug, such as a pharmaceutical composition, refers to the amount that effectively achieves the desired therapeutic or preventative outcome at the necessary dose and time period. Therapeutic effective amounts of drugs can eliminate, reduce, delay, minimize, or prevent adverse effects of disease.

[0051] The term "pharmaceutical composition" refers to a formulation in which the biological activity of the active ingredient contained therein is effective and which does not contain any other ingredients that would have unacceptable toxicity to a subject who would receive the composition.

[0052] "Pharmaceutically acceptable carriers" refer to components in a drug composition other than the active ingredient that are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0053] As used herein, the terms “comprising,” “having,” “including,” or “containing” mean included or open-ended and do not exclude additional, uncited elements or method steps.

[0054] As used in this article, “about” means that a value includes the standard deviation of the error of the apparatus or method used to determine that value.

[0055] As used herein, “or” is defined only as a substitute and “and / or”, but unless expressly stated as a substitute or mutually exclusive of substitutes, the term “or” in the claims means “and / or”.

[0056] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0057] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0058] Unless otherwise stated, all raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Experimental methods in the following examples 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.

[0059] In this invention, wild-type IL-2 is mutated (e.g., the 19th position of SEQ ID NO.1 is mutated from L to R, Q, E, A, H, the 125th position is mutated from C to A, and a hydrophilic polypeptide is added to the C-terminus to obtain a series of IL-2 derivatives, corresponding to IL-2 derivative 1258 (SEQ ID NO.3), IL-2 derivative 1259 (SEQ ID NO.4), IL-2 derivative 1260 (SEQ ID NO.5), IL-2 derivative 1261 (SEQ ID NO.6), and IL-2 derivative 1193 (SEQ ID NO.7), respectively. Alternatively, the 125th position of SEQ ID NO.1 is mutated from C to A, and a hydrophilic polypeptide is added to the C-terminus to obtain IL-2 derivative 1191 (SEQ ID NO.9)). The amino acid positions described in this invention are based on wild-type IL-2 (SEQ ID NO.1).

[0060] In the following examples, the construction and expression of IL-2wt(C125A) (SEQ ID NO.8), IL-2 derivative 1258 (SEQ ID NO.3), and IL-2 mutant 1280 (SEQ ID NO.10) proteins were selected, and then purified and prepared using conventional methods and means, as examples of the present invention.

[0061] Example 1: Plasmid Construction and Expression

[0062] 1. Expression plasmid construction

[0063] Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize genes containing IL-2wt(C125A), IL-2 mutant 1280, and IL-2 derivative 1260. Following the procedures in *Molecular Cloning*, overlap PCR was performed to obtain the target fragment. Then, recombination ligation of the fragment with the universal vector pET41a was performed, followed by transformation, sequencing, and bacterial preservation to obtain plasmids capable of expressing IL-2wt(C125A) and the remaining IL-2 mutants or derivatives.

[0064] 2. Plasmid extraction

[0065] Following the procedures outlined in the Qiagen Mini-prep Kit, plasmids for IL-2wt (C125A), IL-2 mutants, and IL-2 derivatives were prepared.

[0066] 3. Expression of IL-2wt(C125A), IL-2 mutant and IL-2 derivative in Escherichia coli

[0067] The successfully constructed plasmid was transformed into Escherichia coli expression strain BL21(DE3). Single clones were selected and cultured overnight as seed culture. The seed culture was then transferred to a 500 mL Erlenmeyer flask containing Kana-resistant TB medium at a volume ratio of 1:50. The initial OD600 was approximately 0.1. The culture was carried out at 37°C and 220 rpm until the OD600 reached 2.0. IPTG (final concentration 0.5 mmol / L) was added, and the culture was carried out at 37°C and 220 rpm for 4 hours before harvesting the bacteria.

[0068] Expression results were analyzed using SDS-PAGE, such as Figure 2 As shown, the protein was successfully expressed in the form of inclusion bodies (IBs). M represents the marker, T represents the whole-cell lysate, S represents the supernatant of the cell lysate, and P represents the cell lysate precipitate.

[0069] Example 2. Protein refolding, purification, and sample preparation process

[0070] In this invention, all IL-2wt(C125A), IL-2 mutant 1280, and IL-2 derivative 1258 plasmids were transformed into engineered E. coli host bacteria for expression, producing insoluble inclusion bodies. After high-pressure disruption of the bacterial cells and two washings, the inclusion bodies were recovered in preparation for subsequent protein refolding.

[0071] 1. Protein refolding

[0072] Because wild-type IL-2 has poor water solubility and a near-neutral pH, it often requires the addition of large amounts of additives during the refolding process. For example, US20120264817A1, CN111018961A, and EP0268110B1 all mention the addition of additives such as Tween20 and SDS to improve solubility. However, the IL-2 molecule in this invention is modified, resulting in reduced hydrophobicity and pH. Therefore, detergents such as SDS and Tween20 are no longer needed during the refolding process, while still achieving good refolding efficiency.

[0073] The inclusion bodies of the aforementioned IL-2 derivatives (1258, 1259, 1260, 1261, and 1193), after being broken down and washed, were dissolved in 10 times the volume (v / w) of denaturing buffer (8 M Urea, 20 mM Tris, pH 8.5) at room temperature for 20 minutes, followed by the addition of 10 mM DTT and a further 30 minutes of dissolution. The denaturing buffer was then added dropwise to 40 times the volume of refolding buffer (50 mM glycine, 2 M Urea, pH 8.5) containing 20 mM cysteine, and the mixture was stirred overnight at room temperature. The denaturation method for IL-2wt (C125A) and the IL-2 mutant 1280 was the same, but 0.5% SDS was added to the refolding buffer to ensure successful refolding and good refolding efficiency.

[0074] 2. Protein refolding results

[0075] The results are as follows Figure 3 As shown, all IL-2wt(C125A), IL-2 mutant 1280, and IL-2 derivative 1258 successfully renatured under the above conditions, with high renaturation rates. Comparing the renaturation process and results, adding an acidic hydrophilic polypeptide to the C-terminus of IL-2, and mutating the Leu19 position to H, R, Q, E, or A, effectively improves the hydrophilicity of IL-2. Therefore, Tween20 is not needed in the protein renaturation solution, and the protein still renatures well with a high renaturation rate. In contrast, IL-2wt(C125A), IL-2 mutant 1280 (SEQ ID NO:10) without a hydrophilic polypeptide added to the C-terminus but with a Leu19 mutation, and IL-2 derivative 1191 (SEQ ID NO:9) with a hydrophilic polypeptide added to the C-terminus but without a mutation at position 19, require the addition of Tween20 to aid renaturation and achieve higher renaturation rates.

[0076] 3. Protein purification

[0077] The well-renatured IL-2 derivatives 1193, 1258, 1259, 1260, and 1261 were captured directly by GE's Hitrap QFF ion exchange column chromatography.

[0078] The procedure is as follows: Before purification, equilibrate the packing column with 5 column volumes of equilibration buffer (0.02M Tris, pH 8.0); pass the collected supernatant through the column, and then wash the packing column with 2 column volumes of equilibration buffer to remove non-specific binding proteins; wash the packing column with a gradient of 15 column volumes of elution buffer (0.02M Tris, 0.7M NaCl, pH 8.0) and collect the eluent containing the target protein.

[0079] In the collected eluent containing the target protein, except for IL-2wt (C125A), IL-2 mutant 1280, and IL-2 derivative 1191, other proteins can be dissolved completely by adding 1.2M NaCl while stirring. Then, further purification is performed using Phenyl HS FF (GE) to remove DNA, HCP, endotoxins, etc. from the sample. The procedure is as follows: Before purification, equilibrate the packing column with 5 column volumes of equilibration buffer (0.02M Tris, 1.5M NaCl, pH 7.5); pass the collected supernatant through the column, and then wash the packing column with 5 column volumes of 20% elution buffer (0.02M Tris, pH 7.5) to remove some weakly bound contaminating proteins; wash the packing column with a gradient of 5 column volumes of 98% elution buffer (0.02M Tris, pH 7.5), and collect the target protein in the 98% elution buffer. If the purity of some mutants still needs improvement, further purification can be performed by passing them through a molecular sieve under PBS conditions.

[0080] IL-2wt (C125A), as well as IL-2 mutant 1280 and IL-2 derivative 1191, require traditional processing methods, including the introduction of organic reagents and reverse-phase packing materials, to obtain better purity and yield. For example, the first step of ion exchange requires elution with 60% ethanol, and purification is achieved using reverse-phase packing material C8 to obtain high-purity protein.

[0081] All IL-2wt(C125A), IL-2 mutants and IL-2 derivatives were extracted, and 2.5 μg of each final sample was analyzed by SDS-PAGE according to the method described in Molecular Cloning.

[0082] 4. Protein purification results

[0083] The results are as follows Figure 4As shown, all IL-2wt(C125A), IL-2 mutants, and IL-2 derivatives can be purified under the above conditions, and the proteins have the correct molecular weight. Furthermore, during the purification process, it was found that the IL-2 protein modified by this patented invention has an acidic hydrophilic polypeptide added to its C-terminus. Mutations at the Leu19 position, such as H, R, Q, E, and A, effectively improve the hydrophilicity of IL-2, reducing its hydrophobicity and increasing its hydrophilicity. Therefore, the purification process no longer requires the addition of organic reagents or the use of a reverse-phase column. Conversely, the purification of IL-2wt(C125A), IL-2 derivative 1280 without the added hydrophilic polypeptide at the C-terminus, or IL-2 derivative 1191 without the Leu19 mutation requires traditional methods and the use of large amounts of organic reagents, increasing costs and polluting the environment.

[0084] Example 3. Protein Concentration and Stability Test

[0085] 1. Protein Concentration Operation

[0086] The proteins obtained in Example 2 were dialyzed to replace the buffer solution in the samples with the same PBS. Simultaneously, concentration experiments were performed on these proteins to observe changes in their solubility. The specific procedures are as follows:

[0087] All IL-2 derivatives were centrifuged using an Amicon Ultra-15 centrifugal filter (10 kDa molecular weight cutoff) at 4000g and 8°C with an initial volume of 15 mL in a rotating drum. Each rotation lasted 10 min. The concentrate was then removed and mixed thoroughly. Protein changes were observed. After repeated centrifugation and concentration, protein concentration was measured, and protein stability was monitored by SEC-HPLC.

[0088] The instrument used for SEC-HPLC detection was a Waters Acquity UPLC H-Class, with an AdvanceBio SEC 200A column (1.9 μm, 4.6 mm x 150 mm), and the mobile phase was 1 x PBS, 5% IPA. UV280 readings were monitored at room temperature.

[0089] The results of UV280 and stability testing of the concentrated sample are as follows: Figure 5 The stability of each IL-2 derivative was determined by SEC-HPLC after concentration, and the results are shown in Table 1.

[0090] Table 1: Maximum concentration and purity determination of IL-2wt(C125A), IL-2 mutant and IL-2 derivative

[0091]

[0092] 2. Concentration and Stability Results Analysis

[0093] The results are shown in Table 1. Under the same conditions, concentration experiments were conducted on IL-2wt (C125A) and the IL-2 mutant or derivative modified according to this invention. The protein modified according to this invention, with a mutation at the Leu19 site and the addition of a hydrophilic polypeptide at the C-terminus, exhibited decreased hydrophobicity and increased water solubility. Under the same PBS buffer conditions, the protein concentration could be increased to over 8 mg / ml, nearly 20 times that of wild-type IL-2. SEC-HPLC analysis showed that the protein remained stable, maintaining a purity of over 80%, without any polymerization. In contrast, the IL-2 wt (C125A), the IL-2 mutant 1280 without the addition of a hydrophilic peptide at the C-terminus, and the IL-2 derivative 1191 without the Leu19 site mutation, all showed protein concentrations of less than 3 mg / ml after concentration in the same PBS buffer. This indicates that the Leu19 site mutation and the terminal hydrophilic peptide in this invention have a positive synergistic effect on improving the water solubility of IL-2, which is beneficial to increasing the water solubility of IL-2 and lays a good foundation for the formulation preparation of future IL-2 drugs.

[0094] Example 4. Activity test of IL-2 derivatives

[0095] The modified IL-2 of this invention involves a mutation at the Leu19 position and the addition of a hydrophilic polypeptide to the C-terminus. This experiment aims to determine whether these modifications affect the binding of the IL-2 receptor. The binding of the modified protein to the IL-2β and γ-Fc receptors was measured using a Biacore SPR 8K instrument. Furthermore, the cellular bioactivity of the IL-2 derivative was observed using a CTLL2 cell viability assay.

[0096] 1. In vitro activity assay of IL-2 derivatives

[0097] We used Biacore SPR-8K to test the binding strength of different Leu19 mutants to the IL-2β and γ-Fc receptors, as shown in Table 2. The introduction of mutations did not affect the binding strength of IL-2 to the receptors. The specific method is as follows:

[0098] 1) Use Protein-A chip (GE healthcare) to immobilize β and γ-receptors with Fc tags on the chip surface. The immobilization amount is 1000 RU, and the fluidity used is PBS buffer containing 0.05% Tween 20.

[0099] 2) Pass different concentrations of IL-2 derivatives through the solution and detect their response values. The concentration range used includes 0 to 50 nM.

[0100] 3) After the experiment, the data was analyzed using the data processing software that comes with Biacore SPR 8K. The results were obtained using the dynamic fitting method. The specific data are shown in Table 2.

[0101] Table 2: Experimental results on the binding activity of IL-2 derivatives to receptors β and γ-receptors

[0102] SEQ ID NO.9 C125A+ hydrophilic polypeptide 6.71E+05 1.40E-04 2.09E-10 SEQ ID NO.7 L19H,C125A+ hydrophilic polypeptide 6.03E+05 1.71E-04 2.83E-10 SEQ ID NO.3 L19R,C125A+ hydrophilic peptide 1.06E+06 2.16E-04 2.03E-10 SEQ ID NO.4 L19Q,C125A+ hydrophilic peptide 1.19E+06 2.36E-04 1.99E-10 SEQ ID NO.5 L19E,C125A+ hydrophilic peptide 1.88E+06 7.13E-04 3.79E-10 SEQ ID NO.6 L19A,C125A+ hydrophilic peptide 8.99E+05 3.15E-04 3.51E-10

[0103] 2. Detection of CTLL2 cell viability using IL-2 derivatives

[0104] This experiment compared the activity of IL-2 derivatives with that of wild-type IL-2 in the CTLL2 cell line. The experimental steps are as follows:

[0105] 1) Take CTLL2 cells in the logarithmic growth phase, centrifuge at 200g for 5 minutes, resuspend the cells in PBS, centrifuge at 200g for 5 minutes, and discard the supernatant.

[0106] 2) Resuspend the cells in culture medium and perform cell counting. Adjust the cell count to 2 × 10⁶ cells / year. 5 Add 50 μl of each cell per milliliter to a 96-well plate. Dilute the sample to be tested with culture medium to an initial concentration of 30 nM, then perform a 2-fold serial dilution at 10 spots. Add 50 μl of the diluted sample to each well of the 96-well plate.

[0107] 3) Incubate the 96-well plate at 37°C with 5% CO2 for 3 days. After incubation, add 100 μl of Cell Titer Glo (product code G9243, Promega) assay reagent to each well of the 96-well plate, mix well, and then shake on a horizontal shaker at 550 rpm for 3 minutes. After standing at room temperature for 10 minutes, use a microplate reader to detect the chemiluminescence value.

[0108] As shown in Table 3, the EC50 activation of CTLL2 cells by IL-2 derivatives and wild-type IL-2 indicates that the Leu19 mutation, C125A mutation, and C-terminal hydrophilic peptide in IL-2 derivatives had no effect on cell viability. Some IL-2 derivatives even exhibited better biological activity than wild-type IL-2. For detailed results, see [link to table]. Figure 5 And Table 3.

[0109] Table 3: Results of activity assays of wild-type IL-2 and IL-2 derivatives in CTLL2 cells.

[0110] SEQ ID NO.1 Wild-type IL-2 1.327 SEQ ID NO.9 C125A+ hydrophilic polypeptide / 1191 1.104 SEQ ID NO.7 L19H,C125A+ hydrophilic polypeptide / 1193 0.4326 SEQ ID NO.3 L19R,C125A+ hydrophilic peptide / 1258 0.5904 SEQ ID NO.4 L19Q,C125A+ hydrophilic peptide / 1259 0.625 SEQ ID NO.5 L19E, C125A+ hydrophilic peptide / 1260 0.2727 SEQ ID NO.6 L19A,C125A+ hydrophilic peptide / 1261 1.131

[0111] 3. Activity detection results

[0112] From the data above, we can see that the IL-2 derivative modified by this invention patent has similar receptor binding ability and biological activity as wild-type IL-2.

[0113] In summary, the present invention, without affecting the biological activity of the protein, significantly improves its properties by mutating the Leu19 site and adding an acidic hydrophilic polypeptide to the C-terminus, resulting in reduced hydrophobicity and increased water solubility. The modified IL-2 produced by this invention eliminates the need for organic reagents and reverse-phase fillers in production, greatly reducing costs and environmental pollution. Furthermore, the increased water solubility greatly facilitates the later-stage formulation development of IL-2 pharmaceuticals, providing a favorable foundation for formulation formulation and protein concentration improvement.

[0114] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. sequence list <110> Beijing Zhidao Biotechnology Co., Ltd. <120> An IL-2 derivative with improved water solubility <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 133 <212> PRT <213> Human <400> 1 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu His 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 Phe Lys Phe 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 130 <210> 2 <211> 24 <212> PRT <213> Artificial Sequence <400> 2 Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Glu Pro Ala 1 5 10 15 Thr Ser Gly Ser Glu Thr Pro Gly 20 <210> 3 <211> 157 <212> PRT <213> Artificial Sequence <400> 3 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Arg 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 Phe Lys Phe 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 Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro 130 135 140 Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly 145 150 155 <210> 4 <211> 157 <212> PRT <213> Artificial Sequence <400> 4 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Gln 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 Phe Lys Phe 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 Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro 130 135 140 Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly 145 150 155 <210> 5 <211> 157 <212> PRT <213> Artificial Sequence <400> 5 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Glu 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 Phe Lys Phe 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 Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro 130 135 140 Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly 145 150 155 <210> 6 <211> 157 <212> PRT <213> Artificial Sequence <400> 6 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Ala 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 Phe Lys Phe 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 Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro 130 135 140 Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly 145 150 155 <210> 7 <211> 157 <212> PRT <213> Artificial Sequence <400> 7 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu His 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 Phe Lys Phe 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 Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro 130 135 140 Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly 145 150 155 <210> 8 <211> 133 <212> PRT <213> Artificial Sequence <400> 8 Ala Pro Thr 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 Phe Lys Phe 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 130 <210> 9 <211> 157 <212> PRT <213> Artificial Sequence <400> 9 Ala Pro Thr 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 Phe Lys Phe 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 Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro 130 135 140 Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly 145 150 155 <210> 10 <211> 133 <212> PRT <213> Artificial Sequence <400> 10 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu His 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 Phe Lys Phe 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 130

Claims

1. An IL-2 derivative, characterized in that, Compared with wild-type IL-2 SEQ ID NO: 1, the IL-2 derivative has a first amino acid mutation at position 19 (Leu) of the amino acid sequence shown in SEQ ID NO: 1, a second amino acid mutation at position 125 (Cys) of the amino acid sequence shown in SEQ ID NO: 1, and introduces a hydrophilic polypeptide at the N-terminus or C-terminus. The hydrophilic polypeptide sequence is SEPATSGSETPGSEPATSGSETPG. The amino acid sequence of the IL-2 derivative is shown in any one of SEQ ID NO: 3-7.

2. A polynucleotide, characterized in that, The polynucleotide encodes the IL-2 derivative of claim 1.

3. An expression carrier, characterized in that, The expression vector comprises the polynucleotide of claim 2.

4. A host cell, characterized in that, The host cell contains the expression vector of claim 3, or the genome of the host cell is integrated with the polynucleotide of claim 2.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the IL-2 derivative of claim 1 and pharmaceutically acceptable excipients.

6. The use of the IL-2 derivative of claim 1 or the pharmaceutical composition of claim 5 in the preparation of a disease medicament for immunotherapy using IL-2, characterized in that, The diseases mentioned are metastatic renal cell carcinoma, HIV infection, HCV infection, rheumatoid arthritis, and atopic dermatitis.

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