A pegylated il-2 derivative and uses thereof

Abstract

The application discloses a polyethylene glycol (PEG) modified IL-2 derivative and application thereof. By introducing a mutation into an amino acid at the 19th position of a wild type IL-2, introducing a mutation into the 125th position and fusing a hydrophilic short peptide at the end, the hydrophobicity of the IL-2 is reduced, the IL-2 is purified in a water phase in the whole production process, environmental pollution is reduced, cost is lowered and protein stability is increased. Meanwhile, by performing different mutations on the amino acid at the 65th position, the binding activity of the IL-2 to CD25 can be adjusted, and then the optimal CD25 binding strength is obtained. Through further modification, by introducing a cysteine mutation into the hydrophilic short peptide fused at the end, the half-life of the IL-2 is prolonged after PEG modification. The PEG molecule after modification exhibits low toxicity and good anti-tumor effect in animal experiments.

Description

Technical Field

[0001] This invention relates to the field of protein engineering, specifically to a polyethylene glycol-modified IL-2 derivative and its applications. 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. The cysteine ​​at position 125 does not form a disulfide bond with the other cysteine ​​residues, and therefore, in recombinant expression, it is usually mutated to other amino acids, such as Ala or Ser. Post-translational modifications include threonine glycosylation at position 3, and the formation of disulfide bonds between the cysteine ​​residues at positions 58 and 105, forming its essential higher-order structure, primarily composed of four α-helices and some loop sequences (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 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 IL-2 receptors (IL-2R). The IL-2 receptor consists of three subunits: α (CD25), β (CD122), and γ (CD132) receptor subunits. 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, high doses of IL-2 are mainly used to effectively activate effector T cells and NK cells, thereby producing a tumor-killing effect. Humanal desleukin, a recombinant human wild-type IL-2, was approved by the FDA in 1992 for the treatment of metastatic renal cell carcinoma, and in 1998, it was approved for the treatment of metastatic melanoma. However, wild-type IL-2 activates a large number of Treg cells by preferentially binding to the αβγ trimeric receptor on the surface of Treg cells, thereby neutralizing some of the tumor-killing effects of effector T cells and NK cells. Simultaneously, the binding of wild-type IL-2 to α receptors on the surface of endothelial cells can cause side effects such as vascular leakage syndrome. Therefore, one of the key aspects of IL-2 molecular modification is to weaken or eliminate the binding of wild-type IL-2 to α receptors. Furthermore, recent studies have shown that CD8+ cytotoxic T cells also express a small number of α receptors on their surface, and these receptors play an important role in maintaining the vitality of CD8+ cytotoxic T cells. Therefore, completely eliminating the binding of IL-2 to α receptors leads to a decrease in its anti-tumor activity. Balancing the binding activity of IL-2 molecules to α receptors is also one of the key aspects of IL-2 molecule modification.

[0003] In addition, wild-type IL-2 has a short half-life and is quickly metabolized or eliminated in the body. Extending the half-life of IL-2 is one direction for IL-2 molecule modification. Among the IL-2 molecules currently under development, NKTR-214 developed by Nektar Therapeutics is a relatively advanced molecule. It couples an average of six PEG chains near the interface between IL-2 and CD25. This PEG modification effectively blocks its binding to the high-affinity IL-2R receptor, making NKTR-214 more likely to activate CD8-positive memory T cells and NK cells. Sanofi's SAR444245, on the other hand, introduces non-natural amino acids at the interface between the IL-2 protein and CD25, precisely linking PEG chains to the IL-2 surface to block the binding of IL-2 and CD25. However, despite extensive modifications and designs to the IL-2 molecule by numerous pharmaceutical companies and research institutions, current IL-2 drugs under development still suffer from high toxicity and low activity. For example, NKTR-214, developed by Nektar Therapeutics, was administered at a dose of 50 μg / kg in a Phase III clinical trial, only slightly higher than the wild-type IL-2 dose of 35 μg / kg, indicating its continued high toxicity. Subsequent clinical results analysis also revealed that NKTR-214's clinical therapeutic performance was unsatisfactory. Furthermore, wild-type IL-2 has very low water solubility in neutral aqueous solutions, and commercial production typically requires the introduction of organic solvents such as acetonitrile and toxic substances such as SDS (sodium dodecyl sulfate), all of which limit the large-scale preparation and drug formulation development of IL-2.

[0004] Therefore, existing technologies lack IL-2 mutants or derivatives that can simultaneously differentiate the binding activity of CD25, are highly hydrophilic, have low toxicity, and have a long cyclic half-life. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing an IL-2 derivative. While retaining the good activity of the original IL-2, this derivative reduces its hydrophobicity and enhances its hydrophilic properties by introducing mutations at specific sites in wild-type IL-2, introducing a mutation at position 125, and fusing a short hydrophilic peptide at the terminal. This allows for the use of aqueous purification throughout the IL-2 production process, reducing environmental pollution, lowering costs, and increasing protein stability. Simultaneously, by mutating different amino acids at another site, the binding activity of IL-2 to CD25 can be regulated, thereby obtaining the optimal CD25 binding strength. Further modification involves introducing a cysteine ​​mutation into the terminally fused hydrophilic peptide and then performing PEG modification to extend its half-life. The PEG-modified molecule exhibits low toxicity and good antitumor activity in animal experiments.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an IL-2 mutant or derivative wherein the IL-2 mutant or derivative has a first amino acid mutation at position 19 (Leu) and a second amino acid mutation at position 65 (Pro) relative to the amino acid sequence of wild-type IL-2 (SEQ ID NO: 1), and has a substitution of position 125 (Cys) with Ala, and introduces an acidic hydrophilic polypeptide IST at the C-terminus.

[0008] In a preferred embodiment, the first amino acid mutation is selected from the group consisting of amino acid substitutions L19A or L19H.

[0009] In a preferred embodiment, the second amino acid mutation is selected from the group consisting of amino acid substitutions: P65A, P65T, P65S, P65E, or P65Q.

[0010] In a preferred embodiment, the amino acid sequence of the hydrophilic polypeptide IST is as shown in SEQ ID NO:2, specifically SEPATSGSETPGSEPATSGSETPG.

[0011] In a preferred embodiment, the amino acid sequence of the IL-2 mutant or derivative is as shown in any one of SEQ ID NO: 11-15.

[0012] In a second aspect, the present invention provides an IL-2 derivative wherein the C-terminal hydrophilic polypeptide IST of the IL-2 mutant or derivative described in the first aspect undergoes a fourth amino acid mutation, wherein the fourth amino acid mutation, compared with the amino acid sequence SEQ ID NO:2 of IST, is selected from the following substitutions: G19C, G12C, or G7C (numbered starting from the first amino acid in SEQ ID NO:2); the IL-2 derivative further comprises a polyethylene glycol moiety of cysteine ​​linked to the site of the fourth amino acid mutation in the C-terminal hydrophilic polypeptide IST, wherein the polyethylene glycol moiety has a molecular weight between 10 and 40 kDa.

[0013] In a preferred embodiment, the polyethylene glycol portion of the IL-2 derivative has a molecular weight of 20 kDa.

[0014] In a preferred embodiment, the amino acid sequence of the IL-2 derivative is shown in any one of SEQ ID NO: 16-21.

[0015] In a third aspect, the present invention provides a method for increasing the cyclic half-life of the IL-2 mutant or derivative described in the first aspect, the method comprising: (1) mutating a fourth amino acid in the C-terminal hydrophilic polypeptide IST of the IL-2 mutant or derivative described in the first aspect, wherein the fourth amino acid mutation is selected from the following substitutions compared to the amino acid sequence SEQ ID NO:2 of IST: G19C, G12C or G7C (numbered starting from the first amino acid in SEQ ID NO:2); and (2) linking a polyethylene glycol portion to a cysteine ​​residue at the site where the fourth amino acid mutation occurs in the C-terminal hydrophilic polypeptide IST.

[0016] In a preferred embodiment, the polyethylene glycol portion of the IL-2 derivative has a molecular weight between 10 and 40 kDa.

[0017] More preferably, the polyethylene glycol portion of the IL-2 derivative has a molecular weight of 20 kDa.

[0018] In a preferred embodiment, the amino acid sequence of the IL-2 derivative obtained in step (1) is shown in any one of SEQ ID NO: 16-21.

[0019] In a fourth aspect, the present invention provides a polynucleotide that encodes the amino acid sequence of the IL-2 mutant or derivative described in the first or second aspect.

[0020] In a fifth aspect, the present invention provides an expression vector comprising the polynucleotides described in the fourth aspect.

[0021] In a sixth aspect, the present invention provides a host cell comprising the expression vector described in the fifth aspect, or the genome of the host cell having the polynucleotides described in the fourth aspect integrated therein.

[0022] In a seventh aspect, the present invention provides a pharmaceutical composition comprising the IL-2 mutant or derivative described in the first or second aspect and pharmaceutically acceptable excipients.

[0023] In an eighth aspect, the present invention provides the use of the IL-2 mutant or derivative described in the first aspect, the IL-2 derivative described in the second aspect, and the pharmaceutical composition described in the seventh aspect in the preparation of a disease medicament for immunotherapy using IL-2.

[0024] In a preferred embodiment, the disease is cancer, immune disease, human immunodeficiency virus (HIV) infection, hepatitis C virus (HCV) infection, rheumatoid arthritis, atopic dermatitis, and systemic lupus erythematosus, etc.

[0025] In a preferred embodiment, the cancer is treated by stimulating the immune system or by proliferating immune cells.

[0026] Unless otherwise specified, all amino acid numbers in this invention are based on natural IL-2 (SEQ ID NO: 1).

[0027] The beneficial effects of this invention are:

[0028] 1. This invention significantly improves the hydrophilicity of IL-2 mutants or derivatives without affecting molecular activity by introducing a specific point mutation, Leu19, and a hydrophilic polypeptide IST at the end. The solubility in neutral aqueous solution is more than 10 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.

[0029] 2. The modified IL-2 molecule of this invention has significantly improved solubility in neutral water environment due to its increased hydrophilicity, which is beneficial for drug formulation.

[0030] 3. In this invention, the affinity of the modified IL-2 molecule for CD25 varies with the length of the side chain of different amino acids at the P65 mutation site. Mutants with longer side chains exhibit weaker affinity, while those with shorter side chains exhibit stronger affinity. Therefore, this belongs to a class of IL-2 mutants or derivatives whose affinity can be adjusted according to requirements. This invention also found that when the P65 position is mutated to T (threonine), it exhibits both optimal antitumor activity and low toxicity.

[0031] 4. This invention, based on increasing the hydrophilicity of IL-2, introduces a cysteine ​​mutation into the C-terminal hydrophilic peptide IST, and extends its half-life after PEG modification. The PEG-modified IL-2 derivative exhibits low toxicity and good antitumor activity. Attached Figure Description

[0032] Figure 1 SDS-PAGE gel analysis of some IL-2 mutants after expression in E. coli;

[0033] Figure 2 SDS-PAGE gel analysis of some IL-2 mutants after renaturation;

[0034] Figure 3 SDS-PAGE analysis of some IL-2 mutants or derivatives after final purification;

[0035] Figure 4 Cell proliferation experiments of IL-2 mutant and wild-type IL-2 on CTLL2 cells;

[0036] Figure 5 .Activation assay of IL-2 derivatives coupled with PEG in mouse spleen cells;

[0037] Figure 6 Effect of IL-2 derivative 1351-PEG on mouse body weight (3 mg / kg, twice a week);

[0038] Figure 7 The antitumor effect of PEG-conjugated IL-2 derivatives on mice with CT-26 tumor cells; Detailed Implementation

[0039] 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.

[0040] 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.

[0041] Definitions and Explanations:

[0042] 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.

[0043] “Interleukin-2” or “IL-2” refers to any natural IL-2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term covers unprocessed IL-2 as well as any form of IL-2 derived from cells and processed forms. The term also covers naturally occurring IL-2 variants, such as splice variants or allelic variants. An exemplary wild-type human IL-2 amino acid sequence is shown in SEQ ID NO: 1.

[0044] 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 the 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 have a first amino acid mutation at position 19 (Leu) of the amino acid sequence shown in SEQ ID NO: 1, or a second amino acid mutation at position 65 (Pro), or a C125A mutation at position 125, or a fourth amino acid mutation in the C-terminal hydrophilic polypeptide IST of the IL-2 derivative described in the first aspect, wherein the fourth amino acid mutation, compared to the amino acid sequence of IST SEQ ID NO: 2, is selected from the following substitutions: G19C, G12C, or G7C.

[0045] "Derivatives" is intended to be interpreted broadly to include any IL-2-related product. This includes, but is not limited to, human and non-human IL-2 homologues, fragments or truncated forms, fusion proteins (such as those fused with a signal peptide or other active or inactive components, the active component being an antibody or its antigen-binding fragment), modified forms (such as PEGylation, glycosylation, albumin conjugation / fusion, Fc conjugation / fusion, hydroxyethylation, etc.), and conserved modified proteins.

[0046] 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.

[0047] 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.

[0048] 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).

[0049] 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.

[0050] 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.

[0051] General methods

[0052] 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).

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] The IL-2 mutant can be expressed in E. coli without the signal peptide sequence, and the protein is recovered from the inclusion bodies and refolded into its active form.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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)).

[0064] “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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] "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.

[0069] 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.

[0070] 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.

[0071] 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”.

[0072] In this disclosure, "IL-2" can be used interchangeably with "IL2".

[0073] In Examples 1-3 of this invention, wild-type IL-2 was mutated by changing L to R, Q, E, A, H at position 19 of SEQ ID NO.1, changing C to A at position 125, and adding a hydrophilic polypeptide (SEQ ID NO.2) to the C-terminus to obtain a series of IL-2 mutants, corresponding to IL-2 mutant 1258 (SEQ ID NO.3), IL-2 mutant 1259 (SEQ ID NO.4), IL-2 mutant 1260 (SEQ ID NO.5), IL-2 mutant 1261 (SEQ ID NO.6), and IL-2 mutant 1193 (SEQ ID NO.7). Alternatively, by mutating position 125 of SEQ ID NO.1 from C to A, we obtain wild-type IL-2wt(C125A) (SEQ ID NO.8); by mutating position 125 of SEQ ID NO.1 from C to A and adding a hydrophilic polypeptide to the C-terminus, we obtain IL-2 derivative 1191 (SEQ ID NO.9); by mutating position 19 of SEQ ID NO.1 from L to H and position 125 from C to A, we obtain IL-2 mutant 1280 (SEQ ID NO.10). In this invention, the amino acid position numbers are based on wild-type IL-2 (SEQ ID NO.1).

[0074] In addition, in this invention, "hydrophilic polypeptide" or "IST" refers to a polypeptide molecule with strong hydrophilicity. Unless otherwise specified, its specific sequence is shown in SEQ ID NO:2.

[0075] Example 1: Plasmid Construction and Expression

[0076] 1. Expression plasmid construction

[0077] Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize genes containing wild-type IL-2wt (C125A), IL-2 mutant 1280, and IL-2 derivative 1258. Following the procedures in *Molecular Cloning*, overlap PCR was performed to obtain the target fragment. Then, the fragment was ligated into the universal vector pET41a for recombination, transformed, sequenced, and preserved to obtain plasmids capable of expressing IL-2wt (C125A) and the remaining IL-2 mutants or derivatives.

[0078] 2. Plasmid extraction

[0079] Following the procedures outlined in the Qiagen Mini-prep Kit, plasmids for IL-2wt (C125A), IL-2 mutants, and IL-2 derivatives were prepared.

[0080] 3. Expression of IL-2wt(C125A), IL-2 mutant and IL-2 derivative in Escherichia coli

[0081] 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.

[0082] Expression results were analyzed using SDS-PAGE, such as Figure 1 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.

[0083] Example 2. Protein refolding, purification, and sample preparation process

[0084] In this invention, all IL-2wt(C125A), IL-2 mutant 1280, and IL-2 mutant 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.

[0085] 1. Protein refolding

[0086] 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.

[0087] The inclusion bodies of the aforementioned IL-2 mutants (1258, 1259, 1260, 1261, and 1193), after being broken up and washed, were dissolved in 10 times the volume (v / w) of denaturing buffer (8M Urea, 20mM Tris, pH 8.5) at room temperature for 20 minutes, followed by the addition of 10mM DTT and a further 30 minutes of dissolution. The denaturing buffer was then added dropwise to 40 times the volume of refolding buffer (50mM glycine, 2M Urea, pH 8.5) containing 20mM cysteine, and the mixture was stirred overnight at room temperature. The denaturation method for wild-type IL-2wt (C125A) and 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.

[0088] 2. Protein refolding results

[0089] The results are as follows Figure 2 As shown, all IL-2wt (C125A), IL-2 mutant 1280, and IL-2 mutant 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, wild-type 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, all require the addition of Tween20 to aid renaturation and achieve higher renaturation rates.

[0090] 3. Protein purification

[0091] The well-renatured IL-2 mutants 1193, 1258, 1259, 1260, and 1261 were captured directly by GE Hitrap QFF ion exchange column chromatography.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 4. Protein purification results

[0097] The results are as follows Figure 3 As 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 mutant 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.

[0098] Example 3. Protein Concentration and Stability Test

[0099] 1. Protein Concentration Operation

[0100] 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:

[0101] 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.

[0102] 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.

[0103] By detecting the UV280 and stability of the concentrated samples, the stability of each IL-2 derivative after concentration was detected by SEC-HPLC, and the results are shown in Table 1.

[0104] Table 1: Maximum concentration and purity determination of IL-2wt(C125A), IL-2 mutant and IL-2 derivative

[0105]

[0106]

[0107] 2. Concentration and Stability Results Analysis

[0108] 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.

[0109] Example 4. Activity test of IL-2 mutants or derivatives

[0110] 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.

[0111] 1. In vitro activity detection of IL-2 mutants or derivatives

[0112] 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:

[0113] 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.

[0114] 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.

[0115] 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.

[0116] Table 2: Experimental results on the binding activity of IL-2 mutants or derivatives to receptors β and γ-receptors

[0117] 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

[0118] 2. Detection of CTLL2 cell viability using IL-2 mutants or derivatives

[0119] This experiment compared the activity of IL-2 mutants or derivatives with wild-type IL-2 in the CTLL2 cell line. The experimental steps are as follows:

[0120] 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.

[0121] 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.

[0122] 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.

[0123] Table 3 shows the EC50 activation of CTLL2 cells by IL-2 mutants or derivatives and wild-type IL-2. It can be seen that the Leu19 mutation, C125A mutation, and C-terminal hydrophilic peptide in IL-2 derivatives had no effect on cell viability; some IL-2 mutants even showed better biological activity than wild-type IL-2. See the detailed results below. Figure 4 And Table 3.

[0124] Table 3: Results of activity assays of wild-type IL-2 and IL-2 derivatives in CTLL2 cells.

[0125]

[0126]

[0127] 3. Activity detection results

[0128] The data above shows that the IL-2 mutants or derivatives modified by this invention have similar receptor binding capacity and biological activity to wild-type IL-2.

[0129] The IL-2 mutants involved in Examples 5-11 below are based on the IL-2 derivatives obtained in Examples 1-3, with a second amino acid mutation occurring at position 65 (Pro). Simultaneously, a fourth amino acid mutation occurs in the C-terminal hydrophilic polypeptide IST of the IL-2 derivatives obtained in Examples 1-3. Compared with the amino acid sequence SEQ ID NO:2 of IST, the fourth amino acid mutation is selected from the following substitutions: G19C, G12C, or G7C (numbered starting from the first amino acid in SEQ ID NO:2). The IL-2 derivatives also have a polyethylene glycol moiety of cysteine ​​linked to the site where the fourth amino acid mutation occurs in the C-terminal hydrophilic polypeptide IST.

[0130] Example 5: Construction, protein expression, and preparation of IL-2 mutant expression plasmids

[0131] 1. Expression plasmid construction

[0132] Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize wild-type IL-2wt (C125A) and IL-2 mutants 1193 (L19H, C125A, IST), 1267 (L19H, P65E, C125A, IST), 1268 (L19H, P65Q, C125A, IST), 1269 (L19H, P65S, C125A, IST), 1301 (L19H, P65A, C125A, IST), 1302 (L19H, P65T, C125A, IST), and 1270 (L19H, The genes for P65E, C125A, IST-G19C, 1293 (L19H, P65E, C125A, IST-G12C), 1294 (L19H, P65E, C125A, IST-G7C), 1348 (L19A, C125A, IST-G12C), 1349 (L19A, P65T, C125A, IST-G12C), 1350 (L19A, P65S, C125A, IST-G12C), and 1351 (L19A, P65Q, C125A, IST-G12C) were obtained. Following the procedures described in *Molecular Cloning*, overlap PCR was performed to obtain the target fragments. Then, recombination ligation of the fragments with the universal vector pET41a was performed, followed by transformation, sequencing, and bacterial preservation to obtain plasmids capable of expressing the corresponding proteins. Plasmids were prepared for IL-2 wild-type and IL-2 mutants according to the procedures in the Qiagen Mini-prep Kit.

[0133] 2. Expression of Escherichia coli

[0134] The successfully constructed plasmid was transformed into the *E. coli* expression strain BL21(DE3). Single colonies were selected and cultured overnight as seed culture. This seed culture was then transferred at a 1:50 volume ratio to 500 mL of Kana-resistant TB medium in Erlenmeyer flasks. The initial OD600 was approximately 0.1. The culture was maintained at 37°C and 220 rpm until the OD600 reached 2.0. IPTG (final concentration 0.5 mmol / L) was then added, and the culture was continued at 37°C and 220 rpm for 4 hours before harvesting. SDS-PAGE analysis of protein expression showed that all proteins were successfully expressed as inclusion bodies (IBs) at high levels.

[0135] 3. Inclusion body fragmentation and washing

[0136] In this invention, all wild-type and mutant plasmids were transformed into engineered E. coli host bacteria for expression, resulting in insoluble inclusion bodies. The inclusion bodies were then recovered after high-pressure disruption and washing of the bacterial cells.

[0137] 4. Protein preparation

[0138] Inclusion bodies of the same amount of the above-mentioned IL-2 mutant were dissolved in 8M urea solution, and DTT was added to a final concentration of 10mM. After denaturation for 1 hour, the solution was diluted in refolding buffer (20mM Tris-Cl, pH 8.0) and incubated overnight at room temperature. Samples were then extracted for analysis. Purification was performed using anion exchange chromatography Q-FF and gel filtration chromatography Superdex-200. Wild-type IL-2wt (C125A) required additional purification using a C8 reversed-phase column with a flow rate of 10%–80% acetonitrile aqueous solution. Other molecules in this example did not require C8 reversed-phase column purification. After purification, both wild-type IL-2wt (C125A) and the IL-2 mutant showed a purity of over 90% and were relatively stable when analyzed by SDS-PAGE, suitable for further analysis (details are shown in Table 4).

[0139] Table 4: Protein information used in this invention

[0140]

[0141] Example 6. Protein Concentration and Stability Test

[0142] 1. Protein Concentration Operation

[0143] The proteins obtained in Example 5 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:

[0144] All IL-2 mutants were concentrated using an Amicon Ultra-15 centrifugal filter (10 kDa molecular weight cutoff) in a 4000g, 8°C rotating drum for 10 minutes each time. The concentrate was then removed, mixed thoroughly, and protein changes were observed. This process was repeated multiple times until the protein concentration reached or exceeded 5 mg / ml.

[0145] The results of UV280 and stability testing of the concentrated samples are shown in Table 5.

[0146] Table 5: Maximum Concentration and Purity Determination of IL-2wt(C125A) and IL-2 Mutant

[0147]

[0148] 2. Concentration and Stability Results Analysis

[0149] The results are shown in Table 5. Under the same conditions, concentration experiments were conducted on IL-2wt(C125A) and the IL-2 mutant modified according to this invention. The protein modified according to this invention, with the Leu19 mutation and the addition of a hydrophilic polypeptide at the C-terminus, showed decreased hydrophobicity and increased water solubility. Under the same PBS buffer, the protein concentration of both proteins could be increased to over 5 mg / ml, more than 10 times that of wild-type IL-2. In contrast, IL-2wt(C125A), without the addition of a hydrophilic polypeptide at the C-terminus, showed a concentration of approximately 0.53 mg / ml after concentration in the same PBS buffer. This indicates that the Leu19 mutation and the terminal hydrophilic polypeptide in this example have the same effect as in the IL-2 derivatives in Examples 1-3, improving the water solubility of IL-2 and laying a good foundation for future formulation preparation of IL-2 drugs. Furthermore, better protein solubility is beneficial for subsequent PEGylation of the protein.

[0150] Example 7. In vitro binding activity of IL-2 mutants containing different p65 positions against the CD25 receptor

[0151] The modified IL-2 of this invention involves a mutation at position p65 to affect the binding ability of the IL-2 mutant to CD25. This experiment determined the effect of these modifications on the binding of IL-2 to the CD25 receptor. The binding of the modified protein to IL-2CD25 was measured using a Biacore SPR8K instrument.

[0152] The binding strength of different P65 mutants to the CD25 receptor of IL-2 was tested using Biacore SPR-8K, as shown in Table 1. The specific methods are as follows:

[0153] 1) Use a Protein-A chip (GE Healthcare) to immobilize CD25 with an Fc tag onto the chip surface. The immobilization amount is 1000 RU, and the fluidity used is PBS buffer containing 0.05% Tween 20.

[0154] 2) Pass the IL-2 mutant through different concentrations and detect its response value. The concentrations used included 100 nM.

[0155] 3) After the experiment, the binding ability of the IL-2 mutant to CD25 was estimated by comparing the response value of the control molecule 1193 (L19H, C125A, IST) without the P65 mutation. The specific data are shown in Table 6.

[0156] Table 6: Experimental results on the binding activity of wild-type IL-2 and different mutants to receptor CD25.

[0157]

[0158] In in vitro CD25 binding activity experiments of control molecule 1193 (L19H, C125A, IST) without the P65 mutation and different P65 mutants, it was found that the binding strength of IL-2 mutants to CD25 was regulated by the size of the side chain of the amino acid at position 65: when the 65th position was Pro or Ala, which had no side chain or a small side chain, the IL-2 mutant maintained nearly 100% CD25 binding activity; when the 65th position was Ser or Thr, which had a medium-sized side chain, the IL-2 binding activity to CD25 was significantly reduced, maintaining only about 20% of the binding activity; when the 65th position was Glu or Gln, which had a large side chain, the IL-2 mutant almost completely lost its CD25 binding activity. This invention achieves continuous regulation of the binding activity of IL-2 mutants to CD25 through a single amino acid mutation at position 65, which has the advantages of fewer mutation sites and continuous regulation compared to previously reported mutation methods.

[0159] Example 8. Preparation of PEG-modified IL-2 derivatives and in vitro binding activity experiments on CD25 and CD122 receptors.

[0160] To further extend the half-life of the IL-2 mutant, this invention uses PEGylation to modify certain molecules (1270, 1293, 1294, 1348, 1349, 1350, 1351) containing free cysteine ​​residues. The specific method is as follows:

[0161] The IL-2 derivative containing free cysteine ​​was concentrated or diluted to maintain a protein concentration between 0.4 and 1.0 mg / ml, dissolved in 20 mM Hepes, 100 mM NaCl buffer, and the pH was adjusted to 7.0-7.5. 20 kDa mPEG-MAL (synthesized by Xiamen Sinobond Biotechnology Co., Ltd.) was dissolved by weighing in 50 mM disodium hydrogen phosphate solution at pH 7.0 to prepare a 100 mg / ml solution. A 10 mM TCEP solution was prepared and dissolved in ultrapure water.

[0162] Adjust the pH of the purified IL-2 mutant solution to between 7.0 and 7.5 using sodium hydroxide solution. Add 10 mM TCEP solution to reduce the protected cysteine ​​residues in the IL-2 mutant, bringing the final TCEP concentration in the reduction system to between 0.05 and 0.1 mM. Allow the reduction reaction to stand at room temperature for 16-20 hours. Perform mass spectrometry analysis before adding PEG to confirm that the internal disulfide bonds of the skeletal protein are not open, except for the disulfide bonds at the C-terminal mutation sites. Add 100 mg / ml PEG solution to the reduced IL-2 mutant solution to achieve a molar ratio of IL-2 to PEG of 1:10. Stir thoroughly and allow the reaction to stand at room temperature for 30-60 minutes. The sample can then be purified to remove impurities and free PEG.

[0163] The conjugated product was purified using anion exchange chromatography, cation exchange chromatography, or gel filtration chromatography to remove free PEG and obtain the target protein, ultimately achieving near-homogeneous protein purity. After purification, novel PEG-conjugated IL-2 derivatives (1270-PEG, 1293-PEG, 1294-PEG, 1348-PEG, 1349-PEG, 1350-PEG, 1351-PEG) were obtained. All PEG-modified IL-2 molecules were analyzed by iodine-stained SDS-PAGE, and the purity was above 90%.

[0164] To further verify the effect of different PEG modification sites on protein activity, this invention conducted an SPR-based CD122 receptor binding activity assay on PEG-modified proteins 1270-PEG, 1293-PEG, and 1294-PEG. The specific method is as follows: Fc-tagged CD122 receptors were immobilized on the surface of a Protein-A chip (GE Healthcare) at a concentration of 1000 RU. PBS buffer containing 0.05% Tween 20 was used for flow. 50 nM solutions of 1193 and IL-2 derivatives 1270-PEG, 1293-PEG, and 1294-PEG were passed through the buffer, and the response values ​​were measured. After the experiment, the data were analyzed using the data processing software included with Biacore SPR 8K. The specific results are shown in Table 7 below. Compared with the unmodified control molecule 1193, the affinity of the different PEG-modified IL-2 derivatives for the CD122 receptor was slightly decreased, with the decrease being less than 3-fold. Based on the 1293-PEG molecule, we constructed molecules 1348-PEG(L19A,C125A,IST-G12C), 1349-PEG(L19A,P65T,C125A,IST-G12C), 1350-PEG(L19A,P65S,C125A,IST-G12C), and 1351-PEG(L19A,P65Q,C125A,IST-G12C) containing both a P65 mutation and PEG modification. The preparation process is described in Example 5 and in this example.

[0165] Table 7: Experimental results on the binding activity of wild-type IL-2 and different PEG-modified mutants against receptor CD122.

[0166]

[0167] Example 9. Activation of immune cells in mouse spleen by PEG-modified IL-2 derivatives.

[0168] To further screen and verify the activity of the IL-2 mutation designed in this invention, the spleen cells of 6-8 week old C57 mice were used to test the proliferative effects of IL-2 mutants 1348-PEG, 1349-PEG, 1350-PEG, 1351-PEG and control molecule 1193 on CD8+ T cells and Treg cells. The specific operation was as follows: C57 mouse spleens were washed with PBS and the cell number was adjusted to 2×10⁻⁶ cells / year. 6Cells / mL, 1.5 mL / well were seeded into 6-well plates. The test sample was diluted to an initial concentration of 1000 nM, and then serially diluted to a total of 8 concentrations. 1.5 mL / well was added to the seeded cells. Mouse spleen cells and test samples were incubated together at 37°C and 5% CO2 for 7 days.

[0169] Take 1.5 x 10 mm of each sample. 6 Centrifuge the cells at 300g for 5 min, discard the supernatant, add 1 mL of washing buffer, centrifuge at 300g for 5 min, and discard the supernatant again. Resuspend the cells in 100 μL of staining buffer, and add a mixed antibody (CD3, CD4, CD8, CD56, CD127, CD25) to each tube. Mix well, incubate at 4°C in the dark for 30 min, add 1 mL of staining buffer, centrifuge at 300g for 5 min, and discard the supernatant. Resuspend the cells in 400 μL of staining buffer to each tube and perform flow cytometry analysis.

[0170] In mouse spleen cell stimulation experiments, such as Figure 5 As shown, the PEG-modified molecules exhibited varying degrees of decreased ability to stimulate immune cell proliferation compared to the unmodified control molecule 1193, indicating that the PEG-modified IL-2 molecules are milder and may achieve a better dosing window in clinical applications. Furthermore, compared to 1348-PEG without the p65 mutation, 1349-PEG, 1350-PEG, and 1351-PEG with the p65 mutation achieved higher CD8+ / Treg ratios (e.g., at 300 nM), suggesting that these molecules possess better efficacy and lower Treg proliferative activity.

[0171] Example 10: Toxicity test of PEG-modified IL-2 derivative 1351-PEG in mice

[0172] To further verify the low toxicity of the IL-2 mutant designed in this invention, the IL-2 derivative 1351-PEG used in this embodiment was tested for toxicity in mice. A relatively high dosage of 3 mg / kg was used. Specifically, six 6-8 week old C57BL / 6 mice were randomly divided into two groups: a reagent control group (PBS) and a long-acting IL-2 mutant 1351-PEG group, with two mice in each group. The mice were administered the drug via tail vein injection twice a week at a dose of 3 mg / kg. The effects of the drug on the experimental animals and changes in body weight were observed after administration. At the end of the experiment, dissections were performed to examine organs for any visible lesions. The results of the body weight measurement experiment are as follows: Figure 6 As shown.

[0173] Example 11: Tumor growth inhibition experiment of PEG-modified IL-2 derivative in mice

[0174] The IL-2 derivative conjugates used in this example were prepared in Examples 5 or 8. Six 6-8 week old female BALB / c mice were randomly divided into six groups: a reagent control group (PBS), a control IL-2 group (1193), a long-acting IL-2 (1348-PEG) group, and long-acting IL-2 derivatives (1349-PEG) and (1351-PEG) groups. The molecules selected in this example were mainly used to verify the effect of molecules with different CD25 binding abilities on antitumor efficacy. Among them, 1193 and 1348-PEG retained 100% CD25 binding activity, 1349-PEG retained a small amount of CD25 binding activity, and 1351-PEG had almost no CD25 binding activity. In this experiment, five mice in each group were subcutaneously inoculated with CT-26 tumor cells at a rate of 5*10E5 cells in the right shoulder. Starting the day after inoculation, the cells were administered via tail vein injection twice a week, with a dosage of 3 mg / kg per injection for each group. The effects of the drug on the experimental animals were observed after administration, as well as changes in tumor volume in mice. At the experimental endpoint, dissections were performed to examine organs for any visible lesions. The tumor volume detection results in mice are as follows: Figure 7 As shown.

[0175] This animal experiment showed that, in the CT-26 animal model, 1348-PEG and 1349-PEG exhibited the best tumor-suppressive effects. These two molecules retained 100% and a small amount of CD25 binding activity, respectively, both achieving excellent anti-tumor effects. However, mice injected with 1348-PEG showed side effects such as weight loss and lethargy, indicating that while retaining 100% CD25 binding activity resulted in excellent anti-tumor effects, it also brought stronger side effects. No significant side effects such as weight loss were observed in the 1349-PEG group. The control molecules 1193 and 1351-PEG also showed strong anti-tumor activity, but it was weaker than that of 1348-PEG and 1349-PEG, indicating that completely removing CD25 binding activity is not conducive to the efficacy of IL-2 anti-tumor activity. Detailed statistical analysis is shown in Table 8. The tumor volume calculation formula is: Tumor volume TV (mm²) 3 = 0.5 × major axis (mm) × minor axis (mm) 2 Tumor weight inhibition rate (TGI%) = (1 - T / C) × 100%, where T / C = mean tumor weight in the treatment group / mean tumor weight in the control group.

[0176] Table 8. Statistical analysis of the antitumor activity of different IL-2 molecules in the CT-26 mouse model.

[0177]

[0178] 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.

Claims

1. An IL-2 mutant, characterized in that, The amino acid sequence of the IL-2 mutant relative to the wild-type IL-2 (SEQ ID NO:1) is modified by replacing Leu at position 19 with Ala, Pro at position 65 with Thr, and Cys at position 125 with Ala, and then linking an acidic hydrophilic polypeptide IST with the amino acid sequence shown in SEQ ID NO:2 to the C-terminus, wherein Gly at position 12 is replaced by Cys (amino acid numbering starts from the first amino acid in SEQ ID NO:2); the amino acid sequence of the IL-2 mutant is shown in SEQ ID NO:

19.

2. An IL-2 derivative, characterized in that, The IL-2 derivative, relative to the amino acid sequence of wild-type IL-2 SEQ ID NO:1, has Leu at position 19 replaced by Ala, Pro at position 65 replaced by Thr, and Cys at position 125 replaced by Ala, and is linked at the C-terminus to an acidic hydrophilic polypeptide IST with the amino acid sequence shown in SEQ ID NO:2, wherein Gly at position 12 is replaced by Cys (amino acid numbering starts from the first amino acid in SEQ ID NO:2); the IL-2 derivative also has a polyethylene glycol moiety, which is linked to a cysteine ​​residue introduced by the substitution of Gly at position 12 of SEQ ID NO:2 by Cys, and the polyethylene glycol moiety has a molecular weight of 20 kDa.

3. A polynucleotide, characterized in that, The polynucleotide encodes the amino acid sequence of the IL-2 mutant of claim 1.

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

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

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the IL-2 mutant or IL-2 derivative as described in claim 1 or 2 and pharmaceutically acceptable excipients.

7. A method for increasing the cyclic half-life of the IL-2 mutant of claim 1, the method comprising: By linking a 20 kDa polyethylene glycol moiety to the 12th cysteine ​​residue of the C-terminal hydrophilic polypeptide IST of the IL-2 mutant of claim 1, the IL-2 derivative of claim 1 is obtained.

8. The use of the IL-2 mutant of claim 1, the IL-2 derivative of claim 2, or the pharmaceutical composition of claim 6 in the preparation of a medicament for treating cancer, wherein the cancer is metastatic melanoma, metastatic renal cell carcinoma, or colon cancer.

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