Human interleukin-2 variant and use thereof

Abstract

To provide a human interleukin-2 variant and use thereof.SOLUTION: Provided is an IL-2 variant (or a derivative thereof) having eliminated or reduced affinity with a high-affinity receptor IL-2Rα / β / γ and retaining affinity with a medium-affinity receptor IL-2Rβ / γ. Also provided is a recombinant protein / fusion protein comprising an IL-2 variant and a human antibody fragment. An activity of the IL-2 variant is improved, and is close to an activity of a wild human IL-2.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application is a joint venture with a patent application filed on July 30, 2021 (application number 202110868 4095) and a patent application filed on June 1, 2022 (202210621425 9) is claimed as the priority.

[0002] The present disclosure relates to human interleukin-2 (IL-2) mutants having one or more amino acid mutations. The IL-2 variant or derivative thereof is It has lost or reduced affinity for the high-affinity receptors (IL-2Rα / β / γ) and has intermediate affinity The present disclosure also provides a method for the preparation of human IL-2 mutants that maintain their affinity for the IL-2 receptor (IL-2Rβ / γ). Immunoconjugates comprising the antibody, the coding polynucleotide, the vector, the host cell, the pharmaceutical composition, The present invention relates to methods for their preparation, treatment and use.

[0003] Human interleukin-2 (IL-2), also known as T cell growth factor (TCGF), is a The IL-2 gene is located on chromosome 4 (4q27) and contains a total sequence of approximately 7 kb. IL-2 is composed of approximately 133 amino acids and has a molecular weight of approximately 15 kD. Oris Morgan et al., 1976 and 1977, cultures of activated T cells They found that the culture medium could promote T cell proliferation. , and was identified as IL-2.

[0004] In the first in vitro cell experiment, T cells were activated by TCR and CD28. After being transfected, they secrete IL-2 and express the IL-2 receptor (IL-2R) on their cell surface. It was shown that the binding of IL-2 to the IL-2R mediates T cell proliferation and effects on T cells. IL-2 is a molecule that plays a central role in T cell immune responses. In in vivo experiments, after knocking out IL-2 or its receptor, animals were able to It has been found that IL-2 stimulates effector cells (T cells and NK cells) etc.) as well as activating regulatory T cells, thereby suppressing excessive self-immunity. do.

[0005] IL-2 acts through the IL-2R, which is expressed by IL-2Rα (i.e. CD 25), IL-2Rβ (i.e., CD122), and IL-2Rγ (i.e., CD132). The three subunits can form three types of receptors: The high affinity receptor comprises all three subunits IL-2Rα / β / γ and is mesophilic. The high affinity receptor comprises IL-2Rβ / γ, and the low affinity receptor comprises IL-2Rα. IL-2Rβ and IL-2Rγ are required for activation of downstream signaling pathways by IL-2 When IL-2 binds simultaneously to IL-2Rβ and IL-2Rγ, two receptors are activated. The STAT5 subunits form heterodimers to phosphorylate intracellular STAT5, which then phosphorylates the nuclear IL-2Rα enters the IL-2 receptor, thereby inducing the transcription and expression of the corresponding genes. Although not required for signal transduction, it promotes IL-2 binding to IL-2Rβ and IL-2Rγ It is possible to proceed.

[0006] IL-2Rγ is expressed on all immune cells. IL-2Rβ is expressed on CD8+ T cells, It is expressed in NK cells and regulatory T cells, and its expression level increases after T cell activation. 2Rα is persistently highly expressed in regulatory T cells and transiently expressed in activated CD8+ T cells. The expression level is then down-regulated.

[0007] IL-2 is synthesized primarily by activated T cells, especially CD4+ helper T cells. Stimulates cell proliferation and differentiation, and promotes the production of cytotoxic T lymphocytes (CTLs) and peripheral blood lymphocytes. It induces differentiation of lymphocytes into cytotoxic cells and lymphokine-activated killer (LAK) cells, and stimulates T cells. It promotes the expression of cytokines and cytolytic molecules by cells, and promotes the proliferation and differentiation of B cells. It stimulates immunoglobulin synthesis by IL-1 and B cells, as well as natural killer (NK) cells. It stimulates the production, proliferation, and activation of lymphocytes, expanding lymphocyte populations in vivo and promoting the proliferation and proliferation of these cells. The ability of IL-2 to enhance cell effector functions contributes to its antitumor effects, and IL- 2 Immunotherapy is a treatment option for certain patients with metastatic cancer. Currently, high doses of IL-2 It is approved for the treatment of metastatic renal cell carcinoma and malignant melanoma.

[0008] The IL-2 variants disclosed in WO 2009 / 135615 have positions 20, 88, or a mutation at position 126. The L-2 mutant has at least one amino acid sequence at positions 38, 42, 45, 62, 68, or 88. The IL-2 mutant disclosed in U.S. Pat. No. 8,906,356 has one mutation. , with mutations at positions 91 and 126. The IL-2 variants include at least one amino acid sequence at positions 15, 16, 22, 84, 88, or 95. The mutation is also present in one of the genes. The IL-2 mutant disclosed in the specification contains the mutation R38W. Summary of the Invention

[0009] The present disclosure relates to IL-2 variants (or derivatives thereof) having one or more amino acid mutations, The present invention also relates to conjugates of the IL-2 variants, as well as to methods for the use and preparation of the IL-2 variants.

[0010] (IL-2 variants or derivatives thereof) In a first aspect, the present disclosure provides a method for the preparation of IL-2, comprising the steps of: The present invention provides an IL-2 variant (or derivative thereof) that contains one or more amino acid mutations compared to the IL-2 variant.

[0011] "Interleukin-2" or "IL-2" refers to any mammalian cell, including humans. Any natural IL-2 derived from primates, mice, and rodents such as rats may be used. The term includes both unprocessed IL-2 and any processed form of IL-2 derived from cells. The term also includes naturally occurring IL-2 variants, such as splice variants or Allelic variants are also included.

[0012] "Wild-type human IL-2" is a mutant IL-2 with the same amino acid sequence as the wild-type IL-2 at each amino acid position. The term "wild type" refers to an IL-2 variant that is identical to the IL-2 variant except that it has the amino acid sequence of One or more naturally occurring amino acid mutations that do not affect the ability to bind to the IL-2 receptor It is intended to encompass including.

[0013] "Mature wild-type human IL-2" refers to the mature form of wild-type human IL-2. IL-2 further contains an N-terminal signal of approximately 20 amino acids that is not present in the mature IL-2 molecule. Exemplary amino acid sequences of mature wild-type human IL-2 are described, for example, in the INSD C database accession number CAA25742.1, Swiss-Prot P60568 .1, and segment 21-153 of NP_000577.2 in Genbank. In some embodiments, the mature wild-type human IL-2 is expressed as, but not limited to, SE For purposes of recombinant expression, the amino acid sequence shown in Note that an additional methionine M is required at the end of the All sequences with or without M at position 1 are referred to as "mature wild-type human IgG1A ...B1B1B1B1B1B1B1B1B1B1B1B1B1B1B It is to be understood that the term "IL-2" is included within the scope of the term "IL-2."

[0014] In some embodiments, the amino acid mutations embodied in the present application are similar to those of mature wild-type human IL- 2, the region of IL-2 that binds to IL-2Rα (amino acids 26 to 47) position) will be shorter.

[0015] In some embodiments, the amino acid mutations include substitutions, deletions (or truncations), insertions (or (addition), modifications, and any combination thereof. For example, binding of IL-2 To change the properties, one can substitute one amino acid (e.g., one with a different structure and / or chemical properties) An amino acid having a non-natural amino acid sequence may be used to replace another amino acid. The amino acids include the 20 common amino acids. Amino acid mutations can be made by site-directed mutagenesis. using methods known in the art, including gene expression, PCR, gene synthesis, chemical modification, etc. can be generated.

[0016] In some embodiments, the IL-2 variant (or derivative thereof) according to the present disclosure is has reduced affinity for IL-2Rα and has unchanged affinity for IL-2Rβ and / or IL-2Rγ or have increased affinity.

[0017] "Affinity" refers to the sum of non-covalent interactions between the binding site of a molecule and its ligand. Unless otherwise specified, "affinity" as used herein refers to the strength of affinity between members of a binding pair, e.g. For example, affinity refers to the intrinsic binding affinity that reflects the 1:1 interaction between a receptor and a ligand. The rate is usually determined by the dissociation constant (K D ) can be expressed as Each K 解離 and K. 結合 Affinity can be determined by any method known in the art, including the methods described herein. can be measured by standard methods.

[0018] In some embodiments, the IL-2 variant (or derivative thereof) according to the present disclosure is highly It has a reduced affinity for the IL-2Rα / β / γ receptors, but has a medium affinity receptor ( maintain or increase affinity for IL-2Rβ / γ).

[0019] In some embodiments, a "high affinity IL-2 receptor" refers to the receptor γ subunit (γ subunit). The cytokine receptor common subunit γ, also known as γc, or CD132, The receptor β subunit (also known as CD122 or p70), and the receptor α subunit A heterotrimeric form of IL-1, consisting of the CD25 and p55 units (also known as CD25 and p55). Refers to two receptors.

[0020] In some embodiments, an "intermediate affinity IL-2 receptor" is a receptor that is composed of gamma and beta subunits. It refers to the IL-2 receptor that contains only the α subunit and does not contain the α subunit (Olejniczak and and Kasprzak, MedSci Monit14, RA179-189, 2008 reference).

[0021] In some embodiments, the IL-2 variant (or derivative thereof) according to the present disclosure reduces Activation of regulatory T cells (Tregs) and / or activation of Tregs was unaffected or increased and activation of immune effector cells (e.g., T cells, NK cells).

[0022] In some embodiments of the IL-2 variant (or derivative thereof) according to the present disclosure, the mutation is , Tyrosine (Y) at position 31, lysine (K) at position 32, aspartate (A) at position 33 Ragin(N), Proline (P) at position 34, lysine (K) at position 35, and leucine (L) at position 36. N(L), Threonine (T) at position 37, Arginine (R) at position 38, Methionine (M), Leucine (L) at position 40, threonine (T) at position 41, and fluorine (F) at position 42. Phenylalanine (F), Mutations at position 43 (K) and 44 (F) Tyrosine (Y) in Cysteine ​​(C) at position 125, Any one of the positions (consecutive or non-consecutive) (counting from the 1st position of SEQ ID No. 1) Occurs in more than one.

[0023] In some embodiments of the IL-2 variant (or derivative thereof) according to the present disclosure, the mutation is , occurring at one or more of positions 31-32 and 125 (consecutive or discontinuous).

[0024] In some embodiments of the IL-2 variant (or derivative thereof) according to the present disclosure, the mutation is , occurring at one or more of positions 35 to 41 and 125 (consecutive or non-consecutive).

[0025] In some embodiments of the IL-2 variant (or derivative thereof) according to the present disclosure, the mutation is , occurring at one or more of positions 43 to 45 and 125 (consecutive or discontinuous).

[0026] In some embodiments of the IL-2 variant (or derivative thereof) according to the present disclosure, the mutation is , occurring at one or more of positions 31 to 45 and 125 (consecutive or discontinuous).

[0027] In some embodiments of the IL-2 variant (or derivative thereof) according to the present disclosure, the mutation is , 31st to 32nd, 35th to 41st, and 125th (consecutive or non-consecutive) This occurs.

[0028] In some embodiments of the IL-2 variant (or derivative thereof) according to the present disclosure, the mutation is , 35th to 41st, 43rd to 45th, and 125th (consecutive or non-consecutive) This occurs.

[0029] In some embodiments of the IL-2 variant (or derivative thereof) according to the present disclosure, the mutation is , occurring at one or more of positions 31 to 43 and 125 (consecutive or discontinuous).

[0030] In some other embodiments, the compounds are selected from the group consisting of compounds listed in public databases or known literature. When the native sequence is used as the wild-type mature IL-2, the location of the amino acid mutation is the 1st amino acid. It is counted from acid A.

[0031] In some particular embodiments, Gly-Gly-Asn-Pro-Met-His-Gly-Leu-Asp-Gly-Phe- the amino acids at positions 31-45 mutated to Gly (SEQ ID No. 11); The amino acid at positions 31-32 mutated to Gly-Gly, Met-His-Gly-Leu-Asp-Gly (SEQ ID No. 12) Different amino acids at positions 35-41, Met-Gly-Gly-Leu-Gly-Gly (SEQ ID No. 13) Different amino acids at positions 35-43 Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID No. 14) Different amino acids at positions 35-41, The amino acids at positions 43-45 were mutated to Gly; The amino acid at position 125 was mutated to Ala, The present invention provides IL-2 variants comprising any one of the above mutations or a combination thereof.

[0032] In some specific embodiments, the IL-2 variant is (1) YK to GG mutation at positions 31-32 and KLT at positions 35-41 RMLT to MHGLDG mutation and KFY to G mutation at positions 43-45, 1 A mutation of C to A at position 25, or (2) a mutation of amino acids 31-45 to GGNPMHGLDGFG, and Mutation of the amino acid at position 25 to A, is selected from the group consisting of The mutant sequence is SEQ ID No. 2 or 15. When containing the mutation, the IL-2 mutants maintain or increase their affinity for IL-2Rβ / γ. However, it has a reduced affinity for IL-2Rα.

[0033] In some specific embodiments, the IL-2 variant is The mutations at positions 35-41 from KLTRMLT to MHGLDG and at position 125 the amino acid sequence of the mutant is SEQ ID No. 3; When the IL-2 variant contains a mutation selected from the group consisting of: The antibody has a reduced affinity for IL-2Rα while maintaining or increasing affinity for IL-2Rα.

[0034] In some specific embodiments, the IL-2 variant is Mutation of YK to GG at positions 31-32 and A at amino acid position 125 The mutant sequence is SEQ ID No. 4. When the IL-2 variant contains a mutation selected from the group consisting of: The antibody has a reduced affinity for IL-2Rα while maintaining or increasing affinity for IL-2Rα.

[0035] In some specific embodiments, the IL-2 variant is Mutation of KFY to G at positions 43-45 and the amino acid at position 125 to A The mutant sequence is SEQ ID No. 5. When the IL-2 variant contains a mutation selected from the group consisting of: The antibody has a reduced affinity for IL-2Rα while maintaining or increasing affinity for IL-2Rα.

[0036] In some specific embodiments, the IL-2 variant is (1) YK to GG mutation at positions 31-32, (2) a mutation at positions 35-41 of KLTRMLT to MHGLDG; (3) a mutation of the amino acid at position 125 to A; and the mutant sequence is SEQ ID No. 6. When the IL-2 variant contains a mutation selected from the group consisting of: The antibody has a reduced affinity for IL-2Rα while maintaining or increasing affinity for IL-2Rα.

[0037] In some specific embodiments, the IL-2 variant is (1) a mutation at positions 35-41 from KLTRMLT to MHGLDG; (2) a mutation of KFY to G at positions 43–45; (3) a mutation of the amino acid at position 125 to A; and the mutant sequence is SEQ ID No. 7. When the IL-2 variant contains a mutation selected from the group consisting of: The antibody has a reduced affinity for IL-2Rα while maintaining or increasing affinity for IL-2Rα.

[0038] In some specific embodiments, the IL-2 variant is (1) YK to GG mutation at positions 31-32, (2) a mutation of KFY to G at positions 43–45; (3) a mutation of the amino acid at position 125 to A; and the mutant sequence is SEQ ID No. 8. When the IL-2 variant contains a mutation selected from the group consisting of: The antibody has a reduced affinity for IL-2Rα while maintaining or increasing affinity for IL-2Rα.

[0039] In some specific embodiments, the IL-2 variant is The mutations at positions 35–43 from KLTRMLTFK to MGGLGG and at position 125 to A, the mutant sequence being SEQ ID No. 9. When the IL-2 variant contains a mutation selected from the group consisting of: The antibody has a reduced affinity for IL-2Rα while maintaining or increasing affinity for IL-2Rα.

[0040] In some specific embodiments, the IL-2 variant is At positions 35-41, the KLTRMLT to GGGGGG mutation, at positions 43-45, and a mutation at position 125 of the amino acid sequence to A. is SEQ ID No. 10, When the IL-2 variant contains a mutation selected from the group consisting of: The antibody has a reduced affinity for IL-2Rα while maintaining or increasing affinity for IL-2Rα.

[0041] In some specific embodiments, the amino acid sequence of the IL-2 variant (or derivative thereof) is selected from SEQ ID No. 2 to 10.

[0042] [Table 1-1] [Table 1-2] [Table 1-3]

[0043] Naturally expressed human IL-2 has a total of 153 amino acids, with amino acids 1-20 The third acid is the signal peptide. This means that after cleavage of the signal peptide, a total of 133 acids remain. amino acids (i.e., mature human IL-2), and SEQ ID No. 1 in the present disclosure For recombinant expression, artificially produced IL-2 can be expressed as It has an additional methionine at the first position (corresponding to the start codon AUG).

[0044] JPEG2025066741000004.jpg40166

[0045] In some embodiments, the IL-2 variant is in monomeric form.

[0046] In a second aspect, the present disclosure provides a method for the preparation of a medicament for the treatment of cancer, comprising the steps of PEGylation, glycosylation, conjugation to albumin, conjugation to Fc, etc. Modifications selected from the group consisting of conjugation, hydroxyethylation, and de-O-glycosylation Derivatives of IL-2 variants, including the IL-2 variants of the present disclosure, are provided.

[0047] In some embodiments, derivatives of IL-2 variants include all of the IL-2 variants of the disclosure. or a portion thereof, or a functional variant thereof further obtained based on the IL-2 variants of the present disclosure. Derivatives, functional fragments, biologically active peptides, fusion proteins, isoforms or the like and salts thereof, including, for example, IL-2 variants, dimers, trimers, or multimers of IL-2 variants. Fusion proteins containing IL-2 variants, such as IL-2 fusion proteins, are also available. Cosylation, conjugation to albumin, conjugation to Fc, hydroxyethylation, de-O-glycosylation (After transformation, etc.).

[0048] In some embodiments, the derivative of the IL-2 variant is PEGylated (PEG-IL -2).

[0049] In some other embodiments, the PEG-IL-2 variant is a methoxy-PEG-aldehyde. (mPEG-ALD) linker. In certain embodiments, the average molecular weight of PEG is about 5kD to about 50kD, especially 5, 10, 11, 12, 13, 14, 15, 16, 17, and 18 , 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50kD, or about 5 kD to about 40 kD, or about 10 kD to about 30 kD, or about 10 kD to about 3 0 kD, or between about 15 kD and about 30 kD, about 20 kD.

[0050] In certain embodiments, the mPEG-ALD linker is about 5 kDa, about 10 kDa, A PEG molecule having an average molecular weight selected from the group consisting of about 12 kDa and about 20 kDa. In some embodiments, the aldehyde group of mPEG-ALD is acetaldehyde. The aldehyde may be aldehyde, propionaldehyde, or butyraldehyde.

[0051] In one embodiment, the derivative of the IL-2 variant has a length as compared to the mature wild-type human IL-2. It has a serum half-life.

[0052] In some embodiments, the derivative of an IL-2 variant is an antibody or antigen-binding fragment thereof. In some embodiments, the derivative of the IL-2 variant is In a particular embodiment, the IL-2 variant is an IL-2 variant conjugated to the Fc fragment of a human antibody. A variant derivative is an IL-2 mutant conjugated at its C-terminus to the Fc fragment of a human antibody ( That is, the IL-2 variant according to the present application is conjugated to the N-terminus of the Fc fragment of a human antibody. .

[0053] In some cases, the IL-2 variant derivatives may be either homodimeric or heterodimeric. As an example, the Fc fragment may be modified to promote heterodimerization. In some embodiments, the Fc fragment is a knob-into-hole (Knob-into-Hole) modification. The KIH structure introduces a knob structure into the interface of the first Fc fragment. and introducing a hole structure into the interface of the second Fc fragment, and This allows the knob structure to be localized within the hole structure, The knob structure promotes the formation of monodimers and inhibits the formation of homodimers. Small amino acid side chains from the fragment interface are more easily removed, such as tyrosine or tryptophan. The pore structure is constructed by substituting large side chains, whereas the pore structure is constructed by substituting large amino acid side chains. The chain is then separated by a smaller amino acid residue, such as an alanine or threonine residue, at the interface of the second Fc fragment. It is constructed by replacing the amino acid side chains.

[0054] (zygote) In a third aspect, the present disclosure provides a conjugate comprising a first component and a second component, the first The moiety is directly attached (or indirectly attached via a linker) to a second moiety. The first component is an IL-2 variant (or a derivative thereof) of the present disclosure, and the second component is The component is not IL-2 or an IL-2 variant (or a derivative thereof).

[0055] In some embodiments, the IL-2 variant (or derivative thereof) comprises at least one is attached to a second component.

[0056] In some embodiments, the IL-2 variant (or derivative thereof) and the second component are The fusion protein is formed via a peptide bond.

[0057] In certain embodiments, the IL-2 variant (or a derivative thereof) is (or indirectly via a linker) at the carboxyl terminus of

[0058] In certain embodiments, the IL-2 variant (or a derivative thereof) is (or indirectly via a linker) to the amino terminus of

[0059] In some embodiments, the second moiety is an antigen-binding moiety. It refers to a polypeptide molecule that specifically binds to a pitope.

[0060] In some embodiments, the antigen binding component has a moiety attached thereto (e.g., IL-2, its variants or derivatives) to a target site (e.g., a tumor cell or a tumor). The antigen-binding component can be an antibody or an antigen-binding fragment.

[0061] The term "antibody" is used in the broadest sense herein and includes a variety of antibodies as long as they exhibit antigen-binding activity. Antibody structures are included. Antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies, and the like. These include, but are not limited to, specific antibodies (such as bispecific antibodies) and antigen-binding fragments. Antibodies include mouse antibodies, human antibodies, humanized antibodies, chimeric antibodies, and camelid antibodies. It is possible.

[0062] In certain embodiments, the antibody or antigen-binding fragment thereof comprises an antibody heavy chain variable region and and a polypeptide complex comprising an antibody light chain variable region, Fab, Fv, sFv, F(ab') 2. Linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptibodies Antibodies, domain antibodies, and multispecific antibodies (bispecific antibodies, diabodies, tribodies, etc.) The scFv consists of a tetrabody, a tandem di-scFv, and a tandem tri-scFv. is selected from the group.

[0063] In some embodiments, the IL-2 variant (or derivative thereof) binds to two or more antigens. When attached to a synthetic moiety, each antigen-binding component is independently selected from antibodies and antigen-binding fragments. For example, the first antigen-binding moiety can be a Fab molecule and the second antigen-binding moiety can be a s For example, the first antigen-binding component can be an scFv molecule, while In some embodiments, the first and second antigen-binding moieties are also scFv molecules. and the second antigen-binding moiety are independently directed against different antigens or against the same antigen. In some embodiments, the antibody or antigen-binding fragment thereof targets a tumor antigen.

[0064] In some embodiments, the tumor antigen is, for example, a member of the MAGE family (e.g., MAG E-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A 6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE- A11, MAGE-A12, MAGE-Xp2, MAGE-B2, MAGE-Xp3, M AGE-B3, MAGE-Xp4, MAGE-B4, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE ​​family (e.g., G AGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6 , GAGE-7, GAGE-8, GAGE-9), MART-1 / Melan-A, g p75, gp100, DPPIV, ADAbp, cyclophilin, CEA, CAP-1, CAP-2, etv6, aml1, PSA (PSA-1, PSA-2, and PSA-3) , PSMA, T cell receptor, RAGE, LAGE-1, NAG, GnT-V, MUM-1 , CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ra s, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin , γ-catenin, p120ctn, PRAME, NY-ESO-1, CDC27, SSX -1, SSX-2, SSX-1, SSX-4, SSX-5, SCP-1, CT-7 But not limited to these.

[0065] Pharmaceutical Composition In a fourth aspect, the present disclosure provides an IL-2 variant (or a derivative thereof) according to the present disclosure or A pharmaceutical preparation containing the conjugate and, optionally, a pharma- ceutically acceptable diluent, carrier or excipient. A pharmaceutical composition is provided. The pharmaceutical composition can be a lyophilized formulation or an injectable solution.

[0066] In some particular embodiments, the unit dose of the pharmaceutical composition comprises 0.01 wt% to 99 wt% % of an IL-2 variant (or derivative) or conjugate. The amount of IL-2 variant (or derivative thereof) or conjugate contained in a unit dose of the composition is , 0.1-2000mg (e.g., 1-1000mg; 1, 5, 10, 15, 20, 25 , 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 5, 100, 110, 120, 130, 140, 150, 160, 170, 180, 19 0, 200, 250, 300, 350, 400, 450, 500, 550, 600, 65 0, 700, 750, 800, 850, 900, 950, 1000 mg).

[0067] (Polynucleotide) In a fifth aspect, the present disclosure provides a nucleic acid encoding an IL-2 variant (or a derivative thereof) of the present disclosure. The present invention provides a polynucleotide that

[0068] One of skill in the art will understand that nucleotide sequences that code for the same amino acid sequence may vary. Due to codon degeneracy and codon bias in different hosts, different nucleosides may be generated. A sequence of the peptide can encode the same amino acid sequence, and all of these sequences are included within the scope of this disclosure. Can be enjoyed.

[0069] (Expression Vector) In a sixth aspect, the present invention provides an expression vector comprising a polynucleotide according to the present invention. The expression vector may be a eukaryotic expression vector, a prokaryotic expression vector, or a shuttle vector. It may be an expression vector.

[0070] (host cell) In a seventh aspect, the present disclosure provides an expression vector according to the present disclosure, or an I A host cell is provided that expresses the L-2 mutant or that expresses a conjugate according to the present disclosure.

[0071] In some embodiments, the host cell is adapted to express an expression vector comprising a polynucleotide according to the present disclosure. The vector includes a host cell that is transformed or transfected.

[0072] In some embodiments, the host cell is a prokaryotic or eukaryotic cell.

[0073] In some embodiments, the host cell is a bacterial, yeast, or mammalian cell, particularly a Pichia pastoris or Saccharomyces cerevisiae.

[0074] In some particular embodiments, the host cell is a prokaryotic microorganism, such as E. coli.

[0075] In some particular embodiments, the host cell is a eukaryotic cell.

[0076] In some embodiments, plant cells and insect cells expressing glycosylated peptides are Any host cell can be used. Vertebrate cells can also be used as host cells, e.g., in suspension culture. Mammalian cell lines, monkey kidney CV1 cell line (COS-7), human embryonic kidney cell line (293 or 293T cells), baby hamster kidney cells (BHK), and mouse Sertoli cells (TM4 cells), monkey kidney cells (CV1), VERO-76, human cervical cancer cells (HELA), Human kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W13 8), human hepatocytes (Hep G2), mouse breast tumor cells (MMT060562), MR C5 cells, FS4 cells, CHO cells, myeloma cell lines (YO, NS0, P3X63, Sp2 / 0, etc.

[0077] (Methods of Use and Treatment) In an eighth aspect, the present disclosure provides a method for the preparation of a medicament comprising administering to a subject an IL-2 variant (or The present invention also provides uses of the compounds of the present invention, including derivatives thereof, conjugates and pharmaceutical compositions.

[0078] In some embodiments, the present invention relates to the treatment of proliferative and immune disorders, modulation of T cell mediated immune responses, and the like. The present invention provides a method for the treatment of a pulmonary artery disease, comprising administering to a subject a therapeutically effective amount of IL-2 variant (or a derivative thereof) in the treatment of a pulmonary artery disease, and providing ... It will be offered.

[0079] In some embodiments, the proliferative disorder is a tumor or cancer (e.g., a metastatic tumor or cancer). ) or may be a solid tumor.

[0080] In some embodiments, the IL-2 variants (or derivatives thereof), conjugates, or The pharmaceutical compositions are used to treat and stimulate the host's immune system (particularly to stimulate a cell-mediated immune response). It can be used to enhance

[0081] In some embodiments, an enhanced cellular immune response may include, but is not limited to, improved T cell function, improved B cell function, restored lymphocyte function, increased expression of IL-2 receptors, increased T cell responsiveness, increased activity of IL-2 receptors, increased activity of natural killer cells or lymphokine-activated killer (LAK) cells.

[0082] In some embodiments, the IL-2 variants (or derivatives thereof), conjugates, or The pharmaceutical compositions are used to treat proliferative conditions, such as cancer. Non-limiting examples of cancer These include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, Endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, stomach cancer, prostate cancer, blood cancer, skin cancer, These include squamous cell carcinoma, bone cancer, and kidney cancer.

[0083] In some embodiments, the IL-2 variants (or derivatives thereof), conjugates, or or pharmaceutical compositions for use in the abdomen, bones, breasts, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, adrenal thyroid, pituitary, testes, ovaries, thymus, thyroid gland), eyes, head and neck, nervous system (central and peripheral) Treats neoplasms located in the lymphatic system, pelvis, skin, soft tissues, spleen, breast and genitourinary system is used to.

[0084] In some embodiments, the IL-2 variants (or derivatives thereof), conjugates, or The pharmaceutical composition is used to treat a precancerous condition or cancer metastasis. , skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer and head and neck cancer.

[0085] In some embodiments, the IL-2 variants (or derivatives thereof), conjugates, or or the pharmaceutical composition is for treating hypergammaglobulinemia, lymphoproliferative conditions, paraproteinemia, Purpura, sarcoidosis, Sézary syndrome, Waldenström macroglobulinemia It is used to treat glaucoma, Gaucher disease, and histiocytosis.

[0086] In some embodiments, the IL-2 variants (or derivatives thereof), conjugates, or The pharmaceutical compositions are useful for treating autoimmune diseases, transplant rejection, post-traumatic immune responses and infectious diseases. It is used to

[0087] In some embodiments, the autoimmune disease is type I diabetes, rheumatoid arthritis, multiple sclerosis. , chronic gastritis, Crohn's disease, Graves' disease, Bechterev's disease, psoriasis, myasthenia gravis, autoimmune Hepatitis, APECED, Churg-Strauss syndrome, ulcerative colitis, glomerulonephritis, Guillain-Barr syndrome Barre syndrome, Hashimoto's thyroiditis, lichen sclerosus, systemic lupus erythematosus, PANDAS, Urine fever, sarcoidosis, Sjogren's syndrome, stiff man syndrome, scleroderma, Goehner's granulomatosis, vitiligo, autoimmune enteropathy, Goodpasture's syndrome, dermatomyositis, polymyositis The disorder may be selected from the group consisting of inflammation, autoimmune allergies, and asthma.

[0088] In some embodiments, the IL-2 variant (or derivative thereof) is administered in combination with an immunosuppressant. They may be used in combination.

[0089] In some embodiments, the immunosuppressant is a glucocorticoid, azathioprine, Rosporin A, mycophenolate mofetil, tacrolimus, anti-CD3 antibody, anti-CD25 Antibody, anti-TNF-α antibody, methotrexate, cyclosporine, sirolimus, everolimus The compound is selected from the group consisting of cyclophosphamide, fingolimod and cyclophosphamide.

[0090] In some embodiments, a therapeutically effective amount of an IL-2 variant (or derivative thereof) of the present disclosure The conjugate, or pharmaceutical composition is administered to the subject.

[0091] In some embodiments, the subject in need, such as a patient or individual, is typically a human. Which mammal is it?

[0092] In some embodiments, the IL-2 variants (or derivatives thereof), conjugates, or or the pharmaceutical composition is administered at least twice a day, at least once a day, or at least every 48 hours. once, at least once every 72 hours, at least once a week, at least once every 2 weeks at least once a month, at least once every two months, or at least once every three months Administered once to subject.

[0093] In some embodiments, the IL-2 variants (or derivatives thereof), conjugates, or The pharmaceutical composition may be administered via any route, for example, parenteral injection (e.g., subcutaneous injection). It is administered by intravenous injection or intravenous injection. [Brief description of the drawings]

[0094] [Figure 1] 1 is a curve of the interaction between wild-type human IL-2, IL-2 mutants or derivatives thereof and the IL-2Rα receptor. [Diagram 2] 1 is a curve of the interaction between wild-type human IL-2, IL-2 mutants or derivatives thereof and the IL-2Rβ / γ receptor. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0095] The following examples are incorporated to further illustrate the present disclosure. It is not intended to limit the scope of the disclosure.

[0096] Example 1. Recombinant expression and characterization of wild-type human IL-2 and IL-2 mutants or derivatives thereof and preparation) Synthesis, expression and purification of IL-2 variant nucleic acid sequences.

[0097] 1. In this example, wild-type human IL-2 and IL-2 mutants (IL-2-1 to IL-2-9) were ) were expressed separately and purified and prepared using the HPC4 tag at the C-terminus of the molecule. IL-2-10 is a mutant form of IL-2-1 with a human IgG1 Fc sequence added to the C-terminus. This was then purified and prepared.

[0098] The entire gene was synthesized and the coding sequence of each protein was ligated into the expression vector p The final expression vector was then subcloned into cDNA3.1. The accuracy of the assay was confirmed by the determination, and the results were finally transfected into a DH5α clonal line. The plasmid suitable for transfection was extracted by a plasmid extraction kit. Transfect mammalian HEK293 cells using a transfection reagent for transient expression. The proteins were then transfected into the plasmid p53 and purified by affinity chromatography.

[0099] 2. Plasmid Transformation of DH5α 80-100 ng of expression plasmid containing the coding sequence was added to previously prepared DH5α The competent cells were heat-shocked after the addition of the plasmid. The transformants were isolated from the control and transformed with the plasmid pG111.

[0100] The transformed competent cells were added to LB liquid medium, placed in a shaker at 37°C, and incubated for 2 The cells were cultured at 100 rpm for about 30 minutes with shaking. The cultured competent cells were removed from the shaker. Then, a portion of the suspension was pipetted onto a plate containing ampicillin and incubated. The plates were then placed in a centrifuge and incubated at 37°C overnight.

[0101] Select a single clone from a fresh culture plate and incubate it in 2-5 mL of LB medium at 37 °C for 2 h. The culture was inoculated into 200 mL of LB medium at a ratio of 1 / 500. The culture suspension was collected and centrifuged at 37°C for 16 hours. The supernatant was removed.

[0102] 3. Thawing and Passaging HEK293 Cells The medium was pre-warmed to 37°C by setting the water bath at 37°C.

[0103] Remove the HEK293 cells from the liquid nitrogen tank and immediately place them in a 37°C water bath. The mixture was then gently shaken to rapidly thaw (approximately 1 minute).

[0104] Sterilize the outer wall of the tube with 75% ethanol and place the tube in a biosafety cabinet. The cells were transferred to a 15 mL centrifuge tube containing 10 mL of medium and centrifuged at 800 rpm. After centrifugation, the supernatant was removed and the cells were resuspended in a small amount of fresh medium. The cells were then transferred to a culture flask, fresh medium was added, and the flask was washed to disperse the cells evenly. The plate was gently shaken and cell samples were collected for counting and viability detection. ×10 5 ~4×10 5 The cells / mL were controlled and viability was >95%.

[0105] The cells were placed in an incubator at 37°C, 110 rpm, and 5% CO2. 2.0×1 0 6 After culturing the cells for 2-3 days until they reach a density of 100 cells / mL, replenish with fresh medium. Cells were passaged by: and Cell density and viability were monitored.

[0106] 4. Plasmid Transfection of HEK293 Cells One day before transfection, HEK293 cells were cultured at 1 × 10 6 Inoculation density of cells / mL The cells were cultured in 1L suspension at 37°C, 110 rpm, and incubated at 37°C. The cells were cultured at 5% CO2. On the day of transfection, the cell density was increased to 1 × 10 6 cells / mL ~1.5×10 6 Controlled in cells / mL.

[0107] DNA transfection reagent mixture: DNA and transfection reagent are mixed The cells were then added to the transfection buffer, mixed thoroughly, and incubated at 37°C. The mixture of immunization reagents is added to the cells to be transfected and placed in an incubator. The cells were cultured at 37°C, 110 rpm, and 5% CO2 for approximately 4–6 h after transfection. After 1 day, the cell cultures were removed from the incubator and centrifuged to collect the supernatant and cells. Ta.

[0108] 5. Protein Purification After 5 days of incubation, the cell culture medium was harvested and centrifuged, and the supernatant was collected and filtered through a 0.22 μm filter. The samples were permeabilized with 1x PBS, pH 7.4 buffer at 4°C. After dialysis, the samples were subjected to affinity purification using the medium conjugated with HPC4 antibody. The mixture was subjected to gel filtration chromatography using Superdex 200. This was then further purified to obtain a highly purified target protein.

[0109] Example 2. IL-2Rα of wild-type human IL-2 and IL-2 mutants or derivatives thereof (Determination of affinity with The wild-type human IL-2 and its IL-2 mutants or derivatives (IL-2) obtained in Example 1 The binding characteristics of IL-2Rα to IL-2-1 to IL-2-10 were examined using the Octet platform. The Octet platform was used for biolayer interferometry (BLI) ) to detect and analyze interactions between biomolecules.

[0110] 1. Laboratory equipment, reagents and consumables Octet® Octet RED96e System (Men's, California) Law Park, Pall Fortebio Corp. Analysis buffer: SD buffer (PBS(pH7.4)+0.01% BSA+0.02% Tween20), Acetate buffer (10 mM, pH 4.0, 5.0, 6.0), AR2G (Fortebio, Catalog No. 18-5092), 96-well plates (Greiner Bio-One part number 655209).

[0111] 2. The experiment was carried out with reference to the experimental plan below. The entire experiment was completed under the conditions of 30°C, 1000 rpm, and 220 μl / well. The proteins used were IL2-Rα-his and transiently expressed in HEK293 and and IL-2 mutants or derivatives thereof obtained by affinity purification.

[0112] The sample to be analyzed was IL-2Rα, and the dilution buffer was SD buffer. The concentrations of are 500nM, 240nM, 120nM, 60nM, 30nM, 15nM (and and an additional concentration of 1000 nM for some mutants).

[0113] 3. Experimental Data and Processing The experimental data was processed using Data Analysis Software 9.0. One wild-type human IL-2 or an IL-2 mutant (specified) bound to one IL-2Rα Specifically, IL-2-1 to IL-2-10 were selected for fitting. The method of fitting was applied, i.e., the six concentrations were analyzed as a group. The fitting results are shown in Figure 1.

[0114] The experimental data were fitted to a 1:1 binding model to determine whether wild-type human IL-2 or I The affinity Kd values ​​of L2 mutants or derivatives with the receptor IL-2Rα were obtained.

[0115] Binding patterns of wild-type human IL-2 and IL-2 mutants or derivatives thereof to IL-2Rα The data are shown in Figure 1 and Table 2.

[0116] As a result, IL-2-1, IL-2-6, IL-2-8, IL-2-9, and IL- 2-10 does not bind to IL-2Rα and does not bind to IL-2-2, IL-2-3, IL-2-4, IL The interaction between IL-2-5, IL-2-7 and IL-2Rα is reduced to some extent or or was shown to be maintained.

[0117] This suggested the following: (1) The mutation of amino acids 31-32 to GG reduces the binding of IL-2 to IL-2Rα. There was no significant effect on the (2) Mutation of amino acids 43-45 to G inhibits the binding of IL-2 to IL-2Rα. had a certain impact on. (3) The KLTRMLT to MHGLDG mutation at positions 35-41 of IL-2 The effect of 10-HT2A1 on the binding to L-2Rα was not significantly affected. (4) The combination of the above three mutations significantly alters the protein structure, resulting in IL- The binding of IL-2Rα to IL-2Rα was significantly reduced, but no binding between the two was observed under the experimental conditions. It was.

[0118] [Table 2]

[0119] Example 3. IL-2Rβ of wild-type human IL-2 and IL-2 mutants or derivatives thereof / γ affinity determination) IL-2Rβ / of wild-type human IL-2 and its mutants or derivatives in Example 1 The affinity with γ was experimentally detected.

[0120] 1. Laboratory equipment, reagents and consumables Octet® Octet RED96e System, Menlo, California Park, Pall Fortebio Corp. Analysis buffer: SD buffer (PBS(pH7.4)+0.01% BSA+0.02% T ween20), Acetate buffer (10 mM, pH 4.0, 5.0, 6.0), AR2G (Fortebio, Catalog No. 18-5092), 96-well plates (Greiner Bio-One part number 655209).

[0121] For the preparation of IL-2Rβ / γ-Fc heterodimer, IL-2Rβ and IL-2R The γ subunits were cloned and fused to the Fc hole and Fc knob, respectively. IL-2Rβ-Fc-hole and IL-2Rγ-Fc-knob were co-transfected into HEK293 cells The heterodimer was transfected with protein A and molecular sieves Super Purified using dex 200.

[0122] 2. The entire experiment was completed under the following conditions: 30°C, 1000 rpm, 220 μl / well.

[0123] The analyte sample was IL-2Rβ / γ heterodimer, and the dilution buffer was SD buffer. The analyte concentrations were 750nM, 500nM, 250nM, 125nM, and 62. 5nM and 31.3nM.

[0124] 3. Experimental Data and Processing The experimental data were processed using Data Analysis Software 9.0. i.e., one wild-type human IL-2 or IL-2 mutant bound to one IL-2Rβ / γ The antibody or its derivatives (IL-2-1 to IL-2-10) were selected for fitting. A global fitting method was applied, i.e., the six concentrations were grouped. was analyzed.

[0125] The experimental data were fitted to a 1:1 binding model to determine whether wild-type human IL-2 or I The affinity Kd value of the L2 mutant or its derivatives with IL-2Rβ / γ was obtained, and the binding data are shown in Figure 2 and Table 3.

[0126] The results are based on the results of the study using IL-2 mutants or their derivatives (IL-2-1 to IL-2-10) and IL We showed that the interaction between α-2Rβ / γ was not altered.

[0127] [Table 3]

[0128] Example 4. CTLL-2 Cell Lines Responding to Wild-type IL-2, IL-2 Mutants, or Derivatives Cell proliferation assay The biological activity of IL-2 was examined by the increase in the dependent cell line CTLL-2 under different IL-2 concentrations. was detected according to the proliferation rate.

[0129] Cells were cultured in 10% fetal bovine serum and 1% of two antibiotics (penicillin-streptomycin). The cells were incubated at 37°C in 5% CO2 in RPMI 1640 medium containing 100% lysine solution. The cells in logarithmic growth phase were trypsinized, counted under a microscope, and cultured at 1× 10 4 ~5×10 4 The cell suspension was prepared at 100 μl / ml. In three identical wells of a 3-well culture plate, three per plate for each cell type. , 1×10 3 ~5×10 3 Pipette in cells / well and use culture medium as a blank control. 100 μl of the medium was used. Plates were incubated overnight at 37°C.

[0130] Blank, 0.001nM, 0.01nM, 0.1nM, 1nM, 10nM different concentrations Degree groups were set up.

[0131] At 0 and 24 hours, the cells were counted using the Cell Counting Kit-8 (CCK -8) and serum-free minimal essential medium were mixed at a volume ratio of 1:10, and 100 μL was added per well. The cells were added to the test wells and incubated in an incubator with 5% CO2 for 1 hour.

[0132] The absorbance at 490 nm was measured using a microplate reader. The values ​​were recorded.

[0133] The results are shown in Table 4. All IL-2 mutants or their derivatives were CTLL -2 cell proliferation activity, and the mutation affects the IL-2Rβ / γ receptor subunit complex. The results showed that the IL-1 expression level on CTLL-2 cells was not significantly affected by the signal transduction function. The proliferative activity of IL-2-1, IL-2-6, IL-2-8, and IL-2-9 mutants was compared against the positive control. The proliferation activity of the positive control was significantly lower than that of the control. The positive control binds to the IL-2Rα receptor, thereby increasing binding to IL-2Rβ / γ. The inhibitors were potentiated and had higher proliferation activity against CTLL-2 cells.

[0134] IL-2-10 is a mutant IL-2-1 that is linked to the C-terminus of the Fc fragment. The recombinant protein was formed by inducing IL-2 mutants. It is defined as a complex that has a molecular weight sufficient to enhance binding to IL-2Rβ / γ. Therefore, it was found that the proliferation activity of CTLL-2 cells was similar to that of wild-type human IL-2. The data is shown in Table 4.

[0135] [Table 4]

Claims

**Claim 1**: A human IL-2 mutant comprising an amino acid substitution as compared to the mature wild-type human interleukin-2 (IL-2) shown in SEQ ID No. 1, wherein the amino acid residues at positions 35 to 41 of the mature wild-type human IL-2 are mutated to the sequence "MHGLDG" (SEQ ID No. 12), and the amino acid residues at positions 43 to 45 of the mature wild-type human IL-2 are mutated to "G", whereby the amino acid residues at positions 35 to 45 of the mature wild-type human IL-2 are substituted with the sequence "MHGLDGFG", or the amino acid residues at positions 31 to 45 of the mature wild-type human IL-2 are substituted with the sequence "GGNPMHGLDGFG" (SEQ ID No. 11), an IL-2 mutant. **Claim 2** The IL-2 mutant has, as compared to the mature wild-type human IL-2, a reduced affinity for IL-2Rα, and a maintained or increased affinity for IL-2Rβ / γ, the IL-2 mutant according to claim 1. **Claim 3** A derivative of the IL-2 mutant according to claim 1, having a modification selected from the group consisting of PEGylation, glycosylation, conjugation to albumin, conjugation to Fc, hydroxyethylation, and de-O-glycosylation, wherein the PEGylation refers to the binding of PEG at the N-terminus of the IL-2 mutant. **Claim 4**: A conjugate comprising a first component which is the IL-2 mutant according to claim 1, and a second component, wherein the first component directly binds to the second component or indirectly binds via a linker, a conjugate. **Claim 5**: (i) A human IL-2 mutant comprising an amino acid substitution as compared to the mature wild-type human IL-2 shown in SEQ ID No. 1, wherein the amino acid residues at positions 35 to 41 of the mature wild-type human IL-2 are mutated to the sequence "MHGLDG" (SEQ ID No. 12), and the amino acid residues at positions 43 to 45 of the mature wild-type human IL-2 are mutated to "G", whereby the amino acid residues at positions 35 to 45 of the mature wild-type human IL-2 are substituted with the sequence "MHGLDGFG", or the amino acid residues at positions 31 to 45 of the mature wild-type human IL-2 are substituted with the sequence "GGNPMHGLDGFG" (SEQ ID No. 11), an IL-2 mutant; ​ (ii) A derivative of the IL-2 variant defined in (i), wherein the IL-2 variant is modified by PEGylation, glycosylation, conjugation to albumin or Fc, hydroxyethylation, or de-O-glycosylation, and the PEGylation refers to the binding of PEG at the N-terminus of the IL-2 variant; or a derivative of the IL-2 variant; or (iii) A conjugate comprising the IL-2 variant defined in (i) and a second component, wherein the IL-2 variant is directly or indirectly conjugated to the second component via a linker; a conjugate A compound selected from the group consisting of, and A pharmaceutically acceptable diluent, carrier or excipient A pharmaceutical composition containing.

6. (i) A human IL-2 variant containing an amino acid substitution as compared to the mature wild-type human IL-2 shown in SEQ ID No. 1, wherein the amino acid residues at positions 35 to 41 of the mature wild-type human IL-2 are mutated to the sequence "MHGLDG" (SEQ ID No. 12), and the amino acid residues at positions 43 to 45 of the mature wild-type human IL-2 are mutated to "G", whereby the amino acid residues at positions 35 to 45 of the mature wild-type human IL-2 are substituted with the sequence "MHGLDGFG", or the amino acid residues at positions 31 to 45 of the mature wild-type human IL-2 are substituted with the sequence "GGNPMHGLDGFG" (SEQ ID No. 11); an IL-2 variant; (ii) A derivative of the IL-2 variant defined in (i), wherein the IL-2 variant is modified by PEGylation, glycosylation, conjugation to albumin or Fc, hydroxyethylation, or de-O-glycosylation, and the PEGylation refers to the binding of PEG at the N-terminus of the IL-2 variant; or a derivative of the IL-2 variant; or (iii) A conjugate comprising the IL-2 variant defined in (i) and a second component, wherein the IL-2 variant is directly or indirectly conjugated to the second component via a linker; a conjugate A nucleic acid molecule encoding.

7. An expression vector containing the nucleic acid molecule according to Claim 6. **Claim 8** (i) A human IL-2 variant comprising an amino acid substitution as compared with mature wild-type human IL-2 shown in SEQ ID No. 1, wherein the amino acid residues at positions 35 to 41 of the mature wild-type human IL-2 are mutated to the sequence "MHGLDG" (SEQ ID No. 12), and the amino acid residues at positions 43 to 45 of the mature wild-type human IL-2 are mutated to "G", whereby the amino acid residues at positions 35 to 45 of the mature wild-type human IL-2 are substituted with the sequence "MHGLDGFG", or the amino acid residues at positions 31 to 45 of the mature wild-type human IL-2 are substituted with the sequence "GGNPMHGLDGFG" (SEQ ID No. 11); (ii) A derivative of the IL-2 variant defined in (i), wherein the IL-2 variant is modified by PEGylation, glycosylation, conjugation to albumin or Fc, hydroxyethylation, or de-O-glycosylation, and the PEGylation refers to the binding of PEG at the N-terminus of the IL-2 variant; a derivative of the IL-2 variant; or (iii) A conjugate comprising the IL-2 variant defined in (i) and a second component, wherein the IL-2 variant is conjugated to the second component directly or indirectly via a linker; a conjugate A compound selected from the group consisting of A host cell comprising or expressing the same. **Claim 9** For use in the prevention or treatment of a disease selected from the group consisting of a proliferative disease, metastasis of a proliferative disease, and an immune disease, (i) A human IL-2 variant comprising an amino acid substitution as compared with mature wild-type human IL-2 shown in SEQ ID No. 1, wherein the amino acid residues at positions 35 to 41 of the mature wild-type human IL-2 are mutated to the sequence "MHGLDG" (SEQ ID No. 12), and the amino acid residues at positions 43 to 45 of the mature wild-type human IL-2 are mutated to "G", whereby the amino acid residues at positions 35 to 45 of the mature wild-type human IL-2 are substituted with the sequence "MHGLDGFG", or the amino acid residues at positions 31 to 45 of the mature wild-type human IL-2 are substituted with the sequence "GGNPMHGLDGFG" (SEQ ID No. 11); (ii) A derivative of the IL-2 variant defined in (i), wherein the IL-2 variant is modified by PEGylation, glycosylation, conjugation to albumin or Fc, hydroxyethylation, or de-O-glycosylation, and the PEGylation refers to the attachment of PEG at the N-terminus of the IL-2 variant; a derivative of the IL-2 variant; or (iii) A conjugate comprising the IL-2 variant defined in (i) and a second component, wherein the IL-2 variant is directly or indirectly conjugated to the second component via a linker; a conjugate A compound selected from the group consisting of.

10. For use in the treatment of neoplasms located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (such as adrenal gland, parathyroid gland, pituitary gland, testis, ovary, thymus and thyroid gland), eyes, head and neck, nervous system (such as central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, chest or urogenital system, (i) A human IL-2 variant containing an amino acid substitution as compared to mature wild-type human IL-2 shown in SEQ ID No. 1, wherein the amino acid residues at positions 35 to 41 of the mature wild-type human IL-2 are mutated to the sequence "MHGLDG" (SEQ ID No. 12), and the amino acid residues at positions 43 to 45 of the mature wild-type human IL-2 are mutated to "G", so that the amino acid residues at positions 35 to 45 of the mature wild-type human IL-2 are substituted with the sequence "MHGLDGFG", or the amino acid residues at positions 31 to 45 of the mature wild-type human IL-2 are substituted with the sequence "GGNPMHGLDGFG" (SEQ ID No. 11); an IL-2 variant; (ii) A derivative of the IL-2 variant defined in (i), wherein the IL-2 variant is modified by PEGylation, glycosylation, conjugation to albumin or Fc, hydroxyethylation, or de-O-glycosylation, and the PEGylation refers to the attachment of PEG at the N-terminus of the IL-2 variant; a derivative of the IL-2 variant; or (iii) A conjugate comprising the IL-2 variant defined in (i) and a second component, wherein the IL-2 variant is directly or indirectly conjugated to the second component via a linker; a conjugate A compound selected from the group consisting of.

11. The pharmaceutical composition according to claim 5 for use in the treatment of a neoplasm located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (such as adrenal gland, parathyroid gland, pituitary gland, testis, ovary, thymus and thyroid gland), eye, head and neck, nervous system (such as central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, chest or urogenital system.

12. For use in the treatment of a pre-cancerous state or cancer metastasis, (i) a human IL-2 variant comprising an amino acid substitution as compared with the mature wild-type human IL-2 shown in SEQ ID No. 1, wherein the amino acid residues at positions 35-41 of the mature wild-type human IL-2 are mutated to the sequence "MHGLDG" (SEQ ID No. 12), and the amino acid residues at positions 43-45 of the mature wild-type human IL-2 are mutated to "G", so that the amino acid residues at positions 35-45 of the mature wild-type human IL-2 are substituted with the sequence "MHGLDGFG", or the amino acid residues at positions 31-45 of the mature wild-type human IL-2 are substituted with the sequence "GGNPMHGLDGFG" (SEQ ID No. 11); (ii) a derivative of the IL-2 variant defined in (i), wherein the IL-2 variant is modified by PEGylation, glycosylation, conjugation to albumin or Fc, hydroxyethylation, or de-O-glycosylation, and the PEGylation refers to the binding of PEG at the N-terminus of the IL-2 variant; a derivative of the IL-2 variant; or (iii) a conjugate comprising the IL-2 variant defined in (i) and a second component, wherein the IL-2 variant is directly or indirectly conjugated to the second component via a linker; a conjugate which is a compound selected from the group consisting of the cancer is selected from the group consisting of renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer and head and neck cancer; a compound.

13. The pharmaceutical composition according to claim 5 for use in the treatment of a pre-cancerous state or cancer metastasis, wherein the cancer is selected from the group consisting of renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer and head and neck cancer; a pharmaceutical composition.

14. For use in the prevention or treatment of autoimmune diseases, transplant rejection, post-traumatic immune responses and infectious diseases, (i)A human IL-2 variant containing amino acid substitutions as compared to mature wild-type human IL-2 shown in SEQ ID No. 1, wherein the amino acid residues at positions 35 to 41 of the mature wild-type human IL-2 are mutated to the sequence "MHGLDG" (SEQ ID No. 12), and the amino acid residues at positions 43 to 45 of the mature wild-type human IL-2 are mutated to "G", whereby the amino acid residues at positions 35 to 45 of the mature wild-type human IL-2 are substituted with the sequence "MHGLDGFG", or the amino acid residues at positions 31 to 45 of the mature wild-type human IL-2 are substituted with the sequence "GGNPMHGLDGFG" (SEQ ID No. 11), an IL-2 variant; (ii)A derivative of the IL-2 variant defined in (i), wherein the IL-2 variant is modified by PEGylation, glycosylation, conjugation to albumin or Fc, hydroxyethylation, or de-O-glycosylation, and the PEGylation refers to the binding of PEG at the N-terminus of the IL-2 variant, a derivative of the IL-2 variant; or (iii)A conjugate comprising the IL-2 variant defined in (i) and a second component, wherein the IL-2 variant is directly or indirectly conjugated to the second component via a linker, a conjugate A compound selected from the group consisting of

15. For use in enhancing cellular immune responses such as improvement of T cell function, improvement of B cell function, recovery of lymphocyte function, increase in the expression of IL-2 receptor, increase in T cell responsiveness, and increase in the activity of natural killer cells or lymphokine-activated killer (LAK) cells, (i)A human IL-2 variant containing amino acid substitutions as compared to mature wild-type human IL-2 shown in SEQ ID No. 1, wherein the amino acid residues at positions 35 to 41 of the mature wild-type human IL-2 are mutated to the sequence "MHGLDG" (SEQ ID No. 12), and the amino acid residues at positions 43 to 45 of the mature wild-type human IL-2 are mutated to "G", whereby the amino acid residues at positions 35 to 45 of the mature wild-type human IL-2 are substituted with the sequence "MHGLDGFG", or the amino acid residues at positions 31 to 45 of the mature wild-type human IL-2 are substituted with the sequence "GGNPMHGLDGFG" (SEQ ID No. 11), an IL-2 variant; (ii) A derivative of the IL-2 variant defined in (i), wherein the IL-2 variant is modified by PEGylation, glycosylation, conjugation to albumin or Fc, hydroxyethylation, or de-O-glycosylation, and the PEGylation refers to the binding of PEG at the N-terminus of the IL-2 variant; or a derivative of the IL-2 variant; or (iii) A conjugate comprising the IL-2 variant defined in (i) and a second component, wherein the IL-2 variant is directly or indirectly conjugated to the second component via a linker; a conjugate A compound selected from the group consisting of.

16. (a) For use in combination with an immunosuppressive agent such as glucocorticoid, azathioprine, cyclosporin A, mycophenolate mofetil, tacrolimus, anti-CD3 antibody, anti-CD25 antibody, anti-TNF-α antibody, methotrexate, cyclosporine, sirolimus, everolimus, fingolimod or cyclophosphamide; or (b) For use in the manufacture of a medicament for regulating a T cell-mediated immune response; or (c) For use in the manufacture of a medicament for stimulating the immune system of an individual; The IL-2 variant according to claim 1.

17. (a) For use in combination with an immunosuppressive agent such as glucocorticoid, azathioprine, cyclosporin A, mycophenolate mofetil, tacrolimus, anti-CD3 antibody, anti-CD25 antibody, anti-TNF-α antibody, methotrexate, cyclosporine, sirolimus, everolimus, fingolimod or cyclophosphamide; or (b) For use in the manufacture of a medicament for regulating a T cell-mediated immune response; or (c) For use in the manufacture of a medicament for stimulating the immune system of an individual; The derivative of the IL-2 variant according to claim 3.

18. The pharmaceutical composition according to claim 5 for use in the prevention or treatment of a proliferative disease, wherein the proliferative disease is a tumor or cancer such as bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, or kidney cancer; a pharmaceutical composition. The pharmaceutical composition according to claim 5 for use in the prevention or treatment of an immune disease, wherein the immune disease is selected from the group consisting of hypergammaglobulinemia, paraproteinemia, purpura, sarcoidosis, Sézary syndrome, Waldenström macroglobulinemia, Gaucher disease, and histiocytosis, the pharmaceutical composition.

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