CN113831402A

CN113831402A - Human interleukin 2 variant and application thereof

Info

Publication number: CN113831402A
Application number: CN202111247549.7A
Authority: CN
Inventors: 陆雍涛; 刘建光
Original assignee: Xi'an Longteng Jingyun Biotechnology Co ltd
Current assignee: Xi'an Longteng Jingyun Biotechnology Co ltd
Priority date: 2021-07-30
Filing date: 2021-10-26
Publication date: 2021-12-24
Also published as: CN122036911A; CN115057919A

Abstract

本公开涉及一种人白细胞介素2变体及其用途。具体而言，本公开涉及一种IL‑2变体(或其衍生物)，其对高亲和力受体IL‑2Rα/β/γ具有消除或降低的亲和力，并保留对中等亲和力受体IL‑2Rβ/γ的亲和力。The present disclosure relates to a variant of human interleukin 2 and uses thereof. In particular, the present disclosure relates to an IL-2 variant (or a derivative thereof) that has eliminated or reduced affinity for the high-affinity receptor IL-2Rα/β/γ and retains the affinity for the medium-affinity receptor IL-2Rα/β/γ 2Rβ/γ affinity.

Description

Human interleukin 2 variant and application thereof

The present application claims priority from a patent application filed 2021, 7/30 (application No. 2021108684095, a human interleukin 2 variant and uses thereof).

Technical Field

The present disclosure relates to human interleukin-2 (IL-2) variants or derivatives thereof having one or more amino acid mutations. The IL-2 variants or derivatives have an eliminated or reduced affinity for the high affinity receptor (IL-2R α/β/γ) and retain affinity for the medium affinity receptor (IL-2R β/γ). The disclosure also relates to immunoconjugates comprising the human IL-2 variants, encoding polynucleotides, vectors, host cells, pharmaceutical compositions, methods of preparation, and methods of treatment and uses.

Background

Human Interleukin-2 (Interleukin-2, IL-2), also known as T Cell Growth Factor (TCGF). The IL-2 gene is located on chromosome 4 (4q27) and includes a total of about 7kb of sequence. IL-2 is composed of about 133 amino acids and has a molecular weight of about 15 kD. Doris Morgan et al 1976 and 1977, respectively, have found that activated T cell culture media can promote T cell proliferation. The stimulatory factor in the culture broth was purified and identified as IL-2.

Initial in vitro cell experiments showed that T cells, after activation by TCR and CD28, can secrete IL-2 and express the IL-2 receptor (IL-2R) on the cell surface. The combination of IL-2 and IL-2R can cause the proliferation of T cells and the production of effects by T cells. IL-2 is a molecule that plays a central role in T cell immune responses. In vivo experiments have shown that following knock-out of IL-2 or its receptor, the animals develop autoimmunity. IL-2 can activate not only effector cells (such as T cells and NK cells) but also regulatory T cells, thereby suppressing excessive immunity against itself.

IL-2 acts through IL-2R. IL-2R includes three subunits: IL-2R α (i.e., CD25), IL-2R β (i.e., CD122), and IL-2R γ (i.e., CD 132). Three subunits can form three receptor forms: the high affinity receptor comprises all three subunits of IL-2R α/β/γ, and the medium affinity receptor comprises IL-2R β/γ and the low affinity receptor IL-2R α. IL-2R beta and IL-2R gamma are necessary for IL-2 to activate downstream signaling pathways, when IL-2 binds IL-2R beta and IL-2R gamma simultaneously, two receptor subunits form heterodimers, phosphorylate intracellular STAT5, enter the nucleus leading to corresponding gene transcription and expression; IL-2R α is not required for signaling, but can promote the binding of IL-2 to IL-2R β and IL-2R γ.

IL-2R gamma is expressed in all immune cells; IL-2R beta is expressed in CD8+ T cells, NK cells and regulatory T cells, and the expression level is also increased after the T cells are activated; IL-2R alpha is continuously highly expressed in regulatory T cells, and is transiently expressed in activated CD8+ T cells, and then expression level is down-regulated.

IL-2 is synthesized predominantly by activated T cells, especially CD4+ helper T cells. It stimulates proliferation and differentiation of T cells, induces production of Cytotoxic T Lymphocytes (CTLs) and differentiation of peripheral blood lymphocytes into cytotoxic and Lymphokine Activated Killer (LAK) cells, promotes expression of cytokines and cytolytic molecules by T cells, promotes proliferation and differentiation of B cells and immunoglobulin synthesis by B cells, and stimulates production, proliferation and activation of Natural Killer (NK) cells. The ability of IL-2 to expand lymphocyte populations and enhance effector functions of these cells in vivo has led to its anti-tumor effects, and IL-2 immunotherapy has become the treatment of choice for certain metastatic cancer patients. Currently, high doses of IL-2 have been approved for the treatment of metastatic renal cell carcinoma and malignant melanoma.

The IL-2 variants disclosed in WO2009135615 have mutations at positions 20, 88 or 126. WO2012062228 discloses IL-2 variants having a mutation at least one position in 38, 42, 45, 62, 68 or 88. The IL-2 variants disclosed in US8906356 have mutations at positions 91, 126. The IL-2 variants disclosed in US9732134 have a mutation at least one of positions 15, 16, 22, 84, 88 or 95. The IL-2 variants disclosed in US7803361 and US8124066 comprise a R38W mutation.

Disclosure of Invention

The present disclosure relates to IL-2 variants (or derivatives thereof) having one or more amino acid mutations, and conjugates thereof; also relates to uses and methods of making the IL-2 variants.

IL-2 variants or derivatives thereof

In a first aspect, the present disclosure provides an IL-2 variant (or derivative thereof) comprising one or more amino acid mutations compared to mature wild-type human IL-2(SEQ ID No. 11).

"Interleukin-2" or "IL-2" refers to any native IL-2 from any mammal, such as primates (e.g., humans), rodents (e.g., mice and rats). The term encompasses unprocessed IL-2 as well as IL-2 derived from any processed form in a cell. The term also encompasses naturally occurring IL-2 variants (e.g., splice variants or allelic variants).

"wild-type human IL-2" in other respects with IL-2 variants, but in the IL-2 variant at each amino acid position with wild-type amino acid IL-2 form. The term "wild-type" is intended to encompass: comprising one or more naturally occurring amino acid mutations that do not affect the ability to bind to the IL-2 receptor.

"mature wild-type human IL-2" refers to the mature form of wild-type human IL-2. Unprocessed human IL-2 additionally contains a signal peptide of about 20 amino acids from the N-terminus, and lacks the signal peptide in the mature IL-2 molecule. Exemplary mature wild-type human IL-2 amino acid sequences such as but not limited to: database accession number INSDC CAA25742.1, Swiss-Prot P60568.1, Genbank NP 000577.2 sections 21-153. In some embodiments, mature wild-type human IL-2 is the amino acid sequence shown in SEQ ID NO. 11. It should be noted that SEQ ID NO.11 additionally comprises a methionine M at the N-terminus for the purpose of recombinant expression compared to mature human IL-2 in the database. However, the skilled artisan will understand that the presence or absence of a first M falls within the scope of "mature wild-type human IL-2" in the present application.

In some embodiments, the amino acid mutations made herein shorten the length of the region of IL-2 that binds to IL-2Ra (at amino acid positions from about 26 to about 47) as compared to mature wild-type human IL-2.

In some embodiments, the amino acid mutation comprises a substitution, deletion, insertion, modification, and any combination thereof of an amino acid. To alter the binding properties of, for example, IL-2, an amino acid may be substituted for another (e.g., with an amino acid having a different structural and/or chemical property). The substitution of amino acids includes the substitution with unnatural amino acids or 20 standard amino acids. Amino acid mutations (including site-directed mutagenesis, PCR, gene synthesis, chemical modification, etc.) can be generated using methods well known in the art.

In some embodiments, IL-2 variants (or derivatives thereof) according to the present disclosure have reduced affinity for IL-2R α, and maintain affinity for IL-2R β and/or IL-2R γ.

"affinity" refers to the overall strength of a non-covalent interaction between a binding site of a molecule and its ligand. Unless otherwise indicated, "affinity" herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., a receptor and a ligand). Affinity can generally be measured by dissociation constant (K)_D) Expressed as dissociation and association rate constants (K, respectively)_DissociationAnd K_{Bonding of}) The ratio of (a) to (b). Affinity can be measured by methods conventional in the art, including the methods described herein.

In some embodiments, the affinity of an IL-2 variant (or derivative thereof) according to the present disclosure for a high affinity receptor (IL-2R α/β/γ) is reduced or absent, but maintains affinity for a medium affinity receptor (IL-2R β/γ).

In some embodiments, a "high affinity IL-2 receptor" refers to a heterotrimeric form of an IL-2 receptor that consists of a receptor gamma subunit (also referred to as universal cytokine receptor gamma subunit, yc, or CD132), a receptor beta subunit (also referred to as CD122 or p70), and a receptor alpha subunit (also referred to as CD25 or p 55).

In some embodiments, a "medium affinity IL-2 receptor" refers to an IL-2 receptor that comprises only gamma and beta subunits, and no alpha subunits (see Olejniczak and Kasprzak, MedSci Monit14, RA179-189, 2008).

In some embodiments, IL-2 variants (or derivatives thereof) according to the present disclosure have reduced activation of regulatory T cells (tregs), and/or have unaffected or increased activation of immune effector cells (e.g., T cells, NK cells).

In some embodiments, the IL-2 variant (or derivative thereof) according to the present disclosure, wherein the mutation occurs at any one or more positions (continuous or discontinuous) selected from positions 31 to 45 (counting from position 2 of SEQ ID NO: 11):

tyrosine (Y) at position 31, lysine (K) at position 32, asparagine (N) at position 33,

Proline (P) at position 34, lysine (K) at position 35, leucine (L) at position 36,

Threonine (T) at position 37, arginine (R) at position 38, methionine (M) at position 39,

Leucine (L) at position 40, threonine (T) at position 41, phenylalanine (F) at position 42,

Lysine (K) at position 43, phenylalanine (F) at position 44, and tyrosine (Y) at position 45.

In some embodiments, an IL-2 variant (or derivative thereof) according to the present disclosure, wherein the mutation occurs at any one or more of positions 31 to 32 (continuous or discontinuous) (counting from position 2 of SEQ ID NO: 11).

In some embodiments, an IL-2 variant (or derivative thereof) according to the present disclosure, wherein the mutation occurs at any one or more of positions 35 to 41 (continuous or discontinuous) (counting from position 2 of SEQ ID NO: 11).

In some embodiments, an IL-2 variant (or derivative thereof) according to the present disclosure, wherein the mutation occurs at any one or more of positions 43 to 45 (continuous or discontinuous) (counting from position 2 of SEQ ID NO: 11).

In some embodiments, when SEQ ID NO:11 is used as the wild-type mature IL-2, the amino acid mutation sites are counted from position 2 of SEQ ID NO:11 (corresponding to, without consideration of the methionine artificially introduced due to recombinant expression).

In other embodiments, the amino acid mutation sites are counted from amino acid A at position 1 when the wild-type mature IL-2 is a native sequence as described in public databases or in the public literature.

In some particular embodiments, there is provided an IL-2 variant comprising any one or a combination of the following mutations:

mutation at 31-45 th position to Gly-Gly-Asn-Pro-Met-His-Gly-Leu-Asp-Gly-Phe-Gly (SEQ ID No. 9);

mutation of 31-32 th position to Gly-Gly;

the 35 th to 41 th position is mutated into Met-His-Gly-Leu-Asp-Gly (SEQ ID No. 10);

mutation at position 43-45 to Gly.

In some particular embodiments, the affinity of the IL-2 variant for IL-2Ra is reduced and the affinity for IL-2 Rss/γ is maintained when the IL-2 variant comprises a mutation or combination selected from the group consisting of:

YK at 31-32 is mutated into GG; the sequence after mutation is SEQ ID No.3 or 7.

KLTRMLT at position 35-41 is mutated to MHGLDG; the sequence after mutation is SEQ ID No.2 or 6.

KFY at position 43-45 is mutated into G; the sequence after mutation is SEQ ID No.4 or 8.

(1) YK at 31-32 is mutated into GG; and is

(2) KLTRMLT at position 35-41 is mutated to MHGLDG;

the mutated sequence is SEQ ID No.12 or 13.

(1) KLTRMLT at position 35-41 is mutated to MHGLDG; and is

(2) KFY at position 43-45 is mutated into G;

the mutated sequence is: SEQ ID No.14 or 15.

(1) YK at 31-32 is mutated into GG; and is

(2) KFY at position 43-45 is mutated into G;

the sequence after mutation is SEQ ID No.16 or 17.

(1) YK at 31-32 is mutated into GG; and is

(2) KLTRMLT at position 35-41 is mutated to MHGLDG; and is

(3) KFY at position 43-45 is mutated into G;

the sequence after mutation is SEQ ID No.1 or 5.

In some specific embodiments, the amino acid sequence of the IL-2 variant (or derivative thereof) is selected from SEQ ID no: 1-8 and 12-17.

TABLE 1 human IL-2 wild-type and variant sequences

The natural expressed human IL-2 has 153 amino acids, and the amino acids at positions 1-20 are signal peptides. A total of 133 amino acids (i.e., mature human IL-2) after cleavage of the signal peptide, corresponding to positions 2-134 of SEQ ID No.1 of the present disclosure. For recombinant expression purposes, the artificially produced IL-2 additionally carries a methionine in the first position (corresponding to the initiation codon AUG). Thus, the mutation site numbering of the present disclosure is actually counted from the second position of SEQ ID No.1, i.e. counting from amino acid a.

As an example, the expression "amino acids at positions 31-32 are mutated to GG" means that:

sequence MAPTSSSTKKTQLQLEHLLLDLQMILNGINN in human IL-2 mature proteinYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (133 amino acids in total in SEQ ID No. 1), YK at the 31 st position to the 32 th position is mutated into GG.

In some embodiments, the IL-2 variant is a monomeric form.

In a second aspect, the present disclosure provides a derivative of an IL-2 variant comprising an IL-2 variant of the present disclosure, said IL-2 variant having a modification selected from any one of the following, or a combination thereof: pegylation, glycosylation, conjugation to albumin, conjugation to Fc, hydroxyethylation, de-O-glycosylation.

In some embodiments, derivatives of the IL-2 variants include full-length, partial, or further functional derivatives, functional fragments, bioactive peptides, fusion proteins, isoforms, or salts thereof, of the IL-2 variants of the disclosure. For example, a fusion protein comprising an IL-2 variant, a dimer or trimer or multimer of said IL-2 variant. Various modifications of the IL-2 variants (e.g., pegylation, glycosylation, conjugation to albumin, conjugation to Fc, hydroxyethylation, removal of O-glycosylation, etc.).

In some embodiments, the IL-2 variant derivatives are PEGylated (can be expressed as PEG-IL-2).

In other embodiments, the PEG-IL-2 variant comprises a methoxy-PEG-aldehyde (mPEG-ALD) linker. In certain embodiments, the PEG has an average molecular weight of from about 5kD to about 50 kD; specifically 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 kD; or about 5kD to about 40 kD; or from about 10kD to about 30 kD; or between about 10kD and about 30 kD; or between about 15kD and about 20 kD.

In certain embodiments, the mPEG-ALD linker comprises a PEG molecule having an average molecular weight selected from the group consisting of: about 5kDa, about 10kDa, about 12kDa, or about 20 kDa. In certain embodiments, the aldehyde group of mPEG-ALD can be acetaldehyde, propionaldehyde, butyraldehyde, or the like.

In one embodiment, the IL-2 variant derivatives, compared to mature wild type human IL-2 has an extended serum half-life.

Conjugates

In a third aspect, the present disclosure provides a conjugate comprising a first component and a second component; wherein the first component is directly conjugated to (or indirectly conjugated through a linker to) the second component; the first component is an IL-2 variant (or derivative thereof) of the present disclosure; the second component is not IL-2, not an IL-2 variant (or derivative thereof).

In some embodiments, the IL-2 variant (or derivative thereof) is linked to at least one second component.

In some embodiments, the IL-2 variant (or derivative thereof) forms a fusion protein with the second component via a peptide bond.

In some particular embodiments, the IL-2 variant (or derivative thereof) is conjugated to (or indirectly conjugated through a linker to) the carboxy terminus of the second component.

In some specific embodiments, the IL-2 variant (or derivative thereof) is conjugated to (or indirectly conjugated through a linker to) the amino terminus of the second component.

In some embodiments, the second component is an antigen binding component. Antigen binding components refer to: a polypeptide molecule that specifically binds to an epitope.

In some embodiments, the antigen binding component is capable of directing a moiety attached thereto (e.g., IL-2, variants or derivatives thereof of the present disclosure) to a target site (e.g., to a tumor cell or tumor stroma having an antigenic determinant). The antigen binding component may be an antibody or antigen binding fragment.

Antibodies are used herein in the broadest sense and encompass a variety of antibody structures as long as they exhibit antigen binding activity, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments. Antibodies may include murine, human, humanized, chimeric, and camelid antibodies.

In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of: polypeptide complexes comprising an antibody heavy chain variable region and an antibody light chain variable region, Fab, Fv, sFv, F (ab') 2, linear antibodies, single chain antibodies, scFv, sdAb, sdFv, nanobodies, peptidic antibodies (peptibodies), domain antibodies, and multispecific antibodies (bispecific antibodies, diabodies, triabodies, and tetrabodies, tandem di-scfvs, tandem tri-scfvs).

In some embodiments, where an IL-2 variant (or derivative thereof) is linked to more than one antigen binding module, each antigen binding module may be independently selected from antibodies and antigen binding fragments; for example, the first antigen-binding moiety may be a Fab molecule and the second antigen-binding moiety may be a scFv molecule. For example, the first antigen-binding component may be an scFv molecule, while the second antigen-binding component is also an scFv molecule. In some embodiments, the first and second antigen binding modules are independently directed to different antigens or to the same antigen. In some embodiments, the antibody or antigen binding fragment thereof targets a tumor antigen.

In some embodiments, tumor antigens are, for example, but not limited to: MAGE family (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2, MAGE-B2, MAGE-Xp3, MAGE-B3, MAGE-Xp4, MAGE-B4, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4), GAGE family (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-C369), GAGE family (e-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-C368, GAGE-C9), GAGE-1, CAP-72, CAP-P, CAP-1, CAP-P4, CAP-P, CAP-3, CAP-A, CAP-3, CAP-A, CAP-3, and CAP-3, CAP-A3, CAP-3, and CAP-A3, and CAP-3, PSA-2 and PSA-3), PSMA, T cell receptor, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, alpha-fetoprotein, E-cadherin, alpha-catenin (catenin), beta-catenin, gamma-catenin, p120ctn, PRAME, NY-ESO-1, cdc27, SSX-1, SSX-2, SSX-1, SSX-4, SSX-5, SCP-1, CT-7.

Pharmaceutical composition

In a fourth aspect, the present disclosure provides a pharmaceutical composition comprising an IL-2 variant (or derivative thereof), or conjugate according to the present disclosure, optionally comprising a pharmaceutically acceptable diluent, carrier or excipient. The pharmaceutical composition may be a lyophilized formulation or an injectable solution.

In some embodiments, the pharmaceutical composition may contain from 0.01% to 99% by weight of the IL-2 variant (or derivative thereof) or conjugate in a unit dose; or an IL-2 variant (or derivative thereof) or conjugate in an amount of 0.1-2000mg (e.g., 1-1000 mg; 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, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000mg) contained in a unit dose of a pharmaceutical composition.

Polynucleotide

In a fifth aspect, the present disclosure provides a polynucleotide encoding an IL-2 variant (or derivative thereof) of the disclosure.

The skilled artisan understands that nucleotide sequences encoding the same amino acid sequence can differ, and that different nucleotide sequences can encode the same amino acid sequence due to codon degeneracy and codon bias of different hosts, and such sequences are within the scope of the present disclosure.

Expression vector

In a sixth aspect, the present disclosure provides an expression vector comprising a polynucleotide according to the present disclosure. The expression vector may be a eukaryotic expression vector, a prokaryotic expression vector, or a shuttle expression vector.

Host cell

In a seventh aspect, the present disclosure provides a host cell comprising an expression vector according to the present disclosure, or expressing an IL-2 variant according to the present disclosure, or expressing a conjugate according to the present disclosure.

In some embodiments, the host cell comprises (e.g., has been transformed or transfected with) an expression vector comprising a polynucleotide according to the present disclosure.

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

In some embodiments, the host cell is a bacterial, yeast or mammalian cell, and may specifically be pichia pastoris or saccharomyces cerevisiae.

In some embodiments, the host cell is a prokaryotic microorganism (e.g., E.coli).

In some embodiments, the host cell is a eukaryotic cell.

In some embodiments, host cells, such as plant and insect cells, that express glycosylated polypeptides are used. Vertebrate cells can also be used as host cells, for example, suspension grown mammalian cell lines, monkey kidney CV1 line (COS-7), human embryonic kidney line (293 or 293T cells), young hamster kidney cell (BHK), mouse Sertoli (sertoli) cell (TM4 cell), monkey kidney cell (CV1), VERO-76, human cervical cancer cell (HELA), canine kidney cell (MDCK), buffalo rat liver cell (BRL3A), human lung cell (W138), human liver cell (Hep G2), mouse mammary tumor cell (MMT060562), MRC5 cell, FS4 cell, CHO cell, myeloma cell lines (e.g., YO, NS0, P3X63, and Sp 2/0).

Use, method of treatment

In an eighth aspect, the present disclosure provides pharmaceutical uses of IL-2 variants (or derivatives thereof), conjugates, pharmaceutical compositions according to the present disclosure.

In some embodiments, there is provided the use of IL-2 variants (or derivatives thereof) for the treatment of proliferative diseases, immunological diseases, modulating T cell mediated immune responses, stimulating the immune system of an individual.

In some embodiments, the proliferative disease can be a tumor or cancer (e.g., metastatic tumor or cancer), and can be a solid tumor.

In some embodiments, the IL-2 variants (or derivatives thereof), conjugates, pharmaceutical compositions of the present disclosure are useful for therapeutically stimulating the immune system (particularly enhancing cellular immune responses) of a host.

In some embodiments, enhancing a cellular immune response may include, but is not limited to: increased T cell function, increased B cell function, restoration of lymphocyte function, increased IL-2 receptor expression, increased T cell responsiveness, increased natural killer cell activity, or increased lymphokine-activated killer (LAK) cell activity.

In some embodiments, the IL-2 variants (or derivatives thereof), conjugates, pharmaceutical compositions of the present disclosure are used to treat proliferative disorders (e.g., cancer). Non-limiting examples of cancer 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, gastric cancer, prostate cancer, hematologic cancer, skin cancer, squamous cell carcinoma, bone cancer, and renal cancer.

In some embodiments, the IL-2 variants (or derivatives thereof), conjugates, pharmaceutical compositions of the present disclosure are used to treat neoplasms located at: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testis, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, chest, and urogenital system.

In some embodiments, the IL-2 variants (or derivatives thereof), conjugates, pharmaceutical compositions of the present disclosure are used to treat a precancerous condition or cancer metastasis. The cancer is selected from the following: renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer.

In some embodiments, the IL-2 variants (or derivatives thereof), conjugates, pharmaceutical compositions of the present disclosure are used to treat: hypergammaglobulinemia (hypergamma), lymphoproliferative disorders, pathoproteinemia (paraproteemias), purpura (purpura), sarcoidosis, Sezary Syndrome (Sezary Syndrome), Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis (histiocytosis).

In some embodiments, the IL-2 variants (or derivatives thereof), conjugates, pharmaceutical compositions of the present disclosure are used to treat autoimmune diseases, transplant rejection, post-traumatic immune responses, and infectious diseases.

In some embodiments, the autoimmune disease may be selected from: type I diabetes mellitus (type I diabetes mellitus), rheumatoid arthritis (rhematoid arthritis), multiple sclerosis (multiple sclerosis), chronic gastritis (chronic gastritis), Crohn's disease, Basedo disease, Bechterew disease, psoriasis (psoriasis), myasthenia gravis (myasthenia gravis), autoimmune hepatitis (autoimmune hepatitis), APECED, allergic granulomatous vasculitis (Chrorg-Strauss), ulcerative colitis (ulcerative colitis), glomerulonephritis (gloulonephritis), Guillain-Barre syndrome (Guillain-Barre syndrome), Hashimoto thyroiditis (Hashimoto thyroiditis), atrophic lupus erythematosus (systemic sclerosis), rheumatoid arthritis (rheumatoid arthritis), rheumatoid arthritis (rheumatoid arthritis), lupus erythematosus (rheumatoid arthritis), rheumatoid arthritis (rheumatoid arthritis), rheumatoid arthritis, rheumatoid, Scleroderma (scleroderma), Wegener's granulomatosis, vitiligo, autoimmune enteropathy, goodpasture's syndrome, dermatomyositis, polymyositis, autoimmune hypersensitivity, asthma (asthma).

In some embodiments, the IL-2 variant (or derivative thereof) can be used in combination with an immunosuppressive agent.

In some embodiments, the immunosuppressive agent is selected from: glucocorticoids (glucocorticoids); azathioprine (azathioprine); cyclosporin a (cyclosporine a); mycophenolate mofetil (mycophenolatemofetil); tacrolimus (tacrolimus); anti-CD 3 antibodies; anti-CD 25 antibodies; anti-TNF- α antibodies; methotrexate (methotrexate); cyclosporine (cyclosporine); sirolimus (sirolimus); everolimus (everolimus); fingolimod (fingolimod); and cyclophosphamide (cyclophosphamide).

In some embodiments, a therapeutically effective amount of: IL-2 variants (or derivatives thereof), conjugates, pharmaceutical compositions of the present disclosure.

In some embodiments, the subject (e.g., patient or individual) in need of treatment is typically a mammal, e.g., a human.

In some embodiments, the IL-2 variant (or derivative thereof), conjugate, pharmaceutical composition of the present disclosure is administered to a subject at least 2 times daily, at least 1 time every 48 hours, at least once every 72 hours, at least once weekly, at least once every 2 weeks, at least once every month, at least once every 2 months, or at least once every 3 months.

In some embodiments, the IL-2 variants (or derivatives thereof), conjugates, pharmaceutical compositions of the present disclosure are administered by any route. For example, administration is by parenteral injection (e.g., subcutaneous, intravenous).

Drawings

FIG. 1. interaction curves of wild-type human IL-2 and IL-2 receptor alpha.

FIG. 2. interaction curves for IL-2 variants and IL-2 receptor alpha.

FIG. 3 is a graph showing the interaction between wild-type human IL-2 and IL-2 R.beta./gamma..

FIG. 4. interaction curves for IL-2 variants and IL-2R β/γ.

Detailed Description

The present disclosure is further described below with reference to examples, but these examples do not limit the scope of the present disclosure.

Example 1 recombinant expression and production of wild-type human IL-2 and IL-2 variants

Nucleic acid sequences for IL-2 variants were synthesized, expressed and purified.

1. Codon Optimization software MaxCodon Optimization Program V13 was used to optimize the sequences encoding wild-type human IL-2 and variants. Synthesizing a whole gene, and subcloning the coding sequence of the IL2 into an expression vector pcDNA3.1 by a double-enzyme cutting method; the accuracy of the final expression vector was confirmed by enzymatic cleavage and sequencing, and finally transferred to the DH5 alpha clone strain. The plasmid of transfection grade is extracted by a plasmid extraction kit, the plasmid is transfected into mammalian cells HEK293 by a transfection reagent for transient expression, and IL2 protein is purified by affinity chromatography.

2. Plasmid transformation of DH5 alpha

80-100ng of the expression plasmid containing the IL2 coding sequence was aspirated and added to the prepared DH 5. alpha. competent cells. Competent cells after plasmid addition were transformed by heat shock.

And adding the transformed competent cells into an LB liquid culture medium, placing the transformed competent cells into a shaker at 37 ℃, and performing shake culture at 200rpm for about 30 min. The cultured competent cells were taken out, and a part of the suspension was pipetted and applied to a plate containing ampicillin, and placed in an incubator at 37 ℃ for overnight culture.

Single clones were picked from fresh plates and cultured in 2-5mL LB medium at 37 ℃ for 8h at 200 rpm. The strain was inoculated into 200mL of LB medium at a ratio of 1/500 and cultured at 37 ℃ and 200rpm for 16 hours. The cultured bacterial liquid is collected and centrifuged, and the supernatant is removed.

Reviving and passaging HEK293 cells

The water bath was adjusted to 37 ℃; the medium was preheated at 37 ℃.

The HEK293 cells were removed from the liquid nitrogen tank and immediately placed in a 37 ℃ water bath and thawed rapidly with gentle shaking (about 1 min).

Disinfecting the outer wall of the tube by using 75% ethanol, and placing the tube into a biological safety cabinet; the cells were transferred to a 15mL centrifuge tube containing 10mL of medium and centrifuged at 800rpm for 5 min. Removing supernatant from the centrifuged cells, taking a little fresh culture medium to resuspend the cells, and transferring the cells to a culture bottle; adding fresh culture medium, shaking gently to disperse cells uniformly, counting cells, and detecting activity. Make the density controlAt 3X 10⁵To 4X 10⁵cell/mL, viability>95％。

The cells were incubated in an incubator at 110rpm and 37 ℃ with 5% CO 2. The cells are cultured for 2-3 days, and the density reaches 2.0 multiplied by 10⁶at/mL, cells were passaged by supplementing with fresh medium. Cell density and viability assays were monitored.

4. Plasmid transfected HEK293 cell

1 day before transfection, HEK293 cells were suspension cultured to 1L at a seeding density of 1X 10⁶/mL, cultured in an incubator at 110rpm and 37 ℃ with 5% CO 2. On the day of transfection, cell density was controlled at 1X 10⁶To 1.5X 10/mL⁶/mL。

DNA-transfection reagent mixture: DNA and transfection reagents were added to the transfection buffer and mixed well and incubated at 37 ℃. The DNA-transfection reagent mixture was added to the cells to be transfected and placed in an incubator at 110rpm for incubation at 37 ℃ with 5% CO 2. About 4-6 days after transfection, the cell culture was removed, centrifuged, and the supernatant and cells were collected.

5. Protein purification

The cell culture solution after 5 days of culture was centrifuged, and the supernatant was filtered through a 0.22 μm filter. The buffer 1 XPBS pH7.4 was dialyzed at 4 ℃. After dialysis, the target protein was purified by Ni-IDA column.

Example 2 determination of the affinity of wild-type human IL-2 and variant IL-2 for IL-2R α

The binding properties of wild-type human IL-2 and its variant IL-2 obtained in example 1 to IL-2 R.alpha.were examined by Octet. The Octet platform is based on the detection and analysis of biomolecular interactions by the biofilm interference technique (Bio-Layer interference, BLI).

1. Experimental instrument and reagent consumable

Octet RED96e system(Pall Fortebio Corp,Menlo Park,CA)；

Assay Buffer SD Buffer (PBS (pH 7.4) + 0.01% BSA + 0.02% Tween 20);

acetic acid buffer (10mM, pH4.0, 5.0, 6.0);

AR2G (Fortebio, cat # 18-5092);

96-well plates (Greiner Bio-One part No. 655209);

2. the experiment was carried out with reference to the following experimental design, and the whole experiment was carried out at 1000rpm, 30 ℃ and 220. mu.l/well.

The sample to be tested is as follows: IL-2RA (i.e., IL-2 receptor alpha), and the dilution Buffer is SD Buffer;

the concentration range of the substance to be detected is as follows: 1000nM, 500nM, 250nM, 125nM, 62.5 nM.

3. Experimental data and processing

The experimental Data was processed through Data Analysis software 9.0. The fitting model selected a 1:1 model, i.e., 1 wild-type human IL-2 or IL-2 variant (specifically IL-2-1 variant) bound to 1 IL-2 RA. The fitting mode is global; i.e. 5 concentrations were analysed as a group. The fitting results are shown in fig. 1.

The data obtained from the experiments were fitted to a 1:1binding model to give the value of the affinity of the wild-type human IL-2 or IL2 variant for the low affinity receptor IL-2R α in example 1.

According to the interaction graph 1, the experimental data of the interaction kinetics between wild type human IL-2 and IL-2RA shows that the binding signal (Response) value in the experimental data has obvious positive statistical correlation with the increase of the concentration of the analyte. Therefore, the interaction between wild-type human IL-2 and IL-2RA was determined.

According to the interaction graph 2, the experimental data of the interaction kinetics between the IL-2-1 variant and the IL-2RA show that the value of the binding signal in the experimental data has no obvious positive statistical correlation with the increase of the concentration of the analyte. Thus, it is determined that the interaction between the IL-2-1 variant and IL-2RA is reduced or absent.

IL-2-1(-M), IL-2-2, IL-2-3, IL-2-4, IL-2-5, IL-2-6, IL-2-7, IL-2-2(-M), IL-2-3(-M), IL-2-4(-M), IL-2-5(-M), IL-2-6(-M), IL-2-7(-M) and IL-2RA did not interact (curves not shown).

Example 3 determination of the affinity of wild-type human IL-2 and IL-2 variants for IL-2R β/γ

The affinity of wild-type human IL-2 and its variants for IL-2R β/γ in example 1 was examined by Octet experiments.

1. Experimental instrument and reagent consumable

Octet RED96e system(Pall Fortebio Corp,Menlo Park,CA)；

Assay Buffer SD Buffer (PBS (pH 7.4) + 0.01% BSA + 0.02% Tween 20);

acetic acid buffer (10mM, pH4.0, 5.0, 6.0);

AR2G (Fortebio, cat # 18-5092);

96-well plates (Greiner Bio-One part No. 655209).

IL-2R beta and IL-2R gamma subunits are cloned and fused to Fc hole and Fc knob, respectively, for the preparation of IL-2R beta/gamma-Fc heterodimers. IL-2R β -Fc-hole and IL-2R γ -Fc-knob were transfected into HEK293 cells simultaneously. The heterodimer was purified using Protein a and molecular sieve Superdex 200.

2. The whole experiment was carried out at 1000rpm, 30 ℃ and 220. mu.l/well.

The sample to be tested is IL-2R beta/gamma, and the dilution Buffer is SD Buffer. The concentrations of the test substances ranged from 1000nM, 500nM, 250nM, 125nM, 62.5 nM.

3. Experimental data and processing

The experimental Data was processed through Data Analysis software 9.0. Fitting models 1:1 model was chosen, i.e. 1 wild-type human IL-2 or IL-2 variant (IL-2-1) bound to 1 IL-2 R.beta./gamma. The global approach, i.e. 5 concentrations were analyzed as a group. The fitting results are shown in FIG. 3.

The data obtained from the experiments were fitted to a 1:1binding model to derive affinity values for the wild-type human IL-2 or IL2 variant of example 1 for the intermediate affinity receptor IL-2R β/γ.

According to the interaction curve chart 3, the experimental data of the interaction kinetics between the wild type human IL-2 and the IL-2R beta/gamma shows that the numerical value of the combined signal in the experimental data has obvious positive statistical correlation along with the increase of the concentration of the substance to be detected. Therefore, the interaction between wild-type human IL-2 and IL-2R beta/gamma was determined.

According to the interaction graph 4, the experimental data of the interaction kinetics between the IL-2 variant and the IL-2R beta/gamma shows that the numerical value of the binding signal in the experimental data has obvious positive statistical correlation with the increase of the concentration of the analyte. Thus, it was determined that the interaction between the IL-2 variant and IL-2R β/γ was maintained.

IL-2-1(-M), IL-2-2, IL-2-3, IL-2-4, IL-2-5, IL-2-6, IL-2-7, IL-2-2(-M), IL-2-3(-M), IL-2-4(-M), IL-2-5(-M), IL-2-6(-M), IL-2-7(-M) and IL-2 Rbeta/gamma maintained interactions (curves not shown).

Sequence listing

<110> Xian Longteng Jingyun Biotech Co., Ltd

<120> human interleukin 2 variant and use thereof

<130> 310279CG-310195

<150> 2021108684095

<151> 2021-07-30

<160> 17

<170> SIPOSequenceListing 1.0

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Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu

1 5 10 15

His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Gly

20 25 30

Gly Asn Pro Met His Gly Leu Asp Gly Phe Gly Met Pro Lys Lys Ala

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Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu

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Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro

65 70 75 80

Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly

85 90 95

Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile

100 105 110

Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser

115 120 125

Thr Leu Thr

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Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu

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His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr

20 25 30

Lys Asn Pro Met His Gly Leu Asp Gly Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

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Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

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Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

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Ile Ser Thr Leu Thr

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Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu

1 5 10 15

His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Gly

20 25 30

Gly Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro

35 40 45

Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu

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Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His

65 70 75 80

Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu

85 90 95

Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr

100 105 110

Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser

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Ile Ile Ser Thr Leu Thr

130

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Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu

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His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr

20 25 30

Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Gly Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

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Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

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Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

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Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Gly Gly

20 25 30

Asn Pro Met His Gly Leu Asp Gly Phe Gly Met Pro Lys Lys Ala Thr

35 40 45

Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu

50 55 60

Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg

65 70 75 80

Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser

85 90 95

Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val

100 105 110

Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr

115 120 125

Leu Thr

130

<210> 6

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Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Met His Gly Leu Asp Gly Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

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Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Gly Gly

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 8

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Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Gly Met Pro Lys Lys Ala

35 40 45

Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu

50 55 60

Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro

65 70 75 80

Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly

85 90 95

Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile

100 105 110

Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser

115 120 125

Thr Leu Thr

130

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Gly Gly Asn Pro Met His Gly Leu Asp Gly Phe Gly

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Met His Gly Leu Asp Gly

1 5

<210> 11

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<213> Homo sapiens

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Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu

1 5 10 15

His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr

20 25 30

Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro

35 40 45

Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu

50 55 60

Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His

65 70 75 80

Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu

85 90 95

Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr

100 105 110

Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser

115 120 125

Ile Ile Ser Thr Leu Thr

130

<210> 12

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<222>

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Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu

1 5 10 15

His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Gly

20 25 30

Gly Asn Pro Met His Gly Leu Asp Gly Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 13

<211> 132

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<221> VARIANT

<222>

<223> IL-2 variants

<400> 13

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Gly Gly

20 25 30

Asn Pro Met His Gly Leu Asp Gly Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 14

<211> 131

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<221> VARIANT

<222>

<223> IL-2 variants

<400> 14

Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu

1 5 10 15

His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr

20 25 30

Lys Asn Pro Met His Gly Leu Asp Gly Phe Gly Met Pro Lys Lys Ala

35 40 45

Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu

50 55 60

Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro

65 70 75 80

Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly

85 90 95

Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile

100 105 110

Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser

115 120 125

Thr Leu Thr

130

<210> 15

<211> 130

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<221> VARIANT

<222>

<223> IL-2 variants

<400> 15

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Met His Gly Leu Asp Gly Phe Gly Met Pro Lys Lys Ala Thr

35 40 45

Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu

50 55 60

Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg

65 70 75 80

Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser

85 90 95

Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val

100 105 110

Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr

115 120 125

Leu Thr

130

<210> 16

<211> 132

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<221> VARIANT

<222>

<223> IL-2 variants

<400> 16

Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu

1 5 10 15

His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Gly

20 25 30

Gly Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Gly Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 17

<211> 131

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<221> VARIANT

<222>

<223> IL-2 variants

<400> 17

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Gly Gly

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Gly Met Pro Lys Lys Ala

35 40 45

Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu

50 55 60

Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro

65 70 75 80

Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly

85 90 95

Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile

100 105 110

Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser

115 120 125

Thr Leu Thr

130

Claims

1. A human interleukin 2(IL-2) variant, said IL-2 variant comprising an amino acid mutation at one or more of positions 31 to 45 relative to mature wild-type human IL-2;

preferably, wherein the mutation is any one or a combination selected from the group consisting of:

the 31 st to 45 th amino acid residues of the mature wild type human IL-2 are mutated into a sequence shown in SEQ ID No. 9;

the 35 th to 41 th amino acid residues of the mature wild type human IL-2 are mutated into a sequence shown in SEQ ID No. 10;

the 31 st to 32 nd amino acid residues of mature wild type human IL-2 are mutated into GG;

the 43 th to 45 th amino acid residues of the mature wild type human IL-2 are mutated into G.

2. The IL-2 variant of claim 1, wherein the IL-2 variant is selected from the group consisting of seq id no: SEQ ID Nos. 1 to 8, 12 to 17.

3. The IL-2 variant according to any one of claims 1 to 2, wherein:

said IL-2 variant having reduced affinity for IL-2R α; and/or

The IL-2 variant maintains affinity for IL-2R β; and/or

The IL-2 variant maintains affinity for IL-2R γ;

preferably, the IL-2 variant has a reduced affinity for IL-2R α/β/γ and maintains the affinity of the IL-2 variant for IL-2R β/γ.

4. A derivative of an IL-2 variant comprising an IL-2 variant of any one of claims 1 to 3, said IL-2 variant having a modification selected from any one of the following or a combination thereof:

pegylation, glycosylation, conjugation to albumin, conjugation to Fc, hydroxyethylation, de-O-glycosylation;

the pegylation refers to attachment of PEG to the N-terminus of the IL-2 variant;

preferably, the PEG has a molecular weight of from 5kD to 80 kD;

more preferably, the PEG has a molecular weight of from 10kD to 20 kD.

5. A conjugate, comprising:

a first component, and

a second component;

wherein: the first component is directly conjugated to the second component, or indirectly conjugated to the second component through a linker;

the first component is the IL-2 variant of any one of claims 1 to 3 or a derivative of the IL-2 variant of claim 4;

preferably, the second component is an antibody or antigen-binding fragment thereof;

more preferably, the antibody or antigen binding fragment thereof targets a tumor antigen.

6. A pharmaceutical composition comprising:

-the IL-2 variant of any one of claims 1 to 3, or a derivative of the IL-2 variant of claim 4, or the conjugate of claim 5;

-a pharmaceutically acceptable diluent, carrier or excipient.

7. A nucleic acid molecule encoding the IL-2 variant of any one of claims 1 to 3, or the conjugate of claim 5.

8. An expression vector comprising the nucleic acid molecule of claim 7.

9. A host cell comprising or expressing any one selected from the group consisting of:

comprising the expression vector of claim 8,

Expressing the IL-2 variant of any one of claims 1 to 3,

Expressing the conjugate of claim 5;

preferably, the host cell is a prokaryotic or eukaryotic cell;

more preferably, the host cell is selected from any one of: bacterial cells, yeast cells, mammalian cells;

most preferably, the host cell is a Saccharomyces cerevisiae cell or an Escherichia coli cell.

10. Use of any one of the following in the manufacture of a medicament:

the IL-2 variant of any one of claims 1 to 3, a derivative of the IL-2 variant of claim 4, a conjugate of claim 5;

the medicament is for use in the prevention or treatment of any one selected from the group consisting of: proliferative diseases, metastasis of proliferative diseases, immunological diseases;

preferably, the proliferative disease is a tumor or cancer;

preferably, the immune disease is selected from: diabetes, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, eczema, asthma, autoimmune diseases, autoimmune reactions after organ transplantation;

more preferably, the tumor or cancer is selected from: epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, papilloma virus-induced carcinoma, adenocarcinoma, melanoma, sarcoma, teratocarcinoma, lung cancer, metastatic lung cancer, lymphoma, metastatic renal cell carcinoma.