IL-15 muter-Fc / IL-15R alpha subunit-Fc heterodimer and application thereof

Abstract

The invention discloses an IL-15 mutant-Fc / IL-15R alpha subunit-Fc heterodimer and an application of the IL-15 mutant-Fc / IL-15R alpha subunit-Fc heterodimer. The heterodimer protein comprises a protein a and a protein b, wherein the protein a comprises an IL-15 mutant and a first Fc mutant; the protein b comprises an IL-15R alpha subunit sushi structural domain, other partial fragments of the IL-15R alpha subunit and a second Fc mutant; the first Fc mutant and the second Fc mutant are selected from Knob modified Fc or Hole modified Fc, and the modification types of the first Fc mutant and the second Fc mutant are different. Experiments prove that the heterodimer protein provided by the invention can effectively stimulate proliferation of NK cells and T cells, in-vivo experiments show that the growth inhibition of the dimer protein HL-015-9 on tumors is superior to that of ALT-803 under the same injection dosage, and the heterodimer protein HL-015-9 can be used for preparing drugs or preparations for treating tumors and / or virus infection and other diseases.

Description

IL-15 mutant-Fc / IL-15Rα subunit-Fc heterodimer and its use Technical Field

[0001] The present invention belongs to the technical field of medical preparations, and in particular relates to an IL-15 mutant-Fc / IL-15Rα subunit-Fc heterodimer and uses thereof. Background Art

[0002] Human interleukin 15 (IL-15) is a pleiotropic cytokine that activates T cells, B cells, and NK cells and mediates their proliferation and survival. Furthermore, IL-15 can activate, maintain, and expand CD8+ memory T cells without activating regulatory T lymphocytes (Tregs, which have immunosuppressive functions).

[0003] IL-2 was the first cytokine to be identified. It was originally discovered in the supernatant of activated human T cell cultures and is a soluble factor that mediates T cell proliferation. IL-2 is also the first cytokine approved by the FDA for cancer treatment. Although primarily secreted by CD4+ and CD8+ T cells upon antigen stimulation, activated dendritic cells (DCs), mast cells, and NKT cells also produce small amounts of IL-2.

[0004] IL-15 shares some similar functions with IL-2, including stimulating the proliferation of activated T cells, generating cytotoxic effector T cells, and activating and maintaining NK cells. They also promote B cell-induced immunoglobulin synthesis and regulate lymphoid homeostasis. However, unlike IL-2, IL-15 mRNA is expressed in various tissues, including hematopoietic and non-hematopoietic cells such as keratinocytes, neurons, stromal cells, and fibroblasts. However, unlike the widespread expression of IL-15 mRNA, production of mature IL-15 protein is primarily restricted to DCs and monocytes / macrophages.

[0005] IL-15 has a chemotactic effect on T cells: it induces circulating lymphocytes to home to peripheral lymph nodes, inhibits lymphocyte apoptosis, and promotes T cell activation and proliferation, inducing the production of cytotoxic T lymphocytes (CTLs). For CD8+ T cells, IL-15 not only promotes the generation of memory CD8+ T cells but also plays a crucial role in maintaining the number of memory CD8+ T cells in the body. IL-15 also plays a crucial role in the activation and proliferation of NK cells. In mice overexpressing IL-15, the number of NK cells increases significantly, enhancing immune responses. IL-15 also plays a crucial role in the functional maturation of dendritic cells (DCs) and macrophages. In DCs, IL-15 promotes the expression of costimulatory factors and IFN-γ, enhancing their ability to activate CD8+ T cells and NK cells.

[0006] IL-2 and IL-15 are type I four-alpha-helical bundle cytokines that belong to the gamma receptor cytokine family. This group of cytokines shares the same receptor subunit, γc, and exhibits pleiotropic effects in regulating both innate and adaptive immune responses. The receptors for both IL-2 and IL-15 are heterotrimeric, with the exception of the cytokine receptor subunit γc (also known as IL-2Rγ or CD132); they also share a β subunit, here referred to as IL-2 / 15Rβ (also known as CD122). The third and unique receptor subunit for IL-2 and IL-15 is IL-2Rα and IL-15Rα, respectively.

[0007] IL-2 α, β and γ receptors are usually expressed on the surface of a cell. In addition to cell surface expression, IL-2Rα also exists in a soluble form (sIL-2Rα) in certain diseases, including inflammatory diseases, transplant rejection and most malignant diseases.

[0008] IL-15Rα, a unique component of the IL-15 receptor complex, is primarily expressed on monocytes and dendritic cells. Unlike other γc family cytokines (such as IL-2), IL-15, as a cytokine, first binds to IL-15Rα-expressing cells. The IL-15 / IL-15Rα complex is then presented to IL-2 / 15Rβ and γc on activated T cells or NK cells, forming a high-affinity immune synapse. Due to shared receptor subunits (IL-2 / 15Rβγ), IL-2 and IL-15 trigger several similar downstream signaling pathways, including Janus kinases (JAKs) and signal transducers and activators of transcription (STATs). JAK1 interacts with IL-2 / 15Rβ, and JAK3 interacts with γc.

[0009] Despite these similarities, IL-2 and IL-15 also display different functions in vivo, particularly in the adaptive immune response. For example, the development and maintenance of regulatory T cells (Tregs) requires IL-2, which is closely associated with activation-induced cell death (AICD). IL-2 therapy for tumors may cause capillary leak syndrome as a side effect. In contrast, IL-15 does not mediate AICD, but rather inhibits IL-2-induced AICD and does not cause capillary leak syndrome as a side effect. IL-15 is a major force in supporting the persistence of natural killer (NK) cells and memory CD8+ T cells.

[0010] IL-15's therapeutic potential is being continuously explored based on its ability to expand and activate effector lymphocytes in vivo. Furthermore, the role of IL-15 in combination strategies for synergistic immune enhancement is gaining increasing attention. N-803 (Anktiva, ALT-803) is composed of an IL-15 mutant conjugated to the sushi region of IL-15 / IL-15Rα / IgG1Fc fusion protein. Compared to wild-type IL-15, N-803 exhibits favorable pharmacokinetic properties, a longer survival in the body, and enhanced anti-tumor activity.

[0011] At ASCO 2022, ImmunityBio released clinical data showing that the 2-year overall survival rate of N803 combined with Bacillus Calmette-Guérin (BCG) was 99% in 160 patients with BCG-unresponsive non-muscle invasive bladder cancer (NMIBC). In the cohort of patients with carcinoma in situ, there was a complete remission rate of 71% and a median sustained response time of 24.1 months. In the papillary patient cohort, the disease-free survival rate was 53% at 18 months. During the 2-year follow-up, more than 90% of patients avoided cystectomy. For patients with NMIBC who were unresponsive to BCG, the efficacy and safety of N-803+BCG exceeded other existing treatment options. Therefore, the development of better IL-15 drugs has positive significance.

[0012] Invention Disclosure

[0013] The technical problem to be solved by the present invention is how to provide a better IL-15 drug.

[0014] To solve the above technical problems, in a first aspect, the present invention provides a heterodimeric protein comprising protein a and protein b, wherein protein a comprises an IL-15 mutant and a first Fc mutant; protein b comprises an IL-15Rα subunit sushi domain and other partial fragments of the IL-15Rα subunit and a second Fc mutant; the first Fc mutant and the second Fc mutant are selected from Knob-modified Fc or Hole-modified Fc, and the first Fc mutant and the second Fc mutant have different types of Knob modification or Hole modification;

[0015] The IL-15 mutant is a polypeptide having an amino acid sequence selected from any one of SEQ ID No. 18, positions 1-111, SEQ ID No. 15, positions 1-111, SEQ ID No. 14, positions 1-114, SEQ ID No. 17, positions 1-114, SEQ ID No. 16, and positions 1-114 of SEQ ID No. 13;

[0016] The IL-15Rα subunit sushi domain and other partial fragments of the IL-15Rα subunit are polypeptides whose amino acid sequence is SEQ ID No. 2.

[0017] The "sushi region" in the "IL-15Rα subunit sushi domain and other partial fragments of IL-15Rα subunit" mentioned in the present invention mainly refers to the peptide segment whose amino acid sequence is positions 1-65 of SEQ ID No.2.

[0018] The "other partial fragments" in the "IL-15Rα subunit sushi domain and other partial fragments of IL-15Rα subunit" mentioned in the present invention mainly refer to the peptide segment whose amino acid sequence is SEQ ID No. 2, positions 66-78 (DPALVHQRPAPPS).

[0019] Furthermore, the protein a further comprises a linker, which connects the IL-15 mutant and the first Fc mutant, and the linker may be (GGGGS)n, where n is a natural number from 0 to 4. In one embodiment of the present invention, n is 3.

[0020] Furthermore, the protein a includes: the IL-15 mutant, a linker connected to the C-terminus of the IL-15 mutant, and a first Fc mutant connected to the C-terminus of the linker; the protein b includes: the IL-15Rα subunit sushi domain and other partial fragments, and a second Fc mutant connected to the C-terminus of the IL-15Rα subunit sushi domain and other partial fragments.

[0021] In the above heterodimeric protein, the first Fc mutant or the second Fc mutant can be the Fc of antibody IgG1, IgG2, IgG3 or IgG4 or a mutant thereof;

[0022] Furthermore, the first Fc mutant or the second Fc mutant is selected from IgG4 Fc or a mutant thereof.

[0023] Furthermore, if the IgG4 format is used, the serine at position 228 in the hinge region can be mutated to proline to obtain a stable IgG4 Fc mutant.

[0024] In the heterodimeric protein, protein a and protein b are formed by combining via an Fc knob-in-hole. The two peptide chains may have an Fc domain containing a T366W mutation, while the corresponding domain of the other chain contains T366S, L368A, and Y407V mutations; or an Fc domain containing T350V, L351Y, F405A, and Y407V mutations, while the corresponding domain of the other chain contains T350V, T360L, K392L, and T394V mutations; or other combinations of knob-in-holes may be used.

[0025] In one embodiment of the present invention, the amino acid sequence of the knob-modified Fc (second Fc mutant) is SEQ ID No. 9, and its nucleotide sequence is SEQ ID No. 37; the amino acid sequence of the hole-modified Fc (first Fc mutant) is SEQ ID No. 10, and its nucleotide sequence is SEQ ID No. 38.

[0026] In the above heterodimeric protein, the protein a can be any one of A1) to A7):

[0027] A1) a protein having an amino acid sequence of SEQ ID No. 18;

[0028] A2) a protein having an amino acid sequence of SEQ ID No. 15;

[0029] A3) a protein having an amino acid sequence of SEQ ID No. 14;

[0030] A4) a protein having an amino acid sequence of SEQ ID No. 17;

[0031] A5) a protein having an amino acid sequence of SEQ ID No. 16;

[0032] A6) a protein having an amino acid sequence of SEQ ID No. 13;

[0033] A7) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of the protein described in any one of A1) to A6).

[0034] The protein b can be any one of A8)-A10):

[0035] A8) a protein having an amino acid sequence of SEQ ID No. 19;

[0036] A9) a protein having an amino acid sequence of SEQ ID No. 20;

[0037] A10) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of the protein described in A8) or A9).

[0038] Preferably, protein a in the heterodimeric protein is a protein with an amino acid sequence of SEQ ID No.18, and protein b is a protein with an amino acid sequence of SEQ ID No.19.

[0039] To solve the above technical problems, in a second aspect, the present invention provides a biomaterial related to the above heterodimeric protein, wherein the biomaterial is any one of the following:

[0040] B1) a nucleic acid molecule encoding the above-mentioned heterodimeric protein;

[0041] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0042] B3) a recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2);

[0043] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3);

[0044] B5) an animal cell line containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3);

[0045] B6) a plant cell line containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3);

[0046] B7) A host cell that produces the above-mentioned heterodimeric protein.

[0047] To solve the above technical problems, in a third aspect, the present invention provides a product, wherein the active ingredient of the product is the above heterodimeric protein; the use of the product is any one of the following C1) to C6):

[0048] C1) prevention and / or treatment of diseases;

[0049] C2) Activate the body’s immune system;

[0050] C3) Improve or enhance the body's immune function;

[0051] C4) inhibit tumor growth;

[0052] C5) increasing the proliferation activity of NK cells and / or T cells;

[0053] C6) Enhance the killing ability of NK cells against tumor cells (such as K562 cells).

[0054] To solve the above technical problems, in a fourth aspect, the present invention provides a pharmaceutical composition, which consists of the above heterodimeric protein and other drugs.

[0055] Furthermore, the other drugs may be at least one of the following drugs: immune checkpoint drugs, cell engager bispecific antibodies, and cell therapy products.

[0056] To solve the above technical problems, in a fifth aspect, the present invention provides the use of the above heterodimeric protein, the above biomaterial, the above product, or the above pharmaceutical composition in any one of the following D1) to D12):

[0057] D1) Preparation of products for the prevention and / or treatment of diseases;

[0058] D2) Preparation of products for activating the body's immune system;

[0059] D3) Preparation of products that improve or enhance the body's immune function;

[0060] D4) preparing products that inhibit tumor growth;

[0061] D5) preparing a product that increases the proliferation activity of NK cells and / or T cells;

[0062] D6) preparing a product that enhances the killing ability of NK cells against tumor cells (such as K562 cells);

[0063] D7) prevention and / or treatment of diseases;

[0064] D8) Activate the animal's immune system;

[0065] D9) Improve or enhance the body's immune function;

[0066] D10) inhibit tumor growth;

[0067] D11) increasing the proliferation activity of NK cells and / or T cells;

[0068] D12) Enhance the killing ability of NK cells against tumor cells (such as K562 cells).

[0069] In any of the above products or applications, the T cells include CD8+ T cells and / or CD4+ T cells.

[0070] In any of the above products or applications, the organism may be a mammal, including humans and mice.

[0071] To solve the above technical problems, in a sixth aspect, the present invention provides a method for treating a disease, which comprises the following steps: administering the above heterodimeric protein or the above biological material or the above product or the above pharmaceutical composition to a patient so that the patient is treated.

[0072] In any of the above products, applications or methods, the diseases include infectious diseases, tumors, blood diseases, inflammatory diseases and autoimmune diseases.

[0073] The infectious diseases include but are not limited to viral infections (such as smallpox virus infection, HIV infection, HBV infection, etc.), bacterial infections, and fungal infections.

[0074] The tumors include, but are not limited to, melanoma, colorectal cancer, skin cancer, lymphoma, renal cell carcinoma, liver cancer, lung cancer, gastric cancer, and breast cancer.

[0075] The blood diseases include, but are not limited to, anemia, acute myeloid leukemia, myelodysplastic syndrome, and T-cell large granular lymphocytic leukemia.

[0076] The autoimmune diseases include, but are not limited to, multiple sclerosis, psoriasis, rheumatoid arthritis, gastritis, and mucositis.

[0077] In any of the above products, applications or methods, the product may be a drug or a preparation. In actual applications, a carrier material may be added when preparing the drug or preparation.

[0078] The carrier materials include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.), insoluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.).

[0079] These materials can be used to make a variety of dosage forms, including but not limited to tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. It can be a conventional preparation, a sustained-release preparation, a controlled-release preparation and various particulate delivery systems. In order to make unit dosage forms into tablets, various carriers well known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and adhesives, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants , such as dried starch, alginate, agar powder, brown seaweed starch, sodium bicarbonate with citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption enhancers, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further prepared as coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer tablets and multilayer tablets. To prepare the unit dosage form into a pill, a wide variety of carriers known in the art can be used. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginates, sodium lauryl sulfate, methylcellulose, and ethylcellulose. To prepare unit dosage forms as suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, higher alcohol esters, gelatin, and semi-synthetic glycerides. To prepare unit dosage forms as injectable preparations, such as solutions, emulsions, lyophilized powder injections, and suspensions, all diluents commonly used in the art can be used, including water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol may be added to the injection preparation. In addition, conventional cosolvents, buffers, pH adjusters, etc. may also be added. In addition, if necessary, colorants, preservatives, fragrances, flavorings, sweeteners or other materials may also be added to the pharmaceutical preparation.

[0080] The above dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection.

[0081] The beneficial technical effects achieved by the present invention are as follows:

[0082] 1. Through computer-aided design and biological activity screening, the present invention has invented an innovative IL-15 mutant-Fc / IL-15Rα-Fc heterodimeric protein, which exhibits superior biological activity compared to existing similar products, such as the dimeric protein HL-015-9 provided by the present invention. This dimeric protein can effectively stimulate the proliferation of NK cells and T cells and can be used alone or in combination with other drugs (such as immune checkpoint drugs, cell engager bispecific antibodies, or cell therapy products) to achieve better results.

[0083] 2. The heterodimeric protein provided by the present invention can enhance the proliferation activity of CTLL-2 and Mo7e cells.

[0084] 3. The heterodimeric protein provided by the present invention can increase the proliferation activity of NK cells, CD8+T cells, and CD4+T cells.

[0085] 4. The heterodimeric protein provided by the present invention can promote NK cells to kill K562.

[0086] 5. The dimeric protein HL-015-9 provided by the present invention has a better inhibitory effect on tumor growth than ALT-803 at the same injection dose. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 shows the effects of different molecules designed in the first round on the proliferation of Mo7e cells.

[0088] Figure 2 shows the effects of different molecules designed in the first round on the proliferation of CTLL-2 cells.

[0089] FIG3 shows the effect of the second-round designed IL-15 mutant-Fc / IL-15Ralpha fragment-Fc on the proliferation of Mo7e cells.

[0090] Figure 4 shows the effect of the second-round designed IL-15 mutant-Fc / IL-15Ralpha fragment-Fc on the proliferation of CTLL-2 cells.

[0091] FIG5 shows the effects of different IL-15 mutant-Fc / IL-15Ralpha fragment-Fc on NK cell proliferation.

[0092] FIG6 shows the effects of different IL-15 mutant-Fc / IL-15Ralpha fragment-Fc on CD4+ T cell proliferation.

[0093] FIG7 shows the effects of different IL-15 mutant-Fc / IL-15Ralpha fragment-Fc on CD8+ T cell proliferation.

[0094] FIG8 shows the stimulatory effects of different IL-15 mutant-Fc / IL-15Ralpha fragment-Fc on NK cell killing.

[0095] FIG9 shows the efficacy of HL-015-9 in a tumor-bearing mouse model.

[0096] Best Mode for Carrying Out the Invention

[0097] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0098] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0099] Example 1. Construction of expression plasmids for different heterodimeric proteins

[0100] Based on the sequence information of wild-type IL-15 (SEQ ID No. 1) and the sushi domain of the alpha subunit of the IL-15 receptor (SEQ ID No. 2, positions 1-65), different IL-15 / IL-15Ra dimer protein forms were designed. Based on the amino acid sequence information of the designed different IL-15 / IL-15Ra dimer proteins, the mouse IL-2 signal peptide (the amino acid sequence of the signal peptide is shown in SEQ ID No. 39, and its corresponding nucleotide sequence is shown in SEQ ID No. 40) and restriction sites were added. GenScript Biopharmaceuticals constructed different mammalian cell secretory expression plasmids for the IL-15 / IL-15Ra dimer proteins using conventional gene synthesis and molecular construction technologies.

[0101] The following uses the heterodimeric protein HL-015-2 as an example to illustrate the construction process of the eukaryotic cell recombinant expression plasmid:

[0102] GenScript synthesized a DNA molecule with a nucleotide sequence of SEQ ID No. 28 and a DNA molecule with a nucleotide sequence of SEQ ID No. 35. A signal peptide with a nucleotide sequence of SEQ ID No. 40 was added upstream of the DNA molecule with a nucleotide sequence of SEQ ID No. 28 to obtain a new DNA molecule named 28'. DNA molecule 28' was used to replace the fragment between the HindIII and XhoI restriction enzyme recognition sites (a small fragment between the HindIII and XhoI restriction enzyme recognition sites) of the expression vector pCGS3 (Merck), while keeping the other nucleotide sequences of the expression vector pCGS3 unchanged. The resulting recombinant expression plasmid was correctly sequenced and named pCGS3-015-2a. A signal peptide with a nucleotide sequence of SEQ ID No. 40 was added upstream of the DNA molecule with a nucleotide sequence of SEQ ID No. 35 to obtain a new DNA molecule named 35'. The DNA molecule 35' was used to replace the fragment between the BstBI and PacI restriction enzyme recognition sites (the small fragment between the BstBI and PacI restriction enzyme recognition sites) of the recombinant expression plasmid pCGS3-015-2a, while maintaining the remaining nucleotide sequence of the recombinant plasmid pCGS3-015-2a. The resulting recombinant expression plasmid was sequenced and named pCGS3-015-2 after verification. The recombinant expression plasmid expressed the protein with the amino acid sequence of SEQ ID No. 12 and the protein with the amino acid sequence of SEQ ID No. 19, and the two proteins formed a dimer, HL-015-2.

[0103] IL-15-Linker Fc and IL15Ra extracellular regions of different lengths, with or without linker-Fc, were constructed into different heterodimeric molecules via a knob-into-hole connection. The amino acid sequence of the Fc fragment's knob is shown in SEQ ID No. 9, and its nucleotide sequence is SEQ ID No. 37. The amino acid sequence of the hole is shown in SEQ ID No. 10, and its nucleotide sequence is SEQ ID No. 38. The amino acid sequences of the heterodimeric protein HL-015-1 are SEQ ID No. 11 and SEQ ID No. 20, and their nucleotide sequences are SEQ ID No. 27 and SEQ ID No. 36, respectively. The amino acid sequences of the heterodimeric protein HL-015-2 are SEQ ID No. 12 and SEQ ID No. 19, and their nucleotide sequences are SEQ ID No. 28 and SEQ ID No. 35. The amino acid sequences of the heterodimeric protein HL-015-3 are SEQ ID No. 12 and SEQ ID No. 20, and their nucleotide sequences are SEQ ID No. 28 and SEQ ID No. 36, respectively.

[0104] The construction process of HL-015-1 and HL-015-3 recombinant expression plasmids is the same as that of HL-015-2, the only difference being the corresponding replacement of the target coding gene.

[0105] A positive reference was prepared as follows:

[0106] 1) Obtaining the gene encoding the ALT-803 molecule from patent CN103370339A (the ALT-803 molecule is named ALT-803 in the patent and is a dimer composed of proteins with amino acid sequences SEQ ID No. 3 and SEQ ID No. 4);

[0107] 2) Referring to the article Scientific Reports (2018) 8:7675, the P2239 molecule in which IL-15 / IL-15Ra Sushi-Fc is linked by a disulfide bond was named HR-1 (a dimer composed of proteins with the amino acid sequences of SEQ ID No. 5 and SEQ ID No. 6);

[0108] 3) Referring to Scientific Reports (2018) 8:7675, the HRP00018 molecule in which IL-15-Fc and IL-15Ra-sushi domain-Fc are connected by a knob into a hole was named HR-2 (a dimer composed of proteins with amino acid sequences SEQ ID No. 7 and SEQ ID No. 8);

[0109] The sequence of IL-15 in HL-015-1 (positions 1-114 of SEQ ID No. 11) is consistent with the sequence of IL-15 in HR-2 (positions 1-114 of SEQ ID No. 7); the sequence of the IL-15Rα subunit in HL-015-1 (positions 1-78 of SEQ ID No. 20) has 4 more amino acids (sequence: PAPP) than the sequence of IL-15Rα in HR-2 (positions 1-74 of SEQ ID No. 8).

[0110] The IL-15Rα in HL-015-2 has 13 more amino acids (sequence: DPALVHQRPAPPS) than the sushi region (positions 1-65 in SEQ ID No. 6) of the IL-15Rα in ATL-803.

[0111] The difference between HL-015-3 and HL-015-1 is that the IL-15 in HL-015-3 adopts the N72D mutation; and the difference between HL-015-2 and HL-15-03 is that the IL-15Rα and Fc in HL-015-3 have an additional (G4S)3 linker compared to HL-15-2.

[0112] The names of the molecules constructed in the first round of the present invention and their amino acid and nucleotide sequences are shown in Table 1.

[0113] Table 1: Names of molecules constructed in the first round and their amino acid and nucleotide sequences

[0114] Example 2: Protein expression and purification

[0115] According to Thermofisher's ExpiCHO TM The Expression System Kit instructions were used to transfect the recombinant expression plasmids pCGS3-015-1, pCGS3-015-2, pCGS3-015-3, pCGS3-ALT-803, pCGS3-HR-1, and pCGS3-HR-2 described in Example 1, using 200 mL of each recombinant expression plasmid at a plasmid dosage of 0.7 μg / mL. The cells were cultured for 10 days. Cell density increased significantly during the initial stages of culture and then slowly declined. Cell viability remained high, reaching 80% when samples were collected on day 10.

[0116] The supernatant was collected by centrifugation and filtered, and then purified using MabSelect SuRe LX (Cytiva, product number 17-5474-02) (CIP buffer: 0.1 M NaOH, equilibration buffer: 20 mM PB, 0.15 M NaCl, pH 7.0, wash buffer 1: 20 mM PB, 1 M NaCl, pH 7.0, wash buffer 2: 50 mM citric acid-sodium citrate, pH 5.5, elution buffer: 50 mM citric acid-sodium citrate, pH 3.5); the eluate was collected and concentrated, and then purified using Superdex 200 (Cytiva, product number V521276) at a volume of 3% (equilibration and eluent: 20 mmol / L PB, 150 mmol / L NaCl, pH 7.0). The desired peaks were collected, the purity was detected by SEC-HPLC, and the protein concentration was determined by UV spectrophotometry based on the theoretical extinction coefficient of each protein. The heterodimeric proteins HL-015-1 (expressed by the recombinant expression plasmid pCGS3-015-1), HL-015-2 (expressed by the recombinant expression plasmid pCGS3-015-2), HL-015-3 (expressed by the recombinant expression plasmid pCGS3-015-3), ALT-803 (expressed by the recombinant expression plasmid pCGS3-ALT-803), HR-1 (expressed by the recombinant expression plasmid pCGS3-HR-1) and HR-2 (expressed by the recombinant expression plasmid pCGS3-HR-2) were obtained.

[0117] Example 3: Determination of the Effect of Heterodimer Protein on CTLL-2 and Mo7e Cell Proliferation Activity

[0118] Cytokine-dependent CTLL2 cells (Suzhou Vertex Biopharmaceutical Co., Ltd., Catalog No. TCM-C724) were cultured in RPMI 1640 medium supplemented with 200 U / ml IL-2 and 10% fetal bovine serum at 37°C, 5.0% CO₂, until logarithmic phase. Cells were harvested by centrifugation at 1000 rpm for 5 minutes and washed three times with phosphate-buffered saline. The cells were then resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum (assay medium) and plated onto 96-well cell culture plates at a density of 5,000 cells per well. After 4 hours of incubation (cytokine starvation), 10 μl of the heterodimeric protein solutions prepared in Example 2, HL-015-1, HL-015-2, HL-015-3, ALT-803, HR-1, and HR-2, were added to each well. Each heterodimer protein was treated at 11 concentrations: 500 ng / ml, 166.67 ng / ml, 55.56 ng / ml, 18.52 ng / ml, 6.17 ng / ml, 2.06 ng / ml, 0.69 ng / ml, 0.23 ng / ml, 0.076 ng / ml, 0.025 ng / ml, and 0.0085 ng / ml. A negative control (i.e., 0 ng / ml) containing 10 μl of PBS was added to each well. Each concentration was replicated. After 2 days of incubation, CCK8 was added and the cells were incubated for 2 hours at 37°C under 5.0% CO2. Cell growth was monitored by reading the plates at 450 nm and 630 nm. The experiment was repeated three times.

[0119] The assay method for IL-15 / IL-15Ra dimer protein stimulation of Mo7e cell proliferation (Zhejiang Meisen Cell Technology Co., Ltd., Catalog No. CTCC-001-0368) was similar to that for CTLL-2. Mo7e cells were cultured to the logarithmic phase in RPMI 1640 medium supplemented with 8 ng / ml GM-CSF and 10% fetal bovine serum. The cells were harvested and washed, then resuspended in RPMI 1640 medium without GM-CSF and plated into 96-well plates at a density of 20,000 cells per well. After 4 hours of incubation, the heterodimeric proteins HL-015-1, HL-015-2, HL-015-3, ALT-803, HR-1, and HR-2 from Example 2 were serially diluted with PBS to prepare heterodimeric protein solutions, and 10 μl of the heterodimeric protein solution was added to each well. Eight treatments were performed for each heterodimer protein, with the concentrations of the heterodimer protein in these eight treatment wells being 100 ng / ml, 33.33 ng / ml, 11.11 ng / ml, 3.70 ng / ml, 1.23 ng / ml, 0.41 ng / ml, 0.046 ng / ml, and 0.015 ng / ml, respectively. A negative control (i.e., 0 ng / ml) was added to each well, with 10 μl of PBS added. Each concentration was replicated and incubated at 37°C, 5.0% CO₂ for 4 days. After 4 days, CCK8 (20 μl / well) was added and incubated at 37°C, 5.0% CO₂ for 2 hours. Cell growth was then measured at 450 nm and 630 nm. The experiment was repeated three times. The results showed that HL-015-2 had the best ability to stimulate both CTLL-2 and Mo7e cell proliferation (see Figures 1 and 2; the logarithmic values ​​in Figures 1 and 2 are to the base 10). HL-015-1 was superior to HR-2, primarily due to the IL-15Rα subunit in HL-015-1 having four more amino acids than that in HR-2. The IL-15Rα in HL-015-02 also had 13 more amino acids (DPALVHQRPAPPS) in the sushi region (positions 1-65 in SEQ ID No. 6) than that in ATL-803.

[0120] Example 4. Design of IL-15 mutants and acquisition of heterodimeric proteins and comparison of their activities

[0121] Based on the experimental results of Example 2 and Example 3, it was decided to select the IL-15N72D mutation shown in SEQ ID No. 3 and the IL-15Rα-Fc shown in SEQ ID No. 19 to continue the following experiments.

[0122] Using relevant structural software, the structures of IL-15, IL-15Rα, and IL-15Rβ / γ were analyzed to identify amino acid sites that may affect IL-15. Computer modeling and molecular docking were then performed to select amino acid mutations that may enhance IL-15 activity. The corresponding genes were synthesized, and the amino acid mutation sites obtained through screening are shown in Table 2.

[0123] Table 2: IL15 mutants and their amino acid sequences

[0124] A DNA molecule having a nucleotide sequence of SEQ ID No. 34 and a DNA molecule having a nucleotide sequence of SEQ ID No. 35 were synthesized. A signal peptide having a nucleotide sequence of SEQ ID No. 40 was added upstream of the DNA molecule having a nucleotide sequence of SEQ ID No. 34 to obtain a new DNA molecule named 34'. DNA molecule 34' was used to replace the fragment between the HindIII and XhoI restriction enzyme recognition sites (the small fragment between the HindIII and XhoI restriction enzyme recognition sites) of the expression vector pCGS3 (Merck), while the other nucleotide sequences of the expression vector pCGS3 remained unchanged. The resulting recombinant expression plasmid was sequenced and correctly named pCGS3-015-9a. A signal peptide having a nucleotide sequence of SEQ ID No. 40 was added upstream of the DNA molecule having a nucleotide sequence of SEQ ID No. 35 to obtain a new DNA molecule named 35'. The DNA molecule 35' was used to replace the fragment between the BstBI and PacI restriction enzyme recognition sites (the small fragment between the BstBI and PacI restriction enzyme recognition sites) of the recombinant expression plasmid pCGS3-015-9a, while maintaining the remaining nucleotide sequence of the recombinant plasmid pCGS3-015-9a unchanged. The resulting recombinant expression plasmid was sequenced and correctly named pCGS3-015-9. The recombinant expression plasmid pCGS3-015-9 contains the HL-015-9 gene (a DNA molecule with a nucleotide sequence of SEQ ID No. 34 and a DNA molecule with a nucleotide sequence of SEQ ID No. 35), and expresses a protein with an amino acid sequence of SEQ ID No. 18 and a protein with an amino acid sequence of SEQ ID No. 19. The two proteins form the heterodimeric protein HL-015-9.

[0125] The construction method of the recombinant expression plasmids of HL-015-4 to HL-015-8 is the same as that of HL-015-9, the only difference being that the HL-015-9 gene is replaced accordingly to obtain the recombinant expression plasmid pCGS3-HL-015-4 for the HL-015-4 gene (nucleotide sequences are SEQ ID No. 29 and SEQ ID No. 35, respectively), the recombinant expression plasmid pCGS3-HL-015-5 for the HL-015-5 gene (nucleotide sequences are SEQ ID No. 30 and SEQ ID No. 35, respectively), the recombinant expression plasmid pCGS3-HL-015-6 for the HL-015-6 gene (nucleotide sequences are SEQ ID No. 31 and SEQ ID No. 35, respectively), the recombinant expression plasmid pCGS3-HL-015-7 for the HL-015-8 gene (nucleotide sequences are SEQ ID No. 32 and SEQ ID No. 35, respectively), and the recombinant expression plasmid pCGS3-HL-015-8 for the HL-015-4 gene (nucleotide sequences are SEQ ID No. 29 and SEQ ID No. 35, respectively). The recombinant expression plasmid pCGS3-HL-015-8 contains the amino acid sequence of SEQ ID No. 33 and SEQ ID No. 35. pCGS3-HL-015-4 expresses the protein with the amino acid sequence of SEQ ID No. 13 and the protein with the amino acid sequence of SEQ ID No. 19, and the two proteins form the heterodimeric protein HL-015-4. pCGS3-HL-015-5 expresses the protein with the amino acid sequence of SEQ ID No. 14 and the protein with the amino acid sequence of SEQ ID No. 19, and the two proteins form the heterodimeric protein HL-015-5. pCGS3-HL-015-6 expresses the protein with the amino acid sequence of SEQ ID No. 15 and the protein with the amino acid sequence of SEQ ID No. 19, and the two proteins form the heterodimeric protein HL-015-6. pCGS3-HL-015-7 expresses the protein with the amino acid sequence of SEQ ID No. 16 and the protein with the amino acid sequence of SEQ ID No. 19, and the two proteins form the heterodimeric protein HL-015-7. pCGS3-HL-015-8 expresses a protein having an amino acid sequence of SEQ ID No. 17 and a protein having an amino acid sequence of SEQ ID No. 19, and the two proteins form a heterodimeric protein HL-015-8.

[0126] The names of the molecules constructed in the second round of the present invention and their amino acid and nucleotide sequences are shown in Table 3.

[0127] Table 3: Molecular names and their amino acid and nucleotide sequences constructed in the second round

[0128] Recombinant expression plasmids pCGS3-HL-015-4 to pCGS3-HL-015-9 were expressed and protein purified according to the method of Example 2. Activity assays were then performed according to the method of Example 3. The results are shown in Figures 3 and 4 (the logarithmic values ​​in Figures 3 and 4 are based on 10). Among all mutants, HL-015-9 had the strongest activity in stimulating the proliferation of CTLL-2 or Mo7e.

[0129] In the CTLL-2 cell proliferation assay, heterodimeric proteins HL-015-4, HL-015-5, HL-015-6, HL-015-7, HL-015-8, and HL-015-9 were graded and diluted with PBS, respectively, according to the type of dimeric protein, to obtain heterodimeric protein solutions. 10 μl of the heterodimeric protein solution was added to each well. Eight concentrations of each heterodimeric protein were treated, and the concentrations of the heterodimeric protein in these eight treatment wells were 166.67 ng / ml, 55.56 ng / ml, 18.52 ng / ml, 6.17 ng / ml, 2.06 ng / ml, 0.69 ng / ml, 0.23 ng / ml, and 0.076 ng / ml, respectively. The experiment was repeated three times, and a negative control (i.e., 0 ng / ml) was added to each well with 10 μl of PBS. Each concentration was replicated. The remaining procedures were the same as in Example 3.

[0130] In the Mo7e cell proliferation assay, according to the type of dimeric protein, heterodimeric proteins HL-015-4, HL-015-5, HL-015-6, HL-015-7, HL-015-8, and HL-015-9 were gradiently diluted with PBS to obtain heterodimeric protein solutions, and 10 μl of heterodimeric protein solution was added to each well. Each heterodimeric protein was treated with 8 concentrations, and the concentrations of the heterodimeric proteins in these 8 treatment wells were 100 ng / ml, 33.33 ng / ml, 11.11 ng / ml, 3.70 ng / ml, 1.23 ng / ml, 0.41 ng / ml, 0.046 ng / ml, and 0.015 ng / ml, respectively. The experiment was repeated 3 times, and a negative control (i.e., 0 ng / ml) was added to each well with 10 μl of PBS, and duplicate wells were set for each concentration. The remaining operations were the same as in Example 3.

[0131] Example 5: Stimulating effect of heterodimeric protein on proliferation of NK cells, CD8+ T cells, and CD4+ T cells

[0132] NK cells, CD8+ T cells, and CD4+ T cells were isolated from human peripheral blood using NK cell (CD56+, CD3-), CD8+ T cell (CD3+, CD8+), and CD4+ T cell (CD3+, CD8+) isolation kits, respectively. The isolated cells were cultured overnight in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37°C, 5.0% CO2. The cells were harvested and stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) for 15 minutes at 37°C. The cells were then washed by centrifugation and resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum. 10,000 cells per well were plated onto 96-well cell culture plates pre-diluted with PBS to obtain different concentrations of heterodimer solutions containing ALT-803, HL-015-6, and HL-015-9. After 3 days of culture, the number of CFSC-positive cells was analyzed by flow cytometry to determine the effects of different IL-15 / IL-15Rα heterodimers on the growth of different cells. As shown in Figures 5, 6, and 7, HL-015-9 has the strongest activity in stimulating the proliferation of NK cells, CD4+ T cells, and CD8+ T cells.

[0133] In the NK cell proliferation assay, the experiment was divided into three groups based on the type of dimer protein. The concentrations of the dimer proteins ALT-803, HL-015-6, and HL-015-9 after gradient dilution in PBS were 50 ng / ml, 12.5 ng / ml, 3.13 ng / ml, 0.78 ng / ml, 0.20 ng / ml, 0.049 ng / ml, 0.012 ng / ml, and 0.031 ng / ml, respectively. The experiment was repeated three times. A negative control (i.e., 0 ng / ml) was added to each well, with 10 μl of PBS added to each well. Duplicate wells were set for each concentration.

[0134] In the CD4+ cell proliferation assay, the experiment was divided into three groups based on the type of dimer protein. The concentrations of the dimer proteins ALT-803, HL-015-6, and HL-015-9 after gradient dilution in PBS were 50 ng / ml, 12.5 ng / ml, 3.13 ng / ml, 0.78 ng / ml, 0.20 ng / ml, 0.049 ng / ml, 0.012 ng / ml, 0.031 ng / ml, and 0 ng / ml, respectively. The experiment was repeated three times. A negative control (i.e., 0 ng / ml) was added to each well, with 10 μl of PBS added to each well. Duplicate wells were set for each concentration.

[0135] In the CD8+ cell proliferation assay, the experiment was divided into three groups based on the type of dimer protein. The concentrations of the dimer proteins ALT-803, HL-015-6, and HL-015-9 after gradient dilution in PBS were 50 ng / ml, 12.5 ng / ml, 3.13 ng / ml, 0.78 ng / ml, 0.20 ng / ml, 0.049 ng / ml, 0.012 ng / ml, 0.031 ng / ml, and 0 ng / ml, respectively. The experiment was repeated three times. A negative control (i.e., 0 ng / ml) was added to each well with 10 μl of PBS, and duplicate wells were set up for the negative control.

[0136] Example 6: Effect of heterodimeric protein on NK cell killing of K562

[0137] K562 cells (Wuhan Punosai, catalog number CL-0130) were cultured in RPMI 1640 medium containing 5% fetal bovine serum at 37°C and 5.0% CO2 for 48 hours, then BATDA stain was added and incubated at 37°C for 25 minutes. The cells were centrifuged and washed three times with culture medium. The labeled K562 cells were added to a 96-well culture plate at a density of 5,000 cells per well. NK cells that had been co-cultured with culture medium (RPMI 1640 medium containing 5% fetal bovine serum) and heterodimeric proteins ALT-803, HR-1, HL-015-9, HL-015-6, and HL-015-4 for 72 hours were added at a ratio of 1:10 between target cells and effector cells. The target cells and effector cells were incubated at 37°C and 5.0% CO2 for 4 hours, and the supernatant was centrifuged and europium solution was added. The fluorescence value of the solution was detected. NK cells cultured with culture medium alone were used as a control. A blank control group and a maximum release group were also established (the maximum release group consisted of the blank control group supplemented with Triton X at a final concentration of 0.05%, followed by europium detection after incubation). The killing rate was calculated according to the following formula: Killing rate (%) = (mean value of the experimental group - mean value of the blank control group) / (mean value of the maximum release group - mean value of the blank control group). The experimental results are shown in Figure 8. Each IL-15 / IL-15Ra complex significantly enhanced the cytotoxicity of NK cells against K562 cells, with HL-015-9 exhibiting the greatest activity.

[0138] Example 7: In vivo pharmacodynamic evaluation of heterodimeric proteins in animals

[0139] BALB / c mice (7-9 weeks old, female, body weight range 17-23g, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., animal batch number 110011211108488925) were subcutaneously inoculated with CT26 cells (murine colon cancer cell line, cultured in RPMI1640 culture medium containing 10% fetal bovine serum, preserved by Sino-US Crown Biosciences) to establish a subcutaneous transplant tumor model of mouse colon cancer. When the tumor volume reached 60-100 cubic millimeters, the mice were randomly divided into 7 groups for drug administration (the experiment was divided into 7 groups, with 7 tumor-bearing mice in each group). The first day of drug administration was day 1, and the drug administration cycle was day 1, day 4, day 8 and day 11. The drug administration volume was 10 μl / g body weight. The tumor volume was measured twice a week and calculated according to the following formula: Tumor volume (mm3) = 1 / 2×(a×b 2 )(where a represents the major diameter and b represents the minor diameter).

[0140] The heterodimeric protein ALT-803 is dissolved in buffer to obtain a heterodimeric protein ALT-803 solution; the heterodimeric protein HL-015-9 is dissolved in buffer to obtain a heterodimeric protein HL-015-9 solution; wherein the buffer is 0.9% physiological saline for injection.

[0141] The control group was the buffer group, and each mouse was intraperitoneally injected with buffer at 10 μl / g body weight;

[0142] Group 1 was the ALT-803, 0.05 mg / kg group. Each mouse was intraperitoneally injected with 10 μl / g body weight of the heterodimeric protein ALT-803 solution, so that the dosage of the heterodimeric protein ALT-803 was 0.05 mg / kg body weight.

[0143] The second group was the ALT-803, 0.15 mg / kg group. Each mouse was intraperitoneally injected with 10 μl / g body weight of the heterodimeric protein ALT-803 solution, so that the dosage of the heterodimeric protein ALT-803 was 0.15 mg / kg body weight.

[0144] The third group was the ALT-803, 0.6 mg / kg group. Each mouse was intraperitoneally injected with 10 μl / g body weight of the heterodimeric protein ALT-803 solution, so that the dosage of the heterodimeric protein ALT-803 was 0.6 mg / kg body weight.

[0145] The fourth group was the HL-015-9, 0.05 mg / kg group. Each mouse was intraperitoneally injected with 10 μl / g body weight of the heterodimeric protein HL-015-9 solution, so that the dosage of the heterodimeric protein HL-015-9 was 0.05 mg / kg body weight.

[0146] The fifth group was the HL-015-9, 0.15 mg / kg group. Each mouse was intraperitoneally injected with 10 μl / g body weight of the heterodimeric protein HL-015-9 solution, so that the dosage of the heterodimeric protein HL-015-9 was 0.15 mg / kg body weight.

[0147] The sixth group was the HL-015-9, 0.6 mg / kg group. Each mouse was intraperitoneally injected with 10 μl / g body weight of the heterodimeric protein HL-015-9 solution, so that the dosage of the heterodimeric protein HL-015-9 was 0.6 mg / kg body weight.

[0148] The results are shown in FIG9 , which show that under the same concentration conditions, HL-015-9 has a better ability to inhibit tumor growth in model mice than ALT-803 at the corresponding concentration.

[0149] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

[0150] Industrial Applications

[0151] The present invention provides an IL-15 mutant-Fc / IL-15Rα subunit-Fc heterodimeric protein, comprising protein a and protein b. Protein a may be a protein having an amino acid sequence of SEQ ID No. 18, and protein b may be a protein having an amino acid sequence of SEQ ID No. 19. Experiments have demonstrated that the IL-15 mutant-Fc / IL-15Rα subunit-Fc heterodimeric protein and related biomaterials provided herein can effectively stimulate the proliferation of NK cells and T cells. In vivo experiments have shown that the dimeric protein HL-015-9, at equivalent injection doses, inhibits tumor growth better than ALT-803. Therefore, the IL-15 mutant-Fc / IL-15Rα subunit-Fc heterodimeric protein provided herein can be used to prepare drugs or preparations for treating tumors and / or viral infections.

Claims

1. A heterodimeric protein comprising protein a and protein b, wherein protein a comprises an IL-15 mutant and a first Fc mutant; protein b comprises an IL-15Rα subunit sushi domain, other partial fragments of the IL-15Rα subunit, and a second Fc mutant; the first Fc mutant and the second Fc mutant are selected from a Knob-modified Fc or a Hole-modified Fc, and the first Fc mutant and the second Fc mutant have different types of Knob modifications or Hole modifications; The IL-15 mutant is a polypeptide having an amino acid sequence selected from any one of SEQ ID No. 18, positions 1-111, SEQ ID No. 15, positions 1-111, SEQ ID No. 14, positions 1-114, SEQ ID No. 17, positions 1-114, SEQ ID No. 16, and positions 1-114 of SEQ ID No. 13; The IL-15Rα subunit sushi domain and other partial fragments of the IL-15Rα subunit are polypeptides whose amino acid sequence is SEQ ID No.

2.

2. The heterodimeric protein according to claim 1, characterized in that: The protein a further includes a linker, which connects the IL-15 mutant and the first Fc mutant. The linker is (GGGGS)n, where n is a natural number from 0 to 4.

3. The heterodimeric protein according to claim 2, characterized in that: The n is 3.

4. The heterodimeric protein according to any one of claims 1 to 3, characterized in that: The protein a includes: the IL-15 mutant, a linker connected to the C-terminus of the IL-15 mutant, and a first Fc mutant connected to the C-terminus of the linker; the protein b includes: the IL-15Rα subunit sushi domain and other partial fragments, and a second Fc mutant connected to the C-terminus of the IL-15Rα subunit sushi domain and other partial fragments.

5. The heterodimeric protein according to any one of claims 1 to 4, characterized in that: The first Fc mutant or the second Fc mutant is Fc of antibody IgG1, IgG2, IgG3 or IgG4 or a mutant thereof.

6. The heterodimeric protein according to any one of claims 1 to 5, characterized in that: The first Fc mutant or the second Fc mutant is Fc of antibody IgG4 or a mutant thereof.

7. The heterodimeric protein according to any one of claims 1 to 6, characterized in that: The amino acid sequence of the Knob-modified Fc is SEQ ID No.

9.

8. The heterodimeric protein according to any one of claims 1 to 7, characterized in that: The amino acid sequence of the Hole-modified Fc is SEQ ID No.

10.

9. The heterodimeric protein according to any one of claims 1 to 8, characterized in that: The protein a is any one of A1) to A7): A1) a protein having an amino acid sequence of SEQ ID No. 18; A2) a protein having an amino acid sequence of SEQ ID No. 15; A3) a protein having an amino acid sequence of SEQ ID No. 14; A4) a protein having an amino acid sequence of SEQ ID No. 17; A5) a protein having an amino acid sequence of SEQ ID No. 16; A6) a protein having an amino acid sequence of SEQ ID No. 13; A7) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of the protein described in any one of A1) to A6).

10. The heterodimeric protein according to any one of claims 1 to 9, characterized in that: The protein b is any one of A8)-A10): A8) a protein having an amino acid sequence of SEQ ID No. 19; A9) a protein having an amino acid sequence of SEQ ID No. 20; A10) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of the protein described in A8) or A9).

11. A biomaterial related to the heterodimeric protein according to any one of claims 1 to 10, characterized in that: The biological material is any one of the following: B1) a nucleic acid molecule encoding the heterodimeric protein according to any one of claims 1 to 10; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3); B5) an animal cell line containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3); B6) a plant cell line containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3); B7) A host cell that produces the heterodimeric protein according to any one of claims 1 to 10.

12. A product, the active ingredient of which is the heterodimeric protein according to any one of claims 1 to 10; The use of the product is any one of the following C1) to C6): C1) prevention and / or treatment of diseases; C2) Activate the body’s immune system; C3) Improve or enhance the body's immune function; C4) inhibit tumor growth; C5) increasing the proliferation activity of NK cells and / or CD8+ T cells and / or CD4+ T cells; C6) Enhance the killing ability of NK cells against tumor cells.

13. A pharmaceutical composition, comprising the heterodimeric protein according to any one of claims 1 to 10 and other drugs.

14. The pharmaceutical composition according to claim 13, characterized in that: The other drugs may be at least one of the following drugs: immune checkpoint drugs, cell engager bispecific antibodies, and cell therapy products.

15. Use of the heterodimeric protein according to any one of claims 1 to 10, or the biomaterial according to claim 11, or the product according to claim 12, or the pharmaceutical composition according to claim 13 or 14 in any one of the following D1) to D12): D1) Preparation of products for the prevention and / or treatment of diseases; D2) Preparation of products for activating the body's immune system; D3) Preparation of products that improve or enhance the body's immune function; D4) preparing products that inhibit tumor growth; D5) preparing a product that increases the proliferation activity of NK cells and / or T cells; D6) preparing a product that improves the killing ability of NK cells against tumor cells; D7) prevention and / or treatment of diseases; D8) Activate the animal's immune system; D9) Improve or enhance the body's immune function; D10) inhibit tumor growth; D11) increasing the proliferation activity of NK cells and / or T cells; D12) Improve the killing ability of NK cells against tumor cells.

16. The method according to claim 15, characterized in that: Such diseases include infectious diseases, tumors, blood diseases, inflammatory diseases and autoimmune diseases.

17. A method for treating a disease, comprising the steps of administering the heterodimeric protein according to any one of claims 1 to 10, the biomaterial according to claim 11, the product according to claim 12, or the pharmaceutical composition according to claim 13 or 14 to a patient, so that the patient is treated.

18. The method according to claim 17, wherein: Such diseases include infectious diseases, tumors, blood diseases, inflammatory diseases and autoimmune diseases.

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