IL-15 variant-Fc / IL-15R alpha subunit-Fc heterodimers and uses thereof

Abstract

The present invention discloses an IL-15 variant-Fc / IL-15Rα subunit-Fc heterodimer and its use. The heterodimeric protein comprises protein a and protein b, where protein a comprises an IL-15 variant and a first Fc variant, and protein b comprises an IL-15Rα subunit sushi domain, another fragment of the IL-15Rα subunit, and a second Fc variant, the first Fc variant and the second Fc variant being selected from Knob-modified Fc and Hole-modified Fc, and the types of modification between the first and second Fc variants are different. Experiments have demonstrated that the heterodimeric protein provided by the present invention can effectively stimulate the proliferation of NK cells and T cells. In vivo experiments have shown that the dimeric protein HL-015-9 exhibits superior tumor growth inhibition to ALT-803 at comparable injection doses, and can be used to prepare drugs or formulations for treating diseases such as tumors and / or viral infections.

Description

[Technical Field]

[0001] The present invention belongs to the technical field of medical preparations, and specifically relates to IL-15 variant-Fc / IL-15Rα subunit-Fc heterodimers and uses thereof. [Background technology]

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

[0003] IL-2 was the first cytokine identified and was initially found in the culture supernatant of activated human T cells as a soluble factor that induces T cell proliferation. IL-2 was also the first cytokine approved by the FDA for cancer treatment. IL-2 is secreted primarily by CD4+ and CD8+ T cells upon antigen stimulation, but is also produced in small amounts by activated dendritic cells (DCs), mast cells, and NKT cells.

[0004] IL-15 shares several similar functions with IL-2, including stimulating activated T cell proliferation, generating cytotoxic T cells, and activating and maintaining NK cells. It also promotes the induction of immunoglobulin synthesis by B cells and the regulation of lymphoid homeostasis. However, unlike IL-2, IL-15 mRNA is expressed in a variety of tissues, including hematopoietic cells and nonhematopoietic cells, including keratinocytes, neurons, stromal cells, and fibroblasts. However, production of mature IL-15 protein is primarily restricted to DCs and monocytes / macrophages, unlike the widespread expression of IL-15 mRNA.

[0005] IL-15 has a chemotactic effect on T cells. It induces circulating lymphocytes to home to peripheral lymph nodes, inhibits lymphocyte apoptosis, promotes T cell activation and proliferation, and induces the production of cytotoxic T cells (CTLs). IL-15 not only promotes the production 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 an important role in NK cell activation and proliferation. In mice overexpressing IL-15, the number of NK cells significantly increased, leading to enhanced immune responses. IL-15 also plays an important role in the functional maturation of DC cells and macrophages. IL-15 promotes the expression of costimulatory factors and IFN-γ by DC cells, improving their ability to activate CD8+ T cells and NK cells.

[0006] IL-2 and IL-15 are type I four-alpha helix bundle cytokines and belong to the gamma receptor cytokine family. This set of cytokines shares the same receptor subunit, γc, and exhibits pleiotropic functions that regulate innate and adaptive immune responses. The IL-2 and IL-15 receptors are both heterotrimers; in addition to the cytokine receptor subunit γc (also called IL-2Rγ or CD132), they also share a beta subunit, referred to here as IL-2 / 15Rβ (also called CD122). The third subunit of IL-2 and IL-15 is also a unique receptor subunit, IL-2Rα and IL-15Rα, respectively.

[0007] The IL-2 α, β, and γ receptors are normally present on the surface of the same cells, and in addition to being expressed on the cell surface, IL-2Rα is also present in a soluble form (sIL-2Rα) in some diseases, including inflammatory diseases, transplant rejection, and many malignancies.

[0008] IL-15Rα is a unique component of the IL-15 receptor complex, primarily expressed on monocytes and dendritic cells. Unlike other γc family cytokines (e.g., IL-2), IL-15, as a cytokine, first binds to IL-15Rα-expressing cells and then presents the IL-15 / IL-15Rα complex to IL-2 / 15Rβ and γc in activated T cells or NK cells, forming a high-affinity immune synapse. Because IL-2 and IL-15 share the same receptor subunit (IL-2 / 15Rβγ), they trigger multiple similar downstream signaling pathways, including Janus kinases (JAKs), signal transducins, 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 exhibit distinct functions in the body, particularly in the adaptive immune response. For example, IL-2 is required for the development and maintenance of regulatory T cells (Tregs), but IL-2 is closely associated with activation-induced apoptosis (AICD). IL-2-mediated tumor therapy can sometimes cause the side effect of capillary leak syndrome (CAPS). In contrast, IL-15 does not induce CAPS, suppresses IL-2-induced CAPS, and does not cause the side effect of CAPS. IL-15 is a key factor supporting the persistence of natural killer (NK) cells and memory CD8+ T cells.

[0010] Based on the ability of IL-15 to promote and activate effector lymphocytes in vivo, its therapeutic potential is gradually being explored. The role of IL-15 in joint immune-enhancing strategies is also receiving increasing attention. N-803 (Anktiva, ALT-803) combines an IL-15 mutant with an IL-15 / IL-15Rα sushi domain / IgG1 Fc fusion protein. Compared to wild-type IL-15, N-803 has favorable pharmacokinetic properties, a longer in vivo persistence, and stronger antitumor activity.

[0011] Clinical data presented by ImmunityBio at ASCO in 2022 showed that the N803-combined BCG vaccine achieved a 99% two-year overall survival rate in 160 patients with non-muscle-invasive bladder cancer (NMIBC) refractory to BCG. In the patient population with carcinoma in situ, the complete remission rate was 71%, with a median sustained response time of 24.1 months. In the patient population with papillary carcinoma, the 18-month disease-free survival rate was 53%. Over two years of follow-up, over 90% of patients did not undergo cystectomy. For patients with NMIBC refractory to BCG, the therapeutic efficacy and safety of N-803 plus BCG are superior to other conventional treatments. Therefore, the development of better IL-15 drugs is of positive significance. Summary of the Invention

[0012] The technical problem that the present invention aims to solve is how to provide a better IL-15 drug.

[0013] In order to solve the above technical problems, in a first aspect, the present invention provides a method for producing an Fc-antibody comprising: (a) a polypeptide comprising: an IL-15 polypeptide; (b) a polypeptide comprising: an IL-15 polypeptide; (c) a polypeptide comprising: an IL-15 polypeptide; (d) a polypeptide comprising: an IL-15 polypeptide; (e) a polypeptide comprising: an IL-15 polypeptide; (f) a polypeptide comprising: an IL-15 polypeptide; (g) a polypeptide comprising: an IL-15 polypeptide; (h) a polypeptide comprising: an IL-15 polypeptide; (i) a polypeptide comprising: an IL-15 polypeptide; the IL-15 variant is a polypeptide having an amino acid sequence selected from positions 1 to 111 of SEQ ID NO: 18, positions 1 to 111 of SEQ ID NO: 15, positions 1 to 114 of SEQ ID NO: 14, positions 1 to 114 of SEQ ID NO: 17, positions 1 to 114 of SEQ ID NO: 16, and positions 1 to 114 of SEQ ID NO: 13; The present invention provides a heterodimeric protein in which the IL-15Rα subunit sushi domain and another fragment of the IL-15Rα subunit are a polypeptide having the amino acid sequence of SEQ ID NO:2.

[0014] The "sushi domain" in the "IL-15Rα subunit sushi domain and other fragments of the IL-15Rα subunit" described in the present invention mainly refers to a peptide fragment having an amino acid sequence from positions 1 to 65 of SEQ ID NO:2.

[0015] The "other fragment" in the "IL-15Rα subunit sushi domain and other fragments of the IL-15Rα subunit" described in the present invention mainly refers to a peptide fragment having an amino acid sequence of positions 66 to 78 of SEQ ID NO: 2 (DPALVHQRPAPPS).

[0016] Furthermore, protein a further comprises a linker connecting the IL-15 variant and the first Fc variant, 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.

[0017] Furthermore, protein a comprises the IL-15 mutant, a linker linked to the C-terminus of the IL-15 mutant, and a first Fc mutant linked to the C-terminus of the linker, and protein b comprises the IL-15Rα subunit sushi domain and other fragments, and a second Fc mutant linked to the C-terminus of the IL-15Rα subunit sushi domain and other fragments.

[0018] In the heterodimeric protein, the first Fc variant or the second Fc variant may be the Fc of antibody IgG1, IgG2, IgG3, or IgG4, or a variant thereof; Furthermore, said first Fc variant or said second Fc variant is selected from an IgG4 Fc or variant thereof.

[0019] Furthermore, when an IgG4 format is employed, the serine at position 228 in the hinge region can be mutated to proline to obtain a stable IgG4 Fc variant.

[0020] In the heterodimeric protein, protein a and protein b are formed by combining Fc knob-into-hole structures. Of the two peptide chains, the Fc domain of one chain has a T366W mutation, while the corresponding domain of the other chain has T366S, L368A, and Y407V mutations; or the Fc domain of one chain has T350V, L351Y, F405A, and Y407V mutations, while the corresponding domain of the other chain has T350V, T360L, K392L, and T394V mutations. Alternatively, protein a and protein b are formed by another type of knob-into-hole structure.

[0021] In one embodiment of the present invention, the knob-modified Fc (second Fc variant) has an amino acid sequence of SEQ ID NO: 9 and a nucleotide sequence of SEQ ID NO: 37, and the hole-modified Fc (first Fc variant) has an amino acid sequence of SEQ ID NO: 10 and a nucleotide sequence of SEQ ID NO: 38.

[0022] In the heterodimeric protein, the protein a is A1) a protein having the amino acid sequence SEQ ID NO: 18; A2) a protein having the amino acid sequence SEQ ID NO: 15; A3) a protein having the amino acid sequence SEQ ID NO: 14; A4) a protein having the amino acid sequence SEQ ID NO: 17; A5) a protein having the amino acid sequence SEQ ID NO: 16; A6) a protein having the amino acid sequence SEQ ID NO: 13; A7) A fusion protein in which a protein tag is linked to the N-terminus and / or C-terminus of the protein according to any one of A1) to A6); It is one of the following.

[0023] The protein b is A8) a protein having the amino acid sequence SEQ ID NO: 19; A9) a protein having the amino acid sequence SEQ ID NO: 20; A10) A fusion protein in which a protein tag is linked to the N-terminus and / or C-terminus of the protein according to A8) or A9); It may be any one of the above.

[0024] Preferably, protein a in the heterodimeric protein is a protein having the amino acid sequence of SEQ ID NO: 18, and protein b is a protein having the amino acid sequence of SEQ ID NO: 19.

[0025] In order to solve the above technical problem, in a second aspect, the present invention provides a biomaterial associated with the above heterodimeric protein, the biomaterial comprising: B1) a nucleic acid molecule encoding the heterodimeric protein; and B2) an expression cassette comprising the nucleic acid molecule according to B1); B3) a recombinant vector comprising the nucleic acid molecule according to B1) or the expression cassette according to B2); B4) A recombinant microorganism comprising a nucleic acid molecule according to B1), an expression cassette according to B2), or a recombinant vector according to B3); B5) an animal cell line containing a nucleic acid molecule according to B1), an expression cassette according to B2), or a recombinant vector according to B3); B6) a plant cell line containing a nucleic acid molecule according to B1) or an expression cassette according to B2) or a recombinant vector according to B3); B7) a host cell that produces the heterodimeric protein; It is one of the following.

[0026] In order to solve the above technical problems, in a third aspect, the present invention provides a product whose active ingredient is the above heterodimeric protein, and the use of the product is as follows: C1) preventing and / or treating a disease; C2) activating the immune system of the organism; C3) Raising or enhancing the immune function of the organism; C4) Inhibiting tumor growth; C5) Improving the proliferation activity of NK cells and / or T cells; C6) Improving the killing ability of NK cells against tumor cells (e.g., K562 cells); It is one of the following.

[0027] In order to solve the above technical problems, in a fourth aspect, the present invention provides a pharmaceutical composition comprising the above heterodimeric protein and another drug.

[0028] Furthermore, the other agent may be at least one of an immune checkpoint agent, a cell adhesion mechanism bispecific antibody, and a cell therapy product.

[0029] In order to solve the above technical problems, in a fifth aspect, the present invention provides: D1) Preparation of a product for preventing and / or treating a disease; D2) Preparation of a product that activates the organism's immune system; D3) Preparation of products that enhance or enhance immune function of organisms; D4) Preparation of a product that inhibits tumor growth; D5) Preparation of a product that improves the proliferation activity of NK cells and / or T cells; D6) Preparation of a product that improves the killing ability of NK cells against tumor cells (e.g., K562 cells); D7) Preventing and / or treating disease; D8) Activating the immune system of animals; D9) Raising or enhancing the immune function of the organism; D10) Inhibiting tumor growth; D11) Improving the proliferation activity of NK cells and / or T cells; D12) Improving the killing ability of NK cells against tumor cells (e.g., K562 cells); The present invention provides an application of the heterodimeric protein, or the biomaterial, or the product, or the pharmaceutical composition to any one of the following:

[0030] In any of the above products or applications, the T cells comprise CD8+ T cells and / or CD4+ T cells.

[0031] In any of the above products or applications, the organism may be a mammalian organism, including humans and mice.

[0032] In order to solve the above technical problems, in a sixth aspect, the present invention provides a method for treating a disease, comprising the step of administering the above heterodimeric protein, the above biomaterial, the above product, or the above pharmaceutical composition to a patient and treating the patient.

[0033] In any one of the above products, applications or methods, the disease comprises an infectious disease, a tumor, a blood disease, an inflammatory disease and an autoimmune disease.

[0034] The infectious diseases include, but are not limited to, viral infections (eg, smallpox virus infection, HIV infection, HBV infection, etc.), bacterial infections, and fungal infections.

[0035] Such tumors include, but are not limited to, melanoma, colon cancer, skin cancer, lymphoma, renal cell carcinoma, liver cancer, lung cancer, gastric cancer, and breast cancer.

[0036] The hematological diseases include, but are not limited to, anemia, acute myeloid leukemia, myelodysplastic syndrome, and T-cell large granular lymphocytic leukemia.

[0037] Such autoimmune diseases include, but are not limited to, multiple sclerosis, psoriasis, rheumatoid arthropathy, gastritis, and mucositis.

[0038] In any one of the above-described products, applications, or methods, the product may be a drug or preparation. In practical application, a vector material may be further added during the preparation of the drug or preparation.

[0039] The vector materials include, but are not limited to, water-soluble vector materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble vector materials (e.g., ethyl cellulose, cholesteryl stearate, etc.), and enteric vector materials (e.g., cellulose acetate phthalate and carboxymethylethyl cellulose, etc.).

[0040] These materials can be used to produce a variety of dosage forms, including, but not limited to, tablets, capsules, drop-shaped pills, sprays, pills, powders, solutions, suspensions, emulsions, granules, liposomes, patches, lozenges, suppositories, and freeze-dried injections. These may include conventional formulations, sustained-release formulations, and various microparticle drug delivery systems. To produce unit-dose tablets, various vectors known in the art can be widely used. Examples of vectors include diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and aluminum silicate; water, glycerol, polyethylene glycol, ethanol, propyl alcohol, starch slurry, dextrin, rice bran, honey, glucose solution, gum arabic slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, and polyvinylpyrrolidone. Examples of suitable additives include wetting agents and adhesives; disintegrants such as dry starch, alginate, agar powder, brown seaweed starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors such as sucrose, glycerol tristearate, cocoa butter, and hydrogenated oils; absorption enhancers such as quaternary ammonium salts and sodium dodecyl sulfate; and lubricants such as talc powder, silicon dioxide, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be coated, such as sugar-coated tablets, film-coated tablets, and enteric-coated tablets, or double-layered and multi-layered tablets. A wide variety of vectors known in the art can be used to prepare unit-dose pills. Examples of vectors include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, kaolin, and talc powder; adhesives such as gum arabic, gum tiger, gelatin, ethanol, honey, cornstarch, rice paste, and glue; and disintegrants such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methylcellulose, and ethylcellulose.To prepare unit-dose suppositories, various vectors known in the art can be widely used. Examples of vectors include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides. To prepare unit-dose injectable preparations such as solutions, emulsions, lyophilized injections, and suspensions, any diluent commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyisostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. To prepare isotonic injections, an appropriate amount of sodium chloride, glucose, or glycerol can be added to the injectable preparation, and conventional cosolvents, buffers, pH adjusters, and the like can also be added. If necessary, colorants, preservatives, flavorings, flavorings, sweeteners, or other materials can be added to the pharmaceutical formulation.

[0041] The above dosage forms can be administered via injection, including subcutaneous injection, intravenous injection, intramuscular injection, intracavitary injection, and the like.

[0042] The beneficial technical effects obtained by the present invention are as follows:

[0043] 1. In the present invention, through computer-aided design and bioactivity screening, an innovative IL-15 variant-Fc / IL-15Rα-Fc heterodimeric protein has been invented, which has higher biological activity than existing similar products such as HL-015-9. The dimeric protein provided by the present invention can better stimulate the proliferation of NK cells and T cells and can be used alone or in combination with other drugs (e.g., immune checkpoint drugs, cell adhesion molecule bispecific antibodies, or cell therapy products) to achieve better efficacy.

[0044] 2. The heterodimeric protein provided by the present invention can enhance the proliferation activity of CTLL-2 and Mo7e cells.

[0045] 3. The heterodimeric protein provided by the present invention can enhance the proliferation activity of NK cells, CD8+ T cells, and CD4+ T cells.

[0046] 4. The heterodimeric protein provided by the present invention can promote the killing of K562 by NK cells.

[0047] 5. The dimeric protein HL-015-9 provided by the present invention has a superior tumor growth inhibition effect to ALT-803 under the same injection dose. [Brief explanation of the drawings]

[0048] [Figure 1] The proliferation effects of different molecules designed in the first round on Mo7e cells. [Figure 2] The effects of different molecules designed in the first round on the proliferation of CTLL-2 cells. [Figure 3] This shows the effect of the IL-15 mutant-Fc / IL-15Ralpha fragment-Fc designed in the second round on the proliferation of Mo7e cells. [Figure 4] This shows the effect of the IL-15 mutant-Fc / IL-15Ralpha fragment-Fc designed in the second round on the proliferation of CTLL-2 cells. [Figure 5] Effect of different IL-15 mutant-Fc / IL-15Ralpha fragment-Fc on NK cell proliferation. [Figure 6] 1 shows the effect of different IL-15 mutant-Fc / IL-15Ralpha fragment-Fc on CD4+ T cell proliferation. [Figure 7] 1 shows the effect of different IL-15 mutant-Fc / IL-15Ralpha fragment-Fc on CD8+ T cell proliferation. [Figure 8] Stimulation of NK cell killing by different IL-15 mutant-Fc / IL-15Ralpha fragments-Fc. [Figure 9] This shows the efficacy of HL-015-9 in a tumor-bearing mouse model. DETAILED DESCRIPTION OF THE INVENTION

[0049] The present invention will be described in more detail below in conjunction with specific embodiments, but the examples shown are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below do not limit the present invention in any way, but can serve as guidelines for those skilled in the art to make further improvements.

[0050] Unless otherwise specified, the experimental methods in the following examples are all conventional methods and are carried out in accordance with the techniques or conditions described in the literature in this field or the product specifications. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0051] Example 1. Construction of expression plasmids for different heterodimeric proteins Different dimeric IL-15 / IL-15Ra protein forms were designed based on the sequence information of wild-type IL-15 (SEQ ID NO: 1) and the sushi domain of the IL-15 receptor alpha subunit (positions 1-65 of SEQ ID NO: 2). Based on the amino acid sequence information of the different designed IL-15 / IL-15Ra dimeric proteins, a mouse IL-2 signal peptide (the amino acid sequence of the signal peptide is SEQ ID NO: 39, and the corresponding nucleotide sequence is SEQ ID NO: 40) and an enzyme cleavage site were added. GenScript Biotechnology, Inc. used conventional gene synthesis and molecular assembly techniques to construct secretory expression plasmids for different mammalian cells for the IL-15 / IL-15Ra dimeric proteins.

[0052] Hereinafter, the process of constructing a recombinant expression plasmid for eukaryotic cells will be described using the heterodimeric protein HL-015-2 as an example.

[0053] GenScript synthesizes a DNA molecule with the nucleotide sequence SEQ ID NO:28 and a DNA molecule with the nucleotide sequence SEQ ID NO:35. A signal peptide with the nucleotide sequence SEQ ID NO:40 is added upstream of the DNA molecule with the nucleotide sequence SEQ ID NO:28, resulting in a new DNA molecule designated 28'. DNA molecule 28' is used in place of the fragment between the HindIII and XhoI enzyme cleavage recognition sites (the small fragment between the HindIII and XhoI enzyme cleavage recognition sites) of the expression vector pCGS3 (Merck), without changing other nucleotide sequences of the expression vector pCGS3. The resulting recombinant expression plasmid is sequenced and identified as correct, and then designated pCGS3-015-2a. A signal peptide with the nucleotide sequence SEQ ID NO:40 is added upstream of the DNA molecule with the nucleotide sequence SEQ ID NO:35, resulting in a new DNA molecule designated 35'. The DNA molecule 35' was used in place of the fragment between the BstBI and PacI enzyme recognition sites of the recombinant expression plasmid pCGS3-015-2a (the small fragment between the BstBI and PacI enzyme recognition sites), without changing any other nucleotide sequence of the recombinant plasmid pCGS3-015-2a. The resulting recombinant expression plasmid was sequenced and confirmed to be correct, and then named pCGS3-015-2. The recombinant expression plasmid expresses a protein with the amino acid sequence of SEQ ID NO: 12 and a protein with the amino acid sequence of SEQ ID NO: 19, and the two proteins form a dimer HL-015-2.

[0054] IL-15-Linker Fc and different lengths of IL15Ra extracellular region-with or without linker-Fc construct different heterodimeric molecules using a knob into a hole. The Fc fragment has the knob amino acid sequence information shown in SEQ ID NO: 9 and the nucleotide sequence shown in SEQ ID NO: 37, and the hole amino acid sequence information shown in SEQ ID NO: 10 and the nucleotide sequence shown in SEQ ID NO: 38. Here, the heterodimeric protein HL-015-1 has the amino acid sequences shown in SEQ ID NO: 11 and SEQ ID NO: 20 and the nucleotide sequences shown in SEQ ID NO: 27 and SEQ ID NO: 36, respectively; the heterodimeric protein HL-015-2 has the amino acid sequences shown in SEQ ID NO: 12 and SEQ ID NO: 19 and the nucleotide sequences shown in SEQ ID NO: 28 and SEQ ID NO: 35; and the heterodimeric protein HL-015-3 has the amino acid sequences shown in SEQ ID NO: 12 and SEQ ID NO: 20 and the nucleotide sequences shown in SEQ ID NO: 28 and SEQ ID NO: 36, respectively.

[0055] The construction process of the recombinant expression plasmids of HL-015-1 and HL-015-3 was similar to that of HL-015-2, the only difference being the corresponding replacement of the coding gene of interest.

[0056] A positive control is also prepared as follows.

[0057] 1) The coding gene for the ALT-803 molecule (the ALT-803 molecule is named ALT-803 in the patent and is a dimer consisting of proteins with amino acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4) was obtained from patent CN103370339A.

[0058] 2) Referring to the literature 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 consisting of proteins with amino acid sequences of SEQ ID NO: 5 and SEQ ID NO: 6).

[0059] 3) Referring to the literature Scientific Reports (2018) 8:7675, the HRP00018 molecule, in which IL-15-Fc and IL-15Ra-sushi domain-Fc are linked via a knob-into-hole, was named HR-2 (a dimer consisting of proteins with amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 8).

[0060] The sequence of IL-15 in HL-015-1 (positions 1 to 114 of SEQ ID NO: 11) is identical to the sequence of IL-15 in HR-2 (positions 1 to 114 of SEQ ID NO: 7), and the sequence of the IL-15Rα subunit in HL-015-1 (positions 1 to 78 of SEQ ID NO: 20) is four amino acids longer (sequence: PAPP) than the sequence of IL-15Rα in HR-2 (positions 1 to 74 of SEQ ID NO: 8).

[0061] The IL-15Rα in HL-015-2 has 13 more amino acids (sequence: DPALVHQRPAPPS) than the sushi domain of IL-15Rα in ATL-803 (positions 1 to 65 of SEQ ID NO: 6).

[0062] HL-015-3 differs from HL-015-1 in that the IL-15 in HL-015-3 adopts the N72D mutation, but differs from HL-015-2 in that HL-015-3 has an increased (G4S)3 linker between IL-15Rα and Fc compared to HL-15-2.

[0063] 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. [Table 1]

[0064] Example 2: Protein expression and purification According to the instructions for the Thermofisher ExpiCHO® Expression System Kit, the recombinant expression plasmids pCGS3-015-1, pCGS3-015-2, pCGS3-015-3, pCGS3-ALT-803, pCGS3-HR-1, and pCGS3-HR-2 in Example 1 were transfected, respectively. 200 mL of each recombinant expression plasmid was transfected, the amount of plasmid used was 0.7 μg / mL, and the culture time was 10 days. The cell density increased significantly at the beginning of culture and then slowly decreased. The cell viability was high, and when samples were harvested on the 10th day, the cell viability was still 80%.

[0065] The supernatant was collected by centrifugation and filtered under vacuum. The mixture was purified using MabSelect SuRe LX (manufacturer: Cytiva, product number 17-5474-02) (CIP buffer: 0.1 M NaOH, equilibration buffer: 20 mM PB, 0.15 M NaCl, pH 7.0, leaching buffer 1: 20 mM PB, 1 M NaCl, pH 7.0, leaching 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 loaded onto a 3% volume of Superdex 200 (manufacturer: Cytiva, product number V521276) for purification (equilibration and elution buffer: 20 mmol / L PB, 150 mmol / L). The desired peaks were collected in a 5% NaCl (pH 7.0) tube, and the purity was determined by SEC-HPLC. The protein concentrations were then determined by UV spectrophotometer according to the theoretical extinction coefficients 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.

[0066] Example 3: Measurement of the effect of heterodimeric proteins on the proliferation of CTLL-2 and Mo7e cells Cytokine-dependent CTLL2 cells (Suzhou Pinnacle Biopharmaceutical Co., Ltd., product number TCM-C724) were cultured in RPMI 1640 medium supplemented with 200 U / ml IL-2 and 10% fetal bovine serum at 37°C under 5.0% CO2 until logarithmic growth phase. Cells were harvested by centrifugation at 1000 rpm for 5 minutes and washed three times with phosphate-buffered saline. Cells were then resuspended in RPMI 1640 medium containing 10% fetal bovine serum (assay medium) and added to a 96-well cell culture plate at 5000 cells per well. After 4 hours of culture (cytokine starvation), the heterodimeric proteins HL-015-1, HL-015-2, HL-015-3, ALT-803, HR-1, and HR-2 from Example 2 were each gradient diluted with PBS to obtain heterodimeric protein solutions. 10 μL of heterodimeric protein solution was added per well. Eleven treatment concentrations were set for each heterodimeric protein. The heterodimeric protein concentrations in these eleven treatment wells were 500ng / ml, 166.67ng / ml, 55.56ng / ml, 18.52ng / ml, 6.17ng / ml, 2.06ng / ml, 0.69ng / ml, 0.23ng / ml, 0.076ng / ml, 0.025ng / ml, and 0.0085ng / ml, respectively. A negative control (i.e., 0ng / ml) containing 10µl of PBS was added per well. Each concentration was set up in duplicate, and after incubation for 2 days, CCK8 was added and the plate was incubated at 37°C, 5.0% CO2 for 2 hours. The plate was then read at 450nm and 630nm to monitor cell growth. The experiment was repeated three times.

[0067] The test method for stimulating the proliferation of Mo7e cells (Zhejiang Meisen Cell Technology Co., Ltd., product number CTCC-001-0368) with IL-15 / IL-15Ra dimeric protein was similar to that for CTLL-2. Mo7e cells were cultured in RPMI 1640 medium supplemented with 8 ng / ml GM-CSF and 10% fetal bovine serum until logarithmic growth phase. The cells were harvested and washed, then resuspended in RPMI 1640 medium without GM-CSF and added to a 96-well plate at 20,000 cells per well. After 4 hours of culture, the heterodimeric proteins HL-015-1, HL-015-2, HL-015-3, ALT-803, HR-1, and HR-2 from Example 2 were each gradient diluted with PBS to obtain heterodimeric protein solutions, and 10 μl of the heterodimeric protein solution was added per well. Eight treatment concentrations of each heterodimeric protein were prepared. The heterodimeric protein concentrations in the eight 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. A negative control (i.e., 0 ng / ml) was added to each well with 10 μl of PBS. Each concentration was treated with duplicate wells and incubated at 37°C and 5.0% CO2 for 4 days. After 4 days, CCK8 (20 μl / well) was added and the plates were incubated at 37°C and 5.0% CO2 for 2 hours. Cell growth was monitored by reading the plates at 450 nm and 630 nm. Experiments were repeated three times. The results show that HL-015-2 has the best ability to stimulate both CTLL-2 and Mo7e cell proliferation (see Figures 1 and 2, where logarithms are base 10). HL-015-1 is superior to HR-2, and the differences are primarily due to the fact that the IL-15Rα subunit in HL-015-1 contains four more amino acids than the IL-15Rα in HR-2. The IL-15Rα in HL-015-02 also contains 13 more amino acids (DPALVHQRPAPPS) than the sushi domain of the IL-15Rα in ATL-803 (positions 1-65 of SEQ ID NO: 6).

[0068] Example 4: Design of IL-15 mutants, and preparation and activity comparison of heterodimeric proteins Based on the experimental results of Examples 2 and 3, we selected the IL-15 N72D mutation shown in SEQ ID NO: 3 and the IL-15Rα-Fc shown in SEQ ID NO: 19 and decided to continue the following experiments.

[0069] Using relevant structural software, the structures of IL-15, IL-15Rα, and IL-15Rβ / γ were analyzed to find amino acid sites that could affect IL-15. Computer modeling and molecular docking were then performed to select amino acid mutations that could enhance IL-15 activity, and the corresponding genes were synthesized. The amino acid mutation sites obtained through screening are shown in Table 2. [Table 2]

[0070] A DNA molecule having the nucleotide sequence of SEQ ID NO: 34 and a DNA molecule having the nucleotide sequence of SEQ ID NO: 35 were synthesized. A signal peptide having the nucleotide sequence of SEQ ID NO: 40 was added upstream of the DNA molecule having the nucleotide sequence of SEQ ID NO: 34, resulting in a new DNA molecule designated 34'. The DNA molecule 34' was used in place of the fragment between the HindIII and XhoI enzyme cleavage recognition sites (the small fragment between the HindIII and XhoI enzyme cleavage recognition sites) of the expression vector pCGS3 (Merck), without changing other nucleotide sequences of the expression vector pCGS3. The resulting recombinant expression plasmid was sequenced and confirmed to be correct, and then designated pCGS3-015-9a. A signal peptide having the nucleotide sequence of SEQ ID NO: 40 was added upstream of the DNA molecule having the nucleotide sequence of SEQ ID NO: 35, resulting in a new DNA molecule designated 35'. The DNA molecule 35' was used in place of the fragment between the BstBI and PacI enzyme recognition sites of the recombinant expression plasmid pCGS3-015-9a (the small fragment between the BstBI and PacI enzyme recognition sites), without changing any other nucleotide sequence of the recombinant plasmid pCGS3-015-9a. The resulting recombinant expression plasmid was sequenced and confirmed to be correct, and then designated 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, constituting the heterodimeric protein HL-015-9.

[0071] The construction method of the recombinant expression plasmids HL-015-4 to HL-015-8 was the same as that of HL-015-9, with the only differences being the following points. The HL-015-9 gene is correspondingly replaced to obtain recombinant expression plasmids pCGS3-HL-015-4 for the HL-015-4 gene (nucleotide sequences of which are SEQ ID NO:29 and SEQ ID NO:35, respectively), pCGS3-HL-015-5 for the HL-015-5 gene (nucleotide sequences of which are SEQ ID NO:30 and SEQ ID NO:35, respectively), pCGS3-HL-015-6 for the HL-015-6 gene (nucleotide sequences of which are SEQ ID NO:31 and SEQ ID NO:35, respectively), pCGS3-HL-015-7 for the HL-015-7 gene (nucleotide sequences of which are SEQ ID NO:32 and SEQ ID NO:35, respectively), and pCGS3-HL-015-8 for the HL-015-8 gene (nucleotide sequences of which are SEQ ID NO:33 and SEQ ID NO:35, respectively). pCGS3-HL-015-4 expresses the protein whose amino acid sequence is SEQ ID NO: 13 and the protein whose amino acid sequence is SEQ ID NO: 19, and the two proteins form the heterodimeric protein HL-015-4. pCGS3-HL-015-5 expresses the protein whose amino acid sequence is SEQ ID NO: 14 and the protein whose amino acid sequence is SEQ ID NO: 19, and the two proteins form the heterodimeric protein HL-015-5. pCGS3-HL-015-6 expresses the protein whose amino acid sequence is SEQ ID NO: 15 and the protein whose amino acid sequence is SEQ ID NO: 19, and the two proteins form the heterodimeric protein HL-015-6. pCGS3-HL-015-7 expresses the protein whose amino acid sequence is SEQ ID NO: 16 and the protein whose amino acid sequence is SEQ ID NO: 19, and the two proteins form the heterodimeric protein HL-015-7. pCGS3-HL-015-8 expresses the protein whose amino acid sequence is SEQ ID NO: 17 and the protein whose amino acid sequence is SEQ ID NO: 19, and the two proteins constitute the heterodimeric protein HL-015-8.

[0072] 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. [Table 3]

[0073] Expression and protein purification of the recombinant expression plasmids pCGS3-HL-015-4 to pCGS3-HL-015-9 were carried out according to the method in Example 2. Subsequently, activity measurements were carried out according to the method in Example 3. The results are shown in Figures 3 and 4 (logarithms in Figures 3 and 4 are base 10). Of all the mutants, HL-015-9 had the highest stimulatory activity against CTLL-2 or Mo7e proliferation.

[0074] In the CTLL-2 cell proliferation experiment, heterodimeric proteins HL-015-4, HL-015-5, HL-015-6, HL-015-7, HL-015-8, and HL-015-9 were diluted with PBS to obtain heterodimeric protein solutions, and 10 μl of the heterodimeric protein solution was added to each well. Eight treatment concentrations were set up for each heterodimeric protein, with the heterodimeric protein concentrations in the eight treatment wells being 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 performed in triplicate. A negative control (i.e., 0 ng / ml) containing 10 μl of PBS was set up per well, and duplicate wells were set up for each concentration. The remaining procedures were the same as in Example 3.

[0075] In Mo7e cell proliferation experiments, heterodimeric proteins HL-015-4, HL-015-5, HL-015-6, HL-015-7, HL-015-8, and HL-015-9 were diluted with PBS to obtain heterodimeric protein solutions, and 10 μl of the heterodimeric protein solution was added to each well. Eight treatment concentrations were set up for each heterodimeric protein, with the heterodimeric protein concentrations in the 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. Experiments were performed in triplicate. A negative control (i.e., 0 ng / ml) containing 10 μl of PBS was set up per well, and duplicate wells were set up for each concentration. The remaining procedures were the same as in Example 3.

[0076] Example 5: Proliferation-stimulating effect of heterodimeric proteins on NK cells, CD8+ T cells, and CD4+ T cells NK cells, CD8+ T cells, and CD4+ T cells were isolated from human peripheral blood cells using NK cell (CD56+, CD3-), CD8+ T cell (CD3+, CD8+), and CD4+ T cell (CD3+, CD8+) isolation reagent kits, respectively. The isolated cells were cultured overnight in RPMI 1640 medium containing 10% fetal bovine serum at 37°C under 5.0% CO2. The cells were harvested and incubated with 5,6-carboxyfluorescein diacetate succinimidyl ester dye (CFSE) at 37°C for 15 minutes. They were then centrifuged, washed, and resuspended in RPMI 1640 medium containing 10% fetal bovine serum. 10,000 cells per well were added to a 96-well cell culture plate containing various concentrations of heterodimer solutions, which had been diluted with PBS. The heterodimer solutions were designated ALT-803, HL-015-6, and HL-015-9, respectively. After 3 days of culture, the number of CFSC-positive cells was analyzed by flow cytometry to determine the growth effects of different IL-15 / IL-15Rα heterodimers on different cells. As can be seen from Figures 5, 6, and 7, HL-015-9 had the highest stimulatory activity on NK cell, CD4+ T cell, and CD8+ T cell proliferation.

[0077] In the NK cell proliferation experiment, the dimeric proteins were diluted in gradient with PBS to 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, and the experiment was repeated three times. A negative control (i.e., 0 ng / ml) containing 10 μl of PBS was set up per well, and duplicate wells were set up for each concentration.

[0078] In the CD4+ cell proliferation experiments, three experiments were conducted according to the type of dimeric protein. After gradient dilution with PBS, the dimeric proteins ALT-803, HL-015-6, and HL-015-9 were diluted to 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. Experiments were conducted in triplicate. A negative control (i.e., 0 ng / ml) containing 10 μl of PBS was set up per well, and duplicate wells were set up for each concentration.

[0079] For CD8+ cell proliferation experiments, three experiments were conducted, each containing a different dimeric protein. After gradient dilution with PBS, the dimeric proteins ALT-803, HL-015-6, and HL-015-9 were diluted to 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. Experiments were repeated three times. A negative control (i.e., 0 ng / ml) containing 10 μl of PBS was run per well, and negative control duplicate wells were run.

[0080] Example 6. Effect of heterodimeric proteins on NK cell killing of K562 K562 cells (Wuhan Procell, product number CL-0130) were cultured in RPMI 1640 medium containing 5% fetal bovine serum at 37°C under 5.0% CO2 for 48 hours, then BATDA staining solution was added and incubated at 37°C for 25 minutes, centrifuged, and washed three times with medium. The marked K562 cells were added to a 96-well plate at 5,000 cells per well and cultured in medium (RPMI 1640 medium containing 5% fetal bovine serum) at a ratio of 1:10 between target and effector cells. NK cells were co-cultured for 72 hours with 1640 medium (100ng / ml of the heterodimeric proteins ALT-803, HR-1, HL-015-9, HL-015-6, and HL-015-4). The target and effector cells were incubated at 37°C and 5.0% CO for 4 hours. The supernatant was then centrifuged and added to a europium solution. The resulting solution was then assayed for fluorescence intensity. NK cells cultured in medium alone served as the control. A blank control and a maximum release group were also set up. (The maximum release group was prepared by adding 0.05% Triton X to the blank control group, incubating the blank group, and then adding europium for detection.) The killing rate was calculated using the following formula: killing rate (%) = (mean value of experimental group - mean value of blank control group) / (mean value of maximum release group - mean value of blank control group). The experimental results are shown in Figure 8. All of the various IL-15 / IL-15Ra complexes were able to significantly enhance the killing activity of NK cells against K562, with HL-015-9 showing the best activity.

[0081] Example 7: Evaluation of the efficacy of heterodimeric proteins in vivo BALB / c mice (7-9 weeks old, female, weight range 17-23g, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., animal batch number 110011211108488925) were subcutaneously inoculated with CT26 cells (a mouse colon cancer cell line, cultured in RPMI 1640 medium containing 10% fetal bovine serum and stored at the National Institute of Radiological Sciences, China) to establish a subcutaneous colon cancer model. When tumor volumes reached 60-100 mm3, mice were randomly assigned to seven groups (seven tumor-bearing mice per group). The first day of administration was day 1, with treatment cycles of days 1, 4, 8, and 11. The administration volume was 10 μl / g body weight. Tumor volumes were measured twice weekly and calculated using the following formula: tumor volume (mm3). 3 )=1 / 2×(a×b 2 ) (where a represents the major axis and b represents the minor axis).

[0082] The heterodimeric protein ALT-803 was dissolved in a buffer to obtain a heterodimeric protein ALT-803 solution, and the heterodimeric protein HL-015-9 was dissolved in a buffer to obtain a heterodimeric protein HL-015-9 solution, where the buffer was 0.9% saline for injection.

[0083] Here, the control group was the buffer group, and each mouse was intraperitoneally injected with 10 μl / g body weight of buffer. The first group is ALT-803, 0.05 mg / kg group, each mouse is intraperitoneally injected with 10 μl / g body weight of heterodimeric protein ALT-803 solution, the dosage of heterodimeric protein ALT-803 is 0.05 mg / kg body weight; The second group is ALT-803, 0.15 mg / kg group, each mouse is intraperitoneally injected with heterodimeric protein ALT-803 solution at 10 μl / g body weight, the dosage of heterodimeric protein ALT-803 is 0.15 mg / kg body weight; The third group is ALT-803, 0.6 mg / kg group, each mouse is intraperitoneally injected with 10 μl / g body weight of heterodimeric protein ALT-803 solution, the dosage of heterodimeric protein ALT-803 is 0.6 mg / kg body weight; The fourth group is HL-015-9, 0.05 mg / kg group, each mouse is intraperitoneally injected with 10 μl / g body weight of heterodimeric protein HL-015-9 solution, the dosage of heterodimeric protein HL-015-9 is 0.05 mg / kg body weight; The fifth group is HL-015-9, 0.15 mg / kg group, each mouse is intraperitoneally injected with 10 μl / g body weight of heterodimeric protein HL-015-9 solution, the dosage of heterodimeric protein HL-015-9 is 0.15 mg / kg body weight; The sixth group was HL-015-9, 0.6 mg / kg group, in which the heterodimeric protein HL-015-9 solution was intraperitoneally injected at 10 μl / g body weight for each mouse, so that the dosage of the heterodimeric protein HL-015-9 was 0.6 mg / kg body weight.

[0084] The results are shown in Figure 9, which shows 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.

[0085] The present invention has been described in detail above. Those skilled in the art will be able to practice the present invention within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without undue experimentation. Although the present invention has been described with specific examples, it should be understood that the present invention can be further improved. In short, in accordance with the principles of the present invention, this application is intended to include any modifications, uses, or improvements to the present invention that depart from the scope disclosed in this application and include modifications made by ordinary techniques known in the art. Some basic features may be applied according to the scope of the following appended claims.

[0086] Industrial Applications The present invention provides an IL-15 variant-Fc / IL-15R α subunit-Fc heterodimeric protein, wherein the heterodimeric protein comprises protein a and protein b, where protein a may be the protein having the amino acid sequence of SEQ ID NO: 18, and protein b may be the protein having the amino acid sequence of SEQ ID NO: 19. Experiments have demonstrated that the IL-15 variant-Fc / IL-15R α subunit-Fc heterodimeric protein and related biomaterials provided by the present invention can effectively stimulate the proliferation of NK cells and T cells, and in vivo experiments have shown that the dimeric protein HL-015-9 is more effective than ALT-803 in inhibiting tumor growth at equivalent injection doses. Therefore, the IL-15 variant-Fc / IL-15R α subunit-Fc heterodimeric protein provided by the present invention can be used in the preparation of drugs or formulations for treating tumors and / or viral infections.

Claims

1. a protein a and a protein b, wherein the protein a comprises an IL-15 variant and a first Fc variant; the protein b comprises an IL-15Rα subunit sushi domain and another fragment of the IL-15Rα subunit and a second Fc variant; the first Fc variant and the second Fc variant are selected from Knob-modified Fc or Hole-modified Fc, and the type of Knob modification or Hole modification of the first Fc variant and the second Fc variant are different; the IL-15 variant is a polypeptide having an amino acid sequence selected from positions 1 to 111 of SEQ ID NO: 18, positions 1 to 111 of SEQ ID NO: 15, positions 1 to 114 of SEQ ID NO: 14, positions 1 to 114 of SEQ ID NO: 17, positions 1 to 114 of SEQ ID NO: 16, and positions 1 to 114 of SEQ ID NO: 13; A heterodimeric protein in which the IL-15Rα subunit sushi domain and another fragment of the IL-15Rα subunit are a polypeptide having the amino acid sequence of SEQ ID NO:

2.

2. The heterodimeric protein of claim 1, wherein protein a further comprises a linker connecting the IL-15 variant and the first Fc variant, the linker being (GGGGS)n, where n is a natural number from 0 to 4.

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

4. 4. The heterodimeric protein according to claim 1, wherein protein a comprises the IL-15 mutant, a linker linked to the C-terminus of the IL-15 mutant, and a first Fc mutant linked to the C-terminus of the linker, and protein b comprises the IL-15Rα subunit sushi domain and other fragments, and a second Fc mutant linked to the C-terminus of the IL-15Rα subunit sushi domain and other fragments.

5. 5. The heterodimeric protein of claim 1, wherein the first Fc variant or the second Fc variant is an Fc of antibody IgG1, IgG2, IgG3 or IgG4 or a variant thereof.

6. 6. The heterodimeric protein of claim 1, wherein the first Fc variant or the second Fc variant is an Fc of an antibody IgG4 or a variant thereof.

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

9.

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

10.

9. The protein a is A1) a protein having the amino acid sequence SEQ ID NO: 18; A2) a protein having the amino acid sequence SEQ ID NO: 15; A3) a protein having the amino acid sequence SEQ ID NO: 14; A4) a protein having the amino acid sequence of SEQ ID NO: 17; A5) a protein having the amino acid sequence of SEQ ID NO: 16; A6) a protein having the amino acid sequence of SEQ ID NO: 13; A7) A fusion protein in which a protein tag is linked to the N-terminus and / or C-terminus of the protein according to any one of A1) to A6); 9. The heterodimeric protein according to claim 1 , wherein the heterodimeric protein is any one of the following:

10. The protein b is A8) a protein having the amino acid sequence SEQ ID NO: 19; A9) a protein having the amino acid sequence of SEQ ID NO: 20; A10) A fusion protein in which a protein tag is linked to the N-terminus and / or C-terminus of the protein according to A8) or A9); 10. The heterodimeric protein according to claim 1, wherein the heterodimeric protein is any one of the following:

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

12. A product, the active ingredient of which is the heterodimeric protein according to any one of claims 1 to 10, C1) preventing and / or treating a disease; C2) activating the immune system of the organism; and C3) Raising or enhancing the immune function of an organism; and C4) inhibiting tumor growth; and C5) improving the proliferation activity of NK cells and / or CD8+ T cells and / or CD4+ T cells; C6) Improving the ability of NK cells to kill tumor cells; A product that has one of the following uses.

13. A pharmaceutical composition comprising the heterodimeric protein according to any one of claims 1 to 10 and another drug.

14. The pharmaceutical composition of claim 13, wherein the other drug may be at least one of an immune checkpoint drug, a cell adhesion molecule bispecific antibody, and a cell therapy product.

15. D1) Preparation of a product for preventing and / or treating a disease; D2) Preparation of a product that activates the organism's immune system; D3) Preparation of a product that enhances or increases immune function of an organism; D4) Preparation of a product that inhibits tumor growth; D5) Preparation of a product that improves the proliferation activity of NK cells and / or T cells; D6) Preparation of a product that enhances the ability of NK cells to kill tumor cells; D7) preventing and / or treating diseases; D8) activating the immune system of animals; D9) Raising or enhancing the immune function of the organism; D10) inhibiting tumor growth; and D11) Improving the proliferation activity of NK cells and / or T cells; D12) Improving the ability of NK cells to kill tumor cells; 15. The application of a heterodimeric protein according to any one of claims 1 to 10, or a biomaterial according to claim 11, or a product according to claim 12, or a pharmaceutical composition according to claim 13 or 14, to any one of the following:

16. 16. The method of claim 15, wherein the disease comprises an infectious disease, a tumor, a blood disease, an inflammatory disease, and an autoimmune disease.

17. A method for treating a disease, comprising administering to a patient a heterodimeric protein described in any one of claims 1 to 10, a biomaterial described in claim 11, a product described in claim 12, or a pharmaceutical composition described in claim 13 or 14, and treating the patient.

18. 18. The method of claim 17, wherein the disease comprises an infectious disease, a tumor, a blood disease, an inflammatory disease, and an autoimmune disease.

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