IL-15 agonists for cancer treatment

Abstract

The invention provides an IL-15 / IL-15R alpha-Fc fusion protein. The IL-15 / IL-15R alpha-Fc fusion protein comprises an IL-15R alpha-sushi structural domain, an IL-15C terminal structural domain and an IL-15N terminal structural domain, and an Fc domain. Further provided are methods of using such IL-15 / IL-15R alpha-Fc in the treatment of diseases and disorders, such as cancer.

Description

Technical Field

[0001] The present invention relates to interleukin-15 (IL-15) agonists and methods of use thereof. Specifically, the present invention provides an IL-15 / IL-15 receptor α (IL-15Rα)-Fc fusion protein complex comprising an IL-15Rαsushi domain, an IL-15 domain and an Fc domain for use in treating cancer. Background Art

[0002] Cancer immunotherapy is a promising cancer treatment approach. However, it is currently limited by the poor properties of similar drugs, severe dose-limiting toxicity, and disease recurrence. Therefore, there is still a need for an effective composition that can enhance and direct the activity against immune cancer cells with limited toxicity. The present invention provides a novel IL-15 / IL-15Rα-Fc fusion protein that can activate effector immune responses to treat cancer. Summary of the invention

[0003] The present invention provides an IL-15 / IL-15Rα-Fc fusion protein, comprising from N-terminus to C-terminus: (a) an IL-15-Rαsushi domain; (b) an IL-15C-terminal domain; (c) an IL-15N-terminal domain; and (d) an Fc domain.

[0004] In some aspects, the IL-15Rαsushi domain comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 8-11. In some aspects, the IL-15Rαsushi domain comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 8-11.

[0005] In some aspects, the IL-15 C-terminal domain comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, or at least about 98% identical to SEQ ID NO:3 or SEQ ID NO:4.

[0006] In some aspects, the IL-15 N-terminal domain comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, or at least about 98% identical to SEQ ID NO: 5 or SEQ ID NO: 6. In some aspects, the IL-15 N-terminal domain comprises an N72D mutation.

[0007] In some aspects, the Fc domain is an IgG1 Fc domain having an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 12. In some aspects, the Fc domain is an IgG1 Fc domain comprising the amino acid sequence of SEQ ID NO: 12.

[0008] In some aspects, the Fc domain is an IgG4 Fc domain having an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 17. In some aspects, the Fc domain comprises an IgG4 Fc domain of the amino acid sequence of SEQ ID NO: 17.

[0009] In some aspects, the IgG1 or IgG4 Fc domain comprises at least one amino acid substitution. In some aspects, at least one amino acid substitution is N297A (EU numbering). In some aspects, at least one amino acid substitution is L234A, L235A and / or P329G (EU numbering). In some aspects, at least one amino acid substitution is M252Y, S254T, T256E (EU numbering). Or a combination thereof. In some aspects, at least one amino acid substitution is L234A, L235A or a combination thereof.

[0010] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein includes one or more linkers. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein includes a linker between the IL-15C-terminal domain and the IL-15N-terminal domain. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein includes a linker between the IL-15N-terminal domain and the Fc domain. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein includes a linker between the IL-15C-terminal domain and the IL-15N-terminal domain; and a linker between the IL-15N-terminal domain and the Fc domain. In some aspects, the length of one or more linkers is 0, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In some aspects, the length of one or more linkers is 0 or 5 amino acids. In some aspects, one or more linkers consist of an amino acid sequence selected from the group consisting of GG, GS, GGS, GGGS, and GGGGS.

[0011] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with any one of SEQ ID NOs: 20-30. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an amino acid sequence of any one of SEQ ID NOs: 20-30. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein consists of any one of the amino acid sequences of SEQ ID NOs: 20-30.

[0012] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an amino acid sequence having one or more mutations of any one of SEQ ID NOs: 20-30. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an amino acid sequence of SEQ ID NO: 24 having one or more mutations selected from the group consisting of: V3A, V3L, N4A, N4D, N4Q, S7D, S7V, D8T, D8A, K11A, D30N, D61A, D61S, D61N, D61E, E64Q, N65D, I68A, I68V, L69A, L69V, N72E and N72A (the above amino acid positions all correspond to the natural ordering positions of amino acids on SEQ ID NO: 5).

[0013] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 28, and one or more mutations selected from N189Q (referring to the mutation of N at position 189 in SEQ ID NO: 28 to G) and G190A (referring to the mutation of G at position 190 in SEQ ID NO: 28 to A). In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 30, and one or more mutations selected from N189Q and G190A.

[0014] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein binds to IL-2 receptor β and / or IL-2 receptor γ. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein binds to IL-2 receptor β with an affinity of about 0.1 nM to about 25 nM, about 0.5 nM to about 10 nM, or about 1 nM to about 5 nM.

[0015] In some aspects, the present invention provides a polynucleotide encoding the above-mentioned IL-15 / IL-15Rα-Fc fusion protein. In some aspects, the present invention provides an expression vector comprising the polynucleotide. In some aspects, the present invention provides a host cell comprising the polynucleotide or the expression vector.

[0016] In some aspects, the present invention provides a pharmaceutical composition comprising the above-mentioned IL-15 / IL-15Rα-Fc fusion protein and at least one pharmaceutically acceptable carrier or excipient.

[0017] In some aspects, the present invention provides a method for treating cancer, comprising administering an effective amount of the IL-15 / IL-15Rα-Fc fusion protein or a pharmaceutical composition thereof to a subject in need of cancer treatment.

[0018] In some aspects, the cancer is a solid tumor. In some aspects, the cancer is metastatic. In some aspects, the cancer is recurrent or refractory. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1A-Figure 1C An example of a three-dimensional X-ray diffraction (XRD) structure of an IL-15 complex (PDB: 4GS7) is shown. Figure 1A and Figure 1B The combined ribbon structure and surface views of the IL-15 quaternary complex are shown from different angles. Figure 1C The interface of IL-15 and IL-15Rα in the quaternary complex is shown. The dashed line indicates where the linker connects the IL-15 and IL-15Rα domains.

[0020] Figure 2 Exemplary structural designs of IL-15 / IL-15Rα-Fc fusion proteins of the present invention are shown compared to a standard reference structure.

[0021] Figure 3 The binding of IL-15 / IL-15Rα-Fc fusion protein to IL-2Rβ (CD122) is demonstrated.

[0022] Figure 4 The effect of IL-15 / IL-15Rα-Fc fusion protein on NK cell proliferation was demonstrated.

[0023] Figure 5 The effect of IL-15 / IL-15Rα-Fc fusion protein on NK cell cytotoxicity was demonstrated.

[0024] Figure 6The effect of IL-15 / IL-15Rα-Fc fusion protein on NK cell degranulation was demonstrated. NK cells were treated with different concentrations of IL-15 / IL-15Rα-Fc fusion protein overnight. NK cell degranulation was analyzed using anti-CD107a antibody and flow cytometry.

[0025] Figure 7A-7B The effects of IL-15 / IL-15Rα-Fc fusion protein on NK and CD8+ T cell signaling are shown. Fig. 7A Shown is the phosphorylation of STAT5 after primary NK cells were treated with 0.5 nM IL-15 / IL-15Rα-Fc fusion protein overnight. Figure 7B The phosphorylation of STAT5 is shown after primary CD8+ T cells were treated with 0.5 nM IL-15 / IL-15Rα-Fc fusion protein overnight. STAT5 phosphorylation was analyzed using anti-phospho-STAT5 antibody and flow cytometry.

[0026] Fig. 8A Shown are the effects of treating tumor-bearing mice with IL-15 / IL-15Rα-Fc fusion protein for 15 days on tumor growth and metastasis. Figure 8B Shown are the effects of treating tumor-bearing mice with IL-15 / IL-15Rα-Fc fusion protein for 21 days on tumor growth and metastasis.

[0027] Fig.9A Shown are exemplary effects of IL-15 / IL-15Rα-Fc fusion protein treatment on tumor growth in lungs resected from tumor mice compared to controls. Fig. 9B Shown are exemplary effects of IL-15 / IL-15Rα-Fc fusion protein on metastatic tumors compared to controls. Molecule V1 (SEQ ID NO: 31) is a standard IL-15 / IL-15Rα-Fc fusion protein used for reference.

[0028] Fig.10 The Kaplan-Meier survival curves of tumor-bearing mice given different concentrations of IL-15 / IL-15Rα-Fc fusion protein molecule Z1 compared with controls are shown. Molecule V1 is a standard IL-15 / IL-15Rα-Fc fusion protein for reference.

[0029] Fig.11 The data show that compared with the control group, the inhibition rate of B16F10-Luc lung metastasis tumors in the monotherapy group of IL-15 / IL-15Rα-Fc fusion protein molecule Y1 at doses of 0.125, 0.25 and 0.5 mg / kg. Detailed description definition

[0030] The term "a" refers to one or more entities, i.e., may refer to plural referents. Therefore, the terms "a", "one or more", and "at least one" may be used interchangeably herein. In addition, the use of the indefinite article "a" or "an" to refer to "an element" does not exclude the possibility that more than one element is present, unless the context clearly requires that there is one and only one element.

[0031] In this application, the term "about" is used to indicate that the value includes the inherent error variation of the equipment or method used to determine the value, or the variation between the samples tested. Unless otherwise stated or obvious from the context, the term "approximately" means within 10% above or below the reported value (unless the number will exceed 100% or be below 0% of the possible value). When used in conjunction with a numerical range or series, the term "about" applies to the endpoints of the range or each value listed in the series unless otherwise stated. In this application, the terms "about" and "approximately" are equivalent.

[0032] As used herein, the term "sequence identity" refers to the percentage of nucleotides or amino acid sequences in a candidate sequence that are identical to the amino acid residues or nucleotides in a particular (parent) sequence, after the sequences have been aligned and gaps introduced, if necessary, to achieve the maximum percent identity. Alignment for determining percent amino acid sequence identity can be accomplished by various methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Clustal Or Megalign (DNASTAR) software. Smith and Waterman's local homology algorithm (Advances in Applied Mathematics, 2: 482-489 (1981)) provides another approximate alignment method for nucleotide sequences. This algorithm can be applied to amino acid sequences (Dayhoff, Atlas of Protein Sequences and Structure, MO Dayhoff ed., 5 suppl. 3: 353-358, National Biomedical Research Foundation, Washington, DC, USA, and normalized by Gribskov, Nucl. 14 (6): 6745-6763 (1986)) by using the scoring matrix developed by Dayhoff.

[0033] As used herein, the terms "treat", "treatment" or "therapy" and their grammatical variants have the same meaning as those generally understood by those of ordinary skill in the art. In some aspects, these terms may refer to methods for obtaining beneficial or expected clinical results. These terms may refer to slowing down the rate of occurrence or development of a condition, disorder or disease, reducing or alleviating symptoms associated therewith, causing complete or partial regression of the condition, or a combination of any of the above. For the purposes of the present invention, beneficial or expected clinical results include, but are not limited to, alleviation or relief of symptoms, alleviation of the extent of the disease, stabilization of the disease state (such as non-exacerbation), delay or slowing of disease progression, improvement or relief of the disease state, and detectable or undetectable relief (whether partial or complete relief). "Treatment", "treatment" or "therapy" may also mean prolonging survival time relative to expected survival time if not receiving treatment. Therefore, a subject (e.g., a human being) in need of treatment may be a subject who already suffers from a related disease or condition. The terms "treat", "treatment" or "therapy" include inhibiting or reducing the increase in severity of a pathological state or symptom relative to when not receiving treatment, and do not necessarily mean complete cessation of the related disease or condition.

[0034] As used herein, the terms "prevention," "prevention," and "prevention" and grammatical variants thereof refer to methods for preventing the development of a disease or condition or altering its pathology. Thus, "prevention" may refer to preventive measures. For purposes of the present invention, beneficial or expected clinical results include, but are not limited to, preventing or slowing the symptoms, progression, or development of a disease, whether detectable or undetectable. Thus, a subject (e.g., a human being) in need of prevention may be a subject that has not yet suffered from the disease or symptom of interest. The term "prevention" includes slowing the onset of a disease relative to when not receiving treatment, and does not necessarily mean permanent prevention of the disease of interest. Thus, in some cases, "prevention" or "preventing" a condition may refer to reducing the risk of developing the condition, preventing or delaying the onset of symptoms associated with the condition.

[0035] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable diluent" refers to any material, composition or carrier that is physiologically acceptable, i.e., compatible buffers, solvents, dispersion media, coatings, antimicrobial agents, isotonic agents, absorption delaying agents, solid or liquid fillers, excipients, encapsulating materials, etc. The term "pharmaceutically acceptable excipient" refers to an excipient that can be used to prepare a pharmaceutical composition, i.e., generally safe, non-toxic, and has no adverse reactions in biological or other aspects, including excipients that can be used for human medicine and veterinary use.

[0036] The term "therapeutically effective amount" as used herein refers to an amount of a compound (such as IL-15 / IL-15Rα-Fc fusion protein) that achieves the desired biological or therapeutic effect, i.e., an amount that prevents, alleviates or improves one or more symptoms of the disease being treated or prevented.

[0037] As used herein, the term "additional therapy" refers to one or more therapies that may provide a therapeutic effect or benefit to a subject in need thereof. An additional therapy may be a therapeutic agent (such as a chemotherapeutic agent) or a medical device.

[0038] As used herein, the term "subject" refers to any subject, such as a human or non-human mammal, for whom diagnosis, prognosis or treatment is desired. The term "subject" may refer to a human or non-human mammal that is affected by, may be affected by, or is suspected of being affected by a disease. The terms "subject" and "patient" may be used interchangeably herein. In some aspects, the subject is a mammal. Mammals include primates, such as humans, monkeys, chimpanzees, and apes, and non-primates, such as domestic animals, including laboratory animals (such as rabbits and rodents, such as guinea pigs, rats, or mice), household pets, and farm animals (such as cats, dogs, pigs, cattle, sheep, goats, horses, rabbits), and non-domestic animals, such as wild animals, birds, reptiles, fish, etc.

[0039] As used herein, the term "subject in need of treatment" includes subjects who may or will benefit from the methods described herein. Subjects in need of treatment include, but are not limited to, those who already have a disease or condition, those who are susceptible to a disease or condition, those who suspect they have a disease or condition, and those who need to prevent, improve or reverse a disease or condition.

[0040] The term "antigen" refers to a molecule or a portion of a molecule that can be bound by an antibody or antigen-binding fragment thereof, and can also be used to produce antibodies in an animal that can bind to an epitope of the antigen. An antigen may have one or more epitopes.

[0041] The term "activation" refers to the activation of the immune system, such as NK cells and T cells, when treated with the IL-15 / IL-15Rα-Fc fusion protein described herein. The activation of NK cells and T cells by the IL-15 / IL-15Rα-Fc fusion protein described herein can lead to enhanced development, proliferation, survival, cytotoxicity, degranulation and trafficking, thereby enhancing anti-tumor immunity and reducing tumor growth and metastasis.

[0042] The term "cytotoxicity" refers to the ability of an immune cell to kill a target cell. In some aspects, the immune cell is a CD8+T cell, a NK cell, or a NKT cell. In some aspects, the target cell is a tumor cell or a virus-infected cell.

[0043] The term "degranulation" refers to the process by which cells release cytotoxic effector molecules called granules from vesicles. Degranulation of T cells and NK cells results in the release of perforins and granzymes, which kill target cells, such as tumor or virus-infected cells. IL-15 / IL-15Rα-Fc fusion protein

[0044] The present invention relates to interleukin-15 (IL-15) agonists and methods of using the same. Specifically, the present invention provides an IL-15 / IL-15 receptor alpha (IL-15Rα)-Fc fusion protein complex, which includes, from N-terminus to C-terminus: an IL-15Rαsushi domain, an IL-15C-terminal domain, an IL-15N-terminal domain, and an Fc domain. In some aspects, the IL-15 / IL-15 receptor alpha (IL-15Rα)-Fc fusion protein of the present invention can be used to treat cancer.

[0045] IL-15 is a member of the interleukin 2 (IL-2) superfamily and plays a role in the development, survival, proliferation, and activation of multiple lymphocyte lineages, including T cells and NK cells. IL-15 binds and signals through a trimeric receptor composed of a common γ chain, IL-2Rβ, and IL-15Rα. Free IL-15 is present in low levels in the blood and is usually complexed with IL-15Rα, which increases the half-life and bioavailability of IL-15. This specific expression of IL-15 can also improve the activation of T, B, and NK cells, playing a key role in tumor immune surveillance and suppression by early recognition and destruction of malignant cells.

[0046] IL-15 may not induce activation-induced cell death (AICD) of T cells, nor enhance the proliferation, function, or differentiation of immunosuppressive CD4+ T regulatory cells (Tregs). In addition, IL-15 may not induce severe capillary leak syndrome, a toxicity associated with IL-2 therapy, in non-human primates (NHPs) or humans. Thus, IL-15 may activate effector immune cells required for tumor control while reducing associated toxicities.

[0047] Unlike recombinant IL-15, IL-15 / IL-15Rα-Fc fusion proteins may not require trans-expression or cell-to-cell contact to induce IL-15-mediated immune responses. Due to their structure, they may have greater potency, bioavailability, and stability than soluble recombinant IL-15.

[0048] An illustrative sequence of human IL-15 is provided as SEQ ID NO: 1. See also Uniprot Accession No. P40933. MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLY TESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEF LQSFVHIVQMFIN TS(SEQ ID NO:1).

[0049] An illustrative sequence of human IL-15Rα is provided as SEQ ID NO: 2. See also Uniprot Accession No. Q13261. MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLT ECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAI VPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVE MEAMEALPVTWGTSSRDEDLENCSHHL(SEQ ID NO:2). IL-15 domain

[0050] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises one or more IL-15 domains. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an IL-15C-terminal domain. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an IL-15N-terminal domain. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an IL-15C-terminal domain and an IL-15N-terminal domain. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises from N-terminus to C-terminus: an IL-15C-terminal domain and an IL-15N-terminal domain.

[0051] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an IL-15C-terminal domain. In one aspect, the IL-15C-terminal domain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% or at least about 98% sequence identity with SEQ ID NO:3 or SEQ ID NO:4. In some aspects, the IL-15C-terminal domain comprises an amino acid sequence having one or more mutations:3 or SEQ ID NO:4, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations. In some aspects, the IL-15C-terminal domain consists of an amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.

[0052] Exemplary IL-15 C-terminal domains are shown in Table 1 below. Table 1. describe Amino acid sequence SEQ ID NO. IL-15 C-terminal domain 1 NVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 3 IL-15 C-terminal domain 2 TESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 4

[0053] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an IL-15N-terminal domain. In some aspects, the IL-15N-terminal domain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with any one of SEQ ID NOs:5-7. In some aspects, the IL-15C-terminal domain comprises an amino acid sequence comprising any one of SEQ ID NOs:5-7, with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations. In some aspects, the IL-15N-terminal domain comprises one or more mutations of residues selected from the group consisting of: V3, N4, S7, D8, K11, D30, D61, E64, N65, I68, L69, N72 and any combination thereof. In some aspects, the IL-15N-terminal domain includes one or more groups selected from the following residue mutation composition: V3A, V3L, N4Q, N4A, N4D, S7D, S7V, D8T, D8A, K11A, D30N, D61A, D61S, D61N, D61E, E64Q, N65D, I68A, I68V, L69A, L69V, N72E, N72A and any combination thereof. In some aspects, the IL-15N-terminal domain includes N72D mutation. In some aspects, the IL-15N-terminal domain includes D30N mutation, E64Q mutation and N65D mutation. In some aspects, the IL-15N-terminal domain includes N4D mutation and N65D mutation. In some aspects, the IL-15N-terminal domain includes D30N mutation and N65D mutation. In some aspects, the IL-15N-terminal domain comprises an I68A mutation and an L69A mutation. In some aspects, the IL-15N-terminal domain comprises an I68A mutation and an L69V mutation. In some aspects, the IL-15N-terminal domain comprises an I68V mutation and an L69A mutation. In some aspects, the IL-15N-terminal domain comprises an I68V mutation and an L69V mutation. In some aspects, the IL-15N-terminal domain comprises an I68A mutation and an S7V mutation. In some aspects, the IL-15N-terminal domain comprises an I68A mutation and an S7D mutation. In some aspects, the IL-15N-terminal domain comprises an L69A mutation and an S7V mutation. In some aspects, the IL-15N-terminal domain comprises an L69A mutation and an S7D mutation. In some aspects, the IL-15N-terminal domain comprises a D61A mutation and an L69A mutation. In some aspects, the IL-15N-terminal domain comprises a D61A mutation and an I68A mutation. In some aspects, the IL-15 N-terminal domain comprises a D61A mutation and a S7V mutation. In some aspects, the IL-15 N-terminal domain comprises a D61A mutation and a S7D mutation.In some aspects, the IL-15 N-terminal domain comprises a D61N mutation and a L69A mutation. In some aspects, the IL-15 N-terminal domain comprises a D61N mutation and an I68A mutation. In some aspects, the IL-15 N-terminal domain comprises a D61S mutation and an L69A mutation. In some aspects, the IL-15 N-terminal domain comprises a D61S mutation and an I68A mutation.

[0054] In some aspects, the IL-15 N-terminal domain comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. In some aspects, the IL-15 N-terminal domain consists of the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

[0055] Exemplary IL-15 N-terminal domains are shown in Table 2 below. Table 2. IL-15Rα domain

[0056] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an IL-15Rα domain. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an IL-15Rαsushi domain. In some aspects, the IL-15Rαsushi domain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with any one of SEQ ID NOs: 8-11. In some aspects, the IL-15Rαsushi domain comprises any one of SEQ ID NOs: 8-11 amino acid sequences, having one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations. In some aspects, the IL-15Rαsushi domain comprises any one of SEQ ID NOs: 8-11 amino acid sequences. In some aspects, the IL-15Rαsushi domain consists of any one of the amino acid sequences in SEQ ID NOs: 8-11.

[0057] Exemplary IL-15Rα sushi domains are shown in Table 3 below. Table 3. Fc domain

[0058] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an Fc domain. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an IgG1 Fc domain. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an IgG2 Fc domain. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an IgG3 Fc domain. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an IgG4 Fc domain.

[0059] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an IgG1 Fc domain. In some aspects, the IgG1 Fc domain comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 12-16. In some aspects, the IgG1 Fc domain comprises any one of SEQ ID NOs: 12-16 amino acid sequences, with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations. In some aspects, the IgG1 Fc domain comprises any one of SEQ ID NOs: 12-16 amino acid sequences. In some aspects, the IgG1 Fc domain consists of any one of SEQ ID NOs: 12-16 amino acid sequences.

[0060] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an IgG1 Fc domain. In some aspects, the IgG1 Fc domain comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 12. In some aspects, the IgG1 Fc domain comprises an amino acid sequence with one or more mutations in SEQ ID NO: 12, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations. In some aspects, the IgG1 Fc domain comprises the amino acid sequence of SEQ ID NO: 12. In some aspects, the IgG1 Fc domain consists of the amino acid sequence of SEQ ID NO: 12.

[0061] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an IgG4 Fc domain. In some aspects, the IgG1 Fc domain comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 17. In some aspects, the IgG4 Fc domain comprises an amino acid sequence with one or more mutations in SEQ ID NO: 17, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations. In some aspects, the IgG4 Fc domain comprises the amino acid sequence of SEQ ID NO: 17. In some aspects, the IgG4 Fc domain consists of the amino acid sequence of SEQ ID NO: 17.

[0062] Exemplary Fc domains are shown below in Table 4. Mutations in the IgG1 Fc domain are indicated in bold and underlined. Table 4. Linker

[0063] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises one or more linkers. In some aspects, the IgG4 Fc domain consists of the amino acid sequence of SEQ ID NO:17. Herein, the term "linker" refers to a polypeptide sequence that connects two protein domains together. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises a linker. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises two linkers. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises three linkers. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises two linkers. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein does not contain a linker.

[0064] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises one or more linkers comprising at least 1 amino acid. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises one or more linkers comprising at least 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids or more amino acids.

[0065] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises a linker between the IL-15Rαsushi domain and the IL-15C-terminal domain. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises a linker between the IL-15C-terminal domain and the IL-15N-terminal domain. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises a linker between the IL-15N-terminal domain and the Fc domain. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises a linker between the IL-15C-terminal domain and the IL-15N domain, and a linker between the IL-15N-terminal domain and the Fc domain.

[0066] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises one or more linkers, and the one or more linkers include the amino acid sequence of any one of the linkers listed in Table 5. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises one or more linkers, and the one or more linkers comprise the amino acid sequence GS, GG, GGS, GGGS (SEQID NO: 18), GGGGS (SEQ ID NO: 19), or any combination. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises one or more linkers, and the one or more linkers comprise the amino acid sequence GGGGS (SEQ ID NO: 19). In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises one or more linkers, and the one or more linkers consist of the amino acid sequence GGGGS (SEQ ID NO: 19).

[0067] Exemplary linkers are shown in Table 5 below. Table 5. describe Amino acid sequence Linker 1 GS Connector 2 GG Linker 3 GGS Linker 4 GGGS Linker 5 GGGGS Exemplary IL-15 / IL-15Rα-Fc fusion proteins

[0068] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an IL-15Rα-sushi domain, an IL-15C-terminal domain, an IL-15N-terminal domain, and an Fc domain from N-terminus to C-terminus. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an IL-15 receptor α-sushi domain, an IL-15C-terminal domain, a linker, an IL-15N-terminal domain, and an Fc domain from N-terminus to C-terminus. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an IL-15Rα-sushi domain, an IL-15C-terminal domain, an IL-15N-terminal domain, a linker, and an Fc domain from N-terminus to C-terminus. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises, from N-terminus to C-terminus, an IL-15Rα-sushi domain, an IL-15 C-terminal domain, a linker, an IL-15 N-terminal domain, a linker, and an Fc domain.

[0069] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 20-30. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises any one of SEQ ID NOs: 20-30 amino acid sequences, having one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises any one of SEQ ID NOs: 20-30 amino acid sequences. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein consists of any one of SEQ ID NOs: 20-30 amino acid sequences.

[0070] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an amino acid sequence of SEQ ID NO:24, and one or more mutations are selected from the group consisting of V3A, V3L, N4A, N4D, N4Q, S7D, S7V, D8T, D8A, K11A, D30N, D61A, D61S, D61N, D61E, E64Q, N65D, I68A, I68V, L69A, L69V, N72E and N72A.

[0071] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 28. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an amino acid sequence of SEQ ID NO: 28, having one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises an amino acid sequence of SEQ ID NO: 28. The IL-15 / IL-15Rα-Fc fusion protein consists of the amino acid sequence of SEQ ID: 28.

[0072] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises SEQ ID NO:28 having one or more mutations selected from the group consisting of N189Q and G190A.

[0073] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 28 with one or more linkers, wherein the one or more linkers comprise the sequence of amino acids GS, GG, GGS, GGGS, GGGGS.

[0074] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein comprises SEQ ID NO:30 having one or more mutations selected from the group consisting of N189Q and G190A.

[0075] Exemplary IL-15 / IL-15Rα-Fc fusion proteins are shown in Table 6 below. Table 6. Pharmaceutical composition

[0076] In some aspects, the present disclosure provides a pharmaceutical composition comprising an IL-15 / IL15Rα-Fc fusion protein for treating or preventing a disease in a subject. In some aspects, the pharmaceutical composition comprising an IL-15 / IL-15Rα-Fc fusion protein is used to treat cancer.

[0077] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein described herein is formulated as one or more pharmaceutical compositions. In some aspects, the pharmaceutical composition comprises the IL15 / IL-15Rα-Fc fusion protein and a pharmaceutically acceptable carrier, diluent or excipient.

[0078] The pharmaceutical compositions described herein are formulated according to known methods to prepare pharmaceutically useful compositions, wherein the IL-15 / IL-15Rα-Fc fusion protein is combined in a mixture with a pharmaceutically acceptable carrier, diluent or excipient. Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts. For example, Handbook of Pharmaceutical Additives, Second Edition (Editors M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA), Remington's Pharmaceutical Sciences, 20th Edition, pub. Lippincott, Williams & Wilkins, 2000; Handbook of Pharmaceutical Excipients, Second Edition, 1994. The exact nature of the carrier, diluent or excipient depends on the route of administration, which may be oral or may be injection, for example, cutaneous, subcutaneous or intravenous.

[0079] In some aspects, the pharmaceutical compositions described herein are formulated in one of the following dosage forms: intravenous dosage form, intramuscular dosage form, intraperitoneal injection dosage form, subcutaneous injection dosage form, oral dosage form, intranasal dosage form, suppository dosage form, intradermal dosage form or topical dosage form. In some aspects, the pharmaceutical compositions described herein are formulated in intravenous dosage form. In some aspects, the pharmaceutical compositions described herein are formulated in subcutaneous dosage form. In some aspects, the pharmaceutical compositions described herein are formulated in oral dosage form. Treatment methods and uses

[0080] In some aspects, the IL-15 / IL-15Rα-Fc fusion protein or a pharmaceutical composition thereof is used to prevent and / or treat diseases, disorders and conditions in a subject. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein or a pharmaceutical composition thereof is used to prevent or treat cancer in a subject. Preparation method and purification method

[0081] Also provided herein are methods for making and / or purifying the IL-15 / IL-15Rα-Fc fusion proteins described herein. In some aspects, the IL-15 / IL-15Rα-Fc fusion protein is produced by recombinant expression in a host cell. The term "host cell" refers to a cell that can support the replication or expression of a nucleic acid (such as an expression vector) encoding an IL-15 / IL-15Rα-Fc fusion protein. The host cell can be a prokaryotic cell, such as Escherichia coli or a eukaryotic cell (such as a yeast, insect, amphibian, bird or mammalian cell). For example, immortal cell lines such as Sf9, HEK293, CHO-K1, HeLa, etc. are commonly used as host cells. The creation and separation of host cell lines containing nucleic acids or capable of producing IL-15 / IL-15Rα-Fc fusion proteins can be accomplished using standard techniques known in the art.

[0082] In some aspects, these methods include preparing an IL-15 / IL-15Rα-Fc fusion protein encoded by a nucleic acid (e.g., an expression vector). In some aspects, the method includes contacting a host cell with a nucleic acid (e.g., an expression vector) encoding an IL-15 / IL-15Rα-Fc fusion protein. In some aspects, the method includes introducing the nucleic acid into a host cell, such as by transfection, viral transduction (e.g., using a lentivirus or AAV vector), direct microinjection, particle bombardment, etc.

[0083] The IL-15 / IL-15Rα-Fc fusion protein can be produced by culturing the host cells under conditions that express the fusion protein and then recovering the fusion protein. The culture conditions for producing recombinant proteins using various host cells are known to those skilled in the art. In some aspects, the host cells can be maintained in a medium at 95° C. and 5% CO2 for a sufficient period of time to express the fusion protein.

[0084] When recombinant techniques are used to express IL-15 / IL-15Rα-Fc fusion proteins in host cells, it is best to separate (or purify) the desired protein from other components such as host cell factors to obtain a highly pure or substantially homogeneous preparation. Purification can be accomplished by methods well known in the art, such as centrifugation techniques, extraction techniques, chromatography and fractionation techniques (such as size separation by gel filtration, charge separation by ion exchange columns, hydrophobic interaction chromatography, reverse phase chromatography, chromatography on silica gel or cation exchange resins (such as DEAE, etc.), chromatofocusing and Protein A Sepharose chromatography to remove contaminants) and precipitation techniques (ethanol precipitation or ammonium sulfate precipitation). In order to improve the purity of the IL-15 / IL-15Rα-Fc fusion protein, various biochemical purification techniques can be used. Example Example 1: Design and synthesis of IL-15 / IL-15Rα-Fc fusion protein

[0085] We used a structure-based design strategy to synthesize stable interleukin-15 (IL-15) and IL-15 receptor α (IL-15 / IL-15Rα) complexes with unique sequences to enhance therapeutic potential. Structural analysis using the three-dimensional X-ray diffraction (XRD) structure of the IL-15 / IL-15Rα ligand-receptor complex (Figure 1) was used for the rational design of stable IL-15 / IL-15Rα complexes.

[0086] Figure 1A and Figure 1B The combined ribbon structure and surface views of the IL-15 quaternary complex are shown from different angles. Figure 1C The interface of IL-15 and IL-15Rα in the quaternary complex is shown. The dashed line indicates where the linker connects the IL-15 and IL-15Rα domains.

[0087] A minimal stable complex consisting of the IL-15 / IL-15Rαsushi domain was identified. This single polypeptide chain chimeric sequence includes, from N-terminus to C-terminus: the IL-15Rαsushi domain, the C-terminal domain of IL-15, the N-terminal domain of IL-15, and the Fc domain. IL-15 / IL-15Rα-Fc variants were then generated and tested for function and efficacy ( Figure 2 ), which includes four IL-15Rαsushi domains of different lengths ( Figure 2 D1 in ); two different demarcation points separate the IL-15 N-terminal domain from the IL-15 C-terminal domain ( Figure 2 D2 and D3 in ); and different Fc domains ( Figure 2 D4 in ).

[0088] IL-15C-terminal domain and IL-15N-terminal domain ( Figure 2 L1 in IL-15) and between the N-terminal domain and Fc domain ( Figure 2 The length of the linker between IL-15 and IL-15Rαsushi domains also varied. The study found that in the wild-type ligand-receptor complex, the linker length is critical for maintaining the contact between IL-15 and IL-15Rαsushi domains. Finally, we designed single and double point mutations to explore the range of activity of IL-15.

[0089] All variants were cloned and expressed in HEK293T cells. These variants were then tested for biochemical activity, thermal stability and efficacy (Examples 2 and 3). Example 2: In vitro testing of IL-15 / IL-15Rα-Fc fusion protein

[0090] The IL-15 / IL-15Rα-Fc fusion protein expressed in HEK293T cells was purified and its biochemical activity was analyzed for its binding affinity to IL-2Rβ (CD122).

[0091] Surface plasmon resonance (SPR; Biacore system) was used to measure the dissociation constant (KD) between the IL-15 / IL-15Rα-Fc fusion protein and IL-2Rβ. The KD value is a quantitative measurement of the affinity between the receptor and the target ligand. The lower the KD value, the higher the affinity between the receptor (i.e., IL-2Rβ) and the target ligand (i.e., IL-15 / IL-15Rα-Fc).

[0092] The biochemical activity results of IL-15 / IL-15Rα-Fc fusion protein are shown in Figure 3 and Table 7. The KD values ​​ranged from 0.52 nM to 5.21 nM, indicating that the affinity of IL-15 / IL-15Rα-Fc fusion protein for IL-2Rβ increased and decreased. Table 7.

[0093] The molecular properties of the IL-15 / IL-15Rα-Fc fusion protein were evaluated by thermal stability (Table 7) and dynamic light scattering (data not shown).

[0094] A portion of these IL-15 / IL-15Rα-Fc fusion proteins were then selected for further in vitro functional testing. Figure 4 The effects of IL-15 / IL-15Rα-Fc fusion protein on NK cell proliferation and cytotoxicity are shown. NK92 cells are an interleukin-2 (IL-2)-dependent natural killer (NK) cell line used to evaluate the effects of IL-15 / IL15Rα-Fc fusion protein on NK cell proliferation and cytotoxicity. Figure 4 showed that treatment of NK92 cells with increasing concentrations of IL-15 / IL-15Rα-Fc fusion protein increased cell proliferation. Figure 5 showed that treatment of NK92 cells with increasing concentrations of IL-15 / IL-15Rα-Fc fusion protein increased target cell death.

[0095] Then the effect of IL-15 / IL-15Rα-Fc fusion protein on NK cell degranulation was evaluated. NK cells were treated with different concentrations of IL-15 / IL-15Rα-Fc fusion protein overnight. NK cell degranulation was analyzed using anti-CD107a antibody and flow cytometry. Figure 6showed that treatment of NK cells with increasing concentrations of IL-15 / IL-15Rα-Fc fusion protein increased NK cell degranulation compared with untreated NK cells.

[0096] The effects of IL-15 / IL-15Rα-Fc fusion protein on NK and CD8 + T cell signaling were also evaluated in vitro. Fig. 7A Shown is the phosphorylation of STAT5 in primary NK cells treated overnight with 0.5 nM IL-15 / IL-15Rα-Fc fusion protein. Figure 7B Phosphorylation of STAT5 in primary CD8+ T cells treated overnight with 0.5 nM IL-15 / IL-15Rα-Fc fusion protein is shown. STAT5 phosphorylation was analyzed using anti-phospho-STAT5 antibody and flow cytometry. Fig. 7A and Figure 7B It showed that IL-15 / IL-15Rα-Fc fusion protein could increase the activation of NK and CD8+T cells.

[0097] Based on these in vitro test results, IL-15 / IL-15Rα-Fc fusion protein molecules T1, W1, Y1 and Z1 were selected for in vivo testing (Example 3). Example 3: In vivo detection of IL-15 / IL-15Rα-Fc fusion protein

[0098] The ability of IL-15 / IL-15Rα-Fc fusion protein to inhibit tumor growth and metastasis in mice was further tested in vivo.

[0099] Tumor mice were injected with vehicle (negative control), reference molecule V1, molecule W1, molecule Y1 or molecule Z1. Mice were sacrificed on day 15 or day 21 after administration (see Table 1, respectively). Fig. 8A and Figure 8B ). Overall, the treatment with IL-15 / IL-15Rα-Fc fusion protein was well tolerated. Among the four variants tested, molecules W1, Y1, and Z1 had better inhibitory effects on tumor metastasis. The harvested lungs showed different degrees of tumor burden (black), depending on the vector or variant administered (Figure 9).

[0100] The researchers further tested the effect of the IL-15 / IL-15Rα-Fc fusion protein molecule Z1 on the survival rate of tumor-bearing mice. Different doses of the molecule Z1 were injected subcutaneously or intravenously into tumor-bearing mice. Fig.10The results showed that compared with the drug control group, molecule Z1 can improve the survival rate of tumor-bearing mice at all tested doses. Intravenous or subcutaneous injection of molecule Z1 at a dose of 0.25 mg / kg has the greatest effect on mouse survival. Compared with other tested doses, the 0.25 mg / kg dose of molecule Z1 has the greatest effect on mouse survival. Example 4: Animal Model Efficacy - Evaluation of the Efficacy of IL-15 / IL-15Rα-Fc Fusion Protein on Mouse Melanoma Cells B16F10-Luc in the C57BL / 6J Mouse Lung Metastasis Model

[0101] In order to evaluate the efficacy of IL-15 / IL-15Rα-Fc fusion protein in mouse models, mouse melanoma cells B16F10-Luc transfected with Luciferase and C57BL / 6J mice were selected to evaluate the anti-tumor cell metastasis effect of IL-15 / IL-15Rα-Fc fusion protein on mouse melanoma cells B16F10-Luc in the lung metastasis model injected via tail vein in C57BL / 6J mice. A total of 4 groups were designed, and the experimental design, dose, and administration method are shown in Table 8. The weight changes of tumor-bearing mice are shown in Table 9. Table 8. Animal grouping and dosing regimen Table 9. Body weight of tumor-bearing mice (Mean±SEM) Note: *P<0.05VS Group 1, **P<0.01VS Group 1

[0102] Evaluation of the efficacy of IL-15 / IL-15Rα-Fc fusion protein on mouse melanoma cell B16F10-Luc in the lung metastasis model of C57BL / 6J mice - the bioluminescence values ​​of lung metastasis tumors in tumor-bearing mice are detailed in Table 10 and Fig.11 . Table 10. Bioluminescence values ​​of lung metastasis in tumor-bearing mice (Mean ± SEM)

[0103] As shown in Table 10 and Fig.11 As shown, on day 21, the inhibition rate (TGI%) of the monotherapy group of molecule Y1 at 0.125, 0.25 and 0.5 mg / kg on B16F10-Luc lung metastasis was 88%, 87% and 91%, respectively, which were significantly different from the control group (P<0.01). It can be seen that molecule Y1 has good efficacy and safety.

[0104] Together, these results suggest that treatment of mouse cancer models with IL-15 / IL-15Rα-Fc fusion protein inhibits tumor growth and metastasis and improves survival in cancer mouse models. Incorporation by Reference

[0105] All references, articles, publications, patents, patent publications and patent applications cited herein are incorporated herein by reference in their entirety. However, any references, articles, publications, patents, patent publications and patent applications cited herein are not and should not be regarded as an admission or any form of suggestion that they constitute valid prior art or form part of the general knowledge in any country in the world.

Claims

1. A polypeptide comprising, from N-terminus to C-terminus: (a) An IL-15 receptor α-sushi domain; (b) an IL-15 C-terminal domain; (c) an IL-15 N-terminal domain; and (d) An Fc domain.

2. The polypeptide of claim 1, wherein the IL-15 receptor α-sushi domain comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to any one of SEQ ID NOs: 8-11; or further preferably, the IL-15 receptor α-sushi domain comprises an amino acid sequence of any one of SEQ ID NOs: 8-11.

3. The polypeptide of claim 1 or 2, wherein the IL-15 C-terminal domain comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% or at least about 98% identical to SEQ ID NO: 3 or SEQ ID NO: 4; or further preferably, the IL-15 C-terminal domain comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO:

4.

4. The polypeptide according to any one of claims 1 to 3, wherein the IL-15 N-terminal domain comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% or at least about 98% identical to SEQ ID NO: 5 or SEQ ID NO: 6; or further preferably, the IL-15 N-terminal domain comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6; or further preferably, the IL-15 N-terminal domain comprises the N72D mutation.

5. The polypeptide of any one of claims 1 to 4, wherein the Fc domain is an IgG1 Fc domain and the amino acid sequence comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 12; or further preferably, the Fc domain is an IgG1 Fc domain or comprises the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO:

14.

6. The polypeptide of any one of claims 1-4, wherein the Fc domain is an IgG4 Fc domain and the amino acid sequence comprises a sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 17; or further preferably, the Fc domain is an IgG4 Fc domain or comprises the amino acid sequence of SEQ ID NO:

17.

7. The polypeptide of claim 5 or 6, wherein the IgG1 or IgG4 Fc domain comprises at least one amino acid substitution; or further preferably, the at least one amino acid substitution is N297A, L234A, L235A, and / or P329G; or further preferably, the at least one amino acid substitution is M252Y, S254T, T256E, or a combination thereof; or further preferably, the at least one amino acid substitution is L234A, L235A, or a combination thereof.

8. The polypeptide according to any one of claims 1 to 7, wherein the polypeptide comprises one or more linkers.

9. The polypeptide according to claim 8, wherein the polypeptide comprises a linker between the IL-15 C-terminal domain and the IL-15 N-terminal domain; or / and the polypeptide comprises a linker between the IL-15 N-terminal domain and the Fc domain.

10. A polypeptide according to claim 8 or 9, wherein the length of one or more linkers is 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; or further preferably, the length of the one or more linkers is 5 amino acids; or further preferably, the one or more linkers comprise an amino acid sequence selected from the group consisting of GG, GS, GGS, GGGS and GGGGS.

11. The polypeptide of claim 1, comprising an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 20-30.

12. The polypeptide according to claim 1, comprising any one of the amino acid sequences of SEQ ID NOs: 20-30; further preferably, the polypeptide is SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO:

30.

13. The polypeptide according to claim 1, wherein the polypeptide comprises an amino acid sequence in which one or more mutations are made on any one of the amino acid sequences in SEQ ID NOs: 20-30; or further preferably, the polypeptide comprises an amino acid sequence shown in SEQ ID NO: 24, wherein the IL-15 N-terminal domain portion of the sequence has a sequence with one or more mutant amino acids selected from V3A, V3L, N4A, N4D, N4Q, S7D, S7V, D8T, D8A, K11A, D30N, D61A, D61S, D61N, D61E, E64Q, N65D, I68A, I68V, L69A, L69V, N72E and N72A.

14. The polypeptide according to claim 1, wherein the polypeptide comprises an amino acid sequence further comprising N189Q and / or G190A mutations based on the amino acid sequence shown in SEQ ID NO:

28.

15. The polypeptide according to claim 1, wherein the polypeptide comprises an amino acid sequence further comprising N189Q and / or G190A mutations based on the amino acid sequence shown in SEQ ID NO:

30.

16. The polypeptide according to claim 1, wherein the polypeptide consists of any one of the amino acid sequences in SEQ ID NOs: 20-30.

17. The polypeptide according to any one of claims 1-16, wherein the polypeptide binds to IL-2 receptor β and / or IL-2 receptor γ; further preferably, the polypeptide binds to IL-2 receptor β with an affinity of about 0.1 nM to about 25 nM, about 0.5 nM to about 10 nM, or about 1 nM to about 5 nM.

18. A polynucleotide encoding the polypeptide of any one of claims 1-17.

19. An expression vector comprising the polynucleotide of claim 18.

20. A host cell comprising the polynucleotide of claim 18 or the expression vector of claim 19.

21. A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 17 and at least one pharmaceutically acceptable carrier or excipient.

22. A method for treating cancer, comprising administering an effective amount of the polypeptide of any one of claims 1 to 17 or the pharmaceutical composition of claim 21 to a subject in need thereof.

23. The method of claim 22, wherein the cancer is a solid tumor.

24. The method of claim 23, wherein the cancer is metastatic.

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