IL-15 agonists for cancer

Abstract

Provided herein is an IL-15 / IL-15Rα-Fc fusion protein comprising an IL-15 receptor alpha sushi domain, an IL-15 C-terminal domain, an IL-15 N-terminal domain, and an Fc domain. Further provided are methods of using such IL-15 / IL-15Rα-Fc to treat diseases and disorders, such as cancer.

Description

[Technical Field]

[0001] The present invention relates to interleukin-15 (IL-15) agonists and methods of using same. Specifically, the present invention provides an IL-15 / IL-15 receptor alpha (IL-15Rα)-Fc fusion protein complex comprising an IL-15Rα sushi domain, an IL-15 domain, and an Fc domain for the treatment of cancer. [Background technology]

[0002] Cancer immunotherapy is a promising approach for cancer treatment, but is currently limited by poor drug-like properties, severe dose-limiting toxicity, and disease recurrence.Therefore, there remains a need for effective compositions that enhance and induce immune activity against cancer cells with limited toxicity. Summary of the Invention [Problem to be solved by the invention]

[0003] The present invention provides novel IL-15 / IL-15Rα-Fc fusion proteins that activate effector immune responses for the treatment of cancer. [Means for solving the problem]

[0004] The present invention provides an IL-15 / IL-15Rα-Fc fusion protein comprising, from N-terminus to C-terminus: (a) an IL-15 receptor alpha sushi domain, (b) an IL-15 C-terminal domain, (c) an IL-15 N-terminal domain, and (d) an Fc domain. In certain embodiments, the IL-15 receptor alpha sushi domain 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 to any one of SEQ ID NOs: 8 through 11. In certain embodiments, the IL-15 receptor alpha sushi domain comprises the amino acid sequence of any one of SEQ ID NOs: 8 through 11.

[0005] In certain embodiments, the IL-15 C-terminal domain 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%, or at least about 98% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4. In certain embodiments, the IL-15 C-terminal domain comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

[0006] In certain embodiments, the IL-15 N-terminal domain 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%, or at least about 98% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6. In certain embodiments, the IL-15 N-terminal domain comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. In certain embodiments, the IL-15 N-terminal domain comprises an N72D mutation.

[0007] In certain embodiments, the Fc domain is an IgG1 Fc domain comprising 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 to SEQ ID NO: 12. In certain embodiments, the Fc domain is an IgG1 Fc domain comprising the amino acid sequence of SEQ ID NO: 12.

[0008] In certain embodiments, the Fc domain is an IgG4 Fc domain comprising 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 to SEQ ID NO: 17. In certain embodiments, the Fc domain is an IgG4 Fc domain comprising the amino acid sequence of SEQ ID NO: 17.

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

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

[0011] In certain embodiments, 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 to any one of SEQ ID NOs: 20 to 30. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 20 to 30. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein consists of the amino acid sequence of any one of SEQ ID NOs: 20 to 30.

[0012] In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 20 to 30, with one or more mutations. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 24, with 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 correspond to the natural order of the amino acids in SEQ ID NO: 5).

[0013] In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 28 with one or more mutations selected from the group consisting of N189Q (referring to a mutation from N(asparagine)189 to G(glycine) in SEQ ID NO: 28) and G190A (referring to a mutation from G(glycine)190 to A(alanine) in SEQ ID NO: 28). In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 30 with one or more mutations selected from the group consisting of N189Q and G190A.

[0014] In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein binds to IL-2 receptor beta and / or IL-2 receptor gamma 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 certain aspects, the present invention provides a polynucleotide encoding the above-described IL-15 / IL-15Rα-Fc fusion protein. In certain aspects, the present invention provides an expression vector comprising the polynucleotide. In certain aspects, the present disclosure provides a host cell comprising the polynucleotide or the expression vector.

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

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

[0018] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is metastatic. In some embodiments, the cancer has recurred or is refractory to treatment. [Brief explanation of the drawings]

[0019] [Figure 1A] Figure 1 shows an exemplary three-dimensional x-ray diffraction (XRD) structure of an IL-15 complex (PDB: 4GS7). Figure 1A shows the complex ribbon structure and surface view of the IL-15 quaternary complex from different perspectives. [Figure 1B] Figure 1B shows an exemplary three-dimensional x-ray diffraction (XRD) structure of the IL-15 complex (PDB: 4GS7). Figure 1B shows the complex ribbon structure and surface view of the IL-15 quaternary complex from different perspectives. [Figure 1C] Figure 1C shows an exemplary three-dimensional x-ray diffraction (XRD) structure of the IL-15 complex (PDB: 4GS7). Figure 1C shows the interface between IL-15 and IL-15Rα within the quaternary complex. The dashed line indicates where the linker was placed to connect the IL-15 and IL-15Rα domains. [Figure 2] FIG. 1 shows an exemplary cassette design for an IL-15 / IL-15Rα-Fc fusion protein of the invention compared to a standard reference cassette. [Figure 3] FIG. 1 shows the binding of IL-15 / IL-15Rα-Fc fusion protein to IL-2Rβ (CD122). [Figure 4] FIG. 1 shows the effect of IL-15 / IL-15Rα-Fc fusion protein on NK cell proliferation. [Figure 5] FIG. 1 shows the effect of IL-15 / IL-15Rα-Fc fusion protein on NK cell cytotoxicity. [Figure 6]

[0023] Figure 1 shows the effect of IL-15 / IL-15Rα-Fc fusion protein on NK cell degranulation. NK cells were treated overnight with various concentrations of IL-15 / IL-15Rα-Fc fusion protein. NK cell degranulation was analyzed using anti-CD107a antibody and flow cytometry. [Figure 7A] Figure 7 shows the effect of IL-15 / IL-15Rα-Fc fusion protein on NK and CD8+ T cell signaling. Figure 7A shows the phosphorylation of STAT5 in primary NK cells treated overnight with 0.5 nM IL-15 / IL-15Rα-Fc fusion protein. STAT5 phosphorylation was analyzed using an anti-phospho-STAT5 antibody and flow cytometry. [Figure 7B] Figure 7B shows the effect of IL-15 / IL-15Rα-Fc fusion protein on NK and CD8+ T cell signaling. Figure 7B shows the phosphorylation of STAT5 in primary CD8+ T cells treated overnight with 0.5 nM IL-15 / IL-15Rα-Fc fusion protein. STAT5 phosphorylation was analyzed using an anti-phospho-STAT5 antibody and flow cytometry. [Figure 8A] FIG. 8A shows the effect of treatment with IL-15 / IL-15Rα-Fc fusion protein on tumor growth and metastasis in tumor-burdened mice after 15 days. [Figure 8B] FIG. 8B shows the effect of treatment with IL-15 / IL-15Rα-Fc fusion protein on tumor growth and metastasis in tumor-burdened mice after 21 days. [Figure 9A] FIG. 9A shows an exemplary effect of treatment with IL-15 / IL-15Rα-Fc fusion protein on tumor growth in lungs excised from tumor-burdened mice compared to vehicle control. [Figure 9B] Figure 9B shows an exemplary effect of IL-15 / IL-15Rα-Fc fusion proteins on metastasis compared to vehicle controls. Molecule V1 (SEQ ID NO: 31) is a standard IL-15 / IL-15Rα-Fc fusion protein shown for reference. [Figure 10] Figure 1 shows Kaplan-Meier survival curves of tumor-burdened mice administered IL-15 / IL-15Rα-Fc fusion protein molecule Z1 at various concentrations compared to vehicle control. Molecule V1 is a standard IL-15 / IL-15Rα-Fc fusion protein shown for reference. [Figure 11] FIG. 1 shows tumor growth inhibition values ​​(TGI%) for lung metastases in B16F10-Luc mice administered IL-15 / IL-15Rα-Fc fusion protein molecule Y1 at 0.125 mg / kg, 0.25 mg / kg and 0.5 mg / kg compared to vehicle control. DETAILED DESCRIPTION OF THE INVENTION

[0020] definition The term "a" or "an" refers to one or more of that entity, i.e., can refer to multiple referents. Thus, the terms "a," "an," "one or more," and "at least one" are used interchangeably herein. Furthermore, reference to an "element" by the indefinite article "a" or "an" does not exclude the possibility that more than one of that element is present, unless the context clearly requires that there be only one of that element.

[0021] Throughout this specification, the term "about" is used to indicate that a value includes the inherent variation of error for the device or method being used to determine the value or the variation that exists between samples measured. Unless otherwise stated or otherwise clear from the context, the term "about" means within 10% of the reported numerical value (except when such numerical value exceeds 100% or falls below 0% of the possible values). When used in conjunction with a range of values ​​or a series of values, the term "about" applies to the endpoints of the range or each recited value in the series, unless otherwise stated. As used in this application, the terms "about" and "approximately" are used as equivalents.

[0022] 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 amino acid residues or nucleotides in a particular (parent) sequence after aligning the sequences to achieve the maximum percent identity and introducing gaps as necessary. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill of those in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Clustal Omega®, or Megalign (DNASTAR) software. Another approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics, 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using a scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, MODayhoff, ed., 5 suppl. 3: 353-358, National Biomedical Research Foundation, Washington, DC, USA, and standardized by Gribskov, Nucl. Acids Res. 14(6): 6745-6763 (1986).

[0023] As used herein, the terms "treat," "treating," or "treatment," and grammatical variations thereof, have the same meaning as commonly understood by those of skill in the art. In certain embodiments, these terms may refer to an approach for obtaining beneficial or desired clinical results. The terms may refer to delaying the onset or rate of development of a condition, disorder, or disease, reducing or alleviating the symptoms associated therewith, causing complete or partial regression of the condition, or any combination of the above. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, reduction or alleviation of symptoms, whether detectable or undetectable, reduction in the extent of the disease, stabilization of the disease state (e.g., not worsening), delay or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or total). "Treat," "treating," or "treatment" may also mean prolonging survival relative to expected survival if not receiving treatment. Thus, a subject (e.g., a human) in need of treatment may be one already suffering from the disease or disorder in question. The terms "treat," "treating," or "treatment" encompass the inhibition or reduction of the increase in severity of a pathological condition or symptom relative to the absence of treatment, and are not necessarily intended to imply a complete cessation of the associated disease or condition.

[0024] As used herein, the terms "prevent," "preventing," and "prevention," as well as grammatical variations thereof, refer to an approach for preventing the onset of a condition or disease or altering its pathology. Thus, "prevention" can refer to prophylactic or preventative measures. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, the prevention or slowing of disease symptoms, progression, or onset, whether detectable or undetectable. Thus, a subject (e.g., a human) in need of prevention may be one who is not yet afflicted with the disease or disorder in question. The term "prevention" encompasses the slowing of disease onset relative to the absence of treatment and is not necessarily intended to imply permanent prevention of the associated disease, disorder, or condition. Thus, "preventing" or "prevention" of a condition, in certain contexts, can refer to reducing the risk of developing the condition or preventing or delaying the onset of symptoms associated with the condition.

[0025] The phrase "pharmaceutically acceptable carrier" or "pharmaceutically acceptable diluent" refers to any physiologically acceptable material, composition, or vehicle, i.e., compatible buffers, solvents, dispersion media, coatings, antibacterial agents, isotonicity agents, absorption delaying agents, solid or liquid fillers, excipients, encapsulating materials, etc. The phrase "pharmaceutically acceptable excipient" generally means an excipient that is safe, non-toxic, and useful in preparing biologically or otherwise desirable pharmaceutical compositions, and includes excipients that are acceptable for both human pharmaceutical and veterinary use.

[0026] The phrase "therapeutically effective amount," as used herein, refers to the amount of a compound, e.g., an IL-15 / IL-15Rα-Fc fusion protein, that achieves the desired biological or therapeutic effect, i.e., prevents, reduces, or ameliorates one or more symptoms of the recited disease being treated or prevented.

[0027] As used herein, the term "additional therapeutic agent" refers to one or more therapeutic agents that provide a therapeutic effect or benefit to a subject in need of treatment. The additional therapeutic agent may be a therapeutic agent (e.g., a chemotherapeutic agent) or a medical device.

[0028] As used herein, the term "subject" refers to any subject, e.g., a human or non-human mammal, for which diagnosis, prognosis, or treatment is desired. The term "subject" can refer to a human or non-human mammal that is suffering from, likely to suffer from, or suspected of suffering from a disease. The terms "subject" and "patient" are used interchangeably herein. In some embodiments, the subject is a mammal. Mammals include primates such as humans, monkeys, chimpanzees, and apes, as well as non-primates, such as laboratory animals (e.g., rabbits and rodents, e.g., guinea pigs, rats, or mice) and domestic animals, including household pets and farm animals (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits), and non-domestic animals, such as wildlife, birds, reptiles, and fish.

[0029] As used herein, the term "subject in need thereof" includes a subject who may or will benefit from the methods described herein. Subjects in need of treatment include, but are not limited to, subjects who already have a condition or disorder, subjects prone to have a condition or disorder, subjects suspected of having a condition or disorder, and subjects in whom a condition or disorder is to be prevented, ameliorated, or reversed.

[0030] The term "antigen" refers to a molecule or portion of a molecule that can be bound by an antibody or antigen-binding fragment thereof and that can be used in an animal to generate antibodies capable of binding to an epitope of that antigen. An antigen may have one or more epitopes.

[0031] The term "activation," when referring to treatment with the IL-15 / IL-15Rα-Fc fusion proteins described herein, refers to activation of the immune system, such as activation of NK cells and T cells. Activation of NK cells and T cells by the IL-15 / IL-15Rα-Fc fusion proteins described herein results in, for example, increased development, proliferation, survival, cytotoxicity, degranulation, and trafficking. This results in enhanced antitumor immunity, leading to reduced tumor growth and metastasis.

[0032] The term "cytotoxicity" refers to the ability of an immune cell to kill a target cell. In some embodiments, the immune cell is a CD8+ T cell, an NK cell, or an NKT cell. In some embodiments, the target cell is a tumor cell or a virus-infected cell.

[0033] The term "degranulation" refers to the cellular process of releasing cytotoxic effector molecules from secretory vesicles called granules. Degranulation in T cells and NK cells results in the release of perforin and granzymes, which kill target cells, such as tumor or virus-infected cells.

[0034] IL-15 / IL-15Rα-Fc fusion protein 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 comprising, from N-terminus to C-terminus, an IL-15Rα sushi domain, an IL-15 C-terminal domain, an IL-15 N-terminal domain, and an Fc domain. In one embodiment, the IL-15 / IL-15 receptor alpha (IL-15Rα)-Fc fusion protein of the present disclosure is used for the treatment of cancer.

[0035] IL-15 is a member of the interleukin-2 (IL-2) superfamily and functions 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 consisting of the common gamma chain, IL-2R beta (IL-2Rβ), and IL-15 receptor alpha (IL-15Rα). Free IL-15 is present at very low levels in the bloodstream and is often found complexed with IL-15 receptor alpha (IL-15Rα), which improves both IL-15's half-life and bioavailability. This specific presentation of IL-15 can also improve the activation of T cells, B cells, and NK cells, which plays an important role in tumor immune surveillance and inhibition through early recognition and destruction of malignant cells.

[0036] IL-15 may not induce activation-induced cell death (AICD) of T cells or enhance the proliferation, function, or differentiation of immunosuppressive CD4+ T regulatory cells (Tregs). Furthermore, IL-15 may not induce severe capillary leak syndrome, a toxicity associated with IL-2 therapy, in nonhuman primates (NHPs) or humans. Thus, IL-15 may activate effector immune cells necessary for tumor control with less associated toxicity.

[0037] In contrast to recombinant IL-15, the IL-15 / IL-15 receptor alpha (IL-15Rα)-Fc fusion protein may not require trans-representation or cell-cell contact to induce IL-15-mediated immune responses. Due to its structure, the IL-15 / IL-15 receptor alpha (IL-15Rα)-Fc fusion protein may also have greater potency, bioavailability, and stability than soluble recombinant IL-15.

[0038] An exemplary sequence for human IL-15 is provided as SEQ ID NO: 1. See also Uniprot Accession No. P40933. MRISKPHLRS ISIQCYLCLL LNSHFLTEAG IHVFILGCFS AGLPKTEANW VNVISDLKKI EDLIQSMHID ATLYTESDVH PSCKVTAMKC FLLELQVISL ESGDASIHDT VENLIILANN SLSSNGNVTE SGCKECEELE EKNIKEFLQS FVHIVQMFIN TS (SEQ ID NO: 1).

[0039] An exemplary sequence of human IL-15Rα is provided as SEQ ID NO: 2. See also Uniprot Accession No. Q13261. MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYS LYSRERYICN SGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVE MEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO: 2).

[0040] IL-15 domain In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises one or more IL-15 domains. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises an IL-15 C-terminal domain. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises an IL-15 N-terminal domain. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises an IL-15 C-terminal domain and an IL-15 N-terminal domain. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises, from N- to C-terminus, an IL-15 C-terminal domain and an IL-15 N-terminal domain.

[0041] In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises an IL-15 C-terminal domain. In certain embodiments, the IL-15 C-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 to SEQ ID NO:3 or SEQ ID NO:4. In certain embodiments, the IL-15 C-terminal domain comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations. In certain embodiments, the IL-15 C-terminal domain comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. In certain embodiments, the IL-15 C-terminal domain consists of the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.

[0042] Exemplary IL-15 C-terminal domains are shown in Table 1 below.

[0043] [Table 1]

[0044] In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises an IL-15 N-terminal domain. In certain embodiments, the IL-15 N-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 to any one of SEQ ID NOs: 5-7. In certain embodiments, the IL-15 C-terminal domain comprises the amino acid sequence of 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 certain embodiments, the IL-15 N-terminal domain comprises one or more mutations at residues selected from the group consisting of V3, N4, S7, D8, K11, D30, D61, E64, N65, I68, L69, N72, and any combination thereof. In certain embodiments, the IL-15 N-terminal domain comprises one or more mutations selected from the group consisting of 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 certain embodiments, the IL-15 N-terminal domain comprises an N72D mutation. In certain embodiments, the IL-15 N-terminal domain comprises a D30N mutation, an E64Q mutation, and an N65D mutation. In certain embodiments, the IL-15 N-terminal domain comprises an N4D mutation and an N65D mutation. In certain embodiments, the IL-15 N-terminal domain comprises a D30N mutation and an N65D mutation. In certain embodiments, the IL-15 N-terminal domain comprises an I68A mutation and an L69A mutation. In certain embodiments, the IL-15 N-terminal domain comprises an I68A mutation and an L69V mutation. In certain embodiments, the IL-15 N-terminal domain comprises an I68V mutation and an L69A mutation. In certain embodiments, the IL-15 N-terminal domain comprises an I68V mutation and an L69V mutation. In certain embodiments, the IL-15 N-terminal domain comprises an I68A mutation and an S7V mutation. In certain embodiments, the IL-15 N-terminal domain comprises an I68A mutation and an S7D mutation.In certain embodiments, the IL-15 N-terminal domain comprises an L69A mutation and an S7V mutation. In certain embodiments, the IL-15 N-terminal domain comprises an L69A mutation and an S7D mutation. In certain embodiments, the IL-15 N-terminal domain comprises a D61A mutation and an L69A mutation. In certain embodiments, the IL-15 N-terminal domain comprises a D61A mutation and an I68A mutation. In certain embodiments, the IL-15 N-terminal domain comprises a D61A mutation and an S7V mutation. In certain embodiments, the IL-15 N-terminal domain comprises a D61A mutation and an S7D mutation. In certain embodiments, the IL-15 N-terminal domain comprises a D61N mutation and an L69A mutation. In certain embodiments, the IL-15 N-terminal domain comprises a D61N mutation and an I68A mutation. In certain embodiments, the IL-15 N-terminal domain comprises a D61S mutation and an L69A mutation. In certain embodiments, the IL-15 N-terminal domain comprises a D61S mutation and an I68A mutation.

[0045] In certain embodiments, 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 certain embodiments, 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.

[0046] Exemplary IL-15 N-terminal domains are shown in Table 2 below.

[0047] [Table 2]

[0048] IL-15Rα domain In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises an IL-15Rα domain. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises an IL-15Rα sushi domain. In certain embodiments, the IL-15Rα sushi 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 to any one of SEQ ID NOs: 8-11. In certain embodiments, the IL-15Rα sushi domain comprises the amino acid sequence of any one of SEQ ID NOs: 8-11 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations. In certain embodiments, the IL-15Rα sushi domain comprises the amino acid sequence of any one of SEQ ID NOs: 8-11. In one embodiment, the IL-15Rα sushi domain consists of the amino acid sequence of any one of SEQ ID NOs:8 to 11.

[0049] Exemplary IL-15Rα sushi domains are shown in Table 3 below.

[0050] [Table 3]

[0051] Fc domain In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises an Fc domain. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises an IgG1 Fc domain. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises an IgG2 Fc domain. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises an IgG3 Fc domain. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises an IgG4 Fc domain.

[0052] In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises an IgG1 Fc domain. In certain embodiments, the IgG1 Fc 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 to any one of SEQ ID NOs: 12 to 16. In certain embodiments, the IgG1 Fc domain comprises the amino acid sequence of any one of SEQ ID NOs: 12 to 16 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations. In certain embodiments, the IgG1 Fc domain comprises the amino acid sequence of any one of SEQ ID NOs: 12 to 16. In certain embodiments, the IgG1 Fc domain consists of the amino acid sequence of any one of SEQ ID NOs: 12 to 16.

[0053] In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises an IgG1 Fc domain. In certain embodiments, the IgG1 Fc 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 to SEQ ID NO: 12. In certain embodiments, the IgG1 Fc domain comprises the amino acid sequence of SEQ ID NO: 12 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations. In certain embodiments, the IgG1 Fc domain comprises the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the IgG1 Fc domain consists of the amino acid sequence of SEQ ID NO: 12.

[0054] In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises an IgG4 Fc domain. In certain embodiments, the IgG4 Fc 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 to SEQ ID NO: 17. In certain embodiments, the IgG4 Fc domain comprises the amino acid sequence of SEQ ID NO: 17 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations. In certain embodiments, the IgG4 Fc domain comprises the amino acid sequence of SEQ ID NO: 17. In certain embodiments, the IgG4 Fc domain consists of the amino acid sequence of SEQ ID NO: 17.

[0055] Exemplary Fc domains are shown below in Table 4. Mutations in the IgG1 Fc domain are indicated using bold and underlined text.

[0056] [Table 4]

[0057] Linker In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises one or more linkers. As used herein, the term "linker" refers to a polypeptide sequence that links two protein domains together. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises one linker. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises two linkers. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises three linkers. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein does not comprise a linker.

[0058] In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises one or more linkers comprising at least one amino acid, hi certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises one or more linkers comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more amino acids.

[0059] In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises a linker between the IL-15Rα sushi domain and the IL-15 C-terminal domain. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises a linker between the IL-15 C-terminal domain and the IL-15 N-terminal domain. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises a linker between the IL-15 N-terminal domain and the Fc domain. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises a linker between the IL-15 C-terminal domain and the IL-15 N-terminal domain and a linker between the IL-15 N-terminal domain and the Fc domain.

[0060] In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises one or more linkers, wherein the one or more linkers comprise the amino acid sequence of any one of the linkers listed in Table 5. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises one or more linkers, wherein the one or more linkers comprise the amino acid sequence of GS, GG, GGS, GGGS (SEQ ID NO: 18), GGGGS (SEQ ID NO: 19), or any combination thereof. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises one or more linkers, wherein the one or more linkers comprise the amino acid sequence of GGGGS (SEQ ID NO: 19). In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises one or more linkers, wherein the one or more linkers consist of the amino acid sequence of GGGGS (SEQ ID NO: 19).

[0061] Exemplary linkers are shown in Table 5 below.

[0062] [Table 5]

[0063] Exemplary IL-15 / IL-15Rα-Fc Fusion Proteins In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises, from N- to C-terminus, an IL-15Rα sushi domain, an IL-15 C-terminal domain, an IL-15 N-terminal domain, and an Fc domain. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises, from N- to C-terminus, an IL-15Rα sushi domain, an IL-15 C-terminal domain, a linker, an IL-15 N-terminal domain, and an Fc domain. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises, from N- to C-terminus, an IL-15Rα sushi domain, an IL-15 C-terminal domain, an IL-15 N-terminal domain, a linker, and an Fc domain. In one embodiment, 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.

[0064] In certain embodiments, 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 to 30. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 20 to 30 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 20 to 30. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein consists of the amino acid sequence of any one of SEQ ID NOs: 20 to 30.

[0065] In one embodiment, the IL-15 / IL-15Rα-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 24 with 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.

[0066] In certain embodiments, 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 SEQ ID NO: 28. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 28 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 28. In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein consists of the amino acid sequence of SEQ ID NO: 28.

[0067] In one embodiment, the IL-15 / IL-15Rα-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 28 with one or more mutations selected from the group consisting of N189Q and G190A.

[0068] In one embodiment, 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 amino acid sequence GS, GG, GGS, GGGS, or GGGGS.

[0069] In one embodiment, the IL-15 / IL-15Rα-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 30 with one or more mutations selected from the group consisting of N189Q and G190A.

[0070] Exemplary IL-15 / IL-15Rα-Fc fusion proteins are shown in Table 6 below.

[0071] [Table 6] TIFF2025531047000008.tif255157TIFF2025531047000009.tif198170

[0072] Pharmaceutical Composition In certain embodiments, the present invention provides pharmaceutical compositions comprising an IL-15 / IL-15Rα-Fc fusion protein for treating or preventing disease in a subject, hi certain embodiments, the pharmaceutical compositions comprising an IL-15 / IL-15Rα-Fc fusion protein are used for the treatment of cancer.

[0073] In certain embodiments, the IL-15 / IL-15Rα-Fc fusion proteins described herein are formulated as one or more pharmaceutical compositions, hi certain embodiments, the pharmaceutical compositions comprise the IL-15 / IL-15Rα-Fc fusion protein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0074] The pharmaceutical compositions described herein are formulated according to known methods for preparing pharmaceutically useful compositions, whereby the IL-15 / IL-15Rα-Fc fusion protein is combined in admixture with a pharmaceutically acceptable carrier, diluent, or excipient. Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical textbooks. See, for example, Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endecott, New York, USA), Remington's Pharmaceutical Sciences, 20th Edition, pub. Lippincott, Williams & Wilkins, 2000, and Handbook of Pharmaceutical Excipients, 2nd Edition, 1994. The exact nature of the carrier, diluent, or excipient will depend on the route of administration, which may be oral or by injection, e.g., cutaneous, subcutaneous, or intravenous.

[0075] In some embodiments, the pharmaceutical compositions described herein are formulated in one of the following dosage forms: intravenous dosage form, intramuscular dosage form, intraperitoneal dosage form, subcutaneous dosage form, oral dosage form, intranasal dosage form, suppository dosage form, intradermal dosage form, or topical dosage form. In some embodiments, the pharmaceutical compositions described herein are formulated in an intravenous dosage form. In some embodiments, the pharmaceutical compositions described herein are formulated in a subcutaneous dosage form. In some embodiments, the pharmaceutical compositions described herein are formulated in an oral dosage form.

[0076] Treatment Methods and Uses In certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein or pharmaceutical composition thereof is used to prevent and / or treat diseases, disorders, and conditions in a subject, hi certain embodiments, the IL-15 / IL-15Rα-Fc fusion protein or pharmaceutical composition thereof is used to prevent or treat cancer in a subject.

[0077] Preparation and purification methods Also provided herein are methods for producing and / or purifying the IL-15 / IL-15Rα-Fc fusion protein described herein. In certain embodiments, 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 capable of supporting the replication or expression of a nucleic acid (e.g., an expression vector) encoding the IL-15 / IL-15Rα-Fc fusion protein. The host cell can be a prokaryotic cell, such as Escherichia coli (E. coli), or a eukaryotic cell (e.g., yeast, insect, amphibian, avian, or mammalian cell). For example, immortalized cell lines, such as Sf9, HEK293, CHO-K1, and HeLa, are often used as host cells. Creation and isolation of a host cell line containing a nucleic acid or capable of producing the IL-15 / IL-15Rα-Fc fusion protein can be accomplished using standard techniques known in the art.

[0078] In certain embodiments, the method comprises preparing a nucleic acid (e.g., an expression vector) encoding an IL-15 / IL-15Rα-Fc fusion protein. In certain embodiments, the method comprises contacting a host cell with a nucleic acid (e.g., an expression vector) encoding the IL-15 / IL-15Rα-Fc fusion protein. In certain embodiments, the method comprises introducing the nucleic acid into the host cell by, for example, transfection, viral transduction (e.g., using a lentiviral or AAV vector), direct microinjection, particle bombardment, etc.

[0079] The IL-15 / IL-15Rα-Fc fusion protein can be produced by culturing host cells under conditions in which the fusion protein is expressed and recovering the fusion protein. Culture conditions for producing recombinant proteins using various host cells are known to those of skill in the art. In one embodiment, the host cells can be maintained in culture at 95°C in a 5% CO atmosphere for a period of time sufficient to express the fusion protein.

[0080] When an IL-15 / IL-15Rα-Fc fusion protein is expressed in a host cell using recombinant techniques, it is preferable 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 (e.g., size separation by gel filtration, charge separation by ion exchange columns, hydrophobic interaction chromatography, reverse-phase chromatography, chromatography on cation exchange resins such as silica or DEAE, chromatofocusing, and Protein A Sepharose chromatography to remove contaminants), and precipitation techniques (e.g., ethanol precipitation, ammonium sulfate precipitation). Any number of biochemical purification techniques can be used to increase the purity of the IL-15 / IL-15Rα-Fc fusion protein. [Example]

[0081] Example 1 Design and generation of IL-15 / IL-15Rα-Fc fusion protein To improve therapeutic potential, a structure-based design strategy was used to generate stable complexes of interleukin-15 (IL-15) and IL-15 receptor alpha (IL-15 / IL-15Rα) with unique sequence sequences. Structural analysis using a 3D X-ray diffraction (XRD) structure of the IL-15 / IL-15Rα ligand-receptor complex (Figure 1) was used for the rational design of stable complexes of IL-15 / IL-15Rα.

[0082] Figures 1A and 1B show the complex ribbon structure and surface diagram of the IL-15 quaternary complex from different perspectives. Figure 1C shows the interface between IL-15 and IL-15Rα within the quaternary complex. The dashed line indicates where the linker was placed to connect the IL-15 and IL-15Rα domains.

[0083] We identified the minimal stable complex of the IL-15 / IL-15Rα sushi domain. This single polypeptide chain chimeric sequence contains, from N- to C-terminus, the IL-15Rα sushi domain, the IL-15 C-terminal domain, the IL-15 N-terminal domain, and an Fc domain. Next, we generated and tested IL-15 / IL-15Rα-Fc variants for functionality and efficacy (Figure 2). The variants contained four different lengths of the IL-15Rα sushi domain (D1 in Figure 2), two different split points separating the IL-15 N-terminal domain from the IL-15 C-terminal domain (D2 and D3 in Figure 2), and various Fc domains (D4 in Figure 2).

[0084] Additionally, the linker length was varied between the IL-15 C-terminal domain and the IL-15 N-terminal domain (L1 in Figure 2) and between the IL-15 N-terminal domain and the Fc domain (L2 in Figure 2). Linker length was found to be important for maintaining contact between IL-15 and the IL-15Rα sushi domain in the wild-type ligand-receptor complex. Finally, single and double point mutations were engineered to explore the range of IL-15 activity.

[0085] All variants were cloned and expressed in HEK293T cells, and then tested for biochemical activity, thermostability, and efficacy (Examples 2 and 3). Example 2

[0086] In vitro testing of IL-15 / IL-15Rα-Fc fusion protein The IL-15 / IL-15Rα-Fc fusion protein expressed in HEK293T cells was purified and analyzed for biochemical activity characterized by binding affinity to IL-2Rβ (CD122).

[0087] 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 represents a quantitative measure of receptor affinity for a target ligand, with a lower KD value representing a higher affinity of the receptor (i.e., IL-2Rβ) for the target ligand (i.e., IL-15 / IL-15Rα-Fc).

[0088] The results of the biochemical activity of the 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 IL-15 / IL-15Rα-Fc fusion protein conferred a range of both increased and decreased affinity for IL-2Rβ.

[0089] [Table 7]

[0090] The molecular attributes of the IL-15 / IL-15Rα-Fc fusion protein were also evaluated by assessing thermal stability (Table 7) and dynamic light scattering (data not shown).

[0091] A subset of these IL-15 / IL-15Rα-Fc fusion proteins was then selected for further in vitro functional testing. Figure 4 shows the effect of IL-15 / IL-15Rα-Fc fusion proteins on NK cell proliferation and cytotoxicity. NK92 cells, an interleukin-2 (IL-2)-dependent natural killer (NK) cell line, were used to evaluate the effect of IL-15 / IL-15Rα-Fc fusion proteins on NK cell proliferation and cytotoxicity. Figure 4 shows that treatment of NK92 cells with increasing concentrations of IL-15 / IL-15Rα-Fc fusion proteins increased proliferation. Figure 5 shows that treatment of NK92 cells with increasing concentrations of IL-15 / IL-15Rα-Fc fusion proteins increased target cell death.

[0092] Next, IL-15 / IL-15Rα-Fc fusion proteins were evaluated for their effect on NK cell degranulation. NK cells were treated overnight with various concentrations of IL-15 / IL-15Rα-Fc fusion proteins. NK cell degranulation was analyzed using anti-CD107a antibodies and flow cytometry. Figure 6 shows that treatment of NK cells with increasing concentrations of IL-15 / IL-15Rα-Fc fusion proteins increased NK cell degranulation compared to untreated NK cells.

[0093] The effect of IL-15 / IL-15Rα-Fc fusion protein on NK and CD8+ T cell signaling was also evaluated in vitro. Figure 7A shows STAT5 phosphorylation in primary NK cells treated overnight with 0.5 nM IL-15 / IL-15Rα-Fc fusion protein. Figure 7B shows STAT5 phosphorylation in primary CD8+ T cells treated overnight with 0.5 nM IL-15 / IL-15Rα-Fc fusion protein. STAT5 phosphorylation was analyzed using an anti-phospho-STAT5 antibody and flow cytometry. Figures 7A and 7B demonstrate that IL-15 / IL-15Rα-Fc fusion protein increases NK and CD8+ T cell activation.

[0094] 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

[0095] In vivo testing of IL-15 / IL-15Rα-Fc fusion protein The IL-15 / IL-15Rα-Fc fusion proteins were further tested in vivo for their ability to inhibit tumor growth and metastasis in mice.

[0096] Tumor-bearing mice were administered vehicle (negative control), reference molecule V1, molecule W1, molecule Y1, or molecule Z1. Mice were sacrificed 15 or 21 days after administration (Figures 8A and 8B, respectively). Overall, treatment with the IL-15 / IL-15Rα-Fc fusion protein was well tolerated. Of the four variants tested, molecule W1, molecule Y1, and molecule Z1 demonstrated excellent inhibition of tumor metastasis. Harvested lungs revealed varying degrees of tumor burden (black) depending on the vehicle or variant administered (Figure 9A). Other harvested organs showed similar results to the lungs, where molecule Z1 demonstrated excellent reduction in tumor burden compared to the vehicle control and standard reference molecule V1 (Figure 9B).

[0097] The IL-15 / IL-15Rα-Fc fusion protein molecule Z1 was further tested for its effect on survival in tumor-bearing mice. The molecule Z1 was administered subcutaneously or intravenously to tumor-bearing mice at various doses. Figure 10 shows that the molecule Z1 improved the survival rate of tumor-bearing mice at all doses tested compared to the vehicle control. Intravenous or subcutaneous administration of the molecule Z1 at a dose of 0.25 mg / kg demonstrated the greatest effect on survival compared to the other doses tested.

[0098] Overall, these results demonstrate that treatment with IL-15 / IL-15Rα-Fc fusion protein inhibits tumor growth and metastasis and increases survival in a mouse model of cancer. Example 4

[0099] Animal model efficacy: Evaluation of the efficacy of IL-15 / IL-15Rα-Fc fusion protein against mouse melanoma cells B16F10-Luc in a C57BL / 6J mouse lung metastasis model To evaluate the efficacy of the IL-15 / IL-15Rα-Fc fusion protein in a mouse model, luciferase-transfected mouse melanoma cells B16F10-Luc were selected in C57BL / 6J mice, and the effect of the IL-15 / IL-15Rα-Fc fusion protein on the metastatic effect of mouse melanoma cells B16F10-Luc in C57BL / 6J mice was evaluated in an anti-tumor cell metastasis model of lung metastasis via tail vein injection. A total of four groups were designed, and the experimental design, dosage, and administration method are shown in Table 8, and the changes in body weight of the challenged mice are shown in Table 9.

[0100] [Table 8]

[0101] [Table 9]

[0102] Evaluation of the efficacy of IL-15 / IL-15Rα-Fc fusion protein against murine melanoma cells B16F10-Luc in a C57BL / 6J mouse lung metastasis model—Radiance values ​​of lung metastatic tumors from tumor-burdened mice are detailed in Table 10 and FIG. 11.

[0103] [Table 10]

[0104] As shown in Table 10 and Figure 11, on day 21, the tumor growth inhibition values ​​(TGI%) of B16F10-Luc lung metastatic tumors in the monotherapy groups of molecule Y1 at doses of 0.125 mg / kg, 0.25 mg / kg, and 0.5 mg / kg were 88%, 87%, and 91%, respectively, which were all statistically highly significant compared with the tumor growth inhibition values ​​(TGI%) of the control group (P<0.01), indicating that molecule Y1 has good efficacy and safety.

[0105] In conclusion, these results suggest that treatment of mouse tumor models with IL-15 / IL-15Rα-Fc fusion protein inhibits tumor growth and metastasis and further improves survival in mouse tumor models.

[0106] Incorporation by Reference All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, mention of any reference, article, publication, patent, patent publication, or patent application cited herein is not and should not be construed as an admission, or in any way implying, that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Claims

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

2. 2. The polypeptide of claim 1, wherein the IL-15 receptor alpha sushi domain 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 to any one of SEQ ID NOs: 8 to 11, or more preferably, the IL-15 receptor alpha sushi domain comprises the amino acid sequence of any one of SEQ ID NOs: 8 to 11.

3. 3. The polypeptide of claim 1 or 2, wherein the IL-15 C-terminal domain 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%, or at least about 98% sequence identity to SEQ ID NO:3 or SEQ ID NO:4, or more preferably, the IL-15 C-terminal domain comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:

4.

4. 4. The polypeptide of any one of claims 1 to 3, wherein the IL-15 N-terminal domain 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%, or at least about 98% sequence identity to SEQ ID NO:5 or SEQ ID NO:6, or more preferably, the IL-15 N-terminal domain comprises the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6, or more preferably, the IL-15 N-terminal domain comprises an N72D mutation.

5. 5. The polypeptide of claim 1, wherein the Fc domain is an IgG1 Fc domain comprising 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 to SEQ ID NO: 12, or more preferably, the Fc domain is an IgG1 Fc domain comprising the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO:

14.

6. 5. The polypeptide of claim 1, wherein the Fc domain is an IgG4 Fc domain comprising 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 to SEQ ID NO: 17, or more preferably, the Fc domain is an IgG4 Fc domain comprising the amino acid sequence of SEQ ID NO:

17.

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

8. A polypeptide according to any one of claims 1 to 7, comprising one or more linkers.

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

11. 2. The polypeptide of claim 1, comprising 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 to any one of SEQ ID NOs:20 to 30.

12. 2. The polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 20 to 30, or more preferably, the polypeptide consists of the amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO:

30.

13. 2. The polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 20 to 30 with one or more mutations, or more preferably, the polypeptide comprises the amino acid sequence of SEQ ID NO: 24 with one or more mutations in the IL-15N domain 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.

14. 2. The polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 28, having one or more mutations selected from the group consisting of N189Q and G190A.

15. 2. The polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 30, having one or more mutations selected from the group consisting of N189Q and G190A.

16. The polypeptide of claim 1, consisting of any one of the amino acid sequences of SEQ ID NO: 20 to SEQ ID NO:

30.

17. 17. The polypeptide of any one of claims 1 to 16, wherein the polypeptide binds to IL-2 receptor beta and / or IL-2 receptor gamma, more preferably, the polypeptide binds to IL-2 receptor beta 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 to 17.

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

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

21. 18. A pharmaceutical composition comprising a polypeptide according to 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 a polypeptide described in any one of claims 1 to 17 or a pharmaceutical composition described in claim 21 to a subject in need of treatment.

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

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

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