Agonist interleukin-15 complexes and uses thereof

Abstract

The present disclosure provides agonistic IL-15 complexes and methods for making such proteins that have therapeutic uses, as well as methods for treating disease in subjects in need thereof by administering such agonistic IL-15 complexes.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to International Application No. PCT / CN2022 / 116012, filed August 31, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Reference to an electronically submitted sequence listing This application contains a Sequence Listing with filename "069777.11027-6WO2_sequence_listing.xml", created on August 17, 2023, and 10 kb in size. The Sequence Listing is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] The present disclosure relates generally to compositions and methods comprising interleukin-15 (IL-15) agonists, e.g., agonist IL-15 conjugates, which can be used, e.g., in methods of treating disease in a subject in need thereof. [Background technology]

[0004] The cytokine interleukin-15 (IL-15) is a member of the four α-helical bundle family of lymphokines produced by many cells in the body. IL-15 plays a central role in regulating the activity of both the innate and adaptive immune systems, including maintaining memory T cell responses to invading pathogens, inhibiting apoptosis, activating dendritic cells, and inducing natural killer (NK) cell proliferation and cytotoxic activity.

[0005] The IL-15 receptor consists of three polypeptides: the type-specific IL-15 receptor alpha ("IL-15Ra"), the IL-2 / IL-15 receptor beta (or CD122) ("3"), and a common gamma chain (or CD132) ("γ") shared by multiple cytokine receptors. IL-15Ra is expressed by a variety of cell types, but is not necessarily expressed in combination with β and γ. IL-15 signaling has been shown to occur through IL-15Ra, a heterodimeric complex of β and γ, a heterodimeric complex of β and γ, or IL-15RX, a subunit found on mast cells (Ikemizu, Shinji et al. "IL-2 and IL-15 signaling complexes: different but the same." Nature immunology vol. 13, 12 (2012): 1141-2, PMID: 23160210; Briukhovetska, Daria et al. "Interleukins in cancer: from biology to therapy." Nature reviews. Cancer vol. 21, 8 (2021): 481-499, PMID: 3408378).

[0006] Although IL-15 is a soluble protein, endogenous IL-15 is not readily detectable in serum or body fluids. Instead, it occurs primarily as a membrane-bound form expressed or acquired by several types of accessory cells (Fehniger, TA, and MA Caligiuri. "Interleukin 15: biology and relevance to human disease." Blood vol. 97, 1 (2001): 14-32, PMID: 11133738). For example, IL-15 mRNA is detected in both hematopoietic and non-hematopoietic cells, but T cells do not produce IL-15. Instead, IL-15 binds to IL-15Ra and forms a cell surface complex on T cells. IL-15 binds specifically to IL-15Ra with high affinity via a "sushi domain" within exon 2 of the receptor's extracellular domain (Budagian, Vadim et al. "IL-15 / IL-15 receptor biology: a guided tour through an expanding universe." Cytokine & Growth Factor Reviews Vol. 17, 4 (2006): 259-80, PMID: 16815076). After trans-endosomal recycling and remobilization to the cell surface, these IL-15 complexes acquire the ability to activate bystander cells expressing the IL-15Rβγ low-affinity receptor complex, inducing IL-15-mediated signaling via the Jak / Stat pathway. A naturally occurring soluble form of IL-15Ra ("sIL-15Ra") has been identified that is cleaved at a cleavage site within the extracellular domain immediately distal to the receptor's transmembrane domain (Lukic, ML et al. "Lack of the mediators of innate immunity attenuate the development of autoimmune diabetes in mice." Journal of autoimmunity vol. 21, 3 (2003): 239-46, PMID: 14599848). Tumor necrosis factor alpha-converting enzyme (TACE / ADAM17) has been suggested as the protease involved in this process. Summary of the Invention [Problem to be solved by the invention]

[0007] Based on its multifaceted roles in the immune system, various therapies designed to modulate IL-15-mediated functions are being explored. For example, administration of exogenous IL-15 can enhance immune function in patients infected with human immunodeficiency virus (HIV). Despite many advances in understanding the function of IL-15, many challenges remain for IL-15-based therapies, as discussed further below. Therefore, there is strong interest in further developing new IL-15-based therapeutics. [Means for solving the problem]

[0008] The present disclosure generally relates to fusion proteins comprising an interleukin-15 (IL-15) receptor alpha sushi domain fused to an Fc monomer, wherein the IL-15 receptor alpha sushi domain comprises the amino acid sequence of SEQ ID NO:5, and the Fc monomer comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the carboxyl terminus of the IL-15 receptor alpha sushi domain is fused to the amino terminus of the Fc monomer via a linker having the amino acid sequence of GGGGS (SEQ ID NO:7). In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:2.

[0009] Further, the present disclosure generally relates to a fusion protein consisting of the amino acid sequence of SEQ ID NO: 1. Further, the present disclosure generally relates to a fusion protein consisting of the amino acid sequence of SEQ ID NO: 2. Additionally, the present disclosure generally relates to a protein complex comprising a fusion protein described herein and IL-15. In some embodiments, IL-15 comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, IL-15 consists of the amino acid sequence of SEQ ID NO: 3. In some embodiments, IL-15 comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, IL-15 consists of the amino acid sequence of SEQ ID NO: 4.

[0010] Furthermore, the present disclosure generally relates to nucleic acid molecules, e.g., vectors, encoding the fusion proteins described herein. Furthermore, the present disclosure generally relates to host cells comprising nucleic acid molecules, e.g., vectors, encoding the fusion proteins described herein. Additionally, the present disclosure generally relates to methods of producing the fusion proteins described herein, comprising culturing a host cell described herein under conditions sufficient to express the fusion protein and isolating the fusion protein. Furthermore, the present disclosure generally relates to one or more nucleic acid molecules, e.g., one or more vectors, encoding the fusion proteins of the protein complexes described herein and IL-15. Furthermore, the present disclosure generally relates to one or more host cells comprising one or more nucleic acid molecules, e.g., one or more vectors, encoding the fusion proteins of the protein complexes described herein and IL-15. Additionally, the present disclosure generally relates to methods of producing the protein complexes described herein, comprising culturing one or more host cells described herein under conditions sufficient to express the fusion proteins and IL-15, isolating the fusion proteins and IL-15, and optionally assembling the protein complex by combining the isolated fusion proteins and IL-15 in vitro.

[0011] Furthermore, the present disclosure generally relates to pharmaceutical compositions comprising a fusion protein described herein, a protein complex described herein, a nucleic acid molecule described herein, one or more nucleic acid molecules described herein, a host cell described herein, or one or more host cells described herein, and a pharmaceutically acceptable excipient, diluent, or carrier. In some aspects, the pharmaceutical composition comprises a protein complex described herein.

[0012] Furthermore, the present disclosure generally relates to a method of treating a disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical composition described herein. In some embodiments, the method further comprises administering to the subject another therapeutically effective agent. In some embodiments, the other therapeutically effective agent comprises a small molecule compound, a targeted chemotherapeutic agent, a radiotherapeutic agent, or an antibody or antigen-binding fragment thereof. In some embodiments, the other therapeutically effective agent comprises an antibody or antigen-binding fragment thereof that specifically binds to CD20, PD1, PDL1, Her2, EGFR, or c-MET. In some embodiments, the other therapeutically effective agent comprises an anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the disease is a tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the disease is a pathogen infection, e.g., a bacterial infection and / or a viral infection. In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is an inflammatory disease. In some embodiments, the disease is a neurodegenerative disease.

[0013] Furthermore, the present disclosure generally relates to methods of treating a tumor, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition described herein. In some embodiments, the tumor is a solid tumor, such as a carcinoma, sarcoma, or melanoma. In some embodiments, the solid tumor is selected from the group consisting of bladder cancer, lung cancer, breast cancer, colorectal cancer, brain tumor, prostate cancer, melanoma, Merkel cell carcinoma, head and neck cancer, small intestine cancer, squamous cell carcinoma, metastatic solid tumor, or cervical cancer. In some embodiments, the tumor is bladder cancer. In some embodiments, the tumor is lung cancer, e.g., small cell lung cancer. In some embodiments, the tumor is breast cancer. In some embodiments, the tumor is cervical cancer. In some embodiments, the tumor is colorectal cancer. In some embodiments, the method further comprises administering an immunotherapy to the subject. In some embodiments, the immunotherapy comprises an anti-PD-1 antibody or an antigen-binding fragment thereof. In some embodiments, after administration of the pharmaceutical composition, the subject does not experience necrosis in the liver, spleen, lungs, and / or kidneys. In some embodiments, administration of the pharmaceutical composition does not result in a cytokine storm, and in particular, administration of the pharmaceutical composition does not have a significant effect on the in vivo release of the cytokines IL-1β, IL-2, IL-4, IL-10, IL-5, CXCL / KC, and IL-12P / p70. In some embodiments, administration of the pharmaceutical composition does not increase the liver function indicators glutamate transaminase (ALT) and glutathione transaminase (AST). In some embodiments, administration of the pharmaceutical composition does not increase leukocyte release, lymphocyte release, and / or monocyte release.

[0014] Other aspects, features and advantages of the present invention will become apparent from the following disclosure, including the detailed description of the invention and its preferred embodiments, and the appended claims. [Brief explanation of the drawings]

[0015] [Figure 1A-D]FIG. 1A shows a schematic diagram of an IL-15 agonist complex and graphical representations of biolayer interferometry (BLI) results (FIGS. 1B-1D) of the binding of FL115, ALT803, and FL115-WT to IL-15Rβ, respectively, according to an embodiment of the present application, e.g., Example 2. FIG. 1A shows a schematic diagram of an IL-15 agonist complex. FIG. 1B shows a graphical representation of the binding of FL115 to IL-15Rβ. FIG. 1C shows a graphical representation of the binding of ALT803 to IL-15Rβ. FIG. 1D shows a graphical representation of the binding of FL115-WT to IL-15Rβ. Referring to FIGS. 1B-1D, k represents the association rate constant, k represents the dissociation rate constant, and K represents k / k, which indicates the equilibrium constant for binding of the fusion protein to an antigen, reflecting the affinity of the fusion protein.

[0016] [Figure 2A-B]

[0033] Figure 2A shows a graphical representation of the biological activity of FL115 as detected by a CTLL2 cell proliferation assay (Figure 2A) and an M-07e cell proliferation assay (Figure 2B) according to an embodiment of the present application, e.g., Example 3. Figure 2A shows a graphical representation of the proliferation activity of CTLL2 cells, using OD450 measured as a function of the concentration of either FL115-V1 (squares), FL115-V2 (circles), or sFc (inverted triangles). Figure 2B shows a graphical representation of the proliferation activity of M-07e cells, using luminescence measured as a function of the concentration of FL115.

[0017] [Figure 3A-E]Figure 3A shows a graphical representation of the immunostimulatory effect of FL115 on human immune cells according to an embodiment of the present application, e.g., Example 4. Figure 3A shows a graphical representation of a lymphocyte proliferation assay with the percentage proliferative response plotted against the concentration of ALT803 (squares) or FL115 (circles). Figure 3B shows a graphical representation of an immune cell proliferation assay with the absolute cell counts by cell type presented for medium (solid black), ALT803 (solid white), or FL115 (crosshatched pattern), respectively. Figure 3C shows a graphical representation of a cell surface marker activation assay with the CD69 MFI plotted against the concentration of ALT803 CD8+ T cells (squares), FL115 CD8+ T cells (hexagons), ALT803 NK cells (diamonds), FL115 NK cells (stars), FL115 CD4+ T cells (inverted triangles), or ALT803 CD4+ T cells (circles). Figure 3D shows a graphical representation of a granzyme B expression assay with MFI plotted against the concentration of NK cells (circles) or CD8+ T cells (squares). Figure 3E shows a graphical representation of a perforin expression assay with MFI plotted against the concentration of NK cells (circles) or CD8+ T cells (squares).

[0018] [Figure 4] 1 shows a graphical representation of the binding of FL115 to FcRn as measured by surface plasmon resonance (SPR) according to an embodiment of the present application, e.g., Example 5.

[0019] [Figure 5A-E]5A and 5B show graphical representations of the anti-tumor effect of FL115 in an MC38 subcutaneously implanted tumor model according to embodiments of the present application, e.g., Example 6. FIG. 5A shows data obtained for the change in tumor volume over time for FL115 (circles), ALT803 (squares), and PBS (triangles). FIG. 5B shows data obtained for the change in tumor volume over time for FL115. FIG. 5C shows data obtained for the change in tumor volume over time for ALT803. FIG. 5D shows data obtained for the change in tumor volume over time for PBS. FIG. 5E shows data obtained for the change in weight over time for FL115 (circles), ALT803 (squares), and PBS (triangles).

[0020] [Figure 6A-D] 6A and 6B show graphical representations of assays measuring the killing function of NK cells and CD8+ T cells in tumor tissue of an MC38 mouse colon carcinoma subcutaneous tumor model, according to embodiments of the present application, e.g., Example 7. Figure 6A shows the results of measuring GZMB+CD8+ in CD3+ cells for PBS, ALT803, or FL115. Figure 6B shows the results of measuring perforin+CD8+ in CD3+ cells for PBS, ALT803, or FL115. Figure 6C shows the results of measuring GZMB+ in NK cells for PBS, ALT803, or FL115. Figure 6D shows the results of measuring perforin+ in NK cells for PBS, ALT803, or FL115.

[0021] [Figure 7] 7 shows a graphical representation of survival curves for mice in the CT26 mouse colon cancer metastasis tumor model administered with either PBS, ALT803, or FL115, according to embodiments of the present application, e.g., Example 8. Note that the lines representing FL115 and ALT803 overlap in the graphical representation of FIG.

[0022] [Figure 8A-D]8A and 8B show graphical representations of the anti-tumor effects of FL115 in combination with an anti-PD-1 antibody in mice, according to embodiments of the present application, e.g., Example 9. Figure 8A shows results regarding tumor volume over time in CMT167 mice treated with either PBS (circles), FL115 (squares), PD1 (upright triangles), or FL115 + PD-1 (inverted triangles). Figure 8B shows results regarding mouse weight over time in CMT167 mice treated with either PBS (circles), FL115 (squares), PD1 (upright triangles), or FL115 + PD-1 (inverted triangles). Figure 8C shows results regarding tumor volume over time in LLC mice treated with either PBS (circles), FL115 (squares), PD1 (upright triangles), or FL115 + PD-1 (inverted triangles). Figure 8D shows the results for mouse weight over time in LLC mice treated with either PBS (circles), FL115 (squares), PD1 (upright triangles), or FL115 + PD-1 (inverted triangles).

[0023] [Figure 9A-B] 9A and 9B show graphical representations of BLI validation of the affinity of FL115 for CD16a 158V and CD16a 158F according to embodiments of the present application, e.g., Example 10. Figure 9A shows a graphical representation of BLI validation of the affinity of FL115 for CD16a 158F. Figure 9B shows a graphical representation of BLI validation of the affinity of FL115 for CD16a 158V.

[0024] [Figure 10A-D]Figure 10A shows a graphical representation of the effects of FL115 on liver and kidney function in a safety evaluation in mice according to an embodiment of the present application, e.g., Example 11. Figure 10A shows a graphical representation of creatinine (ECRE) levels in mice administered a given dose of PBS, FL115, or ALT803. Figure 10B shows a graphical representation of urea nitrogen (UN) levels in mice administered a given dose of PBS, FL115, or ALT803. Figure 10C shows a graphical representation of glutathione transaminase (AST) levels in mice administered a given dose of PBS, FL115, or ALT803. Figure 10D shows a graphical representation of glutamate transaminase (ALT) levels in mice administered a given dose of PBS, FL115, or ALT803.

[0025] [Figure 11A-D]

[0033] Figure 11A shows graphical representations of the hematological effects of FL115 in mice during safety evaluation according to embodiments of the present application, e.g., Example 11. Figure 11A shows WBC counts in mice administered with given doses of PBS, FL115, or ALT803. Figure 11B shows lymphocyte counts in mice administered with given doses of PBS, FL115, or ALT803. Figure 11C shows monocyte counts in mice administered with given doses of PBS, FL115, or ALT803. Figure 11D shows neutrophil counts in mice administered with given doses of PBS, FL115, or ALT803.

[0026] [Figures 12A-F]12A and 12B show graphical representations of visceral weights of various organs in mice during a safety evaluation of FL115 according to embodiments of the present application, e.g., Example 11. FIG. 12A shows lung weights for mice administered either PBS, FL115, or ALT803. FIG. 12B shows lymph node weights for mice administered either PBS, FL115, or ALT803. FIG. 12C shows spleen weights for mice administered either PBS, FL115, or ALT803. FIG. 12D shows liver weights for mice administered either PBS, FL115, or ALT803. FIG. 12E shows kidney weights for mice administered either PBS, FL115, or ALT803. FIG. 12F shows heart weights for mice administered either PBS, FL115, or ALT803.

[0027] [Figure 13] 1 shows images of H&E results in the 20 mg / kg ALT803 group according to embodiments of the present application, such as Example 11.

[0028] [Figure 14] 1 shows images of histochemistry results of internal organs in mice from various different treatment groups according to embodiments of the present application, e.g., Example 11.

[0029] [Figure 15A-J] FIG. 15 shows a graphical representation of FL115 stimulation of IL-6 (FIG. 15A), IFN-γ (FIG. 15B), TNF-α (FIG. 15C), IL-1β (FIG. 15D), IL-2 (FIG. 15E), IL-4 (FIG. 15F), IL-10 (FIG. 15G), IL-5 (FIG. 15H), CXCL / KC (FIG. 15I), and IL-12P / p70 (FIG. 15J) in mice according to an embodiment of the present application, e.g., Example 12. Open circles represent ALT803 at a dose of 20 mg / kg, diamonds represent ALT803 at a dose of 2 mg / kg, inverted triangles represent ALT803 at a dose of 0.2 mg / kg, upright triangles represent FL115 at a dose of 20 mg / kg, squares represent FL115 at a dose of 2 mg / kg, and circles represent FL115 at a dose of 0.2 mg / kg.

[0030] [Figures 16A-C] Figure 16 shows graphical results of the anti-tumor effect of IL-15 in a subcutaneous mouse bladder cancer model according to an embodiment of the present application, e.g., Example 13. Figure 16A shows a graphical representation of the change in tumor volume over time for mice administered either PBS (circles), 0.2 mg / kg FL115 (squares), 2 mg / kg FL115 (upright triangles), or 20 mg / kg FL115 (inverted triangles). Figure 16B shows a graphical representation of the percentage tumor inhibition over time for mice administered either PBS (circles), 0.2 mg / kg FL115 (squares), 2 mg / kg FL115 (upright triangles), or 20 mg / kg FL115 (inverted triangles). Figure 16C shows a graphical representation of the change in body weight over time for mice administered either PBS (circles), 0.2 mg / kg FL115 (squares), 2 mg / kg FL115 (upright triangles), or 20 mg / kg FL115 (inverted triangles).

[0031] [Figures 17A-C]

[0041] Figure 17 shows data on the anti-tumor effect of IL-15 in an orthotopic model of bladder cancer in mice according to embodiments of the present application, e.g., Example 13. Figure 17A shows the bladder weight of each mouse in four different groups of mice. Figure 17B shows images of the bladders excised from each mouse from the four different groups of mice. Figure 17C shows a graphical representation of the body weight of each mouse from the four different groups of mice.

[0032] [Figure 18A-B] For example, Example 14 shows the results of measuring the half-life of FL115 and ALT803 in mice. Figure 18A shows the serum concentration of FL115 at various time points after administration to mice. Figure 18B shows the serum concentration of ALT803 at various time points after administration to mice. DETAILED DESCRIPTION OF THE INVENTION

[0033] Various publications, articles, and patents are cited or described in the background and throughout the specification, and each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles and the like included in the specification is for the purpose of providing a context for the present invention. Such discussion does not constitute an admission that any or all of these matters form part of the prior art to any invention disclosed or claimed.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings set forth herein. All patents, published patent applications, and publications cited herein are incorporated by reference as if fully set forth herein.

[0035] It should be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0036] Unless otherwise specified, any numerical value, such as a % sequence identity or % sequence identity range described herein, is understood to be modified in all instances by the term "about." Thus, numerical values ​​typically include ±10% of the recited value. For example, a 10 mg dose includes 9 mg to 11 mg. As used herein, the use of numerical ranges expressly includes all possible subranges, all individual numerical values ​​within the range, including integers and fractions of values ​​within such ranges, unless the context clearly indicates otherwise.

[0037] As used herein, the conjunction "and / or" between multiple listed elements is understood to encompass both individual and combined alternatives. For example, when two elements are joined by "and / or," the first alternative refers to the applicability of the first element without the second. The second alternative refers to the applicability of the second element without the first. The third alternative refers to the applicability of the first element and the second element together. Any one of these alternatives is understood to fall within the meaning and therefore meet the requirements of the term "and / or" as used herein. The simultaneous applicability of two or more of the alternatives is also understood to fall within the meaning and therefore meet the requirements of the term "and / or."

[0038] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0039] Throughout the following specification and claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to mean the inclusion of a stated integer or step or group of integers or steps, and not the exclusion of any other integer or step or group of integers or steps. As used herein, the word "comprise" can be interchanged with the words "containing" or "including," or, as sometimes used herein, with the word "having."

[0040] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any of the above terms "comprise," "contain," "include," and "have," whenever used herein in connection with aspects or embodiments of the present invention, may be substituted with the terms "consisting of" or "essentially consisting of" in order to change the scope of the disclosure.

[0041] As used herein, the terms "antibody" and "antibodies" refer to molecules containing an antigen-binding site. Antibodies include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, single-domain antibodies, camelized antibodies, single-chain Fvs (scFvs), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked bispecific Fvs (sdFvs), intrabodies, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies and anti-anti-Id antibodies against antibodies), as well as epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules. Immunoglobulin molecules can belong to any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0042] As used herein, the terms "disease" and "disorder" are used interchangeably to refer to conditions that are affected by IL-15 signaling. In particular, a "disease" and a "disorder" can be a condition, and more particularly, a disease, that is affected by IL-15 signaling.

[0043] The term "agonist" refers to a compound that binds to a receptor and induces an agonistic intracellular response. An agonist mimics the effect of an endogenous ligand, e.g., a hormone, and produces a physiological response similar to that produced by the endogenous ligand.

[0044] As used herein, "fragment crystallizable region" or "Fc" refers to a polypeptide containing the heavy chain constant region, excluding the first domain, CH1, of the constant region. Thus, Fc may include the remaining two domains (CH2 and CH3) of the heavy chain constant region of an IgA, IgD, or IgG antibody, or the remaining three domains (CH2, CH3, and CH4) of the heavy chain constant region of an IgE or IgM antibody. Fc may also include a flexible hinge region linked to the N-terminus of the CH2 domain. In the case of IgA and IgM, Fc may include the J chain. In the case of IgG, Fc contains immunoglobulin domains Cγ2 and Cγ3 and the hinge between Cγ1 and Cγ2. Fc plays multiple roles in dimerization, forming and maintaining the Y-shaped structure of Ig, as well as Fc-mediated effector functions and extended serum half-life. Binding of Fc in IgG to its receptor, Fc gamma receptor (FcγR), triggers antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis, killing and eliminating target cells (e.g., tumor cells). Binding of Fc to serum complement molecules (C1q) initiates the assembly of a membrane attack complex formed by complement cascade proteins, which can destroy target cells, a process known as complement-dependent cytotoxicity (CDC). In addition to mediating effector functions, Fc can also bind to neonatal Fc receptor (FcRn) in a pH-dependent manner, potentially extending the serum half-life of IgG. Furthermore, binding of Fc to immune-related molecules such as Fc receptors can regulate immune responses in vivo. Fc can refer to this region in isolation or in the context of an antibody, antibody fragment, or Fc fusion. Fc can be part of an antibody, Fc fusion, or Fc-containing protein or protein domain. Naturally occurring Fc forms homodimers. Fc variants, non-naturally occurring Fc variants obtained using synthetic biology, are particularly preferred.

[0045] As used herein, "Fc monomer" or "monomeric Fc" refers to an Fc variant that no longer forms homodimers under physiological conditions. Examples of Fc monomers include, but are not limited to, those described in Wang et al., 2017, Front. Immunol., vol. 8, article 1545, WO 2022088484 A1, and U.S. Patent Application Publication No. 20210206847 (the contents of each of which are incorporated herein by reference in their entirety).

[0046] In some embodiments, the Fc monomers of the present disclosure comprise an Fc polypeptide that is only half the molecular weight of a wild-type Fc dimer, retain the FcRn-binding and Protein A / G-binding capabilities of an antibody Fc region, can achieve highly efficient expression in host cells, e.g., prokaryotic host cells, and can exhibit significantly reduced non-specific binding compared to previously developed monomeric IgG1 Fc mutants.

[0047] As used herein, the term "IL-15 functional fragment" refers to a portion of IL-15 that may serve to induce or induce some degree of a biological activity associated with IL-15. Such functional fragments include any mutated IL-15 of any length and / or any truncated version of IL-15 that retains the biological activity of IL-15. In some embodiments, an IL-15 functional fragment comprises or consists of the sequence of SEQ ID NO:3. In some embodiments, an IL-15 functional fragment comprises or consists of the sequence of SEQ ID NO:4. In some embodiments, an IL-15 functional fragment comprises the sequence of any of the IL-15 sequences described throughout this application.

[0048] "Polynucleotide" or "nucleic acid," as used interchangeably herein, refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. "Oligonucleotide," as used herein, refers to a short, generally single-stranded, synthetic polynucleotide that is generally, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of a polynucleotide is equally and fully applicable to oligonucleotides. Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of the nascent RNA transcript is referred to as the transcription direction, and the region of the DNA strand with the same sequence as the RNA transcript that is 5' to the 5' end of the RNA transcript is referred to as the "upstream sequence," and the region of the DNA strand with the same sequence as the RNA transcript that is 3' to the 3' end of the RNA transcript is referred to as the "downstream sequence."

[0049] As used herein, "autoimmune disease" refers to a disease in which the immune system generates an immune response (e.g., a B cell or T cell response) against some of the normal host's antigens (i.e., self-antigens), which then cause tissue damage. Self-antigens can be derived from host cells or from commensal organisms, such as microorganisms that normally colonize mucosal surfaces (called commensals). Autoimmune diseases that affect mammals include, but are not limited to, rheumatoid arthritis, juvenile oligoarthritis, collagen-induced arthritis, adjuvant-induced arthritis, Sjogren's syndrome, multiple sclerosis, experimental autoimmune encephalomyelitis, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis), autoimmune gastric atrophy, pemphigus vulgaris, psoriasis, vitiligo, type 1 diabetes, non-obese diabetes, myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, sclerosing cholangitis, sclerosing sialafilitis, systemic lupus erythematosus, autoimmune thrombocytopenic purpura, Goodpasture's syndrome, Addison's disease, systemic sclerosis, polymyositis, dermatomyositis, autoimmune hemolytic anemia, pernicious anemia, and the like.

[0050] "Viruses" of the present disclosure include, but are not limited to, viruses from the following families: Retroviridae (e.g., human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV)); Picornaviridae (e.g., poliomyelitis virus, hepatitis A virus, hepatitis C virus, enterovirus, human coxsackievirus, rhinovirus, echovirus, foot-and-mouth disease virus); Cadheroviridae (such as virus strains that cause gastroenteritis); Tunicavirus The Virus family (e.g., equine encephalitis virus, rubella virus); the Flaviviridae family (e.g., dengue virus, yellow fever virus, West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, and other encephalitis viruses); the coronavirus family (e.g., coronavirus, severe acute respiratory syndrome (SARS) virus); the Rhabdoviridae family (e.g., vesicular stomatitis virus, rabies virus); the Paramyxoviridae family (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus (RSV)); the Orthomyxoviridae family (e.g., influenza virus, Family Bunyaviridae (e.g., Hantavirus, Sin Nombre virus, Rift Valley fever virus, Bunyavirus, Phlebovirus, and Nairovirus); Family Arenaviridae (e.g., Hemorrhagic fever viruses, Machupo virus, Junin virus); Family Reoviridae (e.g., Reovirus, Orbivirus, and Rotavirus); Family Birnaviridae (e.g., Binnaviridae); Family Hepatoviridae (e.g., Hepatitis B virus); Family Parvoviridae (e.g., Parvovirus); Family Papovaviridae (e.g., Papillomavirus, Polyomavirus, BK virus);Adenoviridae (e.g., most adenoviruses, e.g., adeno-associated viruses); Herpesviruses (e.g., herpes simplex viruses (HSV-1 and HSV-2), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV) and other herpesviruses, e.g., HSV-6); Poxviruses (e.g., variola virus, vaccinia virus, poxvirus); and Irisviruses (e.g., African swine fever virus); Viridans (e.g., Ebola virus, Marburg virus); Caliciviruses (e.g., Norwalk virus) and unclassified viruses (e.g., causative agents of spongiform encephalopathy, causative agents of delta hepatitis (thought to be defective satellites of hepatitis B virus), and astroviruses).

[0051] "Bacteria" of the present disclosure include, but are not limited to, Helicobacter pylori, Borrelia burgdorferi, Legionella pneumophila, Mycobacterium (such as M. tuberculosis, M. avium, M. intracellulare, M. kansaii, and M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, and Streptococcus pyogenes (Group A Streptococcus). Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (Lucky Grass Group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic bacteria), Streptococcus pneumoniae, Pathogenic Campylobacter, Enterococcus, Haemophilus Influenzae, Bacillus anthracis, Corynebacterium diphtheriae diphtheria, Corynebacterium, swine fever virus, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniaepneumoniae, Pasteurella multocida, Bacteroides, Clostridium, Streptomyces malt, Treponema pneumoniae, Treponema pneumoniae Spirochetes, Leptospira or Actinomyces (Actinomyces israelli).

[0052] "Fungi" of the present disclosure include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, or Candida albicans Bacteria (Candida albicans).

[0053] "Parasites" of the present disclosure include, but are not limited to: Plasmodium falciparum or Toxoplasma gondii.

[0054] "Cancer," as used herein, refers to a solid tumor or a blood-borne cancer. Solid tumors of the present disclosure include sarcomas or carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma or another sarcoma, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid tumors, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, bladder cancer, or a tumor of the central nervous system (such as a glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal gland, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, melanoma, neuroblastoma cell tumor, or retinoblastoma). Blood-borne cancers of the present disclosure include leukemias, such as acute leukemia (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic, promyelocytic, myelomononuclear, monocytic and erythroleukemia); chronic leukemia (such as chronic granulocytic (granulocytic) leukemia, chronic granulocytic leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and aggressive forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia or myelodysplasia.

[0055] As used herein, "subject" means any animal, preferably a mammal, most preferably a human, that will be treated or has been treated by a method according to an embodiment of the present disclosure. The term "mammal" as used herein encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs), such as monkeys or apes, humans, and the like, more preferably humans. A human subject may include a patient.

[0056] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. The term also encompasses amino acid polymers that are natural or modified by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Also included within the definition are polypeptides containing one or more analogs of an amino acid, including, for example, but not limited to, unnatural amino acids, and other modifications known in the art. Because the polypeptides of the present disclosure may be based on antibodies or other members of the immunoglobulin superfamily, it is understood that in certain embodiments, a "polypeptide" can exist as a single chain or two or more associated chains.

[0057] As used herein, the terms "treat," "treating," and "treatment," in connection with the administration of a therapy to a subject, refer to the beneficial effect that the subject derives from the therapy, including, but not limited to, a reduction or inhibition of the progression, spread, and / or duration of a disease or disorder, a reduction in the severity or amelioration of a disease or disorder, an amelioration of one or more symptoms of a disease or disorder, and / or a reduction in the duration of one or more symptoms of a disease or disorder that results from the administration of one or more therapies. In specific embodiments, such terms in the context of cancer include, but are not limited to, one, two, or three or more of the following results following administration of a therapy to a subject: (1) reduced tumor or neoplastic growth, (2) reduced tumor formation, (3) eradication, removal, or control of primary localized cancer and / or metastatic cancer, (4) reduced metastatic spread, (5) reduced mortality, (6) increased survival rate, (7) increased length of survival, (8) increased number of patients in remission, (9) reduced hospitalization rate, (10) reduced length of hospitalization, and (11) maintenance of tumor size such that the tumor does not increase in size by more than 10%, or more than 8%, or more than 6%, or more than 4%, preferably the tumor does not increase in size by more than 2%.

[0058] As used herein, the terms "prevent," "preventing," and "prevention," in connection with the administration of a therapy to a subject, refer to the inhibition of the onset or recurrence of a disease or disorder in a subject.

[0059] The phrases "percent (%) sequence identity," or "% identity," or "% identical to," when used in reference to amino acid sequences, describe the number of identical amino acid matches ("hits") in two or more aligned amino acid sequences compared to the number of amino acid residues that make up the entire length of the amino acid sequence. In other words, using alignment, the percentage of amino acid residues that are the same (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of the amino acid sequence) for two or more sequences can be determined when sequences are compared and aligned for maximum correspondence, as measured using sequence comparison algorithms known in the art, or manually aligned and visually inspected. Similar determinations can be made for nucleotide sequences. Thus, sequences compared to determine sequence identity can differ due to amino acid substitutions, additions, or deletions. Suitable programs for aligning protein sequences are known to those of skill in the art. The percentage sequence identity of protein sequences can be determined, for example, by programs such as CLUSTALW, Clustal Omega, FASTA or BLAST, using, for example, the NCBI BLAST algorithm (Altschul SF, et al (1997), Nucleic Acids Res. 25:3389-3402).

[0060] As used herein, a "non-naturally occurring" nucleic acid or polypeptide refers to a nucleic acid or polypeptide that does not exist in nature. A "non-naturally occurring" nucleic acid or polypeptide may be synthesized, treated, fabricated, and / or specifically engineered in a laboratory and / or manufacturing setting. In some cases, a non-naturally occurring nucleic acid or polypeptide may include a naturally occurring nucleic acid or polypeptide that has been treated, processed, or engineered to exhibit properties that were not present in the naturally occurring nucleic acid or polypeptide prior to the treatment. As used herein, a "non-naturally occurring" nucleic acid or polypeptide may be a nucleic acid or polypeptide that has been isolated or separated from the natural source in which it was found, and that lacks covalent bonds with the sequence with which it was associated in the natural source. A "non-naturally occurring" nucleic acid or polypeptide may be produced recombinantly or through other methods, such as chemical synthesis.

[0061] As used herein, the term "operably linked" refers to a connection or juxtaposition in a relationship permitting the components so described to function in their intended manner. For example, a regulatory sequence operably linked to a nucleic acid sequence of interest is capable of directing transcription of the nucleic acid sequence of interest, or a signal sequence operably linked to an amino acid sequence of interest is capable of secreting or translocating the amino acid sequence of interest across a membrane.

[0062] In an attempt to assist the reader of this disclosure, the description is divided into various paragraphs or sections or directed to various embodiments of the present disclosure. These divisions should not be construed as separating the content of one paragraph or section or embodiment from the content of another paragraph or section or embodiment. To the contrary, those skilled in the art will understand that the description has broad application and encompasses all combinations of the various sections, paragraphs, and sentences that may be contemplated. The discussion of any embodiment is meant to be merely exemplary and is not intended to suggest that the scope of the present disclosure, including the claims, is limited to these examples. The present disclosure contemplates the use of any of the applicable components in any combination with any sequence that may be used in the ribonucleic acid molecules of the present disclosure, regardless of whether a particular combination is explicitly described.

[0063] IL-15 and agonist IL-15 complexes Although tumor immune checkpoint inhibitors have proven to be a breakthrough in tumor therapy, the overall efficacy of such treatments is not high, and the objective response rates of monotherapy are mainly in the range of 20% to 30%. Based on an understanding of the tumor microenvironment, many researchers believe that immune activation of NK cells is an important way to solve the problem of the low efficacy of PD-1 monoclonal antibodies.

[0064] Human IL-15, a 12-14 kD cytokine, was discovered by Grabstein et al. in 1994. It may play a role in the body's normal immune response, such as promoting the proliferation of T cells, B cells, and NK cells. IL-15 has been demonstrated to be a useful alternative therapeutic treatment or combination therapy. For example, IL-15 has more skewed NK cell-activating activity than IL-2. Furthermore, IL-2, in addition to the proliferation of killer immune cells, stimulates the proliferation of Tregs, which suppress immune responses and reduce anti-tumor efficacy. In contrast, the specific receptor for IL-15 is IL-15Rα (CD125), which does not activate Treg cells. Therefore, IL-15 has significant advantages over IL-2 as an immune activator. Due to its inherent NK cell-activating ability, IL-15 is expected to become a new generation anti-tumor immune activator for the 70-80% of tumor patients who do not respond to PD-1 monoclonal antibodies, and is expected to have high market potential. As used herein, the term "IL-15" encompasses full-length IL-15 or a functional fragment of IL-15. "IL-15" may be wild-type IL-15 or a mutant IL-15.

[0065] Despite its potential, wild-type IL-15 has an extremely short half-life in vivo, severely limiting its efficacy. Furthermore, IL-15 has a translocation delivery mechanism, and the formation of a complex between IL-15 and IL-15Rα must be considered, posing significant challenges for effective IL-15 therapeutics. In addition to its short half-life and poor pharmacokinetics limiting its clinical application, increased doses of IL-15 can also induce cytokine storms, causing severe toxic side effects and significantly limiting its clinical dose level. Therefore, improved IL-15 therapies are of great interest and value.

[0066] Thus, the present disclosure generally relates to protein complexes, i.e., agonistic IL-15 complexes comprising IL15 and a monomeric IL-15 receptor αSu / Fc fusion protein, and uses thereof. The agonistic IL-15 complexes described herein have a long half-life, exhibit a favorable safety profile, and demonstrate greater antitumor activity compared to other IL-15-based therapies. For example, as demonstrated in the examples below, the in vivo distribution profile of agonistic IL-15 complexes according to embodiments of the present application is flatter than that of a reference (i.e., ALT803), and the C of agonistic IL-15 complexes according to embodiments of the present application is significantly higher. max was not as high as that of the reference and consequently did not promote the large-scale release of cytokines such as IL-6. In the case of agonistic IL-15 complexes such as those described herein, reference ALT803 is a protein complex containing a dimeric IL-15 receptor αSu / Fc fusion protein together with IL15, instead of a monomeric IL-15 receptor αSu / Fc fusion protein together with IL15 (see, e.g., Xu et al., Cancer Res., 2013 May 15;73(10):3075-86).

[0067] In one general aspect, the present application relates to a fusion protein comprising an interleukin-15 (IL-15) receptor alpha sushi domain fused to an Fc monomer, wherein the IL-15 receptor alpha sushi domain comprises the amino acid sequence of SEQ ID NO:5, and the Fc monomer comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the carboxyl terminus of the IL-15 receptor alpha sushi domain is fused to the amino terminus of the Fc monomer via a linker having the amino acid sequence of GGGGS (SEQ ID NO:7), e.g., a linker consisting of the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8. In specific embodiments, the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO:1. In specific embodiments, the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO:2.

[0068] In another general aspect, the present application relates to a protein complex comprising a fusion protein according to an embodiment of the present application and IL-15. In some embodiments, the IL-15 comprises a functional IL-15 fragment having or consisting of the amino acid sequence of SEQ ID NO: 3. In some other embodiments, the IL-15 comprises a functional IL-15 fragment having or consisting of the amino acid sequence of SEQ ID NO: 4.

[0069] In some embodiments, the present disclosure generally relates to an agonistic interleukin-15 (IL-15) complex, wherein the agonistic IL-15 complex agonist comprises an Fc monomer, an IL-15 receptor alpha sushi domain, and an IL-15 functional fragment, wherein the Fc monomer is fused to the IL-15 receptor alpha sushi domain, wherein the Fc monomer-IL-15 receptor alpha sushi domain fusion comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and wherein the IL-15 functional fragment comprises the sequence of SEQ ID NO: 3 or SEQ ID NO: 4. In some aspects, the Fc monomer-IL-15 receptor alpha sushi domain fusion comprises the sequence of SEQ ID NO: 1, and the IL-15 functional fragment comprises the sequence of SEQ ID NO: 3. In some aspects, the Fc monomer-IL-15 receptor alpha sushi domain fusion comprises the sequence of SEQ ID NO: 1, and the IL-15 functional fragment comprises the sequence of SEQ ID NO: 4. In some embodiments, the Fc monomer-IL-15 receptor alpha sushi domain fusion comprises the sequence of SEQ ID NO: 2 and the IL-15 functional fragment comprises the sequence of SEQ ID NO: 3. In some embodiments, the Fc monomer-IL-15 receptor alpha sushi domain fusion comprises the sequence of SEQ ID NO: 2 and the IL-15 functional fragment comprises the sequence of SEQ ID NO: 4.

[0070] In some embodiments, the C-terminus of the IL-15 receptor alpha-sushi domain is linked to an Fc monomer, and the IL-15 receptor alpha-sushi domain is capable of binding to an IL-15 functional fragment. In some embodiments, the C-terminus of the IL-15 receptor alpha-sushi domain is linked to an Fc monomer by a covalent bond. Preferably, the Fc monomer and the IL-15 receptor alpha sushi domain are linked by a linking peptide, more preferably, the linking peptide is GGGGS or (GGGGS)3. A linking peptide refers to a polypeptide chain containing flexible amino acid residues, such as Gly, Ser, Ala, or Thr. The polypeptide chain must be suitable for linking two molecules and have a suitable distance so that they have the correct configuration relative to each other to maintain the desired activity. A suitable length for this purpose includes at least one and no more than 30 amino acid residues. Preferably, the linker is about 1 to 30 amino acids in length, with preferred linker lengths being 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 amino acids. Furthermore, the properties exhibited by the amino acid residues selected for inclusion in the connecting peptide should not significantly affect the activity of the two linked molecules. Thus, the connecting peptide generally does not have a mismatched charge on the two linked molecules, or affect the internal folding of the monomers, or form bonds or other interactions with amino acid residues in one or more of the monomers that would significantly interfere with the binding of the receptor monomer domains. In some embodiments, the connecting peptide contains flexible amino acid residues. For example, connecting peptides can include glycine-serine polymers such as (GS), (GSGGS), (GGGGS), (GGGS), where n is an integer of at least 1, glycine-alanine polymers, alanine-serine polymers, and other flexible connecting peptides known in the art, such as the connecting sequences of Shaker potassium channels and the like.

[0071] In some embodiments, the IL-15 receptor alpha sushi domain of the present disclosure comprises the extracellular domain of IL-15 receptor alpha, starting with the cysteine ​​residue (C1) encoded by the first exon 2 and ending with the fourth exon. Cysteine ​​residue (C4), residues C1 and C4 encoded by sub-exon 2, are all contained in the sushi domain. Also included in the present disclosure are amino acid sequences of the IL-15 receptor alpha sushi domain formed by the substitution, deletion, or addition of one or more amino acid residues and having corresponding activity.

[0072] In some embodiments, the agonist IL-15 complex binds to IL-15 receptor beta (IL-15Rβ). In some embodiments, the agonist IL-15 complex binds to IL-15 receptor beta (IL-15Rβ). In some embodiments, the agonist IL-15 complex binds to IL-15 receptor beta (IL-15Rβ). -9 M or less, 8.00×10 -9 M or less, 7.00×10 -9 M or less, 6.00×10 -9 M or less, 5.00×10 -9 M or less, 4.75×10 -9 M or less, 4.50×10 -9 M or less or 4.47 x 10 -9 In some embodiments, the agonist IL-15 complex binds to IL-15Rβ with a Kd of 9.00×10 or less. In some embodiments, the agonist IL-15 complex binds to FcRn. In some embodiments, the agonist IL-15 complex binds to IL-15Rβ with a Kd of 9.00×10 -6 M or less, 8.00×10 -6 M or less, 7.00×10 -6 M or less, 6.00×10 -6 M or less, 5.00×10 -6 M or less, 4.75×10 -6 M or less, 4.50×10 -6 M or less, 4.25×10 -6 M or less or 4.07 x 10 -6In some embodiments, the agonistic IL-15 complex is capable of binding to FcRn with an affinity equal to or less than M. In some embodiments, the agonistic IL-15 complex is capable of stimulating proliferation of M-07e cells. In some embodiments, the agonistic IL-15 complex is capable of inducing granzyme secretion from tumor-infiltrating killer NK cells and / or CD8+ T cells. Granzymes are a family of serine proteases that induce cell death mediated by a population of cytotoxic lymphocytes (CL) (e.g., cytotoxic T lymphocytes (CTL), natural killer (NK) cells). In some embodiments, the agonistic IL-15 complex is incapable of binding to CD16a 158V or CD16a 158F. In some embodiments, the Fc monomer-IL-15 receptor alpha sushi domain fusion is capable of binding to an IL-15 functional fragment. In some embodiments, the IL-15 functional fragment is an endogenously expressed IL-15 functional fragment. In some embodiments, the IL-15 functional fragment is a mutant IL-15 fragment. In some embodiments, the Fc monomer-IL-15 receptor alpha sushi domain fusion is capable of extending the half-life of IL-15 or an IL-15 functional fragment in vivo.

[0073] Nucleic acid molecules, polynucleotides and host cells The present disclosure generally also relates to nucleic acid molecules encoding the agonistic IL-15 complexes described herein, wherein the Fc monomer-IL-15 receptor alpha sushi domain fusion and the IL-15 functional fragment are encoded by polynucleotides. In some embodiments, the Fc monomer-IL-15 receptor alpha sushi domain fusion and the IL-15 functional fragment are encoded by separate polynucleotides. In some embodiments, the Fc monomer-IL-15 receptor alpha sushi domain fusion and the IL-15 functional fragment are encoded by the same polynucleotide. In some embodiments, the Fc monomer-IL-15 receptor alpha sushi domain fusion and the IL-15 functional fragment are present in the same vector. In some embodiments, the Fc monomer-IL-15 receptor alpha sushi domain fusion and the IL-15 functional fragment are present in separate vectors. In some embodiments, the Fc monomer-IL-15 receptor alpha sushi domain fusion and the IL-15 functional fragment are each operably linked to a different promoter. As used herein, a "vector" refers to a nucleic acid molecule used to transport genetic material into another cell when it is capable of replication and / or expression. Any vector known to those skilled in the art can be used in light of the present disclosure. Examples of vectors include, but are not limited to, plasmids, viral vectors (bacteriophage, animal viruses, and plant viruses), cosmids, and artificial chromosomes (e.g., YACs). Preferably, the vector is a DNA plasmid. The vector may be a DNA vector or an RNA vector. Those skilled in the art can construct the vector of the present application through standard recombinant techniques in light of the present disclosure. The vector of the present application may be an expression vector. As used herein, the term "expression vector" refers to any type of genetic construct containing a nucleic acid encoding a transcribable RNA. Expression vectors include, but are not limited to, vectors for recombinant protein expression, such as DNA plasmids or viral vectors, and vectors for delivery of nucleic acids to a subject intended for expression in the subject's tissue, such as DNA plasmids or viral vectors.It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as, for example, the choice of the host cell to be transformed, the level of expression of protein desired, and the like.

[0074] The vectors of the present application may contain various regulatory sequences. As used herein, the term "regulatory sequence" refers to any sequence that enables, contributes to, or regulates the functional regulation of a nucleic acid molecule, including the replication, duplication, transcription, splicing, translation, stability, and / or transport of a nucleic acid or one of its derivatives (i.e., mRNA) into a host cell or organism. In the context of the present disclosure, this term encompasses promoters, enhancers, and other expression control elements (e.g., polyadenylation signals and sequences that affect mRNA stability).

[0075] In some embodiments of the present application, the vector is a non-viral vector. Examples of non-viral vectors include, but are not limited to, DNA plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, etc. Examples of non-viral vectors include, but are not limited to, RNA replicons, mRNA replicons, modified mRNA replicons, or self-amplifying mRNAs, and closed linear deoxyribonucleic acids, such as linear covalently closed DNA, e.g., linear covalently closed double-stranded DNA molecules. Preferably, the non-viral vector is a DNA plasmid. "DNA plasmid," used interchangeably with "DNA plasmid vector," "plasmid DNA," or "plasmid DNA vector," refers to a double-stranded, generally circular, DNA sequence capable of autonomous replication in a suitable host cell. DNA plasmids used for expression of encoded polynucleotides typically contain an origin of replication, multiple cloning sites, and a selectable marker, which may be, for example, an antibiotic resistance gene. Examples of suitable DNA plasmids that can be used include, but are not limited to, commercially available expression vectors for use in well-known expression systems (including both prokaryotic and eukaryotic systems), such as Escherichia coli pYES2 (Invitrogen, Thermo Fisher Scientific), which can be used for protein production and / or expression in Escherichia coli, and pSE420 (Invitrogen, San Diego, Calif.), which can be used for production and / or expression in Saccharomyces cerevisiae strains of yeast; the MAXBAC® complete baculovirus expression system (Thermo Fisher Scientific), which can be used for production and / or expression in insect cells; pcDNA™ or pcDNA3™ (Life Technologies, Thermo Fisher Scientific), which can be used for high-level constitutive protein expression in mammalian cells; and pVAX or pVAX-1 (Life Technologies, Thermo Fisher Scientific), which can be used for high-level transient expression of a protein of interest in most mammalian cells.The backbone of any commercially available DNA plasmid can be modified to optimize protein expression in a host cell, for example, to invert the orientation of particular elements (e.g., an origin of replication and / or an antibiotic resistance cassette), to replace a promoter endogenous to the plasmid (e.g., a promoter within an antibiotic resistance cassette), and / or to replace a polynucleotide sequence encoding a transcribed protein (e.g., the coding sequence of an antibiotic resistance gene) using conventional techniques and readily available starting materials (see, e.g., Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989)).

[0076] The vectors of the present application, such as DNA plasmids or viral vectors, may contain any regulatory elements necessary to establish the vector's conventional function. Regulatory elements include, but are not limited to, promoters, enhancers, polyadenylation signals, translation stop codons, ribosome binding sequences, transcription terminators, selectable markers, and replication origins. A vector may contain one or more expression cassettes. An "expression cassette" is a portion of a vector that directs the cellular machinery to make RNA and proteins. An expression cassette typically contains three components: a promoter sequence, an open reading frame, and a 3'-untranslated region (UTR) that optionally contains a polyadenylation signal. An open reading frame (ORF) is a reading frame that contains the coding sequence of a protein of interest, from the start codon to the stop codon. As used herein, the term "operably linked" should be interpreted in its broadest reasonable context and refers to the association of polynucleotide elements in a functional relationship. A polynucleotide is "operably linked" when it is placed into a functional relationship with another polynucleotide. For example, a promoter is operably linked to a coding sequence if it affects the transcription of the coding sequence. Any of the components suitable for use in the expression cassettes described herein can be used in any combination and in any order to prepare the vectors of the present application.

[0077] The vector may contain a promoter sequence, preferably within an expression cassette, to control expression. The term "promoter" is used in its conventional sense to refer to a nucleotide sequence that initiates transcription of an operably linked nucleotide sequence. A promoter is located near and on the same strand as the nucleotide sequence it transcribes. Promoters may be constitutive, inducible, or repressible. Promoters may be naturally occurring or synthetic. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters may be homologous promoters (i.e., derived from the same genetic source as the vector) or heterologous promoters (i.e., derived from a different vector or genetic source). For example, if the vector used is a DNA plasmid, the promoter may be endogenous to the plasmid (homologous) or derived from another source (heterologous).

[0078] Examples of promoters that can be used include, but are not limited to, promoters from simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoters, human immunodeficiency virus (HIV) promoters, such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, Moloney virus promoters, avian leukosis virus (ALV) promoters, cytomegalovirus (CMV) promoters, such as the CMV immediate early promoter (CMV-IE), Epstein-Barr virus (EBV) promoter, or Rous sarcoma virus (RSV) promoter. Promoters can also be promoters from human genes, such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. Promoters can also be tissue-specific promoters, such as muscle- or skin-specific promoters (natural or synthetic).

[0079] Additionally, the present disclosure generally relates to host cells comprising the nucleic acid molecules, plasmids, and / or vectors described herein. Host cells of the present disclosure can be any prokaryotic or eukaryotic cell, including, but not limited to, bacterial cells (e.g., Escherichia coli, Bacillus subtilis), insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells (e.g., CHO or BHK cell lines). Other suitable host cells are known to those of skill in the art.

[0080] Pharmaceutical Composition

[0081] Additionally, the present disclosure generally relates to pharmaceutical compositions comprising a fusion protein described herein, a nucleic acid molecule described herein, a plasmid described herein, and / or a host cell described herein, and a pharmaceutically acceptable excipient, diluent, or carrier. In some aspects, the pharmaceutical composition comprises a protein complex described herein. In some aspects, the pharmaceutical composition further comprises one or more anti-PD-1 antibodies or antigen-binding fragments thereof and / or anti-PD-L1 antibodies or antigen-binding fragments thereof.

[0082] Typically, administration of the pharmaceutical compositions and therapeutic combinations of the present disclosure will have a therapeutic goal of generating an immune response against a disease, such as cancer. As used herein, "effective amount" or "therapeutically effective amount" refers to an amount of an agonist IL-15 complex described herein or a composition comprising said complex that is sufficient to induce a desired immune effect or immune response or therapeutic effect in a subject in need thereof. A therapeutically effective amount may be an amount sufficient to induce an immune response in a subject in need thereof. A therapeutically effective amount may be an amount sufficient to generate immunity in a subject in need thereof, for example, to provide a therapeutic effect against a disease, such as cancer. A therapeutically effective amount may vary depending on various factors, such as the subject's physical condition, age, weight, health, etc., the particular application for which protective or therapeutic immunity is to be provided, and the particular disease, e.g., a viral infection, against which immunity is sought. A therapeutically effective amount can be readily determined by one of ordinary skill in the art in light of the present disclosure.

[0083] The compositions and therapeutic combinations of the present disclosure, such as agonist IL-15 conjugates in combination with anti-PD-1 and / or anti-PD-L antibodies or fragments thereof, may also include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers should be non-toxic and not interfere with the efficacy of the active ingredients. Pharmaceutically acceptable carriers may include one or more excipients, such as binders, disintegrants, swelling agents, suspending agents, emulsifiers, wetting agents, lubricants, flavoring agents, sweeteners, preservatives, dyes, solubilizers, and coating agents. Pharmaceutically acceptable carriers may include vehicles, such as lipid (nano)particles. The precise nature of the carrier or other material may depend on the route of administration, such as intramuscular, intradermal, subcutaneous, oral, intravenous, cutaneous, intramucosal (e.g., intestinal), intranasal, or intraperitoneal routes. For liquid injectable formulations, such as suspensions and solutions, suitable carriers and additives include water, glycols, oils, alcohols, preservatives, coloring agents, and the like. For solid oral formulations such as powders, capsules, caplets, gelcaps and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents, etc. For nasal spray / inhalant mixtures, aqueous solutions / suspensions may include water, glycols, oils, emollients, stabilizers, humectants, preservatives, aromatic compounds, flavors, etc. as suitable carriers and additives.

[0084] The compositions and therapeutic combinations of the present application, i.e., those comprising an agonist IL-15 complex, can be formulated in any suitable form for administration to a subject to facilitate administration and improve efficacy, including, but not limited to, oral (enteral) administration and parenteral injection. Parenteral injection includes intravenous injection or infusion, subcutaneous injection, intradermal injection, and intramuscular injection. The compositions of the present application can also be formulated for other routes of administration, such as transmucosal, ocular, rectal, long-acting implant, sublingual, oral mucosal bypassing the portal circulation, inhalation, or intranasal.

[0085] According to embodiments of the present disclosure, compositions and therapeutic combinations for administration will typically include a pharmaceutically acceptable carrier, e.g., a buffer in an aqueous carrier such as buffered saline, e.g., phosphate-buffered saline (PBS). Compositions and therapeutic combinations may also contain pharmaceutically acceptable substances required to approximate physiological conditions, such as pH adjusters and buffering agents. For example, compositions or therapeutic combinations of the present application that include plasmid DNA may contain phosphate-buffered saline (PBS) as a pharmaceutically acceptable carrier.

[0086] Treatment method The present disclosure also generally relates to a method of treating a disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical composition described herein. In some embodiments, the method further comprises administering to the subject another therapeutically effective agent. In some embodiments, the other therapeutically effective agent comprises a small molecule compound, a targeted chemotherapeutic agent, a radiotherapeutic agent, or an antibody or antigen-binding fragment thereof. In some embodiments, the other therapeutically effective agent comprises an antibody or antigen-binding fragment thereof that specifically binds to CD20, PD1, PDL1, Her2, EGFR, or c-MET. In some embodiments, the other therapeutically effective agent comprises an anti-PD-1 antibody or antigen-binding fragment thereof.

[0087] Furthermore, the present disclosure generally relates to a method of treating a disease in a subject in need thereof, the method comprising administering to the subject an effective amount of an agonistic IL-15 complex described herein, a nucleic acid molecule described herein, a plasmid described herein, a host cell described herein, or a pharmaceutical composition described herein. In some embodiments, the method further comprises administering one or more anti-PD-1 antibodies or antigen-binding fragments thereof and / or anti-PD-L1 antibodies or antigen-binding fragments thereof. In some embodiments, the agonistic IL-15 complex and the one or more anti-PD-1 antibodies or antigen-binding fragments thereof and / or anti-PD-L1 antibodies or antigen-binding fragments thereof are administered simultaneously. In some embodiments, the administration of the agonistic IL-15 complex and the one or more anti-PD-1 antibodies or antigen-binding fragments thereof and / or anti-PD-L1 antibodies or antigen-binding fragments thereof is sequential. In some embodiments, the agonistic IL-15 complex is used in combination with a small molecule inhibitor or antibody analog. In some embodiments, the small molecule inhibitor is a targeted chemotherapeutic or radiotherapeutic agent and the antibody analog is an anti-CD20, PD1, PDL1, Her2, EGFR, or c-MET antibody.

[0088] In some aspects, the subject is a mammal, preferably a human. In some aspects, the disease is a tumor. In some aspects, the tumor is a solid tumor. In some aspects, the disease is cancer. In some aspects, the cancer is leukemia, lymphoma, multiple myeloma, malignant melanoma, breast cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, colon cancer, or renal cell carcinoma. In some aspects, the cancer is colon cancer or lung cancer. In some aspects, cancers of the present disclosure include, but are not limited to, lymphoma, blastoma, sarcoma (including liposarcoma), neuroendocrine tumors, mesothelioma, schwannoma, meningioma, adenoma, melanoma, and non-leukemic leukemia or lymphoid malignancies. More specific examples of the above cancers include squamous cell carcinoma (e.g., squamous cell carcinoma), lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma and gastric cancer, gastrointestinal cancer, pancreatic cancer, malignant glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, Cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumors, head cancer, neck cancer, myeloid stromal tumors, giant cell tumor of bone, multiple myeloma, osteolytic bone cancer, central nervous system tumors, brain tumors (glioma, neuroblastoma, astrocytoma, medulloblastoma, ependymoma and retinal neuroblastoma), nasopharyngeal carcinoma, basal cell carcinoma, cholangiocarcinoma, Kaposi's sarcoma, primary liver cancer or endometrial cancer and tumors of the vascular system (angiosarcoma and hemangiopericytoma).

[0089] In some embodiments, the method includes treating a pathogen infection. Pathogens of the present disclosure include, but are not limited to, bacteria, fungi, viruses, and parasites. In some embodiments, the pathogen infection is a bacterial infection and / or a viral infection. In some embodiments, the viral infection includes an infection by an immunodeficiency virus, a smallpox virus, a hepatitis B virus, or any combination of the foregoing. In some embodiments, the viral infection is an HIV infection.

[0090] In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is an inflammatory disease. In some embodiments, the disease is a neurodegenerative disease. In some embodiments, after administration of the agonistic IL-15 complex, the subject experiences no inflammation or mild inflammation in the liver, spleen, lungs, and / or kidneys compared to patients administered ALT803 and / or compared to a negative control. In some embodiments, administration of the agonistic IL-15 complex does not result in a cytokine storm. In some embodiments, administration of the agonistic IL-15 complex has no significant effect on the in vivo release of the cytokines IL-1β, IL-2, IL-4, IL-10, IL-5, CXCL / KC, and IL-12P / p70 compared to patients administered ALT803 and / or compared to a negative control. In some embodiments, administration of an agonist IL-15 complex does not increase the liver function indicators glutamate transaminase (ALT) and glutathione transaminase (AST) compared to subjects administered a negative control and / or compared to subjects administered ALT 803. In some embodiments, administration of an agonist IL-15 complex does not increase leukocyte release, does not increase lymphocyte release, and / or does not increase monocyte release compared to subjects administered a negative control and / or compared to subjects administered ALT 803.

[0091] In some embodiments, the subject receives a dose of 0.1 mg / kg, 0.20 mg / kg, 0.30 mg / kg, 0.40 mg / kg, 0.50 mg / kg, 0.60 mg / kg, 0.70 mg / kg, 0.80 mg / kg, 0.90 mg / kg, 1.00 mg / kg, 1.50 mg / kg, 2.00 mg / kg, 2.50 mg / kg, 3.00 mg / kg, 3.50 mg / kg, 4.00 mg / kg, 4.50 mg / kg, 5.00 mg / kg, 6.0 In some embodiments, the agonist IL-15 complex is administered at 0 mg / kg, 7.00 mg / kg, 8.00 mg / kg, 9.00 mg / kg, 10.0 mg / kg, 12.5 mg / kg, 15.0 mg / kg, 17.5 mg / kg, 20.0 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, or 100 mg / kg. In some embodiments, the agonist IL-15 complex is administered subcutaneously, intramuscularly, or parenterally.

[0092] In some embodiments, the agonistic IL-15 complex is administered to a subject as part of a cellular immunotherapy. In some embodiments, the cellular immunotherapy is CAR-T, TCR-T, DC, CIK, DC-CIK, ECIK, NK, CAS-T, or BiAb-T tumor cell immunotherapy. In some embodiments, administration of the agonistic IL-15 complex induces NK cell and CD8+ T cell function within tumor tissue.

[0093] In some embodiments, the agonist IL-15 complexes described herein can be used to specifically treat conditions including, but not limited to, congestive heart failure (CHF), vasculitis, rosacea, acne, eczema, myocarditis and other myocardial disorders, systemic lupus erythematosus, diabetes, spondylosis, synovial fibroblastic hyperplasia, bone defects, Paget's disease, apraxia, osteopenia, malnutrition, periodontal disease, familial splenic anemia, Langerhans cell histiocytosis, spinal cord injury, acute septic arthritis, osteomalacia, adrenal cortex. Hormonal excess, single bone fibrous dysplasia, multiple bone fibrous dysplasia, periodontal tissue remodeling and fractures, sarcoidosis, bone metastasis / bone pain treatment and malignant hypercalcemia of body fluids, ankylosing spondylitis and other spondyloarthropathies, transplant rejection, viral infections, hematoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (Burkitt's lymphoma, small lymphocytic), lymphoma / chronic lymphocytic leukemia, granuloma fungoides, mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, hairy cell leukemia Lymphoplasmacytic leukemia and lymphoplasmacytic leukemia), lymphoid precursor neoplasms, B-cell acute lymphoblastic non-leukemic leukemia / lymphoma, T-cell acute lymphoblastic non-leukemic leukemia / lymphoma, thymoma, mature T- and NK-cell neoplasms, peripheral T-cell non-leukemic leukemia, mature T-cell non-leukemic leukemia / T-cell lymphoma, large granular lymphocytic leukemia, Langerhans cell histiocytosis, acute myeloid leukemia / myeloma, mature acute myeloid leukemia (AML), differentiated acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia Myeloid leukemia, acute monocytic leukemia, myelodysplastic syndromes, chronic myelodysplasia, chronic myeloid leukemia, osteoporosis, hepatitis, HIV, AIDS, spondyloarthritis, rheumatoid arthritis, inflammatory bowel disease (IBD), sepsis and septic shock, Crohn's disease, psoriasis, scleroderma, graft versus host disease (GVHD), allogeneic islet graft rejection (allogeneic islet graft rejection), hematological malignancies such as multiple myeloma (MM), myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), tumor-associated inflammation, peripheral nerve injury or demyelinating diseases.

[0094] Furthermore, the present disclosure generally relates to methods of treating a tumor, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition described herein. In some embodiments, the tumor is a solid tumor, such as a carcinoma, sarcoma, or melanoma. In some embodiments, the solid tumor is selected from the group consisting of bladder cancer, lung cancer, breast cancer, colorectal cancer, brain tumor, prostate cancer, melanoma, Merkel cell carcinoma, head and neck cancer, small intestine cancer, squamous cell carcinoma, metastatic solid tumor, or cervical cancer. In some embodiments, the tumor is bladder cancer. In some embodiments, the tumor is lung cancer, e.g., small cell lung cancer. In some embodiments, the tumor is breast cancer. In some embodiments, the tumor is cervical cancer. In some embodiments, the tumor is colorectal cancer. In some embodiments, the method further comprises administering an immunotherapy to the subject. In some embodiments, the immunotherapy comprises an anti-PD-1 antibody or an antigen-binding fragment thereof. In some embodiments, after administration of the pharmaceutical composition, the subject does not experience necrosis in the liver, spleen, lungs, and / or kidneys. In some embodiments, administration of the pharmaceutical composition does not result in a cytokine storm, and in particular, administration of the pharmaceutical composition does not have a significant effect on the in vivo release of the cytokines IL-1β, IL-2, IL-4, IL-10, IL-5, CXCL / KC, and IL-12P / p70. In some embodiments, administration of the pharmaceutical composition does not increase the liver function indicators glutamate transaminase (ALT) and glutathione transaminase (AST). In some embodiments, administration of the pharmaceutical composition does not increase leukocyte release, lymphocyte release, and / or monocyte release.

[0095] In addition to the treatment of human disorders, the agonist IL-15 complexes described herein will have significant use in veterinary applications, for example, for the treatment of disorders in livestock such as cattle, sheep, and companion animals such as dogs and cats.

[0096] Production method The present disclosure also generally relates to a method of producing a fusion protein as discussed herein, the method comprising culturing a host cell as described herein under conditions sufficient to express the fusion protein, and isolating the fusion protein.

[0097] Additionally, the present disclosure generally relates to methods of producing a protein complex described herein, comprising culturing one or more host cells described herein under conditions sufficient to express the fusion protein and IL15, isolating the fusion protein and IL15, and optionally assembling the protein complex by combining the isolated fusion protein and IL15 in vitro.

[0098] Furthermore, the present disclosure generally relates to a method of producing an agonistic IL-15 complex, the method comprising: a. transfecting a cell with a nucleic acid molecule comprising an Fc monomer-IL-15 receptor alpha sushi domain fusion and an IL-15 functional fragment; b. culturing the host cell under conditions sufficient to express the Fc monomer-IL-15 receptor alpha sushi domain fusion and the IL-15 functional fragment; c. expressing the Fc monomer-IL-15 receptor alpha sushi domain fusion and the IL-15 functional fragment; d. assembling the agonistic IL-15 complex in vitro; and e. purifying the agonistic IL-15 complex. In some embodiments, the Fc monomer-IL-15 receptor alpha sushi domain fusion and the IL-15 functional fragment are encoded by separate nucleic acid molecules. In some embodiments, the Fc monomer-IL-15 receptor alpha sushi domain fusion and the IL-15 functional fragment are encoded by the same nucleic acid molecule.

[0099] detection kit The present disclosure also generally relates to a detection kit comprising an agonistic IL-15 complex described herein, a nucleic acid molecule described herein, a plasmid described herein, a host cell described herein, or a pharmaceutical composition described herein, optionally used to detect pathogens and / or tumor cells.

[0100] Embodiment Embodiment 1. A fusion protein comprising an interleukin-15 (IL-15) receptor alpha sushi domain fused to an Fc monomer, wherein the IL-15 receptor alpha sushi domain comprises the amino acid sequence of SEQ ID NO:5, and the Fc monomer comprises the amino acid sequence of SEQ ID NO:6.

[0101] Embodiment 2a. The fusion protein of embodiment 1, wherein the carboxyl terminus of the IL-15 receptor alpha sushi domain is fused to the amino terminus of the Fc monomer via a linker, e.g., a glycine-serine polymer, e.g., (GS)n, (GSGGS)n, (GGGGS)n, (GGGS)n (wherein n is an integer of at least 1), a glycine-alanine polymer, or an alanine-serine polymer.

[0102] Embodiment 2. The fusion protein of embodiment 1, wherein the carboxyl terminus of the IL-15 receptor alpha sushi domain is fused to the amino terminus of the Fc monomer via a linker having the amino acid sequence of GGGGS (SEQ ID NO:7).

[0103] Embodiment 3. The fusion protein of embodiment 1, wherein the linker consists of the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8.

[0104] Embodiment 4. The fusion protein of embodiment 3, comprising the amino acid sequence of SEQ ID NO:1.

[0105] Embodiment 5. The fusion protein of embodiment 3, comprising the amino acid sequence of SEQ ID NO:2.

[0106] Embodiment 6. A fusion protein consisting of the amino acid sequence of SEQ ID NO:1.

[0107] Embodiment 7. A fusion protein consisting of the amino acid sequence of SEQ ID NO:2.

[0108] Embodiment 8. A protein complex comprising the fusion protein of any one of Embodiments 1 to 7 and IL-15.

[0109] Embodiment 9a. The protein complex of embodiment 8, wherein the IL-15 comprises full-length IL-15 or a functional fragment of IL-15.

[0110] Embodiment 9b. The protein complex of embodiment 8 or 9a, wherein the IL-15 comprises wild-type IL-15, a mutant IL-15, or a functional fragment thereof.

[0111] Embodiment 9. The protein complex of embodiment 8, wherein the IL-15 comprises the amino acid sequence of SEQ ID NO:3.

[0112] Embodiment 10. The protein complex of embodiment 8, wherein IL-15 consists of the amino acid sequence of SEQ ID NO:3.

[0113] Embodiment 11. The protein complex of embodiment 8, wherein the IL-15 comprises the amino acid sequence of SEQ ID NO:4.

[0114] Embodiment 12. The protein complex of embodiment 8, wherein IL-15 consists of the amino acid sequence of SEQ ID NO:4.

[0115] Embodiment 13. A nucleic acid molecule, e.g., a vector, encoding the fusion protein of any one of embodiments 1 to 7.

[0116] Embodiment 13a. A vector, such as a plasmid, DNA vector, RNA vector, or viral vector, encoding the fusion protein of any one of embodiments 1-7.

[0117] Embodiment 14. A host cell comprising the nucleic acid molecule of embodiment 13.

[0118] Embodiment 14a. A host cell comprising the vector of embodiment 13a.

[0119] Embodiment 15. A method of producing the fusion protein of any one of embodiments 1 to 7, comprising culturing the host cell of embodiment 14 under conditions sufficient to express the fusion protein, and isolating the fusion protein.

[0120] Embodiment 15a. A method of producing the fusion protein of any one of embodiments 1-7, comprising culturing a host cell of embodiment 14a under conditions sufficient to express the fusion protein, and isolating the fusion protein.

[0121] Embodiment 16. One or more nucleic acid molecules, such as one or more vectors, encoding the fusion protein of the protein complex of any one of embodiments 8 to 12 and IL-15.

[0122] Embodiment 16a. One or more vectors, such as one or more plasmids, DNA vectors, RNA vectors or viral vectors, encoding the fusion protein of the protein complex of any one of embodiments 8 to 12 and IL-15.

[0123] Embodiment 17. One or more host cells comprising one or more nucleic acid molecules of embodiment 16.

[0124] Embodiment 17a. One or more host cells comprising one or more vectors of embodiment 16a.

[0125] Embodiment 18. A method for producing a protein complex of any one of embodiments 8 to 12, comprising culturing one or more host cells of embodiment 17 under conditions sufficient to express the fusion protein and IL15, isolating the fusion protein and IL15, and optionally assembling the protein complex by combining the isolated fusion protein and IL15 in vitro.

[0126] Embodiment 18a. A method of producing a protein complex of any one of embodiments 8 to 12, comprising culturing one or more host cells of embodiment 17a under conditions sufficient to express the fusion protein and IL15, isolating the fusion protein and IL15, and optionally assembling the protein complex by combining the isolated fusion protein and IL15 in vitro.

[0127] Embodiment 19. A pharmaceutical composition comprising the fusion protein of any one of embodiments 1 to 7, the protein complex of any one of embodiments 8 to 12, the nucleic acid molecule of embodiment 13, one or more nucleic acid molecules of embodiment 16, the host cell of embodiment 14 or one or more host cells of embodiment 17, and a pharmaceutically acceptable excipient, diluent or carrier.

[0128] Embodiment 20. The pharmaceutical composition of embodiment 19, comprising the protein complex of any one of embodiments 8 to 12.

[0129] Embodiment 21. A method of treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 19 or embodiment 20.

[0130] Embodiment 22 The method of embodiment 21, further comprising administering to the subject another therapeutically effective agent.

[0131] Embodiment 23 The method of embodiment 22, wherein the other therapeutically active agent comprises a small molecule compound, a targeted chemotherapeutic agent, a radiotherapeutic agent, or an antibody or antigen-binding fragment thereof.

[0132] Embodiment 24 The method of embodiment 23, wherein the other therapeutically active agent comprises an antibody or antigen-binding fragment thereof that specifically binds to CD20, PD1, PDL1, Her2, EGFR, or c-MET.

[0133] Embodiment 25. The method of embodiment 24, wherein the other therapeutically active agent comprises an anti-PD-1 antibody or antigen-binding fragment thereof.

[0134] Embodiment 26. The method of any one of embodiments 21 to 25, wherein the disease is a tumor.

[0135] Embodiment 27 The method of embodiment 26, wherein the tumor is a solid tumor.

[0136] Embodiment 28. The method of any one of embodiments 21 to 25, wherein the disease is a pathogen infection, such as a bacterial infection and / or a viral infection.

[0137] Embodiment 29. The method of any one of embodiments 21 to 25, wherein the disease is an autoimmune disease.

[0138] Embodiment 30. The method of any one of embodiments 21 to 25, wherein the disease is an inflammatory disease.

[0139] Embodiment 31. The method of any one of embodiments 21 to 25, wherein the disease is a neurodegenerative disease.

[0140] Embodiment 32. A method for treating a tumor, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of embodiment 20.

[0141] Embodiment 33. The method of embodiment 32, wherein the tumor is a solid tumor, such as a carcinoma, sarcoma, or melanoma.

[0142] Embodiment 34. The method of embodiment 33, wherein the solid tumor is selected from the group consisting of bladder cancer, lung cancer, breast cancer, colorectal cancer, brain cancer, prostate cancer, melanoma, Merkel cell carcinoma, head and neck cancer, small intestine cancer, squamous cell carcinoma, metastatic solid tumors, or cervical cancer.

[0143] Embodiment 35. The method of embodiment 34, wherein the tumor is bladder cancer.

[0144] Embodiment 36 The method of embodiment 34, wherein the tumor is lung cancer, for example small cell lung cancer.

[0145] Embodiment 37. The method of embodiment 34, wherein the tumor is breast cancer.

[0146] Embodiment 38 The method of embodiment 34, wherein the tumor is cervical cancer.

[0147] Embodiment 39. The method of embodiment 34, wherein the tumor is colorectal cancer.

[0148] Embodiment 40 The method of any one of embodiments 32-39, further comprising administering immunotherapy to the subject.

[0149] Embodiment 41. The method of embodiment 40, wherein the immunotherapy comprises an anti-PD-1 antibody or antigen-binding fragment thereof.

[0150] Embodiment 42 The method of any one of embodiments 32 to 41, wherein after administration of the pharmaceutical composition, the subject does not experience necrosis in the liver, spleen, lungs and / or kidneys.

[0151] Embodiment 43. The method of any one of embodiments 32 to 41, wherein administration of the pharmaceutical composition does not result in a cytokine storm, and in particular, administration of the pharmaceutical composition does not have a significant effect on the in vivo release of the cytokines IL-1β, IL-2, IL-4, IL-10, IL-5, CXCL / KC, and IL-12P / p70.

[0152] Embodiment 44. The method of any one of embodiments 32 to 43, wherein administration of the pharmaceutical composition does not increase the liver function indicators glutamate transaminase (ALT) and glutathione transaminase (AST).

[0153] Embodiment 45. The method of any one of embodiments 32-44, wherein administration of the pharmaceutical composition does not increase leukocyte release, lymphocyte release, and / or monocyte release. [Table 1] JPEG2025530778000002.jpg71170 [Example]

[0154] Example 1: Preparation of agonist interleukin-15 conjugates A schematic diagram of the agonist-interleukin-15 complex is shown in Figure 1A. The genes encoding IL15Rα-GGGGS-sFc (SEQ ID NO: 1), IL15Rα-(GGGGS)3-sFc (SEQ ID NO: 2), and the IL15 mutant (SEQ ID NO: 3) were synthesized by Nanjing GenScript Company (Nanjing, GenScript). The genes were cloned into the eukaryotic expression vector PTT (Thermo Fisher). After confirming the sequence of the expression vector, the recombinant protein was transiently expressed using the Expi293 expression system (Thermo Fisher) according to the manufacturer's instructions and purified using Protein G resin (GE Healthcare). The agonist-interleukin-15 complex was purified from the conditioned medium and dialyzed against PBS. The purity of the agonist-interleukin-15 complex was tested by SDS-PAGE, and the protein concentration was measured spectrophotometrically at 280 nm (NanoVue, GE Healthcare). (For specific transformation, expression, and purification methods, see Ying, Tianlei et al. JOURNAL OF BIOLOGICAL CHEMISTRY, 2012. 287(23):19399-19408.)

[0155] Example 2: Binding activity of agonist-interleukin-15 complex to IL-15Rβ The binding activity of the agonist-interleukin-15 complex to its receptor IL-15Rβ was assayed using an Octet-RED (Pall ForteBio) Bio-Layer Interferometry (BLI) instrument. Specifically, 5 μg / mL of IL-15Rβ protein was immobilized on the NI-NTA biosensor in a kinetic buffer (PBS buffer containing 0.02% Tween 20) until saturation (see, e.g., Wang, Chunyu et al., “Engineered Soluble Monomeric IgG1 Fc with Significantly Decreased Non-Specific Binding,” Frontiers in Immunology, vol. 8, pp. 1545-13, 2017, PMID: 29181008; Wang, Chunyu et al., “Design of a Novel Fab-Like Antibody Fragment with Enhanced Stability and Affinity for Clinical Use,” Small Methods, vol. 6, 2(2022): e2100966; and Wang, Chunyu et al., “Design of a Novel Fab-Like Antibody Fragment with Enhanced Stability and Affinity for Clinical Use,” Small Methods, vol. 8, pp. 1545-1545, 2017, PMID: 29181008). (See Vol. 6, 2(2022): e2100966, PMID: 35174992). A baseline was established in kinetic buffer, and the loaded biosensor was immersed for 300 seconds in wells containing serial dilutions of agonist interleukin-15 conjugates. FL115 (also designated FL115-V2), an agonist interleukin-15 conjugate according to an embodiment of the present application, contains a monomeric IL-15 receptor α Su / Fc fusion protein having the amino acid sequence of SEQ ID NO: 2 and a mutant IL15 having the amino acid sequence of SEQ ID NO: 3.Another agonist interleukin-15 complex according to an embodiment of the present application, FL115-WT, contains a monomeric IL-15 receptor α Su / Fc fusion protein having the amino acid sequence of SEQ ID NO:2 and wild-type IL15 having the amino acid sequence of SEQ ID NO:4. The agonist interleukin-15 complex was bound to immobilized IL-15Rβ protein. The IL-15Rβ protein-agonist IL15 complex was then dissociated in kinetic buffer. Affinity was estimated using data analysis software version 8.1 using global data fitted to a 1:1 binding model. The k (association rate constant), k (dissociation rate constant), and K (equilibrium dissociation constant) values ​​were determined by averaging binding curves within a dilution series with an R2 value above the 90% confidence level.

[0156] The results of the assay are shown in Figures 1B to 1D. Referring now to Figures 1B to 1D, the KD of FL115 for IL-15Rβ was 4.47 x 10 -9 M, and the binding constant is 2.77 × 105 M -1 seconds -1 and the dissociation constant is 1.24 x 10 -3 seconds -1 , which showed similar kinetic and affinity characteristics compared to the control ALT803. The results in Figures 1B-1D show that the IL-15 receptor αSu fused to an Fc monomer did not affect the protein interaction, and the binding activity of FL115 to IL-15Rβ was comparable to that of ALT803, an agonistic interleukin-15 complex containing the IL-15 mutant and the IL-15 receptor αSu / Fc fusion protein fused to an Fc dimer.

[0157] Example 3: Cell proliferation test of agonist-interleukin-15 conjugates The biological activity of FL115 was further evaluated using a cell proliferation assay performed using CTLL-2 cells and M-07e cells (megakaryocytes). CTLL-2 cells depend on IL-2 for growth and proliferation, and IL-15 can promote the growth and proliferation of CTLL-2 cells. Therefore, CTLL-2 can be used as a quantification assay for the biological activity of IL-15. The test samples were added to CTLL-2 cells with agonist interleukin-15 complexes and cultured for 24 hours. Their biological activity was analyzed by detecting cell proliferation using the CCK-8 (Cell Counting Kit-8) method. Megakaryocyte M-07e is an IL-15-dependent cell line, and therefore, IL-15 can stimulate their proliferation (see, for example, Meazza, R et al. "Interleukin (IL)-15 induces survival and proliferation of the growth factor-dependent acute myeloid leukemia M-07e through the IL-2 receptor beta / gamma," International Journal of Cancer, Vol. 78, 2 (1998): 189-95, PMID: 9754651; and Finch, DK et al. "Identification of a potent anti-IL-15 antibody with opposing mechanisms of action in vitro and in vivo," British Journal of Pharmacology, Vol. 162, 2 (2011): 480-90, PMID: 20942844). Another agonist interleukin-15 complex according to an embodiment of the present application, FL115-V1, contains a monomeric IL-15 receptor α Su / Fc fusion protein having the amino acid sequence of SEQ ID NO: 1 and a mutant IL15 having the amino acid sequence of SEQ ID NO: 3. M-07e cell line in logarithmic growth phase was washed once with 20 mL of PBS, and then 50 μL of the cell suspension was diluted to a cell concentration of 2.0 × 10 in experimental medium. 550 μL of diluted FL115 (starting working concentration 1.2 μg / mL) was added to each well of a 96-well white plate and mixed with M-07e cells. Two replicate wells were prepared for each concentration gradient point. The 96-well white plate was incubated at 37°C in a 5% CO2 incubator for 48 hours. Fluorescent signals were detected using the CellTiter-Glo Luminescent Cell Viability Assay Kit. Luminescent signal values ​​were detected using a microplate reader.

[0158] The results of the assay are shown in Figures 2A and 2B. Figure 2A shows the proliferation activity of CTLL2 cells in samples treated with FL115-V1 and FL115-V2. As shown in Figure 2B, FL115 significantly stimulated the proliferation of M-07e cells in a dose-dependent manner. It is noteworthy that the proliferation of M-07e cells was more evident as the protein concentration used in the assay increased. The EC50 was 1.894 ng / ml. This example assay provided further evidence of the biological activity of the present agonist-interleukin-15 complex in an in vitro assay.

[0159] Example 4: Immunostimulatory effects of FL115 on human immune cells To evaluate FL115-mediated responses of human immune cells, studies were performed using human PBMCs incubated with FL115. For proliferation assays, CFSE was used to detect PBMC proliferation. CFSE-labeled PBMCs were stimulated with FL115 and ALT803 at concentrations of 100 nM, 10 nM, 1 nM, and 0.1 nM, respectively, in 2e5 plates per well. Cells were incubated for 4 days and then analyzed by flow cytometry to determine cell proliferation based on CFSE. For evaluation of immune cell subsets and activation markers, human PBMCs were cultured with various concentrations of FL115 and ALT803, stained with marker-specific antibodies under appropriate conditions, and analyzed on a FACSVerse flow cytometer (BD Biosciences) using FACSuite software.

[0160] Referring now to Figures 3A-3E, FL115 dose-dependent lymphocyte proliferation was observed in human PBMC cultures, which was better than ALT803 overall (Figure 3A). Treatment with 0.0050 μg / ml of FL115 resulted in a clear expansion in NK cell numbers that was significantly better than ALT803 (Figure 3B). Furthermore, FL115 dose-dependently stimulated the expression of the cell surface activation marker CD69 on NK cells, CD8+ T cells, and CD4+ T cells (Figure 3C). FL115 also induced increased expression of granzyme B and perforin in both human NK cells and CD8+ T cells (Figures 3D and 3E). Collectively, these findings demonstrate that FL115 is capable of activating human immune cells in vitro at concentrations as low as 0.01 nmol / L.

[0161] Example 5: BLI study of FL115 and FcRn In this example, the binding of FL115 to human FcRn was measured using SPR (SPR) and a standard assay protocol. See, for example, Ying, Tianlei et al., "Soluble monomeric IgG1 Fc," The Journal of Biological Chemistry, Vol. 287, 23 (2012): 19399-408, PMID: 22518843; and Ying, Tianlei et al., "Monomeric IgG1 Fc molecules displaying unique Fc receptor interactions that are exploitable to treat inflammation-mediated diseases," mAbs, Vol. 6, 5 (2014): 1201-10, PMID: 25517305). Ligand coupling was performed using a CM5 chip according to the instructions provided with the amino coupling kit. PBS-P+ was used as the buffer. The flow channels of flow cell (Fc) 1 and flow cell 2 were activated with 11.5 mg / ml NHS and 75 mg / ml EDC (1:1) for 420 seconds. The ligand was diluted to 1 μg / mL in NaAc pH 5.0 and then coupled to the activated flow channel Fc2 at a coupling level of 100 RU and a flow rate of 10 μL / min. The Fc1 and Fc2 flow channels were blocked for 420 seconds by injection of 1 M ethanolamine. Binding of human FcRn to FL115 under different pH conditions was assayed using PBS-P+ at pH 6.0 and pH 7.4 as buffers, respectively. FL115 was diluted to 200 μg / mL in PBS-P+ (pH 6.0 or pH 7.4) buffer, which was used as the highest concentration. 200 μg / mL FL115 was diluted two-fold with PBS-P+ (pH 6.0 or pH 7.4) buffer to 0.78 μg / mL. A two-fold gradient of FL115 was injected sequentially into the flow channel at a flow rate of 30 μL / min to bind to the chip-coupled human FcRn. The binding time was set to 90 seconds, and the dissociation time was set to 110 seconds. After dissociation was complete, regeneration was performed with PBS-P+ at pH 7.4.The binding, dissociation, and renaturation were repeated for different concentrations of FL115. Finally, the data were analyzed using a steady-state affinity model selected by the Biacore T200 evaluation software to detect the binding rate of FL115 and human FcRn. FcRn was diluted to a fixed concentration of 1 μg / mL with NaAc pH 5.0, and FL115 diluted in a two-fold gradient with PBS-P+ (pH 6.0 or pH 7.4) buffer was used as the analyte for detection.

[0162] Referring to FIG. 4, the affinity KD value of FL115 for FcRn at pH 6.0 is 4.07×10 -6 M, and at pH 7.4, it did not bind to FcRn (data not shown). The above results demonstrated that the long-acting agonist IL-15 complex FL115 maintains the characteristic pH-dependent FcRn binding of sFc, but exhibits an extended in vivo half-life.

[0163] Example 6: Antitumor effect of FL115 in MC38 subcutaneously transplanted tumor model In this example, the antitumor effect of FL115 was evaluated in a subcutaneously transplanted MC38 (mouse colon adenocarcinoma cell line) tumor model. 5 MC38 mouse colon cancer cells (purchased from Shanghai Jihui Experimental Animal Co., Ltd.) were injected into the right dorsal side of 6-8 week-old female C57BL / 6 mice. Administration of 0.2 mg / kg of FL115, a positive control (ALT803), and a negative control (PBS) was initiated on days 1 and 8 post-inoculation. Tumor size and weight were measured on the indicated days (see Figures 5A-E).

[0164] Referring to Figures 5A-5E, in the MC38 subcutaneously transplanted tumor model, both FL115 and ALT803 had antitumor efficacy, with statistically significant differences compared to the negative control group. In particular, the antitumor effect of FL115 was significantly better than that of the positive control, ALT803. In addition, the weight gain of mice over time (Figure 5E) further confirmed the safety of FL115.

[0165] Example 7: Detection of tumor microenvironment changes induced by FL115 in the MC38 subcutaneous tumor model In this example, changes in immune cells in tumor tissue were evaluated in an MC38 subcutaneous tumor model. 5 C57 mice were subcutaneously inoculated with MC38 tumor cells and randomized into groups on the day after inoculation. Each group of eight mice received intravenous administration of FL115 and ALT803 at a dose of 0.2 mg / kg every seven days for a total of three doses. Mice were sacrificed 24 hours after the third administration, and tumor tissue was excised. Tumors were gently broken into smaller fragments with forceps and digested with 0.2 mg / ml collagenase IV (Sigma-Aldrich) and 0.1 mg / ml deoxyribonuclease I (Sigma-Aldrich) for 1 hour at 37°C. Single-cell suspensions were collected and filtered through a 70 μm nylon mesh, and mouse lymphocytes were isolated using mouse lymphocyte isolation solution (Dacor). Lymphocytes were stained with flow antibodies against surface proteins (purchased from BD), and disrupted membranes were fixed for intracellular proteins (purchased from Sigma-Aldrich) and immediately loaded onto a flow cytometer (purchased from BD) to detect CD8 T cells and NK cells. The results are shown in the figure. The flow assay results for tumors excised from one mouse are shown on the left, and the percentage of immune cells measured in each group of mice (mean ± standard deviation) is shown on the right. Each dot represents a tumor in one mouse.

[0166] Referring to Figures 6A-6D, at the same administration dose (0.2 mg / kg), FL115 induced greater granzyme secretion from tumor-infiltrating killer NK cells and CD8+ T cells than the positive control ALT803 (NK: FL115 (3.65%) vs. PBS (0.30%), P<0.01; CD8+ T: FL115 (18.52%) vs. PBS (8.23%), P<0.05), and induced greater perforin+ CD8+ in CD3+ cells than the positive control ALT803 (NK: FL115 (20.78%) vs. PBS (4.99%), P<0.01; CD8+ T: FL115 (1.75%) vs. PBS (0.14%), P<0.01) (Figures 6A-6D). These results suggest that FL115 antitumor treatment resulted in stronger NK cell and CD8 T cell killing than ALT803 in the tumor microenvironment, demonstrating the stronger antitumor activity of FL115 compared with the positive control, ALT803. Additionally, the effect of FL115 on NK cell activation was significantly different from that of ALT803.

[0167] Example 8: Antitumor effect of FL115 in CT26 metastatic tumor model In this example, FL115 was evaluated in a CT26 metastatic tumor model. 5 CT26 mouse colon carcinoma cells (purchased from Shanghai Jihui Experimental Animal Co., Ltd.) were injected into the tail vein of 6-8 week-old male C57BL / 6 mice. Administration of 0.2 mg / kg of FL115, a positive control (ALT803), and a negative control (PBS) was initiated on days 1, 4, 8, and 11 after injection.

[0168] Referring to Figure 7, in a CT26 mouse colon cancer metastasis tumor model, FL115 had obvious antitumor efficacy, while all mice in the negative control group died during the observation period. In addition, mice in the FL115 group were observed to have good vital signs.

[0169] Example 9: Antitumor effect of FL115 in combination with anti-PD-1 antibody in mice In this example, the antitumor effect of FL115 in combination with an anti-PD-1 antibody (InVivoPlus anti-mouse PD-1 (CD279), clone: ​​RMP1-14, catalog: BP0146-100mg) was evaluated (see, for example, Li, Howard Y et al. "The Tumor Microenvironment Regulates Sensitivity of Murine Lung Tumors to PD-1 / PD-L1 Antibody Blockade," Cancer Immunology Research Vol. 5, 9 (2017): 767-777, PMID: 28819064). Specifically, the ability of FL115 to activate NK cells and enhance the antitumor effect in patients with tumors unresponsive to PD1 monoclonal antibodies was measured. Furthermore, the antitumor activity of FL115 in combination with a PD1 antibody in a non-small cell lung cancer (NSCLC) model was examined to determine whether the two antibodies have a synergistic effect. 5 × 10 51000 lung adenocarcinoma CMT167 cells or LLC cells (purchased from Shanghai Jihui Experimental Animal Co., Ltd.) were injected into the right dorsal side of 6-8 week-old male C57BL / 6 mice (see, for example, Li, Howard Y et al. "The Tumor Microenvironment Regulates Sensitivity of Murine Lung Tumors to PD-1 / PD-L1 Antibody Blockade." Cancer immunology research vol. 5, 9 (2017): 767-777, PMID: 28819064; Tang, Honglin et al. "Inhibition of COX-2 and EGFR by Melafolone Improves Anti-PD-1 Therapy through Vascular Normalization and PD-L1 Downregulation in Lung Cancer." The Journal of Pharmacology and Experimental Therapeutics Vol. 368, 3 (2019): 401-413, PMID: 30591531; and Johnson, Amber M et al. "Cancer Cell-Intrinsic Expression of MHC Class II Regulates the Immune Microenvironment and Response to Anti-PD-1 Therapy in Lung Adenocarcinoma." Journal of Immunology (Baltimore, Md.: 1950) vol. 204, 8 (2020): 2295-2307, PMID: 32179637). Animals in each group were administered FL115, anti-PD1 antibody, or FL115 in combination with anti-PD1 antibody, while a lysis control group (PBS) was set up. Each subject was intravenously administered 10 mg / kg of FL115 and 200 μg of anti-PD1 antibody, with FL115 administered once a week for a total of three doses (D1, D8, D15) and anti-PD1 antibody administered three times (D11, D14, D17).

[0170] 8A-8D, in a C57 mouse model of lung adenocarcinoma CMT167 cells subcutaneously transplanted into the tumor, the relative tumor growth inhibition (TGI) of the FL115-treated group in combination with PD-1 monoclonal antibody was 88.43% compared to the negative control group at 24 days after administration (P<0.0001). No animals were found to have complete tumor regression, and no individual animals were found to be in complete remission. After administration of 10 mg / kg of FL115, the relative tumor growth inhibition (TGI) at 24 days was 79.90% compared to the negative control (PBS) group (P<0.001), with one mouse experiencing complete tumor growth inhibition. After administration of 200 μg of PD-1 monoclonal antibody, the relative TGI at 24 days was -25.37% compared to the negative control group (P>0.05). FL115 combined with PD-1 monoclonal antibody had significantly better tumor inhibition than the PD-1 monoclonal antibody group, with a significant difference (P<0.0001). The FL115 combined with PD-1 monoclonal antibody group had better tumor inhibition than the FL115 group, but the difference was not significant. In a C57 mouse model with subcutaneously implanted tumors from mouse lung cancer LLC cells, the relative tumor growth inhibitor (TGI) of the FL115 combined with PD-1 monoclonal antibody group was 91.12% compared to the negative control group after 28 days of administration (P<0.01), and tumor growth was completely inhibited in two mice. After administration of 10 mg / kg of FL115, the relative TGI was 64.91% compared to the negative control group after 28 days. After administration of FL115 at 10 mg / kg, the relative TGI on day 28 was 64.91% (P<0.05), with one mouse completely inhibited. After administration of PD1 monoclonal antibody at 200 μg, the relative TGI on day 28 was -15.79% compared to the negative control group (P>0.05), with one mouse dying on day 24. The tumor-inhibitory effect of the FL115-treated group in combination with PD-1 monoclonal antibody was significantly better than that of the PD1 monoclonal antibody group, with a significant difference (P<0.05). The tumor-inhibitory effect of the FL115-treated group in combination with PD-1 monoclonal antibody was also better than that of the FL115 group, but the difference was not significant.None of the animals in any treatment group experienced significant weight loss or morbidity during the study.

[0171] In summary, the results showed that the combination of FL115 and anti-PD-1 antibody significantly reduced tumor burden and was effective against both CMT167 cells (a highly metastatic subclone of the lung cancer cell line CMT167 in murine alveolar formation) and Lewis lung carcinoma (LLC) xenograft tumors.

[0172] Example 10: BLI test of the affinity of FL115 to CD16a 158V and CD16a 158F In this example, the affinity of FL115 for CD16a 158V and CD16a 158F was measured using BLI. Previously tested monomeric Fc does not bind to CD16a or CD16b. Generally, CD16a acts as a receptor for activating innate immune cells with killing function, such as NK cells. The receptor has two polymorphic variants: 158V, which has high affinity for IgG1, and 158F, which has low affinity for IgG1. Based on the above, the affinity of FL115 for the Fc receptor CD16a V158 and F158 was evaluated. Specifically, 5 μg / mL of CD16a 158F and 158V proteins were immobilized on an NI-NTA biosensor in a kinetic buffer (PBS buffer containing 0.02% Tween 20) until saturation (see, e.g., Wang, Chunyu et al., “Engineered Soluble Monomeric IgG1 Fc with Significantly Decreased Non-Specific Binding,” Frontiers in Immunology, vol. 81545, November 13, 2017, PMID: 29181008; and Wang, Chunyu et al., “Design of a Novel Fab-Like Antibody Fragment with Enhanced Stability and Affinity for Clinical Use,” Small Methods, vol. 6, 2(2022): e2100966, PMID: 35174992). A baseline was established in kinetic buffer, and the loaded biosensor was immersed in wells containing serial dilutions of FL115 and mutants for 300 seconds. The CD16a antigen-FL115 complex was then dissociated in kinetic buffer. Affinity was estimated using data analysis software version 8.1 using global data fitted to a 1:1 binding model. The k (association rate constant), k (dissociation rate constant), and K (equilibrium dissociation constant) values ​​were determined by averaging binding curves within the dilution series with R values ​​above the 90% confidence level.

[0173] Referring now to Figures 9A and 9B, FL115 did not bind to either CD16a 158V or CD16a 158F, indicating that FL115 has low toxicity.

[0174] Example 11: Safety evaluation of FL115 In this example, the potential toxicity of FL115 and ALT803 was evaluated in BALB / C mice by tail vein administration of different doses (0.2 mg / kg, 2 mg / kg, 20 mg / kg) of FL115 and ALT803 over a 4-week period (once a week, 4 times).

[0175] Referring now to Figures 10A to 10D, the results showed that ALT803 at doses of 2 mg / kg and 20 mg / kg caused significant increases in liver function indicators glutamate transaminase (ALT) and glutathione transaminase (AST) compared to the control group (ALT: 2 mg / kg P<0.0001, 20 mg / kg P<0.0001; AST: 2 mg / kg P<0.0001, 20 mg / kg P<0.0001) and significantly higher increases compared to the FL115 group (ALT: 2 mg / kg P<0.0001, 20 mg / kg P<0.0001; AST: 2 mg / kg P>0.05, 20 mg / kg P<0.0001), both of which suggested substantial liver damage. Furthermore, ALT803 at a dose of 2 mg / kg caused a significant increase in creatinine (ECRE), an indicator of renal function, compared with the FL115 group (P<0.05), and at a dose of 20 mg / kg caused a significant difference in urea nitrogen (UN), an indicator of renal function, compared with both the control group and the FL115 group (P<0.01). These results indicated that ALT803 at 2 mg / kg and 20 mg / kg caused substantial impairment of liver function but had no significant effect on renal function, while FL115 had no significant effect on either the liver or kidney.

[0176] Referring now to Figures 11A to 11D, ALT803 at 20 mg / kg caused all mice to die after 3 days, and at 0.2 mg / kg and 2 mg / kg, ALT803 caused a significant increase in leukocytes in the mice, which was significantly different from the leukocyte release caused by the control group (WBC: 0.2 mg / kg P<0.001; 2 mg / kg P<0.0001) and the leukocyte release caused by the FL115 group (WBC: 0.2 mg / kg P<0.01; 2 mg / kg P<0.0001). Doses of 0.2 mg / kg and 2 mg / kg induced a significant increase in lymphocytes in mice, which was significantly different from the lymphocyte release induced by the control group (lymphocytes: 0.2 mg / kg P < 0.0001; 2 mg / kg P < 0.0001) and the FL115 group (lymphocytes: 0.2 mg / kg P < 0.001; 2 mg / kg P < 0.0001). Doses of 0.2 mg / kg and 2 mg / kg induced a significant increase in monocytes in mice, which was significantly different from the monocyte release induced by the control group (lymphocytes: 0.2 mg / kg P < 0.05; 2 mg / kg P < 0.001) and the FL115 group (lymphocytes: 0.2 mg / kg P < 0.05; 2 mg / kg P < 0.01).

[0177] Referring now to Figures 12A to 12F, unlike the ALT803-administered group, the weighed mice treated with FL115 on day 4 after the fourth administration did not show a significant increase in lung, spleen, and lymph node weights or in visceral weights compared to the control group.

[0178] Referring now to Figure 13, mice in the 20 mg / kg ALT803 group showed no necrosis and mild inflammation in the heart, mild necrosis and severe inflammation in the liver, severe necrosis and severe inflammation in the spleen, moderate necrosis and severe inflammation in the lungs, and mild necrosis and severe inflammation in the kidneys. Figure 14 further shows that mild necrosis was observed in the hearts of mice in the 2 mg / kg ALT803 group compared with the other groups, and mild inflammation was observed in all other groups. Notably, no necrosis was observed in the liver and spleen, and mild inflammation was observed in mice in the 20 mg / kg FL115 group, while mild inflammation was observed in the 2 mg / kg ALT803 group. No necrosis was observed in the lungs, and mild inflammation was observed. No necrosis was observed in the kidneys, and no inflammation was observed in either group. FL115 showed mild inflammation in the liver and spleen except in the high dose group of 20 mg / kg, and showed essentially no significant effect in the low and medium dose groups (0.2 mg / kg and 2 mg / kg). This further supports the high safety of FL115, and confirms that the toxicity of side effects is not significant at the dose-escalation animal level.

[0179] Based on the above results, the ultimate tolerated dose of FL115 in mice was determined to be >20 mg / kg, while ALT803 showed significant side effect toxicity when administered at 2 mg / kg.

[0180] Example 12: Effect of FL115 on in vivo secretion of cytokines in mice In this example, the effect of FL115 on the release of 10 cytokines, including IFN-γ, IL-10, IL-12p70, CXCL1 / KC, IL-1β, IL-2, IL-4, IL-6, IL-5, and TNF-α, in mice was evaluated for its potential risk of inducing a cytokine storm. The assay used chemiluminescence technology based on the Luminex platform. Six-week-old mice were intravenously injected with high and low doses of FL115 or administered a dose of ALT803 as a control. Blood sampling was performed at 0, 2, 4, 6, 8, 24, 48, and 72 hours. Collected mouse serum was analyzed, and the expression of the above 10 cytokines was detected using chemiluminescence detection technology from Shanghai Uninvest Biotechnology Co. The levels of each cytokine secreted in vivo by FL115 and control stimulated mice were graphed using Graphpad software and are shown in Figures 15A-15J.

[0181] Referring to Figures 15A-15J, the results showed that different doses of FL115 had no significant effect on the in vivo release of cytokines IL-1β, IL-2, IL-4, IL-10, IL-5, CXCL / KC, and IL-12P / p70 in mice, but did have some effect on the in vivo release of cytokines IL-6, IFN-γ, and TNF-α in mice, with IL-6 in particular slightly increased after FL115 administration. IL-6 release slightly increased 2 hours after FL115 administration and gradually returned to normal levels after 24 hours. IFN-γ release slightly increased 2 hours after FL115 administration, with release reaching its highest level in the high-dose 20 mg / kg group after 48 hours. TNF-α release slightly increased 2 hours after FL115 administration and gradually decreased to normal levels after 6 hours.

[0182] 15A-15J, the effect of different doses of ALT803 on cytokine release in mice was most pronounced in the high-dose group, where the release of IL-6, IFN-γ, TNF-α, IL-1β, IL-4, IL-10, and CXCL / KC was most significant, inducing a cytokine storm that led to the death of mice in this dose group 96 hours after administration. The effect on IL-6, IFN-γ, IL-2, and CXCL / KC release was also significant in the mid-dose group, but no mouse deaths were observed.

[0183] Example 13: Antitumor effect of FL115 in MB49 mouse bladder cancer model In this example, the antitumor effect of FL115 was evaluated in an MB49 mouse bladder cancer model. 6 MB49 mouse bladder cancer cells (purchased from Beijing Vitalihua Laboratory Animal Technology Co., Ltd.) were injected into the right dorsal side of 6-8 week-old male C57BL / 6 mice. When administered to each mouse in a group of 10 mice labeled D0, the average tumor volume was approximately 60-100 mm. 3 The mice were then administered a total of five doses once a week. The tumor size and weight of the mice were measured on the designated days (see Figures 16A-16C).

[0184] C57BL / 6 mice were inoculated in situ with MB49 (urothelial carcinoma) cells. MB49 cells were harvested during the logarithmic growth phase (third to fourth generations after resuscitation), the culture medium was removed, and the cells were washed twice with DPBS before inoculation (cell viability was measured before and after inoculation). The bladders were pretreated with the bladder irrigation pretreatment solution before inoculation. On day 7 after in situ inoculation (i.e., D7), the cells were randomly grouped according to body weight and administered on the day of grouping. Subsequently, the drug was administered once a week for a total of four doses. The mice were weighed on the designated days and their condition was observed. At the end of the experiment, the bladders of tumor-bearing mice were weighed and photographed (see Figures 17A-C).

[0185] Referring to Figures 16A-16C, in a mouse subcutaneous tumor-bearing model of bladder cancer, FL115 demonstrated highly significant dose-dependent effects (Figure 16A). All mice in the high-dose group (20 mg / kg) achieved complete tumor remission, and the tumor inhibition rate in the medium-dose group (2 mg / kg) was as high as 69.93% (Figure 16B). No significant changes in body weight were observed in any of the mice, demonstrating the high safety of FL115 in vivo (Figure 16C).

[0186] Referring now to Figures 17A-17C, in an orthotopic mouse model of bladder cancer, FL115, both intravenously and intravesically, had highly significant tumor-inhibitory effects, significantly better than those of the BCG group. Animals did not have palpable tumors, i.e., progressive tumors. Compared with the lysate control group, both the bladder-perfused and intravenous FL115 groups significantly reduced tumor burden (bladder weight) on day 21. The mean bladder weight in the bladder-perfused FL115 group was reduced by 63.68%, and the mean bladder weight in the intravenous FL115 group was reduced by 68.92%, both of which had comparable tumor burden inhibition compared to the lysate control group on day 21. The mean bladder weight of intravesical BCG treatment alone was reduced by 13.23%, demonstrating that the tumor-inhibitory effect of FL115 alone, whether administered by bladder instillation or intravenous injection, was significantly better than that of BCG administered via bladder instillation alone (Figures 17A and B). Second, no significant changes in body weight were observed in any of the mice (FIG. 17C), indicating high in vivo safety in FL115 mice.

[0187] Based on the above results, it was determined that FL115 has a low risk of inducing a cytokine storm in mice in preclinical studies.

[0188] Example 14: Serum half-life extension of FL115 The study aimed to evaluate the pharmacokinetic properties of FL115 (eg, FL115-V2) after a single intravenous injection in ICR mice.

[0189] Six- to eight-week-old male ICR mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used in the study. Mice were randomized based on body weight and size. Mice were randomized into four groups, each with five animals. FL115 and the positive control ALT803 were administered intravenously to the four groups at single doses of 0.2 mg / kg and 2 mg / kg, respectively. Serum samples were collected pre-dose (0 h) and 30 min, 1 h, 2 h, 4 h, 8 h, 10 h, 24 h, 48 h, 72 h, and 96 h post-dose. FL115 and ALT803 concentrations were measured using a validated ELISA method. Pharmacokinetic parameters of FL115 and ALT803 were calculated using WinNonLin noncompartmental analysis (NCA).

[0190] As shown in Figures 18A and 18B, the results showed a consistent trend in the changes in serum concentrations in mice in the FL115 and ALT803 groups. After a single intravenous injection of 0.2 mg / kg of FL115 in mice, the serum concentration of FL115 reached its peak 30 minutes after injection, began to decline approximately 2 hours after injection, and was essentially stable after 24 hours. The serum half-life (T 1 / 2 The serum half-life (T) of ALT803 was 11.11 hours. After a single intravenous injection of 0.2 mg / kg of ALT803 in mice, the serum concentration of ALT803 peaked 30 minutes after injection, began to decline approximately 2 hours after injection, and was essentially stable after 72 hours. 1 / 2 ) was 12.66 hours, indicating that FL115 and ALT803 have similar pharmacokinetic properties.

[0191] It will be apparent to those skilled in the art that the present disclosure is not limited to the foregoing examples, but may be embodied in other specific forms without departing from its essential attributes. The examples are therefore to be considered in all respects as illustrative and not restrictive, and reference should be made to the appended claims rather than the foregoing examples, and all changes that come within the meaning and range of equivalency of the claims are therefore desired to be embraced therein.

[0192] It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0193] All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains.

[0194] The present invention illustratively described herein may suitably be practiced in the absence of any element or elements, or any limitation not specifically disclosed herein. Thus, for example, in each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms. The terms and expressions used are used as terms of description, not of limitation, and no exclusion of any equivalents of the shown and described features or portions thereof is intended in the use of such terms and expressions, but it is recognized that various modifications are possible within the scope of the invention as claimed. Furthermore, when features or aspects of the invention are described in terms of a Markush group, those skilled in the art will understand that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. For example, if X is described as selected from the group consisting of bromine, chlorine, and iodine, a claim that X is bromine as well as a claim that X is bromine and chlorine are fully described. Other embodiments are within the scope of the following claims.

Claims

1. A fusion protein comprising an interleukin-15 (IL-15) receptor α-sushi domain fused to an Fc monomer, wherein the IL-15 receptor α-sushi domain comprises the amino acid sequence of SEQ ID NO: 5, and the Fc monomer comprises the amino acid sequence of SEQ ID NO:

6.

2. The fusion protein according to claim 1, wherein the carboxyl terminus of the IL-15 receptor α-sushi domain is fused to the amino terminus of the Fc monomer via a linker having the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO:

8.

3. The fusion protein according to claim 2, comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

4. The fusion protein according to claim 1, comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

5. A protein complex comprising the fusion protein described in claim 1 and IL-15.

6. The protein complex according to claim 5, wherein IL-15 comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO:

4.

7. One or more nucleic acid molecules encoding the fusion protein and IL-15 of the protein complex according to claim 5 or claim 6, for example, one or more vectors.

8. One or more host cells containing one or more nucleic acid molecules as described in claim 7.

9. A method for producing the protein complex described in claim 5 or claim 6, comprising: culturing one or more host cells described in claim 8 under conditions sufficient to express the fusion protein and the IL-15; isolating the fusion protein and the IL-15; and optionally constructing the protein complex by combining the isolated fusion protein and the IL-15 in vitro.

10. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 4, a protein complex according to claim 5 or 6, one or more nucleic acid molecules according to claim 7, one or more host cells according to claim 8, and a pharmaceutically acceptable excipient, diluent, or carrier.

11. A pharmaceutical composition according to claim 10 for use as a pharmaceutical in a method for treating a disease, wherein the method comprises administering the pharmaceutical composition to a subject.

12. The method further comprises administering another therapeutic agent to the subject, The other therapeutic agent may optionally include a small molecule compound, a targeted chemotherapeutic agent, a radiotherapy agent, or an antibody or its antigen-binding fragment. The pharmaceutical composition according to claim 11, further optionally comprising an antibody or antigen-binding fragment thereof that specifically binds to CD20, PD1, PDL1, Her2, EGFR, or c-MET.

13. The pharmaceutical composition according to claim 11, wherein the disease is a pathogenic infection, such as a bacterial infection and / or viral infection, an autoimmune disease, an inflammatory disease, or a neurodegenerative disease.

14. The disease is a tumor, The pharmaceutical composition according to claim 11, wherein the tumor is optionally a solid tumor, such as carcinoma, sarcoma, or melanoma.

15. The solid tumors are selected from the group consisting of bladder cancer, lung cancer, breast cancer, colorectal cancer, brain tumor, prostate cancer, melanoma, Merkel cell carcinoma, head and neck cancer, small intestine cancer, squamous cell carcinoma, metastatic solid tumors, or cervical cancer. The pharmaceutical composition according to claim 14, wherein the lung cancer is optionally, for example, small cell lung cancer.

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