Interleukin-15 fusion protein and methods of use

Abstract

The disclosure provides IL-15 fusion proteins as well as compositions comprising them. The disclosure further provides methods of treating and / or preventing immunodeficiency as well as methods for cancer immunotherapy in a subject, wherein the method includes administering a fusion protein as described herein.

Description

Atty. Docket No.67175WO01 INTERLEUKIN-15 FUSION PROTEIN AND METHODS OF USE RELATED APPLICATIONS

[0001] The present patent application claims the priority benefit of U.S. Provisional Patent Application Ser. No.63 / 471,370, filed June 06, 2023, which is incorporated by reference in its enterity. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (67175WO01_SequenceListing.xml; Size: 26,932 bytes; and Date of Creation: May 30, 2024) is herein incorporated by reference in its entirety. BACKGROUND

[0003] Interleukin-15 (IL-15) is a pleiotropic cytokine produced by many types of cells, including immune cells (e.g., monocytes, macrophages, dendritic cells) and nonimmune cells (e.g. keratinocytes and epithelial cells). IL-15 is involved in diverse immunologic functions and plays an important role in the development, homeostasis, proliferation, and activation of CD8+ T cells, NK cells, NKT cells, and other immune cells (B cells, intestinal intraepithelial lymphocytes, antigen-presenting cells).

[0004] IL-15 receptor has three subunits (polypeptides), the type-specific IL-15R alpha (IL- 15Rα), the IL-2 / IL-15R beta (IL15Rβ), and the common gamma chain (γc). IL-15R has a subunit, IL-15Rα, which binds to IL-15 with picomolar affinity. IL-15 receptor shares a second subunit, the IL-2 / IL-15 receptor β (IL-15Rβ), with IL-2R. The third γc subunit is shared with many other cytokine receptors, including IL-2R, IL-4R, IL-7R, IL-9R, and IL-21R.

[0005] IL-2 is a non-specific T cell growth factor that stimulates the expansion of all subsets of T cells, including CD8+ cytotoxicity T cells, CD4+ helper T cells, regulatory T cells (Treg), and NK cells. It is a secreted cytokine and induces signals through conventional cis-presentation by binding to the preformed IL-2 receptor (IL-2Rαβγc) on the target cell surface. IL-2 displays similar activities to IL-15 in vivo. Recombinant human IL-2 was approved by the FDA for treating melanoma and kidney cancers. When used in conjunction with adoptive T-cell therapy, IL-2 showed impressive tumor regression activities in small percentages of patients. However, 1  Atty. Docket No.67175WO01 patients usually suffered severe dose-limiting side effects (see, e.g., Dutcher, J. P. Current status of interleukin-2 therapy for metastatic renal cell carcinoma and metastatic melanoma. Oncol. Williston Park N 16, 4–10 (2002); Rosenberg, S. A., Yang, J. C., White, D. E. & Steinberg, S. M. Durability of complete responses in patients with metastatic cancer treated with high-dose interleukin-2: identification of the antigens mediating response. Ann. Surg.228, 307–319 (1998).

[0006] IL-15 signaling can be mediated through two mechanisms, trans-presentation and cis- presentation. During trans-presentation, IL-15 is pre-assembled with IL15-Rα subunit in the endoplasmic reticulum (ER) and then shuttled to the cell surface of the antigen-presenting cells (monocytes and dendritic cells), where IL-15 / IL-15Rα complex interacts with the IL-15Rβ / γc subunits of the effector cells (see, e.g., Stonier, S. W. & Schluns, K. S. Trans-presentation: a novel mechanism regulating IL-15 delivery and responses. Immunol. Lett.127, 85–92 (2010)). During cis-presentation, the antigen-presenting cells secrete soluble IL-15 to the intercellular space surrounding the effector cells. The soluble IL-15 binds to the unoccupied IL-15Rα on the effector cell surface. The IL-15 / IL15Rα complex then binds to the IL-15Rβ / γc subunits of the same effector cells. IL-15 exerts its function primarily through trans-presentation rather than cis- presentation in vivo. Trans-presentation allows controlled local delivery of IL-15 and prevents systemic elevation of IL-15, which is detrimental to the homeostasis of lymphocytes and NK cells (see, e.g., Fehniger, T. A. et al. Fatal leukemia in interleukin 15 transgenic mice follows early expansions in natural killer and memory phenotype CD8+ T cells. J. Exp. Med.193, 219– 231 (2001)).

[0007] A variety of IL-15 therapeutics have been developed, given IL-15’s activity in promoting the proliferation and survival of T-cells and NK cells. IL-15 does not regulate Treg numbers nor their activities, and as such, IL-15 is expected to cause fewer side effects when compared to IL- 2. Nevertheless, recombinant human IL-15 (rhIL-15) is known to have a short half-life of about 45 mins (see, e.g., Stoklasek, T. A., Schluns, K. S. & Lefrançois, L. Combined IL-15 / IL- 15Ralpha immunotherapy maximizes IL-15 activity in vivo. J. Immunol. Baltim. Md 1950177, 6072–6080 (2006); and Zhao, M. et al. Development of a recombinant human IL-15 ^sIL- 15Rα / Fc superagonist with improved half-life and its antitumor activity alone or in combination with PD-1 blockade in a mouse model. Biomed. Pharmacother. Biomedecine Pharmacother. 112, 108677 (2019)) requiring frequent rhIL-15 injections (see, e.g., Morre, M., Assouline, B., Rance, I., Gregoire, A. & Breque, C. Glycosylated IL-7, preparation and uses. (2010)) and 2  Atty. Docket No.67175WO01 presenting a challenge for patient compliance. In addition, a large swing in blood IL-15 concentrations between dosages could impact the overall efficacy of the drug.

[0008] Protein fusion formats have been used in an effort to improve pharmacokinetic properties of otherwise short-half-life biologicals, such as IL-15 (e.g., fusion proteins to domains and proteins such as Fc, HSA, and other polypeptides and illustrated in, e.g., Strohl, W. R. Fusion Proteins for Half-Life Extension of Biologics as a Strategy to Make Biobetters. Biodrugs 29, 215–239 (2015)). Among other aspects described herein are novel IL-15 fusion proteins that extend the half-life of the IL-15 protein, demonstrate similar or improved activity, and that are useful in methods for boosting immune response as well as treating and / or preventing disorders and diseases associated with immunodeficiency in a subject in need thereof. SUMMARY

[0009] In an aspect, the disclosure provides a fusion protein comprising at least one of an IL- 15Rα sequence and / or an IL-15 sequence fused to a domain sequence that comprises, in some embodiments, an Fc domain or an albumin protein sequence. In some embodiments, the fusion protein comprises a fusion between the IL-15Rα sequence and the IL-15 sequence, wherein the fusion can be a direct fusion or via a linker sequence. In some embodiments, the fusion protein comprises a first linker sequence between the IL-15Rα sequence and IL-15 sequence, and a second linker sequence between the Fc domain or albumin protein sequence to the IL-15Rα sequence and IL-15 sequence. In some further embodiments, the first and second linkers can comprise an RLI linker or an IgD hinge. In some further embodiments, the IgD hinge is a human IgD sequence. In some further embodiments, the IL-15Rα protein is a human IL-15Rα. In some further embodiments, the IL-15 protein is human IL-15. In some further embodiments, the albumin protein is human serum albumin. In some further embodiments, the IL-15Rα sequence comprises a sushi domain. In some embodiments, the fusion protein comprises a sequence according to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 8.

[0010] In any of the above aspects and embodiments, the IL-15 domain in the fusion protein can exhibit an extended serum half-life relative to recombinant or exogenous human IL-15. In some embodiments, the serum half-life can be extended by at least nine hours. 3  Atty. Docket No.67175WO01

[0011] In any of the above aspects and embodiments, the fusion protein can induce the proliferation of at least one of NK cells (CD3-CD56+), CD8+ T cells (CD3+CD56-CD8+), and NKT cell (CD3+ CD56+) when administered to a subject.

[0012] In any of the above aspects and embodiments, the fusion protein can induce activation of NK cells (CD3-CD56+), CD8+ T cells (CD3+CD56-CD8+), and NKT cells (CD3+ CD56+) when administered to a subject.

[0013] In any of the above aspects and embodiments, the fusion protein can increase lymphocyte counts when administered to a subject.

[0014] In any of the above aspects and embodiments, the fusion protein can increase the total number of splenic CD8+ T cells when administered to a subject.

[0015] In any of the above aspects and embodiments, the fusion protein can increase the total number and cell percentage of splenic NK cells when administered to a subject.

[0016] In any of the above aspects and embodiments, the fusion protein can increase the total number of peritoneal cavity CD8+ T cells when administered to a subject.

[0017] In any of the above aspects and embodiments, the fusion protein can increase the total number and cell percentage of peritoneal cavity NK cells when administered to a subject.

[0018] In some aspects, the disclosure provides a nucleic acid sequence that encodes for any of the fusion proteins and / or SEQ ID NOs as described herein.

[0019] In some aspects, the disclosure provides a recombinant cell comprising the fusion protein according to the aspects and embodiments described herein. In some aspects, the disclosure provides a recombinant cell comprising a nucleic acid sequence encoding any one or more of the fusion proteins according to the aspects and embodiments described herein.

[0020] In some aspects, the disclosure provides a composition comprising the fusion protein in accordance with any of the aspects and embodiments of the disclosure and a pharmaceutically acceptable carrier, diluent, or excipient.

[0021] In some aspects, the disclosure provides a kit comprising the fusion protein in accordance with any of the aspects and embodiments of the disclosure.

[0022] In some aspects, the disclosure provides a method for inducing an immune response in a subject in need of treatment, comprising administering to the subject one or more fusion proteins in accordance with any of the aspects and embodiments of the disclosure. 4  Atty. Docket No.67175WO01

[0023] In some aspects, the disclosure provides a method for treating cancer in a subject in need of treatment, comprising administering to the subject one or more fusion proteins in accordance with any of the aspects and embodiments of the disclosure.

[0024] Thus, in some aspects and embodiments described herein, the IL-15 fusion proteins disclosed herein can be used to treat and / or prevent a disease or disorder in a subject. In some aspects, the IL-15 fusion proteins can stimulate an immune response in a subject in need thereof. In some embodiments, the IL-15 fusion proteins can stimulate white blood cell proliferation, lymphocyte proliferation, and / or lymphocyte differentiation in a subject. In other aspects, the IL- 15 fusion proteins can treat and / or prevent immunodeficiency in a subject. Subjects that can benefit from the disclosed IL-15 fusion proteins include, without limitation, those having cancer (i.e., as a cancer immunotherapy), human immunodeficiency virus, hepatitis B, hepatitis C, lymphopenia, sepsis, and subjects having undergone stem cell, tissue, or organ transplantation. In certain aspects, the IL-15 fusion protein can increase white blood cell counts, increase lymphocyte counts, and / or increase or decrease certain T cell subpopulations in a subject, as discussed herein. The methods disclosed herein comprise administering an IL-15 fusion protein to a subject.

[0025] Additional aspects of the disclosure will be apparent to one of ordinary skill in the art in view of the following description and illustrative examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG.1 is a schematic depicting different designs of IL-15-containing protein constructs, including those as described in the example embodiments of the disclosure.

[0027] FIG.2 illustrates an embodiment of a schematic diagram of the molecular design of illustrative examples of the disclosure, JL19001-1 and JL19001-2. The functional unit of IL- 15R ^ / IL-15 was either fused to the N-terminus of HSA (JL19001-1) or the C-terminus of HSA (JL19001-2).

[0028] FIG.3A and 3B illustrate the differential glycosylation of molecules in accordance with the disclosure. FIG.3A illustrates a schematic diagram of JL19001-1 and JL19001-2 in which the putative N-linked glycosylation site at the C-terminus of IL-15 is depicted. FIG.3B depicts the different migration of JL19001-1 and JL19001-2 on SDS-PAGE under reduced and non- reduced conditions, where PNGase F eliminates the observed size difference (boxed lanes). 5  Atty. Docket No.67175WO01

[0029] FIG.4A depicts in vitro CTLL-2 cell proliferation assays of rhIL-15, JL19001-1, and JL19001-2. FIG.4B depicts in vitro M-07e cell proliferation of rhIL-15, JL19001-1 and JL19001-2 (rhIL-15 serving as a positive control).

[0030] FIG.5A-5D is a schematic model for different sensitivity of rhIL-15 and JL19001 in CTLL-2 vs M-07e cells. The proposed non-limiting model suggests that IL-15 preferentially binds to the high affinity receptors ( ^ ^ ^c) that are more abundant in CTLL-2 cells (A, B), whereas JL19001, with the help of its fusion partner IL-15R ^, preferentially bind to the intermediate affinity receptors ( ^ ^c) that are dominant in M-07e cells (C, D). The differential distribution of IL-15R ^ subunit renders high potency of rhIL-15 in CTLL-2 cells, but not in M- 07e cells. With the pre-existing IL-15R ^ in JL19001, JL19001 may not be favored to bind the high-affinity receptors.

[0031] FIG.6A-6F illustrates a schematic diagram of IL-15 fusion proteins in various formats. FIG.6A illustrates JL19001 also referred as JL19001-2. In this illustrative example, HSA is fused to IL-15R ^ (aa 1-85) via the IgD linker and IL-15 is fused to IL-15R ^ via the RLI linker (linker protein sequences, SGGSGGGGSGGGSGGGGSLQ (SEQ ID NO:13)) FIG.6B illustrates JL19001-3 also referred as pJL177. This illustrative example is similar to JL19001, with the difference being IL-15R ^ (aa 1-85) is replaced with IL-15R ^ sushi domain (aa 1-65). FIG.6C illustrates JL19001-4 also referred as pJL359. This illustrative example is similar to JL19001 with the difference being the IgD linker is replaced with a 3x(G4S) linker. FIG.6D illustrates JL19001-5 also referred as pJL186. This illustrative example is similar to JL19001, with the difference being that the IL-15R ^ domain is removed. FIG.6E illustrates JL19001-6 also referred as pJL185. This illustrative example is similar to JL19001 with the differences being that HSA is replaced with Fc, and that this construct forms a dimer. FIG.6F illustrates the schematic diagram of JL19001-7 also referred as pJL485. In this illustrative example, an IL- 15R ^ sushi domain is fused to the N-terminus of Fc, and there is no linker sequence between the IL-15 and the IL-15R ^ sequences. It is thought that because IL-15R ^ has a very high (picomolar) affinity to IL-15 when the molecules are co-expressed, the IL15 / IL15R ^-Fc complex will assemble readily by the cells.

[0032] FIG.7 depicts the pharmacokinetic profiles after dosing with rhIL-15 (150 ^g / kg), JL19001 (50 ^g / kg), or JL19001 (150 ^g / kg). JL19001 has a longer half-life relative to rhIL-15: 6  Atty. Docket No.67175WO01 rhIL-15 in vivo half-lie is 43.38 min (<1 hour). JL19001 in vivo half-life is 9.7 hours (150 µg / kg) and 11.6 hours (50 µg / kg). Data are analyzed by PKSolver software.

[0033] FIG.8 illustrates flow cytometric surface marker staining of Natural Killer (NK) cells and CD8+ T cells. Dead cells were excluded by 7-AAD, such that only live cells were analyzed in the figure.

[0034] FIG.9 depicts the effects of IL-15 (left panel) and JL19001 (right panel) on the induced proliferation of NK cells (CD3-CD56+), CD8+ T cells (CD3+CD56-CD8+), Non-CD8 T cells (CD3+CD56-CD8-), and NKT cells (CD3+ CD56+). It is noted that the CFSE level was reduced as cells divide.

[0035] FIG.10 depicts the effects of IL-15 (left panel) and JL19001 (right panel) on induced activation of NK cells (CD3-CD56+), CD8+ T cells (CD3+CD56-CD8+), Non-CD8 T cells (CD3+CD56-CD8-), and NKT cells (CD3+ CD56+). It is noted that CD69 is an NK and T cell activation marker. NK cells and T cells upregulate their surface CD69 expression once activated.

[0036] FIG.11 illustrates cell numbers and percentages of different cells in the spleen of mice upon the injection of JL19001.

[0037] FIG.12 illustrates cell numbers and percentages of different cells in the peritoneal cavity of mice upon the injection of JL19001 (T-test values (p-values) are listed in the graphs).

[0038] FIG.13 is a schematic of the experimental design for determining the in vivo efficacy of JL19001 and rhIL-15 (JL19001 at 20, 60 ^g / kg is equivalent to 2.0*10-10, 6.2*10-10mole / kg. rhIL-15 at 12, 36 and 108 ^g / kg is equivalent to 9.3*10-10, 27.9*10-10, 83.7*10-10mole / kg).

[0039] FIG.14 depicts the total numbers of viable cells in the spleen and peritoneal cavity in mice upon the injection of JL19001 or IL-15. The outlier value is excluded from data analysis. The p values of the T-test are listed.

[0040] FIG.15 illustrates the percentages of different cell populations in the spleen in mice upon JL19001 or IL-15 injections. The outlier value is excluded from data analysis. The p values of the T-test are listed.

[0041] FIG.16 depicts the numbers of different cell populations in the spleen in mice following administration with JL19001 or IL-15. The outlier value is excluded from data analysis. The p values of the T-test are listed. 7  Atty. Docket No.67175WO01

[0042] FIG.17 illustrates the percentages of different cell populations in the peritoneal cavity in mice following administration with JL19001 or IL-15. The outlier value is excluded from data analysis. The p values of the T-test are listed.

[0043] FIG.18 depicts the cell numbers of different cell populations in the peritoneal cavity in mice following administration with JL19001 or IL-15. The outlier value is excluded from data analysis. The p values of the T-test are listed. DETAILED DESCRIPTION

[0044] Before continuing to describe the present disclosure in further detail, it is to be understood that this disclosure is not limited to specific proteins, nucleic acids, compositions, or process steps, as such can vary while still falling within the scope of the description provided herein.

[0045] As used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0046] Unless defined otherwise, 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 is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this invention.

[0047] Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, can be referred to by their commonly accepted single-letter codes. A. Fusion proteins

[0048] As used herein, the term “fusion protein” refers to a polypeptide construct generated through the joining and expression of two or more genes encoding distinct polypeptides. The translation of the fusion gene results in a single polypeptide with functional properties derived 8  Atty. Docket No.67175WO01 from each of the original polypeptides. The polypeptides can be fused directly or joined via a linker or hinge.

[0049] Described herein are novel IL-15 fusion proteins. As used herein, the term “interleukin- 15” or “IL-15” protein refers to IL-15 polypeptides or derivatives thereof having substantial amino acid sequence identity (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to a mature, wild-type mammalian IL-15. For example, IL-15 can refer to an amino acid sequence of a recombinant or non-recombinant polypeptide having an amino acid sequence of: i) a native allelic variant of an IL-15 polypeptide, ii) a biologically active fragment of an IL-15 polypeptide, or iii) a biologically active variant or analog of an IL-15 polypeptide. The IL-15 of the fusion protein can be obtained from any mammalian species, or obtained from a recombinant expression system (e.g., yeast, bacteria, etc.). The IL-15 may be glycosylated, partially glycosylated, or non-glycosylated. In some particular embodiments, IL-15 can comprise an amino acid sequence, or functional fragment thereof, as described herein in the listing of sequences (e.g., SEQ ID NO: 7).

[0050] IL-15 signaling is mediated through two mechanisms, trans-presentation and cis- presentation. During trans-presentation, IL-15 is pre-assembled with the IL15-Rα subunit in the endoplasmic reticulum (ER) and then shuttled to the cell surface of the presenting cells, where IL-15 / IL-15Rα complex interact with the IL-15R ^ / ^c subunits of the effector cells. During cis- presentation, the presenting cells secrete soluble IL-15 to the intercellular space surrounding the effector cells. The soluble IL-15 binds to the unoccupied IL-15R ^ on the effector cell surface. The IL-15 / IL15Rα complex then binds to the IL-15R ^ ^ ^c subunits of the same effector cells. IL- 15 exerts its function mostly through trans-presentation rather than cis-presentation in vivo. Trans-presentation allows controlled local delivery of IL-15 and prevents systemic elevation of IL-15, which is detrimental to the homeostasis of lymphocytes and NK cells.

[0051] In various aspects, the disclosure provides a fusion protein that comprises one or both of an IL-15 sequence and / or an IL-15R ^ sequence that is fused to a domain that extends serum half-life (e.g., extends serum half-life relative to IL-15 that is not fused). In some embodiments the fusion comprises an IL-15 sequence that is fused to the domain. In some embodiments the fusion comprises an IL-15R ^ sequence that is fused to the domain. In some preferred embodiments the fusion protein comprises a fusion of an IL-15 sequence and an IL-15R ^ sequence that are fused to the domain (i.e., either as two separate fusions of an IL-15 sequence 9  Atty. Docket No.67175WO01 and an IL15R ^ sequence to the domain, or a single fusion of a protein sequence that comprises a fusion of IL-15 and IL-15R ^ sequences). Thus, in such embodiments, the fusion protein can comprise at least two linked regions, wherein the first region comprises a fusion of an IL-15 sequence and an IL-15R ^ sequence, which is fused to the second region that comprises a domain that extends serum half-life.

[0052] In some embodiments the domain that extends serum half-life can comprise any molecule that can increase the serum half-life of an IL-15 sequence or IL-15R ^ sequence when fused to the domain, when compared to the serum half-life of the non-fused forms of the IL-15 or IL- 15R ^ sequence(s). In some preferred embodiments, the domain comprises an albumin, an antibody Fc region, or a polyethylene glycol (PEG). In some preferred embodiments, the domain comprises an albumin sequence, and in yet other preferred embodiments, the albumin sequence comprises a human serum albumin (HSA) sequence. In some embodiments the domain comprises SEQ ID NO: 11 or SEQ ID NO: 12.

[0053] In accordance with aspects and embodiments of the disclosure, the fusion protein comprises an IL-15 Receptor (IL-15R) sequence (i.e., functional protein or polypeptide fragment). In embodiments, IL-15R refers to IL-15R polypeptides or derivatives thereof having substantial amino acid sequence identity (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to a mature, wild-type mammalian IL-15R sequence. Thus, the IL-15 receptor ("IL-15R") in accordance with some embodiments of the disclosure includes at least one of three subunits, IL-15Rα, IL15Rβ, and common gamma chain ( ^c). IL-15 receptor shares the ^c subunit with many other cytokine receptors, including IL-2R, IL-4R, IL-7R, IL-9R, and IL- 21R. IL-15 receptor shares a second subunit, the IL-2 / IL-15 receptor ^ (IL-15R ^), with IL-2R. The third subunit, IL-15R ^, is unique to IL-15 and binds to IL-15 with picomolar affinity. In some further embodiments, the fusion proteins in accordance with the disclosure comprise an IL- 15R protein or functional fragment thereof, wherein the IL-15R comprises an IL-15Rα sequence. In some particular embodiments, the IL-15Rα can comprise a sushi domain. In yet some further embodiments the IL-15R ^ can comprise an amino acid sequence, or functional fragment thereof, as described herein in the constructs and listing of sequences (e.g., SEQ ID NO: 9 or SEQ ID NO: 10.). 10  Atty. Docket No.67175WO01

[0054] In one aspect, a fusion protein comprises an IL-15Rα protein sequence; a RLI linker; an IL-15 protein sequence; a hinge region; and an albumin protein sequence. In some embodiments, the IL-15Rα protein sequence is a human IL-15Rα sequence. In some embodiments, the IL-15 protein sequence is a human IL-15 sequence. In some embodiments, the albumin protein sequence is a human serum albumin sequence. In some embodiments, the hinge region is derived from a sequence comprising the human IgD hinge region. In some embodiments, the RLI linker is derived from a sequence comprising IL-15 linked to the IL-15Rα sushi domain via a flexible linker and several Fc. In some embodiments, the hinge region covalently links the IL-15 protein, RLI linker, IL-15Rα and the albumin protein.

[0055] As used herein, the term “human serum albumin” refers to a HSA polypeptide or derivative thereof having a substantial amino acid sequence identity to mature, wild-type human HSA, which can be used as carrier protein at least because of its long serum half-life (e.g., up to 21 days). Other carrier proteins, aside from HSA, are also contemplated. In some embodiments, the carrier protein is a heavy chain immunoglobulin constant domain derived from IgA, IgD, IgE, IgG, or IgM. In some embodiments the HSA comprises SEQ ID NO: 12.

[0056] The hinge or linker region can be any amino acid or peptide linker that is generally known and used in the art to generate fusion proteins. Generally, the linker or hinge sequences are used to separate the functional domains of the fusion protein in order to improve their expression, activity, folding, and / or stability. In some embodiments the hinge or linker is derived from the hinge region of an immunoglobulin such as, for example, an IgA, IgD, IgE, IgG, or IgM amino acid sequence. Thus, as used in some embodiments herein, “hinge region” refers to a sequence comprising an amino acid sequence that shares sequence identity or similarity with all or a portion of an immunoglobulin hinge region sequence. Accordingly, a “hinge region” encompasses fragments of the immunoglobulin hinge region that allow the linked polypeptides to achieve a biologically active conformation. Hinge regions in some example embodiments of the disclosure share at least 70-80% sequence identity to a wild-type immunoglobulin hinge region amino acid sequence, preferably, greater than about 90% sequence identity. In some particular aspects, the hinge region comprises a hinge region from, or derived from, human IgD or IgG. Human IgD, which lacks cysteine and is the longest immunoglobulin hinge, provides increased fusion protein flexibility. In some embodiments the linker sequence can comprise a RLI linker sequence (e.g., SEQ ID NO: 13) or another flexible linker sequence 11  Atty. Docket No.67175WO01 comprising glycine (e.g., one or more G4S linker (e.g., G4S, 3x(G4S) or (GGGGS)3, G8, etc.), as generally known in the art and / or as described herein (e.g., SEQ ID NOs: 14-19).

[0057] In some aspects, the fusion proteins disclosed herein can be characterized by one or more of the following structural and / or functional properties.

[0058] In some aspects, a fusion protein comprises a human IL-15Rα (AA1-85), a RLI linker (AA86-105), an IL-15 protein (AA106-219), a hinge region, and an albumin protein.

[0059] In some embodiments, a fusion protein comprises an albumin protein, an IL-15Rα, a hinge region, an IL-15 protein, and a RLI linker. In some embodiments, the C-terminus of HSA (AA1-585) is fused to human IL-15Rα (AA644-728) via the IgD hinge sequence (AA586-643), then fused to the human IL-15 sequence (AA749-862) via a RLI linker (AA729-748).

[0060] In some aspects, a fusion protein comprises an albumin protein, an IL-15Rα sushi domain, a hinge region, an IL-15 protein, and a RLI linker. In some embodiments, the HSA (AA1-585) is fused to human IL-15Rα sushi domain (AA644-708) via the IgD hinge sequence (AA586-643), then fused to the human IL-15 sequence (AA729-842) via a RLI linker (AA709- 728).

[0061] In some aspects, a fusion protein comprises an albumin protein, an IL-15Rα sushi domain, a 3xG4S linker, an IL-15 protein, and a RLI linker. In some embodiments, the HSA (AA1-585) is fused to the human IL-15Rα sushi domain (AA601-665) via a 3x(G4S) linker (AA586-600), then fused to the human IL-15 sequence (AA686-799) via a RLI linker (AA666- 685).

[0062] In some aspects, a fusion protein comprises an albumin protein, an IL-15 protein, and a hinge region. In some embodiments, the HSA (AA1-585) is directly fused to human IL-15 (AA644-757) via the IgD hinge sequence (AA586-643).

[0063] In some aspects, a fusion protein comprises a Fc, an IL-15Rα, a hinge region, an IL-15 protein, and a RLI linker. In some embodiments, human Fc (AA1-231) is fused to human IL- 15Rα (AA290-374) via the IgD hinge sequence (AA232-289), then fused to the human IL-15 sequence (AA395-508) via a RLI linker (AA375-394).

[0064] In some embodiments, human IL-15Rα sushi domain (AA1-65) is directly fused to human Fc (AA66-296).

[0065] In some aspects, a fusion protein comprises an IL-15Rα sushi domain and a Fc. 12  Atty. Docket No.67175WO01

[0066] In still further aspects, the disclosure provides JL19001-1, JL19001-2, JL19001-3, JL19001-4, JL19001-5, JL19001-6, and JL19001-7. In some embodiments, the fusion protein comprises any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 8.

[0067] As one of ordinary skill can appreciate, the sequences disclosed herein can be modified to some degree without compromising the ability of the fusion protein to interact with a target cell receptor, i.e., a receptor on T cells, NK cells, etc. In some aspects, fusion protein sequence variants retain the ability to stimulate cell proliferation and cell differentiation in vivo and / or in vitro. In some aspects, fusion protein sequence variants retain the ability to treat, attenuate, or reduce in severity an immunodeficiency in a subject.

[0068] As used herein, sequence “variants” refer to a fusion protein amino acid sequence comprising at least one amino acid insertion, deletion, and / or substitution, wherein the resulting fusion protein maintains one or more of its functional characteristics as described herein. An amino acid insertion variant is characterized by the insertion of one or more amino acids between two existing amino acids. An amino acid deletion variant is characterized by the deletion of one or more amino acids from the fusion protein sequence. An amino acid substitution is characterized by at least one amino acid in the sequence being replaced by another amino acid. In embodiments relating to substitutions, the amino acid substitution(s) may be a conservative substitution, (i.e., an amino acid from one family of amino acids (acidic, basic, non-polar, and uncharged, based on side chain characteristics, including size) is substituted with an amino acid from the same family).

[0069] In some aspects, the sequence identity between the variant fusion protein sequence and the fusion protein sequences disclosed herein will be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. “Sequence identity” refers to the percentage of amino acid residues that are identical to the sequences being compared. B. Labels, Conjugates, and Moieties

[0070] The fusion proteins disclosed herein can be conjugated to a therapeutic agent, solid support, affinity agent, or a detectable agent. Fusion proteins of the disclosure can be conjugated to labels wherein the fusion protein and / or its associated targets(s) can be detected. Labels 13  Atty. Docket No.67175WO01 include, without limitation, a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme, and a radioisotope.

[0071] In certain aspects, the fusion proteins are conjugated to a fluorophore. The choice of the fluorophore attached to the fusion protein will determine the absorption and fluorescence emission properties of the conjugated fusion protein. Physical properties of a fluorophore label can include, but are not limited to, spectral characteristics (absorption, emission, and stokes shift), fluorescence intensity, lifetime, polarization and photo-bleaching rate, or a combination thereof. All of these physical properties can be used to distinguish one fluorophore from another, and thereby allow for multiplexed analysis. Other desirable properties of the fluorescent label can include cell permeability and low toxicity, for example, if labeling of the fusion protein is to be performed in a cell or an organism (e.g., a living animal).

[0072] In certain aspects, the conjugated label can comprise an enzyme. Enzymes are desirable labels in some embodiments because amplification of the detectable signal can be obtained and result in increased assay sensitivity. The enzyme itself does not produce a detectable response but functions to break down a substrate when it is contacted by an appropriate substrate such that the converted substrate produces a fluorescent, colorimetric or luminescent signal. Enzymes can amplify the detectable signal because one enzyme on a labeling reagent can result in multiple substrates being converted to a detectable signal. The enzyme substrate is selected to yield the preferred measurable product, e.g., colorimetric, fluorescent or chemiluminescence. Such substrates are extensively used in the art and are well known by one skilled in the art and include, for example, oxidoreductases such as horseradish peroxidase and a substrate such as 3,3’-diaminobenzidine (DAB); phosphatase enzymes such as an acid phosphatase, alkaline and a substrate such as 5-bromo-6-chloro-3-indolyl phosphate (BCIP); glycosidases, such as beta- galactosidase, beta-glucuronidase or beta-glucosidase and a substrate such as 5-bromo-4-chloro- 3-indolyl beta-D-galactopyranoside (X-gal); additional enzymes include hydrolases such as cholinesterases and peptidases, oxidases such as glucose oxidase and cytochrome oxidases, and reductases for which suitable substrates are known.

[0073] Enzymes and their appropriate substrates that produce chemiluminescence are suitable for some assays. These include, but are not limited to, natural and recombinant forms of luciferases and aequorins. Chemiluminescence-producing substrates for phosphatases, 14  Atty. Docket No.67175WO01 glycosidases, and oxidases such as those containing stable dioxetanes, luminol, isoluminol, and acridinium esters are additionally useful.

[0074] In another aspect, haptens, such as biotin, are also utilized as labels. Biotin is useful because it can function in an enzyme system to further amplify the detectable signal, and it can function as a tag to be used in affinity chromatography for isolation purposes. For detection purposes, an enzyme conjugate that has an affinity for biotin is used, such as avidin-HRP. Subsequently a peroxidase substrate is added to produce a detectable signal.

[0075] Haptens also include hormones, naturally occurring and synthetic drugs, pollutants, allergens, affector molecules, growth factors, chemokines, cytokines, lymphokines, amino acids, peptides, chemical intermediates, nucleotides, and the like.

[0076] Enzymes and their appropriate substrates that produce chemiluminescence are suitable for some assays. These include, but are not limited to, natural and recombinant forms of luciferases and aequorins. Chemiluminescence-producing substrates for phosphatases, glycosidases and oxidases, such as those containing stable dioxetanes, luminol, isoluminol and acridinium esters, are additionally useful.

[0077] In another aspect, haptens, such as biotin, are also utilized as labels. Biotin is useful because it can function in an enzyme system to further amplify the detectable signal, and it can function as a tag to be used in affinity chromatography for isolation purposes. For detection purposes, an enzyme conjugate that has affinity for biotin is used, such as avidin-HRP. Subsequently, a peroxidase substrate is added to produce a detectable signal.

[0078] Haptens also include hormones, naturally occurring and synthetic drugs, pollutants, allergens, affector molecules, growth factors, chemokines, cytokines, lymphokines, amino acids, peptides, chemical intermediates, nucleotides and the like.

[0079] In certain aspects, fluorescent proteins can be conjugated to the fusion protein as a label. Examples of fluorescent proteins include green fluorescent protein (GFP) and the phycobiliproteins and derivatives thereof. The fluorescent proteins, especially phycobiliprotein, are particularly useful for creating tandem dye labeled labeling reagents. These tandem dyes comprise a fluorescent protein and a fluorophore for the purposes of obtaining a larger stokes shift wherein the emission spectra are farther shifted from the wavelength of the fluorescent protein’s absorption spectra. 15  Atty. Docket No.67175WO01

[0080] In certain aspects, the label is a radioactive isotope. Examples of suitable radioactive materials include, but are not limited to, iodine (121I,123I,125I,131I), carbon (14C), sulfur (35S), tritium (3H), indium (111In,112In,113mIn,115mIn,), technetium (99Tc,99mTc), thallium (201Ti), gallium (68Ga,67Ga), palladium (103Pd), molybdenum (99Mo), xenon (135Xe), fluorine (18F),153SM,177Lu,159Gd,149Pm,140La,175Yb,166Ho,90Y,47Sc,186Re,188Re,142Pr,105Rh, and97Ru.

[0081] In some aspects, drugs (e.g., other active agents) can be conjugated to the fusion protein. For example, a fusion protein can be conjugated to a therapeutic moiety or agent, such as an immunotherapy drug. In some embodiments, the fusion protein can bind to a target cell, and deliver the immunotherapy drug to the cell. Any immunotherapeutic known in the art can be conjugated to the fusion protein. In certain embodiments, drugs and other molecules can be conjugated to a fusion protein via site-specific conjugation. Non-limiting example embodiments of such additional active agents can include antiviral agents, antibiotic agents, antifungal agents, antiparasitic agents, gamma globulin, and the like. Similarly, the fusion protein disclosed herein may be used in combination with one or more other therapeutic interventions that may be used in the treatment of conditions such as an immunodeficiency in a subject. C. Polynucleotides Encoding Fusion Proteins & Recombinant Cells Producing Fusion Proteins

[0082] The disclosure provides methods for producing fusion proteins. In some embodiments relating to methods of producing a fusion protein, a host cell can be transfected with one or more expression vector(s) encoding a fusion protein, and can be cultured under appropriate conditions to allow expression of the polypeptide(s) to occur. The fusion protein can be secreted and isolated from cells and / or cell culture media containing the fusion protein. Alternatively, the fusion protein can be retained in the cytoplasm or in a membrane fraction and the cells harvested and lysed, and the fusion protein subsequently purified and isolated. A cell culture includes host cells, media, and other byproducts. Any suitable media for cell culture can be used in methods of production. Fusion proteins can be isolated from cell culture medium, host cells, or both using common techniques for purifying proteins, including, for example, ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification. In certain aspects, the fusion protein can be produced with a domain (e.g., a His-tag) that facilitates its purification. 16  Atty. Docket No.67175WO01

[0083] A recombinant nucleic acid can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells (yeast, avian, insect, or mammalian), or both. Expression vehicles for production of a recombinant polypeptide include plasmids and other vectors. For instance, suitable vectors include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli. In certain aspects, mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17. In some instances, it can be desirable to express the recombinant polypeptide(s) by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as the ß-gal containing pBlueBac III).

[0084] Techniques for making fusion genes are well known. Essentially, the joining of various nucleic acid fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another aspect, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried 17  Atty. Docket No.67175WO01 out using anchor primers which give rise to complementary overhangs between two consecutive nucleic acid fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).

[0085] In some aspects, an expression vector expressing any of the nucleic acids described above can be used to express a fusion protein in a host cell. For example, a fusion protein can be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.

[0086] Once the expression vector is transferred to a host cell by conventional techniques, the transfected cells are then cultured by conventional techniques to produce a fusion protein. Thus, the disclosure includes host cells containing a polynucleotide encoding a fusion protein, operably linked to a heterologous promoter. In certain aspects, if the fusion protein is encoded from different vectors, the vectors can be co-expressed in the host cell for the expression of the entire fusion protein. In certain aspects, the fusion protein is expressed from a single promoter. In certain aspects, the fusion protein is expressed from multiple promoters. In certain aspects, the fusion protein is encoded on a single vector. In certain aspects, the fusion protein is encoded on multiple vectors.

[0087] Mammalian cell lines available as hosts for expression of fusion proteins are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney 293 cells, and a number of other cell lines. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the fusion protein or portion thereof expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include but are not limited to CHO, HEK293, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any functional immunoglobulin chains), SP20, CRL7O3O, and HsS78Bst cells. 18  Atty. Docket No.67175WO01

[0088] In certain aspects, fusion proteins of the disclosure are stably expressed in a cell line. Stable expression can be used for long-term, high-yield production of recombinant proteins. For example, cell lines that stably express the fusion protein can be generated. Host cells can be transformed with an appropriately engineered vector comprising expression control elements (e.g., promoter, enhancer, transcription terminators, polyadenylation sites, etc.), and a selectable marker gene. Following the introduction of the foreign DNA, cells can be allowed to grow for 1- 2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells that stably integrate the plasmid into their chromosomes to grow and form foci, which in turn can be cloned and expanded into cell lines. Methods for producing stable cell lines with a high yield are well known in the art and reagents are generally available commercially.

[0089] In certain aspects, fusion proteins of the disclosure are transiently expressed in a cell line. Transient transfection is a process in which the nucleic acid introduced into a cell does not integrate into the genome or chromosomal DNA of that cell, but is maintained as an extrachromosomal element, e.g., as an episome, in the cell. Transcription processes of the nucleic acid of the episome are not affected and a protein encoded by the nucleic acid of the episome is produced.

[0090] The cell line, either stable or transiently transfected, is maintained in a cell culture medium and conditions well known in the art that result in the expression and production of a fusion protein. In certain aspects, the mammalian cell culture media is based on commercially available media formulations, including, for example, DMEM or Ham's F12. In other aspects, the cell culture media is modified to support increases in both cell growth and biologic protein expression. As used herein, the terms “cell culture medium,” “culture medium,” and “medium formulation” refer to a nutritive solution for the maintenance, growth, propagation, or expansion of cells in an artificial in vitro environment outside of a multicellular organism or tissue. Cell culture medium can be optimized for a specific cell culture use, including, for example, a cell culture growth medium that is formulated to promote cellular growth or a cell culture production medium that is formulated to promote recombinant protein production. The terms “nutrient”, “ingredient”, and “component” are used interchangeably herein to refer to the constituents that make up a cell culture medium. 19  Atty. Docket No.67175WO01

[0091] Once a fusion protein has been produced, it can be purified by any method known in the art for purification of a protein complex, for example, by chromatography (e.g., ion exchange, affinity, and size column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

[0092] When using recombinant techniques, the fusion protein can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the protein is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Where the protein is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF can be included in any of the foregoing steps to inhibit proteolysis and antibiotics can be included to prevent the growth of adventitious contaminants.

[0093] The composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, hydrophobic interaction chromatography, ion exchange chromatography, gel electrophoresis, dialysis, and / or affinity chromatography either alone or in combination with other purification steps. The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin, SEPHAROSE chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS- PAGE, and ammonium sulfate precipitation are also available.

[0094] Regardless of how a fusion protein is purified, to confirm functional binding activity, binding assays can be performed (before and / or after purification). For example, ELISA assays, including dual ELISA assays can be used. In some aspects, a first binding target is coated on a well, and binding to this target immobilizes the fusion protein. A tagged second binding target is added to the well, and detected. Only fusion proteins that are both immobilized via binding to the first binding target and bound to the second binding target will be detected. 20  Atty. Docket No.67175WO01

[0095] In some aspects, the disclosure provides for recombinant cell lines expressing the fusion protein that can be deposited and maintained with an international depository institution that is authorized under the provisions of the Budapest Treaty (i.e., an International Depositary Authority, IDA). D. Pharmaceutical Formulations

[0096] In certain aspects, the disclosure provides pharmaceutical compositions. Such pharmaceutical compositions can also be compositions comprising a fusion protein as disclosed herein and a pharmaceutically acceptable excipient. In certain aspects, the pharmaceutical compositions of the disclosure are used as a medicament (i.e., in methods of treating or preventing a disease or condition, in a subject in need of treatment or preventative treatment). In some embodiments, pharmaceutical compositions can be compositions comprising a nucleic acid molecule that encodes a fusion protein as disclosed herein.

[0097] In certain aspects, a fusion protein (or a polynucleotide encoding the fusion protein) can be formulated with a pharmaceutically acceptable carrier, excipient or stabilizer, as pharmaceutical compositions. In certain aspects, such pharmaceutical compositions are suitable for administration to a human, or a non-human mammal or animal, via any one or more routes of administration using methods known in the art. The route and / or mode of administration will vary depending upon the desired results. The term “pharmaceutically acceptable carrier” means one or more nontoxic materials that do not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations can routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable preparations can also contain compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration into a human. Other contemplated carriers, excipients, and / or additives, which can be utilized in the formulations described herein include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, protein excipients such as serum albumin, gelatin, casein, salt-forming counterions such as sodium and the like. These and additional known pharmaceutical carriers, excipients and / or additives suitable for use in the formulations described herein are known in the art, e.g., as listed in “Remington: The Science & Practice of Pharmacy”, 21sted., Lippincott Williams & Wilkins, (2005), and in the “Physician’s Desk Reference”, 60th21  Atty. Docket No.67175WO01 ed., Medical Economics, Montvale, N.J. (2005). Pharmaceutically acceptable carriers can be selected that are suitable for the mode of administration, solubility and / or stability desired or required.

[0098] The formulations described herein comprise active agents (i.e., one or more fusion proteins as disclosed herein) in a concentration resulting in a w / v appropriate for a desired dose. In certain aspects, the active agent is present in a formulation at a concentration of about 1 mg / ml to about 200 mg / ml, about 1 mg / ml to about 100 mg / ml, about 1 mg / ml to about 50 mg / ml, or about 1 mg / ml to about 25 mg / ml. In certain aspects, the concentration of the active agent in a formulation can vary from about 0.1% to about 75% by total weight. In certain aspects, the concentration of the active agent is in the range of 0.003 to 1.0 molar.

[0099] When used for in vivo administration, the formulations should be sterile. Formulations can be sterilized by various sterilization methods, including sterile filtration, radiation, etc. In one aspect, the formulation is filter-sterilized with a presterilized 0.22-micron filter. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice as described in “Remington: The Science & Practice of Pharmacy”, 21sted., Lippincott Williams & Wilkins, (2005).

[0100] Therapeutic compositions are encompassed by the pharmaceutical formulations described herein, and can formulated for particular routes of administration, such as oral, nasal, pulmonary, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration. The phrases “parenteral administration” and “administered parenterally” as used herein refer to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0101] The formulations can be in unit dosage form and prepared by any known method. Actual dosage levels of the active ingredients in the pharmaceutical compositions can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient (e.g., “a therapeutically effective amount”). The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions 22  Atty. Docket No.67175WO01 employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. Suitable dosages can range from about 0.0001 to about 100 mg / kg of body weight or greater, for example about 0.1, 1, 10, or 50 mg / kg of body weight, with about 1 to about 10 mg / kg of body weight being suitable.

[0102] In some embodiments of the disclosure, the formulations can be suitable for research use. The concentration of active agent in such formulations, as well as the presence or absence of excipients and / or pyrogens, can be modified or selected based on the particular application and intended use.

[0103] In some aspects, the disclosure provides methods for treating or preventing an immunodeficiency in a subject that comprise the administration of an IL-15 fusion protein as described herein to the subject in need thereof.

[0104] In some aspects, the disclosure provides methods for cancer immunotherapy that comprise administration of an IL-15 fusion protein as described herein to a subject in need thereof.

[0105] In some further aspects of the methods relating to preventing disease, the subject can be at risk for developing lymphopenia or immunodeficiency. By “being at risk” or having an “increased risk”, a subject is identified as having a higher than normal chance of developing lymphopenia or immunodeficiency as compared to the general population. In some embodiments, a subject who has had, or who currently has cancer, HIV, hepatitis B, hepatitis C, sepsis, aplastic anemia, lymphoma, an inherited immune disorder, tuberculosis, renal failure, autoimmune disorders such as lupus, rheumatoid arthritis, and myasthenia gravis, heart failure, or is otherwise considered susceptible to being or becoming immunocompromised has an increased risk of developing lymphopenia or immunodeficiency. At risk subjects can also include subjects receiving chemotherapy, radiation treatment, steroids, aderenocorticotropic hormone, and / or immunosuppressive medications as well as those having undergone stem cell, tissue, and / or organ transplantation.

[0106] The term “immunotherapy” relates to a treatment involving a specific immune response, specifically, stimulation or recovery of immune cell populations. In the context of the present 23  Atty. Docket No.67175WO01 disclosure, terms such as “protect”, “prevent”, “prophylactic”, “preventative”, or “protective” relate to the prevention of the occurrence of or the exacerbation of lymphopenia or immunodeficiency in a subject. Persons having lymphopenia or immunodeficiency, immunocompromised persons, or persons at risk of developing lymphopenia, immunodeficiency, or becoming immunocompromised, as described above, would be considered candidates for immunotherapy. In some embodiments an immunotherapy in accordance with the disclosure comprises an immunotherapy targeted for the treatment of a cancer.

[0107] A prophylactic administration of an immunotherapy, for example, a prophylactic administration of a fusion protein or a composition comprising a fusion protein as disclosed herein, can in certain embodiments protect the recipient from developing lymphopenia or an immunodeficiency. In some embodiments, prophylactic administration can reduce disease progression and / or the severity of the disease or disorder.

[0108] A therapeutic administration of an immunotherapy, for example, a therapeutic administration of a fusion protein or a composition comprising a fusion protein as disclosed herein, can inhibit or attenuate the progression of cancer, or treat or prevent an immunodeficiency (e.g., lymphopenia, arrest or recover loss of white blood cell and / or lymphocyte counts, and / or inhibit, reduce, arrest, or alleviate clinical symptoms of lymphopenia).

[0109] As used herein, “treat”, “treating”, or “treatment” refer to administering a fusion protein or composition as described herein to a subject in order to eliminate, reduce, or attenuate the clinical symptoms of an immunodeficiency; arrest, inhibit, reverse, or slow the progression of an immunodeficiency in a subject.

[0110] The terms “subject”, “individual”, or “patient” are interchangeable, and relate to vertebrates, preferably mammals. For example, mammals in the context of the disclosure are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., as well as animals in captivity such as animals in zoos. The term "animal" as used herein also includes humans. The term "subject" can also include a patient, i.e., an animal, an in particular embodiments, a human having an immunodeficiency or an immunocompromised person.

[0111] The fusion proteins and compositions described herein can be administered via any conventional route, including by injection or infusion. In some embodiments, the administration 24  Atty. Docket No.67175WO01 can be carried out, for example, orally, intravenously, intraperitonealy, intramuscularly, subcutaneously, or transdermally.

[0112] The fusion proteins and the compositions comprising them are administered in effective amounts. An "effective amount" includes an amount that achieves a desired reaction or a desired effect and can be in the form of a single dose or as multiple doses.

[0113] An effective amount of a composition of the disclosure will depend on the severity of the immunodeficiency, the underlying cause (i.e. disease or condition), the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses of the compositions of the disclosure administered can depend on various combinations of such parameters. In embodiments in which an initial amount administered to a patient is insufficient, further administration with higher amounts, more frequent doses, or a different / more localized route of administration can be used.

[0114] In the methods and compositions disclosed herein, an IL-15 fusion protein can be administered to a subject to extend the serum half-life of the IL-15 protein relative to recombinant or exogenous IL-15. F. Kits

[0115] Another aspect of the present disclosure is a kit. In one aspect, a kit comprises any of the compositions or pharmaceutical compositions of a nucleic acid, polypeptide, expression vector, or host cell described above, and instructions or a label directing appropriate use or administration. Optionally, a kit can also include one or more containers and / or a syringe or other device to facilitate delivery or use. The disclosure contemplates that all or any subset of the components for conducting research assays and / or for administering therapeutically effective amounts of a fusion protein can be enclosed in the kit. Similarly, the kit can include instructions for making a polypeptide by, for example culturing a host cell that expresses a nucleic acid that encodes a fusion protein of the disclosure under suitable conditions. By way of additional example, a kit for therapeutic administration of a fusion protein of the disclosure can comprise a solution containing a pharmaceutical formulation of the fusion protein, or a lyophilized preparation of the fusion protein, and instructions for administering the composition to a patient in need thereof and / or for reconstituting the lyophilized product. 25  Atty. Docket No.67175WO01

[0116] The present disclosure also encompasses a finished packaged and labeled pharmaceutical product. This article of manufacture includes the appropriate unit dosage form in an appropriate vessel or container such as a glass vial or other container that is hermetically sealed. In the case of dosage forms suitable for parenteral administration the active ingredient, e.g., an above- described fusion protein, is sterile and suitable for administration as a particulate free solution. In certain aspects, the formulation is suitable for intravenous administration, such as for intravenous infusion to a human or animal.

[0117] In a specific aspect, the formulations of the disclosure are formulated in single dose vials as a sterile liquid. Exemplary containers include, but are not limited to, vials, bottles, pre-filled syringes, IV bags, and blister packs (comprising one or more pills). Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human diagnosis and / or administration.

[0118] As with any pharmaceutical product, the packaging material and container are designed to protect the stability of the product during storage and shipment. Further, the products of the disclosure include instructions for use or other informational material that advise the physician, technician, or patient on how to appropriately prevent or treat the disease or disorder in question. In other words, the article of manufacture includes instruction means indicating or suggesting a dosing regimen including, but not limited to, actual doses, monitoring procedures, etc., and other monitoring information.

[0119] A kit for research purposes can comprise a solution containing a fusion protein or a lyophilized preparation of a fusion protein of the disclosure, wherein the fusion protein binds specifically to one or more targets. The fusion protein can be labeled according to methods known in the art and described herein, including but not limited to labels such as small molecule fluorescent tags, proteins such as biotin, GFP or other fluorescent proteins, or epitope sequences such as his or myc. Similarly, primary antibodies used for detecting the fusion protein can be included in the kit. Primary antibodies can be directed to sequences on the fusion protein or to labels, tags, or epitopes with which the fusion protein is labeled. Primary antibodies can, in turn, be labeled for detection, or, if further amplification of the signal is desired, the primary antibodies can be detected by secondary antibodies, which can also be included in the kit. In 26  Atty. Docket No.67175WO01 some aspects, kits for research use can resemble kits intended for therapeutic uses but further include a label specifying that the kit and its use is restricted to research purposes only. G. Modification and / or Engineering

[0120] The disclosed fusion proteins can also be used to produce therapeutic immunoconjugates, wherein the fusion protein is conjugated with one or more therapeutic agents. For example, fusion proteins as described herein can be used in the production of protein-drug conjugates. Drugs that can be used include, without limitation, large and small molecule active agents such as, for example, immune system modulators, growth factors, (e.g., colony-stimulating factors (CSFs)), antiviral agents, antibiotic agents, antifungal agents, antiparasitic agents, and gamma globulin, and the like. EXAMPLES Example 1: Rational Fusion Protein Design

[0121] Some prior approaches to the design of IL-15 agonists are summarized in FIG.1, which include recombinant proteins of muteins, PEGylated IL-15, heterodimeric IL-15 (hetIL-15) in which IL-15 is co-assembled with IL-15Rα (AA1-175)10, RLI fusions in which IL-15 is linked to the IL-15Rα sushi domain via a flexible linker11,12, a fusion of a single chain antibody (scFv) that binds to human serum albumin (HSA) to deliver IL-15 and IL-12 in one molecule, and several Fc fusion designs. ALT-803 is a co-assembly of IL-15 enhancing mutant N720D with IL-15Rα sushi-Fc. Hengrui’s P22339 engineers an extra disulfide bond between the IL-15 and IL-15Rα sushi-Fc. In Xenco’s heterodimeric Fc fusion design, IL-15 and IL-15Rα sushi domain are fused with Fc-knob and Fc-hole15, respectively.

[0122] Differentiating from all prior fusion designs, the fusions in accordance with the example embodiments of the disclosure and as illustrated in the examples below comprise a fusion of IL- 15 and IL-15Rα (e.g., a sequence comprising AA31-115) which is further fused to human serum albumin (e.g., at the N- and / or C- terminus), all fusions made via one or more linker sequence (e.g., RLI, IgD, G4S, and the like), which format is generally depicted as "JL19001" in FIG.1.

[0123] Briefly, two illustrative fusion molecules were generated, designated as JL19001-1 and JL19001-2, by generating a fusion of IL-15 / IL-15Rα and further fusing that combination to 27  Atty. Docket No.67175WO01 either the N-terminus or the C-terminus of HSA (FIG.2). In these example molecules, the IgD linker is used to connect HSA to IL-15 / IL-15Rα and the RLI linker is used to connect IL-15Rα to IL-15. While the IL-15, IL-15Rα, and albumin sequences can be derived from any mammalian sequences, the particular sequences used in the initial format are all human, which are likely to maintain low immunogenicity. The full-length protein contains 862 amino acids with a calculated molecular weight of 96.16 kDa (see, e.g., FIG.2 and listing of sequences).

[0124] The design of illustrative molecules JL19001-1 and JL19001-2 were shown to provide IL-15Rα and IL-15 as functional units by covalently linking them with a flexible linker. In addition, the fusion of the functional IL-15Rα / IL-15 unit to the carrier HSA protein is effective to extend its half-life in vivo. As shown below, the HSA fusion is demonstrated not only to extend the plasma half-life of IL-15Rα / IL-15 and associated activity in animal studies, it also appears to possess better in vivo efficacy as compared to IL-15 control. Example 2: Production of JL19001

[0125] JL19001 (i.e., SEQ ID NOs.1 and 2) was produced in a bioreactor. Briefly, the JL19001 working cell bank was expanded in the seed culture medium (CD FortiCHO, 25 µM MSX and 1% ACA) every three or four days. After 3 passages, the seed culture was transferred into the production medium (CD FortiCHO and 1% ACA) and inoculated into a bioreactor. The cells were expanded in the bioreactor with CD FortiCHO medium supplemented with 1% ACA for 14 days to produce the JL19001 drug product.

[0126] JL19001 was purified using a four-column process (Octyl, Capto Blue, Capto adhere, Capto Q). Briefly, the culture supernatant was harvested and clarified with a depth filtration step to remove cells and cell debris. The filtrate is then concentrated, adjusted of conductivity, and clarified with another depth filtration step coupled with an absolute filtration with a 0.2 µm filter. JL19001 column purification starts with the Cytiva Octyl Sepharose 4 Fast Flow or Capto Octyl resin as a capture step and partially removes lower molecular weight (LMW) species. JL19001- containing fractions from Octyl FF chromatography are processed through Cytiva Capto Blue resin next, as an intermediate purification step to remove host cell protein (HCP). The JL19001- containing fractions from Blue chromatography are pooled, diafiltered into a phosphate buffer, and incubated at pH 3.5 for 45 minutes to 1 hour to inactivate the virus. After viral inactivation, a mixed-mode column, Cytiva Capto adhere ImpRes, is performed to remove the remaining HSA- 28  Atty. Docket No.67175WO01 related LMW impurities. The JL19001-containing fractions from Capto adhere ImpRes chromatography are purified through an anion exchange column, Cytiva Capto Q to remove JL19001-related higher molecular weight (HMW) species, as well as a mechanism for nucleic acids, endotoxin, and virus removal. The purified product is concentrated and formulated at 5 mg / mL in the formulation buffer as the bulk drug. Example 3: JL19001-1 and JL19001-2 Characterization and Properties

[0127] Analysis of JL19001-1 and JL19001-2 by comparative SDS-PAGE indicates that JL19001-1 may be modified by N-linked glycosylation. JL19001-1 and JL19001-2 migration on both reduced and non-reduced SDS-PAGE is illustrated in FIG.3B, which shows that N- glycosidase F treatment of JL19001-1 effectively reduces its size to that of JL19001-2. It is possible that glycosylation of a sequon at the C-terminus of IL-15 (Asn-Thr-Ser) is glycosylated more efficiently in JL19001-1, where it is internal, than in JL19001-2, where it remains at the C- terminus (FIG.3A). Example 4: JL19001-1 and JL19001-2 Cell Proliferation Activity

[0128] After incubating with JL19001-1 and JL19001-2 for 3 days, CTLL-2 cell proliferation is measured using MTS kit (Promega). As shown in FIG.4A, JL19001-1 appears to be about 5- to 10-fold less potent than JL19001-2 in stimulating CTLL-2 proliferation. Without being bound by theory, it may be that the enhanced glycosylation in JL19001-1 attenuates its activity in stimulating CTLL-2 cell proliferation. In light of this activity, further studies detailed below were performed with the JL19001 molecule identified as JL19001-2, which is simply referred to below as "JL19001" and in the FIGs that illustrate the data resulting from those studies. Example 5: Effect of JL19001 and rhIL-15 in stimulating the growth of CTLL-2 and M-07e Cells

[0129] CTLL-2 cells and M-07e cells are used to determine cytokine dependent growth rate and can respond to recombinant human (rhIL-15). As shown below, JL19001 exhibits different activities on these two cell lines. Table 1 details the EC50of rhIL-15 and the JL19001 molecule. When CTLL-2 cells are used as target cells, the EC50for rhIL-15 is 3.9 pM whereas the EC50for JL19001 is 3636 pM. When M-07e cells are used as target cells, the EC50 of rhIL-15 is 148 pM 29  Atty. Docket No.67175WO01 while the EC50 of JL19001 is 768 pM (FIG.4A). While the data indicates that JL19001 is less potent than rhIL-15 (i.e., about 1000-fold (CTLL-2) and about 5-fold (M-07e) less potent), the relative potency of the molecules in M-07e cells is notable in that, when compared to CTLL-2 cells, rhIL-15 is 35-fold less potent while JL19001 is 5-fold more potent.

[0130] FIG.5 illustrates a model for different sensitivities of rhIL-15 and JL19001 in these two cell lines.  In overview, CTLL-2 cells may express greater amounts of high affinity receptors (αβ ^c) that may result in rhIL-15 preferential binding to those cells relative to M-07e cells, whereas JL19001 with pre-assembled IL-15Rα could preferentially bind to intermediate affinity receptor (β ^c) that are more abundant in M-07e cells. As a result, CTLL-2 cells may give high potency for rhIL-15, but not for JL19001. In contrast, JL19001 is more responsive in M-07e cells which, as discussed above, may be due to greater amounts of intermediate affinity receptor (β ^c). Example 6: IL-15 Fusion Proteins in other Formats

[0131] Several constructs are prepared in an effort to analyze whether the various domains / components of the JL19001 fusion protein may exert an effect on any aspect of JL19001 activity (FIG.6). Construct JL19001-3 (also referred to as pJL177) is constructed to test the effect of an additional 20 amino acids downstream of the sushi domain. Construct JL19001-4 (also referred as pJL359) is constructed by replacing the IgD linker between HSA and IL-15Rα with a 3x(G4S) linker. Construct JL19001-5 (also referred to as pJL186) is constructed by removing the entire IL-15Rα to evaluate whether HSA has a negative effect on IL-15. In addition, two Fc fusion proteins, construct JL19001-6 (also referred as pJL185) and construct JL19001-7 (also referred to as pJL485) were prepared. In construct JL19001-6 (pJL185), IL- 15Rα / IL-15 is fused to the C-terminus of Fc in order to maintain the same configuration of the functional unit as JL19001. In construct JL19001-7 (pJL485), only IL-15Rα is fused to the N- terminus of Fc while IL-15 is separately expressed and provided as a free (i.e., non-fused) molecule. Notably, unlike the JL19001 fusion construct, IL-15 in an Fc fusion is dimeric. Table 1 provides sequence information for several of the exemplary fusion proteins (i.e., "JL19001-X") as well as illustrative sequences of the various fusion protein domains as described herein. Table 1: Amino Acid Sequences for Representative Protein and Peptide Sequences Protein / Peptide Sequence30  Atty. Docket No.67175WO01 NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI SLESGDASIH DTVENLIILA NNSLSSNGNV human IL-15 TESGCKECEE LEEKNIKEFL QSFVHIVQMF INTSAtty. Docket No.67175WO01 (SEQ ID NO: 16) ITCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTSAtty. Docket No.67175WO01 DVMCTAFHDN EETFLKKYLY EIARRHPYFY APELLFFAKR YKAAFTECCQ AADKAACLLP KLDELRDEGK ASSAKQRLKC ASLQKFGERA FKAWAVARLS QRFPKAEFAE VSKLVTDLTKAtty. Docket No.67175WO01 YKFQNALLVR YTKKVPQVST PTLVEVSRNL GKVGSKCCKH PEAKRMPCAE DYLSVVLNQL CVLHEKTPVS DRVTKCCTES LVNRRPCFSA LEVDETYVPK EFNAETFTFH ADICTLSEKE

[0132] Table 2 compares activities of these constructs. Table 2: Comparison of JL19001 potency with IL-15 fusion proteins in other formats EC50 (pM) th  ls  34  Atty. Docket No.67175WO01

[0133] As shown in Table 2, JL19001-3 (also referred to as pJL177) demonstrates potencies similar to JL19001 in both CTLL-2 and M-07e proliferation assays. Accordingly, it follows that the additional 20 amino acids downstream of the sushi domain are likely to not be responsible for attenuated potency of JL19001. Replacing the IgD linker in JL19001 with a 3x(G4S) linker destabilizes JL19001-4 (also referred to as pJL359), compromising its titer (data not shown). Linker replacement does not improve potency as assayed by CTLL-2 proliferation. Fc fusion, compared to HSA fusion, appears to improve the potency for CTLL-2 stimulation by 2-5-fold. The improvement with Fc could come from dimeric format of the fusion protein rather than the fusion partner itself. Significant improvements in potency are observed when IL-15 is expressed without a covalent connection to IL-15Rα, either as HSA fusion (pJL186) or as Fc fusion pre- assembled with IL-15Rα (JL19001-7 / pJL485). While JL19001-5 (pJL186) appears to lose the robustness of expression, its potency appears to be improved by 23-fold. The most dramatic improvement in potency is observed with JL19001-7, in which its potency is improved by over 200-fold, to a level close to rhIL-15. These results suggest that the covalent linking IL-15 to IL- 15Rα accounts for the primary reason that JL19001 appears to have significantly attenuated activity in the CTLL-2 proliferation assay. These data suggest that JL19001 shows lower in vitro potency compared to rhIL-15; however additional data suggests that JL19001 is more potent than rhIL-15 in vivo. Example 7: JL19001 Pharmacokinetics Study

[0134] The pharmacokinetics (PK) profile of two doses of JL19001 (50 ^g / kg and 150 ^g / kg) is illustrated in FIG.7 following intravenous administration to mice. Recombinant human IL-15 (rhIL-15) is administered as a control at 150 ^g / kg. Blood samples are obtained by retro-orbital bleeding at 5 min, 30 min, 1 hr, 2 hr, 4 hr, 8 hr, 24 hr, 72 hr, 96 hr, 120 hr, and 192 hr post- injection. The blood samples are centrifuged (5000 rpm) at 4°C for 15 min to obtain serum. PK values are calculated with PKSolver software and shown in FIG.7.

[0135] At 150 µg / kg, rhIL-15 in vivo half-life is 43.38 min (<1 hour). JL19001 in vivo half-life is 9.7 hours (150 µg / kg) and 11.6 hours (50 µg / kg). These data demonstrate the IL-15 fusion proteins extend the half-life compared to control rhIL-15. Example 8: Effects on T cells and NK cells 35  Atty. Docket No.67175WO01

[0136] The efficacy of JL19001 is evaluated using primary human cells in culture. Human PBMCs are labeled with CellTrace CSFE, and then plated at 100,000 cells / well with rhIL-15 at 10, 1, 0.1 ng / mL, or JL19001 at 1000, 100, 10 ng / mL. After culturing for 4 days, cells are collected, stained with antibodies for CD3, CD8, CD56, CD69 (a T cell activation marker), and analyzed by Attune flow cytometer. Dead cells are excluded by their staining of 7-AAD indicator.

[0137] Cell proliferation is measured by the dilution of CFSE signals. An example of NK cells and CD8+ T cells surface marker staining is illustrated in FIG.8.

[0138] JL19001 and IL-15 have an effect on the proliferation of primary NK and CD8+ T cells. The dilution of CFSE is observed in NK, NKT and CD8+ T cell populations, but reduced in CD8- (mainly CD4+) T cell population as illustrated in FIG.9.

[0139] JL19001 and IL-15 have an effect on the activation of primary NK and CD8+ T cells. The up-regulation of T-cell activation marker CD69 is observed with increasing concentrations of IL-15 and JL19001 in NKT, NK cells and to a lesser extent in CD8+ T cells as illustrated in FIG.10. CD69 expression is diminished in CD8- T cells. Example 9: Delivery of JL19001 to the blood via i.p. injection

[0140] Serum levels of JL19001 are measurable after intraperitoneal (i.p.) injection as listed in Table 3. Mice are given 1250 ^g / kg JL19001 i.p. daily. The blood samples are collected 30 min after JL19001 i.p. injection on day 1 and day 4, sera are prepared, and the concentrations of JL19001 in serum samples are determined using an IL-15 ELISA kit. It was found that JL19001 was distributed to the blood stream quite fast. For example, as short as 30 mins after i.p. injection, 175 ng / mL to 1520 ng / mL JL19001 was detected in the mouse blood. The concentration of JL19001 in the blood is maintained at about 3000 ng / mL on day 4. Table 3: Serum concentrations of JL19001 after i.p. injections Serum JL1900136  Atty. Docket No.67175WO01 Serum JL19001concentration (ng / L)Day 1, Day 4, 3.p. ND, not detectable; n / a, not applicable. JL19001 mouse #5 died after day 4 treatment. There were five mice in each treatment group. Example 10: JL19001 actively increased NK cells and T cells in mouse spleen and peritoneal cavity

[0141] The in vivo efficacy of JL19001 is evaluated using Balb / c mice. Mice are administered JL19001 at daily doses of 20 ^g / kg, 60 ^g / kg, 180 ^g / kg, and 540 ^g / kg i.p. injection, for a period of seven days. Mouse body weight was measured after administration of JL19001 at various concentrations and days. The results are listed in Table 4. Table 4: Daily Body Weight Monitoring MouseInitial body Body Body Body Body Body )37  Atty. Docket No.67175WO01 Initial bod Body Body Body Body Body Group Mousey numberweight (g) weight weight weight weight weight D 2 D 3 D 4 D 5 D 6 g)

[0142] Mouse tolerated JL19001 treatment up to 180 ^g / kg. However, when JL19001 was administered at 540 ^g / kg, mice died after 4 days of treatment.

[0143] After seven days of treatment, surviving mice are sacrificed, the percentages and numbers of CD3+ T cells, CD4+ T cells, CD8+ T cells, and NK cells (CD3-CD335+ cells) in the spleens are determined as depicted in FIG.11.

[0144] FIG.12, depicts the percentages and numbers of CD3+ T cells, CD4+ T cells, CD8+ T cells, and NK cells (CD3-CD335+ cells) in the peritoneal distribution of JL19001 from peritoneal cavity to blood.

[0145] To compare the in vivo activity of JL19001 and rhIL-15, mice were treated with 20 ^g / kg and 60 ^g / kg of JL19001 or 12 ^ ^g / kg, 36 ^ ^g / kg, and 108 µg / kg of rhIL-15, with subsequent measurements of the total splenic cell numbers and total peritoneal cavity cells as depicted in FIG.14. 38  Atty. Docket No.67175WO01

[0146] To compare the in vivo activity of JL19001 and rhIL-15, mice were treated with 20 ^g / kg and 60 ^g / kg of JL19001 or 12 ^ ^g / kg, 36 ^ ^g / kg, and 108 µg / kg of rhIL-15, with subsequent measurement of different splenic cell populations as depicted in FIG.15.

[0147] To compare the in vivo activity of JL19001 and rhIL-15, mice were treated with 20 ^ ^g / kg and 60 ^g / kg of JL19001 or 12 ^ ^g / kg, 36 ^ ^g / kg, and 108 µg / kg of rhIL-15, with subsequent measurements of the numbers of different splenic cell populations as depicted in FIG.16.

[0148] To compare the in vivo activity of JL19001 and rhIL-15, mice were treated with 20 ^ ^g / kg and 60 ^g / kg of JL19001 or 12 ^ ^g / kg, 36 ^ ^g / kg, and 108 µg / kg of rhIL-15, with subsequent measurement of different peritoneal cavity cell populations as depicted in FIG.17.

[0149] To compare the in vivo activity of JL19001 and rhIL-15, mice were treated with 20 ^ ^g / kg and 60 ^g / kg of JL19001 or 12 ^ ^g / kg, 36 ^g / kg, and 108 µg / kg of rhIL-15, with subsequent measurements of the number of different peritoneal cavity cell populations as depicted in FIG.18. Example 11: Comparison of JL19001 vs rhIL-15 in vivo efficacy

[0150] Figure 13 depicts a schematic of the experimental design for determining the in vivo efficacy of JL19001 and rhIL-15. JL19001 at 20, 60 ^g / kg is equivalent to 2.0*10-10, 6.2*10-10mole / kg. rhIL-15 at 12, 36 and 108 ^g / kg are equivalent to 9.3*10-10, 27.9*10-10, and 83.7*10-10mole / kg. Both JL19001 and rhIL-15 increased splenic NK+ cell percentages and numbers (FIG 14-18). The in vivo effects of JL19001 at 60 µg / kg (6.2*10-10mole / kg) are similar to those of rhIL-15 at 108 µg / Kg (83.7*10-10mole / kg), suggesting that JL19001 in vivo activity is ~10-fold higher than rhIL-15 when the same molar amount is administered. Sequence Listing Construct 1 (SEQ ID NO.1) JL19001-1, IL15Rα-IL15-HSA (pJL137) 10 20 30 40 50 60 ITCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS 70 80 90 100 110 120 LKCIRDPALV HQRPAPPSTV TTAGVSGGSG GGGSGGGSGG GGSLQNWVNV ISDLKKIEDL 130 140 150 160 170 180 IQSMHIDATL YTESDVHPSC KVTAMKCFLL ELQVISLESG DASIHDTVEN LIILANNSLS 39  Atty. Docket No.67175WO01 190 200 210 220 230 240 SNGNVTESGC KECEELEEKN IKEFLQSFVH IVQMFINTSE SPKAQASSVP TAQPQAEGSL 250 260 270 280 290 300 AKATTAPATT RNTGRGGEEK KKEKEKEEQE ERETKTPDAH KSEVAHRFKD LGEENFKALV 310 320 330 340 350 360 LIAFAQYLQQ CPFEDHVKLV NEVTEFAKTC VADESAENCD KSLHTLFGDK LCTVATLRET 370 380 390 400 410 420 YGEMADCCAK QEPERNECFL QHKDDNPNLP RLVRPEVDVM CTAFHDNEET FLKKYLYEIA 430 440 450 460 470 480 RRHPYFYAPE LLFFAKRYKA AFTECCQAAD KAACLLPKLD ELRDEGKASS AKQRLKCASL 490 500 510 520 530 540 QKFGERAFKA WAVARLSQRF PKAEFAEVSK LVTDLTKVHT ECCHGDLLEC ADDRADLAKY 550 560 570 580 590 600 ICENQDSISS KLKECCEKPL LEKSHCIAEV ENDEMPADLP SLAADFVESK DVCKNYAEAK 610 620 630 640 650 660 DVFLGMFLYE YARRHPDYSV VLLLRLAKTY ETTLEKCCAA ADPHECYAKV FDEFKPLVEE 670 680 690 700 710 720 PQNLIKQNCE LFEQLGEYKF QNALLVRYTK KVPQVSTPTL VEVSRNLGKV GSKCCKHPEA 730 740 750 760 770 780 KRMPCAEDYL SVVLNQLCVL HEKTPVSDRV TKCCTESLVN RRPCFSALEV DETYVPKEFN 790 800 810 820 830 840 AETFTFHADI CTLSEKERQI KKQTALVELV KHKPKATKEQ LKAVMDDFAA FVEKCCKADD 850 860 KETCFAEEGK KLVAASQAAL GL Human IL‐15R ^ (AA1‐85), RLI linker (AA86‐105), and IL‐15 (AA106‐219) were fused to human HSA (AA278‐862) via  . 40  Atty. Docket No.67175WO01 Construct 2 (SEQ ID NO.2) JL19001-2, HSA-IL15Rα-IL15 (pJL138, also referred as JL19001) 10 20 30 40 50 60 DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHV KLVNEVTEFA KTCVADESAE 70 80 90 100 110 120 NCDKSLHTLF GDKLCTVATL RETYGEMADC CAKQEPERNE CFLQHKDDNP NLPRLVRPEV 130 140 150 160 170 180 DVMCTAFHDN EETFLKKYLY EIARRHPYFY APELLFFAKR YKAAFTECCQ AADKAACLLP 190 200 210 220 230 240 KLDELRDEGK ASSAKQRLKC ASLQKFGERA FKAWAVARLS QRFPKAEFAE VSKLVTDLTK 250 260 270 280 290 300 VHTECCHGDL LECADDRADL AKYICENQDS ISSKLKECCE KPLLEKSHCI AEVENDEMPA 310 320 330 340 350 360 DLPSLAADFV ESKDVCKNYA EAKDVFLGMF LYEYARRHPD YSVVLLLRLA KTYETTLEKC 370 380 390 400 410 420 CAAADPHECY AKVFDEFKPL VEEPQNLIKQ NCELFEQLGE YKFQNALLVR YTKKVPQVST 430 440 450 460 470 480 PTLVEVSRNL GKVGSKCCKH PEAKRMPCAE DYLSVVLNQL CVLHEKTPVS DRVTKCCTES 490 500 510 520 530 540 LVNRRPCFSA LEVDETYVPK EFNAETFTFH ADICTLSEKE RQIKKQTALV ELVKHKPKAT 550 560 570 580 590 600 KEQLKAVMDD FAAFVEKCCK ADDKETCFAE EGKKLVAASQ AALGLESPKA QASSVPTAQP 610 620 630 640 650 660 QAEGSLAKAT TAPATTRNTG RGGEEKKKEK EKEEQEERET KTPITCPPPM SVEHADIWVK 670 680 690 700 710 720 SYSLYSRERY ICNSGFKRKA GTSSLTECVL NKATNVAHWT TPSLKCIRDP ALVHQRPAPP 730 740 750 760 770 780 STVTTAGVSG GSGGGGSGGG SGGGGSLQNW VNVISDLKKI EDLIQSMHID ATLYTESDVH 790 800 810 820 830 840 PSCKVTAMKC FLLELQVISL ESGDASIHDT VENLIILANN SLSSNGNVTE SGCKECEELE 850 860 EKNIKEFLQS FVHIVQMFIN TS HSA (AA1‐585) is first fused to human IL‐15R ^ (AA644‐728) via the IgD hinge sequence (AA586‐643), then fused to Atty. Docket No.67175WO01 Construct 3 (SEQ ID NO.3) JL19001-3, HSA-Sushi-IL15 (pJL177) SEQ ID NO.3 10 20 30 40 50 60 DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHV KLVNEVTEFA KTCVADESAE 70 80 90 100 110 120 NCDKSLHTLF GDKLCTVATL RETYGEMADC CAKQEPERNE CFLQHKDDNP NLPRLVRPEV 130 140 150 160 170 180 DVMCTAFHDN EETFLKKYLY EIARRHPYFY APELLFFAKR YKAAFTECCQ AADKAACLLP 190 200 210 220 230 240 KLDELRDEGK ASSAKQRLKC ASLQKFGERA FKAWAVARLS QRFPKAEFAE VSKLVTDLTK 250 260 270 280 290 300 VHTECCHGDL LECADDRADL AKYICENQDS ISSKLKECCE KPLLEKSHCI AEVENDEMPA 310 320 330 340 350 360 DLPSLAADFV ESKDVCKNYA EAKDVFLGMF LYEYARRHPD YSVVLLLRLA KTYETTLEKC 370 380 390 400 410 420 CAAADPHECY AKVFDEFKPL VEEPQNLIKQ NCELFEQLGE YKFQNALLVR YTKKVPQVST 430 440 450 460 470 480 PTLVEVSRNL GKVGSKCCKH PEAKRMPCAE DYLSVVLNQL CVLHEKTPVS DRVTKCCTES 490 500 510 520 530 540 LVNRRPCFSA LEVDETYVPK EFNAETFTFH ADICTLSEKE RQIKKQTALV ELVKHKPKAT 550 560 570 580 590 600 KEQLKAVMDD FAAFVEKCCK ADDKETCFAE EGKKLVAASQ AALGLESPKA QASSVPTAQP 610 620 630 640 650 660 QAEGSLAKAT TAPATTRNTG RGGEEKKKEK EKEEQEERET KTPITCPPPM SVEHADIWVK 670 680 690 700 710 720 SYSLYSRERY ICNSGFKRKA GTSSLTECVL NKATNVAHWT TPSLKCIRSG GSGGGGSGGG 730 740 750 760 770 780 SGGGGSLQNW VNVISDLKKI EDLIQSMHID ATLYTESDVH PSCKVTAMKC FLLELQVISL 790 800 810 820 830 840 ESGDASIHDT VENLIILANN SLSSNGNVTE SGCKECEELE EKNIKEFLQS FVHIVQMFIN TS HSA (AA1‐585) is first fused to human IL‐15R ^ sushi domain (AA644‐708) via the IgD hinge sequence (AA586‐643), Atty. Docket No.67175WO01 Construct 4 (SEQ ID NO.4) JL19001-4, HSA-IL15Rα-IL15 (pJL359 with 3xG4S linker replacing IgD linker) 10 20 30 40 50 60 DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHV KLVNEVTEFA KTCVADESAE 70 80 90 100 110 120 NCDKSLHTLF GDKLCTVATL RETYGEMADC CAKQEPERNE CFLQHKDDNP NLPRLVRPEV 130 140 150 160 170 180 DVMCTAFHDN EETFLKKYLY EIARRHPYFY APELLFFAKR YKAAFTECCQ AADKAACLLP 190 200 210 220 230 240 KLDELRDEGK ASSAKQRLKC ASLQKFGERA FKAWAVARLS QRFPKAEFAE VSKLVTDLTK 250 260 270 280 290 300 VHTECCHGDL LECADDRADL AKYICENQDS ISSKLKECCE KPLLEKSHCI AEVENDEMPA 310 320 330 340 350 360 DLPSLAADFV ESKDVCKNYA EAKDVFLGMF LYEYARRHPD YSVVLLLRLA KTYETTLEKC 370 380 390 400 410 420 CAAADPHECY AKVFDEFKPL VEEPQNLIKQ NCELFEQLGE YKFQNALLVR YTKKVPQVST 430 440 450 460 470 480 PTLVEVSRNL GKVGSKCCKH PEAKRMPCAE DYLSVVLNQL CVLHEKTPVS DRVTKCCTES 490 500 510 520 530 540 LVNRRPCFSA LEVDETYVPK EFNAETFTFH ADICTLSEKE RQIKKQTALV ELVKHKPKAT 550 560 570 580 590 600 KEQLKAVMDD FAAFVEKCCK ADDKETCFAE EGKKLVAASQ AALGLGGGGS GGGGSGGGGS 610 620 630 640 650 660 ITCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS 670 680 690 700 710 720 LKCIRSGGSG GGGSGGGSGG GGSLQNWVNV ISDLKKIEDL IQSMHIDATL YTESDVHPSC 730 740 750 760 770 780 KVTAMKCFLL ELQVISLESG DASIHDTVEN LIILANNSLS SNGNVTESGC KECEELEEKN 790 IKEFLQSFVH IVQMFINTSAtty. Docket No.67175WO01 Construct 5 (SEQ ID NO.5) JL19001-5, HSA-IL15 (pJL186 without IL-15R ^) 10 20 30 40 50 60 DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHV KLVNEVTEFA KTCVADESAE 70 80 90 100 110 120 NCDKSLHTLF GDKLCTVATL RETYGEMADC CAKQEPERNE CFLQHKDDNP NLPRLVRPEV 130 140 150 160 170 180 DVMCTAFHDN EETFLKKYLY EIARRHPYFY APELLFFAKR YKAAFTECCQ AADKAACLLP 190 200 210 220 230 240 KLDELRDEGK ASSAKQRLKC ASLQKFGERA FKAWAVARLS QRFPKAEFAE VSKLVTDLTK 250 260 270 280 290 300 VHTECCHGDL LECADDRADL AKYICENQDS ISSKLKECCE KPLLEKSHCI AEVENDEMPA 310 320 330 340 350 360 DLPSLAADFV ESKDVCKNYA EAKDVFLGMF LYEYARRHPD YSVVLLLRLA KTYETTLEKC 370 380 390 400 410 420 CAAADPHECY AKVFDEFKPL VEEPQNLIKQ NCELFEQLGE YKFQNALLVR YTKKVPQVST 430 440 450 460 470 480 PTLVEVSRNL GKVGSKCCKH PEAKRMPCAE DYLSVVLNQL CVLHEKTPVS DRVTKCCTES 490 500 510 520 530 540 LVNRRPCFSA LEVDETYVPK EFNAETFTFH ADICTLSEKE RQIKKQTALV ELVKHKPKAT 550 560 570 580 590 600 KEQLKAVMDD FAAFVEKCCK ADDKETCFAE EGKKLVAASQ AALGLESPKA QASSVPTAQP 610 620 630 640 650 660 QAEGSLAKAT TAPATTRNTG RGGEEKKKEK EKEEQEERET KTPNWVNVIS DLKKIEDLIQ 670 680 690 700 710 720 SMHIDATLYT ESDVHPSCKV TAMKCFLLEL QVISLESGDA SIHDTVENLI ILANNSLSSN 730 740 750 GNVTESGCKE CEELEEKNIK EFLQSFVHIV QMFINTS HSA (AA1‐585) is directly fused to human IL‐15 (AA644‐757) via the IgD hinge sequence (AA586‐643). 44  Atty. Docket No.67175WO01 Construct 6 (SEQ ID NO.6): JL19001-6, Fc-IL15R ^-IL15 (pJL185) 10 20 30 40 50 60 EPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF 70 80 90 100 110 120 NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT 130 140 150 160 170 180 ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP 190 200 210 220 230 240 PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GESPKAQASS 250 260 270 280 290 300 VPTAQPQAEG SLAKATTAPA TTRNTGRGGE EKKKEKEKEE QEERETKTPI TCPPPMSVEH 310 320 330 340 350 360 ADIWVKSYSL YSRERYICNS GFKRKAGTSS LTECVLNKAT NVAHWTTPSL KCIRDPALVH 370 380 390 400 410 420 QRPAPPSTVT TAGVSGGSGG GGSGGGSGGG GSLQNWVNVI SDLKKIEDLI QSMHIDATLY 430 440 450 460 470 480 TESDVHPSCK VTAMKCFLLE LQVISLESGD ASIHDTVENL IILANNSLSS NGNVTESGCK 490 500 ECEELEEKNI KEFLQSFVHI VQMFINTS Human Fc (AA1‐231) is first fused to human IL‐15R ^ (AA290‐374) via the IgD hinge sequence (AA232‐289), then  fused to the human IL‐15 sequence (AA395‐508) via a RLI linker (AA375‐394).  45  Atty. Docket No.67175WO01 Construct 7: (SEQ ID NO.7 + SEQ ID NO.8) JL19001-7, IL15 / IL15Rα-Fc JL19001-7 contains two peptides, IL15 (SEQ ID NO.7) and IL15Rα-Fc (SEQ ID NO.8). They are non-covalently linked. They are assembled in the cell because IL15Rα has a picomolar affinity to IL15. SEQ ID NO.7: Sequence of IL15 (in pJL485) 10 20 30 40 50 60 NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI SLESGDASIH 70 80 90 100 110 DTVENLIILA NNSLSSNGNV TESGCKECEE LEEKNIKEFL QSFVHIVQMF INTS SEQ ID NO.8: Sequence of IL15R ^-Fc (pJL485) 10 20 30 40 50 60 ITCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS 70 80 90 100 110 120 LKCIREPKSS DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED 130 140 150 160 170 180 PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA 190 200 210 220 230 240 PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN 250 260 270 280 290 YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG Human IL‐15R ^ sushi domain (AA1‐65) is directly fused to human Fc (AA66‐296).  46  Atty. Docket No.67175WO01 SEQ ID NO.9: IL15R ^ sushi domain Sequence ITCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIR SEQ ID NO.10: IL15R ^ Sequence ITCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGV SEQ ID NO.11: Fc sequence EPKSS DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG   SEQ ID NO.12: HSA sequence DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHV KLVNEVTEFA KTCVADESAE NCDKSLHTLF GDKLCTVATL RETYGEMADC CAKQEPERNE CFLQHKDDNP NLPRLVRPEV DVMCTAFHDN EETFLKKYLY EIARRHPYFY APELLFFAKR YKAAFTECCQ AADKAACLLP KLDELRDEGK ASSAKQRLKC ASLQKFGERA FKAWAVARLS QRFPKAEFAE VSKLVTDLTK VHTECCHGDL LECADDRADL AKYICENQDS ISSKLKECCE KPLLEKSHCI AEVENDEMPA DLPSLAADFV ESKDVCKNYA EAKDVFLGMF LYEYARRHPD YSVVLLLRLA KTYETTLEKC CAAADPHECY AKVFDEFKPL VEEPQNLIKQ NCELFEQLGE YKFQNALLVR YTKKVPQVST PTLVEVSRNL GKVGSKCCKH PEAKRMPCAE DYLSVVLNQL CVLHEKTPVS DRVTKCCTES LVNRRPCFSA LEVDETYVPK EFNAETFTFH ADICTLSEKE RQIKKQTALV ELVKHKPKAT KEQLKAVMDD FAAFVEKCCK ADDKETCFAE EGKKLVAASQ AALGL SEQ ID NO.13: RLI Linker SGGSGG GGSGGGSGGG GSLQ SEQ ID NO.14: IgD Hinge SPKAQASS VPTAQPQAEG SLAKATTAPA TTRNTGRGGE EKKKEKEKEE QEERETKTP SEQ ID NO.15: G4S (3x) GGGGSGGGGS GGGGS 47  Atty. Docket No.67175WO01

[0151] All features disclosed in the specification, including the claims, abstracts, and drawings, and all the steps in any method or process disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Each feature disclosed in the specification, including the claims, abstract, and drawings, can be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0152] It will be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. 48

Claims

Atty. Docket No.67175WO01 CLAIMS We claim:

1. A fusion protein comprising at least one of an IL-15Rα sequence and / or an IL-15 sequence fused to an albumin protein.

2. The fusion protein of claim 1 comprising a fusion between the IL-15Rα sequence and the IL-15 sequence.

3. The fusion protein of claim 1 or claim 2 that comprises a first linker sequence between the IL-15Rα sequence and IL-15 sequence, and a second linker sequence between the albumin protein to the IL-15Rα sequence and IL-15 sequence.

4. The fusion protein of claim 3, wherein the first and second linkers comprise an RLI linker or an IgD hinge.

5. The fusion protein of any of the preceding claims, wherein the IL-15Rα protein is a human IL-15Rα.

6. The fusion protein of any of the preceding claims, wherein the IL-15 protein is human IL- 15.

7. The fusion protein of any of the preceding claims, wherein the albumin protein is human serum albumin.

8. The fusion protein of any of the preceding claims, wherein the IgD hinge is human IgD sequence.

9. The fusion protein of any of the preceding claims, wherein IL-15Rα sequence comprises a sushi domain. 49  Atty. Docket No.67175WO01 10. A fusion protein comprising a sequence according to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:

8.

11. The fusion protein of any one of the preceding claims, wherein the serum half-life of the IL-15 is extended in the fusion protein relative to recombinant or exogenous human IL- 15.

12. The fusion protein of claim 11, wherein the serum half-life is extended by at least nine hours.

13. The fusion protein of any one of the preceding claims, wherein the fusion protein induces proliferation of at least one of NK cells (CD3-CD56+), CD8+ T cells (CD3+CD56- CD8+), and NKT cell (CD3+ CD56+) when administered to a subject.

14. The fusion protein of any one of the preceding claims, wherein the fusion protein induces activation of NK cells (CD3-CD56+), CD8+ T cells (CD3+CD56-CD8+), and NKT cell (CD3+ CD56+) when administered to a subject.

15. The fusion protein of any one of the preceding claims, wherein the fusion protein increases lymphocyte counts when administered to a subject.

16. The fusion protein of any one of the preceding claims, wherein the fusion protein increases the total number of splenic CD8+ T cells when administered to a subject.

17. The fusion protein of any one of the preceding claims, wherein the fusion protein increases the total number and cell percentages of splenic NK cells when administered to a subject.

18. The fusion protein of any one of the preceding claims, wherein the fusion protein increases the total number of peritoneal cavity CD8+ T cells when administered to a subject. 50  Atty. Docket No.67175WO01 19. The fusion protein of any one of the preceding claims, wherein the fusion protein increases the total number and cell percentages of peritoneal cavity NK cells when administered to a subject.

20. A nucleic acid sequence that encodes the fusion protein according to any one of claims 1- 10.

21. A recombinant cell comprising the fusion protein according to any one of claims 1-10 or the nucleic acid sequence according to claim 20.

22. A composition comprising the fusion protein of any one of claims 1-10 and a pharmaceutically acceptable carrier, diluent, or excipient.

23. A kit comprising the fusion protein of any one of claims 1-10.

24. A method for inducing an immune response in a subject in need of treatment, comprising administering to the subject the fusion protein according to any one of claims 1-10 or the composition of claim 22.

25. A method for treating cancer in a subject in need of treatment, comprising administering to the subject the fusion protein according to any one of claims 1-10 or the composition of claim 22. 51

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