Interleukin-15 fusion protein and method of use

Abstract

This disclosure provides IL-15 fusion proteins and compositions comprising IL-15 fusion proteins. This disclosure further provides methods for treating and / or preventing immunodeficiency in subjects, as well as methods for cancer immunotherapy, which include administering fusion proteins as described herein.

Description

[Technical Field]

[0001] Related applications

[0001] This patent application claims priority and interest of U.S. Provisional Patent Application No. 63 / 471,370, filed June 6, 2023, which is incorporated in its entirety by reference. Reference to electronic sequence listings

[0002] The contents of the electronic sequence listing (67175WO01_SequenceListing.xml, size: 26,932 bytes, created: May 30, 2024) are incorporated herein by reference in their entirety. [Background technology]

[0002]

[0003] Interleukin-15 (IL-15) is a pleomorphic cytokine produced by many types of cells, including immune cells (e.g., monocytes, macrophages, dendritic cells) and non-immune cells (e.g., keratinocytes and epithelial cells). IL-15 is involved in diverse immune functions and plays a crucial 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).

[0003]

[0004] The IL-15 receptor has three subunits (polypeptides): type-specific IL-15R-alpha (IL-15Rα), IL-2 / IL-15R-beta (IL-15Rβ), and a common gamma chain (γc). IL-15R includes the subunit IL-15Rα, which binds to IL-15 with picomolar affinity. The IL-15 receptor shares a second subunit, 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.

[0004]

[0005] IL-2 is a nonspecific T cell growth factor that stimulates the proliferation of all subsets of T cells, including CD8+ cytotoxic T cells, CD4+ helper T cells, regulatory T cells (Tregs), and NK cells. IL-2 is a secreted cytokine that binds to pre-formed IL-2 receptors (IL-2Rαβγc) on the surface of target cells, inducing signaling via conventional cis-presentation. IL-2 exhibits similar activity to IL-15 in vivo. Recombinant human IL-2 has been approved by the FDA for the treatment of melanoma and renal cancer. IL-2, when used in combination with adoptive T cell therapy, has shown remarkable tumor regression in a small number of patients. However, patients often suffered from severe dose-limiting side effects (see, for example, Dutcher, JP Current status of interleukin-2 therapy for metastatic renal cell carcinoma and metastatic melanoma. Oncol. Williston Park N 16, 4-10 (2002); Rosenberg, SA, Yang, JC, White, DE & Steinberg, SMDurability 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)).

[0005]

[0006] IL-15 signaling can be mediated through two mechanisms: transpresentation and cis-presentation. During transpresentation, IL-15 pre-associates with the IL15-Rα subunit in the endoplasmic reticulum (ER) and is then transported to the cell surface of antigen-presenting cells (monocytes and dendritic cells), where the IL-15 / IL15Rα complex interacts with the IL-15Rβ / γc subunit of effector cells (see, e.g., Stonier, SW & Schluns, KS, Trans-presentation: a novel mechanism regulating IL-15 delivery and responses. Immunol. Lett. 127, 85-92 (2010)). During cis-presentation, antigen-presenting cells secrete soluble IL-15 into the intercellular space surrounding effector cells. Soluble IL-15 binds to unoccupied IL-15Rα on the surface of effector cells. Subsequently, the IL-15 / IL15Rα complex binds to the IL-15Rβ / γc subunit on the same effector cell. In vivo, IL-15 functions primarily through transpresentation rather than cispresentation. Transpresentation allows for controlled local delivery of IL-15, preventing systemic elevations of IL-15 that are detrimental to lymphocyte and NK cell homeostasis (see, for example, Fehniger, TA 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)).

[0006]

[0007] Because IL-15 has the activity to promote the proliferation and survival of T cells and NK cells, various IL-15 therapeutic drugs have been developed. Since IL-15 does not regulate the number or activity of Treg cells, it is expected to cause fewer side effects compared to IL-2. Nevertheless, recombinant IL-15 (rhIL-15) has a short half-life of approximately 45 minutes (for example, Stoklasek, TA, Schluns, KS & Lefrancois, L. Combined IL-15 / IL-15Ralpha immunotherapy maximizes IL-15 activity in vivo. J. Immunol. Baltim. Md 1950 177, 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). See Pharmacother. 112, 108677 (2019), it requires 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 it is known to be difficult to obtain patient compliance. Furthermore, large fluctuations in blood IL-15 levels between doses can affect the overall efficacy of the drug.

[0007]

[0008] Protein fusion has been used to improve the pharmacokinetic properties of biological agents that normally have short half-lives, such as IL-15 (e.g., fusion proteins to domains and proteins such as Fc and HSA, as well as to other polypeptides, as shown, for example, in Strohl, WRFusion Proteins for Half-Life Extension of Biologics as a Strategy to Make Biobetters. Biodrugs 29, 215-239 (2015)). Among the other embodiments described herein are novel IL-15 fusion proteins that extend the half-life of the IL-15 protein, exhibit similar or improved activity, promote immune responses, and are useful in treating and / or preventing immunodeficiency-related disorders and diseases in subjects requiring such methods. [Overview of the Initiative]

[0008]

[0009] In one aspect, the present 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, which in some embodiments includes an Fc domain or an albumin protein sequence. In some embodiments, the fusion protein comprises a fusion of an IL-15Rα sequence and an IL-15 sequence, and this fusion may 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 the IL-15 sequence, and a second linker sequence between the Fc domain or albumin protein sequence and the IL-15Rα sequence and the IL-15 sequence. In some further embodiments, the first and second linkers may 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 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 aspects, 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.

[0009]

[0010] In any of the above aspects and embodiments, the IL-15 domain in the fusion protein can exhibit an extended serum half-life compared to recombinant or exogenous human IL-15. In some embodiments, the serum half-life can be extended by at least 9 hours.

[0010]

[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 cells (CD3+CD56+) when administered to a subject.

[0011]

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

[0012]

[0013] In any of the above-described aspects and embodiments, when the fusion protein is administered to a subject, it can increase the lymphocyte count.

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

[0013]

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

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

[0014]

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

[0018] In some aspects, the present disclosure provides a nucleic acid sequence encoding any of the fusion proteins and / or SEQ ID numbers described herein.

[0015]

[0019] In some aspects, the present disclosure provides a recombinant cell comprising a fusion protein according to the aspects and embodiments described herein. In some aspects, the present 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.

[0016]

[0020] In some embodiments, the Disclosure provides compositions comprising a fusion protein according to any aspect and embodiment of the Disclosure, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0017]

[0021] In some aspects, the Disclosure provides a kit comprising a fusion protein according to any aspect and embodiment of the Disclosure.

[0022] In some aspects, the Disclosure provides a method for inducing an immune response in a subject requiring treatment, comprising administering one or more fusion proteins according to any aspect and embodiment of the Disclosure.

[0018]

[0023] In some aspects, the Disclosure provides a method for treating cancer in a subject requiring treatment, comprising administering one or more fusion proteins according to any aspect and embodiment of the Disclosure.

[0019]

[0024] Accordingly, in some embodiments and models described herein, the IL-15 fusion proteins disclosed herein may be used to treat and / or prevent diseases or disorders in subjects. In some embodiments, the IL-15 fusion proteins can stimulate an immune response in subjects that require stimulation of an immune response. In some embodiments, the IL-15 fusion proteins can stimulate leukocyte proliferation, lymphocyte proliferation, and / or lymphocyte differentiation in subjects. In other embodiments, the IL-15 fusion proteins can treat and / or prevent immunodeficiency in subjects. Subjects that may benefit from the disclosed IL-15 fusion proteins include, but are not limited to, subjects with cancer (i.e., as cancer immunotherapy), human immunodeficiency virus, hepatitis B, hepatitis C, lymphopenia, sepsis, and subjects that have received stem cell, tissue, or organ transplants. In certain embodiments, as described herein, the IL-15 fusion proteins can increase leukocyte counts, increase lymphocyte counts, and / or increase or decrease certain T cell subpopulations in subjects. The methods disclosed herein include administering an IL-15 fusion protein as a target.

[0020]

[0025] Further aspects of this disclosure will be apparent to those skilled in the art from the following description and examples. [Brief explanation of the drawing]

[0021] [Figure 1]

[0026] This is a schematic diagram illustrating various designs of IL-15-containing protein constructs, such as those described in the exemplary embodiments of this disclosure. [Figure 2]

[0027] This figure shows an embodiment of the schematic diagram of the molecular designs of JL19001-1 and JL19001-2, which are examples of the present disclosure. The functional unit of IL-15Rα / IL-15 is fused to either the N-terminus of the HSA (JL19001-1) or the C-terminus of the HSA (JL19001-2). [Figure 3]

[0028] Figures 3A and 3B illustrate the differences in molecular glycosylation according to this disclosure. Figure 3A is a schematic diagram of JL19001-1 and JL19001-2, showing the presumed N-linked glycosylation site at the C-terminus of IL-15. Figure 3B shows the different migrations of JL19001-1 and JL19001-2 in SDS-PAGE under reducing and non-reducing conditions, where PNGaseF resolves the observed size differences (lanes enclosed in squares). [Figure 4]

[0029] Figure 4A shows the in vitro CTLL-2 cell proliferation assays of rhIL-15, JL19001-1, and JL19001-2. Figure 4B shows the in vitro M-07e cell proliferation assays of rhIL-15, JL19001-1, and JL19001-2 (rhIL-15 is used as a positive control). [Figure 5]

[0030] Figures 5A-5D are schematic models illustrating the differences in sensitivity between rhIL-15 and JL19001 in CTLL-2 cells and M-07e cells. The proposed non-restrictive model shows that IL-15 preferentially binds to the high-affinity receptor (αβγc) abundant in CTLL-2 cells (A, B), while JL19001 preferentially binds to the medium-affinity receptor (βγc) abundant in M-07e cells with the help of its fusion partner, IL-15Rα (C, D). Due to the different distribution of IL-15Rα subunits, the potency of rhIL-15 is higher in CTLL-2 cells but not in M-07e cells. Since JL19001 already contains IL-15Rα, it may not be suitable for binding to the high-affinity receptor. [Figure 6]

[0031] Figures 6A-6F are schematic diagrams of IL-15 fusion proteins in various forms. Figure 6A shows JL19001, also known as JL19001-2. In this example, HSA is fused to IL-15Rα (aa1-85) via an IgD linker, and IL-15 is fused to IL-15Rα via an RLI linker (linker protein sequence, SGGSGGGGSGGGSGGGGSLQ (SEQ ID NO: 13)). Figure 6B shows JL19001-3, also known as pJL177. This example is similar to JL19001, but differs in that IL-15Rα (aa1-85) is replaced by the IL-15Rα sushi domain (aa1-65). Figure 6C shows JL19001-4, also known as pJL359. This example is similar to JL19001, but differs in that the IgD linker is replaced with a 3×(G4S) linker. Figure 6D shows JL19001-5, also known as pJL186. This example is similar to JL19001, but differs in that the IL-15Rα domain has been removed. Figure 6E shows JL19001-6, also known as pJL185. This example is similar to JL19001, but differs in that the HSA is replaced with Fc, and this construct forms a dimer. Figure 6F shows a schematic diagram of JL19001-7, also known as pJL485. In this example, the IL-15Rα sushi domain is fused to the N-terminus of Fc, and there is no linker sequence between IL-15 and the IL-15Rα sequence. When the molecules are co-expressed, IL-15Rα has a very high (picomolecular) affinity for IL-15, so it is thought that the IL15 / IL15Rα-Fc complex can be easily assembled by cells. [Figure 7]

[0032] This figure shows the pharmacokinetic profiles after administration of rhIL-15 (150 μg / kg), JL19001 (50 μg / kg), or JL19001 (150 μg / kg). JL19001 has a longer half-life than rhIL-15; the in vivo half-life of rhIL-15 is 43.38 minutes (<1 hour). The in vivo half-lives of JL19001 are 9.7 hours (150 μg / kg) and 11.6 hours (50 μg / kg). The data were analyzed using PKSolver software. [Figure 8]

[0033] This figure shows flow cytometry surface marker staining of natural killer (NK) cells and CD8+ T cells. Since dead cells have been excluded by 7-AAD, only live cells are analyzed in the figure. [Figure 9]

[0034] This figure shows 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+). Note that CFSE levels decreased as cells divided. [Figure 10]

[0035] This figure shows the effects of IL-15 (left panel) and JL19001 (right panel) on the 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+). Note that CD69 is an NK and T cell activation marker. NK and T cells upregulate surface CD69 expression when activated. [Figure 11]

[0036] This figure shows the cell count and percentage of various cells in the spleen of mice injected with JL19001. [Figure 12]

[0037] This figure shows the cell count and percentage of various cells in the peritoneal cavity of mice injected with JL19001 (the t-test value (p-value) is shown in the figure). [Figure 13]

[0038] This is a schematic diagram of the experimental design to determine the in vivo efficacy of JL19001 and rhIL-15 (20 and 60 μg / kg of JL19001 correspond to 2.0*10⁻¹⁰ and 6.2*10⁻¹⁰ mol / kg, respectively. 12, 36, and 108 μg / kg of rhIL-15 correspond to 9.3*10⁻¹⁰, 27.9*10⁻¹⁰ and 83.7*10⁻¹⁰ mol / kg). [Figure 14]

[0039] This figure shows the total number of viable cells in the spleen and peritoneal cavity of mice injected with JL19001 or IL-15. Outliers have been excluded from the data analysis. The p-values ​​of the t-test are shown. [Figure 15]

[0040] This figure shows the percentage of various cell populations in the spleen of mice injected with JL19001 or IL-15. Outliers have been excluded from the data analysis. The p-values ​​of the t-test are shown. [Figure 16]

[0041] This figure shows the number of various cell populations in the spleen of mice after administration of JL19001 or IL-15. Outliers have been excluded from the data analysis. The p-values ​​of the t-test are shown. [Figure 17]

[0042] This figure shows the percentage of various cell populations in the peritoneal cavity of mice after administration of JL19001 or IL-15. Outliers have been excluded from the data analysis. The p-values ​​of the t-test are shown. [Figure 18]

[0043] This figure shows the cell counts of various cell populations in the peritoneal cavity of mice after administration of JL19001 or IL-15. Outliers have been excluded from the data analysis. The p-values ​​of the t-test are shown. [Modes for carrying out the invention]

[0022]

[0044] Before continuing to provide further details of this disclosure, please understand that this disclosure is not limited to any specific protein, nucleic acid, composition, or process step, and is therefore subject to change as long as it remains within the scope of the descriptions provided herein.

[0023]

[0045] As used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless otherwise clearly indicated by the context.

[0024]

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in the field to which this invention relates. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press serve as general dictionaries for many of the terms used in this invention for those skilled in the art.

[0025]

[0047] In this specification, amino acids may be represented by either the commonly known three-letter or one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides may be represented by commonly accepted one-letter codes.

[0026] A. Fusion protein

[0048] As used herein, the term “fusion protein” means a polypeptide construct produced through the fusion and expression of two or more genes encoding different polypeptides. Translation of a fusion gene yields a single polypeptide possessing the functional properties derived from each of the original polypeptides. Polypeptides may be directly fused or joined via linkers or hinges.

[0027]

[0049] This specification describes novel IL-15 fusion proteins. As used herein, the terms “interleukin-15” or “IL-15” protein mean an IL-15 polypeptide or derivative thereof having substantial amino acid sequence identity (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with mature wild-type mammalian IL-15. For example, IL-15 may mean i) a native allele variant of the IL-15 polypeptide, ii) a biologically active fragment of the IL-15 polypeptide, or iii) the amino acid sequence of a recombinant or non-recombinant polypeptide having the amino acid sequence of a biologically active variant or analog of the IL-15 polypeptide. The IL-15 of the fusion protein may be obtained from any mammalian species or from a recombinant expression system (e.g., yeast, bacteria, etc.). IL-15 may be glycosylated, partially glycosylated, or unglycosylated. In some specific embodiments, IL-15 may include an amino acid sequence or a functional fragment thereof, as described in the sequence listing herein (e.g., SEQ ID NO: 7).

[0028]

[0050] IL-15 signaling is mediated by two mechanisms: transpresentation and cis-presentation. During transpresentation, IL-15 pre-associates with the IL-15-Rα subunit in the endoplasmic reticulum (ER), and is then transported to the surface of the presenting cell, where the IL-15 / IL-15Rα complex interacts with the IL-15Rβ / γc subunit of the effector cell. During cis-presentation, the presenting cell secretes soluble IL-15 into the intercellular space surrounding the effector cell. The soluble IL-15 binds to unoccupied IL-15Rα on the surface of the effector cell. Subsequently, the IL-15 / IL-15Rα complex binds to the IL-15Rβ / γc subunit of the same effector cell. In vivo, IL-15 primarily functions through transpresentation rather than cis-presentation. Transpresentation allows for controlled local delivery of IL-15, preventing systemic elevations of IL-15 that are detrimental to lymphocyte and NK cell homeostasis.

[0029]

[0051] In various embodiments, the disclosure provides a fusion protein comprising one or both of an IL-15 sequence and / or an IL-15Rα sequence fused to a domain that extends the serum half-life (e.g., extends the serum half-life compared to unfused IL-15). In some embodiments, the fusion comprises an IL-15 sequence fused to a domain. In some embodiments, the fusion comprises an IL-15Rα sequence fused to a domain. In some preferred embodiments, the fusion protein comprises a fusion of the IL-15 sequence and the IL-15Rα sequence fused to a domain (i.e., as two separate fusions of the IL-15 sequence and the IL-15Rα sequence to a domain, or as a single fusion of a protein sequence comprising the fusion of the IL-15 sequence and the IL-15Rα sequence). Thus, in such embodiments, the fusion protein may comprise at least two linked regions, the first region comprising the fusion of the IL-15 sequence and the IL-15Rα sequence, which is fused to a second region comprising a domain that extends the serum half-life.

[0030]

[0052] In some embodiments, the serum half-life extending domain may include any molecule that, when fused to the domain, can increase the serum half-life of the IL-15 sequence or IL-15Rα sequence compared to the serum half-life of the unfused form of the IL-15 or IL-15Rα sequence. In some preferred embodiments, the domain includes albumin, an antibody Fc region, or polyethylene glycol (PEG). In some preferred embodiments, the domain includes an albumin sequence, and in yet other preferred embodiments, the albumin sequence includes a human serum albumin (HSA) sequence. In some embodiments, the domain includes SEQ ID NO: 11 or SEQ ID NO: 12.

[0031]

[0053] According to aspects and embodiments of this disclosure, the fusion protein comprises an IL-15 receptor (IL-15R) sequence (i.e., a functional protein or polypeptide fragment). In embodiments, IL-15R means an IL-15R polypeptide or derivative thereof having substantial amino acid sequence identity (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with a mature wild-type mammalian IL-15R sequence. Thus, the IL-15 receptor ("IL-15R") according to some embodiments of this disclosure comprises at least one of three subunits: IL-15Rα, IL-15Rβ, and a common gamma chain (γc). The IL-15 receptor shares the γc subunit with many other cytokine receptors, such as IL-2R, IL-4R, IL-7R, IL-9R, and IL-21R. The IL-15 receptor shares a second subunit, IL-2 / IL-15 receptor β (IL-15Rβ), with IL-2R. A third subunit, IL-15Rα, is specific to IL-15 and binds to IL-15 with picomolar affinity. In some embodiments, the fusion protein according to this disclosure comprises the IL-15R protein or a functional fragment thereof, where IL-15R comprises the IL-15Rα sequence. In some specific embodiments, IL-15Rα may comprise a sushi domain. In some other embodiments, IL-15Rα may comprise an amino acid sequence or a functional fragment thereof (e.g., SEQ ID NO: 9 or SEQ ID NO: 10) as described in the constructs and sequence listings herein.

[0032]

[0054] In one embodiment, the fusion protein comprises an IL-15Rα protein sequence, an 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 obtained from a sequence containing a human IgD hinge region. In some embodiments, the RLI linker is obtained from a sequence containing a flexible linker and IL-15 linked to the IL-15Rα sushi domain via several Fc. In some embodiments, the hinge region is covalently bound to the IL-15 protein, the RLI linker, IL-15Rα, and the albumin protein.

[0033]

[0055] As used herein, the term “human serum albumin” means an HSA polypeptide or derivative thereof having substantial amino acid sequence identity with mature wild-type human HSA and which can be used as a carrier protein at least due to its long serum half-life (e.g., up to 21 days). Other carrier proteins besides HSA have also been considered. In some embodiments, the carrier protein is a heavy-chain immunoglobulin constant domain obtained from IgA, IgD, IgE, IgG, or IgM. In some embodiments, the HSA includes SEQ ID NO: 12.

[0034]

[0056] The hinge or linker region may be any amino acid or peptide linker commonly known and used in the art to generate fusion proteins. Generally, linker or hinge sequences are used to isolate functional domains of fusion proteins to improve their expression, activity, folding, and / or stability. In some embodiments, the hinge or linker is obtained 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 of this specification, “hinge region” means a sequence comprising an amino acid sequence that shares sequence identity or similarity with all or part of an immunoglobulin hinge region sequence. Thus, “hinge region” encompasses a fragment of an immunoglobulin hinge region from which the linked polypeptide can achieve a biologically active conformation. The hinge regions in some exemplary embodiments of this disclosure share at least 70–80% sequence identity, preferably more than about 90%, with the wild-type immunoglobulin hinge region amino acid sequence. In some specific embodiments, the hinge region comprises a hinge region of or derived from human IgD or IgG. Human IgD, being the longest immunoglobulin hinge and cysteine-free, increases the flexibility of the fusion protein. In some embodiments, the linker sequence may comprise an RLI linker sequence (e.g., SEQ ID NO: 13) or another flexible linker sequence containing glycine (e.g., one or more G4S linkers (e.g., G4S, 3×(G4S) or (GGGGS)3, G8, etc.) as commonly known in the art and / or as described herein (e.g., SEQ ID NOs: 14-19).

[0035]

[0057] In some embodiments, the fusion proteins disclosed herein are characterized by one or more of the following structural and / or functional properties:

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

[0036]

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

[0037]

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

[0038]

[0061] In some embodiments, the fusion protein comprises an albumin protein, an L-15Rαsushi domain, a 3×G4S linker, an IL-15 protein, and an RLI linker. In some embodiments, HSAs (AA1-585) are fused to human IL-15Rαsushi domains (AA601-665) via 3×(G4S) linkers (AA586-600), and then fused to human IL-15 sequences (AA686-799) via RLI linkers (AA666-685).

[0039]

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

[0040]

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

[0041]

[0064] In some embodiments, human IL-15Rαsushi domains (AA1-65) are directly fused to human Fc (AA66-296).

[0065] In some embodiments, the fusion protein includes an IL-15Rαsushi domain and an Fc.

[0042]

[0066] In further embodiments, the present disclosure provides JL19001-1, JL19001-2, JL19001-3, JL19001-4, JL19001-5, JL19001-6, and JL19001-7. In some embodiments, the fusion protein includes 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.

[0043]

[0067] As those skilled in the art will understand, the sequences disclosed herein may be modified to some extent without impairing the fusion protein's ability to interact with target cell receptors, i.e., receptors such as those on T cells and NK cells. In some embodiments, the fusion protein sequence variants have the ability to stimulate cell proliferation and differentiation in vivo and / or in vitro. In some embodiments, the fusion protein sequence variants have the ability to treat, alleviate, or reduce the severity of immunodeficiency in a subject.

[0044]

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

[0045]

[0069] In some embodiments, the sequence identity between the mutant fusion protein sequence and the fusion protein sequence disclosed herein is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. “Sequence identity” means the percentage of amino acids identical to the sequence being compared.

[0046] B. Signs, conjugates, and parts

[0070] The fusion proteins disclosed herein may be conjugated to therapeutic agents, solid supports, affinity agents, or detection agents. The fusion proteins disclosed herein may be conjugated to a detectable label in which the fusion protein and / or its associated target may be detected. The label includes, but is not limited to, chromophores, fluorophores, fluorescent proteins, phosphorescent dyes, tandem dyes, particles, haptens, enzymes, and radioisotopes.

[0047]

[0071] In certain embodiments, fusion proteins are conjugated to fluorophores. The selection of fluorophores that bind to the fusion protein determines the absorption and fluorescence properties of the conjugated fusion protein. The physical properties of fluorophore labeling may include, but are not limited to, spectral properties (absorption, emission, and Stokes shift), fluorescence intensity, lifetime, polarization, and photobleaching rate, or a combination thereof. All of these physical properties can be used to distinguish one fluorophore from another, thereby enabling multiplex analysis. Other desirable properties of fluorescence labeling may include, for example, cell permeability and low toxicity when the labeling of fusion proteins is carried out in cells or organisms (e.g., living animals).

[0048]

[0072] In certain embodiments, the conjugated label may include an enzyme. Enzymes are desirable labels because, in some embodiments, they can amplify the detectable signal and improve assay sensitivity. While the enzyme itself does not produce a detectable response, it functions to degrade the substrate upon contact with a suitable substrate, so that the converted substrate produces a fluorescent, colorimetric, or chemiluminescent signal. Since one enzyme on the labeling reagent can yield multiple substrates that are converted into detectable signals, the enzyme can amplify the detectable signal. The enzyme substrate is selected to produce a preferred measurable product, such as colorimetric, fluorescent, or chemiluminescent. Such substrates are widely used in the art and are well known to those skilled in the art, and include, for example, oxidoreductases such as horseradish peroxidase and substrates such as 3,3'-diaminobenzidine (DAB), phosphatases such as acid phosphatase and alkaline phosphatase and substrates such as 5-bromo-6-chloro-3-indolyl phosphate (BCIP), glycosidases such as beta-galactosidase, beta-glucuronidase, or beta-glucosidase and substrates such as 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal), and further enzymes include hydrolases such as cholinesterase and peptidase, oxidases such as glucose oxidase and cytochrome oxidase, and reductases for which suitable substrates are known.

[0049]

[0073] Chemiluminescent enzymes and their suitable substrates are suitable for several assays. These include, but are not limited to, native and recombinant luciferases and aequorin. Chemiluminescent substrates for phosphatases, glycosidases, and oxidases are also useful, including those containing stable dioxetanes, luminols, isoluminols, and acridinium esters.

[0050]

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

[0051]

[0075] Haptens also include hormones, naturally occurring and synthetic drugs, contaminants, allergens, influencing molecules, growth factors, chemokines, cytokines, lymphokines, amino acids, peptides, chemical intermediates, nucleotides, and others.

[0052]

[0076] Chemiluminescent enzymes and their suitable substrates are suitable for several assays. These include, but are not limited to, native and recombinant luciferases and aequorin. Furthermore, chemiluminescent substrates for phosphatases, glycosidases, and oxidases, such as those containing stable dioxetanes, luminols, isoluminols, and acridinium esters, are also useful.

[0053]

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

[0054]

[0078] Haptens also include hormones, naturally occurring and synthetic drugs, contaminants, allergens, influencing molecules, growth factors, chemokines, cytokines, lymphokines, amino acids, peptides, chemical intermediates, nucleotides, and others.

[0055]

[0079] In certain embodiments, the fluorescent protein may be conjugated to a fusion protein as a label. Examples of fluorescent proteins include green fluorescent protein (GFP) and phycobiliproteins and their derivatives. Fluorescent proteins, particularly phycobiliproteins, are particularly useful for generating labeling reagents labeled with tandem dyes. These tandem dyes include a fluorescent protein and a fluorophore for obtaining a larger Stokes shift, and the emission spectrum thereof is further shifted from the wavelength of the absorption spectrum of the fluorescent protein.

[0056]

[0080] In certain embodiments, the label is a radioisotope. Examples of suitable radioactive substances include iodine ( 121 I, 123 I, 125 I, 131 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 111 In, 112 In, 113 mIn, 115 mIn,), technetium ( 99 Tc, 99 mTc), thallium ( 201 Ti), gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 135 Xe), fluorine ( 18 F), [[ID=X]] 153 SM, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, and 97 Ru, including but not limited to these.

[0057]

[0081] In some embodiments, a drug (e.g., other activators) may be conjugated to the fusion protein. For example, the fusion protein may be conjugated to a therapeutic portion or therapeutic agent, such as an immunotherapy drug. In some embodiments, the fusion protein can bind to target cells to deliver the immunotherapy drug to the cells. Immunotherapy drugs known in the art may be conjugated to the fusion protein. In certain embodiments, drugs and other molecules may be conjugated to the fusion protein via site-directed binding. Non-limiting exemplary embodiments of such additional activators may include antiviral agents, antibiotics, antifungal agents, antiparasitic agents, gamma globulins, and the like. Similarly, the fusion proteins disclosed herein may be used in combination with one or more other therapeutic interventions that may be used in the treatment of a condition such as immunodeficiency in question.

[0058] C. Recombinant cells that produce polynucleotides encoding fusion proteins and fusion proteins.

[0082] This disclosure provides a method for producing a fusion protein. In some embodiments relating to the method for producing a fusion protein, host cells may be transfected with one or more expression vectors encoding the fusion protein and cultured under suitable conditions to induce polypeptide expression. The fusion protein may be secreted from the cells and / or isolated from a cell culture medium containing the fusion protein. Alternatively, the fusion protein may be retained in the cytoplasm or membrane fraction, the cells may be collected and lysed, and then the fusion protein may be purified and isolated. The cell culture medium comprises host cells, medium, and other by-products. Any medium suitable for cell culture may be used in the production method. The fusion protein may be isolated from the cell culture medium, host cells, or both using common protein purification techniques such as ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification. In certain embodiments, the fusion protein may be produced with a domain (e.g., a His tag) to facilitate its purification.

[0059]

[0083] Recombinant nucleic acids can be produced by ligating a cloned gene or a portion thereof into a vector suitable for expression in prokaryotic cells, eukaryotic cells (yeast, birds, insects, or mammals), or both. Expression vectors for generating recombinant polypeptides include plasmids and other vectors. For example, suitable vectors include plasmids of the type pBR322, pEMBL, pEX, pBTac, and pUC for expression in prokaryotic cells such as E. coli. In certain embodiments, mammalian expression vectors contain both a prokaryotic sequence that promotes the growth of the vector within bacteria and one or more eukaryotic transcription units that are expressed in eukaryotic cells. Vectors derived from pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg are examples of mammalian expression vectors suitable for transfection into eukaryotic cells. Some of these vectors are modified with bacterial plasmid sequences, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, viral derivatives such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, derived from pREP, and p205) can be used for transient protein expression in eukaryotic cells. Various methods used for plasmid preparation and host organism transformation are well known in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells, as well as general recombination techniques, see Chapters 16 and 17 of Molecular Cloning: A Laboratory Manual, 2nd edition, edited by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press, 1989). In some cases, it is preferable to express recombinant polypeptides using baculovirus expression systems.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 pBlueBac III, which contains β-galactosidase).

[0060]

[0084] Techniques for forming fusion genes are well known. Basically, the joining of various nucleic acid fragments encoding various polypeptide sequences is carried out according to conventional techniques using blunt or staggered ends for ligation, restriction enzyme digestion to provide suitable ends, packing of adherent ends as needed, alkaline phosphatase treatment to avoid undesirable ligation, and enzymatic ligation. In another embodiment, fusion genes can be synthesized by conventional techniques such as automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using anchor primers that create a complementary overhang between two consecutive nucleic acid fragments, which can then be annealed to generate a chimeric gene sequence (see, e.g., *Current Protocols in Molecular Biology*, Ausubel et al., John Wiley & Sons, 1992).

[0061]

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

[0062]

[0086] Once an 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. Therefore, this disclosure includes host cells containing polynucleotides encoding a fusion protein, operably linked to heterologous promoters. In certain embodiments, if the fusion protein is encoded from different vectors, the vectors may be co-expressed in the host cell for complete fusion protein expression. In certain embodiments, the fusion protein is expressed from a single promoter. In certain embodiments, the fusion protein is expressed from multiple promoters. In certain embodiments, the fusion protein is encoded by a single vector. In certain embodiments, the fusion protein is encoded by multiple vectors.

[0063]

[0087] Mammalian cell lines available as hosts for fusion protein expression are well known in the art and include, but are 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., HepG2), human epithelial kidney 293 cells, and many other cell lines, including many immortalized cell lines available from the United States Cell Culture and Cell Line Preservation Center (ATCC). Various host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected so that the expressed fusion protein or any part thereof is precisely modified and processed. For this purpose, eukaryotic host cells with cellular mechanisms for appropriate processing, glycosylation, and phosphorylation of the primary transcript 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 mouse melanoma cell line that does not endogenously produce any functional immunoglobulin chains), SP20, CRL7O3O, and HsS78Bst cells.

[0064]

[0088] In certain embodiments, the fusion protein of this disclosure is stably expressed in a cell line. Stable expression can be used for long-term, high-yield production of recombinant proteins. For example, a cell line that stably expresses the fusion protein may be created. Host cells may be transformed with a appropriately engineered vector containing expression regulatory elements (e.g., promoters, enhancers, transcription termination factors, polyadenylation sites, etc.) and selection marker genes. After introducing the foreign DNA, the cells are grown in fortified medium for 1-2 days and then switched to selective medium. The selection marker in the recombinant plasmid confers resistance to selection, and cells with stably incorporated plasmids into chromosomes are grown to form a foci, which can then be cloned to expand into a cell line. Methods for generating stable cell lines in high yield are well known in the art, and reagents are generally commercially available.

[0065]

[0089] In certain embodiments, the fusion proteins of this disclosure are transiently expressed in cell lines. Transient transfection is the process by which nucleic acids introduced into a cell are not integrated into the cell's genome or chromosomal DNA, but are maintained within the cell as extrachromosomal elements, such as episomes. The transcription process of the nucleic acids in the episome remains unaffected, and proteins encoded by the nucleic acids in the episome are produced.

[0066]

[0090] Stable or transiently transfected cell lines are maintained in cell culture media and conditions well known in the art to induce the expression and production of fusion proteins. In certain embodiments, the mammalian cell culture medium is based on a commercially available medium formulation, such as DMEM or Ham's F12. In other embodiments, the cell culture medium is modified to support both increased cell growth and increased expression of biological proteins. As used herein, the terms “cell culture medium,” “culture medium,” and “medium formulation” mean a nutrient solution for the maintenance, growth, proliferation, or expansion of cells in an artificial in vitro environment outside of a multicellular organism or tissue. Cell culture media may be optimized for specific cell culture applications, including, for example, cell culture growth media formulated to promote cell growth or cell culture production media formulated to promote the production of recombinant proteins. Hereinafter, the terms “nutrient,” “ingredient,” and “component” are used interchangeably to mean the constituent elements of a cell culture medium.

[0067]

[0091] Once the fusion protein is produced, it may be purified by any method known in the art for the purification of protein complexes, for example, by chromatography (e.g., ion exchange, affinity, and size column chromatography), centrifugation, solubility difference, or other standard techniques for protein purification.

[0068]

[0092] When recombinant technology is used, the fusion protein may be produced intracellularly, in the pericellular space, or secreted directly into the culture medium. If the protein is produced intracellularly, the first step is to remove particulate debris, either from the host cell or the lysed fragment, by, for example, centrifugation or ultrafiltration. If the protein is secreted into the culture medium, the supernatant from such an expression system is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration device. Protease inhibitors such as PMSF may be included in either of the aforementioned steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of accidental contaminants.

[0069]

[0093] Compositions prepared from cells may be purified using, for example, hydroxyapatite 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 bound is most often agarose, but other matrices are also 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 protein purification techniques are also available, such as fractionation by ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography with silica, chromatography with heparin, SEPHAROSE chromatography with anion or cation exchange resin (e.g., polyaspartate column), isoelectric focusing, SDS-PAGE, and ammonium sulfate precipitation.

[0070]

[0094] Regardless of the purification method of the fusion protein, a binding assay may be performed (before and / or after purification) to confirm functional binding activity. For example, an ELISA assay such as a dual ELISA assay may be used. In some embodiments, a first binding target is coated into the wells, and binding to this target immobilizes the fusion protein. A tagged second binding target is added to the wells and detected. Only fusion proteins immobilized via binding to the first binding target and bound to the second binding target are detected.

[0071]

[0095] In some aspects, the disclosure provides recombinant cell lines expressing fusion proteins that are deposited and stored in an international depositary body authorized under the provisions of the Budapest Convention (i.e., an International Depository Authority, IDA).

[0072] D. Pharmaceutical preparations

[0096] In certain embodiments, the Disclosure provides a pharmaceutical composition. Such a pharmaceutical composition may also be a composition comprising the fusion protein disclosed herein and pharmaceutically acceptable excipients. In certain embodiments, the pharmaceutical compositions of the Disclosure are used as pharmaceuticals (i.e., in a method of treating or preventing a disease or condition in a subject requiring therapeutic or prophylactic treatment). In some embodiments, the pharmaceutical composition may be a composition comprising a nucleic acid molecule encoding the fusion protein disclosed herein.

[0073]

[0097] In certain embodiments, the fusion protein (or polynucleotide encoding the fusion protein) may be formulated as a pharmaceutical composition with a pharmaceutically acceptable carrier, excipient, or stabilizer. In certain embodiments, such a pharmaceutical composition is suitable for administration to humans, non-human mammals, or animals via one or more routes of administration using methods known in the art. The route and / or mode of administration varies depending on the desired outcome. The term "pharmaceutically acceptable carrier" means one or more non-toxic substances that do not interfere with the efficacy of the biological activity of the active ingredient. Such preparations typically contain salts, buffers, preservatives, a suitable carrier, and optionally other therapeutic agents. Such pharmaceutically acceptable preparations may also contain a suitable solid or liquid filler, diluent, or encapsulating material, which is suitable for administration to humans. Other conceivable carriers, excipients, and / or additives that may be used in the formulations described herein include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, protein excipients such as serum albumin, gelatin, and casein, and salt-forming counterions such as sodium. These and additional known pharmaceutical carriers, excipients, and / or additives suitable for use in the formulations described herein are known in the art, for example, as cited in "Remington: The Science & Practice of Pharmac," 21st edition, Lippincott Williams & Wilkins, (2005) and "Physician's Desk Reference," 60th edition, Medical Economics, Montvale, NJ (2005). A pharmaceutically acceptable carrier suitable for the mode of administration, desired or required solubility, and / or stability may be selected.

[0074]

[0098] The formulations described herein contain an activator (i.e., one or more fusion proteins as disclosed herein) in a concentration that yields a w / v suitable for the desired dose. In certain embodiments, the activator is present in the formulation at concentrations 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 embodiments, the concentration of the activator in the formulation may vary from about 0.1% to about 75% of the total weight. In certain embodiments, the concentration of the activator is in the range of 0.003 to 1.0 mole.

[0075]

[0099] When used for in vivo administration, the formulation must be sterilized. The formulation may be sterilized by various sterilization methods, such as sterile filtration and irradiation. In one embodiment, the formulation is sterilized by filtration through a pre-sterilized 0.22-micron filter. Sterile compositions for injection may be formulated according to conventional pharmaceutical practices, such as those described in Remington: The Science & Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, (2005).

[0076]

[0100] Therapeutic compositions are incorporated into the pharmaceutical formulations described herein and may be formulated for specific routes of administration, such as oral, nasal, pulmonary, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. As used herein, the terms “parenteral administration” and “administered parenterally” typically refer to forms of administration other than enteral and topical administration by injection, and include, but are not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intra-articular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0077]

[0101] The formulation may be in unit dose form and may be prepared by any known method. The actual dose level of the active ingredient in the pharmaceutical composition may be varied to obtain an amount of the active ingredient that is non-toxic to the patient and effective in achieving a desired therapeutic response for a particular patient, composition, and form of administration (e.g., "therapeutic effective dose"). The selected dose level depends on various pharmacokinetic factors such as the activity of the particular composition used, the route of administration, the time of administration, the elimination rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or substances used in combination with the particular composition used, the age, sex, weight, condition, general health, and medical history of the patient being treated, and similar factors well known in the medical field. An appropriate dose may range from about 0.0001 to about 100 mg / kg body weight or more, for example, about 0.1, 1, 10, or 50 mg / kg body weight, but about 1 to about 10 mg / kg body weight is appropriate.

[0078]

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

[0079]

[0103] In some embodiments, the present disclosure provides methods for treating or preventing immunodeficiency in subjects, including the administration of the IL-15 fusion proteins described herein to subjects requiring them.

[0080]

[0104] In some embodiments, the present disclosure provides methods for cancer immunotherapy, including the administration of the IL-15 fusion proteins described herein to subjects requiring them.

[0105] In some further embodiments of methods related to disease prevention, subjects may be at risk of developing lymphopenia or immunodeficiency. “At risk” or “high risk” means that a subject is more likely than usual to develop lymphopenia or immunodeficiency compared to the general population. In some embodiments, subjects who have had or currently have autoimmune diseases such as cancer, HIV, hepatitis B, hepatitis C, sepsis, aplastic anemia, lymphoma, hereditary immune disorders, tuberculosis, renal failure, lupus, rheumatoid arthritis, and myasthenia gravis, heart failure, or who are predisposed to or considered to be immunosuppressed, are at high risk of developing lymphopenia or immunodeficiency. Subjects at risk also include those receiving chemotherapy, radiation therapy, steroids, corticotropic hormones, and / or immunosuppressive therapy, as well as those who have received stem cell, tissue, and / or organ transplants.

[0081]

[0106] The term “immunotherapy” refers to treatments that involve stimulating or restoring a specific immune response, particularly a population of immune cells. In the context of this disclosure, terms such as “protective,” “preventive,” “prophylactic,” “preventive,” or “protective” refer to preventing the onset or exacerbation of lymphopenia or immunodeficiency in a patient. As noted above, patients with lymphopenia or immunodeficiency, immunosuppressed patients, or patients at risk of developing or becoming immunosuppressed with lymphopenia or immunodeficiency are considered candidates for immunotherapy. In some embodiments, immunotherapy according to this disclosure includes immunotherapy targeted at the treatment of cancer.

[0082]

[0107] Prophylactic administration of immunotherapy, such as prophylactic administration of a fusion protein or a composition containing a fusion protein as disclosed herein, can, in certain embodiments, protect the recipient from the development of lymphopenia or immunodeficiency. In some embodiments, prophylactic administration can reduce the progression and / or severity of the disease or disorder.

[0083]

[0108] Therapeutic administration of immunotherapy, such as therapeutic administration of a fusion protein or composition comprising a fusion protein as disclosed herein, can inhibit or mitigate cancer progression or treat or prevent immunodeficiency (for example, in lymphopenia, by preventing or restoring the loss of white blood cell count and / or lymphocyte count, and / or inhibiting, reducing, preventing, or mitigating the clinical symptoms of lymphopenia).

[0084]

[0109] As used herein, “treat,” “treating,” or “treatment” means administering the fusion proteins or compositions described herein to a subject in order to eliminate, reduce, or alleviate the clinical symptoms of immunodeficiency, or to block, inhibit, restore, or delay the progression of immunodeficiency in the subject.

[0085]

[0110] The terms “subject,” “individual,” or “patient” are interchangeable and relating to vertebrates, preferably mammals. For example, mammals in the context of this disclosure include humans, non-human primates, livestock such as dogs, cats, sheep, cattle, goats, pigs, and horses, laboratory animals such as mice, rats, rabbits, and guinea pigs, and domestic animals such as zoo animals. The term “animal” as used herein also includes humans. The term “subject” also includes patients, i.e., animals, in certain embodiments, humans with immunodeficiency or immunosuppressed patients.

[0086]

[0111] The fusion proteins and compositions described herein may be administered via conventional routes such as injection or infusion. In some embodiments, administration may be carried out, for example, orally, intravenously, intraperitoneally, intramuscularly, subcutaneously, or percutaneously.

[0087]

[0112] The fusion protein and the composition containing the fusion protein are administered in an effective dose. The "effective dose" includes the amount necessary to achieve the desired response or effect, and may be in the form of a single dose or multiple doses.

[0088]

[0113] The effective dose of the composition of this disclosure depends on the severity of immunodeficiency, the underlying cause (i.e., disease or condition), individual patient parameters such as age, physiological state, size, and weight, the duration of treatment, the type of accompanying therapy (if any), the specific route of administration, and similar factors. Therefore, the dose of the composition of this disclosure administered may depend on various combinations of such parameters. In embodiments where the initial dose administered to a patient is insufficient, higher doses, more frequent administrations, or further administrations via different / more localized routes of administration may be used.

[0089]

[0114] In the methods and compositions disclosed herein, an IL-15 fusion protein may be administered to a subject in such a manner as to prolong the serum half-life of the IL-15 protein compared to recombinant or exogenous IL-15.

[0090] F. Kit

[0115] Another aspect of the present disclosure is a kit. In one aspect, the kit comprises one of the nucleic acids, polypeptides, expression vectors, or host cell compositions or pharmaceutical compositions described above, and instructions or labels directing appropriate use or administration. Optionally, the kit may also include one or more containers and / or syringes or other devices to facilitate delivery or use. The present disclosure assumes that all or any subset of the components for performing a research assay and / or administering a therapeutically effective dose of the fusion protein may be included in the kit. Similarly, the kit may include instructions for preparing the polypeptide, for example, by culturing host cells expressing the nucleic acid encoding the fusion protein of the present disclosure under appropriate conditions. As an additional example, a kit for the therapeutic administration of the fusion protein of the present disclosure may include a solution containing the 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 and / or for reconstituting the lyophilized product.

[0091]

[0116] The disclosures of the present invention also encompass finished and packaged labeled pharmaceutical products. These products include appropriate unit dosage forms in suitable containers or vessels, such as glass vials or other sealed containers. For dosage forms suitable for parenteral administration, the active ingredient, e.g., the aforementioned fusion protein, is sterile and suitable for administration as a particulate-free solution. In certain embodiments, the formulations are suitable for intravenous administration, such as intravenous infusion into humans or animals.

[0092]

[0117] In certain embodiments, the formulations of the present disclosure are formulated as sterile liquids in single-dose vials. Exemplary containers include, but are not limited to, vials, bottles, pre-filled syringes, IV bags, and blister packs (containing one or more tablets). Optionally, such containers may be accompanied by a notice in the form prescribed by a government agency regulating the manufacture, use, or sale of a medicinal or biological product, which reflects the government agency's authorization for manufacture, use, or sale for the diagnosis and / or administration of human beings.

[0093]

[0118] Similar to pharmaceutical products, packaging materials and containers are designed to ensure the stability of the product during storage and transport. Furthermore, the disclosed product includes instructions for use or other informational materials that advise physicians, technicians, or patients on how to adequately prevent or treat the disease or disorder in question. In other words, the product includes instructions that indicate or suggest actual dosages, including but not limited to administration plans and monitoring procedures, and other monitoring information.

[0094]

[0119] A research kit may include a solution containing a fusion protein or a lyophilized preparation of the fusion protein of this disclosure, the fusion protein specifically binding to one or more targets. The fusion protein may be labeled according to methods known in the art and described herein, and may include, 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, a primary antibody used to detect the fusion protein may be included in the kit. The primary antibody may target a sequence on the fusion protein, or a label, tag, or epitope labeled on the fusion protein. The primary antibody may then be labeled for detection, or, if further amplification of the signal is desired, the primary antibody may be detected by a secondary antibody, and the secondary antibody may also be included in the kit. In some embodiments, a research kit may resemble a kit for therapeutic use, but further include a label indicating that the kit and its use are limited to research purposes only.

[0095] G. Modification and / or Operation

[0120] The disclosed fusion proteins may also be used to generate therapeutic immunoconjugates, in which the fusion proteins are conjugated with one or more therapeutic agents. For example, fusion proteins such as those described herein may be used in the generation of protein-drug conjugates. Available drugs include, but are not limited to, immune system modulators, growth factors (e.g., colony-stimulating factors (CSFs)), antivirals, antibiotics, antifungals, antiparasitic agents, and high and low molecular weight activators such as gamma globulins. [Examples]

[0096] Example 1 rational fusion protein design

[0121] Figure 1 summarizes several conventional approaches to designing IL-15 agonists, including recombinant protein mutain, PEGylated IL-15, and IL-15Rα(AA). 1~175 ) 10 The heterodimer IL-15 (hetIL-15) associated with IL-15, and IL-15 acting as a flexible linker. 11,12 This includes RLI fusions that bind to the IL-15Rαsushi domain via a linkage, fusions of single-chain antibodies (scFv) that bind to human serum albumin (HSA) to deliver IL-15 and IL-12 in a single molecule, and several Fc fusion designs. ALT-803 is a co-assembly of the IL-15 enhanced mutant N720D and IL-15Rαsushi-Fc. Hengrui's P22339 creates an additional disulfide bond between IL-15 and IL-15Rαsushi-Fc. In Xenco's heterodimeric Fc fusion design, the IL-15 and IL-15Rαsushi domains are linked to an Fc-knob and an Fc-hole, respectively. 15 It is fused with it.

[0097]

[0122] Unlike all conventional fusion designs, according to the exemplary embodiments of this disclosure, the fusion shown in the following examples uses IL-15 and IL-15Rα (e.g., AA). 31~115 The fusion comprises a sequence containing (which is further fused to human serum albumin (e.g., N-terminus and / or C-terminus), and the entire fusion is formed via one or more linker sequences (e.g., RLI, IgD, G4S, etc.), the form generally shown as "JL19001" in Figure 1.

[0098]

[0123] Briefly, two exemplary fusion molecules, named JL19001-1 and JL19001-2, were generated by creating an IL-15 / IL-15Rα fusion and then fusing this combination to either the N-terminus or C-terminus of an HSA (Figure 2). In these molecular examples, an IgD linker is used to link the HSA to IL-15 / IL-15Rα, and an RLI linker is used to link IL-15Rα to IL-15. While the IL-15, IL-15Rα, and albumin sequences may be derived from any mammalian sequence, the specific sequences used in the initial form are all human and are thought to maintain low immunogenicity. The full-length protein contains 862 amino acids and has a calculated molecular weight of 96.16 kDa (see, for example, Figure 2 and the sequence listing).

[0099]

[0124] The exemplary molecular designs JL19001-1 and JL19001-2 demonstrated that IL-15Rα and IL-15 can be provided as functional units by covalently binding them via a flexible linker. Furthermore, fusion of the functional IL-15Rα / IL-15 units to a carrier HSA protein is effective in extending their half-life in vivo. As shown below, HSA fusion not only extends the plasma half-life of IL-15Rα / IL-15 and enhances relevant activity in animal studies, but also appears to be more effective in vivo compared to IL-15 controls.

[0100] Example 2 Production of JL19001

[0125] JL19001 (i.e., SEQ ID NOs: 1 and 2) was produced in a bioreactor. Briefly, a JL19001 working cell bank was expanded every 3–4 days in seed culture medium (CD FortiCHO, MSX 25 μM, and 1% ACA). After three passages, the seed cultures were transferred to production medium (CD FortiCHO and 1% ACA) and inoculated into a bioreactor. The cells were expanded in the bioreactor for 14 days using CD FortiCHO medium supplemented with 1% ACA to produce the JL19001 drug.

[0101]

[0126] JL19001 was purified using a four-column process (Octyl, Capto Blue, Capto adhere, Capto Q). Briefly, the culture supernatant was collected and clarified in a deep filtration step to remove cells and cellular residue. The filtrate was then concentrated, its conductivity adjusted, and clarified in another deep filtration step coupled with absolute filtration through a 0.2 μm filter. JL19001 column purification was initiated with Cytiva Octyl Sepharose 4 Fast Flow or Capto Octyl resin as a capture step to partially remove low molecular weight (LMW) species. Next, the JL19001-containing fraction from Octyl FF chromatography was treated with Cytiva Capto Blue resin as an intermediate purification step to remove host cell proteins (HCPs). The JL19001-containing fraction from Blue chromatography was pooled, dialyzed to phosphate buffer, and incubated at pH 3.5 for 45 minutes to 1 hour to inactivate the virus. After virus inactivation, residual HSA-related LMW impurities are removed by mixed-mode column chromatography using Cytiva Capto adhere ImpRes. The JL19001-containing fraction from Capto adhere ImpRes chromatography is purified using an anion exchange column, Cytiva Capto Q, to remove JL19001-related high molecular weight (HMW) species, as well as a mechanism for removing nucleic acids, endotoxins, and viruses. The purified product is concentrated and formulated as a bulk drug at 5 mg / ml in formulation buffer.

[0102] Example 3 Features and characteristics evaluation of JL19001-1 and JL19001-2

[0127] Comparative SDS-PAGE analysis of JL19001-1 and JL19001-2 suggests that JL19001-1 may be modified by N-linked glycosylation. The migration of JL19001-1 and JL19001-2 on reduced and unreduced SDS-PAGE is shown in Figure 3B, demonstrating that N-glycosidase F treatment of JL19001-1 effectively reduces its size to that of JL19001-2. Glycosylation of the C-terminal (Asn-Thr-Ser) sequence of IL-15 may be more efficient in the internal JL19001-1 than in the C-terminal JL19001-2 (Figure 3A).

[0103] Example 4 Cell proliferation activity of JL19001-1 and JL19001-2

[0128] After incubating JL19001-1 and JL19001-2 for 3 days, CTLL-2 cell proliferation was measured using the MTS kit (Promega). As shown in Figure 4A, JL19001-1 appeared to have approximately 5–10 times lower potency in stimulating CTLL-2 proliferation than JL19001-2. While we do not wish to be bound by theory, enhanced glycosylation of JL19001-1 may reduce its activity in stimulating CTLL-2 cell proliferation. Considering this activity, further studies detailed below were conducted using the JL19001 molecule, identified as JL19001-2, which is simply referred to as "JL19001" below and in the figures showing the data obtained from those studies.

[0104] Example 5 The effects of JL19001 and rhIL-15 on stimulating the proliferation of CTLL-2 and M-07e cells.

[0129] CTLL-2 cells and M-07e cells were used to determine cytokine-dependent growth rates and are capable of responding to recombinant human (rhIL-15). As shown below, JL19001 exhibits different activity against these two cell lines. Table 1 shows the EC2 values ​​of rhIL-15 and JL19001 molecules. 50This will be explained in detail. When CTLL-2 cells are used as target cells, rhIL-15 EC 50 It was 3.9 pM, while the EC of JL19001 50 The EC of rhIL-15 is 3636 pM. When M-07e cells are used as target cells, 50 It is 148 pM, EC of JL19001 50 The concentration is 768 pM (Figure 4A). The data show that JL19001 is less potent than rhIL-15 (i.e., approximately 1000 times (CTLL-2) and approximately 5 times (M-07e) less potent), but the relative potency of the molecules in M-07e cells is noteworthy. Compared to CTLL-2 cells, rhIL-15 is 35 times less potent, while JL19001 is 5 times more potent.

[0105]

[0130] Figure 5 shows a model of the different sensitivities of rhIL-15 and JL19001 in these two cell lines. In summary, CTLL-2 cells may express more high-affinity receptors (αβγc) to which rhIL-15 may preferentially bind compared to M-07e cells, while JL19001 containing pre-associated IL-15Rα may preferentially bind to the medium-affinity receptor (βγc) which is abundant in M-07e cells. As a result, CTLL-2 cells show high efficacy against rhIL-15 but not against JL19001. In contrast, JL19001 is more responsive in M-07e cells, likely due to the higher amount of the medium-affinity receptor (βγc) as mentioned above.

[0106] Example 6 Other forms of IL-15 fusion proteins

[0131] Several constructs are prepared to analyze whether various domains / components of the JL19001 fusion protein may affect all aspects of JL19001 activity (Figure 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 to as pJL359) is constructed by replacing the IgD linker between HSA and IL-15Rα with a 3×(G4S) linker. Construct JL19001-5 (also referred to as pJL186) is constructed by removing the entire IL-15Rα to assess whether HSA adversely affects IL-15. Furthermore, two Fc fusion proteins, construct JL19001-6 (also known as pJL185) and construct JL19001-7 (also known as pJL485), were prepared. In construct JL19001-6 (pJL185), IL-15Rα / IL-15 is fused to the C-terminus of Fc to maintain the same configuration as the functional unit of JL19001. In construct JL19001-7 (pJL485), only IL-15Rα is fused to the N-terminus of Fc, but IL-15 is expressed separately and provided as a free (i.e., unfused) molecule. In particular, unlike the JL19001 fusion construct, the IL-15 in the Fc fusion is dimerized. Table 1 provides sequence information for several exemplary fusion proteins (i.e., "JL19001-X") and exemplary sequences of various fusion protein domains described herein.

[0107] [Table 1-1]

[0108] [Table 1-2]

[0109] [Table 1-3]

[0110] [Table 1-4]

[0111]

[0132] Table 2 compares the activity of these constructs.

[0112] [Table 2]

[0113]

[0133] As shown in Table 2, JL19001-3 (also known as pJL177) exhibits similar potency to JL19001 in both CTLL-2 and M-07e proliferation assays. Therefore, it is unlikely that the additional 20 amino acids downstream of the sushi domain are the cause of the weakened potency of JL19001. Replacing the IgD linker of JL19001 with a 3×(G4S) linker makes JL19001-4 (also known as pJL359) unstable and reduces its titer (data not shown). In CTLL-2 proliferation assays, linker replacement did not improve potency. Fc fusion appears to improve CTLL-2 stimulation potency by 2 to 5 times compared to HSA fusion. The improvement in Fc may be due to the dimer form of the fusion protein rather than the fusion partner itself. Significant improvements in potency are observed when IL-15 is expressed as an HSA fusion (pJL186) or as a pre-associated Fc fusion (JL19001-7 / pJL485) without covalent binding to IL-15Rα. JL19001-5 (pJL186) appears to have lost reliable expression, but its potency seems to be improved 23-fold. The most dramatic improvement in potency is observed with JL19001-7, whose potency is improved more than 200-fold to a level close to that of rhIL-15. These results suggest that covalent binding between IL-15 and IL-15Rα is the main reason why JL19001 appears to have significantly reduced activity in the CTLL-2 proliferation assay. These data suggest that JL19001 has lower in vitro efficacy compared to rhIL-15, but additional data suggest that JL19001 has higher in vivo efficacy than rhIL-15.

[0114] Example 7 Pharmacokinetic studies of JL19001

[0134] Figure 7 shows the pharmacokinetic (PK) profiles of two doses (50 μg / kg and 150 μg / kg) of JL19001 after intravenous administration to mice. Recombinant human IL-15 (rhIL-15) was administered as a control at 150 μg / kg. Blood samples were obtained by post-orbital blood collection at 5 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 72 hours, 96 hours, 120 hours, and 192 hours after injection. Blood samples were centrifuged at 4°C for 15 minutes (5000 rpm) to obtain serum. PK values ​​were calculated using PKSolver software and are shown in Figure 7.

[0115]

[0135] At 150 μg / kg, the in vivo half-life of rhIL-15 is 43.38 minutes (<1 hour). The in vivo half-lives of JL19001 are 9.7 hours (150 μg / kg) and 11.6 hours (50 μg / kg). These data indicate that the IL-15 fusion protein has a longer half-life compared to the control rhIL-15.

[0116] Example 8 Effects on T cells and NK cells

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

[0117]

[0137] Cell proliferation is measured by dilution of the CFSE signal. An example of surface marker staining for NK cells and CD8+ T cells is shown in Figure 8.

[0138] JL19001 and IL-15 are effective against the proliferation of primary NK and CD8+ T cells. Dilution of CFSE is observed in NK, NKT, and CD8+ T cell populations, but decreases in CD8-(mainly CD4+) T cell populations, as shown in Figure 9.

[0118]

[0139] JL19001 and IL-15 have an effect on the activation of primary NK and CD8+ T cells. As shown in Figure 10, in NKT and NK cells, upregulation of the T cell activation marker CD69 was observed as the concentrations of IL-15 and JL19001 increased, and this was also observed to a lesser degree in CD8+ T cells. CD69 expression was decreased in CD8- T cells.

[0119] Example 9 Delivery of JL19001 to the blood via IP injection

[0140] Serum levels of JL19001 can be measured after intraperitoneal (ip) injection, as shown in Table 3. Mice are administered JL19001 daily at a dose of 1250 μg / kg via ip. On days 1 and 4, blood samples were collected 30 minutes after ip injection of JL19001, serum was prepared, and the concentration of JL19001 in the serum samples was determined using an IL-15 ELISA kit. JL19001 was found to be distributed into the bloodstream quite rapidly. For example, JL19001 was detected in the blood of mice at concentrations of 175 ng / mL to 1520 ng / mL just 30 minutes after ip injection. The concentration of JL19001 in the blood was maintained at approximately 3000 ng / mL on day 4.

[0120] [Table 3]

[0121] Example 10 In the mouse spleen and peritoneal cavity, JL19001 activity increased NK cells and T cells.

[0141] The efficacy of JL19001 in vivo was evaluated using Balb / c mice. Mice were administered JL19001 by intravenous injection at daily doses of 20 μg / kg, 60 μg / kg, 180 μg / kg, and 540 μg / kg for 7 days. After administration of various concentrations of JL19001 for various durations, the body weight of the mice was measured. The results are shown in Table 4.

[0122] [Table 4]

[0123]

[0142] Mice tolerated JL19001 treatment up to 180 μg / kg. However, when JL19001 was administered at 540 μg / kg, the mice died 4 days after treatment.

[0143] After 7 days of treatment, the surviving mice were euthanized, and the percentages and numbers of CD3+ T cells, CD4+ T cells, CD8+ T cells, and NK cells (CD3-CD335+ cells) in the spleen were determined as shown in Figure 11.

[0124]

[0144] Figure 12 shows 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 the peritoneal cavity into the blood.

[0125]

[0145] As shown in Figure 14, to compare the in vivo activity of JL19001 and rhIL-15, mice were treated with 20 μg / kg and 60 μg / kg of JL19001, or 12 μg / kg, 36 μg / kg, and 108 μg / kg of rhIL-15, and then the total number of spleen cells and peritoneal cells was measured.

[0126]

[0146] As shown in Figure 15, to compare the in vivo activity of JL19001 and rhIL-15, mice were treated with 20 μg / kg and 60 μg / kg of JL19001, or 12 μg / kg, 36 μg / kg, and 108 μg / kg of rhIL-15, and then different spleen cell populations were measured.

[0127]

[0147] As shown in Figure 16, to compare the in vivo activity of JL19001 and rhIL-15, mice were treated with 20 μg / kg and 60 μg / kg of JL19001, or 12 μg / kg, 36 μg / kg, and 108 μg / kg of rhIL-15, and then the number of different spleen cell populations was measured.

[0128]

[0148] As shown in Figure 17, to compare the in vivo activity of JL19001 and rhIL-15, mice were treated with 20 μg / kg and 60 μg / kg of JL19001, or 12 μg / kg, 36 μg / kg, and 108 μg / kg of rhIL-15, and then different peritoneal cell populations were measured.

[0129]

[0149] As shown in Figure 18, to compare the in vivo activity of JL19001 and rhIL-15, mice were treated with 20 μg / kg and 60 μg / kg of JL19001, or 12 μg / kg, 36 μg / kg, and 108 μg / kg of rhIL-15, and the number of different peritoneal cell populations was then measured.

[0130] Example 11 Comparison of in vivo efficacy between JL19001 and rhIL-15

[0150] Figure 13 shows a schematic diagram of the experimental design for determining the efficacy of JL19001 and rhIL-15 in vivo. JL19001 at 20 and 60 μg / kg was 2.0*10 -10 , 6.2*10 -10 This corresponds to moles / kg. rhIL-15 at 12, 36, and 108 μg / kg is 9.3*10 -10 , 27.9*10 -10 , and 83.7*10 -10 This corresponds to moles / kg. Both JL19001 and rhIL-15 increased the percentage and number of NK+ cells in the spleen (Figures 14-18). 60 μg / kg (6.2 * 10 -10The in vivo effect of JL19001 at a dose of mol / kg is 108 μg / kg (83.7 * 10 -10 The effect is similar to that of rhIL-15 (mol / kg), suggesting that when administered at the same molar dose, the in vivo activity of JL19001 is approximately 10 times higher than that of rhIL-15. Array List Structure 1 (Sequence ID 1) JL19001-1, IL15Rα-IL15-HSA (pJL137)

[0131] [ka]

[0132] Human IL-15Rα (AA1-85), RLI linkers (AA86-105), and IL-15 (AA106-219) were fused to human HSA (AA278-862) via IgD hinge sequences (AA220-277).

[0133] [hIL-15Ra]-RLI-[hIL-15]-IgD-[HSA] Structure 2 (Sequence ID 2) JL19001-2, HSA-IL15Rα-IL15 (also known as pJL138, JL19001)

[0134] [ka]

[0135] HSA (AA1~585) first fuses with human IL-15Rα (AA644~728) via IgD hinge sequences (AA586~643), and then fuses with human IL-15 sequences (AA749~862) via RLI linkers (AA729~748).

[0136] [HSA]-IgD-[hIL-15R]-RLI-[hIL-15] Structure 3 (Sequence ID 3) JL19001-3, HSA-Sushi-IL15 (pJL177) Sequence ID 3

[0137] [ka]

[0138] HSAs (AA1-585) are first fused to the human IL-15 Rαsushi domain (AA644-708) via the IgD hinge sequence (AA586-643), and then fused to the human IL-15 sequence (AA729-842) via the RLI linker (AA709-728).

[0139] [HSA]-IgD-[hIL-15Rsushi]-RLI-[hIL-15] Construct 4 (Sequence ID 4) JL19001-4, HSA-IL15Rα-IL15 (pJL359, including 3xG4S linkers instead of IgD linker)

[0140] [ka]

[0141] HSAs (AA1-585) are first fused to the human IL-15 Rαsushi domain (AA1-665) via a 3×G4S linker (AA586-600), and then fused to the human IL-15 sequence (AA686-799) via an RLI linker (AA666-685).

[0142] [HSA]-G4S(3×)-[hIL-15Rsushi]-RLI-[hIL-15] Structure 5 (Sequence ID 5) JL19001-5, HSA-IL15 (pJL186, excluding IL-15Rα)

[0143] [ka]

[0144] HSA (AA1-585) is directly fused to human IL-15 (AA644-757) via IgD hinge sequences (AA586-643). [HSA]-IgD-[hIL-15] Structure 6 (Sequence ID 6): JL19001-6, Fc-IL15Rα-IL15(pJL185)

[0145] [ka]

[0146] Human Fc (AA1~231) is first fused to human IL-15Rα (AA290~374) via IgD hinge sequences (AA232~289), and then fused to human IL-15 sequences (AA395~508) via RLI linkers (AA375~394).

[0147] [Fc]-IgD-[IL-15Ra]-RLI-[hIL-15] Construction 7: (Sequence ID 7 + Sequence ID 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 covalently linked. Because IL15Rα has picomolar affinity for IL15, they associate within the cell.

[0148] Sequence ID 7: Array of IL15 (in pJL485)

[0149] [ka]

[0150] Sequence ID 8: Sequence of IL15Rα-Fc(pJL485)

[0151] [ka]

[0152] The human IL-15Rαsushi domain (AA1-65) is directly fused to human Fc (AA66-296). [IL-15Ra sushi]-[Fc]+[hIL-15] Sequence ID 9: IL15Rαsushi domain sequence

[0153] [ka]

[0154] Sequence ID 10: IL15Rα sequence

[0155] [ka]

[0156] Sequence ID 11: Fc array

[0157] [ka]

[0158] Sequence ID 12: HSA sequence

[0159] [ka]

[0160] Sequence ID 13: RLI Linker

[0161] [ka]

[0162] Sequence ID 14: IgD Hinge

[0163] [ka]

[0164] Sequence ID 15: G4S(3×)

[0165] [ka]

[0166]

[0151] All features disclosed herein, including the claims, abstract, and drawings, and all steps of any disclosed method or process, can be combined in any combination, except for any combination in which at least some of such features and / or steps are mutually exclusive. Each feature disclosed herein, including the claims, abstract, and drawings, can be replaced by an alternative feature that serves the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each disclosed feature is merely an example of a general set of equivalent or similar features.

[0167]

[0152] The present invention has been described in conjunction with its detailed description, but it should be understood that the foregoing description is intended to illustrate the scope of the invention as defined by the appended claims, and not to limit the scope of the invention. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

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

2. The fusion protein according to claim 1, comprising a fusion of an IL-15Rα sequence and an IL-15 sequence.

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

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

5. The fusion protein according to any one of claims 1 to 4, wherein the IL-15Rα protein is human IL-15Rα.

6. The fusion protein according to any one of claims 1 to 5, wherein the IL-15 protein is human IL-15.

7. The fusion protein according to any one of claims 1 to 6, wherein the albumin protein is human serum albumin.

8. The fusion protein according to any one of claims 1 to 7, wherein the IgD hinge is a human IgD sequence.

9. A fusion protein according to any one of claims 1 to 8, wherein the IL-15Rα sequence includes a sushi domain.

10. A fusion protein containing the sequence of any one of the following: 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 according to any one of claims 1 to 10, wherein the serum half-life of IL-15 is prolonged in the fusion protein compared to recombinant or exogenous human IL-15.

12. The fusion protein according to claim 11, wherein the serum half-life is extended by at least 9 hours.

13. The fusion protein according to any one of claims 1 to 12, wherein when administered to a subject, the fusion protein induces the proliferation of at least one of NK cells (CD3-CD56+), CD8+ T cells (CD3+CD56-CD8+), and NKT cells (CD3+CD56+).

14. The fusion protein according to any one of claims 1 to 13, wherein when the fusion protein is administered to a target, it induces the activation of NK cells (CD3-CD56+), CD8+ T cells (CD3+CD56-CD8+), and NKT cells (CD3+CD56+).

15. A fusion protein according to any one of claims 1 to 14, wherein the fusion protein increases the number of lymphocytes when administered to a subject.

16. A fusion protein according to any one of claims 1 to 15, wherein the fusion protein increases the total number of CD8+ T cells in the spleen when administered to a subject.

17. A fusion protein according to any one of claims 1 to 16, wherein the fusion protein increases the total number and cell percentage of NK cells in the spleen when administered to a subject.

18. A fusion protein according to any one of claims 1 to 17, wherein when administered to a subject, the fusion protein increases the total number of peritoneal CD8+ T cells.

19. A fusion protein according to any one of claims 1 to 18, wherein the fusion protein increases the total number and cell percentage of peritoneal NK cells when administered to a subject.

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

21. Recombinant cells comprising a fusion protein according to any one of claims 1 to 10 or a nucleic acid sequence according to claim 20.

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

23. A kit comprising the fusion protein described in any one of claims 1 to 10.

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

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

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