Novel interleukin-15 (IL-15) fusion protein and its use

Abstract

The present disclosure provides novel and improved IL-15 fusion proteins for treating cancer and other disorders. In various embodiments, the fusion protein of the present invention has two functional domains: an IL-15 / IL-15RαSushi domain (also referred to herein as "IL-15 / IL-15RαSushi complex") and an Fc domain, each of which can take different forms and is configured so that IL-15 is fused to the C-terminus of the Fc domain and co-expressed and non-covalently complexed with IL-15RαSushi. Importantly, the fusion protein of the present invention solves several defects observed in the IL-15 therapeutic agents evaluated to date; specifically, the fusion protein shows an extended half-life of IL-15 in vivo and exhibits better preclinical activity than rIL-15 or related cytokine therapeutics.

Description

[0001] Related patent applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 689,051, filed June 22, 2018, which is incorporated herein by reference in its entirety.

[0003] background

[0004] While cancer has traditionally been treated with chemotherapy, radiation therapy, targeted therapies, and surgery, the fifth pillar of cancer treatment, immunotherapy, has emerged in the past decade and has revolutionized the war on cancer. The benchmark for immunotherapy drugs was established by the development of T cell checkpoint (CTLA-4 and PD-1 / PD-L1) inhibitors. These therapies have been shown to effectively expand and reactivate the pool of tumor-specific T cells, leading to objective response rates of up to 50% in patients with certain cancers.

[0005] Recently, interleukin-15 (IL-15), a member of the four-α-helical bundle family of cytokines, has emerged as a candidate immunomodulator for the treatment of cancer. IL-15 binds to its specific receptor IL-15Rα, which is expressed on antigen-presenting dendritic cells, monocytes, and macrophages, and transactivates IL-15Rβ and the shared cytokine receptor γ chain (γ) on responding cells, including T cells and natural killer (NK) cells. c ) to initiate signal transduction. IL-15 shows a wide range of activities and induces the differentiation and proliferation of T cells, B cells and natural killer (NK) cells. IL-15 also enhances CD8 + T cell cytolytic activity and induce persistent antigen-experienced CD8 + CD44 hi Memory T cells. IL-15 stimulates B cell differentiation and immunoglobulin synthesis, and induces dendritic cell maturation. IL-15 does not stimulate immunosuppressive regulatory T cells (Treg). Therefore, it is assumed that increasing IL-15 activity can enhance innate immunity and adaptive immunity and fight tumors, making IL-15 a promising agent for anticancer therapy (Steel et al., Trends in Pharmacological Sciences, 33 (1): 35-41, 2012).

[0006] In the first human phase I clinical trial of intravenous infusion of recombinant human IL-15 in patients with metastatic malignant melanoma, it was reported that IL-15 could be safely administered to patients with metastatic malignant tumors and that administration of IL-15 significantly altered the homeostasis of lymphocyte subsets in the blood, with NK cells and γδ cells being most affected, followed by CD8 memory T cells (Conlon et al., J Clin Oncol., 33(1), 74-82).

[0007] Despite these recent advances in using IL-15 as a cancer immunotherapy agent to enhance immune responses, the effective use of IL-15 as a therapeutic agent remains limited. For example, IL-15 has a short half-life (<40 minutes), resulting in 1) low bioavailability, which hinders its anti-tumor effects in vivo, and 2) the need to administer high doses to achieve therapeutically relevant exposure, which leads to toxicity. Furthermore, it is known that IL-15 is poorly expressed in standard mammalian cell systems.

[0008] There remains an urgent need to provide novel therapeutic agents that are highly effective and safe for the treatment of cancer.

[0009] Disclosure of the Invention

[0010] In one aspect, the present invention provides novel and improved IL-15 fusion proteins for the treatment of cancer. In various embodiments, the fusion proteins of the present invention have two functional domains: an IL-15 / IL-15 receptor alpha (IL-15Rα) component (also referred to herein as an "IL-15 / IL-15Rα complex") and an Fc domain, each of which can take different forms. In various embodiments, the fusion protein is configured such that IL-15 is fused to the C-terminus of the Fc domain or the N-terminus of the Fc domain and is co-expressed and non-covalently complexed with the IL-15Rα domain (see Figures 1B and 1C).

[0011] In various embodiments, the IL-15 fusion protein of the present invention comprises an IL-15 / IL-15Rα complex, wherein the IL-15 domain comprises the sequence of the mature human IL-15 polypeptide as set forth in SEQ ID NO: 2 (also referred to herein as huIL-15 or IL-15 wild-type (wt)). In various embodiments, the IL-15 domain will be an IL-15 variant (or mutant) comprising a sequence derived from the mature human IL-15 polypeptide sequence as set forth in SEQ ID NO: 2. The IL-15 variants (or mutants) are referred to herein using natural amino acids, their positions in the mature sequence, and variant amino acids. For example, huIL-15 "S58D" refers to a human IL-15 comprising an S to D substitution at position 58 of SEQ ID NO: 2. In various embodiments, the IL-15 variant acts as an IL-15 super-agonist, as demonstrated by, for example, increased binding activity to IL-15Rβ and increased functional activity compared to a native IL-15 polypeptide. In various embodiments, the IL-15 variant acts as an IL-15 antagonist, as demonstrated by, for example, binding activity to IL-15Rβ but no functional activity compared to a native IL-15 polypeptide. In various embodiments, the IL-15 variant has increased binding affinity or decreased binding activity to the IL-15Rβγc receptor compared to a native IL-15 polypeptide. In various embodiments, the sequence of the IL-15 variant has at least one (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid change compared to the native IL-15 sequence. Amino acid changes can include changes in IL-15Rβ and / or IL-15Rγ C and / or IL-15Rβγ CIn some embodiments, the amino acid substitution, deletion, or insertion is performed in one or more amino acid domains of the IL-15 that interacts with the polypeptide. In various embodiments, the amino acid change is one or more amino acid substitutions or deletions at positions 30, 31, 32, 58, 62, 63, 67, 68, or 108 of SEQ ID NO: 2. In various embodiments, the amino acid change is a D to T substitution at position 30 of the mature human IL-15 sequence, a V to Y substitution at position 31, an H to E substitution at position 32, an S to D substitution at position 58, a T to D substitution at position 62, a V to F substitution at position 63, an I to V substitution at position 67, an I to F or H or D or K substitution at position 68, or a Q to A or M or S substitution at position 108, or any combination of these substitutions. In various embodiments, the amino acid change is an S to D substitution at position 58 of the mature human IL-15 sequence. In various embodiments, the IL-15 polypeptide comprises an IL-15 variant comprising the S58D mutation of SEQ ID NO: 2.

[0012] In various embodiments, the IL-15 fusion protein of the present invention comprises an IL-15 / IL-15Rα complex, wherein the IL-15Rα comprises an IL-15Rα Sushi domain (SEQ ID NO: 5) or an IL-15Rα extracellular domain (SEQ ID NO: 4) or any binding functional domain of IL-15Rα. In various embodiments, the IL-15Rα domain comprises a sequence that is at least 90% homologous to the sequence set forth in SEQ ID NO: 4. In various embodiments, the IL-15Rα domain comprises a sequence that is at least 95% homologous to the sequence set forth in SEQ ID NO: 4. In various embodiments, the IL-15Rα domain is an IL-15Rα Sushi domain that comprises a sequence that is at least 90% homologous to the sequence set forth in SEQ ID NO: 5. In various embodiments, the IL-15Rα Sushi domain comprises a sequence that is at least 95% homologous to the sequence set forth in SEQ ID NO: 5.

[0013] In various embodiments, the IL-15 fusion protein of the present invention comprises an IL-15 / IL-15RαSushi complex and at least one heterologous protein.

[0014] In various embodiments, the IL-15 fusion protein of the present invention comprises an IL-15 / IL-15Rα complex, wherein IL-15 is fused to the C-terminus or N-terminus of a heterologous protein.

[0015] In various embodiments, the IL-15 fusion protein of the present invention comprises an IL-15 / IL-15Rα-heterologous protein complex in the form of a dimer or a monomer.

[0016] In various embodiments, the heterologous protein is an Fc domain (or a functional fragment thereof). In various embodiments, the Fc domain is selected from the group consisting of: human IgG1 Fc domain, human IgG2 Fc domain, human IgG3 Fc domain, human IgG4 Fc domain, IgA Fc domain, IgD Fc domain, IgE Fc domain, IgG Fc domain and IgMFc domain or any combination thereof. In various embodiments, the Fc domain comprises amino acid changes that cause the Fc domain to have altered complement binding properties or Fc receptor binding properties. Amino acid changes that produce Fc domains with altered complement binding properties or Fc receptor binding properties are known in the art. In various embodiments, the Fc domain sequence for preparing a dimeric IL-15 / IL-15Rα complex-Fc fusion protein is the human IgG1-Fc domain sequence listed in SEQ ID NO: 6. SEQ ID NO: 6 comprises amino acid substitutions that ablate FcγR and C1q binding. In various embodiments, the heterodimeric Fc domain sequence used to prepare a monovalent IL-15 / IL-15Rα complex-Fc fusion protein is the Knob-Fc domain sequence set forth in SEQ ID NO: 7. SEQ ID NO: 7 comprises amino acid substitutions that eliminate binding to FcγRs and C1q. In various embodiments, the heterodimeric Fc domain sequence used to prepare a monovalent IL-15 / IL-15Rα complex-Fc fusion protein is the Hole-Fc domain sequence set forth in SEQ ID NO: 8. SEQ ID NO: 8 comprises amino acid substitutions that eliminate binding to FcγRs and C1q.

[0017] In various embodiments, the IL-15 fusion protein of the present invention comprises an IL-15 / IL-15Rα complex, and the heterologous protein is a full-length non-binding Ab for half-life extension, or a specific antibody or fragment for targeting, multifunctionality and half-life extension.

[0018] In various embodiments, the IL-15 fusion protein of the present invention comprises an IL-15 / IL-15Rα complex, and the heterologous protein is an Ab (monospecific or bispecific) in the form of a full-length IgG or antibody fragment and provides additive or synergistic effects with the IL-15 / IL-15RαSushi complex.

[0019] In various embodiments, the IL-15 fusion protein of the present invention comprises an IL-15 / IL-15Rα complex, and the heterologous protein provides tissue-specific targeting or tumor-specific targeting to increase the local concentration and penetration of IL-15 to the tumor microenvironment, thereby increasing tumor cell killing efficacy and reducing systemic toxicity.

[0020] In various embodiments, the heterologous protein is covalently linked to the IL-15 polypeptide (or functional fragment thereof) of the IL-15 / IL-15RαSushi complex via a polypeptide linker sequence. In various embodiments, the linker can be an artificial sequence of 5, 10, 15, 20, 30, 40 or more amino acids that is relatively free of secondary structure. In various embodiments, the linker is rich in G / S content (e.g., at least about 60%, 70%, 80%, 90% or more of the amino acids in the linker are G or S). In various embodiments, the linker is selected from the group of sequences listed in SEQ ID NOs: 9-12. Each peptide linker sequence can be selected independently.

[0021] In another aspect, the present disclosure provides pharmaceutical compositions comprising an isolated IL-15 fusion protein of the present invention admixed with a pharmaceutically acceptable carrier.

[0022] In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of the present invention. In one embodiment, the subject is a human subject. In various embodiments, the cancer is selected from pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer, or rhabdomyosarcoma.

[0023] In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, the method comprising administering a therapeutically effective amount of a pharmaceutical composition of the present invention in combination with a second therapy selected from the group consisting of: cytotoxic chemotherapy, immunotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiotherapy, stem cell transplantation, cell therapy including CAR-T cells, CAR-NK cells, iPS-induced NK cells, iPS-induced CAR-NK cells, iPS-induced T cells, iPS-induced CAR-T cells or TCR-T cells, and vaccines such as Bacille Calmette-Guerin (BCG). In various embodiments, the combination therapy may include administering to the subject a therapeutically effective amount of an immunotherapy, including, but not limited to, treatment with depleting antibodies directed against specific tumor antigens; treatment with antibody-drug conjugates; treatment with agonistic, antagonistic, or blocking antibodies directed against co-stimulatory or co-inhibitory molecules (immune checkpoints) such as CD276, CD272, CTLA-4, PD-1, PD-L1, CD40, SIRPa, CD47, OX-40, CD137, GITR, LAG3, ICOS, CD27, 4-1BB, TIM-3, B7-H4, Siglec 7, Siglec 8, Siglec 9, Siglec 15, and VISTA; treatment with bispecific T cell-engaging antibodies. treatment with chimeric antigen receptor (CAR)-T cells; treatment with CAR-NK cells; treatment with tumor infiltrating lymphocytes (TIL); treatment with adoptively transferred anti-tumor T cells (ex vivo expanded T cells and / or TCR transgenic T cells); treatment with TALL-104 cells; and treatment with immunostimulants such as Toll-like receptor (TLR) agents (such as TLR4, TLR7, TLR8, TLR9 agonists CpG and imiquimod); and treatment with vaccines such as Bacille Calmette-Guérin (BCG). Calmette-Guerine (BCG) treatment; wherein the combination therapy provides increased effector cell killing of tumor cells, i.e., there is a synergistic effect between the IL-15 / IL-15RαSushi-Fc fusion protein and the immunotherapy when co-administered.

[0024] In another aspect, the present disclosure provides a method for expanding and renewing NK cells and T cells in vitro and in vivo and for maintaining cell survival and half-life in combination with any adoptive transfer NK cell and T cell therapy or CAR-NK and CAR-T therapy.

[0025] In another aspect, the present disclosure provides a method for treating a viral infection in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of the present invention. In one embodiment, the subject is a human subject.

[0026] In another aspect, the present disclosure provides use of an IL-15 fusion protein for the preparation of a medicament for treating cancer.

[0027] In another aspect, the present disclosure provides use of an IL-15 fusion protein for the preparation of a medicament for treating a viral infection.

[0028] In another aspect, the present disclosure provides an isolated nucleic acid molecule comprising a polynucleotide encoding an IL-15 fusion protein of the present disclosure. In various embodiments, the isolated nucleic acid molecule comprises a polynucleotide described herein and further comprises a polynucleotide encoding at least one heterologous protein described herein. In various embodiments, the nucleic acid molecule further comprises a polynucleotide encoding a linker described herein. In various embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NOs: 56-63.

[0029] In another aspect, the present disclosure provides vectors comprising the nucleic acids described herein. In various embodiments, the vectors are expression vectors. In another aspect, the present disclosure provides isolated cells comprising the nucleic acids of the present disclosure. In various embodiments, the cells are host cells comprising the expression vectors of the present disclosure. In another aspect, methods for producing IL-15 fusion proteins are provided by culturing host cells under conditions that promote expression of the protein or polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 depicts several formats of IL-15 / IL-15Rα-Fc fusion proteins of the present invention. (A) IL-15 / IL-15Rα heterodimer Fc fusion format. (B) Monovalent IL-15 / IL-15Rα (non-covalent) Fc fusion format. (C) Bivalent IL-15 / IL-15Rα (non-covalent) Fc fusion format; (D) Monovalent IL-15 (non-covalent) / IL-15Rα Fc fusion protein format. (E) Bivalent IL-15 (non-covalent) / IL-15Rα Fc fusion protein format. For each fusion protein format, the IL-15 / IL-15Rα complex can be located at the C-terminus or N-terminus of the Fc domain; and the IL-15Rα can be an IL-15Rα Sushi domain or an IL-15Rα ECD.

[0032] Figure 2 depicts A) purity and B) monomer percentage of exemplary IL-15 / IL-15Rα (non-covalent)-Fc fusion proteins P-0217, P-0234, and P-0313, as determined by SDS-PAGE and SEC-HPLC, respectively. All three fusion proteins contain an IL-15 / IL-15Rα complex at the C-terminus. P-0217 is a monovalent IL-15 / IL-15Rα (non-covalent) Fc fusion, P-0234 is the dimeric counterpart of P-0217, and P-0313 shares the same fusion configuration as P-0234, but differs only in the S58D substitution in the IL-15 domain.

[0033] Figure 3 depicts SEC chromatograms of several different configurations of IL-15 / IL-15Rα-Fc fusion proteins. Unless otherwise stated, these exemplary fusion proteins all contain an IL-15 / IL-15Rα complex at the C-terminus. P-0162 is a monomeric IL-15 (alone) Fc fusion protein. P-0197 is a monovalent IL-15 / IL-15Rα (non-covalent) Fc fusion, a schematic of which is depicted in Figure 1B. P-0153 is a monomeric IL-15 / IL-15Rα fusion with a heterodimeric Fc fusion format (Figure 1A). P-0167 and P-0198 are the dimeric counterparts of P-0162 and P-0197, respectively. P-0234, P-0220, and P-0223 are all divalent IL-15 / IL-15Rα (non-covalent) Fc fusion proteins (Figure 1C). P-0220 contains the IL-15Rα ECD, and P-0234 contains the IL-15Rα Sushi+ domain. P-0223 differs from P-0234 in that the IL-15 / IL-15Rα complex is attached to the N-terminus of the Fc.

[0034] Figure 4Depicted are the effects of different IL-15 / IL-15RαFc fusion forms on binding activity to IL-15Rβ in an ELISA assay. IL-15Rα is shown to increase the IL-15Rβ binding activity of IL-15 Fc fusion proteins. P-0157 (open circles) is an N-terminal bivalent IL-15 (non-covalent) / IL-15RαSushi Fc fusion protein; P-0153 (closed circles) is a C-terminal IL-15 / IL-15RαSushi heterodimeric Fc fusion protein; P-0162 (closed triangles) is a C-terminal monovalent IL-15 Fc fusion protein without complexed IL-15RαSushi.

[0035] Figure 5 depicts the effect of IL-15Rα on the bioactivity of IL-15 Fc fusion protein. IL-15Rα was shown to enhance the bioactivity of IL-15 Fc fusion protein. The induction of CD69-positive NK cells (Figure 5A) and CD8 T cells (Figure 5B) was measured in an in vitro human PBMC FACS-based assay. P-0197 (open circles) is a C-terminal monovalent IL-15 / IL-15RαSushi (non-covalent) Fc fusion protein; P-0162 (filled circles) is a fusion protein with the same structure as P-0197 but without the complexed IL-15RαSushi.

[0036] Figure 6 depicts the effect of different configurations of IL-15 / IL-15Rα complexes on the biological activity of IL-15 Fc fusion proteins. The induction of CD69-positive NK cells (Figure 6A) and CD8+ T cells (Figure 6B) was measured in an ex vivo human PBMC FACS-based assay. P-0165 (filled circles) is a C-terminal monovalent IL-15 (non-covalent) / IL-15Rα Fc fusion protein; P-0197 (open circles) is a C-terminal monovalent IL-15 / IL-15Rα (non-covalent) Fc fusion protein; P-0153 (open triangles) is a C-terminal IL-15 / IL-15Rα heterodimer Fc fusion protein.

[0037] Figure 7 depicts the effect of linkers on the bioactivity of different forms of IL-15 / IL-15RαFc fusion proteins. The induction of CD69-positive NK cells (Figure 7A) and CD8 T cells (Figure 7B) was measured in an in vitro human PBMC FACS-based assay. P-0165 (filled circles) and P-0166 (open circles) are monovalent IL-15 (non-covalent) / IL-15RαFc fusions with a 15-amino acid rigid linker and a 10-amino acid flexible linker, respectively. P-0197 (filled triangles), P-0207 (open triangles), and P-0217 (stars) are monovalent IL-15 / IL-15Rα (non-covalent) Fc fusion proteins with a rigid linker, a 10-aa GS-rich flexible linker, and a 15-aa GS-rich flexible linker, respectively.

[0038] Figure 8 depicts the effect of N-terminal or C-terminal fusion on the activity of IL-15 / IL-15RαFc fusion proteins. After treatment, the percentage of Ki67-positive CD8 T cells was measured in an ex vivo human PBMC FACS-based assay. (A) P-0218 (filled circles) and benchmark (open circles) are C-terminal bivalent IL-15 (non-covalent) / IL-15RαFc fusion proteins and N-terminal bivalent IL-15 (non-covalent) / IL-15RαFc fusion proteins, respectively. (B) P-0234 (filled triangles) and P-0223 (open triangles) are C-terminal bivalent IL-15 / IL-15Rα (non-covalent) Fc fusion proteins and N-terminal bivalent IL-15 / IL-15Rα (non-covalent) Fc fusion proteins, respectively.

[0039] Figure 9 depicts the effect of IL-15Rα complete ECD or IL-15Rα Sushi domain on the bioactivity of IL-15 / IL-15Rα Fc fusion protein. The induction of CD69-positive NK cells was measured in an ex vivo human PBMC FACS-based assay. (A) P-0234 (filled circles) and P-0220 (open circles) are C-terminal bivalent IL-15 / IL-15Rα (non-covalent) Fc fusion proteins with IL-15Rα Sushi and IL-15Rα complete ECD, respectively. (B) P-0223 (filled circles) and P-0224 (open circles) are N-terminal bivalent IL-15 / IL-15Rα (non-covalent) Fc fusion proteins with IL-15Rα Sushi and IL-15Rα complete ECD, respectively. (C) P-0221 (filled circles) and P-0222 (open circles) are N-terminal monovalent IL-15 / IL-15Rα (non-covalent) Fc fusion proteins with IL-15Rα Sushi and IL-15Rα intact ECD, respectively.

[0040] Figure 10 depicts that the S58D substitution in the IL-15 polypeptide enhances the ability of the IL-15 fusion protein to induce STAT5 phosphorylation on CD8+ T cells (A), CD4+ T cells (B), and NK cells (C). P-0218 (open circles) and P-0314 (closed circles) are bivalent IL-15 (non-covalent) / IL-15Rα Fc fusion proteins comprising IL-15 wild-type and the S58D variant, respectively. P-0234 (open triangles) and P-0313 (closed triangles) are bivalent IL-15 / IL-15Rα (non-covalent) Fc fusion proteins comprising IL-15 wild-type and the S58D variant, respectively.

[0041] Figure 11 depicts that fusion proteins containing the IL-15(S58D) variant exhibit enhanced ability to induce Ki67 expression on CD8+ T cells (A), CD4+ T cells (B), and NK cells (C). P-0218 (open circles) and P-0314 (closed circles) are bivalent IL-15 (non-covalent) / IL-15Rα Fc fusion proteins containing IL-15 wild-type and S58D variant, respectively. P-0234 (open triangles) and P-0313 (closed triangles) are bivalent IL-15 / IL-15Rα (non-covalent) Fc fusion proteins containing IL-15 wild-type and S58D variant, respectively.

[0042] Figure 12 Describe the serum concentration of IL-15 in the mice processed with rhIL-15, benchmark and P-0234 in a 4-day repeated administration study. Female B Balb / C mice were injected with vehicle, rhIL-15 (0.03 mg / kg), benchmark (0.1 mg / kg and 0.5 mg / kg) or P-0234 (0.1 mg / kg and 0.5 mg / kg) daily ip. Terminal blood (terminal blood) was collected 1 hour after the last injection on the 4th day, and serum IL-15 levels were measured using ELISA.

[0043] Figure 13 depicts body weight (A) and % change in body weight from day 0 (B) of Balb / C mice treated with rhIL-15, baseline, and P-0234 during a 4-day repeat dosing study. Data are presented as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's post hoc test. ***p < 0.001 compared to day 0; #p < 0.05 compared to the PBS group.

[0044] Figure 14 depicts the effect of IL-15 compounds on NK cell proliferation and expansion in the peripheral blood of Balb / C mice in a 4-day repeat dosing study. After 4 daily doses, blood was collected and Ki67 was measured and NK cell phenotype analysis was performed by FACS. (A) Percentage of NK cells positive for the proliferation marker Ki67; (B) Percentage of NK cells in the CD3-negative lymphocyte population. Data are expressed as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's post hoc test. ****p < 0.0001, compared to the vehicle group, #p < 0.001 and ##p < 0.01, compared to the baseline of the same dose.

[0045] Figure 15 depicts the effects of IL-15 compounds on the proliferation, expansion, and activation of splenic NK cells in Balb / C mice during a 4-day repeat dosing study. (A) Percentage of splenic NK cells positive for the proliferation marker Ki67; (B) Total number of NK cells in the spleen; (C) Percentage of splenic NK cells positive for CD69. Data are presented as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's post hoc test. ****p < 0.0001, **p < 0.01, *p < 0.05, compared to the vehicle group.

[0046] Figure 16 depicts serum concentrations of P-0313 and benchmark following a single intraperitoneal injection in Balb / C mice. Blood was collected from mice treated with 0.3 mg / kg P-0313 or benchmark at -24 hours (pre-dose) and 1 hour, 4 hours, 24 hours, 72 hours, 144 hours, and 192 hours post-dose. (A) In-house ELISA assay detecting human Fc-IL-15 complex; (B) Commercial ELISA assay detecting human IL-15.

[0047] Figure 17 Depicted are body weights of Balb / C mice over an 8-day period following a single injection of P-0313 and baseline.

[0048] Figure 18 depicts the dose-effect and time-dependent effect of IL-15 / IL-15RαFc fusion protein on Ki67 expression of NK cells (A) and CD8+ T cells (B) after a single injection in Balb / C mice. Blood was collected at -24 hours (before administration) and 1 hour, 4 hours, 24 hours, 72 hours, 144 hours and 192 hours to perform lymphocyte phenotyping and Ki67 measurement by FACS analysis. Data are expressed as mean ± SEM. Statistical analysis was performed by two-way ANOVA test, followed by Tukey's post hoc test. ****p<0.0001, **p<0.01, *p<0.05, compared with the PBS group at their respective time points.

[0049] Figure 19 depicts the dose effect and time-dependent effect of IL-15 / IL-15RαFc fusion protein on the amplification of NK cells (A) and CD8+ T cells (B) in peripheral blood after a single injection in Balb / C mice. Blood was collected at -24 hours (before administration) and 1 hour, 4 hours, 24 hours, 72 hours, 144 hours and 192 hours to perform lymphocyte phenotyping by FACS analysis. Data are expressed as mean ± SEM. Statistical analysis was performed by two-way ANOVA test, followed by Tukey's post hoc test. ****p<0.0001, ***p<0.001, *p<0.05, compared with the PBS group at each time point.

[0050] Figure 20 depicts the inhibition of lung metastasis by P-0313 and benchmark in the mouse CT26 lung metastasis model. 1 day after injection of CT26 cells, vehicle, benchmark (0.3 mg / kg) or P-0313 (0.03 mg / kg and 0.1 mg / kg) 3×Q5D doses were given. Mice were sacrificed on day 16 and microscopy was performed to count lung metastasis nodules. (A) Representative lung photos illustrating metastatic nodules from each treatment group. (B) Lung nodule counts obtained under an optical microscope. Data are expressed as mean ± SEM. Statistical analysis was performed by one-way ANOVA test followed by Tukey's post hoc test. ****p<0.0001, *p<0.05, compared to the PBS group.

[0051] Figure 21 depicts the immunopharmacodynamic profile analysis (profiling) of the CT26 lung metastasis model after treatment with P-0313 or benchmark. 3 days after three Q5D ip injections of P-0313, benchmark, or PBS, the increase in the number of circulating A) NK cells and B) CD8+ T cells per μl of whole blood in CT26-transferred mice was determined by flow cytometry. Data are expressed as mean ± SEM. Statistical analysis was performed by one-way ANOVA test followed by Tukey's post hoc test. ****p<0.0001, ***p<0.001, **p<0.01, compared to the PBS group.

[0052] Figure 22 Depicted are spleen weights in mice treated with P-0313 or a baseline in the CT26 lung metastasis model. Spleens were harvested 3 days after three intravenous injections of IL-15 / IL-15RαFc fusion protein (q5d). Data are presented as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's post hoc test. ****p < 0.0001 compared to the PBS group.

[0053] Figure 23 depicts hepatotoxicity assessment in mice with CT26 lung metastases treated with P-0313 or a baseline. Livers were harvested three days after three Q5D treatments. A) Liver weight; B) Serum ALT level; and C) Serum AST level. Serum ALT and AST levels were determined using commercial ELISA kits. Data are presented as mean ± SEM.

[0054] Figure 24 depicts the anti-tumor efficacy of P-0313 in a subcutaneously established CT26 mouse colorectal tumor model. 5 CT26 cells. When the average tumor volume is ~70mm 3 On day 11, two injections of vehicle (PBS) or P-0313 (0.1 mg / kg or 0.05 mg / kg) were administered every 5 days. (A) Growth curves of CT26 sc tumors. (B) Change in body weight from baseline. Data are expressed as mean ± SEM. Statistical analysis was performed by two-way ANOVA followed by Tukey's post hoc test. **p < 0.0001 compared to the PBS group.

[0055] Figure 25 depicts subcutaneous CT26 tumor growth curves of individual mice receiving (A) vehicle PBS, (B) 0.05 mg / kg P-0313, or (C) 0.01 mg / kg P-0313. n=10 / group.

[0056] Figure 26 depicts the proliferation and expansion of NK cells and CD8 T cells in mice treated with P-0313 in the CT26 mouse colorectal cancer tumor model. After two Q5D treatments starting 11 days after tumor implantation, the increase in Ki67 expression (AB) and the number of circulating cells (per μl whole blood) (CD) of NK cells and CD8+ T cells was determined by flow cytometry on day 19. Data are expressed as mean ± SEM; statistical analysis was performed by one-way ANOVA test followed by Tukey's post hoc test. ****p<0.0001, *P<0.05, compared to the PBS group.

[0057] Figure 27 depicts immunophenotyping of splenic NK cells and CD8 T cells in mice bearing CT26 colorectal cancer tumors treated with P-0313. Following two Q5D treatments starting 11 days after tumor implantation, increases in the number of splenic NK cells (A) and CD8+ T cells (B) were determined by flow cytometry on day 21. Data are presented as mean ± SEM; statistical analysis was performed by one-way ANOVA followed by Tukey's post hoc test. ****p < 0.0001 compared to the PBS group.

[0058] Figure 28 depicts the antitumor efficacy of P-0313 in an unestablished CT26 colorectal tumor model. 5 Three days after implantation of 100 CT26 cells, mice were given vehicle (PBS) or P-0313 (0.1 mg / kg) every five days for a total of five injections. (A) Growth curve of CT26 sc tumors after tumor cell implantation on day 0. (B) Tumor weight on day 25. Data are expressed as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's post hoc test. ***p < 0.001, *p < 0.05, compared with the PBS group.

[0059] Figure 29 depicts spleen weight and percent body weight change in CT26 tumor-bearing mice treated with P-0313. Three days after CT26 tumor cell implantation, mice were given vehicle (PBS) or P-0313 (0.1 mg / kg) every 5 days for a total of 5 injections. (A) Spleen weight at day 25. (B) Percent change in body weight over 25 days. Data are expressed as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's post hoc test. **p < 0.01 compared to the PBS group.

[0060] Means for carrying out the present disclosure

[0061] The present disclosure provides novel and improved IL-15 fusion proteins for the treatment of cancer and other disorders. In various embodiments, the fusion proteins of the present invention have two functional domains: an IL-15 / IL-15RαSushi domain (also referred to herein as an "IL-15 / IL-15RαSushi complex") and an Fc domain, each of which can take different forms and is configured such that IL-15 is fused to the C-terminus or N-terminus of the Fc domain and is co-expressed and non-covalently complexed with IL-15Rα, IL-15RαSushi, or IL-15RαECD (see Figure 1).

[0062] The present disclosure provides IL-15 variants having amino acid substitutions, deletions, insertions, and for use as IL-15 superagonists or antagonists for the treatment of cancer and other disorders.

[0063] The present inventors understand that, in order to extend the circulating half-life of IL-15 or an IL-15 fusion protein and / or increase its biological activity, it would be highly desirable to covalently link IL-15 to the N-terminus or C-terminus of the Fc portion of a human IgG to enhance presentation of IL-15 to its signaling receptors, prevent dissociation of IL-15 from the fusion protein, and limit peak serum concentrations of free IL-15, which are typically associated with side effects of free human IL-15. The present inventors also believe that it would be highly desirable to generate a fusion protein complex comprising the IL-15Rα domain non-covalently bound to IL-15 to more naturally present IL-15 to its signaling receptors. Using the formats of the present invention, the present inventors have demonstrated that one can increase protein expression, reduce immunogenicity, and protect IL-15 from degradation. In various embodiments disclosed or described herein, it is preferred to place the IL-15-IL-15Rα complex as a dimer at the C-terminus to achieve enhanced biological activity and developability, such as increased expression and reduced aggregation. Importantly, the fusion proteins of the present invention address several deficiencies observed with IL-15 therapeutics evaluated to date; specifically, the fusion proteins were shown to extend the half-life of IL-15 in vivo and exhibited superior preclinical activity compared to rIL-15 or related cytokine therapeutics.

[0064] definition

[0065] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues. In various embodiments, "peptide", "polypeptide" and "protein" are amino acid chains whose α carbons are connected by peptide bonds. Therefore, the terminal amino acid at one end (amino terminal) of the chain has a free amino group, while the terminal amino acid at the other end (carboxyl terminal) of the chain has a free carboxyl group. As used herein, the term "amino terminal" (abbreviated as N-terminal) refers to the free α-amino group on the amino acid at the amino terminal of a peptide, or refers to the α-amino group (amino group when participating in a peptide bond) of the amino acid at any other position in the peptide. Similarly, the term "carboxyl terminal" refers to the free carboxyl group on the carboxyl terminal of a peptide, or the carboxyl group of the amino acid at any other position in the peptide. Peptide also includes substantially any polyamino acid, including but not limited to peptide mimetics (peptide mimetic), such as the amino acid connected by ether bonds rather than amide bonds.

[0066] The polypeptides of the present disclosure include polypeptides that have been modified in any manner and for any reason, for example, to: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and (5) confer or alter other physicochemical or functional properties. For example, single amino acid substitutions or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in a naturally occurring sequence (e.g., in portions of the polypeptide outside of the domains that form intermolecular contacts). A "conservative amino acid substitution" refers to a substitution of an amino acid in a polypeptide with a functionally similar amino acid. The following six groups each contain amino acids that are conservative substitutions for each other:

[0067] 1) Alanine (A), serine (S), and threonine (T)

[0068] 2) Aspartic acid (D) and glutamic acid (E)

[0069] 3) Asparagine (N) and glutamine (Q)

[0070] 4) Arginine (R) and Lysine (K)

[0071] 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V)

[0072] 6) Phenylalanine (F), tyrosine (Y) and tryptophan (W)

[0073] A "non-conservative amino acid substitution" refers to a substitution of a member of one of these classes for a member from another class. When making such changes, according to various embodiments, the hydropathic index of the amino acid can be considered. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9) and arginine (-4.5).

[0074] The importance of the hydropathic amino acid index in conferring interactive biological function on proteins is understood in the art (see, for example, Kyte et al., 1982, J. Mol. Biol. 157: 105-131). It is known that certain amino acids can be substituted with other amino acids having similar hydropathic indices or scores and still retain similar biological activity. When making changes based on the hydropathic index, in various embodiments, substitutions of amino acids whose hydropathic index is within ± 2 are included. In various embodiments, those within ± 1 are included, and in various embodiments, those within ± 0.5 are included.

[0075] It is also understood in the art that substitution of similar amino acids can be effectively made based on hydrophilicity, particularly when the resulting biologically functional protein or peptide is intended for use in immunological embodiments, as disclosed herein. In various embodiments, the greatest local average hydrophilicity of a protein (as determined by the hydrophilicity of its adjacent amino acids) correlates with its immunogenicity and antigenicity, i.e., with the biological properties of the protein.

[0076] The following hydrophilicity values ​​are assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 + -.1); glutamic acid (+3.0 + -.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 + -.1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). When making changes based on similar hydrophilicity values, in various embodiments, substitution of amino acids whose hydrophilicity values ​​are within ±2 is included, in various embodiments, those within ±1 are included, and in various embodiments, those within ±0.5 are included.

[0077] Exemplary amino acid substitutions are listed in Table 1.

[0078] Table 1

[0079]

[0080] The skilled artisan will be able to determine suitable polypeptide variants as listed herein using known techniques. In various embodiments, one skilled in the art can identify suitable regions of the molecule that can be altered without destroying activity by targeting regions that are not considered important for activity. In other embodiments, one skilled in the art can identify residues and portions of the molecule that are conserved between similar polypeptides. In additional embodiments, even regions that may be important for biological activity or structure can undergo conservative amino acid substitutions without destroying biological activity or adversely affecting polypeptide structure.

[0081] In addition, one skilled in the art can review structure-function studies that identify residues in similar polypeptides that are important for activity or structure. Based on such comparisons, one can predict the importance of amino acid residues in a polypeptide that correspond to amino acid residues in similar polypeptides that are important for activity or structure. One skilled in the art can choose to substitute chemically similar amino acids for the amino acid residues predicted to be important.

[0082] Those skilled in the art can also analyze the three-dimensional structure and the amino acid sequence relevant to this structure in similar polypeptides.In view of such information, those skilled in the art can predict the arrangement of the amino acid residues of polypeptide in terms of its three-dimensional structure.In various embodiments, those skilled in the art can choose not to carry out radical changes (radical changes) to the amino acid residues predicted on the polypeptide surface, because such residues may participate in important interactions with other molecules.In addition, those skilled in the art can produce test variants comprising single amino acid substitutions at each desired amino acid residue.Then activity assays known to those skilled in the art can be used to screen variants.These variants can be used to collect information about suitable variants.For example, if people find that the change of specific amino acid residues causes destruction, undesirably reduced or inappropriate activity, then variants with such changes can be avoided.In other words, based on the information collected from such routine experiments, those skilled in the art can easily determine to avoid further substituted amino acids therein separately or in combination with other mutations.

[0083] As used herein, the terms "polypeptide fragment" and "truncated polypeptide" refer to polypeptides having an amino-terminal deletion and / or a carboxyl-terminal deletion compared to the corresponding full-length protein. In certain embodiments, the length of the fragment can be, for example, at least 5, at least 10, at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 amino acids. In certain embodiments, the length of the fragment can also be, for example, at most 1000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 450, at most 400, at most 350, at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 25, at most 10, or at most 5 amino acids. A fragment can also comprise one or more additional amino acids at either or both ends, for example, an amino acid sequence from a different naturally occurring protein (e.g., an Fc or leucine zipper domain) or an artificial amino acid sequence (e.g., an artificial linker sequence).

[0084] As used herein, the terms "polypeptide variant," "hybrid polypeptide," and "polypeptide mutant" refer to a polypeptide comprising an amino acid sequence in which one or more amino acid residues are inserted into, deleted from, and / or substituted into an amino acid sequence relative to another polypeptide sequence. In certain embodiments, the number of amino acid residues to be inserted, deleted, or substituted can be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids in length. Hybrids of the present disclosure include fusion proteins.

[0085] A "derivative" of a polypeptide is a polypeptide that has been chemically modified, for example, by conjugation to another chemical moiety such as, for example, polyethylene glycol, albumin (eg, human serum albumin), phosphorylation, and glycosylation.

[0086] The term "% sequence identity" is used interchangeably with the term "% identity" in this article and refers to the level of amino acid sequence identity between two or more peptide sequences or the level of nucleotide sequence identity between two or more nucleotide sequences when using a sequence alignment program. For example, as used herein, 80% identity determined by a defined algorithm means the same as 80% sequence identity, and means that a given sequence is at least 80% identical to another sequence of another length. In certain embodiments, % identity is selected from, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% or greater sequence identity to a given sequence. In certain embodiments, % identity is, for example, in the range of about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95% or about 95% to about 99%.

[0087] The term "% sequence homology" is used interchangeably with the term "% homology" in this article and refers to the level of amino acid sequence homology between two or more peptide sequences or the level of nucleotide sequence homology between two or more nucleotide sequences when compared using a sequence alignment program. For example, as used herein, 80% homology determined by a defined algorithm means the same as 80% sequence homology, and thus the homologues of a given sequence have a sequence homology greater than 80% with respect to the length of the given sequence. In certain embodiments, % homology is selected from, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% or greater sequence homology to a given sequence. In certain embodiments, % homology is, for example, in the range of about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95% or about 95% to about 99%.

[0088] Exemplary computer programs that can be used to determine the identity between two sequences include, but are not limited to, a set of BLAST programs publicly available on the Internet at the NCBI website, such as BLASTN, BLASTX, and TBLASTX, BLASTP, and TBLASTN. See also Altschul et al., J. Mol. Biol. 215: 403-10, 1990 (with particular reference to the published default settings, i.e., parameters w = 4, t = 17) and Altschul et al., Nucleic Acids Res., 25: 3389-3402, 1997. When evaluating a given amino acid sequence relative to amino acid sequences in GenBank protein sequences and other public databases, the BLASTP program is typically used for sequence searching. The BLASTX program is preferably used to search for nucleic acid sequences that have been translated in all reading frames for amino acid sequences in GenBank protein sequences and other public databases. Both BLASTP and BLASTX are run using the default parameters of an open gap penalty of 11.0 and an extended gap penalty of 1.0 and using the BLOSUM-62 matrix. See above.

[0089] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA, 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the minimum total probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences occurs by chance. For example, a nucleic acid is considered similar to a reference sequence if, in a comparison of a test nucleic acid to a reference nucleic acid, the minimum total probability is, e.g., less than about 0.1, less than about 0.01, or less than about 0.001.

[0090] As used herein, the term "heterologous" refers to a non-native or non-natural combination or state, which can be achieved, for example, by replacing existing native components or states with components or states derived from another source. Similarly, expressing a protein in an organism other than the one in which it is naturally expressed constitutes a heterologous expression system and a heterologous protein.

[0091] As used herein, the term "antibody" refers to a protein comprising one or more polypeptides and having specificity for a tumor antigen or for a molecule overexpressed in a pathological state, wherein the polypeptide is substantially or partially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene. Recognized immunoglobulin genes include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as subtypes of these genes and a large number of immunoglobulin variable region genes. Light chains (LC) are classified as κ or λ. Heavy chains (HC) are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. A typical immunoglobulin (e.g., antibody) structural unit comprises a tetramer. Each tetramer comprises two identical pairs of polypeptide chains, each pair having a "light chain" (about 25 kD) and a "heavy chain" (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition.

[0092] As used herein, the term "Fc region" defines the C-terminal region of an immunoglobulin heavy chain, which can be generated by papain digestion of an intact antibody. The Fc region can be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally contains two constant domains, C H2 domain and C H3 domain, and optionally comprising a C H4 Domain. The Fc portion of an antibody mediates several important effector functions, such as cytokine induction, ADCC, phagocytosis, complement-dependent cytotoxicity (CDC), and the half-life / clearance rate of antibodies and antigen-antibody complexes (e.g., the acidic pH of the newborn FcR (FcRn) in the endosome binds to the Fc region of IgG and protects IgG from degradation, thereby contributing to the long serum half-life of IgG). Replacing amino acid residues in the Fc portion to alter antibody effector functions is known in the art (see, e.g., Winter et al., U.S. Patents 5,648,260 and 5,624,821).

[0093] "Polynucleotide" refers to a polymer comprising nucleotide units. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA") and nucleic acid analogs. Nucleic acid analogs include those comprising the following: non-naturally occurring bases, nucleotides that engage with other nucleotides with connections other than naturally occurring phosphodiester bonds, or nucleotides that comprise bases attached by connections other than phosphodiester bonds. Therefore, nucleotide analogs include, for example and without limitation, phosphorothioate, phosphorodithioate, phosphotriester, phosphoramidate, boranophosphate, methylphosphonate, chiral methylphosphonate, 2-O-methyl ribonucleotide, peptide nucleic acid (PNA), etc. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" generally refers to large polynucleotides. The term "oligonucleotide" generally refers to short polynucleotides, generally not more than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (ie, A, T, G, C), this also includes RNA sequences (ie, A, U, G, C) in which "U" replaces "T."

[0094] Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5'-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction. The direction of nucleotide addition from 5' to 3' of a nascent RNA transcript is referred to as the direction of transcription. The DNA strand having the same sequence as the mRNA is referred to as the "coding strand"; the sequence on the DNA strand having the same sequence as the mRNA transcribed from that DNA and located 5' to the 5'-end of the RNA transcript is referred to as the "upstream sequence"; the sequence on the DNA strand having the same sequence as the RNA and located 3' to the 3'-end of the coding RNA transcript is referred to as the "downstream sequence."

[0095] "Complementary" refers to the topological compatibility or matching of the interacting surfaces of two polynucleotides. Thus, the two molecules can be described as complementary, and further, the contact surface features are complementary to each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, or if the first polynucleotide can hybridize with the second polynucleotide under stringent hybridization conditions.

[0096] " vector " is the polynucleotide that can be used to another nucleic acid being connected thereto and is introduced into the cell.A type of vector is " plasmid ", and it refers to that other nucleic acid segment can be connected to linear or circular double-stranded DNA molecule wherein.Another type of vector is viral vector (for example, replication defective retrovirus, adenovirus and adeno-associated virus), and wherein other DNA segment can be introduced in the viral genome.Some vector can be autonomously replicated (for example, bacterial vector and additional mammalian vector (episomal mammalian vector) comprising bacterial replication origin) in the host cell having introduced them.Other vectors (for example, non-additional mammalian vector) are integrated into the genome of the host cell after being introduced into the host cell, and thereby replicate together with the host genome." expression vector " is a type of vector that can guide the expression of the polynucleotide of selection.

[0097] A "regulatory sequence" is a nucleic acid that affects the expression (e.g., the level, timing, or position of expression) of a nucleic acid to which it is operably linked. A regulatory sequence can, for example, exert its effect directly on the regulated nucleic acid, or exert its effect through the action of one or more other molecules (e.g., a polypeptide bound to the regulatory sequence and / or nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Additional examples of regulatory sequences are described in, for example, Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego; Calif. and Baron et al., 1995, Nucleic Acids Res. 23: 3605-06. A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g., the level, timing, or position of expression) of the nucleotide sequence.

[0098] "Host cell" is a cell that can be used to express the polynucleotides of the present disclosure. The host cell can be a prokaryotic organism, such as Escherichia coli (E. coli), or the host cell can be a eukaryotic organism, such as a unicellular eukaryotic organism (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., a human cell, monkey cell, hamster cell, rat cell, mouse cell or insect cell) or a hybridoma. Generally, a host cell is a cell that can be transformed or transfected with a nucleic acid encoding a polypeptide, and the nucleic acid can then be expressed in the host cell. The term "recombinant host cell" can be used to represent a host cell that has been transformed or transfected with a nucleic acid to be expressed. The host cell can also be a cell that comprises nucleic acid but does not express the nucleic acid at a desired level, unless a regulatory sequence is introduced into the host cell so that the regulatory sequence becomes operably connected with the nucleic acid. It should be understood that the term host cell refers not only to specific subject's cells, but also to the offspring or potential offspring of such a cell. Because certain modifications may occur in succeeding generations due to, for example, mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0099] The term "isolated molecule" (wherein the molecule is, for example, a polypeptide or polynucleotide) is a molecule that, by virtue of its origin or source of derivation, (1) is not associated with naturally associated components with which it accompanies it in its natural state, (2) is substantially free of other molecules from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized or expressed in a cellular system different from the cell from which it naturally originates would be "isolated" from its naturally associated components. A molecule may also be rendered substantially free of naturally associated components by isolation using purification techniques well known in the art. Molecular purity or homogeneity can be determined by a number of means well known in the art. For example, the purity of a polypeptide sample can be determined using polyacrylamide gel electrophoresis and staining the gel to visualize the polypeptide using techniques well known in the art. For some purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.

[0100] A protein or polypeptide is "substantially pure," "substantially homogeneous," or "substantially purified" when at least about 60% to 75% of the sample represents a single species of polypeptide. The polypeptide or protein can be monomeric or multimeric. A substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80%, or 90% w / w of the protein sample, more typically about 95% and preferably will be greater than 99% pure. Protein purity or homogeneity can be indicated by many means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualization of individual polypeptide bands by staining the gel with stains well known in the art. For some purposes, higher resolution can be provided by using HPLC or other means well known in the art for purification.

[0101] "Joint" refers to a molecule that connects two other molecules covalently or by ionic bonds, van der Waals forces or hydrogen bonds, for example, a nucleic acid molecule that hybridizes at the 5' end with a complementary sequence and at the 3' end with another complementary sequence to connect two non-complementary sequences. "Cleaving joint" refers to a joint that can be degraded or otherwise cut off to separate the two components connected by the cleavable joint. Cleaving joints are usually cleaved by enzymes, typically peptidases, proteases, nucleases, lipases, etc. Cleaving joints can also be cleaved by environmental factors, such as changes in temperature, pH, salt concentration, etc.

[0102] As used herein, the term "label" or "labeled" refers to the incorporation of another molecule into an antibody. In one embodiment, the label is a detectable marker, such as a polypeptide incorporating a radiolabeled amino acid or attached to a biotinyl moiety that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker or an enzyme activity that can be detected by optical methods or calorimetry). In another embodiment, the label or marker can be therapeutic, such as a drug conjugate or a toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H. 14 C. 15 N. 35 S. 90 Y. 99 Tc, 111 In, 125 I. 131I); fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorophores); enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; a predetermined polypeptide epitope recognized by a secondary reporter (e.g., a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag); magnetic agents, such as gadolinium chelates; toxins, such as pertussis toxin, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione), mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and analogs or homologs thereof. In some embodiments, the label is attached via spacer arms of various lengths to reduce potential steric hindrance.

[0103] The term "immunotherapy" refers to cancer treatments including, but not limited to, treatment with depleting antibodies directed against specific tumor antigens; treatment with antibody-drug conjugates; treatment with agonistic, antagonistic, or blocking antibodies directed against co-stimulatory or co-inhibitory molecules (immune checkpoints) such as CD276, CD272, CTLA-4, PD-1, PD-L1, CD40, SIRPa, CD47, OX-40, CD137, GITR, LAG3, ICOS, CD27, 4-1BB, TIM-3, B7-H4, Siglec 7, Siglec 8, Siglec 9, Siglec 15, and VISTA; treatment with bispecific T cell-engaging antibodies. treatment with chimeric antigen receptor (CAR)-T cells; treatment with CAR-NK cells; treatment with tumor infiltrating lymphocytes (TIL); treatment with adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR transgenic); treatment with TALL-104 cells; and treatment with immunostimulants such as Toll-like receptor (TLR) agonists CpG and imiquimod, as well as treatment with vaccines such as BCG, with the combination therapy providing increased effector cell killing of tumor cells, i.e., there is a synergistic effect between the IL-15 construct and the immunotherapy when co-administered.

[0104] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or composition sufficient to treat a particular disorder, condition, or disease, such as to improve, alleviate, lessen, and / or delay one or more symptoms thereof. With respect to NHL and other cancers or other unwanted cell proliferation, an effective amount includes an amount sufficient to: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow down, and preferably stop, to some extent, the infiltration of cancer cells into peripheral organs; (iv) inhibit (i.e., slow down, and preferably stop, to some extent) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumors; and / or (vii) alleviate to some extent one or more symptoms associated with cancer. An effective amount can be administered in one or more administrations.

[0105] The terms "patient," "individual," and "subject" are used interchangeably and refer to mammals, preferably humans or non-human primates, but also domestic mammals (e.g., canines or felines), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and agricultural mammals (e.g., equines, bovines, swine, ovines). In various embodiments, a patient can be a human (e.g., an adult male, an adult female, an adolescent male, an adolescent female, a male child, a female child) under the care of a physician or other health worker in a hospital, psychiatric care facility, such as an outpatient clinic, or other clinical setting. In various embodiments, a patient can be an immunocompromised patient or a patient with a weakened immune system, including but not limited to patients with primary immunodeficiency, AIDS; cancer patients and transplant patients taking certain immunosuppressive drugs; and patients with genetic diseases that affect the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency). In various embodiments, the patient has an immunogenic cancer, including but not limited to bladder cancer, lung cancer, melanoma, and other cancers reported to have high mutation rates (Lawrence et al., Nature, 499(7457):214-218, 2013).

[0106] "Pharmaceutical composition" refers to a composition suitable for pharmaceutical use in mammals. A pharmaceutical composition comprises a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. "Pharmacologically effective amount" refers to the amount of an agent that effectively produces the expected pharmacological result. "Pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier, vehicle, buffer and excipient, such as phosphate buffered saline solution, 5% dextrose aqueous solution, and emulsions, such as oil / water emulsions or water / oil emulsions, and various types of wetting agents and / or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 21st edition 2005, Mack Publishing Co, Easton. "Pharmaceutically acceptable salts" are salts of compounds that can be formulated for pharmaceutical use, including, for example, metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or salts of organic amines.

[0107] The phrases "administer" or "cause to be administered" refer to actions taken by a medical professional (e.g., a physician) or person controlling a patient's medical care to control and / or permit the administration of the agent / compound in question to a patient. Causing administration can include diagnosing and / or determining an appropriate treatment regimen, and / or prescribing a particular agent / compound to a patient. Such prescribing can include, for example, drafting a prescription form, annotating a medical record, etc. "Causing administration" is also contemplated when describing administration herein.

[0108] "Resistant or refractory cancer" refers to tumor cells or cancers that do not respond to previous anticancer therapies, including, for example, chemotherapy, surgery, radiation therapy, stem cell transplantation, and immunotherapy. Tumor cells can be resistant or refractory at the start of treatment, or they can become resistant or refractory during treatment. Refractory tumor cells include tumors that do not respond at the start of treatment, or tumors that initially respond to treatment for a short period of time but fail to respond to treatment. Refractory tumor cells also include tumors that respond to treatment with anticancer therapy but fail to respond to subsequent rounds of therapy. For the purposes of the present invention, refractory tumor cells also include tumors that appear to be suppressed by treatment with anticancer therapy but recur up to 5 years, sometimes up to 10 years or more, after treatment has stopped. Anticancer therapy can use a single chemotherapeutic agent, radiation alone, targeted therapy alone, surgery alone, or a combination thereof. For ease of description and not limitation, it should be understood that refractory tumor cells are interchangeable with resistant tumors.

[0109] The terms "treat," "treating," and "treatment" refer to a method of alleviating or eliminating a biological disorder and / or at least one of its associated symptoms. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of the symptoms of the disease, disorder, or condition. Additionally, references herein to "treating" include references to curative, palliative, and prophylactic treatments.

[0110] It should be understood that aspects and embodiments of the present disclosure described herein include "consisting of" and / or "consisting essentially of" these aspects and embodiments.

[0111] Reference herein to "about" a value or parameter includes (and describes) variations with respect to that value or parameter itself. For example, reference to "about X" includes a description of "X."

[0112] As used herein and in the appended claims, the singular forms "a," "an," "or," and "the" include plural referents unless the context clearly dictates otherwise. It should be understood that aspects and variations of the present disclosure described herein include "consisting of" and / or "consisting essentially of" these aspects and variations.

[0113] IL-15 / IL-15RαSushi complex

[0114] Interleukin-15 (IL-15) is a cytokine that was identified by two independent groups based on its ability to stimulate the proliferation of IL-2-dependent CTLL-2 T cell lines in the presence of neutralizing anti-IL-2 antibodies (Steel et al., Trends in Pharmacological Sciences, 33(1):35-41, 2012). IL-15 and interleukin-2 (IL-2) have similar biological properties in vitro and share a common receptor (R) signaling component (IL-2 / 15Rβγ c ) is consistent. However, the specificity of IL-15 and IL-2 is provided by unique private α chain receptors, which complete the IL-15Rαβγ and IL-2Rαβγ heterotrimeric high-affinity receptor complex and thereby allow different responsiveness depending on the expressed ligand and high-affinity receptor. Interestingly, both IL-15 transcripts and IL-15Rα transcripts have a much wider tissue distribution than IL-2 / IL-2Rα. In addition, more than one complex post-transcriptional regulatory mechanism strictly controls IL-15 expression. Therefore, based on complex regulation and the different modes of expression of IL-15 and IL-15Rα, the key in vivo function of this receptor / ligand pair may be different from the in vivo function of IL-2 and IL-2Rα. So far, studies examining the biology of IL-15 have identified several key non-redundant roles, such as the importance and function of IL-15 in the development of natural killer (NK) cells, NK-T cells and intestinal intraepithelial lymphocytes. The role of IL-15 during autoimmune processes, such as rheumatoid arthritis, and malignancies, such as adult T-cell leukemia, suggests that dysregulation of IL-15 may lead to deleterious effects on the host (Fehniger et al., Blood, 97:14-32, 2001).

[0115] As used herein, the terms "native IL-15" and "native interleukin-15" in the context of a protein or polypeptide refer to any naturally occurring mammalian interleukin-15 amino acid sequence, including immature or precursor forms and mature forms. Non-limiting examples of GeneBank accession numbers for amino acid sequences of native mammalian interleukin-15 from various species include NP_000576 (human, immature form), CAA62616 (human, immature form), NP_001009207 (Felis catus, immature form), AAB94536 (rattus, immature form), AAB41697 (rattus, immature form), NP_032383 (Mus musculus, immature form), AAR19080 (canine), AAB60398 (macaca mulatta, immature form), AAI00964 (human, immature form), AAH23698 (Mus musculus, immature form), and AAH18149 (human). In various embodiments of the present invention, the native IL-15 is an immature or precursor form of naturally occurring mammalian IL-15. In other embodiments, the native IL-15 is a mature form of naturally occurring mammalian IL-15. In various embodiments, the native IL-15 is a precursor form of naturally occurring human IL-15. In various embodiments, the native IL-15 is a mature form of naturally occurring human IL-15. In various embodiments, the native IL-15 protein / polypeptide is isolated or purified. In various embodiments, the IL-15 domain is derived from the amino acid sequence of the human IL-15 precursor sequence set forth in SEQ ID NO: 1:

[0116]

[0117] IL-15 receptor is a type I cytokine receptor, composed of beta (β) and gamma (γ) subunits shared with IL-2 receptors and alpha (α) subunits that bind IL-15 with high affinity. Full-length human IL-15Rα is a type 1 transmembrane protein with a signal peptide of 32 AA, an extracellular domain of 173 AA, a transmembrane domain of 21 AA, a cytoplasmic tail of 37 AA, and multiple N-connections or O-connected glycosylation sites (Anderson et al., J.Biol Chem, 270: 29862-29869, 1995). It has been previously demonstrated that the natural soluble form of the IL-15Rα chain corresponding to the complete extracellular domain of IL-15Rα acts as a high-affinity IL-15 antagonist. However, in stark contrast to this finding, it has been shown that a recombinant soluble sushi domain of IL-15Rα, which has a large binding affinity for IL-15, acts as a potent IL-15 agonist by enhancing its binding and biological effects (proliferation and prevention of apoptosis) through the IL-15Rβ / γ heterodimer, while the sushi domain does not affect IL-15 binding and the function of the IL-15Rα / β / γ tripartite membrane receptor. These results suggest that such a soluble sushi domain, if naturally produced, may be involved in the trans-presentation mechanism of IL-15 (Mortier et al., J. Biol Chem, 281(3):1612-1619, 2006).

[0118] As used herein, the terms "native IL-15Rα" and "native interleukin-15 receptor α" in the context of a protein or polypeptide refer to any naturally occurring mammalian interleukin-15 receptor α ("IL-15Rα") amino acid sequence, including immature or precursor forms and mature forms and naturally occurring isoforms. Non-limiting examples of GeneBank accession numbers for the amino acid sequences of various natural mammalian IL-15Rα include NP_002180 (human), ABK41438 (cynomolgus), NP_032384 (Mus musculus), Q60819 (Mus musculus), Q13261 (human). In various embodiments, the natural IL-15Rα is an immature form of a naturally occurring mammalian IL-15Rα polypeptide. In various embodiments, the natural IL-15Rα is a mature form of a naturally occurring mammalian IL-15Rα polypeptide. In various embodiments, the natural IL-15Rα is a form of a naturally occurring mammalian IL-15Rα polypeptide. In various embodiments, the natural IL-15Rα is a full-length form of a naturally occurring mammalian IL-15Rα polypeptide. In various embodiments, the natural IL-15Rα is an immature form of a naturally occurring human IL-15Rα polypeptide. In various embodiments, the natural IL-15Rα is a mature form of a naturally occurring human IL-15Rα polypeptide. In various embodiments, the natural IL-15Rα is a form of a naturally occurring human IL-15Rα polypeptide. In various embodiments, the natural IL-15Rα is a full-length form of a naturally occurring human IL-15Rα polypeptide. In various embodiments, the natural IL-15Rα protein or polypeptide is isolated or purified. In various embodiments, the IL-15Rα domain is derived from the amino acid sequence of the human IL-15Rα sequence set forth in SEQ ID NO: 3:

[0119]

[0120] In various embodiments, the native IL-15Rα is an intact extracellular form of a naturally occurring human IL-15Rα polypeptide. In various embodiments, the native IL-15Rα protein or polypeptide is isolated or purified. In various embodiments, the IL-15Rα extracellular domain is derived from the amino acid sequence of the human IL-15Rα sequence set forth in SEQ ID NO: 4:

[0121]

[0122] In various embodiments, the IL-15 fusion protein of the invention comprises an IL-15 / IL-15RαSushi complex, wherein the IL-15 domain comprises the amino acid sequence of the mature human IL-15 polypeptide set forth in SEQ ID NO: 2:

[0123]

[0124] And wherein the IL-15Rα Sushi domain comprises the amino acid sequence of the mature human IL-15Rα polypeptide set forth in SEQ ID NO: 5:

[0125]

[0126] In various embodiments, the IL-15 domain of the IL-15 / IL-15RαSushi complex will be an IL-15 variant (or mutant) comprising a sequence derived from the mature human IL-15 polypeptide sequence listed in SEQ ID NO: 2. Natural amino acids, the positions of natural amino acids in the mature sequence, and variant amino acids are used herein to refer to variants (or mutants) of IL-15. For example, "huIL-15S58D" refers to a human IL-15 comprising an S to D substitution at position 58 of SEQ ID NO: 2. In various embodiments, the IL-15 variant binds to the IL-15Rα polypeptide and acts as an IL-15 agonist or antagonist. In various embodiments, the IL-15 variant with agonist activity has super agonist activity. In various embodiments, the IL-15 variant can act as an IL-15 agonist or antagonist, regardless of its association with IL-15Rα. IL-15 agonists are exemplified by having comparable or increased biological activity compared to wild-type IL-15. IL-15 antagonists are exemplified by reduced biological activity compared to wild-type IL-15 or by the ability to inhibit IL-15 mediated responses. In various embodiments, IL-15 variants bind to IL-15Rβγ with increased or decreased activity. C In various embodiments, the sequence of the IL-15 variant has at least one amino acid change, such as a substitution or deletion, compared to the native IL-15 sequence, such change resulting in IL-15 agonist or antagonist activity. In various embodiments, the amino acid substitution / deletion is located at a position that binds to IL-15Rβ and / or γ CIn various embodiments, the amino acid substitution / deletion does not affect the binding to the IL-15Rα polypeptide or the ability to produce IL-15 variants. Based on the known IL-15 structure, IL-15 and homologous molecules such as IL-2 with a known structure, suitable amino acid substitutions / deletions for producing IL-15 variants can be identified by rational or random mutagenesis and functional assays or other empirical methods provided herein. In addition, suitable amino acid substitutions can be conservative or non-conservative changes and insertions of additional amino acids. In various embodiments, the IL-15 variants of the present invention comprise one or more amino acid substitutions / deletions at positions 30, 31, 32, 62, 63, 67, 68 or 108 of the mature human IL-15 listed in SEQ ID NO: 2. In various embodiments, a D30T ("D30" refers to amino acid "D" and residue position "30" in the native mature human IL-15 sequence, and "T" refers to the amino acid residue substituted at that position in the IL-15 variant), V31Y, H32E, T62D, I68F, or Q108M ​​substitution results in an IL-15 variant with antagonist activity, while an S58D substitution results in an IL-15 variant with agonist activity.

[0127] In various embodiments, the IL-15 domain of the IL-15 / IL-15RαSushi complex will be a human IL-15 variant polypeptide having a deletion from positions 111-114 (SEQ ID NO: 39). In various embodiments, the IL-15 domain of the IL-15 / IL-15RαSushi complex will be a human IL-15 variant polypeptide having a deletion from positions 109-114 (SEQ ID NO: 40). In various embodiments, the IL-15 domain of the IL-15 / IL-15RαSushi complex will be a human IL-15 variant polypeptide having a deletion from positions 108-114 (SEQ ID NO: 41). In various embodiments, the IL-15 domain of the IL-15 / IL-15RαSushi complex will be a human IL-15 variant polypeptide having a deletion from positions 105-114 (SEQ ID NO: 42). In various embodiments, the IL-15 domain of the IL-15 / IL-15RαSushi complex will be a human IL-15 variant polypeptide having a 'GS' insertion after position N95 (SEQ ID NO: 43). In various embodiments, the IL-15 domain of the IL-15 / IL-15RαSushi complex will be a human IL-15 variant polypeptide having a 'GGSGG' insertion after position N95 (SEQ ID NO: 44). In various embodiments, the IL-15 domain of the IL-15 / IL-15RαSushi complex will be a human IL-15 variant polypeptide having a 'GSSGGSGGS' insertion after position N95 (SEQ ID NO: 45).

[0128] Fc domain

[0129] The immunoglobulin of the IgG class is one of the most abundant proteins in human blood. Their circulation half-life can be as long as 21 days. It has been reported that the fusion protein of the Fc region of IgG is combined with the domain of another protein (such as various cytokines and receptors) (see, for example, Capon et al., Nature, 337:525-531, 1989; Chamow et al., Trends Biotechnol., 14:52-60, 1996); U.S. Patent Nos. 5,116,964 and 5,541,087). The prototype fusion protein is a homodimeric protein connected by the cysteine ​​residues in the hinge region of IgG Fc, producing a molecule similar to an IgG molecule, without a heavy chain variable region and CH1 domain and a light chain. The dimer nature of the fusion protein comprising the Fc domain may be advantageous in providing a higher order interaction (i.e., bivalent or bispecific binding) with other molecules. Due to structural homology, Fc fusion proteins exhibit in vivo pharmacokinetic profiles comparable to human IgG of similar isotype.

[0130] The term "Fc" refers to a molecule or sequence comprising the sequence of the non-antigen binding fragment of a complete antibody, whether in monomeric or multimeric form. The original immunoglobulin source of native Fc is preferably human origin, and can be any immunoglobulin. Native Fc consists of monomeric polypeptides, which can be linked to dimers or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between the monomer subunits of native Fc molecules depends on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2) and is in the range of 1 to 4. An example of native Fc is a disulfide-bonded dimer produced by papain digestion of IgG (see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9). As used herein, the term "native Fc" is a general term for monomers, dimers, and multimeric forms. The Fc domain contains binding sites for protein A, protein G, various Fc receptors, and complement proteins.

[0131] In various embodiments, the term "Fc variant" refers to a molecule or sequence that is modified from a native Fc but still contains a binding site for the salvage receptor FcRn. International applications WO 97 / 34631 (published September 25, 1997) and WO 96 / 32478 describe exemplary Fc variants and interactions with salvage receptors and are hereby incorporated by reference. In addition, a native Fc contains sites that can be removed because they provide structural features or biological activities that are not required for the fusion molecules of the present invention. Thus, in various embodiments, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues that affect or participate in (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity when expressed in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0132] The term "Fc domain" includes native Fc and Fc variant molecules and sequences as defined above. Like Fc variants and native Fc, the term "Fc domain" includes molecules in monomeric or multimeric form, whether digested from intact antibodies or produced by recombinant gene expression or by other means.

[0133] In one aspect, the IL-15 fusion protein of the present invention comprises an IL-15 / IL-15RαSushi complex and at least one heterologous protein attached to the IL-15 / IL-15RαSushi complex, wherein the heterologous protein is attached to the IL-15 / IL-15RαSushi complex directly or through a peptide linker sequence to form an IL-15 fusion protein. As used herein, the term "fusion protein" refers to a protein with a heterologous polypeptide attached via recombinant DNA technology. In various embodiments, the heterologous protein is an Fc domain (or a functional fragment thereof), and the resulting fusion protein is an IL-15 / IL-15RαSushi complex-Fc fusion protein. In various embodiments, the IL-15 / IL-15RαSushi complex is fused to at least one polypeptide that confers an extended half-life on the fusion molecule. Such polypeptides include IgG Fc or other polypeptides that bind to neonatal Fcγ / receptors, human serum albumin, or polypeptides that bind to proteins with extended serum half-lives, including IgG, non-IgG immunoglobulins, proteins, and non-protein agents that have increased in vivo half-life due to the presence of IgG constant domains or portions thereof that bind to FcRn, with one or more amino acid modifications that increase the affinity of the constant domain or fragment for FcRn. Such proteins and molecules with increased half-life have the advantage that less amount and / or less dosing frequency are required for the treatment, prevention, or diagnosis of such molecules (see, e.g., U.S. Patent No. 7,658,921). In various embodiments, the Fc domain is selected from the group consisting of: human IgG1 Fc domain, human IgG2 Fc domain, human IgG3 Fc domain, human IgG4 Fc domain, IgA Fc domain, IgD Fc domain, IgE Fc domain, IgG Fc domain, and IgM Fc domain, or any combination thereof. In various embodiments, the Fc domain comprises amino acid changes that result in the Fc domain having altered complement binding properties or Fc receptor binding properties.Amino acid changes that result in Fc domains having altered complement binding properties or Fc receptor binding properties are known in the art.

[0134] In various embodiments, the Fc domain sequence used to prepare the dimeric IL-15 / IL-15Rα complex-Fc fusion protein is the human IgG1-Fc domain sequence set forth in SEQ ID NO: 6:

[0135]

[0136] Wherein SEQ ID NO: 6 contains amino acid substitutions (underlined) that eliminate FcγR and C1q binding.

[0137] In various embodiments, the heterodimeric Fc domain sequence used to prepare the monovalent IL-15 / IL-15Rα complex-Fc fusion protein is the Knob-Fc domain sequence set forth in SEQ ID NO: 7:

[0138]

[0139] Wherein SEQ ID NO: 7 contains amino acid substitutions (underlined) that eliminate FcγR and C1q binding.

[0140] In various embodiments, the heterodimeric Fc domain sequence used to prepare the monovalent IL-15 / IL-15Rα complex-Fc fusion protein is the Hole-Fc domain sequence set forth in SEQ ID NO: 8:

[0141]

[0142] Wherein SEQ ID NO: 8 contains amino acid substitutions (underlined) that eliminate FcγR and C1q binding.

[0143] connector

[0144] In various embodiments, a heterologous protein (e.g., an Fc domain) is covalently linked to the IL-15 polypeptide (or functional fragment thereof) of the IL-15 / IL-15RαSushi complex via a polypeptide linker sequence. In various embodiments, the linker can be an artificial sequence of 5, 10, 15, 20, 30, 40 or more amino acids that is relatively free of secondary structure. In various embodiments, the linker is G / S rich (e.g., at least about 60%, 70%, 80%, 90% or more of the amino acids in the linker are G or S). In various embodiments, the linker is selected from the group consisting of the sequences set forth in SEQ ID NOs: 9-12 and SEQ ID NO: 47. Each peptide linker sequence can be independently selected.

[0145] Examples of novel IL-15 / IL-15RαSushi complex-Fc fusion proteins

[0146] In various embodiments, the IL-15 / IL-15RαSushi heterodimer Fc fusion protein of the present invention (hereinafter also referred to as "P-0153") comprises chain 1 (Hole-Fc-Linker-IL-15) having the amino acid sequence set forth in SEQ ID NO: 13:

[0147]

[0148] wherein the IL-15 domain sequence is underlined and the peptide linker sequence is in bold; and chain 2 (Knob-Fc-Linker-IL-15Rα-Sushi+) having the amino acid sequence set forth in SEQ ID NO: 14:

[0149]

[0150] The IL-15Rα-Sushi+ domain sequence is underlined and the peptide linker sequence is in bold.

[0151] In various embodiments, the IL-15 / IL-15RαSushi heterodimer Fc fusion protein of the present invention (hereinafter also referred to as "P-0156") comprises chain 1 (IL-15-Linker-Hole-Fc) having the amino acid sequence set forth in SEQ ID NO: 15:

[0152]

[0153] wherein the IL-15 domain sequence is underlined and the peptide linker sequence is in bold; and chain 2 (IL-15Rα-Sushi+-Linker-Knob-Fc) having the amino acid sequence set forth in SEQ ID NO: 16:

[0154]

[0155] The IL-15Rα-Sushi+ domain sequence is underlined and the peptide linker sequence is in bold.

[0156] In various embodiments, the IL-15 / IL-15RαSushi heterodimer Fc fusion protein of the present invention (hereinafter also referred to as "P-0155") comprises chain 1 (Hole Fc-Linker-IL-15) having the amino acid sequence set forth in SEQ ID NO: 18:

[0157]

[0158] wherein the IL-15 domain sequence is underlined and the peptide linker sequence is in bold; and chain 2 (Knob-Fc-Linker-IL-15Rα-Sushi+) having the amino acid sequence set forth in SEQ ID NO: 17:

[0159]

[0160] The IL-15Rα-Sushi+ domain sequence is underlined and the peptide linker sequence is in bold.

[0161] In various embodiments, the monovalent IL-15Fc fusion protein of the present invention (hereinafter also referred to as "P-0162") comprises chain 1 (Hole Fc-Linker-IL-15) having the amino acid sequence set forth in SEQ ID NO: 13 and chain 2 (Knob-Fc) having the amino acid sequence set forth in SEQ ID NO: 7.

[0162] In various embodiments, the bivalent IL-15 Fc fusion protein of the present invention (hereinafter also referred to as "P-0167") comprises chain 1 (Hole Fc-Linker-IL-15) having the amino acid sequence set forth in SEQ ID NO: 13 and chain 2 (Knob-Fc-Linker-IL-15Rα-Sushi+) having the amino acid sequence set forth in SEQ ID NO: 55:

[0163]

[0164] The IL-15 domain sequence is underlined and the peptide linker sequence is in bold.

[0165] In various embodiments, the monovalent IL-15 / IL-15RαSushi (non-covalent) Fc fusion protein of the present invention (hereinafter also referred to as "P-0197") comprises chain 1 (Hole-Fc-Linker-IL-15) having the amino acid sequence set forth in SEQ ID NO: 13, chain 2 (IL-15Rα-Sushi+) having the amino acid sequence set forth in SEQ ID NO: 5, and chain 3 (Knob-Fc) having the amino acid sequence set forth in SEQ ID NO: 7.

[0166] In various embodiments, the bivalent IL-15 / IL-15RαSushi (non-covalent) Fc fusion protein of the present invention (hereinafter also referred to as "P-0198") comprises chain 1 (Hole-Fc-Linker-IL-15) having the amino acid sequence set forth in SEQ ID NO: 13, chain 2 (IL-15Rα-Sushi+) having the amino acid sequence set forth in SEQ ID NO: 5, and chain 3 (Knob-Fc-Linker-IL-15) having the amino acid sequence set forth in SEQ ID NO: 55.

[0167] In various embodiments, the monovalent IL-15 / IL-15RαSushi (non-covalent) Fc fusion protein of the present invention (hereinafter also referred to as "P-0201") comprises chain 1 (IL-15-Linker-Hole-Fc) having the amino acid sequence set forth in SEQ ID NO: 15, chain 2 (IL-15Rα-Sushi+) having the amino acid sequence set forth in SEQ ID NO: 5, and chain 3 (Knob-Fc) having the amino acid sequence set forth in SEQ ID NO: 7.

[0168] In various embodiments, the monovalent IL-15 / IL-15RαSushi (non-covalent) Fc fusion protein of the present invention (hereinafter also referred to as "P-0207") comprises chain 1 (Hole-Fc-Linker-IL-15) having the amino acid sequence set forth in SEQ ID NO: 18, chain 2 (IL-15Rα-Sushi+) having the amino acid sequence set forth in SEQ ID NO: 5, and chain 3 (Knob-Fc) having the amino acid sequence set forth in SEQ ID NO: 7.

[0169] In various embodiments, the monovalent IL-15 / IL-15RαSushi (non-covalent) Fc fusion protein of the invention (hereinafter also referred to as "P-0217") comprises Chain 1 (Hole-Fc-Linker-IL-15) having the amino acid sequence set forth in SEQ ID NO: 54:

[0170]

[0171] Wherein the IL-15 domain sequence is underlined and the peptide linker sequence is in bold; chain 2 (IL-15Rα-Sushi+) has the amino acid sequence set forth in SEQ ID NO:5 and chain 3 (Knob-Fc) has the amino acid sequence set forth in SEQ ID NO:7.

[0172] In various embodiments, the monovalent IL-15 / IL-15Rα (non-covalent) complex-Fc fusion protein of the present invention (hereinafter also referred to as "P-0219") comprises chain 1 (Hole-Fc-Linker-IL-15) having the amino acid sequence set forth in SEQ ID NO: 54, chain 2 (IL-15Rα-ECD) having the amino acid sequence set forth in SEQ ID NO: 4, and chain 3 (Knob-Fc) having the amino acid sequence set forth in SEQ ID NO: 7.

[0173] In various embodiments, the monovalent IL-15 / IL-15RαSushi (non-covalent) Fc fusion protein of the present invention (hereinafter also referred to as "P-0221") comprises chain 1 (IL-15-Linker-Hole-Fc) having the amino acid sequence set forth in SEQ ID NO: 19:

[0174]

[0175] Wherein the IL-15 domain sequence is underlined and the peptide linker sequence is in bold; chain 2 (IL-15Rα-Sushi+) has the amino acid sequence set forth in SEQ ID NO:5 and chain 3 (Knob-Fc) has the amino acid sequence set forth in SEQ ID NO:7.

[0176] In various embodiments, the monovalent IL-15 / IL-15Rα (non-covalent) Fc fusion protein of the present invention (hereinafter also referred to as "P-0222") comprises chain 1 (IL-15-Linker-Hole-Fc) having the amino acid sequence set forth in SEQ ID NO: 19, chain 2 (IL-15Rα-ECD) having the amino acid sequence set forth in SEQ ID NO: 4, and chain 3 (Knob-Fc) having the amino acid sequence set forth in SEQ ID NO: 7.

[0177] In various embodiments, the bivalent IL-15 / IL-15RαSushi (non-covalent) Fc fusion protein of the invention (hereinafter also referred to as "P-0234") comprises chain 1 (Fc-linker-IL-15) having the amino acid sequence set forth in SEQ ID NO: 20:

[0178]

[0179] wherein the IL-15 domain sequence is underlined and the peptide linker sequence is in bold; and chain 2 (IL-15Rα-Sushi+) having the amino acid sequence set forth in SEQ ID NO: 5.

[0180] In various embodiments, the bivalent IL-15 / IL-15RαSushi (non-covalent) Fc fusion protein of the invention (hereinafter also referred to as "P-0223") comprises chain 1 (IL-15-Linker-Fc) having the amino acid sequence set forth in SEQ ID NO: 21:

[0181]

[0182] wherein the IL-15 domain sequence is underlined and the peptide linker sequence is in bold; and chain 2 (IL-15Rα-Sushi+) having the amino acid sequence set forth in SEQ ID NO: 5.

[0183] In various embodiments, the bivalent IL-15 / IL-15Rα (non-covalent) Fc fusion protein of the invention (hereinafter also referred to as "P-0220") comprises chain 1 (Fc-linker-IL-15) having the amino acid sequence set forth in SEQ ID NO:20 and chain 2 (IL-15Rα-ECD) having the amino acid sequence set forth in SEQ ID NO:4.

[0184] In various embodiments, the bivalent IL-15 / IL-15Rα (non-covalent) Fc fusion protein of the invention (hereinafter also referred to as "P-0224") comprises chain 1 (IL-15-linker-Fc) having the amino acid sequence set forth in SEQ ID NO:21 and chain 2 (IL-15Rα-ECD) having the amino acid sequence set forth in SEQ ID NO:4.

[0185] In various embodiments, the monovalent IL-15 (non-covalent) / IL-15RαSushi Fc fusion protein of the present invention (hereinafter also referred to as "P-0165") comprises chain 1 (IL-15) having the amino acid sequence set forth in SEQ ID NO: 2, chain 2 (Knob-Fc-Linker-IL-15Rα-Sushi+) having the amino acid sequence set forth in SEQ ID NO: 14, and chain 3 (Hole-Fc) having the amino acid sequence set forth in SEQ ID NO: 8.

[0186] In various embodiments, the monovalent IL-15 (non-covalent) / IL-15RαSushi Fc fusion protein of the present invention (hereinafter also referred to as "P-0166") comprises chain 1 (IL-15) having the amino acid sequence set forth in SEQ ID NO: 2, chain 2 (Knob-Fc-Linker-IL-15Rα-Sushi+) having the amino acid sequence set forth in SEQ ID NO: 22:

[0187]

[0188] wherein the IL-15Rα Sushi domain sequence is underlined and the peptide linker sequence is in bold; and chain 3 (Hole-Fc) having the amino acid sequence set forth in SEQ ID NO: 8.

[0189] In various embodiments, the bivalent IL-15 (non-covalent) / IL-15RαSushi Fc fusion protein of the present invention (hereinafter also referred to as "P-0218") comprises chain 1 (IL-15) having the amino acid sequence set forth in SEQ ID NO: 2, chain 2 (Fc-linker-IL-15Rα-Sushi+) having the amino acid sequence set forth in SEQ ID NO: 23:

[0190]

[0191] The IL-15Rα Sushi domain sequence is underlined and the peptide linker sequence is in bold.

[0192] In various embodiments, the IL-15 / IL-15RαSushi complex will comprise an IL-15 variant having an amino acid sequence selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45.

[0193] In various embodiments, the bivalent IL-15 / IL-15RαSushi (non-covalent) Fc fusion protein of the invention (hereinafter also referred to as "P-0313") comprises chain 1 (Fc-linker-IL-15-S58D) having the amino acid sequence set forth in SEQ ID NO: 46:

[0194]

[0195] wherein the IL-15S58D variant sequence is underlined and the peptide linker sequence is in bold; and chain 2 (IL-15RαSushi) having the amino acid sequence set forth in SEQ ID NO: 5.

[0196] In various embodiments, the IL-15 / IL-15RαSushi complex-Fc fusion protein of the present invention (hereinafter also referred to as "P-0314") comprises chain 1 (Fc-linker-IL-15Rα-Sushi+) having the amino acid sequence set forth in SEQ ID NO: 23 and chain 2 (IL-15S58D) having the amino acid sequence set forth in SEQ ID NO: 24.

[0197] In various embodiments, the monovalent IL-15 / IL-15RαSushi (non-covalent) Fc fusion protein of the invention (hereinafter also referred to as "P-0666") comprises chain 1 (Hole-Fc-Linker-IL-15-S58D) having the amino acid sequence set forth in SEQ ID NO: 48:

[0198]

[0199] Wherein the IL-15 domain sequence is underlined and the peptide linker sequence is in bold; chain 2 (IL-15Rα-Sushi+) has the amino acid sequence set forth in SEQ ID NO:5 and chain 3 (Knob-Fc) has the amino acid sequence set forth in SEQ ID NO:7.

[0200] In various embodiments, the monovalent IL-15 / IL-15RαSushi (non-covalent) Fc fusion protein of the invention (hereinafter also referred to as "P-0667") comprises chain 1 (IL-15-S58D-Linker-Hole-Fc) having the amino acid sequence set forth in SEQ ID NO: 49:

[0201]

[0202] Wherein the IL-15 domain sequence is underlined and the peptide linker sequence is in bold; chain 2 (IL-15Rα-Sushi+) has the amino acid sequence set forth in SEQ ID NO:5 and chain 3 (Knob-Fc) has the amino acid sequence set forth in SEQ ID NO:7.

[0203] In various embodiments, the bivalent IL-15 / IL-15RαSushi (non-covalent) Fc fusion protein of the invention (hereinafter also referred to as "P-0668") comprises chain 1 (IL-15-Linker-Fc) having the amino acid sequence set forth in SEQ ID NO: 50:

[0204]

[0205] wherein the IL-15 domain sequence is underlined and the peptide linker sequence is in bold; and chain 2 (IL-15Rα-Sushi+) having the amino acid sequence set forth in SEQ ID NO: 5.

[0206] In various embodiments, the monovalent IL-15 (non-covalent) / IL-15RαSushi Fc fusion protein of the present invention (hereinafter also referred to as "P-0669") comprises chain 1 (IL-15S58D) having the amino acid sequence set forth in SEQ ID NO: 24, chain 2 (Knob-Fc-Linker-IL-15Rα-Sushi+) having the amino acid sequence set forth in SEQ ID NO: 22; and chain 3 (Hole-Fc) having the amino acid sequence set forth in SEQ ID NO: 8.

[0207] In various embodiments, the monovalent IL-15 (non-covalent) / IL-15RαSushi Fc fusion protein of the present invention (hereinafter also referred to as "P-0670") comprises chain 1 (IL-15S58D) having the amino acid sequence set forth in SEQ ID NO: 24, chain 2 (IL-15Rα-Sushi + -Linker-Knob-Fc) having the amino acid sequence set forth in SEQ ID NO: 51:

[0208]

[0209] wherein the IL-15Rα Sushi domain sequence is underlined and the peptide linker sequence is in bold; and chain 3 (Hole-Fc) having the amino acid sequence set forth in SEQ ID NO: 8.

[0210] In various embodiments, the bivalent IL-15 (non-covalent) / IL-15RαSushi Fc fusion protein of the present invention (hereinafter also referred to as "P-0671") comprises chain 1 (IL-15S58D) having the amino acid sequence set forth in SEQ ID NO: 24, chain 2 (IL-15Rα-Sushi + -Linker-Fc) having the amino acid sequence set forth in SEQ ID NO: 52:

[0211]

[0212] The IL-15Rα Sushi domain sequence is underlined and the peptide linker sequence is in bold.

[0213] polynucleotides

[0214] In another aspect, the present disclosure provides an isolated nucleic acid molecule comprising a polynucleotide encoding the following: IL-15, IL-15 variant, IL-15Rα, IL-15Rα variant, Fc, Fc variant, IL-15-Fc fusion protein, IL-15RαSushi-Fc fusion protein or IL-15 / IL-15RαSushi-Fc fusion protein of the present disclosure. The subject nucleic acid can be single-stranded or double-stranded. Such a nucleic acid can be a DNA or RNA molecule. DNA includes, for example, cDNA, genomic DNA, synthetic DNA, DNA amplified by PCR, and combinations thereof. Genomic DNA encoding the IL-15 / IL-15RαSushi complex is obtained from a genomic library that is available for many species. Synthetic DNA can be obtained by chemically synthesizing overlapping oligonucleotide fragments and then assembling the fragments to reconstruct part or all of the coding region and flanking sequences. RNA can be obtained from a prokaryotic expression vector that directs high-level synthesis of mRNA, such as a vector using a T7 promoter and RNA polymerase. cDNA is obtained from libraries prepared from mRNA isolated from various tissues expressing IL-15. The DNA molecules of the present disclosure include full-length genes as well as polynucleotides and fragments thereof. The full-length gene may also include a sequence encoding an N-terminal signal sequence. Such nucleic acids can be used, for example, in methods for preparing novel IL-15 / IL-15RαSushi-Fc fusion proteins. In various embodiments, the nucleic acid molecules comprise the nucleotide sequences listed in SEQ ID NOs: 56-63.

[0215] In various embodiments, the isolated nucleic acid molecule comprises a polynucleotide described herein and further comprises a polynucleotide encoding at least one heterologous protein described herein. In various embodiments, the nucleic acid molecule further comprises a polynucleotide encoding a linker or hinge joint described herein.

[0216] In various embodiments, the recombinant nucleic acid of the present disclosure can be operably linked to one or more regulatory nucleotide sequences in the expression construct. Regulatory sequences are known in the art and are selected to direct the expression of the IL-15 / IL-15RαSushi-Fc fusion protein. Accordingly, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990). Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcription start and stop sequences, translation start and stop sequences, and enhancers or activation sequences. The present disclosure contemplates constitutive or inducible promoters known in the art. The promoter may be a naturally occurring promoter or a hybrid promoter combining elements of more than one promoter. The expression construct may be present on an episome in the cell, such as a plasmid, or the expression construct may be inserted into a chromosome. In various embodiments, the expression vector comprises a selectable marker gene to allow selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.

[0217] In another aspect of the present disclosure, the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding the IL-15 / IL-15RαSushi complex and operably linked to at least one regulatory sequence. The term "expression vector" refers to a plasmid, phage, virus, or vector used to express a polypeptide from a polynucleotide sequence. Vectors suitable for expression in host cells are readily available and nucleic acid molecules are inserted into the vector using standard recombinant DNA techniques. Such vectors can include a variety of expression control sequences that, when operably linked to a DNA sequence, control the expression of the DNA sequence and can be used in these vectors to express a DNA sequence encoding the IL-15 / IL-15RαSushi-Fc fusion protein. Such useful expression control sequences include, for example, the early and late promoters of SV40, the tet promoter, the early promoter mediated by adenovirus or cytomegalovirus, the RSV promoter, the lac system, the trp system, the TAC or TRC system, the T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter region of lambda phage, the control region of the fd coat protein, the promoter of 3-phosphoglycerate kinase or other glycolytic enzymes, the promoter of acid phosphatases such as PhoS, the promoter of yeast α-mating factor, the polyhedron promoter of the baculovirus system, and other sequences known to control gene expression in prokaryotes or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed and / or the type of protein desired to be expressed. In addition, the copy number of the vector, the ability to control the copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.

[0218] The recombinant nucleic acids of the present disclosure can be produced by ligating the cloned gene or a portion thereof into a vector suitable for expression in prokaryotes, eukaryotic cells (yeast, birds, insects or mammals), or both. Expression vectors for producing recombinant IL-15 / IL-15RαSushi complexes include plasmids and other vectors. For example, suitable vectors include the following types of plasmids: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids for expression in prokaryotes such as Escherichia coli.

[0219] Some mammalian expression vectors include prokaryotic sequences that are beneficial to vector propagation in bacteria and one or more eukaryotic transcription units expressed in eukaryotic cells. PcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfecting eukaryotic cells. Some of these vectors are modified by sequences from bacterial plasmids such as pBR322 to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, viruses such as derivatives of bovine papillomavirus (BPV-1) or derivatives of Epstein-Barr virus (pHEBo, pREP derived and p205) can be used for transient expression of proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems are found in the description of gene therapy delivery systems below. In some cases, it may be desirable to express recombinant polypeptides using a baculovirus expression system. Examples of such baculovirus expression systems include pVL derived vectors (such as pVL1392, pVL1393, and pVL941), pAcUW derived vectors (such as pAcUW1), and pBlueBac derived vectors (such as pBlueBac III containing B-gal).

[0220] In various embodiments, vectors will be designed for producing the subject IL-15 / IL-15RαSushi-Fc fusion protein in CHO cells, such as the pcmv-Script vector (Stratagene, La Jolla, Calif.), the pcDNA4 vector (Invitrogen, Carlsbad, Calif.), and the pCI-neo vector (Promega, Madison, Wis.). It will be apparent that the subject gene constructs can be used to cause expression of the subject IL-15 / IL-15RαSushi-Fc fusion protein in cells proliferating in culture, for example, to produce proteins, including fusion proteins or variant proteins, for purification.

[0221] The present disclosure also relates to host cells transfected with recombinant genes comprising nucleotide sequences encoding the amino acid sequences of one or more subject IL-15 / IL-15RαSushi-Fc fusion proteins. The host cells can be prokaryotic or eukaryotic cells. For example, the IL-15 / IL-15RαSushi complex of the present disclosure can be expressed in bacterial cells such as Escherichia coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.

[0222] Accordingly, the present disclosure also relates to a method for producing a subject IL-15 / IL-15RαSushi-Fc fusion protein. For example, a host cell transfected with an expression vector encoding the IL-15 / IL-15RαSushi complex can be cultured under appropriate conditions that allow the expression of the IL-15 / IL-15RαSushi complex to occur. The IL-15 / IL-15RαSushi complex can be secreted from cells containing the IL-15 / IL-15RαSushi-Fc fusion protein and separated from a mixture of cells and culture medium containing the IL-15 / IL-15RαSushi-Fc fusion protein. Alternatively, the IL-15 / IL-15RαSushi complex can be retained in the cytoplasm or in the membrane fraction, and the cells can be harvested, lysed, and the protein isolated. The cell culture comprises host cells, culture medium, and other by-products. Suitable culture media for cell culture are well known in the art.

[0223] The polypeptides and proteins of the present disclosure can be purified according to protein purification techniques well known to those skilled in the art. These techniques, at one level, involve the crude fractionation of protein fractions and non-protein fractions. After the peptide or polypeptide is separated from other proteins, the peptide or polypeptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). As used herein, the terms "isolated polypeptide" or "purified polypeptide" are intended to refer to a composition that can be separated from other components, wherein the polypeptide is purified to any degree relative to its naturally available state. Thus, a purified polypeptide also refers to a polypeptide that has been separated from the environment in which it may naturally occur. Generally, "purified" will refer to a polypeptide composition that has undergone fractionation to remove various other components, and the polypeptide composition substantially retains its expressed biological activity. When the term "substantially purified" is used, the designation will refer to a peptide or polypeptide composition in which the polypeptide or peptide forms a majority component of the composition, such as about 50%, about 60%, about 70%, about 80%, about 85%, or about 90% or more of the protein in the composition.

[0224] Various techniques suitable for purification will be well known to those skilled in the art. These techniques include, for example, precipitation with ammonium sulfate, PEG, antibodies (immunoprecipitation), or by heat denaturation followed by centrifugation; chromatography, such as affinity chromatography (Protein A column), ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, hydroxyapatite chromatography, hydrophobic interaction chromatography; isoelectric focusing; gel electrophoresis; and combinations of these techniques. As is generally known in the art, it is believed that the order in which the various purification steps are performed can be changed, or that certain steps can be omitted, and still result in a suitable method for preparing a substantially purified polypeptide.

[0225] Pharmaceutical composition

[0226] In another aspect, the present disclosure provides a pharmaceutical composition comprising an IL-15 / IL-15RαSushi-Fc fusion protein mixed with a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are well known and understood by those of ordinary skill in the art and have been widely described (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A.R. Gennaro, Mack Publishing Company, 1990). A pharmaceutically acceptable carrier can be included for the purpose of changing, maintaining or maintaining, for example, pH, osmotic pressure concentration, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or penetration of the composition. Such a pharmaceutical composition can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the polypeptide. Suitable pharmaceutically acceptable carriers include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as PEG-100). agent) (such as caffeine, polyvinyl pyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose or dextrin); proteins (such as serum albumin, gelatin or immunoglobulins); colorants; flavorings and diluents; emulsifiers; hydrophilic polymers (such as polyvinyl pyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerol, propylene glycol); the present invention also provides a pharmaceutical composition comprising: ...

[0227] The main vehicle or carrier in the pharmaceutical composition can be aqueous or non-aqueous in nature. For example, suitable vehicles or carriers can be water for injection, physiological saline solution or artificial cerebrospinal fluid that may be supplemented with other materials common in the composition for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are other exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer of about pH 7.0-8.5 or acetate buffer of about pH 4.0-5.5, which can also include sorbitol or a suitable substitute for sorbitol. In one embodiment of the present disclosure, the composition with the desired degree of purity can be prepared by mixing with an optional formulation agent (formulation agent) (Remington's Pharmaceutical Sciences, the same) to store in the form of a lyophilized block or aqueous solution. In addition, the therapeutic composition can be formulated as a lyophilized product using suitable excipients such as sucrose. The best pharmaceutical composition will be determined by a person of ordinary skill in the art depending on, for example, the intended route of administration, delivery form and desired dosage.

[0228] When parenteral administration is envisaged, the therapeutic pharmaceutical composition may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired IL-15 / IL-15RαSushi complex in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, in which the polypeptide is formulated as a sterile, isotonic solution suitable for storage. In various embodiments, pharmaceutical preparations suitable for injectable administration can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hanks solution, Ringer solution or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. In addition, suspensions of the active compound may be prepared as suitable oily injection suspensions. Optionally, the suspension may also contain a suitable stabilizer or an agent that increases the solubility of the compound and allows the preparation of a highly concentrated solution.

[0229] In various embodiments, the therapeutic pharmaceutical composition can be formulated as for targeted delivery using a colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, pearls, and lipid-based systems, and lipid-based systems include oil-in-water emulsions, micelles, mixed micelles, and liposomes. Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Exemplary phospholipids include lecithin, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine. The targeting of liposomes can also be based on, for example, organ-specificity, cell-specificity, and organelle-specificity and known in the art.

[0230] In various embodiments, oral administration of the pharmaceutical composition is contemplated. Pharmaceutical compositions administered in this form may be formulated with or without carriers commonly used in the formulation of solid dosage forms such as tablets and capsules. In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), one or more therapeutic compounds of the present disclosure may be combined with one or more pharmaceutically acceptable carriers such as sodium citrate or dibasic calcium phosphate. phosphate) and / or any of the following: (1) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and / or gum arabic; (3) humectants such as glycerol; (4) disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) solution retarding agents such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds; (7) wetting agents such as, for example, cetyl alcohol and glyceryl monostearate; (8) absorbents such as kaolin and bentonite; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical composition may also contain a buffer. The solid composition of similar type can also be used as the filler in the soft-filled and hard-filled gelatin capsule using excipients such as lactose (lactose) or lactose (milk sugar) and high molecular weight polyethylene glycol etc.. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to active ingredients, liquid dosage forms can include inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifying agents such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oil (especially, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0231] In various embodiments, it is envisioned that the pharmaceutical composition is topically applied to the skin or to a mucous membrane. Topical preparations can also include one or more of the various agents known as skin or stratum corneum penetration enhancers. Examples of these agents are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methanol or isopropyl alcohol, dimethyl sulfoxide (DMSO) and azone. Other agents can also be included to make the preparation cosmetically acceptable. Examples of these agents are fats, waxes, oils, dyes, fragrances, preservatives, stabilizers and surfactants. Keratin softeners (keratolytic agents) such as those known in the art can also be included. Examples are salicylic acid and sulphur. Formulations for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier and with any preservatives, buffers, or propellants that may be required. In addition to the subject compound of the present disclosure (e.g., IL-15 / IL-15RαSushi-Fc fusion protein), ointments, pastes, creams, and gels can contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0232] Other pharmaceutical compositions contemplated herein for use include formulations comprising polypeptides in formulations for sustained or controlled delivery. In various embodiments, the pharmaceutical composition can be formulated into nanoparticles, as a sustained-release hydrogel, or incorporated into an oncolytic virus. Technologies for preparing various other sustained or controlled delivery vehicles such as liposome carriers, bioerodible microparticles or porous beads, and reservoir injections are also known to those skilled in the art.

[0233] The effective amount of the pharmaceutical composition to be used for treatment will depend on, for example, the treatment background and the treatment goal. It will be understood by those skilled in the art that the appropriate dosage level for treatment will therefore depend in part on the indication, route of administration, and the size (body weight, body surface or organ size) and condition (age and overall health) of the molecule being delivered, the polypeptide being used for and the patient. Accordingly, the clinician can adjust the dosage and change the route of administration to obtain the best therapeutic effect. Typical dosages can depend on the factors mentioned above and are within the range of from about 0.0001mg / kg to about 100mg / kg or more. The polypeptide composition can preferably be injected or administered subcutaneously or intravenously. Long-acting pharmaceutical compositions can be administered once every three to four days, weekly, biweekly, or monthly depending on the half-life and clearance rate of the specific formulation. The frequency of administration will depend on the pharmacokinetic parameters of the polypeptide in the formulation used. Typically, the composition is administered until the dosage for achieving the desired effect is reached. Therefore, the composition can be administered as a single dose or as multiple doses (with the same or different concentrations / dosages) over time or as a continuous infusion. Further improvements in appropriate dosage are routinely carried out. Appropriate dosages can be determined through use of appropriate dose-response data.

[0234] The route of administration of the pharmaceutical composition is according to known methods, such as oral; By intravenous, intraperitoneal, intratumoral, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal vein, intralesional approach, intramedullary, intrathecal, intravesicular, intraventricular, transdermal, subcutaneous or intraperitoneal injection; And intranasal, enteral, local, sublingual, urethra, vaginal or rectal mode; By sustained release system or by implant device. When desired, composition can be administered by bolus injection, or continuously administered by infusion, or administered by implant device. Alternatively or additionally, composition can be administered locally via the implantation of the film, sponge or another suitable material that has been adsorbed or encapsulated thereto by the desired molecule. When using implant device, the device can be implanted in any suitable tissue or organ, and the delivery of the desired molecule can be administered via diffusion, sustained release bolus or continuously administered.

[0235] Therapeutic uses

[0236] The present disclosure provides a method for treating cancer cells in a subject, comprising administering to the subject a therapeutically effective amount (as a monotherapy or in a combination therapy regimen) of an IL-15 / IL-15RαSushi-Fc fusion protein of the present disclosure in a pharmaceutically acceptable carrier, wherein such administration inhibits the growth and / or proliferation of the cancer cells. In particular, the IL-15 / IL-15RαSushi-Fc fusion protein of the present disclosure is useful in treating disorders characterized by cancer. Such disorders include, but are not limited to, solid tumors such as breast cancer, respiratory tract cancer, brain cancer, cancer of the reproductive organs, cancer of the digestive tract, cancer of the urinary tract, eye cancer, liver cancer, skin cancer, head and neck cancer, thyroid cancer, parathyroid cancer, and their distant metastases, lymphomas, sarcomas, multiple myeloma, and leukemias. Examples of breast cancer include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ. Examples of cancers of the respiratory tract include, but are not limited to, small cell lung cancer and non-small cell lung cancer, as well as bronchial adenomas and pleuropulmonary blastomas. Examples of brain cancers include, but are not limited to, brainstem and hypothalamic gliomas, cerebellar and cerebral astrocytomas, neuroblastomas, medulloblastomas, ependymomas, and neuroectodermal and pineal tumors. Tumors of the male / male reproductive organs include, but are not limited to, prostate cancer and testicular cancer. Tumors of the female / female reproductive organs include, but are not limited to, endometrial cancer, cervical cancer, ovarian cancer, vaginal cancer, vulvar cancer, and uterine sarcomas. Tumors of the digestive tract include, but are not limited to, anal cancer, colon cancer, colorectal cancer, esophageal cancer, gallbladder cancer, stomach cancer, liver cancer, breast cancer, pancreatic cancer, rectal cancer, small intestine cancer, and salivary gland cancer. Tumors of the urethra include, but are not limited to, bladder cancer, penile cancer, kidney cancer, renal pelvis cancer, ureteral cancer, and urethral cancer. Eye cancers include, but are not limited to, intraocular melanoma and retinoblastoma. Examples of liver cancer include, but are not limited to, hepatocellular carcinoma (hepatocellular carcinoma with or without fibrolamellar variants), cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixed hepatocellular cholangiocarcinoma. Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell carcinoma, and non-melanoma skin cancer. Head and neck cancers include, but are not limited to, nasopharyngeal carcinoma and lip and oral cancer. Lymphomas include, but are not limited to, AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and central nervous system lymphoma. Sarcomas include, but are not limited to, soft tissue sarcomas, osteosarcomas, malignant fibrous histiocytomas, lymphosarcoma, and rhabdomyosarcomas. Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and hairy cell leukemia.In certain embodiments, the cancer will be one with high expression of TGF-β family members, such as activin A, myostatin, TGF-β, and GDF15, for example, pancreatic cancer, gastric cancer, ovarian cancer, colorectal cancer, melanoma leukemia, lung cancer, prostate cancer, brain cancer, bladder cancer, and head and neck cancer.

[0237] The present disclosure provides a method for treating refractory or drug-resistant liquid or solid tumors by enhancing the therapeutic effect of existing cancer therapeutics as an adjunct.

[0238] The present disclosure also provides a method for treating a viral infection in a subject (including hepatitis A, hepatitis B, hepatitis C, AIDS in HIV infection, human papillomavirus (HPV) infection, genital warts, etc.), comprising administering to a human patient in need thereof an IL-15 / IL-15RαSushi-Fc fusion protein of the present disclosure in a pharmaceutically acceptable carrier, wherein such administration inhibits viral growth and / or replication.

[0239] "Therapeutically effective amount" or "therapeutically effective dose" refers to that amount of the administered therapeutic agent that will relieve to some extent one or more symptoms of the disorder being treated.

[0240] The therapeutically effective dose can be determined by IC 50 Initially estimated from cell culture assays. A dose can then be formulated in animal models to achieve an IC including the IC as determined in cell culture. 50 The circulating plasma concentration range of the drug can be used to more accurately determine the dosage that is useful in humans. The levels in plasma can be measured, for example, by HPLC. The exact composition, route of administration, and dosage can be selected by the individual physician in view of the subject's condition.

[0241] The dosage regimen can be adjusted to provide the optimal desired response (e.g., a therapeutic response or a preventive response). For example, a single bolus injection can be administered, several divided doses (multiple or repeated or maintenance) can be administered over time, and the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic scenario. For ease of administration and consistency of dosage, it is particularly beneficial to formulate parenteral compositions in dosage unit form. As used herein, dosage unit form refers to physically dispersed units suitable as single doses for a mammalian subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect, together with the required pharmaceutical carrier. The specifications of the dosage unit form of the present disclosure will be primarily determined by the unique characteristics of the antibody and the specific therapeutic or preventive effect to be achieved.

[0242] Therefore, it will be understood by those skilled in the art that, based on the disclosure provided herein, dosage and administration regimens are adjusted according to methods well known in the therapeutic field. That is, the maximum tolerated dose can be easily determined, and the effective amount that provides a detectable therapeutic benefit to the subject can also be determined, and the time requirement for applying each agent to provide a detectable therapeutic benefit to the subject can also be determined. Therefore, although certain dosages and administration regimens are exemplified herein, these examples are by no means limiting the dosages and administration regimens that can be provided to the subject when practicing this disclosure.

[0243] It should be noted that dosage values ​​may vary with the type and severity of the condition to be alleviated and may include a single dose or more than one dose. It should also be understood that for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition, and that the dosage ranges listed herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. In addition, the dosage regimen of the compositions of the present disclosure may be based on a number of factors, including the type of disease, the age, weight, sex, medical condition, severity of the condition, route of administration, and the specific antibody used. Thus, the dosage regimen may vary widely but can be routinely determined using standard methods. For example, the dosage may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and / or experimental values. Thus, the present disclosure includes intra-subject dose-escalation as determined by a skilled person. Determining appropriate dosages and regimens is well known in the relevant art and will be understood to be within the skill of the art once the teachings disclosed herein are provided.

[0244] Exemplary, non-limiting daily dosing ranges for a therapeutically or prophylactically effective amount of an IL-15 / IL-15RαSushi-Fc fusion protein of the present disclosure can be 0.0001 to 100 mg / kg body weight, 0.0001 to 90 mg / kg body weight, 0.0001 to 80 mg / kg body weight, 0.0001 to 70 mg / kg body weight, 0.0001 to 60 mg / kg body weight, 0.0001 to 50 mg / kg body weight, 0.0001 to 40 mg / kg body weight, 0.0001 to 30 mg / kg body weight, 0.0001 to 20 mg / kg body weight , 0.0001 to 10 mg / kg body weight, 0.0001 to 5 mg / kg body weight, 0.0001 to 4 mg / kg body weight, 0.0001 to 3 mg / kg body weight, 0.0001 to 2 mg / kg body weight, 0.0001 to 1 mg / kg body weight, 0.0010 to 50 mg / kg body weight, 0.0010 to 40 mg / kg body weight, 0.0010 to 30 mg / kg body weight, 0.0010 to 20 mg / kg body weight, 0.0010 to 10 mg / kg body weight, 0.0010 to 5 mg / kg body weight, 0.0010 to 4 mg / kg body weight, 0.00 10 to 3 mg / kg body weight, 0.0010 to 2 mg / kg body weight, 0.0010 to 1 mg / kg body weight, 0.01 to 50 mg / kg body weight, 0.01 to 40 mg / kg body weight, 0.01 to 30 mg / kg body weight, 0.01 to 20 mg / kg body weight, 0.01 to 10 mg / kg body weight, 0.01 to 5 mg / kg body weight, 0.01 to 4 mg / kg body weight, 0.01 to 3 mg / kg body weight, 0.01 to 2 mg / kg body weight, 0.01 to 1 mg / kg body weight, 0.1 to 50 mg / kg body weight, 0.1 to 40 mg / kg body weight, 0. 1 to 30mg / kg body weight, 0.1 to 20mg / kg body weight, 0.1 to 10mg / kg body weight, 0.1 to 5mg / kg body weight, 0.1 to 4mg / kg body weight, 0.1 to 3mg / kg body weight, 0.1 to 2mg / kg body weight, or 0.1mg / kg body weight, 1 to 50mg / kg body weight, 1 to 40mg / kg body weight, 1 to 30mg / kg body weight, 1 to 20mg / kg body weight, 1 to 10mg / kg body weight, 1 to 5mg / kg body weight, 1 to 4mg / kg body weight, 1 to 3mg / kg body weight, 1 to 2mg / kg body weight or 1 to 1mg / kg body weight. It should be noted that dosage value can change with the type and severity of the situation to be alleviated. It should also be understood that for any particular experimenter, specific dosage regimen should be adjusted over time according to the professional judgment of the people's using of individual needs and administration of compositions or supervision compositions, and the dosage range listed herein is only exemplary and is not intended to limit the scope or practice of the claimed compositions.

[0245] Toxicity and therapeutic index of the pharmaceutical compositions of the present disclosure can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD 50 (a dose lethal to 50% of the population) and ED 50 (the dose that is therapeutically effective in 50% of the population). The dose ratio between the toxic dose and the therapeutically effective dose is the therapeutic index, and the therapeutic index can be expressed as the ratio LD 50 / ED 50 Compositions that exhibit large therapeutic indices are generally preferred.

[0246] The dosing frequency of the IL-15 / IL-15RαSushi-Fc fusion protein pharmaceutical composition depends on the nature of the therapy and the specific disease being treated. Subjects can be treated at regular intervals, such as once a week or once a month, until the desired therapeutic outcome is achieved. Exemplary dosing frequencies include, but are not limited to: once or twice a week without interruption; once or twice a week every other week; once every 2 weeks; once every 3 weeks; once a week without interruption for 2 weeks, then once a month; once a week without interruption for 3 weeks, then once a month; once a month; once every two months; once every 3 months; once every 4 months; once every 5 months; or once every 6 months or once a year.

[0247] Combination therapy

[0248] As used herein, the terms "co-administration," "co-administered," and "in combination with" in reference to an IL-15 / IL-15RαSushi-Fc fusion protein of the present disclosure and one or more other therapeutic agents are intended to mean, and do mean and include, the following: simultaneous administration of such a combination of an IL-15 / IL-15RαSushi-Fc fusion protein of the present disclosure and one or more therapeutic agents to a subject in need of treatment, wherein such components are formulated together into a single dosage form that releases the components to the subject at substantially the same time; substantially simultaneous administration of such a combination of an IL-15 / IL-15RαSushi-Fc fusion protein of the present disclosure and one or more therapeutic agents to a subject in need of treatment, wherein such components are formulated separately from one another into separate dosage forms that are taken by the subject at substantially the same time, whereupon the components are released to the subject at substantially the same time; Such combinations of an RαSushi-Fc fusion protein and one or more therapeutic agents are administered sequentially to a subject in need of treatment, wherein such components are formulated separately from one another into separate dosage forms that are taken by the subject at consecutive times with significant time intervals between each administration, whereby the components are released to the subject at substantially different times; and such combinations of an IL-15 / IL-15RαSushi-Fc fusion protein of the present disclosure and one or more therapeutic agents are administered sequentially to a subject in need of treatment, wherein such components are formulated together into a single dosage form that releases the components in a controlled manner, whereby the components are released to the subject simultaneously, sequentially, and / or overlappingly at the same and / or different times, whereby each part may be administered by the same or different routes.

[0249] On the other hand, the present disclosure provides a method for treating a subject's cancer or cancer metastasis, the method comprising administering a therapeutically effective amount of a pharmaceutical composition of the present invention in combination with a second therapy, wherein the second therapy includes but is not limited to immunotherapy, cytotoxic chemotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiotherapy, and stem cell transplantation. For example, such a method can be used for preventive cancer prevention, prevention of postoperative cancer recurrence and metastasis, and as an adjunct to other conventional cancer therapies. The present disclosure recognizes that the effectiveness of conventional cancer therapies (e.g., chemotherapy, radiotherapy, phototherapy, immunotherapy, and surgery) can be enhanced by the use of the combined methods described herein.

[0250] A large amount of conventional compounds have been shown to have anti-neoplastic activity. These compounds have been used as the medicament in chemotherapy to reduce solid tumors, prevent metastasis and further growth, or reduce the number of the malignant T cells in leukemia or myeloid malignancies. Although chemotherapy is effective in treating various types of malignant tumors, many anti-neoplastic compounds induce undesirable side effects. Shown, when two or more different treatment combinations, treatment can work collaboratively, and allows to reduce the dosage of every kind of treatment, thereby reducing the harmful side effects of every kind of compound produced at higher dosage. In other cases, for treatment, it is that the malignant tumor of refractory nature can respond to the combination therapy of two or more different treatments.

[0251] In various embodiments, a second anticancer agent, such as a chemotherapeutic agent, will be administered to the patient. A list of exemplary chemotherapeutic agents includes, but is not limited to, daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, mechlorethamine, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, bendamustine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin, carboplatin, dapoxetine ... Platinum, oxaliplatin, pentostatin, cladribine, cytarabine, gemcitabine, pralatrexate, mitoxantrone, diethylstilbestrol (DES), fludarabine, ifosfamide, hydroxyureataxanes (such as paclitaxel and docetaxel) and / or anthracycline antibiotics, and combinations of agents such as, but not limited to, DA-EPOCH, CHOP, CVP or FOLFOX. In various embodiments, the dose of such chemotherapeutic agents includes, but is not limited to, about 10 mg / m 2 , 20mg / m 2 , 30mg / m 2 , 40mg / m 2 , 50mg / m 2 , 60mg / m 2 , 75mg / m 2 , 80mg / m 2 , 90mg / m 2 , 100mg / m 2 , 120mg / m 2 , 150mg / m 2 , 175mg / m 2 , 200mg / m 2 , 210mg / m 2 , 220mg / m 2 , 230mg / m 2, 240mg / m 2 , 250mg / m 2 , 260mg / m 2 and 300 mg / m 2 Any of .

[0252] In various embodiments, the combination treatment methods of the present disclosure may also include administering to the subject a therapeutically effective amount of immunotherapy, including, but not limited to, treatment with depleting antibodies directed against specific tumor antigens; treatment with antibody-drug conjugates; treatment with agonistic antibodies, antagonistic antibodies, or blocking antibodies directed against co-stimulatory or co-inhibitory molecules (immune checkpoints) such as CD276, CD272, CTLA-4, PD-1, PD-L1, CD40, SIRPa, CD47, OX-40, CD137, GITR, LAG3, ICOS, CD27, 4-1BB, TIM-3, B7-H4, Siglec 7, Siglec 8, Siglec 9, Siglec 15, and VISTA; treatment with bispecific T cell-engaging antibodies. Treatments such as blinatumomab; treatments involving administration of biological response modifiers (such as IL-2, IL-7, IL-12, IL-21, GM-CSF, IFN-α, IFN-β, and IFN-γ); treatments using therapeutic vaccines such as sipuleucel-T; treatments using dendritic cell vaccines or tumor antigen peptide vaccines; treatments using NK cells; treatments using TCR-T cells; treatments using chimeric antigen receptor (CAR)-T cells; treatments using CAR-NK cells; treatments using iPS-induced NK cells, iPS-induced TCR-T cells, iPS-induced CAR- treatment with T cells or iPS-induced CAR-NK cells; treatment with dendritic cells; treatment with tumor-infiltrating lymphocytes (TIL); treatment with adoptively transferred anti-tumor T cells (expanded ex vivo and / or TCR transgenic); treatment with vaccines such as Bacillus Calmette-Guérin (BCG); treatment with TALL-104 cells; and treatment with immunostimulants such as Toll-like receptor (TLR) agonists CpG and imiquimod; wherein the combination therapy provides increased effector cell killing of tumor cells, i.e., there is a synergistic effect between the IL-15 / IL-15RαSushi-Fc fusion protein and the immunotherapy when co-administered.

[0253] In various embodiments, the combination therapy comprises the simultaneous administration of an IL-15 / IL-15RαSushi-Fc fusion protein and a second composition in the same pharmaceutical composition or in separate pharmaceutical compositions. In various embodiments, the IL-15 / IL-15RαSushi-Fc fusion protein composition and the second composition are administered sequentially, i.e., the IL-15 / IL-15RαSushi-Fc fusion protein composition is administered before or after the administration of the second composition. In various embodiments, the administration of the IL-15 / IL-15RαSushi-Fc fusion protein composition and the second composition is simultaneous, i.e., the administration periods of the IL-15 / IL-15RαSushi-Fc fusion protein composition and the second composition overlap with each other. In various embodiments, the administration of the IL-15 / IL-15RαSushi-Fc fusion protein composition and the second composition is non-simultaneous. For example, in various embodiments, the administration of the IL-15 / IL-15RαSushi-Fc fusion protein composition is terminated and then the second composition is administered. In various embodiments, administration of the second dose of the composition is terminated, and then the IL-15 / IL-15RαSushi-Fc fusion protein composition is administered.

[0254] The following examples are provided to more fully illustrate the present disclosure but should not be construed as limiting the scope of the disclosure.

[0255] Example 1

[0256] Construction, expression and purification of IL-15 / IL-15RαSushi-Fc fusion protein

[0257] All genes were codon optimized for expression in mammalian cells, synthesized and subcloned into a recipient mammalian expression vector (GenScript). Protein expression was driven by a CMV promoter, and a synthetic SV40 polyadenylic acid (polyA) signal sequence was present at the 3' end of the CDS. A leader sequence was engineered at the N-terminus of the construct to ensure appropriate signal transduction and processing for secretion. The fusion protein was produced by co-transfecting suspended HEK293-F cells with a mammalian expression vector using polyethyleneimine (PEI, 25,000 MW linear, Polysciences). If there were two or more expression vectors, the vectors were transfected at a ratio of 1:1. For transfection, HEK293 cells were grown in serum-free FreeStyle TM HEK293 cells were cultured in 293 expression medium (ThermoFisher). For production in 1000 ml shake flasks (maximum working volume 330 mL), HEK293 cells were plated at 0.8 × 10 6The density of individual cells / ml was inoculated and transfected after 24 hours. The expression vector that is 330 μgDNA is mixed with 16.7ml Opti-mem culture medium (ThermoFisher) by total amount. After adding 0.33mg PEI diluted in 16.7ml Opti-mem culture medium, the mixture was vortexed for 15 seconds, and then incubated at room temperature for 10min. Then the DNA / PEI solution was added to the cells and incubated at 37 ℃ with 8% CO2 in an incubator of atmosphere. At the 4th day, sodium butyrate (Millipore Sigma) was added to the cell culture at a final concentration of 2mg / L to help maintain protein expression. After cultivating for 6 days, the supernatant was collected and used for purification by centrifuging at 2200rpm for 20min. The solution was sterile filtered (0.22 μm filter, Corning). Protein A affinity chromatography was used to purify the secreted protein from the cell culture supernatant.

[0258] The secreted protein was purified from the cell culture supernatant using protein A affinity chromatography. The cell culture supernatant was loaded onto a HiTrapMabSelect SuRe 5 ml column (GE Healthcare) equilibrated with 5 column volumes (CV) of phosphate buffered saline pH 7.2 (ThermoFisher). Unbound proteins were removed by washing with 5 CV of PBS pH 7.2 and the target protein was eluted with 25 mM sodium citrate, 25 mM sodium chloride, pH 3.2. The protein solution was neutralized by adding 3% 1 M Tris pH 10.2. The target protein was concentrated using Ultra-15 Ultracel 10K (Merck Millipore), and the buffer was exchanged into PBS, pH 7.2.

[0259] The chromatograms were analyzed by SDS-PAGE and Coomassie (Imperial TM Protein stain (ThermoFisher) was used to analyze the purity and molecular weight of the purified molecules. The aggregate content of the molecules was analyzed on an Agilent 1200 high performance liquid chromatography (HPLC) system using a precast gel system (4%-12% Bis-Tris, ThermoFisher). The samples were injected onto an AdvanceBio size exclusion column ( 4.6 x 150 mm, 2.7 μm, LC column, Agilent).

[0260] The protein concentration of the purified protein samples was determined by measuring the absorbance at 280 nm using a Nanodrop spectrophotometer (ThermoFisher) divided by the molar extinction coefficient calculated based on the amino acid sequence. Endotoxin levels of the purified protein samples were measured using Endosafe nexgen-PTS (Charles River) according to the manufacturer's instructions.

[0261] As an example of the protein profile of isolated IL-15 / IL-15RαFc fusion constructs, SDS-PAGE analysis of P-0217, P-0234, and P-0313 is shown in Figure 2A. P-0217, P-0234, and P-0313 are all IL-15 / IL-15Rα (non-covalent)-Fc fusion proteins containing the IL-15 / IL-15Rα complex at the C-terminus. P-0217 is a monovalent IL-15 / IL-15Rα (non-covalent) Fc fusion, P-0234 is the dimeric counterpart of P-0217, and P-0313 shares the same fusion configuration as P-0234, but differs only in the S58D substitution in the IL-15 domain.

[0262] For both monovalent and bivalent Fc fusions, the IL-15RαSushi+ domain, with a calculated molecular weight of 8.6 kDa, non-covalently associated with IL-15 or IL-15 variants fused to the Fc domain and dissociated under denaturing conditions and migrated as a clear band to the expected position (Figure 2A). The presence of the IL-15Rα-sushi+ band on the gel confirmed the non-covalent association between IL-15 and IL-15Rα during cell culture growth; this association was maintained during protein A purification under low pH elution conditions.

[0263] The size exclusion chromatograms in Figure 2B demonstrate the low aggregation tendency of both fusion forms, as only 1%-2% aggregation was present for all three fusion proteins after the initial protein A capture step without a polishing step. The sharp main peak further demonstrates the tight association of IL-15 and IL-15Rα under native buffer conditions.

[0264] Furthermore, under the same vector and culture conditions, the expression level of the fusion protein was comparable to that of the protein containing only Fc (within a 2-fold difference). The high yield and low aggregation tendency demonstrate the favorable developability profile of monovalent and bivalent IL-15 / IL-15Rα (non-covalent) Fc fusion proteins. Furthermore, amino acid substitutions in IL-15, exemplified by P-0313, did not affect the expression profile of the fusion protein; P-0313 showed almost the same purity and aggregation tendency as its wild-type counterpart P-0234 (Figure 2).

[0265] Example 2

[0266] Purity-focused developability assessment of different fusion protein formats highlights the role of correctly complexed IL-15Rα domains in enhancing the developability profile of fusion proteins

[0267] SEC analysis of purified Protein A samples was used to assess the effects of different fusion formats on protein aggregation propensity and purity. The inventors observed that while protein expression levels may vary between batches due to differences in cell growth, protein aggregation propensity and purity appear to be intrinsic properties associated with a specific protein, with minimal variability between batches, as shown in Figures 3F and 3H for P-0234.

[0268] First, the effect of the complex of IL-15Rα on the protein purity of IL-15-Fc fusion protein was evaluated based on 5 molecules. P-0162 is a C-terminal monomer IL-15 (alone) Fc fusion protein, comprising Hole-Fc-linker 1-IL-15 chain (SEQ ID NO: 13) and an empty Knob-Fc chain (SEQ ID NO: 7). P-0197 is a C-terminal monovalent IL-15 / IL-15Rα (non-covalent) Fc fusion, a schematic diagram of which is depicted in Figure 1B. The only difference between P-0197 and P-0162 is the presence of a free IL-15RαSushi+ domain that is non-covalently complexed with IL-15. P-0153 is a C-terminal monomer IL-15 / IL-15Rα fusion with a heterodimer Fc fusion form, a cartoon of which is shown in Figure 1A. P-0167 and P-0198 are the dimeric counterparts of P-0162 and P-0197, respectively. The size exclusion plots of the five molecules are illustrated in Figures 3A-3E.

[0269] As observed in Figure 3A, the IL-15-Fc monomer fusion without IL-15Rα has a monomer content of 84.5%, and most of the impurities have a lower molecular weight. In contrast, P-0197 contains a monomer content of 98.6% (Figure 3B), and this significant improvement in the protein is clearly promoted by the free IL-15RαSushi+ domain. For the dimeric form, the effect of the free IL-15RαSushi+ domain on the protein quality of the IL-15-Fc fusion protein is further highlighted, which is depicted by the SEC chromatograms of P-0167 and P-0198 in Figures 3D and 3E, respectively. P-0167 contains a wide and irregular peak with shoulders on both sides of the main peak. More notably, P-0167 did not show any in vitro activity in activating NK cells and T cells of fresh human PBMCs, which may be due to incorrect folding of the protein. However, P-0198, which contains the non-covalently bound IL-15RαSushi+ domain in dimeric form, exhibited a sharp main peak with a monomer content of 90.5% (Figure 3E). Somewhat interestingly, if the IL-15RαSushi+ domain is not free but covalently fused to a matching heterodimeric Fc as in P-0153, complexing the IL-15RαSushi+ domain with IL-15-Fc did not produce any improvement in protein purity; instead, the protein sample contained >25% dimers and higher molecular weight soluble aggregates (Figure 3C). Fusion of both IL-15 and IL-15Rα to the Fc domain may create steric constraints that prevent them from interacting in a physiological manner. In summary, the IL-15RαSushi+ domain can significantly improve the purity and biophysical properties of IL-15-Fc fusion proteins, but only if the IL-15Rα domain can associate with IL-15 in a favorable and unrestricted manner.

[0270] Secondly, the impact of IL-15RαECD (SEQ ID NO: 4) versus IL-15RαSushi+ (SEQ ID NO: 5) on the developability of IL-15 / IL-15Rα-Fc fusion proteins was evaluated by comparing P-0234 and P-0220. Both constructs are C-terminal Fc fusions, sharing the same configuration as depicted in Figure 1C, with the IL-15RαSushi+ domain in P-0234 and the IL-15RαECD in P-0220. Their SEC chromatograms are shown in Figures 3F and 3G, respectively. The protein P-0220, which contains the IL-15RαECD domain, not only has a lower purity than its counterpart P-0234 containing IL-15RαSushi+ (88.5% vs. 100%), but is also expressed at a 2.5-fold lower level in the same batch of cells. In short, it is clear that the IL-15Rα Sushi+ domain is a more preferred partner than the IL-15Rα ECD for constructing more developable IL-15 / IL-15Rα-Fc fusion proteins.

[0271] In addition, the effect of the fusion terminus on the developability of the IL-15 / IL-15Rα-Fc fusion protein was also evaluated. P-0234 and P-0223 are dimeric IL-15 / IL-15Rα (non-covalent) Fc fusion proteins, with the IL-15 / IL-15RαSushi complex attached to the C-terminus and N-terminus of Fc, respectively. Their SEC chromatograms (Figures 3H and 3I) showed quite subtle purity differences (98.5% vs. 93.9%). However, the purified P-0223 always contained a broad peak of higher molecular weight substances and a small but noticeable peak containing lower molecular weight impurities, which was essentially absent in P-0234. Despite the small difference, P-0234 with a C-terminal fusion had an indisputably better SEC purity profile, and considering that the two molecules were expressed at comparable levels, P-0234 was the preferred form from a developability perspective.

[0272] In summary, combining the IL-15Rα subunit with the IL-15-Fc fusion can significantly improve expression, purity, and reduce aggregation; and such improvements require that IL-15Rα and IL-15 are correctly associated with each other with minimal spatial constraints. And based on both productivity and purity assessments, the truncated form of the IL-15Rα ECD, IL-15RαSushi+, performed better than the full-length ECD. In addition, placing the IL-15 / IL-15Rα complex at the C-terminus of the Fc is more conducive to obtaining high purity than N-terminal fusion. Therefore, the dimeric IL-15 / IL-15Rα (non-covalent) C-terminal Fc fusion form exemplified by P-0234 integrates all the preferred components and represents a superior form.

[0273] Example 3

[0274] Non-covalent association of IL-15Rα enhances receptor binding and bioactivity of IL-15 / IL-15RαFc fusion protein

[0275] IL-15 binds to its specific receptor IL-15Rα with high affinity, and both are expressed on antigen-presenting cells. The association of IL-15Rα with IL-15 trans-presents IL-15 to responsive lymphocytes (including NK cells, T cells, and B cells) via IL-15Rβ and γ C The formation of the ligand-trimeric receptor complex (IL-15-IL-15Rαβγ) initiates an intracellular signaling cascade, leading to downstream biological effects. The complexity of IL-15 receptor biology makes it challenging to design optimal IL-15 fusion proteins to achieve a conformationally effective ligand-trimeric receptor signaling complex.

[0276] We hypothesized that covalent attachment of IL-15 to the N-terminus or C-terminus of the Fc portion of human IgG would enhance in vivo half-life compared to non-covalent association of IL-15 with an IL-15RαFc fusion protein. We also hypothesized that the IL-15Rα domain is required for the IL-15 Fc fusion protein to enhance its interaction with the intermediate-affinity IL-2Rβγ receptor and promote the formation of the high-affinity ligand-trimeric receptor signaling complex. Furthermore, we proposed that non-covalent association of the IL-15Rα domain with the IL-15 Fc fusion protein preserves the native association of IL-15 and IL-15Rα and the optimal conformation for IL-15 trans-presentation. Furthermore, we hypothesized that the generation of such a fusion protein complex would be feasible due to the extremely high binding affinity between the IL-15 and IL-15Rα domains.

[0277] Different configurations of IL-15 Fc fusion proteins containing the IL-15 / IL-15Rα domain complex were constructed. The binding activity of the fusion proteins to the IL-15Rβ subunit was determined by measuring CD69 expression on human CD8 and NK cells, and their biological activity in stimulating lymphocyte activation was analyzed. An exemplary structural diagram of the fusion protein is shown in Figure 1.

[0278] Binding activity was tested using an ELISA assay. Briefly, Nunc Maxisorp (ThermoFisher) plates were coated overnight with huIL-15Rβ-6His at 1 μg / well (100 μl / well) in bicarbonate buffer pH 9.4 (ThermoFisher) at 4°C. After washing three times with PBS / 0.05% Tween 20, the plates were incubated with SuperBlock (300 μl / well) for 2 hours at room temperature to block nonspecific binding. After washing, IL-15 compounds (each serially diluted 3-fold with blocking buffer) were added to the plates (100 μl / well) and incubated at room temperature for 1 hour. After washing, Fc fusions were detected by incubating with a goat anti-human IgG Fc secondary antibody conjugated to horseradish peroxidase (HRP) (100 μl / well) diluted 1:5000 in blocking buffer (ThermoFisher) at room temperature for 1 hour. After washing, TMB substrate (ThermoFisher) (100 μL / well) was added. The plate was sealed and incubated at room temperature in the dark for 5-20 min. The reaction was stopped by adding 2N sulfuric acid (Ricca Chemical) (50 μL / well) and the absorbance was read at 450-590 nm.

[0279] Biological activity was determined by measuring the induction of CD69 expression on human NK cells and CD8 T cells. CD69 is a cell surface glycoprotein that is induced early in the process of lymphocyte activation. An in vitro human peripheral blood mononuclear cell (PBMC) assay was established to analyze the number / percentage of NK cells or CD8 T cells expressing CD69 after IL-15 treatment. Specifically, human PBMCs were isolated from buffy coats purchased from the Blood Oklahoma Institute by Ficoll-Hypaque centrifugation. Purified human PBMCs were treated with serial dilutions of each IL-15 test compound and incubated at 37°C for 48 hours. Cells were collected by centrifugation at 300G and resuspended in FACS buffer. After blocking Fc receptors by adding human TruStain FcX (1:50 dilution), cells were stained with anti-human CD56-FITC antibody, anti-human CD69-PE antibody, and anti-human CD8-APC antibody (1:50 dilution). After incubation with antibodies for 30 minutes at room temperature, cells were collected and washed, resuspended in FACS buffer, and prepared for flow cytometry analysis. CD69 expression on CD56+ NK cells and CD8+ T cells was determined, and data are expressed as the % of CD69-positive cells in the gated population.

[0280] P-0157 is an N-terminal bivalent IL-15 (non-covalent) / IL-15RαSushi Fc fusion protein; P-0153 is a C-terminal IL-15 / IL-15RαSushi heterodimeric Fc fusion protein; P-0162 is a C-terminal monovalent Fc-IL-15 fusion protein without complexed IL-15RαSushi. ELISA binding assays showed that the incorporation of the IL-15RαSushi domain significantly increased the binding strength of IL-15 Fc fusion proteins (P-0157 and P0153) to IL-15Rβ compared to the fusion protein without complexed IL-15Rα (P-0162). Figure 4 ), indicating the important role of IL-15Rα in promoting the interaction between the fusion protein and the receptor. In addition, compared with IL-15 non-covalently bound to the IL-15RαFc fusion protein (P-0157), the receptor binding activity was reduced when both IL-15 and IL-15Rα were covalently conjugated to Fc (P-0153) in the form of heterodimers ( Figure 4 ), suggesting that conformationally restricted fusion forms negatively impact receptor binding activity.

[0281] Consistent with the binding capacity, the incorporation of IL-15Rα also increased the biological activity of the IL-15 fusion protein compared to the fusion protein without IL-15Rα. P-0197 is a C-terminal monovalent IL-15 / IL-15Rα (non-covalent) Fc fusion protein, and P-0162 shares the same structure with P-0197 but does not contain IL-15RαSushi. Compared with P-0162, P-0197 showed a 10-fold and 6-fold increased potency in inducing CD69-positive NK cells (Figure 5A) and CD8 T cells (Figure 5B), respectively, which is attributed to the inclusion of IL-15Rα.

[0282] Finally, the biological activities of three C-terminal monovalent IL-15 / IL-15RαFc fusion proteins were compared. P-0165 is an Fc fusion protein with IL-15Rα fused to the Fc domain and non-covalently bound IL-15; P-0197 is a reverse arrangement with IL-15 fused to the Fc domain and non-covalently bound IL-15Rα; P-0153 has a heterodimeric structure with both IL-15 and IL-15Rα fused to the Fc domain. As shown in Figure 6, in terms of inducing CD69-positive NK cells (Figure 6A) and CD8 T cells (Figure 6B), the fusion proteins with IL-15 or IL-15Rα non-covalently complexed showed better efficacy than the fusion proteins with both IL-15 / IL-15Rα covalently and heterodimerically fused to Fc. Our data suggest that optimal conformational association between IL-15 and IL-15Rα is crucial for the IL-15 fusion protein to bind to its receptor and exert its biological activity. Increased conformational constraints, such as in heterodimeric Fc-fusion formats, negatively impact biological activity.

[0283] Example 4

[0284] Effect of linkers on the activity of IL-15 / IL-15RαFc fusion protein

[0285] Selecting an appropriate linker to connect protein domains is crucial in fusion protein engineering. Peptide linkers not only provide spatial distance between fusion protein domains and allow them to fold independently, but also directly influence the structural stability and functional properties of the fusion protein. Here, we examined the effects of linker flexibility and length on the bioactivity of an IL-15 / IL-15RαFc fusion protein.

[0286] As previously described, bioactivity was determined by measuring the induction of CD69 expression on human NK cells and CD8 T cells in an ex vivo human PBMC FACS-based assay. P-0165 and P-0166 are monovalent IL-15 (non-covalent) / IL-15Rα Fc fusions with a 15-amino acid rigid linker and a 10-amino acid flexible linker, respectively. P-0197, P-0207, and P-0217 are monovalent IL-15 / IL-15Rα (non-covalent) Fc fusion proteins with a rigid linker, a 10-amino acid GS-rich flexible linker, and a 15-amino acid GS-rich flexible linker, respectively. The results showed that the rigidity or length of the peptide linkers connecting Fc and IL-15 (P-0197, P-0207, and P-0217) or connecting Fc and IL-15Rα (P-0165 and P-0166) did not affect the biological activity of the tested fusion proteins (Figures 7A and 7B).

[0287] Example 5

[0288] Effect of valence on the activity of IL-15 / IL-15Rα fusion protein

[0289] Most Fc fusion proteins for therapeutic use are homodimeric because IgG1 Fc naturally homodimerizes due to disulfide bonds formed in the hinge region. Dimeric proteins offer advantages in terms of avidity, stability, quantity, size, and functionality. However, Fc engineering can produce monomeric Fc fusion proteins. This change can affect biological activity, pharmacokinetics, side effects, or reduce the size of the dimeric protein to facilitate tissue penetration. To evaluate the effect of valency on the biological activity of IL-15 / IL-15RαFc fusion proteins, monomeric and homodimeric forms with different IL-15 / IL-15RαFc fusion configurations were constructed and tested for their biological activity.

[0290] As previously described, bioactivity was determined by measuring the induction of CD69 expression on human NK cells and CD8 T cells in an ex vivo human PBMC FACS-based assay. The results showed that across all fusion formats tested, the homodimeric form of the IL-15 / IL-15RαFc fusion protein exhibited approximately a 2-fold enhancement in bioactivity compared to the respective monomeric counterparts (Table 2), suggesting that dimeric valency can provide functional advantages.

[0291] Table 2

[0292] Effect of the valence of IL-15 / IL-15RαSushi complex on the biological activity of IL-15 / IL-15Rα fusion protein

[0293]

[0294] Example 6

[0295] Effects of N-terminal Fc fusion or C-terminal Fc fusion on the biological activity of IL-15

[0296] Fc fusion proteins can be constructed by placing the IL-15 / IL-15Rα complex (connected by a spacer linker) at the N-terminus or C-terminus of the Fc. The optimal scaffold is determined by whether the fusion protein is correctly folded and expressed and whether biological activity is retained. The IL-15 / IL-15RαFc fusion protein is generated by attaching the IL-15 / IL-15Rα complex (separated by a linker) to the N-terminus or C-terminus of the Fc. The IL-15 / IL-15Rα complex also has different configurations in the case of C-terminal fusion and N-terminal fusion. P-0218 is a C-terminal bivalent IL-15 (non-covalent) / IL-15RαFc fusion protein, and the benchmark is the N-terminal counterpart of P-0218 (which additionally contains the N72D substitution in IL-15). P-0234 is a C-terminal bivalent IL-15 / IL-15Rα (non-covalent) Fc fusion protein, and P-0223 is the N-terminal counterpart of P-0234.

[0297] The biological activity of the fusion protein was determined by measuring Ki67 expression in the nuclei of NK cells and CD8 T cells after treatment with the IL-15 compound. IL-15 is a potent lymphocyte growth factor that stimulates the proliferation and differentiation of NK cells, T cells, and B cells. Ki67 is a nuclear protein that is induced in all active phases of the cell cycle (G1, S, G2, and M), but not in the resting phase (G0), and is therefore a marker of cell proliferation.

[0298] An in vitro human PBMC assay was established. Briefly, purified human PBMCs were treated with serial dilutions of the IL-15 test compound and incubated at 37°C for 3 days. Every 2 days, 50% of the culture medium was supplemented with fresh culture medium and test compound. On the 3rd day, the cells were washed once with FACS buffer (1% FBS / PBS) and first stained with Fc blocking agent and surface marker antibodies (including anti-human CD56-FITC, anti-human CD8-APC and anti-human CD4-Percp-cy5.5 (1:50 dilution)). After 30 minutes of incubation and washing, the cell pellet was fully resuspended with 200 μl / well of 1X Foxp3 fixation and permeabilization working solution and incubated in the dark at room temperature for 30 minutes. After centrifugation, 200 μl of 1X permeabilization buffer was added to each well for another wash. The cell pellet was resuspended in permeabilization buffer with anti-human Ki67-PE (1:10 dilution). After incubation at room temperature for 30 minutes, cells were collected and washed, resuspended in FACS buffer, and prepared for flow cytometric analysis. Data are expressed as the % of Ki67-positive cells in the gated population.

[0299] As shown in Figure 8, C-terminal Fc fusions (P-0218 and P-0234) consistently demonstrated stronger induction of Ki67-positive CD8 T cells or CD8 T cell proliferation compared to their N-terminal Fc fusion counterparts (benchmark and P-0223) (Figures 8A and 8B). The data indicate that the C-terminus of Fc is the preferred site for attachment of the IL-15 / IL-15Rα complex and allows for the preservation of biological activity.

[0300] Example 7

[0301] Effect of receptor-α domain selection on the activity of IL-15 / IL-15RαFc fusion protein

[0302] IL-15 binds to the extracellular domain (ECD) of IL-15Rα, and this binding is primarily caused by a conserved protein binding motif called the Sushi domain. For the construction of fusion proteins, short and truncated forms of IL-15Rα may be desirable to reduce size and structural complexity. To ensure binding specificity and affinity, all or part of the ECD domain that confers binding to IL-15 (the Sushi domain with an additional 12AA) is constructed into the fusion protein, and functional activity is determined.

[0303] P-0234 and P-0220 are C-terminal dimeric IL-15 / IL-15Rα (non-covalent) fusion proteins, in which the IL-15Rα is sushi and the intact ECD, respectively. P-0223 and P-0224 are N-terminal dimeric IL-15 / IL-15Rα (non-covalent) Fc fusion proteins, in which the IL-15Rα is sushi and the intact ECD, respectively. P-0221 and P-0222 are N-terminal monovalent IL-15 / IL-15Rα (non-covalent) Fc fusion proteins, in which the IL-15Rα is sushi and the intact ECD, respectively. The results showed that the fusion proteins non-covalently complexed with the IL-15Rα sushi domain were more effective in inducing CD69-positive NK cells than those complexed with the intact IL-15Rα ECD, regardless of whether the fusion format was N-terminal or C-terminal, dimer or monomeric (Figure 9). The data indicate that the IL-15RαSushi domain is more suitable than the complete ECD for constructing IL-15 / IL-15RαFc fusion proteins and confers an optimal conformation for the interaction of IL-15 with signaling receptors.

[0304] Example 8

[0305] Binding activity of IL-15 mutants and their fusion proteins to IL-15Rβ

[0306] When searching for IL-15 agonists, superagonists, or antagonists, deletions, insertions, or point mutations are introduced into the human IL-15 peptide sequence at the contact interface between IL-15 and receptor β or γ. The variants are introduced into different forms of IL-15 / IL-15RαFc fusion proteins, and the binding activity to IL-15Rβ is quantified by enzyme-linked immunosorbent assay (ELISA) as previously described.

[0307] Table 3 shows the IL-15Rβ binding activity of C-terminal IL-15 variants / IL-15Rα heterodimer Fc fusion proteins. The IL-15 variants have amino acid deletions, insertions, or point mutations introduced into the human IL-15 peptide. Compared with the full-length wild-type IL-15 fusion protein, truncating 3 amino acids from the C-terminus of IL-15 retains the binding activity of the fusion protein to IL-15Rβ, while further truncating 6 or 9 amino acids from the C-terminus of IL-15 causes the binding activity of the fusion protein to gradually decrease. Inserting GS of different lengths after N95 causes the IL-15Rβ binding activity of the fusion protein to decrease. Single point mutations (Q108S and Q108A) and combined mutations (Q108S, D30T, V31Y, H31E) at position 108 largely retain the Rβ binding ability of the wild-type fusion protein.

[0308] Table 4 shows the IL-15Rβ binding activity of monomeric IL-15 variant (non-covalent) / IL-15RαFc fusion proteins containing a single amino acid substitution at position 58, 62, 63, 67, or 68 of the human IL-15 domain. The Fc fusion protein P-0185 comprising the IL-15(I67V) variant exhibited similar binding activity to IL-15Rβ as the wild-type fusion protein. P-0182 is a fusion protein comprising an IL-15 variant having an amino acid substitution (S58D) from serine to aspartic acid at position 58, which exhibited a 4-fold increase in binding to IL-15Rβ compared to the wild-type fusion protein. Substitutions at positions 62, 63, and 68 of the IL-15 peptide resulted in varying degrees of reduction in the IL-15Rβ binding activity of the fusion protein.

[0309] Table 5 shows the IL-15Rβ binding activity of dimeric IL-15 variants / IL-15Rα (non-covalent) Fc fusion proteins, wherein human IL-15 comprises a single amino acid substitution at position 58 or 68 or an amino acid insertion after N95. Similarly, as observed in P-0182 (Table 4), P-0313, comprising the same IL-15 variant with S58D, exhibited a 2-fold enhancement in binding to IL-15Rβ compared to its corresponding wild-type fusion protein P-0234. The data reinforce the view that the S58D substitution in the IL-15 peptide can modulate IL-15 as a superagonist due to enhanced receptor binding activity.

[0310] In summary, IL-15 variant Fc fusion proteins were created and identified to have different IL-15Rβ binding activities. Some IL-15 variants showed reduced ability to bind to IL-15Rβ than their wild-type counterparts (Tables 3-5). Some variants, such as P-0173, P-0179, P-0180, P-0181, P-0185, retained binding activity to IL-15Rβ similar to that of the wild-type (Table 3). Single point mutations in which the serine at position 58 in the human IL-15 domain was replaced with aspartic acid provided enhanced binding activity of the fusion proteins (P-0182 and P-0313) to IL-15Rβ (Tables 4 and 5).

[0311] Table 3

[0312]

[0313] Table 4

[0314]

[0315] Table 5

[0316]

[0317] Example 9

[0318] Functional activity of IL-15 variant / IL-15RαFc fusion protein

[0319] The functional activity of Fc fusion proteins of IL-15 variant / IL-15Rα complexes in stimulating lymphocyte activation was evaluated. An ex vivo human PBMC assay was established as previously described to analyze the number / percentage of CD8 T cells expressing the lymphocyte activation marker CD69.

[0320] P-0234 is a C-terminal dimeric IL-15 / IL-15RαSushi (non-covalent) Fc fusion protein that was optimized by a combination of preferred configurations, including covalent attachment of IL-15 to Fc, C-terminal fusion, dimeric valence, and non-covalent IL-15RαSushi complexes. P-0313 is the S58D counterpart of P-0234. P-0313 shares the same fusion configuration as P-0234, but differs only in the S58D substitution in the IL-15 polypeptide. Previously, P-0313 exhibited increased IL-15Rβ binding activity compared to its wild-type counterpart, P-0234 (Table 5). Consistent with the enhanced binding activity to IL-15Rβ, the variant P-0313 containing the S58D mutation also exhibited increased potency in inducing CD69-positive T cells (Table 6), confirming that P-0313 exhibits superagonist activity. Interestingly, two IL-15 variant fusion proteins (P-0179 and P-0181) that bound to IL-15Rβ comparable to the wild-type fusion protein (Table 3) showed a complete loss of their ability to induce CD69-positive CD8 T cells (Table 6), suggesting that these two IL-15 variants may have impaired binding to γ C "These variants, including P-0179 and P-0181, can act as primary negative antagonists to block endogenous IL-15 function," said study senior author Dr.

[0321] Additional Fc fusion proteins of the IL-15 variant / IL-15RαSushi complex were also tested in the CD69 assay, and their bioactivity was either retained or reduced compared to the respective wild-type fusion proteins (Tables 6 and 7).

[0322] Table 6

[0323]

[0324] *NA, not applicable, too low to be quantified

[0325] Table 7

[0326]

[0327] *NA, not applicable—too low to quantify

[0328] Example 10

[0329] Signaling activity of IL-15(S58D) / IL-15RαFc fusion protein

[0330] The IL-15(S58D) variant exhibits increased binding activity to IL-15Rβ and enhanced ability to stimulate CD69-positive lymphocytes. The current study examined the signaling activity of the IL-15(S58D) / IL-15Rα fusion protein in stimulating intracellular phosphorylation of signal transducer and activator of transcription 5 (pSTAT5) in NK cells and T cells.

[0331] In the in vitro human PBMC assay after IL-15 compound treatment, STAT5 phosphorylation was determined by intracellular FACS analysis. Briefly, purified human PBMCs were treated with serial dilutions of IL-15 test compounds and incubated at 37°C for 15 minutes. At the end of the treatment, the cells were washed once with FACS buffer (1% FBS / PBS) and incubated at 37°C for 15 minutes in 150 μl / well preheated Cytofix fixation buffer. The fixed cells should be washed again and resuspended in 150 μl / well precooled Perm buffer II at 4°C for 30 minutes. After blocking Fc receptors by adding human TruStain FcX (1:50 dilution), the cells were stained with anti-human CD56-FITC, anti-human pSTAT5-PE, anti-human CD8-APC, and anti-human CD4-Percp-cy5.5 (1:50 dilution). After incubation with antibodies for 45 minutes at room temperature, cells were collected and washed, resuspended in FACS buffer, and prepared for flow cytometric analysis. Data are expressed as the % of pSTAT5-positive cells in the gated population.

[0332] The S58D mutation was introduced into two forms of Fc fusion proteins: a bivalent IL-15 (non-covalent) / IL-15Rα Fc fusion and an IL-15 / IL-15Rα (non-covalent) Fc fusion. P-0218 and P-0314 are C-terminal dimeric IL-15 (non-covalent) / IL-15Rα Fc fusion proteins containing wild-type IL-15 and the S58D variant IL-15, respectively. P-0234 and P-0313 are C-terminal dimeric IL-15 / IL-15Rα (non-covalent) Fc fusion proteins containing wild-type IL-15 and the S58D variant IL-15, respectively. In CD8 T cells (Figure 10A), CD4 T cells (Figure 10B), and NK cells (Figure 10C), regardless of fusion configuration, IL-15 (S58D) variant fusion proteins (P-0314 and P-0313) exhibited approximately a 2-fold increase in the ability to stimulate STAT5 phosphorylation compared to their respective wild-type fusion proteins (P-0218 and P-0234). The data demonstrate that the S58D substitution in the IL-15 peptide results in superagonist activity of various IL-15 proteins.

[0333] Example 11

[0334] Cell proliferation activity of IL-15(S58D) / IL-15-RαFc fusion protein

[0335] After observing an increased ability to bind to IL-15Rβ, stimulate STAT5 phosphorylation, and induce CD69 expression, the ability of the IL-15(S58D) variant Fc fusion protein to stimulate cell proliferation compared to the wild-type fusion protein was tested by measuring Ki67 expression in NK cells and CD8 T cells. Human PBMCs were treated with increasing doses of the IL-15 fusion molecule, and Ki67 expression was determined by intracellular FACS analysis gating on the CD56+ NK cell population and CD8+ T cell population, as previously described.

[0336] Similar to what was observed for STAT5 phosphorylation shown in (Figure 10), the fusion protein of the IL-15(S58D) variant also exhibited a 2-fold increase in the ability to stimulate Ki67 expression in CD8+ T cells (Figure 11A) and CD4+ T cells (Figure 11B) as well as CD56+ NK cells (Figure 11C) compared to the wild type. These data reinforce that the introduction of the S58D mutation into the IL-15 domain provides an enhancement of a range of biological activities, including receptor binding, intracellular signaling, activation of cell surface markers, and cell proliferation.

[0337] Example 12

[0338] A 4-Day Repeated Dosing Study of P-0234 Compared to rhIL-15 and Benchmark in Mice

[0339] The IL-15 / IL-15RαFc fusion protein exhibits strong binding to IL-15Rβ in vitro and ex vivo, inducing intracellular signaling cascades and stimulating the proliferation of NK and CD8 T lymphocytes. Here, we examined the serum exposure of various IL-15 compounds in mice and their effects on the proliferation and expansion of mouse NK cells. The proteins tested included recombinant human native IL-15 (rhIL-15), P-0234 (a C-terminal bivalent IL-15 / IL-15Rα (non-covalent) Fc fusion protein), and a benchmark compound (an N-terminal bivalent IL-15 (non-covalent) / IL-15Rα Fc fusion protein containing the N72D mutation in IL-15).

[0340] 7-week-old female Balb / c mice are received from Charles River Laboratory and adapted to at least 7 days in the institution before being studied. Mice are given IL-15 compounds of equal molar doses of ip injection every day for 4 days. Processing includes vehicle, 0.03mg / kg rhIL-15 (40pmol / kg), 0.1mg / kg and 0.5mg / kg benchmark (40pmol / kg and 200pmol / kg) and 0.1mg / kg and 0.5mg / kg P-0234 (40pmol / kg and 200pmol / kg). There are 5 mice in each group. Body weight is recorded every day before and during the treatment. Mice are put to death 1 hour after the last injection, and terminal blood is collected by cardiac puncture.

[0341] Heparinized whole blood and spleen are collected to carry out NK cell phenotypic analysis and Ki67 intracellular staining.After lysis red blood cell and with the anti-mouse CD16 / CD32 (1:50 dilution) blocking Fc receptor of purification, to the NK cell surface marker staining (including anti-mouse CD3-FITC and anti-mouse CD49b-APC (1:50 dilution)) of mononuclear blood cell and splenocyte in single cell suspension, at room temperature in the dark for 30 minutes.For intracellular Ki67 staining, with the 1X Foxp3 fixation / permeabilization working solution of 200ul / hole, cell precipitation is fully resuspended, and incubated in the dark for 30 minutes at room temperature. Wash cell with the 1X permeabilization buffer solution of 200ul, and with the anti-mouse CD16 / CD32 (1:50 dilution) blocking Fc receptor of purification.Then for NK cell colony, use Ki67-PE and anti-mouse CD3-FITC and anti-mouse CD49b-APC to dye cell (1:50 dilution). After 30 min of incubation, cells were collected, washed, resuspended in FACS buffer, and prepared for flow cytometry analysis. Statistical analysis was performed by one-way ANOVA with Tukey's post hoc multiple comparisons in GraphPad Prism software.

[0342] Figure 12 The serum concentration of IL-15 is shown 1 hour after the last injection of IL-15 compound. IL-15 was measured by a commercial ELISA kit for detecting IL-15 according to the manufacturer's instructions (R&D systems; Cat# DY247). After 4 daily doses of the compound given at equimolar doses, the cumulative serum concentration of IL-15 was highest in mice treated with P-0234, intermediate in mice treated with the benchmark, and lowest in mice treated with rhIL-15 ( Figure 12 ). Comparing the baseline and P-0234 administered at 0.1 mg / kg and 0.5 mg / kg, P-0234 consistently demonstrated serum concentrations that were 6-fold and 4-fold higher than the baseline. The mean serum concentrations of IL-15 were: 3.2 ± 0.6 (ng / ml) for rhIL-15 (0.03 mg / kg dose), 13 ± 8 and 121 ± 36 (ng / ml) for the baseline compound (0.1 mg / kg and 0.5 mg / kg doses, respectively), and 72 ± 14 and 443 ± 57 (ng / ml) for P-0234 (0.1 mg / kg and 0.5 mg / kg doses, respectively). The higher serum exposure suggests that P-0234 may exhibit a longer in vivo half-life and serum retention compared to the baseline and rhIL-15.

[0343] Although there was no difference in total body weight between treatment groups, mice treated with the higher dose of the benchmark lost nearly 6% of their body weight over the 4 days of treatment (Figures 13A and 13B). The effect was statistically significant compared to baseline values ​​on day 0 and the vehicle group, indicating that potential dose-limiting toxicity was observed with the benchmark compound, but not P-0234.

[0344] All IL-15 compounds tested increased the percentage of Ki67 positive NK cells in peripheral blood (Figure 14 A), demonstrating enhanced NK cell proliferation. However, the significant increase in the percentage of NK cell numbers in CD3 negative peripheral blood lymphocytes was observed only in mice treated with P-0234 at two test dose levels (Figure 14 B). Although an increase in NK cell percentages was also observed in mice treated with rhIL-15 and lower doses of the benchmark, the impact did not reach statistical significance (Figure 14 B). Interestingly, a decline in the number of NK cells in peripheral blood was observed in mice treated with higher doses of the benchmark (Figure 14 B). Such a reverse pharmacodynamic dose-response suggests toxicity consistent with the observed weight loss in this group.

[0345] The effects of IL-15 compounds on lymphocyte proliferation and amplification were also examined in the lymphoid organ spleen. Similar to what was observed in peripheral blood, all IL-15 compounds increased spleen Ki67 positive NK cells compared to vehicle (Figure 15A). Only low-dose benchmarks and P-0234 significantly increased the total number of NK cells in the spleen (Figure 15B). Similarly, the reverse dose-response of the benchmark to spleen NK cell amplification was observed (Figure 15B), and this was related to the high and lasting CD69 expression on the spleen NK cells measured 4 days after termination (Figure 15C). Data show that benchmark compounds may overstimulate NK cells and cause cell consumption. For IL-15 non-covalently bound to IL-15RαFc fusion protein, IL-15 may dissociate from the fusion complex and cause lymphocyte overstimulation, cell consumption, toxicity and weight loss.

[0346] Example 13

[0347] Pharmacokinetic and pharmacodynamic effects of IL-15 / IL-15RαFc fusion protein after single injection in mice

[0348] A dose-response study of P-0313 (a C-terminal bivalent IL-15 (S58D) / IL-15Rα (non-covalent) Fc fusion protein) was conducted in Balb / C mice following a single injection. The effects on peripheral blood lymphocyte proliferation and expansion were monitored over time. Furthermore, the pharmacokinetics and pharmacodynamics (PK / PD) of P-0313 were compared with those of a benchmark (an N-terminal bivalent IL-15 (non-covalent) / IL-15Rα Fc fusion protein containing the N72D mutation in IL-15) following a single injection.

[0349] The female balb / c mice of 7 week age receives from Charles River laboratory, and adapts to at least 7 days in institution, is studied then.In time 0, use vehicle, benchmark (0.3mg / kg) or P-0313 (0.01mg / kg, 0.03mg / kg, 0.1mg / kg and 0.3mg / kg) to mice ip.In-24hr (before administration) and injection back 1 hour, 4 hours, 24 hours, 72 hours, 120 hours and 192 hours blood drawing.Before processing and during processing, record body weight every day.Every group comprises 5 mice.

[0350] Heparinized whole blood was used for immunophenotyping and the volume was recorded. After lysing red blood cells using BD pharm lysis buffer, the total number of surviving mononuclear blood cells was counted by trypan blue dead cell exclusion method and Ki67 intracellular staining was performed. The cell pellet was fully resuspended with 200ul / well 1X Foxp3 fixation / permeabilization working solution and incubated in the dark for 30 minutes at room temperature. After centrifugation, 200ul of 1X permeabilization buffer was added to each well for another wash. After blocking Fc receptors with purified anti-mouse CD16 / CD32 (1:50 dilution), cells were stained with anti-mouse CD3-FITC, Ki67-PE, anti-mouse CD49b-APC and anti-mouse CD8-Percpy5.5 (1:50 dilution). After incubation for 30 minutes, cells were collected and washed, resuspended in FACS buffer, and analyzed by flow cytometry. Statistical analysis was performed by one-way ANOVA with Tukey's multiple comparisons in GraphPad prism software.

[0351] Two different ELISA assays were used to measure the serum concentration of the compound. An internal ELISA assay was developed to measure IL-15 and Fc complexes, while a commercial ELISA assay only measured IL-15 (both the capture antibody and the detection antibody reacted with human IL-15). For the internal ELISA assay, maxisorp plates were coated with anti-IL-15 antibodies (R&D systems MAB647) at 4°C overnight. The plates were blocked with SuperBlock. Standards and samples of varying dilutions were applied to the plates and incubated at room temperature for 1 hour. Active compounds were detected using anti-human IgG Fc-HRP and Ultra TMB substrate solution detection signals. Values ​​were calculated using GraphPad Prism interpolation from nonlinear regression curve fitting.

[0352] Both compounds were detectable in the serum for the first 24 hours with comparable serum concentrations. Peak concentrations were observed 4 hours after ip administration. At 72 hours, only P-0313 remained measurable, while the benchmark became undetectable in all three mice (Figures 16A-16B). Similar results were obtained using two different ELISA assays, confirming that P-0313 had a pharmacokinetic profile superior to the benchmark. This result confirmed the observations shown in previous Example 12, i.e., P-0234 (IL-15 / IL-15Rα (non-covalent) Fc fusion protein) also exhibited serum exposure higher than the benchmark. These data strongly support that, in terms of the extension of the IL-15 in vivo half-life, the IL-15 / IL-15Rα (non-covalent) Fc fusion configuration is superior to the configuration of the IL-15 (non-covalent) / IL-15RαFc fusion protein.

[0353] No significant changes in body weight were observed in any of the treatment groups ( Figure 17 ).

[0354] In mice treated with P-0313, a dose-dependent increase in Ki67 expression was observed in NK cells and CD8 T cells (Figures 18 A and 18 B). The effect reached a peak at 72 hours and lasted to 120 hours for the benchmark, and further extended to 192 hours (Figure 18 A) for P-0313, indicating that P-0313 is longer than the effect of the benchmark. In addition, P-0313 showed similar Ki67 induction when the dosage was 3-10 times lower than the dosage of the benchmark, indicating that P-0313 is more effective than the benchmark (Figures 18 A and 18 B). At a 10-fold low dosage, a significant response of NK cells was observed compared to CD8 T cells, indicating that NK cells are more sensitive to P-0313 processing than CD8 T cells.

[0355] Consistent with the increase of observed cell proliferation marker Ki67, a dose-dependent amplification of NK cells and CD8+ T cells in the blood was observed in the P-0313 treatment group (Figures 19 A and 19 B). Cell amplification was observed at 72 hours and peaked at 120 hours. P-0313 at 0.01mg / kg, 0.03mg / kg, 0.1mg / kg and 0.3mg / kg dosages increased NK cells by 4 times, 15 times, 50 times and 163 times from baseline (Figure 19 B), and at 0.1mg / kg and 0.3mg / kg dosages, CD8 T cells were also increased by 10 times and 50 times (Figure 19 B) from baseline. In contrast, the benchmark of 0.3mg / kg dosage only expanded peripheral NK cells by 28 times and expanded CD8 T cells by 12 times (Figures 19 A and 19 B).

[0356] In conclusion, P-0313 exhibited superior pharmacokinetics and pharmacodynamics to the benchmark compound for NK cell and CD8 T cell proliferation and expansion.

[0357] Example 14

[0358] Effect of IL-15 / IL-15RαFc fusion protein on the inhibition of lung metastasis of colon cancer in mice

[0359] To investigate the anti-metastatic efficacy and immune response of IL-15 / IL-15Rα-Fc fusion protein in tumor models, 1x10 5 Individual mouse colon cancer cells CT26-WT (ATCC CRL-2638) were intravenously injected into female balb / C mice (10-12 week old). The next day, every five days (day 1, day 6, day 11 after cell transplantation) were injected with 0.03mg / kg or 0.1mg / kg of P-0313 or the benchmark compound of 0.3mg / kg by iv injection. Vehicle (PBS) was included as a negative control, and each group comprised 8 mice. On the 15th day, blood samples were collected and lymphocyte phenotyping and liver enzyme measurement were performed. On the 16th day, all mice were put to death and tissue harvested. Lungs were expanded with 15% India ink and decolorized in Fekete solution (10% formaldehyde, 5% glacial acetic acid and 60% ethanol). Under an optical microscope, the lung tumor nodules of the whole lung were counted, and the anti-metastatic effect was represented by the different numbers of tumor nodules between the treated group and the vehicle control.

[0360] To investigate immune responses, peripheral blood was collected from mice in heparin-treated tubes on day 15, and the blood volume used for the assay was recorded for each mouse. After erythrocyte lysis with BD pharm lysis buffer, the total number of viable mononuclear blood cells was counted by trypan blue dead cell exclusion and used for intracellular staining for immune cell phenotyping and Ki67 proliferation analysis as previously described. After cell fixation, permeabilization, and antibody staining, cells were collected, washed, resuspended in FACS buffer, and analyzed by flow cytometry.

[0361] Figure 20 A shows the representative photos of the lung from each group, to illustrate lung nodules. Microscopy counting and quantitative (Figure 20 B) are carried out to lung metastasis lesions. As shown in Figure 20, the benchmark molecule given with 0.3mg / kg has suppressed lung metastasis, and lung nodule count reduces by 84%, confirming that activation IL-15 approach effectively prevents the formation and growth of lung metastasis. Remarkably, using IL-15 / IL-15Rα-Fc compound P-0313 with 3 times of low dosage (0.1mg / kg) causes the complete inhibition of lung metastasis development, and in all 8 mice processed, observe zero nodules (Figure 20 A and Figure 20 B). P-0313 is superior to benchmark in suppressing the formation and growth of lung metastasis, and this is consistent with the pharmacokinetic and pharmacodynamic effects of the enhanced previously demonstrated (embodiment 12 and embodiment 13). Compared to the benchmark (which contains IL-15 non-covalently linked to the Fc chain via the IL-15RαSushi domain), P-0313 (whose IL-15 portion is covalently linked to the Fc domain) demonstrated a significant increase in IL-15 serum half-life (Example 13). P-0313 administered at 0.03 mg / kg also reduced lung metastasis with an inhibitory effect of ∼35% (Figures 20A and 20B). This observation further highlights that P-0313 is effective at a much lower dose than the benchmark and highlights the importance of the biodistribution and bioavailability of the IL-15 / IL-15Rα-Fc complex for its anti-cancer effects in vivo.

[0362] After three repeated Q5D dosing, the expansion of both NK cells and CD8+ T cells in the peripheral blood remained significantly elevated in P-0313-treated mice (Figures 21A and 21B), which was associated with a significant increase in spleen weight in this treatment group ( Figure 22 In contrast, only a very slight expansion of CD8+ T cells was observed in the group treated with 0.3 mg / kg baseline; no increase in the number of circulating NK cells was observed compared to the control group (Figures 21A and 21B). However, spleen weight was significantly increased in the baseline treated group ( Figure 22The data suggest that after repeated dosing, expanded lymphocytes may migrate to lymphoid tissue for storage, or that cell depletion may occur. Since all three treatment groups showed anti-tumor effects, the data suggest that CD8+ T cells or NK cells may be the effector subsets involved in the anti-tumor effect. However, the complete eradication of lung metastases observed in the group treated with 0.1 mg / kg of P-0313 suggests that the strongest inhibition of tumor growth was induced by the actions of both NK cells and CD8+ T cells.

[0363] Liver weight and serum concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured to assess treatment-related hepatotoxicity. As depicted in Figures 23A-23C, there was no increase in liver weight, ALT levels, or AST levels in any treatment group compared to the vehicle group. The data indicate that the potent anti-tumor effect of the IL-15 / IL-15RαFc fusion protein was not associated with hepatotoxicity.

[0364] Example 15

[0365] Effects of IL-15 / IL-15RαFc fusion protein on the growth of established CT26 solid tumors in mice

[0366] To further investigate the antitumor efficacy and immune response of IL-15 / IL-15RαFc fusion protein in established tumor models, female Balb / C mice (10-12 weeks old) were injected subcutaneously in the right flank with 1×10 5 On day 11, when the average tumor volume was 70 mm 3 Mice were randomly divided into three groups (n=10 / group) and received an intraperitoneal injection of vehicle (PBS) or P-0313 (0.1 mg / kg or 0.05 mg / kg) on ​​the same day of randomization. An additional intraperitoneal injection of each test agent was performed on day 16 (2 doses in total). Tumors were measured three times a week using a caliper, and tumor volume was calculated as follows: volume = 0.5 x (width) 2 x (length). To study immune responses, non-terminal peripheral blood was collected in heparin-treated tubes on day 19. On day 21, all mice were sacrificed for tissue harvest.

[0367] As shown in Figure 24 A, the mice that PBS handles develops large subcutaneous tumor rapidly. P-0313 treatment mice with 0.1mg / kg or 0.05mg / kg are roughly equal in terms of delaying tumor growth (Figure 24 A). Tumor growth curves (Figure 25 A-Figure 25 C) of each individual mouse have been drawn to all three treatment groups. Obviously, mice respond well to the treatment of P-0313, and demonstrate delayed and synchronous tumor growth inhibition, particularly in the initial stage (Figure 25 A-Figure 25 C) of two test dose groups. On the 21st day after tumor inoculation, the average tumor volume in the mice treated with PBS was 820mm. 3 In comparison, the mean tumor volume in mice treated with both doses of P-0313 was 410 mm 3 ( FIG. 25A , **P<0.01; one-way ANOVA and Tukey post hoc test) Notably, initially, P-0313 at the higher dose (0.1 mg / kg) showed a greater reduction in tumor burden than the lower dose group, but the difference gradually diminished with treatment.

[0368] Over the course of the 21-day study, P-0313-treated mice exhibited similar weight gain as PBS-treated mice ( FIG. 24B ), indicating that P-0313 was well tolerated at both doses tested and was not associated with significant toxicity.

[0369] Next, we examined the effects of P-0313 on CD8 T cells and NK cell colonies in peripheral blood and spleen. P-0313 was administered to tumor-bearing mice and induced strong NK cells and CD8 T cell proliferation (Figure 26 A and Figure 26 B) and dose-dependent amplification (Figure 26 C and Figure 26 D) of NK cells and CD8 T cells, and similar immune cell responses (Example 13) were observed in non-tumor-bearing mice. In these two lymphocyte colonies, a higher multiple of change (~100 times for the 0.1 mg / kg administration group and ~38 times for the 0.05 mg / kg administration group) was observed in NK cells. CD8+ T cells expanded ~5.4 times at 0.1 mg / kg dosage, and expanded ~2.7 times for the 0.05 mg / kg administration group.

[0370] P-0313 also enhanced the expansion of both NK cells and CD8+ T cells in the spleen, as in peripheral blood (Figures 27A and 27B), but the amplitude / fold change in the spleen was less obvious. A higher fold change was observed in NK cells (~10 times for the 0.1 mg / kg administration group and ~8 times for the 0.05 mg / kg administration group). CD8+ T cells expanded ~2.7 times at the 0.1 mg / kg dose and only slightly expanded for the 0.05 mg / kg administration group.

[0371] Taken together, these data demonstrate that P-0313 treatment significantly delays and inhibits solid tumor growth, and that this antitumor effect is associated with the proliferation and expansion of cytotoxic NK cells and CD8 T cells in tumor-bearing mice, consistent with the overall immunomodulatory properties of IL-15. Because P-0313 contains an Fc region lacking effector function, the antitumor activity of P-0313 in vivo is not due to direct killing of tumor cells, but rather to robust activation of cytotoxic CD8+ T cells and NK cells, leading to an effective immune response against tumor cells.

[0372] Example 16

[0373] Effects of IL-15 / IL-15RαFc fusion protein on the growth of unestablished CT26 solid tumors in Balb / C mice

[0374] Similar studies were performed in an unestablished CT26 tumor model to confirm the antitumor efficacy of P-0313. 5 Three days after subcutaneous implantation of tumor cells with CT26 cells, mice received intraperitoneal injections of vehicle (PBS) or P-0313 (0.1 mg / kg) every five days for a total of five injections. Mice were killed on day 25 and tumors were measured 2-3 times per week.

[0375] Similar to what was observed in the established CT26 tumor model (Example 14), P-0313 demonstrated significant inhibition of tumor growth (Figure 28A) and a significant reduction in solid tumor mass (Figure 28B). For 5 repeated doses, mice treated with P-0313 showed a moderate increase in spleen weight (Figure 29A) and no significant reduction in body weight gain (Figure 29B), indicating that P-0313 was well tolerated.

[0376] Overall, these data demonstrate that P-0313 is an effective immunotherapeutic agent against solid and liquid tumors, as well as tumor cell metastases, with a well-tolerated safety profile.

[0377] All articles and methods disclosed and claimed herein can be prepared and performed without undue experimentation in view of the present disclosure. Although the articles and methods of the present disclosure have been described according to preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the articles and methods without departing from the spirit and scope of the present disclosure. It will be apparent to those skilled in the art that all such variations and equivalents, whether existing or developed later, are considered to be within the spirit and scope of the present disclosure as defined by the appended claims. All patents, patent applications, and publications mentioned in the specification indicate the level of ordinary technicians in the field to which the present disclosure belongs. For all purposes, all patents, patent applications, and publications are incorporated herein by reference in their entirety, and to the extent that each individual publication is individually and specifically indicated to be incorporated herein by reference in its entirety for any and all purposes. The present disclosure exemplarily described herein can be suitably practiced in the absence of any one or more elements not specifically disclosed herein. It will be understood, therefore, that although the present disclosure has been particularly disclosed by preferred embodiments and optional features, those skilled in the art may seek modifications and variations of the concepts disclosed herein, and that such modifications and variations are considered to be within the scope of the present disclosure as defined by the appended claims.

[0378] Sequence Listing

[0379] The nucleic acid and amino acid sequences listed in the accompanying Sequence Listing are shown using the standard letter abbreviations for nucleotide bases and the single-letter code for amino acids as set forth in 37 CFR 1.822.

[0380] SEQ ID NO: 1 is the amino acid sequence of human IL-15 precursor.

[0381] SEQ ID NO: 2 is the amino acid sequence of the mature form of human IL-15.

[0382] SEQ ID NO: 3 is the amino acid sequence of human IL-15R.

[0383] SEQ ID NO: 4 is the amino acid sequence of the extracellular domain of human IL-15Rα.

[0384] SEQ ID NO: 5 is the human IL-15Rα sushi domain + amino acid sequence.

[0385] SEQ ID NO: 6 is the amino acid sequence of human IgG1-Fc.

[0386] SEQ ID NO: 7 is the Knob-Fc amino acid sequence.

[0387] SEQ ID NO: 8 is the Hole-Fc amino acid sequence.

[0388] SEQ ID NOs: 9-12 are the amino acid sequences of various peptide linkers.

[0389] SEQ ID NO: 13 is the amino acid sequence of Hole-Fc-Linker 1-IL-15 chain.

[0390] SEQ ID NO: 14 is the amino acid sequence of Knob-Fc-Linker 1-IL-15Rα-Sushi+ chain.

[0391] SEQ ID NO: 15 is the amino acid sequence of IL-15-Linker 4-Hole-Fc chain.

[0392] SEQ ID NO: 16 is the amino acid sequence of IL-15Rα-Sushi+-Linker 4-Knob-Fc chain.

[0393] SEQ ID NO: 17 is the amino acid sequence of Knob-Fc-Linker 2-IL-15Rα-Sushi+ chain.

[0394] SEQ ID NO: 18 is the amino acid sequence of Hole-Fc-Linker 2-IL-15 chain.

[0395] SEQ ID NO: 19 is the amino acid sequence of IL-15-Linker 3-Hole-Fc chain.

[0396] SEQ ID NO: 20 is the amino acid sequence of Fc-linker 3-IL-15 chain.

[0397] SEQ ID NO: 21 is the amino acid sequence of IL-15-Linker 3-Fc chain.

[0398] SEQ ID NO: 22 is the amino acid sequence of Knob-Fc-Linker 2-IL-15Rα-Sushi+ chain.

[0399] SEQ ID NO: 23 is the amino acid sequence of Fc-linker 2-IL-15Rα-Sushi+ chain.

[0400] SEQ ID NOs: 24-45 are the amino acid sequences of various IL-15 variant polypeptides.

[0401] SEQ ID NO: 46 is the amino acid sequence of Fc-linker 3-IL-15S58D chain.

[0402] SEQ ID NO: 47 is the amino acid sequence of the peptide linker.

[0403] SEQ ID NO: 48 is the amino acid sequence of Hole-Fc-Linker 3-IL-15-S58D chain.

[0404] SEQ ID NO: 49 is the amino acid sequence of IL-15-S58D-Linker 3-Hole-Fc chain.

[0405] SEQ ID NO: 50 is the amino acid sequence of IL-15-S58D-Linker 3-Fc chain.

[0406] SEQ ID NO: 51 is the amino acid sequence of IL-15Rα-Sushi+Linker 2-Knob-Fc chain.

[0407] SEQ ID NO: 52 is the amino acid sequence of IL-15Rα-Sushi+Linker 2-Fc chain.

[0408] SEQ ID NO: 53 is the amino acid sequence of Hole-Fc-Linker 1-IL-15-S58D chain.

[0409] SEQ ID NO: 54 is the amino acid sequence of Hole-Fc-Linker 3-IL-15 chain.

[0410] SEQ ID NO: 55 is the amino acid sequence of Knob-Fc-Linker 1-IL-15 chain.

[0411] SEQ ID NOs: 56-63 are nucleotide sequences encoding various IL-15 / IL-15Rα-Fc fusion chains.

[0412] SEQ ID NOs: 64 and 65 are the amino acid sequences of the two polypeptide chains of interest.

[0413] Sequence Listing

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

Claims

1. An isolated interleukin-15 (IL-15) fusion protein complex, comprising: (1) an IL-15 polypeptide covalently linked to an Fc domain via a peptide linker; and (2) an IL-15 receptor α (IL-15Rα) domain non-covalently linked to the IL-15 polypeptide, wherein the IL-15 polypeptide is an IL-15 variant polypeptide of SEQ ID NO: 2 in which S at position 58 is substituted with D; wherein the IL-15Rα domain is an IL-15 receptor α Sushi (IL-15RαSushi) domain consisting of the amino acid sequence set forth in SEQ ID NO:

5.

2. The isolated IL-15 fusion protein complex of claim 1, wherein the IL-15 polypeptide is covalently linked to the C-terminus of the Fc domain.

3. The isolated IL-15 fusion protein complex of claim 1, wherein the IL-15 polypeptide is covalently linked to the N-terminus of the Fc domain.

4. The isolated IL-15 fusion protein complex according to any one of claims 1 to 3, wherein the Fc domain is selected from the group consisting of a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, an IgA Fc domain, an IgD Fc domain, an IgE Fc domain, an IgG Fc domain, and an IgM Fc domain. 5 . The isolated IL-15 fusion protein complex according to claim 1 , wherein each Fc domain is an Fc domain having a silent effector function and / or having a half-life extending function.

6. The isolated IL-15 fusion protein complex according to any one of claims 1 to 3, wherein the amino acid sequence of the Fc domain is selected from the group consisting of the amino acid sequences set forth in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO:

8.

7. The isolated IL-15 fusion protein complex according to claim 1, wherein the peptide linker is selected from the group consisting of sequences listed in SEQ ID NOs: 9-12.

8. An isolated IL-15 fusion protein complex, comprising: (1) two IL-15 polypeptides, the two IL-15 polypeptides being covalently linked to two Fc domains via a peptide linker; and (2) two IL-15Rα domains, the two IL-15Rα domains being non-covalently linked to each IL-15 polypeptide, wherein the IL-15 polypeptide is an IL-15 variant polypeptide, the IL-15 variant polypeptide being SEQ ID NO: 2 in which S at position 58 is substituted with D; wherein the IL-15Rα domain is an IL-15 receptor αSushi (IL-15RαSushi) domain, the IL-15RαSushi domain consisting of the amino acid sequence set forth in SEQ ID NO:

5.

9. The isolated IL-15 fusion protein complex of claim 8, wherein the two IL-15 polypeptides are covalently linked to the C-termini of the two Fc domains.

10. The isolated IL-15 fusion protein complex of claim 8, wherein the two IL-15 polypeptides are covalently linked to the N-termini of the two Fc domains.

11. The isolated IL-15 fusion protein complex according to any one of claims 8 to 10, wherein the Fc domain is selected from the group consisting of a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, an IgA Fc domain, an IgD Fc domain, an IgE Fc domain, an IgG Fc domain, and an IgM Fc domain.

12. The isolated IL-15 fusion protein complex according to any one of claims 8 to 10, wherein each Fc domain is an Fc domain having a silent effector function and / or having a half-life extending function.

13. The isolated IL-15 fusion protein complex according to any one of claims 8 to 10, wherein the amino acid sequence of the Fc domain is selected from the group consisting of the amino acid sequences set forth in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO:

8.

14. The isolated IL-15 fusion protein complex according to claim 8, wherein the peptide linker is selected from the group consisting of sequences listed in SEQ ID NOs: 9-12.

15. A pharmaceutical composition comprising the isolated IL-15 fusion protein complex according to any one of claims 1 to 14 in admixture with a pharmaceutically acceptable carrier.

16. Use of the pharmaceutical composition of claim 15 in the preparation of a medicament for treating colon cancer or colon cancer metastasis in a subject.

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